The Subfertility Handbook: A Clinician's Guide

This helpful and practical handbook will be an essential resource for all clinicians dealing with problems of subfertility. It provides a comprehensive approach to infertility, starting with the initial history taking and dealing with the diagnostic techniques of ultrasound and surgery, as well as all forms of therapy, incuding ovulation induction, infertility surgery, andrology, donor insemination and reproductive technologies. The authors are experienced members of the Monash University affiliated Reproductive Medicine Clinics - one of the world's pioneering centres in IVF technology. They have distilled a wealth of expertise and 'hands on' experience in this area to provide an invaluable guide to all those involved in the treatment of infertility, from gynaecologists to physicians and postgraduate students specializing in reproductive medicine.

The subfertility handbook: a clinician's guide

Special thanks to Lee McLaren for her help with the preparation of the typescript. (G.T.K.)

The subfertility handbook: a clinician's guide Edited by

GABORT. KOVACS Department of Obstetrics and Gynaecology, Box Hill Hospital, Monash University, Melbourne

CAMBRIDGE UNIVERSITY PRESS

PUBLISHED BY THE PRESS SYNDICATE OF THE UNIVERSITY OF CAMBRIDGE

The Pitt Building, Trumpington Street, Cambridge CB2 1RP, United Kingdom CAMBRIDGE UNIVERSITY PRESS

The Edinburgh Building, Cambridge CB2 2RU, United Kingdom 40 West 20th Street, New York, NY 10011-4211, USA 10 Stamford Road, Oakleigh, Melbourne 3166, Australia ©Cambridge University Press 1997 This book is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 1997 Printed in the United Kingdom at the University Press, Cambridge Typeset in Times 11/14 pt [VN] A catalogue recordfor this book is available from the British Library Library of Congress Cataloguing in Publication data The subfertility handbook: a clinician's guide /edited by Gabor T. Kovacs. p. cm. ISBN 0-521-56016-0 (hb) 1. Infertility-Handbooks, manuals, etc. I. Kovacs, Gabor, MRCOG, FRACOG. [DNLM: 1. Infertility-diagnosis. 2. Infertility-therapy. WP570S941 1997] RC889.S83 1997 618.1'78-dc21 DNLM/DLC for Library of Congress 97-9495 CIP Every effort has been made in preparing this book to provide accurate and up-to-date information which is in accord with accepted standards and practice at the time of publication. Nevertheless, the authors, editors and publisher can make no warranties that the information herein is totally free from error, not least because clinical standards are constantly changing through research and regulation. The authors, editors and publisher therefore disclaim all liability for direct or consequential damages resulting from the use of the material contained in this book. The reader is strongly advised to pay careful attention to information provided by the manufacturer of any drugs or equipment that they plan to use. ISBN 0 521 56016 0 hardback

Contents

List of contributors

ix

1 Introduction Jim Tsaltas

1

2

The first interview with an infertile couple Nick Lolatgis and Gabor T. Kovacs

9

3

Assessment of the female partner Bruce Downing

17

4

Assessment of the male partner Peter J. Fuller and Henry G. Burger

40

5 Treatment options for male subfertility Gordon Baker and David M. de Kretser

50

6

Management of the woman with chronic anovulation Anthony Lawrence and David Healy

69

7

Cervical factor, unexplained subfertility and artificial insemination with husband sperm Gabor T. Kovacs and Beverley Vollenhoven

83

In-vitro fertilization: indications, stimulation and clinical techniques James McK. Talbot and Mark Lawrence

88

8

vn

viii

List of contents 9

The role of gamete intrafallopian transfer Carl Wood

10 The use of assisted reproductive technology for the treatment of male infertility Robert I. McLachlan

109

124

11 The use of donor insemination Gabor T. Kovacs

139

12 The donor egg programme John Leeton

151

13 Endometriosis Jim Tsaltas and David Healy

163

14 The role of ultrasound in subfertility Victor Hurley and Maria Leoni

176

15 The role of surgery in infertility Carl Wood

187

16 Laboratory techniques Peter Jackson and Jennifer Burden

220

17 The results of assisted reproductive technology Vivien MacLachlan

235

18 Infertility counselling Janet Anderson and Rita Alesi

249

Index

269

Contributors

Rita Alesi

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Janet Anderson

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Gordon Baker

Prince Henry's Institute of Medical Research Reproductive Medicine Clinic Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168

Jennifer Burden

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Henry G. Burger

Prince Henry's Institute of Medical Research Reproductive Medicine Clinic Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 David M. de Kretser

Prince Henry's Institute of Medical Research Reproductive Medicine Clinic Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168

IX

List of contributors Bruce Downing

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Peter J. Fuller

Prince Henry's Institute of Medical Research Reproductive Medicine Clinic Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 David L. Healy

Department of Obstetrics and Gynaecology Monash University Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 Victor Hurley

Monash Ultrasound for Women 89 Bridge Road Richmond Victoria Australia 3121

Peter Jackson

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Gabor T. Kovacs

Department of Obstetrics and Gynaecology Level 3, Box Hill Hospital Nelson Road Box Hill Melbourne Victoria Australia 3128 Anthony Lawrence

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Mark Lawrence

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 John Leeton

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121

List of contributors Maria Leoni

Monash Ultrasound for Women 89 Bridge Road Richmond Victoria Australia 3121 Nick Lolatgis

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Vivien MacLachlan

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121 Robert I. McLachlan

Prince Henry's Institute of Medical Research Reproductive Medicine Clinic Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 James McK. Talbot

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121

XI

Jim Tsaltas

Department of Obstetrics and Gynaecology Monash University Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 Beverley Vollenhoven

Department of Obstetrics and Gynaecology Monash University Monash Medical Centre 246 Clayton Road Clayton Victoria Australia 3168 Carl Wood

Monash IVF 89 Bridge Road Richmond Victoria Australia 3121

1 Introduction JIMTSALTAS

Infertility is a problem that affects many couples. Most adults have life plans that include children. When efforts to have children are unsuccessful, feelings of helplessness, frustration and despair are common. It is often at this point that many couples seek help from a Clinician. The Clinician's role is not only to help define the couple's problem, but also to be sympathetic and considerate to their emotional needs at this most difficult time. An understanding of the epidemiology, as well as of the historical aspects of the treatment of infertility, will be extremely helpful in achieving this goal. Infertility is defined as the state in which a couple, desirous of a child, cannot conceive after 12 months of unprotected intercourse (Mueller and Daling, 1989; Thonneau et al, 1991). This is taken as being abnormal as 90% of couples will conceive within that time (Tietze, 1956; 1968). Infertility is either primary, when no pregnancy has ever occurred, or secondary, where there has been a pregnancy, regardless of the outcome (Thonneau et al, 1991). The ratio of patients presenting with primary and secondary infertility has remained remarkably stable with 67-71% of patients with infertility presenting with primary infertility and 29-33% presenting with secondary infertility (Hull et al, 1985; Templeton, Fraser and Thompson, 1991; Thonneau etal, 1991). To understand infertility better, it is important to appreciate the epidemiological term, fecundity (Spira, 1986; Jansen, 1993). The fecundity of a couple is measured by their fecundability, i.e. the monthly probability of conception without the use of any contraception (Jansen, 1993). A couple is subfecund when there is an involuntarily long interval between births or until first conception. When the fecundability of the couple is zero, for whatever reason, the couple is infecund, or sterile (Jansen, 1993). The rate of sterility is believed to be 3-5% but the true incidence is unknown (Spira, 1986; Jansen, 1993). The reason for this may be that more couples have been inquiring about

2

J. Tsaltas

infertility and requesting medical assistance in order to have a child. There has been no general increase in the prevalence of infertility, and the increase in demand for treatment appears to have been generated by greater expectations (Aral and Cates, 1983; Templeton, Fraser and Thompson, 1991). This in turn has been partly generated by the increased media focus on the new medical procedures and technologies available for achieving pregnancy. The fecundability rate amongst young couples who discontinue contraception in order to become pregnant is quoted to range from 25% to 36% (Cramer, Walker and Schiff, 1979; Harlap and Baras, 1984; Spira, 1986). For many years, the incidence of infertility was thought by many to be 10%, but a number of excellent recent studies give a clear indication as to the true figure, which ranges from 13.5% to 18.4% (Hull et al, 1985; Thonneau et al, 1991), and translates to one in seven women. These studies have demonstrated that infertility is a real health problem because of its prevalence (Thonneau et al, 1991). Clinicians, both in general practice and in specialist gynaecological practice, will regularly encounter couples presenting with these problems. In women, the main causes of infertility - accounting for 50% of cases - are ovulation disorders and tubal damage (Spira, 1986; Thonneau et al, 1991). Some of the rarer causes of infertility include endometriosis, hyperprolactinaemia and genital tract disorders (Thonneau et al, 1991). In men, the main causes of infertility are oligospermia, asthenozoospermia, teratospermia and azoospermia, which account for 20-25% of cases (Thonneau et al, 1991). In 18% of couples, no cause is found (Spira, 1986), and 3% of couples in this group will conceive each month without assisted reproductive techniques (Barnea, Holford and Mclnnes, 1989; Hull et al, 1985; Deaton et al, 1990). Combined causes of infertility can be found in 10-30% of couples (Hull et al, 1985; Thonneau et al, 1991; Jones and Toner, 1993). It is therefore very important to investigate both partners, and one must never assume that infertility is exclusively a female or a male problem. Female risk factors of infertility

A number of factors have been identified as increasing the risk of becoming infertile, especially in women. Two of the major risk factors associated with female infertility are pelvic inflammatory disease and sexually transmitted diseases. The number of reported cases of the former has been increasing over the past two decades (Curran, 1980; Westrom, 1980). Damaged fallopian tubes are the major cause of infertility, and two organisms, Neisseria gonorrhoea and Chlamydia trachomatis, are responsible for the majority of cases

Introduction

3

(Mueller and Daling, 1989). The risk of becoming infertile increases with each successive episode of pelvic inflammatory disease and increases with the severity of each episode (Westrom, 1975; 1980). Pelvic inflammatory disease can be a silent process, particularly when due to Chlamydia trachomatis. Its long term effects are the same as pelvic inflammatory disease, which is symptomatic and requires medical intervention (Rosenfeld et ai, 1983). The risk of pelvic inflammatory disease is increased in women who contract a sexually transmitted disease when using an IUD. Pelvic surgery, including surgery for appendicitis (Mueller and Daling, 1989), is associated with an increased risk of infertility (Thonneau et ah, 1991), although this risk may be reduced by the increasing use of laparoscopic surgery which has a lower postoperative adhesion rate. Ectopic pregnancy is associated with an increased risk of tubal infertility (Mueller et ah, 1987), but this may be due to the pre-existing conditions that lead to the ectopic pregnancy rather than to the surgery itself. Induced abortion, if followed by infection, may lead to tubal disease and infertility. However, in well-designed studies, there appeared to be no increased risk of tubal infertility in a population for whom abortions were performed safely under appropriate sterile conditions with proper medical supervision (Daling et ah, 1985). Cigarette smoking is becoming more prevalent amongst teenage girls and young adult women. Recent controlled studies suggest that women who smoke have an increased risk of infertility due to tubal disease and to abnormalities of cervical mucus (Phipps, Cramer and Schiff, 1987). The relationship between age and fertility is not so clear cut. Many women are now delaying the age at which they have theirfirstchild, and it appears that fertility may reduce with increase in age, particularly for women over 35 years (Spira, 1986). Male risk factors of infertility

There are also a number of risk factors associated with male infertility. Sexually transmitted diseases are an important risk factor for male as well as for female infertility, most commonly those caused by Neisseria gonorrhoea and Chlamydia trachomatis. These organisms can cause changes in semen quality and, if left untreated, an infection may result in blockage of the vas deferens or seminal vesicles (Megory et al., 1987). Mumps orchitis is rare in men in their reproductive years, but approximately 30% of men with orchitis will become azoospermic. The presence of a varicocele, a history of inguinal hernia surgery, and vesicular damage due to torsion or trauma may all lead to infertility. The relationship of male infertility with cigarette smoking is controversial. There are conflicting reports from studies, some showing an increased

4

J. Tsaltas

risk of infertility (Wentz, 1986) and others showing no adverse effects from smoking (Vogt, Heller and Borelli, 1986). Emotional experiences and infertility

Infertility is a major life crisis for most couples. People usually assume that they are fertile and when they want to conceive, they will be able to. The emotional experience passes through several phases, beginning with disbelief and denial, moving into frustration and, only after a considerable time, to acceptance (Jones and Toner, 1993). The physician who sees couples with infertility needs to communicate to them an awareness of their emotional crisis. It is difficult to ascertain from the literature what couples' expectations are, but from our own experience at Monash IVF, we find them to be extremely high. With constant media coverage of medical 'miracles' - test tube babies - most couples expect instant success. It is important that the treating physician, in conjunction with a specially trained counsellor, goes through both the positive aspects of assisted reproductive technology as well as some of the negative aspects with which each couple may have to deal. Couples will feel pressure, frustration, and often loneliness as their friends begin to have children. They feel that they are failures. They become tired, feel shocked and dismayed. Issues in the household often take on a greater importance than they would in 'normal' circumstances when couples are not under this stress. Couples may also feel a loss of control, particularly when enrolled in an in-vitro fertilization (IVF) programme. With time and counselling, a number of these hurdles can be overcome. Couples become more realistic, they often show a greater ability to empathize with other people's problems and realize that not every aspect can be controlled (Stens, 1989; Lewis, 1989; Goldman, 1989; Bohm, 1989; Stuart, 1989; Winkler, 1989; Lene, 1989; Oberauer, 1989; Kozolanka, 1989; Domar etai, 1992). Historical aspects of infertility: A brief overview

The emotional rollercoaster which is associated with infertility has been with us for as long as the problem has existed. The treatment of this problem, however, has changed dramatically over the past 50 years, and many of these changes have culminated in the modern techniques used for assisted reproduction. It has been in the second half of this century that the most dramatic advances in the management of the infertile couple have been made. Even in the 1950s, certain clinicians recognized the contributions that veterinary medicine could make to the field of human reproduction. At this time, there were

Introduction

5

four separate areas of progress in the management of infertility. Firstly, the ability to diagnose tubal patency (Rubin, 1950); secondly, the study of spermatozoa, which included the establishment of standards of normal semen analysis, artificial insemination using donated or husband's sperm and cryopreservation of sperm (Page and Houlding, 1951). The first guidelines were set down for donor insemination, and involved using donors who were syphilis negative, free of disease and whose mental behaviour was of unquestionable normality (Shields, 1950). The third area of progress involved the increase in the state of knowledge regarding sex hormones and, finally, there was an improvement in the methods used to determine the timing of ovulation. The phenomenon of Spinbarkeit was described and was used to determine ovulation and to improve the results of artificial insemination (Cohen, Stein and Kay, 1956). The first wedge resections of polycystic ovaries were described (Stein and Leventhal, 1935; Stein and Cohen, 1939) and gonadotrophins were extracted from a human menopausal era. In the 1970s, artificial insemination using frozen, banked, donor sperm steadily became more widely practised. In 1951, Hellman described delicate instruments and suture materials to aid in tubal surgery, and these were widely adapted in the 1960s. However, these techniques were not to become successful until the 1970s, when microsurgical techniques were established. Microsurgical techniques which had initially been developed for the repair of blood vessels, were applied to tubal surgery. Research began into applying these techniques to tubal anastomoses with significant improvements on the previously poor results (Paterson and Wood, 1974; Gomel, 1977). At the same time, laparoscopy was being used for diagnostic purposes, and it was really at this time that the era of minimally invasive surgery began (Smith and Dillon, 1970). All these changes occurred virtually hand-in-hand with the development of IVF, and dramatically lessened the need for tubal reconstructive surgery. The first successful live birth through IVF was reported by Steptoe and Edwards in 1978. At the same time as all of these new reproductive technologies were developing, marked advances were being made in laboratory techniques as radio-immunoassays were developed for human chorionic gonadotrophin (hCG) and luteinizing hormone (LH) as well as oestrogen. Radio-immunoassay to detect prolactin was also developed and the aetiology of the amenorrhoea-galactorrhoea syndrome became clear. It was at this stage that bromocriptine also became available and revolutionized treatment (Jewelewicz and Zimmerman, 1978). Gonadotrophin-releasing hormone (GnRH) was also identified in the early 1970s, and is now one of the most commonly used drugs for ovulation induction and its analogues for ovarian suppression.

6

J. Tsaltas

In the 1980s, there was a surge in the technological advance for the clinical practice of endocrinology and infertility. During this decade, IVF became a practical and available treatment for infertility. Initially, oocyte retrieval was performed laparoscopically, but this has now been superseded by transvaginal ultrasound guided oocyte pick up. Other techniques such as gamete intrafallopian transfer (GIFT) and IVF using natural cycles were developed and now offer more options to the IVF patient. The last 50 years of progress in managing the infertile couple have provided options where no treatment would have been available in the past. The development of procedures, techniques, drugs and laboratory assays which has occurred during this time has laid the foundation for modern fertility management. Help can now be offered where none was available before (Chen and Wallach, 1994).

Conclusion

Infertility is such a common problem that nearly every general practitioner and gynaecologist will be confronted by it. An understanding of its prevalence and epidemiology, as well as an appreciation of the emotional strain that couples experience during this difficult time, are important. They will not only help with the medical aspects of treatment, but will allow patients to be treated with respect, dignity and kindness through this extremely difficult and stressful period in their lives. References Aral, S.O. and Cates, W. 1983. The increasing concern with infertility. Journal of the American Medical Association 250: 2327-31. Barnea, E.R., Holford, T.R. and Mclnnes, D.R.A. 1989. Longterm progress of infertile couples with normal basic investigations: A life table analysis. Obstetrics and Gynecology 66: 24-6. Bohm, C. 1989. The insemination circus. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 75-81. London: Pandora Press. Chen, S.H. and Wallach, E.E. 1994. Five decades of progress in management of the infertile couple. Fertility and Sterility 62: 665-85. Cohen, M.R., Stein, I.F.S. and Kay, B.M. 1956. Optional time for therapeutic insemination: Spinnbarkeit as the preferred criterion. Fertility and Sterility 7: 141-54. Cramer, D.W., Walker, A.M. and Schiff, I. 1979. Statistical methods in evaluating the outcome of infertility therapy. Fertility and Sterility 32: 80-6. Curran, J. 1980. Economic consequences of pelvic inflammatory disease in the United States. American Journal of Obstetrics and Gynecology 7: 848-51. Daling, J.R., Weiss, N.S., Vogt, L., Spadoni, L.R., Soderstrom, R., Moore, D.E. and

Introduction

1

Studel, B.V. 9185. Tubal infertility in relation to prior induced abortion. Fertility and Sterility 43: 389-94. Deaton, J.L., Gibson, M., Blackmer, K.M., Nakajima, S.T., Budger, G.J. and Brunsted, J.R. 1990. A randomized, controlled trial of clomiphene citrate and intrauterine insemination in couples with unexplained infertility or surgically corrected endometriosis. Fertility and Sterility 54: 1083-8. Domar, A. D., Seibel, M., Broome, A., Friedman, R. and Zuttermeister, P.C. 1992. The prevalence and predictability of depression in infertile women. Fertility and Sterility 58: 1158-63. Goldman, A. 1989. The production of eggs and the will of God. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 67-74. London: Pandora Press. Gomel, V. 1977. Tubal reanastomosis by microsurgery. Fertility and Sterility 28: 59-65. Harlap, S. and Baras, M. 1984. Conception - waits in fertile women after stopping oral contraceptives. International Journal of Fertility 29: 73-80. Hellman, L.M. 1951. The use of polyethylene in human tubal plastic operations. Fertility and Sterility 2: 498-504. Hull, M.G.R., Glazener, C.M.A., Kelly, N.J., Conway, D.I., Foster, P.A., Hinton, R.A., Coulson, C , Lambert, P.A., Watt, E.M. and Desai, K.M. 1985. Population study of causes, treatment, and outcome of infertility. British Medical Journal 291: 1693-7. Jansen, R.P.S. 1993. Relative infertility: Modelling clinical paradoxes. Fertility and Sterility 59: 1041-5. Jewelewicz, R. and Zimmerman, E.A. 1978. Current management of the amenorrhoea-galactorrhoea syndrome. Fertility and Sterility 29: 597-603. Jones, H.W. and Toner, J.P. 1993. The infertile couple. The New England Journal of Medicine 329: 1710-15. Kozolanka, K. 1989. Giving up: The choice that isn't. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 121-9. London: Pandora Press. Lene, K. 1989. We are not just eggs but human beings. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 101-110. London: Pandora Press. Lewis, M. 1989. Nowhere for me to be. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 19-27. London: Pandora Press. Megory, E., Zuckerman, H., Shohom, Z. and Lunenfeld, B. 1987. Infection and male fertility. Obstetric and Gynecology Survey 42: 283-90. Mueller, B.A. and Daling, J.R. 1989. Epidemiology of infertility. Extent of the problem - risk factors and associated social changes. In Controversies in Reproductive Endocrinology and Infertility, ed. M.R. Soules, pp. 1-13. New York: Elsevier. Mueller, B.A., Daling, J.R., Weiss, N.S., Moore, D.E., Spadoni, L.R. and Soderstrom, R.M. 1987. Tubal pregnancy and the risk of subsequent infertility. Obstetrics and Gynecology 69: 722-6. Oberauer, B. 1989. Baby making in Australia. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 111-20. London: Pandora Press. Page, E.W. and Houlding, F. 1951. The clinical interpretation of 1000 semen analyses

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/. Tsaltas

assay applicants for sterility studies. Fertility and Sterility 2: 140-51. Paterson, P. and Wood, C. 1974. The use of microsurgery in the reanastomosis of the rabbit fallopian tube. Fertility and Sterility 25: 757-61. Phipps, W.R., Cramer, D.W. and Schiff, I. 1987. The association between smoking and female infertility. Fertility and Sterility 48: 377-82. Rosenfeld, D.L., Seidman, S.M., Bronson, R.A. and Scholl, G.M. 1983. Unsuspected chronic pelvic inflammatory disease in the infertile female. Fertility and Sterility 39: 44-8. Rubin, I.C. 1950. Thirty years of progress in beating infertility. Fertility and Sterility 1: 389-406. Shields, F.E. 1950. Artificial insemination as related to the female. Fertility and Sterility 1:271-80. Smith, B.D. and Dillon, T.F. 1970. Laparoscopy. Fertility and Sterility 21: 193-200. Spira, A. 1986. Epidemiology of human reproduction. Human Reproduction 1: 11115. Stein, I.F. and Cohen, M.R. 1939. Surgical treatment of bilateral polycystic ovaries amenorrhoea and sterility. American Journal of Obstetrics and Gynecology 38: 465-80. Stein, I.F. and Leventhal, M.L. 1935. Amenorrheoa associated with bilateral polycystic ovaries. American Journal of Obstetrics and Gynaecology 29: 181-91. Stens, K. 1989. Give me children, or else I die. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 11-18. London: Pandora Press. Steptoe, P.C. and Edwards, R.G. 1978. Birth after the re-implantation of a human embryo. (Letter.) Lancet 2: 336. Stuart, A. 1989. Is it, want it? I just don't know. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 82-89. London: Pandora Press. Templeton, A., Fraser, C. and Thompson, B. 1991. Infertility - epidemiology and referral practice. Human Reproduction 6: 1391—4. Thonneau, P., Marchard, S., Tallee, A., Ferial, M., Ducot, B., Lansac, J., Lopes, P., Tabaste, J. and Spira, A. 1991. Incidence and main courses of infertility in a resident population (1850000) of three French regions (1988-1989). Human Reproduction 6: 811-16. Tietze, C. 1956. Statistical contributions to the study of human fertility. Fertility and Sterility 1:^-95. Tietze, C. 1968. Fertility after the discontinuation of intrauterine and oral contraception. International Journal of Fertility 13: 385-9. Vogt, H-J., Heller, H.D. and Borelli, S. 1986. Sperm quality of healthy smokers, ex-smokers, and never-smokers. Fertility and Sterility 45: 106-10. Wentz, A.C. 1986. Cigarette smoking and infertility. Fertility and Sterility 46: 365-7. Westrom, L. 1975. Effect of acute pelvic inflammatory disease on fertility. American Journal of Obstetrics and Gynecology 121: 707-13. Westrom, L. 1980. Incidence, prevalence and trend of acute pelvic inflammatory disease and its consequences in industrialised countries. American Journal of Obstetrics and Gynecology 138: 880-92. Winkler, U. 1989. He called me number 27. In Infertility: Women Speak Out about their Experiences of Reproductive Medicine, ed. Renate D. Klein, pp. 90-100. London: Pandora Press.

2 The first interview with an infertile couple NICK LOLATGIS and GABOR T. KOVACS

The first principle in consultations for subfertility is that the problem should be considered a 'couple problem' and the couple should be seen together if at all possible. Secondly, it should be remembered that couples often feel very stressed at this time. They also often feel that it is not fair that they should be singled out with a fertility problem whereas all their friends manage to conceive with ease. There are often pressures from parents, especially as siblings may be producing grandchildren. It is therefore important for the clinician to be understanding during the consultation. A good starting point is to inquire about the couple's occupations. This is an area about which they both know more than the doctor, and therefore can discuss it confidently. Having broken the ice, one should then move on to the general history, such as for how long they have been having unprotected intercourse, their ages, and any previous history of fertility. It is then logical to concentrate on the female partner's medical/menstrual history. Her age of menarche, initial cycles, the use of the oral contraceptive pill, other methods of contraception, and subsequent and current menstrual cycle lengths, and the duration of menstruation should all be recorded. Any abnormal intermenstrual bleeding should be identified, for example endometriosis is often associated with premenstrual spotting. Other symptoms of ovulation such as mid-cycle pain or spotting, and premenstrual symptoms should be asked about. Regular painful cycles, especially if associated with premenstrual symptoms, usually suggest ovulatory cycles. It is also worthwhile inquiring whether the woman experiences the mucus changes associated with ovulatory cycles: classically the 'dry' days just after menstruation, with the quantity of mucus increasing until just prior to ovulation, when the secretions are copious and egg-whitey. Just after ovulation, the secretions dramatically change and become less stretchy and opaque like glue.

10

N. Lolatgis and G. T. Kovacs

Other relevant points in the female's history include any previous pregnancies, miscarriages or terminations. Her past medical history and surgical history should also be ascertained, especially any history of pelvic inflammatory disease or appendicitis, or surgery on pelvic organs. Any medications and allergies as well as the family history are also relevant. The woman's current weight and any change (10% or more within the last 12 months) should be noted. The body mass index (weight in kilograms divided by height in metres squared) should be calculated. Details of smoking, alcohol intake and exercise pattern complete the female history. With respect to the male partner, the history is less complicated. Again, occupation needs to be explored, with the possibility of any noxious influences. Any history of surgery on the testes or scrotum, infections or trauma medications, as well as alcohol, nicotine or other drug intake need to be identified. Questions should also be asked about family history, physical activity and social habits. Finally, the couple's coital habit needs to be explored. It may be best to leave this to the end of the history taking, by which time they have got to know the clinician a bit better. It is best to commence with frequency of intercourse, especially determining if timing may coincide with ovulation, but this is often difficult with hindsight, and may be best done prospectively with a basal temperature chart. Then one should inquire about the quality of intercourse. It should be ascertained whether the male has any difficulty with erections, whether penetration is difficult or painful, the use of any lubricants, and the adequacy of ejaculation. If excessive semen is lost after intercourse, this may suggest inadequate penetration. Generally women say that they have to insert a tissue into their panties afterwards; loss of larger quantities of semen is excessive. One should then proceed with examination. For the female, a general examination including blood pressure, breast and abdomen should be performed, and any abnormalities noted. Particular care should be taken to note any abdominal scars. The vulva and vagina should be inspected before a bimanual examination to size the uterus and note any abnormalities or tenderness. Any nodules in the pouch of Douglas or afixedretro verted uterus would suggest endometriosis. Cervical cytology should also be performed. Some clinicians would take cervical swabs to detect the presence of Chlamydia trachomatis, and sometimes for mycoplasma/ureaplasma, although the value of the first and the significance of the latter two are uncertain. When examining the male, one should concentrate on his genitalia. The size of the testicles, presence of vas, and the detection of any swellings such as

11

The first interview with an infertile couple Jan.

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Figure 2.1

Basal body temperature charts.

hernia or varicose veins should be recorded. Examination of the prostate for size and tenderness may be indicated. At the conclusion of the history and examination of both partners, the couple should be a little more comfortable, and time should be taken to explain what is involved with conception, and what the management plan will

12

N. Lolatgis and G. T. Kovacs

Table 2.1. Instructions for recording temperature Charting your temperature is useful in that it can indicate if ovulation has occurred. The first day of your period is Day 1 of your cycle. You will need: a thermometer - mercury or digital a temperature chart a clock a pen. How to take your temperature.

Mercury thermometer 1. Make sure you shake down the mercury the night before. 2. Take your temperature at about the same time each morning. 3. When you wake in the morning, before getting out of bed, talking, or having a cup of tea, TAKE YOUR TEMPERATURE. Put the thermometer in your mouth, under your tongue, and leave it there for three minutes. 4. After three minutes, remove the thermometer, note the reading, and mark it on your temperature chart. Digital thermometer Follow instructions 3 and 4 from above but remove it when it bleeps. Note: If you have had a restless night, make sure that you have been asleep a minimum of two hours before taking your temperature. 5. Mark your temperature with a dot (•) in the middle of the relevant square of temperature on the vertical scale and date on the horizontal scale. 6. Join the consecutive dots with a line. 7. If you miss a day, leave that day blank. 8. On each day of bleeding place an X in the third top square. Any bleeding between periods should be recorded with a 'B' and spotting with an 'S'. 9. On days when you have intercourse place a circle (O) around the dot.

be. It is helpful to explain the female anatomy (the vagina, uterus, tubes and ovaries) and that there are three main fertility parameters. First, the right number of normal sperm have to be placed in the right place at the correct time; secondly, oocytes (eggs) have to be released; and thirdly, the female organs have to be healthy enough to collect the egg from the ovary and transport it downwards, to facilitate the passage of sperm from the vagina to

Thefirstinterview with an infertile couple the fallopian tube, to aid fertilization, and then to transport the early embryo to the uterine cavity. To investigate these three parameters, we would recommend starting a basal body temperature chart (Figure 2.1, Table 2.1) to indicate if and when ovulation occurs, as well as the timing of intercourse. We would request a serum progesterone in the estimated mid-luteal phase, together with a prolactin estimation. In the absence of regular periods, serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels should also be measured, and thyroid function tests should be performed. Rubella immunity should also be confirmed when blood tests are being taken. The male partner should be requested to have a semen analysis, following two to three days of abstinence, on a masturbated specimen delivered promptly to the laboratory. The semen analysis is discussed in detail in Chapter 4. If ovulation appears to be occurring and the semen quality is reasonable, with adequate timing of intercourse, the next assessment should be that of the female pelvis. Although a pelvic ultrasound examination may give some information, a laparoscopy is usually indicated. The other alternative, of X-ray visualization of the tubes by hysterosalpingography, is rarely carried out. Falloposcopy, a relatively recent technique to visualize the inside of the fallopian tubes, may also be recommended. These techniques are discussed in more detail in Chapter 3. This sort of management plan usually aids a couple with subfertility. They may have further questions, and should be encouraged to write these down and bring them back to the next appointment. The results of investigations would be discussed at the next interview and, depending on the findings, a management plan decided upon. Further reading

Wood, E.C. and Kovacs, G.T. 1996. Infertility: All your Questions Answered. Melbourne: Hill of Content.

13

14

N. Lolatgis and G. T. Kovacs Appendix A U.R.! Given Names

MONASH MEDICAL CENTRE Surname

FEMALE INFERTILITY ASSESSMENT

AGE

LZU

Date of Birth

Age

Medical Officer

I |

HEIGHT

Religion

Sex

Marital Status

II II

BODY MASS INDEX I

INFERTILITY: Primary

Duration

I months

PAST OBSTETRIC HISTORY:.

MENSTRUAL HISTORY: Menarche Menstrual I I Bleeding

I

I

Regularity 'remenstru I Premenstrual Symptoms

Dysmenorrhoea

I

P.I.D.

TUBAL FACTORS: I.U.D. Abdominopelvic Surgery

>

GENERAL FACTORS: Medical Disease Employment/Environmental Factors COITAL HISTORY: Frequency

|

. Smoker Knowledge of I fertile period I

Dyspareunia

I I

m

z

Tl

m

CONTRACEPTIVE HISTORY:

3D EXAMINATION FINDINGS: General Examination .

Secondary sexual characteristics:

Breast Development

>

to to

Pelvic Examination

m

Basal body INVESTIGATIONS: Temperature Chart

Oestradiol L.H.

LZH

FINDINGS: Ovulatory status . Documented by Tubal Assessment Documented by

L

Male Factor: Yes L J

No L J

MANAGEMENT PLAN:

Specify.

to

w

m

z

H

15

The first interview with an infertile couple

Appendix B

MALE

MEDICAL

REPRODUCTIVE MEDICINE CLINIC A JOINT ACTIVITY OF

ASSESSMENT

PHIMR

Doctor:

Date:.

URNo.:

Surname:

DOB

Partner's Surname:.

DOB

Marital status: |

| Married

Infertility:

Q

Previous fertility:

Husband:

IRD

|

| Delacto

Primary

Duration

I

| Secondary

(monlhs)

Duration.

(months)

Wile:

Past Medical history:

Diabetes Sinopulmonary Malignancy Recent pyrexia Other

• • • •

• • • •

Details:

Past Surgical history: N

Y

|

|

[

| Maldescent ol testes: L

.Treatment (yr).

|

|

|

| Varicocele ligation:

L

.Date:

|

|

|

| Vasectomy:

Date.

.Reversal:

1

I I

I Genital surgery/injury: ...

I

j I

j General surgery:

Infectious disease: N

STDQ

Q

Epididymitis |

NY

Y

N

Y

|

|

|

Orchits •

NY

•

UTI •

•

Comments: Medications/Drugs: Tobacco .

Alcohol

Environmental/occupational: Coital History: Libido Ejaculation.

Frequency: . Timing of Int.:

/wk Random |

Erections:. Fertile phase |

|

Comments:.

(cont).

16

N. Lolatgis and G. T. Kovacs

EXAMINATION: General:

Androgen status:. Genital:

Gynaecomastia:.

Penis

Scrotum .

Testes Epididymis Vasa Varicocele

Comments:.

INVESTIGATIONS:

LH:

FSH:

Testosterone:

Semen analyses (enclose if not our labs)

Testicular biopsy: Other:

CASE SUMMARY/PLAN:

DIAGNOSIS:

Q

Normal

|

Principal

1.

Secondary

1. 2. 3

| Abnormal

PHL.

3 Assessment of the female partner BRUCE DOWNING

The perception of subfertility varies with the individual and encompasses many aspects including the need for investigation and the speed of investigation. An older patient may warrant full investigation after six months of unprotected coition even though the diagnosis of infertility cannot be made until there have been 12 months of unprotected coition. A couple presenting for routine consultation may say that they wish to attempt conception yet may have a history, symptoms, or signs that strongly suggest difficulties in procreation. They should undergo appropriate investigations early. The investigation of the infertile couple requires an organized approach that encompasses andrology, endocrinology and pelvic assessment so that appropriate pathways of therapy can be planned. Counselling

The establishment of an optimal doctor-patient relationship is paramount in the management of the infertile couple. Time spent initially in understanding the patients' perceptions of their infertility in terms of their real chance of having a child of their own and in understanding past events or current lifestyle practices that they believe will inhibit their chances of pregnancy, is crucial. Assessment of the patients' understanding of their own anatomy and physiology is important, with guidance being given about appropriate references for the patients to read. High levels of stress are recognized in patients undergoing investigation and treatment for infertility (Downey et al, 1989; Wright, Duchesne and Sabourin, 1991), although men score less disturbance than their female partners on scales of anxiety, depression, hostility, cognitive disturbance, stress and self-esteem (Wright et al., 1991). Specialized infertility counsellors are now readily available, attached to most infertility clinics, and should be 17

18

B. Downing

utilized by practitioners who believe patients have unrealistic expectations, are obsessed, depressed, or allow their infertility to rule their lives. Many female patients experience the feeling of a loss of control over a very important aspect of their life - the inability lovingly to create a child, passing on their genes, the fulfilment of pregnancy and breast feeding, and the loss of the potential to be a parent. General questions that often create significant anxiety should be discussed and include the age of both the man and woman, the coital frequency, effects of cigarette smoking as well as exercise, diet and weight change, in addition to oral drugs and chemicals that may be taken. Fertility declines with the increasing age of both the man and woman, with the chance of pregnancy declining significantly between groups of women aged less than 35 as compared to those over 35 (Schwartz and Mayaux 1982). Of women younger than 25, 75% had achieved pregnancy within six menstrual cycles, whereas women older than 35 had achieved pregnancy after six menstrual cycles in only 25% of cases. McLeod and Gold (1953) reported that intercourse more than three times per week maximized the chance of conception. There is now clear evidence that smoking is associated with a reduced chance of pregnancy as well as increasing the likelihood of both ectopic pregnancy and spontaneous miscarriage (Stillman, 1989). Cigarette smoke affects the peristalsis of the fallopian tube in addition to affecting the immune system. All patients planning to embark on achieving pregnancy should be strongly encouraged to cease smoking and be referred to skilled counsellors working in this area. An association between exercise and oligo/amenorrhoea is well established, particularly in patients who have a history of irregular periods. Although there is no trial that supports exercise as a cause of infertility, there is evidence that weight change is associated with infertility. Bullen, Skrinar and Beitins (1985) showed that weight change rather than exercise was necessary for menstrual variations. There is evidence that weight loss in the obese patient correlated well with pregnancy achievement; however, a systematic approach of a multidisciplinary, non-threatening nature was required.

Clinical practices to reduce stress

Attention should be paid to the effects of stress, and techniques should be utilized to reduce it. These include: 1. outline a full management plan, 2. outline a timeframe,

Assessment of the female partner 3. 4. 5. 6.

outline all options, empower the patients to make their choice, provide educational material, explore lifestyle modification. The referring letter

This letter should contain pertinent details regarding historical facts that pertain to both the patients' infertility and other relevant medical conditions and should convey the referring doctor's assessment of relevant social and economic factors as well as the results of any investigations carried out. Medical and surgical history

A general medical and surgical history can be obtained by the patient completing a questionnaire prior to consultation, which should include the following. 1. Personal information: names, address, contact phone numbers and details regarding privacy of calls and the most appropriate time for contact. 2. Date of birth (age), marital status, occupation, religion - both birth and current - and academic background. 3. Details of health insurance and duration of current cover. 4. Address, phone, fax and e-mail numbers for the referring doctor and the patient's family doctor. 5. Consumption, both past and present, of alcohol and cigarettes. 6. General health: allergies to drugs or dressings, past history of blood transfusions or difficulties with anaesthesia/analgesia; any question of bleeding tendencies. 7. Details of any current medication including vitamin supplements (all patients hoping to achieve pregnancy should be recommended folic acid 0.5 mg. per day). 8. Any of the following diseases should be acknowledged and amplified by the patient. Cardiovascular: rheumatic fever, cardiac murmur, high blood pressure, chest pain, thrombosis, varicose veins, anaemia. Respiratory: asthma, bronchitis. Urinary: infection, poor control. Gastrointestinal: ulcers, irritable bowel syndrome, per anal bleeding. Hormone disease: diabetes, thyroid. Infectious diseases: hepatitis, AIDS. Nervous disorders: epilepsy, depression.

19

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Monthly probability of pregnancy

Figure 3.1 Modelling the distribution of fecundability in a young population. A mean of 0.2 and coefficient of distribution of 0.6 are assumed. Relative infertility (RI) is set below the 5th percentile; the ellipse indicates the 2-3% of the population with complete infertility or sterility (S). N = normal fecundability. (Published by courtesy of Robert Jansen.) 9. Previous surgery. General surgery including: intra-abdominal surgery (appendicectomy, cholecystectomy, bowel surgery), back surgery, thyroid surgery, renal surgery. Gynaecological surgery including: ovarian surgery, tubal surgery, uterine surgery (myomectomy, endometrial ablation, septum resection), ectopic pregnancy removal, as well as caesarean section and dilatation and curettage. Pain is often an element in the history and should be correlated with the physical findings. Any areas highlighted can be assessed in greater depth. Special attention should be paid to diseases that are associated with increased anaesthetic or pregnancy risk as well as those that have ramifications for fertility. The couple should be asked why they present at this particular time for their infertility management. They should understand that only 2% or 3% of couples have complete infertility and no chance of pregnancy. The graph in Figure 3.1 shows the monthly probability of pregnancy, known as fecundability. The patients need to understand that they are most likely to have relative infertility and that in most cases investigations will ascertain the cause or causes. Each cause on its own may not be sufficient to prevent pregnancy; however, multiple causes have a cumulative effect in reducing the chance of pregnancy. The most important factors are the duration of infertility and the time left to achieve a pregnancy. The life-table graph in Figure 3.2 models the accumulating probability of

21

Assessment of the female partner 1.0 0.9 CD Q. 0.8 c 0.7 o t 0.6 o Q. O 0.5 Q. 0.4 O) 0.3 0.2 0.1 0

•fsr = 0.046

c

CO

D)

= 0.030

-fsr

0

3

6

9

12

15

18 21 Months

24

27

30

33

= 0.007

36

Figure 3.2 Modelling the accumulating probability of pregnancy over 3 years from mean 1st month of fecundabilities (fi) of 0.2, 0.05 and 0.01. Right-hand figures are the residual mean fecundability of each group for month 37 (f37). (Published by courtesy of Robert Jansen.)

pregnancy over three years and is a useful method of judging the outcome of infertility treatment. This graph assumes all causes for complete infertility have been excluded and shows the average fecundability of those not pregnant after three years, with fecundability decreasing from 0.3 to 0.046, equivalent to a disease that caused a 75% reduction in fecundability. Diseases that reduce fecundability - such as peritubal adhesions, mild endometriosis, reduced semen parameters and others- when combined, result in a much greater reduction of fertility as the decreases are multiplied. The multiplication of factors is shown in Table 3.1 and an understanding by the patients of the significance of combined minor factors can help answer the question often asked: 'Why are we not achieving a pregnancy?' As a result of improving technology, the percentage of patients with idiopathic infertility is declining. Furthermore, many patients who have absolute infertility are now able to achieve pregnancy via in-vitro fertilization employing the intracytoplasmic technique with sperm retrieved directly from the vas, epididymis or testis. The infertility history should be obtained during a consultation with both partners at which it is possible to gain some insight into their relationship. On occasion it may be necessary to advise against pursuing infertility treatment until the couple has sought counselling, often a hard decision for the consultant to make, but necessary, however, to reduce turmoil in the future. At some point it is often helpful to interview both partners separately in order to discuss sensitive issues that they may not feel free to raise in front of their partner. These issues may include previous pregnancies, anxieties about cop-

22

B. Downing

Table 3.1. Modelling the effect of multiple, minor abnormalities on fecundability and on mean time to pregnancy. (Published by courtesy of Robert Jansen.) Number of factors

Mean fecundability

Pregnant in 2 years (%)

Pregnant in 3 years (%)

Mean years to pregnancy

0 1 2 3

0.2 0.05 0.01 0.002

93.6 63.8 20.7

96.7 75.5 28.9

4.7

6.9

0.3 3 7 40

ing with the responsibility of a family and financial considerations as well as other aspects of the person's psychological, medical or surgical past. If the couple has sought infertility management elsewhere, the reason for change should be reviewed in the hope of avoiding a similar situation. Usually the disquiet is the result of poor communication and misinterpretation, and discussion of previous difficulties at the time of the first consultation may result in the couple changing their expectations or deciding not to continue in your care. Infertility history

The discussion of a patient's infertility should be carried out after more general issues have been explored, so that the patient feels at ease and is less likely to overlook issues that may be significant. The history should cover the following points. 1. Duration of infertility (relate to the age of the patient). 2. Previous pregnancies - include for each pregnancy: time taken to conceive duration of pregnancy abortion (spontaneous/induced) outcome-delivery (vaginal/abdominal). 3. Any event in the patient's past that they believe could be causative. 4. Past history: intra-abdominal surgery (non-gynaecological, gynaecological endometriosis, ovarian, uterine, tubal) other surgery, accidents (fractured pelvis) past use of intra-uterine devices and duration and use of other contraceptive measures. 5. Menstrual history:

Assessment of the female partner

6.

7.

8.

9. 10.

menarche - onset age, regularity and pain pain in relation to the menstrual cycle - temporal pattern in relation to bleeding and ovulation, character and intensity, duration, precipitating and alleviating factors. Intercourse: libido current and past, infertility stress effect frequency and relation to ovulation pain, superficial/deep, cyclical relation, aversion and vaginismus lubricant/douche use semen retention. Toxic hazards: occupational (radiation, chemicals) Social (alcohol, cigarettes, recreational drugs, spas, saunas). Psychosocial aspects: factors precipitating the desire for pregnancy now psychosexual response to infertility the quality of the marriage social and cultural pressures for childbearing ethical and moral concerns. Other gynaecological diseases including endometriosis, pelvic inflammatory disease and recurrent ovarian cysts. Body habitus change: change in fat distribution, change in hair distribution, change in pigmentation.

The history enables a focus of probabilities in terms of the likelihood of the underlying cause. However, in many situations more than one cause exists. Physical examination must encompass both general and pelvic examination, which will help establish the most appropriate, cost-effective investigations. General examination

The general examination starts when the patient walks into the consulting room - observations of body habitus, skin health, hirsutism, gait, posture and mental alertness are all relevant. The patient's weight and height are measured so that the body mass index (BMI - weight in kilograms/height in metres squared) can be calculated. If the BMI is less than 19, anorexia may be present as well as hypogonadotrophic hypogonadism. Blood pressure, if abnormal, warrants assessment prior to further management. If the pulse is low, this should correlate with the exercise history and, if rapid, may indicate anxiety or thyroid disease.

23

24

B. Downing

Table 3.2. Tanner staging of breast and pubic hair development Tanner stage Breast changes

Pubic hair changes

1 2 3

Papillary elevation Elevation of breast bud and papilla Further enlargement of breast tissue and papilla

4

Elevation of the papilla and areolar unit from the breast; papilla is at or above the equator of the breast Areolar is recessed into the breast Terminal hair covering the labia and/or papilla is below the equator of majora, mons pubis, and inner thigh the breast

5

Lanugo-type hair Dark, terminal hair on labia majora Terminal hair covering the labia majora and spreading to the mons pubis Terminal hair covering the labia majora and mons pubis fully

Examination of the head and neck should look for exophthalmos, facial hirsutism, acne, temporal balding, abnormal pigmentation, thyroid enlargement and a Buffalo hump at the lower neck (Cushing's syndrome). Chest examination should include assessment of breast development, the presence of galactorrhoea and peri-areolar hirsutism, and the Tanner staging of breast development should be noted (Table 3.2). The breasts should be examined for lumps and the patient investigated by mammography, ultrasonography and biopsy as indicated. The cardiorespiratory system should be assessed with appropriate referral if abnormal. Abdominal examination should exclude evidence of ascites, enlarged liver or spleen and look at the distribution of body fat (centripetal and Buffalo hump in Cushing's syndrome) and for evidence of pink striae which may be present, often laterally on the flanks or axillae (Cushing's syndrome). An assessment of hair and pigment distribution should be made with reference to the Tanner staging of pubic hair development (Table 3.2). Peripheral examination should look for varicose veins, oedema, bruising and evidence of good perfusion. If the patient appears to have a low BMI and poor grooming, needle puncture sites should be looked for and appropriate questions asked. Muscle wasting and difficulty rising from a squatting position are hallmarks of Cushing's syndrome.

Gynaecological examination

The abdominal examination involves careful palpation of the lower abdomen for masses arising from the pelvis - these may be ovarian, uterine or, rarely,

Assessment of the female partner

25

tubal in origin. A note of faeces in the descending colon should be made and questions asked in relation to bowel habit. Areas of tenderness should be noted. If the patient has complained of pain, it is helpful at this point in the examination to ask her to map on her abdomen and pelvis the areas where she experiences the pain. The patient can be given a pain map to complete for documentation, noting changes in pain in relation to the menstrual cycle. The site of pain can then be correlated with physical findings. Striae will have already been noted, along with hair distribution according to Tanner. The vulva is inspected for evidence of normal anatomy, with particular reference to cliteromegaly (where the shaft of the clitoris is greater than 1 cm), labial fusion, imperforate hymen and vaginal agenesis. Very rarely will patients achieve reproductive age without a diagnosis of imperforate hymen or vaginal agenesis, though shy, withdrawn women may do so. Inspection of the vagina will note a vaginal septum and the nature of the vaginal folds with particular reference to their flattening, pallor and lack of secretions (common in oestrogen deficient states). Inspection of the cervix will note the contour (size and evidence of laceration), epithelium (ectocervical/ endocervical or atypical) and evidence of normal cervical secretions for that stage of the menstrual cycle. Bimanual examination of the uterus and adnexa will reveal the size and shape of the uterus as well as its mobility. A uterus may appear to be enlarged secondary to pregnancy, fibroids, adenomyosis, multiparity, or due to obesity or retroversion, making assessment difficult. Vaginismus may also make it impossible to examine the pelvis adequately. The adnexa are examined for evidence of thickening of the utero-sacral ligaments (this may represent endometriosis of the utero-sacral ligaments); enlarged ovaries may be felt and their mobility and tenderness noted. Any undue pelvic tenderness should be noted and questions asked in relation to the persistence of pain on physical examination in the past or pain with intercourse or on defaecation or micturition. The investigative pathway

The investigation assesses two main areas - those of ovulation and pelvic viscera normality. Ovulation is assessedfirstbecause it is less invasive and does not have the potential for morbidity and the rare mortality that can be associated with assessment of uterine, ovarian and tubal normality. Assessment of ovulation The assessment of ovulation is complex, involving history, examination and

26

B. Downing Table 3.3. Assessment ofovulation 1. 2. 3. 4. 5. 6. 7. 8.

Regular menstrual cycle Pre-menstrual molimina Mid-cycle mucous changes Basal body temperature Endometrial biopsy Plasma progesterone Assessment of plasma or urinary LH Ultrasound follicle tracking, follicle growth, endometrium, blood flow

investigation (Table 3.3). A patient with a menstrual cycle of between 21 and 35 days, with bleeding lasting from 2 to 7 days, who experiences midcycle stretchy mucus and premenstrual breast fullness is likely to be ovulatory. Of couples who present with infertility, 20% to 25% will have disorders of ovulation. Magyar et ah (1979) showed that a history of regular (bleeding the same number of days + / - 2 each cycle), cyclic (interval the same number of days + / - 2 each cycle), and predictable (moliminal symptoms present) menses indicates ovulation is present in approximately 98% of women.

Symptoms ofovulation Premenstrual mastalgia is a progesterone effect manifest by enlargement of the breasts (secondary to both glandular proliferation and retention of fluid by the breast) associated with increased sensitivity of the nipples, areolar and breast tissue to physical trauma - the patient may find it uncomfortable to turn over in bed and sometimes a bra needs to be worn at night for comfort. Midcycle pain in either iliac fossa can also be indicative of ovulation. However, it may not be accurate in terms of defining when ovulation is occurring, as pain may be due to any of the following causes: stretching of the ovarian capsule as the follicle enlarges before ovulation (these symptoms may be exacerbated particularly when there are peri-ovarian adhesions and symptoms will be maximal before ovulation); bleeding from the ovulating follicle may irritate peritoneal surfaces causing pain; and pain may occur secondary to bleeding into the follicle after ovulation, causing further stretching of the ovarian capsule. Midcycle mucus may be obvious to some patients who notice their mucus change from scant, thick and viscous in nature to a clear, stretchy, lubricative mucus which, if viewed microscopically, is seen to be thin, acellular, with ferning present and has an alkaline pH. The latter mucus is due to high

Assessment of the female partner

27

oestrogen levels whereas the thick, scant mucus is due to progesterone domination - these changes were recognized by Billings and are used both to aid and avoid conception. The patient who wishes to learn about this technique should be referred to the book by Billings and Westmore (1988). It is necessary for the patient to become aware of her own mucus; ultimately, she should become sufficiently experienced and testing will only be necessary for three or four days in a regular cycle. In the future it will be possible to assess the mucus for enzymes present using dipsticks and other techniques (Trefes, Vincenzini and Vann, 1986) and this will greatly simplify the technique.

Investigations ofovulation These include the measurement of basal body temperature (BBT), endometrial biopsy, ultrasound follicle tracking, progesterone assay and luteinizing hormone (LH) assay. Basal body temperature The basal body temperature rises by 0.3 to 0.5 degrees during the luteal phase. This is a progestogenic effect on the hypothalamus and follows the LH peak by 2 days. It is important that the temperature is taken immediately on waking after an uninterrupted night's sleep. Unfortunately this technique only retrospectively determines ovulation. Endometrial biopsy Noyes and colleagues (1950) studied the histopathology of the endometrium throughout the menstrual cycle, defining the microscopic picture of the endometrium with the oestrogen and progesterone levels. This enables dating of the endometrium with reference to the expected onset of the next menstrual period. Luteal phase deficiency The standard for the diagnosis of luteal phase deficiency (LPD) requires strict adherence to the guidelines outlined by Noyes and colleagues (1950). 1. Each sample must be obtained from the anterior fundal wall of the uterus. 2. Ideally, biopsies should be obtained 11 to 13 days after ovulation. 3. Specimens may be accurately evaluated in relation to either ovulation events or subsequent menses. 4. 'Lag' of three or more days could be considered abnormal. 5. A luteal phase of less than 11 days should be considered abnormal.

28

B. Downing

6. The diagnosis of LPD should be based on abnormal findings in at least two cycles. Noyes (1956) assessed the uteri of 100 hysterectomy specimens and reported that secretory endometrium showed uniformity of development. Three per cent of the infertile population were found by Jones and Pourmand (1962) to have luteal phase defects. The diagnosis requires two consecutive endometrial biopsies as 20% of patients have a single out of phase biopsy (Rosenberg, Luciano and Riddick, 1980). Progesterone assay Elevated serum progesterones greater than 30 ng/ml are regarded as presumptive evidence of ovulation, though clearly it is no proof that the egg actually leaves the follicle to embark on its journey down the tube. Luteinized unruptured follicles (LUF) have a recognized association with infertility. Usually the progesterone is measured about day 21 of a cycle, roughly the peak of the progesterone rise. If the progesterone is above basal levels and the patient has an irregular cycle, further questioning should determine when the commencement of menstruation occurred, as the patient may have ovulated and be at one extreme, either the ascent or descent of progesterone value. Serial weekly progesterones may be necessary. Peak progesterone should be above 30 ng/ml about day 21 of the cycle or a series of three progesterone levels on alternate days in the midluteal phase should be greater than 15 ng/ml (Abraham, Maroulis and Marshall, 1974). Luteinizing hormone assay Plasma or urinary LH assay will show a rise approximately 36 hours prior to ovulation. Urinary reagent strips are now available which enable patients to test their own urine with convenience, on a regular basis, with greater than 85% accuracy. The testing should be carried out at the same time every day, starting two to three days before expected ovulation. A colour change predicts ovulation in 12 to 24 hours. Unfortunately this technique is expensive, but is particularly useful for timing artificial insemination. Ultrasound With the development of vaginal ultrasound and its application to IVF, it is clear that there are ultrasound features that closely relate to ovulation. The preovulatory follicle grows at a rate of 2 to 3 mm per day (Renaud, Macler and Dervain, 1980) and is 17 to 25 mm at the time of ovulation (O'Herlihy, de Crespigny and Lopata, 1980). There is a linear correlation between the oestro-

Assessment of the female partner gen level and the size and number of follicles; a disparity correlates with a reduction in the number of eggs collected in IVF and a reduced chance of fertilization. Pregnancy is associated with follicles of larger size at the time of ovulation and usually those greater than 20 mm (Marinho, Hassan and Goessens, 1982). After ovulation, features of the development of a corpus luteum are present. Ultrasound features of ovulation include a reduction in the size of the follicle associated with loss of definition of the follicular wall, together with the presence of fluid in the pouch of Douglas and even multiple echoes in the follicle. Ultrasound assessment is usually carried out one or two days before expected ovulation and repeated one or two days after ovulation. Ultrasound examination of the architecture and thickness of the endometrium is used in the assessment of the infertile patient. A difference exists between the endometrium in the follicular phase as compared with the luteal phase: endometrial thickness at the time of ovulation of less than 6 mm is associated with a reduced pregnancy rate; a thickness of 9 mm would appear to be ideal, whereas a thickness of greater than 13 mm is associated with increased spontaneous miscarriage (Dickey, Olar and Curole, 1992). Doppler ultrasound is now being used to assess the blood flow to the uterus and endometrium. Rather than depend on a single test, a combination is preferable. A BBT together with ultrasound tracking and luteal phase progesterone is an excellent means of ovulatory assessment. Assessment of abnormalities of ovulation

Abnormalities of ovulation must be assessed for their cause. The direction of assessment is formulated from the history and examination findings. Medical diseases must be assessed and treated by the appropriate medical specialist. The obese patient requires long-term counselling, employing a group approach with psychologist, health maintenance nurse and fitness/lifestyle trainer after full endocrine assessment. There is considerable risk involved in achieving pregnancy with BMI greater than 30, both from the patient's viewpoint and in terms of obstetric management. There is good reason to withhold treatment until the patient has lost weight. Investigation of the obese patient 1. Check BMI. 2. Duration of obesity: Short term: assess psychological stress, changes in eating pattern and

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exercise (is there an injury reducing mobility?), full blood examination, thyroid function tests, liver function tests, renal function tests, (query tests for Cushing's). Long term: onset as a teenager - consider polycystic ovaries (PCO), particularly if hirsutism is present as well as irregular or absent menses. Polycystic ovarian disease Polycystic ovarian disease will be described in detail elsewhere. However, its diagnosis is important as it is associated with abnormalities of ovulation and luteal phase defects. The response of the polycystic ovaries to ovulatory stimulants is less predictable and great care must be exercised. Diagnosis depends on an ultrasound examination demonstrating greater than 10 to 15 follicles in each ovary, raised androstenedione and testosterone levels, with an LH: FSH ratio greater than 2:1. Galactorrhoea In the presence of galactorrhoea, the prolactin level is almost invariably elevated, and if this elevation is twice the upper limit of normal for the laboratory, both the follicular and luteal phases are usually abnormal and often oligomenorrhoea or amenorrhoea is present. An elevated prolactin can be caused by a number of factors which include pituitary tumours diagnosed by computerized tomography scan (CT) or magnetic resonance imaging (MRI), the latter being more accurate but also more expensive. Hypothalamic tumours such as craniopharyngiomas, gliomas, pinealoma or hamartomas may also influence prolactin secretion, as may rarer infiltrative diseases such as sarcoid and tuberculosis. Drugs acting on the central nervous system may also stimulate prolactin secretion. These include cimetidine, dopamine receptor agonists, metaclopromide, antihypertensives, alpha-methyl dopa, clonidine, reserpine, anxiolytics, antidepressants and antipsychotics. Questions relating to medication should clearly be part of the history, in addition to questions relating to thyroid function and polycystic ovarian syndrome. Prolactin can also be elevated in sleep, nipple stimulation, acute stress, intercourse, exercise and with pregnancy and lactation. When galactorrhoea is present in association with oligomenorrhoea or amenorrhoea, the treatment is bromocriptine titrated to the plasma prolactin level. Hirsutism Patients who have increased hair growth are likely to have increased androgen secretion.

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Non-androgenic This may be racial and familial in origin and can also occur in the perimenopausal period, thus premature menopause may need to be considered. Hirsutism is also common in hypothyroidism and acromegaly and with porphyria and drugs such as phenytoin, diazoxide and glucocorticoids. Androgenic Androgenic causes include sources of adrenal origin such as occur with congenital adrenal hyperplasia, Cushing's syndrome or adrenal tumour. Ovarian sources of androgens are elevated in polycystic ovarian syndrome, hyperthecosis or functional tumour and may also occur in intersex where an ovotestis may be present. Enquiries should cover medication with androgens (body building) and danazol. Increased 5-alpha reductase skin activity may also be associated with hirsutism but will not interfere with ovulation. The clinical history should encompass the onset and duration of symptoms, the pattern of hair growth, presence of acne, voice change, alterations in menstrual history and details regarding the growth spurt and other endocrine diseases. Recent weight change, medication and family history should be considered as well as any cosmetic surgery undertaken. Investigations should include plasma testosterone and, if greater than 200 ng/dl, an androgenproducing tumour of either adrenal or ovarian origin should be sought and ultrasound, CT or MRI scan arranged. With ovarian sources of androgens, the dehydroepiandrosterone sulphate (DHEAS) is usually elevated. If greater than 700 /*g/dl, adrenal hyperfunction or neoplasia should be considered. Cushing's syndrome Pituitary hypersecretion of adrenocorticotrophic hormone (ACTH) causes adrenal hyperplasia with a high cortisol and 17-hydroxy progesterone-suppressing GnRH gonadotrophin. Screening tests for Cushing's syndrome include a dexamethasone suppression test (Liddle, Estep and Kendall, 1959). Seventy-five percent of patients with Cushing's syndrome have oligomenorrhoea or amenorrhoea. Assessment of pelvic viscera Clinical pelvic examination will alert the physician to gross abnormalities, by noting both abnormal masses attached to or separate from the uterus or ovaries, or reduced mobility of these structures. Abnormal discharge or undue tenderness may raise the question of pelvic inflammatory disease, endometriosis or ovarian cysts.

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Ultrasound examination Ultrasound examination is a necessary part of the assessment of all infertile patients. It is best carried out vaginally as the transducer is much closer to the organ being assessed and therefore better images are obtained. Abdominal ultrasound of the pelvis or renal tract is used with a full bladder when vaginal access is denied (anxiety, age, virgo-intacta) or there are historical or physical findings that raise the question of a congenital abnormality or recurrent urinary tract infection. The kidneys and ureters should be assessed. Ultrasound examination may detect fibroids causing uterine cavity distortion, a uterine septum, other abnormalities of the uterine cavity, an unacceptably thin uterine scar (caesarean section, myomectomy), the presence of ovarian cysts (functional - if in doubt rescan the patient in four to six weeks), endometriotic, serous or mucinous (septa with solid areas may be present) cystadenomas, cystic ovarian tumours (colour Doppler will show abnormally elevated blood flow - common in malignancy) or solid ovarian tumours. In addition, features may be suggestive of loculated fluid collections or hydrosalpinges. The shape of the uterus is determined, looking for the presence of fibroids, polyps or adenomyosis that may distort the cavity. The direct role of adenomyosis in infertility is unclear. Hydrosalpinges are now known to reduce the chance of pregnancy in in-vitro fertilization (Anders et al, 1994) and can be diagnosed on ultrasound and their progress or resolution followed. Their removal laparoscopically or tubocornucal clipping is now a consideration - a further application of both ultrasound and falloposcopy in assessment of damage. Hysterosalpingography The hysterosalpingogram (HSG) may be used to assess uterine and tubal status as a front line investigation in all patients with normal ovulation whose partners have normospermia, who have been infertile for less than two years and have no past history or physical findings to suggest pelvic inflammatory disease. If a couple is over 35, then investigation should include laparoscopy, falloposcopy and hysteroscopy as the time left to achieve spontaneous pregnancy is more limited. Investigation earlier than one year is warranted if the couple has a past history of pelvic inflammatory disease, appendicitis, ectopic pregnancy, post-partum curettage, pelvic surgery, proven chlamydia, recurrent spontaneous abortions or abnormal uterine bleeding. Salpingitis isthmica nodosa can only be diagnosed by HSG, and more detail about the extent of Asherman's syndrome can be gained by HSG than by hysteroscopy. Until falloposcopy, the site of tubal obstruction could only be

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ascertained accurately by this technique. The HSG ascertains the anatomy of the uterine cavity, excluding significant distortion as well as outlining the uterine tubes so the normality of ampullary folds can be noted. The procedure is inexpensive when carried out in the x-ray department; however, it can be painful if partial or complete obstruction leads to high pressures necessary to minimize the false positive seen in cornual obstruction. Pregnancy is the only true test of tubal function. Oil-based contrast media are recommended for HSG unless the likelihood of obstruction is high, as oil can be associated with granuloma formation. An HSG should not be carried out when a patient is bleeding because of the risk of vascular spread of contrast and the presence of clot within the uterus causing artifactual impression of polyps or fibroids. Contraindications include a history of pelvic infection (which is often exacerbated by the procedure) and pelvic tenderness. The radiologist must be trained in gynaecology or the gynaecologist should perform the procedure. Complications include iodine allergy, pelvic infection, granuloma formation - especially with oily contrast media and embolization, though the latter has not been shown to be of great significance. The radiation dose to the ovaries is in the range of 500-1000 mrad, as shown by Shirley (1971). The hysterosalpingogram is able to demonstrate uterine and tubal abnormalities. Uterine abnormalities may include hypoplasia, cavity distortion if congenital secondary to a fusion defect or acquired secondary mucus polyps, intra cavity fibroids or intrauterine adhesions secondary to scarring following delivery or curettage. The tubal lumen is assessed from the tubal ostia to fimbria, noting the pressure required to get dye flow and whether the dye flows in a continuous fashion or has apparent areas of hold up. The dye should be uniform and not patchy and may outline diverticulae and polyps. Longitudinal folds should be seen, particularly in the ampulla and dye be seen to spill freely into the peritoneal cavity with no distension of the ampulla (hydrosalpinx). If this investigation is abnormal, then laparoscopy, falloposcopy and hysteroscopy should be performed. Hysterosalpingography has the disadvantage of being unable to visualize the fimbriae, ovaries, peri-ovarian or tubal adhesions or confirm endometriosis. Mild endometriosis has now been shown to reduce fertility. Hysteroscopy The hysteroscope enables the direct visualization of the uterine cavity and cervical canal and is usually carried out by a rigid telescope, 4 mm outside diameter (OD), which may require cervical dilatation when in its sheath.

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Flexible fiberoptic hysteroscopes are available, but orientation is more difficult. More recently, 2 mm hysteroscopes have become available which will permit utilization in the consulting room, reducing costs. Indications for use primarily include abnormal uterine bleeding, abnormal hysterosalpingographic findings and those patients having anaesthesia for laparoscopy. Up to 60% of abnormalities of the uterine cavity may be missed by hysterosalpingography, thus hysteroscopy has an important place. Contraindications include heavy bleeding as views are obstructed and CO2 or fluid embolism more likely, active pelvic infection, carcinoma of the cervix or endometrium and cervical stenosis. Ultrasound guidance may be of assistance in negotiating a tight cervical canal. Visualization of the uterine cavity will localize endometrial abnormalities that may include mucus polyps, fibroid polyps, congenital septa and acquired intrauterine adhesions as well as local areas of hypoplasia or malignancy. Hysteroscopy is usually combined with laparoscopy. Complications include cervical trauma, CO2 embolism (Valle, 1995) if flow rates are high or intrauterine pressures are more than 100-150 mmHg, and uterine perforation. Laparoscopy Laparoscopy, the gold standard for pelvic assessment, enables direct visualization of the peritoneal cavity, usually through a 10 mm telescope inserted at the umbilicus. Considerable head down tilt may be required to remove bowel from the pelvis for complete viewing. The 2 mm microlaparoscope (Downing and Wood, 1995a) can be used here for diagnostic purposes. However, if endometriosis is a consideration or laparoscopic surgery is to be undertaken, a standard 10 mm laparoscope should be used. Laparoscopy enables visualization of the pelvic viscera so peritubal or periovarian adhesions can be identified and an assessment can be made of the health of the ampulla and fimbriae. The site and extent of adhesion should be noted, along with the extent of tubal serosal surface involved and the density of the adhesion - whether filmy and translucent or dense and thick with little tubal mobility. Thick adhesions involving large areas of tubal serosal surface are more difficult to separate and more likely to recur. Adhesions reduce fertility by reducing the movement of the fimbriae over the ovarian surface and by inhibiting normal muscle segmentation that assists gamete and embryo transport. The fimbriae should be freely mobile, with full access to the whole ovarian surface and no agglutination of the folds or fimbriae themselves. It may be necessary to use a probe to pass into the ampulla or fill the pouch of Douglas for underwater

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viewing to complete the assessment. If hydrosalpinges are present, their diameter should be noted, as should the thickness of the wall. A thick fibrotic wall and mucosal adhesions have a poorer prognosis for pregnancy than a thin membranous wall (Brosens, 1995). Observation following surgery to reopen the tubes shows good regeneration of both cilia and secretory function. The uterine size and shape is noted and correlated with the hysteroscopic findings, particularly in relation to congenital anomalies, i.e. degrees of septation or uterine horn anomalies. Acquired abnormalities include scarring from surgery or pregnancy and adenomyosis or fibroids. The latter two may cause obstruction of the interstitial portion of the uteric tube. Endometriosis may be diagnosed and great care must be taken to look for the morphologic types which are now recognized and initially documented by Jansen (Jansen and Russell, 1986). All visualized endometriosis should be treated at the initial laparoscopic surgery by ablating with the laser or diathermy or by surgical excision so that no abnormal (including fibrotic) tissue remains. If severe, follow-up antiendometriotic drug therapy should be considered. The testing of tubal patency has until now been achieved by passing dye (methylene blue) via a cervical cannula and laparoscopically observing its flow down the tube and out through the fimbria. Distension of the tube is noted along with sites of hold up of the passage of dye. Clearly a tube that is partially obstructed may be reported as patent, yet significant disease may be present within its lumen. This has been one of the limitations of this investigation and the hysterosalpingogram, until the development of the falloposcope. Falloposcopy

Falloposcopy enables the human fallopian tube lumen and wall to be assessed. This technique, developed by Kerin et al. (1990), has both diagnostic and therapeutic application. It allows the direct visualization of the inside of the human fallopian tube using a 0.5 mm fibreoptic flexible telescope passed through the cervix, across the uterine cavity and through the tubal ostia into the tube. A laparoscopy is carried out at the time of the falloposcopy so the pelvis can be fully assessed. Usually by the time the laparoscopic surgeon achieves an intra-abdominal view, the falloposcope is at the ampulla. The forceps are used to hold the tube in the longitudinal axis of the falloposcope in order to obtain better images. The linear everting catheter balloon is rolled out ahead of the telescope until resistance is encountered or the full extent of the balloon

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utilized (10 cm). When resistance is encountered, direct visualization is carried out to see the nature of the obstruction. The microlaparoscope (Imagyn) was used in later cases to reduce patient trauma (Downing and Wood, 1995a). Both antegrade and retrograde visualization can be used, with the Imagyn falloposcope being tracked out ahead of the balloon, thus avoiding possible artefact secondary to the tube being dilated by the balloon. Diagnostic application Fluid irrigation causes the tube to distend and contract due to the elasticity in a healthy tubal wall when flushing fluid is turned on and off. Fibrotic stenoses are identified when passage of either the telescope or linear everting catheter causes longitudinal traction on the tube with increased resistance felt as a tactile response by the operator. Mucus, debris, blood (ectopic pregnancy) and fine and dense fibrous adhesions are seen within the tube, with agglutination of folds in the outer isthmus and ampulla clearly noted. The normal folded epithelium is noted to become flattened and featureless with increasing severity of damage. The normal vascular pattern of the isthmus and ampullary folds is seen to be reduced and in some patients only fibrotic scar tissue remains. The site of the tubal damage is clearly identified. The transition from normal tube to damage of tube may occur over 3-5 mm, most commonly in the isthmus. Purely falloposcopic assessment of the fimbria is difficult without the aid of a laparoscope. The salpingoscope provides the best assessment of the ampulla because of its better resolution due to its rod lens system. The following observations are made at each region of the tube, interstitial, isthmus and ampulla. Wall elasticity: normal abnormal Mucosa: normal flat adhesions Colour: normal pale Diameter: normal

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stenosis hydrosalpinx Patency: normal obstruction: telescope dye

Therapeutic application A study of 200 patients was carried out, all of whom had infertility of greater than two years duration, 80 with unexplained infertility, 37 with adhesions, 12 with endometriosis, other patients having failed to achieve pregnancies following IVF or microsurgery. The Imagyn linear everting balloon catheter system was used in all cases studied. The procedure was performed under general anaesthesia. Falloposcopy was undertaken in association with laparoscopy in all patients and the total time averaged was 33 minutes. Twenty-four per cent of patients achieved a pregnancy spontaneously; 35% of those with severe tubal disease were excluded. Of patients subsequently undergoing GIFT, 46% became pregnant. The ectopic pregnancy rate was 10%. One patient is pregnant after microsurgical anastomosis of local tubal damage identified falloposcopically. Patients with unexplained infertility have a 60% chance of tubal luminal disease being identified. The telescope, by visualization of the area of stenosis, could be used to push through the narrowing and the balloon guided over the telescope then inflated to dilate the stenosis. No perforation occurred in a normal fallopian tube. Luminal tube disease is associated with ectopic pregnancy, and disease in one tube, particularly secondary to infection, is usually mirrored in the contralateral tube. Seven out of 27 patients with a past history of ectopic pregnancy had normal tubes. All eight patients who had ectopic pregnancies after falloposcopy had luminal tubal disease, all but two with abnormalities in more than one site, flat avascular epithelium being the main characteristic. Only two of the patients did not have a past history of ectopic pregnancy. Twenty of the 42 patients who achieved intrauterine pregnancy did so within three months. Thirty-eight tubes were assessed in these patients: only two had bilateral normal tubes, with a total of nine tubes being falloposcopically normal. Twenty had abnormalities of the isthmus, 13 of the interstitium portion with 8 ampullary abnormalities. The most common abnormality was fibrosis of the isthmus, with flat avascular epithelium coming second. Of the 38 tubes, 16 had disease in more than one site. The isthmus is the most common site of disease and filmy, web-like ad-

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hesions are the most common. If localized, resection and anastomosis of the healthy tube may be an option, and balloon tuboplasty has a significant post surgery pregnancy rate. Flat, pale epithelium had a worse prognosis, especially related to ectopic pregnancy. Of 200 patients who have undergone falloposcopy, 47 have achieved spontaneous natural pregnancy, all but four within a year. Falloposcopy will become an integral part of the investigation of the infertile patient and will have a therapeutic benefit in those with mild tubal disease. References Abraham, G.E., Maroulis, G.B. and Marshall, J.R. 1974. Evaluation of ovulation and corpus luteum function using measurements of plasma progesterone. Obstetrics and Gynaecology 44: 522.

Anders, N.A., Zhou, Y., Fan, J.M. and Karsten, P. 1994. Low implantation rate after in-vitro fertilisation in patients with hydrosalpinges diagnosed by ultrasonography. Human Reproduction 9: 1935-8. Billings, E. and Westmore, A.C. 1988. The Billings Method- Controlling Fertility without Drugs or Devices. Melbourne: Anne O'Donovan Ltd. Brosens, I.V.O. 1995. Classifications of Distal Tubal Defects: The One that could be Practicalfor Physicians and Usefulfor Patients. References Gynaecology Obstetrique - Numero Special Congres Vichy - IFFS 1995 Experts Conference, Satellite Symposium, Tuboperitoneal Infertility and Ectopic Pregnancy. Bullen, B.A., Skrinar, G.S. and Beitins, I.Z. 1985. Induction of menstrual disorders by strenuous exercise in untrained women. New England Journal of Medicine 312: 1349-53. Dickey, R.P., Olar, T.T. and Curole, D.N. 1992. Endometrial pattern and thickness associated with pregnancy outcome after assisted reduction. Human Reproduction 7: 418. Downey, J., Yingling, S., McKinney, M. and Husami, N. 1989. Mood disorders, psychiatric symptoms and distress in women presenting for infertility evaluation. Fertility and Sterility 52: 425-32. Downing, B.G. and Wood, C. 1995a. Experience with a new microlaparoscope 2 mm in external diameter. Australia and New Zealand Journal of Obstetrics and Gynecology 35 :202-4. Downing, B.G. and Wood, C. 1995b. Predictive value of falloposcopy. References Gynecology Obstetrique - Numero Special Congres Vichy - IFFS 1995. Experts Conference - Satellite Symposium. Tuboperitoneal Infertility and Ectopic Pregnancy. Jansen, R.P.S. and Russell, P. 1986. Nonpigmented endometriosis: Clinical, laparoscopic, and pathologic definition, American Journal of Obstetrics and Gynaecology 155: 1154-9. Jones, G.E.S. and Pourmand, K. 1962. An evaluation of aetiological factors and therapy in 555 private patients with primary infertility. Fertility and Sterility 13: 398. Kerin, F.J., Daykhovsky, L., Grundfest, W.S. and Surrey, E.S. 1990. Falloposcopy: A microendoscopic transvaginal technique for diagnosing and treating endotubal disease incorporating guide wire cannulation and direct balloon tuboplasty. Journal of Reproductive Medicine 35: 606-12.

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Liddle, G.W., Estep, H.L. and Kendall, J.W. Jr 1959. Clinical approach of a new test of pituitary reserve. Journal of Clinical Endocrinology and Metabolism 19: 875. Magyar, D.M., Boyers, S.P., Marshall, J.R. and Abraham, G.E. 1979. Regular menstrual cycles and premenstrual molimina as indicators of ovulation. Obstetrics and Gynaecology 53:411. Marinho, A.O., Hassan, N. and Goessens, K.V. 1982. Real-time pelvic ultrasonography during the peri-ovulatory period of patients attending an artificial insemination clinic. Fertility and Sterility 37: 633. McLeod, J.M. and Gold, R.Z. 1953. The factor in fertility and infertility: VI. Semen quality and certain other factors in relation to ease of conception. Fertility and Sterility A: 10-33. Noyes, R.W. 1956. Uniformity of secretory endometrium: study of multiple sections from 100 uteri removed at operation. Fertility and Sterility 7: 103-09. Noyes, R.W., Hertig, A.T. and Rock, J. 1950. Dating the endometrial biopsy. Fertility and Sterility 1: 3-25. O'Herlihy, C, de Crespigny, L.C.H. and Lopata, A. 1980. Preovulatory follicular size: a comparison of ultrasound and laparoscopic measurements. Fertility and Sterility 34: 24. Renaud, R.L., Macler, J. and Dervain, I. 1980. Echographic study of follicular maturation and ovulation during the normal menstrual cycle. Fertility and Sterility 33: 272. Rosenberg, S.M., Luciano, A.A. and Riddick, D.H. 1980. Luteal phase defect: the relative frequency of and encouraging response to, treatment with vaginal progesterone. Fertility and Sterility 34: 17. Schwartz, D. and Mayaux, M.J. 1982. Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands. New England Journal of Medicine 307: 404—06.

Shirley, R.L. 1971. Ovarian radiation dosage during hysterosalpingogram. Fertility and Sterility 22: 83-5. Stillman, R.J. (ed.) 1989. Smoking and reproductive health. Seminars in Reproductive Endocrinology!: 291-348. Trefes, C, Vincenzini, M.T. and Vann, P. 1986. Changes in enzyme levels in human cervical mucous during the menstrual cycle. International Journal of Fertility 3: 59. Valle, R.F. 1995. Diagnostic hysteroscopy. In Infertility Evaluation and Treatment, ed. W.R. Keye, R.J. Chang, R.W. Rebar and M.R. Soule, pp. 370. Pennsylvania: W.B. Saunders. Wright, J., Duchesne, C. and Sabourin, S. 1991. Psychosocial distress and infertility: men and women respond differently. Fertility and Sterility 55: 100-08.

4 Assessment of the male partner PETER J. FULLER and HENRY G. BURGER

Introduction

Though this chapter will deal in detail with the clinical assessment of the male partner in an infertile couple, it is necessary to emphasize (as described in Chapter 2) the importance of treating the couple rather than just the individual. There is no doubt that subfertility frequently occurs in both partners and the effects tend, on the basis of a number of studies, to be synergistic rather than additive. Therefore both partners in any couple with subfertility must be assessed fully and a strategy of investigation and management planned that takes account of them both. Thus the assessment of the male should occur in parallel with the assessment outlined in Chapter 3 for the female partner. With the recent exciting developments in assisted reproductive technologies, particularly as applied to male subfertility (as outlined in Chapter 10), there is a temptation to take a minimalistic approach to the assessment of the male partner and to rely simply on the semen analysis followed by a decision as to whether to proceed to in-vitro fertilization, perhaps with sperm microinjection. It is important to recognize that there are uncommon but potentially treatable and/or preventable causes of male subfertility which should be identified before proceeding to more invasive and expensive technologies. The couple must be made fully aware of the options available to them, including the likely success rates, complications and expenses involved. As outlined in Chapters 2 and 3, it is also important not to view the problem of subfertility in complete isolation from other medical problems. A general history and examination are therefore essential parts of the assessment. This, of course, is familiar to the clinician and will not be pursued further except where it is relevant to specific causes of subfertility. In this chapter we will emphasize the key role of the history and examination in identifying potentially treatable causes of subfertility in the male and thereby directing further investigations and management as appropriate. 40

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History

The initial assessment will involve a complete history from the couple, including the duration of their infertility and also whether there is any history of previous fatherhood, both of which influence the prognosis. Testicular factors should be sought as a history of testicular maldescent, with perhaps corrective surgery, may be present. In the patient with a history of maldescent, and particularly of cryptorchidism associated with abdominal or inguinal testes, it is important to emphasize the potential risk of malignancy which may occur in the dysgenetic gonad. Such patients should be encouraged to follow a course of self-examination in much the same way as women should examine their breasts on a regular basis to detect early breast cancer. A history of trauma or of swelling, pain or penile discharge may suggest other relevant testicular factors, such as torsion of the testes or orchitis of either bacterial or viral origin, including mumps orchitis. If symptoms of infection are present, a specific history of sexually transmitted diseases may be elicited. Previous lower abdominal surgery involving the testes and/or inguinal region may be relevant, e.g. a past history of vasectomy with or without attempted reversal. Sexual function should be established with respect to potency, frequency and timing of intercourse and the occurrence of normal vaginal intercourse. Where there is any doubt about sexual function, issues of pubertal development and growth might also need clarification. Where delayed, absent or incomplete pubertal development has occurred, central causes such as a pituitary tumour or a craniopharyngioma should be considered. Gonadal dysgenesis associated particularly with chromosomal abnormalities must be considered; Klinefelter's syndrome is the most common of these, occurring at a frequency of 1 in 1000 live male births. The first presentation of this condition may be one of infertility. Possible exposure to environmental factors should be determined, which may be obvious from the general medical history or from lifestyle factors. Thus excessive exercise, very frequent exposure to saunas or other sources of extreme and constant heat and/or extreme stress should be identified. Medical treatment with a range of agents which are known to impair or inhibit spermatogenesis should also be identified. These agents include sulphasalizine, alkylating agents, cyclophosphamide, procabazine, cis-platinum, cimetidine and nitrofurantoin. In addition, exposure to environmental toxins such as environmental oestrogens, insecticides, chlorinated hydrocarbons, cadmium, mercury and arsenic should be noted. A history of heavy nicotine abuse, alcohol intake or other substance abuse including, of course, anabolic steroids and so-called recreational drugs such as

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marijuana and cocaine - should be sought. All these agents have, to varying degrees, been associated with subfertility. The most extreme are the anabolic steroids which are associated with hypogonadotrophic hypogonadism. As part of the general assessment, the presence of acute intercurrent illness should be sought. It is well established that serious acute and especially febrile illnesses may be associated with a transient but reversible impairment of spermatogenesis. It may take up to six months for complete recovery. The presence of specific chronic illnesses should be excluded. Diabetes mellitus may be associated with problems of impotence and retrograde ejaculation. Cystic nbrosis is frequently, if not always, associated with congenital absence of the vas. Congenital absence of the vas in the absence of clinical cystic fibrosis has recently been shown in many cases to be an attenuated form of cystic fibrosis, with at least one of the known mutations in the CF genes being present. Inflammatory bowel disease may be associated with subfertility, in part due to the therapeutic agents used. Recurrent chest or sinus infection should be noted as there is an association with subfertility through cystic fibrosis- so-called Young's syndrome and, indeed, the much more uncommon Kartageneer's syndrome. Pituitary disease may be a cause of secondary hypogonadism, and malignancy is often associated with impaired spermatogenesis. The presence of malignancy, particularly of haematogenous malignancy in any younger male, is often an indication for storage of semen, but the quality of the semen obtained at presentation is frequently found to be extremely poor and associated with a very low fertilization rate.

Examination

As already mentioned, a general physical examination is a very important component of the initial assessment in order to exclude the various chronic diseases outlined above. Thus, stigmata of liver disease, evidence of respiratory disease or the presence of lymphadenopathy may alert the clinician to important underlying illnesses whose manifestation, at this presentation, is infertility. The degree of androgenization of the patient should be assessed. The clinician should look particularly for the presence of a eunuchoid body habitus as well as assessing the degree of muscular development, beard growth, pubic and auxiliary hair. The frequency of shaving can be a useful guide to the adequacy of beard growth. The degree of androgenization varies with both familial and ethnic background and this may need to be taken into account in making an objective assessment. The presence of anosmia or hyposmia should be sought if hypogonadism is present, to establish the diagnosis of Kalmann's syndrome, a rare but well-described cause of hy-

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pogonadotrophic hypogonadism. In the course of the general physical examination, the presence of gynaecomastia and/or galactorrhoea should be sought, which may be a guide to the presence of a prolactinoma, although the former is much more likely to indicate Klinefelter's syndrome. The specific part of the examination is the genital examination. The penis should be examined with regard to size and structure. The site of the meatus should be determined and evidence of induration sought. Scrotal swellings may be identified on inspection, including the presence of a varicocele and there may be evidence of scars resulting either from trauma or surgery. Palpation of the scrotal contents is all important. Testicular volumes should be determined objectively using an orchidometer, and the texture of the testes, particularly their consistency, should be determined. A testicular volume at or below 12 ml strongly suggests the possibility of seminiferous tubule failure or, rarely, hypogonadotrophic hypogonadism. The presence of any lumps or irregularities, particularly hard irregularities, should be noted. The presence and the quality of both vasa along their length within the scrotum should be determined. Similarly, the posterior margin of the testis should be felt to define the epididymis clearly, looking particularly for abnormal thickening or nodularity as well as whether they appear to befilled.The presence of a varicocele is said to occur in 5-20% of the male population. It remains controversial as to whether it is or is not an important cause of subfertility. (This issue is addressed more fully in Chapter 5.) A large varicocele will be best detected by inspection. Smaller varicoceles can be detected by palpation, which may be enhanced by increasing intra-abdominal pressure either through cough or a valsalva manoeuvre. Standing the patient and examining in the upright position may further enhance the detection of a varicocele. Varicoceles should be classified into grades 1 to 3 on the basis of whether only a cough impulse is present (grade 1), the lesion is palpable (grade 2) or is large enough to be visible (grade 3). Varicoceles generally occur on the left side or sometimes bilaterally. The presence of an isolated right-sided varicocele is said to be associated with an increased incidence of renal malignancy and thus, when found, an intravenous pyelogram (IVP) should be performed. Finally, a rectal examination should be performed, looking for evidence of prostatitis as manifest by a tender or indurated prostate. In addition, other prostate pathology, in particular prostatic malignancy, may be detected at this time. Investigations

The following is a list of general and more specific tests which may be relevant in the investigation of male subfertility. Clearly not all should be applied to

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P. J. Fuller and H. G. Burger Table 4.1. Semen analyses parameters Normal range Ejaculate volume pH Sperm concentration Motility Viability Morphology Inflammatory cells Antibodies (immunobead test)

> 2.0 ml 7.2-7.8 > 20 million/ml > 50% progressively motile > 75% sperm excluding dye < 85% abnormal forms < 1 million cells/ml < 20% sperm binding to bead

patients, and the selection will be heavily guided by the findings on history and examination. We have grouped the various tests rather arbitrarily into andrological, biochemical and genetic. The first investigation is the semen analysis, which needs to be performed in a competent, experienced laboratory as many of its subtleties are not dealt with adequately by routine, non-specialist pathology laboratories. Issues of standardization, quality control and the use of some of the newer computing techniques are important. It is important to pay particular attention to proper collection of the specimen. The specimen optimally should be fresh: if transported any distance, it should be maintained at close to body temperature and should be collected into a sterile plastic specimen container. A suitable period of abstinence prior to the collection (generally about 48-72 hours) is recommended. Long periods of abstinence are not helpful. Two baseline semen analyses at least three weeks apart are recommended and if these tests are contradictory, a further analysis may be necessary. Many patients have significant fluctuations in their semen quality. It is important that the entire ejaculate is collected as most of the motile sperm are found in the first part of the ejaculate. Specific parameters that should be determined in the semen analysis are detailed in Table 4.1. Motility should be graded according to World Health Organization (1992) criteria. Assessment of morphology has a significant subjective component; however, where an experienced operator applies clearly defined WHO criteria, there is reasonable reproducibility. Antibodies can be detected on sperm using either a direct or indirect immunobead test. A high concentration of antisperm antibodies, particularly clustered on the sperm heads, can be a significant cause of infertility. This is discussed further in Chapter 5. Antisperm antibodies may also be detected in serum. The strongest correlate with fertility remains the sperm count. The fertilizing potential tends to fall exponentially as

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the number decreases: thus sperm counts in the range of 10-20 million per ml may be relatively fertile where the female partner is fully fertile but may be a significant contributor to the infertility if there is also impairment in the female partner.

Biochemistry Baseline hormone investigations should often include measurement of serum testosterone, FSH and LH levels. Testosterone levels may be low in approximately 30% of patients with severe seminiferous tubule damage where presumably the process has spread beyond the tubules to involve the Leydig cells. This may have implications for future therapy for the patient with respect to androgen therapy. A low testosterone may also result from an elevated serum prolactin, and hypogonadism with or without infertility is frequently the presentation of a prolactinoma in the male. The FSH level is used as a guide to the integrity of spermatogenesis. Conventional wisdom suggests that a significant elevation of the FSH is pathognomonic of primary disorders of spermatogenesis. However, recent reports have suggested that this is not always the case: normal levels of FSH may be found in men with severe seminiferous tubule damage and moderately elevated levels may be found in men with obstructive azoospermia. Marked increases in FSH, particularly associated with small testicular volumes, often indicate the presence of Klinefelter's syndrome. A modest increase in the FSH level does not preclude some degree of spermatogenesis. Conversely, a normal FSH does not ensure spermatogenesis, though it may be used in support of an obstructive cause for oligoazoospermia, particularly in the presence of normal testicular volumes. The range for FSH values is rather wide, reflecting some discordance and heterogeneity in the molecular forms making the correlation between immunoreactive and bioreactive FSH not as tight as for less complex hormonal molecules. Elevated FSH levels in men with seminiferous tubule failure have been postulated to result from decreased secretion of the testicular peptide, inhibin, which has a direct negative feedback effect on FSH secretion. Currently available inhibin assays have not confirmed this postulate, but the recent development of an assay specific to inhibin B has provided evidence that it is this molecule which is the relevant feedback signal in the male. Finally, a range of biochemical investigations may be important in seeking non-testicular non-endocrine causes of subfertility, and thus liver function tests, serum ferritin and a full blood may be important.

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A karyotype is indicated when the diagnosis of seminiferous tubule failure leading to severe oligoazoospermia is made. A range of abnormalities may be present. The most common is the 47 XXY genotype of Klinefelter's syndrome. The finding of Klinefelter's syndrome makes fertility impossible for the male except in the rare case of a mosaic Klinefelter's. In the case of Klinefelter's syndrome, where the female partner seeks pregnancy, the only remaining option is donor insemination, as discussed in Chapter 11. The male partner should not, however, be neglected. Often patients with Klinefelter's syndrome, some even presenting at this late stage, will have low or low-normal testosterone levels. They will benefit from androgen replacement therapy, and indeed some clinicians would recommend all patients with Klinefelter's syndrome be treated with androgen replacement therapy irrespective of their initial testosterone level. In addition, such patients arguably have an increased risk of breast cancer relative to the rest of the male population: the majority of cases of male breast cancer occur in patients with Klinefelter's syndrome. Nevertheless, it is still rare in this subgroup and it is not clear whether teaching breast self-examination is necessarily useful. Other genetic causes of infertility have been described, though most are rare. The exception is Kallmann's syndrome where, at least in the X-linked form, mutations of the KALIG-1 gene have been described (Bhasin, de Kretser and Baker, 1994). The condition results from a failure of migration of the GnRH neurons from the olfactory placode into the hypothalamus. The presence of anosmia in a patient with hypogonadotrophic hypogonadism would strongly argue for this diagnosis. Further evidence may be gained from an MRI scan of the hypothalamus; however, routine genetic testing is not currently available. In patients with congenital absence of the vas, there is now strong evidence for the presence of one or more cystic fibrosis mutations. Most appear to be compound heterozygotes (Chillon et al., 1995). Whilst genetic testing will not alter the diagnosis, it may be important in terms of counselling and further management as these patients are potentially fertile using the assisted reproductive technologies described in Chapter 10. Recently, several groups have described microdeletions of the Y chromosome associated with azoospermia (Bhasin, de Kretser and Baker, 1994), and in one series approximately 10% of azoospermic males had a deletion of the so-called DAZ (deleted in azoospermia) gene (Reijo et al., 1995). Again, this test is not yet in routine use but it is conceivable that it may become widely available in the near future. Some of these men have small numbers of spermatogonia and thus, with the rapid advances in artificial reproductive technologies, particularly microinjection,

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the possibility of using the sperm and perhaps transmitting the genetic defect is a major concern. Finally, there is a collection of rare genetic causes of azoospermia including mutations of the FSH receptor, oestrogen resistance, androgen insensitivity syndromes, etc. Specialized investigations

More sophisticated testing may be required, subject to the results of the various investigations already described. Sperm function studies Formal assessment of the ability of sperm to move through donor mucus, the so-called mucus penetration test, may be performed as a crude measure of sperm function. This will, for instance, be dramatically impaired in the presence of antisperm antibodies and may be a useful monitor for therapy (World Health Organization, 1992). The test can also be extended to include the wife's mucus by doing a Kremer crossover mucus penetration test where donor mucus, donor sperm, husband's sperm and wife's mucus are used in a crossover design to test for compatibility (World Health Organization, 1992). The scientific basis for this test is a little obscure. A similar result may be achieved with a post-coital test where mucus is collected after intercourse to establish whether viable and mobile sperm are present. Occasional men appear to have a condition of epididymal necrospermia where, for reasons that are not yet clear, degeneration of sperm occurs in what appears to be a hostile epididymal environment. Paradoxically, semen quality may improve with frequent ejaculation. Finally, post-ejaculation urine may be collected to look for evidence of retrograde ejaculation. This is said to occur most commonly in disorders of autonomic neuropathy such as seen in some diabetic patients, patients with multiple sclerosis or after bladder neck surgery. It is important to identify as the sperm can potentially be collected and used for insemination. Further investigations of sperm function may include hamster zona binding studies, but they have limited clinical utility. Swim-up studies may be performed, often as part of a work-up prior to IVF (see Chapter 16). Where there is a suggestion of an obstructive cause, a rectal ultrasound may be useful in diagnosing the presence of a hydrocele, ejaculatory duct obstruction, prostatic utricle, the absence of the seminal vesicles, the site of ectopic testes and also in determining the presence or absence of other forms of prostatic disease. A CT scan or, perhaps preferably, an MRI scan of the

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pituitary may be relevant where pituitary disease with secondary hypogonadism is suspected. Where testicular pathology is present, a testicular biopsy may be warranted. Particularly in the patient with azoospermia associated with normal testicular volumes and a normal or modestly elevated FSH, it is important to establish the aetiology of the azoospermia, looking particularly to identify an obstruction or the presence of low numbers of sperm which may be adequate for intracytoplasmic sperm microinjection. Traditionally, testicular biopsy has been performed as an open procedure, allowing an adequate wedge of tissue, usually from both testes, to be obtained. Some would argue that because the biopsy comes from the surface of the testes there may be some sampling artefact; however, the quality of the subsequent histology is usually high. If varicocele surgery is being performed, a strong case can be made for taking testicular bipsy at the same time. More recently there has been a trend towards use of a needle biopsy under local anaesthetic (Mallidis and Baker, 1994). This should be performed by an experienced operator who undertakes the procedure regularly. This technique is limited by the small number of tubules that are obtained as well as disruption of the normal architecture and the relationships within the testes. Nevertheless, in many cases a clear diagnosis is obtained without recourse to a general anaesthetic and a more invasive procedure. The finding of small numbers of sperm in biopsies from azoospermic patients with high FSH levels which have been suitable for intracytoplasmic sperm microinjection means that this area is very much in a state of flux at present (Tournaye et ai, 1995). Assessment of testicular pathology with respect to infertility requires the skills of a specialist pathologist with experience in andrology and it is our experience that assessment by general pathologists is often unsatisfactory. Finally, the newer reproductive technologies, including in-vitro fertilization and sperm microinjection, provide a powerful in-vitro test of sperm function. IVF fertility rates correlate well both with morphology of the sperm and also with the presence or absence of normal zona binding. The above list of investigations is both daunting and potentially expensive! It is important to proceed through a logical series of steps, drawing upon investigations as indicated by the previous investigations. We would normally recommend an initial assessment with two semen analyses and a testosterone and FSH level. The subsequent tests will then follow depending on those results. Obviously the important thing is to identify a correctable cause; once correctable causes have been excluded, then one can argue that the critical next step is the recourse to an in-vitro assisted reproductive technology.

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References Bhasin, S., de Kretser, D.M. and Baker, H.W.G. 1994. Clinical review 64: Pathophysiology and natural history of male infertility. Journal of Clinical Endocrinology and Metabolism 79: 1525—9. Chillon, M., Casals, T., Mercier, B., Bassas, L., Lissens, W., Silber, S., Romey, M.C., Ruiz-Romero, J., Verlingue, C. and Claustres, M. 1995. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. New England Journal of Medicine 332: 1475-80. Mallidis, C. and Baker, H.W.G. 1994. Fine needle aspiration biopsy of the testis. Fertility and Sterility 61: 367-75. Reijo, R., Yi Lee, T., Salo, P., Alagappan, R., Brown, L.G., Rosenberg, M., Rozen, S., Jaffe, T., Straus, D., Hovatta, O., de la Chapelle, A., Silver, S. and Page, D.C. 1995. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genetics 10: 383-93. Tournaye, H., Camus, M., Goossens, A., Liu, J., Nagy, P., Silber, S., Van Steirteghem, A.C. and Devroey, P. 1995. Recent concepts in the management of infertility because of non-obstructive azoospermia. Human Reproduction 10: 115-19. World Health Organization 1992. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 3rd edn. Cambridge: Cambridge University Press. Further reading Baker, H.W.G. 1994. Male infertility. Endocrinology and Metabolism Clinics of North America 23: 783-93. Baker, H.W.G. 1995. Male infertility. In De Groot's Textbook of Endocrinology, 3rd edn, ed. L.J. De Groot, pp. 2404-33. Philadelphia: W.B. Saunders. Burger, H.G. and Baker, H.W.G. 1987. The treatment of infertility. Annual Review of Medicine 38: 29-40. Nieschlag, E. 1993. Care for the infertile male. Clinical Endocrinology 38: 123-33. Skakkebaek, N.E., Giwercman, A. and de Kretser, D. 1994. Pathogenesis and management of male infertility. Lancet 343: 1473-9.

5 Treatment options for male subfertility GORDON BAKER and DAVID M. de KRETSER

Approximately 15% of couples are involuntarily infertile. In the United Kingdom it has been estimated that 24% of women will consult a doctor about infertility at some stage during their lives (Greenhall and Vessey, 1990). Surveys produce variable frequencies for male disorders causing infertility, probably because of the use of different definitions of male and female infertility and variations in the thoroughness of investigation but the lowest estimates are about 25% (Comhaire et al., 1987). Commonly, both partners have defects which contribute to the infertility. In this chapter we outline a method of management of male infertility and emphasize the effectiveness or otherwise of treatments. Usually, clinical evaluation, semen analyses and other investigations (as indicated below) allow each man's problem to be assigned to one of three therapeutic groups: untreatable sterility, treatable conditions, or subfertility. Subfertility is most common, and treatable conditions are rare (Baker, 1995). Conditions causing untreatable sterility

Conditions causing severe primary seminiferous tubule failure with persistent azoospermia are untreatable (Table 5.1). The azoospermia must be shown to be persistent otherwise natural pregnancies may occur, albeit at a very low rate. In addition, if any live sperm can be obtained, these can be cryopreserved for intracytoplasmic sperm injection (ICSI) (Tournaye et al., 1995). The men may have a history of events causing the testicular failure such as torsion of the testes or cytotoxic drug therapy, or an association such as orchidopexies for bilateral undescended testes. Features of deficient androgen production or action may be present such as low libido and defects of sexual performance, or loss or failure of development of secondary sex hair. Eunuchoidal proportions are an indication that androgen deficiency predated puberty. Severe spermato50

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Table 5.1. Causes of primary seminiferous tubule failure Klinefelter's syndrome Non-XXY chromosomal disorder with germ cell arrest or other defect of spermatogenesis DAZ or other Yq microdeletion or mutation Other single gene defect such as androgen receptor defect Undescended testes past or current Post-orchitis, mumps and other causes Post-torsion of testes Post traumatic - testicular injury, orchidectomy Post chemotherapy or irradiation genie disorders are often associated with reduced testicular size: less than 15 ml in westernized adults by orchidometry. Serum FSH levels are often elevated and aid in the differential diagnosis of obstructive azoospermia but there are some exceptions. Normal FSH may be seen with severe spermatogenic disorders, particularly germ cell arrest, and, less frequently, high FSH may be seen with minimal or no impairment of spermatogenesis (Hausere? al., 1995). Estimates of FSH, LH and testosterone are also valuable for confirming androgen deficiency and distinguishing primary from secondary testicular failure in which the gonadotrophin levels are not elevated. A karyotype is performed for confirmation of Klinefelter's syndrome or identification of rarer chromosomal disorders associated with severe spermatogenic disorders. In general, this investigation, which is expensive, is most useful in men with testicular volumes of less than 8 ml. Androgen receptor defects are found in some patients with normal male phenotype and otherwise idiopathic primary seminiferous tubule disorders (McPhaul et al., 1993). In about another 15% of men with these disorders, microdeletions are being discovered by polymerase chain reaction using Y-specific sequence tag sites in region 6 of the long arm of the Y chromosome (Vogt et al., 1992; Bhasin et al., 1994; de Kretser, 1995; Reijo et al., 1996; Najmabadi et al., 1996). An open reading frame in the distal part of region 6 with an RNA-binding protein motif has been named the deleted in azoospermia (DAZ) gene. It is likely that testing for these genetic causes of spermatogenic disorders will become routine in the near future as they may be transmitted by ICSI (Reijo et al, 1996). Testicular biopsies are useful to demonstrate the severity of the spermatogenic defect as it is now possible to use elongated spermatids for ICSI. In the past, patients with severe structural defects affecting all sperm such as absent acrosomes, pin head sperm, absent dynein arms, other cilial defects, and sperm

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defects which blocked specific stages of the fertilization process were also untreatably sterile, but now it may be possible to obtain fertilization and pregnancies with ICSI (Harari et al, 1995). However, it is likely that some of these (immotile cilia syndrome, absent acrosomes) are due to as yet unidentified gene mutations and the use of ICSI to achieve fertilization may result in transmission of genetic defects. Couples in whom spermatozoa or maturing germ cells cannot be obtained require counselling to enable them to accept that the sterility is untreatable. Other alternatives for having a family such as adoption or donor insemination should be suggested. Treatable conditions

Treatable causes of male infertility are listed in Tables 5.2 to 5.6. These conditions are individually uncommon and, although effective treatments exist, success is variable. Male genital tract obstructions Clinical evaluation of the man is particularly important for the diagnosis of genital tract obstruction. There may be a history of scrotal trauma or surgery, epididymitis, chronic cough and sputum production or other illness which suggests the cause (Table 5.2). Measurement of testicular size with an orchidometer and careful palpation of the epididymis are necessary to confirm the presence and normality of the head, body and tail of the epididymis and the scrotal portion of the vas. Miscellaneous anomalies may cause confusion, including retroversion of the testis so that the vas is anterior to the spermatic cord. Cysts in the head, body or tail of the epididymis are usually incidental. Ultrasound of the testis may be useful if clinical examination is equivocal or difficult because of a tight scrotum or tense hydrocele. Semen analysis may assist definition of the level of obstruction. With bilateral congenital absence of the vas (BCAV) or ejaculatory duct obstruction, the semen volume, pH and fructose are low because of the absence of seminal vesicle secretion. Men with suspected genital tract obstruction should be screened for sperm antibodies as they are often present with cauda epididymal and vasal blocks and, less frequently, with other obstructions. Sperm antibodies are a negative prognostic factor for pregnancy after surgery. Serum FSH levels are usually normal with genital tract obstruction and an elevated FSH suggests a spermatogenic disorder. Cystic fibrosis gene studies are important with bilateral congenital absence of the vas. More than 50% of

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Table 5.2. Causes of male genital tract obstruction Intratesticular obstruction - idiopathic, post-inflammatory Caput epididymal obstruction - Young syndrome, cysts, iatrogenic Corpus and cauda epididymal obstructions - post-inflammatory Vasal obstruction - vasectomy, other iatrogenic, trauma, post-inflammatory Bilateral congenital absence of the vas - CF gene mutations, idiopathic Ejaculatory duct obstruction - prostatic cysts, post-inflammatory Unilateral congenital absence of vas - with ipsilateral renal tract malformation Multisite obstructions - post-inflammatory Caucasian men with BCAV are heterozygous for delta F508 or other common CF gene mutations and are presumed to be compound heterozygotes (Anguiano et ai, 1992). The 5T allele has been recognized recently to reduce mRNA production for the CF transmembrane regulator and contribute to BCAV in a proportion of patients (Chillon et al., 1995). Couples at high risk of having a child with CF are counselled before ICSI and offered preimplantation or prenatal diagnosis. Testicular biopsies are performed to confirm adequate spermatogenesis before or at the time of surgery. Most men with genital tract obstruction have persistent azoospermia, and testicular size, serum FSH levels and testicular histology are all normal. However some have partial or intermittent obstructions and others have combined obstructions and spermatogenic disorders. Surgery for cauda epididymal and vasal blocks, including vasectomy reversal, produce moderately successful results, with perhaps 70% of men achieving reasonable sperm output and approximately half of these producing a pregnancy within one year of the operation (Southwick and Temple-Smith, 1988). Adverse factors in this group are the presence of sperm antibodies, preexisting reduced fertility and long duration of obstruction. Restenosis of the anastomosis sites may occur in up to one third of patients. Sperm collected from the body or tail of the epididymis also appear to produce reasonable fertilization rates with standard IVF. In contrast, surgery for the more common conditions with obstructions in the caput epididymis, including vasoepididymostomy and implantation of artificial spermatoceles, produces poor results and has largely been replaced by microepididymal sperm aspiration (MESA) or testicular biopsy and IVF. Results of standard IVF procedures using sperm collected from the head of the epididymis were poor until ICSI became available. ICSI produces excellent results with sperm collected by MESA, from epididymal cysts or from seminiferous tubules obtained by needle aspiration biopsy of testes. About 70% of the oocytes fertilize and 40% of the couples obtain a continuing pregnancy (Harari et al., 1995).

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The general management for possible genital tract obstruction - azoospermia or severe oligospermia, normal testicular size and normal FSH - is to explain that there is a possibility (30%) that the clinical picture may result from severe seminiferous tubule failure. Results of surgery depend on the level of obstruction. Vasovasostomy or vasoepididymostomy may be performed for vasectomy reversal and cauda or low corpus epididymal obstructions with fair to good the results (Southwick and Temple-Smith, 1988). Patients with high epididymal or intratesticular blocks are best served by MESA and ICSI. Semen collected from the epididymis can be stored frozen and used later for further attempts at ICSI/IVF. Because pregnancy cannot be guaranteed, some couples may prefer to use donor insemination. If reconstructive surgery is to be undertaken, the surgeon should perform scrotal exploration to determine that the vasa are patent by syringing or performing vasograms, confirm that there are sperm present in the epididymal tubule, and perform a testicular biopsy. The microsurgical vasoepididymostomy should be performed with single tubule to vas anastomosis. If possible, sperm should be collected for cryopreservation in case the operation is unsuccessful. The aspirated epididymal or vasal fluid can be diluted in culture medium containing 50% human serum and then treated in the same way as semen. If the sperm are to be collected by MESA, then this should also be performed with the dissecting microscope and the epididymal tubule should be repaired after collecting the sperm so that the procedure can be performed again if necessary (Silber et ah, 1990). Sperm autoimmunity

Sperm autoimmunity is the commonest medically treatable condition seen in men presenting with infertility. The condition can be associated with genital tract obstruction, with other autoimmune disorders in the patient or his family, with orchitis, unilateral or bilateral, or without such associations (Table 5.3). It is characterized by the presence of antibodies to sperm in blood, seminal plasma and on the surface of spermatozoa, and is associated with variable semen quality, ranging from azoospermic to normal, and severely impaired sperm-cervical mucus penetration. Sperm antibodies can be present in fertile men and in infertile men at low levels which are insufficient to impair fertility further (Baker et al, 1983). The immunobead test is the best screening procedure for sperm antibodies. It involves mixing washed sperm suspensions with polyacrylamide microbeads covalently coated with antihuman immunoglobulin antibodies (World Health Organisation, 1992). If the sperm have antibodies bound to the surface, then

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Table 5.3. Causes and associations of sperm autoimmunity Idiopathic Association with organ specific autoimmune diseases, e.g. Hashimoto, thyroiditis Genital tract obstruction, e.g. vasectomy Orchitis, unilateral or bilateral the immunobeads will bind to the sperm. Both IgG and IgA immunobeads should be used. The proportion of motile sperm with beads attached and the predominant patterns of binding to the sperm - head, tail, tail tip or all over are determined by microscopy. A positive test is where more than 50% of motile sperm have beads attached. Usually there is greater than 70-80% IgA immunobead binding to the sperm heads in men with clinically significant sperm autoimmunity. The sperm-mucus penetration test is most useful for assessing the significance of positive sperm antibody tests. If there is no sperm-mucus penetration, then the prognosis for a pregnancy without treatment is very poor (less than 0.5% per month). On the other hand, if sperm-mucus penetration is not completely blocked, the man should not be regarded as having sperm autoimmunity and other causes of the couple's infertility should be sought (Baker et al, 1983). If sperm antibody levels are high and there is zero sperm-mucus penetration, treatment with prednisolone in immunosuppressive doses has been shown in controlled trials to produce a significant improvement in fertility (Hendry et al, 1990). Prednisolone can be given in a dose of 50 mg/day, continuously for four tofivemonths, or in intermittent regimens of escalating doses from 20 mg to 75 mg per day, daily for 10 to 14 days, starting at the beginning of the wife's menstrual cycle. The major problem with these treatments is the risk of serious side-effects, particularly aseptic necrosis of the femoral head. This may occur in up to 1% of men treated and the risk may be enhanced by heavy alcohol intake. Patients must be made aware of the potential for serious side effects and the relatively limited success of the treatment. Pregnancy rates during prednisolone therapy are about 5% per month, so that about 25% of couples conceive during a four to five-month course. Positive prognostic factors appear to be a normal female partner, a fall in antibody levels and improvement in semen and mucus penetration. Surgery for genital tract obstruction or unilateral orchitis may reduce sperm antibody levels. Cryopreservation of semen during prednisolone treatment may allow addi-

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Table 5.4. Causes of gonadotrophin deficiency or suppression Kallmann's syndrome Idiopathic isolated gonadotrophin deficiency Associated with other pituitary hormone deficiencies, e.g. growth hormone Pituitary tumour, hypophysectomy, trauma Hyperprolactinaemia, tumour, drugs Haemochromatosis, both primary and secondary to repeated transfusions Suppression with starvation, anorexia, exercise, illness, stress Suppression with drugs, e.g. androgens

tional pregnancies to be obtained by artificial insemination by husband (AIH) and avoid the need for further courses of glucocorticoids. Stored semen may also be used for IVF. Where there are female factors or relative contraindications to glucocorticoids in the man, prednisolone 50 mg/day can be given for four to eight weeks before IVF. Although this has not been confirmed to be successful by controlled clinical trials, the results appear to be beneficial. Performing standard IVF with high level antibodies without prednisolone treatment is rarely successful, although occasional patients seem to have a satisfactory result (Clarke et al., 1985). Sperm autoimmunity can also be treated by ICSI without prior reduction of the antibody levels.

Gonadotrophin deficiency Gonadotrophin deficiency is a very rare cause of male infertility but it is treatable with gonadotrophin or pulsatile gonadotrophin-releasing hormone therapy. Most patients seen with gonadotrophin deficiency had the condition diagnosed because of the delayed puberty (Table 5.4). Commonly these men have congenital disorders of gonadotrophin-releasing hormone production, such as Kallmann's syndrome or combined pituitary hormone deficiencies. Occasional patients have gonadotrophin deficiency from pituitary tumours, trauma or haemochromatosis. Suppression of gonadotrophin secretion by extraneous steroids, other drugs or illness is occasionally found in men presenting with infertility. Ill or starving men, and athletes in negative energy balance, may have low testosterone levels, low libido and subtle signs of androgen deficiency, but sperm are present in the semen although sperm motility is usually reduced and semen volume low. This is referred to as the fertile eunuch syndrome and may result from isolated LH deficiency. The fertile eunuch syndrome may also be seen with hyperprolactinaemia. If possible, underlying conditions causing gonadotrophin suppression

Treatment options for male subfertility should be treated, hyperprolactinaemia suppressed and additional pituitary hormone deficiencies replaced. Treatment of gonadotrophin deficiency with intramuscular chorionic gonadotrophin (for replacement of LH), 1500 to 6000 units per week in one to three doses, is sometimes successful in inducing spermatogenesis on its own, especially in those patients who developed gonadotrophin deficiency after puberty. Although hCG is commonly prescribed as injections three times per week, Padron et al. (1980) have shown that a single weekly injection provides identical patterns of testosterone. Treatment with hCG alone is rarely successful in Kallmann's syndrome. Most patients require combined chorionic gonadotrophin and FSH, usually 75-300 units intramuscularly on alternate days or three times per week. FSH is given as urinary menopausal gonadotrophin or recombinant human FSH. The treatment should be monitored by measurement of testicular volume and semen analysis. Measurements of testosterone and LH (hCG) may be worthwhile to check compliance with the injection regimen. If the testes do not continue to increase in size over a period of three to six months, the dose of FSH could be doubled. If androgen effects are inadequate then increased doses of hCG up to 5000 units three times a week may be necessary. More than 50% of gonadotrophin deficient men have children with this regimen, but the response is often slow over months or years. Men with previous undescended testes, very small testes (less than 3 ml) and those who do not comply closely with the treatment protocol generally do poorly. The responders tend to do better with each repeated treatment. Pregnancies usually occur while semen analysis results are well below normal and it is rare to be able to store semen of good quality (Burger and Baker, 1984). Pulsatile administration of gonadotrophin releasing hormone can also be effective in men with intact pituitary glands. If no pregnancy occurs after sperm have been present in the semen for a reasonable time (6-18 months), IVF could be tried. Coital disorders

Coital disorders are an infrequent but important cause of male infertility (Table 5.5). The cause is usually obvious, such as spinal cord injury, but it may be unknown or psychological in origin. A recent deterioration in sexual performance may be the first symptom of a serious illness such as diabetes or pituitary tumour. Impotence is the inability to obtain erections sufficient for sexual intercourse. It may be a temporary or intermittent problem associated with the anxiety of discovering the infertility. Selective impotence with attempted intercourse while spontaneous and nocturnal erections continue to occur with

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Table 5.5. Coital disorders Impotence - neural, psychogenic, drug related, vascular Failure of ejaculation - neural, idiopathic, drugs Retrograde ejaculation - neural, idiopathic Low libido, low frequency intercourse, idiopathic, psychogenic

unchanged frequency also points to a psychological cause. This is clearly different from a persistent and complete inability to achieve any erections. Treatment with antihypertensive and psychotropic drugs may be associated with impotence. Failure of ejaculation occurs commonly with serious spinal cord injuries or pelvic nerve damage. It may also occur without other illness and then is usually associated with continued nocturnal emissions. The major tranquillizers such as thioridazine will inhibit ejaculation in some patients. Retrograde ejaculation occurs when the bladder neck fails to contract at the time of ejaculation so that most or all of the ejaculate passes into the bladder. This is easily confirmed by having the post ejaculatory urine examined for the presence of spermatozoa. It is usually associated with serious autonomic nerve damage from injuries or diabetic autonomic neuropathy. Some antihypertensive medications may also produce retrograde ejaculation. On occasions, it may be partial so that a small amount of fluid appears at the urethral orifice at the time of ejaculation, and in these instances the differential diagnosis is androgen deficiency, BCAV or ejaculatory duct obstruction. A number of patients with infertility have a low frequency of intercourse (less than twice per month). There may be some relationship difficulties, stress from the infertility, ambivalence about having a family or low libido. Encouraging these couples to have intercourse regularly at the midcycle period when ovulation is expected will often result in a natural pregnancy despite there being other pathology such as endometriosis and oligospermia. Treatment with drugs to improve sexual performance is not often successful. Obviously androgens should not be used as they will suppress spermatogenesis. Cholinergic antihistamines have been used to treat retrograde ejaculation with variable success. Advice to alter coital technique to improve performance may be helpful in occasional patients. For example, attempting intercourse with a full bladder may result in antegrade ejaculation in some patients with retrograde ejaculation and failure of ejaculation. The general approach to patients with sexual disorders is to diagnose and treat (where possible) serious underlying illnesses. If sexual performance cannot be improved, artificial insemination of the wife is possible if good

Treatment options for male subfertility quality sperm are available. If, on the other hand, semen quality is poor, then IVF with ICSI may be more efficient. If semen can be obtained relatively simply and safely at home, the couple may be taught to inseminate the wife with a syringe. If home insemination is not possible, artificial insemination can be performed with fresh or frozen sperm in the clinic. If adequate nocturnal emissions can be obtained for freezing, then these can be used. Some patients may have normal semen which was collected before the coital disorder developed or it may have been collected in the acute phase of spinal cord injury. Sperm may be prepared for insemination from the urine of men with retrograde ejaculation (Mahadevan, Leeton and Trounson, 1981). We advise patients to drink 300 ml of water every three hours during the day for two days before the ejaculation and to take alkylinizing effervescent tablets (ural) with each glass of water. On the morning of the insemination, they take two ural tablets with 600 ml of water and come to the laboratory within one hour. The pH and osmolality of the urine is checked and the man is asked to fast or drink more water to obtain urine osmolality between 300 and 600 mosmol/1. Then the man ejaculates and passes urine immediately afterwards. The urine is centrifuged and the sperm washed and resuspended in culture medium containing 5% human serum before insemination. While there is some possibility that a pregnancy will occur with insemination of even a few motile sperm, better results are obtained with high motile sperm concentration and we would generally not think it worthwhile attempting insemination with less than 1 M/ml motile sperm. If the semen quality is poor then it could still be cryopreserved for ICSI. Electroejaculation (EE) is a useful technique for obtaining sperm from men with a variety of sexual difficulties. Tamponading the bladder neck with a balloon catheter to prevent retrograde ejaculation improves the efficiency of this technique (Lim et ai, 1994). It is mainly used for men with spinal cord injuries but can also be used with general or spinal anaesthesia in able bodied men with ejaculatory disorders. In some patients, EE does not work and this may be because of a spermatogenic disorder or because of lower motor neuron lesions that are not responsive to the electric current. Under these circumstances it may be possible to obtain sperm by aspiration of the vas or epididymis or by biopsy of the testes. Reversible toxin or drug exposures While many factors can potentially affect spermatogenesis and sperm function, it is in fact rare to find a reversible element in most patients presenting

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G. Baker and D. M. de Kretser Table 5.6. Reversible illness or toxin exposure Illness - acute, chronic, febrile, other Antispermatogenic agents, e.g. salazopyrin Recreational drugs, e.g. alcohol Heat, e.g. frequent saunas

with infertility. Despite this, it is most important to check for these factors very carefully (Table 5.6). Any acute illness can temporarily impair sperm production and semen quality. Often several months are required for recovery from such episodes in view of the 70-day period required for spermatogonial precursors to proceed to spermatozoa. Similarly, exposure to heat from sauna baths or perhaps even summer temperatures may impair sperm production. This may be more important in outdoor workers and drivers. Severe chronic illness of any sort can be associated with impairment of spermatogenesis, which may recover with return of good health. It is possible that rapid changes in weight, either loss or gain, may also be associated with transient impairment of spermatogenesis. Drugs which affect gonadotrophin secretion or sexual performance are covered in other sections. The main antispermatogenic drugs having effects directly on the testes are therapeutic agents for cancer. Many of these will produce cessation of spermatogenesis which may or may not recover, depending on dose, duration and individual susceptibility. Salazopyrin, used for chronic inflammatory bowel conditions and arthritis, also has a moderately severe antispermatogenic effect due to the sulphapyridine. In some patients, fertility does not appear to be greatly impaired but in many men semen quality falls substantially, with oligospermia, low sperm motility and increased abnormal morphology, particularly with large sperm heads. The semen may also have a yellow colour. Recovery is usually rapid and pregnancies occur several months after stopping the drug and transferring to some other agent to control the underlying illness. Recreational drugs such as alcohol, marijuana and narcotics also impair testicular function in a variable way, but occasional patients are seen who stop using these agents and have dramatic improvements in semen quality. Androgen and anabolic steroids are commonly abused by sportsmen. There have been reports of infertility associated with calcium channel blockers that appear to have a specific effect on inhibiting the sperm capacitation, but these reports are controversial.

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Table 5.7. Associations of male subfertility of uncertain therapeutic relevance Varicocele Non-specific genital tract inflammation Hormonal/nutritional imbalance Adverse lifestyle factors Coital techniques

SubfertUity

Most men seen for infertility have sperm present in the semen ranging in number from very low to normal, with or without abnormalities of motility and morphology. They may or may not have associated conditions such as varicocele or evidence of genital tract inflammation (Table 5.7). Often the semen impairment and the degree of infertility are not closely related, probably because of the interaction of other factors such as female fertility. This lack of relationship is highlighted by the finding of fertile men in the general community with severely abnormal semen analyses. Also, trials of male contraceptive agents which suppress sperm production suggest that very low sperm concentrations, less than 1 to 3 M/ml, must be achieved before pregnancy rate is reduced. In contrast, investigation of the causes of failure of fertilization with standard IVF has revealed previously unsuspected causes of male infertility (Liu and Baker, 1992; 1994). Most patients with persistent failure of fertilization have specific sperm defects, particularly high proportions of sperm with subtle defects of morphology only detectable by using strict criteria of morphological normality. Other defects of the fertilization process have also been discovered, such as inability to undergo the acrosome reaction after the sperm bind to the surface of the zona pellucida (Liu and Baker, 1994). Most of these patients have otherwise normal semen quality and in the past would have been regarded as having idiopathic infertility. Provided the treatable and reversible conditions covered above have been excluded, it is uncertain whether any therapeutic intervention in this group is successful. Although many treatments have been considered in the past, their effectiveness is controversial. Few controlled trials have been conducted and most of those that have are too small or of suboptimal design and have produced conflicting results (Baker, 1986). Therapeutic trials for this problem are difficult to conduct because of the limited knowledge of the pathological mechanisms of defective sperm production. Additional problems are the day-to-day variability in semen analysis results which leads to regression to the

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mean. The couples in this group are not absolutely sterile, and natural pregnancies occur without treatment, for example 30% in one year and 45% by two years (Baker, 1995). The effectiveness of ICSI also makes difficult the design and implementation of therapeutic trials in which natural fertility is the endpoint.

Varicocele

Varicoceles are common and related to infertility in that they are more frequent in men seeking treatment for infertility than in fertile controls, semen quality is worse in men with varicoceles than in those without, and there is often a degree of left testicular atrophy associated with a varicocele that is clearly apparent from the disparity between the sizes of the right and left testes. However, while there is good evidence that varicoceles can impair testicular function, the evidence that treatment of the varicocele improves semen quality and fertility is arguable. Previous observational follow-up studies suggested treatment had no effect on semen quality or fertility (Baker et al, 1985). Recently, small controlled trials have produced discrepant results, but one showed some benefit in improved semen quality and increased pregnancy rates (Madgar et al., 1995; Nieschlag et al., 1995). However, the improvement in pregnancy rate is somewhat limited and may not be clearly related to the improvement in semen quality. Another small trial showed no benefit of treating the varicocele. It is possible that men with a single functional left testis may benefit from treatment of a varicocele (Hendry, 1992). However, overall, it is not possible to select which men with varicoceles and male infertility will have a predictable improvement in the semen quality and fertility after treatment of the varicocele.

Non-specific genital tract inflammation Men with defective sperm quality may have evidence of inflammation in the semen with increased leucocyte numbers or change in consistency of the semen as a result of the alteration in accessory sex gland function. The frequency of this problem seems to vary considerably from group to group. As with varicocele, the effectiveness of treatment is controversial, with most controlled trials having shown no beneficial effect. However, there remain individual patients with prostatitis who have dramatic improvements in semen quality following treatment, suggesting that the inflammation may have produced partial obstruction or specific impairment of sperm motility. There is good

Treatment options for male subfertility experimental evidence that polymorphonuclear leucocytes can damage sperm by reactive oxygen species generation (Aitken and Baker, 1995). If antibiotic therapy is to be used in men with male infertility, the agents must be chosen carefully because a number, such as the penicillins, are ineffective because they do not enter prostatic fluid very well and others, such as nitrofurantoin and sulphonamides, might have antispermatogenic effects. If a specific pathogen can be isolated from the semen or prostatic fluid, then specific therapy should be given. Broad spectrum agents such as doxycycline, erythromycin and noroxin may be used in other situations. Hormone methods The possibility of stimulating spermatogenesis or improving the function of sperm with hormone treatment such as follicle-stimulating hormone, clomiphene and low-dose androgens has been considered for many years. Also testosterone rebound therapy was in vogue for some time. This involved giving testosterone in high doses to suppress gonadotrophin secretion and spermatogenesis for three to four months in the hope that there would be a rebound increase in sperm output following the treatment. Controlled trials of some of these agents have shown no beneficial effects. Vitamins and nutritional supplements Vitamins C and E and zinc are commonly used for male infertility by natural therapists. While dietary deficiencies of these agents and other vitamins, such as pyridoxine and vitamin B12, can impair sperm production, infertility caused in this way must be extremely uncommon. It is unlikely that treatment would have a beneficial effect when given in replacement doses unless there was a clinically significant deficiency. Recently, there have been controlled trials of treatment with antioxidants, including vitamin E, which do suggest some beneficial effect on sperm function (Lenzi et ai, 1994; Kessopoulou et al., 1995). However, further confirmatory trials are required before their clinical use could be recommended. Lifestyle factors There is a perception that modern living and perhaps environmental pollution contribute to poor semen quality. There are also reports that would tend to implicate cigarette smoking and coffee intake with impaired sperm function, but these have not been confirmed in all studies. However, smoking does

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reduce fertility in women (Howe et al., 1985). Severe stress is also anecdotally associated with spermatogenic suppression. Occasionally individuals are seen who seem to have been exposed to severe stress, for example war, and who have poor semen quality which recovers over a number of months. Whether this is incidental or related to removal of stress or improved nutrition is difficult to determine. Testicular hyperthermia is claimed to be a clinically important cause of sperm defects by some workers, and cooling the testis is recommended, although this has also not been studied adequately. A pragmatic approach is to advise couples to moderate their lifestyle, to avoid stress and illness where possible and, most importantly, to continue to have intercourse regularly at the time of ovulation.

Artificial insemination with husband's semen Artificial insemination with husband's semen (AIH) has been used in the hope of improving pregnancy rates in couples with mild to moderate semen abnormalities. However, except where there is a coital disorder, this is probably ineffective and the pregnancy rates are probably the same as are achieved by sexual intercourse. Sperm preparation as for in vitro fertilization and intrauterine insemination also appears to be of no benefit. Ovulation induction to increase the number of oocytes ovulated probably does increase the pregnancy rate, but also the multiple pregnancy rate (Crosignani and Walters, 1994). In countries where IVF is not very expensive, most would prefer not to use this approach because of the uncontrolled risk of multiple pregnancy.

General management of male subfcrtility

Prognosis for natural pregnancy Studies of the outcome of large groups of patients seen for infertility reveal several important prognostic factors for natural pregnancy (Baker et al. 1985; Baker, 1995). In general, the better the semen quality, the higher the pregnancy rate; the longer the duration of infertility, the lower the pregnancy rate; and the older the wife, the lower the pregnancy rate. Couples with previous pregnancies have about double the pregnancy rate of couples in whom other factors are the same but who have no previous pregnancy. Female fertility disorders also have an adverse impact on the outcome, particularly ovulatory disorders and tubal disease. Patients under the age of 30, with relatively short durations of infertility and semen quality which is only mildly to moderately impaired may have pregnancy rates between 30% and 60% in one year. On the

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other hand, if the female is older than 35 years, the duration of infertility is greater than five years or the sperm concentration is less than 1 M/ml, the couple have a relatively low chance of producing a natural pregnancy in one year (less than 20%). These figures can be used to help couples understand their likelihood of achieving natural pregnancies. Couples with a reasonable chance of a natural pregnancy should continue trying longer than those with a lower chance because of advanced female age, long duration of infertility or particularly poor semen quality. IVF will give the latter groups a greater chance than they have of conceiving naturally. The availability of ICSI for severe sperm problems has largely overcome the problems with low fertilization rates from semen defects. With ICSI, fertilization rates are about the same as obtained with standard IVF with normal semen. The pregnancy rates with ICSI are variable, being high with genital tract obstructions and somewhat lower with sperm defects, and lower still with severe oligospermia where it is not certain that the sperm are alive or where only spermatids can be obtained with difficulty from testicular tissue (Harari et al, 1995).

General approach A general approach to the management of patients with subfertility is as follows (Table 5.8). The prognosis for a natural pregnancy is estimated. The doubtful value of the various empirical therapies is mentioned. The couple is appraised of the alternatives of remaining childless or choosing adoption or donor insemination if a pregnancy does not occur within a reasonable period. The timing of intercourse to maximize the chances of conception is discussed. Investigation of the female is reviewed and remediable factors dealt with. IVF offers extra hope to many couples in this group and the miracle of ICSI has enabled a number of couples with severe sperm defects to have their own children, although unfortunately many are unsuccessful. Improved understanding of the nature and causes of sperm defects remains an important goal so that effective treatments and preventative strategies can be developed. Table 5.8. General management of male subfertility Discuss prognosis Discuss doubtful value of medical therapies Discuss alternatives of donor insemination, adoption and accepting childlessness Review investigation and treatment of the female Review timing of coitus Consider IVF/ICSI

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References Aitken, J.A. and Baker H.W.G. 1995. Seminal leukocytes: passengers, terrorists or good Samaritans? Human Reproduction 10: 1736-9. Anguiano, A., Oates, R.D., Amos, J.A., Dean, M., Gerrard B., Stewart, C, Mather, T.A., White, M.B. and Milunsky, A. 1992. Congenital bilateral absence of the vas deferens, a primary genital form of cystic fibrosis. Journal of the American Medical Association 267: 1794-7. Baker, H.W.G. 1986. Requirements for controlled therapeutic trials in male infertility. Clinical Reproduction and Fertility 4: 13-25. Baker, H.W.G. 1995. Male infertility. In Endocrinology, 2nd edn, ed. L.J. de Groot, pp. 2404-33. Philadelphia: W.B. Saunders. Baker, H.W.G., Burger, H.G., de Kretser, D.M., Hudson, B., Rennie, G.C. and Straffon, W.G.E. 1985. Testicular vein ligation and fertility in men with varicoceles. British Medical Journal 291: 1678-80. Baker, H.W.G., Clarke, G.N., Hudson, B., McBain, J.C., McGowan, M.P. and Pepperell, R.J. 1983. Treatment of sperm autoimmunity in men. Clinical Reproduction and Fertility 2: 55-71. Bhasin S., de Kretser D.M. and Baker H.W.G. 1994. Pathophysiology and natural history of male factor infertility. Journal of Clinical Endocrinology and Metabolism 79: 1525-9. Burger, H.G. and Baker, H.W.G. 1984. Therapeutic considerations and results of gonadotropin treatment in male hypogonadotropic hypogonadism. Annals of the New York Academy of Sciences 438: 447-53. Chillon, M., Casals, T., Mercier, B., Bassas, L., Lissens, W., Silber, S., Romey, M.C., Ruiz-Romero, J., Verlingue, C, Claustres, M., Nunes, V., Ferec, C. and Estivill, X. 1995. Mutations in the cysticfibrosisgene in patients with congenital absence of the vas deferens. New England Journal of Medicine 332: 1475-80. Clarke, G.N., Lopata, A., McBain, J.C., Baker, H.W.G. and Johnston, W.I.H. 1985. Effect of sperm antibodies in males on human in vitro fertilization IVF. American Journal of Reproductive Immunology and Microbiology 8: 62-6. Comhaire F.H., de Kretser D.M., Farley T.M. and Row P.J. 1987. Towards more objectivity in diagnosis and management of male infertility. International Journal of Andrology (Supplement) 7: 1-53. Crosignani, P.G. and Walters, D.E. 1994. Clinical pregnancy and male subfertility; the ESHRE multicentre trial on the treatment of male subfertility. Human Reproduction 9: 1112-18. de Kretser, D.M. 1995. The potential of intracytoplasmic sperm injection ICSI to transmit genetic defects causing male infertility. Reproduction Fertility and Development 1: 137-42. Greenhall E. and Vessey M. 1990. The prevalence of subfertility: a review of the current confusion and a report of two new studies. Fertility and Sterility 54: 978-83. Harari, O., Bourne, H., McDonald, M., Richings, N., Speirs, A.L., Johnston, W.I.H. and Baker, H.W.G. 1995. Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility. Fertility and Sterility 64: 360-8. Hauser, R., Temple-Smith, P.D., Southwick, G.J. and de Kretser, D.M. 1995. Fertility in cases of hypergonadotropic azoospermia. Fertility and Sterility 63: 631-6. Hendry W.F. 1992. Effects of left varicocele ligation in subfertile males with absent or atrophic right testes. Fertility and Sterility 57: 1342-4. Hendry, W.F., Hughes, L., Scammell, G., Pryor, J.P. and Hargreave, T.B. 1990.

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Comparison of prednisolone and placebo in subfertile men with antibodies to spermatozoa. Lancet 335: 85-8. Howe, G., Westhoff, C , Vessey, M. and Yeates, D. 1985. Effects of age, cigarette smoking, and other factors on fertility: findings in a large prospective study. British Medical Journal 290: 1697-700. Kessopoulou, E., Powers, H.J., Sharma, K.K., Pearson, M.J., Russell, J.M., Cooke, I.D. and Barratt, C.L. 1995. A double-blind randomized placebo cross-over controlled trial using the antioxidant vitamin E to treat reactive oxygen species associated male infertility. Fertility and Sterilility 64: 825-31. Lenzi, A., Picardo, M., Gandini, L., Lombardo, F., Terminali, O., Passi, S. and Dondero, F.T.I. 1994. Glutathione treatment of dyspermia: effect on the lipoperoxidation process. Human Reproduction 9: 2044-50. Lim, T.C., Mallidis, C , Hill, ST., Skinner, D.J., Carter, P.D., Brown, D.J. and Baker, H.W.G. 1994. A simple technique to prevent retrograde ejaculation during assisted ejaculation. Paraplegia 32: 142-9. Liu, D.Y. and Baker, H.W.G. 1992. Tests of human sperm function and fertilization in vitro. Fertilility and Sterility 58: 465-83. Liu, D.Y. and Baker, H.W.G. 1994. Disorderd acrosome reaction of sperm bound to the zona pellucida: a newly discovered sperm defect with reduced sperm-zona pellucida penetration and reduced fertilization in vitro. Human Reproduction 9: 1694-700. Madgar, I., Weissenberg, R., Lunenfeld, B., Karasik, A. and Goldwasser, B. 1995. Controlled trial of high spermatic vein ligation for varicocele in infertile men. Fertility and Sterility 63: 120-4. Mahadevan, M., Leeton, J.F. and Trounson, A.O. 1981. Noninvasive method of semen collection for successful artificial insemination in a case of retrograde ejaculation. Fertility and Sterility 36: 243-7. McPhaul, M.J., Marcelli, M., Zoppi, S., Griffin, J.E. and Wilson, J.D. 1993. Genetic basis of endocrine disease 4. The spectrum of mutations in the androgen receptor gene that causes androgen resistance. Journal of Clinical Endocrinology and Metabolism 76: 17-23. Najmabadi, H., Huang, V., Yen, P., Subbarao, M.N., Bhasin, D., Banaag, L., Naseeruddin, S., de Kretser, D.M., Baker, H.W.G., McLachlan, R.I., Loveland, K.A., and Bhasin, S. 1996. Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy. Journal of Clinical Endocrinology

and Metabolism

81:1347-52.

Nieschlag, E., Hertle, L., Fischedick, A. and Behre, H.M. 1995. Treatment of varicocele: counselling as effective as occlusion of the vena spermatica. Human Reproduction 10: 347-53. Padron, R.S., Wischiesen, J., Hudson, B., Burger, H.G. and de Kretser, D.M. 1980. Prolonged biphasic response of plasma testosterone to single intramuscular injections of human chorionic gonadotropin. Journal of Clinical Endocrinology and Metabolism 50: 1100-4. Reijo, R., Alagappan, R.K., Patrizio, P. and Page, D.C. 1996. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 347: 1290-3. Silber, S.J., Ord, T., Balmaceda, J., Patrizio P. and Asch, R.H. 1990. Congenital absence of the vas deferens. The fertilizing capacity of human epididymal sperm. New England Journal of Medicine 323: 1788-92.

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Southwick, G.J. and Temple-Smith, P.D. 1988. Epididymal microsurgery: current techniques and new horizons. Microsurgery 9: 266—77'. Tournaye, H., Camus, M., Goossens, A., Liu, J., Nagy, P., Silber, S., Van Stierteghem, A.C. and Devroey, P. 1995. Recent concepts in the management of infertility because of non-obstructive azoospermia. Human Reproduction 10, supplement 1: 115-119. Vogt, P., Chandley, A.C, Hargreave, T.B., Keil, R., Ma, K. and Sharkey, A. 1992. Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to AZF, a human spermatogenesis gene. Human Genetics 89: 491-6. World Health Organisation 1992. Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction, 3rd edn. Cambridge: Cambridge University Press. 1992.

Management of the woman with chronic anovulation ANTHONY LAWRENCE and DAVID HEALY

Chronic ovulatory disorders are a major cause of infertility, together with tubal disease and male factor problems. It holds therefore that women with chronic anovulation are likely to present with infertility. Appropriate investigations will define various subgroups of patients with chronic anovulation. Ovulation induction will be appropriate in most cases, specifically tailored to individual requirements within these subgroups. Other barriers to conception may co-exist, and it is important that both partners be seen and assessed as part of the infertility work-up.

Diagnosis of anovulation

The diagnosis of chronic anovulation requires an assessment of ovulation as well as establishing the likely cause of anovulation. Historically, ovulatory symptoms include regular (21-35-day) menstrual cycles with Mittelschmirz pain at midcycle, awareness of cervical mucus changes through the cycle, classically described as runny 'egg-white' mucus at ovulation and becoming thicker and opaque in the second half of the cycle. In women thought not to be ovulating, pointers to causes of anovulation may include menopausal symptoms in those with ovarian failure, weight loss/stress in hypothalamic dysfunction, obesity, hirsutism in polycystic ovarian disease, and in hyperprolactinaemia/galactorrhoea, visual disturbances, headache and drug ingestion. Physical examination is often non-contributory for cause, although it does afford the opportunity to assess the breasts and perform a pelvic examination, including a pap smear - important in women planning pregnancy. The body mass index should be determined. Obese women are more resistant to treat69

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ment and obesity is a significant risk factor for pregnancy. Other issues, such as advanced maternal age, smoking and the impact of diseases such as diabetes in pregnancy, are addressed. The side-effects of planned treatment, particularly multiple pregnancy and ovarian hyperstimulation syndrome, are explained. Patients must be rubella immune prior to initiating ovulation induction. The only absolute evidence of ovulation is pregnancy. A presumptive diagnosis of ovulation is retrospective and based on a serum progesterone estimation greater than 30 nmol/1 seven to ten days prior to commencement of menstruation in a non-conceptual cycle. The progesterone measurement correlates well with the histology on endometrial biopsy. Several progesterone levels may need to be assessed in patients with irregular cycles to ensure one is well timed. Elevated progesterone levels may be seen in patients with premature luteinization of the dominant follicle. Clearly, patients with infrequent menses or amenorrhoea can be presumed to be anovulatory following exclusion of the rare Asherman's syndrome. Infrequent menses reflects infrequent ovulation. Ovulation induction will therefore enhance the probability of pregnancy by increasing the frequency of ovulation. Physiology An approach to the investigation and management of the anovulatory woman requires a broad understanding of the complex menstrual endocrine interrelationships involving the hypothalamic-pituitary-ovarian axis. Pulsatile release of GnRH from the hypothalamus maintains basal levels of gonadotrophin production from the anterior pituitary. Ovarian steroid production falls as the corpus luteum from the previous cycle regresses. The level of FSH rises during this early follicular phase, stimulating follicle growth which in turn actively secretes oestradiol. The level of LH rises as well during this follicular phase, also stimulating ovarian steroid production. Ovulation occurs when a critical level of oestradiol secretion is reached. Peaks of FSH and LH occur at this time. A clearly demonstrable LH surge occurs via a positive feedback loop. The LH surge initiates ovulation. Oestradiol levels fall in the preovulatory phase. The corpus luteum actively secretes oestradiol and progesterone in the luteal phase. Investigation and categorization of anovulation

Estimation of gonadotrophins, including prolactin, and vaginal ultrasound scan enable patients to be assigned to one of five anovulation subgroups.

Management of the woman with chronic anovulation Thyroid function should also be assessed. Although a very uncommon cause of anovulation, thyroid disorders will require specific therapy. A semen analysis will determine the potential fertility of the male partner. Although unlikely, it is important to exclude the possibility of pregnancy prior to commencing treatment. Hypergonadotrophic hypogonadism Elevated FSH and LH to within the menopausal range indicates ovarian failure, a natural occurrence at the end of reproductive life. Approximately 10% of all patients in the reproductive age group with secondary amenorrhoea have FSH levels in the postmenopausal range. Premature menopause will occur in 1% of women before the age of 40. This may be obvious or more subtle 'occult ovarian failure'. These patients, for all practical purposes, will not respond to ovulation induction and should be considered for a donor oocyte programme. The rare resistant ovary syndrome, diagnosed by the presence of primordial follicles on full-thickness ovarian biopsy, usually does not respond to gonadotrophin stimulation, and these patients will be candidates for a donor oocyte programme. Unpredictable spontaneous ovulation may occur in this group over years (O'Herlihy, Pepperell and Evans, 1980) but is of no practical significance in those patients presenting with infertility. Hypogonadotrophic hypogonadism Very low levels of pituitary gonadotrophin production secondary to hypothalamic suppression of GnRH release will result in suppression of ovarian ovulatory activity. Causes such as a significantly reduced BMI (less than 19 kg/m2) should be addressed as spontaneous resumption of ovulation may occur. Psychiatric disorders such as anorexia nervosa may present as chronic anovulation. Kallmann's syndrome and isolated deficiency of gonadotrophin production fall within this subgroup. Hypogonadotrophic patients are profoundly oestrogen deficient and will not bleed following a progestogen challenge. Apart from infertility concerns, as with patients with premature menopause, long-term risks of osteoporosis and accelerated cardiovascular disease will need to be addressed with oestrogen replacement. Eugonadotrophic hypogonadism This is a common subgroup of anovulatory women. These patients are often

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oligomenorrhoeic compared to the first two subgroups. There is a functional disturbance of the hypothalamic-pituitary-ovarian axis. Basal oestradiol levels are normal and patients will usually respond to a progestogen challenge. Polycystic ovarian disease

The commonest clinical entity of women with chronic anovulation as defined by high-resolution vaginal ultrasound scan presents a diverse clinical picture. Ultrasound scan criteria are: (a) ovarian volume greater than 8 cm3 (b) more than eight ovarian cysts 2-8 mm in diameter (c) echogenic ovarian stroma. Patients usually have one or more markers, either clinical - hirsutism/acne, obesity - or biochemical-elevated LH or LH: FSH ratio, elevated concentrations of testosterone or androstenedione. Adams et al. reported a 57% incidence of polycystic ovarian disease (PCOD) in patients with chronic anovulation (Adams, Poison and Franks, 1986). Hyperprolactinaemia

Elevated prolactin levels suppress pulsatile secretion of GnRH and also LHmediated oestradiol production. Treatment and monitoring

The aim of ovulation induction is the development of a single dominant follicle, subsequent ovulation leading to a singleton pregnancy. This physiological approach contrasts with assisted reproductive technology procedures where multiple codominant folliculogenesis is induced to maximize oocyte retrieval. Best quality oocytes or embryos can then be chosen to transfer to maximize conception with acceptable multiple pregnancy rates and reduced risk of ovarian hyperstimulation syndrome. Clomiphene citrate

Anovulatory women with either eugonadotrophic hypogonadism or polycystic ovarian disease are suitable for ovulation induction using clomiphene citrate. This drug has been available for several decades and should be considered asfirst-linetherapy in these anovulatory subgroups. Clomiphene's principal pharmacological action is thought to be at the level of the hy-

Management of the woman with chronic anovulation pothalamus, stimulating production and release of GnRH. Clomiphene has weak biologic oestrogenic activity, occupying oestradiol receptors and blocking the true endogenous feedback signal. It may act to reduce oestradiol intracellular receptors. FSH and LH production is enhanced, with the direct effect of ovulation induction. Patients with hypogonadotrophic hypogonadism already have a lowered oestrogen feedback signal and would not be expected to respond reliably. Treatment should commence in the early follicular phase. The initial dose in eugonadotrophic patients is 50 mg. Those patients with polycystic ovarian disease may be particularly sensitive and response at 25 mg should be assessed. In patients who are amenorrhoeic or severely oligomenorrhoeic, treatment commences following an induced withdrawal bleed (Provera 10 mg per day for ten days). Patients likely to respond to clomiphene citrate therapy will have endogenous oestradiol production and should therefore bleed following progestogen challenge. Treatment may commence in the absence of a withdrawal bleed, following exclusion of pregnancy in patients who are amenorrhoeic or severely oligomenorrhoeic, without impacting adversely on pregnancy rates. Timing of treatment, however, is based on the menstrual bleed and simplifies management. The commencement dose of clomiphene is administered daily for five days. To avoid confusion, day one is considered the first day of full menstrual flow if this occurs before 6 p.m. Premenstrual spotting, common in patients with luteal phase inadequacy, should be ignored. There is no significant difference in pregnancy rates or incidence of multiple pregnancy based on commencement day, though theoretically selection of the dominant follicle occurs from day five and we would therefore favour a day five to nine schedule. A basal body temperature chart provides a pictorial representation of the treatment cycle, though monophasic patterns do occur in ovulatory cycles. Instructions for treatment may be written directly onto the chart to minimize confusion. Ovulation usually occursfiveto ten days after the last clomiphene tablet, and serum progesterone estimation as a marker for ovulation is determined at approximately day 21. Intercourse is permitted on alternate days but is best timed day 10-16. Clomiphene dosage should be increased by 50 mg per day to a maximum of 200 mg per day in successive anovulatory cycles. Patients with an elevated BMI are often more resistant to ovulation induction, requiring higher dosage to induce ovulation. The majority of clomiphene pregnancies are achieved in the first three ovulatory treatment cycles and the likelihood of pregnancy after six ovulatory cycles is low. As clomiphene therapy is simple and low risk, it is reasonable to

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withhold tubal assessment by laparoscopy in patients who give no clear history of factors linked to tubal disease, until at least three ovulatory nonconceptual cycles. Conception rates per ovulatory cycle are close to that of the normal fertile population and ovulation rates approach 80%. There is an approximately 6% incidence of twins; higher multiples are rare. Numerous investigators have reported that clomiphene adversely affects cervical mucus quality and may delay endometrial maturation. This anti-oestrogenic effect is due to competition with oestradiol for receptor sites. One hypothesis to explain the achieved conception rate is that the increased levels of oestradiol often achieved in clomiphene induced cycles may counterbalance this anti-oestrogenic property. In refractory polycystic ovarian disease patients, studies suggest in the majority a failure of follicular maturation (Poison et al., 1989). Hypotheses to explain this resistance to clomiphene therapy include diminished FSH biologic activity or FSH effect modulation at the ovarian level by paracrine factors (Franks et al., 1988). PCOD patients have an altered endocrine milieu with elevated levels of local androgens. This may increase resistance to clomiphene therapy. In patients with high, normal or elevated testosterone or dehydroepiandrosterone, the use of steroid therapy (dexamethasone 0.5 mg nocte) to suppress adrenal androgen production has been shown to be worthwhile. In those resistant PCOD patients with evidence of follicular development but failure of the LH surge, the addition of 5000 iu of hCG may trigger ovulation. The timing of hCG administration is best assessed using vaginal ultrasound measurements of follicle growth combined with serum oestradiol levels. Side-effects of clomiphene therapy are generally minor and well tolerated and include hot flushes, abdominal bloating and discomfort, nausea and vomiting, and, rarely, visual disturbances. Serious side-effects such as ovarian hyperstimulation syndrome (see below) and large ovarian cyst formation are much less common, although more likely with PCOD patients. A case of haemorrhage into the anterior pituitary during pregnancy following clomiphene ovulation induction has been reported (Nagulesparan and Roper, 1978). The incidence of side-effects is not dose related. At the review appointment following completion of thefirsttreatment cycle, the basal body temperature pattern combined with progesterone estimation will determine whether ovulation has occurred. A sustained temperature rise in an ovulatory cycle is suggestive of pregnancy. This can be confirmed with a serum beta-hCG estimation. Side effects should be assessed and patient compliance with therapy determined. Clinical examination is usually of little value

Management of the woman with chronic anovulation unless symptoms suggest a complication of a functional ovarian cyst. Most ovarian cysts will resolve prior to the next treatment cycle. There is no evidence that patients need to break between successive treatment cycles. Human menopausal gonadotrophin

Patients refractory to clomiphene therapy will be candidates for human menopausal gonadotrophin (hMG) ovulation induction. This preparation, obtained from the urine of menopausal women, has been available worldwide since 1963 (Crooke et al., 1963). Human menopausal gonadotrophin contains 75 iu FSH/75 iu LH. Recombinant gonadotrophins are becoming widely available. In contrast to clomiphene, hMG acts by direct ovarian stimulation. The therapeutic range is narrow and careful monitoring is required to avoid risks of high multiple pregnancy and ovarian hyperstimulation syndrome. Even so, there was a 13% incidence of twins in our series. The hMG preparations are distinct from human pituitary gonadotrophins (hPG) (Gemzell, Diczfalusy and Killinger, 1958) in use until the mid-1980s, and now directly linked to Creutzfeld-Jakob disease (Lazarus, 1985). Patients must be aware of the risks of hMG use and the need for close monitoring to optimize treatment. The patient/nurse/medical practitioner link at Monash provides a model for patient management. This ovulation induction (01) clinic co-ordinates patient care through a dedicated 01 nurse overseen by specialist endocrinologists. The nurse practitioner's role encompasses an information discussion interview prior to commencing treatment, where issues including the role and responsibility of the 01 clinic and its staff, treatment and its effects and possible side effects and costs are discussed. Each day patients are seen, ultrasound and blood tests are organized, results graphed and discussed with the endocrinologists on duty and patients informed of their treatment progress and dosage changes. The 01 nurse's direct link to patients provides ongoing support, counselling, information and encouragement. Various protocols have been devised for hMG ovulation induction. The Monash protocol (see Table 6.1) aims to provide tight control of ovarian stimulation through smaller increments in dosage based on oestradiol and ultrasound monitoring. Care is needed to ensure development of a small number of dominant follicles while avoiding stimulation of a large number of intermediate follicles thought to contribute to ovarian hyperstimulation syndrome (OHSS) development. Patients may have treatment in successive cycles, but close monitoring is still ongoing in each cycle as response to drug stimulation may differ. Average duration of hMG treatment is five to ten days. Patients with PCOD are often particularly sensitive, and treatment may need

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Table 6.1. hMG ovulation induction protocol All women commence treatment after a spontaneous or progestogen-induced withdrawal bleed. Baseline ultrasound scan day 2-5 of cycle. Ovarian cysts >20 mm, ultrasound scan repeated after one week. Women with normal BMI (20-26 kg/m2) commence treatment with hMG 75 iu daily. Women with BMI >26 kg/m2 commence treatment with hMG 112 iu. This commencement dosage regime is for the first treatment cycle. In subsequent cycles, the commencement dose is determined by previous response. Serum oestradiol assayed every second day after four days till level 600 pmol/1, then daily. hMG dose continued for five days; if there is no doubling in serum oestradiol concentration, dose increased by 1/2 ampoule. Further increase in hMG dosage atfive-dayintervals by 1/2 ampoule based on oestradiol response. Repeat ultrasound scan when serum oestradiol >740 pmol/1. On day of ultrasound scan, assay oestradiol, progesterone, luteinizing hormone. Assuming follicle growth of 2 mm/day, subsequent ultrasound scan may be arranged for expected day of hCG administration. hCG administered that evening if ultrasound scan confirms the presence of one to three follicles with mean diameter > 14 mm. Cycle abandoned if more than three follicles present > 14 mm; hCG withheld and no intercourse for one week. Aim for serum E2 approximately 1200 pmol/1 at hCG administration. No hMG on this day, assay oestradiol, progesterone, luteinizing hormone. Intercourse on the evening of hCG administration and subsequent three evenings. If receiving donor insemination, insemination to take place on the first of two mornings after hCG administration. Oestradiol and progesterone estimations on day three, six, nine post hCG. 1000 iu hCG support in luteal phase on days three and six if no evidence of hyperstimulation and day nine if progesterone does not reach 30 nmol/1 or has fallen below 20 nmol/1. If treatment unsuccessful, baseline ultrasound scan performed day two to four of the next cycle prior to recommencing treatment. If no period two weeks after hMG administration, assay oestradiol, progesterone and jShCG.

Management of the woman with chronic anovulation

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to be extended over two to three weeks to produce a single dominant follicle gradually. Laparoscopic tubal assessment is indicated after three ovulatory non-conceptual cycles and is a prerequisite prior to ovulation induction attempts in patients at risk for tubal disease. Patients achieving a pregnancy require ultrasound scanning at six weeks' gestation to determine the foetal number and also location as there is a reported link to ectopic pregnancy (McBain et al., 1980). Patients with PCOD are at particular risk of multifollicular development and ovarian hyperstimulation syndrome related to elevated levels of LH and androgens leading to premature luteinization, follicular atresia and possible meiotic disturbance (Stanger and Yovich, 1985). The use of pure FSH would seem theoretically sound and preferable (Anderson et al., 1989), but trials to date have shown no significant decrease in multifollicular growth and ovarian hyperstimulation syndrome in conventional dose regimes (Kelly and Jewelewicz, 1990). Dale et al. propose a low-dose FSH protocol in order to find the threshold dose for unifollicular maturation. Their incidence of ovarian hyperstimulation syndrome was very low, although 17% of cycles were cancelled due to multifollicular development (Dale et al., 1993). FSH does not appear to confer any significant benefit over hMG in cycles where judicious dose increase follows careful monitoring of response. Nor is pulsatile GnRH analogue therapy as efficacious as hMG ovulation induction in polycystic ovarian syndrome (Saffan and Seibel, 1992). Pituitary desensitization with GnRH analogues to convert PCOD patients to a hypogonadotrophic state followed by hMG or FSH ovarian stimulation does not avoid a multifollicular response (Tanbo et al., 1990), and the additional weeks of pretreatment required for desensitization appear unwarranted. Schoot et al. reviewed the available trial data and noted no clear advantage of the addition of GnRH analogues to hMG or FSH ovulation induction (Schoot et al, 1992). High concentrations of LH in the late follicular phase in patients with PCOD have been thought to be a key factor in the higher spontaneous abortion rate seen in pregnant patients with polycystic ovarian disease compared with the background population (Homburg et al., 1988). Co-treatment with GnRH agonists prior to hMG ovulation induction has been shown to be worthwhile (Homburg et al., 1993), although McClure et al. found in their retrospective study no evidence of a relationship between serum LH levels and pregnancy outcome and, by contrast, that age and basal serum oestradiol were predictive of pregnancy outcome (McClure et al., 1993). The cumulative pregnancy rates for patients with PCOD and other forms of clomiphene citrate-resistant ovulation treated between 1985 and 1991 at the

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S>

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1 30 o

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•PCOD patients n = 141 ° All other patients n =126

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0 1 2 3 4 5 6 7 8 Cycles of human gonadotrophin therapy

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Figure 6.1 Cumulative pregnancy rates in PCOD and other patients receiving hMG therapy. Prince Henry's Institute of Medical Research and the Monash Department of Obstetrics and Gynaecology are presented in Figure 6.1. Therapy with hMG is clearly highly successful in achieving ovulation and pregnancy. Patients who fail to conceive in six ovulatory cycles should be considered for assisted reproductive technology, either IVF or GIFT. Those patients who fail to ovulate are also candidates for assisted reproductive technology, while resistant PCOD patients may respond to ovarian drilling (Gjonnaess, 1984). Bromocriptine Elevated prolactin levels are a common disorder in patients with chronic anovulation. This anovulatory subgroup presents as infertility, with menstrual disturbances ranging from oligomenorrhoea of varying severity through to secondary amenorrhoea. Approximately 22% of women with secondary amenorrhoea have elevated prolactin levels (Pepperell, 1978). One-third of patients with hyperprolactinaemia will have demonstrable galactorrhoea. Pulsatile prolactin secretion from the pituitary is under tonic inhibitory control from hypothalamic dopamine production. Elevated prolactin inhibits GnRH release and also LH-mediated oestradiol production by the ovary. Patients with hyperprolactinaemia demonstrated on two occasions should have pituitary imaging once hypothyroidism and drugs associated with hyperprolactinaemia have been excluded. MRI is the imaging technique of choice

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but is expensive, and coned views of the pituitary fossa or CT scan will be sufficient in most clinical settings. It is important to exclude the presence of a macroadenoma as rapid tumour expansion may occur during pregnancy. Macroadenomas may require a treatment combination of surgery, irradiation and drug therapy - parenteral and vaginal routes are available. Most patients will ovulate at a treatment dose of 5 mg to 10 mg per day, the majority conceiving within six ovulatory cycles (Pepperell, McBain and Healy, 1977). Some patients may require the addition of clomiphene citrate once prolactin levels have normalized should ovulation fail to occur. Bromocriptine may be discontinued in pregnancy in patients with microadenomas with minimal risk of significant tumour growth. This therapy may be continued in pregnancy if there is a macroadenoma, particularly with suprasellar extension. There is no evidence of teratogenicity. Pulsatile gonadotrophin releasing hormone (GnRH)

Patients with hypogonadotrophic hypogonadism will be suitable for ovulation induction using pulsatile GnRH analogue therapy. This patient subgroup has a disturbance of GnRH release and GnRH treatment replaces this functional loss. This drug must be administered as a pulse dose via a miniaturized portable infusion device (Leyendecher, Wildt and Hansmann, 1980) either intravenously or subcutaneously to avoid down regulation of pituitary receptors. As with hMG ovulation induction, close monitoring of treatment via a nurse/patient/practitioner link will be required to optimize management. Nursing staff oversee pump refilling, needle insertion and changing. Pulsatile GnRH stimulates pituitary gonadotrophin release, initiating and maintaining normal menstrual cycling, with intact positive and negative feedback loops. This physiological approach importantly reduces the incidence of ovarian hyperstimulation syndrome. The multiple pregnancy rate theoretically should be that of the normal background fertile population, though it has been reported as higher in some series (Braat et al., 1989). Disadvantages include cost and inconvenience of the delivery units and the risk of endocarditis with the intravenous route of administration. Subcutaneous GnRH has produced a normal conception rate in our 01 clinic patients. Ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome is a serious and potentially life-threatening iatrogenic complication of ovulation induction complicated by ovarian

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enlargement and third space fluid accumulation with intravascular volume depletion and haemoconcentration. Mild, moderate and severe categories are recognized in various classifications. Patients with PCOD are particularly sensitive to the development of OHSS, perhaps related to increased numbers of intermediary follicles. Women who achieve pregnancy are also at increased risk (Tulandi, Mclnnes and Arronet, 1984). The incidence of severe OHSS following ovulation induction with exogenous gonadotrophins is estimated between 0.25% and 0.9% (RCOG Guidelines No. 5, 1995). The biochemical basis is yet unproved, although evidence exists linking vascular endothelial growth factor to increased capillary permeability in this syndrome (McClure et al., 1994). Serious complications include development of ascites, pleural and pericardial effusions, thromboembolism, renal impairment and adult respiratory distress syndrome. Principles of management are symptomatic relief, correction of haemoconcentration, prevention of thromboembolism and maintenance of cardiorespiratory and renal function (RCOG Guidelines No. 5, 1995). The reader is referred to the RCOG Guidelines for detailed management of this syndrome. References

Adams, J., Poison, D.W. and Franks, S. (1986). Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. British Medical Journal 293: 355-9. Anderson, R.E., Gragun, J.M., Chang, R.J., Stanczyk, F.Z. and Lobo, R.A. 1989. A pharmodynamic comparison of urinary follicle stimulating hormone and human menopausal gonadotrophin in normal women and polycystic ovary syndrome. Fertility and Sterility 52: 216-20. Braat, D.D.M., Ayalon, D., Blunt, S.M., Bogchelman, D., Coelingh Brennink, J.J.T., Handelsman, D.J., Heinemann, M.J., Lappohn, R.E., Lorijn, R.H.W., Rolland, R., Willemsen, W.M.P. and Schoemaker, J. 1989. Pregnancy outcome in luteinizing hormone-releasing hormone induced cycles: a multicentre study. Gynaecological Endocrinology 3: 35—44.

Crooke, A.C., Butt, W.R., Palmer, R.F., Morris, R., Edwards, R.L. and Anson, C.J. 1963. Clinical trial of human gonadotrophins. 1. The effect of pituitary and urinary follicle stimulating hormone and chorionic gonadotrophin on patients with idiopathic secondary amenorrhoea. Journal of Obstetrics and Gynaecology British Commonwealth 70: 604. Dale, P.O., Tanbo, T., Lunde, O. and Abyholm, T. 1993. Ovulation induction with low-dose follicle-stimulating hormone in women with the polycystic ovary syndrome. Acta Obstetrica Gynaecologica Scandanavica 72: 43-6. Franks, S., Mason, H.D., Poison, D.W., Winstron, R.M.L., Margara, R., and Reed, M.J. 1988. Mechanism and management of ovulatory failure in women with polycystic ovary syndrome. Human Reproduction 3: 531-4. Gemzell, C.A., Diczfalusy, E. and Killinger, K.G. 1958. Clinical effects of human pituitary follicle-stimulating hormone (FSH). Journal of Clinical Endocrinology

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and Metabolism 18: 1333-48. Gjonnaess, H. 1984. Polycystic ovarian syndrome treated by ovarian electrocautery through the laparoscope. Fertility and Sterility 42: 20-5. Homburg, R., Armar, N.A., Eshel, A., Adams, J. and Jacobs, H.S. 1988. Influence of serum luteinizing hormone concentrations on ovulation, conception and early pregnancy loss in polycystic ovary syndrome. British Medical Journal 297: 10246. Homburg, R., Levy, T., Berkovitz, D., Farcht, J., Feldberg, D., Ashkenazi, J. and Ben-Rafael, Z. 1993. Gonadotrophin-releasing hormone agonist reduces the miscarriage rate for pregnancies achieved in women with polycystic ovarian syndrome. Fertility and Sterility 59: 527-31. Kelly, A.C. and Jewelewicz, R. 1990. Alternate regimes for ovulation induction in polycystic ovarian disease. Fertility and Sterility 54: 195-201. Lazarus, L. 1985. Suspension of the Australian Human Pituitary Hormone Programme. Medical Journal of Australia 143: 57-9. Leyendecher, G., Wildt, L. and Hansmann, M. 1980. Pregnancy following chronic intermittent administration of GnRH by means of a portable pump - a new approach to the treatment of hypothalamic amenorrhoea. Journal of Clinical Endocrinology and Metabolism 51: 1214-16. McBain, J.C., Evans, J.H., Pepperell, R.J., Robinson, H.P., Smith, M.A. and Brown, J.B. 1980. An unexpectedly high rate of ectopic pregnancy following the induction of ovulation with human pituitary and chorionic gonadotrophin. British Journal of Obstetrics and Gynaecology 87: 5-9. McClure, N., Healy, D.L., Rogers, P.A.W., Sullivan, J., Beaton, L., Haning, R.V.J., Connolly, D.J. and Robertson, D.M. 1994. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet ii: 235-6. McClure, N., McDonald, J., Kovacs, G.T., Healy, D.L., McLoud, P.I., McQuinn, B. and Burger, H.G. 1993. Age and follicular phase estradiol are better predictors of pregnancy outcome than luteinizing hormone in monotropin ovulation induction for anovulatory polycystic ovarian syndrome. Fertility and Sterility 59: 729-33. Nagulesparan, M. and Roper, J. 1978. Haemorrhage into the anterior pituitary during pregnancy after induction of ovulation with clomiphene. British Journal of Obstetrics and Gynaecology 85: 153-5. O'Herlihy, C , Pepperell, R.J. and Evans, J.H. 1980. The significance of FSH elevation in young women with disorders of ovulation. British Medical Journal 281: 1447— 9. Pepperell, R.J. 1978. In Clinical Investigation and Assessment of Amenorrhoea. Proceedings of the 6th Asia and Oceania Congress of Endocrinology, Singapore, Vol. 2, p. 389. Singapore: University of Singapore. Pepperell, R.J., McBain, J.C. and Healy, D.L. 1977. Ovulation induction with bromocriptine (CB 154) in patients with hyperprolactinaemia. Australian & New Zealand Journal of Obstetrics and Gynaecology 2: 522. Poison, D.W., Kiddy, D.S., Mason, H.D. and Franks, S. 1989. Induction of ovulation with clomiphene citrate in women with polycystic ovary syndrome; the difference between responders and non-responders. Fertility and Sterility 51: 30-4. RCOG Guidelines No. 5, 1995. London: Royal College of Obstetrics and GynaecologySaffan, D.S. and Seibel, M.M. 1992. Value of subcutaneous and intravenous pulsatile gonadotrophin releasing hormone in polycystic ovary disease. Journal of Repro-

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ductive Medicine 37: 545-51. Schoot, D.C., Pijlman, B., Stijnen, T. and Fauser, B.C. 1992. Effects of gonadotrophin releasing hormone agonist addition to gonadotrophin induction of ovulation in polycystic ovary syndrome patients. European Journal of Obstetrics, Gynecology and Reproductive Biology 45: 53-8. Stanger, J.D. and Yovich, J.L. 1985. Reduced in vitro fertilisation of oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. British Journal of Obstetrics and Gynaecology 92: 385-90. Tanbo, T., Dale, P.O., Kjekshus, E., Haug, E. and Abyholm, T. 1990. Stimulation with human menopausal gonadotrophin versus follicle-stimulating hormone after pituitary suppression in polycystic ovarian syndrome. Fertility and Sterility 53: 798-803. Tulandi, J., Mclnnes, R.A. and Arronet, G.H. 1984. Ovarian hyperstimulation syndrome following ovulation induction with HMG. International Journal of'Fertility 29\ 113-17.

Cervical factor, unexplained subfertility and artificial insemination with husband sperm GABOR T. KOVACS and BEVERLEY VOLLENHOVEN

Thefirstrecorded AIH in the human was allegedly performed by Doctor John Hunter during the late 18th century. His nephew, Sir Everard Home, reported a normal pregnancy and delivery as a result of the procedure in the wife of a London linen merchant (Finegold, 1980). A new wave of scientific interest in AIH emerged during the 1970s. This was probably stimulated by the emerging attention paid to subfertility, and the inability of any preparations consistently to improve semen quality in subfertile men. In 1978, the First International Symposium on Artificial Insemination Homologous and Male Subfertility was held in Bordeaux, France, and was attended by many distinguished clinicians from all over the world (Emperarire and Audebert, 1978). Subsequently, the indications for AIH have been classified intofivegroups. 1. Mechanical problems in the male, i.e. impotence, hypospadias, premature ejaculation, or retrograde ejaculation. 2. Mechanical problems in the female such as vaginismus or prolapse. 3. 'Cervical hostility'. 4. Impaired semen quality - of volume, concentration, motility, morphology or the presence of antibodies. 5. Unexplained subfertility.

Mechanical problems in the male

If there is inability by the male to deposit semen at the top of the vagina, conception is unlikely to occur. By artificially depositing semen at the exocervix during the fertile phase, the problem is easily overcome and pregnancy results. In the case of retrograde ejaculation, the semen needs to be recovered from the urine after ejaculation and, to prevent acid damage, the urine needs 83

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to be alkalinized in advance (Mahadevan, Leeton and Trounson, 1981). The prognosis for AIH is excellent in these situations when there is only a mechanical problem to overcome. Mechanical problems in the female

Severe vaginismus is usually psychogenic, and requires prolonged therapy. It is sometimes an alternative for AIH to be undertaken with a fine cannula and syringe. This often results in conception, but of course the underlying problem still exists. Similarly, in the case of severe uterine prolapse, the mechanics of introducing sperm into the cervix may not be possible by intercourse, but can be overcome by insemination. Severe obesity is a similar indication for AIH. Cervical hostility

The role of cervical factor has been debated since Marion Sims described the postcoital test (Sims, 1866). In this test, a specimen of cervical mucus was examined some hours after intercourse, and if the presence of motile sperm was detected, three conclusions could be drawn. First, that the man was not barren; second, that the cervix was in the right position to catch the semen; and third, that the secretions were not inimical to the sperm. If none of the sperm were moving, it was concluded that the secretions were hostile, and AIH into the uterine cavity was recommended. Unfortunately, although case reports for success have been reported, no properly controlled trials have been carried out to show the value of this treatment. Another form of sperm-mucus interaction testing was the slide contact test of Miller and Kurzrok (1932). In this test, cervical mucus is placed on a microscope slide and a drop of semen is placed alongside and covered with a coverslip so that contact occurs between the two specimens. The migration of sperm into the mucus is then observed under the microscope. A more sophisticated sperm-mucus interaction test was described by Kremer in 1965. In this in-vitro test, a sample of cervical mucus is aspirated into a capillary tube, which is then inserted into a reservoir of semen, producing a 'sperm penetration meter'. The migration of sperm along the mucus column is then observed, and the distance of sperm migration over a given time, the density of sperm penetration at different places, and the velocity of the spermatozoa are assessed as parameters of mucus penetration. A further refinement of the Kremer test is to test both the male and female partner with control mucus/sperm to define whether the problem is due to sperm or mucus. The value of the postcoital test (PCT) was questioned (Kovacs, Newman

Cervicalfactor, unexplained subfertility and AIH and Henson, 1978) when it was found that several normal couples had an 'abnormal' PCT. Furthermore, even if cervical hostility does exist, its treatment is debatable. Clinicians who believe that the cervix is purely a filter advocate intrauterine AIH as a way of overcoming cervical hostility. Those who believe in the 'reservoir' theory would argue that bypassing the cervix does not achieve anything. Suffice it to say that although there are case reports of successful AIH, there are no controlled trials to show that AIH is of benefit if the cause of subfertility is thought to be related to cervical hostility. In 1996, a couple consisting of a regularly ovulating female with patent fallopian tubes and a male partner with semen parameters in the normal range with a history of subfertility, can be diagnosed as an 'idiopathic subfertility'. The treatment offered to such a couple is assisted reproductive technology (ART) in the form of IVF or GIFT. If tests of sperm-mucus interaction are carried out and cervical hostility is suspected, in the absence of the proven value of AIH, the treatment that can be offered is ART. Thus, as the result of mucus-sperm interaction testing does not change the clinical management, there is probably little sense in carrying out such tests. As the cervical factor is not part of our routine investigations, the Kremer test is only utilized as part of the assessment of the significance of male antisperm antibodies in the seminal fluid (Kremer, 1965; see Chapter 4). Subnormal semen quality and AIH

The use of AIH in cases of suboptimal sperm quality is based on the principle that if there are insufficient sperm in the ejaculate, or if they are submotile, injecting them through the cervix into the uterine cavity may make it easier for them to get to the ovum in the fallopian tube, and result in conception. Again, although there are many case reports, there is no properly controlled prospective trial to show that it is effective. In a detailed review of intrauterine insemination (IUI) (Allen et al., 1985), it was concluded that IUI did not appear to improve pregnancy rates significantly for men with oligospermia. The application of sperm preparation techniques called sperm washing in conjunction with IUI used for in-vitro fertilization showed promising initial results in a randomized trial (Kerin et al., 1984), albeit on a fairly small sample. However, two prospective randomized trials of washed sperm IUI both showed no increase in conception rates when compared to intercourse (Hughes, Collins and Garner, 1987; Ho et al, 1989). The next ray of sunshine was the combination of controlled ovarian hyperstimulation (COH) in conjunction with IUI (Dodson et al, 1987). This technique enabled the possibility of stimulating the development of several

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oocytes, and placing in the uterine cavity large numbers of motile sperm, to try to imitate GIFT where there is an increase in the number of gametes (both male and female) in the fallopian tubes. A retrospective study of 751 cycles of treatment in 322 couples by Chaffkin and colleagues in 1991 found that the combination of IUI and COH resulted in per cycle fecundity of 19.6% versus 6.3% for stimulation alone, and 3.4% for IUI. When the patients were analysed with respect to diagnostic groups of male factor, cervical factor, endometriosis or unexplained fertility, the same trend was observed. A study by Friedman and colleagues (1991) suggested that the per cycle fecundity drops to insignificant levels after the fourth cycle, and they recommend that only three or at most four cycles of this treatment be administered. It is therefore concluded that three cycles of AIH with IUI may be a reasonable alternative for couples with unexplained subfertility, mild to moderate abnormalities of semen quality as long as more than one million motile sperm can be isolated (Dodson and Haney, 1991), mild endometriosis and unexplained subfertility, with success rates intermediate between IVF and natural intercourse. It is essential that IUI be combined with COH using gonadotrophins; clomiphene citrate is of no benefit. Any more than three cycles of treatment are not indicated. If pregnancy has not resulted, ART procedures need to be undertaken. Risks of COH/IUI The commonest complication of COH is multiple pregnancy with rates of between 25% and 30% (Wallach 1991). The risk of ovarian hyperstimulation syndrome (OHS) requiring hospitalization occurs in about 1%. The use of intrauterine catheterization may lead to pelvic inflammatory disease, but the incidence of this is unknown, due partly to the difficulty of diagnosis. Finally, ectopic pregnancy rates of up to 5% have been reported (Dodson and Haney, 1991). References Allen, N.C., Herbert, CM., Maxson, W.S., Rogers, B.J., Diamond, M.P. and Wentz, A.C. 1985. Intrauterine insemination: a critical review. Fertility and Sterility 44: 569-80. Chaffkin, L.M., Nulsen, J.C., Luciano, A.A. and Metzger, A.A. 1991. A comparative analysis of the cycle fecundity rates associated with combined human menopausal gonadotropin hMG and intrauterine insemination IUI versus either hMG or IUI alone. Fertility and Sterility 55: 252-7. Dodson, W.C. and Haney, A.F. 1991. Controlled ovarian hyperstimulation and intrauterine insemination for the treatment of infertility. Fertility and Sterility 55: 457-67.

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Dodson, W.C., Whitesides, D.B., Hughes, C.L., Easley, H.A. and Haney, A.F. 1987. Superovulation with intrauterine insemination in the treatment of infertility: a possible alternative to gamete intrafallopian transfer and in vitro fertilization. Fertility and Sterility, 48: 441. Emperarire, J.C., Audebert, A. 1978. Proceedings of First International Symposium on Artificial Insemination Homologous and Male Subfertility, Bordeaux, France. Finegold, J.W. (ed.) 1980. Artificial Insemination with Husband Sperm, pp. 4-6. Springfield: Charles C Thomas. Friedman, A.J., Juneau-Norcross, M., Sedensky, B., Andrews, N., Dorfman, J. and Cramer, D.W. 1991. Life table analysis of intrauterine insemination pregnancy rates for couples with cervical factor, male factor, and idiopathic infertility. Fertility and Sterility 55: 1005-07. Ho, P.C., Poon, I.M.L., Chan, S.Y.W., Wang, C. 1989. Intrauterine insemination is not useful in oligoasthenospermia. Fertility and Sterility 51: 682-5. Hughes, E.G., Collins, J.P., Garner, P.R. 1987. Homologous artificial insemination for oligoasthenospermia: a randomised controlled study comparing intracervical and intrauterine techniques. Fertility and Sterility 48: 278-81. Kerin, J.F.P., Peek, J., Warnes, G.M., Kirby, C, Jeffrey, R., Matthews, CD. and Cox, L.W. 1984. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet 1: 533-5. Kovacs, G.T., Newman, G.B. and Henson, G.L. 1978. The postcoital test: What is normal? British Medical Journal 1:818. Kremer, J. 1965. A simple sperm penetration test. InternationalJournal of Fertility 10: 209-15. Mahadevan, M., Leeton, J.F. and Trounson, A.O. 1981. Noninvasive method of semen collection for successful artificial insemination in a case of retrograde ejaculation. Fertility and Sterility 36: 243-7. Miller, E.G., and Kurzrok, R. 1932. Biochemical studies of human semen. American Journal of Obstetrics and Gynecology 24: 19-26. Sims, J.M. 1866. Clinical Notes on Uterine Surgery with Special References to the Management of the Sterile Condition. London: Robert Hardwicke. Wallach, E.E. 1991. Gonadotrophin treatment for the ovulatory patient - the pros and cons of empiric therapy for infertility. Fertility and Sterility 55: 478-80.

8 In-vitro fertilization: indications, stimulation and clinical techniques JAMES McK. TALBOT and MARK LAWRENCE

Edwards and Steptoe first described the technique for in-vitro fertilization (IVF) and embryo transfer (ET) in 1976 and the subsequent births of two normal babies in 1978 (Steptoe and Edwards, 1978). Since then, the success rate of the system has been improved (to 30%) by the use of fertility drugs to provide more oocytes and prematuration to mature the oocytes before fertilization (Trounson et al, 1981). The techniques are now used in 53 countries throughout the world. In 1993, the results of 492 units from all over the world were collected from national surveys and registers. Since 1985, more than 53 635 women had been treated and 34 316 babies had been born from 224473 treatment cycles, following more than 160518 transfer cycles. Only about 65-75% of all resulting pregnancies attained live births. The remainder ended with spontaneous abortions (26%), or ectopic pregnancies (5.54%). The multiple pregnancy rate (22%) was higher than the normal population and contributed to higher rates of preterm deliveries and perinatal mortality. No increased incidence of chromosomal alterations and malformations were noted during the years (2.25%). Since the birth of the first IVF baby, tremendous developments have occurred regarding the indications for assisted reproductive technology (ART). For example, the dramatic development concerning male infertility which initially was considered to involve a small fraction of patients benefiting from IVF, now, with the development of intracytoplasmic sperm injection (ICSI), involves up to 35% of started cycles. However, a substantial group of patients still remain unhelped. There is a reduction in success rates in women between the ages of 35 and 39 years, and a further reduction in women older than 40 years of age. There is also doubt about the cost-benefit of ART versus conventional therapy for subfertility. In women with unexplained infertility, hMG super ovulation treatment is as successful, less expensive, and carries a smaller risk than the surgical approach

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of ART. However, IVF is more expensive than three cycles of hMG and intrauterine insemination (IUI). The indications for treatment were broadened over the years and the procedure became a final step for diagnosis and treatment of unexplained infertility. Use of gonadotrophin-releasing hormone agonists (GnRHA) has improved pregnancy rates, reduced blood sampling and prevented natural ovulation. The disadvantages of stimulated cycles include higher risks of multiple pregnancy and of hyperstimulation, and side effects of the drugs. IVF/ET has become a relatively safe procedure but should be used only when indicated. To obtain the best results, quality control should be regulated by professional and/or public associations and further research is necessary in order to improve success rates. Natural cycles or immature egg collection (IOC) may become alternatives to the use of the stimulated cycle. Multiple pregnancies may be reduced, by reducing the number of eggs or embryos transferred. Embryo freezing has made an important contribution to overall pregnancy rates by enabling patients to use excess eggs and embryos. The social and legal concerns resulting from the use of frozen embryos requires new ethical and legal consideration. Donor eggs have made a contribution to achieving pregnancy in women with absent or repeated poor-quality eggs and have increased the chance of conception in women over the age of 40. Micromanipulation of sperm and eggs has enabled fertilization and conception when sperm are defective in quantity and quality. Sampling of cells in early embryos enables genetic diagnosis and may be used in selecting chromosomally normal embryos in IVF procedures or in couples at risk of recessive genetic disease. In summary, ART has developed over a decade to become useful for couples with infertility which cannot be cured by simple treatments. The birth rates are comparable to natural conception and the incidence of congenital malformations is not increased. The costs and complexities of treatment have been reduced, which in turn has reduced the stress and social inconvenience of therapy. Problems related to the birth risk of multiple pregnancy and the use of the stimulated cycle are being reduced as new techniques for severe male infertility and the detection of genetic abnormalities in the embryo have been introduced.

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Tubal infertility Tubal infertility is defined as persistent bilateral tubal obstruction, absence of tubes or tubal damage which has resulted in a period of infertility of more than 12 months' duration. Possible therapy of tubal infertility includes hysterosalpingogram, chromatography at the time of laparoscopy, selective transcervical tubal cannulation and falloposcopy. Each of their effects is restricted to the mechanical defect that may involve the tubal pathology. Tubal surgery, when indicated, allows for natural conception and more than one pregnancy can be achieved as a result (Smalldrudge and Tait, 1993). It should be considered the first-line treatment for proximal tubal disease, reversal of sterilization and fimbrial adhesions. In vitro fertilization should be offered to those with severe distal tubal disease and severe endometriosis. It may also be indicated in patients who have not conceived within one year following microsurgery. In patients with patent tubes following microsurgery, gamete intrafallopian transfer may be performed. More clinical experience can solve the dilemma of whether it is beneficial to perform GIFT or to offer IVF primarily to the patient. Between January 1987 and December 1990 at the Jones Institute for Reproductive Medicine, the most common indications for IVF/ET were tubal factors (57%) (Seard and Jones, 1992). Much of the discussion about the management of tubal disease has centered on the cost of ART versus tubal surgery. Most authors agree that the cost of IVF is less than, or the same as, that of tubal surgery. ART is the preferred method for women suffering from multiple tubal obstruction, after bilateral salpingectomy, or with extensive and dense pelvic adhesions. Check et al. (1994) compared the cumulative probability of pregnancy after multiple IVF cycles by age and cause of infertility. The three-month cumulative probabilities of pregnancy based on life-table analysis were 33% in women with tubal factor who were younger than 35 years of age, 25% in women with tubal factor who were older than 35 years of age, 30% for women with multiple factors who were younger than 35 years of age, and 14% for women with multiple factors who were older than 35 years of age. This study demonstrates the significance of the effect of age and infertility factors on pregnancy and delivery rates.

Male infertility Male infertility has become as common as tubal factors as an indication for IVF/ET. Initially, male subfertility was treated using GIFT when couples had

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not responded to any other method of treatment, including intrauterine insemination, and patent tubes were present (Wiedemann and Hepp, 1989). However, the results show that the pregnancy outcome after GIFT in couples with severe male infertility is significantly lower than that following GIFT with normal sperm. With the establishment of ICSI for couples with male infertility, most causes of male infertility can now be treated. The majority of infertile males have oligospermia and/or low sperm motility and are subfertile rather than sterile. In men with azoospermia, sperm cells obtained from the epididymis or by testicular biopsy may prove satisfactory for the ICSI technique. The exceptional results of subzonal injection of the oocyte (SUZI), and more particularly ICSI, now raise the prospect of pregnancy being possible even when there are low numbers of sperm in the ejaculate, and bring to treatment focus some azoospermic men and men without antegrade ejaculate. The advance in clinical results from SUZI and ICSI over earlier zona drilling and zona binding techniques appears to be giving better implantation potential to the embryos. Technical factors critical for achieving high rates of fertilization and pregnancy include the use of standardized ICSI pipettes, the immobilization of sperm before injection, and the aspiration of a minimal amount of ooplasm before reinjection with the sperm. Intracytoplasmic sperm injection is superior to other micromanipulation methods for alleviating male infertility. Recently, it has been shown to be superior in terms of fertilization rates and pregnancy rates when compared with SUZI. However, there has been some concern regarding the abnormal fetal karyotypes. Van Steirteghem et al. (1993) have reported that a single spermatozoon was injected into the ooplasm of oocytes with a fertilization rate of 64.2%, and this was not influenced by sperm morphology or motility. Total and clinical pregnancy rates of 49.6% and 39.2%, respectively, per ET were reported. This new development raises scepticism regarding the applicability of conventional semen parameters. If normal embryos can be produced by ICSI, doubts regarding such theories as a natural selection process and the correlation of sperm head morphology and quality of the DNA arise. In conclusion, only ten years after Yovich and Stanger stated in 1984 that only a small fraction of oligospermic men benefitted from IVF, the results of this treatment for male infertility had improved dramatically. Endometriosis

The aetiology of infertility in endometriosis is unclear, and a multitude of factors are involved. In advanced (stage III and IV) endometriosis, endomet-

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riosis and pelvic adhesions distort pelvic anatomy and mechanically interfere with the reproductive process. In stage I and II disease, the mechanism of infertility is less clear. These effects are relative and individually variable, and some women with endometriosis have no fertility problems. A variety of mechanisms through which limited endometriosis may lower fertility have been postulated. Ovulatory dysfunction, the prevalence of which would seem to be similar in endometriosis and in the infertile population in general. Alterations in gamete/embryo transport. Antifertility effects of the peritoneal fluid, which may affect sperm motility/survival, fertilization, gamete interaction and early embryo development. Peritoneal macrophages causing increased sperm phagocytosis, decreased motility and impaired ability to penetrate the egg. An anti-implantation effect related to evidence for antigen-antibody reaction in the uterine cavity of women with endometriosis which may be the mechanism of infertility and recurrent miscarriages. Most of the mechanisms implicated in endometriosis should be corrected by IVF/ET. Aspiration of the eggs with ultrasound-guided needles, in-vitro fertilization, early embryonic development in the laboratory, and embryo transfer into the female reproductive system should correct problems caused by ovulatory dysfunction, abnormal fertilization, failure of early embryonic development or abnormal ovum pick-up/transport. One would, therefore, expect IVF/ET pregnancy rates to be comparable in patients with and without endometriosis. There is no agreement in the literature regarding the benefits of medical pretreatment of endometriosis before the IVF cycles. The most common medical pretreatments were either danazol or GnRHA.

Ovulatory disorders

Disorders of ovulation resistant to treatment by ovulation induction with either clomiphene citrate or urinary gonadotrophins may respond to IVF/ET. The most common disorder of ovulation which may prove resistant is PCOS. The presenting symptom of patients with PCOS is often infertility due to chronic oligo-ovulation or anovulation, and the restoration of ovulatory function assumes paramount importance. Clomiphene citrate is the first line of treatment for chronic anovulation that

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accompanies PCOS. However, if it fails, then conventional gonadotrophin therapy is indicated. Unfortunately, this therapy is associated with an increased spontaneous abortion rate, multiple gestation rate and OHSS. Although today's readily available monitoring technology (ultrasonography) and rapid serum oestrogen measurements make gonadotrophin therapy safer in terms of multiple gestation and OHSS, they are both time intensive and expensive and these complications lead to a high rate of cycle cancellation. The use of IVF/ET in patients with PCOS enables: controlled ovarian stimulation in conjunction with GnRH A and down regulation (Hombergef ai, 1993) aspiration of all ovarian follicles present the option of freeze/thawing all resultant embryos and subsequent transfer in a non-stimulation cycle the resultant decrease in the incidence of OHSS. In summary, IVF/ET is being increasingly used in association with ovulatory disorders and in particular PCOS. However, despite the high pregnancy rate from transfer of embryos, there is a high first trimester abortion rate as well. Unexplained infertility

Unexplained infertility can be defined to include those couples with more that two years of infertility with no abnormalities on repeated investigation of the fallopian tubes, ovulation, luteal phase, cervical mucus, semen, semen-mucus interaction or intercourse. However, it should be noted that there are many couples who have minor abnormalities but no adequate explanation for their inability to conceive. The duration of the infertility and the age of the patient are particularly important. It should also be noted that a large number of patients enter IVF programmes as 'idiopathic infertility' but that during subsequent investigations and treatment, the cause of the infertility may become apparent. Therefore IVF/ET has resulted in the diagnosis and treatment of idiopathic infertility. Many empirical modalities have been suggested for the treatment of couples with unexplained infertility. These include IUI, steroid and antibiotic therapy, bromocriptine, ovulation induction, combined treatments and ART (Navot et al., 1988). The ESHERE multicentre trial was designed to compare the effectiveness of superovulation alone with superovulation with either IUI, GIFT, IVF or intraperitoneal insemination. The pregnancy rates obtained in the trial were in excess of rates reported for untreated couples, and the mean pregnancy rates for the four invasive methods were similar. However, the application of

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ART increased the chance of pregnancy by nearly two times beyond that obtained by superovulation alone.

Immunological infertility

Immunological factors may operate at almost every step in the human reproductive process. Some of the cases of immune infertility could result from destruction of gametes by antisperm antibodies or by preventing embryo cleavage and early development. The mechanism by which the presence of antisperm antibodies interferes with human reproduction has never been documented clearly and there is no absolute proof that immunity to sperm can cause subfertility. Theoretically, ART should bypass the early stages of fertilization, during which sperm are exposed to the antisperm antibodies within the female genital tract, and alleviate subfertility related to female immunological infertility. Recent investigations report that female patients with significant levels of antisperm antibodies in the serum had similar fertilization rates after IVF when compared with patients with no significant levels and suggest that females' antisperm antibodies may not hinder fertilization in vitro. In this particular group of conditions, ART has not yet caused a dramatic change and the benefits derived from it remain unclear.

Failed donor insemination

Patients who have failed to become pregnant using donor insemination (DI) and who have been fully investigated to exclude other causes of infertility may be treated by IVF/ET. The decision to proceed to IVF is usually made following six failed DI treatment cycles. However, as the success rate per ART treatment cycle is three times higher than that per cycle of DI treatment, social considerations, including the distance the patient has to travel or work commitments, have to be taken into consideration. If, on further investigation of the female partner, new or unidentified causes of infertility are found, then IVF may be indicated at an earlier stage.

Donation of eggs and embryos

It is technically feasible to transfer donated eggs from one woman to another. The baby would be the result of the husband's sperm, a donated egg and the environment of the wife's uterus during the pregnancy and birth.

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The indications for donation are: absent ovaries strip ovaries failed hormonal induction of ovulation inherited genetic diseases transmitted through the female premature ovarian failure poor quality oocytes from female with repeated failure of IVF patients after surgical castration and after x-ray or chemotherapy. The establishment of pregnancy utilizing oocyte or embryo donation is not adversely affected by the chronological age of the recipient, implying that the age related decline in fertility is primarily related to oocyte ageing and not to loss of endometrial receptivity (Sauer et al, 1994). Also, prior exposure to chemotherapy may alter endometrial integrity and lead to greater pregnancy wastage in women receiving donated embryos. This modality of treatment raised serious legal, social, religious and ethical issues such as the maximum age of treatment and donor selection. Transfer ofsexed embryos Some diseases are sex linked, e.g. haemophilia and muscular dystrophy, and only affect male offspring. When it is known that either the male or female children are almost certain to have the disease, the transfer of embryos of the unaffected sex will eliminate the risk. Surrogacy Surrogacy may be indicated for women who have had a hysterectomy or severe uterine malformation but have retained their ovaries. Couples in this situation have requested IVF using their own oocytes and the husband's spermatozoa, and transfer of the embryo to a surrogate woman who would carry the pregnancy and give birth. The child would then be given to the infertile couple. Summary Assisted reproductive technology is currently used for a wide range of indications and has become an acceptable tool in the treatment of subfertile couples. However, controversy still exists concerning the effectiveness of ART compared with conventional treatment for various conditions that cause subfertility. The effectiveness of IVF in terms of pregnancy rates is demonstrable for patients with severe bilateral tubal disease and male infertility. For patients with other causes of infertility, the differences in pregnancy rates do not reach

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statistical significance. The exact role of ART in the management of PCOS, immunological infertility and endometriosis remains to be determined. Stimulation regimes Introduction

The stimulation protocols currently in use at Monash IVF will be described. The aim of all stimulation protocols is to induce the development of a follicle cohort in both ovaries. A number of equidominant follicles should ideally be produced. The success rate in any one IVF cycle has been clearly shown to be increased from 4.2% in a natural cycle to 17% in a stimulated cycle where more than one embryo is transferred (Trounson and Wood, 1993). Trounson and colleagues of the Monash Group showed in 1981 that an increased number of eggs at the time of oocyte retrieval was directly linked to a significant improvement in pregnancy rates. Modifications to stimulation protocols are continually being reviewed. They are designed with the following aims. To prevent premature luteinization. To prevent spontaneous ovulation. To improve endometrial response. To allow greaterflexibilityfor ambulatory patient management, with reduced cycle monitoring, and the giving of injections at home. The protocol needs to be tailored to each individual patient in order to maximize the prospect of success whilst minimizing possible complications. The patient's history, diagnosis and, particularly, previous performance in a stimulated assisted reproductive technology cycle will need to be scrutinized before deciding on the appropriate treatment in a treatment cycle. Emphasis is given to a team approach in the making of decisions regarding stimulation. Usually the patient's clinician, liaising with the nurse practitioner directly involved with the patient's care, and the embryologist, if necessary in consultation with a specialist endocrinologist, will meet to decide the most appropriate protocol. Therapeutic agents

Varied protocols of controlled ovarian hyperstimulation have been developed within different centres. They are all based on the use of the agents described. Ongoing modifications will occur depending on the patient's response, newer agents and results of treatment.

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Clomiphene citrate Clomiphene citrate acts in a negative feedback system at pituitary level to displace oestrogen, thereby causing an increase in endogenous gonadotrophin secretion. Evidence of a hypothalamic site of action with an increase in pulsatile release of GnRH has been reported (Kerin et al., 1985). Clomiphene citrate has been used successfully in ART protocols, allowing multiple synchronous follicular development and therefore multiple oocyte collection. The major disadvantages of its administration are cycle cancellation, due to a premature LH rise and therefore ovulation before egg collection is scheduled, and asynchronous growth of follicles, with a likelihood of few oocytes being recovered. A possible negative influence on endometrial receptivity due to anti-oestrogenic properties has been described. To avoid the patient's spontaneous ovulation, hCG is given to induce a timed ovulation for ovum pick-up. Intense monitoring is required to pinpoint the timing of hCG administration accurately. This involves, at the minimum, twice daily estimates of plasma oestradiol, LH and progesterone to detect a possible premature LH surge. Patients who commence spontaneous ovulation will have their treatment cycle cancelled, unless ovulation minus two hours falls within the theatre schedule for oocyte recovery. Purified FSH Purified FSH, derived by recombinant DNA technology, has a theoretical advantage of enhancing synchronous follicular development. The elimination of exogenous LH should theoretically improve oocyte quality, although this advantage has not been proven in clinical trials comparing hMG to FSH (Lavy et al, 1988). The reduction in spontaneous LH surge in patients undergoing purified FSH stimulatory cycles has been shown to be statistically significant. Exogenous gonadotrophin dosage A starting dose is arbitrarily determined. Influencing factors are the patient's age and past history (Table 8.1). Gonadotrophin-releasing hormone agonists Gonadotrophin-releasing hormone agonists have had a marked influence on controlled ovarian hyperstimulation. Their use has significantly improved pregnancy rates per treatment cycle (Hughes et al, 1992). The agonist Buserelin in combination with hMG, was first used in 1984 for improved folliculogenesis in an IVF programme (Porter et al., 1984). The mechanism of

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J. McK. Talbot and M. Lawrence Table 8.1. Influence of patient's age and history on starting dosage of gonadotrophin Dose (iu) Age < 37 37- 40 > 40 History PCO Poor response, 3-5 oocytes Normal response, > 5 oocytes Minimal response, < 3 oocytes OHSS BMI > 30, add 75 iu to starting dose.

150 225 300 75 300 150 450 75

action is a reversible suppression of pituitary gonadotrophin secretion thereby enhancing the efficiency of exogenous gonadotrophin administration (Fleming et al, 1982). Follicular phase LH level fluctuations are one of the main factors in the variable ovarian response to controlled stimulation. High oestradiol levels may cause a premature LH surge, thereby causing premature follicular luteinization (Howies et al., 1986). The use of GnRH agonists removes these unwanted variables. They may be administered by daily subcutaneous injection (Lucrin), nasal spray (Buserelin) or by a sustained release injectable (Zoladex). They are given in varied combinations with exogenous gonadotrophins. The protocol of GnRH agonist together with hMG is the benchmark for controlled ovarian hyperstimulation in nearly all major IVF programmes. A number of protocols have been developed. GnRHA/hMG - short 'boost' protocol. GnRHA/hMG - long 'down regulation' protocol. Clomiphene citrate - 'Minimal stimulation' protocol. Natural - 'unstimulated'. Immature. At Monash IVF, GnRH agonists are administered in two protocols depending on the patient's previous response to controlled ovarian hyperstimulation, oocyte quality and age. The protocols differ in timing of administration during the patient's own menstrual cycle and the length of administration of the GnRH agonist, before commencing exogenous gonadotrophin therapy. The short follicular phase protocol (called Boost or Flare) takes advantage of the

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initial stimulatory phase of endogenous gonadotrophin secretion and suppression of LH in the late follicular phase (Barriere et al, 1987). Exogenous gonadotrophin therapy commences the day following GnRH agonist administration and has an additive effect to the patient's own endogenous FSH rise. The long luteal phase protocol (called down regulation) commences on day 21 of the patient's cycle. Exogenous gonadotrophin is commenced when the oestradiol, progesterone and LH levels indicate pituitary and ovarian suppression. The midluteal phase administration of GnRH agonist causes more prompt and more profound ovarian suppression (Smitz, Ron-El and Tarlatzis, 1992). The literature is varied in terms of the pregnancy rates when the two protocols are compared. On balance, it seems that the long down regulation protocol affords significantly better results (Tan, Kingsland and Campbell, 1990; Padose/a/., 1991). At Monash IVF, the short protocol is used as the routine because of its greater patient acceptability, convenience and cost effectiveness. The long protocol is presently reserved for patients who have had a previous poor response to stimulation, are aged over 40 or who are having an intracytoplasmic microinjection of their oocytes. Boost stimulation regime In this short protocol, the GnRH agonist Leuprolide (Abbott Pharmaceuticals, Sydney) is commenced in a dose of 0.75 mg subcutaneously on day two of the cycle, continuing as a single daily injection or Synarel (Syntex, Sydney) two sniffs per day of nasal spray until the day prior to hCG administration. Administration of hMG commences on day three of the cycle until the day prior to hCG injection. The dose is adjusted according to ovarian response. When adequate criteria have been satisfied, hCG 5000 IU is given intramuscularly and oocyte retrieval performed 36 hours later. Down regulation This protocol commences on day 21 of the cycle. Leuprolide 1.0 mg is given each day and E2, P4, and LH levels are measured at weekly intervals until down regulation occurs. Down regulation is confirmed when oestradiol is < 180pmol/l, LH < 2 IU, progesterone < 2 mmol. Administration of hMG commences after down regulation occurs. It continues and hCG is given as for the boost stimulation protocol. Leuprolide 0.5 mg per day is given during the stimulatory phase. Minimal stimulation This stimulation cycle is recommended for patients who either wish to avoid

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the possible hyperstimulatory side-effects of exogenous gonadotrophins or who have exceeded the limit of Government rebatable gonadotrophin-stimulated cycles. Clomiphene citrate is commenced on day two of the menstrual cycle. The first day of bleeding prior to 6 p.m. is taken as day one. A dose of 50 mg twice daily is continued forfivedays. hCG 5000 iu is given when the leading follicle has reached 18 mm or more as measured by vaginal ultrasound scan. The oocyte pick-up is timed at 34 hours post hCG injection. The possibility of spontaneous ovulation prior to this necessitates an ultrasound scan before oocyte recovery on the day of admission. Natural cycle No drugs are given to these patients. The aim is to retrieve the oocyte in a normally ovulating patient. The limitation of this protocol is that, at most, only one oocyte can be collected and it is possible that ovulation may occur prior to egg pick-up. Immature oocyte collection Patients may have a number of immature oocytes collected from developing follicles. These oocytes are then matured in vitro and fertilized, and embryos are cultured and subsequently replaced in the standard manner. As yet, success has been poor and research continues into technique enhancement. If clinical success is achieved, immature oocyte collection will enable a number of oocytes to be collected from an unstimulated patient.

Cycle management Monitoring the treatment cycle The monitoring of follicular response to superovulation requires a combination of parameters. Specifically, there needs to be vaginal ultrasound assessment of follicular size and number, and measurements of plasma oestradiol, LH and progesterone levels need to be ascertained. At Monash IVF, monitoring of follicular activity has been simplified. Biophysical and biochemical measurements are made on days eight and ten of the treatment cycle. Occasionally a third monitoring is required to time hCG administration accurately, particularly in minimal stimulation cycles. The interpretation of results is made by the team each afternoon. The patient's history and current treatment are scrutinized together with the current results. Decisions concerning changes in management, cycle cancel-

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lation, alteration in drug dosage, timing of hCG and oocyte recovery are conveyed promptly to the patient. Natural cycle management Although this treatment protocol is drug free, the likelihood of missed ovulation necessitates intense monitoring. The management is summarized as follows. 1. Ultrasound on day ten. 2. Oestradiol, progesterone and LH measurements daily from day ten, with twice-daily measurements near the expected time of ovulation. 3. hCG 3000IU is given when the leading follicle is greater or equal to 18 mm and the plasma oestradiol is greater than 500 pmol/1. 4. Oocyte recovery is 34 hours post hCG injection. 5. A vaginal ultrasound is performed prior to theatre admission. Criteria for cancellation The following are guidelines for Lucrin-FSH protocols. 1. 2. 3. 4.

Abnormal findings seen on ultrasound scan. Five or fewer follicles or oestradiol less than 3000 pmol/1. Falling oestradiol levels despite increased stimulation. Oestradiol greater than 25 000 pmol/1. If this hyperstimulation occurs, consider continuing GnRH agonist down regulation until the ovaries are quiescent. Treatment of a cancelled cycle

There are patients with inadequate follicle numbers and patent tubes whose treatment cycle may be corrected to controlled ovarian hyperstimulation. 1. hCG 5000 iu when follicles estimated to be 18-20 mm. 2. Intercourse for patients with normal sperm, 24 hours after hCG. 3. Intrauterine insemination for male factor patients 34 hours post hCG injection. 4. hCG boosters (1000 iu) on days three, six and nine post intercourse or IUI. hCG (5000 iu) is administered to patients when each of the following criteria are satisfied: the oestradiol level is rising appropriately, there is a cohort of at least three follicles of 18-20 mm, E2 > 3000 pmol. The oocyte retrieval is timed thirty six hours post hCG administration.

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Egg pick-up is timed 36 hours after hCG administration in a stimulated cycle and 34 hours post hCG in a natural cycle. This timing allows the maximum number of preovulatory oocytes to be retrieved. It also allows greaterflexibilityin timing of cases for theatre. Oocyte retrieval for IVF Over the past 15 years, the technique for oocyte retrieval has evolved from being a laparoscopic procedure, lasting an hour or more, to a procedure involving ultrasound-guided needle aspiration and taking approximately 20 minutes. The laparoscopic approach involved a full relaxant general anaesthetic. Follicles were punctured and fluid aspirated using a stainless steel needle of 23 cms length with an internal diameter of 2.0 mm and an external diameter of 2.2 mm. The problems encountered in this technique, apart from the morbidity associated with laparoscopy, included the obese patient where entry into the abdomen was difficult, adhesions making visualization unclear, bleeding within and from the follicle due to direct trauma, and follicular leakage. Lenz and Lauritsen (1982) reported the first use of ultrasound-guided percutaneous follicle aspiration, with an oocyte recovery rate of 50% per patient. The needle used had an internal diameter of 0.6 mm and was therefore less traumatic. The next advancement and refinement came with the switch to ultrasoundguided needle aspiration per vaginum. Quite apart from the benefits to a patient of this less invasive procedure, the number of eggs retrieved at oocyte collection began to increase. This was because ultrasound affords greater accuracy in needle-tip localization, and also follicles within the ovary which would normally have been missed by the blind searching of the laparoscopic needle could be clearly seen by ultrasound, and directly aspirated. In summary, vaginal ultrasound needle follicle aspiration affords a dual benefit to the patient when compared to laparoscopy in terms of a reduced morbidity and ease of aspiration of previously unrecognized follicles within the ovary. The following description is of the current methodology of oocyte retrieval. It is applicable to all cases. The technique of collection is not influenced by the stimulation protocol nor by whether the patient is to undergo IVF or GIFT. Equipment At Monash IVF, the ultrasound machine used is an Acuson model number 128 (Acuson Computed Sonography, Mountainview, California). It has

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clear image enhancement and allows visualization of follicles as small as 2 mm. Although not normally aspirated for oocyte pick-up in routine cases, these follicles are significant in the collection of immature oocytes. It is important that the ultrasound image clearly shows the major structures to be avoided at the time of needle aspiration. In particular, the relationship of the ovary to blood vessels, uterus and loops of bowel must be noted to enable a trajectory for the needle to be selected so that its path through the pelvis is atraumatic. The ultrasound probe used for vaginal oocyte recovery is a 5 MHz vaginal probe (Acuson model EV519). Preparatory to oocyte collection it is disinfected with chlorhexadine 0.5% for a minimum often minutes and covered with a latex condom. A specially adapted needle guide is fixed to the probe. It is important that all parts of the system have smooth, rounded edges rendering them completely atraumatic. A 17-gauge disposable single-channel aspiration needle, 31 cm in length (William Cook Pty Ltd, Australia), is used. The bevel of the needle is set at 45 degrees. It has an ultrasound-identifiable mark on the tip to aid visualization. Before use it is inspected to ensure that it is sharp, thus minimizing the risk of tearing of a follicle when introduced. Teflon tubing, continuous from the needle bevel to a silicone bung, delivers the aspirate into 10 ml tissue culture tubes. A simple system of heating blocks placed in a specially adapted test tube holder with space for a Pyrex container offlushingmedium and a glass syringe has been devised. This allows the temperature of the system to remain constant at 37 degrees Celsius. The importance of accurate temperature control cannot be overemphasized. A sufficient length of Teflon tubing also runs from the silicone bung to the aspiration pump connection. The pump is a foot-pedal-operated Kmar-2000 vacuum regulator (William Cook Pty Ltd, Australia). It allows the vacuum pressure to be selected accurately. For routine follicle aspiration, a pressure of minus 17 kPa is chosen. The technique of oocyte recovery For routine IVF cases the patient is given a light (neurolept) anaesthetic. Usually a combination of Fentanyl, Midazolam and Propofol is administered. In the lithotomy position, appropriate drapes are applied. No chemical disinfection of the vagina is performed. Disinfecting agents have been shown to be potentially toxic and could well compromise embryo development. The vaginal ultrasound probe with sterile covering and needle guide attached is introduced. A general inspection of the pelvis is made. Particular attention is

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given to the position of the ovaries in relation to blood vessels, bowel and the uterus. The approximate size and number of follicles are assessed on each side. An appropriate trajectory path for the needle through the pelvis is selected. The aspirating system is checked to be operational by sucking a small amount of culture medium through the needle into a culture tube. This tube is then discarded. Follicles are aspirated in a sequential fashion from largest to smallest. It is important to aspirate the largest follicles first as this will reveal the previously hidden smaller follicles within the ovary. In most cases it is not necessary to withdraw the needle from the ovary, but rather to change its position within the ovary to go from one follicle to the next. Occasionally the needle may block due to blood and debris. If this occurs it should be withdrawn. A reverse flush down the needle into a culture tube is usually all that is required to clear it. A needle wash is performed if the needle is withdrawn between follicles or when moving to the other ovary. Culture tubes are examined almost immediately by an embryologist. Oocytes can be quickly identified. The surgeon is able to watch the progress of the collection by means of a camera connected to the embryologist's microscope and relayed to a monitor in the operating theatre. Individual follicle flushing has been largely superseded by the vaginal technique where all follicles are visualized and drained. In cases where folliculogenesis has been suboptimal and only a few follicles are present, follicle flushing may be required. An appropriately sterilized glass syringe with needle tip, filled with flushing medium (Herpes buffered culture solution), is inserted into the flared end of the Teflon tubing coming from the aspiration needle. The approximate follicular volume is inserted and then aspirated. This procedure is repeated up to six times to retrieve the oocyte.

Embryo transfer

The transfer of embryos into the uterine cavity, although possibly the most straightforward part of the IVF process, is nonetheless one of the most important steps. The gentle, atraumatic introduction of embryos into the intrauterine environment is essential for a successful outcome. Embryos are transferred on either the second or third day after oocyte recovery. The different timings are selected to avoid embryo transfer on Sunday. There is no difference in pregnancy rates between the days of transfer. The pregnancy rate is significantly higher if more than one embryo is replaced. Embryo quality is a significant determining factor for successful pregnancy. The maximum number of embryos replaced at Monash IVF is

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three. In certain circumstances, to reduce the probability of triplets, the number of embryos replaced is limited to two. Specifically, patients who are aged less than 38 years and have more than three embryos scoring 7.0 have a two-embryo transfer. There have been trials involving various catheter designs for embryo transfer. Criteria for successful catheter design include the following. Ease of handling. Passage through an undilated cervix. Non embryo toxic. Appropriate markings for depth of insertion within the uterus. Catheter tip detectable on ultrasound. The catheter currently used at Monash IVF is the K-Pet Catheter. It comprises an outer catheter, which acts as a sleeve to guide the cannula containing the embryos into the uterus. The outer catheter is made of polyurethane with a polypropylene hub. The inner embryo cannula is made of Teflon with a silicone hub. It measures 30 cm in length and has a diameter of 1 mm. The outer catheter is introduced through the cervix to the internal os by means of a metal stilette, which affords rigidity andflexibility.At the internal os, the outer sheath is advanced and the stilette removed. Technique of transfer The embryologist checks the identification of the patient and confirms the number of embryos to be transferred. The patient is admitted to the operating room on the day surgery unit. The embryo transfer is performed without anaesthesia. The patient is placed in a slight Trendelenberg position with her feet resting on elevated lithotomy poles, giving approximately 30 degrees of hip flexion. The embryos are shown to the patient and her partner via a monitor connected to the microscope in the embryology laboratory. A bivalve speculum is inserted and the cervix exposed. Mucus is cleansed from the cervix using peanut swabs soaked in culture medium. No antiseptic is used. The outer sleeve of the K-Pet catheter is introduced into the cervix and advanced to the internal os. The guide wire is removed and the sleeve advanced to the 5cm mark. After correct placement of the sleeve the embryologist is signalled to load the cannula.

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J.McK. Talbot and M. Lawrence Technique of loading the embryo cannula

0.02 ml of air is drawn into a 1 ml syringe and the cannula attached. A 3 /il column of media is introduced into the cannula, 2 /A of air is then introduced. The embryos in culture medium (3 /il) are drawn into the cannula followed by 2 /A of air. The tip of the cannula is sealed by dipping it into media and is pulled back into its sterile packaging and taken into the operating theatre. The surgeon then introduces the cannula via the catheter sleeve into the uterus to the 5 cm mark. The embryos are released into the uterus by gentle pressure on the plunger. Lutealphase support

Luteal phase support is given to patients who have been on GnRH agonist therapy and is necessary because the GnRH agonists cause a short luteal phase due to LH suppression. hCG injections (1000IU) are given intramuscularly on days three, six and nine post egg pick-up. There is no firm evidence suggesting the need for routine use of luteal phase support in patients who have had induction of ovulation with hMG or hMG and clomiphene citrate. Despite this, hCG as above or progesterone pessaries (100 mg three times daily) have sometimes been prescribed for patients following embryo transfer until the day of the pregnancy test (day 16 post egg pick-up). The hCG is replaced by progesterone pessaries (100 mg tds) if the risk of hyperstimulation is significant: E2 > 15 000 pmol/L or > 20 oocytes collected. References Barriere, P., Lopes, P., Boiffard, J-P., Pousset, C, Quentin, M., Sagot, P., L'Hermite, A., Lenat, M.F. and Charbonnel, B. 1987. Use of GnRH analogies in ovulation induction for in vitro fertilisation: benefit of a short administration regimen. Journal In Vitro Fertilisation and Embryo Transfer. 4: 64-5. Check, J.H., Laurie, D., Callan, C, Baker, A. and Benefer, K. 1994. Comparison of the cumulative probability of pregnancy after in vitro fertilization embryo transfer by infertility factors and age. Fertility and Sterility 6: 257-61. Fleming, R., Adam, A.H., Barlow, D.H., Black, W.P., MacNaughton, M.C. and Coutts, J.R.T. 1982. A new systematic treatment for infertile women with abnormal hormone profiles. British Journal of Obstetrics and Gynaecology 89: 80-3. Homberg, R., Berkowitz, D., Levy, T., Felberg, D., Ashkanazi, J. and Ben-Rafael, Z. 1993. In-vitro-fertilization and embryo transfer for the treatment of infertility associated with poly cystic ovary syndrome. Fertility and Sterility 6: 858-63. Howies, CM., Macnamee, M.C, Edwards, R.G., Goswamy, R. and Steptoe, P.C.

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1986. Effect of high tonic levels of luteinising hormone on outcome of in vitro fertilisation. Lancet 2: 521. Hughes, E.G., Federkow, D.M., Daya, S., Sagle, M., DeKoppel, P. and Collins, J. 1992. The routine use of gonadotrophin-releasing hormone agonists prior to in vitro fertilisation and gamete intrafallopian transfer: a meta-analysis of randomised controlled trials. Fertility and Sterility 58: 888-96. Kerin, J., Liu, J., Phillipou, G. and Yen, S. 1985. Evidence for a hypothalamic site of action of Clomiphene Citrate in women. Journal of Clinical Epidemiology and Metabolism 61: 265-8. Lavy, G., Pellicer, A., Diamond, M. and De Cherney, A. 1988. Ovarian stimulation for in vitro fertilisation and embryo transfer, human menopausal gonadotrophin versus pure human follicle stimulating hormone: a random prospective study. Fertility and Sterility 50: 74-8. Lenz, S. and Lauritsen, J. 1982. Ultrasonically guided percutaneous aspiration of human follicles in local anaesthesia: a new method to collect oocytes for in vitro fertilisation. Fertility and Sterility 38: 673-7. Navot, D., Mausher, S.Z.J., Rehninger, S., Liu, H.C., Veek, L.L., Kreiner, D. et al. 1988. The value of in-vitro-fertilization for the treatment of unexplained infertility. Fertility and Sterility 49: 854-7. Pados, G., Tarlatzis, B.C., Bontis, H., Lagos, S., Papadimas, J., Spanos, E. and Mantalenakis, S. 1991. Ovarian stimulation with Buserelin/HMG/HCG: prospective study of short vs long protocol. Human Reproduction Abstract 7th annual • meeting of ESHRE, Paris 28-30 June 1991, pp. 364-5. Porter, R.N., Smith, W., Craft, I.L., Abdulwahid, N.A. and Jacobs, H.S. 1984. Induction of ovulation for in vitro fertilisation using Buserelin and gonadotrophins. Lancet 2: 1284-5. Sauer, M.V., Paulsmer, R.J., Ary, B.A. and Lobo, R.A. 1994. Three hundred cycles of oocyte donation at the University of Southern California; assessing the effect of age and infertility diagnosis on pregnancy and implantation rates. Assisted Reproduction 11: 922-6. Seard, M.A. and Jones, H.W.J. 1992. Indications of in-vitro-fertilization; changing trends; The Norfolk Experience. Annals of Medicine Singapore 21: 4459-70. Smalldrudge, J. and Tait, J. 1993. The results of tubal surgery in the treatment of infertility in Wellington 1986-90. New Zealand Medical jJournal 14: 106, 124-6. Smitz, J., Ron-El, R. and Tarlatzis, B.C. 1992. The use of gonadotrophin releasing hormone agonists for in vitro fertilisation and other assisted procreation techniques: Experience from three centres. Human Reproduction 7, Suppl. 1: 49-66. Steptoe, P.C. and Edwards, R.G. 1978. Birth after reimplantation of a human embryo. Lancet ii: 366. Tan, S.L., Kingsland, C. and Campbell, S. 1990. The use of Buserelin in in vitro fertilisation - A comparison between the long and short protocols of administration. Gynaecology and Endocrinology A, (Suppl. 2): Abstract No. 107. Trounson, A.O., Leeton, J.F., Wood, E.C., Webb, J. and Wood, J. 1981. Pregnancies in humans by fertilization in vitro and embryo transfer in controlled ovulatory cycles. Science 212: 681-2. Trounson, A.O. and Wood, E.C. 1993. IVF and related technology. The present and the future. Medical Journal of Australia 1, 58: 853-7. Van Steirteghim, A.C., Nagy, Z., Jons, H., Liu, J., Staaesser, C. and Smitz, J. 1993. High implantation rates after intracytoplasmic sperm injection. Human Reproduction?,: 1061-6.

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Weidemann, R. and Hepp, H. 1989. Gamete intrafallopian transfer in male subfertility. Human Reproduction 4: 409-11. Yovich, J.L. and Stanger, J.D. 1984. The limitation of in vitro fertilization from males with severe oligospermia and abnormal sperm morphology. In Vitro Fertilisation and Embryo Transfer 1: 172-9.

9 The role of gamete intrafallopian transfer CARL WOOD

History (Perone, 1991) 1979

1980

1983

1983 1984 1986 1988

Shettles reported a successful pregnancy following placement of eggs in the fallopian tube at the time of sterilization reversal, artificial insemination preceding the surgical procedure. Kreitman and Hadjec showed that low oocyte transfer and mating resulted in 5 of 31 monkeys becoming pregnant. The transfer was proximal to the site of tubal ligation. Abate reported the first pregnancy in a patient following laparoscopic sperm and oocyte transfer to the fallopian tube. The Lancet refused publication of the letter. Tesarik obtained one birth following oocyte and sperm transfer to the tube after tubal repair in nine patients. Asch reported several pregnancies from laparoscopic gamete transfer; egg retrieval was by laparoscopy. Dewraz and Blacklege transferred fertilized oocytes to the tube after in-vitro fertilization. Jansen showed that pregnancy could be obtained by vaginal insemination of gametes into the fallopian tube. Physiological basis

The understanding of ovulation, fertilization, embryo cleavage and corpus luteum function is significant in terms of understanding conception. The mechanisms of sperm transport through the female genitalia towards the site of fertilization in the tubes and of fimbriae oocyte pick-up are still obscure. It is possible that these mechanisms, which cannot be evaluated by means of clinical or laboratory tests at present, are impaired in some of the unexplained cases of infertility. 109

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Idiopathic infertility and mild or moderate endometriosis, which anatomically do not affect the tubes and/or ovaries, are common diagnostic groups in infertility. The reason for the infertility in these patients is not clear. It has been suggested that the sperm cannot reach the tubal fertilization site due to a defect in sperm transport mechanism. An alternative explanation is that there is an impairment of oocyte pick-up by the fimbriated end of the tube at the time of ovulation. There is some evidence to support these assumptions: elevated levels of thromboxane B2 and 6 keto-prostaglandin Fl have been found in the peritoneal fluid of infertile women with endometriosis as compared with patients without endometriotic lesions. These prostaglandin products may act on the tubal smooth musculature and interfere with tubal motility, i.e. tubal transport and tubal oocyte pick-up mechanisms. Others have suggested an ovum-capturing inhibition factor in the peritoneal fluid of women with endometriosis that may be responsible for fimbrial failure to pick up the oocyte during ovulation. It has been shown that tubal pick-up of the oocyte may be as low as 63%, even in highly fertile women, and that it may be even lower in cases of unexplained infertility and endometriosis. If the assumption is correct that in some cases of unexplained infertility and endometriosis there is an impaired ovum pick-up mechanism and/or failure of sperm to reach the tubal fertilization site, then the direct placement of gametes, oocytes and sperm into the fallopian tube by the GIFT technique should be able to overcome these impairments. Selection of patients

Patients with infertility who have one normal tube and in whom simpler treatments have failed may be considered for the GIFT procedure. Hysteroscopy, falloposcopy, laparoscopy and vaginal ultrasound may be required to check the suitability of the tubes for the GIFT procedure. The tubes may be assessed by laparoscopic hydrotubation, measurement of tubal opening pressure, distal salpingoscopy, falloposcopy and tubal cannulation (Kerin, 1992). These tests exclude tubal blockage, stenosis and abnormality of the endosalpinx. Previous ectopic pregnancy is acceptable if there is one normal tube. Previous failed tubal microsurgery is associated with reduced pregnancy rates with the GIFT procedure and increased risks of ectopic pregnancy. Apart from tubal disease, contraindications to GIFT include endometriosis, stage III and IV, active infection and intrauterine cavity abnormalities. Patients over 40 years have reduced success rates and donor oocytes may be offered. After two failed GIFT cycles, repeat hysteroscopy is indicated as abnormal-

The role of gamete intrafallopian transfer ities which may affect infertility have been found in 22% of patients (Kirsop et al, 1991). Patients may return to GIFT or IVF, depending upon the nature of the abnormality. Falloposcopy will further improve the selection of patients for the GIFT procedure as abnormalities such as polyps, synechiae and epithelial flattening or tubal dilations may be detected (Kerin, 1992). It is uncertain how severe endosalpingeal abnormalities have to be to contraindicate the procedure. Such abnormalities may prevent pregnancy or increase the risk of ectopic pregnancy. Pregnancy and ectopic pregnancy ratios in patients having GIFT after minor tubal abnormalities have been found by falloposcopy will determine the significance of these changes and subsequently improve the selection of patients. Cumulative pregnancy rates for the GIFT procedure show little decrease in pregnancy rates up to the sixth treatment. At Monash IVF, cumulative pregnancy rates derived from 1205 cycles show that 75-80% of women under 40 years would conceive after four cycles and only 26.8% of those over 40 years would conceive after four cycles. Such data may be biased as patients having difficulty in responding to ovarian stimulation, few eggs collected or poor oocyte quality may be discouraged from repeated treatments. Nevertheless, patients with reasonable response to ovarian stimulation and oocyte collection can be encouraged to continue treatment. Laparoscopic GIFT

At present most GIFT procedures are done by laparoscopy under general anaesthetic. The use of 100% carbon dioxide to induce a pneumoperitoneum has not been found deleterious to gametes when compared to low concentrations of carbon dioxide (5%) in air - pregnancy rates being the same in the two groups (Khan et al, 1989). GIFT is usually performed as a day procedure. Local anaesthesia is used in some centres, intravenous sedation and narcotics being combined with infiltration of local anaesthesia into the lower abdomen. Spinal anaesthesia has also been used successfully. Pneumoperitoneum may cause upper abdominal and shoulder pain so that the volume of the pneumoperitoneum is reduced to a minimum volume when local anaesthesia is used. Epidural anaesthesia has the same limitations, anaesthesia being limited to the lower abdomen. Variations in clinical surgical technique have been shown to influence pregnancy rates so that attention to the details of technique is important (Isherwood 1990). Pregnancy rates have also been shown to be affected by clinical ability (Brady, Leeton and Osborn, 1989).

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Figure 9.1 Accessory tubal opening. Patency demonstrated up to blind end at 3 cm.

A television monitor with a camera attached to the laparoscope enables visualization by theatre staff and video recording of the procedure. Tubal handling is minimized by improving access using a 30-degree Trendelenburg tilt and by aspirating blood if pooling is excessive. The end of the tube is held with a blunt holding forceps. Both tubes are inspected and the most normal tube chosen. Large sacculations and accessory ostia are more common in unexplained infertility, and may be causal in this respect (Isherwood, 1990), so that the tube without such changes is chosen for transfer. Even in the presence of accessory tubal ostia, the pregnancy rate for GIFT is the same as in other patients (Isherwood, 1990). Presumably any effect of accessory ostia on fertility is overcome by placing gametes beyond the accessory opening. Accessory tubes are less common (Figure 9.1). The success of the GIFT procedure is the same whether one or two tubes are used (Osborn, 1989; Penzias et ah, 1991). The use of a single tube has the advantage of placing the best eggs into the best tube, and reducing the time involved in handling gametes. It may not be justified to avoid using the left tube because access is sometimes more difficult in the presence of the sigmoid colon. The exposure of gametes to low temperature can be reduced by the

The role of gamete intrafallopian transfer embryologist working in a humidicrib in the operating room, so that the time between aspiration of gametes into the catheter and their installation into the fallopian tube can be less than 30 seconds. Before the gametes are transported from the humidicrib, a curved plastic guide catheter is placed into the fallopian tube. It may be helpful to pass the catheter at least 3 cm into the tube to exclude distal stenosis. The distance from the end of the tube for gamete injection may depend on the local tubal anatomy. It is important to pass the gamete catheter beyond accessory ostia or large sacculations. More pregnancies have occurred when gametes were placed beyond 4 cm in a single tube (69.6%) than when they were placed between 3 and 4 cm (41.2%) (Yee et al., 1989). The study sample of 57 was too small to achieve statistical significance. It is possible that a deeper placement of gametes would reduce the risk of tubal expulsion. The outer guide catheter should be withdrawn from the tube before the gamete catheter is emptied, partly to reduce any sucking effect when it is withdrawn and to enable visualization of the centimetre markings on the gamete catheter. The gamete catheter is slowly withdrawn, egress of fluid from the tube checked for, and the end of the tube held vertically for one minute before release. Vaginal GIFT

Vaginal GIFT has been performed by transcervical tubal catheterization using hysteroscopy or vaginal ultrasound guidance, 'blind' as a manual procedure, or by falloposcopy (Lucenae/a/., 1989, 1990; Possatie/a/., 1991; Ferraioloe? al., 1991). The procedure has the obvious advantages of not requiring anaesthesia or laparoscopy. Success rates vary. Blind tubal transfer resulted in seven clinical pregnancies from 25 successful tubal cannulations (18%), three abortions and one ectopic pregnancy occurring (Ferraiolo et al, 1991). Similar results have been achieved by hysteroscopy: seven pregnancies in 26 patients (27%), in association with two miscarriages (Possati et al., 1991). Using vaginal ultrasound, five pregnancies were reported in 16 cycles (31%) by Lucena et al. (1989). Although these reports of small numbers of patients with three different techniques are satisfactory, other centres have found difficulty in attaining similar results. The problems of vaginal GIFT may be related to endometrial trauma, inner tubal trauma, uncertainty in siting the placement of gametes in the tube, whose length varies from 8 to 18 cm, and possible loss of gametes from the outer or inner end of the tube when the gametes are misplaced. Vaginal ultrasound and hysteroscopy provide evidence that catheterization has been successful and the

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patient often notices discomfort on the side in which the tube has been catheterized. Hysteroscopy is more often uncomfortable than ultrasound or blind tubal catheterization. The site of placement of gametes in the tube may not be critical as one group has obtained satisfactory pregnancy rates by depositing the gametes in the inner half of the tube. Gametes may move to the ampulla regardless of the site of placement or, alternatively, gametes flushed from a tube may be propelled several centimetres in an outward direction by fluid flow. Falloposcopy has been suggested as a method to perform GIFT by Kerin and Bauer (Kerin, 1992). It has several advantages: tubal cannulation is done under vision, the inside of the tube can be checked for abnormalities, and the site of gamete deposition can be confirmed. One possible disadvantage is that the diameter of the falloposcope is larger than the Cook catheter used for vaginal GIFT. A pregnancy has already resulted following falloposcopic GIFT. Culdoscopic GIFT

Six pregnancies have been reported in 12 patients using culdoscopy for the GIFT procedure (Diamond et al., 1992). This is encouraging as culdoscopy has the advantage of requiring less anaesthesia and no abdominal incisions when compared with laparoscopy. Most gynaecologists are unfamiliar with the technique of culdoscopy: access to the fimbria is aided by uterine manipulation. Number of oocytes transferred

Pregnancy rates increase with the number of oocytes transferred, as does the multiple pregnancy rate. The occurrence of triplets or higher order multiple pregnancy is associated with unwanted conceptions and increased risks of physical and mental defects in survivors from severe prematurity. One method of resolving this problem is to transfer large numbers of eggs - five or more and then pursue fetal reduction if three or more conceptions result. Abortion with early transvaginal ultrasound-guided fetal reduction at seven to eight weeks occurred in one of 18 procedures, which may be lower than fetal reduction later in pregnancy (Itskovitz-Eldor et al, 1992). The disadvantage of early fetal reduction is that spontaneous fetal reduction is common in early pregnancy. Among 26 triplet conceptions, 12 singleton births, 12 twin births and 2 miscarriages resulted without fetal reduction (Blumenfeld et al, 1992). Fetal reduction may only be indicated in quadruplets and quintuplets, unless there are other obstetric factors increasing the risk of preterm birth.

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Table 9.1. Pregnancy rates for GIFT procedures related to number and selection of eggs transferred No of eggs transferred Percentage pregnancy rates Selected eggs transferred Percentage pregnancy rates All eggs transferred Multiple pregnancies

1

2

3

4+

37 (70)

33(351)

29 (343)

35(48)

12(81)

16(112)

19(125)

32 (27)

18

20

34

1

Numbers in brackets. The alternative to fetal reduction is to reduce the number of oocytes transferred to the tube. In women with more than two eggs collected, transferring the best two eggs has not reduced the pregnancy rate (29%) per GIFT procedure (Table 9.1; Freeman and Osborn, 1989). This avoids the risk of triplets. Women with up to three eggs collected can have all their eggs transferred as multiple pregnancy is less common than in women having five or more eggs collected.

GIFT combined with laparoscopic, diagnostic or operative procedures

In most cases, laparoscopic GIFT is done separately to diagnostic or operative laparoscopy, the suitability and possible need for operative procedures preceding the definitive laparoscopy for GIFT. Combining diagnostic and operative endoscopy with the GIFT procedure has the advantages of avoiding one or two laparoscopies, reducing the duration of infertility, and reducing patients' costs, stress and time off work. The possible disadvantages include the reduction of pregnancy success rates, the uncertain timing of oocyte pick-up for patients having the GIFT procedure after a combined operative GIFT laparoscopy, and the need for the surgeon to have more diverse endoscopy skills. Grindoff et al explored the combined use of laparoscopy for diagnostic, operative, and the GIFT procedure in 33 women (Grindoff et al., 1990). In 13 of these women, the diagnostic procedure led to immediate GIFT or IVF. In the remaining 19, adhesiolysis or electrocautery for endometriosis was carried out before the GIFT procedure. The pregnancy rate was similar in the operative group - 5 of 19 (26%) - to that in the non-operative group - 4 of 13 (31%). The pregnancy rates were identical for the IVF and GIFT procedures. Corson has reached the same conclusion: that treatment of endometriosis does not affect the success rate of GIFT (Corson et al., 1991).

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GIFT was combined with adhesiolysis in 208 patients with non-endometriotic adhesions (al-Shawaf et al., 1990a). The pregnancy rate in the 134 patients suitable for adhesiolysis was 38.8%, being similar to the 74 patients not suitable for surgery (40.5%). The pregnancy rate was lower for those operated on with stage III and IV adhesions, and the ectopic pregnancy rate increased 3.5 times in the operative group. It was concluded that GIFT was an option in patients having adhesiolysis, particularly with stage I and II adhesions. GIFT has been less successful in association with tubal microsurgery, three pregnancies occurring in 26 patients, of which only one was ongoing (Mardesic et al, 1989). Ectopic pregnancy is more common after previous tubal surgery, occurring in 10 of 30 patients (33%) (al-Shawaf et al, 1990b). It may be disadvantageous to perform tubal surgery in association with GIFT or use GIFT after previous failed tubal surgery. However, GIFT has been successful in association with fimbriectomy reversal, four of four patients becoming pregnant (Novy et al., 1991). It is difficult to know whether the addition of GIFT is important as fimbriectomy reversal has an average success rate (22-55%), depending on the width of the ampulla. One normal pregnancy and birth has been reported after injecting gametes by needle into a tube blocked at the fimbrial end (Formigli et al., 1990), an unusual procedure which may have had the advantage of avoiding operative trauma or adhesive disease secondary to previous surgery. The same technique has been used in a patient who repeatedly failed with IVF in association with Asherman's syndrome, the blocked tube leading to a normal upper uterine cavity on one side (Formigli et al., 1990). GIFT after previous ectopic pregnancy

Previous ectopic pregnancy may not affect the success rate of the GIFT procedure (Guirgis and Craft, 1992). Twenty-eight pregnancies resulted from 111 GIFT procedures in women with a previous ectopic pregnancy (25.2%). Only one ectopic pregnancy resulted. This ectopic pregnancy rate is lower than that in spontaneous pregnancy after a previous ectopic pregnancy (7-10%). Using the non-ectopic tube in a GIFT procedure may reduce the risk of repeat ectopic pregnancy from spontaneous conception. GIFT in association with intrauterine and ectopic pregnancy

Five ectopic pregnancies have been reported in association with intrauterine pregnancy in 601 clinical IVF pregnancies, an incidence of 0.83% (Li et al.,

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1992). After laparoscopic surgical intervention, all five intrauterine pregnancies resulted in live births.

GIFT and bicornuate uteri

Thirty GIFT procedures have been reported in association with bicornuate uteri and ten pregnancies resulted (33%), so that fertility is unaffected in these circumstances (Guirgis and Shrivastav, 1990). One ectopic pregnancy and one miscarriage occurred; three premature births resulted, which is similar to the rate of premature birth after natural conception in such patients (10-15%). GIFT in association with donor eggs

GIFT may be useful in donor egg programmes (Borrero et al, 1989). Eleven of 19 (58%) patients with premature ovarian failure became pregnant using incremental hormone replacement therapy up to 100 days and GIFT gamete procedure. GIFT in association with surrogacy

A successful pregnancy has been reported using another woman's tubes for the deposition of gametes, thenflushingembryos from the uterus and transferring them to the patient's uterus (Formigli et al, 1989). The patient had failed tubal microsurgery and repeated failure of IVF. GIFT surrogacy has the disadvantages of the risk of ectopic pregnancy in the surrogate, or incomplete uterine flushing risk with a resultant unwanted uterine pregnancy, both of which may deter potential surrogates. A variation of GIFT

Fallopian replacement of eggs with delayed intrauterine insemination (FREDI) is based on the assumption that the sperm in the ususal GIFT procedure are placed with eggs when the eggs may not be fully mature. The eggs are placed in the fallopian tube and when they are judged to be mature, after several hours, high intrauterine insemination is performed (Leung et al., 1989). Eight pregnancies from 23 attempts were reported in 1989 (35%). The same group have reported high pregnancy rates combining the usual GIFT procedure with intracervical and intrauterine insemination performed after GIFT: 27 pregnancies in 56 GIFT procedures (48%) (Tucker et al, 1989). The in-vitro preparation of sperm for GIFT is rigid in most centres and the

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addition of sperm to the female genital tract may provide an additional or preferable source of sperm capable of fertilizing eggs. A controlled trial of this method is warranted. Effectiveness of the GIFT procedure

GIFT has been found to be nearly ten times as successful as no treatment in 76 couples who had unexplained infertility for three years. The cumulative pregnancy rate after three GIFT procedures was 52%, resulting in a monthly fecundability of 0.17, while the monthly fecundability with no treatment before and after the GIFT procedures over 24 months was 0.02 (Murdock et al, 1991). Randomized controlled trials would be better undertaken in groups randomized so that one group received two GIFT treatments while the other group waited untreated over the duration of the two treatment cycles. The success rate of GIFT depends upon both the number of eggs transferred and the quality of sperm (Guzick et al, 1989). Logistic regression of sperm and egg factors can predict the probability of pregnancy as between 4% and 59%. Three or more mature eggs increase the chance of pregnancy by 3.8 times, while sperm motility less than 30% decreases the chance by 0.34. Failure of three or more supernumerary oocytes to fertilize in vitro in association with a GIFT procedure greatly increases the chance that pregnancy will not occur (Cushnahan and Osborn, 1989). In a collaborative study of 80 GIFT procedures, the pregnancy rates were endometriosis 38%, idiopathic 35% and male factor 18% (Diedrich and Bauer, 1992). Alam et al. have calculated the cumulative pregnancy rate from one GIFT procedure with cryopreservation of embryos. Fifty-one of 97 patients had embryos frozen, half of whom have had the embryos transferred. The cumulative pregnancy rate was 52.2% (Alam et al, 1993). Except for the study of Tanbo et al. (Tanbo, Dale and Abyholm, 1990), GIFT has resulted in higher pregnancy rates than IVF (Table 9.2; Tanbo et al., 1990; Murdoch et al, 1991; Wessels et al, 1992). This is also true for national data in Australia. Where IVF has similar success rates to GIFT, IVF will be the preferred procedure as it avoids laparoscopy and general anaesthesia. For the infertility centres where GIFT is more successful than IVF, GIFT would be suitable for infertility patients with one normal tube. It requires less laboratory service and is less sensitive to problems of quality control of embryology techniques. In reported studies, zygote intrafallopian transfer (ZIFT) and tubal embryo stage transfer (TEST) have produced results equal to or better than GIFT (Tanbo et al, 1990; Pool et al, 1990; Hammitt et al, 1990; Table 9.2). It is

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Table 9.2. Use oflVFand GIFT in Australia Year

IVF

Live birth rate (%)

GIFT

Live birth rate (%)

1989 1990 1991 1992 1993

7228 7153 7353 7564 7709

9.0 10.1

2700 2783 4372 4342 4215

28.9 20.8 19.5 20.4 20.8

7.8 8.5 9.5

difficult to interpret these data as ZIFT and TEST only occur in patients with successful fertilization. Patients with failed fertilization are included in GIFT data but are excluded from TEST or ZIFT. The idea of combining GIFT and IVF resulted from the desire to determine if fertilization occurred in vitro, particularly in patients with male infertility. Because the average number of eggs collected per pick-up is now nine, fertilization can usually be determined in the GIFT procedure by using the excess eggs for IVF. In centres where GIFT is better than IVF, there seems little point in using oocytes for IVF. It is better to proceed to GIFT and check fertilization with accessory ooctyes. Transferring embryos to the tube or uterus after IVF yields similar results, so the tube may not be more advantageous than the laboratory in some centres (Tothefa/., 1992). In male infertility a retrospective study showed GIFT was superior to TEST. A controlled trial confirmed this result, the pregnancy rate being significantly higher for GIFT (25%) than for TEST (13%) (Calderon et ai, 1991). In a Monash IVF study, microinjected oocytes resulted in more pregnancies when placed immediately in the tube - microinjection intrafallopian transfer (MIFT) 7/28 (25%) - than when fertilized in vitro and transferred later to the tube or uterus - 8/66 (12%). ZIFT and TEST may be used if fertilization cannot be checked by GIFT with excess oocytes, or if real pregnancy rates, including patients with failed fertilization in vitro, were significantly higher than GIFT. Such centres would be expected to have higher pregnancy rates for IVF than for GIFT. Comparison of GIFT with intrauterine insemination and superovulation

Intrauterine insemination alone has a low success rate of 1-6.6%, but in association with superovulation (SO) the pregnancy rate per cycle ranges from 1.5% to 20% (Kaplan et al, 1989; Abyholm et al, 1992; Mills et al., 1992). Adding intraperitoneal insemination to IUI and SO, a pregnancy rate of 29.3% has been reported (Abyholm et al., 1992). It is difficult to compare SO

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alone with SO and IUI, but the two most favourable reports of SO and IUI had pregnancy rates of 18% and 20% (Mills et al, 1992; Hull et al., 1992), while the two most favourable reports of SO alone were 9.7% and 8.9% (Wessels et al, 1992; Abyholm et al, 1992). The number of cycles included for each method may alter results as pregnancy rates may decline with increased number of treatments. Apart from the one report of combined IUI, IPI and SO pregnancy rates with SO ± IUI are much less than for GIFT (Iffland et al, 1991). Because of the increased cost of GIFT it may save money for both the patient and health service to use IUI ± SO prior to GIFT. Because of the variation in success rates for SO alone or with IUI, it may be preferable for centres to establish their own success rates in controlled trials before deciding how to use the simpler techniques. GIFT has been done in association with natural cycles but the clinical pregnancy rate is low- six pregnancies in 48 treatments (13%), which may not be high enough to justify laparoscopy. If vaginal GIFT becomes more successful, then natural cycle GIFT may become more popular.

Current status of GIFT

The GIFT procedure became more common in Australia between 1991 and 1993. The live-birth rate was three times higher for GIFT than IVF in 1989 (see Table 9.2) and has remained double the IVF rate between 1990 and 1993. In-vitro fertilization pregnancy rates have increased to 20-25% in several units in Australia and GIFT is being used less often in such units. The role of GIFT will mainly be in units where IVF cannot achieve comparable pregnancy rates, where laboratory facilities are not available for IVF, where IVF is more expensive than GIFT, and where Roman Catholic hospitals do not permit the use of the IVF procedure. References Abyholm, T., Tanbo, T., Dale, P.O. and Magnus, O. 1992. In vivo fertilization procedures in infertile women with patent fallopian tubes: a comparison of gamete intrafallopian transfer, combined intrauterine and intraperitoneal insemination, and controlled ovarian hyperstimulation alone. Journal of Assisted Reproductive Genetics 9(1): 19-23. Alam, V., Weckstein, L., Ord, T., Stone, S., Balmaceda, J.P. and Asch, R.H. 1993. Cumulative pregnancy rate of the GIFT and cryopreservation of embryos. Human Reproduction 8(4): 559-62. al-Shawaf, T., Ah-Moye, M., Fiamanya, W., Smith, B. and Craft, I. 1990a. Gamete intra-fallopian transfer in non-endometriotic pelvic adhesions. Human Reproduction 5(4): 434-8. al-Shawaf, T., Serhal, P., Fiamanya, W., Harper, J. and Craft, I. 1990b. Transfal-

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lopian tube embryo transfer: successful pregnancy outcome. Journal of In Vitro Fertilisation and Embryo Transfer 7(6): 337—40. Blumenfeld, Z., Dirnfeld, M., Abramovici, H., Amit, A., Bronhtein, M. and Brandes, J.M. 1992. Spontaneous fetal reduction in multiple gestations assessed by transvaginal ultrasound. British Journal of Obstetrics and Gynaecology 99(4): 333-7. Borrero, C , Remohi, J., Ord, T., Balmaceda, J.P., Rojas, F. and Asch, R.H. 1989. A program of oocyte donation and gamete intra-fallopian transfer. Human Reproduction 4(3): 275-9. Brady, T., Leeton, J. and Osborn, J.C. 1989. Relationship between clinical and technical ability on GIFT pregnancy rates. Annual Meeting of American Fertility Society, Washington DC. Calderon, I., Fuscaldo, G., Yates, C , Azuma, K., Osborn, J. and Wood, C. 1991. GIFT versus TEST for male infertility, a prospective randomised trial. Proceedings of the American Fertility Society, 48th Annual Meeting, New Orleans, Abstract. Corson, S.L., Batzer, F.R., Gocial, B., Daly, D.C., Eisenberg, E., Huppert, L.C. and Maislin, G. 1991. Surgical treatment of endometriosis at the time of gamete intrafallopian transfer. Journal of Reproductive Medicine 36(4): 274-8. Cushnahan, L. and Osborn, J.C. 1989. Failed fertilisation of supernumery oocytes in GIFT is predictive of a poor outcome. Annual Meeting of American Fertility Society, Washington DC. Diamond, E., Yemini, M., Mukaida, T., Sutin, R. and Veniszee, A. 1992. Culdoscopic gamete tubal transfer: a new approach to gamete intrafallopian tubal transfer. Fertility and Sterility 57(5): 1114-6. Diedrich, K. and Bauer, O. 1992. Indications and outcomes of assisted reproduction. In: Ballieres Clinical Obstetrics & Gynaecology, June 6(2), pp. 373-88. Ferraiolo, A., Croce, S., Anserini, P., Remorgida, V., Lanera, P., Capitanio, G.L. and de Cecco, L. 1991. 'Blind' transcervical transfer of gametes in the fallopian tube: a preliminary study. Human Reproduction 6(4): 537-40. Formigli, L., Pagano, M., Formigli, G., Stangalini, A., Belotti, G. and Roccio, C. 1990. Gamete intrafallopian transfer in a woman with tubal occlusion. A case report. Journal of Reproductive Medicine 35(1): 58-60. Formigli, L., Pagano, M., Roccio, C , Stangalini, A., Belotti, G., Coglitore, M.T. and Formigli, G. 1989. Surrogate human fallopian tubes for overcoming tubal infertility. Human Reproduction 4(4): 416-7. Freeman, L. and Osborn, J.C. 1989. How many oocytes should be transferred at GIFT? Annual Meeting of American Fertility Society, Washington DC. Grindoff, P.R., Hall, J.L., Nelson, L.M. and Stillman, R.J. 1990. Efficacy of assisted reproductive technology during diagnostic and operative infertility laparoscopy. Obstetrics and Gynecology 75(2): 299-301. Guirgis, R.R. and Craft, L. 1992. Gamete intrafallopian transfer in women who had ectopic pregnancy previously. Obstetrics and Gynecology 79(4): 586-8. Guirgis, R.R. and Shrivastav, P. 1990. Gamete intrafallopian transfer (GIFT) in women with bicornuate uteri. Journal of In Vitro Fertilisation and Embryo Transfer 7(5): 283-4. Guzick, D.S., Balmaceda, J.P., Ord, T. and Asch, R.H. 1989. The importance of egg and sperm factors in predicting the likelihood of pregnancy from gamete intrafallopian transfer. Fertility and Sterility 52(5): 795-800. Hammitt, D.G., Syrop, C.H., Hahn, S.J., Walker, D.L., Butkowski, C.R. and Donovan, J.F. 1990. Comparison of concurrent pregnancy rates for in-vitro fertiliz-

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ation - embryo transfer, pronuclear stage embryo transfer and gamete intrafallopian transfer. Human Reproduction 5(8): 947-54. Hull, M.G., Eddomes, H.A., Fahey, V, Abuzeid, M.I., Mills, M.S., Cahill, D.J., Fleming, C.F., Wardle, P.G., Ford, W.C. and McDermott, A. 1992. Expectations of assisted conception for infertility. British Medical Journal 304: 1465-9. Iffland, C.A., Reid, W., Amso, N., Bernard, A.G., Buckland, G. and Shaw, R.W. 1991. A within-patient comparison between superovulation with intra-uterine artifical insemination using husband's washed spermatozoa and gamete intrafallopian transfer in unexplained infertility. European Journal of Obstetrics and Gynaecological Biology 39(3): 181-6. Isherwood, P.J. 1990. Gamete intrafallopian transfer in women with accessory tubal ostia. British Journal of Obstetrics and Gynaecology 97: 542-8. Itskovitz-Eldor, J., Drugan, A., Levron, J., Thaler, I. and Brandes, J.M. 1992. Transvaginal embryo aspiration - a safe method for selective reduction in multiple pregnancies. Fertility and Sterility 58(2): 351-5. Kaplan, C.R., Olive, D.L., Sabella, V., Asch, R.H., Balmaceda, J.P., Riehl, R.M., Groff, T.R., Burns, W.N. and Schenken, R.S. 1989. Gamete intrafallopian transfer vs superovulation with intrauterine insemination for the treatment of infertility. Journal of In Vitro Fertilisation and Embryo Transfer 6(5): 298-304. Kerin, J.F. 1992. Nonhysteroscopic falloposcopy: a proposed method for visual guidance and verification of tubal cannula placement for endotuboplasty, gamete and embryo transfer procedures. Fertility and Sterility 57(5): 1133-5. Khan, I., Devroey, P., Van den Bergh, M., Camus, M., Wistanto, A., Staessen, C, Smitz, J. and Van Steirteghem, A. 1989. The effect of pneumoperitoneum gases on fertilization, cleavage and pregnancy in human in-vitro fertilization and gamete intra-fallopian transfer. Human Reproduction 4(3): 323-6. Kirsop, R., Porter, R., Torode, H., Smith, D. and Saunders, D. 1991. The role of hysteroscopy in patients having failed IVF/GIFT transfer cycles. Australian and New Zealand Journal of Obstetrics and Gynaecology 31 (3): 263-4. Leung, C.K., Leon, M.K., Chan, Y.M., Wong, C.J., Chan, H.H. and Tucker, M.J. 1989. Fallopian replacement of eggs with delayed intrauterine insemination (FREDI): an alternative to gamete intrafallopian transfer (GIFT). Journal of In Vitro Fertlisation and Embryo Transfer 6(3): 129-33. Li, H.P., Balmaceda, J.P., Zouves, C, Cittadini, E., Casas, P.F., Johnston, I. and Asch, R.H. 1992. Heterotopic pregnancy associated with gamete intra-fallopian transfer. Human Reproduction 7(1): 131-5. Lucena, E., Paulson, J.D., Ruiz, J., Asmar, P., Mendoza, J.C., Ortiz, J.A., Gomez, M., Arango, A., Lucena, C. and Lucena, A. 1990. Vaginal gamete intrafallopian transfer. Experience with 14 cases. Journal of Reproductive Medicine 35(6): 645-7. Lucena, E., Ruiz, J.A., Mendoza, J.C., Ortiz, J.A., Lucena, C, Gomez, M. and Arango, A. 1989. Vaginal intratubal insemination (VITI) and vaginal GIFT, endosonographic technique: early experience. Human Reproduction 4(6): 658-62. Mardesic, T., Laitl, J., Jirasek, J.E., Stroufova, A. and Presl, J. 1989. Gamete intrafallopian transfer (GIFT) as a complement to the tubal microsurgery. Zentralblat Gynakologie 111(5): 276-80. Mills, M.S., Eddowes, H.A., Cahill, D.J., Fahy, U.M., Abuzeid, M.I., McDermott, A. and Hull, M.G. 1992. A prospective controlled study of in-vitro fertilization, gamete intra-fallopian transfer and intrauterine insemination combined with superovulation. Human Reproduction 7(4): 490-4. Murdoch, A.P., Harris, M., Mahroo, M., Williams, M. and Dunlop, W. 1991. Gamete

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intra-fallopian transfer (GIFT) compared with intrauterine insemination in the treatment of unexplained infertility. British Journal of Obstetrics and Gynaecology 98(11): 1107-11. Novy, M.J., Hickok, L.R., Patton, P.E., Craemer, M.J. and Wolf, D.P. 1991. Pregnancy after fimbriectomy reversal: results of microsurgery augmented by gamete intrafallopian transfer and embryo transfer. Fertility and Sterility 56(6): 1166-8. Osborn, J.C. 1989. Unilateral transfer of oocytes at GIFT does not result in an increased pregnancy rate compared to bilateral transfer. Annual Meeting of American Fertility Society, Washington DC. Penzias, A.S., Alper, M.M., Oskowitz, S.P., Berger, M.J. and Thompson, I.E. 1991. Comparison of unilateral and bilateral tubal transfer in gamete intrafallopian transfer (GIFT). Journal of In Vitro Fertilisation and Embryo Transfer 8(5): 276-8. Perone, N. 1991. Gamete intrafallopian transfer (GIFT): historic perspective [editorial]. Journal of In Vitro Fertilisation and Embryo Transfer 8(1): 1-4. Pool, T.B., Ellsworth, L.R., Garza, J.R., Martin, J.E., Miller, S.S. and Atiee, S.H. 1990. Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2 year study. Fertility and Sterility 54(3): 482-8. Possati, G., Seracchioli, R., Melega, C, Pareschi, A., Maccolini, A. and Flamigni, C. 1991. Gamete intrafallopian transfer by hysteroscopy as an alternative treatment for infertility. Fertility and Sterility 56(3): 496-9. Tanbo, T., Dale, P.O. and Abyholm, T. 1990. Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in-vitro fertilization, gamete intrafallopian transfer and tubal embryo stage transfer. Human Reproduction 5(3): 266-70. Toth, T.L., Oehninger, S., Toner, J.P., Brzyski, R.G., Acosta, A.A. and Muasher, S.J. 1992. Embryo transfer to the uterus or the fallopian tube after in vitro fertilization yields similar results. Fertility and Sterility 57(5): 1110-13. Tucker, M.K., Chan, Y.M., Chan, S.Y., Wong, C.J., Mao, K.R., Leong, M.K. and Leung, C.K. 1989. The use of human follicular fluid in gamete intra-fallopian transfer. Human Reproduction 4(8): 931-6. Wessels, P.H., Cronje, H.S., Oosthuizen, A.P., Trumpelmann, M.D., Grobler, S. and Hamlett, D.K. 1992. Cost-effectiveness of gamete intra-fallopian transfer in comparison with induction of ovulation with gonadotrophins in the treatment of female infertility: a clinical trial. Fertility and Sterility 57(1): 163-7. Yee, B., Rosen, G.F., Chacon, R.R., Soubra, S. and Stone, S.C. 1989. Gamete intrafallopian transfer: the effect of the number of eggs used and the depth of gamete placement on pregnancy initiation. Fertility and Sterility 52(4): 639-44.

10 The use of assisted reproductive technology for the treatment of male infertility ROBERT I. McLACHLAN

Introduction

Male infertility affects one man in 20 and contributes with equal frequency to infertile unions. In many men it is not yet possible to improve the quality of ejaculated sperm, which has led to the widespread use of assisted reproductive techniques to resolve infertility. These techniques are now widely discussed in the lay press and as a result the population is far more knowledgeable and willing to discuss male infertility. Accordingly, it is essential that all clinicians involved in reproductive medicine be able to discuss the options for fertility using ART. It is important to recognize that ART does not represent a true treatment for male infertility as it does not provide for an increase in sperm quality and improved prospects for natural conception. However, it does provide a powerful means of bypassing the infertility associated with poor sperm quality and provides pregnancy rates similar to those of normal couples having unprotected intercourse (approximately 20-25% pregnancy per cycle). Despite severely reduced sperm number, motility and quality, ART permits fertilization by providing a high concentration of sperm to come into close proximity with the egg and even to be injected into the egg cytoplasm if the need arises. The prospects for pregnancy in severe male infertility have improved markedly since the advent of ICSI as the number and quality of sperm no longer limit the chance of success. Indeed, it has been suggested that the assessment of men with infertility requires no more than a semen analysis to ensure that some sperm are available for injection. This is a dangerous proposition as specific treatments are available for many types of male infertility, and good medical practice demands that therapeutic interventions should be aimed toward natural conception. Furthermore, clinical assessment of all infertile men is essential to examine for related health issues such as testicular cancer 124

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and the need for androgen replacement therapy. Thus it is a serious error to regard the performance of a semen analysis as completing the investigation of the male. Background fertility rates in male infertility are better than widely appreciated (Baker et al., 1985) and the decision of when to enter an ART programme is a complex one, involving issues such as the duration of infertility, the age and reproductive health of the female, psychosocial aspects and the severity of the sperm defect. Modern approaches to semen analysis and sperm function testing now provide a basis for counselling about natural and ART outcomes.

Selection of ART for male factor patients

In general, the mild-moderate male factor patients are treated with the simplest ART procedures as these are the least expensive and have established safety records, whereas new sophisticated treatments, such as ICSI, are reserved for severe cases for whom there is no alternative. When planning the first treatment cycle, a 'best guess' of the ART method must be based on the quality of the semen as assessed using the WHO criteria (World Health Organization, 1992) and/or of the washed sample (see below). The number, motility profile and morphology are all correlated with the likelihood of fertilization, with morphology being the strongest predictor. In addition, more sophisticated tests of sperm function can be used to predict ART outcomes (Liu and Baker, 1992a, 1992b). Other factors directing the selection of the initial ART approach include female factors (e.g. tubal disease precluding the use of GIFT), and the expertise and preference of the treating unit. In second and subsequent cycles, the experience of the previous cycles is an invaluable guide in deciding either to continue with the existing approach, if results have been good, or to change to a more sophisticated method following poor or failed fertilization outcomes.

Types of ART in male factor infertility

All ART treatments involve the collection of eggs and sperm followed by the facilitation of their union by coculture in vitro and the transfer of embryos (IVF), or the placement of the gametes immediately into the fallopian tube wherein fertilization occurs (GIFT). The treatment strategy outlined below represents the approach used by our unit. However, variations on the theme will be found in all ART programmes. The first step is to assess the quality of sperm available for insemination.

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Methods have been developed for the selection of the most motile and normally shaped sperm from the ejaculate. The most widely applied method involves the centrifugation of sperm through a gradient which retards the passage of all but the most motile sperm, which can then be washed free of the gradient material into culture media to give a 'washed' sample ready for insemination (Bolton and Braude, 1984). Such centrifugation/sedimentation kits are commercially available (e.g. Sperm Prep, Fertility Technologies, USA). These washed samples have enhanced motility and morphology profiles and consequently improved fertilization rates. Sperm motility stimulants, such as pentoxifylline, have been reported to improve the motility profile and rate of acrosomal loss in asthenospermic samples, although whether there is an improvement in fertilization rates remains controversial (Tournaye et ai, 1993a). Such agents must be washed out of the sample prior to insemination as there is evidence that they can retard embryo development (Tournaye et al., 1993b). The washed sperm preparation is assessed in terms of the total number of motile sperm, the percentage of sperm showing rapid (type A) or total motility (type A+B+C), and the sperm morphology (World Health Organization, 1992). A trial wash of semen is useful when deciding whether the patients are suitable for (i) GIFT (requiring a minimum of 1.5 million motile sperm) as opposed to conventional, and (ii) microdrop insemination (requiring a minimum of 50000 motile sperm) as opposed to ICSI which has essentially no lower limit. Due to the inherent variability of human ejaculates, such trial washes do not necessarily predict what will be available on the day of egg pick-up, making it necessary that units have someflexibilityin allowing a last minute change of treatment. Most units provide two basic approaches, namely conventional or microdrop IVF for mild-moderate male factor problem and microinjection for severe cases. Some units, including ours, also offer the GIFT procedure for mild cases as it has provided excellent results over many years. When considering the outcome of such programmes, it is important to define the parameters used. In this review, success is described as clinical pregnancy rate per egg collection. A clinical pregnancy is one in which a fetal heart is detected at six weeks' gestation. As the miscarriage rate in ART treatments is approximately 20%, the couples should appreciate that the chance of delivering a live baby is derived by multiplying thesefiguresby 0.8.

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Conventional IVF Technique Conventional IVF is available in all IVF programmes and involves the insemination of eggs in a standard culture well (about 0.6 ml volume) with motile sperm at concentrations between 200000 and 1 000000 per ml. Fertilization is assessed one day later and one to three embryos are transferred transvaginally (at the four to eight-cell stage) on day two or three. Suitable patients The ejaculates of most men with mild-moderate reductions in sperm number (counts 5-19 million) and/or motility (20-49% total motility) will provide a washed semen sample with sufficiently high numbers (more than 500000) of motile sperm. Anticipated results Fertilization rates (FR) of 40-50% would be expected and as a result clinical pregnancy rates of 20-25% per cycle. One cautionary note is that men with more than 90% abnormal forms in the ejaculate do poorly with this form of IVF (FR < 30%) irrespective of the other parameters. An attempt at microdrop insemination using high insemination concentrations (Hammit, 1993) may be tried initially, although ICSI is recommended as first-line treatment if more than 95% abnormal forms are present. Microdrop insemination Many units prefer to perform inseminations in small-volume culture wells (about 50 jul) as it is proposed that they offer advantages over conventional IVF by requiring fewer sperm and by promoting improved embryo development. Microdrop insemination is simple to perform and provides good results in mild-moderate male factor infertility, with clinical pregnancy rates of approximately 25% per cycle. As few as 50 000 motile sperm are required from the washed sample, meaning that quite severe semen defects can be successfully treated. The same constraints of morphology apply as for conventional IVF. Gamete intrafallopian transfer Technique Eggs (one to three) are placed together with motile sperm (500000-1 500000) into the fallopian tube on the same day as egg collection.

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Suitable patients In mild male factor or idiopathic infertility, large numbers of motile sperm can be obtained for GIFT. Ejaculate quality criteria are the same as those for conventional IVF: sperm number 5-19 million, and/or motility 20-49% total motility. If doubt exists as to their suitability for GIFT, a trial wash is useful in demonstrating the ability to obtain the requisite number of motile sperm. Anticipated results There are two principle reasons why GIFT is not widely practised. 1. Fertilization is not observable, meaning that if pregnancy does not occur, it is impossible to assess whether fertilization has failed to occur because of some significant sperm and/or egg problem, or whether in fact early pregnancy loss has occurred. This information could well influence the choice of the treatment in the next cycle. 2. There is a need for a laparoscopy for the GIFT transfer. Many units claim similar results with conventional IVF without the need to resort to GIFT. Nonetheless, we have continued to use this approach and obtain a pregnancy rate of around 25% per cycle.

Intracytoplasmic sperm injection - the new technology for male infertility The procedure involves the direct injection of a single sperm into the egg cytoplasm, thus the term 'intracytoplasmic sperm injection' (Palermo et al., 1992). No development in ART in the past decade has had a greater impact than ICSI, which has revolutionized the management of severe male infertility by providing couples with success rates equal to those of other IVF couples, and by permitting pregnancies in couples once thought untreatable. While the technique sounds straightforward, it was once thought technically impossible and certain to cause irreparable damage to the egg. Technical advances in micromanipulation and the diligence of the Belgian group have now lead to the rapid development and acceptance of ICSI. Some technical aspects of the procedure will be reviewed, followed by the clinical success rates and an outline of issues for the future of ICSI. The development of the ICSI procedure In-vitro fertilization programmes had for years struggled to achieve fertilization and pregnancy in couples with the common problem of very low sperm

The use of assisted reproductive technology numbers and/or motility (oligoasthenospermia). In other couples with less severe, or even no apparent, male factor problems, failed fertilization was presumed to occur as a result of defects in sperm function (e.g. egg attachment or penetration) or egg defects. Occasionally the use of high numbers of motile sperm in the IVF culture well would achieve success, but often fertilization and pregnancy rates remained disappointing. The injection of a few sperm beneath the outer layer of the egg (subzonal microinjection) was the first significant breakthrough for such couples (Ng et al., 1988). However, the SUZI technique was limited to the use of motile sperm, a low fertilization rate (approximately 20% of eggs injected) and frequently by the problem that more than one sperm would fertilize the egg, such polyspermic fertilizations being non-viable. Other approaches have included partial zona dissection in which a region of the zona pellucida is dissolved with a directed stream of weak acid thereby facilitating sperm access to the egg outer membrane (Cohen et al., 1991). Again, poor fertilization rates relative to ICSI and a tendency toward polyspermy limited this approach. The ICSI technique first came to general awareness at a Serono Symposium in Adelaide, South Australia, in December 1992, when Professor Van Steirteghem made it clear that the process in fact damaged few eggs and that fertilization and embryo development rates were far superior than those obtained using SUZI, while, predictably, polyspermy was avoided (Van Steirteghem et al., 1993). Shortly afterwards, several centres confirmed these results and SUZI was abandoned by all clinics which had the resources (financial and technical) to perform ICSI. Whether this decision will ultimately be shown to be premature remains to be seen. It has been strongly argued that further assessments of the safety of ICSI should have been obtained prior to such widespread application (De Jonge and Pierce, 1995). Primarily due to lack of such resources, some programmes continue to use SUZI or the related micromanipulation techniques such as partial zona dissection. Clinical application of the ICSI technique The technique was first used for couples with either severe male factor infertility or unexplained failure to fertilize with conventional IVF. Severe male factor infertility is generally defined as combinations of oligoasthenospermia and/or sperm showing severe defects of shape (teratospermia). ICSI has now been extended to new patient groups with infertility problems previously considered untreatable. Patient categories considered for ICSI are similar across all IVF programmes; those applying at Monash IVF are shown in Table 10.1.

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Table 10.1. Indications for intracytoplasmic 1.

2. 3.

4. 5.

6.

7.

8.

9.

sperm injection

Severe oligoasthenospermia with insufficient sperm for conventional IVF. This group includes men with extremely low sperm numbers, e.g. less than 0.2 million/ml. Unexplainedfailure or poor fertilization in previous IVF cycles, i.e. possible defects in sperm function. Severe structural abnormalities of sperm (teratospermia). Fertilization rates using conventional or microdrop IVF techniques are reduced when teratospermia rates of >90% are present (defined as per the strict criteria, see Chapter 4) and exceedingly low when rates > 96% Immotile sperm, e.g. inherited disorders of the sperm tail for which no other IVF technique is effective. Necrospermia. Sperm can undergo necrosis during epididymal transit either spontaneously (so called 'epididymal necrospermia') or occasionally following surgical or infective damage (e.g. post-vasectomy reversal). Such ejaculates have a high proportion of immotile and non-viable sperm resulting in poor results using conventional IVF. Sperm autoimmunity in which antibodies impair sperm motility or egg binding. High levels of antibodies directed to the head often render conventional IVF ineffective. Epididymal or testicular sperm obtained at surgery from men with a range of congenital or acquired blockages to sperm outflow. Such sperm are often immotile and incapable of fertilization with conventional IVF. Sperm obtained by electroejaculation or from urine in cases of retrograde ejaculation, e.g. in patients with diabetes or paraplegia. These sperm are often in low number or have poor motility or vitality. Azoospermia and testicular failure. Very recently, some groups have advocated the use of open testicular biopsy to recover sperm from men with severe spermatogenic problems and azoospermia.

The ICSI technique The procedure involves the production of exceedingly fine glass pipettes (external diameter approximately 7 ;um) into which a single sperm is drawn, tail first (Figure 10.1). The egg is held in place by a larger holding pipette (on the left) while the injection pipette is gradually inserted through the zona (the pale hallow) into the cytoplasm. A critical aspect of the procedure is the aspiration of a small amount of egg cytoplasm into the injection pipette to ensure that it is within the cytoplasm (McLachlan et ai, 1995). The sperm is then released and the pipette withdrawn. The egg shows considerable deformation during this process but resumes its normal structure within minutes.

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Figure 10.1 Single sperm ready for microinjection into the cytoplasm of a mature oocyte.

Results from ICSI The Belgian group have the most extensive experience and recently reported on 2853 cycles involving 1953 couples (Van Steirteghem et al., 1995). The overall normal fertilization rate achieved was 70%, embryos were transferred in 91% of couples and a positive serum hCG was obtained in 34% of cycles. A similar result was found with sperm obtained at surgery from the testis or epididymis. The excellent results have established that the outlook for severe male infertility is now similar to that of couples with other types of infertility such as tubal disease. Two additional points to be made are: (i) it is remarkable that overall only 3% of couples failed to achieve fertilization, with as yet no specific subgroup being resistant, and (ii) similar pregnancy rates are obtained irrespective of the infertility diagnosis - ICSI appears 'non-selectively' effective. Recent results at Monash IVF over the past six months involving 295 egg collections show 73.7% FR in 2308 eggs. Over 90% of patients received an embryo transfer, with a clinical pregnancy rate per cycle of 23.1%. The true success of such programmes needs to take into account pregnancies arising from the later transfer of excess embryos frozen at ICSI cycles. In our

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programme, about 25% of couples have excessive embryos frozen and the pregnancy rate in cycles involving the transfer of these embryos is about 20% per cycle. Overall, it is realistic to indicate to couples that their chance of taking home a baby after a single cycle of treatment is approximately 18%, but the inclusion of the transfer of frozen embryos would increase this to around 22%. In keeping with the broad applicability of ICSI, this outlook can also be given to patients with extremely low sperm counts, epididymal and testicular sperm. The latter require further discussion. Epididymal sperm (including congenital absence of the vas) In many cases of obstructive azoospermia, only epididymal sperm can be obtained at explorative or corrective surgery. As a result of their poor motility and relative immaturity, no ART procedure other than ICSI provides acceptable fertilization and pregnancy rates (Silber et al., 1994, 1995; Nagy et al., 1995). Since microsurgical techniques are required for epididymal sperm retrieval, it is desirable to avoid the need for repeated surgery. This goal can be achieved by combining epididymal sperm aspiration and cryopreservation with multiple aliquots being made for later ICSI treatment cycles if required. Indeed, Devroey et al. (1995b) demonstrated in a small series that cryopreservation of supernumerary epididymal spermatozoa was a successful method to optimize the chance of success in patients with obstructive azoospermia as fresh and frozen spermatozoa resulted in similar fertilization and pregnancy rates. We have recently reported on 59 consecutive ICSI cycles using cryopreserved epididymal spermatozoa in which we observed a somewhat lower than expected normal fertilization rate (approximately 50%), yet embryo transfer and pregnancy rates (around 20% per cycle) similar to those of couples undertaking ICSI using ejaculated sperm for spermatogenic defects (Holden et al., 1997). Sperm vitality prior to freezing was an important predictor of outcome as fertilization rates were poor with values below 20%, presumably reflecting degeneration within the epididymis. The cryopreservation approach therefore does not significantly compromise treatment outcomes yet avoids the need for both repeated surgery and the co-ordination of egg retrieval and sperm aspiration. We concluded that the cryopreservation of epididymal sperm should be routinely performed during scrotal exploration for obstructive azoospermia as 'insurance' against persistent azoospermia and as an effective alternative to repetitive microsurgery or the use of testicular biopsy to obtain testicular sperm. Congenital absence of the vas (CAV) requires particular mention. This condition may be unilateral or bilateral and is a common cause of obstructive azoospermia, having an incidence of 1:2500 in the population. This congeni-

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tal defect in Wolffian duct differentiation is associated with a high frequency of cystic fibrosis (CF) gene defects, with approximately 70% being heterozygotes for CF gene coding region mutations. Recently it has been suggested that many CAV men are compound heterozygotes, with lesions in the coding region and/or in the introns (Chillon et ah, 1995), leading to the suggestion that bilateral CAV (BCAV) and CF are extremes of the same disease spectrum. The implication is that when the use of surgically recovered sperm from CAV men is being considered, thorough genetic screening of the spouse is also essential to reduce the risk of conception of a child with the full-blown CF phenotype.

Testicular sperm for obstructive azoospermia In some cases, sperm can only be obtained directly from testicular tissue. Such cases include: (i) surgically irremediable obstruction in the proximal epididymis or rete testis as a result of previous infection, surgery or congenital abnormality; and (ii) patients electing to use the testicular biopsy to obtain sperm rather than scrotal explorative surgery. There are many reasons for the decision, including the cost of such surgery, concerns about time off work or a reluctance to reverse a vasectomy. Sperm can be readily collected from a small fragment of seminiferous tubule obtained using the fine needle biopsy approach (Mallidis and Baker, 1994) and this procedure can be repeated at each egg collection. It is wise to perform a trial biopsy prior to ICSI in all cases just to be certain that spermatogenesis is proceeding normally and therefore that sperm will be available at egg pick-up. This is recommended because occasionally one sees even vasectomized men, with previously excellent fertility, in whom few if any sperm are obtainable, presumably reflecting an underlying spermatogenic problem. Excellent results have been reported using testicular sperm (Silber
Testicular sperm in cases of necrospermia The presence of a high proportion of dead sperm in the ejaculates is associated with poor fertilization using conventional IVF. Such cases can be idiopathic necrospermia or follow trauma to the genital tract, e.g. post infective or following vasectomy reversal. ICSI using viable sperm obtained by testicular biopsy offers excellent prospects for fertilization (Tournaye et ai, 1996). Overview of ICSI approach The revolution in male infertility treatment provided by ICSI requires further analysis under the headings of basic biology, safety and genetic implications, monitoring of programmes and future applications.

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ICSI addresses basic physiological questions ICSI has directly answered several important physiological questions. 1. It has established that the maturation of sperm in the epididymis, while necessary for normal fertilization in vivo, is not essential for fertilization following microinjection. Sperm motility is not essential for fertilization. 2. Recent data suggest that the upper part of the neck of the sperm, containing the centriole, is needed for early embryonic development. 3. Even structurally abnormal sperm appear to fertilize normally and to produce normal offspring, i.e. sperm structure does not relate to the genetic material it contains (with some reservations, see below). Nonetheless, new questions have arisen as a result of ICSI, such as: why don't 100% of eggs fertilize? In many of these failed-fertilization eggs, it is apparent that the normal decondensation of the sperm head fails and/or the migration/ fusion of the two pronuclei does not occur. What mechanisms control these important functions? A great deal remains to be understood in these processes.

Safety of ICSI There has been concern expressed from the outset about the potential for adverse outcomes granted that ICSI avoids all natural selective processes, with often severely abnormal sperm being selected. However, to date the data have been most reassuring. In 423 children conceived by ICSI, the congenital malformation rate (3.3%) was no greater than that for naturally occurring pregnancies, while the obstetric outcomes were similar to other forms of ART (Wisanto et al., 1995). The Belgian group have also reported 385 chromosomal assessments (by amniocentesis or chorionic villus biopsy) and detected three clinically concerning abnormalities (primarily Klinefelter's syndrome). The investigators concluded that there was not a significant increase in chromosomal abnormalities in ICSI offspring. However, both they and ourselves continue to recommend prenatal chromosomal assessment. Whether there is an increased risk of sex chromosome aneuploidy remains to be established. It must be emphasized that definitive information about the risk of congenital abnormalities will not be known until a very much larger number of births have been recorded. In both Australia and Europe (the latter under the auspices of ESHRE), data bases are being established to permit long term follow-up studies. Of necessity, such studies will need to continue well into the next century to follow both the general and reproductive health of these children.

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Genetic basis of male infertility A specific issue of great importance to ICSI programmes is the risk of similar infertility problems in the offspring. Recent data suggest that as many as 20% of azoospermic and severely oligospermic men have microdeletions of the Y chromosome (Reijo et al. 1995). It is presumed that genes involved in spermatogenesis are to be found in these regions. One such gene was recently described as the 'deleted in azoospermia' or DAZ gene (Reijo et al. 1995). The current polymerase chain reaction (PCR) based screening methods may fail to detect other defects in other as yet unidentified spermatogenic genes, such as single base mutations, misplaced stop codons or small deletions. Thus it is very likely that other abnormalities will eventually be found in the remainder. As the male is the only source of the Y chromosome for a male child, it seems likely that defects in sperm production will be transmitted. This outcome is not certain as it is possible that the autosomal genes may also be involved in spermatogenesis and accordingly that the maternal chromosomes may have an ameliorating effect. Clearly this issue needs to be studied in detail with long-term follow-up studies correlating genetic profiles with sperm quality. At present, ICSI is being used for the more severe male factor couples, but with its high fertilization and pregnancy rates comes the question as to whether it should be used in milder cases. On the other hand, recent data suggest that ICSI may also allow fertility in azoospermic men with testicular failure (see below). What concerns should one have regarding these new applications? It must be recognized that within the umbrella term of 'male factor infertility' there are likely to be many as yet undefined specific genetic conditions. How will these individual conditions be studied in order to determine whether there are problems in embryo development, pregnancy rates and clinical outcomes peculiar to particular types of infertility? This is especially true in the less common sperm defects where current experience is very limited. Azoospermic men with testicular failure Recent data have suggested that ICSI can be used in this group, which comprises about 1:150 men. The findings of azoospermia, reduced testicular volumes and an elevated serum FSH level, are classic in primary seminiferous tubule failure, with such couples generally being advised either to abandon hope of fertility or consider donor insemination. The relationship between serum FSH and testicular morphology has never been straightforward, for example it is well known that approximately 15% of azoospermic men show-

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ing a pattern of arrested germ cell or even the Sertoli cell only syndrome will have a normal serum FSH. Nonetheless, it has been well accepted that the finding of a high FSH (i.e. more than twice the upper limit of normal) almost certainly indicates the absence of mature sperm. The usefulness of serum FSH measurement in the management of severe male infertility has recently been called into question by two observations. First, successful surgical correction of obstructive azoospermia was shown despite the presence of high serum FSH preoperatively (Hauser et al., 1995). Secondly, viable sperm could be extracted from open testicular biopsies from approximately 50% of azoospermic men with testicular failure and used for ICSI yielding fertilization and pregnancy rates similar to those of ejaculated sperm (Tournaye et al., 1995; Devroey et al., 1995a). Even the apparent absence of sperm on a diagnostic open biopsy cannot be assumed to indicate that no sperm will be obtained when a biopsy is taken at the time of ICSI, perhaps because the latter involves the processing of the entire samples rather than just a few cross-sectional examinations. The implication is that there are small foci of complete spermatogenesis persisting even in severely damaged testis, and that the number of sperm produced is so low that no sperm are identified in the ejaculate, perhaps due to their reabsorption during epididymal transit. Is it now appropriate to advise all azoospermic patients, irrespective of their physical and endocrinefindings,of the possibility of using this technique as an alternative to donor insemination? The answer to this question will depend on whether these initial findings can be widely confirmed. In addition there are significant issues of safety and monitoring which must be resolved before this new approach can be widely applied. It is well established that azoospermic men have high rates of chromosomal abnormality (approximately 20%: Kjessler, 1974) such that karyotypic analysis should be performed prior to consideration for ICSI. Conclusion

The pastfiveyears have seen a profound change in the applicability of ART to male factor infertility. Using ICSI, fertility is possible in any man with any sperm available anywhere in the genital tract. Many would argue that our ability to achieve fertilization and apparently normal births far outstrips our understanding of the basic processes involved. Developments in the genetics of male infertility now promise to redress this imbalance and perhaps will lead to a true treatment of male infertility. The initial impression of the safety of ICSI needs to be revisited continually to ensure that the children are normal and

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that couples are properly counselled prior to participation in what still must be regarded as a clinical research programme. References Baker, H.W.G., Burger, H., de Kretser, D.M. and Hudson, B. 1985. Relative incidence of etiological disorders in male infertility. In: Male Sexual Functioned. R.S. Santen and R.S. Swerdloff, pp. 341-72, New York: Marcell Dekker. Bolton, V.N. and Braude, P.R. 1984. Preparation of human spermatozoa for in vitro fertilisation by isopycnic centrifugation on self-generating density gradient. Archives of Andrology 13: 167-76. Chillon, M., Casals, T., Mercier, B., Bassas, L., Lissens, W., Silber, S., Romey, M.C., Ruiz-Romero, J., Verlingue, C. and Claustres, M. 1995. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. New England Journal of Medicine 332: 1475-80. Cohen, J., Alikani, M., Maker, H.E., Adler, A., Talansky, B.E. and Rosenwaks, Z. 1991. Partial zona dissection or subzonal insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertility and Sterility 56: 696-706. De Jonge, C. and Pierce, J. 1995. Intracytoplasmic sperm injection - what kind of reproduction is being assisted? Human Reproduction 10: 2518-28. Devroey, P., Liu, J., Nagy, Z., Goosens, A., Tournaye, H., Camus, M., Van Steirteghem, A. and Silber, S. 1995a Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Human Reproduction 10: 1457-60. Devroey, P., Silber, S., Nagy, Z., Liu, J., Tournaye, J., Joris, H., Verheyen, G. and Van Steirteghem, E. 1995b. Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen-thawed epididymal spermatozoa. Human Reproduction 10: 903-06. Hammitt, D.G. 1993. Treatment of male-factor infertility in vitro insemination with high concentrations of motile sperm. Seminars in Reproduction and Endocrinology 11:72-82. Hauser, R., Temple-Smith, P.D., Southwick, G.J. and de Kretser, D.M. 1995. Fertility in cases of hypergonadotrophic azoospermia. Fertility and Sterility 63: 631-6. Holden, C , Fuscaldo, G., Jackson, P., Cato, A., Southwick, G., Hauser, R., TempleSmith, P. and McLachlan, R.I. 1997. Frozen-thawed epididymal spermatozoa for intracytoplasmic sperm injection. Fertility and Sterility 67: 81-7. Kjessler, B. 1974. Chromosomal constitution and male reproductive failure. In: Male Fertility and Sterility, ed. R.E. Mancini and L. Martini, pp. 231-47, New York: Academic Press. Liu, D. and Baker, H.W.G. 1992a. Tests of human sperm function and fertilization in vitro. Fertility and Sterility 58: 465-83. Liu, D. and Baker, H.W.G. 1992b. Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding and fertilization rates in vitro. Fertility and Sterility 58: 1178-84. Mallidis, C. and Baker, H.W.G. 1994. Fine needle aspiration biopsy of the testis. Fertility and Sterility 61: 367-75. McLachlan, R.I., Fuscaldo, G., Rho, H., Poulos, C , Dalrymple, J., Jackson, P. and Holden, C.A. 1995. Clinical results from intracytoplasmic sperm injection at Monash IVF. Reproduction and Fertility Development 7: 247-53.

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Nagy, Z., Liu, J., Cecile, J., Silber, S., Devroey, P. and Van Steirteghem, A. 1995. Using ejaculated fresh and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertility and Sterility 63: 808-15. Ng, S-C, Bongso, A., Ratnam, S.S., Sathanathan, H., Chan, C.L.K., Wong, P.C., Hagglund, L., Anandakumar, C, Wong, Y.C. and Goh, V.H.H. 1988. Pregnancy after transfer of sperm under the zona. Lancet 2: 790. Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. 1992. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340: 17-18. Reijo, R., Lee, T-Y., Salo, P., Alagappan, R., Brown, L.G., Rosenberg, M., Rozen, S., Jaffe, T., Straus, D., Hovatta, O., de la Chapelle, A., Silber, S. and Page, D.C. 1995. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genetics 10: 383-93. Silber, S., Nagy, Z., Liu, J., Godoy, H., Devroey, P. and Van Steirteghem, A. 1994. Conventional IVF versus ICSI intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration (MESA). Human Reproduction^: 1705— 09. Silber, S., Nagy, Z., Liu, J., Tournaye, H., Lissens, W., Ferec, C. and Van Steirteghem, A. 1995. The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Human Reproduction 10:2031-43. Tournaye, H., Camus, M., Goossens, A., Liu, J., Nagy, P., Silber, S., Van Steirteghem, A. and Devroey, P. 1995. Recent concepts in the management of infertility because of non-obstructive azoospermia. Human Reproduction 10: 115-19. Tournaye, H., Janssens, R., Camus, M. et al. 1993a. Pentoxifylline is not useful in enhancing sperm function in cases with previous in vitro fertilization failure. Fertility and Sterility 59: 210-15. Tournaye, H., Liu, J., Nagy, Z., Verheyen, G., Van Steirteghem, A. and Devroey, P. 1996. The use of testicular sperm for intracytoplasmic sperm injection in patients with necrozoospermia. Fertility and Sterility 66: 331-4. Tournaye, H., Van der Linden, M., Van den Abbeel, E., Devroey, P. and Van Steirteghem, A. 1993b. Effect of Pentoxifylline implantation and post-implantation development of mouse embryos in vitro. Human Reproduction 8: 1948-54. Van Steirteghem, A.C, Liu, J., Joris, H., Nagy, Z., Janssenswillen, C, Tournaye, H., Derde, M.P., Van Assche, E. and Devroey, P. 1993. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Human Reproduction 8: 1055— 60. Van Steirteghem, A.C, Tournaye, H., Van der Elst, J., Verheyen, G., Liebaers, I. and Devroey, P. 1995. Intracytoplasmic sperm injection three years after the birth of the first ICSI child. Human Reproduction 10: 2527-8. Wisanto, A., Magnus, M., Bonduelle, M., Liu, J., Camus, M., Tournaye, H., Liebaers, I., Van Steirteghem, A.C. and Devroey, P. 1995. Obstetric outcome of 424 pregnancies after intracytoplasmic sperm injection. Human Reproduction 10: 2713-18 World Health Organization 1992. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 3rd edn. Cambridge: Cambridge University Press.

11 The use of donor insemination G A B O R T . KOVACS

It is estimated that of the 50-80 million couples who are infertile worldwide, there is a significant male component in about one-third. The only effective therapy for these couples has been the use of reproductive technology and, with the development of microinjection any man who has got some sperm can potentially be treated (see Chapter 10). Unfortunately many couples who attempt reproductive technology will not become pregnant. Other couples do not have the financial resources to enter these programmes, and some others are morally opposed to the techniques involved. Furthermore, there are men who have got no sperm at all and these couples are candidates for donor insemination. The use of donor sperm to produce a pregnancy for couples where the male is infertile has been practised for many centuries. It can be carried out without medical intervention if a donor known to the couple donates sperm and the couple self-inseminate. The disadvantage of this method is that there is only minimal screening and there is a risk of transmitting sexually transmitted diseases. The next level of sophistication came when the donors were recruited by medical practitioners and the semen was used 'fresh' to inseminate the wife of the infertile male. The problems of using fresh semen are again that matching is limited, screening is not possible and there is a risk of transmitting infectious agents. In the 1990s only sperm that has been frozen, screened and quarantined should be used. At the end of the quarantine period the donor can be retested and, if negative, the semen can then be released. The advantage of this is that the 'window' period for seroconversion is covered. There are several other advantages to using frozen sperm. First, the collection of semen is separated from the time of treatment. It is also possible to screen the donor for genetic, medical and social factors. The quality of each specimen can be ascertained and only those that are suitably fertile are then used. As stated 139

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above, this also allows for semen to be quarantined until seroconversion, so various infectious diseases can be excluded from the donor. Although the first suggestion that sperm may be frozen and subsequently used for artificial insemination was made by Mantegazza in 1886, it was not until 1938 that Jahnel reported sperm survival after freezing. However, it was the work of Polge, Smith and Parkes (1949) that showed that sperm survival improved significantly when a cryoprotectant in the form of glycerol was added to the semen prior to freezing. The first human pregnancy with sperm cryostorage was reported in 1954 when a series of four pregnancies obtained using this method was described (Bunge, Keettel and Sherman, 1954). The next major development in sperm cryopreservation was a two stage freezing and storage method using liquid nitrogen, which was described by Sherman and colleagues in 1962. This method was simple, efficient, easily reproducible and combined both freezing and storing. It described how the semen and glycerol need to be frozen in nitrogen vapour at 16°C-25°C per minute and then stored in liquid nitrogen at -196°C. The first pregnancy resulting from this technique was described in 1963 (Sherman, 1963), with four healthy births in 1964 (Perloff, Steinberger and Sherman, 1964). The State of the Art was reviewed by Sherman in 1973 (Sherman, 1973). He pointed out that human sperm were relatively durable to temperature shock and nitrogen vapour was confirmed as the preferable method of freezing, in aliquots of 0.25-1 cc in either glass containers or straws. Thirdly, he concluded that liquid nitrogen storage at -196°C is the best method, and glycerol the best cryoprotectant. He reported on a pregnancy which resulted in a normal birth from semen that had been stored for ten years. He pointed out that there was no evidence that either freezing or storing of spermatozoa induced any genetic changes. He concluded that the use of frozen stored semen for donor insemination was no longer experimental but accepted therapy. He reported on a survey of 15 centres worldwide which had been carrying out this treatment, with a total of 686 conceptions, over 500 normal children being delivered, with a congenital abnormality rate of only 1.2% and a miscarriage rate of 7.3%. The treatment received widespread publicity when the first International Workshop on Artificial Insemination and Sperm Cryopreservation was held in France in April 1979. Thirty-seven countries presented their data at this meeting (David and Price, 1980). The method of sperm cryopreservation and its use in artificial insemination have changed little in the last 15 years. However, there have been changes in the monitoring of ovulation, and also many of the social attributes have altered quite rapidly in the last decade.

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Indications for donor insemination

The most common indication for donor insemination is male sterility or male subfertility. With the development of in-vitro fertilization, many men with subfertile semen analysis can now produce children, but there will still be large numbers opting for the use of donor sperm. Some men with sterility due to obstruction will also be able to produce pregnancies by techniques of sperm aspiration from the testicle and subsequent microinjection of their partner's oocytes. Nevertheless, those men who have primary testicular failure and do not produce sperm will remain sterile. The choice for these couples is to opt for donor insemination, to explore the possibility of adopting a child, or to choose a child-free existence. Another indication for the use of donor sperm is for the elimination of a potential genetic disease. Couples who have produced a child with a disease transmitted by recessive genes will have a 1:4 chance of having a recurrence. If the couple do not wish to risk another child being born with the same disease, the use of donor sperm can virtually eliminate this possibility. About 1% of couples who seek donor insemination are in this category. Another possible reason for the use of donor semen is when the woman is Rhesus negative and has been Rhesus immunized with a positive partner. If that partner's sperm is replaced by sperm from a Rhesus-negative donor, then the risk of immunization does not exist. With the better treatment of Rhesusnegative women and the use of anti-D, this is again a less frequent indication for donor insemination.

Selection of donors

When donor insemination programmes first commenced, most donors were medical students or medical staff. The first guidelines which were established with regard to choosing donor sperm were at the Third Australian National AID Workshop in Adelaide in 1979, when minimum recommendations for.the screening of potential donors were established. These included a family history of inherited disease, and a personal history of physical, mental or psychological disability. In 1979, serology was recommended for a blood group and Rhesus factor, and syphilis screening. Bacteriological tests, such as culture for gonococcus in at least the first donation, was also recommended. It was also suggested that Mediterranean donors be screened for thalassaemia and Jewish donors for Tay-Sachs. The physical characteristics of the donor, including his height, weight, build, hair and eye colour and complexion, as well as his racial origins, must always be recorded.

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Subsequently, it has been recommended that donors are uniformly screened for hepatitis B and C, as well as for HIV III. Because of the potential 'window' period for a seroconversion after HIV infection, it was recommended that after collection the semen should be quarantined for a minimum of six months, at which time the donor should be retested. Only if the second test for HIV is negative can the semen be released. Although there is some uncertainty about how long after an HIV infection seropositivity can occur, most people infected seem to become positive within about six weeks. It was therefore felt that having a six-month quarantine would introduce an adequate safety factor so that, should donor sperm be collected during the 'window' period, the donor should become positive before the end of his quarantine period. The first reported transmission of HIV through stored donor semen came from Sydney in 1985 (Stewart et al., 1985) when four women who had been treated between 1983 and 1984 were infected by an HIV-positive bisexual donor. Up to 1996, a total of only 12 women worldwide have been identified as contracting HIV through donor insemination, two in Canada (Aranata et al., 1995) and six from the United States. Most of the women from North America were identified through 'look-back' studies. It has subsequently been calculated that the risk of transmission from an infected donor per insemination is 0.5% (confidence level 0.16%-1.37%) (Bignall, 1995). There have been no reports of HIV transmission through donor insemination since universal screening and quarantine were introduced in the mid 1980s. Screening for other infections has also been suggested, including for cytomegalovirus (CMV), herpes simplex virus (HSVI and HSVII) and Chlamydia trachomatis. It was initially hoped that freezing and thawing may eliminate potential pathogens from the semen, but studies have shown that it does not completely inactivate HSV or other viruses (Moore et al., 1989). Cytomegalovirus has also been isolated from semen which had been frozen for nine months (Hammitt, Aschenbrenner and Williamson, 1988). Some workers have suggested that only donors who are negative for CMV antibodies should be accepted. However, as 40% of potential donors have antibodies, this would significantly deplete the source of potential donors. The compromise situation has been suggested that the CMV status of the donor should be recorded and negative donors should be used for women who are CMV negative, and positive donors for women who already have antibodies. However, the infectivity of men who are seropositive is probably only sporadic and occasional. Barratt (1993) has cultured 400 semen samples from ten men who were seropositive for CMV without a single culture being positive for the virus. A

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study carried out in our institution is 1983 (McGowan et al., 1983) showed that all the five men who had positive semen cultures for CMV or herpes had subnormal semen analysis, and also an increase in white cells in the semen. An easy screening test may therefore be to exclude men with high white cell counts, although some of them might have already excluded themselves by having subnormal semen quality. There is uniform agreement that serological testing for Treponemapallidum should be undertaken, as well as for hepatitis B virus and HIV antibodies. It is also usual practice to test for Neisseria gonorrhoea in the first sample and then randomly. Testing for Chlamydia trachomatis is not usually undertaken as it cannot be simply done from a semen specimen but needs to involve a urethral swab which is painful and time consuming. Whereas some authorities have recommended screening for Mycoplasma hominis and Ureaplasma urealyticum, screening populations in Melbourne has shown that nearly half the women are positive for Ureaplasma and almost a quarter for Mycoplasma and therefore these organisms can almost be regarded as normal variants. In summary, the screening of donors is undertaken to minimize the risk of passing on genetic or infectious disease. The recruiting process should therefore include elements of history, examination and special tests. From the history anyone should be excluded who is at high risk from HIV or other sexually transmitted diseases (STDs). Anyone with current genital symptoms or a previous history of STD should also be excluded. Preferably, men who are in stable, monogamous relationships should be selected as donors. Finally, in order to eliminate genetic disease, a list of recognized hereditary diseases should be used as a checklist (Table 11.1). Examination should be undertaken to exclude any genital disease, genital warts or urethral discharge. Any abnormality of the testicles should also be noted. Blood tests should include blood group and Rh factor and infection screens, as a minimum, should include HIV titres, syphilis, hepatitis B and C and maybe CMV screening. Selection of donors with regard to sperm quality is also a complicated area. Each laboratory should draw up its own minimal criteria for accepting men as semen donors. These would include semen concentration, normal sperm morphology and both initial and post-thaw motility after a test freeze. Although there are now more sophisticated tests of the fertilizing capacity of semen, such as staining for the acrosome reaction, the hampster oocyte penetration test, the hypo-osmotic swelling test and detailed analysis of sperm movement, including cervical mucus sperm penetration, their results and cost make them impractical for use in standard donor selection. It is recommended that standard semen analysis criteria are used and, as a method of quality control,

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G. T. Kovacs Table 11.1. Inheritable diseases/traits Thalassaemia (blood disorder) Colour blindness Tay-Sachs (blindness, child death) Haemophilia (blood disorder) Mental retardation Huntington's (involuntary movements) Diabetes (sugar digestion troubles) Sickle cell trait (blood disorder) PKU (special diet needed to prevent cretinism) Alkaptonuria (urine disorder) Eczema (skin rash) Cysticfibrosis(lung disease, thick mucus) Asthma (breathing problems) Epilepsy (fits) Glaucoma (eye disorder) Multiple sclerosis Parkinson's (shakes) Spina bifida (badly formed backbone) Any allergies, e.g. hay fever, or from eating certain food, taking certain medication, or after contact with certain materials etc.

each donor's potency factor be constantly reviewed. This can be expressed as the ratio of pregnancies per insemination. If a particular donor does not live up to the expectations and has a significantly lower fertility ratio than the other donors in the centre, then he should be discarded (Johnson et ai, 1994). Pretreatment assessment

The diagnosis of male sterility is often undertaken by a practitioner who is not the one who will be providing donor insemination services. At the time of telling the couple the diagnosis, this initial practitioner should explain the options available, such as child-free existence, adoption or the use of donor sperm. Preferably, an overview of donor insemination should be given and some detailed written information should be available for distribution. The couple will then have the chance to read about donor insemination before being referred to the insemination clinic. On the first consultation in the donor insemination clinic, the clinician should explain to the couple what donor insemination entails, including the selection and screening of donors, donor matching, the time at which treatment has to be given, the chance of success, and also the legal and social issues involved. It is also advisable (compulsory in

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some states) for the couple to be seen by a counsellor to discuss the social issues in detail, especially with respect to the effect on the child. If the couple then decide to proceed with donor insemination, treatment can be organized. In women who have no risk factors, minimum investigations need to be undertaken. Rubella immunity should be confirmed, blood group and Rh ascertained for matching purposes, and a menstrual history taken. For women who menstruate regularly there is probably no need for hormone measurements, although a baseline prolactin may be worthwhile to exclude hyperprolactinaemia. Tests of tubal patency are not undertaken unless there is a previous history suggesting a risk factor. Some units request a couple of basal temperature charts to confirm ovulation and enable a guesstimate to be made of the likely time for ovulation. It is now universal practice to determine the best time for insemination on the basis of LH assays. Although these were initially performed on blood samples, they can now easily be carried out with home monitoring using urinary kits (Clear Plan, Parke Davis, NSW). Inseminations are timed for the day after the LH peak, which should coincide approximately with the time of ovulation. The method of insemination varies. Some centres use special artificial insemination guns modified from bovine artificial insemination, whereas others use a simple syringe and needle apparatus. There are numerous syringe attachments that have been developed for artificial insemination and none has been proven superior to any other. Other centres use cervical caps for inseminations. Most centres now only inseminate on the fertile day with a single insemination. Result of inseminations

A reasonable conception rate in most units should be about 10% per cycle. However, a better way to express the chance of success is by lifetable analysis. A typical lifetable is shown in Figure 11.1. These are the results of over 1000 courses of insemination at Prince Henry's Institute (modified from Kovacs et al, 1988). The prognosis for outcome varies with the age of the recipient (Figure 11.2: Kovacs et al, 1988). Should pregnancy fail to occur within three or four cycles of insemination, it is important to exclude any pelvic pathology and therefore diagnostic laparoscopy would be recommended. Once 12 cycles of insemination have been undertaken, the chance of subsequent pregnancy becomes quite small. It may then be preferable to look at other options, such as donor sperm GIFT.

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0 60 c 50 a> o ai 40 Q.

> 30 20 10 0

2

4

6

8 10 12 14 16 18 20 22 24 Cycles of treatment

Figure 11.1 The outcome of donor insemination. Life table pregnancy rate of over 1000 couples. (From Kovacs et al., 1988. Reproduced with permission from the British Journal of Obstetrics and Gynaecology.) 70 Under 25 O 26 to 30 years

60

31 to 35 years

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I 40 Q.

a>

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Figure 11.2 The outcome of treatment analysed with respect to recipient's age.

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Social aspects

When donor insemination was first used there was an air of secrecy. Children were not told about the method of their conception and couples were advised to pretend that the child was their own. With the development of reproductive technology, donor insemination has become much more accepted and now it is far more usual for children to be told about their origin than it used to be. In some countries the identity of the donor now has to be released to the children when they reach 18 years of age, thus introducing a very open approach to donor insemination. This has been the situation in Sweden for some time and a similar system is about to be introduced in Australia in the State of Victoria. Books have been written to help couples tell their children about their method of conception, for example How I Began (Paul, 1988) and My Story (Infertility Research Trust, 1991). Both these books present quite a helpful way of explaining to children about the concept of donor sperm and the method of donor insemination.

The legal situation

The legal situation is different not only in every country but, within Australia, in every state. The concepts that have to be covered by laws are as follows. Firstly, any child born as the result of donor insemination is the legal child of the father and the mother who consented to treatment. Secondly, the donor has no rights or responsibilities to the child. Thirdly, issues with regard to access of information need to be controlled. The most liberal point of view is that the identities of both the donor and recipient should be available to the other, but more conservative points of view give access to non-identifying information only. As yet, there are no studies to compare the outcome for children who have information compared to children who do not.

Follow-up of children conceived by donor insemination

The initial report on children conceived by donor insemination was undertaken in Japan in 1968. Iizuka and his colleagues reported on 54 children conceived from donor sperm and compared their physical and mental development (Iizuka, Swada and Nishina, 1968). Similar data on 30 and 75 families were also presented from France in 1979, in a descriptive fashion, with reassuring results. The first Australian study of 50 children conceived by donor insemination was by Clayton and Kovacs in 1982 and concerned children between the ages of one and three years. This study found no

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apparent major abnormalities in obstetric, paediatric or emotional development. The first American study was reported in 1990 when 362 families were studied for milestones such as rolling over, pulling to stand, walking alone, first spoken words and phrases. The study group was not significantly different from the general population. When school performance was analysed, the percentages of children who were gifted and of those with a learning disability were similar to those in the community (Amuzu, Laxova and Sander, 1990). Thefirstcontrolled study that was undertaken to follow the development of children conceived by donor insemination was from our institute in Melbourne in 1993 (Kovacs et al.,1993). Twenty-two children who were conceived using the donor insemination programme were followed up at between six and eight years of age and assessed with a psychosocial test called the Achenbach Child Behaviour Checklist. On comparing the figures for children conceived by donor insemination to controls who were conceived naturally or other controls who were adopted, there was no significant difference between the three groups. In a more recent study from Sydney (Durna et al., 1995), 354 couples who had undergone treatment with donor sperm were studied. They had quite a good response rate of 78%, and more than three quarters of the couples said the treatment had a positive effect on their marriages. The marriage breakdown in this group was only 3.6%, which is far less than in the general community. Another detailed study was reported from London in 1995 in which Golombok and colleagues compared 45 families conceived with donor sperm to 41 IVF families and 43 naturally conceived families, as well as 55 families with an adopted child. The study was carried out on children between four and eight years of age, and a battery of tests included a questionnaire to be completed by the teacher if the child was at school. The factors assessed included marital and psychiatric state of the parents, the quality of parenting and the children's emotions, as well as behaviour and relationships. The quality of parenting for children conceived with donor insemination and IVF was superior to that of naturally conceived children. There were no significant differences in families whose children were conceived using donor sperm or IVF. One could thus conclude from the available evidence that using donor sperm to complete a family when the husband is sterile is a very realistic option with a good prognosis. References Amuzu, B., Laxova, R. and Sander, S. 1990. Pregnancy outcome, health of children, and family adjustment after donor insemination. Obstetrics and Gynaecology 75: 899-905.

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Arenata, M.R.G., Masiola, L., Eller, A., O'Neil, L., Ginsberg, M.M., Bursaw, M., Marik, J., Friedman, S., Sims, C.A., Rekart, M. and Collie, F. 1995. HIV transmission through donor artificial insemination. Journal of the American Medical Association 273: 854-8. Barratt, C.L.R. 1993. Donor recruitment, selection and screening. In Donor Insemination, ed. C.L.R. Barratt and I.D. Cooke, pp. 3-11. Cambridge: Cambridge University Press. Bignall, J. 1995. HIV infection from donor semen. (News item) Lancet 345: 7914. Bunge, R.G., Keettel, W.C. and Sherman, J.K. 1954. Clinical use of frozen semen. Report of 4 cases. Fertility and Sterility 5: 520-9. Clayton, C.E. and Kovacs, G.T. 1982. AID offspring. Initial follow-up study of 50 couples. Medical Journal of Australia 1: 338-9. David, G. and Price, W.S. 1980. Human Artificial Insemination and Sperm Cryopreservation. London, New York: Plenum Press. Duma, E.M., Bebe, J., Leader, L.R., Steigrad, S.J. and Garrett, D.G. 1995. Donor insemination: effects on parents. The Medical Journal of Australia 163: 248-51. Golombok, S., Cook, R., Bish, A. and Murray, C. 1995. Families created by the new reproductive technologies: quality of parenting and social and emotional development of the children. Child Development 64: 285-98. Hammitt, D.C., Aschenbrenner, B.S. and Williamson, R.A. 1988. Culture of cytomegalovirus from frozen-thawed semen. Fertility and Sterility 49: 554-7. Iizuka, R., Swada, Y. and Nishina, Ohi. M. 1968. The physical and mental development of children born following artificial insemination. International Journal of Fertility 13: 24-32. Infertility Research Trust 1991. My Story. University Department of Obstetrics and Gynaecology, Jessop Hospital for Women. Johnson, R.C., Kovacs, G.T., Lording, D.H. and Baker, H.W.G. 1994. Correlation of semen variables and pregnancy rates for donor insemination: a 15-year retrospective. Fertility and Sterility 61: 355-9. Kovacs, G., Baker, G., Burger, H., deKretser D., Lording, D.W. and Lee, J. 1988. AID with cryopreserved semen: a decade of experience. British Journal of Obstetrics and Gynaecology 95: 354-60. Kovacs, G.T., Mushin, D., Kane, H. and Baker, H.W.G. 1993. A controlled study of the psychosocial development of children conceived following insemination with donor semen. Human Reproduction 8: 788-90. McGowan, M.P., Hayes, K., Kovacs, G.T. and Leydon J.A. 1983. Prevalence of Cytomegalovirus and Herpes simplex virus in human semen. International Journal of Andrology 6: 331-6. Moore, D.E., Ashley, R.L., Zarutskie, P.W., Coombs, R.W., Soules, M.R. and Corey L. 1989. Transmission of genital herpes by donor insemination. Journal of the American Medical Association 261: 3441-3. Paul, J. (ed.) 1988. How I Began. The Story of Donor Insemination. The NSW Infertility Social Workers Group. The Fertility Society of Australia. Melbourne: Ambassador Press. Perloff, W.H., Steinberger, E. and Sherman, J.K. 1964. Conception with human spermatozoa frozen by nitrogen vapour technic. Fertility and Sterility 15: 501. Polge, C , Smith, A.V. and Parkes, A.S. 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164: 166-4. Sherman, J.K. 1963. Banks for frozen stored human spermatozoa. Proceedings II International Congress of Genetics 1: 273.

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Sherman, J.K. 1973. Synopsis of the use of frozen human semen since 1964: State of the art of human semen banking. Fertility and Sterility 24: 397-412. Stewart, G.J., Tyler, J.P.P., Cunningham, A.L., Driscoll, G.L., Barr, J.A., Gold, J. and Lamont, B.J. 1985. Transmission of human T-cell lymphotropic virus the III (HTLV-III) by artificial insemination by donor. Lancet ii: 581-5.

12 The donor egg programme JOHNLEETON

Introduction

The concept of egg or embryo donation is not new as the first successful embryo transfer was performed in a rabbit over 100 years ago. The first successful embryo transfer in a human was reported by Buster et al. in 1983. Following the successful development of IVF technology after 1980, donated eggs could be fertilized in vitro and then transferred to recipient endometria. Thefirstpregnancy resulting from the transfer of an in-vitro-fertilized donated egg was reported by Trounson et al. in 1983. This pregnancy ended in spontaneous abortion, but the same group achieved the first successful invitro-fertilized donor egg pregnancy in the following year (Lutjen et al., 1984). These early cases primarily involved recipients who had developed premature ovarian failure. More recently, the indications for egg donation have extended to women with an inherent risk of passing on a serious genetic defect to their offspring and to women aged over 40 years who have failed to conceive on routine IVF programmes. The most recent indication relates to postmenopausal women aged over 50 years requesting pregnancy. Donor egg (DE) programmes have developed rapidly in most developed countries over the last ten years. Most established IVF programmes include donor egg procedures, and over 250 donor egg programmes operate in the US, performing over 2500 transfers annually. The number of donor egg transfers will increase significantly in most major centres during the next few years. Early donor egg pregnancies were achieved in recipients undergoing a sequential regimen of hormone replacement therapy mimicking the natural menstrual cycle. A more flexible and adaptable protocol was developed using afixedhormonal dose with a variable time length oestrogen replacement cycle that allowed programmed synchrony between donor and recipient cycles. Further developments in reproductive technology have enhanced the efficiency of donor egg procedures. These include the transition of egg collection 151

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from a surgical to a non-surgical procedure, the use of gonadotrophin-releasing hormone agonists to facilitate synchrony between donor and recipient cycles, and the active recruitment of egg donors to promote the quantity, quality and availability of donor eggs.

Donor egg recipients Indications 1. The main indication for egg donation was originally for ovarian failure, due to congenital absence, bilateral oophorectomy or premature menopause. 2. Later, women who carry a high risk of transfer of a genetic disease to their offspring were included. 3. Most recently, a growing number of women who fail to conceive after multiple attempts on routine IVF programmes, usually associated with increasing age, are requesting donor egg treatment, which many studies have shown will increase their pregnancy prognosis from approximately 5% per IVF cycle to 30-40% per DE cycle. There is also a small but growing number of younger women with consistently poor quality embryos who fail to conceive on IVF programmes but who can readily conceive with donor egg cycles. This has introduced the new concept of an 'egg impairment syndrome' which can represent one possible cause of idiopathic infertility. 4. Finally, the small but growing number of post-menopausal women over 50 years old seeking pregnancies from donor eggs do not appear to have a biological limiting factor, and pregnancy rates and outcome appear to be comparable to younger age groups. No absolute upper age limits for donor egg recipients have been set. The author's personal choice of limitation lies within the natural upper limits of spontaneous pregnancy, i.e. mid fifties. The deciding factor should primarily depend upon the possible psychosocial effects on the child as well as the physical and emotional health of the recipient couple.

Screening 1. Hormone status. A hormonal profile (E2, P4, LH, FSH, prolactin) is made

to assess ovulatory function. 2. Vaginal ultrasonic scan. Ultrasound scanning is the best way to check the endometrial cavity for its appearance, measurement and response to hor-

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mone replacement therapy. It also checks possible pelvic pathology in both the uterus and ovaries. Rubella immunity must be confirmed. Hepatitis B antigen, hepatitis C antibody, HIV screening are done on both the recipient and partner. Blood group and Rhesus factor for both recipient partners to exclude or treat possible rhesus immunization following delivery. Semen analyses. Cases of severe oligospermia may now be managed by ICSI with donor eggs. Counselling. All recipient couples should have at least one and preferably two counselling sessions to explore and check for any possible factor that could preclude any long-term successful outcome. In situations of known egg donor treatments, it is useful to conduct interviews of the donor and recipient couples both together and separately from each couple. Counselling sessions will also include discussions concerning confidentiality of the egg donation process and the parents' attitudes regarding telling the child about their conception at a later date. Screening for identifying recipients at risk for heart disease, hypertension and diabetes, especially in those aged over 40 years, must be thorough. A written consent form, outlining the success rates, risks and implications of an egg donor pregnancy, should be signed by the recipient couple. Egg donors

Egg donors may be recruited from the following sources. 1. Infertile patients donating excess eggs during a routine IVF treatment cycle. 2. Known donors, i.e. fertile women known to the recipient who donate eggs to the latter, usually for altruistic reasons. 3. Anonymous donors responding to advertisements who will usually donate for financial compensation. 4. Women undergoing a concomitant gynaecological procedure, e.g. laparoscopic sterilization. Most large DE programmes accept donors from all the above categories, although category 4 has been universally found to be the least successful. Direct known donation is banned in France and some institutional ethics committees have also disallowed this procedure (1). Known egg donation has the advantages of known identities between donor and recipient and enhances the chance of arranging synchrony and transfer of fresh embryos. It may,

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however, offer a greater risk of disharmony and disputation between donor and recipient following an inappropriate outcome. The risk is minimized with adequate counselling and information. The author favours category 2 and has found no problems in this group. Payment for egg donors is common in the US, with fees of $US 1000-2000 per egg collection. Payment of donors is disallowed by statute legislation in some countries, including Australia. A world wide shortage of egg donors exists, but the most successful recruitment is found in the US where donors are paid. Such donors may become known or remain anonymous to the recipient. Age of donors It is prudent but not essential that egg donors should be aged 21 or more years. Upper age limits should not be rigidly set, but many studies have confirmed that successful outcomes are significantly related to egg donor age, and decrease after the age of 35 years. Women over the age of 35 years may become egg donors, but their recipients should understand that their pregnancy chance is reduced and the abnormal pregnancy rate is increased. The 'ideal' egg donor is aged 21-35 years, has had at least one child, has completed her family, and remains in a stable relationship. A summation of these factors will reduce the potential risk of regret. All combined factors are infrequently found in each donor, but age would be the most critical. Screening of donors 1. Tests for ovulation. These include a full hormone profile (E2, P4, LH, FSH, prolactin), and a vaginal ultrasonic baseline scan may also confirm ovulation in the secretory phase. 2. Ultrasonic vaginal scan to check ovarian morphology and accessibility for egg collection. 3. Blood group, especially rhesus factor, as DE pregnancies resulting from rhesus-positive donors will need anti-D immunization for the mother if she is Rh negative. 4. Tests for infective risks. These include testing for HIV, hepatitis B Ag, hepatitis C Ab, syphilis and gonorrhoea. It is important to have all donor's partners checked for HIV, hepatitis B Ag, and hepatitis C Ab as well. 5. Mandatory quarantine storage of donor embryos. In view of the minimal but potential risk of HIV infection despite negative serological testing, as is practised by all donor insemination clinics today, some DE clinics are deep-freezing all donor embryos for three to six months before final trans-

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fer. This practice is acceptable if frozen-thawed embryo transfer pregnancy results are satisfactory, but unacceptable if they are low. In the latter situation, the transfer of fresh donor embryos is permissible as long as adequate information regarding the potential risks is given and an appropriate consent form is signed by the recipients. Sperm antibodies should be excluded in the donor before using her serum in the IVF culture medium. Screening for genetic risk factors. The degree of sophistication in locating possible genetic risk factors remains low and relies mainly on a full family and medical history. Most DE programmes have developed their own closed-ended detailed questionnaires which include a full listing of the physical and mental health of the donor and her first-degree family members. A medical history related to second- and third-degree relatives should also be recorded. Routine cytogenic testing of donors is controversial and not generally supported because of its high cost coupled to low yields. Carrier testing should be made for those genes that exist in high frequency in specific ethnic groups, e.g. ^-thalassaemia for southern Europeans and Tay-Sachs disease for Ashkenazi Jews. Counselling. All gamete donors should have at least one and preferably two extended counselling sessions for both the donor and her partner to check for attitude and motivation of their donation, and to exclude any possible risk factors that may lead to regret for their action at a later date. A written consent form, confirming full understanding of the egg donation procedure and its implications, must be signed by all donors. Preparation of the endometrium for egg donation Anovulatory cycles

The first successful pregnancy in a woman without ovarian function was achieved using hormone replacement therapy (HRT) designed to mimic the oestradiol (E2) and progesterone (P4) levels found in periphereal plasma during a spontaneous menstrual cycle. Major changes in HRT have developed since that time. The single most significant advance in HRT for donor egg recipients was the introduction of the variable length E2 replacement (Serhal and Craft, 1987). This approach has eliminated most of the problems associated with synchronization between donor and recipient cycles by providing a window of at least 14 days for transfer to the recipient. Today there is considerable variation in both the dose and route of administration of both exogenous E2 and P4 in HRT regimens. We have found successful pregnancy rates from HRT cycles in women under 40 years of age

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with Progynova (oestradiol valerate) tablets 4 mg daily supplemented with progesterone vaginal pessaries 300 mg daily, beginning on the day of the donor egg collection, despite relatively low levels of circulating plasma P4 and poor-volume endometrial development (Leeton et al, 1989). A reduced implantation rate in women aged over 40 years has been reported in several studies (Meldrum et al, 1992). A significant increase in delayed secretory maturation in women aged over 35 years has also been found. These studies suggest an ageing effect upon the uterus to support nidation and pregnancy. This delay in secretory maturation may be overcome by increasing P4 dosage in women aged over 40 years who are receiving donated eggs. Although unproven, it is generally accepted that egg recipient women over 40 years should be treated with HRT dosages of P4 with supplementary progesterone (50-100 mg/day) injections. Under these conditions, preliminary trials of egg donation to post-menopausal women aged over 40 years have reported success rates similar to those seen in younger recipients for both implantation and pregnancy. An important consideration in deciding the dose and route of administration of HRT for endometrial preparation lies in the difference between E2 and P4 concentrations in the uterine versus the peripheral circulation. Studies on the Rhesus monkey have shown that a countercurrent flow exchange mechanism has a major physiological effect on raising uterine P4 blood levels at least ten times higher than peripheral circulation. There is good evidence that a similar exchange mechanism also occurs in the human. These studies therefore suggest that monitoring of the E2 and P4 levels in the peripheral circulation may not give an accurate indication of the steroid levels delivered to the endometrium. Vaginal ultrasonic scanning of the endometrium on the day of embryo transfer gives a more objective understanding of endometrial development. Recently, E2 replacement therapy has been achieved for endometrial preparation using transdermal oestradiol preparations. Steingold et al. (1991) compared the biochemical effects of transdermal with oral E2 administration when used in dosages appropriate for endometrial maturation. Both regimens gave good plasma E2 profiles. They showed, however, that the relatively high dose of transdermal E2 did not significantly alter the hepatic parameters studied, suggesting that this route of E2 administration may have less adverse hepatic effects. Further studies on this route of E2 administration are indicated. Preparation of endometrium in ovulatory cycles Ovulating women receiving the above HRT on a natural cycle have been found in some studies (including our own) to have a significant increase in

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breakthrough bleeding and decrease in pregnancy rates compared to women with ovarian failure. For these reasons it is generally accepted that these cycles should be initially down-regulated with GnRHA, followed by the above HRT.

Trial HR T cycles

Many programmes favour a trial cycle prior to treatment cycles to check for an adequate endometrial response and an absence of uterine bleeding or general side-effects. Endometrial response is best judged on the ultrasonic endometrial appearance rather than on peripheral hormone levels. A preliminary endometrial biopsy is helpful, but today is no longer necessary. A trial cycle can be costly and time wasting but does confer the advantages of compliance and effectiveness of endometrial maturation and the chance to perform a trial catheter transfer.

HRT dosages

Oestrogen replacement therapy is given by most clinics as Progynova tablets 4-6 mg/day in fixed divided doses. The window for donor egg transfers is wide and has yet to be defined. Workers have reported pregnancies following follicular phases ranging from 5 to 35 days (Navot et al, 1991). Progesterone replacement therapy can be administered as P4 vaginal pessaries, sublingual P4 tablets, dermal patches or intramuscular injections. We have found P4 vaginal pessaries in divided doses of 300 mg/day to give adequate endometrial maturation in most cases. This may be supplemented in cases of inadequate endometrial response or in older women with intramuscular Proluton injections 50-100 mg daily. In summary, morphological studies of donor egg recipient's endometria following HRT have shown a wide variability in response, while failing to identify appearances or features that are mandatory for successful implantation. Results suggest that endometrial conditions for successful implantation may not be tightly regulated in the human, opening up the possibility of further clinical developments and simplifications in HRT protocols for donor egg recipients.

Endocrinology of early pregnancy in women without ovarian function

Successful pregnancies in this group of women have conclusively shown that pregnancies without a corpus luteum can develop normally. With the excep-

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tion of E2 and P4 substitution, there are no other ovarian factors whose absence would be detrimental to continuation of these pregnancies. In early pregnancies, the E2 and P4 levels directly reflect the HRT. Our studies with oral oestradiol valerate (Progynova) 4 mg daily and P4 pessaries (300 mg daily) have shown significantly higher levels of plasma E2 with lower levels of plasma P4 when compared with natural pregnancy data. Despite these discrepancies, the plasma /?hCG levels and pregnancy outcome were comparable to natural pregnancy results. Relaxin is a hormone derived from the corpus luteum and is not found in pregnant women without ovarian function. Donor egg pregnancies in women without ovarian function can undergo spontaneous labour and vaginal delivery. It would appear that relaxin is not essential for vaginal delivery, although its absence may be a contributing factor to these women's higher caesarean section rate of delivery. Hormone replacement therapy in early donor egg pregnancies

It is generally accepted that adequate E2 and P4 support is necessary in early donor egg pregnancies in women without ovarian function. When using relatively low doses of E2 and P4 support, Calderon et al. (1991) showed that placental steroidogenesis, as represented by endogenous /ftiCG levels was present as early as two weeks after embryo transfer. Significant levels of endogenous E2, P4 and /?hCG are found by the eighth gestational week and there would appear to be no benefit to continuing HRT beyond this date. However, many clinics throughout the world are reluctant to discontinue replacement treatment at this time, and continue routine supportive measures well into the second trimester of pregnancy. A safe practical approach to this problem is the weekly reduction of E2 and P4 support, beginning approximately at the sixth week of gestation. A check hormone assay on plasma E2 and P4 24-48 hours after this hormone change would decide whether this reduction in hormone therapy is acceptable, as evidenced by continuing or rising levels of plasma E2, P4 and /JhCG. There appears to be little evidence that continuing HRT in DE pregnancies beyond ten weeks gestation is of benefit, including for those pregnant women who bleed vaginally or abort beyond that date. Results of donor egg programmes

There are many factors involved in pregnancy rates achieved through IVF techniques, including DE pregnancies. These include the age and selection of

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donors, choice of recipients, timing and amount of HRT, clinical and laboratory techniques including embryo cryopreservation, and the number of embryos transferred. The last factor is probably the most significant of all in determining pregnancy rates. Although a wide range of successful DE pregnancy rates have been reported by many clinics, several conclusions can be drawn from their results. 1. Pregnancy rates of DE pregnancies are equal to and possibly better than those of conventional IVF pregnancies. 2. Transfer of fresh embryos has a higher success rate than transfer of cryopreserved embryos. 3. The younger the egg donor, the higher success rate. 4. The age of the recipient does not appear to be a significant factor in determining pregnancy rates. 5. Pregnancy rates are related directly to the number of embryos transferred. This number ranges between three and six in the US and two and three in Australia and most European countries.

Follow-up of donor egg pregnancies

Follow-up data on DE pregnancies are still inadequate as few large series have been reported. The first clinical follow-up of 43 DE pregnancies was reported by the Monash IVF group (Leeton et al., 1992). It showed the perinatal loss, premature birth rate and low-birth-weight infants to be comparable to natural pregnancy data. A further report of 120 DE pregnancies by the same group in 1995 has shown similar perinatal results (Leeton, Rombauts and Withers, 1995). Of greater concern has been the maternal risk of developing complications during pregnancy and delivery, especially in relation to hypertension and pre-eclampsia. A high incidence of the latter was reported by early workers, but could be explained by the relatively high number of multiple pregnancies in older women with their known risks of pre-eclampsia. The 1995 Monash IVF study reported a 22% rate of pre-eclampsia in 79 singleton pregnancies, which was comparable to 17% in a control group. This study also reported a 2% incidence of hypertension during DE pregnancies. To date, follow-up data would suggest that maternal risks in DE pregnancies are directly related to both the age of the recipient and the incidence of multiple pregnancy and are unrelated to the donor egg situation itself. Previous studies have demonstrated in natural pregnancies that gestational diabetes, hypertension, placental complications and caesarean section rate are

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increased in older women. The 1995 Monash IVF study reported a caesarean section rate of 81% in 91 deliveries. This high rate of abdominal delivery is due to several factors, including increased maternal age, possibly the absence of circulating relaxin hormone, and a natural obstetric concern about the importance of the pregnancy. The reluctance to disallow vaginal delivery in all cases is unfounded, and a more conservative approach to vaginal delivery is justified on the basis of routine obstetric criteria. Donor embryo programmes

The growing number of cryopreserved human embryos in most IVF programmes, particularly in the US, offers the possibility of future embryo donations to infertile recipient couples. The success rate of these programmes will directly reflect the pregnancy rates from frozen-thawed embryos at that clinic. These results are usually significantly lower than fresh embryo transfer rates. Donor embryo programmes also have the additional disadvantage in relation to donor egg programmes of absence of the male genes from the infertile woman's husband. This era of growing waiting lists for donor egg treatments will indirectly promote the acceptance of cryopreserved donor embryos. Routine HRT preparations as described previously for donor egg recipients can be given to recipients without ovarian function. In ovulating recipients, the transfer of donor embryos may be made during a natural cycle by timing the embryo transfer at approximately three days after the spontaneous LH surge. Legal aspects of donor egg programmes

The potential for egg donation has shattered several fundamental concepts in reproduction. There no longer exists a finite age limit to bear a child. Furthermore, a woman who bears a child may not be the genetic parent and, alternatively, with the development of IVF surrogacy, a woman who does not become pregnant may in fact be the genetic parent. It is obvious that our traditional laws to protect the rights of parents and children are inadequate today. The rights of all parties involved in egg donation procedures require adequate information to allow all participants the opportunity for informed consent concerning significant risks involved, protection from the transmission of infectious or genetic diseases, and an adequate amount of nonidentifying information about the genetic 'parent'. The last-mentioned includes records of the egg donor's physical characteristics, including blood group and Rhesus factor, her detailed family history, to eliminate possible

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genetic disease transmission, and a knowledge of her general educational, professional and lifestyle status. Child rearing has always been considered by liberal societies to be a personal matter that should not be controlled by government legislation. Similarly, artificial reproductive technologies, including gamete donation, should not be restricted by statute legislation as changes are occurring rapidly in both the development of these technologies and society's attitude towards them. Some form of legislation is necessary; all Australian states and some US states have enacted statute legislation to protect donor gamete programmes by declaring the gamete recipient the legal parent and conferring no legal rights or responsibilities to the donor towards the resulting child. Further laws will be passed to protect all parties in the donor gamete programmes, but adequate protection today cannot be guaranteed. It is imperative that the legal and medical professions work on these problems together in order to protect all parties involved in artificial reproductive technologies in the future.

Future strategies in donor egg programmes

The overwhelming problem facing most DE programmes today lies in the difficulty of recruiting adequate egg donors. Current strategies of media advertising with or without financial incentives have had limited success. There is a need further to reduce the 'invasiveness' of multiple injections and blood tests as well as their potential long-term side-effects. In this regard it is appropriate to offer minimal stimulation protocols of clomiphene citrate to egg donors, despite their known associated reduced pregnancy rates for the recipients. Three potential developments could greatly increase the supply of donor eggs in the future if proved successful. 1. Development of the immature egg collection programme whereby unripe eggs are collected between 8 and 12 days of a non-stimulated cycle. Successful pregnancies have been reported by Chay et al. (1991), but success rates remain very low and the procedure must be considered experimental at this stage. 2. Cryopreservation of eggs. To date, no successful pregnancies have been reported although good embryo development has been found following in-vitro fertilization of frozen-thawed eggs. 3. Collection of fetal or cadaveric eggs. Although these avenues for collection are scientifically possible, at present they both remain unacceptable for ethical reasons.

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References Buster, J.E., Bustillo, M., Thorneycroft, I.H., Simon, J.A., Boyers, S.P., Marshall, J.R., Louw, J.A., Seed, R.W. and Seed, R.G. 1983. Non-surgical transfer of in vivo fertilized donated ova tofiveinfertile women. Lancet 2: 223. Calderon, I., McClure, N., Azuma, K., MacLachlan, V., Leeton, J., de Kretser, D., Sapiro, M. and Healy, D. 1991. The endocrinology of early donor oocyte pregnancy in patients with no ovarian function. Endocrine Society of Australia, Sydney 68. Chay, K.Y., Koo, J.J., Choi, D.H., Han, S.Y. and Yoon, T.K. 1991. Pregnancy after in vitro fertilisation of human follicular oocytes collected from unstimulated cycles, their culture in vitro, and their transfer in a donor oocyte programme. Fertility and Sterility 55: 109-13. Leeton, J., Calderon, I., Burden, J., Azuma, K. and Renou, P. 1992. Maintenance of pregnancy and obstetric outcome in donor egg pregnancies. Reproduction and Fertility Development 4: 713-18. Leeton, J., Rogers, P., Cameron, I., Caro, C. and Healy, D. 1989. Pregnancy results following embryo transfer in women receiving low-dose variable length estrogen replacement therapy for premature ovarian failure. Journal of In Vitro Fertilization and Embryo Transfer 6: 232-5. Leeton, J., Rombauts, L. and Withers, R. 1995. Obstetrical outcome of 120 consecutive donor egg pregnancies. Annual Scientific Meeting of the Fertility Society of Australia 62 (Abstract). Melbourne: FSA. Lutjen, P., Trounson, A., Leeton, J., Findlay, J., Wood, C. and Renou, P. 1984. The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature (London) 307: 174-5. Meldrum, D.R., Marr, B., Stubbs, C, Wisot, A., Yeo, L. and Hamilton, F. 1992. Impaired uterine receptivity in infertile women over age 40 having oocyte donation and correction with increased progesterone replacement. Reproduction and Fertility Development 4: 689-93. Navot, D., Scott, R.T., Droesch, K., Veeck, L., Liu, H.C. and Rosenwaks, Z. 1991. The window for embryo transfer and the efficiency of human conception in vitro. Fertility and Sterility 55: 114-18. Serhal, P. and Craft, I. 1987. Simplified treatment for ovum donations. Lancet 1: 687-8. Steingold, K., Matt, D. and De Ziegler, D. 1991. Comparison of transdermal to oral estradiol administration on hormonal and hepatic parameters in women with premature ovarian failure. Journal of Clinical Endocrinology and Metabolism 73: 275-80. Trounson, A., Leeton, J., Besanko, M., Wood, C. and Conti, A. 1983. Pregnancy established in an infertile patient after transfer of a donated embryo fertilized in vitro. British Medical Journal 286: 835-8.

13 Endometriosis J I M T S A L T A S and D A V I D H E A L Y

Endometriosis is one of the most frequently encountered gynaecological diseases. Its aetiology, physiology and treatment remain controversial. This chapter covers the diagnosis, aetiology, assessment and treatment options for this disorder and discusses its role in infertility and prognosis after treatment. Incidence The true incidence of endometriosis remains unknown, despite a number of studies that have tried to estimate it. One study stated that 10-15% of all premenopausal women had this disorder (Hasson, 1976); another found it in 10-15% of women undergoing diagnostic laparoscopy for pain, infertility and dysmenorrhoea. The latter study also looked at women undergoing laparoscopic sterilization and found that only 2-5% had endometriosis (Strathy, Molgaard and Coulman, 1982). It also looked at women with infertility alone and found that 30-40% of women who underwent laparoscopy for infertility had endometriosis. The conclusion was that endometriosis had to be related in some way to infertility as either an association or a cause or effect. Pathogenesis Before pathogenesis can be discussed, it is important to have a uniformly accepted definition of the disease. The gold standard in diagnosis is the presence of histologically documented menstrual glands and stroma outside the uterus. It also helps if there is evidence of menstrual cyclicity with haemosiderin-laden macrophages. Coelomic metaplasia This was the first widely considered theory of the pathogenesis of endometrio163

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sis. According to this proposal, in response to certain unspecified stimuli, cells may undergo a metaplastic process which changes their character and physiological function. It appears that the cells change from peritoneal mesothelium to endometrium. This is currently not a widely held theory for the aetiology of endometriosis (Merrill, 1966). Embryonic cell arrest It has been speculated that embryonic cell arrest could explain the presence of ectopic endometrium. This theory is not widely supported and is believed to be improbable (Batt and Smith, 1989). Transplantation of exfoliated endometrium This is the most widely accepted theory for the pathogenesis of endometriosis. Transplantation of endometrium occurs through transtubal dissemination, lymphatic and vascular channels as well as by iatrogenic deposition. Transtubal dissemination appears to be the most common method of dissemination, with most endometriosis being found on the peritoneal surfaces of the pelvis and the pelvic viscera. Retrograde menstruation is almost a universal phenomenon in women (Blumenkrantz et ai, 1981; Halme et ah, 1984). To add weight to this theory, vital endometrial cells have been demonstrated in the fallopian tube (Geist, 1979) and endometrial cells have been noted in the peritoneal fluid from culdocentesis and laparoscopy (Jenkins, Olive and Haney, 1986). Genetics A familial association has long been suspected in the development of endometriosis. Endometriosis can be demonstrated in approximately 7% of first-degree female relatives of infected individuals (Lamb, Hoffman and Nicholis, 1986), and has also been shown to be present in 2% of second degree female relatives. The pattern appears to be one of polygenetic or multifactorial inheritance. The only racial differentiation which has been demonstrated to date is amongst Japanese women, who have a higher incidence of endometriosis as compared to women of other races (Miyazawa, 1976). Retrograde menstruation is the predominant mechanism in the development of endometriosis. It appears that women who have short cycle lengths (less than 27 days) and longer menstrual flow (more than 7 days) appear to have twice the risk of developing endometriosis compared with women who have longer cycle lengths and shorter durations of flow (Cramer et al., 1986).

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Clinical aspects

The symptoms in patients with endometriosis are extremely variable, and depend on the site of the ectopic endometrium. However, it is apparent that the extent of the disease is not necessarily related to the intensity of the symptoms. Often, endometriosis can be an incidental finding during surgery or at the time of diagnostic laparoscopy particularly in patients who present with infertility. Often, the more classical symptoms should alert a practitioner to the possibility of endometriosis: dysmenorrhoea, lower abdominal pelvic pain, dyspareunia, infertility, menstrual irregularity associated with pelvic pain, and acute abdomen secondary to a complication to an endometrioma, for example rupture or torsion of the cyst. Other less common symptoms include cyclical tenesmus and rectal bleeding. Diarrhoea and colonic obstruction are rare but have been seen with endometriosis. Cyclical haematuria and pain have been reported, as has ureteric obstruction. Cyclical pain and bleeding and cyclical haemoptysis have also been noted in surgical scars. A careful examination should be performed. Abdominal examination should look for the presence of a pelvic mass and include a careful assessment of sites of tenderness. Vaginal examination should involve assessment for adnexal masses and, in particular, nodularity in the pouch of Douglas and thickening of the uterosacral ligaments, which are all evidence of endometriosis. It is often easier to feel a nodule in the pouch than to see it. This careful assessment of the retrocervical erectile vaginal septum will help in planning management. Diagnosis

The only definitive way of confirming endometriosis is to visualize deposits and histologically confirm the presence of endometriotic tissue by performing a biopsy of suspicious lesions. Laparoscopy is the gold standard in achieving this. Various descriptions are used to paint a picture of what endometriosis looks like. The terminology used to describe peritoneal endometriosis includes: 'powder-burn', punctured black lesions vascularised glandular papule vesicular lesions marked vascularisation red, 'flame-like' lesions petechial peritoneum

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distorted peritoneum: yellow, brown or white scarred peritoneum peritoneal defects or 'windows' subovarian adhesions At the time of laparoscopy, not only can the diagnosis be made, but the disease can be classified and treated. Classification

In patients with endometriosis, surgical findings range from microscopic disease to a frozen pelvis (Nisolle et al., 1990), and a language was needed to help communicate the findings of each laparoscopy. The American Fertility Society (AFS) endometriosis classification system was devised, and the AFS was the first to try to include a complete and simple description of the surgical findings. It went on to become the international language of endometriosis (American Fertility Society, 1979) and, having undergone a number of modifications, is now used in the form devised in 1985 (American Fertility Society, 1985). Why classify? A classification has a number of objectives. It is a common language, allowing all those who treat and see the disease to communicate easily (Candiani, 1986). It can be used to provide guidelines for treatment, to look at the prognosis of patients with fertility after treatment - the likelihood of the relief of pelvic pain and the recurrence rate. Obviously a classification is both used as a research tool and has applications in clinical practice. The staging of endometriosis should be performed at the time of laparoscopy. In the future, the use of operative laparoscopic techniques will be essential, particularly when the clinician is confronted with a pelvis which is difficult to visualize because of extensive adhesions and other anatomical difficulties. A thorough laparoscopic evaluation includes the following steps. 1. Placing the patient in steep Trendelenberg position, taking care to push the bowel and the omentum over the top of the pelvic brim. 2. Always inspect the pelvis in an ordered fashion. 3. Inspect the anterior and posterior cul-de-sac. 4. Tubes and ovaries should be mobilized and inspected for tubal and ovarian adhesions. 5. It is always important to inspect the ovarian hilum and to look at the anterior ovarian surface. 6. The broad ligament should then be inspected. 7. Enlarged ovaries and/or ovarian cysts should be routinely punctured to look for additional endometriomas.

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8. It is important to inspect the cavity of an endometrioma prior to biopsy and suitable treatment. 9. Always inspect the upper abdomen.

The future Is there a marker for 'endometriosis activity'? It is most likely that the actual consequences of endometriosis, such as infertility and chronic pelvic pain are more closely related to the activity of the disease than to its apparent severity. Hopefully, a biochemical or pathological marker will be available in the future to help in the evaluation of this disease. Currently, Cal25 is used as a marker to try to track it. Although the disease process of endometriosis is still poorly understood, it is hoped that future classification systems will not only involve a visual factor, but will incorporate a pathophysiological relationship with infertility or pelvic pain and the activity of the disease.

Endometriosis and infertility

Endometriosis in association with infertility remains a frustrating yet common clinical situation encountered by every gynaecologist. With the advent of laparoscopy, particularly in the evaluation of infertility, the frequency of diagnosis of endometriosis has increased (Petersohn and Behrman, 1970; Hasson, 1976; Drake and Grunert, 1980; Strathy et al, 1982). These studies reported an incidence of endometriosis in infertile couples between 20% and 50%. Current clinical practice appears to be to treat endometriosis either medically or surgically, but one of the most controversial areas in its management concerns the question: if there is no mechanical damage to the ovaries or tubes, does endometriosis have a causal relationship with infertility? The answer has not yet been truly established. One way to examine such a relationship would be to determine the true prevalence of the disease in both fertile and infertile couples. Unfortunately, this is not ethically possible as it would require laparoscopy of totally asymptomatic women.

Potential mechanisms of endometriosis associated infertility

Mechanical damage There would appear to be unanimous agreement that endometriosis, especially moderate to severe disease, can cause significant damage to the tubes, ovaries and surrounding peritoneum, which can obviously result in tubal

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occlusion andfimbrialdamage. However, unlike pelvic infection, endometriosis does not appear to cause damage to the tubal epithelium, and therefore surgical treatment can markedly improve fertility rates. No mechanical damage Tubal dysfunction Peritoneal fluid prostaglandin, synthesized by ectopic endometrium, may have an adverse impact on fertility by causing tubal dysfunction which could alter sperm, oocyte or embryo transport, reducing the likelihood of conception. There have been a number of studies which have shown elevated levels of prostaglandins in peritoneal fluid of infertile women with endometriosis (Drake et al, 1981, Baadawy et al, 1982), but other studies do not show this (Dawood, Khan-Dawood and Wilson, 1984). It has been shown in some studies that prostaglandins are not found to be contraceptive. The analogues of prostaglandins do not appear to alter fallopian tube transport of human oocytes (Croxatto et al, 1978). Therefore, the evidence to support this theory of infertility is difficult to accept at this time. The follicle

Endometriosis may alter follicular function, either by altering the growth of the follicle or by changing the timing or the quality of follicular rupture. It has been suggested that subtle ovulatory defects are present in infertile women who have endometriosis. However, there is as yet no convincing evidence that endometriosis causes anovulation or luteal phase deficiency (Pittaway et al., 1983). It is believed that the luteal phase of women with endometriosis is normal, and to date there is no evidence to refute this. During the peak of its popularity, the luteinized unruptured follicle syndrome - where a mature dominant follicle converts into a corpus luteum in the absence of active rupture of the capsule and release of the oocyte - was believed to be more common in women with endometriosis. Several investigators have found no association between an apparently unruptured and luteinized follicle and endometriosis (Koninckx et al, 1980; Dhort et al, 1984). Immunity To date, there are no convincing data that suggest an alteration in cell mediated immunity secondary to endometriosis is responsible for the reduction in fertility, or even for the disease itself. Studies in this area are still in their

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infancy. Further progress awaits a comprehensive hypothesis of how it would influence the likelihood of developing endometriosis or infertility.

Peritonealfluidinflammation

Leucocytes normally exist in the peritoneal fluid (Haney, Muscato and Weinberg, 1981). Their function is believed to be to remove sperm and any bacteria ascending through the genital tract. Infertile women with endometriosis have a high number of leucocytes in their peritoneal fluid as compared with fertile women. The increase is primarily due to activated macrophages (Haney et al., 1981; Syrop and Halme, 1987). There is an increase in the volume of the inflammatory exudate, the number of macrophages, the degree of activation of the macrophages, and the concentration of macrophage secretory products. Activated peritonealfluidleucocytes have the potential to impede endometrial cell implantation and mediate infertility either by direct cellular toxicity or through their secretion of proteolytic enzymes and cytokines. It appears that peritoneal fluid exudate can have adverse effects on the reproductive process, impeding it on a number of levels. This can occur by phagocytosis of sperm, decreased sperm motility, altered sperm/egg interactions, failure of oocyte capture by the fimbriae, and altered embryo development (Muscato, Haney and Weinberg, 1982; Chacho et al, 1986; Burke, 1987). Treatment outcomes in infertile patients with endometriosis

Which patients should be treated? How effective is therapy? What are the outcomes? These are questions which are constantly asked when considering the treatment of patients with infertility and endometriosis. There appears to be a definite benefit when treating patients with stage 3 or 4 disease. In most of these cases, there is mechanical distortion of the pelvic tissues which interferes with fertility. Surgery improves anatomical relationships and will enhance fertility rates (Olive and Lee, 1986). It can be performed either by traditional laparotomy or using advanced operative laparoscopy techniques. Both techniques appear to offer good results (Candiani et al., 1991). It is important not just to diathermy or ablate the endometriotic tissue, but actually to remove it. Excision of endometriotic tissue is probably the most effective method of treatment (Wheeler and Malinak, 1987; Redwine, 1991). One of the areas that continues to be marred by controversy concerns which therapeutic approach to take when the only findings are endometriotic implants without distortion of the pelvic viscera. Currently, medical or surgical treatment appears to offer no benefit over no treatment at all. The first

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prospective randomized and controlled study appeared in 1982. It compared the use of danazol for six months and no treatment for six months (Seibel et al, 1982), and showed no statistical difference in the pregnancy rates between the two groups. This was also found by Hull et al. in 1987, who used medroxyprogesterone acetate in their treatment group. Nor has any difference been shown when comparing medical management, no treatment and ablation of minimal endometriosis (Chong, Keene and Thornton, 1990). An argument for treatment was to prevent any probable progression of the disease. This was initially supported by a number of retrospective studies which showed an almost 100% progression in disease. It was argued by these practitioners that all endometriosis should be treated in order to prevent progression (Jansen and Russell, 1986). However, prospective studies show that endometriosis comes and goes according to its own biological cycle (D'Hooghe et al., 1992), particularly when mild (Koninckx and Martin, 1994). Surgery should only be performed if there are symptoms which can be directly attributed to the endometriosis, i.e. pain or deep dyspareunia (Martin, 1995). In summary, despite immense interest and sophisticated diagnostic techniques, little is known about the relationship between endometriosis and infertility and, in particular, about the mechanisms of infertility in couples with minimal endometriosis. Neither medical nor surgical therapy has been demonstrated to improve the likelihood of pregnancy in patients where there is no distortion of the pelvic anatomy. Currently, therapy should only be recommended if there is any distortion of the pelvic anatomy or if symptoms such as pain are present as well as infertility. Endometriosis: medical therapy

An alternative to surgical treatment is medical treatment of endometriosis, and debate exists about which is better. Often a combination of both techniques is used. The argument by endoscopists is that treatment can occur at the time of laparoscopy and side-effects of medical therapy can be avoided. Those in favour of medical therapy say that it avoids the complications of surgery, in particular the risk of postoperative adhesions. The main drugs which are used for medical management of endometriosis are: medroxyprogesterone acetate danazol gestrinone GnRH agonists

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Studies show similar efficacy in controlling symptoms and in laparoscopic resolution when comparing all of the above medications (Telimaa et al, 1987; Thomas and Cooke, 1987; Fraser et al, 1991). The choice of treatment often depends upon the individual practitioner, the patient and the side-effect profile of each particular drug. Medical therapy is effective in relieving the symptoms of the disease in 80% of women treated, although there is a gradual return of symptoms in 30-60% of patients after one year of follow-up (Henzel et al, 1988; Fedele et al., 1989; D'Mowski, et al, 1989; D'Lugi et al, 1990). It appears that medical therapy is not currently effective in eliminating the disease and should be used as an adjunct to surgery or to control symptoms temporarily. Endometriosis: surgery

Why operate? Unfortunately, as discussed above, medical therapy will not eliminate endometriosis: there is significant reduction in AFS scores following medical therapy but the score never reaches zero. In 1987, Evers demonstrated by laparoscopy carried out two months after the cessation of therapy that there was no significant difference in the size and number of implants when compared to the initial score (first-look laparoscopy). This study showed there was a swift recurrence after ceasing medical therapy (Evers, 1987). Surgical treatment can be individualized, depending on the presenting symptoms and reproductive goals of the patient. In women with stage 1 and 2 disease who have somatic complaints, surgical treatment at the time of diagnosis is highly recommended and offers the greatest likelihood of relief. Most surgeons would treat these women laparoscopically. Treatment can be either by ablation or excision (Sutton and Hill, 1990). Surgery is the treatment of choice in women with stage 3 and 4 disease, whether the presenting symptoms are pain or infertility. It can be performed either laparoscopically or by laparotomy, depending on the skills of the individual surgeon. Laparoscopy offers many advantages over laparotomy and will probably become the treatment of choice for most gynaecological procedures that are now performed by laparotomy (Gomel, 1989), particularly for endometriosis. The objectives of conservative surgery are to resect macroscopic endometriosis and adhesions completely, minimize tissue handling, and restore the normal tubo-ovarian relations. Complete haemostasis is important and thorough irrigation should be performed to remove deposited fibrin (Nezhat, Crowgy and Nezhat, 1989). Unfortunately, conservative surgery is not always the answer for endometriosis. Ablative surgery should be considered when the patient has intractable pelvic pain that has not responded to conservative

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therapy. The precise choice of the excisional surgery depends on a number of factors, such as the age of the patient, her main symptoms, the site of major endometriotic involvement and, most importantly, the woman's childbearing wishes. Radical surgery will involve one or a combination of the following procedures: salpingo-oophorectomy, hysterectomy with excision of any deeply infiltrating endometriosis (Martin, 1988; Daniell, Krentz and Gurley, 1991; Redwine, 1991; Reich, 1992).

Conclusion Endometriosis is a complex disease. Its pathogenesis, natural history and definitive treatment are still controversial. Further research into its origins and randomized control trials for its management, particularly when associated with infertility, are required. Currently, it appears that surgical management, with excision of the disease using a laparoscopic approach, is the best form of treatment. References American Fertility Society 1979. Classification of endometriosis. Fertility and Sterility 32:633. American Fertility Society 1985. Revised American Fertility Society classification of endometriosis. Fertility and Sterility 43: 351-2. Baadawy, S.Z.A., Marshall, L., Gabal, A.A. and Nusbaum, M.L. 1982. The concentration of 13, 14-dihydro-15 ketoprostaglandin F2 and prostaglandin E2 in peritoneal fluid of infertile patients with and without endometriosis. Fertility and Sterility 38: 166-70. Batt, R.E. and Smith, R.A. 1989. Embryonic theory of histogenesis of endometriosis in peritoneal pockets. Obstetrics and Gynecology Clinics of North America 16: 15-28. Blumenkrantz, M.J., Gallagher, N., Bashmore, R.A. and Tenhkoft, H. 1981. Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstetrics and Gynecology 57: 667-70. Burke, R.K. 1987. Effect of peritoneal washings from women with endometriosis on sperm velocity. Journal of Reproductive Medicine 32: 743-6. Candiani, G.B. 1986. The classification of endometriosis: historical evolution, critical review and present state of the art. Acta Europacea Fertilization 17: 85-92. Candiani, G.B., Vercellini, P., Fedele, L., Bianchi, S., Vendola, N. and Candiani, N. 1991. Conservative surgical treatment for severe endometriosis in infertile women: Are we making progress? Obstetrical and Gynaecological Survey 46: 490-8. Chacho, K.J., Stronkowski-Chacho, M., Andressen, P.J. and Scommegna, A. 1986. Peritoneal fluid in patients with and without endometriosis; prostonoids and macrophages and their effect on the spermatozoa penetration assay. American Journal of Obstetrics and Gynecology 154: 1290-9. Chong, A.P., Keene, M.E. and Thornton, N.C. 1990. Comparison of three modes of

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treatment for infertility patients with minimal pelvic endometriosis. Fertility and Sterility 53: 407-10. Cramer, D.W., Wilson, E., Stillman, R.J., Berger, M.J., Beliste, S., Schiff, I., Albrecht, B. and Gibson, M. 1986. The relation of endometriosis characteristics, smoking and exercise. Journal of the American Medical Association 225: 1904-8. Croxatto, H.B., Ortiz, M.E., Guiloff, E., Ibarra, A. and Salvatierra, A. 1978. Effect of 15s-15-methyl prostaglandin F2 on human oviductal motility and ovum transport. Fertility and Sterility 30: 408-14. D'Hooghe, T.M., Bamba, C.S., Isahakia, M. and Koninckx, P.R. 1992. Evolution of spontaneous endometriosis in the Baboon (Papio amibis, Papio cynocephalis) over a 12-week period. Fertility and Sterility 58: 409-12. D'Lugi, A.M., Miller, J.D., Knittle, J. and Lupren Study Group 1990. Lupren depot (leuprelide acetate for depot suspension) in the treatment of endometriosis: a randomised placebo-controlled double blind study. Fertility and Sterility 42: 408-15. D'Mowski, W.P., Radwanski, E., Binor, Z., Tummon, I. and Pepping, P. 1989. Ovarian suppression induced with buserelin or danazol in the management of endometriosis: a randomised comparative study. Fertility and Sterility 51: 395400. Daniell, J.F., Krentz, D.R. and Gurley, L.D. 1991. Laser laparoscopic management of large endometriomas. Fertility and Sterility 55: 692-5. Dawood, M.Y., Khan-Dawood, F.S. and Wilson, L.Jr. 1984. Peritoneal fluid prostaglandins and prostanoids in women with endometriosis, chronic pelvic inflammatory disease and pelvic pain. American Journal of Obstetrics and Gynecology 141: 391-5. Dhort, M., Serreyn, R., Duvivier, P., Varluchene, E., De Boever, J. and Vanderkerckhove, D. 1984. Ovulation stigma and concentration of progesterone and estradiol in peritoneal fluid: relation with fertility and endometriosis. Fertility and Sterility 41: 872-7. Drake, T.S. and Grunert, G.M. 1980. The unsuspected pelvic factor in the infertility investigation. Fertility and Sterility 34: 27-31. Drake, T.S., O'Brien, W.F., Ramwell, P.W. and Metz, S.A. 1981. Peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F2 in endometriosis. American Journal of Obstetrics and Gynecology 140: 401-4. Evers, J.L.H. 1987. The second-look laparoscopy for evaluation of the result of medical treatment of endometriosis should not be performed during ovarian suppression. Fertility and Sterility 47: 502-4. Fedele, L., Bianchi, S., Viezzoli, T., Arcaini, L. and Candiani, G.B. 1989. Gestinone versus danazol in the treatment of endometriosis. Fertility and Sterility 51: 781-5. Fraser, I.S., Shearman, R.P., Jansen, R.P.S. and Sutherland, P.D. 1991. A comparative treatment trial of endometriosis using the gonadotrophin-releasing agonist, nafarelin and the synthetic steroid, danazol. Australian and New Zealand Journal of Obstetrics and Gynaecology 31:158-63. Geist, S.F. 1979. The viability of fragments of menstrual endometrium. American Journal of Obstetrics and Gynecology 125: 751-4. Gomel, V. 1989. Operative laparoscopy: time for acceptance. Fertility and Sterility 52: 1-11. Halme, J., Hammond, H.G., Hulka, J.F., Raj, S.G. andTalbert, M. 1984. Retrograde menstruation in healthy women and in patients with endometriosis. Obstetrics and Gynecology 64: 151-4.

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Haney, A.F., Muscato, J.J. and Weinberg, J.B. 1981. Peritoneal fluid cell population in infertility patients. Fertility and Sterility 35: 696-8. Hasson, H.M. 1976. Incidence of endometriosis in diagnostic laparoscopy. Journal of Reproductive Medicine 16: 135-6. Henzel, M.R., Corson, S.L., Moghissi, K., Buthram, V.C., Berquist, C. and Jacobson, J. 1988. Administration of nasal nafarelin as compared with oral danazol for endometriosis. New England Journal of Medicine 318: 485-7. Hull, M.E., Moghissi, K.S., Magyar, D.F. and Hayes, M.F. 1987. Composition of different treatment modalities of endometriosis in infertile women. Fertility and Sterility 47: 40-4. Jansen, R.P.S. and Russell, P. 1986. Non pigmented endometriosis: clinical, laparoscopic and pathologic definition. American Journal of Obstetrics and Gynecology 155: 1154-9. Jenkins, S., Olive, D.C. and Haney, A.F. 1986. Endometriosis: Pathogenic implications of the anatomic distribution. Obstetrics and Gynecology 67: 325-8. Koninckx, P.R., Ide, P., Vanderbrouche, W. and Brossens, I.P. 1980. New aspects of pathophysiology of endometriosis and associated infertility. Journal of Reproductive Medicine 24: 257-60. Koninckx, P.R. and Martin, D.C. 1994. Treatment of deeply infiltrating endometriosis. Current Opinions in Obstetrics and Gynecology 6: 231—4. Lamb, K., Hoffman, R.G. and Nicholis, T.R. 1986. Family traits analysis: A case control study of 43 women with endometriosis and their best friends. American Journal of Obstetrics and Gynecology 154: 596-601. Martin, D.C. 1988. Laparoscopic and vaginal colpotomy for excision of infiltrating cul-de-sac endometriosis. Journal of Reproductive Medicine 33: 806-8. Martin, D.C. 1995. Pain and infertility - a rationale for different treatment approaches. British Journal of Obstetrics and Gynaecology 102, Supplement 12: 2-3. Merrill, J.A. 1966. Experimental induction of endometriosis across Millepore filters. American Journal of Obstetrics and Gynecology 94: 780-4. Miyazawa, K. 1976. Incidence of endometriosis among Japanese women. Obstetrics and Gynecology 48: 407-9. Muscato, J.J., Haney, A.F. and Weinberg, J.B. 1982. Sperm phagocytosis by human peritoneal macrophages: a possible cause of infertility in endometriosis. American Journal of Obstetrics and Gynecology 144: 503-10. Nezhat, C , Crowgy, S.R. and Nezhat, F. 1989. Video laparoscopy for the treatment of endometriosis associated infertility. Fertility and Sterility 51: 237—40. Nisolle, M., Paindaveine, B., Bourdon, A., Berliere, M., Casanas-Roux, F. and Donnez, J. 1990. Histologic study of peritoneal endometriosis. Fertility and Sterility 53: 984-8. Olive, D.L. and Lee, K.L. 1986. Analysis of sequential treatment protocols for endometriosis-associated infertility. American Journal of Obstetrics and Gynecology 154: 613-9. Petersohn, E.P. and Behrman, S.J. 1970. Laparoscopy of the infertile patient. Obstetrics and Gynecology 36: 363-7. Pittaway, D.E., Maxson, W., Danniell, J., Herbert, C. and Wentz, A.L. 1983. Luteal phase defects in infertility patients with endometriosis. Fertility and Sterility 39: 712-13. Redwine, D.B. 1991. Conservative laparoscopic excision of endometriosis by sharp dissection. Life table analysis of reoperation and persistent or recurrent disease. Fertility and Sterility 58: 628-34.

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Reich, H. 1992. Laparoscopic hysterectomy. Surgical Laparoscopy and Endoscopy 2: 85-8. Seibel, M.M., Berger, M.J., Weinstein, F.G. and Taymer, M.D. 1982. The effectiveness of danazol on subsequent fertility in minimal endometriosis. Fertility and Sterility 38: 534-7. Strathy, J.H., Molgaard, C.A. and Coulman, C.B. 1982. Endometriosis and infertility: A laparoscopic study of endometriosis among fertile and infertile women. Fertility and Sterility 38: 667-72. Sutton, C. J.G. and Hill, D. 1990. Laser laparoscopy in the treatment of endometriosis. A 5-year study. British Journal of Obstetrics and Gynaecology 97: 901-5. Syrop, C.H. and Halme, J. 1987. Cyclic changes of peritoneal fluid parameters in normal and infertile patients. Obstetrics and Gynecology 69: 416-19. Telimaa, S., Puolakka, J., Ronnberg, L. and Kauppila, A. 1987. Placebo controlled comparison of danazol and high dose medroxy progesterone acetate in the treatment of endometriosis. Gynaecological Endocrinology 1: 13-23. Thomas, E.J. and Cooke, I.D. 1987. Impact of gestrinone on the course of asymptomatic endometriosis. British MedicalJournal 294: 1117-19. Wheeler, J.M. and Malinak, C.R. 1987. Recurrent endometriosis. Contributions in Gynecology and Obstetrics 16:13-21.

14 The role of ultrasound in subfertility VICTOR HURLEY and MARIA LEONI

Introduction

Ultrasound techniques play an important role in all stages of the management of a couple suffering from subfertility. Ultrasound is used to investigate both partners for causes of subfertility. It is also important in the monitoring of follicular development in spontaneous and stimulated ovarian cycles and in the guiding of procedures which form part of assisted reproductive treatments. Complications of ART treatments such as ovarian hyperstimulation syndrome are diagnosed and the therapy used in treating them is assessed. When a pregnancy occurs, the number, location and viability of the fetuses are established and as pregnancy advances, normal morphological development is confirmed. The extensive role that ultrasound plays in subfertility has become possible since the advent of transvaginal transducers. These transducers produce high resolution images, often providing information of histological detail, which was not possible using previous transabdominal, transvesical techniques. Transvaginal imaging is a dynamic investigation, effectively an extension of the bimanual gynaecological examination. Mobility of structures and areas of pelvic tenderness can be precisely defined. Doppler ultrasound (an ultrasound technique which measures blood flow) is able to provide measurements of organ vascularity which in turn provide important physiological and pathological insights into aspects of subfertility assessment and treatment.

The ultrasound investigation of the subfertile couple

A transvaginal ultrasound examination is an integral part of the initial investigation of the woman in a subfertile couple. It is complimentary to diagnostic laparoscopy and hysteroscopy, often revealing significant pathology not de176

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Table 14.1. Pathology that may be detected during transvaginal ultrasound examination Ovarian Endometriosis Polycystic ovarian disease Ovarian cysts Uterine Fibroids Endometrial/cervical polyps Asherman's syndrome Endometritis Congenital abnormalities Adenomyosis 'Forgotten' IUCDs Adnexal Hydro/pyosalpinges Pelvic collections

tected by these methods. The examination is best performed in the early proliferative phase of the menstrual cycle when ovaries are less active and there is less risk of confusing pathology with physiological cysts, haematomas or corpora lutea. At this stage the endometrial cavity is easier to image and subtle pathology such as small endometrial polyps may be detected, which may be missed if the examination is performed in the secretory phase of the cycle when the endometrium is thick and obscures fine detail. Table 14.1 lists pathology which may be detected during such an investigation. Endometriosis Endometriosis is a factor in subfertility in 10-15% of patients. Ovarian endometriomas can usually be detected by transvaginal ultrasound examinations and have a typical 'ground-glass' echo texture (Figure 14.1). Small ovarian endometriomas, centrally positioned in the ovarian stroma, are sometimes diagnosed which escape detection at the time of diagnostic laparoscopy. If there is uncertainty, examinations in a subsequent menstrual cycle are useful. Functional cysts and haematomas will usually disappear or alter their appearance, while endometriomas remain unchanged. Occasionally, peritoneal masses of endometriosis may be seen but in most cases ultrasound cannot reliably detect endometriosis in areas other than the ovaries. Peritoneal endometriosis may be suspected when pelvic tenderness is encountered during

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Figure 14.1 An ovary containing two endometriomas with the typical ground-glass echo appearance associated with this condition.

ultrasound examination or when fixation of pelvic structures is noted. The fixed, tender, retroverted uterus is a commonfindingin patients with endometriosis. Polycystic ovarian disease Polycystic ovarian disease is commonly associated with disorders of ovulation, being present in 15-20% of women presenting for investigation of subfertility. The condition is diagnosed ultrasonographically when there are more than ten primordial follicles in the ovarian stroma and the ovarian volume is increased (Figure 14.2). Ovarian cysts All types of ovarian cysts may be detected at the time of an ultrasound examination. Most will be incidental findings with little influence on fertility. Few have characteristic ultrasound appearances, and specific diagnoses will only be established with surgical removal. Occasionally, persistent simple ovarian cysts may produce oestrogens which reduce fertility. These can be effectively treated using ultrasound guided drainage techniques.

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Figure 14.2 The ultrasound appearance of a polycystic ovary. Fibroids

Fibroids or leiomyomata will usually be an incidental finding in a subfertile woman, but occasionally submucosal or interstitial fibroids may be diagnosed which may have significant implications for fertility and maintenance of pregnancy. The technique of uterine irrigation or sonohysteroscopy (see below) is invaluable as an adjunct to transvaginal ultrasound in the accurate diagnosis of such pathology. The relationship offibroidsto the normal uterine architecture, particularly the cavity, is not a fixed one. Fibroids may move in and out of the uterine cavity during myometrial contractions.

Polyps Small endometrial and cervical polyps are a common finding in normal fertile women and it is uncertain whether they are of significance in subfertile women (Figure 14.3). They have a characteristic echogenic, circumscribed appearance but may sometimes be difficult to distinguish from small submucosal fibroids. Uterine irrigation or sonohysteroscopy is useful in establishing the diagnosis. Where they are detected at an initial assessment ultrasound examination, it is wise to perform a repeat examination prior to surgical removal as many will disappear following menstruation.

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Figure 14.3 This ultrasonogram displays a small echogenic polyp in the fundal region of the endometrial cavity. The uterus is retroverted, with the fundus to the right. A Sherman's syndrome

Intrauterine adhesions are characterized ultrasonographically by disruption of the endometrial cavity line and multiple echogenic foci adjacent to the cavity. The patient will usually have a history of septic abortion or multiple instances of curettage.

Endometritis The ultrasound features of this condition are non-specific. Suspicion of endometrial inflammation may be raised when the uterus is enlarged and tender and the endometrial cavity line is blurred, with the normally clear demarcation between endometrium and myometrium lost. Fluid in the uterine cavity is an irregularly observed finding.

Congenital uterine abnormalities Congenital uterine abnormalities are common in fertile as well as in subfertile women, occurring in 3% of the population. Abnormalities of Mullerian

The role of ultrasound in subfertility fusion, such as the ageneses or hypoplasias, rarely present with infertility, being more commonly seen in the clinical context of primary amenorrhoea. The influence of bicornuate and septate abnormalities on fertility is uncertain; endometriosis and pelvic adhesions are said to be more common in patients with these abnormalities. Some association between unexplained infertility and uterine abnormalities has been observed, with successful outcomes achieved following metroplasty procedures. Transvaginal ultrasound, particularly in conjunction with uterine irrigation, can usually identify and classify the different types of congenital uterine abnormalities. Adenomyosis Adenomyosis is a common condition in which deposits of endometrium develop in the myometrium. Formerly known as endometriosis interna, its affect on fertility is uncertain. The diagnosis of adenomyosis using ultrasound techniques is difficult as the features are often non specific. Features described include: myometrial mottling, cystic or calcine areas in the myometrium, diffuse uterine enlargement, and hypervascularity of the myometrium. Hydro / pyosalpinges The characteristic ultrasound appearance of a hydrosalpinx or pyosalpinx is that of a tubular adnexal cystic structure with distinctive invaginations in its walls. The presence of particulate matter within the cyst usually indicates the presence of inflammatory material. Pelvic collections Loculated fluid collections in the pelvis are often detected ultrasonographically in patients with acute or chronic pelvic inflammatory disease. Monitoring follicular development Ultrasound techniques may be used to monitor follicular development in both natural and stimulated cycles. Natural cycles The normal ovarian and uterine physiological events associated with the menstrual cycle and ovulation can be observed ultrasonographically. Examin-

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ations performed in the late proliferative and mid-secretory phases of the menstrual cycle can confirm the recruitment, maturation, rupture and luteinization of the ovarian follicle and appropriate endometrial preparation. The dominant follicle becomes recognizable by day five to seven following menstruation. It then grows 2-3 mm per day, reaching a maximum diameter of 13-25 mm at the time of ovulation. The most common mean diameter at ovulation is 20-25 mm. The endometrium develops from a thin line immediately following menstruation into a typical triple-layered proliferative phase appearance measuring 5-15 mm at the time of ovulation. An ultrasound examination at day 10-12 can confirm these normal features. Rarely, if serial examinations are performed, the cumulus oophorus may be imaged immediately prior to ovulation. The events occurring in the secretory phase of a normal unstimulated menstrual cycle are best observed at around day 20-22. At that time the development of internal echoes, cyst wall thickening and peripheral vascularity characterize the appearances of the corpus luteum and provide evidence of ovulation. Concurrently the endometrium becomes 'filled in' and develops an echogenic texture, while increasing in thickness to 15-20 mm. Free fluid in the pouch of Douglas provides further evidence of ovulation. Attempts to predict the precise timing of spontaneous ovulation using ultrasound monitoring of follicle growth have proved disappointing as there is too wide a range of periovulatory follicular sizes. Other described signs such as the 'double contour' sign of granulosa cell layer separation are similarly unreliable. Abnormal ovulation

The diagnosis of the luteinized unruptured follicle syndrome is established if the dominant follicle it still present 36 hours after a luteinizing hormone surge. Stimulated ovarian cycles

Anovulatory women can be induced to produce a follicle using clomiphene citrate, hMG and GnRH anologues. Serial ultrasound monitoring of follicular development is usually carried out from day eight on a daily or every second day basis until the maturing follicle reaches 18-20 mm. Human chorionic gonadotrophin is then administered to induce ovulation. This may be withheld if a large number of follicles are maturing to avoid the problems of multiple pregnancy and OHSS. Ovulation usually occurs 36-48 hours following hCG administration, allowing optimum timing of intercourse or insemination when artificial insemination is being performed.

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Assisted reproductive techniques

Ultrasound may be used either alone or in conjunction with oestradiol assays to assess the development and maturation of follicles and in timing oocyte retrieval in IVF and GIFT cycles. Where ultrasound alone is used, daily examinations are performed from day nine until the leading follicle reaches 18-24 mm. Human chorionic gonadotrophin is then administered and oocyte retrieval scheduled for 36-48 hours later. If oestradiol assays are utilized, a level of 400 pg/ml per follicle greater than 14 mm in diameter is expected. The timing of oocyte retrieval is determined by the number of follicles, their sizes and the correlation with oestradiol levels (Smith et al, 1980). Uterine receptivity

The endometrial thickness at the time of embryo transfer has been thought to be associated with pregnancy outcome. Certainly no patients become pregnant if the thickness is 4 mm or less. Where IVF is being utilized, consideration should be given to cancellation of the cycle and embryo freezing if the endometrium is so thin. Attempts at classifying appearances of the endometrium and correlating them to pregnancy outcome have been less successful. Ultrasound guided procedures

A number of ultrasound guided needling procedures have developed, all of which utilize a biopsy guide attached to the transvaginal transducer. The needles are inserted under direct ultrasound vision, with guide lines on the machine monitor corresponding with the biopsy guide. The needle may be inserted manually or with an automated puncture device. The needle used must have an inner diameter of at least 1 mm to avoid damage to the oocyte and an echogenic tip to optimize visualization. The length required varies according to the guide and transducer but is typically 25-35 cm. Needles can be manufactured to be extremely sharp, which means minimal discomfort is felt by the patient during the procedures. Light general or local anaesthetic is usually all that is required. Oocyte retrieval, ovarian cyst aspiration and ectopic pregnancy needling and methotrexate instillation are all performed using this technique. If the ovary is inaccessible transvaginally, ultrasound guided aspiration may also be performed transabdominally. Embryo transfer

Embryo transfer may be carried out under direct ultrasound vision (Hurley et

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al., 1991). After insertion of the transfer catheter, the transvaginal transducer is positioned in the vagina immediately below the cervix. The catheter tip can then be manoeuvred under direct vision into an optimum position at the fundal limit of the cavity. This technique is particularly helpful in patients in whom cervical cannulation is difficult or where congenital or acquired uterine cavity abnormalities are present. In cases where it is not possible to cannulate the cervix, transfer may be effected by guiding a transfer needle transmyometrially through the lateral vaginal fornix or transabdominally utilizing needle guide systems. Transcervical catheterization of the fallopian tubes Fine plastic catheters can be guided into the fallopian tubes under direct ultrasound vision via the cervix (Jansen and Anderson, 1987). These catheters may be used to establish tubal cornual patency, particularly where doubt exists following radiological or laparoscopic investigations of tubal patency. The passage of such catheters may even relieve tubal blockage. Gametes and embryos may also be transferred into the fallopian tubes using this technique as an alternative to the more invasive laparoscopic methods. Hysterosonographyy'uterine irrigation Sterile saline may be introduced into the uterine cavity to enhance diagnosis of suspected cavity pathology. Where polyps or submucosal fibroids are suspected, the diagnosis may be established with certainty and appropriate therapy planned. Pregnancy assessment Transvaginal ultrasound is used to examine early pregnancy development in the subfertile woman, whether the conception is spontaneous or as a result of ART. It is used to confirm normal pregnancy location, number and viability. Ectopic pregnancy occurs in 5% of all pregnancies conceived via ART and heterotopic intra and ectopic pregnancies are also more common. The combination of transvaginal ultrasound and quantitative j?-hCG assays is the method of choice for diagnosing ectopic pregnancies. Multiple pregnancies occur in 20% of all ART pregnancies. Spontaneous abortion is more common in pregnancies conceived via ART and ultrasound may be used frequently throughout the first trimester to establish normal development. Neural tube defects and some cardiac anomalies (transposition of the great

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arteries) are said to be more frequently seen in pregnancies conceived via ART. A transvaginal ultrasound examination at 12 weeks will diagnose most neural tube defects and at 18 weeks will provide the best opportunity to detect cardiac defects.

Multiple pregnancy reduction High-order multiple pregnancies are more frequent following infertility treatments, particularly those involving the use of gonadotrophins. Couples may seek reduction of the number of fetuses to improve the obstetric outcome and diminish the psychological impact of such pregnancies. This procedure is carried out using ultrasound-guided needling; usually transabdominally; typically at 10-12 weeks' gestation. Potassium chloride is injected into the thorax of the fetus via an 18-20-gauge needle. The procedure carries a risk of total pregnancy loss of approximately 5% (Evans et al, 1993).

Complications of ART Pelvic pain and abdominal distension commonly occur after ART cycles. Transabdominal and transvaginal ultrasound examinations can be used to diagnose haemorrhage into a residual cyst or the peritoneal cavity.

Ovarian hyperstimulation syndrome This potentially life-threatening condition occurs after ovarian stimulation, particularly where gonadotrophins are used. It is characterized by enlarged multicystic ovaries and fluid in the body cavities. Ascites and pleural effusions, seen in the severe forms, may lead to respiratory compromise and death if not diagnosed and treated effectively. Transabdominal ultrasound is the method used to diagnose, classify and monitor the treatment of OHSS.

Transrectal ultrasound assessment of male infertility The prostate gland, seminal vesicles, urethra, vas deferens and ejaculatory ducts may be examined using transrectal transducers. Congenital abnormalities such as the absence of the seminal vesicles or vas deferens or obstructive lesions of the tubular system may be diagnosed. Cysts of these structures and stone formation are sometimes seen in the infertile male at the time of transrectal ultrasound (Kuligowska, 1994).

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References Evans, M.I., Dommergues, M., Wapner, R.J. et al. 1993. Efficacy of transabdominal multifetal pregnancy reduction: collaborative experience among the world's largest centres. Obstetrics and Gynaecology 82: 61-5. Hurley, V.A., Osborn, J.C., Leoni, M.A. and Leeton, J. 1991. Ultrasound guided embryo transfer; a controlled trial. Fertility and Sterility 55: 559-62. Jansen, R.P.S. and Anderson, J.C. 1987. Catheterisation of the fallopian tubes from the vagina. Lancet 2: 309-10. Kuligowska, E. 1994. Transrectal ultrasonography in diagnosis and management of male infertility. In Imaging in Infertility and Reproductive Endocrinology, ed. R. Jaffe, R.A. Pierson and J.S. Abramowicz, pp. 217-29. Philadelphia: J.B. Lippincott. Smith, D.H., Picker, R.H., Sinosich, M. and Saunders, D.M. 1980. Assessment of ovulation by ultrasound and estradiol levels during spontaneous and induced cycles. Fertility and Sterility 33: 387-90.

15 The role of surgery in infertility CARL WOOD

Introduction

In 270 infertility patients seen between 1990 and 1991 in a private gynaecological practice, two-thirds had pelvic disease requiring surgery (personal series). Sixty percent of the patients became pregnant subsequent to surgery for pelvic adhesions, endometriosis, tubal blockage, fibroids, adenomyosis, and uterine intracavity polyps, fibroids or adhesions. In contrast, only 12% of male infertility can be treated by surgery (Hudson, Baker and de Kretser, 1980). Infertility surgery rarely requires laparotomy, most procedures being performed by laparoscopy, hysteroscopy and, more recently, falloposcopy. The last three techniques are used both to diagnose and treat the cause of infertility. Diagnostic laparoscopy is being simplified by the development of finer laparoscopes, the new 2-mm-diameter micro laparoscope requiring much smaller incisions and less anaesthesia and allowing quicker patient recovery (Downing and Wood, 1995). Tubalsurgery

A variety of procedures may be performed to overcome tubal infertility. These include fimbrioplasty, salpingostomy, salpingo-ovariolysis, tubotubal anastomosis, uterotubal anastomosis, and tubal cannulation. The principles of tubal surgery include the following. 1. Reduction of tissue trauma by atraumatic techniques using fine instruments and keeping tissue surfaces moist. 2. Complete haemostasis reduces the risk of adhesions. 3. Abnormal tissue such as thick adhesions should be excised. 4. Tissue planes must be realigned in the normal anatomical position.

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5. Magnification enables the surgeon to define the anatomy and pathology more precisely and facilitates the application of the above principles. It also enables the surgeon to use fine instruments and fine sutures.

Salpingo-ovariolysis

Adhesions which cover the tubes and/or ovaries may interfere with conception. Hydrotubation is performed to confirm the tubes are patent. The adhesions may coexist with intratubal lesions, causing distal occlusion. Salpingoovariolysis may be the only procedure performed or may be the first step for other reconstructive procedures such as tubotubal anastomosis. The adhesive layers are grasped and retracted to identify and expose them. The adhesions are cut close to the tube or ovary. Fine adhesions can be cut but broad adhesions should be exised otherwise they will reattach. Sharp scissors, electrosurgery with a microelectrode or laser energy can be used to cut the adhesions. Haemostasis can be obtained by microbipolar forceps or monopolar electrodes. If adhesions are dense, a plane of dissection is commenced by a small incision and then blunt-ended scissors or hydrodissection are used to open the plane. The adhesion is grasped at its midpoint and, with gentle traction, its attachments are usually exposed. A small incision is made over a translucent area to identify structures lying beneath the adhesion. The pneumoperitoneum pressure is reduced as previously unidentified bleeding may then commence. It may be helpful to examine the fimbria under fluid as the anatomy of the fimbria and filmy adhesions are better seen. Once haemostasis and adhesiolysis have been completed, 1-2 litres of buffered physiologic solution is left in the pelvic cavity. Fimbrioplasty

Agglutination of the fimbriae results in constriction and a varying degree of patency. The ampulla distends before the escape of dye solution from the tube. The point of narrowing may be outside the fimbriae when a fibrous layer covers them. Stenosis may also be prefimbrial, the fimbriae having a normal appearance and the fluid dilating the tube at the ampulla in the prefimbrial portion of the tube. Periadnexal adhesions are frequent and must be removed. Fimbrial stenosis is overcome by one of two methods. The tube is distended by hydrotubation and 3-mm alligator-jawed forceps are introduced, opening the jaws to break down the adhesions gently. The alternative method, which is preferable, is to use fine scissors to break down the adhesions, taking advan-

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tage of the magnification provided by the laparoscope placed close to the operation site. If there is prefimbrial stenosis, an incision is made along the densest scar tissue as this would not contain normal tube. If no definite fibrotic scar can be identified, then an incision along the antemesenteric border is preferable as less bleeding is likely. The incision runs from the distal fimbria past the constriction ring of the ampulla.

Salpingostomy A new opening can be made if the tube is completely blocked. The operation is not worthwhile unless the lining of the tube has functional integrity. This can be determined by falloposcopy or distal salpingostomy. Atraumatic forceps are used to grasp the end of the tube close to the occluded portion. Several incisions are made in the avascular areas until hydrotubation confirms adequate patency. After the first incision has been made, the choice of further incisions can best be made by looking at the inside surface of the tube to select further avascular areas for the incision. This prevents division of normal mucosal folds. Fixing the edges of the stoma has been advocated but is not required to maintain patency and may reduce the natural mobility of the tube.

Results of reconstructive surgery Results of procedures mainly depend on the extent of the tubal damage and the adequacy of the surgical procedure. Other infertility factors would also reduce the chance of success. An intrauterine pregnancy rate of 59% was reported by Gomel in 1977 for salpingo-ovariolysis. The published intrauterine pregnancy rates vary between 21% and 62% and the tubal pregnancy rates between 4% and 7.5% (Gomel, 1977). A multicentre, controlled, randomized study showed the pregnancy rates were 45% amongst the surgical group and only 16% in the untreated control group after a period of two years (Tulandi, Collins and Burrows, 1990). The reported pregnancy rates for fimbrioplasty vary between 26% and 50% (Gomel and Taylor, 1992). The results of laparoscopic salpingostomy vary between 18% and 29% and ectopic pregnancy rates between 3% and 11% (Gomel, 1977) - the former may be less than those obtained by microsurgery although comparable cases have not been studied. The advantage of the laparoscopic approach is that it can be done at the time of diagnostic laparo-

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scopy and is associated with reduced discomfort and cost, prompt recovery and the avoidance of a second operation. Tubotubal anastomosis

This operation is most often performed to reverse previous sterilization but is sometimes undertaken for a localized cornual or midtubal occlusion. It is unlikely that the results of the microsurgical procedures of minilaparotomy can be matched by those of laparoscopy. Microsurgical repair is relatively easy to perform through minilaparotomy, patients being discharged one to two days after surgery. Falloposcopy of the proximal segment for anastomosis may be an advantage in determining the length of tube to be excised prior to anastomosis. Although laparoscopic techniques for tubotubal anastomosis have been described, and in some cases performed extremely well, the technique is difficult and has not become used routinely by laparoscopic surgeons. A minilaparotomy technique involves the use of 8/0 sutures, placement of interrupted sutures in the musculature and epithelial layers, the accurate alignment of the two segments, and reinforcement with a second seromuscular layer to sustain physical strength of the suture line. In patients having tubotubal anastomosis for blockage associated with previous tubal disease, it is even more important to determine the tubal health outside the area of blockage. The falloposcope is inserted from the vagina to check the inner tube proximal to the block, and then reinserted into the outer end, gaining access from the abdomen. This can be done by introducing the falloposcope with the assistance of the laparoscope at the outer end, and through the vagina, to inspect the inner end of the tube proximal to the block. Although failure of tubal anastomosis is best treated by IVF, a small number of patients have had a successful second sterilization reversal. This can be done if the first sterilization has involved the isthmic portion of the tube and sufficient length remains to perform a good anastomosis. Endometriosis

In a non-randomized study, Tulandi and Mouchawar found that laparoscopic coagulation of minimal and mild endometriosis (stage I and II, revised American Fertility Society classification) was superior to no treatment (Tulandi and Mouchawar, 1991). In a more conclusive study, Nowroozi et al. found the pregnancy rate after cauterization of mild endometriosis (60.9%) was significantly higher than after expectant management (18.5%) (Nowroozi et al., 1987; Table 15.1). These studies suggest that removal of endometriosis im-

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Table 15.1. Randomized trial of laparoscopic electrocoagulation and no therapy in endometriosis. The average duration of infertility was 26 months, and patients had undergone normal fertility investigations. Patients with pelvic adhesions were excluded

Fulguration of endometriotic implants No treatment

Number

Number pregnant within 8 months

69 54

42 (60.8%)* 10(18.5%)

*p<0.001. Data from Nowroozi et al. (1987).

Table 15.2. Comparison ofpregnancy rates following laparoscopic electrocautery or laser treatment of mild to moderate endometriosis Number of studies Laparoscopic coagulation 6 Laparoscopic CCb laser 4

Total number Period of of patients follow-up 316 126

9-15 months 6-12 months

Range of pregnancy rates (0/25-75 22-65

Data from Reich, McGlynn and Salvat (1991).

proves pregnancy rate. It is not clear whether this effect is due to the cure of pain and more regular coitus or to the removal of antifertility effects of endometriotic peritoneal lesions. Several studies have shown that conservative surgical treatment of endometriosis by laparoscopy is similar, if not superior, to treatment by laparotomy (Olive and Martin, 1987; Nezhat, Cromgey and Nezhat, 1989). The conservative surgery consisted of freeing adhesions and ablation of implants, endometriomas and uterosacral ligaments. Laparoscopic electrocoagulation has also been compared to medical treatment with danocrine for six months in moderate endometriosis. Pregnancy rates after seven months were similar for danocrine (39%) and electrosurgery (44%) (Seiler, Cigwani and Ballard, 1986). In ten studies, comparison of pregnancy rates following laparoscopic electrocautery and CO2 laser for the treatment of mild to moderate endometriosis shows no difference (Reich, McGlynn and Salvat, 1991; Table 15.2). The period of follow-up was sometimes longer in the coagulation studies, although

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this factor may not be important because most pregnancies occur in the first nine months after surgery. Argon lasers can selectively vaporize pigmented endometriotic lesions while preserving normal tissue. The pregnancy rate after laparoscopic argon laser treatment of endometriosis is similar to that of other methods (33.9%) (Keye, Hanso and Astin, 1987). A controlled study may be more informative, although it is difficult for a surgeon to be equally skilled in electrocautery and laser surgery. Surgical excision by scissors has not been compared to the use of electrocautery or laser. It may be important for the surgeon to choose the technique found to be most safe and effective in his or her experience. It is probable that electrosurgery is as effective as laser. Peritoneal lesions Diagnosis

The appearance of endometriosis may be varied and it may be difficult to detect. Biopsy studies show that at least 7% of lesions are missed and the extent of lesions is underestimated in 50% of cases (Martin, Armic and El Sehg, 1990). Experienced laparoscopists, when compared to histological biopsy, have a diagnostic accuracy of 86%, while less experienced surgeons have an accuracy of only 41% (Martin et al., 1989). The limitations of laparoscopy diagnosis are emphasized by thefindingthat scanning electron microscopy of normal-appearing peritoneum has shown endometriosis in up to 25% of patients (Murphy, Green and Babbie, 1986). Occult endometriosis has been confirmed by histological diagnosis in normal-appearing peritoneum of 6% of patients without endometriosis, and of 13% of patients with endometriosis (Nissole, Caspanas-Roux and Donnez, 1988). Magnification at laparoscopy may assist in diagnosis. The working distance of the laparoscope determines the degree of magnification, ranging from shrinkage of 0.6 at 5 cm to magnification of 8-10 at 3 mm from the peritoneum (Murphy et al, 1986). The colour and appearance of the lesions vary - white scarred, strawberrylike red, red flame, red patches, clear vesicles, peritoneal defects, yellow-brown patches, brown, black puckering and adhesions. Biopsy of lesions shows that 81% of white, opaque and red-flame lesions are endometriosis and 67% of glandular lesions are endometriotic (Cook and Rock, 1993). Endometriosis is found less frequently in yellow-brown (47%) and peritoneal (45%) defects (Jansen and Russell, 1986). Non-pigmented lesions are more common than pigmented lesions, which increases the difficulty in visual diagnosis, particularly if the gynaecologist is unaware of the significance of non-pigmented disease. Peritoneal defects occur in 7% of the normal population and in 28% of patients

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with endometriosis (Chatman and Zbella, 1986). Biopsy at the edge of the defect most often detects the associated endometriosis. Pigmentation and infiltration of lesions increase with age (Redwine, 1987), suggesting that in some patients endometriosis may be a progressive disease. Nissole et al. suggest that the early stage of the disease is associated with clear glandular and red flame appearances, but after resolution of the inflammatory response and bleeding, reduced vascularity and scarring produce yellow-brown or black lesions, and adhesions (Nissole et al., 1988). Because no lesion is certainly endometriosis, it is preferable to biopsy at least two peritoneal lesions to establish the diagnosis. The biopsy can be done with a punch or scissors. Scissors allow 'tailoring' of the biopsy to increase its area or depth. Scissors with attached monopolar electrocautery forceps save time, should bleeding be associated with the biopsy. Conditions which may mimic endometriosis and may be determined by biopsy are inflammation, chronic infection (actinomycosis or tuberculosis), granuloma, foreign body reaction, endosalpingiosis and implants from an ectopic pregnancy, dermoid cyst or neoplasia. Laparoscopic removal of peritoneal lesions at the time of diagnosis has several advantages: avoidance of drug therapy or laparotomy, rapid recovery from treatment, and possible reduced duration of infertility. Possible complications of operative laparoscopy include damage to the bowel, ureter or blood vessels, gas embolism, haemorrhage or infection. While these complications are very uncommon (less than 1%), they are more dangerous than the more common side-effects of drug therapy. Diagnostic laparoscopy precedes the use of drug therapy and shares the same risks of operative laparoscopy, excluding ureteric damage. The incidence of complications in association with diagnostic laparoscopy may be lower than with operative laparoscopy. Technique of removal

Endometriotic lesions may be removed by electrocautery, laser, ultrasoundgenerated vibrational energy or excision. Excision may be carried out by disposable Endoshear scissors with an attached monopolar electrode. These scissors are effective, and can be rotated externally to access various angles in the pelvic cavity, thus reducing arm movement. The scissors may be used with 70-100 W of cutting or 50 W of coagulating monopolar current. Reusable scissors, blunt ended with an attached monopolar electrode, are also effective, the quality of the instruments improving over the last few years. Sharp scissors are dangerous because of the possibility of bowel or vessel puncture. When scissors are used, the peritoneum over the lesion is elevated, which determines the mobility and depth of the

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lesion. The lesion is excised with a small margin of surrounding normal peritoneum (2-5 mm), depending upon the size of the lesion and the proximity of surrounding lesions. If multiple lesions occur in a defined area, it may be preferable to excise a patch of peritoneum. Occult (microscopic lesions) occur in 13% of patients with endometriosis and this frequency may be higher with increased numbers of visible lesions. Complete stripping of the peritoneum in the pouch of Douglas and lateral pelvic walls below the ovaries has been described by Redwine (Redwine, 1991) and practised by the author. In a study of 12 patients having the peritoneum covering the pouch of Douglas removed, 8 had a subsequent laparoscopy. No adhesions occurred, although the new peritoneum was sometimes opaque and less mobile. The same procedure has been performed on the lateral pelvic wall below the ovary infivepatients and only one of the four reviewed by laparoscopy developed significant adhesions between the ovary and pelvic wall. Redwine prefers non-laser resection of endometriosis and has achieved a low five-year recurrence rate of endometriosis using this method (Redwine, 1991). Monopolar scissors are versatile for resection of endometriosis: 'allowing various cutting or coagulation surfaces, sharp or blunt dissection, fulguration, ease of tissue handling and tactile feedback'. The advantages of scissor excision are that local or block removal of peritoneum is feasible, the depth of the lesion can be easily gauged as thermal damage does not blur the appearance of the lesions, associated bleeding can be dealt with by scissor diathermy, and the peritoneal removal is quick and the method is cheap compared to the use of laser. Damage to adjacent vital structures, large blood vessels or ureter is unlikely because the scissors are not penetrating tissue or obscuring adjacent structures by opacity caused by thermal coagulation. Other advantages of scissor excision compared to laser and electrocautery are the absence of smoke, the absence of tissue carbon formation which may cause granuloma, and the availability of all tissue for histological examination (Table 15.3). Scissor excision may have the disadvantage of removing excess peritoneum (a dubious fault because of the frequency of microscopic lesions) or being more difficult to perform. Electrocautery may be used to vaporize or coagulate endometriotic lesions. Vaporization may be achieved by high-density cutting current using a needle electrode but may be difficult to control because of penetration into tissue. Coagulation can be achieved by coagulation current touching the surface with a spoon orflatelectrode. A roller ball may be used for block removal of a large area of peritoneum. The correct current intensity is determined by gradually increasing current from zero until superficial whitening occurs in an area of peritoneum distant from vital structures. Because of electrical and thermal

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Table 15.3. Advantages of scissor diathermy removal of peritoneal lesions Facilitates local or block removal of peritoneum Clearly defines depth and extent of lesions Bleeding easily controlled Rapid and cheap method Reduces risk of damage to adjacent structures as no penetration or opacification of adjacent tissue Availability of all tissue for histology

penetration into tissue, lesions over the ureter, bladder or bowel are best mobilized by grasping forceps and pulled or dissected away from vital structures before electrocautery is applied. Fulguration is achieved by spray coagulation using a spoon or flat electrode held 1-2 mm from the tissue surface and 50-70 W coagulation current. There is little penetration of tissue because the energy is lost when sparking occurs between the electrode and tissue surface and a superficial coagulum forms. This type of electrocautery can be used over a large area of endometriosis but has two disadvantages. The area affected by the spray coagulation may be difficult to define accurately and, if the electrode accidentally touches the tissue surface, deep penetration of the current may occur. It is useful if surface bleeding from veins occurs over an area where individual vessels could not be easily identified, e.g. the bladder, as a superficial coagulum forms quickly which is not removed when the electrode moves away from the target area. Bipolar electrocautery forceps limit the spread of current between the electrodes at least until the tissue between the probes has coagulated. Continuing cautery beyond this time may result in electrical and thermal energy spreading beyond the forceps. Bipolar forceps are available which emit a noise indicating coagulation is complete. In their absence, reduction of current using an ammeter, or the cessation of bubbles and tissue shrinkage may indicate effective coagulation. Bipolar electrocoagulation has the advantage of closing vessels up to 2 mm in diameter and is useful in treating endometriotic nodules or flat lesions attached or close to blood vessels or vital organs, e.g. on the lateral pelvic wall, bladder or rectum. The ability of electrocautery to deal with blood vessels is one advantage over laser. One disadvantage of bipolar forceps is that the size of the prongs makes precise definition of the area treated difficult. Microbipolar forceps may be more useful but are only able to coagulate vessels less than 1 mm in diameter. Laser has been used in a variety of ways to deal with endometriotic lesions.

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Using the CO2 laser at a lower density of 2500-5000 W/cm2 with single or repeat pulses of 0.5-1.0 sec limits coagulative penetration of tissue to 0.1-0.2 mm. Vaporization of tissue is achieved by using a higher-power density superpulse. Care in the use of the foot piece and adequate speed of movement across the tissue are required to avoid deeper penetration. As endometriotic lesions are greater than 3 mm in depth in 60% of patients, vaporization excision is often required. The use of laser close to blood vessels has the disadvantage that opening a vessel wall may predispose to gas embolus. The CO2 jet is sometimes set close to the end of the laser rod. Laser has the advantage of precision in delivering thermal energy, but has the disadvantage of increased costs and complexity of equipment. Improvements in the development of monopolar scissors and electrocautery-delivering systems have minimized the advantages of laser. Peritoneal lesions on the colon may be treated by CO2 laser superpulse at low power (5-10 W) moved rapidly across the surface. If muscle is penetrated, 3/0 or 4/0 absorbable suture closure is indicated as it is difficult to gauge the depth of muscle penetration. Ureteric dissection

Ureteric dissection may be required when peritoneal lesions extend over the ureter, endometriomas are close to the ureter, the ovary is fixed to the lateral pelvic wall, or the ureter is obscured by scar tissue and hysterectomy or oophorectomy is required. Ureteric dissection is easiest when the ureter can be seen. The ureter can be grasped with non-traumatic ureter-grasping forceps, the distal end of the forceps having a gap to enclose but not compress the ureter. Blunt-ended scissors can then be used to open the peritoneum lateral to the course of the ureter and free the ureter sufficiently on its lateral margins to trace its course or separate it from the area of endometriosis. The ureter should not be freed of vascular attachments. Endometriomas attached to the ureter have been freed by laparoscopic surgery (Wood and Maher, 1993). If the ureter cannot be grasped, the peritoneum is elevated and opened over a small area adjacent to the ureter to facilitate subperitoneal dissection. Scar tissue may bind the ureter to the pelvic wall. If the ureter cannot be seen or is enclosed in dense scar tissue, dissection is only possible after ureteric catheterization. Ureteric stenosis may be overcome by removal of scar tissue or endometriomas constricting the ureter. Stents may be placed in the ureter for three to four weeks to ensure healing of the ureter and reduce the risk of re-stenosis. Postoperative intravenous pyelography will enable the patency and diameter of the ureter to be checked four to six weeks after surgery.

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Ovarian endometrioma The diagnosis of endometriomas is usually made on the basis of the occurrence of unilateral (ovarian) midcycle pain and, less often, premenstrual and menstrual pain (Wood, Maher and Hill, 1992). High-resolution vaginal ultrasound has an accuracy of 80% in the detection of endometriomas (Wood et al., 1992). Definitive diagnosis is made at laparoscopy after inspection (66%), mobilization (20%), and needling of the ovary (14%) (Wood et al, 1992). Laparoscopic technique is important in making the diagnosis. In women with an enlarged ovary, particularly if endometriosis is present elsewhere in the pelvis, needling of the ovary is required to exclude the presence of an endometrioma. Small endometriomas may be present in a normal-sized ovary; ultrasound may assist in their diagnosis. Removal of an endometrioma is preferable to medical therapy (Canis, 1993) or simple drainage (Donnez, Nissole and Wayembergh, 1989). The medical cure rate has ranged from 12% to 45% and the surgical cure rate from 70% to 80% (Canis, 1993). The best cure rates after one year have followed careful excision of the endometrioma (90%), with only 4% of patients still having pain, and 50% achieving pregnancy (Wood et al, 1992). Technique of removal Surgery initially involves mobilization of the ovary and drainage of the endometrioma. The operation is easily achieved using three accessory incisions: two being used to hold the ovary or the edges of the endometrioma, and one for the instrument used to excise the endometrioma. One 5-mm accessory incision may be avoided by using a long Verres needle for ovarian stabilization through a 1-mm stab incision. The needle can be used for holding, moving or squeezing the ovary. Ports on both sides of the abdomen allow sufficiently wide angles to retract the ovary medially away from the lateral pelvic wall or bowel, and to open the endometrioma enabling laparoscopic access and visualization inside the ovary when dealing with large endometriomas. The endometrioma is incised over the point of maximum curvature by electrocautery, laser or scissors. Electrocautery has the advantage of containing bleeding over the line of the incision while scissors, in the absence of thermal injury, allow an excellent view of the ovarian capsule and endometrioma wall. In about half the endometriomas, an initial incision for drainage is not required as the endometrioma has formed between the pelvic wall and ovary or between the irregular surfaces of the ovary. The endometrioma drains

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naturally after mobilization of the ovary. Drainage is achieved by suction irrigation to reduce and dilute spillage of the chocolate-like material most often found inside the endometrioma. A suction irrigator should be inside the abdomen at the time of ovarian mobilization to reduce spillage. Dilution of thick material may be required before the suction is effective. The wall of the endometrioma is best removed. Simple drainage of the endometrioma has proven unsuccessful. The edge of the endometrioma wall may be seen easily beneath the ovarian capsule but sometimes requires sharp scissor dissection to define it. Electrocautery and laser systems may blur this dissection. Once the edge of the endometrioma wall is clasped with the tooth forceps, it can be slowly peeled out of the ovary (the 'orange peel technique'), cutting any adhesion or blood vessels remaining between the wall of the endometrioma and the ovary. Electrocautery has the advantage of dealing with bleeding. Monopolar scissors combine the advantage of scissors and electrocautery. Dissection of a large endometrioma can be completed with the laparoscope inside its cavity. Suction irrigation and intracavity laparoscopy enable complete removal of the endometrioma wall, as frequently small segments do not peel off and can easily be left inside the ovary. Sometimes the wall of the endometrioma will not peel away from the ovary so that scissor, laser or electrocautery excision is required. Scissor dissection is more likely to cause bleeding, so monopolar scissors are preferable. Thermal injury of the ovary is reduced by using the scissors with back-up diathermy only to control bleeding. If the surgical plane for excision is not clear because of excessive scar or bleeding, which is unusual, the wall of the endometrioma may be coagulated or vaporized by electrocautery or laser. The obvious disadvantage is that thermal injury may occur to adjacent ovarian tissue, reducing follicle population in the ovary. Sometimes bleeding requires the use of bipolar forceps to reduce the spread of coagulation into the adjacent ovary. Suture of the ovary may also be used to control bleeding, using a 3/0 absorbable suture with a needle large enough to compact the ovarian tissue. Internal or external tying methods may be suitable. Oophorectomy is preferable if no normal ovarian tissue is found after removal of a large endometrioma. Oophorectomy may be considered also if the endometrioma has been recurrent or the ovary is adhered to the lateral pelvic wall by very rigid scar tissue and the ovary fragments when dissected. Endometriomas up to 12 cm in diameter have been removed by the above methods.

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Two-stage technique An alternative is to use a two-stage procedure (Donnez et al., 1989). At the initial laparoscopy, the endometrioma is drained and GnRH analogues are used for 8-12 weeks to reduce the size and vascularity of the endometrioma further. At a second laparoscopy, the endometrioma is removed and the GnRH analogues are continued for another 8-12 weeks. This method makes the surgery easier and possibly more effective, although a controlled study has not yet demonstrated this. It has the disadvantages of increased costs, the use of expensive drugs and two operations; the two operations and the side-effects of the drugs increase patient morbidity and progress toward the resolution of infertility is delayed. The satisfactory results of single surgical treatment after one year - 95% pain free, 50% pregnancy rate for stage 3 and 4 endometriosis, and 10% recurrent endometrioma rate (Wood et al., 1992) - make it difficult to establish that the two-stage surgical/medical therapy is more effective. The single surgical treatment may be best modified to include a second laparoscopy at three or six months to treat patients who are not pregnant, still have pain or have ultrasound evidence of a persistent endometrioma. The most common sequelae after the treatment of endometriomas are adhesions (40% of patients), which may warrant further laparoscopic surgery in those patients with persistent pain or infertility (Wood et al., 1992). Antiadhesive regimes have demonstrated some efficacy in controlled trials so that Interceed, in the absence of bleeding surfaces, may be used to wrap around the ovary or to be placed on the lateral pelvic wall after treatment of an ovarian endometrioma. If the surgical surfaces are still moist with blood, 1-2 litres of buffered solution may be instilled into the peritoneal cavity, or Goretex membrane stapled onto the pelvic wall below the ovary. Laparoscopic surgery has been shown to be as effective as laparotomy in the removal of endometriomas in one uncontrolled trial (Wood et al., 1992). Tough adhesions, ovarian pain and ovarian removal were more common after laparotomy and ovarian suture. A controlled trial using laparoscopic surgery with and without suture after ovarian cystectomy has shown adhesions are more common when sutures are used (Nezhat and Nezhat, 1991). The absence of sutures avoids tissue reaction. The process of ovulation has not been associated with ovarian adhesion formation so that the ovary, at least at ovulation, has the ability to heal open wounds repeatedly without significant scarring. Other operative techniques may also reduce the risk of adhesions. Ovarian epithelial damage can be avoided by using retraction rather than grasping forceps. Damage to the ovarian surface can be minimized by draining the endometrioma first and then removing the capsule of the endometrioma

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from the inside rather than cutting normal ovary to excise the lesion. The ovarian wound edges fall together and the opening in the ovary approximates that following natural ovulation. Ovarian cystoscopy Brosens has explored the use of ovarian cystoscopy to investigate cysts which are not malignant and to avoid unnecessary ovarian surgery (Brosens, 1993). Non-malignant cysts can simply be aspirated, providing inspection of the lining of the cyst and biopsy show no evidence of malignancy or endometriosis. A 5-mm cystoscope is placed inside the cyst and fluid irrigated to inspect the cyst. Haemorrhagic follicular cysts or a cystic corpus luteum can be differentiated from an endometrial cyst and biopsies can be taken to confirm this. Argon laser surgery has been achieved inside endometrial cysts on localized active areas of endometriosis seen within the cyst; up to one-third of resected endometriomas show only fibrous tissue with pigmentation. Surgery of endometriomas can be minimized to cause less tissue and ovarian damage. Twenty patients treated in this manner have shown no recurrence after one year. Localized endometrioma ablation may simplify the surgical treatment of endometriomas. There may still be a need to reduce large endometriomas to reduce ovarian size and the risk of adhesion formation. Removal of the ovary The occurrence of very large endometriomas, recurrent endometriomas in the same ovary, severe ovarian fixity unrelieved by previous surgery, the uncertain coexistence of malignancy within the endometrioma, and the need to cure endometriosis and retain the uterus to pursue donor egg IVF, may each indicate ovarian removal. Sutures, bipoloar coagulation, staples and endoloops have been used for ovarian removal with endometriosis. Sutures require careful opening of the peritoneum below and lateral to the infundibulopelvic ligament to avoid the ureter and vascular damage. Once achieved, it is more reliable than other methods of haemostasis, as bleeding may need repeated attention when using bipolar forceps, staples or endoloops. External ties of size 2 absorbable sutures with a sliding knot techique using the Clark Reich knot pusher are reliable. The results of laparoscopic ovarian removal for endometriosis are satisfactory (Wood, personal series), particularly for adhesive disease, 13 of 15 remaining pain free for two years and achieving pregnancy. The recurrence of endometriosis in two of the five patients following ovarian removal because of severe endometriosis suggests a repeat laparoscopy may be offered three months after oophorectomy to check for recurrence of endometriosis.

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Pouch of Douglas

Treatment options for pain or infertility secondary to cul-de-sac obliteration include ovarian suppression therapy with danocrine or GnRHA, or surgery. For current infertility or the preservation of fertility, reconstructive surgery can be considered by laparotomy, microsurgery or laparoscopy, depending on the skill and experience of the surgeon. Endometriosis in the pouch of Douglas may differ from other pelvic endometriosis by a tendency to be infiltrative, resistant to medical therapy, difficult to access by surgery and occurring in older women (Koninckx, Mueleman and Demeyere, 1991). Nodular endometriotic lesions develop in the uterosacral ligaments, the vagina and the anterior rectal wall. Medication may be less effective because of the larger size of the lesions and the reduced vascularity associated with the frequent occurrence of scarring in the pouch of Douglas. Laser and electrocautery are limited by the difficulty in gaining access to the recto vaginal space and in determining the extent of the lesions as the anatomy and pathology become distorted by the scarring. The rectum may be attached to the cervix, uterine body and lateral pelvic wall. Surgical dissection and excision allow more accurate removal of the pathology (Redwine, 1993). Scarring in the pouch of Douglas is often assumed to signify a healed area of endometriosis. Biopsy of scarred lesions often shows active endometriosis (Reich, personal communication). Diagnosis

Menstrual pain is present in most patients and deep coital pain in nearly all patients (Wood, Maher and Hill, 1993a). The coital pain sometimes persists for several hours after intercourse, is worse on one or other side, and is sometimes lessened by changing coital position. Most patients have pain on defaecation, which is marked or restricted to the time of menstruation; bowel habits also are altered at menstruation (Wood et ah, 1993a). Bladder and ovulatory pain are less common. Tenderness is present on palpating the pouch of Douglas and can often be localized to one or both uterosacral ligaments. Palpation can reproduce the pain of coitus so that clinical localization of the disease is possible and accurate. Nodularity in the pouch of Douglas may be detected, but may be masked by the pain associated with pelvic examination. Transcervical uterine biopsy may be useful as it will determine whether or not pouch of Douglas endometriosis is an extension of adenomyosis (Wood, Maher and Hill, 1993b). Hysterectomy and recto vaginal dissection would be more appropriate when adenomyosis and pouch of Douglas endometriosis coexist.

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The operative techniques may include excision of the peritoneum over the pouch of Douglas, removal of the uterosacral ligaments and, if the cul-de-sac is obliterated, rectal mobilization and excision of rectal or vaginal lesions. Removal ofperitoneum Endometriotic peritoneal lesions which are red or superficial are lasered or cauterized. If they are extensive, an elliptical incision is made around the lesion, which is elevated by blunt or hydro-dissection before excision. Sometimes the whole of the peritoneum over the pouch of Douglas is removed. Removal of the uterosacral ligaments Uterosacral ligaments are removed when scarring, nodules or multiple superficial lesions are present. The course of the ureter is identified and the uterosacral ligament is retracted medially at the point of attachment to the uterus. Scissors with a monopolar electrode and electrocautery with a unipolar spoon or CO2 laser (superpulse 10-20 W) are used to free the ligament from the uterus and surrounding tissue. The vaginal and rectal probes display these organs during the procedure. If the rectum is adherent to the ligaments, the rectum is mobilized first. Excision of the uterosacral ligaments includes surrounding peritoneum, the area removed depending upon the extent of the disease. Cul-de-sac obliteration and dissection A mechanical bowel preparation is used orally the afternoon before surgery. This produces a brisk, self-limiting diarrhoea that rapidly empties the bowel without disrupting electrolyte balance. A 2-g dose of cefoxitin sodium is given intravenously at the commencement of surgery. The anterior rectum is first freed to expose the loose areolar tissue of the rectovaginal septum prior to excising the visible and deep palpable nodules of flbrotic endometriosis. The rectum may require mobilization from the uterus, cervix, vagina and uterosacral ligaments. When the rectum is attached to the uterus, careful dissection is required. Blunt-ended scissors are used to open the peritoneum at the junction between the rectum and vagina or where it is mobile if the anatomy is not clear. Sometimes hydro-dissection is used to open a tissue plane between the rectum and surrounding tissue. The fluid creates cleavage planes in the areas of least resistance, where further incision and dissection are safe in connective tissue separated from the rectum. This dissection is aided by the uterine, vaginal and rectal probes displaying these organs. A blunt curette is used to antevert the uterus and display the cul-de-sac. A sponge on a ring forceps dents the posterior vaginal fornix and a rectal probe

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or handle end of a curette displays the rectum. Blunt or scissor dissection may be unsafe as an initial technique to open the pouch of Douglas as the mechanical force applied may open the rectum when it is diseased and adherent to the cervix. Deep fibrotic, often nodular, endometriotic lesions are excised from the uterosacral ligaments, upper posterior vagina (the location of which is continually confirmed by the sponge in the posterior fornix) and posterior cervix. The dissection on the outside of the vaginal wall proceeds using hydrodissection, electrosurgery or scissors. Usually an endopelvic fascial layer, infiltrated with endometriosis, is identified, and after this layer is excised, the soft, pliable, upper posterior vaginal wall is uncovered. It is difficult accurately to distinguish fibrotic endometriosis from the posterior cervix at the cervicovaginal junction; a lesion may completely penetrate the vaginal wall. Dissection is then performed accordingly, with removal of all visible endometriotic lesions. Lesions extending totally through the vagina are treated with en-bloc laparoscopic resection from the cul-de-sac to posterior vaginal wall; the pneumoperitoneum is maintained with a sponge in the vagina or a peritoneal elevator - either a Laparofan or Maher metal elevator. The posterior vaginal wall is closed either vaginally or laparoscopically. Endometriotic nodules infiltrating the anterior rectal muscularis are excised, usually with the surgeon or an assistant's finger in the rectum just beneath the lesion. Deep rectal muscularis defects are always closed with suture. A 3/0 or 4/0 absorbable suture is tied outside the peritoneal cavity and then slipped downward. At the close of each procedure, the peritoneal cavity is irrigated extensively with lactated Ringer's solution. Rectal integrity is checked by a Betadine enema and sigmoidoscopy is done to check for narrowing. The results of laparoscopic dissection of the pouch of Douglas are good both in subsequent pregnancy (74%) (Reich et al, 1991) and in relief of coital and rectal pain (92%) (Wood et al., 1993a). If the endometriosis has penetrated deeply into the rectal walls, a fullthickness repair of the rectal wall is made using 3/0 absorbable sutures. These sutures can be placed at laparoscopy or at laparotomy. If there is extensive involvement of the rectal wall, anterior resection of the rectum may be required. This may be facilitated by laparoscopic rectal dissection and mobilization, the resection being done by Endo EEA stapling of the bowel, or by laparotomy. If the lesion is extensive and low in the rectum, after rectal mobilization the nodule may be prolapsed through the anus and a resection done external to the peritoneum. Reich has successfully performed a rectal resection laparoscopically for endometriosis using an Endo EEA staple, plac-

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ing ties around the rectum to suture the staple rod in place (Reich, personal communication). The operative advantages of a laparoscopic approach to cul-de-sac obliteration are easy intraoperative access to the rectum and vagina and a magnification source that is easier to manipulate than an operating microscope. Obliteration of the cul-de-sac secondary to endometriosis can be treated effectively laparoscopically by cul-de-sac dissection with excision of deep fibrotic endometriosis and restoration of cul-de-sac anatomy, resulting in the resolution of infertility and pelvic pain. The benefits to the patient are the avoidance of either major abdominal surgery, with its related morbidity, or ovarian suppressive therapy, which prohibits fertility during its administration and does not appear to penetrate deep infiltrating endometriotic lesions. Adenomyosis Hysterectomy is the usual treatment offered to patients with a suspected diagnosis of adenomyosis, which is based on the combination of menorrhagia, dysmenorrhoea and an enlarged uterus in the absence of fibromyomata. The clinical diagnosis of adenomyosis may be unreliable when compared to the histological diagnosis in excised uteri. Vaginal ultrasound and percutaneous or transcervical myometrial biopsy enable the preoperative diagnosis of adenomyosis (Wood et al., 1993b). Conservative surgery, endometrial resection, myometrial reduction by electrocautery, excision of localized areas of adenomyosis, or wedge resection of extensive areas of myometrium may enable palliative or curative treatment without hysterectomy. Diagnosis

Vaginal ultrasound may show mottled areas in the myometrium. This is a non-specific sign and may also be associated with myomatosis or no histological abnormality. Colour Doppler may show areas of reduced bloodflowin the presence of adenomyosis (Wood, Hurley et al., 1993). Percutaneous myometrial biopsy has been performed as an outpatient procedure under ultrasound control. Local anaesthesia is used in the abdominal wall above the symphysis pubis and a biopsy gun with an attached biopsy needle is fired into myometrial areas showing mottling, reduced blood flow or a discrete mass. Up to three biopsies are taken. Apart from some pain, no complications have occurred (Wood, Hurley et al., 1993). Transcervical myometrial biopsy is performed at the completion of endometrial resection with an electrocautery loop used with cutting current of

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100 W. Bleeding is controlled by coagulation 70 W using a loop or ball electrode. Myometrial biopsy can be done routinely at the time of endometrial resection. The presence of adenomyosis may alter the prognosis for symptom relief after surgery and also warns the patient of possible future menstrual symptoms.

Laparoscopic reduction of adenomyosis Laparoscopic myometrial reduction has been achieved by electrocautery using a Corson needle electrode, 50 W coagulation at 1-3 cm depth (Wood, Hurley et al, 1993). The procedure is assisted by the preliminary injection of a 1 in 50 dilution of vasopressin into the myometrium. Patients with localized changes on ultrasound or the external appearance of abnormal areas of myometrium at laparoscopy may be suitable for the procedure. Because of the uncertain effects of leaving large areas of necrotic myometrium which may release toxic substances into the circulation, areas treated have been limited to 5 x 3 cm. Laparoscopic excision of adenomyosis or adenomyoma Adenomyoma can be excised by laparoscopy as the abnormal area of myometrium can be denned by a change of contour, consistency or colour of the myometrium. The border between normal and abnormal myometrium may be irregular, with close tissue connection, and a capsule is usually absent. Excision is more difficult and slower than removing a myoma. A spoon electrode with cutting 100 W and coagulation 70 W, Endoshear scissors with an attached monopolar electrode and bipolar forceps may all be used to remove the abnormal area. Ultrasound-generated vibrational energy may also be used and has the advantage of the absence of smoke. Vasopressin may be useful to reduce bleeding, which is not usually severe. A myoma screw, 5 or 10 mm, enables manipulation of the adenomyoma during dissection. The adenomyoma cavity is closed by sutures. The irregularity of the base of the cavity makes suture more difficult and although large adenomyomas (greater than 6 cm) have been removed laparoscopically, repair of the cavity may be more easily achieved by minilaparotomy. When present, diffuse adenomyosis may involve nearly all of the uterus. Two patients have requested uterine conservation when the uterus was 16 cm and 20 cm in length. Laparoscopic excision of the abnormal areas was abandoned after three hours because of persistent bleeding, the slowness of the dissection, and the uncertainty that any uterus would be conserved. The operations were completed by laparotomy, when 80-90% of the myometrium was excised and the uterus repaired.

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Laparoscopic wedge excision of adenomyotic areas has been completed successfully when the uterine size is 12 cm or less. Endometrial resection In patients with dysmenorrhoea, menorrhagia and abnormal areas of myometrium on ultrasound, endometrial resection has been performed if the predominant symptom is menorrhagia. Myometrial biopsy is performed to confirm the diagnosis of adenomyosis. An electrocautery loop electrode has been used to remove up to 1 cm of myometrium, where adenomyosis is most severe, but this may increase the risk of bleeding and uterine perforation. The suspected or proven diagnosis of adenomyosis prior to surgery enables the discussion of alternative treatments: endometrial resection, myometrial reduction by cautery or laser, myometrial excision, or hysterectomy. Until larger numbers of patients with adenomyosis have been treated by endometrial resection, myometrial reduction and myometrial excision, the success rates of these procedures cannot be accurately estimated. Loffer agrees that the success rate of endometrial resection is less in women with adenomyosis (Loffer, 1988). Because endometrial resection is so simple a procedure compared to hysterectomy, many patients would wish to try it before hysterectomy, even if the success rate is only 50%. Adenomyosis reduction by electrocautery requires further exploration. The procedure is simple and the risk of bleeding and adhesions may be less than with excisional surgery. Excisional surgery is simple in the presence of adenomyomas as the diseased area is readily identified. Preoperative use of danocrine or GnRH analogues may reduce the uterine blood flow and bleeding at the time of surgery. Vaginal ultrasound and myometrial biopsy are important initial investigations in developing new strategies for treating adenomyosis. Improvement of the conservative surgical procedures for adenomyosis may further reduce the need for hysterectomy in this condition. Fibromyomas

Approximately 30% of women with myomas have been reported to have menstrual abnormalities (Vollenhoven, Lawrence and Healy, 1990). Prior to the advent of endometrial ablation, myomas accounted for 21.7% of the 30 000 hysterectomies performed in Australia in 1988. Fibroids are more common in obese women who show a high conversion of androgens to oestrogens in peripheral adipose tissue. They have a lower incidence in women who take an oral contraceptive and also in those who

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smoke cigarettes (Ross, Pike and Vessey, 1986). Although the oral contraceptive pill induces an oestrogenic state, it is constant, and because follicular growth is suppressed, the normal peak plasma concentrations of oestradiol do not occur. Presumably it is these peak plasma concentrations of oestradiol which are important in stimulating fibroid growth. Cigarette smoking is known to be potentially anti-oestrogenic (Goldman and Cramer, 1990). Furthermore, there is an increased risk of fibroids with increasing parity (Lumsden, West and Baird, 1987). Submucous fibroids frequently cause distortion of the uterine cavity and this is assumed to be the cause of the menstrual problems and infertility. Only 40% of women having hysterectomy to remove a fibroid uterus with the subjective complaint of menorrhagia had submucous fibroids diagnosed at operation (Lumsden et al., 1987; 1989; West et al., 1987). It now seems clear that heavy menstrual loss may be associated with fibroids at any site. If a woman wishes to maintain fertility, myomectomy is the surgical treatment of choice, and may be performed by laparotomy or laparoscopy. A survey of the literature suggests that 80% of patients so treated achieve symptomatic relief, which supports the concept that fibroids cause menstrual problems (Buttram and Reiter, 1981). Further support for this idea comes from a recent study of hysteroscopic myomectomy where the surrounding endometrium was left intact (Derman, Rehnstrom and Neuwirth, 1991). Menorrhagia was relieved in over 80% of subjects. In a small study of hysteroscopic myomectomy in which ten women had their fibroids partially removed (only the portion protruding into the cavity was removed), success was claimed in eight cases, the remaining two requiring conventional myomectomy (Michlewitz and Reindollar, 1988). It is not necessary to remove all the fibroid tissue to relieve menorrhagia and it also suggests that distortion of the uterine cavity is a vital factor. GnRH agonists Gonadotrophin-releasing hormone agonists induce hypo-oestrogenism and a decrease in fibroid size (Filicori, Hall and Loughlin, 1983; West, Lumsden and Lawson, 1987). Endometrial atrophy also occurs, leading to amenorrhoea, which is beneficial in anaemic women as a significant rise in haemoglobin occurs (Candiani, Vercellini and Fedele, 1990). Regrowth of fibroids occurs after cessation of treatment, and in those with dysfunctional uterine bleeding, menorrhagia recurs rapidly (Shaw and Fraser, 1984). However, in spite of this, a significant number of women claim a 'carry over' benefit of this treatment and many are able to avoid hysterectomy, particularly in the older age group

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(West, Lumsden and Baird, 1992). The principal disadvantage of the GnRH agonist is the presence of postmenopausal side-effects which occur to some extent in the majority of treated women. They may be relieved partially by the addition of other hormonal preparations. When goserelin is given alone for three months and medroxyprogesterone acetate (MPA) is then added in combination with it, the initial fibroid shrinkage is maintained (Friedman, Barbieri and Doubilet, 1988). Preoperative GnRH analogues reduce menorrhagia, restore haemoglobin level, allow elective surgery, permit the patient to bank her own blood, reduce the size of incisions, may convert open procedures to laparoscopic or vaginal, reduce arterial blood flow to the myoma, reduce operative blood loss and may facilitate myoma removal (Lumsden et al, 1987; West et al, 1987; Candiani et al, 1990; West et al., 1992). A bdominal myomectomy Myomectomy has been performed via an abdominal incision for many years. Since myomas are often multiple and can be deep in the myometrium, they can be difficult to reach and remove through one incision (Maheux, 1989). Some are too large to be removed easily. Major technical concerns relating to the procedure are minimization of blood loss, prevention of postoperative adhesions (which can impact on fertility) and accurate reconstruction of uterine anatomy (Benagiano, Morini and Primiero, 1992). Myomas can recur after myomectomy. A recurrence rate of 15% has been reported based on several studies, with 10% of patients requiring subsequent treatment (Buttram and Reiter, 1981). A ten-year study reported a recurrence rate of 27% over this period (Candiani et al, 1991). Both genetic predisposition and undetected, small myomas have been suggested as reasons for the appearance of myomas after myomectomy (Candiani et al, 1991). For those women who wish to preserve their fertility, myomectomy is an alternative to hysterectomy in the treatment of myomas. The procedure is suggested in women who wish to delay conception and have a myomatous uterus of 12 weeks' gestational size or larger, or one that is growing rapidly. Abdominal myomectomy is not performed in the majority of women with myomas, due to increased technical difficulty compared with hysterectomy and the possibility of myoma recurrence. Blood loss and morbidity are higher after myomectomy than after hysterectomy (Gardner and Shaw, 1992). A planned myomectomy may be changed to hysterectomy if there is a very large number of myomas, the myoma cannot be removed because of the absence of a capsule preventing easy enucleation, excessive bleeding occurs at

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myomectomy, myosarcoma is suspected, or myomectomy involves the removal of the endometrium. The ratio of hysterectomy to myomectomy in women with fibroids is 5:1 in Australia. Myomas may contribute to infertility in some cases. Pregnancy rates of 40-59% following myomectomy have been reported (Buttram and Reiter, 1981; Starks, 1988; Smith and Uhlir, 1990). Neither age of the patient nor number and size of myomas removed appears to influence postoperative pregnancy rates. Other than those already mentioned, complications of abdominal myomectomy are those of any abdominal surgery. Little information is available about its morbidity and mortality. Average lengths of hospital stay of four to five days for myomectomy have been reported as being consistent with most laparotomies (Smith and Uhlir, 1990; Hutchins, 1992). Laparoscopic myomectomy Laparoscopic myomectomy has been used mainly for pedunculated and subserous fibroids (Table 15.4). Subserous fibroids may be removed by coagulating the pedicle with bipolar or monopolar electrosurgery and transecting it with scissors. Intramuralfibroidsmay be removed by incising the capsule with a monopolar hook or spoon electrode, enucleating the fibroid by grasping it with forceps and peeling it out of its bed, then dissecting it with forceps and scissors. The myoma is stabilized by a myoma screw. Bleeding points may then be closed, as in normal open myomectomy, by one or two layers of 3/0 sutures or one absorbable suture, tying the knots inside the abdomen with forceps or by the extracorporeal knotting technique. The fibroid should be removed from the abdomen by enlarging the umbilical or suprapubic incision, by posterior colpotomy or by morcellation with a tissue punch. Alternatively, the fibroid may be pulled up against the abdominal wall with forceps and cut into several pieces with a scalpel inserted after enlarging the incision to allow removal through a small incision (minilaparotomy) or through an 11-mm diameter cannula. McLucas (1991) has described a combined laparoscopic and vaginal approach to posterior wall fibroids. The fibroid was prepared for myomectomy laparoscopically and the operation completed under direct vision through a colpotomy incision. Dubuisson (1991) presented a series of 58 patients who underwent myomectomy between January 1990 and June 1991, 51 by laparoscopic surgery. He used sutures in 34 cases, and of the seven patients on whom he performed

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Table 15.4. Results of studies on laparoscopic myomectomy

Characteristics Number of patients in study* Number of myomas removed Further treatment (%) hysterectomy Complications (%) haemorrhage other Operating time (minutes) Average length of hospital stay (days)

Dubuisson etal. (1991)§

Daniell anc1 Nezhat et al. Gurley(1991)§(1991)

Goldfarb (1992)

43

17

154

75

92

>20

347

t

n.a.

0.0

0 0 25-240

0 0 30-180

2.8

<1.0

1.3

0.0

0.6 5.2J 50-190

0 1.3 25-30

0.8

n.a.

*Follow-up varies within as well as between studies, from 2 months to 3 years. Rather than being resected, myomas were pre-operatively shrunk by drug treatment, then coagulated with a Nd: YAG laser. Total shrinkage was 50-90%. •Hysterectomy in one patient. §Cited in Hirsch (1993). Adapted from Hirsch (1993) Technologies for the Treatment of Menorrhagia and Uterine Myomas. Health Technology Series, Australian Institute of Health and Welfare, with permission. f

second-look laparoscopy, there were adhesions in only one. No patient had complications and the average hospital stay was 2.8 days. Nezhat and colleagues (Nezhat, Nezhat and Silfen, 1991) reported 154 patients having laparoscopic myomectomy, one patient requiring postoperative laparotomy. Indentations of the uterus were noticed when sutures were not used. They then used a combined laparoscopy-minilaparotomy for the large myomas, which reduces operating time, facilitates enucleation of the myoma and suturing, and reduces blood loss. Results were comparable to those achieved by open myomectomy or laparoscopic myomectomy. Adhesions are common after myomectomy, and Hyskon and Interceed have not been found to be effective when compared to no anti-adhesive regime (Surrey, 1993). Suturing Goretex membrane over the scar has proven effective when compared to controls (Tulandi, Murphy and Rock, 1993). The Goretex membrane is removed by laparoscopy six weeks after surgery. Laparoscopic-minilaparotomy has been used to speed surgery and ensure firm suture of the myoma cavity. It enables the use of an abdominal elevator and a 3-cm incision in the low abdomen (Wood and Maher, 1993).

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Laparoscopic myoma reduction Goldfarb (Goldfarb, 1992) and Wood et al. have used laser and electrosurgery to reduce myomas laparoscopically without removing them. This may avoid two of the major hazards of myomectomy, excessive blood loss and postoperative adhesions. Myomectomy may be associated with significant blood loss (500 ml on average), the risk of blood transfusion (41%), or the need to proceed to undesired hysterectomy because of difficulty controlling haemorrhage (Gardner and Shaw, 1992). Nezhat demonstrated few adhesions: 3% when the myoma cavity was not sutured after laparoscopic myomectomy, compared to 45% when the cavity was sutured (Dubuisson, 1991). Adhesions may occur in 40% or more of patients having myomectomy (Buttram and Reiter, 1981). The absence of sutures may lead to indentations and weakness of the uterine scar (Nezhat et al., 1991). The frequency of conception in 1143 women wanting children after myomectomy was only 40%, suggesting postoperative adhesion formation may be one factor limiting their fertility (Buttram and Reiter, 1981). Hysteroscopy is done prior to myoma reduction to exclude the presence of intracavity myomas. The operation is done by laparoscopy, one accessory incision in the lower abdomen being for a myoma screw holding the myoma and another accessory incision being for the Corson or Goldfarb bipolar electrocautery needle and the suction irrigator. The myoma is first injected with 1 in 50 vasopressin and the myoma electrocoagulated using a 50-W coagulation current. The needle is inserted deep into the myoma at 1-cm intervals over its surface. Blanching occurs up to 1 cm from the point of needle insertion. The myometrium outside the myoma capsule is also coagulated. One or two litres of a buffered solution, Ringer's or Hartmann's, are left in the peritoneal cavity at the completion of the operation. Myomas between 2 and 8 cm have been obliterated by electrocoagulation (Tulandi, Murphy and Rock, 1993). The average reduction in diameter is about 50%. The absence of bleeding and adhesions at the electrocoagulation site suggests the method may have advantages when compared to myomectomy. All the myomas successfully treated were both subserous and intramural. Myomas unsuccessfully treated are often intramural. It may be difficult to target myomas which are not protruding from the uterine surface. The scar formed at the surgical site of electrocoagulation appeared healthy and myometrial thickness was not reduced. However, the strength of the scar cannot be estimated.

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Further testing of electrocoagulation in the treatment of subserous myomas up to 8 cm in diameter is suggested. The possible advantages may be the reduction in blood loss, the decreased chance of blood transfusion, the lower risk of conversion to hysterectomy and postoperative adhesion formation, and maintenance of fertility. Endoscopy and assisted reproductive technology Role of endoscopy in avoiding ART

The role of minimally invasive surgery in avoiding ART was demonstrated in 275 patients treated between 1990 and 1991 who presented with primary or secondary infertility. Forty-five percent achieved pregnancy as a result of endoscopic surgery to treat endometriosis, ovarian disease or pelvic adhesions, and intrauterine synechiae, fibroids, or polyps. Forty percent of patients proceeded to ovulation induction or ART, and the remaining patients had laparotomy surgery, abandoned treatment, or conceived spontaneously. Laparoscopy in the selection of patients for ART

Laparoscopy is of use in the selection of patients for IVF, GIFT, or microsurgery. Patients who have significant tubal disease are selected for IVF. Patients with prolonged infertility and with a normal tube, particularly if the lining is checked by falloposcopy, are suitable for GIFT. Patients who have had two or three failed GIFT treatments may have a review falloposcopy to determine whether minor tubal damage has reduced their chance of succeeding by this method. Minor tubal changes, such as reduction of vascularity and fine adhesions, may be compatible with gamete or embryo transport. Treatment of such patients by the GIFT programme, without acting on the information obtained by falloposcopy, may determine the significance of such changes, either in preventing conception or increasing the risk of ectopic pregnancy. Some patients who have previously had no known cause for their infertility have been satisfied to discover after falloposcopy that they have tubal disease and may be better suited to IVF. Combined GIFT and infertility surgery

In most cases, laparoscopic GIFT is carried out separately from diagnostic or operative laparoscopy; the suitability and possible need for operative procedures preceding the definitive laparoscopy for GIFT. Combining diagnostic and operative endoscopy with the GIFT procedure has the advantages of avoiding one or two laparoscopies, reducing the duration of infertility, and

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reducing patients' costs, stress, and time off work. The possible disadvantages include the reduction of pregnancy success rates, the uncertain timing of oocyte pick-up for patients having the GIFT procedure after a combined operative GIFT laparoscopy, and the need for the surgeon to have more diverse endoscopy skills. Grindoff and colleagues (1990) explored the combined use of laparoscopy for diagnostic, operative and GIFT procedures in 32 women. In 13 women the diagnostic procedure led to immediate GIFT or IVF. In the remaining 19 women, adhesiolysis or electrocautery for endometriosis was carried out before the GIFT procedure. The pregnancy rate was the same in the operative group - 5 (26%) out of 19 - as in the non-operative group - 4 (31%) out of 13. The pregnancy rates were identical for the IVF and GIFT procedures. Corson and colleagues (1991) have reached the same conclusion that coincident treatment of endometriosis does not affect the success rate of GIFT. Gamete intrafallopian transfer was combined with adhesiolysis in 208 patients with non-endometriotic adhesions (al-Shawaf et al., 1990a). The pregnancy rate in the 134 patients suitable for adhesiolysis was 38.8%, being similar to the 74 patients not suitable for surgery, 35.6%. The pregnancy rate was lower for those operated on with stage III and stage IV adhesions, and the ectopic pregnancy rate increased 3.5 times in the operative group. It was concluded that GIFT was an option in patients having adhesiolysis, particularly with stage I and stage II adhesions. Gamete intrafallopian transfer has been less successful in association with tubal microsurgery: three pregnancies occurring in 26 patients, of which only one was ongoing (Mardesic et al., 1989). Ectopic pregnancy is more common after previous tubal surgery, occurring in 10 (33%) out of 30 patients (alShawaf et al, 1990b). It may be disadvantageous to perform tubal surgery in association with GIFT, or use GIFT after previous failed tubal surgery. Gamete intrafallopian transfer has been successful in association with fimbriectomy reversal, four out of four patients becoming pregnant (Novy et al., 1991). It is difficult to know whether or not the addition of GIFT is important, as fimbriectomy reversal has an average success rate (22-55%), depending on the width of the ampulla. Immature oocyte collection In-vitro fertilization and GIFT have been performed by vaginal ultrasound needle puncture of small follicles. The usual technique of immature egg collection involves the aspiration of fluid from follicles between 15 and 25 mm in diameter. The techniques for immature egg collection requires aspiration of

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eggs from follicles between 3 and 10 mm (Trounson, Wood and Kansche, 1994). There have been changes in ultrasound sensitivity and needle design to assist oocyte collection from small, immature follicles. Oocytes were recovered using high-resolution vaginal ultrasonography (Acuson, Melbourne, Victoria, Australia) to visualize the small follicles. The aspiration pressure used in the needle was reduced from the usual 15.0 kPa for recovery of mature oocytes to 7.5 kPa as this improved recovery of immature oocytes in preliminary developmental studies. A special needle with increased rigidity, a shortened bevel, and ultrasound stippling close to the tip was used to access the small follicle. The needle was always introduced into the follicle with the bevel facing downwards in order to prevent loss of follicular fluid, which would occur if an outward-facing bevel passed at an angle into the follicle. Needle blockage was common as stroma and blood vessels were traversed more often than in ovaries containing mature follicles. This necessitated multiple flushing of the needle with culture medium. The mobility of the ovary made puncture of small follicles more difficult in some polycystic ovarian syndrome patients, so laparoscopic oocyte recovery was substituted in these circumstances. The ovary was fixed by holding instruments introduced through two 5-mm incisions in the lateral part of the lower abdomen. As follicles are most often superficial in the ovary, small follicles containing immature oocytes appear through the laparoscope as grey-blue areas of 2-5 mm on the surface of the ovary. All the follicles visible by ultrasound were penetrated by the needle and the contents aspirated. Follicles in close vicinity were punctured one after another before removing the needle and aspiration of pH-buffered and heparinized culture medium to clear the follicular contents from the needle and attached tubing. The needle was reintroduced into the ovary to recover follicular contents from another sector of the ovary and the procedure was repeated until all the follicles were needled. The follicular aspirates were sent to a laboratory where the eggs were identified and matured. If this technique can be applied to all patients, stimulation of the ovaries with the associated side-effects and complications may be avoided in IVF patients. Only a few babies have resulted from this technique, which requires further development. Laparoscopic treatment of ectopic pregnancy following ART Ectopic pregnancy occurs in 5% of patients having GIFT or IVF. The use of sensitive vaginal ultrasound and quantitative assessment of plasma hCG have allowed early diagnosis and almost universal treatment of ectopic pregnancy

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by operative laparoscopy. Salpingotomy and salpingectomy are the most common procedures required. The presence of tubal rupture or an interstitial pregnancy was thought to contraindicate the use of the laparoscopic method. Improvement in suction irrigation and the use of vasopressin and reliable bipolar endocoagulation have allowed the treatment of tubal rupture or an interstitial tubal ectopic pregnancy. It is not certain if the use of methotrexate will replace the endoscopic treatment of ectopic pregnancy. Methotrexate does avoid anaesthesia and laparoscopy, and the results are similar to those of the laparoscopic method. It has been shown to be equally effective to laparoscopic surgery in a prospective, randomized, controlled trial, and also may result in considerable cost savings in 30% of patients judged suitable for its use (Croinin and Washington, 1993). Methotrexate may be less effective when the plasma hCG levels are higher. Although there is no evidence that methotrexate has long-term effects on the oocytes, there are still some concerns expressed about its possible long-term effects.

References al-Shawaf, T., Ah-Moye, M., Fiamanya, W., Smith, B. and Craft, I. 1990a. Gamete intra-fallopian transfer in non-endometriotic pelvic adhesions. Human Reproduction 5: 434-8. al-Shawaf, T., Serhal, P., Fiamanya, W., Harper, J. and Craft, I. 1990b. Transfallopian tube embryo transfer: successful pregnancy outcome. Journal of In Vitro Fertilization and Embryo Transfer!: 337-40. Benagiano, G., Morini, A. and Primiero, F.M. 1992. Fibroids: overview of current and future treatment options. British Journal of Obstetrics and Gynaecology 99 (Supplement 7): 18-22. Brosens, I. 1993. Endo-ovarian surgery for ovarian endometriomas. In Endoscopic Surgery for Gynaecologists, ed. C. Sutton and M. Diamond, pp. 142-6. London: Saunders. Buttram, V.C. and Reiter, R.C. 1981. Uterine leiomyomata: etiology, symptomatology and management. Fertility and Sterility 36: 433-45. Candiani, G.B., Fedele, L. and Parazzini, F. 1991. Risk of recurrence after myomectomy. British Journal of Obstetrics and Gynaecology 98: 385-9. Candiani, G.B., Vercellini, P. and Fedele, L. 1990. Use of goserelin depot, a gonadotropin-releasing hormone agonist, in the treatment of menorrhagia and severe anaemia in women with leiomyomata uteri. Acta Obstetrica et Gynaecologica Scandinavica 69: 413-15. Canis, M. 1993. Surgery of the Ovary. Third Meeting of the International Society of Gynaecological Endoscopists, June 24-6, Washington. Chatman, D.L. and Zbella, E.A. 1986. Pelvic peritoneal defects and endometriosis: future observations. Fertility and Sterility 46: 711-14. Cook, A.S. and Rock, J.A. 1993. Diagnosis of endometriosis: laparoscopic appearances. In Endoscopic Surgery for Gynaecologists, ed. C. Sutton and M. Diamond, p. 202. London: Saunders. Corson, S.L., Batzer, F.R., Gocial, B., Daly, D.C., Eisenberg, E., Huppert, L.C. and

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Maislin, G. 1991. Surgical treatment of endometriosis at the time of gamete intrafallopian transfer. Journal of Reproductive Medicine 36: 274-8. Croinin, M.D. and Washington, A.E. 1993. Cost of ectopic pregnancy management, surgery versus methotrexate. Fertility and Sterility 60: 963-9. Derman, S.G., Rehnstrom, J. and Neuwirth, R.S. 1991. The long term effectiveness of hysteroscopic treatment of menorrhagia and leiomyomas. Obstetrics and Gynecology 77: 592^4. Donnez, J., Nissole, M. and Wayembergh, M. 1989. CO2 laser laparoscopy in peritoneal endometriosis and on ovarian endometrial cysts. In Operative Laparoscopy and Hysteroscopy, ed. J. Donnez, pp. 53-78. Leuven: Nauweberts. Downing, B. and Wood, C. 1995. Initial experience with a new microlaparoscope 2mm in external diameter. Australian and New Zealand Journal of Gynaecology. Dubuisson, J.B. 1991. Laparoscopic Myomectomy. Presented at the X Journees Aquitaines de Perfectionnement en Reproduction Humaine, Bordeaux. Filicori, M., Hall, D.A. and Loughlin, J.S. 1983. A conservative approach to the management of uterine leiomyomata: pituitary desensitisation by a luteinizing hormone-releasing hormone analogue. American Journal of Obstetrics and Gynecology 147: 726-7. Friedman, A.J., Barbieri, R.L. and Doubilet, P.M. 1988. A randomised double-blind trial of gonadotropin releasing hormone agonist (leuprolide) with or without medroxyprogesterone acetate in the treatment of leiomyomata uteri. Fertility and Sterility 49: 404-9. Gardner, R.L. and Shaw, R.L. 1992. GnRH agonists and blood loss at surgery. In Uterine Fibroids. A Time for Review, ed. R. Shaw, pp. 123-34. Carnforth, UK: Parthenon. Goldfarb, H.A. 1992. Nd YAG laser laparoscopic coagulation of symptomatic myomas. Journal of Reproductive Medicine 37: 636-8. Goldman, M.B. and Cramer, D.W. 1990. The epidemiology of endometriosis. In Current Concepts in Endometriosis, ed. D.R. Chadra and V.C. Buttram, pp. 00-00. New York: Alan R Liss. Gomel, V. 1977. Salpingostomy by laparoscopy. Journal of Reproductive Medicine 18: 265-7. Gomel, V. and Taylor, P.J. 1992. In vitro fertilization versus reconstructive tubal surgery. Journal of Assisted Reproduction and Genetics 9: 306-09. Grindoff, P.R., Hall, J.L., Nelson, L.M. and Stillman, R.J. 1990. Efficacy of assisted reproductive technology during diagnostic and operative infertility laparoscopy. Obstetrics and Gynecology 75: 299-301. Hudson, B., Baker, H.W.G. and de Kretser, D.M. 1980. The abnormal semen sample. In The Infertile Couple, ed. R.J. Pepperell, B. Hudson and C. Wood, p. 93. Edinburgh: Churchill Livingstone. Hutchins, F.L. 1992. Myomectomy after selective preoperative treatment with a gonadotropin releasing hormone analogue. Journal of Reproductive Medicine 37: 699-702. Jansen, R.P.S. and Russel, P. 1986. Non pigmental endometriosis: clinical, laparoscopic and pathological distribution. American Journal of Obstetrics and Gynecology 155: 1154-9. Keye, W.R., Hanso, L.W. and Astin, M. 1987. Argon laser therapy of endometriosis: a review of 92 consecutive patients. Fertility and Sterility 47: 208-12. Koninckx, P.R., Mueleman, C. and Demeyere, S. 1991. Suggestive evidence that pelvic endometriosis is a progressive disease whereas deeply infiltrating endomet-

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riosis is associated with pelvic pain. Fertility and Sterility 55: 759-65. Loffer, F. 1988. Laser ablation of the endometrium. Obstetrics and Gynecology Clinics of North America 15: 77-89. Lumsden, M.A., West, C.P. and Baird, D.T. 1987. Goserelin therapy before surgery for fibroids. Lancet 1: 36-7. Lumsden, M.A., West, C.P., Hillier, H. and Baird, D.T. 1989. Tamoxifen acts as an oestrogen agonist in women treated by LHRH agonist (Zoladex) and prevents shrinkage of uterine fibroids. Fertility and Sterility 52: 924-9. Maheux, R. 1989. Treatment of uterine leiomyomata: past, present and future. Hormone Research 39 (Supplement 1): 125-33. Mardesic, T., Laitl, J., Jirasek, J.E., Stroufova, A. and Presl, J. 1989. Gamete intrafallopian transfer (GIFT) as a complement to tubal microsurgery. Zentralblat Gyndkologie 111: 276-80. Martin, D.C., Armic, R. and El Sehg, F.A. 1990. Histologic diagnosis of endometriosis. Journal of Gynecologic Surgery 6: 275-9. Martin, D.C., Hubert, C.P., Vander Zwagg, R. and El Zekg, F.A. 1989. Laparoscopic appearances of peritoneal endometriosis. Fertility and Sterility 51: 63-7. McLucas, B. 1991. Myomectomy via Colpotomy with Laparoscopic Assistance. Presented at the II Biennial Meeting of the International Society for Gynaecologic Endoscopy, Barcelona. Michlewitz, H. and Reindollar, R.H. 1988. Hysteroscopic myomectomy using hysteroscopic guidance. Proceedings of the XII World Congress of Gynaecology and Obstetrics, Rio de Janeiro, Abstract, p. 661. Congress Press. Murphy, A.A., Green, W.R. and Babbie, D. 1986. Unsuspected endometriosis diagnosed by scanning electron microscope in visually normal peritoneum. Fertility and Sterility 46: 522-4. Nezhat, C, Cromgey, S. and Nezhat, F. 1989. Videolaseroscopy for the treatment of endometriosis associated infertility. Fertility and Sterility 51: 237-40. Nezhat, C. and Nezhat, F. 1991. Postoperative adhesion formation after ovarian cystectomy. Abstracts of American Fertility Society Meeting 0-012, p. 86. Washington, DC. Nezhat, C, Nezhat, F. and Silfen, S.L. 1991. Laparoscopic myomectomy. International Journal of Fertility 36: 275-80. Nissole, M., Caspanas-Roux, F. and Donnez, J. 1988. Histologic study of ovarian endometriosis after hormonal therapy. Fertility and Sterility 49: 423-6. Novy, M.J., Hickok, L.R., Patton, P.E., Craemer, M.J. and Wolf, D.P. 1991. Pregnancy after fimbriectomy reversal: results of microsurgery augmented by gamete intrafallopian tube transfer and embryo transfer. Fertility and Sterility 56: 1166-8. Nowroozi, K., Chase, J.S., Check, J.N. and Wu, C.H. 1987. The importance of laparoscopic coagulation of mild endometriosis in infertile women. International Journal of Fertility 32: 442—4.

Olive, D.L. and Martin, D.C. 1987. Treatment of endometriosis associated with infertility with CO2 laser laparoscopy: the use of one and two parameter exponential models. Fertility and Sterility 48: 18-23. Redwine, D.B. 1987. Age related evaluation in colour appearance of endometriosis. Fertility and Sterility 48: 1062-3. Redwine, D.B. 1991. Laparoscopic excision of endometriosis (LAPEX) by sharp dissection. In Laparoscopic Appearance of Endometriosis, 2nd edn, Vol. I, ed. D.L. Martin, pp. 9-19. Memphis: Rescuge Press.

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Redwine, D. 1993. Non laser reduction of endometriosis. In Endoscopic Surgery for Gynaecologists, ed. C. Sutton and M. Diamond, pp. 220-8. London: Saunders. Reich, H., McGlynn, F. and Salvat, J. 1991. Laparoscopic treatment of cul-de-sac obliteration secondary to retrocervical deep fibrotic endometriosis. Journal of Reproductive Medicine 36: 516-22. Ross, P.K., Pike, M.C. and Vessey, M.P. 1986. Risk factors for uterine fibroids: reduced risk associated with oral contraceptives. British Medical Journal 293: 359-63. Seiler, J.C., Cigwani, C. and Ballard, L. 1986. Laparoscopic cauterization of endometriosis for infertility: a controlled study. Fertility and Sterility 46: 1098-100. Shaw, R.W. and Fraser, H.M. 1984. Use of a superactive luteinizing hormonereleasing hormone (LHRH) agonist in the treatment of menorrhagia. British Journal of Obstetrics and Gynaecology 91:913-16. Smith, D.C. and Uhlir, J.K. 1990. Myomectomy as a reproductive procedure. American Journal of Obstetrics and Gynecology 162: 1476-82. Starks, G.C. 1988. CO? laser myomectomy in an infertile population. Journal of Reproductive Medicine 33: 184-6. Surrey, C. 1993. Adhesive Formation after Myomectomy. Abstract of Biennial Meeting of International Society of Gynecologic Endoscopists, Washington, DC. Trounson, A., Wood, C. and Kansche, A. 1994. In vitro maturation of human oocytes recovered by ultrasonic follicular aspiration from unstimulated anovulatory and ovulatory polycystic ovarian (PCO) patients and their fertilization and developmental capacity including a pregnancy after embryo transfer. Fertility and Sterility 62: 353-62. Tulandi, T., Collins, J.A. and Burrows, E. 1990. Treatment-dependent and treatmentindependent pregnancy among women with periadnexal adhesions. American Journal of Obstetrics and Gynecology 162: 354—7. Tulandi, T. and Mouchawar, M. 1991. Treatment dependent and treatment independent pregnancy among women with minimal and mild endometriosis. Fertility and Sterility 56: 790-1. Tulandi, T., Murphy, A.A. and Rock, J.A. 1993. Effects of Gore-tex surgical membrane in post myomectomy adhesions. Abstracts of the American Fertility Society Meeting, Montreal, Canada, p. 178. Vollenhoven, B.J., Lawrence, A.S. and Healy, D.L. 1990. Uterine fibroids: a clinical review. British Journal of Obstetrics and Gynaecology 97: 285-98. West, C.P., Lumsden, M.A. and Baird, J.T. 1992. Goserelin (Zoladex) in the treatment of fibroids. British Journal of Obstetrics and Gynaecology 99 (Supplement 7): 27-30. West, C.P., Lumsden, M.A. and Lawson, S. 1987. Shrinkage of uterinefibroidsduring therapy with goserelin (Zoladex): a luteinizing hormone-releasing hormone agonist administered as a monthly subcutaneous depot. Fertility and Sterility 48: 45-51. Wood, C, Hurley, V., Fortune, D. and Leoni, M. 1993. Percutaneous ultrasound guided needle uterine biopsy. Medical Journal of Australia 158(7): 458-60. Wood, C. and Maher, P. 1993. Laparoscopic removal of endometrioma attached to the ureter. Australian and New Zealand Journal of Surgery 63: 735-6. Wood, C, Maher, P. and Hill, D. 1992. Diagnosis and surgical management of endometriomas. Australian and New Zealand Journal of Obstetrics and Gynaecology^!): 161-3. Wood, C, Maher, P. and Hill, D. 1993a. Laparoscopy removal of endometriosis in

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the Pouch of Douglas. Australian and New Zealand Journal of Obstetrics and Gynaecology 33(3): 295-9. Wood, C , Maher, P. and Hill, D. 1993b. Biopsy diagnosis and conservative surgical treatment of adenomyosis. Australian and New Zealand Journal of Obstetrics and Gynaecology 33(3): 319-21.

16 Laboratory techniques PETER JACKSON and JENNIFER BURDEN

The embryology laboratory is a key component of any in-vitro fertilization programme. It is there thatfirstlythe oocytes and sperm and then the embryos are nurtured during the IVF process. In this chapter the physical requirements, procedures and techniques will be discussed, with mention of the different options as appropriate. The actual process of IVF involves a number of steps: 1. 2. 3. 4. 5. 6. 7.

collection of oocytes preparation of semen samples insemination of the oocytes checking for fertilization culturing the embryos transfer of the embryos embryo cryopreservation and storage.

As a result of its function, the laboratory must be appropriately located, sized and equipped. However, there are a number of different ways of setting up the laboratory. Since the first development of IVF, three methods of providing embryology services have evolved: the static dedicated laboratory, the mobile laboratory, and the transport IVF laboratory. The static dedicated laboratory is usually located within the immediate vicinity of where oocyte collection and embryo transfer are performed. The mobile laboratory is a fully equipped laboratory capable of setting up a service completely independently of the parent unit. The transport IVF laboratory is a combination of the preceding two situations. Oocyte collections are performed at a site distant from the parent laboratory and the oocytes are transported to the main laboratory for the remainder of the IVF process. As well as the main laboratory, a number of support systems are required. 220

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Unless culture media are to be purchased, an appropriate room is required for their preparation. Areas for cleaning surgical and laboratory instruments to a nonembryo toxic standard. A room for sperm production. An area for long-term storage of cryopreserved embryos. A place for data collection and analysis. By their very nature, some of these functions should ideally be performed in separate areas, while others are compatible and can be performed in the same area if need be. Most laboratories have to make some compromises due to space constraints and therefore an outline of the most usual practice will be given. The functions of media preparation and instrument cleaning are usually combined in the same room, or perhaps in adjacent rooms if space is available. If both functions are to be performed in the same room, it should be clearly divided into clean and dirty areas and the separation of work processes strictly adhered to. Sperm production on demand can be stressful, and on occasions the clinical nature of the process can lead to an incomplete ejaculation occurring - or worse still an inability to produce a sample at all. The sperm production room should be just that, not a public toilet or room which may be used for other purposes. The man needs to feel that he is in a secure, private environment. There should be a toilet and hand basin within the room, or at the very least close by. As many couples like to be together at this stage of the treatment, the room should contain a sofa or sofa-bed fitted with removable and washable covers. Video facilities and erotic literature are helpful. The room should be close to the laboratory and equipped with a system for alerting the embryologist that the sperm sample is ready for collection. Some men are happier producing the semen sample in the privacy of their own home. If the semen analysis is normal and sperm antibodies are not present, patients are often allowed to produce outside the hospital, providing they are able to deliver the sample to the laboratory within one hour of production. Long-term embryo storage brings with it the responsibility of looking after a couple's hopes and dreams for (potential) offspring, possibly for extended periods. Monash IVF currently stores something in the order of 2000 embryos. This necessitates that the embryos be kept in a secure location that is easily accessible to the laboratory. The embryos can be stored within the laboratory or in a nearby room. There should be provision for electronic

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monitoring of the liquid nitrogen levels and a system of alerting staff to low liquid nitrogen levels as well as a security alarm. Additionally, manual monitoring should be incorporated into the daily routine. The liquid nitrogen bulk store should also be located close to the laboratories, preferably with easy access provided to enable the supplier to access the bulk liquid nitrogen storage vessel with minimal inconvenience to staff. The majority of the embryologists' work - namely, sperm preparation, in-vitro fertilization, oocyte and embryo culture, micromanipulation, embryo assessment, freezing and thawing - should be performed in an appropriately equipped laboratory. The primary requirement for the embryology laboratory is a clean environment close to the site of oocyte collection and embryo transfer - usually an operating theatre. At Monash IVF there are three laboratories: two static laboratories built as dedicated units within day-surgery complexes, and a highly successful mobile laboratory which services a rural satellite programme. The dedicated laboratories share the filtered air supply to the theatre and the emergency power supply. They are cleaned and serviced as part of the theatre suite. The satellite programme is serviced by a fully equipped mobile laboratory which visits a number of separate sites during the year, all of which have different facilities and physical relationships between theatre and the room used by the mobile laboratory. However, the common theme between all the satellite locations is that the bulk of the non-theatre work (i.e. everything except the oocyte collections and embryo transfers) is performed within horizontal laminar flow cabinets which ensure a clean work environment for the gametes and embryos. Oocyte collection is a team effort, requiring the skills of both surgeon and embryologist, and should preferably be performed in an operating theatre. If general or neurolept anaesthesia is to be used, a theatre will be required with the associated support of nurses and anaesthetic staff. Many units use only local anaesthesia and thus in this situation a theatre is not considered to be an absolute requirement. Whatever the case, it is essential that the embryologist is close to the oocyte collection, preferably in the same room, although communication from an adjacent room via a hatchway is an acceptable alternative. Similarly, the embryologist needs to be close to the patient at the time of the embryo transfer. It is not acceptable for the embryo transfer catheter to be loaded some distance away and carried to the patient because of the potential for cooling of the embryos and pH changes of the medium within the catheter. Oocyte and embryo culture

Culture methods may vary slightly between different clinics, but four factors

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are fundamental to successful culture of human oocytes and embryos: the correct osmolarity, pH and temperature must be maintained at all times and sterility of the culture system ensured through strict adherence to high standards of aseptic technique.

pH The pH must be maintained between 7.2 and 7.4. This is commonly achieved using media containing sodium bicarbonate which is buffered against CO2 by culturing in an atmosphere of 5% CO 2 :5% 02:90% N 2 or 5% CO2 in air. The lower O2 tension employed by using the 5:5:90 gas mixture is useful in reducing the potential for production of excess free oxygen radicals. Indicators such as phenol red are used to monitor the pH. The phenol red can be incorporated into the culture medium, or phenol-red-free media can be used for culture and a phenol red indicator tube or droplet incubated alongside the culture tubes/drops to provide pH monitoring.

Temperature The microtubule structure within the human egg is extremely thermolabile and has been shown to be irreparably damaged by exposure to temperatures below 30°C (Pickering et al., 1990). To assist with temperature maintenance during procedures we have temperature controlled stages on all microscopes and warming plates in the laminar flow cabinets. Incubators should be set to operate within a maximum deviation of half a degree of the set point of 37°C.

Sterility All media are sterilized by filtration through 0.22-micron filters and supplemented with penicillin and streptomycin. Strict aseptic handling technique should be employed at all times. Infections of culture media occasionally occur and can most often be traced to infected semen.

Osmolarity The normal osmolarity for media used in human IVF is 285 mosmol/kg +/- 5 mosmol/kg. Steps should be taken to reduce the loss of water from the culture system by culturing in atmospheres of high relative humidity or in drops under oil.

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Media used for human IVF can be classified into two types: simple and complex. Simple media are balanced salt solutions such as Earles or Tyrodes supplemented with sodium pyruvate as an energy source, a protein constituent, e.g. maternal serum, and antibiotics (penicillin G and streptomycin). They are buffered with sodium bicarbonate and carbon dioxide (5%). These media are in common use throughout the IVF community and give acceptable results in terms of fertilization and pregnancy rates. However, they are not specifically designed for embryological culture. Complex media have also been used and were originally designed to simulate the metabolic requirements of cell cultures within tissue culture laboratories. Some of these were subsequently modified for use in human embryology. These media comprise several components - generally an inorganic salt component, an amino acid component, a vitamin component, an energy component and a buffer component. Other additions such as fatty acids and buffers such as HEPES have also been used. The most important constituent of any culture medium is the water. This can be purchased or prepared on site using water purification systems such as the Millipore system. Whatever its source, the water must be as pure as possible, being free of dissolved organic (especially toxins) and inorganic (especially heavy metals) substances. Arguably, the protein source is the next most important component of the culture medium. Traditionally, maternal serum has generally been used as the protein source but its composition is highly variable, even within the same patient. The ability of proteins to chelate heavy metals can mask toxic effects, leading to subtle damage to the embryo. Serum adds a large variety of unknown compounds to the culture medium, many of which may not actually be beneficial, and completely alters the composition of the medium, making media comparisons difficult. Alternatives to using maternal serum are blood transfusion grade human serum albumin (HSA) or bovine serum albumin (BSA). However, due to the occurrence of bovine spongyform encephalopathy (BSE), it is essential that the supplier's actual source of albumin be identified if BSA is to be used. At the time of writing, the authors understanding is that BSA prepared from bovine serum of New Zealand origin was free of BSE. Culture media should always be checked for final pH (7.2-7.4), osmotic pressure (285 +/-5 mosmol/kg) and presence of endotoxins prior to use. The quality control of media and their constituents is a difficult but pertinent issue. There is currently no single test sufficiently sensitive to evaluate the suitability of media or their constituent compounds intended for use in human embryo culture systems. The mouse embryo culture test (Gardner and Lane, 1993) and

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the sperm survival test are the most common bioassays in use and both can be relatively insensitive to subtle toxic effects and are really only capable of detecting serious defects in culture media. To optimize the sensitivity of the test we use mice embryos and restrict the protein enriched culture period to the one-cell stage. Thereafter, to avoid possible masking effects by protein, the embryos are cultured in protein free media to identify potential blocks that may occur at the two cell stage. Ultimately the most sensitive test of a particular medium's capabilities is its performance under actual human embryo culture conditions. Culture techniques Broadly speaking, there are two culture systems in use. One system utilizes oil (called the closed system) while the other does not (called the open system). Variations in the implementation of both systems have evolved over the years. However, the principles behind and factors for and against each system remain the same. The closed culture system The oil or closed culture system comprises culturing oocytes or embryos in small volumes, such as microdrops (5-100 /A), placed under mineral oil or light liquid paraffin oil, usually in a small petri dish. The petri dish can be incubated either on the incubator shelf or enclosed within a small desiccator. The latter method is preferable in that each petri dish is enclosed within its own vessel, which has beenfloodedwith 5% CO2:5% O2:90% N2 before being sealed, and is therefore not subject to atmospheric fluctuations every time the incubator door is opened, as dishes cultured on the incubator shelf would be. In busy laboratories or where incubators are over-utilized CO2 levels within the incubator can fluctuate markedly and may not reach the correct level during the working day due to continuous door opening. Therefore culturing within sealed vessels, preferably one per patient, is an obvious way of avoiding this common problem. The main function of the oil is to prevent evaporation of the microdrops, thus retaining the correct osmotic pressure, while enabling the oocytes or embryos to be rapidly examined or handled. It also acts as a bacteriostatic interface between the drops and the environment. It does not prevent loss of CO2 from the medium; it merely serves to slow the rate of loss. This can lead to a false sense of security as CO2 is lost first from the oil overlying the medium. The indicator colour of the microdrop does not begin to change until CO2 has diffused from the medium droplet into the oil overlay, which very often

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happens after the dish has been returned to the incubator and of course goes unobserved. Furthermore, once CO2 has been lost from the oil and the medium and the dish have been returned to the correct CO2 atmosphere, the oil then retards the recovery phase of the microdrop by a factor of about 2. Therefore it is essential that the microdrops are under 5% CO2 at all times except when the dish is actually required for use, and that time limits (usually 2 minutes) should be employed to restrict the actual time that the microdrops are at risk. In practice, this is easy to achieve in the hands of experienced embryologists who are able to work at speed. The microdrop/oil system is a very convenient and easy system that facilitates immediate access to the oocytes or embryos. A number of oocytes or embryos (but not all of a patient's ) can be cultured in the same dish and critical microscopic examination of the oocytes or embryos performed rapidly without having to remove them from the culture vessel. As the oil itself can be highly toxic, it is essential that it is obtained from a reputable supplier and carefully tested prior to use. The open culture system Culturing without oil in the open system avoids the potential problems associated with the use of substandard oil, but is generally more cumbersome. In our laboratories, embryos that are not cultured under oil are cultured in 5-ml tubes. The advantages of this system are that each oocyte or embryo is stored in its own tube, that the properly capped tube maintains the correct pH more effectively outside the incubator, and the tubes are easily and safely transported. However, each time an oocyte or embryo is to be manipulated - such as at fertilization check or to critically evaluate embryos - it needs to be removed from the tube. In order to prevent pH changes while the embryos are out of the tube, we use a handling medium containing HEPES buffer. This also enables the embryologist to spend more time evaluating the embryo. There is no bacteriostatic interface between the oocytes or embryos and the atmosphere, although if the correct technique is employed infections from this source should be rare. Needless to say, as oocytes are cultured separately, times for labelling and preparation of the tubes are considerably longer than for dishes where a number of oocytes are cultured together. However, individual cultures offer a high level of security in that only one oocyte or embryo is handled at a time. Due to the larger numbers of sperm used for insemination in the tube (100 000 per tube versus 3000-5000 per microdrop for normal sperm), a pH gradient can form as the sperm settle to the bottom of the tube. Additionally there may be a higher concentration of free oxygen radicals in the tube compared to the microdrops. Open dishes have also been used but are

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Laboratory techniques Follicular phase tracking Ovulation induction (hCG)

Pre OPU

\ Sperm production —•- Sperm preparation \ Oocyte pick-up (OPU) and gamete intrafallopian transfer (GIFT)

Day 0

Insemination

}

Fertilization check

} Day1

\ Embryo culture Embryo transfer \ Embryo cryopreservation

Day 2 or 3

Figure 16.1 An outline of the IVF process. even less satisfactory in that they are highly prone to pH changes and evaporation. It is important to note that both tubes (open system) and microdrops (closed system) have been used successfully and at Monash IVF we employ both techniques to suit specific circumstances. Outline of the IVF process

The work of the embryologist is the culmination of a series of events that were set in train the day the couple presented to their infertility clinician. The details of these events are set out elsewhere in this book and need not be reviewed here. The embryologist's main brief is to identify and take responsibility for the oocytes aspirated by the IVF clinician at the oocyte recovery, with the intention of providing optimal conditions for the creation of embryos which will be returned to the patient or frozen for subsequent use by the patient. The process is summarized in Figure 16.1. Oocyte collection The surgical aspects of the oocyte collection procedure have been detailed elsewhere. Once the embryologist receives the aspirated follicular fluid or follicular flushing, it is transferred to a large petri dish and examined for the

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presence of an oocyte under low-power magnification using a stereomicroscope. In the absence of an oocyte, the presence of granulosa cells indicates that a follicle rather than a cyst has been punctured. Further examination of granulosa cell morphology will enable some preliminary indication of the follicle's state of maturity to be ascertained. Information regarding the maturation status of the follicle should be communicated to the surgeon. At Monash IVF this is achieved in two ways: by having the embryologist isolating the oocytes in the theatre, and by the use of a video camera and monitor positioned to allow the surgeon to observe the contents of the petri dish on the monitor. Once an oocyte is identified this is immediately communicated to the surgeon. While the surgeon moves on to the next follicle, the embryologist removes the oocyte from the follicular fluid using a Pasteur pipette and washes it in clean culture medium buffered with HEPES or non-HEPES-buffered medium kept under a flow of 5% CO2. Once washed, the oocyte is transferred to its culture vessel, either tube or microdrop. Upon completion of the oocyte collection, the oocytes are placed in an incubator and left undisturbed until the time of insemination, usually three to six hours later. If the patient is having GIFT rather than IVF, the oocytes and sperm are loaded into a catheter to be passed into the fimbrial end of the fallopian tube where the gametes will be deposited. Oocyte assessment During the oocyte collection, data regarding the number and quality of oocytes are recorded. An indication of the quality of the oocyte can be ascertained from the appearance of the oocyte cumulus mass (OCM) and the granulosa cells accompanying the oocyte. The oocyte can be graded into the following categories: mature, immature, post mature, atretic, degenerate, damaged. The condition of the cytoplasm can also be noted for factors such as vacuoles, excessive granularity or organelle concentration, cytoplasmic degeneration, zona pellucida thickness and density, as well as mechanical defects caused during the oocyte aspiration or the cumulus removal procedure. Oocytes with expanded cumulus masses can be examined by tilting the dish and allowing the cumulus mass to flatten out or by removing medium from the microdrop, causing the drop to flatten and squashing the cumulus mass, thus exposing the oocyte. However, it is seldom necessary to employ these techniques for routine IVF. If intracytoplasmic sperm injection (ICSI) is to be performed, the oocytes will have their cumulus masses removed using the

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enzyme hyaluronidase. At this stage, an accurate assessment of nuclear maturity can be gained as well as an idea of cytoplasmic condition. This is not usually done for routine IVF. Several workers have developed indirect scoring criteria for evaluation of the oocyte. These are based on the morphology of the cumulus mass and corona radiata cells surrounding the oocyte. Comparisons of actual oocyte maturity between the indirect and direct methods show a high error rate. This was observed after the advent of microsurgical insemination techniques when it became necessary to remove the OCM to facilitate the microsurgical procedure. It has been noted by various workers that many oocytes initially scored as mature based on the OCM morphology are not at the metaphase II stage (Fishel et al, 1990). The majority of non-metaphase II oocytes were at the germinal vesicle breakdown (GVBD) stage, while a small proportion were germinal vesicle (GV) oocytes. In our unit the OCMs of oocytes undergoing ICSI are removed one to two hours after oocyte recovery and the injection is performed two to four hours later. It has been noted that many oocytes scored as GVBD earlier in the day have extruded a polar body by the time of the injection. This equates to roughly the same time that oocytes undergoing normal IVF would be inseminated. So, although the error rate is high for the indirect scoring system, it is adequate for routine IVF purposes where identification of overtly immature, postmature, degenerate or atretic oocytes is all that is required. Sperm evaluation and preparation

In order for fertilization to occur, it is necessary for spermatozoa to undergo capacitation and the acrosome reaction. Capacitation involves the alteration of the outer membranes of the spermatozoa exposing underlying zona pellucida receptors and is an essential precursor to the development of the acrosome reaction. The acrosome reaction is dependent upon an influx of calcium which leads to fusions of the inner and outer acrosomal membrane causing the release of the various acrosomal hydrolytic enzymes, such as hyaluronidase and acrosin, amongst others. The main function of the acrosome reaction is to render the sperm capable of binding to the oocyte zona pellucida, to facilitate penetration of the zona pellucida and subsequent fusion with the egg membrane. Seminal fluid contains many substances which prevent the induction of capacitation and it is therefore necessary to remove the sperm from the seminal plasma in order to initiate the capacitation process in vitro. Simple washing by centrifugation is sufficient to achieve this. However, this is insuffi-

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cient to separate the motile sperm from the leucocytes, cellular debris and non-motile sperm contained within the semen, all of which can have deleterious effects on the motile sperm population through release of reactive oxygen species (ROS) and other degradation substances. Washing the semen by centrifugation creates a sperm pellet at the bottom of the tube. If the supernatant medium is removed and replaced with fresh medium carefully layered over the pellet, the motile sperm can then be left to swim up into the fresh supernatant medium and recovered. Sperm recovery yields tend to be low using this technique. An alternative method, used by a number of clinics, incorporates the use of a suspension of silica particles coated with polyvinylpyrrolidone, called Percoll (Pharmacia, Uppsala, Sweden). Various concentrations or gradients are used and are created by layering the different concentrations (usually 45% and 90% in our laboratory) of Percoll (Pharmacia, Uppsala, Sweden) to create a discontinuous or stepped density gradient. Semen is then layered on top of the gradient and centrifuged through it. Debris, non-motile sperm, sperm with severe morphological abnormalities and round cells are separated from the morphologically normal motile sperm population at Percoll densities corresponding to their own. The viable sperm are able to pass through the 90% layer and collect in a pellet at the bottom of the tube. This ensures that highly motile sperm populations are created free of unwanted cells and cellular debris. The sperm are then removed from the 90% Percoll fraction by dilution of the sperm pellet with culture medium and centrifugation. Insemination Insemination of the oocytes is performed three to six hours after oocyte pick-up for IVF. There are a number of insemination methods in use, depending on the culture system and the quality of the sperm sample. These range from mixing the gametes in tubes or microdrops (a single oocyte and up to several hundreds of thousands of sperm) to ICSI where an oocyte is injected with a single sperm. For normal sperm it is not necessary to inseminate with more than 100 000 motile sperm per millilitre and some units use concentrations as low as 50 000 motile sperm per millilitre if the sperm is considered to be normal. Appropriate numbers of sperm are simply added to the tube or microdrop containing the oocyte to ensure the appropriate final sperm concentration. Alternatively, a suspension containing the appropriate number of sperm can be prepared, new drops or tubes set up, and the oocytes transferred into the sperm suspension.

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For moderately oligozoospermic samples, the insemination concentration can be increased to relatively high levels (up to 500000 motile sperm per millilitre, particularly if severe teratozoospermia is also present) but this it at the expense of increased levels of reactive oxygen species within the culture medium and the possible detrimental effect these might have on the oocytes. High numbers of sperm can also increase the rates of polyspermy, particularly if the sperm morphology has not been critically assessed. The advent of ICSI has led to a reduction in the use of high insemination techniques. Fertilization rates are higher with ICSI, and the detrimental effects of high levels of free radical reactive oxygen species is avoided. Intracytoplasmic sperm injection was developed by the group of Van Steirteghem in Brussels to assist fertilization for the most severe forms of male factor infertility which hitherto were untreatable by normal IVF methods. The technique involves denuding the oocyte of cumulus cells by enzymatic and mechanical means. This is followed by the capture of a viable sperm and its subsequent injection into the oocyte cytoplasm. Sperm are immobilized prior to injection by disrupting the membranes of the sperm midpiece using a microneedle. The needle itself is a feat of engineering in miniature, made from glass and being about 7 n wide (OD), bevelled, with a sharp point being pulled after softening the glass bevel. The sperm head (+/- 5 jU wide) should fit snugly inside the microneedle, almost like a piston within a cylinder, to ensure absolute control of the injection process and to minimize the volume of medium injected with the sperm into the oocyte cytoplasm. The ICSI technique represents a major breakthrough in the treatment of the infertile male. It is a highly successful method and is now in relatively widespread use. Fertilization and pregnancy rates are at least as good as those achieved with normal sperm in the in-vitro fertilization system. The technique has enabled the treatment of men hitherto thought to be untreatable, such those with virtual azoospermia and 'Sertoli cell only syndrome'. Tiny foci of spermatogenesis have been found following testicular biopsy, and sperm have been successfully extracted from the testicular tissue and used for ICSI with subsequent fertilization and embryo development occurring. The most common application of this technique is used for the severe male factor patient (i.e. severe oligo-, astheno- or teratozoospermic male) and for apparently normospermic men for whom in-vitro fertilization has failed to occur. Fertilization evaluation Some 16 to 20 hours after insemination the oocytes are examined to determine if normal fertilization has occurred. The observation of two pronuclei and two

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polar bodies is considered consistent with normal fertilization. In practice, two polar bodies are not always clearly visible as one or both may have fragmented. Observation of more than two pronuclei is indicative of abnormal fertilization; commonly three pronuclei are seen in these cases, although higher numbers are observed less frequently. The occurrence of an extra pronucleus is usually due to fertilization by two sperm but can also be attributed less commonly to fertilization by a diploid sperm or, alternatively, penetration of a diploid oocyte by a haploid sperm. These multipronucleate oocytes are not suitable for transfer and should be excluded from further culture. Examination of the oocyte cytoplasm is facilitated by mechanical removal of the corona radiata cells by gently pipetting the oocyte up and down a finely drawn glass pipette. Once the presence of normal fertilization has been established, the oocytes are returned to culture for a further 24 hours in fresh medium. Embryo assessment for embryo transfer and freezing Embryo transfer is normally performed two days after oocyte pick-up or 44-48 hours after insemination. The embryos are assessed using developmental and morphological criteria and assigned a score. Variations in the exact scoring method exist between units but fundamentally all embryo assessment methods in routine use have two components - rate of development and embryo morphology - as the basis for the scoring system. Embryo morphology in particular is prone to observational inconsistency and subjectivity. Methods of assessing embryo quality using physiological criteria such as pyruvate or glucose utilization are being developed but currently remain the province of the researcher and are not available for use in routine IVF programmes. However, despite the qualitative nature of current scoring methods, it is still possible to establish crude correlations between embryo quality and outcomes such as the incidence of pregnancy and ability to survive freezing and thawing. Determination of the number of embryos to transfer should be made in consultation with the patients' clinician and based on factors such as embryo quality, age of the patient, previous pregnancy or the number of previously unsuccessful transfers, and the risk of multiple pregnancy (particularly highorder multiple pregnancies). Generally, young women, particularly those undergoing their first or second cycle, should be restricted to having a maximum of two embryos transferred. Older women or those with a demonstrated repeated failure to achieve a pregnancy are suitable for three (or higher in exceptional circumstances) embryo transfers. In our unit, similar scoring criteria apply to the selection of embryos for

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freezing as apply to the selection of 'good' embryos for transfer. A score of seven out of a total achievable score often, based on a combined developmental and morphological scoring system, is the minimum for suitability for freezing. Embryos are frozen in 0.25-ml straws on day two or day three, using propanediol and sucrose as cryoprotectants, and stored in liquid nitrogen at -196°C. Embryo transfer The surgical details of the embryo transfer technique are described elsewhere in this book. From the embryologist's point of view, once the outer sheath of the embryo transfer catheter has been satisfactorily located within the cervix, the embryologist loads the inner catheter with the required number of embryos. It is important to ensure that the embryos are located near the tip of the catheter to minimize the volume of medium injected with the embryos. After the catheter has been withdrawn, it is examined to ensure that no embryos have been retained. Laboratory records

All records should clearly show patient identity, date of birth, address and unit or treatment number, preferably by using self-adhesive labels. The clinical or scientific data to be recorded for each patient will vary according to the needs of particular laboratories but should at the very least include the following: number of eggs recovered and inseminated; sperm analysis parameters and insemination numbers, including method (i.e. ICSI or IVF); number of embryos formed and their quality; number of embryos transferred or frozen; and the outcome of treatment, including number of embryos implanting. Any special procedures such as assisted hatching should also be noted. At Monash IVF, we use separate colour-coded laboratory record forms for different procedures such as IVF, ICSI and frozen embryo transfer. Where possible, duplicate records should be maintained. We use two systems, a manual system and a computerized system. Computer back-ups are performed daily and back-up discs or tapes are kept in a fire-proof safe. Record storage systems should be sufficiently secure to ensure that patient confidentiality is maintained.

Checking systems and security

Finally, a word on checking systems and general security in embryology laboratories. Given the disastrous consequences for parents and child of any

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mistake occurring within the embryology laboratory, adequate checking systems must be in place. Patient's name bands should always be checked by at least two people, namely the doctor and embryologist performing the procedure. All vessels, including waste vessels, should be clearly labelled with the name and number of the patient and preferably colour coded. The name on the culture vessels should be witnessed prior to commencing any procedure. Each time a gamete or embryo is transferred to a new vessel, the names on both the old and new containers should be checked by a co-worker. The straws and goblets containing embryos frozen for long-term storage should be clearly and individually labelled with permanent marker capable of withstanding temperatures of -196°C. The recording system for long-term embryo storage needs to be accurately maintained, with absolute attention to detail, especially the location of the embryos. Regular tank inventory audits must be undertaken to ensure accuracy of the records. Duplicate storage records (including computer back-ups) should be maintained and kept in a separate place. Quality assurance

In addition to the quality control steps performed on culture media, systems should also be in place to ensure that there is adequate quality assurance (QA) monitoring of the equipment. Daily and weekly schedules recording key physical parameters of equipment function such as temperature and CO2 checks and records of cleaning programmes should be maintained. A daily shut down checklist detailing all the main daily tasks should be used as a safeguard to ensure that all tasks have been completed and that preparations for the next day's work have been carried out. As tasks are completed, the person responsible initials the checklist. This is an essential aid in a busy laboratory where a large number of staff are involved in the daily routine. References Fishel, S.B., Jackson, P., Antinori, S., Johnson, J., Lisi, F., Chiariello, F., Versaci, C. and Lisi, R. 1990. Sub-zonal insemination (SUZI) for the alleviation of infertility. Fertility and Sterility 54: 828-35. Gardner, D.K. and Lane, M. 1993. Embryo culture systems. In Handbook of In Vitro Fertilisation, ed. A. Trounson and D. Gardner, p. 102. Boca Raton: CRC Press. Pickering. S., Braude, P.R., Cant, R.M., Currie, R.M. and Johnson, M.H. 1990. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertility and Sterility 54: 102-08.

17 The results of assisted reproductive technology VIVIEN M A C L A C H L A N

Introduction

The initial success of ART was achieved by using non-stimulated cycles with single-embryo transfers, but the low conception rates associated with nonstimulated cycles led to the development of ovarian stimulation protocols. These protocols allowed for the development of multiple follicles and the collection of more than one oocyte. This in turn made multiple embryo transfers possible, producing a higher pregnancy rate than with non-stimulated cycles. However, in addition to improving a patient's chances of success with ART, multiple embryo transfers resulted in a marked increase in the incidence of multiple gestations. The worldwide results of ART report a multiple pregnancy rate in the range of 15-40% of the total pregnancies. This compares with the rate of spontaneous twins (of about 1.0%) and that of triplets (0.01% of natural pregnancies).

Reporting ART success rates

Success rates are dependent upon individual variables, including patient age, cause of infertility, duration of infertility, ovarian reserve, number and maturity of eggs retrieved, success or failure of fertilization and cleavage in vitro, number of oocytes or embryos transferred, number of embryos cryopreserved, and adequacy of the luteal phase after transfer. With conception rates dependent upon such a multitude of parameters, predicting results is a difficult and often unreliable process. Assisted reproductive technology clinics present their results in a variety of ways, many of which are misleading. All success rate definitions include a numerator that reports a measure of the number of implantations, pregnancies or live births, and a denominator that includes the number of treatments 235

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Table 17.1. Potential numerators and denominators in reporting success rates Numerators (measure ofpregnancy) Live infants: the number of live infants Live delivery: delivery that includes one or more living infant Viable uterine pregnancy: detection of a viable intrauterine gestation sac at ultrasound examination Clinical pregnancy: one or more gestation sacs present when examined by ultrasound or evidence of pregnancy seen at surgery Implantations: number of gestation sacs identified by ultrasound. Viable intrauterine implantations: number of viable intrauterine gestation sacs identified by ultrasound Denominator (measure of treatments) Transfer procedure: the number of oocyte or embryo transfer procedures Oocytes or embryos transferred: the number of oocytes or embryos transferred in one procedure Oocyte collections: the number of procedures in which oocyte collection is attempted Cycles initiated: the number of treatment cycles initiated with the aim of proceeding to oocyte collection

or procedures that occur for each pregnancy. The most widely used numerators and denominators to report ART success rates are listed in Table 17.1. Choice of both a clearly defined numerator and denominator is crucial. The three procedures in in-vitro fertilization and embryo transfer - ovarian stimulation, oocyte collection and ET - make up one IVF/ET cycle. Each of the three procedures has been used as the denominator in definitions of pregnancy success rates. For couples embarking on ART, the chances of success are more accurately reflected by the number of deliveries resulting in one or more live infants. Clinical pregnancy rates are commonly used by ART clinics as an internal monitor of their own success rates. The clinical pregnancy rates include blighted ova and ectopic pregnancies. Some clinics also include biochemical or preclinical pregnancies in pregnancy rates. These 'pregnancies' are classified by the detection of an elevated serum or urine hCG measurement. The inclusion of these 'pregnancies' in pregnancy rates is misleading and should be avoided. Another outcome of interest to ART clinics is the implantation rate which is defined as the number of gestation sacs identified per transferred oocyte or embryo. This rate gives an indication of the efficiency of the IVF/ET or GIFT protocols and may prove useful in identifying the most successful

The results of assisted reproductive technology therapeutic regimens for establishing successful pregnancies with low risk of multiple gestations. The choice of denominator is even more problematic than that of the numerator and varies from one clinic to another. The number of treatment cycles initiated, oocyte collection procedures and oocyte or embryo transfer procedures are all important clinical and research measures of the efficiency of each step of an ART treatment cycle. It is important that all cycles that have been initiated are included. Cancellation, for whatever reason and at whatever stage in the cycle, should be acknowledged, not disregarded, in the calculation of the success rate. It is likely that a woman who indicated little chance of a successful outcome in her first few treatment cycles would be discouraged from seeking further treatment. Confusion, or lack of agreement, arises as to the definition of a stimulation procedure as supernumerary embryos may be cryopreserved and transferred in a later cycle so that more than one ET procedure may result from the same stimulation procedure. Hence, pregnancy rates per stimulated cycle for different clinics may or may not include the addition of pregnancies that result from the transfer of the remaining cryopreserved embryos. Also, in treatment cycles where all embryos are cryopreserved for later transfer because the patient is considered at risk of ovarian hyperstimulation syndrome, the IVF/ET process will not be completed until a later menstrual cycle. These are examples of reasons why some clinics justify different ways of reporting ART success rates per stimulated treatment cycle, but as a result comparisons of success rates between clinics can be misleading. The number of live deliveries per 100 oocyte or embryo transfer procedures is now frequently used as a measure of pregnancy success rate in ART clinics. There are, however, certain limitations to this definition. One limitation is that a measurement based on the number of procedures rather than on the number of couples undergoing IVF/ET means that the success rate will be biased towards those couple who are less successful at becoming pregnant with IVF/ET. Couples with less success undergo more cycles before achieving pregnancy or discontinuing therapy. Dependent observations are another problem. The probability of live delivery per ET procedure is likely to be similar for each of several IVF/ET cycles in the same couple (dependent observations), whereas the probability may be different for single cycles from each of several different couples (independent observations). Without couples' specific data, therefore, calculating P values or confidence limits around estimated differences in pregnancy success rates is generally inappropriate. For example, conducting statistical comparisons of clinical pregnancy rates between ART clinics would be inappropriate because

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the data are not provided by the number of cycles and pregnancy outcomes per couple, thus we cannot adjust for the possibility that multiple cycles can be contributed by the same couple. Couples' specific data, however, are more expensive and burdensome to collect than clinic-specific data. Another concern is the fact that reporting pregnancy success as the number of live deliveries per ET procedure is an assumption that the probability of pregnancy is the same in all cycles. Whether the probability of live delivery actually remains the same with each successive IVF/ET in the same couple is unclear. Theoretically, the probability of success might increase with additional cycles if the therapeutic protocol has been adjusted from knowledge gained in earlier cycles, or it might decline if additional cycles provide further evidence that a couple will not respond to IVF/ET therapy. Some reports of ART or other infertility treatments have used life-table analysis with cumulative pregnancy rates over several monthly cycles as one approach to this concern (Tan et ai, 1992; Kovacs, 1993). In these reports, the life-table analysis shows a cumulative pregnancy rate after six cycles of between 56% and 58%. One of the problems in reporting cumulative probability of pregnancy is that there has been no standard definition used for an ART treatment cycle. For example, some studies have included all cycles initiated with the aim of proceeding to an oocyte collection, other studies include all cycles in which oocyte collection has been attempted, and others include only cycles where a transfer has occurred. To complicate matters further, some studies include cycles irrespective of the stimulation protocol used, whilst others evaluate cycles in which the same protocol has been used throughout. There has been further criticism of the use of life-table analysis to estimate the success rates for ART. It is assumed that in standard life-table analysis, such as survival after operations, those patients who are lost to follow-up have the same chance of further survival as those who remain under surveillance. This method of analysis is usually used to follow up a single intervention (such as an operation) rather than a series of interventions. When life-table analysis is applied to ART, it is assumed that the couples who drop out while still not pregnant would therefore have had the same chance of success as those who remained in the programme. In fact, those who remain in the programme after two or three cycles may have a higher chance of success compared with those who have left the programme possibly because of a low chance of success. If, however, life-table analysis of cumulative pregnancy rates and live birth rates was stratified according to prognostic indicators (such as age, duration of infertility and ovarian reserve), some of the criticisms of the life-table approach could be addressed. There have been several reports of life-table analysis showing, for example, the significance of female age and ovarian

The results of assisted reproductive technology reserve (Scott et al., 1995) and age and infertility etiology (Dor et ai, 1996) on cumulative pregnancy rates. Since many women nowadays have repeated ART treatment cycles, the cumulative conception rate for a given number of treatment cycles is a far more useful estimate of success than the pregnancy rate per cycle of treatment. The cumulative pregnancy rate or life-table analysis for the presentation of ART results allows for the follow-up of a cohort for different periods and also allows for dropouts. It is, to date, the best way of giving the couple realistic knowledge of the probability of ultimate success. Results of ART in Australia and New Zealand

A report of ART activities in Australia and New Zealand is prepared each year by the Australian Institute of Health and Welfare National Perinatal Statistics Unit and the Fertility Society of Australia. The report of assisted conception cycles in 1992 and 1993 was combined (Lancaster, Shafir and Huang, 1995). It contains a summary of the results of treatment of infertility by assisted conception in all ART clinics in Australia and New Zealand. There were 28 clinics listed in the 1992/1993 report: nine clinics in New South Wales, four in Victoria, three in Queensland, two in South Australia, three in Western Australia, one in Tasmania, one in Canberra and five in New Zealand. The report includes data on IVF, GIFT and the newer techniques of microinsemination that are used to treat male infertility, but excludes other treatment of infertility by artificial insemination or by ovulation induction without IVF or GIFT. The ART clinics report summary data on treatment cycles occurring during each calendar year. The data include the number of cycles commenced and the number progressing to the stage of oocyte retrieval, embryo transfer, clinical pregnancy and live births. Each ART clinic reports separate results for IVF with uterine transfer of fresh embryos, IVF with tubal transfer of fresh embryos, GIFT, transfer of frozen/thawed embryos (FET), transfer of donated oocytes to recipients (DO/REC) and the various techniques of sperm microinsemination. Also provided are tabulated data on the age distribution, causes of infertility, drugs used to stimulate ovulation, and the number of embryos or oocytes transferred for women treated by IVF and uterine transfer, IVF and tubal transfer, and GIFT. Each unit provides individual patient data for clinical pregnancies. The data include details as for the summary data plus additional data such as the pregnancy outcome for each gestation sac, obstetric complications, method of delivery, sex and birthweight of each baby, condition at birth and any congenital malformations or abnormalities.

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Table 17.2. Clinical pregnancy and live birth rates

Treatment

Year

Pregnancies per 100 transfers mean (*range)

Live births per 100 transfers mean (*range)

IVF uterine transfer IVF fallopian transfer GIFT

1992 1993 1992 1993 1992 1993 1992 1993 1992 1993

14.7(9.7-18.6) 16.2(11.1-28.6) 17.0 21.6 27.7(19.5-35.6) 27.9 (20.0-35.0) 13.5(6.6-18.9) 13.5(6.7-18.4) 16.6 19.6

10.2(4.5-12.9) 11.6(6.5-20.0) 12.3 15.5 20.8(12.5-28.8) 20.9(11.2-26.5) 9.8 (4.0-14.7) 9.6(5.3-13.3) 12.7 14.0

FET DO/REC

*Range: only results for clinics with at least 100 transfers shown. From Lancaster et al. (1995).

All the Australian and New Zealand data presented in this chapter are taken directly from the cited publication by Professor Paul Lancaster and colleagues. Pregnancy and live birth rates for the Australian and New Zealand ART clinics

The clinical pregnancy rates and live birth rates for the years 1992 and 1993 are shown in Table 17.2. Only data for clinics where at least 100 transfers were performed for each treatment are included in the range data. Nonetheless, there were still marked variations in both the pregnancy and live birth rates among the individual ART clinics. The ranges of pregnancy and live birth rates shown in Table 17.2 have the important limitation of being summary data that do not reflect the variation in patient characteristics from one clinic to another. The heterogeneity of patient populations probably explains some of the differences seen among the ART clinics. Clinics that specialize in treating the most difficult infertility patients, for example, may have low summary rates that are nevertheless superior to those of other clinics treating the same type of patients. One way to improve the usefulness of these rates would be to report them stratified by clinical and demographic characteristics that predict pregnancy success. No consensus exists as to the explanation for a possible increase in overall success with the GIFT procedure compared with IVF/ET. During 1992 and 1993, the patient profiles encompassed different age distributions - a higher

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Table 17.3. Causes of Infertility for IVF/ETand

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GIFT cycles in 1992 and 1993

Causes of infertility

IVF/ET (%)

GIFT (%)

Tubal only Other female only Male factors only Multiple causes Unexplained

45.9 11.1 17.8 15.2 10.0

5.2 24.8 18.8 12.2 39.0

From Lancaster et al. (1995). percentage of GIFT patients were less than 35 years of age (62.3 % versus 57.3 % for IVF). There were also differences in the diagnostic categories (Table 17.3). Patients who had a GIFT cycle had an increased incidence of a single female cause of infertility such as cervical factor, ovulation defects or endometriosis, and an increased incidence of unexplained infertility. It is common for patients with unexplained infertility to begin ART treatment with GIFT and, if unsuccessful after a few cycles change the subsequent procedures to IVF/ET. It is possible that these patients may have a reduced chance of pregnancy and hence a lower overall IVF/ET success rate. All these factors need to be considered carefully when comparing the success rates of ART procedures. For a true comparison of IVF/ET with GIFT, or indeed any other form of therapy such as stimulation protocols, one needs evidence in the form of randomized controlled trials. In properly designed randomized, controlled trials, power calculations are performed to determine the minimum number of cases in each arm to show significant differences between study parameters. Randomization should eliminate differences in the clinical or demographic characteristics of the patients in each of the groups, making for a valid comparison between the parameters under investigation. There are many problems in the construction of such trials. One of the problems is recruiting sufficient patients to complete a trial. For example, if a clinic was to investigate whether ICSI and ET resulted in a 5% improvement in live birth rate over the standard IVF/ET for women over the age of 37 years, the total sample size required would be at least 1320. It would not be possible for a single clinic to perform such a study within a reasonable time frame. If a study was performed to compare, for example, IVF/ET with GIFT, the results may show no overall benefit of one therapy over the other but, on closer analysis, may reveal subsets of patients who have improved results with one of the forms of treatment. Unless such studies are performed, one cannot make valid conclusions or decisions based on summary data.

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V. MacLachlan I B Preg/admit I

I Preg/OPU

—•— Preg/transfer

<29

29-31

32-34

35-37

38-40

41-43

>44

Maternal age groups

Figure 17.1 Clinical pregnancy rates by maternal age, 1994-5.

Maternal age Women who became pregnant after IVF or GIFT in 1992 and 1993 were generally older than the mothers of all babies born in Australia. In 1992, 27.2% of IVF pregnancies were among women aged 35-39 years, compared with 26.1% of GIFT pregnancies and 9.7% of all Australian births. In that year, the proportion of mothers aged 40 years and over was 5.1% for IVF, 4.6% for GIFT and 1.6% for all births in Australia. It is widely accepted that the probability of a pregnancy after ET is affected by maternal age (Figure 17.1). The number of cycles discontinued due to poor follicular development is also effected by maternal age, rising from around 10% in women less than 29 years to around 35% in women over the age of 44 years. Number ofoocytes collected The average number of oocytes collected by laparoscopy or ultrasound guidance has increased since the mid-1980s. For the 1992 and 1993 IVF/ET and GIFT conception cycles, the mean number of oocytes collected was 11. The increase in the number ofoocytes collected has paralleled the introduction of GnRH agonists to ART stimulation protocols, beginning in the late 1980s. Number of embryos j oocytes transferred There has been a continuing increase in the proportion of IVF pregnancies resulting from the transfer of two embryos and also a decline in the proportion

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c £

2

5

••<>•••• I V F

-•-GIFT

Q. 1

0 1 1979-85 1986 1987 1988 1989 Year

I

1990

1991 1992 1993

Figure 17.2 Incidence of triplet pregnancies, IVF and GIFT, 1979-93 (Lancaster et al., 1995).

of IVF pregnancies resulting from the transfer of four or more embryos. In 1992, only 3.5% of pregnancies followed transfer of four or more embryos, and the percentage of 1.8% was even lower in 1993. Since 1988, the mean number of embryos transferred in IVF pregnancies has declined to 2.4 in 1993. In 1992 and 1993, most GIFT pregnancies followed transfer of three or fewer oocytes. There were relatively fewer pregnancies resulting from transfer of four or more oocytes than in previous years, and an increased trend in pregnancies after transfer of two oocytes. The mean number of oocytes transferred in GIFT pregnancies has declined from 4.0 during 1979-85 to 2.8 in 1993. It can be assumed that the reduction in the mean number of embryos or oocytes transferred has been the main contributing factor in decreasing the incidence of triplet pregnancies (Figure 17.2). Spontaneous abortion

Spontaneous abortion occurred in 22.0% and 20.9% of IVF pregnancies in 1992 and 1993 respectively, similar to the rates in recent years. The incidence of spontaneous abortion increased with maternal age (Figure 17.3). Among 236 pregnancies conceived after microinsemination in 1990 to 1993, there were 52 (22.0%) spontaneous abortions. The spontaneous abortion rate of 19.3% for GIFT pregnancies in 1993 was the lowest rate in any year since GIFT was first used in 1985. Ectopic pregnancies

The proportion of ectopic pregnancies occurring after IVF/ET declined from 5.9% in 1991 to 4.9% in 1992 and 4.0% in 1993. Ectopic pregnancy was also

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V. MacLachlan 70 r | IVF

60-

I GIFT

5

g ° £ 40 2 c o 30 20 10 <25

25-29

30-34

35-39

40-44

45+

Total

Maternal age groups

Figure 17.3 Spontaneous abortions by maternal age groups, IVF and GIFT pregnancies, 1979-93 (Lancaster et ai, 1995). less likely in GIFT pregnancies in 1992 and 1993 than in previous years, occurring in 2.6% in 1992 and in 3.1% in 1993.

Selective reduction of fetuses

Selective reduction of fetuses is performed in early pregnancy to abort severely malformed fetuses in multiple pregnancies and to reduce multiple births. In 1992 and 1993, selective reduction was performed in five IVF and seven GIFT pregnancies. Fetal reduction had previously been performed in two pregnancies in 1988, one in 1989, one in 1990, and nine in 1991. The indication for fetal reduction was a chromosomal abnormality in three pregnancies. In two triplet GIFT pregnancies that were reduced to twin pregnancies, spontaneous abortions of the remaining fetuses occurred. Spontaneous abortion of the remaining fetus also occurred after selective reduction of a twin pregnancy for trisomy 21.

Preterm birth rates For viable pregnancies of at least 20 weeks' gestation the proportion of preterm births of less than 37 weeks' gestation in 1992-93 was 21.8% after IVF/ET and 25.2% after GIFT (Table 17.4). In singleton pregnancies, the proportion of preterm births was 14.3% and 11.5% for IVF/ET and GIFT, respectively. These proportions were more than double the preterm rate of 6.9% in all Australian pregnancies in 1992. The preterm birth rates for twins and triplets are shown in Table 17.4.

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Table 17.4. Preterm birth rates for IVF/ETand GIFT, 1992-1993

IVE/ET GIFT

Singleton (%)

Twin(%)

Triplet (%)

Total (%)

14.3 11.5

54.0 60.3

89.5 97.9

21.8 25.2

From Lancaster et al. (1995).

Multiple pregnancies Multiple pregnancy occurred in 17.6% of IVF pregnancies in 1992 and 17.3% in 1993 - slightly more than the rate of 16.0% in 1991, but less than in other years. The multiple pregnancy rate for GIFT was higher - 27.1% in 1992 and 24.2% in 1993. Twin pregnancies accounted for 15.9% of IVF pregnancies in 1992 and 15.7% in 1993. Twins occurred in 23.5% of GIFT pregnancies in 1992 and 21.5% in 1993 - similar to the rate in recent years. Triplet pregnancies accounted for 1.7% of IVF pregnancies in 1992 and 1.5% in 1993. The triplet rate for GIFT decreased from 3.5% in 1992 to 2.4% in 1993 - the lowest rate for any year since GIFT was first used in 1985. The incidence of triplet pregnancy for IVF/ET and GIFT for 1979-1993 is shown in Figure 17.2. In 1992-1993, multiple pregnancy occurred in 12.1% of pregnancies resulting from transfer of frozen embryos.

Method of delivery As in previous years, caesarean rates were higher for multiple than for singleton IVF pregnancies. In 1992-93, the caesarean rate was 37.3% for singleton pregnancies, 53.5% for twin pregnancies, and 94.7% for triplet pregnancies. The caesarean rates showed a trend to increase with advancing maternal age from 21.4% at 20-24 years to 22.8% at 25-29 years; 41.3% at 30-34 years; 37.3% at 35-39 years; and 62.5% at 40 years and over. For all Australian singleton births in 1992, the caesarean rates were 17.8% at 20-24 years, 20.3% at 25-29 years, 23.3% at 30-34 years, 28.3% at 35-39 years, and 34.2% for women aged 40-44 years. The caesarean birth rates for GIFT pregnancies showed similar trends to those for IVF pregnancies.

Birthweights The mean birthweight and the incidence of low birthweight (less than 2500 g) for infants born after IVF and GIFT in 1992 and 1993 differed considerably

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Table 17.5. Birthweights (grams) of at least 20 weeks' gestation Singleton

IVF/ET 3220 3185 GIFT All Australian births 3384

Twin

Triplet

Total

2287 2277 2396

1761 1595 1615

2909 2748 3368

From Lancaster et al. (1995).

Table 17.6. Incidence of low birthweight (less than 2500 grams) Singleton (%) IVF/ET 12.1 GIFT 11.1 All Australian births 5.0

Twin (%)

Triplet (%)

Total (%)

56.5 58.8

92.1 93.6

27.2 33.8

From Lancaster et al. (1995).

from the birthweights for all Australian births in 1992. The mean birthweight of IVF infants was 2909 g, 2748 g for GIFT births and 3368 g for all Australian births (Table 17.5). The high incidence of multiple births accounted for much of this difference (Table 17.6). The incidence of low birthweight was similar for all maternal age groups. Perinatal mortality Perinatal deaths including fetal deaths (stillbirths) of at least 20 weeks' gestation and neonatal deaths of liveborn infants occurring within 28 days of birth for IVF and GIFT were above those of all Australian births. For ART births, the rate was 32.8 and 29.0 per 1000 births for IVF and GIFT births, respectively. For singleton IVF births, the rate was 23.8 and for GIFT, 9.4 per 1000 births. The overall perinatal death rate for IVF and GIFT births was higher in multiple births. For all Australian births of at least 20 weeks' gestation, the perinatal death rate was 12.0 per 1000 births in 1992. Congenital malformations Among the Australian and New Zealand IVF and GIFT live births, stillbirths and induced abortions of at least 16 weeks' gestation conceived in 1985 to 1993, the incidence of major congenital malformations was 2.5% for IVF and

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Table 17.7. Major congenital malformations, 1985-93 Outcome

Singleton

Multiple

Total

IVF GIFT IVF GIFT

6388 3409 185(2.9%) 99 (2.9%)

3419 2548 62(1.8%) 59 (2.3%)

9807 5957 247 (2.5%) 158(2.7%)

Total births Congenital malformations

From Lancaster et al. (1995). 2.7% for GIFT pregnancies (Table 17.7). The incidence of major congenital malformations for pregnancies conceived in 1990 to 1993 by microinsemination procedures (220 pregnancies) was 3.2%.

Discussion In Australia we restrict the number of embryos and oocytes transferred to two or three at a time, except in exceptional circumstances. The results for the United States and Canada for 1992 and 1993 show a 34% multiple birth rate for both IVF/ET and GIFT. This was much higher than the multiple birth rate of 17% for IVF and 25% for GIFT during the same period in Australia and New Zealand. There is increasing concern amongst neonatologists that it is the initial number of implantations that causes birth problems as much as the multiple births. Data have suggested that live births after selective embryo reduction are still associated with decreased birthweight and gestational age at delivery. Furthermore, fetal growth of selective embryo reduction pregnancies seems to be impaired independent of the gestational age at which delivery occurs. It is for these reasons that at Monash IVF, prognostic indicators of pregnancy success, such as the women's age, indication for ART and duration of infertility, are considered when determining the number of embryos or oocytes to be transferred. The efficiency of human reproduction remains a subject of debate despite the published studies that deal with this topic, but it is a vital 'control' against which to compare ART results. Fertility rates in presumably fertile populations are difficult to ascertain due to the complexity and cost of conducting such studies. In one report it was estimated that the peak normal fertility per cycle is only approximately 33% in the first month, but falls quickly, settling to approximately 5%. The average is estimated to be only 20-25% per cycle. It could be said that the results of ART compare favourably when judged against these estimations.

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References Dor, J., Seidman, D.S., Ben-Shlomo, I., Levran, D., Ben-Rafael, Z. and Mashiach, S. 1996. Cumulative pregnancy rate following in-vitro fertilization: the significance of age and infertility aetiology. Human Reproduction 11: 425-8. Kovacs, G. 1993. The likelihood of pregnancy with IVF and GIFT in Australia and New Zealand. The MedicalJoumal of Australia 158: 805-7. Lancaster, P., Shafir, E. and Huang, J. 1995. Assisted Conception, Australia and New Zealand, 1992 and 1993. Sydney: AIHW National Perinatal Statistics Unit. Assisted Conception Series: No. J. Scott, R.T., Opsahl, M.S., Leonardi, M.R., Neall, G.S., Illions, E.H. and Navot, D. 1995. Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age. Human Reproduction 10: 1706-10. Tan, S.L., Royston, P., Campbell, S., Jacobs, H.S., Betts, J., Mason, B. and Edwards, R.G. 1992. Cumulative conception and livebirth rates after in-vitro fertilisation. Lancet 339: 1390-4.

18 Infertility counselling J A N E T A N D E R S O N and RITA ALESI

In many countries, legislation and the requirements of regulatory authorities impact on the provision of infertility counselling services in assisted reproduction clinics. Our experience is based on providing an infertility counselling service that is governed by legislation that was introduced in 1984 (Infertility Medical Procedures Act), in Victoria, Australia. Under this legislation all patients and donors must attend counselling prior to commencing treatment, and counselling must be available freely to all patients at all times. Counsellors have therefore become an integral part of the treatment team and have had the opportunity of gaining very wide experience of the types of issues with which patients are grappling. Counsellors have also had to grapple with issues such as defining the purpose of the initial counselling session, refining the interview to best meet the needs of the patients, and developing ways to raise the awareness of psychological issues with other staff. It is with this background that we have written this chapter. The aim of this chapter is to explore why infertility counselling is important and necessary. The psychological experiences of patients and the issues that arise for them are explored, and consideration is given to different types of psychological counselling and how counselling can benefit patients. Some recommendations are made for clinicians who need to understand the psychological experiences of their patients and work closely with the counsellor. The chapter also deals with the often complex issues faced by recipients and donors of genetic material and the role of the counsellor in helping them to deal with those issues. The experiences and needs of donor conceived offspring are also considered. Definition of counselling

Infertility counselling can now be regarded as an area of specialization for counsellors (Jennings, 1995). The importance of counselling has been re249

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inforced by reports such as the Warnock Report in England (Warnock, 1985) and the Waller Report in Australia (1984) which have recommended that counselling be freely available within clinics. Some jurisdictions have legislated to ensure that counselling is available to all patients, and in some cases it is a pretreatment requirement to attend counselling. However, the use of the word counselling is confusing as it is a generic term that can be defined in differing ways. For example, many clinicians would consider they carry out counselling when they discuss options and treatments with patients, although this is generally regarded as distinct from psychological counselling (Neuberg, 1995). Acknowledging the lack of clarity of the term counselling, the Australian National Bioethics Consultative Council (1990) report on counselling within clinics identified four types of counselling: information provision facilitation of decision making (especially in relation to the use of donor gametes) emotional support therapeutic counselling. While staff from different disciplines (including counselling) may provide information and assistance with decision making, support and therapeutic counselling were seen as the areas of expertise of mental health professionals. Nursing staff also provide support for patients, but their expertise and availability for extended counselling are limited. Therefore, it is necessary to make a distinction between the counselling that occurs as part of the clinicianpatient or nurse-patient relationship, and the counselling that occurs with a professionally trained counsellor. Psychological implications of infertility

Developmental^, parenthood represents the attainment of mature adulthood. As such, it satisfies the need to develop as an adult and to demonstrate independence and creativity. Savage summarizes the importance, psychologically, of parenthood: 'Childbearing preserves, biologically, the continuation of the species. Psychologically, the child is also, in part, a completion of the parents.' (Savage, 1989: p.33). The inability to attain parenthood can be seen as an interference in the progression of adult development. Infertility frustrates the deep need to reproduce one's self and create the next generation. If the individual is to mature and develop, the interference in adult development that infertility represents must be overcome (Leon, 1990). Viewed in this way, the intense

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drive that many patients have to pursue treatment becomes understandable. However, given that for some couples treatment will not succeed, the task becomes how to resolve the interference to adult development on a psychological level without having a child. How this can be resolved is a difficult question that will be considered at more length in the section on general counselling for infertility. The central experience of infertility is of loss and emptiness. Patients often present with overwhelming feelings of sadness, despair, anger and guilt. Because their responses are similar to those of the bereaved, and because infertility represents a loss, patients are often described as grieving. However, the grief response in infertility is complicated by several factors. Firstly, in infertility there is no tangible loss and no discrete event that signifies the loss. What is lost is the potential to reproduce, and the hopes and fantasies associated with future fertility. It is much harder to grieve for something that was never realized and never experienced than it is to grieve for a person who was known and loved. Patients often report a sense of unreality as they experience grief yet their daily lives continue exactly as before. What has changed is their view of their future and of themselves as parents, not the reality of their lives. Secondly, the loss of fertility is often not regarded as a significant loss by people who have no experience of it. Many patients report experiences of feeling hurt when others have made thoughtless remarks about their infertility (Callan and Hennessey, 1988). They often develop a defensive facade so that their grief is hidden and others cannot see their pain and vulnerability. Therefore, infertility patients often do not have access to the social support that is more readily available to the recently bereaved. Thirdly, infertility treatment offers hope that the infertility may be overcome and that a pregnancy may occur. Therefore, the loss is uncertain and the grieving cannot proceed. Emotional reactions to the loss are experienced in a cyclical manner. Crucial points at which the perception of loss is heightened are at the time of diagnosis, at the time of menstruation and after an unsuccessful treatment. At other times there may be renewed hope and optimism. Such extremes of emotion are often cited by patients as particularly difficult, and infertility is commonly described as being on an emotional rollercoaster. These unique features of grief in infertility patients have implications for the provision of psychological services. Because of the cyclical nature of the grief, it may be very difficult to proceed with straightforward grief counselling. People are more likely to seek counselling at times of distress and likely to terminate counselling when their mood lifts, only to re-present at a later time. The psychologist therefore needs to work within this pattern of shifting

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moods. Support and empathy can be provided at times of sadness, but the development of further insight and reflection about the meaning of infertility for the patient's life and relationship may not be possible. However, over time, if a pregnancy does not occur, there may be opportunities to address these issues. It is important for the psychologist to be sensitive to which issues patients are ready to address, and not to pursue issues they are not comfortable dealing with. Infertility patients are also likely to use denial as a coping mechanism (Leon, 1990). The existence of treatments that may overcome their infertility means they can avoid the grief of infertility by investing hope in the next cycle of treatment. If this continues for too long, it can become maladaptive, with the couple caught in a chronic cycle of hope and disappointment. They may have little energy left for other activities, and experience a loss of purpose in life apart from their infertility treatment. Such patients can be sensitively invited to consider their situation and can be encouraged to set a limit on what they are prepared to endure. However, in practice it can be extremely difficult to raise these issues in the clinical setting because of the strength of the couple's defences, and the fact that the use of denial as a coping mechanism means they are unlikely to seek counselling. The isolation experienced by the infertile means that the relationships they form with the treatment team can be very strong. Their need for empathy and support is great and their contact with the team may be thefirsttime they have received this. Acceptance of and support for those experiencing strong emotions can be very therapeutic and lead to self-awareness and self-acceptance, but it can also be harder to move on and leave the safe and supportive environment of the clinic. Psychological consequences of infertility Low self esteem The impact of infertility on self esteem is debilitating. It threatens to undermine the sense of being a competent adult and creates feelings of guilt, shame and worthlessness (Mahlstedt, 1985). This is especially damaging if the self concept is based entirely on the capacity to reproduce. Societal attitudes to the infertile and negative media reports of IVF programmes may also reinforce feelings of difference and stigmatization. Identity issues If a person's self-concept is based on having children, infertility is a threat to their identity (Becker, 1990). Successful resolution of infertility relies on

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forming a self-concept that includes a view of self as worthwhile, competent and childless. This can usually only be achieved after a period of confusion and disintegration that can be perceived as a crisis by the individual. The search for other roles and alternative sources of self-esteem is usually difficult. The fulfilment of having children is unlikely to be replaced wholly by anything else, but rather a number of different things. Major changes in life direction are considered in the search for alternatives, and this can often mean reconsidering the future of the marriage relationship. When the future of the relationship was always thought of as including children, the same search for identity and alternative sources of meaning applies to the relationship. The individual's search for a new identity and the relationship's search for a new identity are interrelated. The two may occur in parallel or separately, but both need to be negotiated. The threat of childlessness to the survival of the relationship may be the motivation for seeking infertility treatment.

Stress reactions Individual

Overt symptoms that often motivate couples to seek counselling include difficulty sleeping and concentrating, irritability and social withdrawal, lack of pleasure in previously pleasurable activities, lack of motivation and lethargy. Difficulties at work and in relationships with others may result. More severe psychological difficulties may be experienced, including eating disorders, drug and alcohol use. The loss of control and uncertainty about the future often result in heightened anxiety, sometimes including panic attacks. Anxiety is a particular problem at times of waiting for treatment to begin or waiting for the end of the menstrual cycle to ascertain whether pregnancy has occurred. Feelings of helplessness and loss of control are intensified at these times. The possibility of clinical depression should also be considered. Depression should be distinguished from the normal feelings of distress that arise. If these do not resolve with time, or change in intensity in response to external events, it is more likely that independent treatment for depression will be necessary.

Relationship

Stress reactions also affect the relationship. Communication difficulties, particularly in relation to infertility, are common. Gender differences in identifying infertility as a problem and coping strategies can lead to problems. Women are often the first to express concern about fertility and initiate investigation. Men are often privately less concerned and are confident that

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pregnancy will occur (Mahlstedt, MacDuff and Bernstein, 1987). They are therefore not as open to discussion about the meaning of infertility for them as they do not acknowledge that it is a real possibility that they may not have children. Their coping strategies are more likely to involve ignoring the problem and denying its emotional results. Women are more likely to want to discuss their emotions and explore the impact of infertility. An unresponsive partner and lack of support from friends or family can create feelings of frustration and isolation for the woman. Conflict can occur and lack of communication can create distance between the partners. If infertility is not resolved quickly, the man may begin to recognize the real possibility that the couple may not have children and begin to feel the same isolation and distress that the woman felt earlier. However, she may have moved on from these feelings and may have already started to develop alternative ways of coping and deriving satisfaction from life. It may be difficult for her then to acknowledge the man's feelings and provide support for him. Sexual difficulties are also common (Gervaise, 1993). Sexuality and fertility are extremely closely linked. Sexual difficulties can arise from the earliest times of infertility investigation. Timing sex to coincide with the fertile period is often extremely stressful and interferes significantly with enjoyment and spontaneity. As infertility persists, sex is seen as having only one purpose: to achieve a pregnancy. The only sexual activity may be occurring around ovulation. As enjoyment lessens, interest in sex declines, intensifying the difficulties. Sexual difficulties can also reinforce feelings of failure and inadequacy for both partners.

Psychological counselling for infertility Pretreatment counselling Infertility counselling is an integral part of the treatment procedures in our clinic. In Victoria, Australia, pretreatment counselling is legally mandated for all couples receiving treatment and all donors and their partners. This means that a routine part of the pretreatment preparation is a consultation with the psychologist. The content of this consultation is flexible so that the concerns that a couple has can be expressed and dealt with. However, there are four aims that the psychologist hopes to achieve. Informed consent The counsellor does not have total responsibility for ensuring that the patients are giving informed consent to the procedures they are undertaking. However,

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as a team member, the counsellor can complement the process. A brief overview of the treatment procedures can be a natural starting point for the discussion. Any misunderstandings a couple has about the treatment, or about the biology of reproduction, will be obvious and can be addressed at this time, either directly or by referral to another staff member. Decision making Patients are required to make decisions about their treatment which can be ethically difficult. The option to freeze excess embryos is available to patients provided they indicate whether they would donate or dispose of the embryos if they died, were divorced, or were unable or unwilling to use them. The couple needs to understand the psychological and emotional implications of these decisions and come to agreement about the decision. Realistic expectations Couples who have been trying to conceive for several years are often extremely hopeful about the prospects of success when they commence treatment. While it is important that they remain positive, the counsellor should help them to develop a realistic idea of the chances of success, and educate them about the cumulative success rates so that they develop a broader view of their treatment than just focusing on the first cycle. The counsellor should not have the sole responsibility for doing this. The clinic literature and the information provided by the clinician should also address success rates realistically. If the counsellor is the only member of the team to refer to the fact that the treatment may not succeed, his or her relationship with the patients will be damaged, and he or she will be perceived by the patients as excessively negative. Psychological prepara tion An important aim of the counselling interview is to ensure that the couple is psychologically prepared for the treatment. This involves being aware of the possibilities of failure at the stimulation stage, egg pick-up, fertilization and implantation. The couples will also be encouraged to consider what part of the cycle will be most stressful for them, and what coping strategies may be useful. It is also important to consider what may be helpful for the husband and wife individually as well as jointly. A useful way to start the discussion may be to consider what has helped them to cope with their infertility in the past and what has been difficult for them. Compulsory counselling prior to treatment has both advantages and disadvantages. It can unfortunately contribute to patients' feelings of being out of control if they object to the fact that it is compulsory. They can sometimes be

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angry and view the interview as an invasion of privacy. While this does not occur very frequently, it does need to be dealt with in a sensitive way that acknowledges the couple's feelings. The compulsory counselling also tends to be very factually based, and perceptions of the nature of counselling and its potential to alleviate distress can be distorted because of this experience. However, the positive effects of counselling are that problems can be detected at an early stage and action can be taken. This may be as simple as correcting misunderstandings, or may require the team to be alerted to the fact that a patient is psychologically vulnerable. Problems that have a direct bearing on fertility can also be revealed during counselling. These include alcohol abuse and eating disorders. Perhaps the most compelling reason to have counselling as a requirement is that patients rate it as very helpful. Approximately threequarters of patients surveyed at our clinic described the pretreatment counselling as beneficial, and it is logical to infer they would be prepared to attend again if they needed to. Brief supportive counselling

As previously described, distress is to be expected when people are having fertility problems and are undergoing treatment aimed at achieving a pregnancy. It is appropriate to consider how counselling might alleviate this natural distress. If there are no compounding problems, it is common for patients to undertake brief supportive counselling. This may consist of up to ten consecutive sessions, or may consist of one or two sessions at times of maximum distress. It is not uncommon for patients to seek help regularly when, for example, a pregnancy does not occur after a treatment, or during treatment for a brief period, and not return until the next acute episode. In either case, the strategies employed by the counsellor closely follow Dershimer's (1990) description of informal counselling for the bereaved. The main components are outlined below. Empathic listening Because infertility is a socially unacknowledged loss, patients often have a great need to talk about their experiences. Facilitating the expression of fears, hopes, disappointments, anger and sadness can be a very powerful experience. It is important that the disclosure and expression of strong emotions are met with empathy and acceptance. This may be the first time that the patient has been able to express his or her feelings without being judged and made to feel inadequate and guilty because of them. Since his or her partner is also grappling with infertility, the person who may have provided support during

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other difficulties may not be available. If the couple present together, it may be the first time they have heard their partner speak freely about their pain. Hopefully, this will facilitate further communication between them and increase their understanding and tolerance of each other. The value of listening and sharing the patients' experiences should not be underestimated. Neither should the amount of energy and skill it requires to listen carefully and respond appropriately. Patients are often exquisitely sensitive to any indication that they are being judged, and every response that is given must be carefully thought through. The temptation to provide solutions and suggestions should be resisted until patients have been encouraged to express their feelings fully. Education Patients can derive benefit from education about normal emotional reactions to infertility. The hidden nature of infertility can result in people who are distressed feeling as if they are going crazy. They will frequently ask whether what they are experiencing is normal, and even if they don't ask, the counsellor should provide some reassurance about their normality. If patients have met with a disapproving response to their distress from their partners or others they are close to, this issue may need to be addressed by reviewing the losses of infertility and the difficulties of grieving those losses. Reading material is often beneficial, but one of the most powerful experiences is attendance at a support group. Contact with others who are also infertile can restore a sense of normality, and begin to break down isolation and feelings of helplessness. Problem solving The problems of everyday life are compounded by infertility. Patients frequently request help with a range of problems such as what to tell their employer about their frequent absences, how to respond to genuine questions about whether they have children, and how to preserve a relationship with a sister or friend who is pregnant and handle the intense emotions which arise when the baby is born. The confusion felt by the patient at a time of emotional distress can make these problems seem overwhelming. Systematically working out and evaluating the options can help the patient feel more in control. If the couple attends counselling, they may present relationship problems that need to be solved. Frequently, communication is a problem, with one or both partners feeling they cannot communicate their needs. Training in communication and conflict-resolution skills can resolve some of the problems, especially if the relationship is strong and the problems have been precipitated by infertility.

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The aim of therapy for infertility includes helping patients to move away from their identity as infertile patients and establish a different identity. The aim is to incorporate their infertility into a new identity that also includes many other characteristics that contribute to a positive self concept. The transition is likely to be long and painful and at times confusing. It may involve a deep questioning of the purpose of life and a re-evaluation of what contributes a sense of meaning to life. The concept of resolution is useful, but can be confusing as well. Resolution of infertility implies that it is possible to reach a point where infertility is not associated with emotional pain and distress. However, it may be more realistic to acknowledge that there will always be pain associated with infertility, but the intensity of the pain diminishes over time. The aim is for the pain of infertility to be accepted and incorporated into life so that it does not have the acute quality that it did in the earlier stages of diagnosis and treatment. Anton (1992) views resolution as a process that involves ten key steps. These are summarized below: 1. Acknowledging and experiencing the loss by talking about what the loss represents. 2. Understanding the loss. 3. Surviving the loss and letting go of feelings of being a victim. 4. Letting go of blame. 5. Talking to significant others and breaking down the silence surrounding infertility. 6. Using available resources, such as support groups, and establishing contact with people in a similar situation. 7. Finding different ways to nurture. 8. Re-establishing contact with children. 9. Maximizing the advantages of child-free living. 10. Embracing the quest for spiritual wholeness and the search for meaning in life. It is appropriate to undertake all or some of these steps in infertility therapy. Only after some steps have been taken to accept the loss that infertility represents can energy be directed towards other activities. In our experience, this process can be spread over many years, and it is frequently only some time after finishing treatment that patients present for therapy. The time delay presents a problem insofar as patients do not consider themselves entitled to infertility therapy at the clinic where they received treatment in the past. They

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may also prefer to seek therapy in a more emotionally neutral setting. It is therefore important that infertility counselling is freely available in the community and that clinics recognize that patients may require counselling after they have finished treatment.

Considerations for clinicians Communication with patients Treatment for infertility can extend over a long period of time, and can also ultimately be unsuccessful. It is therefore extremely important that the relationship with the clinician is positive. This is more likely to allow the patient to feel satisfied with what has happened in treatment, and to move on if treatment is ultimately unsuccessful. Treat the couple The decision to have a child is a couple's decision. The decision to proceed with consulting a specialist and to receive infertility treatment is also a couple's decision, and it is desirable that both the husband and wife are involved in consultations and decision making. This may not be possible at every consultation. However, it may be a warning sign if one of the partners is less involved. It may indicate lack of commitment to the treatment, or that the issues are too painful for the partner to become too involved. It may also lead to detachment and lack of knowledge, which may impact negatively on the marital relationship. Acknowledge limitations It is important that the limitations of treatment are acknowledged. Success cannot be guaranteed and this should be explicitly stated so that patients develop a realistic idea of what to expect, and also to help them to adjust in the event that success is not achieved. Acknowledging treatment limitations does not have to be excessively negative. Rather, positivity can be balanced with realism.

Raise difficult issues in a sensitive way The issues that need to be considered by infertile couples are often emotionally difficult, especially if they are in shock and still trying to come to terms with their situation. Allow space and time for them to absorb what is being said,

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and explicitly acknowledge that what you are saying may be difficult for them to hear. Psychological referrals

Early identification of problems and referral to a psychologist for specialist counselling are essential. The chronic and stressful nature of infertility means that problems can compound and affect the marital relationship and all areas of the patients' lives. A referral from the clinician is more likely to result in the patient attending counselling, and gives the clear message that the emotional well being of the patient is equally important as their physical well-being. However, psychological referral is not necessary for all patients. Consideration of the following risk factors may help in identifying those who may be helped by counselling. Riskfactors A previous history of psychological difficulties, especially depression or anxiety, may indicate underlying vulnerability. Infertility and infertility treatment are stressful enough to precipitate psychological difficulties, particularly if there is an underlying vulnerability. Patients who have come from dysfunctional or abusive families are especially at risk. Low self-esteem, guilt and self-blame are common reactions to abuse and these may leave the individual with few resources to deal with infertility. In addition, people who have experienced dysfunctional families have often learned to cope by concentrating on planning their own future family and the potential to create the love and security they did not experience in their own family. Infertility will therefore be a significant loss. As discussed previously, people for whom fertility is central to their identity may not cope well with infertility treatment. Those who have a range of activities which are satisfying and which provide them with a sense of mastery and accomplishment may be more resilient. Maladaptive emotional reactions may indicate potential problems while undergoing treatment. Anger is a normal reaction to infertility and thwarted hopes. However, excessive anger that is directed at the treatment team is maladaptive because it alienates those who are trying to help, and makes it difficult to reach out to the patient. Couples who have had very many treatments with no success, and those who proceed despite a very poor prognosis may be using denial to cope with the overwhelming emotions associated with infertility. Again, this may be viewed as a maladaptive reaction if it is preventing the couple from progressing to another stage of life.

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The distress of losing a pregnancy is compounded when there has been a history of infertility. Feelings of hopelessness and failure may be overwhelming. A referral for counselling following pregnancy loss should always be considered. It can take several months or more to grieve a pregnancy loss, and further treatment without success can also compound the feelings of loss. The more treatment a couple undertakes without success, the greater the risk they will suffer adverse psychological consequences. We recommend that all patients be offered a follow-up counselling interview after three unsuccessful cycles. This session can function to allow them to review their experiences and any difficulties they have experienced, and often includes a consideration of whether they will continue with treatment. Often further sessions are booked to allow more in-depth discussion of the issues. Counselling in donor programmes: the psychological and emotional aspects Donor issues

There are a number of important issues that need to be addressed in counselling. Informed consent Prior to consenting to become a donor, the donor and, if appropriate, his or her partner need to be fully informed of the treatment procedures. This includes an understanding of the drug therapy and its function, the risks, the possible side-effects, the success rates and the consent forms they are required to complete. The donor needs to be informed of his or her rights, the rights of the recipient couple and those of any children conceived through this process. A major concern for many people is the accessibility of identifying information. Current Victorian legislation provides for donor-conceived children to have access to such information when they become 18 years of age. There may also be the opportunity to make contact with their donor. An important consideration is the emotional impact this type of contact may have on all the individuals involved. The donor needs to consider whether such contact would be considered as an intrusion and a threatening experience rather than a positive and welcomed one. Psychological aspects of the donation process An important focus of counselling is the psychological and emotional aspects of the donation process. Firstly, it is important to ascertain the donor's

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motivations behind the decision to donate, and to ensure that not only are they genuine but also carefully thought through with regard to the risks and possible long-term implications (medically and psychologically). The maturity and insightfulness of the individual are taken into consideration as prognostic factors in their being able to cope with any difficulties which may arise from this action in the future. Ensuring that their motivations for donating are altruistic rather than a compensating experience for some other psychological distress is important when making an assessment of their ability to cope with the experience, and the type of impact it may have on their psychological well-being. The counsellor has a responsibility to protect the rights of donors by ensuring they are giving informed consent and their psychological state is such that they will be able to cope with the demands involved. Of equal importance is protecting the rights of the recipients, and thus it is important to ensure that donors are psychologically stable as well as medically fit and healthy. This involves a comprehensive clinical process of reviewing their psychosocial development and an assessment of their psychiatric history. Psychological assessment of donors

The complex role of psychologically screening donors evolved in our practice mainly in relation to anonymous donors. Known donors are chosen by the recipients, who take responsibility for their choice. A number of factors suggested that thorough assessment of anonymous donors was warranted. Firstly, it was unsatisfying from a clinical perspective that the time allotted to interviewing donors was not sufficient to gain a full clinical evaluation of their psychological profile. This was seen as more important for anonymous donors who have not usually had the strongly motivating experience of observing a close friend or relative struggling with infertility. Secondly, research conducted on the expectations of donor recipients (Alesi and Anderson, 1995) revealed that patients actually had high expectations regarding the screening of donors. The recipients involved in the study considered that the following factors should be included in the screening of the donors: medical history current physical and mental health status HIV/AIDS status sexually transmitted diseases educational level psychiatric illnesses

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illicit drug use alcoholism criminal record This type of feedback indicated that patients viewed psychological variables as important in the assessment of the donors. The initial evaluation can be done in one session taking approximately two to three hours. The first stage of the interview usually involves fulfilling the informed consent requirements and exploring issues relevant to becoming a donor. A full history is taken, exploring the potential donor's early childhood development through to adulthood, including educational, psychological and social development. Family history and interactions in significant relationships are investigated, including screening for psychiatric illnesses and behavioural disorders. The Minnesota Multiphasic Personality Inventory-2 (Butcher et ai, 1989) is administered to detect the presence of any psychiatric illnesses and to gain a greater insight into personality traits of the individual. Other standard assessment questionnaires may be used as required and, if further information is needed, a second interview can be scheduled. The need to assess donors should be balanced by an awareness that donors are voluntary and therefore do not get paid for their services, and the number of donors is already low. Therefore, a psychologically threatening and timeconsuming evaluation process may deter people from donating. It is our experience that the evaluation can be carried out in a non threatening manner. Known and anonymous donor issues If the donor is part of an open arrangement whereby they are known or related to the recipient, there are very important issues that require analysis in the counselling process. Firstly, it is vital to determine the nature of the arrangement and to ensure that it is a voluntary decision, not one resulting from subtle expectations that may be experienced by the donor as coercive. Secondly, the dynamics of the relationship between the donor and recipient couple require exploration. The arrangement can involve the wider family network, including potential grandparents, aunts, uncles, cousins. Thought needs to be given to how the child will be received into the family and viewed by others. Potential conflicts about how children should be raised, what they will be told about their conception, and what relationship they will have with the donor need to be considered. A discussion of the expectations of all parties and potentially difficult times for both the donor and recipient couple can be very beneficial. The aim is to facilitate discussion and the process of openness and honesty, especially regarding emotions.

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The question of whether the arrangement and genetic origins of a donor child are to remain a secret between the immediate parties involved or to be shared openly with family and friends is often difficult for all parties. The potentially damaging impact of children finding out about their donor origins from someone other than their parents, or during a time of family breakdown or bereavement, should be emphasized. The impact of secrecy should be explored, with the aim of raising the awareness and insight of the participants involved to the potential implications and consequences of their decisions. The impact of secrecy The literature on adoption and infertility treatment deals extensively with the secrecy that has often surrounded the genetic origins of an adopted or donorconceived child. A review of the literature indicates that there are some common elements in the issues raised, but also some very diverse ones which are particular to conception through donor gametes. One of the primary concerns about the presence of a secret in a family is how the existence of the secret can distort or influence the communication process between members of that family (Imber-Black, 1993). A further concern is the effect of the secret on the relationship between parents and children: 'Since the presence of a toxic secret can inhibit communication, a family's ability to solve problems or to confront normal developmental issues, may be seriously impaired' (Imber-Black, 1993: 13). The psychological pressure that is involved in maintaining a secret within a family may then affect the family's overall pattern of communication. To try to avoid the issue and any potential conflict that may arise if the child should ask questions, the parents and other family members may tell deliberate lies or withhold information. If the child should somehow discover the information from another source, trust and the credibility of family relationships may be eroded. Imber-Black (1993) suggests that for those who are the keepers of the secret, symptoms of guilt and anxiety may be experienced. This may develop as a result of the individual fearing disclosure and thus monitoring conversations to ensure that secrecy is maintained. Those who are excluded from the secret (i.e. the child) can also experience anxiety as they sense the interpersonal tension in their environment, which can even affect their sense of belonging within the family. One of the important differences raised in the literature about secrecy in adoption versus secrecy in infertility is the fact that in the disclosure of adoption the issue of infertility is not directly raised (Cook et al, 1995). In the disclosure of the genetic origins of a donor child, an explanation about the conception process and thus one parent's infertility is acknowledged and

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perhaps raised for discussion. This may elicit a number of responses from the parent in question, particularly re-awakening concerns about his or her own infertility. For example, feelings of depression, fear of rejection, lack of sexual potency may be re-experienced many years later. The confinements of secrecy may be rationalized as a way of protecting the child from the stigma of being different, when in reality it may be a form of denial, protecting the parents from openly acknowledging their infertility and the child's genetic origins. These are complex psychological mechanisms underlying human behaviour and the intellectual processes involved in decision making. Psychological defences often operate at an unconscious level to protect the individual from emotionally threatening stimuli. It is well recognized in the literature that the experience of infertility is one which challenges the psyche and those aspects of the ego that are central to the functioning of human nature, such as selfesteem, identity and sexuality. The parents of a child conceived through donor gametes face the additional complexities associated with being a social parent. Although there may be ethical and psychological advantages to being open with children regarding their genetic origins, serious consequences could ensue if parental communication is inadequate or if the information is revealed in a damaging way such as in anger (Mahlstedt and Greenfeld, 1989; Papp, 1993). Research and information from studies of adoption can be used to inform the recipients of how to approach the issue of telling their child in the most supportive way. Access to further counselling once they have become parents is most important to provide the support when it is needed most. Preparation of the donor parent needs to be reinforced and supported at various levels, not just at pretreatment counselling. During the early stages when the clinician makes the diagnosis and proposes a treatment plan, his or her opinion and support can be important influences in preparing couples for the issues they will be facing. They would also help the couple to maintain a realistic perspective on their situation. On a societal level, some thought needs to be given to the provision of services for families as it is likely that parents who have donor-conceived children will require support at various stages of their family's development. It is possible that support at later stages will be as important or more important in terms of the family's functioning than the counselling they receive at the time of accessing treatment. The recipients: the psychological issues

In becoming a recipient of an embryo or gamete donation, the recipient couple has to come to terms with a number of issues before undertaking any treat-

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ment. It is the role of the counsellor to assist the wife and husband individually and as a couple to come to terms with their infertility. An important process is the resolution of the grief associated with the infertile partner not being able to have his or her own genetic child. Infertile couples often experience a range of emotional reactions. Feelings of anger, grief, sadness, lowered self-esteem and self-blame are quite common reactions whether they have experienced prior IVF treatment or not. Repeated failed treatment cycles, miscarriage or stillbirth can further compound the experience of grief and disappointment. Coming to terms with these issues prior to starting a donor programme can help the couple become better prepared to deal with the new demands and issues associated with being recipients of donor gametes or embryos. If a couple conceives a child through an anonymous donation, there are other issues to review. It is important to raise the possibility that the child may be curious about the donor, and may even initiate a search for the donor. This possibility can be experienced as emotionally threatening by the parents, eliciting fears that the child will reject them for the genetic parent. One way of helping the recipients address this issue is to assist them to shift their perspective and look upon the importance of their role in this entire transaction. If it is a female infertility problem, the recipient woman will be the gestational mother and thus have the opportunity to bond with her child from the moment of conception as well as throughout the pregnancy. The same principle applies if it is a male factor infertility problem. Even though the sperm was donated, the husband will be responsible for supporting his wife throughout the pregnancy and providing for their child. Another important aspect of counselling recipients is helping them to reframe their concept of the role of parenthood. In our society a great deal of importance is placed on the genetic link between parent and child. The emphasis is shifted to examining the importance of the actual relationship that can develop between parent and child, including the type of nurturing that is involved at an emotional and intellectual level. This helps to bring the focus back to a consideration of the parents' social influence and the input they will have into the child's psychological development, rather than making the assumption that a satisfying and effective parent-child relationship would occur automatically if there were a genetic link. The issues for donor-conceived individuals

Reproductive technology and the culture that has developed in infertility clinics focus primarily on the welfare and interests of the infertile couple. In recent years a new voice has emerged: the voice of the donor-conceived

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individuals seeking acknowledgment and the right to access information about their genetic heritage. The rights of offspring to access information about the donors who contributed to their genetic make-up are only being recognized very slowly. However, legislation in some jurisdictions has acknowledged that they should be accorded these rights. For example, the Infertility (Medical Procedures) Act 1984 (Victoria) has required that detailed identifying information about donors be kept in a central register. The Infertility Treatment Act (1995) (Victoria) supersedes the earlier act and ensures that children will have the right to access this information at the age of 18, whether or not the donor agrees to being identified. Counselling must be available to all parties at this time. The new Act also emphasizes that the welfare and interests of the child should be considered when planning treatment. While it will be some years before the effects of the legislation will be experienced, the message is clear - that the interests of the child should be represented. There are still many offspring who will not benefit from this legislation and who may require counselling to deal with the secrecy that surrounded their birth, and the impossibility of discovering who their genetic parents were. An American, Bill Cordray, has argued persuasively for the rights of donor-conceived children. He was himself conceived as a result of artificial insemination with donor sperm, and presented a seminar in Melbourne, Australia in 1993, entitled 'Let the offspring speak'. His interest and research in the area were promoted by his own experiences of finding out as an adult that he was not genetically related to the father who had brought him up. Cordray's belief is that a people's connections to their past (genetic origins) are necessary elements for the development of their identity and for the growth of self-esteem and psychological health. He also acknowledges the fear of rejection that motivated his parents to conceal the circumstances of his conception from him: 'My last few years with my Dad were closer than I had ever expected. I know that if he had not had to fear my feelings about him we would have been closer from the start. I never would have rejected a person who trusted me with his pain. I am still mourning for the loss of a full relationship that was denied out of uninformed fear.' (Cordray, 1993: p. 13). The experiences of people conceived through donated gametes are only just beginning to be documented. However, those experiences should inform the counselling that is carried out in donor programmes. One of the biggest problems for counsellors and patients has been the lack of follow-up information about families. Now that some voices are being heard, the isolation and secrecy that have accompanied infertility, and particularly donor programmes, can begin to break down. It is likely that counselling services in the

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future will need to respond to the needs of families as they develop, and the needs of donor-conceived offspring. References Alesi, R. and Anderson, J. 1995. Screening of Gamete Donors: The Recipients' Expectations. Unpublished manuscript. Anton, L.H. 1992. Never to be a Mother: A Guide for All Women Who Didn't - or Couldn't - Have Children. New York: Harper Collins. Australian National Bioethics Consultative Council 1990. Issues Paper on Infertility Counselling.

Becker, G. 1990. Healing the Infertile Family. New York: Bantam. Butcher, J., Dahlstrom, W., Graham, J., Tellegen, A. and Kaemmer, B. 1989. Minnesota Multiphasic Personality Inventory-2 MMPI-2: Manualfor Administration and Scoring. Minneapolis: University of Minnesota Press. Callan, V. and Hennessey, J. 1988. Emotional aspects and support in in vitro fertilization and embryo transfer programs. Journal of In Vitro Fertilization and Embryo Transfer 5: 290-95. Cook, R., Golombok, S., Bish, A. and Murray C. 1995. Keeping secrets: A study of parental attitudes toward telling about donor insemination. American Journal of Orthopsychiatry 65: 549-59. Cordray, A. W. 1993. Let the offspring speak: The need for a sense of self-identity and the impact of secrecy on families who adopt through donor insemination with anonymous fathers. Seminar presentation. Dershimer, R.A. 1990. Counselling the Bereaved. New York: Pergamon Press. Gervaise, P. 1993. The psychological impact of infertility: the separation of procreation and recreation. The Canadian Journal of Human Sexuality 2: 104-06. Imber-Black, E. 1993. Secrets in Families and Family Therapy. New York: W.W. Norton. Jennings, S.E. 1995. Infertility Counselling. London: Blackwell. Leon, I.G. 1990. When A Baby Dies. Psychotherapy for Pregnancy and Newborn Loss. New Haven, London: Yale University Press. Mahlstedt, P.P. 1985. Psychological components of infertility. Fertility and Sterility 43: 335-46. Mahlstedt, P.P. and Greenfeld, D.A. 1989. Assisted reproductive technology with donor gametes: The need for patient preparation. Fertility and Sterility 52: 908-14. Mahlstedt, P. MacDuff, S. and Bernstein, J. 1987. Emotional factors and the IVF and ET process. Journal of In Vitro Fertilization and Embryo Transfer 4: 232-6. Neuberg, R. 1995. Infertility counselling: A consultant's personal view. In Infertility Counselling, ed. S.E. Jennings, pp. 231-3. London: Blackwell. Papp, P. 1993. The worm in the bud: Secrets between parents and children. In Secrets in Families and Family Therapy, ed. E. Imber-Black, pp. 66-85. New York: W.W. Norton. Savage, J. 1989. Mourning Unlived Lives. A Psychological Study of Childbearing Loss. Illinios: Chiron Publications. Waller Report 1984. Committee to Consider Social, Ethical and Legal Issues arising from In Vitro Fertilisation. Parliament of the State of Victoria, Melbourne. Warnock, M. 1985. A Question of Life. Oxford: Blackwell.

Index

abortion infertility and, 3 selective reduction of fetuses, 244 spontaneous, 243 adenomyosis, 181 treatment of, 204-6 adhesions, 34, 116 anovulation categorization of, 70-2 diagnosis of, 69-70 egg donation and, 155-6 early pregnancy endocrinology, 157-8 IVF and, 92-3 physiology of, 70 treatment of, 72-9 see also ovulation assessment; ovulation induction artificial insemination with husband's sperm, 64, 83-6 subnormal semen quality and, 85-6 see also intrauterine insemination Asherman's syndrome, 180 assessment of female partner, 17-38 pelvic viscera, 31-8 falloposcopy, 35-8 hysterosalpingography, 32-3 hysteroscopy, 33-4 laparoscopy, 34-5 ultrasound, 32 referring letter, 19 see also examination; history taking; ovulation assessment assessment of male partner, 40-8 history taking, 10,41-2 investigations, 43-7 biochemistry, 45 genetic tests, 46-7, 51 see also examination assisted reproductive technology (ART) indications for, 88-9 male infertility and, 125-37 patient selection, 125 sperm selection methods, 126

results of birthweights, 245-6 congenital malformations, 246-7 ectopic pregnancies, 243-4 in Australia and New Zealand, 239-47 live birth rates, 240-1 maternal age, 242 method of delivery, 245 multiple pregnancies, 245 number of embryos/oocytes transferred, 242-3 number of oocytes collected, 242 perinatal mortality, 246 pregnancy rates, 240-2 preterm birth rates, 244-5 reporting of success rates, 235-9 selective reduction of fetuses, 244 spontaneous abortion, 243 see also individual techniques azoospermia, 50-1, 53-4 ICSIand, 132-3, 135-6 basal body temperature charting, 11-13 ovulation assessment, 27 bicornuate uteri, GIFT and, 117 birth rates, 240-1 preterm, 244-5 birthweights, 245-6 body temperature see basal body temperature boost stimulation regime, 99 breast development, 24 bromocriptine, 78-9 cervical hostility, 84-5 Chlamydia trachomatis, 2-3 cigarette smoking, 3, 18 clomiphene citrate, 72-5, 97 coelomic metaplasia, 163-4 coital disorders, 57-9 congenital absence of the vas, 42, 52-3 ICSIand, 132-3 congenital malformations, 246-7

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Index

congenital uterine abnormalities, 180-1 controlled ovarian hyperstimulation (COH) regimes, 96-102 boost stimulation regime, 99 cancellation, 101-2 cycle management, 100-1 down regulation, 99 immature oocyte collection, 100 minimal stimulation, 99-100 natural cycle, 100 therapeutic agents, 96-9 risks of, 86 with IUI, 85-6 see also ovulation induction counselling, 249-68 brief supportive, 256-7 considerations for clinicians, 259-61 definition of, 249-50 donor programmes and, 261-8 donor issues, 261-5 issues for donor-conceived individuals, 266-8 recipient issues, 265-6 female partner, 17-18 pretreatment counselling, 254-6 psychological referrals, 260-1 therapeutic, 258-9 culture media, 224-5 Cushing's syndrome, 31 cysticfibrosis,42, 52-3, 133 cysts, ovarian, 178 cystoscopy, 200 cytomegalovirus (CMV), donor insemination and, 142-3 delivery, method of, 245 dietary supplements, 63 donor insemination (DI), 139-48 donor selection, 141-4 failed, as indication for IVF, 94 follow-up of children, 147-8 history of, 139-40 indications for, 141 legal aspects, 147 outcome of, 145-6 pretreatment assessment, 144-5 social aspects, 147 see also counselling down regulation, 99 drug exposure, spermatogenesis and, 59-60 ectopic pregnancy, 3, 243-4 GIFT and, 116-17 laparoscopic treatment following ART, 214-15 egg donation, 94-5 donor characteristics, 153-5 age, 154 screening, 154-5 early pregnancy endocrinology in anovulatory women, 157-8 HRTand, 158 endometrium preparation, 155-7 anovulatory cycles, 155-6

ovulatory cycles, 156-7 follow-up of, 159-60 future strategies, 161 GIFT and, 117 history of, 151-2 indications for, 152 legal aspects, 160-1 recipients of, 152-3 results of, 158-9 see also counselling ejaculation electroejaculation, 59 failure of, 58 retrograde, 58 embryo culture, 222-7 closed culture system, 225-6 culture media, 224-5 open culture system, 226-7 embryo donation, 94-5, 151, 160 embryo storage, 221-2,232-3 embryo transfer (ET), 88-9,104-6, 151,233 embryo assessment, 232-3 number of embryos transferred, 242-3 sexed embryos, 95 technique, 105-6 ultrasound use, 183—4 see also in-vitro fertilization embryology laboratory, 220-2 checking systems, 233-4 laboratory records, 233 quality assurance, 234 security, 233-4 embryonic cell arrest, 164 endometriomas diagnosis, 197 removal techniques, 197-200 endometriosis, 35, 110, 163-72, 190-204 as indication for IVF, 91-2 classification of, 166-7 clinical aspects, 165 diagnosis of, 165-6 genetics, 164 incidence of, 163 infertility and, 167-70 treatment outcomes, 169-70 medical therapy, 170-1 pathogenesis, 163-4 surgical treatment, 171-2, 190-204 ovarian endometriomas, 197-200 peritoneal lesions, 192-6 pouch of Douglas, 201-4 removal of ovary, 200 ultrasound examination, 177-8 endometritis, 180 endometrium biopsy, ovulation assessment, 27 preparation for egg donation, 155-7 anovulatory cycles, 155-6 ovulatory cycles, 156-7 endoscopy, 212-15 see also laparoscopy eugonadotrophic hypogonadism, 71 -2

Index examination female partner, 23-5 first interview, 10 gynaecological examination, 24-5 male partner, 42-3 first interview, 10-11 see also assessment of female partner; assessment of male partner fallopian replacement of eggs with delayed intrauterine insemination (FREDI), 117-18 fallopian tubes, transcervical catheterization, 184 falloposcopy, 35-8 diagnostic application, 36-7 therapeutic application, 37-8 fecundability, 1-2,20-2 fertile eunuch syndrome, 56 fertilization evaluation, 231-2 fibroids see fibromyomas fibromyomas, 178,206-12 abdominal myomectomy, 208-9 GnRH agonist treatment, 207-8 laparoscopic myoma reduction, 211-12 laparoscopic myomectomy, 209-10 fimbrioplasty, 188-9 follicle stimulating hormone (FSH) controlled ovarian hyperstimulation, 97 levels in male partner, 45, 51 follicular development, ultrasound monitoring, 181-2 follicular function, endometriosis and, 168 galactorrhoea, 30 gamete intrafallopian transfer (GIFT), 109-20, 127-8 bicornuate uteri and, 117 combined use of, 115-16,212-13 comparison with intrauterine insemination and superovulation, 119-20 culdoscopic GIFT, 114 current status of, 120 ectopic pregnancy and, 116-17 effectiveness of, 118-19 egg donation and, 117 fallopian replacement of eggs with delayed intrauterine insemination (FREDI), 117-18 history of, 109 laparoscopic GIFT, 111-13 number of oocytes transferred, 114-15, 242-3 patient selection, 110-11 physiological basis, 109-10 surrogacy and, 117 vaginal GIFT, 113-14 see also assisted reproductive technology genital tract inflammation, male, 62-3 genital tract obstructions, male, 52-4 ICSI and, 132-3 gonadotrophin deficiency, 56-7 gonadotrophin releasing hormone (GnRH) anovulation treatment, 79

271 GnRH agonists controlled ovarian hyperstimulation, 97-9 fibromyoma treatment, 207-8 gynaecological examination, 24-5 hirsutism, 30-1 history taking female partner, 9-10 infertility history, 22-4 medical and surgical history, 19-22 first interview, 9-13 male partner, 10, 41-2 see also assessment of female partner; assessment of male partner HIV, donor insemination and, 142, 143 hormone replacement therapy (HRT), egg donation and, 155-7, 158 human menopausal gonadotrophin (hMG) anovulation treatment, 75-8 controlled ovarian hyperstimulation, 98-9 hydrosalpinges, 32, 181 hypergonadotrophic hypogonadism, 71 hyperprolactinaemia, 72, 78 hypogonadotrophic hypogonadism, 71 hysterosalpingography, 32-3 hysteroscopy, 33-4 hysterosonography, 184 identity issues, 252-3 immature oocyte collection, 100 immunological infertility, 94 endometriosis and, 168-9 sperm autoimmunity, 54-6 impotence, 57-8 in-vitro fertilization (IVF), 88-9, 127 indications for, 90-6 luteal phase support, 106 oocyte retrieval, 102-4 process of, 220, 227-33 embryo assessment, 232-3 embryo transfer, 233 fertilization evaluation, 231-2 insemination, 230-1 oocyte assessment, 228-9 oocyte collection, 227-8 sperm preparation, 229-30 see also assisted reproductive technology; controlled ovarian hyperstimulation; embryo transfer infertility, 1-2 causes of, 2 emotional experiences and, 4 endometriosis and, 167-70 treatment outcomes, 169-70 female risk factors, 2-3 historical aspects, 4-6 immunological, 94, 168-9 male ART and, 124-37 as indication for IVF, 90-1 genetic basis, 135 risk factors, 3-4

272

Index

infertility, male (com.) subfertility, 61-5 transrectal ultrasound assessment, 185 psychological consequences of, 252-3 psychological implications of, 250-2 tubal, 90 unexplained, 93-4 untreatable sterility, 50-2 see also individual causes of infertility informed consent, 254-5 donors, 261 intracytoplasmic sperm injection (ICSI), 91, 128-36,231 development of, 128-9 epididymal sperm, 132-3 indications for, 129-30 physiological questions and, 134 results of, 131-2 safety of, 134 genetic basis of infertility and, 135 technique, 130-1 testicular failure and, 135-6 testicular sperm, 133 intrauterine insemination (IUI), 85-6 risks of, 86 with superovulation, 119-20 see also artificial insemination with husband's sperm; donor insemination Kallmann's syndrome, 46, 56, 57 Klinefelter's syndrome, 46, 51 laboratory records, 233 laparoscopy, 34-5 combined use of with GIFT, 115,212-13 ectopic pregnancy treatment following ART, 214-15 laparoscopic GIFT, 111-13 myoma reduction, 211-12 myomectomy, 209-10 role in patient selection for ART, 212 libido, low, 58 live birth rates, 240-1 luteal phase deficiency (LPD), 27-8 luteal phase support, 106 luteinized unruptured follicles (LUF), 28 luteinizing hormone assay, 28 macroadenoma, 79 maternal age, 242 menstrual cycle history taking, 9 see also natural cycle protocol microdrop insemination, 127 microepididymal sperm aspiration (MESA), 53-4 minimal stimulation, 99-100 miscarriage, 243 multiple pregnancies, 245 reduction, 185 myomas see adenomyosis; fibromyomas

myomectomy abdominal, 208-9 laparoscopic, 209-10 natural cycle protocol, 100 management of, 101 ultrasound monitoring, 181-2 necrospermia, 133 Neisseria gonorrhoea, 2, 3 nutritional supplements, 63 obesity, ovulation assessment and, 29-30, 69-70 oocyte culture, 222-7 closed culture system, 225-6 culture media, 224-5 open culture system, 226-7 oocyte donation see egg donation oocyte insemination procedure, 230-1 see also intracytoplasmic sperm injection oocyte retrieval, 102-4, 222 immature oocyte collection, 100 oocyte assessment, 228-9 technique, 213-14, 227-8 orchitis, 3 ovarian cysts, 178 cystoscopy, 200 ovarian hyperstimulation syndrome (OHSS), 79-80 ultrasound diagnosis, 185 ovarian stimulation see controlled ovarian hyperstimulation; ovulation induction ovary, removal of, 200 ovulation assessment, 25-31 abnormalities of ovulation, 29-31 history taking, 9 investigations, 27-9 symptoms of ovulation, 26-7 ultrasound monitoring, 28-9, 181-2 see also anovulation ovulation induction, 72-9 bromocriptine, 78-9 clomiphene citrate, 72-5 hMG, 75-8 pulsatile GnRH, 79 see also controlled ovarian hyperstimulation ovulatory disorders see anovulation pelvic collections, 181 pelvic inflammatory disease, 2-3 perinatal mortality, 246 peritoneal fluid inflammation, 169 polycystic ovarian disease (PCOD), 72 clomiphene citrate treatment and, 74 hMG treatment and, 75-8 IVF and, 92-3 ovulation assessment and, 30 ultrasound examination, 178 polyps, 179 postcoital test, 84-5 pouch of Douglas, 201-4 pregnancy rates, 240-2

273

Index preterm birth rates, 244-5 progesterone assay, 28 pyosalpinges, 181

superovulation (SO), 119-20 surrogacy, 95 GIFT and, 117

relaxin, 158 retrograde ejaculation, 58

Tanner staging, 24 testosterone levels, 45 toxin exposures, 59-60 tubal embryo stage transfer (TEST), 118-19 tubal infertility, 90 endometriosisand, 168 see also tubal surgery tubal patency, 35 tubal surgery, 187-90 fimbrioplasty, 188-9 results of, 189-90 salpingo-ovariolysis, 188 salpingostomy, 189 tubotubal anastomosis, 190

salpingo-ovariolysis, 188 salpingostomy, 189 self esteem, low, 252 semen analysis, 44-5 specimen collection, 44, 221 sperm function studies, 47-8 seminiferous tubule failure, 50-1 smoking, 3, 18,63 sperm acrosome reaction, 229 autoimmunity, 54-6 capacitation, 229 function studies, 47-8 preparation for IVF, 229-30 selection methods for ART, 126 specimen collection, 44, 221 spermatogenesis, toxin and drug exposure and,

59-60 spontaneous abortion, 243 sterility, untreatable, 50-2 see also infertility stillbirths, 246 stress, 17-18,64 reactions, 253-4 reduction of, 18-19 subfertility, male, 61-5 hormonal treatment, 63 lifestyle factors, 63-4 nutritional supplementation and, 63 prognosis for natural pregnancy, 64-5 see also infertility subzonal injection of the oocyte (SUZ1), 91, 129

ultrasound, 32, 176-85 assisted reproductive techniques and, 183-4 follicular development monitoring, 181-2 investigations, 176-81 male infertility assessment, 185 multiple pregnancy reduction, 185 ovarian hyperstimulation syndrome diagnosis, 185 ovulation assessment, 28-9, 181-2 pregnancy assessment, 184-5 uterus bicornuate, GIFT and, 117 congenital abnormalities, 180-1 receptivity, ultrasound assessment, 183 uterine irrigation, 184 varicocele, 3, 62 vitamins, male subfertility treatment, 63 zygote intrafallopian transfer (ZIFT), 118-19