The Art and Science of Assisted Reproductive Techniques

The Art and Science of Assisted Reproductive Techniques (ART) The Art and Science of Assisted Reproductive Techniques...

Author: Gautam N. Allahbadia | Rita Basuray Das

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The Art and Science of Assisted Reproductive Techniques (ART)

The Art and Science of Assisted Reproductive Techniques (ART) Editors Gautam N Allahbadia MD DNB Scientific Director, Rotunda-Center for Human Reproduction Mumbai, India Consultant, Rotunda-Virk Center for Human Reproduction Jalandhar, India Consultant, Rotunda-Hygeia Center for Human Reproduction Srinagar, India Consultant, Rotunda-Southend Center for Human Reproduction New Delhi, India Consultant, Bombay Hospital and Medical Research Centre Mumbai, India

Rita Basuray Das PhD Medical Liaison Executive Department of Reproductive Health North America Serono USA

Assistant Editor Rubina Merchant PhD Embryologist, Rotunda-Center for Human Reproduction Mumbai, India

Foreword by Bruno Lunenfeld MD FRCOG FACOG (hon) POGS (hon) Faculty of Life Sciences Bar -Ilan University Ramat Gan 52900 Israel

LONDON AND NEW YORK A PARTHENON BOOK

© 2003 GN Allahbadia and RB Das First published in India in 2003 by

Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India. EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi 110 002, India Phones: 23272143, 23272703, 23282021, 23245672 m\, Fax: +91–011–23276490 e-mail: [email protected], Visit our website: http://www.jaypeebrothers.com/ This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to http://www.ebookstore.tandf.co.uk/.” First published in the United Kingdom by Taylor & Francis in 2004. Exclusively distributed worldwide (excluding the Indian Subcontinent) by Martin Dunitz, a member of the Taylor & Francis Group. Tel.: +44 (0) 20 7583 9855 Fax.: +44 (0) 20 7842 2298 E-mail: [email protected] Website: http://www.dunitz.co.uk/ This edition published in the Taylor & Francis e-Library, 2005. All right reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or othrwise, without the prior permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. Although every effort has been made to ensure that drug doses and information are presented accurately in this publication, the ultimate responsibility rests with the prescribing physician. Neither the publishers nor the authors can be held responsible for errors or for any consequences arising from the use of information contained herein. For detailed presceibing information or instructions on the use of any product or procedure dicussed herein, please consult the prescribing information or instructional material issued by the manufacturer. A CIP record for this book is available from the British Library. ISBN 0-203-64051-9 Master e-book ISBN

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dedicated to our spouses Swati and Sumit

Foreword

Prof Bruno Lunenfeld, MD, FRCOG FACOG (hon) POGS (hon) Faculty of Life Sciences, Bar -Ilan University Ramat Gan 52900 Israel The “conquest of infertility” is an incredible achievement, it is a victory of human will, endurance and technology. However as we have entered the new millennium, new challenges are arising in relation to scientific, ethical and humanitarian aspects in infertility management. However, in spite of our increasing knowledge and skills many questions remain unanswered and new concerns and problems constantly arise. In the early nineties, ICSI was still an experimental therapeutic approach yet in the meantime it has revolutionized the treatment of male infertility worldwide. Testicular sperm extraction for ICSI and cryopreservation techniques are important milestones in assisted reproductive technology while in vitro maturation of germinal cells the intracytoplasmic injection of round and elongated spermatids are still in the experimental stage. In the last few years new compounds such as recombinant gonadotropins (FSH LH and hCG) and GnRH antagonists came into use for ovulation induction and controlled ovarian stimulation which improved safety, allows for individualized adjusted therapies and make treatment more patient friendly. Pre-implantation genetic diagnosis is improvingbut most genetic implications of assisted reproduction are still under debate. Oocyte donation is now widely practiced, but the moral, ethical and judiciary issues of Oocyte sharing spreading from country to country and surrogacy by embryo transfer is are still under debate. There is also an important public demand for information and transparency regarding pregnancy and fetal outcome after ART. The relative excessive rate of high order multiple pregnancies with negative effects on mother and child has still to be accepted by all as a serious complication. More efforts are needed to reduce multiple pregnancy rate as well as the occurrence of hyperstimulation and its consequences. Those involved in reproductive medicine must, on the one hand, make all possible efforts to enable conception to all couples desiring it and, on the other hand, make the utmost to try to avoid maternal and neonatal complications such as the hyperstimulation syndrome, multiple pregnancy and premature delivery. These are formidable goals and this book attempts with theoretical considerations, discussions and newer insights into many aspects within the broad aspect of assisted reproduction to become an important tool for those interest in the field of reproductive medicine It covers the basics of reproductive endocrinology, theoretical and practical aspects of ovarian stimulation and modern understanding and management of PCOS. The book

describes recent developments in drug research with reference to both “gonadotropins, GnRH antagonists and newer delivery systems. Besides useful sections on Endoscopy, Third Party Reproduction, and Implantation a chapter dedicated to stem cell culture and replication written by prominent scientists can be found. This is a truly comprehensive book for the Obstetrician and Gynecologist who is yearning for knowledge in the field of ART. This book has brought together leading medical and scientific experts who describe in a clear and concise manner the “how, why and therefore” of ART. It has been written to be readable and usable by research fellows, who want to get an insight into the technical developments, by a clinical and scientific team who want to know the A to Z of setting up an ART program, as well as “veterans” in the field who want an up-to-date review on the newest techniques and advances.

Bruno Lunenfeld

Preface

In his famous preface to Cromwell, Victor Hugo pointed out that one seldom inspects the cellar of a house after visiting its salons nor examines the roots of a tree after eating its fruit. The readers of this book will judge it by the substance of its contents and its style, not by a pretext. If the guest has returned several times, then surely he knows that the cellar is well stocked. Having said this, it is reasonable to ask why prefaces and forewords are written and are they ever read? My contribution is intended to present the objectives of this book. Within the last two decades, the amount of information generated by theoretical and clinical research in the field of reproductive physiology and infertility has increased so enormously that it has become virtually unmanageable. To be professionally efficient, however, both the research worker and the clinician must constantly keep abreast with recent developments and discoveries. The different chapters in the book cover a wide range of reproductive medicine and science. They represent the cutting-edges of our discipline and are written by acknowledged experts in the field. The editors demonstrated an exceptional capacity to select contributors who are at the forefront of their disciplines, thereby ensuring the freshness of each chapter. To convey the essence of a rapidly growing field in a single book, it is necessary to be selective in both the extent and depth of coverage. This is especially true for a book designed for students, researchers and practitioners of medicine. This book covers the “classical problems” of reproductive medicine, including stimulation protocols, ART procedures, implantation, cryopreservation, endocrinological disorders, male factor, endoscopy and ultrasonography. Chapters on third party reproduction, PGD, unsolved problems such as treatment of poor responders, treatment of the older patients and endometriosis, and the future of infertility therapy provide important and recent information, as well as food for thought. I sincerely hope that young doctors and experienced clinicians, medical students and teachers, as well as scientists working in the field of reproductive medicine and endocrinology, will find this book interesting and helpful. Moreover, I sincerely hope that this overview will stimulate new work and a continuous dialogue between basic scientists and clinicians, between gynecologists and andrologists, and between immunologists, endoscopic surgeons and psychologists. Without doubt, teams of highly specialized scientists and clinicians must teach reproductive medicine, and infertile couples should be

treated as a unit by specialists with a broad scientific basis and clinical experience in the diagnosis and therapy of both male and female infertility. I would like to express my appreciation to all the authors for their valuable contributions. They produced a book that is not only of critical substance but is also a real pleasure to read. Dusseldorf, Germany Hugo C Verhoeven MD

List of Contributors

Vida Acosta MD TS Andrology Laboratory Services Inc Chicago, Illinois, USA Ashok Agarwal PhD HCLD Center for Advanced Research in Human Reproduction Infertility, and Sexual Function Urological Institute and Department of Obstetrics & Gynecology Cleveland Clinic Foundation Cleveland, Ohio 44195, USA Richard A Ajayi MRCOG Consultant Gynaecologist and Director The Assisted Conception Unit, The Bridge Clinic Limited Victoria Island, Lagos, Nigeria Erdal Aktan MD Ege IVF Center, Izmir-Turkey Gautam N Allahbadia MD DNB Scientific Director, Rotunda-Center for Human Reproduction Mumbai, India Consultant, Rotunda-Virk Center for Human Reproduction Jalandhar, India Consultant, Rotunda-Hygeia Center for Human Reproduction, Srinagar, India Consultant, Rotunda-Southend Center for Human Reproduction, New Delhi, India Consultant, Bombay Hospital and Medical Research Centre Mumbai, India Swati G Allahbadia MD Associate Professor, Department of Obstetrics and Gynecology Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai, India

Najar Amso PhD FRCOG Senior Lecturer and Consultant Gynaecologist University Hospital of Wales Heath Park, Cardiff, Wales Claus Yding Andersen MSc DMSc Laboratory of Reproductive Biology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Carlo De Angelis MD Professor, Chief-Center for Minimally Invasive Therapy Department of Gynecological Sciences and Perinatology University of Rome, “La Sapienza” Policlinico Umberto I, Rome, Italy Monica Antinori MD Resident Fellow, Center for Minimally Invasive Therapy Department of Gynecological Sciences and Perinatology University of Rome, “La Sapienza” Policlinico Umberto I 00161 Rome, Italy Gerardo Ardiles MD Department of Reproductive Medicine Copiapo, Arica Regional Hospital, Chile Foad Azem MD Racine IVF Unit Department of Obstetrics and Gynecology Tel-Aviv Sourasky Medical Center Affiliated to Sackler School of Medicine University of Tel-Aviv, Israel Ramesh B MD DGO FCPS DICOC DFP, Diploma in Gynecological Endoscopy Consultant Gynaecologist and Endoscopic Surgeon Bangalore, India Antonio Barbaro ASIC Associazione per lo studio dell’infertilita di coppia Roma Viale Aventino 61, Italy Sudip Basu MD DNB MRCOG MRCPI Research Fellow and Senior Registrar Cardiff Assisted Reproduction Unit University Hospital of Wales, Heath Park Cardiff, Wales Micha Baum MD IVF Unit Department of Obstetrics and Gynecology Sheba Medical Center, Tel-Hashomer, Israel 52621 and Sackler School of Medicine Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel

Asha Baxi MS MRCOG Disha Fertility and Surgical Centre Indore (MP), India Angela Beaten PhD Chief Scientist, Isis Clinic, Waikato Hospital Hamilton, New Zealand Claudio A Benadiva MD IVF Laboratory Director The Center for Advanced Reproductive Services Associate Clinical Professor, University of Connecticut Farmington, Connecticut, USA Alexandra Bermúdez MD Associate of Reproducciòn y Genètica Hospital Angeles del Pedregal Universidad La Salle, Mèxico City, Mexico Jaydip Bhaumik MBBS DGO MS DNB MRCOG Staff Grade Doctor Department of Obstetrics and Gynaecology Princess of Wales Hospital Bridgend, Wales Zeev Blumenfeld MD Associate Professor, Reproductive Endocrinology Department of Obstetrics and Gynecology Rambam MedCtr Technion-Faculty of Medicine Haifa, Israel Andrea Borini MD Director, Tecnobios Procreazione Bologna, Italy Bijit Chowdhury DGO MD Reader, Dept. of Obstetrics and Gynecology Vivekananda Institute of Medical Sciences Kolkata, India Makolm Clarke BAppSc (Medical Science) MBA (International business) Senior Management Systems Assessor, Australia Silvio Cuneo MD Associate of Reproducciòn y Genètica Hospital Angeles del Pedregal, Universidad La Salle Co-Director of Centro de Fertilidad Humana Mèxico City, Mexico Ben-Yosef Dalit Sara Racine IVF Unit, Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Israel

Sajal Datta MD FICMCH Assistant Professor, Dept. of Obstetrics and Gynecology Vivekananda Institute of Medical Sciences Kolkata, India Aygiil Demirol MD Clinic IVF Center, Ankara, Turkey Sandra K Dill AM Executive Director, ACCESS Australia Infertility Network Chair, International Consumer Support for Infertility (ICSI) Parramatta NSW 2124, Australia Jehoshua Dor MD IVFUnit Department of Obstetrics and Gynecology Sheba Medical Center Tel-Hashomer, Israel and Sackler School of Medicine Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel Dov Feldberg MD Professor and Acting Chairman Department of Obstetrics and Gynecology Tel-Aviv University School of Medicine, Rabin Medical Center Tel-Aviv, Israel Benjamin Fisch MD PhD Department of Obstetrics and Gynecology Rabin Medical Center Petah Tiqva and Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel Simon Fischel CARE (Centres for Assisted Reproduction) Park Hospital, Sherwood Lodge Drive Arnold, Nottingham, UK Ester Polak de Fried MD CER Medical Institute, Director Argentine Soc. of Sterility and Fertility (SAEF), President IFFS, Treasurer, Humboldt Buenos Aires, Argentina Goral N Gandhi MSc Laboratory Director, Rotunda-Center for Human Reproduction Mumbai, India Hossein Gholami Gynecologist President of Associazione per lo Studio dell’Infertilità di coppia (ASIC) ASIC Center for Human Reproduction Rome, Italy

Alfredo Góngora MD Director of Centro de Fertilidad Humana Mexico City, Mexico Mirudhubashini Govindarajan FRCS (Canada) Director, Women’s Center Assisted Reproductive Technology Center Head, Dept. of Obstetrics and Gynecology Sri Ramakrishna Hospital Coimbatore, India Krishnendu Gupta DGO MD FICMCH FICOG Professor, Department of Obstetrics and Gynecology Vivekananda Institute of Medical Sciences Kolkata, India Timur Gürgan MD Professor and Director Division of Reproduction Endocrinology and Infertility Faculty of Medicine University of Hacettepe, Ankara, Turkey Scientific Director Women’s Health, Infertility and Genetic Research Center Ankara, Turkey Alfonso Nájar Gutiérrez MD Director of Reproducciòn y Genètica Hospital Angeles del Pedregal, Universidad La Salle Mèxico City, Mexico Rafael C Haciski MD FACOG Diplomate Amer, Board of Obstetrics and Gynaecology Baltimore, Md, USA Hugh C Hensleigh PhD HCLD Center for Applied Reproductive Science Johnson City Tennessee, USA Vaclav Insler MD FRCOG Professor (Emeritus) of Obstetrics and Gynecology Hebrew University Hadassah Medical School Jerusalem, Israel Milica Ivanovic BS BA Andrology Laboratory Services Inc Chicago, Illinois, USA Ariel Jaffa MD Ultrasound Unit Department of Obstetrics and Gynecology Lis Maternity Hospital, Tel Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Tel-Aviv University Tel-Aviv, Israel

CAM Jansen MD PhD Department of IVF, Obstetrics and Gynaecology Reinier de Graafgroep, loc Diaconessenhuis Fonteynenburghlaan 5 Voorburg, The Netherlands RS Jeyendran DVM PhD Andrology Laboratory Services Inc Chicago, Illinois, USA Department of Physical Medicine and Rehabilitation North Westem Medical School Chicago, Illinois, USA Sucheta Jindal MD Dept. of Obstetrics and Gynaecology Basildon Hospital Nethermayne, Basildon Essex SS16 5NL, UK Padma Rekha Jirge MRCOG Scientific Director Sushrut-Assisted Conception and Endogynaecology Centre Kolhapur, India Otto Kabdebo MD Dr Wilhelm Krüsmann Frauenklinik München, Germany Kaushal Kadam MD Clinical Associate Rotunda-Center for Human Reproduction Mumbai, India Semra Kahraman MD Associate Professor Director of Ýstanbul Memorial Hospital ART and Reproductive Genetics Center Ýstanbul Memorial Hospital, Ýstanbul Turkey Vishvanath C Karande MD FACOG President, Medical Director Karande and Associates Director, In Vitro Fertilization Program Center for Human Reproduction Chicago, Illinois, USA Shashank R Karekar MSc Embryologist, IVF Unit, Phadnis Clinic Pvt Ltd Pune, India Ran Keidar MD Ultrasound Unit Department of Obstetrics and Gynecology Lis Maternity Hospital,

Tel Aviv Sourasky Medical Center, and the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Sarah L Keller MD Department of Obstetric and Gynaecology Reproductive Endocrinology Washington University School of Medicine St. Louis, MO, USA Prashant L Kharche Embryologist IVF Unit, Phadnis Clinic Pvt Ltd Pune, India Ashok Khurana MD Senior Fetustician, Consulting Gynaecological Ultrasonologist and Medical Director, The Ultrasound Lab New Delhi, India Frank M Köhn MD Department of Dermatology and Allergology Technical University Munich, Germany Vijay Kulkarni MS Consultant Andrologist Dr Kulkarni’s Clinic Mumbai, India Alka Kumar Consultant Hysteroscopic Surgeon, Anil Hospital Jaipur, India Atul Kumar Consultant Hysteroscopic Surgeon, Anil Hospital Jaipur, India TC Anand Kumar DSc FAMS FASc Professor and Chairman Hope Infertility Clinic Pvt Ltd Bangalore, India MS Lakshmi DNB Consultant at Assisted Reproductive Technology Center, Sri Ramakrishna Hospital Coimbatore, India Joseph B Lessing MD Professor, Sara Racine IVF Unit Department of Obstetrics and Gynecology Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Israel

Jacob Levron MD IVF Unit, Department of Obstetrics and Gynecology Sheba Medical Center, Tel-Hashomer, Israel 52621 and Sackler School of Medicine Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel Jin Ho Lim MD President, Maria Infertility Hospital Seoul, Korea Franco Lisi Clinical Director of Servizio di Riproduzione Assistita Clinica Villa, Europa Rome, Italy Bruno Lunenfeld MD FRCOG FACOG (hon) POGS (hon) Professor, Faculty of Life Sciences, Bar-Ilan University Ramat Gan, Israel Eitan Lunenfeld MD Professor, Fertility and IVF Unit Department of Obstetrics and Gynecology Soroka Medical Center Faculty of Health Sciences, Ben Gurion University of the Negev Beer Sheva, Israel Sonia Malik DGO MD Clinical Director Southend-Rotunda Center for Human Reproduction Holy Angels Hospital New Delhi, India Aniruddha Malpani MD Malpani Infertility Clinic Mumbai, India Anjali Malpani MD Malpani Infertility Clinic Mumbai, India Roy Mashiach MD Department of Obstetrics and Gynaecology Chaim Sheba Medical Center 52621 Tel-Hashomer, Israel, and Sackler School of Medicine Tel-Aviv University Tel-Aviv, Israel Shlomo Mashiach MD Department of Obstetrics and Gynaecology Chaim Sheba Medical Center, 52621 Tel-Hashomer, Israel and Sackler School of Medicine, Tel-Aviv University Tel-Aviv, Israel Professor and Head of IVF Department “Assuta” Medical Center Tel-Aviv Recumbant of The Chair for the Investigation and Research of Fetal

Anomalies, The Medical Faculty of the “Tel-Aviv University”, Israel Jayant G Mehta PhD Scientific Director Institute of Reproductive Medicine and Women’s Health (A Unit of Madras Medical Mission) Chennai, India Nwora A Melie Chief Embryologist, The Assisted Conception Unit The Bridge Clinic Limited, Victoria Island Lagos, Nigeria Rubina Merchant PhD Embryologist, Rotunda-Center for Human Reproduction Mumbai, India Deepak Modi Malpani Infertility Clinic, Jamuna Sagar, SBS Road, Colaba Mumbai, India Javaid Muġloo MD Medical Director Rotunda-Hygeia Center f or Human Reproduction Srinagar, Jammu and Kashmir, India Jane M Nani MD FACOG Director of Research and Education Advanced Reproductive Health Centers Ltd, North Chicago, Illinois, USA Nico Naumann MD Associazione per lo Studio dell’Infertilità di coppia (ASIC) Viale AVentino Rome, Italy Fernando Neuspiller MD. Department of Reproductive Medicine Cer Medical Institute Buenos Aires, Argentina Raoul Orvieto MD Department of Obstetrics and Gynecology Rabin Medical Center Petah Tiqva and Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel Kemal Ozgur MD Clinical Director, Antalya IVF Center Antalya, Turkey Mandakini Parihar Hon. Associate Professor of Obstetrics and Gynecology KJ Somaiya Medical College, Mumbai Director, Mandakini IVF Centre, Mumbai, India

Avinash Phadnis MD Director, IVF Unit, Phadnis Clinic Pvt Ltd Pune, India Anil B Pinto MD Fellow/Clinical Instructor Advanced Assisted Reproductive Technologies Program Department of Obstetrics and Gynaecology Reproductive Endocrinology Washington University School of Medicine St Louis, MO, USA Luca Dal Prato Tecnobios Procreazione, Center for Reproductive Health Bologna, Italy Elizabeth Puscheck MD Division of Reproductive Endocrine and Infertility Department of Obstetrics and Gynecology Wayne State University Medical School Detroit, Michigan, USA Odem R Randall MD Associate Professor and Division Director Advanced Assisted Reproductive Technologies Program Department of Obstetrics and Gynaecology Reproductive Endocrinology Washington University School of Medicine St Louis, MO, USA PM Rijnders MSc Embryologist and Laboratory Director Department of IVF, Reinier de Graafgroep Voorburg, The Netherlands Leonardo Rinaldi BIOGENESI, Casa di Cura Villa Europa all’EUR Via Eufrate 27 Eur, Rome, Italy Nirmala Sadasivam MD DGO Consultant Gynaecologist and Infertility Specialist Maruthi Medical Centre and Hospital Erode, Tamil Nadu, India Ramadan Abdou Saleh MD Center for Advanced Research in Human Reproduction Infertility, and Sexual Function Urological Institute and Department of Obstetrics and Gynecology Cleveland Clinic Foundation, Cleveland, Ohio, USA

Reeti Sahni MD Consultant, Department of Radiology Indraprastha Apollo Hospital New Delhi, India Wolf B Schill MD Center of Dermatology and Andrology, University of Giessen Germany David W Schmidt MD Associate Professor Department of Obstetrics and Gynecology The University of Connecticut Health Center Farmington, Connecticut, USA The Center for Advance Reproductive Services Farmington, Connecticut, USA Daniel S Seidman MD Associate Professor, Department of Obstetrics and Gynecology Chaim Sheba Medical Center Tel-Hashomer, Israel Rupin Shah MS MCh (Urology) Consultant Andrologist and Microsurgeon Lilavati Hospital and Bhatia General Hospital Mumbai, India Rakesh K Sharma PhD Center for Advanced Research in Human Reproduction Infertility, and Sexual Function Urological Institute and Department of Obstetrics and Gynecology Cleveland Clinic Foundation Cleveland, Ohio, USA Siya S Sharma MD DNB DGO MD DNB DGO MICOG MNAMS Assistant Professor, Department of Obstetrics and Gynaecology Kasturba Medical College Manipal, India Pankaj Shrivastav MD DGO FRCOG Deputy Director, Dubai Gynecology and Fertility Centre Dubai, UAE Tali Silberstein MD Fertility and In Vitro Fertilization Unit Department of Obstetrics and Gynecology Soroka University Medical Center and the Faculty of Health Sciences Ben Gurion University of the Negev Beer Sheva, Israel

Kuldeep Singh MD Consultant Ultrasonologist The Ultrasound Lab New Delhi, India Ved Prakash Singh MBBS FRANZCOG MD Consultant in Reproductive Medicine Gynaecology and Obstetrics ISIS Clinic, Fertility Associates and Waikato Hospital Hamilton, New Zealand Rakesh Sinha MD DGO Clinical Fellowship in Endoscopy (UK) Diploma in Endoscopy (Germany) Gynaecological Endoscopic Surgeon Bombay Hospital, Lilavati Hospital Sir HN Hospital, Bhatia Hospital Bombay Endoscopy Society and Centre for Minimally Invasive Surgery Research Co Pvt Ltd Exclusive Laser Center for Women, Mumbai, India Ruslan V Sobolev PhD Head of Reproductive Department Odessa State Medical University Odessa, Ukraine Weon Young Son PhD Research Director, Maria Infertility Hospital Seoul, Korea Caner Sonmez MD Antalya-IVF, Antalya, Turkey Michael Spitz Andrology Laboratory Services Inc Chicago, Illinois, USA Nona Morgan Swank RNC BSN IVF Nurse Coordinator The Infertility and Reproductive Medicine Centre Barnes-Jewish Hospital at Washington University School of Medicine St. Louis, MO, USA Radha Syed MD FACOG Department of Obstetrics and Gynaecology Division of Endoscopy St Vincents Hospital, Staten Island St Vincents Catholic Medical Centers Staten Island, NY, USA and Staten Island University Hospital, Staten Island, New York, USA

István Szabó MD Medical University of Pécs Department of Obstetrics and Gynecology Pécs, Hungary Yona Tadir MD Professor, Obstetrics and Gynecology Director of Clinical Research Beckman Laser Institute, University of California, Irvine and Ramat Marpe Hospital Tel-Aviv University Tel-Aviv, Israel Samuel S Thatcher MD PhD Center for Applied Reproductive Science Johnson City, Tennessee, USA Alan Thornhill PhD Malpani Infertility Clinic Mumbai, India Katherine E Tucker PhD HCLD Scientific Director, Department of IVF Reinier de Graafgroep, Voorburg, The Netherlands Michael J Tucker PhD Scientific Director, Georgia Reproductive Specialists Atlanta, Georgia, USA Acharya Umesh MD Consultant in Reproductive Medicine South West Centre for Reproductive Medicine Ocean Suite-Level 6, Derriford Hospital Plymouth, UK Thankam R Varma PhD FRCS FRCOG Medical Director, Institute of Reproductive Medicine and Women’s Health (A Unit of Madras Medical Mission) Chennai, India Attila Vereczkey MD Nyiro Gyula Hospital, Department Infertility and Assisted Reproductive Techniques Budapest, Hungary Surendra Pal Singh Virk MSc(Hons) MISPAT Lab Director and Biologist Rotunda-Virk Center for Human Reproduction, Virk Hospital Jalandhar, India Sanjay Wagle MD MRCP Physician and Critical Care Specialist Bombay Hospital and Medical Research Centre Bombay Hospital Mumbai, India

Lars Grabow Westergaard MD DMSc Odense University Hospital and Fertility Clinic Trianglen Copenhagen, Denmark Daniel B Williams MD Associate Professor and Director Advanced Assisted Reproductive Technologies Program Department of Obstetrics and Gynecology Reproductive Endocrinology Washington University School of Medicine St. Louis, MO, USA Igal Wolman MD Ultrasound Unit in Obstetric and Gynecology Lis Maternity Hospital Tel-Aviv, Israel Beatriz Xoconostle PhD Chief of Molecular Biotechnology Department CINVESTAV—IPN Mèxico City, Mexico San Hyun Yoon PhD Manager, Department of IVF Research Maria Infertility Hospital Seoul, Korea Valery N Zaporozhan PhD Rector of Odessa State Medical University Chairman of Obstetrics and Gynecology Department Odessa State Medical University Odessa, Ukraine

Contents

Section 1 INTRODUCTION 1. Advent of Medically Assisted Reproductive Technologies (MART) in India TC AnandKumar 2. The Endocrinology of ART Zeeυ Blumenfeld 3. Efficient Classification of Infertility Vaclaυ Insler, Bruno Lunenfeld 4. Modern Work-up of Infertility Krishnendu Gupta, Sajal Datta, Bijit Chowdhury

3

8 22 33

Section 2 STIMULATION STRATEGIES 5. Ovulation Induction with Tamoxifen Citrate 42 Padma Rekha Jirge, Acharya Umesh 6. Defining the Poor Ovarian Response before Controlled Ovarian 51 Hyperstimulation Daυid W Schmidt, Claudio A Benadiva 7. Aromatase Inhibitors—Their Role in the Treatment of Infertility 71 Pankaj Shrivastav 8. Urinary Human FSH Versus Recombinant Human FSH 76 Eitan Lunenfeld, Tali Silberstein 9. Stimulation Strategies for Complex IVF Patients 83 Franco Lisi, Leonardo Rinaldi, Simon Fischel 10. Programming the Cycle with Oral Contraceptives Antecedent to the use 95 of Antagonists Nico Naumann, Hossein Gholami 11. Agonists Versus Antagonists: Physiology to Clinical Success 101 Ester Polak de Fried, Fernando Neuspiller, Gerardo Ardiles

12. Microdose GnRH for the Stimulation of Low Responders CAM Jansen, KE Tucker 13. The Role of GnRH Antagonist in the Management of Poor Responders Hossein Gholami, Nico Naumann, Antonio Barbaro 14. Alternative Approaches to Ovarian Stimulation and Triggering of Ovulation Gautam N Allahbadia, Kaushal Kadam, Swati G Allahbadia, Avinash Phadnis 15. Role of LH in Stimulation Protocols for ART Lars Grabow Westergaard, Claus Yding Andersen 16. Role of hMG-HP in Stimulated Cycles for ART Gautam N Allahbadia, Kaushal Kadam, SPS Virk 17. Luteal Phase Support Valery N Zaporozhan, Ruslan V Soboleυ 18. Severe OHSS: A Critical Care Physician’s Point of View Sanjay Wagle 19. Ovulation Induction: Surgical Approach Attila Vereczkey, Otto Kabdebo, István Szabó

112 118 124

160 170 185 196 201

Section 3 POLYCYSTIC OVARY SYNDROME (PCOS) 20. Polycystic Ovary Syndrome: An Update Jane M Nani 21. In Vitro Oocyte Maturation Ved Prakash Singh 22. Polycystic Ovary Syndrome: Genetics and Health Consequences Sudip Basu, Najar Amso, Jaydip Bhaumik

221 234 237

Section 4 ART PROCEDURES 23. Vaginal Oocyte Retrieval Gautam N Allahbadia, Goral N Gandhi, Kaushal Kadam 24. Gamete Intrafallopian Transfer (GIFT) Andrea Borini, Luca Dal Prato 25. Zygote Intrafallopian Tube Transfer (ZIFT): Patient Selection is the Key to Beneficial Utilization Daniel S Seidman 26. Fallopian Tube Sperm Perfusion Gautam N Allahbadia, Swati G Allahbadia, Sonia Malik, Javaid Mugloo 27. Use of Lasers in ART: Clinical Applications and Potential Research Tools Yona Tadir

251 267 277

283 292

28. Blastocyst Transfer: One Step Further in the Quest for the Magic Bullet 306 Jacob Levron, Micha Baum, Jehoshua Dor, Daniel S Seidman Section 5 LABORATORY ISSUES 29. Semen Analysis for Clinical Interpretation Elizabeth Puscheck, RS Jeyendran 30. Fundamentals of Sperm Processing Techniques RS Jeyendmn, Vida Acosta, Milica Ivanovic 31. Sperm Separation Techniques: Comparison and Evaluation of Gradient Products KE Tucker, CAM Jansen 32. Prediction of ART Outcome in Male Factor Infertility Patients by a New Semen Quality Score Ashok Agarwal, Rakesh K Sharma 33. Why Should We Assess Oocyte and Embryo Morphology? KE Tucker, CAM Jansen 34. Benefits and Drawbacks of Extended Embryo Culture CAM Jansen, PM Rijnders, KE Tucker 35. Really, Just How Important is the Level of Room Lighting in the IVF Laboratory on Embryo Development? KE Tucker, CAM Jansen 36. The Mouse Embryo Bioassay: Is It the “Gold Standard” for Quality Control Testing in the IVF Laboratory? KE Tucker, CAM Jansen 37. Human Oocyte and Embryo Cryopreservation Michael J Tucker 38. In Vitro Maturation: Future Clinical Applications Jin Ho Lim, Weon Young Son, San Hyun Yoon 39. Oxidative Stress and DNA Damage in Human Sperm: The Cleveland Clinic Story Ashok Agarwal, Ramadan Abdou Saleh 40. Quality Management in an Assisted Reproductive Therapy Environment Malcolm Clarke

314 327 335

341

364 373 380

387

393 407 419

446

Section 6 CONTEMPORARY THOUGHTS 41. How to Improve Success Rates in IVF? Anjali Malpani, Aniruddha Malpani 42. Current Immunological Assays: Are they Enough to Uncover the Supposed Immune Causes for Assisted Reproduction Failure? Aygül Demirol, Erdal Aktan, Timur Gürgan

454 461

43. Endometriosis and ART Ved Prakash Singh, Angela Beaten 44. Infertility: Is there Success after Forty? Daniel B Williams, Anil B Pinto 45. Inheritance of Infertility Mirudhubashini Goυindarajan, MS Lakshmi 46. Sperm Separation Mandakini Parihar 47. Ovarian Tissue Cryopreservation in Cancerous Patients: State of the Art Zeev Blumenfeld 48. Molecular Biology Applied to ART Silυio Cuneo, Alexandra Bermúdez, Alfredo Góngora, Beatriz Xoconostle, Alfonso Nájar Gutiérrez 49. Assisted Reproductive Technologies in Human Immunodeficiency Virus (HIV) Sero-discordant Couples: Practice, Prognosis and Future Prospects Richard A Ajayi, Nwora A Melie

466 472 481 491 503

524

533

Section 7 THIRD PARTY REPRODUCTION 50. Gestational Surrogacy Anil B Pinto, Nona Morgan Swank 51. Oocyte Donation Siya S Sharma, Sucheta Jindal 52. Oocyte-Sharing Programs Gautam N Allahbadia, Goral N Gandhi, Prashant L Kharche, Shashank R Karekar, Aυinash Phadnis 53. Germinal Stem Cells: Culture and Replication Jayant G Mehta, Thankam R Varma 54. Cytoplasmic and Nuclear Transfer: New Life for an Old Egg? Hugh C Hensleigh, Samuel S Thatcher

541 553 561

571 587

Section 8 IMPLANTATION 55. The Endometrium and Implantation Anil B Pinto, Daniel B Williams, Odem R Randall 56. Modulators of Endometrial Receptivity: A Molecular Symphony Rafael C Haciski 57. Endometrial Preparation for Patients Undergoing Frozen-Thawed Embryo Transfer Cycles Raoul Orυieto, Benjamin Fisch, Doυ Feldberg

598 609 623

58. Pathophysiology of Implantation Failure in IVF Jayant G Mehta, Thankam R Varma 59. Recurrent Implantation Failures: The Preferred Therapeutic Approach Sonia Malik 60. Repeated Pregnancy Loss (RPL): Is Investigation Important? Asha Baxi 61. Antiphospholipid Antibodies in ART Gautam N Allahbadia, Sonia Malik, SPS Virk

629 642 654 659

Section 9 CRYOPRESERVATION 62. Cryopreservation of Oocytes and Embryos Foad Azem, Ben Yosef Dalit, Joseph B Lessing 63. Cryopreservation of Human Spermatozoa Frank M Köhn, Wolf B Schill

674 684

Section 10 ENDOSCOPY AND ART 64. Fertility Following Laparoscopic Surgery and Hysteroscopic Surgery B Ramesh, Nirmala Sadasiυam 65. The Role of Hysteroscopy in the Management of Infertility Carlo De Angelis, Monica Antinori 66. Modern Management of the Ischemic Black-Blue Twisted Adenexa Roy Mashiach, Shlomo Mashiach, Daniel S Seidman 67. Fertiloscopy: A New Technique and an Alternative to Conventional Laparoscopy in Infertility Radha Syed 68. Hysteroscopic Assessment of Selective Tubal Pressures and Tubal Cannulation by Air Bubble Stents Atul Kumar, Alka Kumar 69. Intrauterine Adhesions Rakesh Sinha

694 710 728 734

749

753

Section 11 ULTRASONOGRAPHY 70. Update on Ultrasound Guided Embryo Transfer 765 Vishυanath C Karande 71. Cervical Mucus Evaluation by Transvaginal Ultrasonography: A Novel 777 Approach Ran Keidar, Ariel Jaffa, Igal Wolman 72. MultiFetal Pregnancy Reduction 785 Ashok Khurana, Reeti Sahni, Sonia Malik, Kuldeep Singh

Section 12 THE MALE FACTOR 73. Non-obstructive Azoospermia: Predictive Criteria for Sperm Retrieval Vijay Kulkarni 74. Epididymal and Testicular Sperm Retrieval Rupin Shah 75. Microdeletions in the Y-Chromosome and Male Infertility Vida Acosta, Michael Spitz, RS Jeyendran 76. Isolated Teratozoospermia—ICSI or IUI Kemal Ozgur, Caner Sonmez 77. Hormone Substitution in Male Infertility Frank M Köhn, Wolf B Schill

792 798 806 812 821

Section 13 PREIMPLANTATION GENETIC DIAGNOSIS (PGD) 78. Preimplantation Genetic Diagnosis in Cases with Abnormal Gamete Cell Morphology Semra Kahraman 79. Preimplantation Genetic Diagnosis (PGD) for Sex Selection Aniruddha Malpani, Anjali Malpani, Alan Thornhill, Deepak Modi

827

838

Section 14 PRESENT AND FUTURE OF INFERTILITY 80. Present and Future of Infertility Therapy Bruno Lunenfeld 81. Patient Support in the ART Program Rubina Merchant 82. Politics, Partnerships and IVF Sandra K Dill 83. IVF Success from a Clinician’s Viewpoint: How to Get it, How to Keep it? Sarah L Keller, Anil B Pinto, Daniel B Williams 84. Bandwagon IVF: All for One and One for All KE Tucker, CAM Jansen Index

844 856 871 881

892

904

SECTION 1 Introduction

CHAPTER 1 Advent of Medically Assisted Reproductive Technologies (MART) in India TC Anand Kumar INTRODUCTION In vitro fertilization and embryo transfer leading to a successful pregnancy was well established in experimental animals during the early part of the 20th century. It was such findings that led to the publication of early science fiction “Brave New World’ by Aldous Huxley in the 1930’s. In the Brave New World, Huxley envisaged a society in which babies were artificially procreated. Babies were ‘tailor made’ to fulfill specific tasks (was this a forecast of ‘Designer babies’ that appeared in the latter part of the 20th and early part of he 21st centuries?). Fiction often precedes true events. The world’s first ‘artificially created’ human baby, Louise Brown, was born on 28th of July 1978 by a process of removing an oocyte from the ovaries of a woman, fertilizing it in a petri dish with the husband’s sperm, leaving the fertilized egg in the petri dish for a short period until it divided and replacing into the woman’s womb leading to a live birth. This entire process has now come to be known as in vitro fertilization and embryo transfer (IVF-ET) as carried by the original investigators, Patrick Steptoe and Robert Edwards.1 This led to the birth of the world’s first ‘test-tube baby’, a very popular terminology that is nevertheless a misnomer as testtubes are not used for in vitro fertilization. Following this success, a number of newer techniques were subsequently developed by others and given different terminologies. Briefly, these techniques aim to assist barren couples to parent a child through Medically Assisted Reproductive Technologies (MART). Infertility is considered a curse in India and infertile couples often face social ostracism. Any method that improves the chances of barren couples bearing a child is therefore a very attractive proposition to Indians. While the world was experimenting with MART, India was not far behind as would be evident in the narrative given below. The aim of this chapter is to highlight events that led to the firm establishment of MART in India. INDIA’S FIRSTTEST-TUBE BABY Exactly 67 days after the birth of the world’s first test-tube baby an Indian team, led by Subhas Mukherjee (physiologist) Sunit Mukherjee (cryobiologist) and Bhattacharya (a gynecologist) announced to the world, through the press and other media, the birth of

The art and science of assisted reproductive techniques

4

‘Durga’ conceived following the transfer of an embryo produced by fertilizingm vitro an oocyte aspirated from the mother, and transferring the embryo back to the mothers womb.2–4 Subhas Mukherjee was essentially a loner, much dedicated to his work and kept much of his work confidential as he was not sure of the outcome of his efforts. Following his announcement, his uninformed colleagues and others subjected him to great humiliation. He was ridiculed, professionally harassed by the authorities and he was ultimately driven to commit suicide. Mukherjee however, left behind copious notes that have recently been collated and published in Kolkata. He also wrote an ‘official’ note to the Government of West Bengal describing the procedure he followed in some detail. This note was written at the request of the Government of West Bengal. He also published a very short note describing the procedure he followed. However, much of his work was not published as Mukherjee wanted to repeat his studies several times to confirm his finding. However, he was encouraged to publicize his achievement through the press and television and later at the Indian Science Congress when the world’s first test tube baby was born. His announcement elicited some very sharp inquiries by the Government which setup a ‘Star-Chamber’ committee to verify his claims. The committee did not have any expertise in the field of human reproduction to appreciate his contributions; it only ridiculed his claims and humiliated him at a public meeting in Kolkata. The Government of West Bengal asked him to submit a report of his work. A copy of this report, signed by all the three investigators on 19 October 1978, is available amongst the personal papers of Subhas Mukherjee. He, along with his colleagues, published a note in the Journal of Cryogenics 1979; 3:80 on how to freeze embryos and recover them for intrauterine transfer at a later stage.5 His presentation at the Indian Science Congress in 1978 was reported in the New Scientist and his work received global publicity.6 The information presented, gathered over a period of a few years by the author here is based on his note to the Government, and his short publication. It was gathered through personal interviews with Kanupriya (the real name of “Durga”), her parents and some of the surviving people who were associated with the work, and a sworn affidavit from the parents stating their personal experience.7 Mukherjee had informed the parents that since the mother’s tubes were blocked, he was going to attempt a novel way of getting her pregnant that involved taking out eggs from the mother and fertilizing them outside her body and replacing the embryo back into her womb. He also informed them that he was not sure of the outcome or even if the child would be normal. The parents agreed to try out anything but insisted that the whole matter kept confidential as they did not wish to be socially ostracized for having subjected themselves to an experiment that resulted in the birth of an abnormal child. When the baby girl was born they gave it a pseudonym—‘Durga’ In other words, this was a case where the patients’ informed consent was obtained much before treatment was started. The girl, whose actual name is Kanupriya, is now a young lady and, at the moment of writing this document, is a student of Business Management in Pune. Novel Techniques used by the Kolkata team based on the Report submitted by Mukherjee to the West Bengal Government (1978) and the Article published in the Journal of Cryogenics (1979)

Advent of Medically Assisted Reproductive Technologies (MART) in India

5

There are many significant differences between the techniques used by the British and Indian teams as described below: • Mukherjee was the first to successfully use human menopausal gonadotropins (hMG) for ovulation stimulation in an IVF program to ensure the availability of multiple ovarian follicles for aspiration. hMG is now routinely used all over the world in IVF Programs and the credit for its first time use has been given to Howard Jones (USA).8 Indeed Mukherjee’s colleagues still have the old boxes containing gonadotropins manufactured by SERONO, that were routinely used by him. • Mukherjee was the first to approach the ovaries via the vaginal route by posterior colpotomy. The transvaginal route is now the most widely used approach to the ovaries for follicular aspiration under ultrasonographic guidance. • Mukherjee was the first person to have succeeded in freezing and thawing human embryos using a reagent (DMSO) that is now very commonly used for freezing embryos. The Australian team, headed by Trounson is credited for having first made this discovery in the 1980’susingDMSO.9 • Mukherjee was the first to have aspirated oocytes in a stimulated cycle, fertilize them in vitro and freeze the embryos in that cycle; recover, thaw and transfer them into the uterus during the following, natural cycle. This procedure has since been used successfully and independently by several other clinics. One must remember that the world was not yet ready to accept the reality of initiating life outside the body Each and every pioneering work in this field faced great criticism once the work was reported starting from the British team, to the American and Australian teams. It is therefore not surprising that Mukherjee’s colleagues also looked down upon Mukherjee’s work; they had absolutely no idea of what was possible in the ‘Brave New World’ that had just dawned. The fate of Mukherjee is rather tragic. His public ridicule and humiliation by his colleagues, harassment by the West Bengal Government, led him to make the ultimate sacrifice with his life. India’ Second Test Tube Baby—The ICMR’s Institute for Research in Reproduction (IRR) in Bombay undertook a project to produce a baby through in vitro fertilization and embryo transfer. The reason for undertaking such a project was to acquire skills in handling human gametes; gain an understanding of the physiological deficiencies causing infertility as such knowledge could lead to the development of better contraceptives. It was also thought necessary to have a method of reversing infertility caused by tubal sterilizations under the Family Planning Program in such rare instances where women, who have lost their child born before sterilization, desire to have another baby. Under the advice of the Scientific Advisory Committee of the Institute, a project was mounted under the leadership of Professor TC Anand Kumar in 1982. He gathered a team comprising biologists from the IRR and a gynecologist from one of the collaborating institutions from the neighborhood, the King Edward Memorial (KEM) hospital. The protocol for undertaking this work was drawn up by the IRR based on what was possible. The entire project at the IRR was fully funded by the Indian Council of Medical Research. Patients with blocked tubes, as diagnosed by laparoscopy, were selected for IVF. Ovulation was induced with clomiphene citrate and hMG; ovarian response monitored by rapid estimations of estradiol levels at the IRR in daily serial blood samples. Semen analysis was carried out at the IRR according to the WHO semen


6

analysis manual. The oocytes were aspirated in the KEM hospital, rapidly transported in a ‘warm’ (37°C) thermacool box to IRR, which was just a few hundred yards away from the hospital, where the biologists at the IRR carried out all the in vitro culture work, including the processing of semen. Resultant embryos were transferred into the patient’s uterus at IRR. Conception following in vitro fertilization and embryo transfer led to the birth of Harsha on the 6th of August 1986. This birth of Harsha did not go unheeded and without criticisms. The press gave ample coverage of the event. The Indian Parliament asked the ICMR to verify the claims made by IRR and whether the project was approved by the ICMR. The IRR had not only obtained the Scientific Advisory Committee’s approval but had also obtained the institutional ethics committee’s approval in accordance with the ICMR’s Guidelines. It was because of such transparency in the IRR’s work, by which all the members of the Scientific Advisory Committee had witnessed growth of embryos and their transfer into the patients uterus that eventually got pregnant, that the ICMR was able to substantiate IRR’s claim and answer any criticism. However, because the details of Subhas Mukherjee’s work were not widely known, Harsha was termed as India’s first ‘scientifically’ documented test tube baby. The scientific documentation was initially published in the Bulletin of the Indian Council of Medical Research.10 The birth of the first GIFT baby and the first baby born after embryo donation soon followed the birth of Harsha through the efforts of IRR and the KEM Hospital. MART had at last arrived in India and accepted by the public. Mushrooming of IVF clinics in India occurred thereafter. Concluding Statement The birth of Louise Brown in the UK and Durga in India raised many controversies ranging from disbelief to outright criticisms. Some libelous charges were made by the Press against Edwards and Steptoe that were successfully challenged in the court in favor of the scientists. In India too, not only did Mukherjee f ace criticism but even the work carried out in Bombay was questioned by Parliament and even ridiculed by some. All these are of the past. Today, over a million babies have been born the world over. Making babies is big business both commercially as well as in opening out new therapeutic modalities. A whole range of therapies are predicted to emerge from embryonic stem cells used for tissue or even organ repair in conditions such a diabetes, Parkinsonism, Alzheimer’s, broken spinal cord, cardiovascular disorders and bone damage. The source of stem cells is spare and surplus embryos produced through MART. As with every technological innovation, there is a good and a bad side to MART. Infertile couples especially in India are a gullible lot and are prepared to go to any extent just to have a child. There are also an equal number of capricious infertility clinics run by untrained staff, ill equipped and making tall claims on their success rates. Recognizing this state of affairs, the Indian Council of Medical Research and the National Academy of Medical Sciences, have drawn up Guidelines for the Accreditation, Supervision and Regulation of Infertility Clinics in India. This Draft is intended to be a prelude to legislation.

Advent of Medically Assisted Reproductive Technologies (MART) in India

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REFERENCES 1. Steptoe PC, Edwards RG. Birth after the pre-implantation of a human embryo. Lancet 1978; ii:366. 2. Staff Reporter, Amrita Bazar. October 6, 1978; 1–7,. 3. Staff Reporter, Statesman. October 6, 1978. 4. Staff Reporter, Statesman, October 17, 1978. 5. Mukerji S, Mukherjee S, Bhattacharya SK. Indian J of Cryogenics 1978; 3:80. 6. Jayaraman KS. New Scientist 1978; 80:159. 7. Anand Kumar TC. Architect of India’s first test tube baby: Dr. Subhas Mukerji (16 January 1931 to 19 July 1981) Curr Sci 1997; 72:526–31. 8. Jones W Jr, Jones GS, Andrews M et al. The program of in vitro fertilization at Norfolk. Fertil Steril 1982; 38:14. 9. Trounson AO, Mohr LR. Human pregnancy following cryopreservation, whawing and transfer of an eight-cell embryo. Nature 1982; 305:707–09. 10. Anand Kumar TC. ICMR Bulletin 1986; 16.

CHAPTER 2 The Endocrinology of ART Zeev Blumenfeld The technique of in υitro fertilization (IVF), originally devised by Edwards and Steptoe,1 involves the combination of three major disciplines: reproductive endocrinology, surgery, and embryology2 A reproductive endocrinologist experienced in ovulation induction with an understanding of the underlying physiology of follicular development and oocyte maturation is indispensable.2 Successful use of methods of stimulation to insure the retrieval of multiple mature oocytes requires such knowledge for both drug or hormone administration and for patient monitoring to ensure retrieval of oocytes which are properly matured.2 After unsuccessfully using stimulated cycles for many years, Edwards and Steptoe were successful in achieving two normal births following IVF and embryo transfer (ET) of oocytes recovered during natural cycles.3,4 They have attributed the breakthrough success to the use of spontaneous, unstimulated cycles. They believed then, that ovarian hyperstimulation, particularly with human menopausal gonadotropins (hMG), caused “abnormal follicular steroid production and a derangement of the luteal phase”.4 Since those early days of IVF, a better understanding of the endocrinology of ART has lead to the controlled ovarian stimulation (COS) using gonadotropins in an attempt to maximize the efficiency of one IVF cycle, by generating many embryos for present and future use. The last two decades have been accompanied by a plethora of information on various endocrine aspects of ovulation induction and COS for IVF/ART, such as increased LH concentrations, using GnRH agonists and antagonists, urinary hMG versus recombinant FSH and others.5–10 Increased endogenous LH levels are common in patients with PCOS. It is this feature which is thought to result in the reduced conception rates and increased miscarriage rates in both natural and assisted conception cycles in these patients.6,11 To establish unifollicular development in these patients while minimizing the risk of complications, a number of strategies have been employed. Concerns about the adverse effects of increased LH levels in many PCOS patients have led to suggestions that the use of purified urinary-derived FSH (u-FSH) preparations (or those with a relatively low LH content) may confer a clinical benefit over hMG preparations. In the meta-analyses performed, a significant reduction in the incidence of OHSS (OR 0.33; 95% CI 0.16–0.65) was found for u-FSH compared to hMG.6 The beneficial effect of FSH was demonstrated only where no analogue was used (OR 0.20; 95% CI 0.08–0.46). In the largest research clinical trial (109 cycles available for analysis),12 both treatment groups appeared to be well matched for the duration of infertility (5.6 vs 6.3 years) and BMI (29.3 vs 28.4) and the mean numbers of ampoules used per group were similar (26.6 vs 23.6). In this study,12 other main differences with u-

The Endocrinology of ART

9

FSH were a significant reduction in the number of developing follicles and lower oestradiol levels. The reduced incidence of OHSS was 6% (4/68) of u-FSH cycles compared with 37% (15/41) of hMG cycles. While the relatively wide confidence intervals mean that smaller additional benefits of u-FSH on other outcomes cannot be excluded, the trends are not suggestive of this. Thus, despite theoretical advantages, the only benefit of u-FSH preparations compared to traditional hMG preparations appears a reduced risk of OHSS. This benefit needs to be balanced against the additional financial costs involved. More expensive recombinant FSH (r-FSH) products have been introduced for both ovulation induction6,10 and controlled ovarian stimulation in IVF,7 but no direct comparisons with the other gonadotrophin preparations in PCOS are available. The clinical value of further additional expense remains therefore unknown.6 A further strategy that has been used to improve clinical outcome in PCOS is the adjunctive use of a GnRH-a during ovulation induction with gonadotropins. Concomitant use of a GnRH-a may be able to prevent premature luteinisation during follicular development and thereby increase cycle fecundity (per cycle started). During the later stages of follicular maturation in the normal ovulatory cycle, plasma FSH and LH are maintained at low concentrations by the negative feedback effects of oestradiol and inhibin until the preovulatory surge. At this stage the dominant follicle has grown from approximately 10 mm in diameter to 17–25 mm,6,13 and the plasma oestradiol concentration has increased to approximately 900 pmol/L. The positive feedback surge of LH has dynamic effects upon the follicle by reducing luteinisation and ovulation. Accordingly this event needs to be precisely timed to coincide with follicle maturity. At the ovarian level the consequences of a premature LH surge may not be restricted to an early rise in circulating progesterone or premature luteinisation.14 Evidence for this supposition comes from in υitro fertilization programmes where elevated follicular phase LH concentrations have been shown to be associated with poorer oocyte and embryo quality15 and lower pregnancy rates.16 As discussed earlier, ovulation induction with gonadotrophins often results in multiple follicular growth in PCOS patients. The resultant supraphysiological levels of oestradiol may have both positive and negative feedback effects.17 When luteinisation is induced in immature follicles (diameter 30% of cycles and none in the suppressed group. This suggests that the major benefit of concomitant GnRH-a administration may be in the prevention of premature luteinisation rather than correction of the metabolic disorder of PCOS. This may result in an improvement of the effective ovulation rate by allowing direct clinical control of the timing of luteinisation and ovulation. Another important potential benefit of the administration of a GnRH-a with gonadotropins for ovulation induction is the possible reduction in miscarriage, thought to be due to elevated LH levels.6,11,20 Unfortunately, only one research clinical trial gave information on the rates of miscarriage.21 This study reported the miscarriage rates from a total of only 12 pregnancies, thereby limiting any conclusions to be drawn. Overall, the significantly higher overstimulation rate plus the additional inconvenience and cost of concomitant GnRH-a administration may not justify their routine use in the absence of improved pregnancy rates for in-vivo ovulation induction, whereas their beneficial effect in preventing premature LH surge in IVF cycles has been proven. With the development of GnRH-antagonists for the use in ovulation induction and IVF,9 there will be a need to examine similar questions as their effects on the hypothalamic pituitary axis are similar to those following prolonged use of a GnRH-a. A further potential strategy for improving the efficacy of gonadotropin stimulation is by modifying their administration. Under physiological circumstances gonadotropins are released from the pituitary gland in a pulsatile manner every 1 to 2 hours.22 Following intramuscular administration of gonadotropins peak serum concentrations are achieved within one hour of injection, gradually declining until the next injection.6,23 Therefore, to reflect a more physiological pattern of release, pulsatile subcutaneous gonadotropins have been considered as an alternative to intramuscular administration. Three studies looked at the efficacy of pulsatile subcutaneous regimens,24–26 but they provided limited data with only 148 cycles in total. No significant difference for any of the outcomes studied were found but CI remains wide.6 A further strategy used has been that of alternate day versus daily administration of intramuscular gonadotropins.12,26 For this purpose, these studies used a starting dose of 2 ampoules per day for the daily injection subjects and 4 ampoules every second day for the alternate day injection subjects.6 Thus, in overall equal time periods the same quantity of gonadotropins was being administered. If equally or more effective, the rationale is that this could reduce the inconvenience to the patient. The incidence of local side effects would be likely to be diminished because of the lower number of injections. In addition, if these injections were being administered by medical or nursing staff, further time savings would be possible. Unfortunately, limited data restrict the ability to make reliable


11

conclusions but for the main outcomes there was no significant difference between regimens demonstrated.6 Brown27,28 was the first to demonstrate that if the FSH dose was increased in very small amounts it was possible to find a threshold for a single follicle to develop. It was also shown that in the same individual the difference in dose between f ailing to obtain a response and stimulation of follicular development may be as little as 20%.28 Furthermore, it has been shown in monkeys that once enough FSH has been given for selection of the dominant follicle, the dose can be decreased and the follicle will go on to ovulate6 This has more recently led to the development of step-up and step-down protocols.10 The low-dose step-up regimen usually employs a starting dose of 0.5–0.75 of an ampoule (37.5–50 IU), increased only after 14 days if there is no response and then only by half an ampoule every 7 days. Treatment cycles using this approach can be quite long, up to 28–35 days, but it is thought that this may reduce the risk of multiple follicular growth. Another advantage of the step-up protocol may be a less demanding monitoring frequency than with conventional administration. At the beginning of induction, due to slower follicular development, weekly assessment is usually sufficient. As multifollicular development is less common and progresses less rapidly the number of monitoring sessions may also be reduced. The data available for the comparison of lowdose step-up protocols with standard regimens was very limited and no significant differences were found for the outcomes of interest.6 While a number of tentative conclusions can be drawn from the meta-analyses of the 14 included research clinical studies, they need to be interpreted with caution.6 Overall, the methodological quality of the trials was fairly poor. For example, the method of randomization was rarely specified and blinding almost never performed. There were many trials with a cross-over design with its inherent problems. Equally patients (or cycles) were often excluded from analysis after randomization without further details being given so that an intention to treat analysis was impossible. Other concerns can be made in terms of the patient group being studied. Patient heterogeneity in PCOS is well recognized. In addition, the definition of clomiphene-resistance is also variable, encompassing both patients who may have failed to conceive following successful induction of ovulation and also patients who may not have ovulated at all. There was also some variation in the treatment regimens being administered but the principles and protocols of treatment were similar. In many studies only a limited number of the outcomes of interest were reported.6 Three recent publications have debated on the important clinical question: Do GnRH antagonists lower embryo implantation?29–31 Concerns have been raised regarding possible adverse effects of GnRH antagonists on extrapituitary reproductive cells and organs-the ovarian cells, oocyte, embryo, and endometrium.30,31 These concerns are based on numerous in vitro studies suggesting decreased biosynthesis of growth factors caused by local action of GnRH antagonists.29–33 Specifically by decreasing the biosynthesis of growth factors, GnRH antagonists may compromise key events in the reproductive process. This in turn may result in low implantation rate during assisted reproductive technologies (ART). This reasoning is based on a large body of evidence that documents the ubiquitous existence of GnRH receptors in cells and tissues associated with human reproduction.29–33


12

By now, in all the clinical studies with the GnRH antagonists (independent of the dose used; 0.25 mg/day or 3.0 mg as depot), LH has been successfully suppressed, a lower amount of FSH was required, patient satisfaction was highly rated (no histamine release) and ovarian hyperstimulation syndrome (OHSS) was cut by half, when compared with patients treated with the GnRH agonist.29,30 Furthermore, no significant differences in the number of oocytes retrieved, fertilization rates and embryo quality between patients treated with GnRH agonist or GnRH antagonist, were found.29–31 However, a trend toward a decrease in oestradiol concentrations, pregnancy rates and ongoing pregnancies seem to suggest that implantation rates per transferred embryo are reduced in cycles stimulated with the use of GnRH antagonist (29–33). The decrease in oestradiol concentrations in cycles of controlled ovarian stimulation using GnRH antagonists and recombinant FSH may have been overcome by the addition of LH via hMG administration. Although the results were not statistically significant (with 0.25 mg or 3 mg of GnRH antagonist), these parameters were aggravated in a dose-dependent manner. For example, implantation rates varied from 1% to 20% when 2.00 or 0.25 mg/day of the GnRH antagonist was administered, respectively.29,30 Cytokines, growth factors, and their receptors have been detected in both preimplantation and periimplantation embryos, the fallopian tube, and uterine endometrium.29,33 Moreover, their role in embryo development, endometrial preparation, and the implantation process is now well documented.29,33 Several groups have shown that improved embryo morphology development, and hatching as well as better implantation rates can be obtained after embryo co-culture on feeder layers of human oviductal cells and sequential oviductal-endometrial cells. Therefore, oviductal cells in the feeder layer for embryo cultures might produce factors that possess direct or indirect embryotropic activity.29,33 However, these embryotropic factors, their regulation, and their potential function in-vivo are as yet undiscovered, and their influence on the gametes and embryo has not yet been explored.29,33 There is ample evidence that a variety of human tissues, such as the endometrium,29,30,33 ovary29,30,33 testis, and myometrium, express extrahypothalamic GnRH that is immunologically, biologically, and chemically identical to the hypothalamic hormone. Furthermore, the presence of GnRH receptor in cumulus-oocyte complexes and preimplantation embryos at different developmental stages has been established.29,30,33 Several GnRH agonists have been shown to have a direct action on these peripheral receptors both in-vivo and in vitro and, consequently, to mediate a stimulatory effect on spontaneous contraction of human myometrium and fallopian tubes, as well as to enhance fertilization, pre-implantation embryonic development, and implantation.33 Fertilization, pre-implantation embryonic development, and implantation are a complex series of steps that, under normal circumstances, begin in the fallopian tube, before the blastocyst reaches the uterine cavity and attaches to the maternal endometrium. To complete this enigmatic process, there is an embryonic-maternal dialogue, in which the embryo and the maternal reproductive tract induce changes in each other to promote embryonic development and endometrial receptivity.29,30,33 It has been recently hypothesized33 that an interaction between the embryo and the maternal reproductive tract via the GnRH system may be playing an important role during fertilization, pre-implantation embryonic development, and the implantation


13

process. Indeed, Casan et al33 recently provided evidence at both the mRNA and protein levels that GnRH is produced in the human fallopian tube during the luteal phase of the menstrual cycle at the same time that spermatozoa and oocytes are deposited communally in the oviduct to promote their union and nurture the resultant zygotes and early embryos. Therefore, the presence of immunoreactive GnRH in the ephitelial cells of the human oviduct during the luteal phase may play a paracrine role in fertilization and early cleavage stage embryonic development (29, 33). This hypothesis is supported by experimental evidence that GnRH has a direct stimulatory effect on both fertilization and early embryonic development.33 Previous reports in human and other species have demonstrated that GnRH and GnRH agonist enhance in vitro fertilization.33 Accordingly, GnRH increased zona binding ability when human sperm was incubated with these peptides.33 Moreover, oocytes fertilized in medium containing GnRH had a higher cleavage rate than controls not receiving the hormone. On the other hand, incubating the sperm and/or oocytes with a GnRH antagonist ablated the stimulatory effects of GnRH on in vitro fertilization.33 Therefore, these effects seem to be mediated by the presence of specific receptors for GnRH in both oocytes and spermatozoa.33 The data from in vitro culture of pre-implantation embryos exposed to GnRH agonist and antagonist suggest that GnRH may play a significant role in early embryonic development.33 Thus, GnRH and its agonist seem to enhance embryonic development, whereas GnRH antagonist has a detrimental effect. Further, GnRH antagonist is able to completely block early embryonic development, and the reversal of this effect by the agonist in a dose-dependent fashion suggests a specific receptormediated effect, rather than a non-specific or toxic effect.33 It seems, therefore, that the possible embryo-maternal dialogue occurs at the level of the endosalpinx through the GnRH system, being enhanced by GnRH and its agonists and inhibited by GnRH antagonists.30,33 The clinical observations of higher pregnancy rates after GIFT and ZIFT, as compared to in vitro fertilization/embryo transfer, support this hypothesis.33 The trend towards lower pregnancy rates in ART cycles using antagonists may be merely the result of the learning curve, as suggested by Kol31 in a recent debate. However, one cannot ignore the possible adverse effects of the antagonists at one, or more, of the levels where GnRH receptors were identified: ovary, tube, endometrium, or others.29 In the future, it may well prove that the pregnancy rates of ART cycles using antagonists may be as good as those achieved by the agonists, and the beneficial effects of lowering the number of FSH ampoules, shortening the stimulation period, and minimizing the risk of OHSS are real advantages. Until then we should walk very carefully and not abandon the agonists for the antagonists; rather, we should conduct prospective comparative studies that may refute or validate the present speculations that the beneficial in vivo effect of the GnRH agonists on fertilization, early embryonic development, and implantation in both human and animals may be inhibited by the antagonists.29,30,33


14

Activin/Inhibin in Folliculogenesis and Reproduction Activin-A may function as an autocrine or paracrine regulator of follicular f unction in the human and primate ovary.34–36 Activin binding sites were found in rat granulosa cells at a density of about 4000 binding sites/cell. Activin may decrease progesterone secretion, both basal and hCG-stimulated, may decrease aromatase activity in granulosa cells, both basal and FSH-stimulated, may decrease androgen synthesis, and inhibit the P450 cytochrome 17α activity and mRNA, thus decreasing the function of the 17-αhydroxylase/17,20 lyase steroidogenetic enzymes.34–36 Spencer et al34 have found that activin/inhibin subunits mRNA (α, βA, and (βB) in the human adrenal cortex, both fetal and adult, may be possibly regulated by ACTH. Activin suppressed the fetal zone and increased the ACTH-induced shift in the cortisol/dehydroepiandro-sterone sulfate ratio.34 Activin has been found to promote Graafian follicle growth, apoptosis, and ovulation and to block meiosis at metaphase I in the adult rat.37–40 The paracrine effect of inhibin on folliculogenesis was evaluated by injecting Rhinhibin-A directly into the mature rat ovaries.40 The diameter of the ovarian follicles increased in the Rh-inhibin-A treated group of rats as compared to controls, suggesting a direct or indirect effect in folliculogenesis.40 Inhibin may regulate follicular maturation, particularly in immature follicles, by stimulating theca cell androgen production, through the “two cell-two gonadotropin” theory35–41 The dose, time, route of administration, stage of follicular maturation, presence of neutralizing binding proteins, availability of specific receptors, and the relative ratio between activin and inhibin, may all contribute to the dynamics of cellular response and affect the final folliculogenetic result.40 On the other hand, others41 have concluded that inhibin is an unlikely factor to play a significant role in dominant follicle feedback actions, since antral follicles contribute equally to the ovarian immunoreactive-inhibin secretion. However, inhibin concentration did not differ in blood draining the ovary bearing the dominant follicle compared to the contralateral gonad.41 It has been speculated41 that inhibin-B, being secreted by the recently recruited cohort of follicles in response to FSH, may possibly limit the duration of the FSH rise, thus narrowing the “FSH window” of follicular recruitment, through negative feedback at the pituitary level, a mechanism crucial for monofollicular development.41 Alak et al42 have monitored the breakdown of the germinal vessicle (GV), progression to metaphase II (MII) and fertilization in vitro (IVF) of rhesus oocytes, recovered by oophorectomy, after 48 hours of culture with inhibin-A and/or activin-A. Activin-A alone (100 ng/mL) stimulated GV-breakdown (GVBD), whereas both GVBD and MII development was significantly enhanced by inhibin and activin coincubation.42 Follistatin abolished the stimulatory effect of activin and that of inhibin/activin coincubation. Exposure to inhibin and activin significantly increased the IVF of MII oocytes from 25% to 68%,42 suggesting that inhibin and activin are potent stimulators of primate oocyte maturation and possibly also fertilization. In contrast to the reported antagonistic effects of inhibin and activin in different target organs and diverse gonadal and extragonadal cell types, it seems that regarding oocyte maturation and fertilization inhibin and activin function synergistically with each other.42 What are the possible explanations to the different mechanisms of action? It may be hypothesized that the receptors for activin/inhibin in the oocyte- cumulus-corona complex (OCCC) may be specific for only the common b subunit.42 Indeed, activin


15

receptor II subtype has been reported to be expressed in the rat oocyte.42,43 Another possible explanation is that one peptide (inhibin/activin) works at the cumulus cell level, whereas the other acts on the oocyte itself, 42 Also, the exogenous peptides may alter the dynamics of the receptor mRNA expression in the different compartments of the follicle (theca cell, granulosa cell, cumulus cell, or oocyte) with resulting alteration of the different cells and oocyte function and maturation.42,43 Obviously, further research is needed to answer these questions and resolve the various possibilities. Is the maturation effect mediated through the cumulus cells or is it directly on the oocytes? Preliminary data indicate that rh-activin enhances the maturation of denuded monkey oocytes, and that activin binds to ovulated oocyte corona cumulus complex (OCCC).43 Usually, in vitro maturation is monitored by nuclear maturation alone since GVBD and polar body extrusion are easily observed.43 However, Alak et al.’s study42 suggests a beneficial effect on cytoplasmic maturation as well, since activin and/or inhibin elevated the fertilization of mature (MII) oocytes to 50–68% versus 17–32%, as previously reported for the macaque’s unstimulated oocytes. Indeed, in a previous study, in human, Cha et al 44 have achieved a fertilization rate of 32–81% of human unstimulated oocytes, after incubation with follicular fluid, known to contain inhibin and activin, and following in vitro maturation and fertilization one successful pregnancy was generated. More recently,45 it was also shown that activin Ahas accelerated meiotic maturation of human oocytes and has modulated granulosa cell steroidogenesis in vitro. In human ovaries, immunohistochemical and in situ hybridization studies have revealed that the expression patterns of the α, βA-, and (βB inhibin subunits and follistatin are modulated during folliculogenesis,46 suggesting that a dynamic but tightly regulated pattern of inhibin, activin, and follistatin biosynthesis occurs during follicular maturation.47–50 Furthermore, animal studies indicate that both inhibin and activin can modulate follicular development.37,40 Additional in vitro studies indicate that both activin and inhibin can influence maturation of the enclosed oocyte. For example, activin or inhibin treatment of cumulus oocyte complexes from various species was shown to enhance their attainment of meiotic competence,45,48,50 whereas activin facilitated the developmental competence of bovine cumulus-oocyte complexes, an effect that was reversible by follistatin.49 Thus, the tightly regulated pattern of inhibin, activin, and follistatin biosynthesis during follicular development may influence the coordinated processes of oocyte and follicle maturation. The α-subunit of inhibin is produced in vast excess over the amount necessary to produce dimeric inhibin.51 This results in various forms of monomeric α-subunit in follicular fluid and serum, including the full-length precursor protein and a form containing a short segment of the pro-region disulfide linked to mature α-subunit, creating a 26-kD peptide known as pro-αC.50,52,53 Although the physiologic significance of free α-subunit is not known, these proteins may inhibit FSH binding to its receptor,54 influence follicle development,50,55 or inhibit post-cleavage development of bovine embryos derived from cumulus-oocyte complexes matured and fertilized in υitro.56 Taken together, these results suggest that free α-subunit, dimeric inhibins, activin, and/or follistatin may influence human follicle and oocyte development.50


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To test this hypothesis, the mRNA for the inhibin-activin-follistatin system and hormone concentrations were recently examined in the granulosa cells and follicular fluids retrieved by follicular aspiration from patients undergoing IVF.51 The results of this study.51 indicate that some α-subunit mRNA biosynthesis is associated with normal oocyte and follicle maturation but that excessive α-subunit is associated with lower quality embryos. In addition, levels of both follistatin and progesterone were higher in follicles with more mature oocytes.51 None of the analyzed hormones were associated with oocyte or embryo quality.51 Of note, granulosa-cell inhibin α-subunit mRNA levels were associated with several aspects of oocyte maturation and competence. Significantly higher inhibin α-subunit mRNA levels were associated with more mature, higher grade, and successfully fertilized oocytes, suggesting that inhibin α-subunit protein, either alone or as dimeric inhibin, is produced in follicles that contain mature, healthy oocytes. However, inhibin α-subunit mRNA levels were significantly higher in poorer-quality embryos that were not developing properly. This later observation suggests that too much inhibin α-subunit can detrimentally affect the developmental competence of oocytes, as manifested by poorerquality embryos.50,51 These associations are consistent with recent observations that treatment with purified pro-αC protein, a processed form of inhibin α-subunit, inhibited development of bovine embryos in vitro but had no effect on oocyte fertilization or cleavage.56 Furthermore, these observations suggest that the positive association between inhibin α-subunit mRNA levels and oocyte maturation, quality and fertilization that were observed in human follicles might be related more to activities of dimeric inhibin proteins that do not contain pro-αC, whereas the negative association of inhibin α-subunit mRNA levels and embryo quality might be due to production of monomeric pro-αC proteins, which cannot be exclusively measured at this time.51 Taken together, these results suggest that some inhibin α-subunit biosynthesis is necessary for oocyte maturation but that excess inhibin α-subunit, particularly in the form of monomeric pro-αC protein, is deleterious to embryo development.51 In addition to its effect on oocyte developmental competence, monomeric inhibin αsubunit proteins may influence granulosa-cell responsiveness to FSH because immuno afftnity purified natural and recombinant inhibin α-subunit proteins inhibited FSH binding to its receptor.54 Furthermore, immuno neutralization of α-inhibin in sheep with antibodies to the precursor region led to an increase in circulating FSH levels and in the number of developing follicles but caused almost complete absence of follicles >1 mm in diameter.55 When viewed together with the results of this recent study,51 these observations suggest that monomeric inhibin α-subunit is directly or indirectly related to development or maturation of oocytes and follicles and that higher concentrations of inhibin α-subunit may be deleterious to oocyte and follicle maturation. If indeed the α-inhibin precursor, pro-αC protein, is an FSH-receptor binding competitor, antagonizing its bioactivity, follicles with high pro-αC levels would be resistant to FSH, shedding a new prospective on the pathogenesis of the idiopathic premature ovarian failure (POF) and the socalled “gonadotropin-resistant ovary” syndrome in young women.54,57 Alternatively, follicles with more dimeric inhibin (α-β), not possessing the FSH-receptor binding competitor activity of the a-subunit, might be more sensitive to FSH and therefore proceed to dominance and ovulation. On the basis of


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these assumptions and hypotheses one may view the complex process of folliculogenesis as a balance interplay between activin, inhibin and their subunits.50 Activin may have a crucial role in acquiring receptivity of the undifferentiated granulosa cells to FSH. Therefore, at the beginning of the folliculogenetic process, the primordial, uncommitted follicles need a β-activin milieu in order to proceed to the stage of primary follicles committed to growth and differentiation. Those small follicles exposed to an excess of α-subunit prevaillance will become atretic whereas those who will acquire the ability to differentiate and grow will be gradually exposed to increasing influence of α-β inhibin dimer and in parallel a gradual decrease in the β-β activin dimer influence.50,58 The increasing concentrations of inhibin will, in an endocrine mechanism, suppress the pituitary FSH release enabling for the selection of one dominant follicle which has acquired by no w the ability of continuous growth and development in-spite of lower FSH concentrations, and concomitantly will restrict the growth of the nondominant follicles.37,58 The increased need for androstenedione, produced by the theca cells, as a substrate for oestradiol production by the granulosa cells, according to the “two cell-two gonadotropin” theory is met by the increasing inhibin concentrations. The action of activin to increase FSH receptors and granulosa cell responsiveness to FSH at the early stage of folliculogenesis, in an autocrine or paracrine manner, is significantly diminuated or even shut off at the selection and dominance periods of the mid- and late follicular phase, thus preventing from additional follicles to reach the dominant stage. Moreover, increased activin concentrations in the mid-or late follicular phase may induce follicular atresia.50,58 The possible detrimental effect of the pro-αC protein/ α-inhibin precursor extrapolates on several important unavoidable questions, speculations, and future endeavours:

1. Are the levels of α-inhibin precursors/pro-αC really increased in young women with unexplained POF? 2. If indeed, this increased α-inhibin precursors are involved in the pathophysiologic process of POF, one may suggest a possible mechanism for the reported beneficial effect of the GnRH-agonist/glucocorticosteroids/hMG co-treatment in POF.50,57 The GnRH-agonist promoted decrease in the endogenously high FSH levels, may bring about a decrease in the inhibin subunit production by the granulosa cells, thus enabling a temporary release from the detrimental effect of α-inhibin precursor activity as an FSH-receptor binding competitive inhibitor.50 The concurrent administration of exogenous FSH or hMG may thus induce folliculogenesis, and in some cases even ovulation and conception.50,57 Of course, this speculative explanation awaits future scientific substantiation. 3. The pro-αC/α-inhibinprecursor may have a possible prognostic followup significance in patients with POF to possibly monitor the effect of various hormonal manipulative treatments.57


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The precise physiologic role of inhibin α-subunit and, in particular, the forms of inhibin α-subunit protein that may influence oocyte and follicle maturation merits further investigation.50 ACKNOWLEDGEMENT The help of Dr Marina Ritter, Mrs Batia Navar and Architect Ruth Blumenfeld, is thankfully acknowledged. REFERENCES 1. Steptoe PC, Edwards RG, Purdy JM. Clinical aspects of pregnancies established with cleaving embryos grown in vitro. Br J Obstet Gynaecol 1980; 87:757. 2. Jones GS. Update on in vitro fertilization. Endocrine Reviews 1984; 5:62. 3. Steptoe PC, Edwards RG. Birth after the reimplantation of the human embryo. Lancet 1978; 2:366. 4. Blankstein J, Mashiach S, Lunenfeld B. In vitro fertilization and embryo transfer; in: “ovulation induction and in vitro fertilization”, year book medical publishers, Inc. Chicago, 1986, p. 155. 5. Blumenfeld Z. Ovulation induction by gonadotropins; in: “Manual of Ovulation Induction”, ed. Gautam Allahbadiah, Rotunda Press, Mumbay, India, 2000, 27–45. 6. Nugent D, Vandekerckhove P, Huges E, Arnot M, Lilford R. Gonadotropin therapy for ovulation induction in subfertility associated with polycystic ovary syndrome. The Cochrane database of systematic reviews 2001; 2:1–22. 7. Out H, Driessen S, Mannaerts B, Coelingh Bennick H. Recombinant follicle- stimulating hormone (follitropin beta, Puregon) yields higher pregnancy rates in in vitro fertilization than urinary gonadotropins. Fertil Steril 1997; 68:138–42. 8. Franks S, Hamilton-Fairley D. Ovulation induction: gonadotropins. In: Reproductive Endocrinology, Surgery, and Technology: EY Adashi, JA Rock, and Z (Ed). Rosenwaks, Lipincott-Raven Publishers, Philadelphia, 1996; 1207–23. 9. Reissmann T, Felberbaum R, Diedrich K, Engel J, Comaru-Schally A, Schally A. Development and applications of luteneizing hormone-releasing hormone antagonists in the treatment of. infertility: an overview. Hum Reprod 1995; 10:1974–81. 10. van Santbrink E, Fauser B. Urinary follicle stimulating hormone for normogonadotropic clomiphene-resistant infertility: Prospective, randomized comparison between low-dose step-up and step-down dose regimens. J Clin Endocrinol Metab 1997; 82:3597–602. 11. Balen A, Tan S, Jacobs H. Hypersecretion of luteinising hormone—a significant cause of subfertility and miscarriage. Br J Obstet Gynaecol 1993; 100:1082–9. 12. McFaul P, Traub A, Sheridan B, Leslie H. Daily or alternate-day FSH therapy in patients with polycystic ovarian disease resistant to clomiphene citrate treatment. Int J Fertil 1989; 34:194–8. 13. Hackeloer B, Fleming R, Robinson H, Adam A, Coults J. Correlation of ultrasonic and endocrinological assessment of follicular development. Am J Obstet Gynecol 1979; 135:122–8. 14. Fleming R, Coults J. Induction of multiple follicular growth in normally menstruating women with endogenous gonadotropin suppression. Fertil Steril 1986; 45:226–30. 15. Stanger J, Yovich J. Reduced in vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol 1985; 92:385–93.


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16. Howles C, MacNamee M, Edwards R, Goswamy R, Steptoe P. Effect of high tonic levels of LH on outcome of in vitro fertilization. Lancet 1986; ii:521–2. 17. Fleming R, Coults J. LHRH analogues for ovulation induction, with particular reference to polycystic ovary syndrome. Ballier’s Clin Obstet & Gynaecol 1988; 2:677–87. 18. Fleming R, Black W, Coults J. Effects of LH suppression in polycystic ovary syndrome. Clin Endocrinol 1985; 23:683–8. 19. Homburg R, Eshel A, Kilborn J, Adams J, Jacobs H. Combined luteneizing hormone releasing hormone analogue and exogenous gonadotrophins for the treatment of infertility associated with polycystic ovaries. Hum Reprod 1990; 5:32–5. 20. Homburg R, Amar N, Eshel A, Adams J, Jacobs H. Influence of serum luteneizing hormone concentrations on ovulation, conception and early pregnancy loss in polycystic ovary syndrome. Br Med J 1988; 297:1024–6. 21. Bachus K, Hughes C, Haney A, Dodson W. The luteal phase in polycystic ovary syndrome during ovulation induction with human menopausal gonadotrophin with and without leuprolide acetate. Fertil Steril 1990; 54:27–31. 22. Yen S, Tsai C, Naftolin F, Vandenberg G, Ajabor L. Pulsatile patterns of gonadotrophin release in patients with and without ovarian function. J Clin Endocrinol Metab 1972; 34:671–5. 23. Kemmann E, Brandeis V, Shelden R, Nosher J. The initial experience with the use of a portable pump in the delivery of human menopausal gonadotropins. Fertil Steril 1983; 40:448–53. 24. Rossmanith W, Sterzik K, Wolf A. Initial experiences with subcutaneous pulsatile human menopausal gonadotropin administration: successful induction of ovulation in patients with polycystic ovarian disease. Int J Fertil 1987; 32:460–6. 25. Quartero H, Dixon J, Westwood O, Hicks B, Chapman M. Ovulation induction in polycystic ovarian disease by pure FSH (Metrodin): A comparison between chronic low-dose pulsatile administration and i.m. injections. Hum Reprod 1989; 4:247–9. 26. McFaul P, Traub A, Thompson W. Treatment of clomiphene citrate-resistant polycystic ovarian syndrome with pure follicle-stimulating hormone or human menopausal gonadotropin. Fertil Steril 1990; 53:792–7. 27. Brown J. Pituitary control of ovarian function-concepts derived from gonadotrophin therapy. Aus NZ J Obstet Gynaecol 1978; 18:47–54. 28. Brown J, Evans J, Adey F, Taft H, Townsend L. Factors involved in the induction of fertile ovulation with human gonadotrophins. J Obstet Gynaecol Brit Comm 1969; 76:289–307. 29. Blumenfeld Z. Gonadotropin-releasing hormone: a change for the better. Editor’s Corner. Fertil Steril 2001; 76. 30. Hernandez E. Embryo implantation and GnRH antagonists;—Embryo implantation: the Rubicon for GnRH antagonists. Hum. Reprod. 2000; 15:1211–16. 31. Kol S. Embryo implantation and GnRH antagonists; GnRH antagonists in ART: lower embryo implantation? Hum. Reprod. 2000; 16. 32. Dickson SE, Fraser HM. Inhibition of early luteal angiogenesis by gonadotropin-releasing hormone antagonist treatment in the primate. J. Clin. Endocrinol. Metab. 2000; 85:2339–44. 33. Casan EM, Raga F, Bonilla-Musoles F, Polan ML. Human oviductal gonadotropin-releasing hormone: possible implications in fertilization, early embryonic development, and implantation. J. Clin. Endocrinol. Metab. 2000; 85:1377–81. 34. Spencer SJ, Rabinovici J, Mesiano S, Goldsmith PC, Jaffe RB. Activin and inhibin in the human adrenal gland. Regulation and differential effects in fetal and adult cells. J Clin Invest 1992; 90:142–9. 35. Yen SSC. The human menstrual cycle: neuroendocrine regulation. In: Yen SSC, Jaffe RB, Barbieri RL (eds.), “Reproductive Endocrinology”, 4th ed., Philadelphia: W.B. Saunders Comp., 1999; pp. 191–217.


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36. Yeh J, Adashi EY. The ovarian life cycle. In: Yen SSC, Jaffe RB, Barbieri RL (eds.), “Reproductive Endocrinology”, 4th ed., Philadelphia: W. B. Saunders Comp., 1999; pp. 173–4. 37. Woodruff TK, Mather JP. Inhibin, activin and the female reproductive axis. Annu Rev Physiol 1995; 57:219–44. 38. DePaolo L, Bicsak T, Erickson G, Shimasaki S, Ling N. Follistatin and activin: Apotential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 1991; 198:500–12. 39. Woodruff TK. Regulation of cellular and system function by activin. Biochemical Pharmacology 1998; 55:953–63. 40. Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JR Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 1990; 127:3196–205. 41. Fauser BC, Van-Hensden AM. Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev 1997; 18:71–106. 42. Alak BH, Smith GD, Woodruff TK, Stouffer RL, Wolf DP. Enhancement of primate oocyte maturation and fertilizatin in vitro by inhibin A and activin A. Fertil Steril 1996; 66:646–51. 43. Cameron V, Nishimura E, Mathews L, Lewis K, Sawchendo P, Vale W. Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis. Endocrinology 1994; 134:799–808. 44. Cha KY, Koo JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from non-stimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55:109–13. 45. Alak BM, Coskun S, Friedman CI, Kennard EA, Kim MH, Seifer DB. Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertil Steril 1998; 70:1126–30. 46. Roberts VJ, Barth S, El-Roeiy A, Yen SS. Expression of inhibin/ activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J Clin Endocrinol Metab 1993;77:1402–10. 47. Yamoto M, Minami S, Nakano R, Kobayashi M. Immunohisto-chemical localization of inhibin/activin subunits in human ovarian follicles during the menstrual cycle. J Clin Endocrinol Metab 1992; 74:989–93. 48. Sadatsuki M, Tsutsumi O, Yamada R, Muramatsu M, Taketani Y Local regulatory effects of activin A and follistatin on meiotic maturation of rat oocytes. Biochem. Biophys Res Commun 1993; 196:388–95. 49. Silva CC, Knight PG. Modulatory actions of activin A and follistatin on the developmental competence of in vitro matured bovine oocytes. Biol Reprod 1998; 58:558–65. 50. Blumenfeld Z, Ritter M. Inhibin, activin, and follistatin in human fetal pituitary and gonadal physiology. Ann NY Acad Sci 2001; 935 (in press). 51. Fujiwara T, Lambert-Messerlian G, Sidis Y, et al. Analysis of follicular fluid hormone concentrations and granulosa cell mRNA levels of the inhibin- activin-follistatin system: relation to oocyte and embryo characteristics. Fertil Steril 2000; 74:348–55. 52. Robertson DM, Giacometti M, Foulds LM, et al. Isolation of inhibin alpha subunit precursor proteins from bovine follicular fluid. Endocrinology 1989; 125:2141–9. 53. Schneyer AL, Mason AJ, Burton LE, Ziegner JR, Crowley WF. Immunoreactive inhibin alpha subunit in human serum: implications for RIA. J Clin Endocrinol Metab 1990; 70:1208–12. 54. Schneyer AL, Sluss PM, Whitcomb RW, Martin KA, Sprengel R, Crowley WF, Jr. Precursors of alpha-inhibin modulate FSH receptor binding and biological activity. Endocrinology 1991; 129:1987–99.


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55. Dhar A, Salamonsen LA, Doughton BW, Brown RW, Findlay JK. Effect of immunization against the amino-teminal peptide (alpha N) of the alpha 43-subunit of inhibin on follicular atresia and expression of tissue inhibitor of matrix metalloproteinase (TIMP-1) in ovarian follicles of sheep. J Reprod Fertil 1998; 114:147–55. 56. Silva CC, Groome N, Knight PG. Demonstration of a suppressive effect of inhibin a subunit on the developmental competence of in vitro matured bovine oocytes. J Reprod Fertil 1999; 115:381–8. 57. Blumenfeld Z, Halachmi S, Peretz BA, Shmuel Z, Golan D, Makler A. Premature ovarian failure—the prognostic application of autoimmunity conception after ovulation induction. Fertil Steril 1993; 59:750–5. 58. Mather JP, Woodruff TK, Krummen L. Paracrine regulation of reproductive function by inhibin and activin. Proc Soc Exp Biol Med 1992; 201:1–15.

CHAPTER 3 Efficient Classification of Infertility Vaclav Insler, Bruno Lunenfeld CLASSIFICATION OF INFERTILITY INTO SPECIFIC GROUPS Infertility is almost never a physically debilitating disease. It is, ho we ver, a serious psychological burden and a social disadvantage, thus presenting a serious medical problem. Since infertility is a collective presenting symptom to many different diseases, an instrument facilitating classification of infertile couples into different groups must be applied. The list of the main parameters used for classification of infertility is virtually endless. Classification may be based on demographical or sociological parameters, on main symptoms or on possible late sequelae. The most frequently used assessment of infertility has been founded on establishment of pathophysiological mechanisms. Indeed, this traditional method has been for many years taught in medical schools and succesfully practiced in many branches of medicine. No wonder that it was almost automatically applied to infertility. This led to construction of a scheduled, rather extensive, diagnostic sequence which was applied in each case in order to arrive at a diagnosis as exact as possible before application of any therapy (Fig. 3.1). The main elements of this sequence were detailed anamnesis; accurate general physical and genital examination of both partners; hormonal sonographic and other assessments of ovulation; examination of mechanical parameters by hysterosalpingography and/or laparoscopy and hysteroscopy; at least two semen fluid analyses; post coital test and a whole array of special examinations if considered indicated or required. For specific disturbances such as amenorrhea or azoospermia additional diagnostic schemes were utilized. Following establishment of diagnosis, the appropriate treatment was determined and applied. If no pregnancy ensued, partial or full repetition of the diagnostic sequence

Efficient Classification of Infertility

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Fig. 3.1: The traditional classification of infertility according to pathophysiological mechanisms was exercised, again followed by a therapeutic span, this time using also advanced reproductive technologies such as in vitro fertilization (IVF) or gamete intrafallopian transfer (GIFT) or zygote intrafallopian transfer (ZIFT, TET). The duration of this diagnostic/therapeutic cadence was between 30 and 60 months. This relatively long delay in obtaining pregnancy has not been accepted by the modern achievement society. Two additional prominent impediments to this traditional approach have been observed: (1) a large percentage of infertile cases have been finally diagnosed as unexplained (or idiopathic) infertility and (2) in over 50% of the population seen at the majority of infertility clinics in industrialized countries, small fertility disorders such as mild oligo-terato-asthenospermia (OTA), or probable peritubal adhesions or oligoovulation and/or corpus luteum deficiency or mild endometriosis are simultaneously present (combined or multifactorial infertility). An extensive literature survey reported the incidence of idiopathic infertility to be between 1.4 to 50 percent.1 Obviously, treatment of these two entities, i.e. unexplained infertility and combined infertility is empirical and thus exact diagnosis is of academic importance only. It has also been proven that, regardless of the cause of infertility, the most relevant single parameter for success of infertility therapy is the age of the female partner.2–6 Thus, in women 35 years old (or older) all diagnostic and therapeutic sequences must be applied in an extremely concise and efficient manner. Since in many centers this age group has increased steadily in recent years, the routine diagnosis and treatment protocols must take this parameter into serious consideration.


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WHYIS A NEW APPROACH TO MANAGEMENT OF INFERTILITY NEEDED? Within the last 50 years, a tremendous progress has been made in the theoretical knowledge of physiology of reproduction and effective therapeutic modalities were introduced making successful treatment of different types of infertility a realistic proposition (Table 3.1).

Table 3.1: Highlights in development of modern treatment of infertility 1953 Clinical use of urinary hormone assays (gonadotropins and steroids) 1959 Availability of human pituitary and urinary gonadot-ropins for clinical research 1960 Attempt at classification of patients suitable for gonadotropin therapy 1961 Introduction of clomiphene citrate for clinical research 1961 Birth of the first baby following hMG treatment 1965 Wide scale clinical use of gonadotropins and clomiphene 1968 Introduction of progesterone challenge test 1970 Routine use of hormonal radioimmunoassays 1970 Availability of native GnRH for clinical testing 1972 Introduction of prolactin assays 1974 Introduction of prolactin inhibiting agents in therapy 1979 Application of sonography for measurement of ovarian follicles 1979 Birth of Louise Brown, after in vitro fertilization 1982 Introduction of purified FSH into clinical use 1985 Use of GnRH analogues in combination with gonadotropins for induction of ovulation (superovulation) 1986 Wide scale clinical use of advanced reproduction technologies (ART) 198 Pronounced impact of modern society trends on clinical practice 1990 Wide scale use of embryo cryopreservation in IVF clinics 1992 Introduction of micromanipulation (ICSI) for treatment of male infertility 1992 Birth of first babies following induction of ovulation with recombinant FSH 1994 Birth of first child after ICSI 1996 Molecular biology enables detailed understanding of hormones-receptors interaction 1998 Application of GnRH antagonist for reducing premature LH peak during induction of superovulation 1999 Attempts at cryopreservation of ovarian slices and of oocytes

Approximately during the same time a substantial change in structure, function and aspirations of society has taken place, accompanied by significant alteration of the society’s expectations from medical services and of traditional patient-doctor relationship. The stratified, inert, traditional community was replaced by the modern, mobile and fluid consumer society constantly exposed to communication explosion and re-modeled by mass media. The modern individual exists in and must adjust to an achievement society in which he lives. Achievement society requires that success is not only obtained but also shown in


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public, because the peer conception of achievement determines the status of each member of the society. This type of community does not tolerate losers. Mishap in any area including health is perceived as failure and diminishes the individuars social standing. Thus, infertility, in addition to the natural human and psychological burden represents also a serious social handicap. It should also be remembered that industrialization of society has changed family life patterns and reproductive habits. Financial needs, “equal opportunities” and market demands have motivated women to enter the labor force. Since society does not provide sufficient services for working mothers and their babies, many women delay conception according to their professional career and start producing children well after the age of 30. The percentage of patients aged 40 or more increases constantly and some IVF centers practically provide reproduction on demand rather than infertility treatment. In the setting of modern achievement society, infertile couples expect to obtain pregnancy within a rather short period of treatment and demand an application of the most sophisticated new therapeutic modalities virtually from the beginning. Medicine is a profession providing distinctive services to the society. The substance of these services and also their style must be constantly changed according to the requirements of the society we are serving. Considering the real developments in reproductive medicine and the demands of society to achieve success as quickly as possible, the time has come to reorganize the diagnostic and therapeutic sequences applied in infertile couples. THE PROPOSED NEW CLASSIFICATION OF INFERTILITY Fertility is the result of an array of factors interplaying in a well balanced, additive or synergistic manner. This means that a “malfunction” of one factor can be compensated by an optimal expression of other factors. An oligoovulating woman with a “hyperfertile” male partner may not have a fertility problem, whereas a woman with mild endometriosis with a hypofertile male partner may be diagnosed as infertile. (Fig. 3.2). Since the whole process is so complicated and accurate diagnosis of the degree of impediment of each factor is so cumbersome and costly, it seems that in today’s consumer society it may be more practical and economical to diagnose the infertile couple according to the available treatment modalities. This single-focused approach could help in optimizing management and results of infertility treatment. It certainly could not be considered a full-scope classification of male or female infertility based on nosological entities and physiological mechanisms. It is certainly not meant to replace existing diagnoses such as Kallman’s Syndrome, Polycystic Ovarian Disease (PCOD), Testicular Feminization Syndrome, Pelvic Inflammatory Disease (PID), endometriosis, Varicocoele or Klinefelter Syndrome.


26

Fig. 3.2: Fertility or infertility as a result of balancing of different fertility factors in both partners Some of these entities, if diagnosed, should be managed independently of treatment of infertility. Sometimes, treatment of infertility should be preceded or supported by additional measures such as surgical or medical treatment of endometriosis, uterine fibroids, ligation of varicose spermatic veins or treatment of genital infections. Considering the above reasons we devised a new comprehensive diagnostic sequence aiming at relatively early classification of infertile couples into groups fitting to specific therapeutic modalities presently available. This sequence consists of: • anamnesis • general physical and genital examination of both partners • semen analysis (including a “swim up test) • basic hormonal tests (FSH, LH, prolactin, testosterone, dehydroepiandrosterone sulfate and TSH) • hysterosalpingography (or laparoscopy) and, if indicated, hysteroscopy (see Fig. 3.3).


27

Fig. 3.3: The proposed new classification of infertillty based on available treatment modalities Remarks 1. If anamnesis and/or genital examination of the female raises a suspicion of outfow tract abnormalities (e.g. Rokitansky-Kuster-Hauser Syndrome) exact diagnosis must be made using appropriate tests. 2. Obstructive azoospermia must be distinguished from non-obstructive azoospermia so that the means of sperm retrieval (by PESA, TESA or TESE) can be decided upon. This simple diagnostic sequence is completed within 2–3 months and allows the classification of infertile couples into 6 main groups: i. Hypogonadotropic amenorrhea ii. Hypergonadotropic amenorrhea (ovarian failure) iii. Hyperprolactinemia iv. Severe mechanical infertility (tubal obstruction) v. Severe male infertility (azoospermia or severe OAT syndrome), and vi. Multifactorial subfertility which also includes some monofactorial situations such as anovulation or PCOD or mild endometriosis. The multifactorial infertility group will obviously be numerically the largest (Fig. 3.4). Specific therapeutic modalities exist for each of the aforementioned diagnostic categories (Fig. 3.5). Patients with hypogonadotropic amenorrhea are efficiently treated by gonadotropin substitution therapy, the ovulation rate being over 90% and cumulative pregnancy rate per patient approximately 80% after 4 months in women below the age of 35.7 Another


28

treatment modality for this group is pulsatile application of gonadotropin releasing hormone. The efficacy of this therapy is equal to that obtained by gonadotropins,

Fig. 3.4: The incidence of different infertility types however, for some unexplained reasons, this treatment is less frequently used. Women with hypergonadotropic amenorrhea (ovarian failure) should be forthwith offered ovum donation. This therapy may result in a pregnancy rate between 31.1% and 61%.8–10 Women with severe mechanical infertility are treated with IVF-ET and may expect a cumulative pregnancy rate to be approximately 75% after six treatment cycles.11–12 In most infertility centers tubal microsurgery is offered only to patients in whom infertility is the result of previous tubal sterilization procedures. Couples with severe male infertility are offered either artificial insemination using donor sperm (AID) or IVF combined with intracytoplasmic sperm injection (ICSI). The cumulative pregnancy rate of the former procedure is in the range of 70–87% after six treatment cycles.5–13 It is still to early to fully asses the efficacy of IVF combined with ICSI. Van Steirteghem and his collaborators already reported a clinical pregnancy rate of 35.8% per cycle in 1993. It is apparent that in every busy infertility clinic most cases will eventually be classified as multifactorial subfertility (this diagnosis also including couples previously categorized under “Unexplained infertility” or “Transient ovulatory disturbances” or “Possible PCOD”). In this group of patients the following management sequence is employed: Women are treated with clomiphene citrate combined with intrauterine insemination (IUI) for at least 3 consecutive apparently ovulatory cycles. If pregnancy is not obtained, controlled ovarian hyperstimulation (COH), preferrably using pure FSH preparations, accompanied by intrauterine insemination with appropriately prepared husband’s sperm is carried out for 3–4 cycles. If no pregnancy ensues, at this stage the


29

couple is offered advanced reproductive technologies (ART) such as gamete intrafallopian transfer (GIFT),

Fig. 3.5: Treatment modalities available for different types of infertility zygote intrafallopian transfer (ZIFT) or IVF-ET (if specifically indicated combined with ICSI procedure). Clomiphene citrate is a powerful ovulation inducer. A recent meta-analysis of four randomized placebo controlled studies summarized that clomiphene showed odds ratio for ovulation 6.82 and for pregnancy 3.42 as compared to placebo.14 Gonadotropin therapy is the best means for induction of superovulation as shown repeatedly in IVF cycles. Controlled ovarian hyperstimulation (COH) is therefore a logical proposal for enhancing the probability of conception in women with infertility. In the Multifactorial Subfertility group, fertility of the male partner may also be decreased and thus COH in this group should always be combined with IUI. Indeed, COH+IUI treatment modality has been shown to be much more effective in obtaining pregnancy than either COH or IUI alone in couples with unexplained or moderate male infertility.15,17 Khalil et al pointed out that the pregnancy rate following IUI is very low if the total motile sperm count is less than 5 million.18 Other authors even proposed to use 10 million motile sperm count as a threshold for deciding whether to apply COH+IUI or to utilize IVF as a first-line therapy.19 Analysis of literature and personal experience indicate that 3 cycles of clomiphene therapy with IUI will produce a pregnancy rate of approximately 30 percent;20 3 months of gonadotropin therapy combined with IUI should result in a conception rate of at least 40%;21–23 and 3 cycles of IVF-ET (if required combined with ICSI) may be expected to produce a pregnancy rate of at least 40%.11 Thus, from one hundred infertile couples


30

belonging to the large group of “Multifactorial Subfertility” 75 have a fair chance of achieving pregnancy after 3 months of diagnostic procedures and 12 months of intensive treatment (Fig. 3.6). This is a sequence of events conforming to the demands of modern consumer society.

Fig. 3.6: Results of proposed therapy in one hundred theoretical couples diagnosed with multifactorial infertility The economy of infertility treatment must also be seriously considered. According to Guzick et al the cost of pregnancy achieved by clomiphene+IUI is $ 10,000, by gonadotropin superovulation+IUI $ 17,000 and by IVF $ 50,000, respectively24 A study from the Netherlands18 calculated that in couples with unexplained infertility the cost per birth was $ 5110 and 14,679 when COH+IUI or IVF was used.25 Zayed and his co-workers estimated that in United Kingdom a live birth required an investment of 1,923 £ when COH+IUI was the treatment modality and 4,611 £ when IVF was applied.26 It must be stressed that the proposed management scheme is appropriate for the majority of patients presenting themselves in fertility clinics. It does not purport to cover the extremities of the diagnostic arch. It certainly does not intend to replace knowledge, expertise and common sense. REFERENCES 1. Bettendorf G. The normal infertile couple. In: Infertility: Male and Female. V Insler, B Lunenfeld (Eds), Churchill Livingstone, Edinburgh London Madrid Melbourne and New York, (1st edn), 1986; 332–47.


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2. Templeton A, Morris J, Parslow B. Factors affecting the outcome of IVF treatment. Gynecol Endocrinol 1996; 10 (Suppl 1):24. 3. Rabau E, Lunenfeld B, Insler V. The treatment of fertility disturbances with special reference to the use of human gonadotropins. In: Fertility Disturbances in Men and Women. Ch A Joel (Ed), S.Karger, Basel 1971; 508–40. 4. Tan SL, Royston P, Campbell S et al. Cumulative conception and livebirth rates after in-vitro fertilisation. Lancet 1992; 339:1390–94. 5. Shenfield F, Doyle P, Valentine A, Steele SJ, Tan SL. Effects of age gravidity and male infertility status on cumulative conception rates following artificial insemination with cryopreserved donor semen: analysis of 2,998 cycles of treatment in one centre over 10 years. Hum Reprod 1993; 8:60–64. 6. Lipitz S, Rabinovici J, Goldenberg M, Bilder D, Dor J, Mashiah S. Complete failure of fertilization in couples with mechanical infertility: implications for subsequent in vitro fertilization cycles. Fertil Steril 1994; 6:863–66. 7. Insler V, Lunenfeld B: Human gonadotropins, in Infertility: Male and Female, V Insler, B Lunenfeld (Eds), Churchill Livingstone, Edinburgh London Madrid Melbourne New York and Tokyo (2nd edn), 1993; 387. 8. Navot D, Bergh PA, Williams MA, Garrisi GJ, Guzman I, Sandler B et al. Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet 1991; 337:1375–77. 9. Yaron Y, Botchan A, Amit A, Kogossowski A, Yovel I, Lessing JB. Endometrial receptivity: the age-related decline in pregnancy rates and the effect of ovarian function. Fertil Steril 1993; 60:314–18. 10. Feinman M, Sher G, Massaranni G, Vaught L, Andreyko J, Salem R et al. High fecundity rates in donor oocyte recipients and in-vitro fertilization surrogates using parenteral oestradiol valerate. Hum Reprod 1993; 8:1145–47. 11. Cohen J, deMouzon J, Lancaster P. VHIth World Congress on in vitro Fertilization and Alternate Assisted Reproduction, Kyoto, September 1993, World Collaborative Report 1991. 12. Testart J, Plachot M, Mandelbaum J, Salat-Baroux J, Frydman R, Cohen J: World collaborative report on IVF-ET and GIFT: 1989 results. Hum Reprod 1992; 7:362–69. 13. Glezerman M. Artificial insemination. In: Infertility: Male and Female: V Insler, B Lunenfeld (Eds), Churchill Livingstone, Edinburgh London Madrid Melbourne New York and Tokyo (2nd edn), 1993; 643–58. 14. Hughes E, Collins J, Vandekerckhove P. Clomiphene citrate for unexplained subfertility in women. Cochrane Database Syst Rev CD 2000; 0000057. 15. Arcaini L, Bianchi S, Baglioni A, Marchini M, Tozzi L, Fedele L. Superovulation and intrauterine insemination vs. superovulation. Alone in the treatment of unexplained infertility. A randomized Study. J Reprod Med 1996; 41:614–18. 16. Hughes EC. The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: a meta-analysis. Hum Reprod 1997; 12:1865–72. 17. Zeyneloglu HB, Arici A, Olive DL, Duleba AJ. Comparison of intrauterine insemination with timed intercourse in superovulated cycles with gonadotropins: a meta-analysis. Fertil Steril 1998; 69:486–91. 18. Khalil MR, Rasmussen PE, Erb K, Laursen SB, Rex S, Westergaard LG. Homologous intrauterine insemination. An evaluation of prognostic factors based on a review of 2473 cycles. Acta Obstet Gynecol Scand 2001; 80:74–81. 19. Van Voorhis BJ, Barnett M, Sparks AET, Syrop CH, Rosenthal G, Dawson J. Effect of the total motile sperm count on the efficacy and cost-effectiveness of intreauterine insemination and in vitro fertilization. Fertil Steril 2001; 75:661–8. 20. Lunenfeld B, Insler V, Glezerman M. Diagnosis and treatment of functional infertility, (3rd edn), Blackwell Wissenschaft, Berlin, 1993; 56.


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21. Irianni FM, Ramey J, Vaintraub MT, Oehninger S, Acosta AA. Therapeutic intrauterine insemination improves with gonadotropin ovarian stimulation. Arch Androl 1993; 31:55–62. 22. Nulsen JC, Walsh S, Dumez S, Metzger DA. A randomized and longitudinal study of human menopausal gonadotropin with intrauterine insemination in the treatment of infertility. Obstet Gynecol 1993; 82:780–86. 23. Aboulghar MA, Mansour RT, Serour GI, AminY, AbbasAM, Salah IM. Ovarian superstimulation and intrauterine insemination for the treatment of unexplained infertility. Fertil Steril 1993; 60:303–6. 24. Guzick DS, Sullivan MW, Adamson GD, Cedars MI, Falk RJ, Peterson EP et al. Efficacy of treatment for unexplained infertility. Fertil Steril 1998; 70:207–13. 25. GoverdeAJ, McDonnel J, Vermeiden JP, Schats R, Rutten FF, Schoemaker J. Intrauterine insemination or in-vitro fertilization in idiopathic subfertility and male subfertility: a randomized trial and cost-effectiveness analysis. Lancet 2000; 355:13–18. 26. Zayed F, Lenton EA, Cooke ID. Comparison between stimulated in vitro fertilization and stimulated intrauterine insemination for the treatment of unexplained and mild male factor infertility. Hum Reprod 1997; 12:2408–13.

CHAPTER 4 Modern Work-up of Infertility Krishnendu Gupta, Sajal Datta, Bijit Chowdhury INTRODUCTION Infertility is a global issue and has become a major healthcare concern. The evaluation of the infertile couple, usually starts at our office. Taking 10–15 percent of the reproductive age group of the population to be infertile, India with more than a billion population at the present time, has an extremely high number of infertile couples. The number of infertility clinics for ART has increased ten-fold, if not more, in the last decade. Contemporary therapy in the treatment of infertility is progressing faster than any other field of medicine. The treatment for infertility is tedious, time consuming, costly and often, without success. Keeping this in mind, the treating clinician must acknowledge the couple’s frustrations and fears in their zeal to have a child. Hence, we need to be extremely sympathetic and provide adequate counselling to these childless couples. PRACTICAL WORK-UP: CURRENT SCENARIO Before examination of the woman, thorough counselling with respect to the pros and the cons of the available modalities of treatment, their results and outcome, need to be clearly explained to them. Once they are motivated, only then should the actual work-up start. Of course, in the initial visit itself, a thorough history-taking of the couple should be undertaken, both individually and together. Individual history-taking, often provides information which would normally have been missed/ hidden in a joint interview. The Usual Work-up History and Physical Examination Female history • Menstrual: Helps to assess the ovulatory status by regularity and predictability • Contraceptive use: Previous history of IUCD use (can lead to endometritis and endosalpingitis), injectable contraceptives (can delay ovulation for long periods) • Sexual: Infrequent coital frequency, vaginismus and/ or severe dyspareunia can delay fertility; Presence of active STDs (HIV, HPV infection etc) need to be ruled out


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• Medical: Chronic diseases like hypothyroidism, uncontrolled diabetes, hyperandrogenemia can cause anovulation; Genital tuberculosis can lead to infertility; Addiction to drugs, alcohol and/or nicotine requires evaluation • Surgical: Previous abdominal/pelvic surgery can alter the tubo-ovarian function by adhesion formation • Previous infertility treatment • Past obstetric interference (in cases of secondary infertility) • Family: Genetic and endocrinological problems; premature ovarian failure/menopause. Examination • General physical height and weight of the woman is recorded to assess the body mass index (BMI); weight change is more significant than absolute weight; body shape and stature is assessed to exclude Turner’s syndrome and testicular feminization syndrome, particularly if present with primary amenorrhea; hair distribution also needs to be looked into to exclude hirsutism or hypoandrogenism. • Breast Poor development in the presence of primary amenorrhea usually indicates hypoestrogenism; The presence or absence of galactorrhea should be looked into. • Abdominal A thorough examination may reveal some systemic disorder/pelvic pathology • Local (enlargement of the clitoris >2 cm or glans diameter >1 cm is abnormal and is a sign of virilism), speculum and vaginal examination to exclude any obvious pelvic pathology. Occasionally, recto-vaginal examination may be required if endometriosis is suspected. Male history • Occupation: Men working near sources of heat like blast furnaces, coal burners, etc, may be predisposed to altered spermatogenesis (due to rise in the core temperature in the body) • Sexual: Coital practice, premature/retrograde ejaculation, change in libido • Medical: High fever >38°C (may suppress spermato genesis over a period of six months); X-ray exposure to the groin; testicular injury/torsion/tumor; mumps/ tubercular orchitis; epididymitis, prostatitis, repeated UTI, penile discharge; diabetes, neurological disease, antihypertensive therapy (can lead to impotence); sulfasalazine therapy for Crohn’s disease or ulcerative colitis (severely affects the semen count and motility); Other drugs like cimetidine, nitrofurantoin, spirono-lactone, niradozole, colchicines can alter spermatogenesis; dependance on drugs, alcohol and/or nicotine should also be evaluated • Surgical: Undescended testes, varicocele, herniorrhaphy vasectomy Examination • General: Gross overweight has been found to be associated with reduced testicular volume, suggesting impairment of spermatogenesis; Klinefelter’s syndrome needs to be excluded (disproportionate long limb length in relation to trunk length, gynecomastia); signs of hypoandrogenism.

Modern Work-up of Infertility

35

• Abdominal: To exclude any hernia. • Genital organs to exclude undescended testes, testicular atrophy, testicular consistency (soft testes are nearly always associated with impaired spermatogenesis), small penile size, varicocele, hypospadias, phimosis, urethral discharge.

EVALUATION OF THE MALE Semen Analysis Semen analysis of the husband is most often the first step in the evaluation of the infertile couple, and is concurrently assessed with the woman’s ovulatory status. Definitive information with regard to motility, morphology and sperm concentration can be obtained from the ejaculate. Semen analysis is relatively simple to perform, inexpensive and noninvasive, and often reflects the problem attributable to man. A single normal semen analysis need not be repeated. In the event of an abnormal result, it should be repeated in 2 to 3 months because spermatogenesis requires 60 to 72 days. The common parameters of semen analysis1 with their respective normal values are given in Table 4.1.

Table 4.1 Parameter

Normal range

Ejaculate volume >2.0 ml pH 7.2–7.8 Sperm concentration >20 million/ml Motility >50% progressively motile Viability >75% sperm excluding dye Morphology 80 pg/mL was found in 10 percent of women aged 38 to 42 years who had a day 3 FSH within normal range. No live births occurred in these women.5 This study concluded that obtaining a basal E2 added further documentation of the favorable or unfavorable fertility potential in older women. Clomifene Citrate Challenge Test Clomifene citrate challenge test (CCCT) determines the FSH levels in conjunction with the use of clomifene citrate (CC). It is considered a more accurate predictor of ovarian reserve than the day 3 FSH level alone. It may be indicated in women suspected of having premature ovarian failure or early menopause. Some clinicians routinely recommend the CCCT in all women over age 35.6 100 mg of CC is taken orally on days 5 to 9 of a woman’s menstrual cycle. FSH is measured on day 3 and day 10. If the FSH is greater than 15 mIU/mL on either day, it is considered abnormal and indicative diminished ovarian reserve. It is felt that the CCCT picks up the more subtle difference in ovarian reserve than the FSH. It is also more expensive and time-consuming, and involves the taking of medication.

Modern Work-up of Infertility

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Table 4.2 Test

Indications for request

When to test

Normal findings

Progesterone Shortened luteal phase (

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