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

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‘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

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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

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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.

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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-

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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 <17 mm) failure of normal oocyte release is frequently observed.18 Protracted treatment with a GnRH-a reduces the pituitary secretion of LH and ovarian steroid production in women with PCOS.6,18 The consequences of LH suppression in patients with PCOS depend upon the duration of therapy By the time the LH is fully suppressed (after 12–15 days) the oestradiol has fallen to postmenopausal levels but the ovarian androgens have only been normalised.6,18 A higher overstimulation rate (OR 3.15; 95% CI 1.48–6.70) with the addition of a GnRH-a to gonadotropins was observed.6 The higher overstimulation rate was mainly accounted for by the study where 19 of 57 patients with analogue down-regulation and overstimulation compared with 5 of 65 with gonadotropin alone.19 This suggests that in this clinically homogenous group of patients with intact pituitary function, the higher overstimulation rate was independent of endogenous gonadotropins. The authors also analyzed the number of ampoules used in cycles in which overstimulation occurred and found them to be no higher than in those in which uniovulation and/ or pregnancy occurred, suggesting that differential sensitivity of the ovary exists in similar PCOS patients. There was a tendency to an increased pregnancy rate with GnRH-a

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administration. This was accounted for mainly by the study17 in which only the first cycle of treatment was randomized to treatment with hMG±GnRH-a. Of 38 patients treated with hMG alone, 5 conceived (13%) and of 40 with hMG and buserelin 14 conceived (35%).17 The same study commented on the follicular growth profiles in 13 patients who had failed to conceive in the first cycle and had subsequently had treatment in the opposite arm. They found no difference in the rates of growth or the numbers of small, medium or large follicles observed at any stage, i.e. that the dynamics of follicle growth were unchanged by LH suppression. In patients having up to 6 cycles of treatment, those cycles without GnRH-a suppression showed premature luteinisation in >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

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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

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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

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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

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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

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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

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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.

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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).

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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

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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

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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

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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.

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• 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 <85% abnormal forms Inflammatory <1 million cells/ml Antibodies (immunobead test) <20% sperm binding to bead

Semen collection technique and transport has a significant influence on the sperm concentration. Abstinence of at least 48 hours (72 hours preferable) must be maintained before semen collection by masturbation. Caution should be exercised while collecting the semen, so that the first portion of the ejaculate is not lost, which if inadvertently done, will give a false lowered count. Routine semen analysis does not assess actual sperm function, such as the capacity of the sperm to penetrate and fertilize the ovum, hence the necessity of sperm function tests. Sperm Function Tests The sperm function tests usually performed (depending on the capability of the laboratory/andrologist) are hypoosmotic swelling (HOS) test (for sperm vitality); hyaluronate migration test (for sperm migration); hyperactivation assay or sperm surface lectin binding (assessment of capacitation); triple-stain technique or fluorescent lectin labeling (assessment of the acrosome reaction); nuclear chromatin decondensation (NCD)

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test, reactive oxygen species production, sperm-zona binding test (ZBT) or zona-free hamster egg penetration test: HEPT (for fertilizable capacity).2 Should the semen analysis come repeatedly abnormal, the following tests can be performed: a. Examination of the post-orgasm urine in males with a suspicion of retrograde ejaculation b. Measurement of serum FSH to discriminate between hypergonadotropic and normo- or hypogonadotropic hypogonadism (in azoospermia or severe oligozoospermia) c. Measurement of serum testosterone in presence of clinical signs of hypoandrogenism d. Measurement of serum prolactin in cases of decreased libido and penile erection e. Scrotal thermography in presence of abnormal spermatozoa f. Doppler ultrasonography of the scrotum in cases of suspect thermographic findings g. Imaging of hypothalamo-pituitary region in patients with frank hyperprolactinemia or gonadotropin deficiency h. Testicular biopsy in patients with unexplained azoospermia, normal testicular volume and normal FSH; Obstructive azoospermia is diagnosed if the ejaculate has no sperm and the testicular biopsy reveals a full spermatogenic complement in most of the seminiferous tubules.

EVALUATION OF THE FEMALE Assessment of Ovulatory Factor Ovulatory dysfunction is said to be the commonest cause of female infertility (40%). The ovulatory status can be assessed by the following methods: a. Clinical: regular predictable period is often associated with ovulation b. Imaging: transvaginal sonography (TVS) for folliculometry (serial scanning of the ovaries to measure the growth of the follicles and observe the day of rupture) with the synchronous development of the endometrium (triple line endometrium in the proliferative phase followed by homogenous pattern in the secretory phase) c. Biochemical: serum progesterone estimation is suggestive of ovulation, if a level >18 nmol/L is found between day 20–24 of a 28-day cycle. Three consecutive cycles should be studied in patients with irregular menses before reaching a conclusion as to the ovulatory status of the patient. d. Endometrial biopsy (EB): when performed in the luteal phase of the cycle and the histology report show secretory changes, luteinization of the follicle which usually follows ovulation is confirmed, but, can occur in the absence of the release of the ovum (unruptured luteinized follicle: LUF). Proper dating of the endometrium is also important to rule out luteal phase defect (lag of >2 days). EB can be taken either as an OPD procedure or during invasive procedures like laparoscopy. e. Oυulation predictor kit (OPK): OPK provides a relatively accurate means of ovulation prediction. OPK monitors the level of LH in the urine. During a regular menstrual cycle, the LH level will peak before ovulation (LH surge) which stimulates the extrusion of the ova.

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f. Other methods: Not used routinely in the modern work-up of infertility • Basal body temperature (BBT) chart • Billing’s cervical mucus method. Assessment of Tubal and Peritoneal Factor a. Hysterosalpingography (HSG) demonstrates both tubal patency as well as the normalcy of the internal uterine cavity contour. It is easy to perform, can be done as an OPD procedure and can detect the site of block. It is most often, the first line investigation to assess the tubal factor. It is performed in the proliferative phase of the cycle, usually between day 6–10. b. Laparoscopy it is the “gold standard” for assessing pathology in the peritoneal cavity (like endometriosis, adhesions), uterus, fallopian tubes and ovaries. It is often combined with chromopertubation (dye test for tubal status), endometrial biopsy and hysteroscopy. Laparoscopy is usually performed in the late secretory phase (or, premenstrual) of the menstrual cycle. c. Sonosalpingography is a more non-invasive method of assessing tubal patency using sterile normal saline under TVS guidance d. Two-dimensional hysterosalpingo-contrast sonography (2D-HyCoSy) similar to sonosalpingography, but performed with an enhancing media (ECHOVIST-200). In addition to evaluation of the uterine cavity, the echo-enhancing agent clearly delineates the fallopian tubes till it escapes through the fimbrial ends into the peritoneal cavity. Recently, a pilot study was conducted using 3D-HyCoSy3 as an outpatient procedure to assess infertile women with a high degree of success, with several advantages over the conventional X-ray HSG. e. Falloposcopy the interior of both fallopian tubes can be assessed with the introduction of a falloposcope. This procedure can detect the exact level of block, assess the ciliary action and tubal rugosity. f. Three-dimensional power Doppler imaging (3D-PDI)4 this relatively new sonographic technique has been studied to view the entire length of the fallopian tube. Compared to the 2D-HyCoSy, the scanning time was less with less contrast being required for the procedure. Assessment of the Uterus a. Hysteroscopy: by directly visualizing the uterine cavity with the help of a hysteroscope (in addition to an evaluation of the cervix and the endocervical canal with a colpomicrohysteroscope), any distortion of the endometrium or uterus can be detected. Should there be any pathology like polyps or submucus fibroids, they can be removed at the same sitting. b. HSG: provides an opportunity to study the interior of the uterine cavity to detect any irregularity of the uterine contour and/or “filling defects” which may suggest the presence of polyps, submucus fibroids or intrauterine synechiae (Asherman’s syndrome).

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c. Ultrasound: transabdominal sonography (TAS) is a very useful tool in detecting structural anomalies of the uterus such as fibroids. The presence of a tubal hydrosalpinx, polycystic ovaries, ovarian cysts or tumors can readily be detected. As mentioned earlier, the use of the transvaginal probe greatly enhances greater and more detailed visualization of the endometrium and adnexa. Evaluation of the Cervix and Vagina a. Wet mount: this simple yet often neglected test should be routinely performed on all infertility patients during the initial physical examination, along with a Pap smear, if not done within one year. If WBCs are present, a chlamydia/gonorrhea culture should be done. Vaginal cultures need not be part of the initial work-up unless history reveals potential past or present exposure to sexually transmitted disease (STDs). b. A thorough evaluation of the cervix should be done at the initial examination to rule out any erosion, polyps, growths or tears. Incompetence should also be ruled out. Hormonal Evaluation Cyclical and predictable menstrual cycles within 25 to 32 days almost always ensure ovulation on a regular basis. It probably also assumes normalcy of several hormones such as FSH, LH, prolactin and androgens, that are commonly ordered in infertility and that are commonly requested for during an infertility evaluation. The commonly performed hormonal tests with the probable scientific basis are given in Table 4.2. Another hormone which has recently been found to be a useful indicator of cycle outcome related to age in conjunction with basal FSH is a day 3 measurement of estradiol (E2). An elevated E2 level of >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.

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Table 4.2 Test

Indications for request

When to test

Normal findings

Progesterone Shortened luteal phase (<11 days) Midluteal Day 21 or 7 days 10–15 ng/mL Monophasic BBT Unexplained after BBT rise or LH surge infertility Endometrial Shortened luteal phase (<11 days) 11–13 days after BBT rise or Endometrium biopsy Unexplained infertility LH surge or 1–3 days before within 2 days of next expected menses cycle day Prolactin Irregular menses Luteal phase Luteal phase (around day 20) <20 ng/mL Values defect Headache/blurred vision >25 ng/mL Galactorrhea Unexplained indicates a CT infertility scan TSH Manifestations that may suggest Any point during menstrual Laboratory hyperthyroidism or cycle variations often hypothyroidism present a Hyperprolactinemia problem!! LH/FSH ratio Indicators of PCOD present such Secretory phase Day 3–5 <3:1 as hirsutism, oligoamenorrhea, acne, obesity FSH History suggestive of premature Proliferative/follicular Day 3, <15–20 mIU/mL ovarian failure or menopause also day 10, if clomifene such as hot flushes, irritability, or citrate is used irregular menses BBT: Basal body temperature; CT: Computer tomography; FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; PCOD: Polycystic ovary disease

Counselling Three questions obsess infertile patients. “Am I ever going to be pregnant?”, “Am I losing my mind?”, and even more frightening, “Is the distress that I feel preventing me from getting pregnant?”. It has long been a common idea that infertility is “all in the mind”, so it is not surprising that patients fear they are the cause of their own infertility. We, as clinicians, must take it upon ourselves to not only break these myths and misconceptions but communicate effectively, guide the couple and support them through this mental, physical and financial ordeal that they often need to endure. Kinsberg7 listed the following recommendations for physicians treating infertile patients: 1. Educate and inform couples at each visit about their diagnosis, prognosis, and treatment options, even if it seems redundant 2. Acknowledge and normalize the emotional aspects of these treatment options 3. Encourage couples not to rush their decision about treatment 4. Encourage couples to consult with a mental health professional.

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CONCLUSIONS The subject of what should be considered routine testing in the initial investigation of the infertile couple, and who should care for the infertile couple, is controversial. Many clinicians still order an extensive number of tests during the work-up. An attempt has been made to highlight some of the accepted investigations in the modern era, in the light of the tremendous advances that have been made in the field of reproductive medicine. As primary care providers for our women, it is us gynecologists’ who must take it upon ourselves, to provide the much needed emotional support in addition to the required work-up and treatment of the often “hapless” infertile couples. REFERENCES 1. Fuller PJ, Burger HG. Assessment of the male partner. In Kovacs G (Ed): The subfertility handbook: a clinician’s guide. Cambridge University Press, 1997. 2. Mortimer D. Semen Analysis. In Mortimer D (Ed): Practical laboratory andrology. New York: Oxford University Press, Inc 1994. 3. Kiyokawa K, Masuda H, Fuyuki T et al. Three-dimensional Hysterosalping-Contrast Sonography (3D-Hy-CoSy) as an outpatient procedure to assess infertile women: a pilot study. Ultrasound Obstet Gynecol 2000; 16:648–54. 4. Sladkevicius P, Ojha K, Campbell S, et al. Three-dimensional power doppler imaging in the assessment of fallopian tube patency. Ultrasound Obstet Gynecol 2000; 16:644–47. 5. Buyalos RP, Daneshmand S, Brzechiffa B. Basal estradiol and follicle-stimulating hormone predict fecundity in women of advanced reproductive age undergoing ovulation induction therapy Fertility and Sterility 1997; 68:272–77. 6. Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertility and Sterility 1995; 63:1–12. 7. Kingsberg SA. Assisted reproductive techniques and male factor infertility: psychological perspectives on the treatment recommendations of IUI, IVF, and ICSI. Syllabus from the course Male Infertility: The Medical and Psychological Team Approach to Treatment, sponsored by the American Society of Reproductive Medicine, 1996.

SECTION 2 Stimulation Strategies

CHAPTER 5 Ovulation Induction with Tamoxifen Citrate Padma Rekha Jirge, Acharya Umesh Ovulation induction is the process of promotion of follicular growth and development culminating in ovulation. Introduction of gonadotrophins, followed by antiestrogens has revolutionized the practice of reproductive endocrinology. Introduction of clomiphene citrate in the 1960s has been a major therapeutic breakthrough.1 This brought into reality the novel notion of chemical initiation of ovulation with a synthetic compound as opposed to hormonal ovulation induction by a naturally occurring hormone.2 This was followed by synthesis and clinical use of other compounds of the same family. The most widely used of these antiestrogens is tamoxifen citrate (ICI 46, 474). Being in clinical use since 1970s, it now has a firm place as an adjuvant in the management of breast cancer and prevention of its recurrence in the contralateral breast. This has largely overshadowed its efficacy as an ovulation inducing agent. CLINICAL PHARMACOLOGY Tamoxifen (TMX) is a nonsteroidal triphenylethylene structurally similar to clomiphene citrate (CC), both of which bear structural resemblance to the diethylstilbestrol (DES) molecule. They all have three phenyl groups linked together and attached to one other radical. However, unlike CC, tamoxifen does not exhibit geometric isomerism and is exclusively a trans isomer. Neither CC nor TMX are true anti-estrogens, as they have partial agonistic activity as well. They are pharmacologically classified as partial agonists with antagonist action.3 Pharmacokinetics and Metabolism In common with all the triphenylethylene compounds, TMX has a long biological halflife of 7 days due to its liphophilic nature. 99% of the drug is protein bound. In

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Fig. 5.1: Diagrammatic representation of structure of oestradiol and diethylstilboestrol and triphenylethylene anti-oestrogens. (From ref. 32) addition, the parent drug and its phenolic metabolite undergo enterohepatic recirculation. About six weeks are required to clear the drug from the body. The principal metabolite of TMX is N-desmethyl-tamoxifen, which accumulates in the plasma because of a long half-life of 14 days. It is also hydroxylated to 4hydroxytamoxifen.4 Though a minor component of plasma, its affinity for estrogen receptor is 50–80 times higher than tamoxifen, making it pharmacologically significant. When administered in short courses, however, the parent drug is believed to be responsible for most of the actions.5

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Mode of Action When used as an ovulation-inducing agent, the most easily demonstrable action of TMX is exerted through the hypothalamus in the form of increasing circulating levels of LH and FSH. By competitively blocking the estrogen receptors, it releases the hypothalamus from the negative feedback effects of endogenous oestrogen, thereby increasing the activity of LHRH pulse generator.6,7 The consequent rise in pituitary gonadotrophins particularly FSH stimulates the follicular growth. In addition there is evidence that TMX has a direct effect on the pituitary and the ovary. Rise in circulating oestradiol level during follicular phase followed by a rise in circulating progesterone in the luteal phase are in part due to its direct action on the ovary. This effect is noted, both when TMX is used for ovulation induction,8–10 and as a long-term treatment in premenopausal women with breast cancer11 or uterine fibroids12 Tamoxifen exhibits target tissue specificity in that it is antiestrogenic at one site and estrogenic at another. This is evidenced by its effect on the ovaries, uterine endometrium and cervical mucus. Although it has an agonist effect on the ovary leading to folliculogenesis, the antagonistic effect on the endometrium may have important clinical implications. However, it is interesting to note that, in spite of being a purer antiestrogen than CC, it is reported to have a favorable effect on the cervical mucus.13–15 This could be due to the very high levels of serum oestradiol during therapy, which may overcome the antiestrogenic effect. However, these encouraging results have not been reproduced by others.16–18 This could be related to the individual variation in the end organ sensitivity to the drug and to the etiology of infertility, rather than the direct effect of the drug. At the cellular level, the target tissue specificity can be explained by the following mechanisms19: 1. Presence of two types of estrogen receptors (ER)-ERα and ERβ. 2. Different cells may have different intracellular environments that determine whether an antiestrogen behaves as an agonist or antagonist. 3. Presence of certain elements within the genes interacting with the tamoxifen-ER complex, which could have an effect on how an antiestrogen acts.

PHYSIOLOGICAL EFFECTS Endocrine Effects Short term administration of TMX in normogonadotrophic, normoprolactinaemic anovulatory women results in a rise in pituitary gonadotrophins, in particular FSH, thus promoting follicular growth. The subsequent increase in the circulating estrogen levels leads to ovulation in subfertile women. This is followed by the luteotrophic effect, as evidenced by the elevation of the circulating levels of progesterone. In addition, TMX reduces the midcycle peaks of prolactin.20 Curiously, despite the oestrogenic potency of TMX being lower than that of CC, the potentially ovotoxic rise in serum LH during the early follicular phase is less pronounced for TMX than CC.20

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Bones and Lipids It is well known that TMX maintains bone density in postmenopausal women. In contrast, it is shown to induce slight but significant bone loss in premenopausal women.21 However, when administered in short courses for ovulation induction, it is unlikely to have any such effect. TMX has an estrogenic effect on serum lipid profile and leads to a decrease in lowdensity lipoprotein level. Interestingly, HDL levels increased by estrogen therapy are unaffected by tamoxifen therapy. Once again, it is unlikely to have such beneficial effect when used in short courses for ovulation induction. CLINICAL APPLICATIONS Indications Tamoxifen has been extensively used in the management of breast carcinoma. CC, another triphenylethylene, is firmly established as the primary agent for ovulation induction in anovulatory women. The use of TMX for ovulation induction has been overshadowed by these factors and the data available in this regard is limited. However, the evidence so far shows that TMX is equally effective as, or marginally superior to, CC in inducing ovulation in normogonadotrophic, normoprolactinaemic anovulatory women. With TMX, the ovulation rate in these women is 57–95%, which is comparable to that with CC.6,22–24 There is some evidence that TMX is effective in inducing ovulation in women with polycystic ovarian disease resistant to CC.25,26 In the only study comparing a combination of CC and TMX with CC alone, a higher incidence of ovulation was noted in the combination arm.27 There is little doubt that adequate circulating estrogen is necessary for ovulation induction with TMX as is the case with CC Women with hypothalamic anovulation are generally not suitable for treatment with TMX, although it can be used empirically in such women, as they may respond to TMX in preference to CC.28 The available data regarding pregnancy in the anovulatory women treated with TMX is reassuring. The pregnancy rate of 20–35% per cycle is similar to that reported with CC.22–24 This is indeed similar to pregnancy rate in spontaneous cycles. The incidence of miscarriage following TMX therapy is similar to that following CC22 in this group of women. There are no data regarding the incidence of multiple pregnancies with TMX. However, ovulation initiation with TMX is likely to produce fewer follicles than with CC. Both TMX and CC lead to higher levels of circulating oestradiol and progesterone compared to natural cycles. However, the rise seen in CC treated cycles is higher than in TMX cycles, suggesting multiple follicular development with CC.10,13 Hence, the incidence of twins and higher order multiple pregnancies is unlikely to be higher than that following administration of CC, which is approximately 10%.

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Table 5.1: Data showing comparative rates of ovulation and pregnancy with TMX and CC Study

No. of patients No. of cycles Oυulatory cycles Pregnancy rate TMX CC TMX CC TMX(%) CC(%) TMX(%) CC(%)

Boostanfar 46 et al 2001 (20) Messinis et al 46 1982 (21)

40

113

91

46

89

89

50 (44.2) 41 (45.1) 50 (56.2) 56 (62.9)

10 (20.0) 6 (14.6) No data

Tamoxifen may be a useful alternative to CC for ovulation induction in anovulatory women, especially in those who are intolerant to CC. It may be preferable to CC in obese anovulatory women.29 It may have a role in CC resistant anovulation prior to hormonal or surgical therapy. The combination therapy appears attractive for those women resistant to a single agent. Use of higher doses of either drug and hence the untoward effects can thus be avoided. However, this requires proper evaluation prior to implementing in clinical practice. It is generally believed to have an antiestrogenic effect on the endometrium. However, this has not been widely documented, as many of the above studies have not used ultrasonography for monitoring ovulation. Whether it has any beneficial role in women with profound antiestrogenic effect on endometrium from CC would be an interesting aspect to evaluate. The very limited data available is in favor of TMX.15 As discussed earlier, the effect of TMX on cervical mucus quality appears to be variable. However, in women with abnormal cervical mucus score with CC therapy, a trial with TMX would be worthwhile prior to proceeding to higher forms of stimulation or intrauterine insemination.

Table 5.2: Indications for TMX therapy in subfertile women Primary indications: • Alternative to CC in normogonadotrophic, normoprolactinaemic • anovulation. (Those with side effects to CC/obese women) CC resistant anovulation. • ?? In combination with CC in anovulatory women. • Subfertile women with abnormal cervical mucus score • Subfertility associated with luteal phase defect Secondary indications: • Ovarian stimulation prior to IUI • Controlled ovarian stimulation for IVF

By virtue of its luteotrophic effect, TMX is a suitable alternative to CC in women with luteal phase dysfunction.9 In this subgroup of women, both TMX and CC are equally effective in achieving pregnancy. However, there is contradictory evidence regarding the incidence of miscarriage in these women.30,31 Hence, until further evaluated, TMX should be reserved for those women in whom CC has been unsuccessful or associated with side effects.

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In addition, as with CC, it may be suitable for use in controlled ovarian stimulation for the purpose of intrauterine insemination (IUI) and in vitro fertilization (IVF). TMX administration just prior to HCG administration has not shown any untoward effect on the oocyte quality, embryo cleavage or on hormone profile.32 Dose, Administration and Monitoring A detailed clinical assessment is important prior to commencement of treatment. Pelvic ultrasonography to exclude ovarian cysts is advisable. Endocrinological evaluation should be performed to exclude thyroid, adrenal and other causes of anovulation. Severe male factor problems should be excluded in order to avoid the indiscriminate use of the anti-estrogens. The same holds true for assessing tubal factor, if pregnancy is not achieved despite ovulation. The initial dose is 20 mg per day for 5 days, starting from the second or third day of a natural or induced menstrual bleed. The dose can be increased stepwise 40 mg, with a maximum of 60 mg if there is no response to lower dose regimes. The lack of response to a particular dose in a single cycle warrants an increment in the dose. There is no additional advantage in increasing the dose once an ovulatory dose is reached. The response to treatment can be assessed in various ways. Initial cycles require minimal monitoring, as lack of efficacy will be determined relatively rapidly. The simplest method is recording basal body temperature. Urinary LH testing during midcycle or measuring circulating progesterone level during the midluteal phase provides further confirmation of ovulation. Serial transvaginal ultrasonography helps to monitor follicular growth and ovulation. This is particularly useful when higher doses are used, or when there is concern regarding the antiestrogenic effect on endometrium or to time HCG administration to facilitate ovulation in the presence of mature unruptured follicles. Use of ultrasono graphy helps in identification of ovarian cyst formation, so that further treatment can be suitably delayed. The safe and reasonable duration of treatment with TMX has never been investigated. However, treatment should be reviewed after six ovulatory cycles. Also, any risks associated with long-term treatment have not been defined. The RCOG evidence-based guidelines recommend one year’s use of anti-estrogens prior to assisted conception.33 Contraindications A history or presence of liver disease, occurrence of visual symptoms such as dark spots or flashes of light and loss of visual acuity, preexisting ovarian cysts or substantial posttreatment residual ovarian enlargement, and abnormal genital tract bleeding are the rare but absolute contraindications to TMX administration. The most important of all the contraindications is pregnancy. Inadvertent administration of TMX can happen if lack of a menstrual period is mistaken for treatment failure and a subsequent cycle commenced. In such circumstances, neither TMX nor progesterone should be administered before excluding a pregnancy.

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Side Effects In common with other antiestrogens, TMX administration can be associated with nausea, abdominal discomfort, hot flushes and more serious problems such as thrombocytopenia, thromboembolic disease, corneal opacities and retinopathy have been all reported. The side effects are generally less pronounced than with CC and the severe adverse reactions have only been reported with continuous therapy for malignant disease.34 However, information is lacking in its short term use for ovulation induction. Ovarian hyperstimulation following ovulation induction with TMX is rare and usually mild. Endometrial hyperplasia, and even a modest increase in the incidence of endometrial carcinoma, are noted only with long-term therapy with TMX.35 Whether it causes endometrial carcinoma, or facilitates its early detection, is unclear at present. The data is reassuring regarding its use for periods of up to two years.36 The only confirmed ovarian pathology associated with TMX therapy is formation of benign ovarian cysts. There is concern regarding association between ovarian granulosa tumours and ovulation inducing agents. The evidence at present is reassuring and suggests nulliparity and anovulatory infertility to be linked with these tumours rather the use of any specific ovulation-inducing drug.37 There is no increase in the incidence of ovarian malignancy when TMX is used for up to two years.36 There is no report of hepatocellular carcinoma with TMX in humans although this has been reported in rats.38,39 There is no reported cases to suggest that TMX is teratogenic in humans. Animal studies using supraphysiological doses of TMX have led to the development of multiple genital tract abnormalities in fetuses, similar that following in utero exposure to diethylstilboestrol.40 Similar abnormalities are noted when TMX is administered to newborn animals. Clinically such a situation can only arise when a pregnant or breastfeeding woman is given TMX. However, in view of their similarity to DES, it is prudent to exercise caution and avoid exposure of fetuses and neonates to any antioestrogens.34 Conclusion Tamoxifen is chemically similar to the most commonly used drug for ovulation induction, clomiphene citrate. However, there are clinically important differences in the actions of these two agents owing to the difference in their structure and their variable target tissue specificity. The understanding of anti-oestrogens is far from complete. Although TMX has been in clinical use for more than two decades, it has only been sporadically used for initiation of ovulation. The available data suggests that it is a suitable, cost effective and safe alternative to CC in anovulatory infertility In addition, it may be successful in those who are resistant to CC. Randomised controlled trials are needed to evaluate its role and to define its place in the management of subfertile women.

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REFERENCES 1. Adashi EY. Ovulation induction: Clomiphene Citrate. In: Adashi EY, Rock JA, Rosenwaks Z (Eds). Reproductive Endocrinology, Surgery and Technology. Lippencott-Raven 1996; 1:1181– 1206. 2. Greenblatt RB. Chemical induction of ovulation. Fertil Steril 1961; 12:402 3. Jordan CV. Estrogen receptor antagonists. Reproductive Endocrinology, Surgery and Technology 1996; 1:527–45 4. Jordon VC, Collins MM, Rowsby L, Prestwich G. A monohydroxylated metabolite of tamoxifen with potent anti-oestrogenic activity J Endocrinol 1977; 75:305–16. 5. Ruenitz PC, Bagley JR. Comparative fates of clomiphene and tamoxifen in the immature female rat. Drug Metab Disp 1985; 13:582–86. 6. Gerhard I, Runnebaum B. Comparison between tamoxifen and clomiphene therapy in women with anovulation. Arch Gynecol 1979; 227(4):279–88. 7. Chosin T, Talto F. Endocrine profiles in tamoxifen—induced ovulatory cycles. Fertil Steril 1983; 40:23–30. 8. Tajima C: Luteotropic effects of tamoxifen in infertile women. Fertil Steril 1984; 42:223–27. 9. Tajima C: Endocrine profiles in tamoxifen-induced conception cycles. Fertil Steril 1984; 42:548–53. 10. Senior BE, Cawood ML, Oakey RE, McKiddie JM, Siddle DR. Clin Endocrinol 1978; 8:381– 89. 11. Jordon VC, Fritz NF, Langan-Fahey SM, Thompson M and Tormey DC Alteration of endocrine parameters in premenopausal women with breast cancer during long term adjuvant therapy with tamoxifen as a single agent. J Natl Cancer Inst 1991a; 83:1488–91. 12. Lumsden MA, West CP, Baird DT. Tamoxifen prolongs luteal phase in premenopausal women but has no effect on the size of uterine fibroids. Clin Endocrinol 1989; 31:335–43. 13. Roumen FJ, Doesburg WH, Rolland R: Treatment of infertile women with a deficient postcoital test with two antiestrogens: clomiphene and tamoxifen. Fertil Steril 1984; 41:237–243. 14. Annapurna V, Dhaliwal LK, Gopalan S. Effect of two antiestrogens, clomiphene citrate and tamoxif en, on cervical mucus and sperm cervical mucus interaction. Int J Fertil Womens Med 1997; 42:215–18. 15. Allahbadia GN, Allahbadia SG. Evaluatio of endometrium using transvaginal sonography in clomiphene citrate versus tamoxifen stimulated cycles. In Allahbadia GN, ed. Transvaginal Sonography in Infertility. Rotunda Medical Technologies (P) Ltd 1998; 97–108. 16. Elstein M, Fawcett GM. Effects of the anti-oestrogens, clomiphene and tamoxifen, on the cervical factor in female infertility. Ciba Found Symp 1984; 109:173–79. 17. Tepper R, Lunenfeld B, Shaler J, Ovadia J, Blankstein J. The effect of Clomiphene citrate and tamoxifen on cervical mucus. Acta Obstet Gynecol Scand 1988; 67:311–14. 18. Acharya U, Irvine DS, Hamilton MP, Templeton AA. The effect of three anti- estrogen drugs on cervical mucus quality and invitro sperm-cervical mucus in ovulatory women. Hum Reprod 1993; 8:437–41. 19. Macgregor JI, Jordan VC. Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 1998; 50:151–96. 20. Groom GV, Griffiths K. Effect of anti-androgen tamoxifen on plasma levels of luetinizing hormone, follicle stimulating hormone, prolactin, oestradiol and progesterone in normal premenopausal women. J Endocrinol 1976; 70:421–28. 21. Powels TJ, Hickish T, Kanis JA, Ashley S. Effect of tamoxifen on bone mineral density measured by dual-energy X-ray absorptiometry in healthy premenopausal and postmenopausal women. J Clin Oncol 1996; 14:78–84.

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22. Boostanfar R, Jain JK, Mishell DR Jr., Paulson RJ. A prospective randomized trial comparing clomiphene citrate with tamoxifen citrate for ovulation induction. Fertil Steril 2001; 75(5):1024–26. 23. Messinis IE, Nillius S J. Comparison between tamoxifen and clomiphene for induction of ovulation. Acta Obstet Gynecol Scand 1982; 61(4):377–79. 24. Ruiz-Velasco V, Rosas-Arceo J, Matute MM. Chemical inducers of ovulation: Comparative results. Int J Fertil 1979; 24(1):61–64. 25. Borenstein R, Shoham Z, Yemini M, Barash A, Fienstein M, Rozenman D. Tamoxifen treatment in women with failure of clomiphene citrate therapy. Aust N Z J Obstet Gynaecol 1989 May; 29(2):173–75. 26. Gulekli B, Ozaksit G, turhan NO et al: Tamoxifen: an alternative approach in clomiphene resistant polycystic ovarian syndrome patients. J Pak Med Assoc 1993; 43(5):89–90. 27. Suginami H, Kitagawa H, Nakahashi N, Yano K, Matsubara K. A clomiphene citrate and tamoxifen citrate combination therapy: a novel therapy for ovulation induction. Fertil Steril 1993; 59(5):976–79. 28. Weseley AC, Melnick H. Tamoxifen in clomiphene resistant hypothalamic anovulation. Int J Fertil 1987; 32(3):226–28. 29. Boostanfar R, Abbassi D, Tourgeman D, Saadat P, Jain JK, Mishell DR, Paulson RJ. The comparative efficacy of tamoxifen and clomiphene citrate in obese and non-obese anovulatory women: a prospective randomized trial. Fertil Steril 2002; 77(3):S20–21. 30. Buvat J, Buvat-Herbaut M, Marcolin G. Anti-estrogens as treatment of female and male infertility. Horm Res 1987; 28:219–29. 31. Wu CH. Less miscarriage in pregnancy following treatment of infertile patients with luteal phase dysfunction as compared to clomiphene treatment. Early Pregnancy 1997; 3(4):303–05. 32. Fisk NM, Templeton AA, Papdopoulos GC. Lack of effect of high-dose antiestrogen on the maturation and in vitro fertilization of human oocyte. Hum Reprod 1989; 4:584–87. 33. The Management of infertility in secondary care, National Evidence based guidelines. Royal College of Obstetricians and Gynaecologists, 1998. 34. Fox R. Anti-oestrogen therapy in polycystic ovarian syndrome. In: Shaw RW, ed. Advances in Reproductive Endocrinology: Polycystic Ovaries—A disorder or a symptom? Parthenon 1991; 3:149–64. 35. Morrow M, Jordan VC. The tamoxifen trial for breast cancer: clinical issue. In: DeVita VT, Hellman S, Rosenberg Sa, eds. Cancer Prevention. Philadelphia: Lippencott 1992; 1–10. 36. Cook LS, Weiss NS, Schwartz SM, White E, McKnight B, Moore DE, Daling JR. Populationbased study of tamoxifen therapy and subsequent ovarian, endometrial, and breast cancers. J Natl Cancer Inst 1995; 87:1359–64. 37. Unkila-Kallio L, Tiitinen A, Wahlstrom T, Lehtovirta P, Leminen A. Reproductive features in women developing ovarian granulosa cell tumour at a fertile age. Hum Reprod 2000; 15(3):589–93. 38. Dragan YP, Nawaysir E, Fahey S, Vaughan J, McCague, Jordan VC, Pitot HC. The effect of tamoxifen and two of its nonisomerizable fixed-ring analogs on multistage rat hepatocarcinogenesis. Carcinogenesis 1996; 17:585–94. 39. Muhleman K, Cook LS, Weiss NS. The incidence of hepatocellular carcinoma in US white women with breast cancer after introduction of tamoxifen in 1977. Breast Cancer Res Treat 1994; 30:201–04. 40. Cunha GR, Taguchi O, Namiawa R, Nishizuka Y, Robboy SJ. Teratogenic effects of clomiphene, tamoxifen and diethylstilbestrol on the developing human female genital tract. Hum Pathol 1987; 18:1132–43.

CHAPTER 6 Defining the Poor Ovarian Response before Controlled Ovarian Hyperstimulation David W Schmidt, Claudio A Benadiva OVERVIEW The management of the “poor ovarian responder” in controlled ovarian hyperstimulation (COH) at in vitro fertilization (IVF) centers around the world has been a long-standing challenge. Despite advances in ovarian stimulation protocols and IVF laboratory techniques, a significant proportion of subfertile patients will be prone to a poor ovarian response with COH. Although there is not a clear, universal definition of the “poor responder” patient, they tend to represent about 10 percent of patients undergoing treatment in the form of ART (assisted reproductive technology),1 and may be as much as 18 percent at some larger centers.2 This lack of standardized criteria to define poor ovarian response contributes to the difficulty in properly identifying these patients. Most authors define the poor ovarian response in patients that develop less than four mature oocytes by the time of human chorionic gonadotropin (hCG) administration, or a peak estradiol (E2) of less than 500 pg/ml during IVF.2 Other IVF centers may loosely define a poor ovarian responder as any patient having undergone a previous IVF cycle with a poor stimulation outcome. A flaw of this classification system is that it is retrospective in nature. While careful review of previous IVF cycles is crucial in determining future COH protocols, it would be preferable to identify these patients prior to initiating ovarian stimulation. With the expense and resources involved with IVF, the optimal goal of defining the poor ovarian response, is to do so before the patient is treated. Many screening tests have been proposed over the years. Some of these tests have been static serum markers, while others have been dynamic tests evaluating ovarian reserve. It is important to realize that many of the tests have looked at different outcomes; some have measured ovarian response while others have looked at pregnancy rates. The goal of the ideal ovarian screening test, or combination of tests, should be to determine if a woman should attempt IVF with her own oocytes, and to identify which patients will require more aggressive COH protocols. The age related decline in fecundity is a well-accepted process with a steady reduction in the pool of primordial follicles in the ovarian cortex.3 Studies have shown that this onset of diminished ovarian reserve is variable but starts well before menopause, ranging from 5–6 years up to 10 years before the climacteric.4,5 For this reason it may be important to screen subfertile patients of all ages, in order to detect the subset of younger patients with diminished ovarian reserve. Proper classification of the “poor ovarian responder” before treatment begins allows the clinician to appropriately counsel the patient on accurate prognosis and realistic chances of pregnancy. Although some of these tests are experimental and lack prospective randomized studies, some are useful adjuncts in determining proper treatment protocols for the poor ovarian responder. Ovarian reserve testing is critical for women considering IVF in order to assure that their

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time, money and emotions are not wasted in cases where it has little or no hope of success. Methods of Assessing Ovarian Reserve Chronological Age It is well established that a woman’s advancing age is directly correlated with lower ovarian response to ovarian stimulation and to declining pregnancy prognosis.4,6 For the most part a woman’s age is a good general indicator of ovarian reserve;7 ho wever, because of its high variability, it is not a reliable means of screening subfertile patients considering reproductive assistance. The basic premise for chronological age as a general assessment of a woman’s reproductive potential stems from the work of Richardson et al, who showed a correlation with follicular depletion and diminished oocyte quality.5 Faddy et al looked specifically at the concentration of primordial follicles in the ovarian cortex with advancing age. During embryonic development, the number of oogonia peaks by 20 weeks of gestation at approximately 7 million oocytes.8At birth this number declines to 1 million germ cells and by puberty only 250,000 to 300,000 remain. Although the store of germ cells in any individual female determines the eventual onset of menopause, the decline in reproductive potential may precede irregular menses by as much as 10 years.3 This age related decline in fecundity is exemplif ied by cultures that prohibit the use of contraception, such as the Hutterite women. This sect of women in western Canada and the United States has no motive to limit the number of children they have; therefore, the true age related trends in fertility can be analyzed. Tietze saw a decline in f ertility at the age of 34 in this group, with the last pregnancy for women at an average age of 41. At age 34, 11 percent of the women ceased having children and by 39 years old, 33 percent of the women had no further pregnancies.9 After the age of 45, pregnancy was extremely rare. Studies reviewing pregnancy rates in donor insemination cycles also lend support to this phenomenon of age related decline in fecundity.10,11 In these populations coital frequency does not play a role in fertility rates, and for the most part this sample population represents a general population of women with representative fertility rates. less than 25 years old. This declined to 57 percent in Virro et al found a 94 percent pregnancy rate in patients women between the ages of 36 and 40. More recent molecular research has indicated a genetic etiology for the age related diminished ovarian reserve. In addition to the decline in number of oogonia with age, there is evidence to show there is also a decline in oocyte quality with increasing maternal age. First shown in mice with DNA fragmentation,12 the rates of chromosomal abnormalities were found to be significantly higher in older women when their unfertilized oocytes were analyzed following IVF.13 A significantly higher amount of dissociated chromatids was found in older women. For women less than 34 the rate of genetic aberrations was 24 percent. Between the ages of 35 and 39, the rate was 52 percent and in women 40 years and older the rate was 95.8 percent. Advancing maternal age can adversely affect implantation rates14 and increase the risk of miscarriage. The consistently higher pregnancy rates in young donor oocyte cycles

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however, suggests that the non-ovarian effects of aging on the female reproductive system are less significant than the aforementioned decline in oocyte quality and quantity.15,16 Some studies have looked at the outcomes of IVF based on age alone. Dew et al measured ovarian response, fertilization rates, pregnancy rates and outcomes in 779 women who underwent IVF. Three age subgroups were compared, and the age group over 40 showed extremely poor outcomes. Other retrospective studies have shown similar results in older IVF patients.17,18 One of the larger, more recent studies looked at IVF outcomes in 762 patients prospectively. They found a stronger impact of diminished ovarian reserve in patients compared to the effects of their chronological age in terms of implantation, clinical pregnancy and live birth rates.19 Specifically, patients with diminished ovarian reserve were recruited to show that this has a more significant impact on IVF outcomes than age alone. When the need to assess reproductive potential goes beyond the general fertile population, the chronological age as a predictor becomes inaccurate. Scott et al analyzed pregnancy rates in the general infertility population with respect to both age and ovarian reserve. They showed that age alone was a fairly reasonable predictor of fecundity in patients with normal ovarian reserve, but that it was a poor prognostic indicator in patients with any degree of diminished ovarian reserve.20 Other studies have also looked specifically at IVF populations and have found age alone to be a poor prognostic tool.21,22 In summary, patients presenting for IVF treatment cannot be counseled on the basis of age alone. Alarge number of these patients may have some degree of diminished ovarian reserve regardless of age and require more accurate prognostic tests before treatment is initiated. Cycle Day 3 FSH Levels Many studies have shown the prognostic utility of the basal FSH assessment in predicting ovarian response to COH and pregnancy rates. Although the initial studies of serum FSH values did not correlate pregnancy outcomes, they were useful in establishing the cycle day 3 serum FSH test in ovarian reserve screening. Korenman and Sherman first documented elevations of FSH levels in women in their mid-thirties even in the face of normal menstrual cycles.23,24 These elevations occur first in the early follicular phase. Many studies have looked at the day 3 FSH levels, but none to date have looked at them in the general inf ertile population. They have all been used to predict IVF outcomes in some manner. Although most studies have assessed the serum FSH concentrations on day 3 of the cycle, one study by Hansen et al compared values on day 2 to 5 and found no statistical variation between these days in women with regular menstrual cycles.25 Thus some flexibility in this timing of the screening test may exist if an accurate cycle day is obtained. The first study demonstrating the utility of early cycle FSH screening in detecting a potential poor ovarian responder was by Muasher et al.26 The study looked at pregnancy rates and basal FSH levels while comparing gonadotropin releasing hormone (GnRH) stimulation test results with the ovarian response to gonadotropins. The number of patients was small, but other studies would follow to define a clear prognostic role of FSH levels before IVF The largest study to date is a retrospective study of 758 IVF cycles in 441 patients that showed a distinct decline in pregnancy rates with increased basal FSH concentrations:

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day 3 FSH levels above 15 mIU/ml showed a significant decline in pregnancy rates, and very few pregnancies were seen with levels above 25 mIU/ ml. Patients with a low basal FSH concentration <15 mIU/ ml, had an ongoing pregnancy rate of 17 percent. Moderately elevated FSH levels between 15 and 24.9 mIU/ml correlated with an ongoing pregnancy rate of 9.3 percent. Ongoing pregnancy rates of only 3.6 percent were seen in patients with basal levels3 25 mIU/ml. A follow up study at the same institution with 1,478 IVF cycles also showed basal FSH levels provided significantly better prognostic value in both pregnancy rates and cancellation rates over chronological age.27 One caveat to the basal FSH assessment for ovarian reserve is its potential to fluctuate widely from cycle to cycle. This is particularly noted in women with diminished ovarian reserve. In a study by Brown et al, if a patient was under the age of 40 and had a day 3 FSH level <20 mIU/ml, then her chance of having an “abnormal” level above 20 mIU/ml in any given month during that year to follow was only 15 percent. If the patient was greater than 40 years old, her chance of an abnormal value in that following year increased to 75 percent.28 Two studies have looked at the importance of fluctuating basal FSH levels in determining IVF prognosis. Scott et al studied a group of 28 women undergoing IVF who had an elevated FSH value in one cycle and a normal value in another. The outcome measures of ovarian response to stimulation, number of oocytes retrieved, and the fertilization rates did not differ between the two cycles.29 Regardless of the FSH value, the patient responded as a “poor responder” in both cycles. This indicated that once a woman develops a fluctuating day 3 FSH value, she has already shown a diminished ovarian reserve and will behave as such. It also shows that there is limited value in serial screening of day 3 FSH values awaiting an “optimal value” before treatment, since outcomes are poor once the FSH value has begun to show higher variabiliiy.2 This information however is useful for choosing an aggressive stimulation protocol for this subset of patients. Alater study in 1996 looked at pregnancy rates in IVF patients according to the number of abnormal basal FSH values that were obtained.30 If the FSH value was> 20 mIU/ml in the IVF treatment cycle, no pregnancies were seen. In patients with a “normal value” (<20 mIU/ ml in this study) and no other previously elevated values, a pregnancy rate of 16.5 percent was seen. If the FSH value was normal, but there was one previously elevated FSH concentration 3 20 mIU/ml, then the pregnancy rate dropped to 5.6 percent. FSH levels 3 25 mIU/ml at any time resulted in no pregnancies. More recent studies have also correlated a high risk of persistently elevated basal FSH values in patients with an initial value>12 mIU/ ml, with more than 50 percent remaining elevated in a subsequent cycle.31 This underlies the importance of fluctuating FSH values in counseling patients considering IVF treatment. Based on the published information, it appears that even if the FSH value is in the “normal range” just before an intended IVF cycle and even if the stimulation appears adequate, successful pregnancy rates remain poor with a history of elevated basal FSH serum levels. It is worth mentioning that the day 3 FSH ovarian reserve screen holds its prognostic value for patients with one ovary. A comparison of FSH levels in these patients to those with two ovaries, showed statistically similar ovarian responses to gonadotropins, pregnancy rates and delivery rates after controlling for the higher basal FSH levels initially found in the patients with one ovary.32

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With these serum assessments of ovarian reserve, it is important to stress that each IVF center must define their own critical values due to the large variation in what is considered “abnormal”. This variance is in part due to the interassay difference between laboratories with the various hormonal assays that are utilized. Hershlag et al showed as much as a twofold interassay difference in the FSH and E2 concentrations between five different laboratories.33 Other studies have confirmed the interassay variation.34 It is essential for each IVF center to establish their own norms or adopt those of a similar assay from a larger, representative IVF center. Follicular Phase Inhibin Levels The precise physiological basis of follicular FSH serum screening is not entirely understood. It had been theorized that the FSH levels are directly related to inhibin levels, but early studies failed to show an expected variance in early basal inhibin levels as FSH levels fluctuated.35 More recent research has differentiated inhibin A and inhibin B with more specific assays than previous ones, which were only able to measure total inhibin concentrations.22 These more sensitive assays were able to detect a significant decline of inhibin B levels in the early follicular phase with increasing serum FSH levels.36 We know that inhibin is an inhibitor of FSH secretion, and that the two dimers have different patterns in the menstrual cycle first described by Groome et al.37 Specifically, the inhibin B concentrations have been shown to be lower in the early follicular phase, and decrease further with increasing FSH concentrations and increasing age.36 Granulosa cells are believed to secrete both dimers; with inhibin A being secreted in the luteal phase and inhibin B predominately in the follicular phase.37 Inhibin A is seen to increase in the late follicular phase after the rise in serum E2 and is believed to be secreted by the dominant follicle.38 Hence, inhibin A is thought to be a marker of follicular maturity and decreases with age, which may be reflective of the fewer granulosa cells in older women.39 Inhibin B concentrations increase during the late luteal phase and early follicular phase. With its peak in the mid-follicular phase,40 Inhibin B has been postulated to represent the quantity or quality of the developing follicles in that cycle.38 As direct products of the granulosa cells, these two dimers theoretically might better reflect ovarian reserve as a marker of secretory capacity and follicle number. Although these original studies characterized the patterns of dimeric inhibin levels in the menstrual cycle and noted various trends in their levels with age, the clinical utility of these serum markers in predicting ovarian reserve has been controversial.41 The studies of inhibin B levels have certainly all shown corresponding lower day 3 levels as FSH values increase, but is this just a reflection of its inhibitory action of FSH secretion, or does this serum assay provide more useful information on IVF outcome? Two initial studies investigated this question and showed an independent predictive value of early serum inhibin B levels for IVF outcomes. Seifer et al showed that inhibin B levels on cycle day 3 can be used as a direct measure of ovarian reserve, with concentrations <45 pg/ml correlating with lower E2 responses and fewer oocytes retrieved.42 Higher IVF cancellation rates and lower clinical pregnancy rates were also seen in women with day 3 inhibin B levels <45 pg/ml. Balasch et al compared the predictive value of chronological age to inhibin B and FSH levels and found day 3 inhibin B levels to be a better predictor of IVF cancellation than age.22 The day 3 inhibin B level may also be useful in detecting women with diminished

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ovarian reserve who have normal day 3 FSH values. Researchers have shown that decreases in inhibin B often precede serum FSH changes as ovarian reserve declines.43,44 More recent studies have failed to show a clinical benefit in measuring basal inhibin B for predicting IVF outcome.38,45–47 It has some utility as a marker for ovarian reserve, but it’s value in predicting IVF pregnancy outcome has not been well defined. Peñarrubia et al recently showed in a retrospective study that day 5 inhibin B levels measured during treatment cycles correlated well with lack of ovarian response, but not with pregnancy outcome.48 A cut-off value of 141 pg/ml was used. This study looked at the dynamic changes in the serum inhibin B concentrations during IVF stimulation, which may be a more useful clinical marker of ovarian response. More recently, the added prognostic value of inhibin B levels during COH for IVF, has been confirmed in a randomized, prospective clinical trial.49 Day 5 inhibin B levels were measured af ter 4 days of stimulation and were found to correlate with the number of mature follicles> 14 mm, number of oocytes retrieved, and number fertilized. Women with levels <400 pg/ml had poorer outcomes in all of the aforementioned IVF outcome parameters, compared to those with levels >400 pg/ml. This study also showed a beneficial role in early detection of either the poor responder for cancellation, or the hyperresponder for reduction of medication dose. The study suggested that serum day 5 inhibin B levels <100 pg/ml may be an indication for cancellation of that cycle, and that levels >1000 pg/ml may warrant reduction in the gonadotrophin dose and close monitoring for ovarian hyperstimulation syndrome (OHSS).49 The current drawback of inhibin B levels used clinically is that there is not yet a universal assay standard since both human recombinant and follicular fluid standards are being used.48 Thus, the values of inhibin B should be used as guidelines along with other clinical or laboratory assessments of ovarian response. The additional information that this serum marker provides however, appears to be useful in counseling patients during COH with an early assessment of ovarian stimulation. It may aid in early IVF cancellation avoiding further cost and therapy, as well as being useful in dictating future management strategies. Serum Estradiol Levels The additional prognostic value of basal E2 levels in predicting IVF outcomes has remained controversial. Basal E2 values are beneficial in screening for the potential poor ovarian responder in the context of a “normal” FSH value. It has been shown that a day 3 E2 level can vary as much as 40 percent compared to day 2 or 4 values, while the FSH value only shows an 18 percent variance between these days.28 Thus, while the FSH value alone is a more accurate predictor of ovarian reserve, the E2 level has value in interpreting the FSH results. Because of the negative feedback of elevated E2 levels on FSH secretion, a “normal” value of FSH on day 3 of a cycle may be falsely low in the face of elevated E2 levels. As previously mentioned, the early follicular phase E2 level can vary widely between days 2 to 4 and elevated levels may be present due to an early recruitment or development of a dominant follicle. This early luteal recruitment may occur when a diminished cohort of follicles produces less inhibin.50 It is possible that the higher E2 level might suppress FSH levels into the “normal” range even when a patient has diminished ovarian reserve. Elevated follicular phase E2 levels may also be seen in the perimenopause.51 Regardless

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of age, elevated day 3 E2 levels and FSH levels have also been associated with an increased risk of recurrent pregnancy loss, likely due to diminished ovarian reserve.52 Two prominent studies have been published looking at the role of day 3 E2 values for prognosis in ovarian stimulation and pregnancy rates in IVF. Licciardi et al assessed the ongoing pregnancy rates for patients with day 3 E2 levels <30 pg/ml, E2>75 pg/ml, and intermediate values. No pregnancies occurred when E2 values exceeded 75 pg/ml, and pregnancies were the highest in the <30 pg/ml group.53 The authors suggested that an E2 determination with day 3 FSH assessment was superior to either test alone. Controversy stemmed from this study because when FSH values are controlled for, there is no difference in pregnancy rates in women with increased or normal E2 values.2 A study by Smotrich et al with 225 patients undergoing IVF demonstrated a lower pregnancy rate in women with day 3 E2 levels ≥80 pg/ ml.54 No pregnancies occurred with E2 levels ≥100 pg/ ml on day 3. Higher cancellation rates were also seen with E2 ≥80 pg/ml, and this occurred independently of day 3 FSH values. An additional prospective study of 231 patients undergoing first attempt IVF cycles was performed that confirmed the added prognostic benefit of elevated E2 levels in a subset of patients with normal basal FSH values.55 An elevated FSH value alone retained its predictive value for poor ovarian response, but in the “normal” FSH group a difference in cancellation rate was seen (56%, versus 13% in patients with low E2). The investigators however, used a discriminatory FSH value of 17 mIU/ml, which is higher than most ART centers allow today. Further studies are required to accurately define threshold values for day 3 E2 levels and how to interpret them in conjunction with the day 3 FSH results. Serum markers such basal FSH: LH ratios have not been shown to be of an added benefit over other serum markers in predicting pregnancy outcomes in IVF. One study showed however, that a high FSH: LH ratio >3 (despite normal FSH values) may help identify the poor ovarian responder.56 Controlling for age and number of embryos transferred, this study also noted significantly fewer oocytes retrieved, lower implantation rates and poorer clinical pregnancy rates in women with a higher day 3 ratio preceding COH. Clomiphene Citrate Challenge Test Although the aforementioned serum markers can be easily obtained and are useful in assessing baseline ovarian reserve, they are inferior in detecting those patients with diminished ovarian reserve who have normal day 3 test results. This is due in part, to the intercycle variability of these markers which somewhat limits the value of the tests. The clomiphene citrate challenge test (CCCT) was originally described by Navot et al in 1987 as a means of assessing ovarian reserve in women 35 years of age or older.57 It has been validated by several other studies since then, and argued that it is a more reliable predictor of diminished ovarian reserve than FSH values alone when predicting response to COH.58 The test checks hormone levels on the 3rd and 10th day of a patient’s cycle in which 100 mg of clomiphene citrate has been taken orally from days 5 through 9. This test is a dynamic assessment of the ovarian reserve indirectly. It evaluates the hypothalamic-pituitary-ovarian axis with the premise that women with normal ovarian reserve will have enough metabolic activity from a cohort of developing follicles to overcome the impact of clomiphene citrate and be able to suppress the FSH value to the

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normal range by day 10.27 A more recent investigation has suggested that decreased inhibin B productionby the granulosa cells of women with an abnormal CCCT leads to the elevated FSH value seen on cycle day 10.59 The CCCT detects as many as 2–3 times more women with diminished ovarian reserve than the day 3 FSH value alone.60,61 The CCCT combines the day 3 FSH and E2 prognostic values with the dynamic ovarian response seen by day 10. The day 3 or day 10 FSH value is considered normal when it is £ 9.6 mIU/ml. Values between 10 and 15 mIU/ ml are considered indeterminate and pregnancy is possible, but lower pregnancy rates are seen and more aggressive stimulation protocols may be required. Patients with day 3 or day 10 FSH values ≥17 mIU/ml with a CCCT rarely become pregnant and exhibit higher miscarriage rates.62 It is important to obtain E2 values on both days to place the FSH values in context on day 3 as previously described. The E2 levels drawn on day 10 help to identify patients that are unresponsive to clomiphene citrate, such as those with hypothalamic amenorrhea. Hoftman et al suggested that cycle day progesterone levels ≥1.1 ng/ml with the CCCT might be indicative of a short follicular phase and poor reproductive performance.63 Other speculated markers of the CCCT, such as progesterone-to-E2 ratio, have not been shown to be useful in detecting diminished ovarian reserve.64 It is important to understand that the CCCT lacks positive predictive value. It is up to 94 percent accurate57 in detecting patients with diminished ovarian reserve who might have otherwise gone undetected with a normal day 3 FSH value; however, a normal CCCT does not necessarily predict the successful outcome of patients undergoing COH for IVF. There is still an age related decline in fecundity even when the CCCT yields normal results.27 Another drawback with the CCCT is that there is a potential for some intercycle variation in the cycle day 10 FSH values. This was demonstrated in a study by Hannoun et al,65 which found a significant variation in 75 percent of the patients, although the variation did not correlate well with the potential to achieve a pregnancy. Multiple studies have shown the predictive value of the CCCT in determining diminished ovarian reserve in patients undergoing COH.21,58,66,67 In the general infertility population, Scott et al studied 236 patients prospectively and confirmed the negative predictive value of the test with pregnancies occurring in 43 percent of patients with a normal CCCT, but in only 9 percent of patients with abnormal results. Pregnancy rates were significantly decreased even when controlling for age.60 An interesting finding of this long-term study however, was the very high incidence of abnormal tests in patients with unexplained infertility. This represented the largest category of patients with an abnormal CCCT (38%) and was not related to age.27,60 Therefore, the CCCT may be particularly beneficial in screening patients with a diagnosis of “unexplained infertility”, older patients ≥ 35, and certainly patients that are suspected to have diminished ovarian reserve regardless of their age. Other Dynamic Ovarian Reserve Tests Another dynamic test of ovarian reserve described in the literature is the GnRH-a Stimulation Test (GAST). Originally proposed by Padilla et al, the test evaluates the change in serum E2 levels between cycle day 2 and 3 after 1 mg of subcutaneous leuprolide acetate is administered. Four different patterns of E2 levels were noted. Patients with E2 elevations by day 2 and declines by day 3 had better implantation and

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pregnancy rates than those patients with either no rise in E2, or persistently elevated E2 levels.68 Winslow et al showed a trend in increased pregnancy rates with an initial increased estrogen response, but the number of oocytes was not statistically higher in the group with a normal test result.69 From these initial studies, the prognostic value of the GAST fcr differentiating patients with normal and diminished ovarian reserve seemed limited. A subsequent study five years later looked at the effect of 0.3 mg of buserelin acetate on FSH levels before and 2 hrs after administration during the first day of ovarian stimulation in IVF cycles.70 The study did show a correlation in the sum of the two FSH concentrations with stimulation outcome, but it did not provide any additional prognostic value over basal FSH values alone. The most recent comparison of the GAST with other tests of ovarian reserve found the test to be the least sensitive, and less accurate than all the other tests.61 The additional expense of the test over other methods also limits its practical use in most IVF centers. The GAST has not been evaluated in non-IVF populations and perhaps further studies are needed before it is accepted as a standard test of ovarian reserve. The exogenous FSH ovarian reserve test (EFORT) is another dynamic test that has been described. Originally, the test was developed to improve the predictive value of day 3 FSH values in COH for IVF.71 Specifically, the E2 level is recorded on cycle day 3 before and 24 hours af ter the administration of 300 IU of purified FSH. It was postulated that the dynamic increase in E2 ≥30 pg/ml would be predictive of a good response in a subsequent IVF cycle. The study measured ongoing pregnancy rates and cancellation rates in a total of 52 cycles. When both the basal day 3 FSH value was £ 11 IU/l, and the D E2 was ≥30 pg/ml, the EFORT provided significant improvement in predicting ovarian stimulation outcome over the predictive value of basal FSH alone.71 Fabregues et al attempted to reproduce the findings of the previous study with a slight variation of the EFORT.72 E2 levels were measured 5 days after 300 IU of FSH were administered, failing to show a prognostic benefit for IVF outcome. Finally, other investigators have evaluated other markers, such as inhibin B levels, that may have prognostic value for IVF outcome when combined with the EFORT.73 In a retrospective study including only “poor” and “good” responders, Dzik et al found a significant prognostic value of the 24-hour change in inhibin B serum levels with the EFORT. The “poor response” group showed a 2.4-fold-less increase in inhibin B levels (70%) as compared with the “good responders” (167%). Thus, the authors propose that this provocative serum marker may be useful in identifying poor ovarian responders before IVF. Large, prospective studies should be performed including patients with a broader range of ovarian response to IVF before this EFORT marker becomes standardized. Sonographic Assessment Using transvaginal ultrasonography, a woman’s measured ovarian volume has been proposed as a useful predictor of ovarian response before COH. The ovarian volume does not vary much in women of normal ovarian reserve. An average volume of 5.8 cm3 is noted by the age of 18.74 Several studies have shown an association with ovarian volume and the outcome of COH for IVF.75– 79 Syrop and colleagues suspected a poorer ovarian response in women with smaller ovarian volumes, and they correlated ovarian volumes

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measured on cycle day 23 in 188 women with their COH parameters and cycle outcomes.75 The volumes of both ovaries were calculated using the ellipsoid formula π/6 (length×height×width), which simplifies to 0.526×length×height×width.75 They found that age correlated very poorly with ovarian volume. Both the patient’s age and the total ovarian volume were independently predictive of cycle cancellation, peak E2 concentration, number of oocytes retrieved, and number of embryos obtained. The volume of the smallest ovary was predictive of these parameters in addition to pregnancy rates. They concluded that ovarian volume might be an important, noninvasive, predictor of ovarian reserve. The test is simple to perform and shows little inter-observer variation.80 A recent study by Frattarelli et al found that the mean ovarian diameter measured in the largest sagital plane is also useful. A comparison showed it to be a quick, yet reliable estimate of the measured ovarian volume.81 Assessment of ovarian volume sonographically can be a useful modality in identifying and counseling patients that may have a poor ovarian response before they undergo COH. This hypothesis was confirmed by a prospective clinical trial by Lass et al.77 Excluding polycystic ovarian syndrome patients, 140 women were studied, and a single ovarian volume of <3 cm3 was significantly predictive of higher IVF cancellation rates (>50%) compared with patients who’s smallest ovarian volume was >3 cm3.77 Single ovarian volumes <3 cm3 were found in patients of all ages. These patients required more ampoules of gonadotropins during stimulation, had poorer follicular development and yielded fewer oocytes.77 The authors advocated transvaginal ovarian volume assessment for all patients before undergoing IVF treatment as a simple, cost-effective means of identifying poor ovarian responders. Both of these previous studies assessed ovarian volumes in the late luteal phase, prior to COH utilizing a long luteal leuprolide protocol and subsequent gonadotropin stimulation. A study by Sharara et al showed no significant difference in the ovarian volumes, nor the mean number of antral follicles noted between the initial assessment on day 21, the day leuprolide acetate (LA) was started, and the day of starting gonadotropins.82 Desensitization of the pituitary with LA seems to have no effect on overall ovarian volume measurements. Thus, whether ovarian volume assessment is done in the luteal phase or early follicular phase, it still retains significant prognostic value. One caveat to the assessment of ovarian volume is that the ovaries should not contain cysts or large follicles (only follicles <10–15 mm were allowed in all of these studies). Syrop et al also conducted a study looking at the predictive value of ovarian volume while controlling for smoking status and history, which had not been controlled for in previous studies. With age and smoking status accounted for, the investigators found ovarian volume to be a better measure of ovarian reserve than basal FSH values.79 The same investigators recently analyzed the response of COH in patients with an ovarian volume <3 cm3. They switched to a more aggressive microdose GnRH agonist stimulation protocol when a diminished ovarian volume was detected.83 Patients with a decreased volume in the smallest ovary required higher gonadotropin doses, more days of stimulation and had a lower yield of oocytes. When this more aggressive stimulation plan was used (versus a standard long luteal agonist protocol), implantation and pregnancy rates were comparable to those who had larger ovarian volumes. This suggests that intervention with more aggressive stimulation plans may benefit patients with decreased ovarian volumes, if they are identified prior to undergoing treatment.

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The antral follicle count (AFC) assessed early in the follicular phase is an additional ultrasound modality that can be a useful predictor of IVF success. Several studies have shown the AFC to correlate with the number of oocytes collected in a future IVF cycle as well as with cancellation rates.78,84 The first investigators to assess the AFC did so in 31 healthy women between the ages of 22 and 42. They established a 60 percent decline in the number of antral follicles 2 mm or larger over this timeframe.85 This study suggested that the AFC might be representative of ovarian reserve. Other studies have applied this measure of ovarian reserve to IVF outcome. Tomas et al measured both ovarian volumes and the AFC (follicles 2–5 mm) before starting COH for IVF. Three groups were established: “inactive ovaries” with <5 follicles in both ovaries, “normal ovaries” with 5–10 follicles total, and “polycystic ovaries” with >15 follicles counted. Ovarian volume did correlate with the AFC, but the number of small follicles present before ovarian stimulation was a better predictor of IVF outcome than ovarian volume or age alone.78 Lass et al established a correlation between ovarian volume and follicular density in women ≥35 years of age,76 but it appears that women with a diminished AFC respond poorly to ovarian stimulation, even when ovarian volumes are equal to women with normal AFC’s.86 Chang et al measured the AFC on cycle day 1 or 2 and classified the patients into three groups according to the number of antral follicles visualized by transvaginal ultrasound: < 4, between 4–10, and >10 follicles.84 The group with an AFC of <4 had no pregnancies, showed the highest day 3 FSH concentrations, had the highest cancellation rates, and required the most ampoules of FSH during stimulation. In a recent prospective study, Laszlo et al compared the AFC to the traditional basal serum markers of ovarian reserve. Asingle ultrasonographer assessed AFC’s during the early follicular phase without any pituitary suppression. The AFC was the single best predictor for poor ovarian response in women undergoing their first IVF cycle.87 Ovarian volume, chronological age, and basal E2 values did not help predict IVF outcomes. This underscores the importance of looking beyond chronological age and FSH values in assessing candidates for IVF Although the advent of transvaginal sonography has improved our ability to detect poor ovarian responders through volumetric assessment and AFC’s, there are still limitations. Some investigators have advocated the use of three dimensional (3D) ultrasound, suggesting that it might be superior for ovarian volume measurement,88,89 and more sensitive in detecting smaller antral follicles.84,86 Kupesic and Kurjak recently compared pretreatment 3D ultrasound ovarian measurements and the total AFC prospectively with subsequent ovulation induction parameters. The 3D AFC had superior predictive value for a favorable IVF outcome over peak E2 on hCG administration day, total ovarian volume and chronological age.90 The advent of 3D ultrasound technology has also helped revisit some previous conclusions drawn from older studies using twodimensional (2D) ultrasonography regarding the actual predictive value of ovarian volume. The recent findings of Schild et al using 3D transvaginal ultrasound for predicting IVF success, failed to predict conception in women with unilateral ovarian volumes £ 3 mL3, as was shown in earlier studies with 2D ultrasound.91 On the other hand, 3D ultrasound has strengthened the claim that the AFC assessed prior to IVF has significant prognostic value. The advances in 3D ultrasound technology have lead to its important role in research, however it is still relatively new and not yet in widespread

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clinical use. The images can take a bit longer to generate which may limit its use in a practical sense, particularly in high volume IVF centers. FUTURE DIRECTIONS As the technology improves and becomes more widely available, a time may come when 3D ultrasound becomes a universal prognostic tool in all IVF centers. In addition, other applications of transvaginal ultrasound are under investigation, such as color power angiography to assess follicular blood flow and predict the development of healthy oocytes.92 Pulsed color Doppler has shown that the intraovarian pulsatility index (PI) is significantly lower in FSH-treated patients compared with spontaneous cycles, suggesting that multiple follicular development is related to a reduction in the impedance of perifollicular blood flow.93 A few studies have tested the predictive value of intraovarian blood flow for IVF. Tekay et al failed to find any difference in flow with IVF patients who subsequently became pregnant versus those who did not.94 Balakier and Stronell were able to find a strong correlation between follicular size in women undergoing COH and their peak perif ollicular velocity and resistance index, but this did not correlate to the maturity of the oocytes.95 Kupesic has shown a correlation in the ovarian stromal flow index and number of mature oocytes retrieved in an IVF cycle and pregnancy rates.90 The addition of computer-aided programs to 3D transvaginal ultrasonography has also been investigated recently. Fanchin et al revisited the question of whether endometrial echogenic patterns in the late follicular phase predict IVF-ET outcomes. There have been conflicting answers to this question from previous studies, most likely due to differences in embryo quality that had not been controlled for, and from the intra-observer variability in the assessment of the endometrial pattern.96–98 Computer programs that analyze the endometrial echogenicity digitally may remove any variation in human assessment, and may improve its prognostic value in IVF cycles.99 Another group of researchers has applied computer-aided analysis to the 3D sonographic assessment of endometrial volume during IVF cycles, in hopes of reducing the range of error by traditional means.100 They found that a virtual organ computer-aided analysis (VOC AL) improved accuracy of ascertained endometrial volumes, but its importance in predicting IVF outcomes, or identifying poor IVF candidates, requires further clinical studies. The advances in cellular and molecular biology techniques have improved our current serological markers of ovarian reserve. They have also given future prospect for other markers being studied. The future of identifying the poor ovarian responder before COH may lie in these new molecular ovarian markers. Gonadotropin surge-attenuating factor (GnSAF) is an ovarian factor not yet well characterized. It is involved in the ovarianpituitary axis, reducing responsiveness of the pituitary to GnRH without affecting LH or FSH secretion.101 Patients with low ovarian reserve may have less GnSAF production, and this may be involved with the premature luteinization that occurs more frequently in these patients.101,102 A preliminary study has shown that poor ovarian response patients have significantly lower circulating levels of GnSAF and a significantly blunted GnSAF rise following FSH stimulation.101 GnSAF levels are however, still investigational at this point due to the lack of an available immunoassay.

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Molecular advances are also being used to study other ovarian factors, such as vascular endothelial growth factor (VEGF) and their receptors. It appears that a delicate balance between VEGF and its soluble tyrosine receptor, sVEGFR-1, is essential for an adequate ovarian response to gonadotropin stimulation.103 An initial study has found an excess of sVEGFR-1 in patients with poor ovarian response to COH correlating with reduced conception.103 Further development in this field is required before this test becomes a clinically useful marker of poor ovarian response. CONCLUSIONS Identifying the ‘poor ovarian responder’ before IVF has its obvious benefits. It allows for appropriate counseling of patients that may not proceed with COH and IVF treatment, but rather consider donor-oocyte IVF or adoption. Pretreatment detection of the ‘poor responder’ however, is not a diagnosis of sterility, and pregnancies can still occur. It is essential that patients with diminished ovarian reserve be identified early, ideally before therapy is initiated, so that appropriately aggressive treatment protocols may be chosen. Discussion of the various treatment strategies for optimizing COH and IVF in these patients is beyond the scope of this chapter, but obviously the specific management options play as much of a role in IVF outcomes as does the proper identification of the ‘poor responder’. Many of the current ovarian reserve modalities have been reviewed, but it is critical to understand the importance of a careful history of the patient who is about to consider COH and IVF. The history may reveal clues of diminished ovarian reserve. For example, a history of current or past smoking has been shown in several studies to reduce the ovarian reserve, reduce ovarian volume, lead to poor ovarian response to stimulation at earlier ages, and predict higher IVF cancellation rates.75,79,104 A history suggestive of severe pelvic and peri-ovarian adhesions may also be significant, as several studies have correlated poorer folliculogenesis related to impeded diffusion of exogenous gonadotropins to the follicles during COH, and lower pregnancy rates.105–107 The history of a patient’s previous ovarian response to stimulation is also paramount in their successful treatment. Although this chapter has focused on markers of ovarian reserve prior to COH, a history of previous poor response can be a significant predictor of future IVF outcome and guide management decisions. For instance, women with a low number of retrieved oocytes (0–3) at their first IVF cycle are more likely to reach menopause at an earlier age,108 and aggressive subsequent management may be indicated. Ideally, effective serum markers and sonographic assessment of ovarian reserve should allow for appro priate management of the ‘poor responder’. Thus, repeated futile attempts of IVF can be avoided in poor candidates, or aggressive management of IVF candidates showing diminished ovarian reserve can be pursued early A realistic chance of pregnancy should be discussed with the patient. Markers detecting diminished ovarian reserve do not necessarily correlate with chances of obtaining a pregnancy. Age alone is only a general predictor of ovarian reserve in the general population, and a combination of the aforementioned tests should be used to screen patients with subfertility before embarking on ahy IVF treatments. Currently, a combination of basal FSH, E2, inhibin B levels, and sonographic AFC may provide the best prognostic value for future IVF

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outcomes. Independently, each has limited sensitivity and specificity for outcome predictions. Our university based IVF center manages approximately 1,000 IVF cycles each year. In our hands, an initial transvaginal AFC and ovarian volume have been useful in identifying the potential ‘poor responder’ with the parameters mentioned previously. We combine this sonographic evaluation with basal day 3 FSH, LH, and E2 values in our pretreatment assessment (Fig. 6.1). The CCCT is used selectively in patients that yield a ‘normal’ FSH value <10, but are suspected of having diminished ovarian reserve. Because IVF centers may have various transvaginal sonographic equipment and expertise, and

Fig. 6.1: Overview of ovarian reserve assessment serum markers of ovarian reserve may be subject to interassay variability, each ART program must determine what tests and what range of values are predictive of a poor ovarian response in their own IVF patients. REFERENCES 1. Pellicer A et al. Outcome of in vitro fertilization in women with low response to ovarian stimulation. Fertil Steril 1987; 47:812–15. 2. Scott RT, Jr. Evaluation and treatment of low responders. Semin Reprod Endocrinol 1996; 14(4):317–37.

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3. Faddy MJ, Gosden RG, Gougeon A et al. Accelerated disappearance of ovarian follicles in midlife: implications for forecasting menopause. Hum Reprod 1992; 7:1342–46. 4. Alrayyes S, Fakih H, Khan I. Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection. Fertil Steril 1997; 68(1):123–27. 5. Richardson SJ, Senikas V, Nelson JF. Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J Clin Endocrinol Metab 1987; 65:1231– 37. 6. Dew JE, Don RA, Hughes GJ, Johnson TC, Steigrad SJ. The influence of advanced age on the outcome of assisted reproduction. J Assist Reprod Genet 1998; 15(4):210–14. 7. Jacobs SL, Metzger DA, Dodson WC, Haney AF. Effect of age on response to human menopausal gonadotropin stimulation. J Clin Endocrinol Metab 1990; 71:1525–1530. 8. Baker TG. A quantitative and cytological study of germ cells in human ovaries. Proc R Soc Lond Biol J 1963; 158:417–33. 9. Tietze C. Reproductive span and rate of reproduction among Hutterite women. Fertil Steril 1957; 8:89–97. 10. Van Noord-Zaadstra BM, Looman CWN, Alsbach H et al. Delaying child-bearing: effect of age on fecundity and outcome of pregnancy. Br Med J 1991; 302:1361–65. 11. Virro MR, ShewchukAB. Pregnancy outcome in 242 conceptions after artificial insemination with donor sperm and effects of maternal age on the prognosis of successful pregnancy. Am J Obstet Gynecol 1984; 148:518–24. 12. Fujino X, Ozaki K, Yamamasu S, Ito F, Matsuoka I, Hayashi E et al. DNA fragmentation of oocytes in aged mice. Hum Reprod 1996; 11:1480–83. 13. Lim AST, Tsakok MFH. Age-related decline in fertility; a link to degenerative oocytes? Fertil Steril 1997; 68:265–71. 14. Spandorfer S, Chung P, Kligman I, Liu H, Davis O, Rosenwaks Z. An analysis of the effect of age on implantation rates. J Assist Reprod Genet 2000; 17(6):303–6. 15. Scott RT, Rosenwaks Z. Oocyte donation. In: JJ S (Ed): Obstetrics and gynecology. Philadelphia: JB Lippincott Co, 1990; 1–14. 16. Rotsztejn DA, Asch RH. Effect of aging on assisted reproductive technologies (ART): experience from oocyte donation. Semin Reprod Endocrinol 1991; 9:272–79. 17. Roest J, van Heusden AM, Mous H, Zeilmaker GH, Verhoeff A. The ovarian response as a predictor for successful in vitro fertilization treatment after the age of 40 years. Fertil Steril 1996; 66(6):969–73. 18. Hanoch J, Lavy Y, Holzer H, Hurwitz A, Simon A, Revel A et al. Young low responders protected from untoward effects of reduced ovarian response. Fertil Steril 1998; 69(6):1001–4. 19. El-Toukhy T, Khalaf Y, Hart R, Taylor A, Braude P. Young age does not protect against the adverse effects of reduced ovarian reserve-an eight year study. Hum Reprod 2002; 17(6): 1519– 24. 20. Scott RT, Opsahl MS, Leonardi MR et al. Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age. Hum Reprod 1995; 10:1706– 10. 21. Toner JP, Philput CB, Jones GS et al. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991; 55:784–91. 22. Balasch J, Creus M, Fabregues F, Carmona F, Casamitjana R, Ascaso C et al. Inhibin, folliclestimulating hormone, and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin—releasing hormone agonistgonadotropin treatment. Am J Obstet Gynecol 1996; 175(5):1226–30. 23. Pearlstone AC, Fournet N, Gambone JC et al. Ovulation induction in women of age 40 and older: the importance of basal follicle-stimulating hormone level and chronological age. Fertil Steril 1992; 58:674–79. 24. Sherman BM, Krorenman SG. Hormonal characteristics of the human menstrual cycle throughout reproductive life. J Clin Invest 1975; 55:699–706.

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25. Hansen LM, Batzer FR, Gutman JN et al. Evaluating ovarian reserve: follicle stimulating hormone and oestradiol variability during cycle days 2–5. Hum Reprod 1996; 11:486–89. 26. Muasher SJ, Oehninger S, Simonetti S, Matta J, Ellis LM, Liu H-C. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988; 50:298–307. 27. Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril 1995; 63(1):1– 11. 28. Brown JR, Liu H-C, Sewitch KF, Rosenwaks Z, Berkeley AS. Variability of day 3 folliclestimulating hormone levels in eumenorrheic women. J Reprod Med 1995; 40(9):620–24. 29. Scott RTJ, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 folliclestimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril 1990; 54:297–302. 30. Martin JSB, Nisker JA, Tummon IS et al. Future in vitro fertilization pregnancy potential of women with variably elevated day 3 follicle-stimulating hormone levels. Fertil Steril 1996; 65:1238–40. 31. Lass A, Gerrard A, Abusheikha N, Akagbosu F, Brinsden P. IVF performance of women who have fluctuating early follicular FSH levels. J Assist Reprod Genet 2000; 17(10):566–73. 32. Khalifa E, Toner JP, Muasher SJ, Acosta AA. Significance of basal follicle-stimulating hormone levels in women with one ovary in a program of in vitro fertilization. Fertil Steril 1992; 57:835–39. 33. Herslag A, Lesser M, Montejusco D, Lavy G, Kaplan P, Liu H-C et al. Interinstitutional variability of follicle stimulating hormone and estradiol levels. Fertil Steril 1992; 58:1123–26. 34. Lambalk CB, De Koning CH. Interpretation of elevated FSH in the regular menstrual cycle. Maturitas 1998; 30:215–20. 35. Hughes EG, Robertson DM, Handlesman DJ, Hayward S, Healy DL, de Kretser DM. Inhibin and estradiol responses to ovarian hyperstimulation: effects of age and predictive value for in vitro fertilization outcome. J Clin Endocrinol Metab 1990; 70:358–64. 36. Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE, Soules MR. Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular levels of dimeric inhibin A and B in spontaneous menstrual cycles. J Clin Endocrinol Metab 1996; 81:2742–45. 37. Groome N, Illingworth P, O’Brien M, Pai R, Rodger FE, Mather JP et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996; 81(4):110–14. 38. Hall JE, Welt CK, Cramer DW. Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum Reprod 1999; 14:409–15. 39. Santoro N, Adel T, Skurnick JH. Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil Steril 1999; 71:658–62. 40. Danforth DR, Arbogast LK, Mroueh J et al. Dimeric inhibin: a direct marker of ovarian aging. Fertil Steril 1998; 70:119–23. 41. Bancsi LF, Broekmans FJ, te Velde ER. Predictive value of serum inhibin B for ART outcome? Fertil Steril 1997; 68:947–48. 42. Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner AC, Blazar AS, Berk CA. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril 1997; 67:110–14. 43. Seifer DB, Scott RTJ, Bergh PA, Abrogast LK, Freidman CI, Mack CK et al. Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone. Fertil Steril 1999; 72:63–65. 44. Welt CK, McNicholl DJ, Taylor AE, Hall JE. Female reproductive aging is marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab 1999; 84:105–11. 45. Corson SL, Gutman JN, Batzer FR, Wallace H, Klein N, Soules MR. Inhibin-B as a test of ovarian reserve for infertile women. Hum Reprod 1999; 14:2818–21.

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46. Ravhon A, Lavery S, Michael S, Donaldson M, Margara R, Trew G et al. Dynamic assays of inhibin B and oestradiol following buserelin acetate administration as predictors of ovarian response in IVF. Hum Reprod 2000; 15:2297–2301. 47. Creus M, Penarrubia J, Fabregues F, Vidal E, Carmona F, Casamitjana R et al. Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum Reprod 2000; 15(11):2341–46. 48. Penarrubia J, Balasch J, Fabregues F, Carmona F, Casamitjana R, Moreno V et al. Day 5 inhibin B serum concentrations as predictors of assisted reproductive technology outcome in cycles stimulated with gonadotrophin- releasing hormone agonistgonadotrophin treatment. Hum Reprod 2000; 15(7):1499–504. 49. Fawzy M, Lambert A, Harrison RF, Knight PG, Groome N, Hennelly B et al. Day 5 inhibin B levels in a treatment cycle are predictive of IVF outcome. Hum Reprod 2002; 17(6):1535–43. 50. Kligman I, Rosenwaks Z. Differentiating clinical profiles: predicting good responders, poor responders, and hyperresponders. Fertil Steril 2001; 76(6):1185–90. 51. Santoro N, Rosenberg-Brown J, Adel T et al. Characterization of reproductive hormonal dynamics in the perimenopause. J Clin Endocrinol Metab 1996; 81:1495–1501. 52. Trout SW, Seifer DB. Do women with unexplained recurrent pregnancy loss have higher day 3 serum FSH and estradiol values? Fertil Steril 2000; 74(2):335–37. 53. Licciardi FL, Liu H-C, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995; 64:991–94. 54. Smotrich D, Widra E, Gindoff P, Levy MJ, Hall JL, Stillman RJ. Prognostic value of day 3 estradiol on in vitro fertilization outcome. Fertil Steril 1995; 64:1136–40. 55. Evers JL, Slaats P, Land JA, Dumoulin JC, Dunselman GAJ. Elevated levels of basal estradiol17B predict poor response in patients with normal basal levels of follicle stimulating hormone undergoing in vitro fertilization. Fertil Steril 1998; 69(6):1010–14. 56. Barroso G, Oehninger S, Monzo A, Kolm P, Gibbons WE, Muasher SJ. High FSH: LH ratio and low LH levels in basal cycle day 3: impact on follicular development and IVF outcome. J Assist Reprod Genet 2001; 8(9):499–505. 57. Navot D, Rosenwaks Z, Mergalioth EJ. Prognostic assessment of female fecundity. Lancet 1987; 332:645–47. 58. Tanbo T, Dale PO, Lunde O, Norman N, Abyholm T. Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated folliclestimulating hormone levels. Fertil Steril 1992; 57(4):819–24. 59. Hofmann GE, Danforth DR, Seifer DB. Inhibin-B: the physiologic basis of the clomiphene citrate challenge test for ovarian reserve screening. Fertil Steril 1998; 69(3):474–77. 60. Scott RT, Leonardi MR, Hofmann GE et al. A prospective evaluation of clomiphene citrate challenge test screening in the general infertility population. Obstet Gynecol 1993; 82:539–45. 61. Gulekli B, BulbulY, OnvuralA. Accuracy of ovarian reserve tests. Hum Reprod 1999; 14:2822– 26. 62. Hofmann GE, Khoury J, Thie J. Recurrent pregnancy loss and diminished ovarian reserve. Fertil Steril 2000; 74(6):1192–95. 63. Hofmann GE, Scott RT, Horowitz GM, Thie J, Navot D. Evaluation of the reproductive performance of women with elevated day 10 progesterone levels during ovarian reserve screening. Fertil Steril 1995; 63(5):979–83. 64. Hofmann GE, Khoury J, Michener C. Elevated serum progesterone-to-estradiol ratio during gonadotropin stimulation for intrauterine insemination or in vitro fertilization is not associated with diminished ovarian reserve. Fertil Steril 2002; 78(1):47–50. 65. Hannoun A, Abu Musa A, Awwad J et al. Clomiphene citrate challenge test: a cycle to cycle variability of cycle day 10 follicle stimulating hormone level. Clin Exp Obstet Gynecol 1998; 25:155–56.

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66. Loumaye E, Billion J-M, Mine J-M, Psalit I, Pensis M, Thomas K. Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril 1990; 53:295–301. 67. Tanbo T, Dale PO, Abyholm T, Stokke KT. Follicle-stimulating hormone as a prognostic indicator in clomiphene cycles for in vitro fertilization. Hum Reprod 1989; 4(6):647–50. 68. Padilla SL, Smith RD, Garcia JE. The Lupron screening test: tailoring the use of leuprolide acetate in ovarian stimulation for in vitro fertilization. Fertil Steril 1991; 56:79–83. 69. Winslow KL, Toner JP, Bryzyski RG, Oehninger SC, Acosta AA, Muasher SJ. The gonadotropin-releasing hormone agonist stimulation test—a sensitive predictor of performance in the flare-up in vitro fertilization cycle. Fertil Steril 1991; 56:711–17. 70. Galtier-Dereure F, De Bouard V, Picto MC, Vergnes C, Humeau C, Bringer J et al. Ovarian reserve test with the gonadotrophinreleasing hormone agonist buserelin: correlation with in vitro fertilization outcome. Hum Reprod 1996; 11:1393–98. 71. Fanchin R, de Ziegler D, Olivennes F, Taieb J, Dzik A, Frydman R. Exogenous follicle stimulating hormone ovarian reserve test (EFORT): a simple and reliable screening test for detecting ‘poor responders’ in in vitro fertilization. Hum Reprod 1994; 9(9):1607–11. 72. Fabregues F, Balasch J, Creus M et al. Ovarian reserve test with human menopausal gonadotropin as a predictor of in vitro fertilization outcome. J Assist Reprod Genet 2000; 17:13–19. 73. Dzik A, Lambert-Messerlian G, Izzo VM, Soares JB, Pinotti JA, Seifer DB. Inhibin B response to EFORT is associated with the outcome of oocyte retrieval in the subsequent in vitro fertilization cycle. Fertil Steril 2000; 74(6):1114–17. 74. Ivarson SA, Nillson KO, Persson PH. Ultrasonography of the pelvic organs in prepubertal and postpubertal girls. Arch Dis Child 1983; 58:352–54. 75. Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel predictor for assisted reproduction. Fertil Steril 1995; 64:1167–71. 76. Lass A et al. Follicular density in ovarian biopsy of infertile women: a novel method to assess ovarian reserve. Hum Reprod 1997; 12:1028–31. 77. Lass A, Skull J, McVeigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in vitro fertilization can predict poor response. Hum Reprod 1997; 12(2):294–97. 78. Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in vitro fertilization. Hum Reprod 1997; 12(2):220–23. 79. Syrop CH et al. Ovarian volume may predict assisted reproductive outcomes better than follicle stimulating hormone concentration on day 3. Hum Reprod 1999; 14:1752–56. 80. Higgins RV, Van Nagell JRJ, Woods CH, Thompson EA, Kryscio RJ. Interobserver variation in ovarian measurements using transvaginal sonography. Gynecol Oncol 1990; 39:69–71. 81. Frattarelli JL, Levi AJ, Miller BT. A prospective novel method of determining ovarian size during in vitro fertilization cycles. J Assist Reprod Genet 2002; 19(1):39–41. 82. Sharara FI, Lim J, McClamrock HD. The effect of pituitary desensitization on ovarian volume measurements prior to in vitro fertilization. Hum Reprod 1999; 14(1):183–85. 83. Sharara FI, McClamrock HD. Use of microdose GnRH agonist protocol in women with low ovarian volumes undergoing IVE. Hum Reprod 2001; 16(3):500–3. 84. Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. Use of the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril 1998; 69(3):505–10. 85. Reuss ML et al. Age and ovarian follicle pool assessed with transvaginal ultrasonography. Am J Obstet Gynecol 1996; 174:624–27. 86. Pellicer A, Ardiles G, Neuspiller F et al. Evaluation of the ovarian reserve in young low responders with normal basal FSH levels of follicle stimulating hormone using three dimensional ultrasonography. Fertil Steril 1998; 70:671–75.

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87. Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002; 77(2):328–36. 88. Brunner M, Obruca A, Bauer P, Feichtinger W. Clinical application of volume estimation based on three-dimensional ultrasonography. Ultrasound Obstet Gynecol 1995; 6:358–61. 89. Tulandi T, Watkin K, Tan SL. Reproductive performance and three-dimensional ultrasound volume determination of polycystic ovaries following laparoscopic ovarian drilling. Int J Fertil Women’s Med 1996; 42:436–40. 90. Kupesic S, Kurjak A. Predictors of IVF outcome by three-dimensional ultrasound. Hum Reprod 2002; 17(4):950–55. 91. Schild RL, Knobloch C, Diminished ovarian reserven C, Fimmers R, van der Ven H, Hansmann M. The role of ovarian volume in an in vitro fertilization programme as assessed by 3D ultrasound. Arch Gynecol Obstet 2001; 265(2):67–72. 92. Sladkevicius P, Campbell S. Advanced ultrasound examination in the management of subfertility. Curr Opin Obstet Gynecol 2000; 12(3):221–25. 93. Stringini FAL, Scida PAM, Parri C et al. Modifications in uterine and intraovarian artery impedance in cycles of treatment with exogenous gonadotropins: effects of luteal phase support. Fertil Steril 1995; 64:76–80. 94. TekayA, Martikainen H, Jouppila P. Blood flow changes in uterine and ovarian vasculature, and predictive value of transvaginal pulsed colour Doppler ultrasonography in an in vitro fertilization programme. Hum Reprod 1995; 11:1589–91. 95. Balakier H, Stronell RG. Color doppler assessment of folliculogenesis in in vitro fertilization patients. Fertil Steril 1994; 62:1211–16. 96. Coulam CB, Bustillo M, Soenksen DM, Britten S. Ultrasonographic predictors of implantation after assisted reproduction. Fertil Steril 1994; 62:1004–10. 97. Oliveira JB, Baruffi RL, Mauri AL, Petersen CG, Campos MS, Franco JGJ. Endometrial ultrasonography as predictor of pregnancy in an in vitro fertilization programme. Hum Reprod 1993; 8:1312–15. 98. Check JH, Nowroozi K, Choe J, Lurie D, Dietterich C. The effect of endometrial thickness and echo pattern on in vitro fertilization outcome in donor oocyte-embryo transfer cycle. Fertil Steril 1993; 59:72–75. 99. Fanchin R, Righini C, Ayoubi JM, Olivennes F, de Ziegler D, Frydman R. New look at endometrial echogenicity: objective computer-assisted measurements predict endometrial receptivity in in vitro fertilization- embryo transfer. Fertil Steril 2000; 74(2):274–81. 100. Bordes A, Bory AM, Benchaib M, Rudigoz RC, Salle B. Reproducibility of transvaginal three-dimensional endometrial volume measurements with virtual organ computer-aided analysis (VOCAL) during ovarian stimulation. Ultrasound Obstet Gynecol 2002; 19:76–80. 101. Martinez F, Barri PN, Coroleu B, Tur R, Sorsa-Leslie T, Harris WJ et al. Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles. Hum Reprod 2002; 17(3):634–40. 102. ShulmanA, GhetlerY, BaythY, Ben-Nun I. The significance of an early (Premature) rise of plasma progesterone in In vitro Fertilization cycles induced by a ‘Long Protocol’ of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J Assist Reprod Genet 1996; 13:207–11. 103. Neulen J, Wenzel D, Hornig C, Wunsch E, Weissenborn U, Grunwald K et al. Poor responderhigh responder: the importance of soluble vascular endothelial growth f actor receptor 1 in ovarian stimulation protocols. Hum Reprod 2001; 16(4):621–26. 104. El-Nemr A, Al-Shawaf T, Sabatini L, Wilson C, Lower AM, Grudzinskas JG. Effect of smoking on ovarian reserve and ovarian stimulation in in- vitro fertilization and embryo transfer. Hum Reprod 1998; 13(8):2192–98.

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105. Mahadevan MM, Wiseman D, Leader A et al. The effects of ovarian adhesive disease upon follicular development in cycles of controlled stimulation for in vitro fentilization. Fertil Steril 1985; 44:489–92. 106. Nagata Y, Honjou K, Shindou M et al. The effects of periovarian adhesions in human IVF-ET programs. J. Fertil. Implant. 1997; 14:54–57. 107. Nagata Y, Honjou K, Sonoda M, Makino I, Tamura R, Kawara-bayashi T. Peri-ovarian adhesions interfere with the diffusion of gonadotrophin into the follicular fluid. Hum Reprod 1998; 13(8):2072–76. 108. de Boer EJ, den Tonkelaar I, te Velde ER, Burger CW, Klip H, van Leeuwen FE. A low number of retrieved oocytes at in vitro fertilization treatment is predictive of early menopause. Fertil Steril 2002; 77(5):978–85.

CHAPTER 7 Aromatase Inhibitors—Their Role in the Treatment of Infertility Pankaj Shrivastav The understanding of how drugs work at the molecular level sometimes allows their use for indications quite diverse from the primary one. Aromatase Inhibitors represent such a group of drugs. Though these drugs have been known for over 2 decades, it is only recently that they have been applied to such diverse indications as breast cancer, infertility and endometriosis. To understand how this has come about, it is imperative that the physiological role of the enzyme aromatase is understood. Several human tissues produce estrogen and each of these contain the enzyme aromatase, which catalyses the conversion of C19 steroid to estrogen.1 In normally cycling women, the seat of primary production of estrogen and thus of aromatase expression, is the granulosa cells of the ovary. The aromatase activity in the ovarian granulosa cells is directly under the control of the gonadotrophin FSH, through a pathway involving cyclic AMP and a host of other factors. Two of these, steroidogenic factor-1 (SF-1), and cAMP response element binding protein (CREB), bind to the promoter of CYP19 gene, which is also known as aromatase P450, and this leads to the production of the protein known as aromatase.2,3 Once the production of the aromatase protein has begun, this automatically leads to a conversion of the androgenic precursors produced in the theca cells to oestradiol. This event primarily takes place in the granulosa cells of ovulatory, premenopausal women. There are other extra ovarian sources of estrogen production. In the postmenopausal woman, high levels of aromatase activity is present in the adipose tissue fibroblasts and skin fibroblasts.4 Adipose tissue fibroblasts comprise a major source of estrogen in postmenopausal women. The aromatase activity in adipose tissue is only present in the undifferentiated fibroblast and not in any significant quantity in the mature adipocytes.4,5 These fibroblasts use as their substrate, androstenedione, which is produced in the adrenal cortex. The androstenedione is aromatized in the fibroblasts of the adipose tissue to oestrone, which is further converted to oestradiol at the same site.1 The realization, that by inhibiting the enzyme aromatase, estrogen production levels would be suppressed in both the ovarian granulosa cells and the undifferentiated fibroblast of the adipose tissue, led to the introduction of aromatase inhibitors to combat estrogen responsive breast cancer. Initially, these compounds were used as a second line modality in patients who had not responded to Tamoxifen, but with the realization that they appear to be more effective than Tamoxifen, it is possible that they may replace Tamoxifen as the first-line of treatment in the follow-up of women with breast cancer.6 The estrogen synthesized in the granulosa cells of premenopausal women exerts a negative feedback effect at the pituitary and hypothalamic levels, thereby suppressing the production of FSH. It was then realized that if estrogen production could be suppressed

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by suppressing the enzyme aromatase, the negative feedback exerted by estrogen at the hypothalamic/pituitary level would be abolished, thereby leading to an increased production of gonadotrophins. It is this understanding that has led to the introduction of aromatase inhibitors in the management of anovulatory infertility. Use of Aromatase Inhibitors in Infertility The first line treatment for women with anovulatory infertility is anti-estrogens and the most commonly utilized drug in this group is clomiphene citrate. However, in women with polycystic ovarian disease, 20–25% of women are resistant to clomiphene citrate and fail to ovulate with this medication. Even in women who do ovulate, there is a discrepancy between the ovulation rate and conception rate, and women who conceive following the use of clomiphene citrate are reported to have a higher incidence of miscarriages.7,8 It is postulated that prolonged use of clomiphene citrate, due to its antiestrogenic action, leads to a long lasting depletion of estrogen receptors (ER). Clomiphene citrate has a long half-life and accumulates in the body. This may lead to a detrimental effect on the development of the endometrium (an estrogen responsive site) and also on the quality of cervical mucus. At present, women who fail to respond to clomiphene citrate are put on gonadotrophin preparations, which though successful in inducing ovulation, subject the women to an increased risk of massive multifollicular development. This may lead to the ovarian hyperstimulation syndrome (OHSS) and consequently, to multiple pregnancy Aromatase inhibitors would form a useful second line modality of treatment in patients who do not ovulate/conceive following clomiphene citrate. Mitwally and Casper.9 were the first to recommended the use of aromatase inhibitors in women who do not ovulate with clomiphene citrate. Aromatase inhibitors mimic the action of clomiphene citrate in inducing ovulation but without the associated depletion of estrogen receptors. These authors have recommended the use of an aromatase inhibitor, Letrozole in an oral dose of 2.5 mg. daily from the 3rd to the 7th day of the menstrual cycle. This would inhibit estrogen secretion from the granulosa cells, which would lead to the release of the hypothalamic/pituitary axis from the negative feedback action of estrogen, leading to an increased secretion of gonadotrophins and consequently to ovarian follicular development. They succeeded in inducing ovulation and obtaining pregnancies in women who had previously failed to respond to clomiphene citrate. Aromatase inhibitors reach peak plasma concentrations in 4–8 hours and have relatively short half-life of 45 hours.9 There is complete bioavailability of the medication on oral administration. An additional benefit of the use of aromatase inhibitors is that they are not associated with the anti estrogenic action of clomiphene citrate on peripheral tissues, such as endometrium and cervical mucus. In women with polycystic ovarian disease, who did not respond to clomiphene citrate, Mitwally and Casper9 reported ovulation in 75% of cycles when Letrozole was used, and a clinical pregnancy rate of 17% per cycle. In women who responded to clomiphene citrate but developed very poor quality endometrium, Mitwally and Casper9 reported an improved thickness of the endometrium, which they believed would help in implantation. The estrogen levels in women on aromatase inhibitors was found to be 2–3 times lower than that reported in clomiphene

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citrate cycles, but despite this, the endometrium thickness was greater in the aromatase inhibitor cycle.9 The same authors9 have also suggested an alternative hypothesis for the action of Letrozole-they postulate that it may act locally in the ovary itself, increasing the follicular sensitivity to FSH. As the conversion of intra ovarian androgens to estrogen is blocked, there is an accumulation of androgens within the ovary. Data from primates10 suggests that increased levels of androgens in the ovary have a stimulatory effect on follicular growth, which implies that androgens promote follicular growth by amplifying the effects of FSH. The ability of aromatase inhibitors to induce ovulation without any deleterious effect on endometrial development and thickness may also allow these compounds to be used in women undergoing frozen-thawed embryo replacement cycles. There is a subset of women who do not produce endometrium of optimal thickness in either natural cycles, or cycles stimulated with clomiphene citrate. It would be interesting to see whether aromatase inhibitors may help these women to produce an endometrium more conducive to implantation. The Role of Aromatase Inhibitors in the Treatment of Endometriosis Current medical modalities for the treatment of endometriosis (GnRH Agonists, oral contraceptives, Danazol and medroxyprogesterone acetate), all have major limitations in their effectiveness. Most of these treatments are unsatisfactory due to the incomplete eradication of pain. Moreover, their action appears to be temporary with the return of symptoms and the disease, once the treatment is discontinued. Recent investigations,11 into the metabolic activity within the ectopic endometrial tissue, have shown that large amounts of estrogen could be synthesized locally within the endometriotic deposits. This locally produced estrogen can not be blocked by the use of GnRH Agonists or Danazol. Additionally oestradiol, which is produced in peripheral sites i.e., undifferentiated adipose and skin fibroblasts, may be one of the reasons why these compounds are not completely effective in the suppression of symptoms in women with endometriosis. Noble et al,12 have reported the abnormal presence of aromatase in ectopic endometrial tissue and this may be responsible for the excessive local production of oestradiol. It is interesting to note that aromatase is not present in normal intrauterine endometrium. Prostaglandins are abundantly present in ectopic endometrial tissue and contribute to the symptoms associated with endometriosis. Noble et al,13 have reported an induction of high levels of aromatase activity by prostaglandin PGE2 in endometriotic tissue. The induction of aromatase activity in endometriotic tissue would lead to an increased local production of oestradiol. This in turn would lead to induction of cyclo-oxygenase type 2 (COX-2) which is the rate-limiting enzyme for prostaglandin E2 biosynthesis.14 The prostaglandin E2 would then lead to further stimulation of aromatase production, thereby establishing a positive feedback cycle. This understanding of the molecular events in ectopic endometrial deposits have led to the first report of the use of aromatase inhibitors in treating endometriosis. Takayama et al,15 used 1 mg daily of anastrozole (an aromatase inhibitor) in a woman with post menopausal endometriosis of the vaginal cuff who had failed to respond to hysterectomy and bilateral salpingooophorectomy, subsequent resection of the endometriotic deposits

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and treatment with megesterol acetate. With the use of anastrozole, there was a rapid decrease in pelvic pain and the endometric implants at the vaginal apex rapidly regressed in size. It was thought that the benefit of the treatment was due to the inhibition of the aromatase activity in the endometriotic tissue, and the consequent lowering of estrogen levels. This in turn would have led to suppression of COX-2 expression, which would diminish the formation of prostaglandin E2, thereby leading to amelioration of symptoms. One significant side effect of the treatment was a bone loss of about 6.2% over a 9-month period. In future, it is possible that aromatase inhibitors may be used in conjunction with GnRH Agonists in the management of patients with severe endometriosis. This may lead to an increased symptom free duration for the patient after discontinuation of treatment. One benefit of aromatase treatment alone in the management of endometriosis is that it does not inhibit ovulation and this may be of significance to inf ertile women with endometriosis who are desirous of conceiving. Aromatase inhibitors are relatively new additions to our armamentarium in the treatment of infertility, but they do hold potential in the two indications that I have outlined above. Further investigations and randomized trials will determine whether they have a role to play, if any, in the medical management of inf ertility. REFERENCES 1. Simpson ER, Mahwendroo MS, Means GD et al. Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Rev 1994; 15:342–55. 2. Michael MD, Kilgore MQ, Morohashi KI et al. Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 1995; 270:13561–566. 3. Michael MD, Michael LF, Simpson ER. A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell. Endocrinol., 1997; 134:147–56. 4. Ackerman GE, Smith ME, Mendelson CR et al. Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J. Clin. Endocrinol Metab., 1981; 53:412–17. 5. Price T, Aitkin J, Head J, et al. Determination of aromatase cytochrome P450 messenger RNA in human breast tissues by competitive polymerase chain reaction (PCR) amplification. J Clin Endocrinol Metab 1992; 74:1247–1252. 6. Harper-Wynne C, Dowsett M. Recent advances in the clinical application of aromatase inhibitors. J. Steroid Biochem Mol Biol 2001; 76(1–5):179–86. 7. Franks S, Adams J, Mason H, Polson D. Ovulatory disorders in women with polycystic ovary syndrome. Clin Obstet Gynecol 1985; 12:605–32. 8. Hull MGR. The causes of infertility and relative effectiveness of treatment. In: Templeton AA, Drife JO. editors. Infertility: London: Springer-Verlag, 1992; 33–62. 9. Mitwally, Mohamed FM MD, Casper, Robert FMD. Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate. Fertility and Sterility, 2001; 75:305–309. 10. Weil SJ, Vendola K, Zhou J, Adesanya OO, Wang J, Okafor J, et al. Androgen receptor gene expression in the primate ovary: Cellular localization, regulation, and functional correlations. J Clin Endocrinol Metab 1998; 83:2479–85.

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11. Serdar E Bulun1, Khaled M Zeitoun2, Kazuto Takayama3 and Hironobu Sasano3 Molecular basis for treating endometriosis with aromatase inhibitors. Human Reproduction Update 2000; 6(5):413–18. 12. Noble LS, Simpson ER, Johns A et al: Aromatase expression in endometriosis. J. Clin. Endocrinol. Metab., 1996; 81:174–79. 13. Noble LS, Takayama K, Putman JM et al. Prostaglandin E2 stimulates aromatase expression in endometriosis-derived stromal cells. J Clin Endocrinol Metab 1997; 82:600–606. 14. Huang JC, Dawood MY, Wu KK. Regulation of cyclooxygenase-2 gene in cultured endometrial stromal cells by sex steroids. Proceedings of 52nd Annual Meeting of the American Society for Reproductive Medicine 1996; 1:5. 15. Takayama K, Zeitoun K, Gunby RT et al. Treatment of severe postmenopausal endometriosis with an aromatase inhibitor. Fertil. Steril, 1998; 69:709–13.

CHAPTER 8 Urinary Human FSH Versus Recombinant Human FSH Eitan Lunenfeld, Tali Silberstein INTRODUCTION The use of exogenous gonadotrophins to induce ovulation is a well established practice for the management of infertility.1 From the late 1950s, these were prepared using urine obtained from menopausal women (human menopausal gonadotrophin, hMG),2 and an international reference standard was established with such preparations.3 However, the early preparations were of low purity and specific activity (8 IU/mg protein), and contained both follicle stimulating hormone (FSH) and luteinizing hormone (LH).4 Although the development of immunopurification techniques during the 1980s5,6 enabled the production of urinary FSH (u-hFSH) preparations from which LH had been removed and with increased specific activity (e.g. Metrodin, Ares-Serono, Geneva, Switzerland, 100–150 IU/mg protein; Metrodin HP, Ares-Serono, Geneva, Switzerland, 9000 IU/mg protein), the increasing demand for assisted reproductive techniques (ART) during the same period required the collection of ever larger volumes of urine. For example, the annual production of Metrodin HP requires 60 million litres of urine to be collected from approximately 300,000 donors.1,4 Although these relatively pure urinary FSH preparations proved significantly more potent than other hMG preparations, they have since been discredited because of their batch to batch inconsistently. Thus, there was a clear need for a safe, reliable and cost-effective source of FSH. Recombinant Human FSH (r-hFSH) Preparations The application of recombinant DNA technology finally made possible the production of a pharmaceutical FSH preparation, resulting in the approval of r-hFSH (follitro pin alpha, Gonal F, Ares-Serono, Geneva, Switzerland) in 1995. Follitropin alpha is produced by transfecting Chinese hamster ovary cells with the genes for the a and b subunits of human FSH.7,8 The secretory products of these cells undergo a six-step purification process to give a final preparation which is highly pure biochemically (> 99% FSH), with high specific activity (approximately 10,000 IU/mg protein) and which is structurally identical to native human pituitary FSH.4 Unlike u-hFSH, however, follitropin alpha shows a low level of oxidation and/or degradation; typically less than 10%, compared with 30–40% for u-hFSH. A second r-hFSH preparation, follitropin beta (Puregon/Follistim, NV Organon, Oss, The Netherlands), is also available. The follitropin alpha and follitropin beta molecules are almost indistinguishable structurally and biochemically, except for some minor

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differences in the percentage of oxidized/degraded isoforms. In addition, it has been demonstrated that, when administered on an equivalent IU basis, they identical ovarian stimulation characteristics.9,10,11 In view of the similarities clinical efficacy, data obtained from studies of both r-hFSH preparations discussed together in the remainder of this review.

recently produce of their will be

r-hFSH Induces Follicular Development The clinical utility of r-hFSH was first demonstrated in 1992, when two case reports12,13 described the successful use of r-hFSH in combination with a gonadotrophinreleasing hormone (GnRH) agonist to stimulate multiple follicular development and oestradiol secretion in patients undergoing in vitro fertilization and embryo transfer (IVF-ET). The retrieved oocytes were fertilized and pregnancies were achieved. These preliminary observations were subsequently confirmed in clinical trials which investigated the efficacy of r-hFSH in ART in combination with different GnRH agonists in a long protocol.14,15 Comparison of r-hFSH with u-hFSH in ART The clinical development programs for both r-hFSH preparations included key studies which compared the efficacy and safety of r-hFSH in ART with that of u-hFSH. Most of these studies employed protocols which involved pituitary desensitization, and these are discussed below. The studies which have compared the efficacy and tolerability of r-hFSH and u-hFSH are summarized in Table 8.1. All these studies have demonstrated that r-hFSH is at least as effective as u-hFSH in promoting follicular development in ART.16,20

Table 8.1: Summary of characteristics of studies comparing recombi- nant human folliclestimulating hormone (r-hFSH) with urinary human follicle-stimulating hormone (u-hFSH) r-hFSH u-hFSH p-υalue Total IU r-hFSH Study Group (16) O’Dea et al (17) Hedon et al (18) Out et al (19) Frydman et al (20) Days of treatment r-hFSH Study Group (16) O’Dea et al (17) Hedon et al (18) Out et al (19) Frydman et al (20) Number of embryos

2270 2498 2265 2138 2070

2095 2123 p=0.00 2213 p=0.75 2385 p<0.000 3052

9.9 10.0 10.2 10.7 11.7

9.4 9.0 p=0.00 10.3 p=0.83 11.3 p<0.000 14.5 p<0.000

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5.0 5.6 3.7 3.1 5

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6.1 6.4 p=0.36 4.0 p=0.69 2.6 p<0.00 3.5 p<0.000

Preliminary observations by O’Dea et al17 indicated that the number of follicles >14 mm diameter and clinical pregnancy rates per completed cycle were comparable for women receiving leuprolide and either u-hFSH or r-hFSH, but that pain at the injection site was less frequent among the r-hFSH-treated patients. These findings were supported by the first multicentre, prospective, randomized clinical trial comparing r-hFSH and u-hFSH in women undergoing IVF-ET.16 In this small study, patients received either intramuscular injections of u-hFSH (n= 63) or subcutaneous injections of r-hFSH (n=60) following down-regulation with intranasal buserelin in a long protocol, and the results confirmed that r-hFSH was as effective and well tolerated as u-hFSH. In a second, small, assessor-blind multicentre trial, a total of 99 women were randomized in a 3:2 ratio to receive either subcutaneous r-hFSH (n=60) or intramuscular u-hFSH (n=39), and down-regulation was achieved with daily subcutaneous injections of triptorelin.18 Although the diff erences between the two treatments did not reach statistical significance, trends were observed towards a higher number of retrieved oocytes (9.7 vs 8.9), higher serum oestradiol levels, (7551 vs 5514 pmol/l), and higher ongoing pregnancy rates per cycle (30.2% vs 17.4%) and per transfer (34.0% vs 18.8%) among women who received r-hFSH compared with those treated with u-hFSH. Three patients (5%) who received r-hFSH were hospitalized for ovarian hyperstimulation syndrome (OHSS), and there was no OHSS in the u-hFSH group. A large, prospective, multicentre study, involving a total of 1027 patients who were treated with intranasal buserelin provided evidence for the superior efficacy of r-hFSH compared with u-hFSH.19 Among patients receiving subcutaneous r-hFSH (n=615), a significantly higher number of oocytes were retrieved (10.8 vs 8.9, adjusted for centre effect; p<0.0001), with a lower total dose of FSH (2138 vs 2385 IU; p<0.0001) over a shorter treatment period (10.7 vs 11.3 days; p<0.0001) compared with those who were treated with intramuscular u-hFSH (n=412). The number of high quality embryos was also significantly greater among the women who received the recombinant product (3.1 vs 2.6; p=0.003), but the implantation rates and clinical pregnancy rates per attempt and per transfer did not differ between the two groups. However, when frozen embryo cycles were included in the analysis, ongoing pregnancy rates were significantly higher in the rhFSH group (25.5%) than in the u-hFSH group (20.4%; p<0.05). Finally, the incidence of OHSS was similar in the two groups (3.2% with r-hFSH and 2.0% with u-hFSH) and no anti-FSH antibodies were detected in the women who received r-hFSH. In a double blind, randomized study, Frydman et al20 compared the results in 139 patients who received r-hFSH to 130 patients who received u-hFSH. Among patients receiving r-hFSH, there was a significantly higher mean number of oocytes and mean number of embryos obtained. With r-hFSH, significantly fewer FSH treatment days were required than with u-hFSH. No significant diff erence was found in live born children. These studies all confirmed the observation that r-hFSH alone can successfully induce multiple follicular growth,14 even in pituitary down-regulated cycles with very low

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endogenous LH activity. Furthermore, they provided evidence that r-hFSH was more effective than u-hFSH in inducing multiple ovulation for IVF-ET.21 This is supported by the findings of a meta-analysis of three prospective multicentre, randomized, comparative trials of r-hFSH with u-hFSH and hMG.22 Such analyses are particularly useful when the results from individual studies appear to show effects with similar trends, yet lack the power to demonstrate statistical significance.23 The meta-analysis found that the ongoing pregnancy rate (at least 12 weeks after embryo transfer per started cycle) was higher for r-hFSH (22.9%) than for urinary gonadotrophins (17.9%), and that the 5% treatment difference was statistically significant in favour of r-hFSH (p= 0.044).22 Furthermore, when the replacement of cryopreserved embryos was also included in the analysis, the treatment difference increased to 6.4% (p=0.011). Finally, it is noteworthy that two studies which compared r-hFSH and u-hFSH for ART in cycles without pituitary down-regulation have also found that the recombinant products are more effective than urinary gonadotrophins.24,25 Comparison of r-hFSH and Highly Purified u-hFSH The efficacy and safety of r-hFSH for inducing multiple follicular development in ART have also been compared with highly purified u-hFSH (u-hFSH HP), a product with specific activity which approaches that of r-hFSH and which can be injected subcutaneously The first such study was a prospective, randomized, assessor-blind, two centre trial, which showed that r-hFSH (n=119) is more effective than u-hFSH HP (n=114) in inducing follicular development in women undergoing ovarian stimulation for IVF, including intracytoplasmic sperm injection (ICSI), and who were down-regulated with intranasal buserelin in a long protocol.26 The mean number of oocytes retrieved, which was the primary endpoint for the study, was significantly higher among the women who received r-hFSH (12.2±5.5) than in those who were treated with u-hFSH HP (7.6±4.4; p<0.0001). In addition, the number of days of FSH treatment (11.0± 1.6 vs 13.5±3.7; p<0.001) and the number of ampoules of FSH used (21.9±5.1 vs 31.9±13.4; p<0.0001) were significantly lower in the r-hFSH group compared with the u-hFSH HP group. Among patients treated using ICSI (63 per group), no differences in embryo maturation were observed between the two groups. However, the mean number of embryos obtained was higher in the patients receiving the recombinant product. Although there were no significant differences between r-hFSH and u-hFSH treatment in the pregnancy rate per started cycle (45% and 37%, respectively) or per embryo transfer (48% and 47%, respectively), the number of cryopreserved embryos was significantly higher in the rhFSH group (3.2) than in the u-hFSH group (p<0.0001). Thus, there was a potentially higher chance of conceiving from a single cycle of r-hFSH treatment compared with uhFSH. Finally, the two treatments were well tolerated, and the incidence of OHSS was 5.1% in the r-hFSH group and 1.7% in the u-hFSH group. More recently, the preliminary results of the first double-blind, randomized comparison of subcutaneous injections of r-hFSH and u-hFSH HP in women undergoing ART have become available.27 This study has also shown that the mean number of oocytes recovered (11.0 ±5.9 vs 8.8±4.8; p=0.0001) and the mean number of embryos obtained (5.0±3.7 vs 3.5±2.9; p=0.0002) were both significantly higher among the

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women treated with r-hFSH (n=130) compared with those who received u-hFSH HP (n=116). In addition, the duration of treatment was significantly less in the r-hFSH group (11.7±1.9 days) than in the u-hFSH HP group (14.5+3.3 days; p=0.001), and the r-hFSH treated patients used significantly fewer ampoules of 75 IU FSH (27.6±10.2 vs 40.7±13.6; p= 0.0001). However, the study did not have sufficient power to detect any difference in ongoing clinical pregnancy rates. Both treatments were well tolerated, and the incidence of OHSS was less than 1.0% in both groups. Another randomized, single blind, multicentere, multinationale study done by Schats et al31 support the conclusion that r-hFSH is more effective than highly purified u-hFSH in inducing multiple follicular development. In this study 496 women were randomized, 232 and 231 in the r-hFSH and HP u-hFSH groups respectively. The duration of FSH treatment was significantly shorter with r-hFSH than with u-hFSH, and significantly fewer ampoules were required. More follicles ≥10 mm in diameter, and more oocytes retrieved with r-hFSH. No statistical difference in pregnancy rate was found between groups, although patients receiving r-hFSH had a higher pregnancy rate per cycle than patients given u-hFSH. In a recent cost effective analysis,28 derived from a published meta analysis, comparing recombinant to highly purified u-hFSH Ola et al found that the increment in cost spent to gain an extra clinical pregnancy became progressively more with advancing age as thus as the clinical pregnancy fall. CONCLUSIONS Taken together, and notwithstanding their differences in design and treatment regimens, most of the studies which have compared r-hFSH with either u-hFSH or u-hFSH HP have demonstrated the superior efficacy of the recombinant product for the induction of superovulation in women undergoing ART. In particular, r-hFSH treatment has been associated with a greater number of embryos obtained following administration of a lower total dose of FSH compared with the urinary gonadotrophins. Two recent studies have demonstrated the desirability of having a large number of oocytes available to ensure a successful outcome to ART. An analysis of their database by the Human Fertilization and Embryology Authority29 in the UK has demonstrated that the number of embryos in culture is a major predictor of pregnancy outcome: women who had more than four embryos in culture had a significantly higher chance of achieving pregnancy than those with four embryos or less, regardless of age. Furthermore, for women with more than four embryos in culture, and who subsequently had either two or three embryos transferred, the incidence of pregnancy was similar irrespective of the number of embryos transferred. Similarly Scholtes and Zeilmaker30 found that the chance of having at least one blastocyst to transfer was significantly increased if more than four oocytes were collected from the woman. If the woman had the ‘ideal’ number of 10–15 oocytes available for collection, the chance of blastocyst transfer was 80%. Thus, by increasing the number of oocytes available compared to uhFSH treatment, the use of r-hFSH to induce superovulation may help to increase the chances of pregnancy in couples undergoing ART

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To conclude: Recombinant FSH offers potential benefits over urinary FSH particularly in terms of purity and reliability of production causing less local and systemic allergic reactions. It should also be noted that, in young good responding patients r-hFSH treatment results in a higher number of embryos than are obtained with urinary gonadotrophins, thus enabling good embryos that are not needed for immediate transfer to be cryopreserved and transferred later if needed. REFERENCES 1. Lunenfeld B, Lunenfeld E. Gonadotropic preparations-lessons learned. Fertil Steril 1997; 67:812–14. 2. Lunenfeld B. Treatment of anovulation by human gonadotrophins. J Int Fed Gyn and Obst 1963; 1:153. 3. WHO Expert Committee on Biological Standardization, Human Menopausal Gonadotrophins. World Health Organization Technical Report Series. Geneva: World Health Organization, 1963. 4. Howles CM, Wikland M. The use of recombinant human FSH in vitro fertilization. In: Shohan, Z Howles CM, Jacobs HS (Eds). Female infertility therapy: Current practice. London: Martin Dunitz, 1999:103–14. 5. Eshkol A, Lunenfeld B. Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human menopausal gonadotrophin (HMG). Part III. Acta Endocrinologica 1967; 54:919. 6. Donini P, Puzzuoli D, D’Alessio I, Lunenfeld B, EshkolA, Parlow AF. Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human postmenopausal gonadotrophin (HMG): Part I. Acta Endocrinologica 1966; 52:169. 7. Howles CM. Genetic engineering of human FSH (Gonal-F). Hum Reprod Update 1996; 2:172– 91. 8. Chappel S, Kelton C, Nugent N et al. Expression of human gonadotrophins by recombinant DNA methods. In: Genazzi, A.R., Petraglia, E, eds. Proceedings of the 3rd World Congress on Gynecological Endocrinology. Carnforth: Parthenon Publishing, 1992; 179–84. 9. Brinsden P, Akagbosu F, Gibbons L et al. Gonal-F vs Puregon: results of a randomized, assessor-blind, comparative study in women undergoing ART. 14th Annual Meeting of the European Society for Human Reproduction and Embryology, Gothenburg, 1998: abstract. 10. Sargeant S. A study to evaluate the ease of use and tolerability by patients of gonadotropins old and new. British Fertil Society Annual General Meeting, Sheffield Abstract F9 1998. 11. Brinsden P, Akagbosu F, Gibbons LM et al. A comparison of the efficasy and tolerability of two recombinant human follicle-stimulating hormone preparations in patients undergoing in vitro fertilization-embryo transfer. Fertil Steril 2000; 73:114–16. 12. Germond M, Dessole S, Senn A et al. Successful in vitro fertilization and embryo transfer after treatment with recombinant human FSH. Lancet 1992; 339:1170. 13. Devroey P, Van Steirteghem A, Mannaerts B, Coelingh Bennink, H. Successful in vitro fertilization and embryo transfer after treatment with recombinant human FSH. Lancet 1992; 339:1170–71. 14. Devroey P, Mannaerts B, Smitz J et al. Clinical outcome of a pilot efficacy study on recombinant human follicle-stimulating hormone (Org 32489) combined with various gonadotrophin releasing hormone agonist regimens. Hum Reprod 1994; 9:1064–69. 15. Reddy R, Al-Oum M, Ledger W et al. An alternate day, stepdown regimen using Gonal-F® (rhFSH) in IVF: a UK multicentre study. Hum Reprod 1996; 11:130–31.

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16. Recombinant Human FSH Study Group. Clinical assessment of recombinant human folliclestimulating hormone in stimulating ovarian follicular development before in vitro fertilization. Fertil Steril 1995; 63:77–86. 17. O’Dea L, Loumaye E, Liu H. A randomised, comparative, multicenter clinical trial of recombinant and urinary human FSH in in vitro fertilization and embryo transfer. Fertil Steril. 1993; Suppl: S50–S51 Abstracts No. 0–106. 18. Hedon B, Out HJ, Hughes JN et al. Efficacy and safety of recombinant FSH (Puregon) in infertile women pituitarysuppressed with triptorelin undergoing in vitro fertilisation: a prospective, randomised, assessor-blind, multicentre trial. Human Reprod. 1995; 10:3102–06. 19. Out HJ, Mannaerts BMJL, Driessen SGAJ et al. A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle-stimulating hormone (Puregon versus Metrodin) in in vitro fertilization. Hum Reprod 1995; 10:2534–40. 20. Frydman R, Howeles CM. And Truong F. A double-blind, randomized study to compared recombinant human follicle stimulating (FSH; Gonal-F) with highly purified urinary FSH (Metrodin HP) in women undergoing assisted reproductive techniques including intracytoplasmatic sperm injection. Hum Reprod 2000; 3:520–25. 21. McDonough PG. The coming of wonders (Editorial comment). Fertil Steril 1997; 68:138–42. 22. Out HJ, Driessen SGAJ, Mannaerts BMJL, Coelingh Bennick, HJT Recombinant folliclestimulating hormone (follitropin beta, Puregona) yields higher pregnancy rates in in vitro fertilization than urinary gonadotrophins. Fertil Steril 1997; 68:138–42. 23. D’Agostinho RB, Weintraub M. Meta-analysis: a method for synthesizing research. Clin Pharmacol Ther. 1995; 58:605–16. 24. Jansen CAM, Van Os MC. Puregon without analogs: an oxymoron. Gynecol Endocrinol 1996; 10(Suppl):34. 25. Strowitzki T, Kentenich H, Kiesel L et al. Ovarian stimulation in women undergoing in vitro fertilization and embryo transfer using recombinant human follicle stimulating hormone (GonalF) in non-down-regulated cycles. Hum Reprod 1995; 10:3097–3101. 26. Bergh C, Howles CM, Borg K et al. Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum Reprod 1997; 12:2133–39. 27. Frydman R, Avril C, Camier, B et al. A Double-blind, randomised study comparing the efficacy of recombinant follicle stimulating hormone (r-hFSH/Gonal-F) and highly purified FSH (uhFSH HP/Metrodin HP) in inducing superovulation in women undergoing assisted reproductive techniques (ART). 14thAnnual Meeting of the European Society for Human Reproduction and Embryology, Gothenburg, 1998: abstract. 28. Ola B, Papaioartnou S, Afnan MA et al. Recombinant or urinary follicle-stimulating hormone? A cost-effectiveness analysis derived by particularizing the number needed to treat from published meta-analysis. Fertil Steril 2001; 75:1106–10. 29. Human Fertilisation and Embryology Authority Sixth Annual Report 1997. London: Human Fertilisation and Embryology Authority, 1997. 30. Scholtes MCW, Zeilmaker GH. Blastocyst transfer in day-5 embryo transfer depends primarily on number of oocytes retrieved and not on age. Fertil Steril. 1998; 69:78–83.

CHAPTER 9 Stimulation Strategies for Complex IVF Patients Franco Lisi, Leonardo Rinaldi, Simon Fischel INTRODUCTION The first successful birth after in υitro fertilization (IVF) was achieved in a natural, unstimulated cycle.1 However, by the beginning of the 1980’s it became apparent that multiple follicular stimulation was an important tool for better outcome results after IVF: pregnancy rates improved with the use of ovulationinduction techniques, mainly as a result of transferring severalembryos.2–4 Urinary extracts of gonadotrophins from postmenopausal women were available for use in the human since the 1960’s5–6 at either FSH: LH ratio of 1:1 (prepared as human menopausal gonadotrophin; HMG) or later in the preparation of FSH HP (high purity) with only traces of LH (<0.1 IU LH/1000 IU FSH). During the 1990s, recombinant DNAtechnology permitted the introduction of rFSH, a preparation totally devoid of LH activity. First reported the use of luteinizing hormone-releasing hormone (LHRH) analogues for assisted conception.7 It was considered that ‘down-regulation’ (internalisation of receptors) of the pituitary would eliminate premature luteinizing hormone (LH) surges, which were of ten weak or erratic as a result of combined follicular stimulation with clomiphene citrate and gonadotrophin.8 Furthermore, by preventing the spontaneous LH surge, not only was the risk of premature ovulation reduced, but the advantage of a synchronised cohort of oocytes providing more metphase II oocytes resulted. There are two extremes among the “Complex IVF Patients”, in terms of follicular stimulation: a. the high (or hyper-) responders with a risk of ovarian hyperstimulation syndrome and b. the poor responders with reduced follicular recruitment and poor prognosis for IVF outcome.

POOR RESPONDER PATIENTS Definition of Poor Responder Low ovarian response to stimulation occurs in ~9–24 percent of patients9 and still represents one of the most intractable problems of infertility/IVF treatment. This group of patients is heterogeneous, consisting of young patients with early depletion of primordial follicles, or patients with ‘resistant ovary syndrome’ in whom follicles are still present in

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the ovaries although they fail to respond to stimulation. In the latter group, a large dosage of FSH is administrated in an attempt to recover a reduced number of oocytes. An idealovarian stimulation strategy has yet to be defined for these patients. In Europe and North America, women are delaying childbearing, and the number of patients who are defined as poor responders referred to IVF centres has increased in recent years. Older patients with a poor ovarian response of prolonged duration present a significant group of patients. In recent years, assisted reproductive technologies have given us a better understanding of the events that precede menopause.10 It is now realized that an accelerated decline of ovarian function begins much earlier than previously thought, most likely in the midthirties.11 At around this time the total remainingnumber of follicles in the ovaries has been shown to be near 25,00012 with a rapid loss over time, as well as qualitative changes in the remaining oocytes/follicles including a high incidence of aneuploidy. Prediction of Response Predicting ovarian response to stimulation is of paramount importance to the patient as well as inthe organization of a successful in υitro fertilization (IVF) programme. Early follicular phase serum concentrations of FSH and estradiol are generally recognized as markers for ovarian reserve; raised serum levels of FSH13 or estradiol14 are predictive of a poor response to ovarian stimulation and consequently a reduced pregnancy rate in IVF. Higher pregnancy rates per attempt have been reported in patients with low based FSH levels (less than 15 mIU/mL) than those with moderate levels (15 to 24.9 mIU/ml), both of which were higher than those with excessively elevated FSH levels (greater than 25 mIU/ ml).13 The basal FSH levelreasonably accurately reflects ovarian reserve. Other predictors of ovarian response include baseline ultrasound measurement of the ovarian volume,15 the ovarian antral follicle count,16and the maximum velocity of ovarian stromal blood flow measured with colour and pulsed Doppler.17,18 Several tests of functional reserve of the ovaries can be used to predict a low response to standard protocols.19 These include luteinizing hormone (LH), oestradiol, the clomiphene citrate challenge test, and inhibin B concentrations.19–21Other tests include the day 3 FSH: LH ratio, gonadotrophin-releasing hormone agonist (GnRHa) stimulation test, and the exogenous follicle stimulating hormone ovarian reserve test.22,23 However, despite the validity of all these tests and there still remain patients who respond poorly to stimulation normal tests of ovarian reserve. Poor ovarian response may, therefore, not be entirely predictable until a patient has failed stimulation under a standard stimulation protocol. DIFFERENTGnRH ANALOGUE PROTOCOLS There is no agreement on the “perfect protocol” to be used in poor responders and many different opinions have been expressed by numerous investigators.24 reviewed the different stimulation protocols that have been proposed: i. varying the dose or the day of cycle for initiating stimulation with gonadotrophins;25 doubling the HMG dose in the course of an IVF treatment cycle is not effective in enhancing ovarian response in poor responders. This is in accordance with current

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theories on follicular growth, which state that follicular recruitment occurs only in the late luteal and early follicular phase of the menstrual cycle. Poor responders do not benefit from commencing recombinant human FSH therapy in the luteal phase. ii. pituitary desensitization with a GnRHa in the luteal phase of the previous cycle, followed by stimulation with a high dose of gonadotrophins (‘long protocol’). iii. initiating GnRHa and gonadotrophins together in the follicular phase (‘flare up protocor’) has offered limited and discordant results.26 iv. co-treatment with oestrogens, growth hormone or birth control pills, offered little benefit.27–29 v. using clomiphene citrate for stimulation is also largely unsuccesful; Awonuga and Nabi,30 concluded that IVF using long-protocol buserelin/hMG is more successful than using clomiphene citrate stimulation. However, this advantage may not be significant in those women with a previous poor response to buserelin/hMG. vi. natural cycle IVF might still offer advantages to poor responder patients as there seems to be a group who have regular menstrual cycles, respond inadequately to stimulation yet produce normal oocytes in the spontaneous cycle.31 An encouraging number of pregnancies have been achieved by IVF during natural cycles in poor responders to ovarian stimulation,32 but there remains the risk of premature ovulation. Pregnancy rates of 18.8% in natural IVF cycles vs 0 in stimulated cycles have been obtained in the same group of patients. This may not be the first approach than can be considered in IVF but it should be offered as an alternative af ter two failures in ovarian response using classical protocols of stimulation. All of the above strategies have met with only limited success. Hence, when deciding upon a stimulation protocol, it is mandatory to test ovarian reserve prior to stimulation in an effort to predict those who will respond poorly to standard stimulation. Every centre should establish criteria to screen out patients with severe ‘egg factor’24 where the chances of live-birth are practically zero (e.g. a day 3 FSH concentration repeatedly >12 mIU/ ml). These patients should not undergo stimulationand should instead be recommended no treatment or offered oocyte donation in countries where this technique is available. Patients with an anticipated low response should be stimulated with one of the protocols discussed above. This may often be more of a ‘trial and error’ approach rather than predicted the right strategy. Patients who fail to respond at all would be candidates for oocyte donation. ROLE OF LH IN MULTIPLE FOLLICULAR STIMULATION FOR IVF Human FSH for clinical use was first extracted from pituitary glands and the first pregnancies were reported in 1960.33–34 Subsequently, urinary extracts of gonadotrophins from postmenopausal women were shown to be safe and effective and became the standard for 30 years at either FSH: LH ratio of 1:1 (prepared as human menopausal gonadotrophin; HMG) or in the preparation of FSH HP (high purity) with only traces of LH (<0.1 IU LH/1000IU FSH). During the 1990s, recombinant DNA technology permitted the introduction of rFSH, a preparation totally devoid of LH activity, for clinical use:35–37 Recombinant FSH r(FSH) is well tolerated without sideeffects, its

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plasma half-life is similar to that of the native hormone and no anti-recombinant FSH antibodies have been noted. Recombinant LH (rLH) has been available for clinical trials since 1993 and was first used in addition to rFSH to induce ovulation in female hypopituitary-hypogonadotropic patients as an alternative to conventional HCG for ovulation induction reported that rFSH did not facilitate sufficient steroidogenesis even in large follicles, and addition of low dose rLH was required to induce adequate oestradiol secretion for characteristic mucus secretion, endometrial growth and pregnancy. However, the amount of rLH required to support rFSH-induced follicular development is still not clear. It has been argued that during GnRH agonist pituitary suppression (‘down-regulation’) sufficient endogenous LH exists for adequate follicular recruitment and development,38 “cycle performance”39,40 and preimplantation development;41 while excessive concentrations of LH have been deemed detrimental.42–44 However, other studies on inclusion of exogenous LH in follicular stimulation protocols have demonstrated no specific affect on blastulat formation, but a significant effect on embryo implantation and pregnancy rates.45–47 Laml et al,48 studied a group of six normogonadotrophic female ‘poor responders’ patients and recommended the supplementation of rLH to rFSH for follicular stimulation. Apparent benef its were demonstrated in a pilot study of 12 patients who had been poor responders in 17 cycles of treatment, supplementing rFSH stimulation with rLH.49 Twelve patients (17 cycles: 10 ICSI and 7 IVF) who, during follicular stimulation with rFSH, required >3000 IU to reach follicular maturity (group A) returned for subsequent cycles supplemented with rLH (group B). The latter group of patients had a mean basal FSH measured on day 3 of a natural cycle of 12.2 mIU/ml (range 7–19) and a mean age of 36.1 years. Down-regulation was as in the first cycles and was induced with triptoreline 0.1 mg (Ipsen, Paris, France) subcutaneously from the mid-luteal phase of the previous cycle (day 21) for 3 weeks before starting rFSH stimulation. On day 7 of stimulation, 75IU rLH (Luveris; Serono, Geneva, Switzerland) was administered to supplement the ongoing rFSH administration until the day of HCG administration (10,000 IU; Profasi, Serono). There was a significant increase in the percentage of feritilization in group B (86.0%) compared with group A (60.9%), and a trend towards an increased percentage of cleavage and grade 1 score for embryo morphology in group B patients. Due to the increased percentage of fertilization, however, more embryos were available for patients in group B when compared with patients in group A. In group A, one clinical pregnancy was obtained compared with six in group B. A later study50 evaluated the use of rLH supplementation in an unselected group of 122 IVF patients using recombinant rFSH and rLH for follicular stimulation (Group A), and compared the data to a group of 331 patients using rFSH only during the same period (Group B): in both groups down regulation was induced with Triptoreline 0.1 mg (Ipsen, France) subcutaneously from the mid-luteal phase of the previous cycle (day 21) for 3 weeks before starting gonadotrophin stimulation. The number, size and rate of follicle development were monitored daily during days 7–15 of stimulation with no difference observed in the presence or absence of exogenous rLH. No difference was observed in fertilization rate or the mean number of embryos available for transfer. No significant differences were scored between the proportion of grade 1 embryos or grades 1 and 2 combined, or embryo cleavage. The implantation rate showed a positive trend for patients

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receiving rLH. We also examined E2:M2 ratio and implantation rates for the subset of patients who had LH values <1.0 IU (n=22), those patients whose basal FSH was >10 IU (n=25) and patients requiring >2500 IU FSH (n=38). There was a significant increase in the implantation rate of Group A patients when down regulation LH was <1.0 IU and in patients who recived more than 2500 IU of exogenous FSH. GnRH antagonists have recently become available in clinical practice. They have been shown to prevent a premature LH surge during ovarian stimulation by binding competitively to the GnRH receptors of the gonadotrophic cells and leading to an immediate arrest of the gonadotrophin secretion.51 Two protocols were initially developed: the Lubeck protocol and the French protocol. In the Lubeck protocol the GnRH antagonist is administered starting from the 6th or 7th day of ovarian stimulation until the day of HCG at a daily dose of 0.25 mg sc.52 In the French protocol a single dose (3 mg sc) of the GnRH antagonist is administered on the 6th or 7th day of stimulation and repeated after 48–96 hours if HCG has not been given.53 Applying GnRH antagonists for ovulation induction in assisted conception in an unselected population has resulted in a dramatic reduction in the duration of therapy and in a reduction of the amount of gonadotrophin needed for ovulation induction, whereas doubts still remains about the effect on pregnancy rates and ovarian hyperstimulation syndrome (OHSS).54 It is expected that with greater experience in using the antagonist, clinicians will be able to finely tune its use with regard to the regimens of administration and selection of patients.

Table 9.1: Implantation rates and E2: M2 ratio for patients in Groups A and B when down-regulation LH <1.0 IU, or Basal FSH >10 IU or rFSH administration >2500 IU Down Reg LH <1.0 (Mean±SEM) Group A E2:M2 Ratio 1121.9±78.6 Implantation 4/26(15.4%) Rate *P<0.05

Group B 918.5±85.2 0/38*

Basal FSH >10 IU (Mean±SEM)

FSH >2500 (Mean±SEM)

Group A 990.8±30.6 2/21(9.5%)

Group A Group B 792.9±17.6 897.2±19.3 5/71(7.0%) 10/14(71,4%)

Group B 626.6±41.4 0/19

The management of ‘poor responder’ patients in IVF has always been tackled by clinicians in different ways, such as decreasing the amount or varying the timing of GnRH agonist administered, as in microdose GnRH agonist flare up regimens.55–58 A lack of uniformity in definition of the poor responder and of prospective randomized trials make a valid interpretation from the literature very difficult. Of the varied strategies proposed, those that seem to be more uniformly beneficial are microdose GnRH-agonist flare and late luteal phase initiation of a short course of low-dose GnRH-agonist discontinued before controlled ovarian stimulation. But no single regimen of GnRH agonist will benefit all poor responders.59 In fact, with the discovery of GnRH receptors in the human ovary someinvestigators assume that GnRH agonists might have a direct and deleterious effect on the ovary which is especially important for poor responder patients.8,60 In the light of these findings, there is a tendency towards the total elimination

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of GnRH agonists, while increasing the gonadotrophin dosage for poor responders with diminished ovarian reserves, who are already suppressed. A few studies have been published regarding the use of GnRH antagonist in poor responders. The use of cetrorelix (GnRH antagonist; Asta Medica, Frankfurt, Germany) used in conjunction with clomiphene citrate and gonadotrophin for ovarian stimulation in a group of patients considered to be ‘difficult responders’ to conventional GnRH analogue and gonadotrophin protocols was assessed 18 poor responders (24 cycles) with no live birth in 23 previous IVF cycles with GnRH agonists were stimulated with a a daily dose of clomiphene citrate 100 mg for 5 days and gonadotrophin injections from cycle day 2. Cetrorelix 0.25 mg/ day was started when the leading follicle reached 14 mm. Seventeen cycles were completed (70.2%) and four out of the completed 17 cycles resultedin a clinical pregnancy (23.5% per completed cycle). Two pregnancies ended in miscarriage while the other two resulted in two twin live births with a live birth rate of 11.8 percent per completed cycle. On the basis of the concerns about the use of GnRH agonists for poor responder patients,61 compared two ovarian stimulation protocols in which no GnRH agonists were used. In all, 40 patients with a poor response in previous treatment cycles were included. They were divided into two groups: Group I (n=20) received ovarian stimulation for 20 cycles, without the addition of either GnRH agonist or antagonist; while Group II (n=20) patients received ovarian stimulation for 20 cycles, including the administration of a GnRH antagonist (Cetrorelix, 0.25 mg daily) during thelate follicular phase. There was no statistically significant difference between the groups for mean age, duration of infertility baseline FSH concentration, cancellation rate, number of ampoules of gonadotrophin used, number of mature oocytes retrieved, oestradiolconcentrations on the day of injection of human chorionic gonadotrophin (HCG), fertilization rate, number of embryos transferred, and finally clinical pregnancy and implantation rates. The same authors62 in a prospective randomized trial studied a total of 48 poor responder patients described from previous cycles which were included and grouped into two: Group I consisted of 24 patients in 24 cycles in which leuprolide acetate (40 µg s.c. per day) was initiated on cycle day 2 followed by exogenous gonadotrophins on cycle day 3; Group II consisted of 24 patients in 24 cycles in which ovarian stimulation included gonadotrophin-releasing hormone (GnRH) antagonist administration (cetrorelix, 0.25 mg daily during late follicular phase). While only the oestradiol concentrations on the day of HCG were lower in Group II compared with Group I, the clinical pregnancy and implantation rates among groups still did not show any significance. The efficacy of GnRH antagonist ‘Cetrorelix’ in poor responders was compared with the standard long protocol:63 21 poor responders who underwent ICSI were treated with Cetrorelix according to the multiple-dose protocol and were compared with 21 poor responders treated according to the long protocol. The use of GnRH antagonist in a multiple dose protocol gave a pregnancy rate of 14.3 percent, which was in the range expected for patients with poor response, but with shorter treatment duration and with fewer ampoules of gonadotropins as compared with the use of a GnRH agonist protocol in a depot formulation. Lisi et al (unpublished) also assessed the use of Cetrorelix 0.25 mg daily during late follicular phase in a group of 38 patients with poor response identified by either baseline FSH concentrations of >15 mIU/ml, and/or a poor response in at least two previous IVF attempts; mean age in the group was 41, 6±4, 3 years. In 84

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percent (32 out 38) of cases egg recovery was successful and 1, 8±2, 1 oocytes were collected. In 71 percent (23 out of 32) of cases a transfer was performed and 2±1, 4 embryos were transferred. One pregnancy was established and is ongoing (4%), which is within in the expected range for this group of patients. Therefore, from current data we conclude that GnRH antagonist use for poor responders is of potential use and awaites further evaluation.59 HIGH RESPONDER PATIENTS OHSS is a dangerous complication of controlled ovarian hyperstimulation (COH) for IVF; its frequency is 0, 5–2 percent in the general IVF population, but higher in PCOS or PCOS-like patients.64 Moreover patients at high risk for OHSS undergoing COH for IVF with classic ovulation induction protocols for IVF (which provides GnRH agonist down regulation followed by gonadotrophins) may show a decrease in oocyte and embryo quality in spite of a high number of oocytes collected.65 More recently protocols using gonadotrophins and GnRH antagonists have been extensively used with some evidence of reduced risk for OHSS in unselected population.54 Craft treated 7 patients at high risk, having PCO and a history of OHSS.8 The treatment protocol involved a daily dose of clomiphene citrate 100 mg for 5 days, gonadotrophin injections from cycle day 2 and Cetrorelix 0.25 mg/day when the leading follicle reached 14 mm. The outcome was favourable compared to previous treatment with GnRH agonists: fewer oocytes wereproduced (10.2 versus 14.5 oocytes/cycle), using a lower dose of gonadotrophin (170 versus 189 IU/oocyte) and resulted inone ongoing pregnancy. No patients experienced OHSS. On the basis of this preliminary report, Lisi et al (unpublished) recently investigated the results of a trial of mild controlled ovarian hyperstimulation (COH) in a high OHSS risk group of patients. In the group of 8 patients there were ultrasonographic features of PCO, all had already undergone COH for IVF with a long protocol (pituitary down regulation followed by a maximum of 150 UI of rFSH), having had at least one cycle cancelled for high risk of OHSS (>40 follicles seen at ultrasound and/or 3500 pg/ml of E2 on the day of HCG) and with 4 out of the 8 previously completed IVF cycles giving rise to clinical OHSS. The patients underwent 8 cycle of COH with clomiphene citrate 100 mg per day from the 2nd to the 6th day of the cycle and a starting dose of 75 U. I. of rFSH per day from the 5th day of the cycle, followed by the administration of GnRh antagonist 0, 25 mg/day starting at follicular size of 14 mm. Ovulation was induced by administration of 10.000 UI of HCG. Fertilization was achieved in 5 cases with IVF and in 3 cases with ICSI. All luteal phases were supplemented with progesterone 50 mg i.m. per day. No cycles were cancelled. The mean E2 on the day of hCG was 1562±880 pg/ml.; 8.7 (±3, 7) oocytes per patient were collected, 4.6 (±1) embryos per patient were observed on the 2nd day of culture and 2.7 (±1) embryos per patient were transferred into the uterus. Three clinical pregnancies were achieved (37.5% per cycle). One delivered at 34 weeks, 1 ongoing at 11 weeks, 1 aborted (6 weeks). No severe or moderate OHSS were noted, even in those who became pregnant. We concluded that the use of a mild protocol of stimulation in patients at high risk for OHSS, undergoing COH for IVF might be a useful tool in reducing the risk of OHSS without compromising the chance of pregnancy.

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Our experience of using clomiphene citrate-gonadotrophin-cetrorelix for ‘hyper responders’ suggests that this protocol will be of value for patients at the risk of OHSS, since a lower dose of gonadotrophins may be found effective in stimulating an acceptable number of follicles. But, following on from the data by Craft et al8 and our experience a controlled randomized study is now required to confirm the preliminary observations. Koland Itskovitz-Eldor66 have suggested using the use of gonadotrophin-releasing hormone (GnRH) agonists instead of HCG to trigger ovulation (a single s.c. injection of 0.2 mg triptorelin) and reduce the prospect of OHSS. This strategy was first introduced by Itskovitz et al,67–68 and has been used ever since with excellent results in terms of OHSS prevention. This strategy is not applicable in ovarian stimulation cycles in which pituitary down-regulationis induced by GnRH agonists. However, in non down-regulated or in GnRH antagonist-based cycles this approach is reported to prevent any clinically significant OHSS.68–71 The future will see a variety of options for reducing the risk of OHSS and maximising the opportunity for pregnancy in ‘hyper responding’ patients, but the introduction of regimes should be evidence-based using a carefully planned and controlled strategy. REFERENCES 1. Steptoe P, Edwards R. Birth after the reimplantation of a human embryo (Letter). Lancet ii:1978; 336. 2. Lopata A, Brown JB, Leeton JF et al. In vitro fertilization of preovulatory oocytes and embryo transfer in infertile patients treated with clomiphene and human chorionic gonadotrophin. Fertil Steril 1978; 20:27–35. 3. Testart J, Belaisch-Allart J, Frydman R. Relationship betweenembryo transfer results and ovarian response and in vitro fertilization rate: analysis of 186 human pregnancies. Fertil Steril 1986; 45:237–43. 4. Wood C, McMaster R, Rennie G et al. Factors influencing pregnancy rates following in vitro fertilization and embryo transfer. Fertil Steril 1995; 43:245–50. 5. Donini P, Puzzuoli D, D’Alessio I, Lunenfeld B, Eshkol A, Parlow AR Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human post-menopausal gonadotrophin (HMG). II. Preparation of biological apparently pure FSH by selective binding of the LH with an anti-HGG serum and subsequent chromatography. Acta Endocrinol (Copenh) 1966; 52:186–98. 6. Donini P, Puzzuoli D, D’Alessio I, Lunenfeld B, EshkolA. Parlow AFPurification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human postmenopausal gonadotrophin (HMG). Separation of FSH and LH by electrophoresis, chromatography and gel filtration procedures. Acta Endocrinol (Copenh) 1966; 52:169–85. 7. Fleming R, Adam AH, Barlow DH et al. A new systematic treatment for infertile women with abnormal hormone profiles. Br J Obstet Gynaecol 1982; 89:80–83. 8. Craft I Gorgy A, Hill J et al. Will GnRH antagonist provide new hope for patients considered difficult responders to GnRH agonist protocols? Hum Reprod 1999; 14:2959–62. 9. Keay SD, Liversedge NH, Mathur RS, Jenkins JM. Assisted conception following poor ovarian response to gonadotrophin stimulation. Br J Obstet Gynaecol 1997; 104:521–27. 10. Seifer D, Naftolin F. Moving toward an earlier and better understanding of perimenopause. Fertil Steril 1998; 69:387–88. 11. Bopp B, Seifer D. Oocyte loss and the perimenopause. Clin. Obstet. Gynaecol 1998; 41:898– 11.

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12. Faddy M, Gosden R, Gougeon A, Richardson S, Nelson J. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992; 7:1342–46 13. Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989; 51:651–54. 14. Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995; 64:991–94. 15. Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 1995; 64:1167–71. 16. Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in vitro fertilization. Hum Reprod 1997; 12:220–23. 17. Zaidi J, Barber J, Kyei-Mensah A, Bekir J, Campbell S, Tan SL. Relationship of ovarian stromal blood flow at the baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 1996; 88:779–84. 18. Engmann L, Sladkevicius P, Agrawal R, Bekir JS, Campbell S, Tan SL. Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil. Steril 1999; 71:22–29. 19. Sharara FI, Scott RT (Jr), Seifer D. The detection of diminished ovarian reserve in infertile women. Am J Obstet Gynecol 1998; 179:804–12. 20. Loumaye E, Billion JM, Mine JM et al. Prediction of individual response to controlled ovarian hyper stimulation by means of a clomiphene citrate challenge test. Fertil Steril 1990; 53:295– 301. 21. Noci I, Biagiotti R, Maggi M et al. Low day 3 luteinizing hormone values are predictive of reduced response to ovarian stimulation. Hum Reprod 1998; 13:531–34. 22. Fanchin R, de Ziegler D, Olivennes F et al. Exogenous follicle stimulating hormone ovarian reserve test (EFORT): a simple and reliable screening test for detecting ‘poor responders’ in in vitro fertilization. Hum Reprod 1994; 9:1607–11. 23. Mukherjee T, Copperman AB, Lapinski R et al. An elevated day three follicle-stimulating hormone: luteinizing hormone ratio (FSH: LH) in the presence of a normal day 3 FSH predicts a poor response to controlled ovarian hyperstimulation. Fertil Steril 1996; 65:588–93. 24. Karande Vishvanath and Gleicher Norbert. A rational approach to the management of low responders in in vitro fertilization: Opinion. Human Reproduction 1999; 14:1744–48. 25. Karande VC, Jones GS, Veeck LL, Muasher SJ. High-dose follicle-stimulating hormone stimulation at the onset of the menstrual cycle does not improve the in vitro fertilization outcome in low-responder patients. Fertil Steril 1990; 53:486–89. 26. Olivennes F, Righini C, Fanchin R, Torrisi C, Hazout A, Glissant M, Fernandez H, Frydman E A protocol using a low dose of gonadotrophin-releasing hormone agonist might be the best protocol for patients with high follicle-stimulating hormone concentrations on day 3. Human Reproduction: Oxford University Press, 1996; 11:1169–72. 27. Gonen Y, Jacobsen W, Casper RF. Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil Steril 1990; 53, 282–87. 28. Dor J, Seidman DS, Amudai E, Bider D, Levran D, Mashiach S. Adjuvant growth hormone therapy in poor responders to in vitro fertilization: a prospective randomized placebo-controlled double-blind study Human Reproduction 1995; 10:40–43. 29. Russell JB. Pre-cycle estrogen treatment and poor responders. Assist Reprod Rev 1995; 5:82– 89. 30. Awonuga AO, Nabi A. In vitro fertilization with low-dose clomiphene citrate stimulation in women who respond poorly to superovulation. J Assist Reprod Genet 1997; 14:503–7

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31. Lindheim S, Vidali A, Ditkoff E et al. Poor responders to ovarian hyperstimulation may benefit from an attempt at natural-cycle oocyte retrieval. J Assist Reprod Genet 1997; 14:174–76. 32. Bassil S, Godin PA, J Donnez. Outcome of in vitro fertilization through natural cycles in poorresponders. Human Reproduction,1999; 14:1262–65. 33. Gemzell CA, Dicfaluzy E, Tillinger KG. Human pituitary follicle-stimulating hormone I: clinical effect of a partly purified preparation. CIBA Foundation Colloquia in Endocrinology 1960; 13:191–200. 34. Lunenfeld B, Sulimovici S, Rabau E et al. 1962 L’induction de 1’ovulation dans les amenorrhees hypophysaries par un traitment de gonadotrophins urinaries menopausique et de gonadotrophines. 35. Germond M, Dessole S, SennA et al Successful in vitro fertilisation and embryo transfer after treatment with recombinant human FSH. Lancet 1992; 339:1170. 36. Mannaerts B, Shoham Z, Schoot D et al. Single dose pharmacokinetics and pharmacodynamics of recombinant human follicle-stimulating hormone (Org 32489) in gonadotropin deficient volunteers. Fertility and Sterility 1993; 59:108–14. 37. Porchet HC, Le Cotonnec JY, Canali S et al. Pharmacokinetics of recombinant human follicle stimulating hormone after intravenous, intramuscular, and subcutaneous administration in monkeys, and comparison with intravenous administration of urinary follicle stimulating hormone. Drug Metabolism and Disposition 1993; 21:144–50. 38. Daya S, Gunby J, Hughes EG, Collins JA, Sagle MA. Follicle-stimulating hormone versus human menopausal gonadotrophin for in vitro fertilization cycles: a meta-analysis. Fertil Steril 1995; 64:347–54. 39. Sills ES, Levy D, Moomjy M, McGee M, Rosenwaks Z. A prospective randomized comparison of ovulation induction using highly purified follicle-stimulating hormone alone and with recombinant human luteinizing hormone in in vitro fertilization. Human Reprod 1999; 14(9):2230–35. 40. Balasch J, Vidal E, Peaarrubia J, Casamitjana R, Carmona F, Creus M, et al. Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Hum Reprod 2001; 16:1636–43. 41. Fleming R, Lloyd F, Herbert M, Fenwick J. ÿ Griffiths T, ÿMurdoch A. Effects of profound suppression of luteinising hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Hum Reprod 1998; 13:1788–92. 42. Chappel S, Howles C. Reevaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. Hum Reprod 1991; 6:1206–12. 43. Yamashita T, Ishimaru T, Fujishita A, Kawano M, Yamabe T. Influence of serum follicle stimulating hormone to luteinising hormone ratio during Buserelin acetate-induced pituitary desensitisation on ovarian response to exogenous gonadotrophins in an in vitro fertilisation and emryo transfer programme. Hum Reprod 1996; 11:1615–19. 44. Lui X, Andoh K, Mizunuma H, Kamijo T, Kikuchi N, Yamada K, Ibuki Y. Effects of recombinant human FSH (rhFSH), urinary purified FSH (uFSH), and hMG on small preantral follicles and tertiary follicles from normal adult and androgen-sterilised female mice. Fertil Steril 2000; 73:372–80. 45. Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilisation programmes. Fertil Steril 1999; 72:604–09. 46. Filicori M, Cognigni GE. Clinical Review 126: Roles and novel regimens of lutenising hormone and follicle-stimulating in ovulation induction. J Clin Endocrinol Metab 2001; 86:1437–41. 47. Levy DP, Navarro JM, Schattman GL, Davis OK, Rosenwaks Z. The role of LH in ovarian stimulation. Exogenous LH: let’s design the future. Hum Reprod 2000; 15:2258–65.

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48. Laml T, Obruca A, Fischl F et al. Recombinant luteinizing hormone in ovarian hyperstimulation after stimulation failure in normogonadotropic women. Gynecological Endocrinology 1999; 13:98–103. 49. Lisi F, Rinaldi L, Fishel S, Lisi R, Pepe GP, Picconeri MG, et al. Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study. Reproductive Biomedicine online webpaper 2001/226 on web 15; 2001. 50. Lisi F, Rinaldi L, Fishel S, Lisi R, Pepe GP, Picconeri MG, Campbell A. Use of Recombinant FSH (GONAL F) and Recombinant LH (Luveris) for multiple follicular stimulation in an unselected group of patients undergoing in vitro fertilization. Reproductive Biomedicine Online 2002; 5(2):104–08. 51. Diedrich K, Diedrich E, Santos E. Suppression of the endogenous LH-surge by the GnRH antagonist Cetrorelix during ovarian stimulation. Hum Reprod 1994; 9:788–91. 52. Albano C, Smitz J, Camus M. et al. Comparison of different doses of GnRH antagonist cetrorelix durino controlled ovarian hyperstimulation. Fertil Steril 2000; 67:917–22. 53. Olivennes F, Alvarez S., Bouchard P et al. The use of a GnRH antagonist (cetrorelix) in a single dose protocol in IVF-ETy: a dose finding study of 3 vs 2 mg. Hum Reprod. 1998; 13:2411–14. 54. Al-Inany H and Aboulgar M. GnRH antagonists in assisted reproduction: a Cochrane review Hum Reprod 2002; 17:874–85. 55. Scott RT, Navot D. Enhacement of ovarian responsiveness with microdoses with GnRh agonist during ovulation induction for in vitro feertilisation. Fertil Steril 1994; 61:880–85 56. Schoolcraft W, Schlenker T, Gee M et al. Improved controlled hyperstimulation in poor responder in vitro fertilisation patients with a microdose FSH flare, GH protocol. Fertil Steril. 1997; 67:93–97. 57. Surrey ES, Bower J, Hill DM et al. Clinical and endocrine effect of a microdose o GnRH agonistflare regimen administered to poor responders who undergo in vitro fertilisation. Fertil Steril 1998; 69:419–24. 58. Padilla SL, Dugan K, Maruschak Y et al. Use of flare-up protocol with high doses of hFSH and hMG for in vitro fertilization in poor responders Fertil Steril 1996; 65:796–99. 59. Surrey ES, Schoolcraft WB. Evaluating strategies for improving ovarian response of the poor responders undergoing assisted reproductive techniques. Fertil Steril 2000; 73:667–76. 60. Leung PCK. GnRH receptor and potential action in human ovary. Gynaecol. Endocrinol 1999; 13:10. 61. Akman MA, Erden HF, Tosun SB, et al Addition of GnRH antagonist in cycles of poor responders undergoing IVF. Hum Reprod 2000; 15:2145–47. 62. Akman MA, Erden HF, Tosun SB, et al. Comparison of agonistic flare-up-protocol and antagonistic multiple dose protocol in ovarian stimulation of poor responders: results of a prospective randomized trial. Hum Reprod 2001; 16:868–70. 63. Nikolettos N, Al-Hasani S, Felberbaum Re et al. Gonadotropin-releasing hormone antagonist protocol: a novel method of ovarian stimulation in poor responders. Eur J Obstet Gynecol Reprod Biol 2001; 97:202–7 64. Forman RG. Severe OHSS-an acceptabble price? Hum Reprod 1999; 11:2687–88. 65. Agakbosu F, Marcus S, Abusheikha N et al. Does ovarian hyperstimulation syndrome affect the quality of oocytes? Hum Repro 1998; 9:2583–84. 66. Kol S, Itskovitz-Eldor J. Severe OHSS. Yes, there is a strategy to prevent it! Hum Reprod 15:2266–67. 67. Itskovitz J, Boldes R, Barlev A et al. The induction of LH surge and oocyte maturation by GnRH analogue (buserelin) in women undergoing ovarian stimulation for in vitro fertilization. Gynecol Endocrinol 1988; 2:165 68. Itskovitz J, Boldes R, Levron J et al. Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist. Fertil Steril 1991; 56:213–20.

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69. Lewit N, Kol S, Manor D, Itskovitz-Eldor J. The use of GnRH analogs for induction of the preovulatory gonadotropin surge in assisted reproduction and prevention of the ovarian hyperstimulation syndrome. Gynecol Endocrinol 1995; (Suppl.4):13–17. 70. Lewit N, Kol S, Manor D, Itskovitz-Eldor J. Comparison of GnRH analogues and HCG for the induction of ovulation and prevention of ovarian hyperstimulation syndrome: a case-control study Hum Reprod 1996; 11:1399–1402. 71. van der Meer S, Gerris J, Joostens M, Tas B. Triggering of ovulation using a gonadotrophinreleasing hormone agonist does not prevent ovarian hyperstimulation syndrome. Hum Reprod 1993; 8:1628–31.

CHAPTER 10 Progmmming the Cycle with Oral Contraceptives Antecedent to the use of Antagonists Nico Naumann, Hossein Gholami INTRODUCTION To improve results in assisted reproductive techniques the individually adapted treatment for each patient is paramount. The combination of several drugs should enhance the results and an ‘old’ drug like a combined oral contraceptives (COC) can easily provide benefit to a ‘new’ drug like the GnRH-antagonist. GnRH-ANTAGONIST Release of FSH and LH by the pituitary gland is regulated through the pulsatile emission of gonadotropin-releasing hormone (GnRH). The inhibition of the action of GnRH in sex-hormone-dependent diseases or luteinization during assisted reproduction was first obtained by GnRH-agonists and more recently by GnRH-antagonists.1 GnRH-agonists have been used for several years and the associated disadvantages like flare-up in the short protocol or the need for long down regulation are well known. Unlike GnRH-agonists, GnRH-antagonists do not stimulate the release of gonadotrophins in the first few days but suppress it in an immediate and reversible way by blocking the GnRH receptors in the hypothalamus.2 There are only two registered antagonists in use, Cetrorelix (Serono), and Ganirelix (Organon),3 with principally two types of protocols involving a single dose subcutaneous administration of 3 mg starting on day 8 of stimulation.4–6 The multiple dose regimen starts on day six of FSH stimulation with four to five days of daily injections. To date the minimal dose is 0.25 mg and the duration depends on the monitored effect on LH. Interestingly, the multiple dose regimen has been found to necessitate shorter stimulation with a smaller amounts of FSH.7–8 WHAT IS THE ORAL CONTRACEPTIVE? The oral contraceptive pill is either a combined preparation of ethynyl estradiol (EE) and progestin, or progestin only The commonly used combined oral contraceptives (COC) contain a varying dose of 20–30 mg of EE and a varying dose of a progestin. The administration of a COC suppresses the natural menstrual cycle by inhibiting follicular

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maturation and ovulation. This is achieved by the positive feedback of estrogens to the hypothalamus blocking the surge of FSH and LH,9 thereby maintaining the endometrium thin. However, the suppression of follicular growth is not 100 percent. In 1995 Teichmann10 showed that suppression depends on the dosage of EE and 30 mg is more likely than 20 mg to suppress early follicular growth. The 21-day COC is of ten associated with f ollicular growth during the week of drug-free week. van Heusden and Fauser (1999)11 and jain et al (2000)12 described follicles of 10–12 mm, which could have matured and reached ovulation in the drug-free interval. Blocking ovulation with a COC over many years causes a lower turnover of the ovarian follicular resources.13 Administration of norethisterone 10 mg daily is likely to block the release of FSH and LH. Due to their low dosage, today’s progestin-only pills do not act on the hypothalamus. Their contraceptive effect is related to the influence on the cervical mucus and endometrium. Since the introduction of the COC in the 1960s its use has been extended. There are several known benefits such as the positive ‘side-effects’ on endometriosis, dysmenorrhoea, bleeding irregularities, polycystic ovaries and acne. Effect on Endometriosis Hormonal treatment of endometriosis aims at changing the endocrine condition that allows endometriotic growth that is low in oestradiol and/or high in progestin. The latter alone or in combination with oestradiol, blocks ovulation and in some doses induces amenorrhoea. At the same time the ectopic endometrium becomes atrophied and intraperitoneal inflammation is reduced. Therefore, an insufficient supply or the absence of ‘food’ to the endometriosis keeps the disease ‘on hold’, but surgery is thought to be the only means by which the extent of endometriosis can be reduced. The antidysmenorrheic effect is subsequent to this process of ‘starving’ the endometriosis. Alternative hormonal treatments like danazol or gonadotrophin-releasing hormone analogues have shown no better results and once they are stopped the previous endometriosis and its symptoms return. Effect on Bleeding Pattern Menstrual bleeding disorders like menorrhagia, dysfunctional bleeding and irregular cycles have different effects on a woman’s fertility. Very short cycles indicate that the follicles are unlikely to mature. Hence ovulation is often absent and at best unpredictable. The absence of ovulation prevents the endometrium from maturing and bleeding irregularities can follow from this, from intermittent spotting to prolonged bleeding. Prolonged cycles indicate irregularities in follicle maturation and amenorrhoea inhibits endometrial maturation. Severe PCO syndrome with year-long amenorrhoea can induce endometrial atrophy. The COC facilitates a harmonious bleeding pattern once every four weeks and facilitates ovarian quiescence whilst suppressing follicle maturation. At the same time the

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endometrium develops to a minimum degree of maturation and, af ter long-term use a COC facilitates the immediate reestablishment of a spontaneous cycle when the COC is stopped. In premature ovarian failure the uterus will be kept in such condition that minimum endometrial thickness can be achieved. Effect on Polycystic Ovaries PCO syndrome is defined morphologically and endocrinologically. The classical feature of 2–8 mm cysts in a ‘necklace’ pattern is associated with a higher ovarian diameter. The administration of a COC over several months reduces the number and size of cysts and the ovarian volume. Further COC positively influences the endocrine situation by reducing the production of androgens which are responsible for the PCO symptoms of seborrhoeic acne and hirsutism. Arise in sex hormone binding globulin (SHBG) facilitates reduction of free testosterone, but there have been adverse effects with norgestrel-containing COCs.14 At the same time women with PCO and obesity have been shown to develop insulin intolerance with COCs containing cyproterone acetate and being overweight increases the risk of venous thrombolic disease.15 Not least, the uterus itself benefits from COCs as they provide protection from endometrial hyperplasia and the risk of endometrial cancer. Ovarian Cysts and COC Intra-ovarian fluid containing spaces over 30 mm are defined as ovarian cysts and because of hormonal activity their incidence is highest during the reproductive years. Apart from benign cystic tumours like teratomas or epithelial cysts of serous or mucinous nature, cysts can result from unruptured follicles or corpus luteum. When there is no ovulation the follicle can persist and even grow. Thereby the persisting follicle becomes a functional cyst which might be reabsorbed at a later stage or persist and interfere with ovarian stimulation. The flare-up effect of GnRH-agonists is known to be co-responsible for the occurrence of ovarian cysts but can be avoided by long-down regulation or pretreatment with oral contraceptives. Most ovarian cysts, and especially those resulting from the flare-up effect of GnRHagonists, produce oestradiol which stimulates endometrial growth, another undesired effect that often causes the cancellation of an IVF cycle.16 Biljan (1998)17 reported that oral contraception given prior to starting an IVF cycle reduces the time required to suppress pituitary activity by GnRH-agonists and the amount of FSH stimulation. Some clinics recommend pretreatment with oral contraceptives as part of a standard protocol. Poor Responders During the use of GnRH-agonists their flare-up effect has been exploited so as to obtain better response in poor responders.18 Poor responders further benefit from pretreatment by oral contraceptives. Down-regulation with a COC enhances the ovarian response and later FSH stimulation due to the suppression of ovarian activity Women who have

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undergone IVF cycles of poor response, with regard to the number of follicles produced and the number of retrieved oocytes, should undergo one to three cycles of COC prior to further treatment.19 Good Responders Even women known to respond well to FSH stimulation may obtain benefits from oral contraceptives prior to a treatment cycle, as women with PCO syndrome can be prevented from responding in an exaggerated way with the elevated risk of ovarian hyperstimulation syndrome (OHSS).20 The oral contraceptive is administered for 25 days and in the last five days overlaps with leuprolide acetate subcutaneously prior to starting FSH on day three of the cycle. In terms of risk for OHSS, the cancellation rate and pregnancy rate were reported to be better than without the oral contraceptive preparation. Use of COC Prior to GnRH-Antagonist The benefits of oral contraception in ART can be exploited when given prior to the cycle with GnRH-antagonist. The indications and benefits are many or a combination of the following: 1. suppression of ovarian activity and its hormonal interference with FSH administration in IVF by blocking the GnRH receptor in the hypothalamus. 2. batching patients into groups that start FSH stimulation at the same time, making it more feasible for the IVF clinic to program their work. 3. individual timing of the start of the IVF-cycle. 4. inhibition of early follicular growth and resulting cysts. 5. enhancement of ovarian response in poor responders. 6. reduction of cancellation rate. 7. reduction of incidence of OHSS. Protocol The commonly used protocol consists of administration of an oral contraceptive one cycle prior to the ovarian stimulation cycle. We recommend a COC with a minimum of 30 mg of EE, from cycle day 1 to 11, combined with desogestrel 0.15 mg from days 11 to 21. Exclusion criteria are any past history of risk factors for thrombosis or impaired liver function. Once the COC has been stopped, withdrawal bleeding will occur after a few days. If the withdrawal bleeding has to be postponed for individual planning or batching a group of patients, the combined administration of EE and Progestin is continued without interruption. When withdrawal bleeding occurs, LH and oestradiol have to be controlled to ensure that ovarian activity is suppressed. A vaginal scan can check for the presence of cysts and a thin endometrium, and only then can controlled ovarian hyperstimulation (COH) start with the administration of FSH. The first monitoring takes place on day five of stimulation by ultrasound scan and hormone assays for E2 and LH. This monitoring is repeated every second day. The

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administration of the antagonist starts when the leading follicle reaches 12 mm. The 0.25 mg dosage allows a better individual control of the LH-surge rather than a single dose of 3 mg. Hence a daily dose of 0.25 mg (Ganirelix acetate, Organon Italia Spa) is given until the final follicular maturation including the day of HCG administration. REFERENCES 1. Felberbaum RE, Diedrich K in Lunefeld B. GnRhAnalogues. The state of the ART at the Millenium The Parthenon Publishing Group, New York, pp. 47–63 The use of GnRh antagonist in assisted reproduction technologies. 2. Edwards RG. Time to revolutionize ovarian stimulation. Hum Reprod 1996; 11:917–19 3. The Ganirelix Dose-Finding Study Group Hum reprod. A double-blind, randomized, dosefinding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist ganirelix to prevent premature luteinizing hormone surges in women undergoing ovarian stimualtion with recombinant follicle stimulating hormone 1998; 13:3023–31. 4. Albano C. Comparison of different doses of gonadotropin-releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67:917–22. 5. Janssens RM et al. Dose-finding study of triptorelin acetate for prevention of a premature LH surge in IVF: a prospective, randomized, double-blind, placebo-conrolled study, Hum Reprod 2000; 15:2333–40. 6. Olivennes F. GnRH antagonist (cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose finding study of 3 versus 2 mg, Hum Reprod 1998; 13:2411–14. 7. Borm G. Treatment with Gonadotrophine releasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient. Results of a controlled, randomized, multicentre trial. Hum Reprod 2000; 15:1490–98. 8. Fluker M. The North American Ganirelix Study Group. Efficacy and safety of ganirelix acetate (Antagon/Orgalutran) versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil Steril 2001; 75:38–45. 9. Swerdloff RS, Odell WD. Serum luteinizing and follicle stimulating hormone levels during sequential and non-sequential contraceptive treatment of eugonadal women. J Clin Endocrinol 29:157–163. 10. Teichmann AT. The influence of the dose of EE in oral contraception on follicle growth. Gynecol. Endocrinol. 1995; 9(4):299–303. 11. van Heusden AM, Fauser BC BCJM. Activity of the pituitary-ovarian axis in the pill-free interval during use of low-dose combined oral contraceptive. Contraception, 1999; 59:237–43. 12. Jain JK et al. Comparison of ovarian follicular activity during treatment with a monthly injectable contraceptive and a low-dose oral contraceptive. Contraception, 2000; 61:195–98. 13. Crosignani P. Ovarian and endometrial function during hormonal contraception. Hum Reprod 2001; 16(7):1527–35. 14. van der Vange N et al. Effects of seven low-dose combined oral contraceptives on sex-hormone binding globulin, corticosteroid binding globulin, total and free testosterone, Contraception. 1990; 41:345–52. 15. Morin-Pappunen LC et al. Endocrine and metabolic effects of metoformin versus ethinyl estradiol-cyproterone acetate in obese women with polycystic ovary syndrome: a randomized study. J Clin Endocrinol Metab 2000; 85, 3161–68. 16. Pistofidis G, Tsirigotis M. Presentation at ESHRE 2002. Akeso Fertility Centre Adrianiou and Sohou St 115 Athens Greece. The microdose analogue protocol: A protocol for all?

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17. Biljan MM. Effects of pretreatment with an oral contraceptive on the time required to achieve pituitary suppression with gonadotropin-releasing hormone analogues and on subsequent implantation and pregnancy rates. Fert Steril 1998; 1063–9. 18. Schoolcraft W et al. Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle-stimulating hormone flare, growth hormone protocol. Fert Steril 1997; 67(1):93–97. 19. Al-Mizyen E. Does pretreatment with progestogen or OCP in low responders followed by the GnRHa flare protocol improve the outcome of IVF-ET? J Assist Reprod genet 2000; 17(3):140– 6. 20. Damario MA. Dual suppression with oral contraceptives and gonadotrophin releasing-hormone agonists improves in vitro fertilization outcome in high responder patients. Hum Reprod 1997; 12:2359–65.

CHAPTER 11 Agonists Versus Antagonists: Physiology to Clinical Success Ester Polak de Fried, Fernando Neuspiller, Gerardo Ardiles INTRODUCTION Although in vitro fertilisation (IVF) was first successfully performed with an oocyte retrieved from a natural cycle, the vast majority of IVF/ICSI cycles now employ controlled ovarian (hyper)stimulation (COH). In simple terms, the purpose of COH is to maximise the number of fertilisable oocytes obtainable at the time of oocyte retrieval. Despite close monitoring of follicular growth (via ultrasound and serum estradiol determinations), 10–25 percent cycles were cancelled due to premature luteinizing hormone (LH) surges. During the early to middle 1980s, clinicians started to employ GnRH agonists to achieve a state of hormonally selective temporary hypophysectomy, thereby denying any chance of premature LH surge. Gonadotropins-releasing hormone (GnRH) is a peptide composed of 10 amino acids that was first isolated and characterized in 1971.1 It is produced in the hypothalamus by neurones located predominantly in the arcuate nucleus and liberated into capillaries of the portal system of the pituitary in a pulsatile way. In response, pulsatile release of LH and FSH occurs from the pituitary gland. In humans, the frequency of pulses is between 70 and 220 minutes. GnRH binds to specific, transmembrane receptors on the gonadotrophic cells in the pituitary2 This leads to increased synthesis of LH and FSH as well as the calcium-dependent release of gonadotropins. These events are mediated by second messengers including inositol phosphate, leukotrienes and protein kinase C.2 The pulsatile release of GnRH is essential to the response of the gonadotrophs. Continuous delivery of GnRH to the pituitary for a long period of time leads to the inhibition of LH and FSH release. Soon after the identification of the GnRH stmcture, agonist analogs (GnRHa) were synthesized. GnRHa have a 100 to 200 times higher binding affinity for GnRH receptors than the native molecule.4 Due to their long half-life (1–6 hours), these agents provide continual rather than pulsatile signalling, and thus, daily administration results in a biphasic response over time. GnRHa initially induce the liberation of large amounts of LH and FSH from the pituitary, and an increase in the number of GnRH receptors (up-regulation, flare-up effect). However, within a short time (1–2 weeks), daily administration leads to internalization of the agonist/receptor complex, and a decrease in the number of receptors (down-regulation). In addition, there is an uncoupling of the second messenger cascade from the few available receptors. This process has been called “desensitisation,” and it renders the pituitary refractory to the stimulatory effect of GnRH. These phenomena represent the basis for the clinical use of the agonises, which have been efficient in

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disorders in which suppression of gonadotropins and ovarian steroids is desired. The pituitary blockade is completely reversible upon cessation of therapy, and normal menstrual cycle is re-established within 3 to 6 weeks. It is important to emphasize that the suppressive effects of continuous treatment with the agonises are always preceded by an initial stimulatory phase, in which LH and FSH are secreted in supraphysiological amounts.4 Within a period of about 12 hours, this “flare-up” effect leads to a 5-fold increase of FSH, a 10-fold rise in LH, and a 4-fold elevation of estradiol (E2). Postmenopausal E2 levels are commonly reached after 21 days of treatment. The antagonists bind competitively to the receptors, and thereby prevent endogenous GnRH from exerting its stimulatory effects on the pituitary cells.5 The structural modifications of GnRH antagonists allow binding to occur without activation of the intracellular secondary messenger events. Due to the allergic side effects, ranging from local erythema and induration to anaphylactoid reactions, GnRH antagonists were not available for clinical use until recently. Both compounds, ganirelix and cetrorelix, have modifications at positions 1, 2, 3, 6, and 10, although ganirelix, but not cetrorelix, also shows a modif ication at position 8.5 Median terminal half-lives ranging from 5 to 60 hours have been reported for cetrorelix single-dose administration.6–7 The elimination half-life of ganirelix after single and multiple-dose administration is 13 to 16 hours.8–9 Both compounds can be administered subcutaneously (SC), and they seem to be equipotent regarding gonadotropins suppression, presenting full suppression within 4 to 8 hours after administration. GnRH ANTAGONISTS Before GnRHa became available, approximately 18 percent of stimulated cycles within an IVF program were cancelled due to premature LH surges. By using the GnRHa to prevent LH surges via gonadotrope GnRH receptor down-regulation and desensitisation, this percentage decreased to about 2 percent, and concomitantly the fertilisation and pregnancy rates (PRs) increased (Felberbaum RE, 2000). Several treatment schedules are currently in use, including the so-called “long protocol,” which produces pronounced pituitary suppression before exogenous gonadotropins administration, by administering the GnRHa in the luteal phase of the pretreatment cycle, and the “short” and “ultrashort” protocols, in which the flare-up effect is used along with the down-regulation that later ensues. In this sense, the long protocol is generally the most effective and is most often used at present. However, it has some disadvantages, 1. An extended treatment period with the agonist is necessary before gonadotropins are suppressed. 2. Due to the pronounced suppression of FSH secretion by GnRH agonists, higher doses of gonadotropins are needed to achieve follicular maturation. 3. The highest dose of gonadotropins used has been postulated to increase the risk of ovarian hyperstimulation syndrome;11 4. As suppression of gonadotropins secretion is still present 10 to 12 days after agonist administration has been stopped, corpus lutetium function is impaired, and therefore, luteal phase supplementation is mandatory;12–13 and finally,

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5. GnRHa administration can be associated with side effects related to hormonal depletion (such as hot flashes and vaginal dryness). One important advantage in the long GnRHa protocol is the fact that cycles can be completely controlled in terms of gonadotropins stimulation starting dates, once suppression is achieved. Protocols 14

In 1991, Ditkoff et al reported that a GnRH antagonist (Nal-Glu) when administered for a short period of time is capable of suppressing the ovulation-inducing midcycle LH peak. This study demonstrated that a GnRH antagonist could temporarily prevent an LH surge, and this now has become the goal of using the clinically available GnRH antagonists, cetrorelix and ganirelix, in ovulation induction protocols for IVF. There are two cetrorelix treatment protocols currently available: multiple and single dose. Single or dual injections in IVF have been studied extensively by Olivennes et al.15– 17 In their initial study, a dose of 5 mg of cetrorelix was administered when plasma E2 levels were between 150 and 200 pg/ml per follicle of ~14 mm. A second injection would be performed 48 hours later if the follicles were not ready for maturation by hCG. The success of this dosing schedule suggested that increasing the interval and decreasing the dose might be beneficial. In their subsequent study, a 3-mg dose of cetrorelix was administered on day 8 of the stimulation cycle and a second injection was administered 72 hours later, if hCG was not given in that interval. This protocol was capable of preventing LH surge in all of the 11 patients studied. Recently, a single dose of 3 mg of cetrorelix was administered to 115 patients by the same authors on day 7 of hMG stimulation unless their E2 level was below 400 pg/ml, in which case the injection was delayed.17 If hCG administration was not performed within 4 days of cetrorelix administration, a daily injection of 0.25 mg was given until hCG administration. Eight percent of the women were given one additional dose of 0.25 mg of cetrorelix, and 2 percent received two additional doses of 0.25 mg. None of the 115 patients experienced an LH surge in this regimen. Recently, Rongieres-Bertrand et al18 added a single injection of cetrorelix to prevent premature LH surge in natural cycles. When plasma E2 concentrations reached 100 to 150 pg/ml, with a leading follicle between 12 and 14 mm in diameter, a single injection of 0.5 mg (n19 cycles) or 1 mg (n25 cycles) of cetrorelix was administered, and repeated 72 hours later if ovulation was not triggered in the meantime. LH surges were not detected in any of the patients. Daily administration of different doses of cetrorelix was another approach tried by several groups.19–26 Albano et al24 compared two different dosages of Cetrorelix. Twenty-four patients received 0.5 mg of Cetrorelix per day whereas 45 patients received 0.25 mg of Cetrorelix per day. The clinical PRs (38% in the 0.5-mg/day group, and 27% in the 0.25-mg/ day group) were not statistically different. Felberbaum et al19 and Albano et al26 have reported two large clinical trials using this dose. In their prospectively randomized study (2000), 188 patients received 0.25 mg of Cetrorelix (S.C.) daily, starting from day 6 of the hMG treatment, and 85 patients received daily doses of buserelin administered intranasally. Ultimately, 96 percent of patients in the cetrorelix group, and 91 percent in the buserelin group reached the day of hCG injection. In the cetrorelix group, three patients (1.6%) had a premature LH rise with a concomitant progesterone rise after having started cetrorelix administration. In their subsequent study,19 a total of 346 women were administered

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cetrorelix. Ninety-six percent reached the day of hCG administration, and 94 percent underwent oocyte retrieval. Only three cases of elevated LH with increased progesterone secretion were observed after initiation of cetrorelix administration, reflecting an incidence of 0.9 percent. The minimal effective dose of ganirelix, successful in preventing premature LH surges, has been suggested to be 0.25 mg/day (de Jong D, 2001). However, when daily ganirelix administration (0.25 mg/day) has been compared with a long GnRH protocol, the incidence of LH rises has been slightly higher in the ganirelix group27,31 There is some evidence that high LH concentrations during the follicular phase of ovarian stimulation cycles have a negative impact on fertilisation and implantation rates.30,31 Albano et al have suggested that the interruption of LH rises affects the quality and/or maturity of the oocytes and they have stressed the importance of early administration of the GnRH antagonist. In summary, single and daily dose administrations of Cetrorelix and daily doses of Canirelix were able to reliably prevent the onset of premature LH surges. Comparing the single versus daily regimen, when the reported median duration of cetrorelix treatment of 5 days is considered, and the total dose of Cetrorelix is calculated (1,25 mg), we can assume that daily administration uses less per total dose than a single administration (3 mg). Cycle Characteristics As prestimulation suppression of endogenous gonadotropins does not occur, the issue of whether less ampoules of exogenous gonadotropins can be used has been investigated. For regimens using a single administration of cetrorelix, Olivennes et al17 have reported that the mean number of hMG ampoules (27.7±4.2) was lower than the mean number of vials administered in a similar population treated with GnRHa (38.3±15.4). When administration of a single 3-mg dose of cetrorelix (126 patients) was compared with the administration of a depot of triptorelin (43 patients),17 the days of stimulation, number of ampoules administered, and serum E2 levels on the day of hCG administration were significantly lower in the cetrorelix group. In protocols using daily administration of cetrorelix, the mean numbers of hMG ampoules were between 26 and 36 considering different studies. In the largest of these studies,19 0.25 mg of cetrorelix per day was administered to a total of 346 women, and the median duration of Cetrorelix treatment was 5 days (mean 5.7 days), and a mean of 25.2 ampoules of hMG were given over a mean of 10.4 days. The mean number of follicles with a diameter >20 mm was 2,4. Daily ganirelix administration (0.25 mg/day) also has been compared with long protocols of buserelin and leuprolide acetate.28,30,31 Median total gonadotropins dose was lower in the ganirelix group in these studies. In conclusion, in prospectively randomized studies,17,26,28,30,31 in which third generation antagonists were compared with a GnRHa long protocol, it has been shown that antagonists decrease the total number of gonadotropins ampoules, shorten the stimulation period, and decrease E2 levels on the day of hCG administration.

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Luteal Phase Luteal phase deficiency is frequently observed in patients undergoing COH with a GnRHa. The prolonged pituitary suppression has been suggested to be an etiologic f actor for early luteolysis.32 In contrast, because of the rapid recovery of the pituitary gonadotrophs after discontinuation of the antagonist and the early evidence that shows no disturbance of the luteal phase with the use of the antagonist, Nal-Glu, it has been speculated that luteal phase supplementation is unnecessary in cycles associated with GnRH antagonists.14 Albano et al25 did not support the luteal phase in their first six patients. However, as all these patients showed bleeding in the midluteal phase, luteal support was added to the protocol for all subsequent subjects. No pregnancy occurred in these six initial patients. It has been documented that serum LH concentrations decreased after the preovulatory hCG injection in all patients. However, a progressive increase in LH was observed after day 7, reaching normal values. It has been suggested that hCG administration exerts a direct effect on the pituitary, and is responsible for this decrease in LH in the early luteal phase. Albano et al12 concluded that corpus luteum function remains impaired in cycles that are stimulated with cetrorelix, hMG, and hCG. Tavaniotou et al33 investigated the effects of GnRH antagonists on luteal phase by comparing patients stimulated with hMG and Cetrorelix with patients stimulated with hMG only, for IVF. Luteal phase has been supported in both of these groups with hCG. Luteal phase serum LH concentrations were low but similar between groups. They concluded that suppressed LH concentrations may notbe attributed solely to the GnRH antagonist administration, but hCG may be partially responsible as well. De Jong et al29 have also attempted to eliminate exogenous luteal phase support. When compared with a control group of regularly cycling women, significantly lower luteal phase LH and FSH concentrations were observed in the group of treated patients who did not conceive. However, as expected, luteal phase serum E2 and progesterone levels in these patients were high compared with the control group. Theoretically, it may be that substitution of hCG by recLH or a GnRH agonist would not disrupt the H-P-O axis in the same manner as a large dose of hCG does, and this may avoid the need for luteal phase supplementation in GnRH antagonist cycles. IVF-ET Clinical PRs (per transfer) of up to 55 percent have been reported in IVF studies, which were conducted with gonadotropins and GnRH antagonists. Results of Prospective randomized trials can be observed in(Tables11.1 and 2). In these studies, the number of embryos was also higher in the GnRHa group (4.5 vs 6.0 and 5.4 vs 7.5).17–26 Recently, Akman et al44 have compared the results of IVF-ET for poor responders stimulated with gonado tropins alone, and gonadotropins plus Cetrorelix. Adding Cetrorelix did not improve the mean oocyte number nor the proportion of mature oocytes retrieved.

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In conclusion, Cetrorelix has resulted in fewer mature oocytes and embryos than GnRHa, but this decrease did not affect pregnancies per transfer. Daily Ganirelix administration (0.25 mg/day) has also been compared with a long GnRHa protocol. In comparison with GnRHa treatment, ganirelix treatment resulted in one preovulatory follicle less and, consequently, one to three cumulus-oocyte complexes (COC) less were recovered at oocyte retrieval. Fertilisation rates and the number of good quality embryos were comparable between groups. Implantation and PRs tended to be lower in the ganirelix group. The study on the outcome of freeze-thaw cycles using cryopreserved embryos in stimulation cycles during the above-mentioned dose-finding study has suggested that there is no direct negative effect of Ganirelix on the quality of oocytes and embryos, and high doses of Ganirelix do not adversely affect the potential of embryos to establish clinical pregnancy in freeze-thaw cycles.34 However, a direct effect on the endometrium by relatively high doses cannot be excluded, in as much as human endometrial GnRH receptors have been identified.35–36 These findings brought to question the role of GnRH antagonists at the cellular level in endometrium and extrapituitary tissues.3

Table 11.1 Premature LH surge Oocytes number Antagonist n/N Agonist n/N Antagonist mean Agonist mean Albano 2000 European Orgal. 2000 European Mid-East 2001 Olivennes 2000 North American

3/198 13/463 1/226 0/126 2/198

1/95 3/237 0/111 1/43 0/99

6.0 9.1 7.9 9.2 11.6

10.6 10.4 9.6 12.8 14.1

Table 11.2 Clinical Pregnancy rate Miscarriage Antagonist n/N (%) Agonist n/N (%) Antagonist Agonist Albano 2000 European Orgal. 2000 European Mid East 2001 Olivennes 2000 North American

42/198 (21.2) 101/463 (21.8) 73/226 (32.3)

22/95 (23.1) 67/237 (28.2) 40/111 (36.0)

7/42 1/101 3/73

2/22 6/67 3/40

26/126 (20.6) 66/198 (33.3)

11/43 (25.5) 36/99 (38.3)

4/26 5/66

3/11 0/36

Nikolettos et al45 have compared the cryopreservation outcome of human oocytes obtained by COH with hMG and cetrorelix with the outcome of oocytes obtained by COH with hMG and triptorelin; the results were similar in both groups in terms of implantation and pregnancy per transfer rate. Potential Benefits of GnRH Antagonists One of the major risks of COH for IVF is that of OHSS. In prospective randomized studies, it has been shown that antagonists cause less OHSS than the long GnRHa

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protocol.38 Itskovitz-Eldor et al39 have suggested that a GnRH antagonist protocol, with a single injection of GnRHa to mimic the midcycle LH surge, prevents OHSS in high responders. Human chorionic gonadotropins was withheld in eight patients, who underwent COH with rec-FSH and concomitant Ganirelix treatment, and who were considered to have an increased risk of developing OHSS. In these patients, ovulation was triggered with a single injection of 0.2 mg of triptorelin. After GnRHa injection, endogenous serum LH and FSH surges were observed with median peak values of 219 and 19 ILJ/liter, respectively. The mean number of oocytes obtained was 23.4, 83 percent of which were mature. None of the patients developed any signs or symptoms of OHSS. Four clinical pregnancies (17% per transfer) were achieved from fresh and frozen ETs.39 Their preliminary results suggest that this regimen may prove to be useful, however, their overall PR was disappointing. Side Effect Profile Because the histamine releasing potential and subsequent severe local and systemic allergic reactions had been a problem with the use of earlier GnRH antagonists, transdermal skin tests were performed in the preclinical studies of cetrorelix and ganirelix.40–42 However, no major systemic adverse reactions were reported in these trials,42 and therefore, in clinical use, this is not required. In a recent clinical study19 where a total of 346 women undergoing IVF have been treated with Cetrorelix, none of them had to be cancelled due to allergic, anaphylactoid, local hypersensitivity, or other adverse reactions. In recent controlled studies, lower rates of drug-related adverse reactions and injection site reactions have been reported in the Ganirelix group than in the GnRHa group. Recently, Ludwig et al43 have reported pregnancy, birth, and follow-up data (up to 2 years of age) from the world’s largest cohort of children born after IVF using cetrorelix. They have analysed 208 pregnancies after fresh ETs resulting in 163 deliveries of 209 live-born children and 23 pregnancies after frozen ETs resulting in 16 deliveries of 18 live-born children. Ectopic and heterotopic pregnancies were ~4.5 percent. They have also reported delivery rates per clinical pregnancy of 78 percent and 70 percent in fresh and frozen ET cycles, respectively Ludwig et al43 have also shown that children born f rom IVF cycles using Cetrorelix had no increased risk of malformations. They have concluded that cetrorelix has no detrimental effect on the pregnancy course of women or on the birth characteristics and developmental competence of children. CONCLUSIONS GnRH antagonists are now clinically available for use in ART cycles. Their advantages in this setting have been clearly elucidated by many authors. The studies reviewed herein indicate that they are an acceptable alternative to the use of GnRHa in IVF cycles. These studies have documented that the doses are both efficient in preventing LH surges and non detrimental to the IVF success rates. Additional use in clinical settings versus research protocols will help refine their use in specific patient subpopulations. In

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conclusion, the GnRH antagonist fixed protocol facilitates a short and simple protocol for ovarian stimulation in assisted conception. However, in view of the available data, there is a small but statistically significant lower pregnancy rate that requires counselling subfertile couples before recommending a change from GnRh agonists to antagonists. REFERENCES 1. Conn PM, Crowley WF Jr. Gonadotropin-releasing hormone and its analogues. N Engl J Med 1991; 24:93–103. 2. Huckle WR, Conn PM. Molecular mechanism of gonadotropin releasing hormone action. II. The effector system. Endocr Rev 1988; 9:387–95. 3. Hernandez ER. Embryo implantation and GnRH antagonists: Embryo implantation: The Rubicon for GnRH antagonists. Hum Reprod 2000; 15:1211–16. 4. Reissmann T, Felberbaum R, Diedrich K et al. Development and applications of luteinizing hormone-releasing hormone antagonists in the treatment of infertility: An overview. Hum Reprod 1995; 10:1974–81. 5. Felberbaum R, Diedrich K. Ovarian stimulation for in vitro fertilization/ICSI with gonadotrophins and gonadotrophin-releasing hormone analogues: Agonists and antagonists. Hum Reprod 1999; 14:207–21. 6. Erb K, Klipping C, Duijkers I et al. Pharmacodynamic effects and plasma pharmacokinetics of single doses of cetrorelix acetate in healthy premenopausal women. Fertil Steril 2001; 75:316– 23. 7. Duijkers IJ, Klipping C, Willemsen WN et al. Single and multiple dose pharmacokinetics and pharmacodynamics of the gonadotrophin-releasing hormone antagonist Cetrorelix in healthy female volunteers. Hum Reprod 1998; 13:2392–98. 8. Oberye JJ, Mannaerts BM, Huisman JA et al. Pharmacokinetic and pharmacodynamic characteristics of ganirelix (Antagon/ Orgalutran). Part II. Dose-proportionality and gonadotropin suppression after multiple doses of ganirelix in healthy female volunteers. Fertil Steril 1999; 72:1006–12. 9. Oberye JJ, Mannaerts BM, Kleijn HJ et al. Pharmacokinetic and pharmacodynamic characteristics of ganirelix (Antagon/ Orgalutran). Part I. Absolute bioavailability of 0.25 mg of ganirelix after a single subcutaneous injection in healthy female volunteers. Fertil Steril 1999; 72:1001–05. 10. Ben-Rafael Z, Lipitz S, Bider D et al. Ovarian hyporesponsiveness in combined gonadotropinreleasing hormone agonist and menotropin therapy is associated with low serum follicle stimulating hormone levels. Fertil Steril 1991; 55:272–75. 11. Rizk B, Smitz J. Ovarian hyperstimulation syndrome after superovulation using GnRH agonists for IVF and related procedures. Hum Reprod 1992; 7:320–27. 12. Albano C, Grimbizis G, Smitz J et al. The luteal phase of nonsupplemented cycles after ovarian superovulation with human menopausal gonadotropin and the gonadotropin releasing hormone antagonist Cetrorelix. Fertil Steril 1998; 70:357–59. 13. Whelan JG 3rd, Vlahos NF. The ovarian hyperstimulation syndrome. Fertil Steril 2000; 73:883–96. 14. Ditkoff EC, Cassidenti DL, Paulson RJ et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991; 165:1811–17. 15. Olivennes F, Fanchin R, Bouchard P et al. The single or dual administration of the gonadotropin-releasing hormone antagonist Cetrorelix in an in vitro fertilization-embryo transfer program. Fertil Steril 1994; 62:468–76.

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16. Olivennes F, Alvarez S, Bouchard P et al. The use of a GnRH antagonist (cetror.elix) in a single dose protocol in IVF-embryo transfer: A dose finding study of 3 versus 2 mg. Hum Reprod 1998; 13:2411–14. 17. Olivennes F, Belaisch-Allart J, Emperaire JC et al. Prospective, randomized, controlled study of in vitro fertilization embryo transfer with a single dose of a luteinizing hormone releasing hormone (LH-RH) antagonist (cetrorelix) or a depot formula of an LH-RH agonist (triptorelin). Fertil Steril 2000; 73:314–20. 18. Rongieres-Bertrand C, Olivennes F, Righini C et al. Revival of the natural cycles in in vitro fertilization with the use of a new gonadotrophin-releasing hormone antagonist (cetrorelix): Apilot study with minimal stimulation. Hum Reprod 1999; 14:683–88. 19. Felberbaum RE, Albano C, Ludwig M et al. Ovarian stimulation for assisted reproduction with HMG and concomitant midcycle administration of the GnRH antagonist cetrorelix according to the multiple dose protocol: A prospective uncontrolled phase III study. Hum Reprod 2000; 15:1015–20. 20. Diedrich K, Diedrich C, Santos E et al. Suppression of the endogenous luteinizing hormone surge by the gonadotrophinreleasing hormone antagonist cetrorelix during ovarian stimulation. Hum Reprod 1994; 9:788–91. 21. Felberbaum R, Reissmann T, Kupker W et al. Hormone profiles under ovarian stimulation with human menopausal gonadotropin (hMG) and concomitant administration of the gonadotropin— releasing hormone (GnRH)-antagonist cetrorelix at different dosages. J Assist Reprod Genet 1996; 13:216–22. 22. Ubaldi F, Albano C, Peukert M et al. Subtle progesterone rise after the administration of the gonadotrophin-releasing hormone antagonist cetrorelix in intracytoplasmic sperm injection cycles. Hum Reprod 1996; 11:1405–07. 23. Albano C, Smitz J, Camus M et al. Hormonal profile during the follicular phase in cycles stimulated with a combination of human menopausal gonadotrophin and gonadotrophin releasing hormone antagonist (cetrorelix). Hum Reprod 1996; 11:2114–18. 24. Albano C, Smitz J, Camus M et al. Comparison of different doses of gonadotropin-releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67:917–22. 25. Albano C, Smitz J, Tournaye H et al. Luteal phase and clinical outcome after human menopausal gonadotrophin/gonadotrophin releasing hormone antagonist treatment for ovarian stimulation in in vitro fertilization/intracytoplasmic sperm injection cycles. Hum Reprod 1999; 14:1426–30. 26. Albano C, Felberbaum RE, Smitz J et al. Ovarian stimulation with HMG: Results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group. Hum Reprod 2000; 15:526–31. 27. Akman MA, Erden HF, Tosun SB et al. Comparison of agonistic flare-up-protocol and antagonistic multiple dose protocol in ovarian stimulation of poor responders: Results of a prospective randomized trial. Hum Reprod 2001; 16:868–70. 28. The European and Middle East Orgalutran Study Group. Comparable clinical outcome using the GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH surges in women undergoing ovarian stimulation. Hum Reprod 2001; 16:644–51. 29. de Jong D, Macklon NS, Eijkemans MJ et al. Dynamics of the development of multiple f ollicles during ovarian stimulation for in vitro fertilization using recombinant folliclestimulating hormone (Puregon) and various doses of the gonadotropin releasing hormone antagonist ganirelix (Orgalutran/Antagon). Fertil Steril 2001; 75:688–93. 30. The North American Ganirelix Study Group. Efficacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil Steril 2001; 75:38–45.

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31. European Orgalutran Study Group, Borm G, Mannaerts B. Treatment with the gonadotrophinreleasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe, and convenient: Results of a controlled, randomized, multicentre trial. Hum Reprod 2000; 15:1490–98. 32. Smitz J, Erard P, Camus M et al. Pituitary gonadotrophin secretory capacity during the luteal phase in superovulation using GnRH-agonists and HMG in a desensitization or flare-up protocol. Hum Reprod 1992; 7:1225–29. 33. Tavaniotou A, Albano C, Smitz J et al. Comparison of LH concentrations in the early and midluteal phase in IVF cycles after treatment with HMG alone or in association with the GnRH antagonist cetrorelix. Hum Reprod 2001; 16:663–67. 34. Kol S, Lightman A, Hillensjo T et al. High doses of gonadotrophin-releasing hormone antagonist in in vitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles. Hum Reprod 1999; 14:2242–44. 35. Dong KW, Marcelin K, Hsu MI et al. Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. Mol Hum Reprod 1998; 4:893–98. 36. Raga F, Casan EM, Kruessel JS et al. Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod 1998; 59:661–69. 38. Ludwig M, Felberbaum RE, Devroey P et al. Significant reduction of the incidence of ovarian hyperstimulation syndrome (OHSS) by using the LHRH antagonist cetrorelix (Certrotide) in controlled ovarian stimulation for assisted reproduction. Arch Gynecol Obstet 2000; 264:29–32. 39. Itskovitz-Eldor J, Kol S, Mannaerts B. Use of a single bolus of GnRH agonist triptorelin to trigger ovulation after GnRH antagonist ganirelix treatment in women undergoing ovarian stimulation for assisted reproduction, with special reference to the prevention of ovarian hyperstimulation syndrome: Preliminary report: short communication. Hum Reprod 2000; 15:1965–68. 40. Nelson LR, Fujimoto VY, Jaffe RB et al. Suppression of follicular phase pituitary-gonadal function by a potent new gonadotropin-releasing hormone antagonist with reduced histaminereleasing properties (ganirelix). Fertil Steril 1995; 63:963–69. 41. Fujimoto VY, Monroe SE, Nelson LR et al. Dose-related suppression of serum luteinizing hormone in women by a potent new gonadotropin-releasing hormone antagonist (Ganirelix) administered by intranasal spray Fertil Steril 1997; 67:469–73. 42. Reissmann T, Schally AV, Bouchard P et al. The LHRH antagonist cetrorelix: A review. Hum Reprod Update 2000; 6:322–31. 43. Ludwig M, Riethmuller-Winzen H, Felberbaum RE et al. Health of 227 children born after controlled ovarian stimulation for in vitro fertilization using the luteinizing hormone-releasing hormone antagonist cetrorelix. Fertil Steril 2001; 75:18–22. 44. Akman MA, Erden HF, Tosun SB et al. Addition of GnRH antagonist in cycles of poor responders undergoing IVF. Hum Reprod 2000; 15:2145–47. 45. Nikolettos N, Al-Hasani S, Felberbaum R et al. Comparison of cryopreservation outcome with human pronuclear stage oocytes obtained by the GnRH antagonist, cetrorelix, and GnRH agonists. Eur J Obstet Gynecol Reprod Biol 2000; 93:91–95. 46. Belchetz PE, Plant TM, Nakai Y et al. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 1978; 202:631–33. 47. Bouchard P, Fauser BC. Gonadotropin-releasing hormone antagonist: New tools vs. old habits. Fertil Steril 2000; 73:18–20. 48. de Jong D, Macklon NS, Fauser BC. A pilot study involving minimal ovarian stimulation for in vitro fertilization: Extending the “follicle-stimulating hormone window” combined with the gonadotropin-releasing hormone antagonist cetrorelix. Fertil Steril 2000; 73:1051–54.

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49. Mettler L, Brandenburg K. Cetrotide confirmatory trial of cetrorelix/ 0.25 mg in 26 women undergoing ovarian stimulation withrecombinant follicle stimulating hormones for IVF, ICSI, and embryo transfer. Clin Exp Obstet Gynecol 2000; 27:103–05. 50. Al-Inany H, Aboulghar M. GnRH antagonist in assisted reproduction: a Cochrane review. Hum Reprod 2002; 17(4):874–85.

CHAPTER 12 Microdose GnRH for the Stimulation of Low Responders CAM Jansen, KE Tucker OVERVIEW Gonadoptrophin-releasing hormone (GnRH) agonists have initially been introduced in the field of reproductive medicine on an ‘off label use’ basis without preceding proper dose finding studies. As a result doses that were required for the original indication (i.e. prostate carcinoma) were used, begging the question whether these schemes would not lead to LH levels that were too low for the maintenance of proper follicle growth in some patients. During the era of follicle stimulation with HMG-a combination of FSH and LH (or hCG)- this aspect might stay unnoticed as there will always be a sufficient exogenous administration to sustain follicle growth, but this may change for some patients since the wide scale use of recombinant FSH devoid of any LH activity. Some authors have appropriately questioned the dosing for GnRH agonists: they have coined the term “micro dose”, but one should question this term as it may well be that what is considered normal may actually be a “macro dose.” Dose finding studies have only recently been performed, but uncontrolled studies with lower dosage have suggested an improvement in response for certain specific groups such as previous low responders. However none of these studies are prospective, randomized and controlled. So far, although it is possible that certain patients may benefit from lower dosages, there still is no proper scientific basis for either attitude. Therefore there is an urgent need for randomized controlled trials. Methods and Results The first to suggest the benefit of GnRH agonists for prevention of premature luteinization in reproductive medicine was Richard Fleming as early as 1986.1 Several dosages with several GnRH agonists are used, most commonly buserelin, nafarelin, triptorelin and leuprolide. Originally buserelin was used universally as this was the first and only available product. The subsequent increase in the use of leuprolide (mainly in the USA) and triptorelin (mainly in Europe and many other countries) at the expense of buserelin was more the result of the promotion policy, or the lack thereof by the pharmaceutical companies, rather than of scientifically founded data. Interestingly, the large-scale use of depot formulas seems mainly to have been confined to certain countries such as France and Israel. In these countries the fear of a possible long-term influence of depot preparations seems to have been less prominent. In addition there is insufficient evidence to compare the different GnRH agonists in terms of efficiency and efficacy. Most prospective randomized studies compare one

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agonist with another, without any preceding necessary dose finding studies. The only real dose-finding study was initiated by Dr Mak and performed at the Free University in Amsterdam, in a prospective, double blind placebo controlled (unique in this field), randomized study. It was clear that much lower doses, even up to 5 µg/day of triptorelin could suffice in obtaining pituitary desensitization2. In a further study there turned out to be no influence on pregnancy rates between any of the dosages used (placebo, 15, 50 and 100 µg/day), and even the placebo led to a similar pregnancy rate3. Additional details of this study have been published previously4 ‘Microdose’ Protocols in IVF The first to suggest that some patients might benefit from micro dose GnRH protocols (40 µg per day instead of the usual 1000 µg leuprolide acetate) was Richard Scott5. However in this study the subsequent response with the microdose protocol was compared to the previous response with the standard GnRH dose, leaving the study open to the criticism that this may be due to the phenomenon of ‘regression to the mean’. The same holds for a similar study of almost identical design that was published later6. In addition in this second study, where patients received 80 µg leuprolide per day the previous low responders also received Growth Hormone (GH) as supplementation in the subsequent treatment, thus having different stimulation schemes in both arms. A third study is open to the same criticism: cycle analysis was performed before and after the micro dose treatment in poor responders whilst patients were subsequently divided according to their age (above or below 40 years).7 The ‘Poor Responder’ In all studies concerning micro dose GnRH the group of patients was emphasized that pose the greatest challenge to all of us, i.e. the so-called ‘poor responders’. In an overview of the proposed stimulation regimes in poor responders it was first concluded that so far there is no uniform definition of the word ‘poor’ responder.8 Various authors use different definitions of the word, ranging from the number of follicles on ultrasound, (ranging from 2 to 5), the maximal E2 level (ranging from 100–660 pg/ml) on the day of hCG, the mean daily gonadoptrophin used, the total number of ampoules and so on. In addition it was concluded that there is no RCT to show that the micro dose GnRH is of benefit to poor responders, that at best only some patients might benefit from it, and that in any case it is not possible to identify this patient in advance. In another retrospective analysis between the standard midluteal long protocol and a micro dose of 40 µg daily dose of leuprolide it was concluded that except for a significant increase in cancellation rate in the micro dose group there were no significant differences in the other variables such as the oocyte yield or the pregnancy rate9. In one study the ovarian volume before the start of stimulation was taken into account, and results were compared in the different groups. However the stimulation scheme was not randomized: patients with a small volume (less than 3 cc) received a micro dose agonist scheme, whilst women with a normal volume had a standard dose agonist scheme.10 In addition in this study there was a surprisingly large group with very small ovaries: 28 % of all patients had a mean volume of less than 3 cc, which means (with the ellipsoid formula of

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D1 ×D2×D3×0.526 or 0.523 depending on the publication) a mean size of the complete ovary of less than 18 mm. The authors suggested that the ovarian response would have been less if a standard dose was used, citing previous literature.11,12 However in only one of these there was a decrease in oocyte numbers in small ovaries and in neither of these there was a decrease in pregnancy rate. Furthermore the incidence of small ovaries (less than 3 ml) ranged between 9 and 12 percent in these studies, a considerably lower incidence than the present study. It has recently been suggested that patients that do not respond to the stimulation with recombinant FSH under GnRH suppression may be ‘rescued’ by switching over to HMG13. In this study patients that did not respond to the combination of a GnRH agonist and recombinant FSH were randomized to either an increase in rec FSH or to stimulation with the same original dose of HMG. The oocyte yield as well as the oestradiol concentration was significantly higher in the HMG group. The authors ascribe this to the lack of sufficient LH, needed for adequate follicle growth that can be overcome by the administration of exogenous LH, but then one might equally expect a favorable effect from a decrease in the dosage of the GnRH agonist. Similarly Westergaard et al14 have described an increase in miscarriage rates in patients that showed very low LH levels (below 0.5 IU/l in a sensitive assay with a detection limit of 0,05 IU/l) after GnRH down regulation in comparison to those with higher LH levels14. This suggests that indeed there is a minimum concentration of LH necessary for adequate follicle growth. In the past, when stimulation was performed with hMG this was a moot point as exogenously administered LH (and/or hCG) was present in the hMG formulation (mandatory 1:1— FSH: LH ratio by the pharmacopoeia) which was always sufficient to allow for proper folliculogenesis. In this study the GnRH dose was decreased considerably after down regulation was achieved from 0.5 mg to 0.2 mg buserelin per day when ovarian stimulation was commenced with recombinant FSH. The philosophy behind this dose decrease is that one may do with less GnRH agonist; however LH levels were not measured in the first days after dosage decrease. It has been published that LH levels may fall to undetectable levels after midfollicular cessation of the agonist thereby compromising the sustenance of follicle growth.15 Ideally before the introduction of any medication, dose finding studies should be performed to establish the minimum effective dose to achieve any effect with a minimum in complications and cost. This has never taken place with GnRH agonists. Their rapid and widespread introduction was due to the instant universal recognition of their ability to prevent luteinization and ovulation before follicle aspiration. This peculiar type of drug introduction was only possible because these substances were already available, as mentioned previously. As such it is surprising that it has taken longer than 10 years after their entry into the field of ART before the first proper dose-finding studies for COH in IVF were performed. This study is characterized by two peculiar aspects: one semantic, and one methodological. Firstly, the implantation rate (IR) is defined as others would define the clinical pregnancy rate/ET (i.e. the presence of at least one gestational sac per transfer).

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Table 12.1: Results of a dose-finding study with the GnRH agonist, triptorelin. ITT: intention to treat; Started: cycles commenced; ET: Embryo transfer; Cl preg: Clinical pregnancy rateerroneously called implantatlon rate; Ong: Ongoing pregnancy rate Dose Placebo 15 mg/d 50 mg/d 100 mg/d

ITT Start ET Cl preg Ong 60 60 60 60

59 56 56 58

46 55 55 55

10 8 10 13

5 8 9 7

This means that whenever the term IR is mentioned it should actually be PR. Secondly, an LH peak is defined in this study as a doubling from baseline with one later value higher than the doubled one. This means, according to the authors, that if baseline level is 3IU/l, a rise to 7IU/l, and then to 8 IU/l the next day, depicts a LH peak. This, as such, cannot be considered to be a full-blown LH surge, necessary to result in all changes prior to and after ovulation (i.e. resumption of meiosis and adequate luteinization) and it is known that during follicular stimulation such small LH peaks can be present. In the placebo group, even after these small LH elevations, a follicle puncture was performed, without additional HCG supplementation. It is, therefore, not surprising that there were surprisingly few transfers in the placebo group, and this may also explain the high miscarriage rate The so-called ‘normal’ dose was based on the initial assumption that the degree of down regulation needed to be as complete as possible. A meta-analysis of several prospective and randomized studies comparing down-regulated and non-down-regulated treatments as first choice in IVF shows that a significant difference in pregnancy rates exists between these two types of stimulation protocols.16 It should be noted, however, that the differences are due to exceptionally low results in some studies in the nonanalogue group, much lower than previously published in comparable studies performed by these same investigators. Nevertheless, the use of GnRH analogues has become ubiquitous in an incredibly short time span, mainly because of the market availability and quite possibly, for doctors’ convenience. The use of GnRH analogues minimizes the need for careful follicle monitoring, as even inaccurate measurements are not usually penalized by premature LH surges. The rapid acceptance of these drugs however has occurred in spite of the increased risk of the ovarian hyperstimulation syndrome (OHSS). The magnitude of this risk is more difficult to quantify than pregnancy rate, mainly because of its lower incidence. Many prospective randomized studies do not mention this feature; in addition there is a publicationbias in reporting side effects and almost a total publication deficit with regard to the mortality from this syndrome. A Medline and Embase search revealed only one two cases resulting from the complications of the OHSS.17

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Determination of Down Regulation Often a serum estradiol level is used to confirm adequate down regulation and is usually below 0.2 nmol/l. It should be noted, however, that present Enzyme Immuno Assays are not always appropriate, as there is a wide scatter in the low range.18 The intra-assay variation in the low range where the cut-off level is present may be in the range of 25%, This means that when one measures E2 by EIA, one in four patients with sufficient down-regulation and E2 below 0.5 nmol/l still seemingly have too high an E2 level. LH levels can be measured as well, but many assays have a cut-off level of 1IU/l and do not allow for the determination of the minimum LH necessary to optimize follicle growth, as well as granulosa-cell estradiol synthesis after stimulation with recombinant FSH. CONCLUSIONS ‘Micro-dose’ GnRH protocols are mainly suggested for the so-called ‘poor responder’ patients. However as proper dose finding studies are lacking there is no proper use of the word, and the so-called ‘normal dose’ may well turn out to be ‘macro- dose’. None of the studies are randomized, and it now seems that the GnRH antagonists have become the newest hype in the treatment of ‘poor responders’. It will soon become evident that these as well will not be the panacea we are all looking for, and that most poor responders will just have a limited stock of recruitable follicles available, regardless of whatever one decides to choose as stimulation regimen. REFERENCES 1. Fleming R, Coutts JR. Induction of multiple follicular growth in normally menstruating women with endogenous gonadotropin suppression. Fertil Steril 1986; 45:226–30. 2. Janssens RMJ, Vermeiden JPW, Lambalk CB, Schats R, Schoemaker J Gonadotrophin-releasing hormone agonist dose-dependency of pituitary desensitization during controlled ovarian hyperstimulation in IVF. Hum Reprod 1998; 13:2386–91. 3. Janssens RMJ, Lambalk CB, Vermeiden JPW, Schats R, Bernards JM, Rekers-Mombarg LTM, Schoemaker J. Dose- finding study of triptorelin- acetate needed for prevention of a premature LH surge in OIVF: Aprospective, randomised, double blind, placebo controlled study. Hum Reprod 2000; 15:2333–40. 4. Jansen CAM, Tucker KE, GnRH agonist dose and ovulation induction outcome Proceedings of the WCOI 2000. 5. Scott RT, Navot D. Enhancement of ovarian responsiveness with microdoses of gonadotropinreleasing hormone agonist during ovulation induction for in vitro fertilization. Fertil Steril 1994; 61:880–85. 6. Schoolcraft W, Schlenker T, Gee M, Stevens J, Wagley L. Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose folliclestimulating hormone flare, growth hormone protocol. Fertil Steril 1997; 67:93–97. 7. Surrey ES, Bower J, Hill DM, Ramsey J, Surrey MW. Clinical and endocrine effects of a microdose GnRH agonist flare regimen administered to poor responders who are undergoing in vitro fertilization. Fertil Steril 1998; 69:419–24. 8. Surrey ES, Schoolcraft WB. Evaluating strategies for improving ovarian response of the poor responder undergoing assisted reproductive techniques. Fertil Steril 2000; 73:667–76

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9. Leondires MP, Escalpes M, Segars JH, Scott RT Jr, Miller BT. Microdose follicular phase gonadotropin-releasing hormone agonist (GnRH-a) compared with luteal phase GnRH-a for ovarian stimulation at in vitro fertilization. Fertil Steril 1999; 72:1018–23. 10. Sharara FI, McClamrock HD. Use of microdose GnRH agonist protocol in women with low ovarian volumes undergoing IVF. Hum Reprod 2001; 16:500–3. 11. Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 1995; 64:1167–71. 12. Lass A, Skull J, McVeigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in vitro fertilization can predict poor response. Hum Reprod 1997; 12:294–97. 13. De Placido G, MolloA, Alviggi C, Strina I, Varricchio MT, Ranieri A, Colacurci N, Tolino A, Wilding M. Rescue of IVF cycles by HMG in pituitary down-regulated normogonadotrophic young women characterized by a poor initial response to recombinant FSH. Hum Reprod 2001; 16:1875–79 14. Westergaard LG, Laursen SB, Andersen CY. Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction. Hum. Reprod. 2000; 15:1003–08. 15. Cedrin-Durnerin I, Bidart JM, Robert P, Wolf JP, Uzan M, Hugues JN. Consequences on gonadotrophin secretion of an early discontinuation of gonadotrophin-releasing hormone agonist administration in short-term protocol for in vitro fertilization. Hum Reprod 2000; 15:1009–14. 16. Hughes EG, Fedorkow DM, Daya S. The routine use of gonadotropin releasing hormone agonist prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992; 58:888. 17. Cluroe AD, Synek BA fatal case of ovarian hyperstimulation syndrome with cerebral infarction. Pathology 1995; 27:344–46. 18. Tucker KE, Rietdijk AM, Postma T, van de Water C, Jansen CAM,. The accuracy of enzyme immunoassays of oestradiol for the determination of ovarian reserve and the degree of downregulation. Hum Reprod 2000; (Suppl 15):230.

CHAPTER 13 The Role of GnRH Antagonist in the Management of Poor Responders Hossein Gholami, Nico Naumann, Antonio Barbaro INTRODUCTION Low ovarian response to stimulation occurs in 9–24 percent of patients1 and still represents one of the most intractable problems of inf ertility treatment. The management of poor responders in IVF has always been a big problem and the ideal approach has still to be formulated. The poor response to stimulation with gonadotrophin is often age-related and the ovarian reserve declines with age but it can also occur in younger women. However, the rate of this decline seems to vary among individuals and depends on the clinical history and various environmental and genetic factors. Severe endometriosis, pelvic inflammatory disease, ovarian surgery, various systemic illnesses, chemotherapy and smoking are all well known factors affecting the ovarian reserve.2–3 Also compromised endocrine, paracrine and autocrine signals can result in altered communication between the granulosa cells and the oocytes, which result in abnormal nuclear and cytoplasmic maturation with the oocyte.4 Definition of Poor Responders There is no standard definition of poor response to ovarian stimulation. The original definition of poor response to ovarian response was based only on low oestradiol concentrations. They stimulated patients with 150 IU of human menopausal gonadotrophin (HMG) IM and defined poor responders as those patients who had a peak oestradiol concentration of <300 pg/ml (1101.3 pmol/l). The definition of “poor response” varies from one group to another. Serafini et al5 defined poor responders as those who produced less than three follicles despite adequate ovarian stimulation. Jenkis et al6 considered four follicles as their cut-off point. This definition generally implies failure to achieve a certain number of mature follicles or a certain estrogen level in relation to the amount of ovarian stimulation that has been given.1 It is possible that women who do not respond well to a relatively low dose of gonadotrophin will response better to a higher dose, but it has been shown that increasing the dose beyond a certain level rarely improves the outcome.7 Despite these differences in definition “poor responders” represent a heterogeneous group of patients who can be divided clinically into two main groups. The first group includes those with low ovarian reserve as reflected by their high basal follicle

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stimulating hormone (FSH), and the second group includes women with normal ovarian reserve who are inherently low responders to gonadotropin stimulation.8 Also older patients (>40 years) present a poor response in ovarian stimulation, with prolonged duration and also increased cost of treatment. Assessment of Ovarian Reserve Test of functional reserve of ovaries can of ten be used to predict low response to standard protocols, various tests have been developed to assess ovarian reserve. These include day 3 FSH,9–10 ovarian volume, the clomiphene citrate challenge test, Inhibin B,11–12 the GnRH agonist stimulation test,13 antral follicle count,14 measurement of ovarian stroma blood flow with colour-Doppler,15 and using the information from an previous IVF cycle in order to predict an individuars reproductive potential.16 Stimulation Protocol Several stimulation protocols have been proposed to try and improve outcomes in poor responders. These include: i. natural cycle IVF; NOIC natural oocyte intravaginal culture;17 ii. using clomiphene citrate for stimulation;18 iii. co-treatment with oestrogens, growth hormone or birth control pills;18 iv. varying the dose or the day of cycle for initiating stimulation with gonadotrphins; v. pituitary desensitization with a GnRHa in the luteal phase of the previous cycle, followed by stimulation with a high dose of gonadotrophins “long protocol”,5 and vi. initiating GnRHa and gonadotrophins together in the follicular phase “flare protocol.” All of these strategies have met with only limited success. Synthetic GnRH and its Analogs The hypothalamic decapeptide GnRH was first isolated and synthesized in 1971 by Amos et al.19 Its half-life is 2–4 minutes. Rapid cleavage of the bonds between amino acids (6– 7, 7–8, and 9–10) leads to its rapid metabolism. Substitution of amino acids at the 6– and 10 positions strengthens the bonds, leading to longer half-lives. These amino acidsubstituted new synthetic analogs display increased receptor binding and prolonged receptor occupation, resulting in downregulation of receptors and desensitization of the pituitary, leading to hypogonadism. GnRH analogs induce a hypoestrogenic state that is comparable to oophorectomy. During suppression, serum concentrations of oestradiol, FSH, LH, and progesterone decrease significantly, serum testosterone decreases slightly, and serum prolactin concentration is not affected. Substitution of amino acids in position 1, 2, 3, 6, or 10 results in an antagonistic analog. Since the new synthetic peptides are very different from the native decapeptide, receptor occupation leads to an immediate antagonistic response, resulting in hypogonadism. GnRH antagonist was developed together with GnRH-agonist20 but the clinical use was limited because of allergic side effects. However, the development of ‘third generation’ GnRH antagonist appears to have overcome this problem.21

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Cetrorelix (SB-75; Ac-D-nal (2), D-Phe (4cl), D-pal (3), D-Cit6, D-Ala 10 GnRH) is one of these third generation antagonists and has been promising in research to delay LH surge with a single injection dose of 3 mg or as low dosage (0, 25 mg) daily given in the late follicular phase. Use of GnRH Analogue The multif ollicular development is used in IVF-embryo transfer previa down regulation with GnRH agonist regimen to prevent a premature LH surge.22 Aperiod of 7–14 days are required for pituitary down-regulation. It has been reported that some women require a longer period of GnRH treatment to achieve adequate down-regulation.23 Initial gonadotrophin flare response risks be exaggeration of ovarian androgen and oestradiol responses upon institution of GnRH therapy.24 In contrast, the administration of GnRH antagonist, Cetrotide given in single dose of 3 mg in the late follicular phase, or given as low dosage (0, 25 mg) daily, on day 6 of gonadotrophin injection produce an immediate decrease in endogenous gonadotrophin concentrations. The timing of human chorionic gonadotrophin (HCG,) is based on leading follicular diameter, serum E2 and LH concentration. Ultrasound-guided vaginal oocyte retrievals in anesthesia is scheduled 35 h after HCG(10000 IU) administration.

Fig. 13.1: Protocol treatment for women undergoing ovarian stimulation using multiple dose Cetrorelix (Cetrotide) to suppress LH secretion. OPU=oocyte pickup The luteal phase is supported with progesterone-in-oil (50 mg im daily) on the day af ter oocyte retrieval and continued only for 7 days. The rapid effect of GnRH antagonist offers the potential and convenient treatment and suppression of a premature LH surge in women undergoing ovarian hyperstimulationprotocols.

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Fig. 13.2: Protocol treatment for women undergoing ovarian stimulation using single dose Cetrorelix (Cetrotide 3 mg) to suppress LH secretion. OPU=oocyte pickup A disadvantages of this shorter GnRH antagonist regimen is that the timing of oocyte retrieval is outside the clinician’s control. DISCUSSION The introduction of GnRH antagonists to clinical practice may be a new hope for the poor responder patients. There is conflicting data in the literature on the ovarian effects of GnRH agonists and the antagonists. Both are used successfully in ovarian stimulation protocols for preventing the premature LH surges in poor responder women. Recently various reports have appeared in the literature documenting their success.25–26 Craft et al evaluated the effects of GnRH antagonist use in 31 IVF/gamete intraFallopian transfer (GIFT) cycles of difficult responder.27 Among them, 18 patients (24 cycles) were poor responders. Although this study did not reach statistical significance, the cycle cancellation was less compared with the previous agonist protocol, more oocytes at a lower dose of FSH were produced and two live births resulted. GnRH antagonists, Cetrorelix and Ganirelix, have both been approved for ovarian stimulation to prevent a premature LH surge. Different studies which compared GnRH agonist with the new antagonist treatment found no significant differences concerning the most important goals, e.g. pregnancy rate, fertilization and quality of oocytes. Our experience of using gonadotrophin (Gonal F)-Cetrorelix for poor responder suggests that this protocol will be of value for some patients who have not responded well to GnRH agonist and gonadotrophins in term of follicologenesis and pregnancy rate, that could be explained by an alteration in ovarian functions as direct effects of GnRH agonist on steroidogenesis in granulosa cells and inhibition of progesterone secretion by GnRH agonist in luteal phase. In addition, GnRH antagonist given in late follicular phase

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during ovarian stimulation will prevent the premature LH surges while not causing any suppression in the early follicular phase which is a critical period for IVF success in patients with decreased ovarian reserves. REFERENCES 1. Keay SD, Liversedge NH, Mathur RS, Jenkins JM. Assisted conception following poor ovarian response to gonadotrophin stimulation. Br J Obstet Gynaecol 1997; 104:521–25. 2. Wardle PG, McLaughlin EA, Ray BD, McDermott A, Hull MG. Endometriosis and ovulatory disorder: reduced fertilization in vitro compared with tubal and unexplained infertility Lancet 1985; 2:236–37. 3. Lass A, Skull J, Mc Veigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before human menopausal gonadotrophin superovulation for in vitro fertilization can predict poor response. Hum Reprod 1997; 12:294–97. 4. Eppig J. Intracomunication between mammalian oocytes and companion somatic cells. Bioessays 1991; 13:569–74. 5. Serafini P, Stone B, Kerin J et al. An alternative approch to controlled ovarian hyperstimulation in poor responders with gonadotrophin releasing hormone analog. Fertil Steril 1988; 49:90–95. 6. Jenkins JM, Davies DW, Devonport H et al. Comparison of poor responders with good responders using a standard buserelin/ human menopausal gonadotrophin regime for in vitro fertilization. Hum Reprod 1991; 6:918–21. 7. Out HJ, Braat DD, lintsen RM, Gurgan T, Bukulmez O, Gokmen O, Keles G, Caballero P, Gonzalez JM, Ferbegues F et al. Increasing the daily dose of recombinant follicle stimulating hormone (Puregon) does not compensate for the age-related decline in retrievable oocyte after ovarian stimulation. Hum Reprod 2000; 15:29–35. 8. Lashen H, Ledger W, Lopez-Bernal, Barlow D. Poor responders to ovulation induction: is proceeding to in vitro fertilization worthwhile? Hum Reprod 1999; 14:964–69. 9. Scott R, Hofmann G. Prognostic assessment of ovarian reserve. Fertil Steril 1995; 63:1–11. 10. Sharif K, Elgendy M, Lashen H, Afnan M. Age and basal stimulating hormone as predictors of in vitro fertilization outcome. Br J Obstet Gynaecol 1998; 105:107–12. 11. Seifer D, Lambert-Messerlian G, Hogan JW, Gardiner A, Blazar A, Brek C. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril 1997; 67:110–14. 12. Eldar-Geva T et al. Serum inhibin B levels measured early during FSH administration for IVF may be of value in predicting the number of oocytes to be retrieved in normal and low responders. Hum Reprod 2002; 17:2331–37. 13. Galtier-Dereure F, De Bouard V, Picot Vergnes C, Humeau C, Bringer J, Hedon B. Ovarian reserve test with gonadotrophin-releasing hormone agonist buserline: correlation with in vitro fertilization outcome. Hum Reprod 1996; 11:1393–98. 14. Chang MY, Chang Ch-H, Hsieh TT et al. Use the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril 1998; 69:505–10. 15. Engmann L, Sladkevicius P, Agrawal R et al. Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil Steril 1999; 71:22–29. 16. Van Rysselberge M, Puissant F, Barlow P, Lejeune B, Delvigne A, Leroy F. Fertility prognosis in IVF treatment of patient with cancelled cycles. Hum Reprod 1989; 4:663–66. 17. Gholami G-Hossein et al. Natural oocyte intravaginal culture (NOIC). Atti 6th International Congress of Geographic Medicine; Shiraz, Iran 1993; 44–45. 18. Gonen Y, Jacobsen W, Casper RF. Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil Steril 1990; 53:282–87.

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19. Amoss M, Burgus R, Blackwell R, Vale W, Fellows R, Guilemin R. Purification, aminoacid composition and N-terminus of the hypothalamic luteinizing hormone-releasing factor (LRF) of bovin origin. Biochem Biophys Res Commum 1971; 44:205–08. 20. Hahn DW, McGuire JL, Vale WW, Rivier J. Reproductive/ endocrine and anaphylactoid properties of an LHRH antagonist (ORF-12860). Life Sci. 1985; 37:505–14. 21. Felberbaum RE, Diedrich K. The use of GnRH antagonist in assisted reproduction technologies. In Lunefeld B (Ed), GnRH Analogus. The state of the Art at the Millenium. The Parthenon Publishing Group, New York 1999; 47–63. 22. Huirne JAF, Lambalk CB, Janssens R, Schoemaker J. GnRH agonists versus antagonist, where are we today? In: Ben-Rafael Z, Schoham Z (Eds), The first congress on controversies in Obstetrics, Gynecology and Infertility. Monduzzi Editore, Bologna, Italy, 1999; 77–81. 23. Tanbo T, Abyholm T, Magnus O, Henriksen T. Gonadotropin and ovarian production in polycystic ovarian syndrome during suppression with a gonadotropin-releasing hormone agonist. Gynecol Obstet Invest 1989; 28:147–51. 24. Suikkari AM, MacLachlan V, Montaldo J et al. Ultrasonographic appearance of polycistic ovaries is associated with exaggerated ovarian androgen and Oestradiol responses togonadotrophin-releasing hormone agonist in women undergoing assisted reproduction treatmen. Hum Reprod 1995; 10:513–19. 25. Frydman R, Cornel C, de Ziegler D et al. Sponataneous luteinizing hormone surges can be reliably prevented by the timely administration of a gonadotrophin releasing hormone antagonist(Nal-Glu) during the late follicular phase. Hum Reprod 1992; 7:930–33. 26. Diedrich K, Diedrich E, Santos E et al. Supression of endogenus LH-surge by the GnRH antagonist cetrorelix during ovarian stimulation. Hum Reprod 1994; 9:788–91. 27. Craft I, Gorgy A, Hill J et al. Will GnRH antagonists provide a new hope for patients considered difficult responders to GnRH agonist protocols? Hum Reprod 1999; 14:2959–62.

CHAPTER 14 Alternative Approaches to Ovarian Stimulation and Triggering of Ovulation Gautam N Allahbadia, Kaushal Kadam, Swati G Allahbadia, Avinash Phadnis OVARIAN STIMULATION Introduction Ovulation induction is a frequently utilized therapeutic procedure for the management of inf ertility. The goals of ovulation induction depend on the medical condition of each couple, and can be grouped in two major categories. Firstly, procedures conducted to restore ovulation in patients with menstrual and ovulatory disorders. The preferred approach in this condition is to use clomiphene citrate, and, when this drug fails, pulsatile gonadotropin-releasing hormone (GnRH) or low-dose gonadotropins. These latter are indicated because of the efficacy and reduced risk of ovarian hyperstimulation and/or multiple conception associated with their use.1,2 Secondly, stimulation of multiple folliculogenesis in normal women undergoing assisted reproductive procedures (ART). Numerous follicles/oocytes are necessary for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) and thus, full dose gonadotropin regimens are indicated in these subjects. The introduction of gonadotropin-releasing hormone analogues (GnRH-a) into assisted reproduction technique (ART) protocols some 12 years ago greatly contributed to the success of modern IVF treatment. These medications induce pituitary desensitization, thereby suppressing premature endogenous LH surges, reducing cancellation rates, improving the overall number of oocytes retrieved, and improving implantation rates.3,4 Patients with oligomenorrhoea and/or polycystic ovarian disease often respond inappropriately to exogenous gonadotrophins or pulsatile GnRH. On the other hand, patients with profound depression of gonadotropin secretion (e.g. primary hypogonadotropic amenorrhea) are the best candidates for induction of ovulation and show a lower incidence of complications (ovarian hyperstimulation). Fleming et al5,6 gave intranasal buserelin (100 mcg five times daily) for 2–3 weeks to eight women with oligomenorrhoea and elevated concentrations of luteinizing hormone (LH) and androgen. Human menopausal gonadotrophins (hMG) were then administered and buserelin was continued until ovulation was induced with exogenous human chorionic gonadotropin (hCG). Ovulation was achieved in 12 cycles and seven out of the eight patients became pregnant. A spontaneous preovulatory LH surge did not occur in these cycles and no signs of premature luteinization of follicles were noticed. Pretreatment of patients with a GnRH agonist, particularly when polycystic ovarian

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disease is present, allows reduction of ovarian responsiveness and control of spontaneous ovulation. In addition to classical ovulation induction, this approach may be of particular interest in patients undergoing IVF as it may synchronize follicular development and prevent early follicular luteinization.7 Types and Characteristics of Different Protocols Treatment Protocols with GnRH-a and Gonadotropins Four major protocols that combine exogenous gonadotropins and GnRH agonists are currently employed. They can be given as biodegradable implants, injections or nasal sprays and pulsatile injections, as used with native gonadotropin-releasing hormone(GnRH).8 The use of depot injections of GnRH agonist gives results similar to those obtained with daily injections.9 Longprotocol This is the most traditional and widely-employed protocol. It consists of GnRH agonist administration started in the mid-luteal phase of the cycle preceding gonadotropin ovulation induction and continued until hCG administration. This regimen appears to provide improved clinical results (greater number of preovulatory follicles and embryos, increased pregnancy rates) both over the use of gonadotropins alone10,11 and other combined regimens.11,12 Patient scheduling is also optimal with the long regimen. Hence, long regimens are currently the most widely-used regimens in ART programmes. Short Protocol This protocol consists of the administration of GnRH agonist starting in the early follicular phase of the ovulation induction cycle concomitantly with exogenous gonadotrophins and until hCG administration.13 The rationale of this regimen is to combine exogenous gonadotrophins with the rise of endogenous gonadotrophins obtained with early follicular phase GnRH agonist administration. This type of administration may provide some economic advantages in terms of reduced GnRH agonist and gonadotropin dose; however, the poorer clinical outcome of patients treated in this manner has led to a progressive decline in the use of this regimen. Preparatory Protocol This protocol consists of GnRH agonist administration limited to the mid- and late luteal phase of the cycle preceding gonadotropin-induced ovulation.14 It was recently proposed that pituitary suppression may outlast GnRH agonist administration and still prevent a spontaneous pre-ovulatory LH surge during gonadotropin administration. Experience with this regimen is still very limited and additional studies will be required to confirm that endogenous LH suppression is achieved throughout the follicular phase.

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Ultrashort Regimen This consists of an early follicular phase administration of GnRH agonist started concomitantly with exogenous gonadotrophins and discontinued after only 3 days. This regimen was initially introduced for the treatment of poor responders.15 As with the preparatory regimen this scheme is based on the assumption that suppression of the midcycle LH surge can be obtained through a very short course of GnRH agonist. Reasonably good results have been reported with this regimen16 but its use is still limited. Long Protocol Using a Monthly Depot Preparation This constitutes administering a long acting GnRH depot preparation either in the follicular phase or midluteal phase in the previous month. The down regulation effect lingers on for 28–60 days after an intramuscular or subcutaneous injection. Some studies have previously demonstrated the efficacy of one dose of subcutaneous goserelin17–20 for pituitary suppression, before ovarian hyperstimulation in women undergoing IVF. Tapanainen et al17 evaluated 49 patients that underwent IVF, using goserelin for pituitary suppression, starting at the luteal phase for a long protocol treatment, that were compared to 51 women using buserelin. The only observed difference was a higher number of ampoules needed for follicular maturation in the goserelin group. The results observed by Oyesanya et al18 however have demonstrated that patients that used goserelin needed less time to reach downregulation. Porcu et al21 did not find any difference in the time to reach desensitization when comparing depot (leuprorelin) and daily (buserelin) GnRHa. Some authors22 have described higher follicular recruitment and oocyte retrieval when GnRHa was commenced in the early follicular phase. A recent study comparing daily leuprolide acetate versus depot goserelin demonstrates that both routes of GnRHa have similar effects for pituitary suppression and ovulation induction in assisted reproductive treatment.23 The authors concluded that the long-acting GnRHa is an excellent option, as only a single subcutaneous dose is necessary, decreasing the risk of the patient to forget its use and therefore interference with the down regulation and, most important, it does not interfere in the patient’s quality of life.23 Results with Various Protocols The mechanisms of therapeutic action of GnRH agonists when combined with exogenous gonadotropins are complex, controversial, and still not completely understood. In addition to practical considerations regarding the greater ease of scheduling GnRH agonist-suppressed patients for ovulation induction and assisted reproduction procedures, the most relevant endocrine effect of GnRH agonists is the abolishment of the endogenous preovulatory LH surge.24 In the pre-agonist era, the untimely LH surge was associated with premature ovulation and/ or follicular luteinization and, thus, treatment cancellation occurred in a large number of cycles (>30%). An additional advantage of the combined use of GnRH agonists and exogenous gonadotrophins is the greater follicle yield, according to some commentators, than can be achieved using gonadotropin only protocols.11,12 One possible explanation of this effect is a reduction of intraovarian androgens. GnRH agonists profoundly suppress LH concentrations and consequent gonadal steroidogenic activity. Intraovarian androgens

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enhance follicular atresia.25 Monkey ovaries in flare-up GnRH agonist cycles (a regimen thatis characterized by elevated serum androgen concentrations).11 show greater follicular atresia and fewer dissociated granulosa layer follicles.26 Conversely, long GnRHa protocols, both when associated with pulsatile GnRH27 and with gonadotrophins11,28 show lower follicular phase serum androgen concentrations. Thus, lower intraovarian follicular phase concentrations could reduce follicular atresia and promote the development of a greater number of ovarian follicles. On the other hand, careful monitoring of ultra-short treatments before HMG is administered, can avoid luteinizing-degenerating corpora lutea inluteal cysts. Arandomized double-blind trial with the addition of growth hormone to GnRH agonist protocols showed no advantage to most patients in terms of follicle numbers and pregnancies.29 In addition to lowering cycle cancellation rates30,31 and increasing follicle number in long protocols,11,31 GnRH agonists may be effective in improving the number and viability of embryos,31,32 and pregnancy rates.10,32,33,34 However, the issue of whether GnRH agonists actually improve assisted reproduction success rates is still controversial.30,34,35 Some patients with inadequate response to ovulation induction may additionally profit from GnRH agonist supplementation.36,37 However, response to gonadotrophins in polycystic ovarian syndrome (PCOS) does not seem to be improved,30 and pajdents with premature ovarian failure remain unresponsive.38 Luteal phase deficiencies also arise from these forms of ovarian stimulation.39 As previously indicated, long GnRH agonist protocols appear to be superior to flareup protocols12,40,41 and are more widely used in assisted reproduction centres. However, good clinical results have also been reported with the flare-up regimen42,43,44 and it should be remembered that long regimens generally require a more prolonged stimulation and the administration of greater dosages of exogenous gonadotrophins. Long regimens, thus, tend to be more expensive, as more GnRH agonist and gonadotropin are administered. Ultrashort protocols have also been found to be effective,16,45 but the endogenous LH surge may not always be preventable with these regimens.15,44 Hsieh et al recently compared pituitary down-regulation with half-dose LA depot (1.88 mg sc, group 1) versus LA (0.5 mg/d sc, group 2) which was started on menstrual days.21–23,46 A total of 289 patients in group 1 and 158 in group 2 were included. There were no statistically significant differences between the two groups in baseline concentrations of E(2) and FSH, concentrations of E (2), FSH, and LH during hCG administration, gonadotropin dosage, the number of oocytes retrieved, the number of oocytes fertilized and embryos transferred, and pregnancy rates. The authors concluded that single half-dose LA depot offers a useful alternative for pituitary suppression in ovarian stimulation for IVF.46 A recent Thai trial47 studied the effects of depot leuprorelin on the IVF cycle and was done on nine couples. A single intramuscular injection of depot leuprorelin was given to the woman a couple days before ovulation. Seven days after ovulation, the serum progesterone level was measured and showed the same normal level as the natural ovulatory cycle. The progesterone levels varied from 12.59 to 96.0 ng/ml. On day three of the menstruation, the hormonal profiles showed a complete pituitary and ovarian suppression. FSH, LH and estrogen levels were less than 4.1 mIU/ml, 2.8 mIU/ml and 9.4 pg/ml respectively. The hMG stimulation took 11 days on average (9–15 days). A hundred and two oocytes were retrieved and among these there were 86 mature oocytes

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(84.3%). All oocytes were inseminated despite prematurity and resulted in 82.35 per cent fertilization. Normal fertilization occurred in 77.45 per cent (79/102). Good embryos developed in 58.23 per cent (46/79). No more than three embryos were transferred. Four women conceived, among them there was a set of twins. The implantation rate was 44.44 per cent (4/9). One abortion was found in the early first trimester. The take home baby rate was 33.33 per cent (3/9).47 Dada et al also conclude that with nafarelin and leuprorelin, stimulation with gonadotrophins may begin after 2 weeks of suppression and that long-acting GnRHa are as effective as short-acting analogues with no detrimental effects on the luteal phase.48 Moreover, some studies on the use of GnRH agonists point to the high pregnancy rates attained with clomiphene citrate/hMG stimulation which are equivalent to those gained with GnRH agonists.49 Indeed, a call has been made for a resolution of the protocols used for ovarian stimulation, with a stress on the advantages of milder forms or even natural cycles.50 Finally GnRH agonists have been used to replace hCG to provide an endogenous preovulatory LH surge during ovulation induction.51 Controlled trials have revealed that these treatments are just as effective.52 This approach may reduce the occurrence of ovarian hyperstimulation. However, additional luteal phase support with hCG or gonadal steroids is required in these regimens53 and this approach is not compatible with GnRHa administration in the follicular phase. The combined use of GnRH agonists and gonadotrophins does not appear to be associated with a substantial difference in complications when compared with more traditional ovulation induction regimens. As more follicles tend to mature, at least in the long regimens, a greater risk of ovarian hyperstimulation may exist.54 The recent introduction of GnRH antagonists has led to renewed interest in the use of GnRH agonists to induce final oocyte maturation. In a randomized multicenter study, the efficacies of two different GnRH agonists were compared with that of hCG for triggering final stages of oocyte maturation after ovarian hyperstimulation for in vitro fertilization.55 Ovarian stimulation was conducted by recombinant FSH (Puregon), and the GnRH antagonist Ganirelix (Orgalutran) was coadministered for the prevention of a premature LH rise. Luteal support was provided by daily progestin administration. Frequent blood sampling was performed at midcycle in the first 47 eligible subjects included in the current study, who were randomized for a single dose of 0.2 mg triptorelin (n=17), 0.5 mg leuprorelin (n=15), or 10,000 IU hCG (n=15). Serum concentrations of LH, FSH, E2, and progesterone (P) were assessed at variable intervals. LH peaked at 4 h after both triptorelin and leuprorelin administration, with median LH levels of 130 and 107 IU/liter (P<0.001), respectively. LH levels returned to baseline after 24 h. Subjects receiving hCG showed peak levels of 240 IU/liter hCG 24 h after administration. Arise in FSHto 19 IU/liter (P< 0.001) was noted in both GnRH agonist groups, 8 h after injection. Within 24 h, the areas under the curve for LH and FSH were significantly higher (P<0.001) in both GnRH agonist groups compared with that for hCG. E2 and P levels were similar for all groups up to the day of oocyte retrieval. Luteal phase areas under the curve for P and E2 were significantly elevated (P<0.001) in the hCG group. The mean (SD) numbers of oocytes retrieved were 9.8 5.4, 8.7 4.5, and 8.3 3.3; the percentages of metaphase II oocytes were 72 percent, 85 percent, and 86 percent; and fertilization rates were 61 percent, 62 percent, and 56 percent in the triptorelin, leuprorelin, and hCG group, respectively (P=NS for all three

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comparisons). These findings support the effective induction of final oocyte maturation in both GnRH agonist groups. The authors concluded that, after treatment with the GnRH antagonist ganirelix for the prevention of premature LH surges, triggering of final stages of oocyte maturation can be induced effectively by a single bolus injection of GnRH agonist, as demonstrated by the induced endogenous LH and FSH surge and the quality and fertilization rate of recovered oocytes.55 Moreover, corpus luteum formation is induced by GnRH agonists with luteal phase steroid levels closer to the physiological range compared with hCG.55 This more physiological approach for inducing oocyte maturation may represent a successful and safer alternative for in υitro fertilization patients undergoing ovarian hyperstimulation. Chromosomal anomalies are not increased56 and cryopreserved embryos are not different in GnRH agonistexposed patients.57 Abortion rates appear to be lower33 or unaffected29 by GnRH agonists. A lower rate of early miscarriage appears to be particularly evident in endometriosis patients undergoing IVF af ter 6 months of GnRH agonist treatment.58 Althoughit was suggested that exposure to GnRH agonist in monkeys during early pregnancy may be associated with abortion and infant abnormalities,59 accidental GnRH agonist administration in human pregnancy is usually uneventful. Depot GnRH agonist use that provides measurable drug concentrations in early pregnancy is not associated with an increased abortion rate.60 Why do We Need Alternative Approaches to Ovarian Stimulation Protocols? Most women undergoing IVF are normo-ovulatory but are exposed to pituitary desensitization (down-regulation) and exogenous gonadotrophins for follicular stimulation to enhance the chances of a successful outcome following their IVF treatment. Women who had regular cycles might become poor responders to exogenous follicular stimulation; and some women with unexplained secondary infertility might produce poor quality eggs and embryos. We are still in the search for that perf ect ovarian strtnulation protocol combining both GnRH analogs and gonadotropins that will give us an “adequate” number of oocytes; these oocytes should be of good quality resulting in embryos with a very good morphological score with a high implantation potential. The resulting pregnancies should carry to term as a result of the optimal uterine and endocrinological environment that has resulted as a consequence of using that “perfect” stimulation protocol. In the quest for that perfect stimulation protocol, we must imbibe knowledge of the use of GnRH analogs and gonadotropins in different endocrine milieus such as “poor responders” and from natural and artificially stimulated hypogonadotropic hypogonadic states. Until the introduction of recombinant technology, first using recFSH and, more recently recLH, urinary products containing a variety of other substances were used. The regulation of folliculogenesis and ovulation in the natural cycle is controlled by an exquisite orchestration of hormonal events, involving various feedback mechanisms within endocrine and paracrine systems, and diurnal variation regulated via the hypothalamic-hypophyseal-ovarian axis. The use of exogenous, even partially purified, urinary products provides little opportunity to study the effects of a particular

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gonadotropin in isolation. The combined use of down-regulation by gonadotropinreleasing hormone (GnRH) analogues and exogenous recombinant gonadotrophins has provided an opportunity to investigate the requirement for FSH and LH, alone or combined, during follicular stimulation. The biochemistry, pharmacokinetics and pharmacodynamics of recombinant gonadotrophins have been thoroughly discussed elsewhere.61,62,63,64 An increasing number of studies demonstrate that both FSH and LH from mid-phase folliculogenesis are essential for optimal physiological function; furthermore, especially in patients who are profoundly down-regulated, LH appears to be essential;65–73 although this has been disputed.66 Previously, the use of recLH had been examined in conjunction with recFSH for follicular stimulation after down-regulation in patients requiring excessive recFSH (>2500 IU).74,75 In these patients, the addition of recLH appeared to improve embryo quality, implantation and delivery rates. A possible increase in the implantation rate was observed in all patients given recLH, but there was a significant effect of the use of recLH on implantation in patients with peripheral LH concentrations <1.0 IU/l at down-regulation.76 However, it has been postulated that GnRH agonist down-regulation might result in profound suppression of peripheral LH, impairing adequate estradiol synthesis70,77,78 resulting in decreased fertilization rates and increasing the risk of early pregnancy loss.73,77 This has been disputed in a recent study by.66 These authors used receiver-operating characteristics (ROC) to examine serum LH on day 7 of stimulation as a prognosticator of IVF outcome af ter downregulation and stimulation with rFSH. Balasch et al66 found no difference in the median and range of LH values for conception and non-conception cycles, pregnancy and miscarriage; they concluded that there was no requirement for exogenous LH supplementation. Part of the difference between the conclusions of these authors and those of previous studies can be attributed to the type of GnRH agonist and its degree of hydrophobicity, which correlates to the degree of pituitary desensitization.79,80 Balasch et al66 used leuprolide acetate compared with buserelin, as used by Fleming et al81 and Westergaard et al.73 Balasch et al66 also suggest that their ROC curve analysis approach reduces inaccuracies in overall quantification of outcome data that might be affected by the prevalence of a particular condition. It is debatable whether this approach is of more value, but it should be considered in future prospective analyses. Clinical situations arise in which LH is naturally inactive or absent, such as Kallmann’s syndrome. In these patients, treatment with purified recFSH results in multiple follicular development but deficient ovarian steroidogenesis; however, there is disagreement regarding whether the presence or absence of LH induces fewer preovulatory follicles,82 or makes no difference.83 It is clear that there is a range of heterogeneity in the case of hypogonadotropic hypogonadism syndromes, hence the various published discrepancies when measuring serum and follicle estradiol concentrations, inhibin concentrations, endometrial thickness, reoccurrence of ovulation, rate of fertilization and embryo cryo-survival rates 65,82,83,84 However, in previous and more recent studies in a specific group of patients who previously required excessive recFSH to achieve follicular stimulation,74,75 the improved outcome results indicated a subset of patients that might benefit from LH inclusion. This was confirmed in a recent study in three patients requiring >2500 IU FSH to complete stimulation, in whom five out of seven embryos implanted if recLH was administered,

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compared with those in group B, who required >2500 IU FSH.76 The authors concluded that if the patient is profoundly down-regulated in terms of LH concentrations <1.0 IU/ 1 IU, or if excessive amounts of FSH (>2500 IU) have been required to complete stimulation, supplementation with LH appears to be beneficial. Therefore, it is important to continue investigations to establish whether there are groups of patients who, for hitherto unknown reasons, would benefit from either supplementation of LH during follicular stimulation or from decreasing the intensity of downregulation in the stimulation protocols. A good outcome for assisted reproductive techniques (ART) depends on many factors and a sufficient number of oocytes retrieved is one of the most important factors in performing IVF-ET or TET. Women whose ovarian reserve is too poor to retrieve enough mature oocytes have to suffer more, pay more, and have less hope of having their own children. Poor ovarian response to gonadotropins usually results in poor results in ART. This condition becomes more common as the women grow older. These patients often have a diminished ovarian reserve as demonstrated by an elevated FSH and estradiol levels as well as diminished inhibin-B production on menstrual cycle day 3. FSH elevations in the early follicular phase signal declining ovarian reserve; this occurs even when the patient continues to have regular menses.85 In previous studies, researchers have shown that patients with abnormal day 3 testing have a poor prognosis when undergoing IVF.86 Although intercycle fluctuation in basal gonadotropin levels may be seen, once a woman is found to have an elevated basal FSH, the ovarian response is diminished even in subsequent treatment cycles in which her basal FSH is normal.87 Pellicer et al88 have shown that granulosa-luteal cells obtained from older women at follicular aspiration for IVF have significantly reduced ability to secrete immunoreactive inhibin in vitro. This relative lack of inhibin production permits the rise of FSH levels despite normal estradiol production. Recently, it was reported that women with low serum day 3 Inhibin-B levels demonstrate a poorer response to ovulation induction and are less likely to conceive a clinical pregnancy through ART relative to women with high day 3 inhibin-B.89 Often these patients are referred to oocyte donation programs. However, not all patients wish to abandon attempts of conception with their own eggs and therefore more “aggressive” protocols havebeen devised. Several approaches have been advocated in treating the low responder. These have included increasing the dosage of gonadotropins, decreasing the luteal leuprolide dosage, clomiphene citrate based protocols and flare-up protocols. Winslow et al further described a GnRH-a Stimulation Test (GAST).90 In this test, the day 3 estradiol response following the subcutaneous administration of 1 mg of leuprolide acetate on day 2 was ascertained. The estradiol increase was an indicator of ovarian response to stimulation in flare-up cycles. There were significant correlations between estradiol and peak estradiol levels, number of mature oocytes retrieved, and pregnancy rates. When there was no estradiol rise after the leuprolide administration, the response was significantly lower and the pregnancy rate was zero.90 To overcome the problem of poor ovarian response some modifications of the COH protocol were developed. Ibrahim et al91 reported that growth hormone may augment the effect of gonadotropins in poor responders when combined with the gonadotropins. However, Dor et al found that administration of growth hormone did not improve the ovarian response in a randomized placebo-controlled study92 Another way to increase the ovarian response is to increase the dosage of gonadotropins. In some cases there may be a

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correlationbetween the dosage of gonadotropins and the response to ovarian stimulation. However, it does not definitely increase the numbers of follicles and the pregnancy outcome. Land and colleagues reported that in a group of poor responders, high-dose HMG injections increased the numbers of follicles and retrieved oocytes. But there was no significant increase in the numbers of embryos. The pregnancy outcome was still low.93 Combining clomiphene citrate and gonadotropins was once considered an alternative in COH for poor responders, not only because of the possible effect on ovarian stimulation but also because of the relative economic benefit. However, the outcome in terms of numbers of oocytes, synchronization of follicular growth. fertilization rates, and implantation rates was not better than COH with a combination of gonadotropins and GnRHa. But the combination of clomiphene citrate with gonadotropins is still considered an alternative in poor responders.94 COH with long GnRHa protocols with daily GnRHa administration from the midluteal phase of the previous menstrual cycle through the whole course of ovarian stimulation can be roughly divided into two major aspects: down-regulation on pituitary function and ovarian hyperstimulation by exogenous gonadotropins. Pituitary down-regulation is achieved by daily doses of GnRHa and its purpose is to prevent the effects of endogenous gonadotropins released by the pituitary gland, and thus to make each follicle grow “homogeneously” that is, to achieve synchronization of follicular growth. However, it is now recognized that long-term use of GnRHa will also decrease the ovarian response to gonadotropins. In patients with poor ovarian reserve, the negative effects of long term GnRHa may cause a poor outcome with COH. This negative effect of GnRHa also increases the doses of exogenous gonadotropins needed to achieve an adequate ovarian response and therefore increases the cost of COH. Restriction of the dose of GnRHa has been considered since the mid-1990s. Feldberg discovered that minidose GnRHa will increase the E2 level, and increase the number of oocytes retrieved, and also the pregnancy and implantation rates.95 The flare-up effect of GnRH agonist has been established in the COH cycle since the late 1980s. It is suggested that short-term GnRHa administration in the follicular phase of menstruation stimulates the ovary by releasing gonadotropin from the pituitary gland. Many studies suggest the use of short GnRHa flare-up protocols instead of long GnRHa protocols in poor responders.96–98 Surrey concluded that the improved effect of flare-up protocols may be due to enhanced release of early follicular phase endogenous FSH without concomitant deleterious rises in androgen levels or corpus luteum rescue.99 The flare-up protocol also minimizes the doses of GnRHa toprevent the suppression of ovarian response to gonadotropins. Many centers use the flare-up protocols as the standard protocols in poor responders. Essentially about 20–40 mcg leuprolide acetate was given twice a day starting on Menstruation Days 1, 2 and 3 to promote the flareup effect and also to minimize the ovarian suppression. The results were encouraging although the prob-lem of premature LH surge, asynchronous growth of follicles, and deterioration of oocyte function are still not resolved.99–101 Schachter and colleagues assessed the efficacy of a protocol involving the discontinuation of the GnRH analogue at the mid-phase of ovarian stimulation for IVF in patients with a previous poor response.102 The study design was a prospective casecontrol evaluation compared with same patient’s previous performance. Thirty-six patients enrolled in an IVF program were treated in two consecutive cycles. The first with

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a standardized protocol utilizing midluteal administration of Nafarelin (N) 600 mcg/d continued throughout the stimulation phase with human menopausal gonadotropin (hMG) until follicles of 20 mm were identified by transvaginal ultrasound (Standard group). Patients with a poor response in the Standard cycle were treated in the subsequent cycle with N and hMG initially in a similar manner, then N was stopped after 5 days of hMG stimulation (N-stop group). All clinical and laboratory aspects of treatment were done in a similar fashion in both cycles, each patient acting as her own control. Peak estradiol (E2) and number of aspirated oocytes were increased in the N-stop cycle (+16.9% and +28%, respectively), but insignificantly so. The percent of cleaving embryos was significantly increased by 27.9 percent (p=0.03) in the N-stop cycle, as embryo morphology was improved by 22 percent (p=0.02). The efficacy of gonadotropin treatment was enhanced in the N-stop cycle, as expressed by a 32.5 percent increase in oocytes retrieved per hMG ampoule administered (p=0.04). Three cycles of 36 were cancelled during the N-stop cycle, whereas only one was cancelled in the standard protocol cycle. Of the 36 patients, 7 conceived in the N-stop protocol and 5 are ongoing pregnancies. The authors concluded that discontinuation of GnRH-a during ovarian stimulation for IVF has a benef icial effect on both E2 and oocyte production.102 Embryo cleavage rates and morphology were significantly improved; this may be due to improved oocyte quality, which may have been responsible for achieving pregnancies.102 The efficacy of gonadotropin treatment was enhanced when GnRH-a was discontinued. These results hint that GnRH-a may have a direct negative effect on folliculogenesis and oocytes, which is apparent especially in poor responder patients. However, other reports indicate that GnRH-a may be responsible for some direct adverse effects on ovarian function, especially steroidogenesis.103,104 Oocyte quality was also found to be dependent on GnRH-a concentration in vitro so that high levels of GnRH-a inhibited fertilization and lower levels actually enhanced fertilization.105–106 It might be concluded, therefore, that ovarian responsiveness to gonadotropin stimulation could actually improve if the GnRH-a effect was rescinded during stimulation. The recovery of pituitary sensitivity after discontinuation of GnRH-a is variable, depending on patients age, dose, type of GnRHa and mode of administration, and the degree of ovarian stimulation, especially estradiol levels. Although some patients were able to release LH after stopping GnRH-a in as little as 3 days, and primate data demonstrate that the ref ractory phase does not last more than 6 days,107 the average pituitary recovery time after cessation of nasally administered GnRH-a was f ound to be 7–10 days108 and 28–30 days after a depot preparation.109 Discontinuing GnRH-a in midstimulation phase would allow this additional time for enhanced stimulation while the pituitary was still refractory. GnRH agonist administration is virtually universal in modern IVF stimulation protocols, enabling ovarian stimulation without interference by endogenous pituitary gonadotropin secretion. The addition of these medications has greatly increased ART success rates and as such are associated with extremely low cancellation rates and practically no side effects. Notwithstanding these facts, evidence has accumulated in recent years that GnRH agonists have extrapituitary effects, especially at various sites in the genital tract, in a number of mammalian species.109,110 GnRH receptors, GnRH receptor mRNA, and receptor binding have been found in endometrial, myometrial, endosalpingeal,111 and placental cells.112 GnRH has been found to have receptors in

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ovarian granulosa-both follicular phase and luteal cells113,114 and theca cells, and in fact have been found in oocytes.115 GnRH production in endometrial cells may have an important role in implantation and in the endometrialembryonic “cross-talk”.111,116 The physiology of such receptors is still enigmatic although, as the systemic levels of GnRH are very low, it is more than likely that GnRH (or GnRH-like peptides) produced locally have a direct or indirect paracrine/autocrine role in regulation of ovarian function.117–20 In vitro studies in mice105 demonstrated that fertilization rates in the presence of low concentrations of GnRH-a were enhanced, demonstratinga direct effect of GnRH-a on oocytes in an in vitro culture system. The direct effect of GnRH and its agonists on ovarian steroidogenesis is especially important in the context of ART for poor-responder patients. Some studies failed to show a clear-cut effect of GnRH-a on steroid production in vitro121,122 whereas others reported a stimulatory effect at low concentrations of GnRH.103 Gaetje104 found a dose dependent inhibition of FSH-induced granulosa cell estradiol production by Decapeptyl (Dtriptorelin) as opposed to control cultures. Parinaud and colleagues103 found that GnRH-a (Buserelin) added to luteal phase granulosacell cultures inhibited LH induced progesterone synthesis. These studies convincingly argue for an inhibitory, negative direct effect of GnRH-a on ovarian folliculogenesis or follicular function, in patients with normal (non-PCOS) ovaries. Numerous studies have investigated ways to overcome or bypass the direct GnRH-a effect on the ovary Some authors have tried to reduce GnRH-a doses or to stop its administration during exogenous gonadotropin stimulation to improve ovarian responsiveness, although the results have been contradictory. Higher estradiol levels were achieved with lower doses of gonadotropins, increasing cost-effectiveness, but PR’s were unchanged.123,124 Several groups have tried discontinuation protocols with varying success. Hazout108 utilized a 7-day triptorelin protocol starting on Cycle day 2. When compared with the classic triptorelin 3.75 mg depot form, the discontinuation protocol yielded more embryos per cycle despite markedly decreased hMG requirements, and the cancellation rate was quite low at 2.3 percent. Pantos and colleagues125 described their experience with discontinuing subcutaneous Buserelin 0.5 mg/d after 10 days starting in the mid-luteal phase. There were no differences in estradiol levels, hMG dose, or oocytes recovered, although the PR in the discontinuationgroup was almost twice that of the standard protocol (35.2% vs. 19.4%). There were no cancellations in the discontinuation group, despite up to 12 days without GnRH-a. Sungurtekin and Jansen126 similarly showed that LH levels were suppressed for 11 days after discontinuation of leuprolide acetate. Fujii et al127 also studied two groups of patients in a discontinuous GnRH-a protocol, initiating down-regulation in the mid-luteal phase and discontinuing the analogue at Cycle day 7. Interestingly, these authors f ound that in the discontinuous protocol more hMG was needed, but less oocytes were f ertilized and the cancellation rate was very high (35%). These patients were selected at random from the general IVF population and were not identified as being “poor responders.” Faber et al128 stopped administration of leuprolide at the onset of menses, in conjunction with high dose gonadotropin therapy. Pregnancy rates were improved in this protocol in a group of 182 low responders. Only 1.2 percent of cycles were cancelled (1/80) because of premature elevation of LH.

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Another study129 examined the positive relationship between delay of hMG initiation after depot form GnRH-a and ovarian response and pregnancy rates, this seems to indicate that initiation of gonadotropin stimulation in the presence of reduced concentrations of GnRH-a improves ovarian response and implantation rates. A report from China described a total of 100 women undergoing ovarian stimulation with gonadotropin-releasing hormone agonist (GnRHa) and a human menopausal gonadotropin (hMG) for in-vitro fertilization (IVF) participating in this randomized comparative study.9 Leuprolide acetate at a dose of 0.5 mg/day s.c. (n =52, group I), or low-dose leuprolide acetate depot at a dose of 1.88 mg s.c. (n=48, group II), was started on days 21–23 of the cycle. Stimulation with 225 IU/day hMG was started after pituitary desensitization had been achieved. The luteal phase was supported by human chorionic gonadotropin (hCG) i.m. injection. There were 11 pregnancies (21.2%) after the use of leuprolide acetate and 12 pregnancies (25.0%) in those given leuprolide acetate depot; no statistical difference existed between these two groups. Their conclusion was that a s.c. low-dose leuprolide acetate depot injection may offer a useful alternative for pituitary suppression in ovarian stimulation for IVF.9 Porcu et al compared the effects of depot and daily forms of GnRH analogs in one hundred seventeen patients undergoing IVF who were randomized between two treatment groups.109 Pituitary desensitization was obtained in group 1 (60 patients) with a single IM injection of leuprorelin (3.75 mg), and in group 2 (57 patients) with buserelin (0.3 mg SC twice daily). In a subgroup of 10 patients (5 for the depot form and 5 for the daily form) several GnRH tests were performed to investigate pituitary desensitization. No differences were found in the time to reach desensitization. Resumption of pituitary activity occurred in 7 days with the daily form and in about 2 months with the depot form. No significant differences were found in the stimulation pattern, oocyte quality, percentage of fertilization. The pregnancy rate per. transfer was slightly, but not significantly, better in the depot group (29.4% vs 25.9%). Implantation rate (11.9% vs. 12.3%) and the percentage of miscarriages (26.6% vs 28.5%) were similar. They concluded that the Depot and daily forms of GnRH analogs are equally effective in superovulation induction for IVF but considering improved patient compliance and preference, depot forms are more advantageous.109 Recently, Dirnfeld and associates examined the effect of a GnRH-a midluteal “stop” protocol in a randomized study.130 One cycle of 40 in the study group was cancelled because of premature LH elevation. A trend towards more oocytes obtained in the study group was found only in those patients previously designated as normal-FSH poor responders; in the general IVF population, no significant differences were demonstrated between the “stop” and the conventional protocol. Conversely, Pinkas and coworkers demonstrated a greater yield of oocytes and subsequently more available embryos for transfer with their “stop” protocol, with no premature LH surges.131 Improved morphology, if all other factors are comparable, is most likely due to improved oocyte quality which might be attributable to the change in stimulation protocol. Improved oocyte quality and increased fertilization/cleavage rates may also be attributed to lower in vitro levels of GnRH-a in the follicular fluid in the study cycle as opposed to the standard cycle, in accordance with in vitro findings by Yang et al.105 It is surmised that discontinuation of the GnRH-a during gonadotropin stimulation rescinds an inhibitory effect of the GnRH-a on the ovary, enabling an improved ovarian response. This improvernent includes both a more efficacious gonadotropin effect on the

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ovary, allowing growth of more follicles, and a follicular environment that supports the development of better quality oocytes. It is possible that the discontinuation of GnRH-a also had a beneficial influence on implantation, as GnRH has been found to play a role in embryo-endometrial communication.132 There are a number of reasons why the long protocol of administration of GnRHa is superior to the short protocol and has therefore remained the gold standard. It has been well demonstrated that the initial agonistic action of the GnRH-a results in increases in LH concentrations to preovulatory surge levels. This may lead to rescue of the corpus luteum, luteinization of immature follicles as shown by a rise in the serum P levels,133 and an increase in thecal androgen levels that may reduce folliculogenesis.134 Exposure of the developing follicle to inappropriately high levels of LH may be particularly severe in patients in whom the return to baseline levels of LH takes longer than average, for example, in those who have polycystic ovarian disease or who form cysts as a result of agonist administration.135 It has also been shown that the degree of LH suppression is more variable when the short protocol is used.136 In fact, some studies have suggested that when the short protocol is used, 5 to 10 percent of cycles may be complicated by a premature surge of LH.137 In one recent study in which GnRH-a was commenced on day 2 and hMG on day 5 of the cycle,138 it was found that the best results were obtained in those cases in which there was a prompt elevation of the serum E2 concentrations followed by a fall by cycle day 4 to 6. In the 20 percent of cycles in which the serum E2 concentration showed a prompt and persistent rise through cycle day 5, implantation rates and PRs were significantly lower. Little information on the fluctuations of LH in the early follicular phase was given in that study, but the results suggest that once follicular growth occurs, exposure to the agonistic phase of GnRH-a is probably inimical. The results of that study138 also suggest that it is not so much that pituitary desensitization before administration of gonadotropins is unnecessary but rather that some patients achieve pituitary desensitization very rapidly so that by the time the active phase of follicular growth occurs, the levels of LH are already at basal levels. There is a considerable amount of data supporting the adverse fertility effects of exposure to high LH concentrations.134,139,140 It is associated with an increased incidence of infertility and miscarriage139 and failure to conceive despite ovulation.140,141 It has been suggested that to optimize results with short protocols, US examination of the ovaries and measurement of the serum P concentration should be performed before initiation of GnRH-a therapy so that if an ovarian cyst is visualized or the serum P concentration is elevated, gonadotropin administration can be delayed until ovarian inactivity is demonstrated.142 It has also been suggested that daily measurements of the serum E2 concentration should be performed so that if they do not fall promptly, the long protocol could be used in subsequent cycles.138 Both these approaches negate one of the major advantages of GnRH-a therapy in comparison with ovarian stimulation using CC and hMG, namely, the simplicity of use and the reduction of monitoring afforded; for example, cysts seen in long GnRH-a cycles can be safely ignored.143 Given the fact that the short protocol produces no significant financial savings, it would appear that the long protocol is to be preferred for use in IVF.

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Alternative Approaches to Ovarian Stimulation Protocols Long-term GnRHa suppresses the ovarian response but an inadequate GnRHa dose may result in insufficient pituitary down-regulation and may cause spontaneous LH surge. We were not therefore in favor of “stop” protocols.” The second lesson learnt was to decrease the total GnRHa dose. We did this in two phases- the first phase was a GnRHa microdose protocol that we used for a 115 consecutive IVF cases. The second phase was to use a depot preparation in one third the prescribed dose 15 days before onset of gonadotropin stimulation for a minimum of 100 consecutive cases (in progress). This meant we used Injection Lupride Depot in the dose of 1.25 mg (Lupride Depot 3.75 mg, Inca Division, Sun Pharma, India) in each stimulatory cycle for IVF-ET; all the other medications and monitoring protocols remaining the same. In the first phase, to reduce the total GnRHa dose we considered decreasing the daily GnRHa doses. We used 250 mcg leuprolide acetate (Lupride, Inca Division, Sun Pharma, India) instead of the conventional 1.0 mg daily subcutaneous injection to maintain pituitary down-regulation. In addition to the change in daily GnRHa dose the time course was also changed as compared to the above “stop” protocols. Leuprolide acetate was not stopped at menstruation onset to shorten the duration of GnRHa administration; instead this microdose was continued like in the conventional long protocol. The net effect was to decrease the total dose of GnRHa in the complete stimulatory cycle. The result, more mature oocytes per cycle, confirmed good ovarian response and good follicular synchronization. We conducted a study to assess the efficacy of the microdose (0.12 mL=250 mcg) short-acting leuprolide acetate, luteal phase long protocol in normal responding women undergoing/VF/ICSI (UnpubUshed data). From January 2001 to March 2002, all normal responding women who had undergone IVF/ICSI and had used urinary hMG/Po-FSH and this modified microdose leuprolide acetate long protocol (n=115) were included in this retrospective analysis. The indications for the Assisted Reproductive Techniques were Male Factor (n= 31), Unexplained Infertility (n=18), Tubal Factor (n= 36), Endometriosis (n=11), PCOS (n=5) and Combined Factor (Male and Female) (n=14). The patients ages ranged from 20–41 years. Pituitary down regulation was considered to have been achieved if E2 levels were below 50 pg/mL and a urinary gonadotropin preparation was started with a dose of 225 IU/day (3×75 IU ampoules) for 6 days. Ovarian response was assessed by ultrasound on day 7, and the gonadotropin dose adjusted as necessary. Human Chorionic Gonadotropin (hCG), 10,000 IU, was administered intramuscularly when the leading follicles were between 16–18 mm. Oocyte Retrieval occurred 34–36 hours after hCG administration. The mean age of the patients was 30.23+/−4.81 years and the ages ranged from 20–41 years. The mean number of days of stimulation were 10.36+/−1.96 and this was similar across all the indications for Assisted Reproductive Techniques. The mean number of gonadotropin ampoules used was 46.79+/−19.55 whereas the mean amount of drug in IU was 3576.08.+/−1460.5. The mean number of follicles produced was 13.26+/−6.63 while the mean number of eggs retrieved was 12.42+/−6.73. The mean fertilization rate was 64.43+/−22.71 and the mean embryo cleavage rate was 87.28+/−21.03. The mean number of embryos transferred in our study was 5.42+/−2.96. There were 29 clinical ongoing pregnancies with a clinical pregnancy rate (CPR) of 25.22 percent. Using the microdose Lupride luteal phase long protocol, gave us good fertilization rates, cleavage rates and an acceptable ongoing clinical

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pregnancy rate. Microdose Lupride luteal phase long protocol has the added patient advantage of lower cost since the pituitary suppression is not profound and thus lesser ampoules of gonadotropins are consumed. We have initiated the one third Lupride depot protocol from July 2002 onwards and the preliminary results seem comparable to the microdose protocol. OVULATIONTRIGGERING Introduction Ovulation has exclusively been triggered with Human Chorionic Gonadotropin (hCG) since the earlier times of follicular stimulation with Pregnant Mare Serum Gonadotropins (PMSG). hCG was chosen in regard of its LH-like effect, when isolated or purified human LH was not available. hCG, however, is not the physiologic hormone for ovulation triggering and shows many discrepancies in pharmacokinetics and bioavailability with LH, accounting for the permanent risk for ovarian hyperstimulation syndrome (OHSS) following hCG administration. Human recombinant LH should become freely available in the coming years, but it is at present possible to trigger ovulation in hMG—stimulated patients with their own pituitary LH, using a short-acting GnRH agonist; even in antagonist down regulated cycles.55 Literature shows that this method of triggering ovulation in in-vitro fertilization (IVF) cycles as well as in non IVF cycles results in a satisfactory ovulatory process and pregnancy rates comparable to those observed following hCG administration.144 More over, triggering ovulation with endogenous LH considerably reduces the risks for OHSS, and perhaps for multiple pregnancies. Optimum posology for each GnRH agonist available remains to be evaluated to minimize the occurrence of short luteal phases f ollowing ovulation triggering with endogenous LH.145 Ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies are the two main complications of ovulation induction using gonadotropins. Withholding an ovulatory dose of human chorionic gonadotropin (hCG) remains the safest option for prevention of both complications. However, this policy frustrates both patient and physician, wastes time and money due to cancelled treatment, and results in cancellation of a high proportion of cycles that would not have progressed to clinical OHSS. As gonadotropin releasing hormone analogs (GnRH-a) elicit surges of endogenous luteinizing hormone and follicle stimulating hormone, GnRH-a may be an acceptable substitute for hCG to salvage treatment cycles in patients thought to be at risk for OHSS or multiple pregnancy.146,147 In patients treated with gonadotropins, the luteinizing hormone (LH) surge is usually absent or attenuated; therefore, the admirdstration of human chorionic gonadotropin (hCG) is required to induce oocyte maturation and ovulation.148,149 Whereas acute exposure of hCG appropriately may replace the LH surge for the induction of these periovulatory events, it remains to be determined whether hCG exposure alters the normal patterns of the final stage of follicular development, oocyte maturation, and corpus and luteum function.150,151 Although similar in action to LH, hCG, because of its longer half-life (>24 hours versus 60 minutes)152,153 does not provide a physiologic stimulus that is identical to the

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endogenous LH surge.154–56Furthermore, by contrast with the spontaneous normal menstrual cycle, where both LH and follicle-stimulating hormone (FSH) are secreted at midcycle, administration of hCG results in an increase in LH activity only. Because of its longer half-life compared with that of LH, hCG administration to hMG-treated patients results in a sustained luteotropic effect, development of multiple corpora lutea, and supraphysio logic levels of estradiol (E2) and progesterone (P) throughout the luteal phase. In patients with excessive responses to gonadotropin stimulation, this sustained luteotropic stimulation may result in ovarian hyper-stimulation syndrome (OHSS), the most serious complication related to gonadotropin therapy.157Excessive levels of circulating E2 have been implicated in the relatively high rates of implantation failure and early embryonic loss in stimulated cycles.158,159 Until recently, hCG was the only effective therapy available for the induction of oocyte maturation and ovulation in stimulated cycles. The use of gonadotropin-releasing hormone (GnRH) to trigger a mid-cycle LH surge and ovulation is ineffective because it elicits a transient LH surge for only a few hours, which is physiologically insufficient to initiate meiotic maturation of the oocytes and to trigger ovulation. Previous attempts to trigger ovulation with repeated injections or infusion of GnRH in anovulatory patients after hMG treatment yielded variable results 160–164. Unlike hMG and GnRH-agonist, which is associated with luteal phase dysfunction, hMG and GnRH offers an alternative due to the ability of hCG luteal support and rescue, providing the E2 levels are not dangerously increased.165 The potent GnRH analogue (GnRHa) induces a sustained release of LH from the pituitary gland that may last for 24 hours. This initial “flare-up” effect is followed by pituitary desensitization to further GnRH stimulation.166–169 In 1988, Itskovitz et al reported preliminary results demonstrating the efficacy of one or two GnRHa injections to trigger a sustained preovulatory LH/FSH surge that effectively induced oocyte maturation in patients undergoing ovarian stimulation for the purpose of IVF/ET.170,171 They have also shown that injection of GnRHa instead of hCG provides, for the first time a means by which the development of OHSS in patients at high risk for having this syndrome reliably can be prevented.170,171 However, GnRH-a administration can induce short luteal phase. This defect may be ascribed to the pituitary desensitization rather than to a direct effect on corpus luteum. Luteal phase support is needed to prevent luteal phase deficiency.172 Allahbadia et al173examined the efficacy of SC Leuprolide Acetate in inducing ovulation and a normal luteal phase during unstimulated, Clomiphene Citrate (CC) stimulated and Gonadotropin Stimulated superovulation cycles for Intrauterine Insemination (IUI). All the patients recruited for this prospective study had undergone a routine infertility evaluation, which includes their baseline hormonal profiles, a hysterosalpingogram, diagnostic laparoscopy and a semen analysis of their partner. Thirty cycles of Intrauterine Insemination were included in this study Ten cycles were unstimulated cycles (Group A), 10 cycles were CC stimulated (100 mg/ day from days 5 to 9) (Group B) and Ten cycles were Gonadotropin Stimulated Cycles (5 cycles hMG stimulated +5 cycles PoFSH stimulated) (Group C). Administration of 2 mg SC Leuprolide Acetate (Injection Lupride, Inca Division, Sun Pharma, India) (1mg×2 injections, 12 hours apart) was used for induction of midcycle LH surge in all cases. Two IUIs were performed 28 and 40 hours after the SC GnRHa injection. Luteal phase support

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was given in the form of Injection hCG 10,000 IU, 3 and 6 days after follicular rupture in all three groups. Assessment of luteal phase duration was done to estimate the adequacy of the luteal phase. Serial ultrasounds were also done to determine the incidence of luteinized unruptured f ollicle syndrome. All thirty treatment cycles had adequate luteal phases as assessed by luteal phase duration. Ongoing pregnancies occurred in all three groups. They concluded that Leuprolide acetate given SC is as effective as hCG in inducing ovulation and achieving pregnancies in unstimulated and superovulated IUI cycles.173 The Spontaneous LH/FSH Surge Follicle-enclosed oocytes are arrested in the prophase of the first meiotic division until the midcycle LH/FSH surge. The surge initiates a cascade of events that results in germinal vesicle breakdown and re-initiation of meiosis, luteinization of the follicular wall, and eventually, ovulation. The duration of the normal midcycle LH surge is 48.7+/−9.3 hours.174 Its onset occurs abruptly. The normal LH surge can be divided into three phases, a rapidly ascending limb (14 hours), a peak plateau phase (14 hours), and a long descending phase (20 hours). The rate of increase and decrease in the LH concentration is greater than that of FSH. Serum E2 levels reach a peak at about the time of the onset of the LH surge and then decline rapidly. The circulating P level increases exponentially, beginning 12 hours after the onset of the LH surge. It then plateaus for approximately 24 hours preceding ovulation. After follicular rupture (36 hours after LH surge onset), a second rise in the P level and a continuous fall in the E2 concentration are observed, reflecting an acute shift in ovarian steroidogenesis in f avor of P and the beginning of the luteal phase. The temporal relationship between the LH surge and human oocyte maturation in vivo throughout the human preovulatory period has been studied by Seibel et al.175 If an oocyte was harvested more than 18 hours after the onset of the LH surge, resumption of meiosis had occurred. Twenty-eight to 38 hours after the onset of the LH surge, preovulatory oocytes in metaphase II were obtained. Others demonstrated that 14 hours of elevated LH did not elicit the normal periovulatory events in follicles of stimulated monkeys.176However, the majority of the oocytes retrieved 27 hours after hCG injection had reentered meiosis. These studies in humans and monkeys suggest that the threshold duration for the LH surge levels required to reinitiate meiosis appears to be 14–18 hours. To obtain metaphase II oocytes at the time of follicle aspiration, a LH surge of more than 28 hours appears to be required.175 The threshold amplitude of the midcycle LH surge required for human oocyte maturation and other periovulatory events is not known. Studies in rats suggest that only 5 percent of the normal LH surge amplitude is necessary for oocyte maturation, whereas 85 percent of the surge is required for ovulation, suggesting that the threshold of LH exposure varies for different periovulatory events.177 No comparable information is available for primates. An endogenous LH/FSH surge occurs infrequently or is attenuated in women treated with gonadotropins, despite persistently elevated levels of Estradiol.148 Therefore, the administration of hCG is needed to induce oocyte maturation and ovulation. It has been suggested that nonsteroidal factors, gonadotropin inhibin surgeinhibiting factor and

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inhibin, present in follicular fluid, are secreted from the ovary and block the surge mode of LH and FSH secretion induced by either a bolus of E2 or GnRH.178,179However, as discussed subsequently, worldwide data show that GnRHa injection can over come this block and elicit a LH/FSH surge in ovarian-stimulated patients that is comparable in magnitude to that of the normal menstrual cycle.180–185 The GnRHa-lnduced LH/FSH Surge Several regimens for the induction of a preovulatory LH/ FSH surge with GnRHa have been reported both for ovulation induction and IVF and were found to be effective in triggering oocyte maturation and ovulation. These include single or repeated injections of GnRHa (100–500 mcg) given either subcutaneously or intranasally172,186–193 The injection of GnRHa results in an acute release of LH and FSH. The serum LH and FSH levels rise over 4 and 12 hours, respectively, and are elevated for 24–36 hours. The amplitude of the surge is similar to that seen in the normal menstrual cycle, but by contrast with the natural cycle, the surge consists only of two phases: a short ascending limb (>4 hours) and a long descending limb (>20 hours)194. The Luteal Phase Patients given GnRHa to trigger endogenous LH surge have an apparently normal follicular-luteal shift in ovarian steroidogenesis but have lower circulating luteal E2 and P levels than do patients injected with an ovulatory dose of hCG25. Some of these patients have early luteolysis and a short luteal phase 174, 187. The longer duration of plasma hCG elevation compared with the briefer GnRHa-induced LH elevation may result in higher luteal phase E2 and P levels. After ovulation, the corpus luteum is dependent on Pituitary LH.195,196 It is also possible, therefore, that the prolonged down-regulation of pituitary GnRH receptors after a midcycle injection of high-dose GnRHa results in reduced LH support for the developing corpora lutea, reduced steroidogenesis, and early luteolysis. hCG luteal support is a useful tool to overcome the luteal phase inadequacy that characterizes GnRHa-triggered cycles after gonadotropin stimulation.197 In patients of PCOS, various workers have supplemented the luteal phase with progesterone and have confirmed luteal phase adequacy.198,199 Further research is needed to study the function of the corpus luteum throughout the luteal phase and early pregnancy and the requirements for luteal support in ovarian-stimulated patients in whom oocyte maturation and ovulation were induced by GnRHa. All current protocols use high-dose GnRHa (100–500 mcg). The minimal effective dose of GnRHa required to trigger an endogenous midcycle LH surge sufficient to induce oocyte maturation and ovulation, without signif icantly affecting the normal development and function of the corpus luteum, remains to be established. Benefits and Limitations The currently available data suggest that GnRHa is an effective alternative to hCG for use in IVF and IUI cycles or for the induction of ovulation in anovulatory women.200–202

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Pregnancy rates in cycles in which oocyte maturation was induced by GnRHa are similar to the rates observed in hCG cycles. The use of GnRHa instead of hCG for ovulation induction has several potential advantages. Whereas the role of the midcycle LH surge in oocyte maturation, luteinization of the granulosa theca cells, and follicle rupture is well established, it is not known whether the concurrent midcycle FSH surge plays any physiologic role in these periovulatory events in primates. The presence of a midcycle FSH surge is not obligatory because apparently normal oocyte maturation and ovulation do occur after administration of hCG. In rats, however, a role for FSH in the maturation of the oocyte-cumulus complex has been demonstrated.203 It is not known whether the use of GnRHa, because of its release of endogenous FSH, has any advantage over the use of hCG, which has no FSH-like activity. A potential advantage for the use of GnRHa instead of hCG for ovulation induction stems from the short (24–36 hours) duration of the LH surge induced by GnRHa, which provides a more physiologic ovulatory stimulus than the extended surge (approximately 6 days) associated with hCG. This time-limited stimulus can be restricted to the few follicles that are more mature, and thus a lower frequency of multiple pregnancies could be expected in patients undergoing ovarian stimulation for the purpose of ovulation induction. This GnRHa-induced LH surge is associated with lower luteal E2 and P than that seen after hCG injection. Luteal phase support and the desired concentrations of E2 and P could be managed more accurately, thus avoiding the excessive levels of circulating estrogens and, theoretically, improving the chance for implantation and increasing pregnancy rates in stimulated cycles.158,159 As discussed previously, GnRHa therapy has been found to be effective for preventing the development of OHSS in patients at high risk for having this syndrome.171 It should be noted that GnRHa would not be effective for triggering an adequate LH surge in women with a low gonadotropic LH reserve (e.g., hypothalamic hypogonadism) or in cycles where GnRHa downregulation was used to prevent a spontaneous LH surge or early luteinization whereas it can be used for cycles with GnRH antagonists. A GnRH-agonist can successfully induce an LH surge even after GnRH antagonist administration204. However, the effect of the antagonist on the quality of the GnRH-a-induced LH surge as well as the oocyte quality remain to be evaluated. Prevention of OHSS An important benefit emerging from the use of GnRHa, rather than hCG, for ovulation induction, is the ability of this therapeutic regimen to prevent OHSS, the most serious complication related to gonadotropin therapy The full-blown clinical syndrome is characterized by ovarian enlargement with multiple functioning luteal cysts, increased vascular permeability, third-space accumulation of fluid, hemoconcentration, and oliguria. Cases of renal failure, hypovolemic shock, thromboembolism, adult respiratory distress syndrome, and even death have been reported.157 The pathogenesis of OHSS is not known, but it clearly is related to the existence of multiple functioning corpora lutea and to the sustained luteotropic effects of endogenous or exogenous hCG. Until recently, there has been no means by which OHSS could be prevented because withholding hCG administration results in failure to ovulate and conceive. Follicle

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aspiration and elective cryopreservation of all embryos to minimize the risk of OHSS in IVF patients at high risk of having OHSS does not eliminate the syndrome.205 In 78 women with serum E2 levels greater than 3,500 pg/ ml (mean approximately 5,000 pg/ml on the day of hCG injection), after pituitary downregulation with buserelin and ovarian stimulation with hMG, all their embryos were cryopreserved, and buserelin was continued in the luteal phase. Twenty-one women (27%) had OHSS, and six had the severe form of the syndrome. In 1988, Itskovitz et al suggested that midcycle injection of high-dose GnRHa (buserelin, 500 mg×one dose or 500 mg×two doses, 8 hours apart) is effective, not only for the induction of oocyte maturation and ovulation, but also for the prevention of OHSS in ovarian-stimulated patients.170,171 More studies are required to determine the efficacy and safety of midcycle GnRHa administration in reducing the risk of OHSS in patients with exaggerated response to gonadotropin therapy The current data strongly suggest that the use of GnRHa in place of hCG permits, for the first time, ovarian stimulation without the risk of OHSS. Ovulation Triggering Using Choriogonadotropin Alfa In ART and ovulation induction cycles, exogenous hCG plays an important role because of its structural similarity to LH. In the normal ovulatory cycle, a surge of LH induces final maturation of the ovarian follicles and leads to ovulation. Because hCG and LH share the same binding sites, the use of hCG as a therapeutic substitute for LH, to mimic the preovulatory surge, has become almost universal in ART. As such, the dosing and timing of the hCG injection, along with excellent patient communication are critical steps toward a successful outcome. In 1987, Abdalla et al investigated three incremental doses of hCG in an attempt to identify the lowest effective dose associated with optimal ART outcomes.206 Although 2000 IU was associated with sub-optimal results, there were no significant differences between 5,000 and 10,000 IU in terms of egg recovery, fertilization and pregnancy outcome. Since then, the most common hCG dose used in the United States for ovulation triggering has been 10,000 IU. Scott et al207 found that oocytes retrieved from follicles >15 mm had a high probability for being mature. The optimal time interval between the hCG injection and oocyte retrieval has also been investigated. Mansour et al208 confirmed that the percent of metaphase II oocytes was highest when the time interval was not less than 36 hours. Recently, arecombinant version of hCG became commercially available in the USA. This product, Ovidrel or recombinant Choriogonadotropin Alfa, shares many of the advantages seen with other recombinant products: it is a well characterized protein with exceptionally high specific activity, purity and batch-to-batch consistency.209 A number of prospective, controlled, randomized, comparative trials have been undertaken with recombinant Choriogonadotropin Alfa in ART and OI.210–212 In the specified study population, two doses of r-hCG, 250 and 500 mcg, and 10,000 Units USP urinary hCG were evaluated. All three treatment groups demonstrated equivalent efficacy as measured by the number of oocytes retrieved: 13.6, 14.6 and 13.7 for 250 mcg, 500 mcg and 10,000 IU hCG, respectively. However, the 500 mcg dose was associated with a significantly higher rate of OHSS (9.0%) when compared to the 250 mcg dose (3.2%).211

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In clinical practice, with an unselected patient population, outcomes with recombinant Choriogonadotropin Alfa compared to urinary hCG have been comparable.210– 212 Schoolcraft et al213 reported their initial results with rechCG earlier this year. Compared to u-hCG, the number of metaphase II oocytes were 13.8±6.8 vs. 12.7±6.4 for r-hCG and u-hCG, respectively. Fertilization rates were also similar at 74.0 percent and 74.3 percent for r-hCG and u-hCG, respectively. Ongoing pregnancy rates of 55.9 percent and 56.3 percent for r-hCG and u-hCG were also reported.213 Doody compared their outcomes with 434 u-hCG cycles from March 2000 through December 2001.209 The mean number of oocytes retrieved with u-hCG and r-hCG were 12.98 and 13.78, respectively. In ICSI cycles, the mean number of metaphase II oocytes was 9.61 and 10.49 with u-hCG and r-hCG, respectively. Fertilization and pregnancy rates were also similar at 69.7 percent and 45.0 percent compared to 66.5 percent and 41.4 percent, respectively.209 Ovulation Triggering Using recLH It is likely that treatment with gonadotropins derived from human urine will soon be replaced for induction of ovulation and ovarian stimulation for in-vitro fertilization by the use of wholly biosynthetic preparations. Recently Laboratories Serono has developed a highly purified recLH (Luveris™) suitable for therapeutic use. The availability of a large amount of a highly pure recLH preparation with potential uses in humans allows for the first time precise assessment of the physiological role of LH in reproduction and development of its potential use as a pharmaceutical agent. Alarge enough dose by single injection could mimic the LH surge more closely than by use of chorionic gonadotropin (hCG), which lasts much longer.214 In natural menstrual cycles, final follicular maturation, ovulation, and corpus luteum formation are induced by the mid-cycle Luteinizing hormone (LH) surge. Infertility therapy includes stimulation of follicular development by administration of human follicle stimulating hormone (FSH) followed by an injection of urine-derived human chorionic gonadotropin (hCG) to mimic the endogenous LH surge. hCG is used because it shares biological properties with LH and is easier to obtain than LH, But hCG has longer pharmacodynamic activity than LH, which could be disadvantageous in patients at high risk of ovarian hyperstimulation syndrome (OHSS).215–216 Recombinant DNA technology has made it possible to develop a form of LH that is structurally and functionally very similar to the natural hormone. recLH in appropriate high dosage can mimic the preovulatory surge required to induce final follicular and oocyte maturation and a recent report showed that a single administration of recLH can induce adequate final follicular maturation, corpus luteum formation, and lead to a viable pregnancy without clinical and ultrasound signs of OHSS in a patient with a high response to FSH.217 The administration of GnRHa instead of hCG, to produce an endogenous LH surge, has been proposed to prevent OHSS in women at risk.173 The use of recombinant human LH may provide another physiological route to stimulate follicular rupture and final oocyte maturation, while at the same time helping to prevent OHSS in women at risk; this notion is based on its short half life; which is similar to that of human LH.218–219 A future potential use of recLH will be its replacement of hCG as the ovulatory trigger, as has been demonstrated in non-human primates220 and the recLH may mimic

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the physiological endogenous LH surge more closely than hCG.221 This may help prevent OHSS,215 possibly as a result of very different half lives of natural and recombinant LH (measured in hours);222 compared with hCG (measured in days). Also, in a study by Romeu et al,221 the implantation rate was significantly higher with recLH versus hCG possibly because of the better embryo quality produced by recLH. A recent clinical study214 tested 4 doses of recLH, 5,000 IU, 15,000 IU, 30,000 IU and 15,000 IU+10,000 IU. Two hundred and fifty-nine patients were enrolled, of whom 129 received recLH and 121 were administered 5,000 IU u-hCG. All 250 patients were included in the safety and efficacy analysis. The primary efficacy endpoint was to compare the number of oocytes recovered 34–38 hours after u-hCG/recLH administration. The results revealed that 5,000 IU, 15,000 IU, 30,000 IU and 15,000 IU+10,000 IU of recLH are equivalent to 5,000 IU u-hCG for inducing final follicular maturation, allowing oocyte recovery. In the safety assessment, there were no significant differences in the incidence or type of adverse events between the u-hCG and recLH treatments.214 Regarding the evaluation of OHSS, a, clear dose response relationship was observed, a single dose of 5,000 IU, 15,000 IU or 30,000 IU appeared safer than 15,000+10,000 IU of recFSH and 5,000 IU of u-hCG. In the 5,000 IU, 15,000 IU and 30,000 IU recLH groups the size of the ovaries, the proportion of patients developing ascites and the total renin levels were statistically significantly lower than in patients treated with 5,000 IU u-hCG. The safety profile of the 15,000+10,000 IU recLH group is clearly poorer, and closer to that of u-hCG. The study demonstrated the clinical efficacy of recLH in inducing final follicular maturation and early luteinization in IVF patients.214 These findings support the hypothesis that the use of recLH as shorter lasting and therefore more physiological surrogate surge would be beneficial in terms of reducing the risk of OHSS. A single dose between 15,000 and 30,000 IU provides the best efficacy/safety ratio.214 In another study223 where ovulatory patients were pretreated with GnRH agonist (n=250) and stimulated with recFSH, doses ranging between 5000 and 30 000IU recLH have been used to trigger final follicular maturation and luteinization prior to IVF and embryo transfer. All doses tested were shown to be as effective as 5000IU hCG to trigger cumulus and oocyte maturation and early luteinization. Mid-luteal phase ovarian volume increase, serum renin concentration and fluid accumulation in the abdominal cavity were positively correlated with the dose of recLH used to trigger final follicular maturation.223 Recent Advances The recent introduction of GnRh antagonist protocols 69,224,225 has offered new opportunities of using GnRH agonists to trigger ovulation and preventing OHSS due to the mechanism of action of GnRH antagonists, i.e. competitive inhibition and relatively short duration of action. Studies in monkeys226 have clearly demonstrated that although tonic gonadotropins remain suppressed under GnRH antagonist treatment, acute LH release can be elicited in a GnRH challenge test. In small scale studies in humans, it was demonstrated227 that under GnRH antagonist treatment the pituitary retains its responsiveness to GnRH, while others228 showed in stimulation cycles for IUI that ovulation can be triggered by GnRH agonist after GnRH antagonist treatment. From these preliminary reports, it can be deduced that the pituitary response to GnRH or GnRH

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agonist is preserved during stimulation protocols including GnRH antagonists. The extent of pituitary suppression in this type of protocol is GnRH antagonist dose dependent. Under the reported minimal effective dose of GnRH antagonists (0.25 mg, daily), ovulation can be safely and effectively triggered with a GnRH agonist. A new treatment option for patients for patients undergoing ovarian stimulation is the GnRH antagonist protocol, with the possibility of using 0.2 mg Triptorelin to trigger ovulation in patients at high risk for developing OHSS was recently reported.229 All patients had at least 20 follicles >/=11 mm and/or serum estradiol at least 3000 pg/mL. The rapid fall of estrogen concentrations after ET, the lack of midluteal estradiol-peak, as well as the absence of free pelvic fluid suggest that triggering of final oocyte maturation with GnRH agonist leads to a more physiological luteal phase estradiol and progesterone concentrations. So far, four clinical pregnancies have been achieved from the embryos obtained from these cycles, including the first birth following this approach. The introduction of GnRH agonist induced triggering of ovulation in GnRH antagonist protocols would offer additional benefits to all patients; i.e. both high and normal responders, although the efficacy and safety of such new treatment regimens needs to be established in comparative, randomized studies. REFERENCES 1. Filicori M, Flamigni C, Dellai P. Treatment of anovulation with pulsatile gonadotropin-releasing hormone: prognostic factors and clinical results in 600 cycles. J Clin Endocrinol Metab 1994; 79:1215–20. 2. Franks S, Gilling Smith C. Advances in induction of ovulation. Curr Opin Obstet Gynecol 1994; 6:136–40. 3. Hugues JN, Cedrin Dunerin I. Revisiting gonadotropin releasing hormone agonist protocols and management of poor ovarian responses to gonadotrophins. Hum Reprod Update 1998; 4:83– 101. 4. Filicori M, Cognigni GE, Arnone R. Role of different GnRH agonist regimens in pituitary suppression and the outcome of controlled ovarian hyperstimulation. Hum Reprod 1996; 11(Suppl 3):123–32. 5. Fleming R, Adam AH, Barlow DH. A new systematic treatment for infertile women with abnormal hormone profiles. Br J Obstet Gynaecol 1982; 89:80–83. 6. Fleming R, Haxton MJ, Hamilton MR Successful treatment of infertile women with oligomenorrhoea using a combination of an LHRH agonist and exogenous gonadotrophins. Br J Obstet Gynaecol 1985; 92:369–73. 7. Porter RN, Smith W, Craft IL. Induction of ovulation for in-vitro fertilisation using buserelin and gonadotrophins. (Letter). Lancet 1984; 2:1284–85. 8. Leyendecker G, Wildt L. Follicular phase gonadotropin releasing hormone agonist and human gonadotropins: a better alternative for ovulation induction in in vitro fertilization. J Reprod Fertil 1983; 69:397–99. 9. Tsai HD, Chen CM, Lo HY, Chang CC. Subcutaneous low dose leuprolide acetate depot versus leuprolide acetate for women undergoing ovarian stimulation for in-vitro fertilization. Hum Reprod 1995; 10:2909–12. 10. Hughes EG, Fedorkow DM, Daya S. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992; 58:888–96.

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11. Filicori M, Flamigni C, Cognigni GE. Different gonadotropin and leuprorelin ovulation induction regimens markedly affect follicular fluid hormone levels and folliculogenesis. Fertil Steril 1996; 65:387–93. 12. Tan SL, Kingsland C, Campbell S et al. The long protocol of administration of gonadotropinreleasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril 1992; 57:810–14. 13. Macnamee MC, Howles CM, Edwards RG, Taylor PJ, Elder KT. Short term luteinizing hormone releasing hormone agonist treatment: Prospective trial of a novel ovarian stimulation regimen for in vitro fertilization. Fertil Steril 1989; 52:264–69. 14. Pantos K, Meimeth Damianaki T, Vaxevanoglou T, Kapetanakis E. Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilization program. Fertil Steril 1994; 61:709–13. 15. Howles CM, MacNamee MC, Edwards RG. Flare Protocol. Lancet 1986; 2:521–22. 16. Ron El R, Herman A, Golan A et al. Ultrashort gonadotropin-releasing hormone agonist (GnRH-a) protocol in comparison with the long-acting GnRH-a protocol and menotropin alone. Fertil Steril 1992; 58:1164–68. 17. Tapanainen J, Hovatta O, Juntunen K, Martikainen H, Ratsula K, Tulppala M et al. Subcutaneous goserelin versus intranasal buserelin for pituitary down-regulation in patients undergoing IVF: Arandomized comparative study. Hum Reprod 1993; 8:2052–55. 18. Oyesanya OA, Teo SK, Quah E, Abdurazak N, Lee FY, Cheng WC. Pituitary down-regulation prior to in-vitro fertilization and embryo transfer: Acomparisonbetween a single dose of Zoladex depot and multiple daily doses of suprefact. Hum Reprod 1995; 10:1042–44. 19. Filicori M, Flamigni C, Cognigni G, Dellai P, Arnone R, Falbo A et al. Comparison of the suppressive capacity of different depot gonadotropin-releasing hormone analogs in women. J Clin Endocrinol Metab 1993:77:130–33. 20. Dhont M, Onghena A, Coetsier T, De Sutter P. Prospective randomized study of clomiphene citrate and gonadotrophins versus goserelin and gonadotrophins for follicular stimulation in assisted reproduction. Hum Reprod 1995; 10:791–96. 21. Porcu E, Filicori M, Dal PL, Fabbri R, Seracchioli R, Colombi C et al. Comparison between depot leuprorelin and daily buserelin in IVF. J Assist Reprod Genet 1995; 12:15–19. 22. Ron El R, HermanA, GolanA, Van der Venet H, Caspi E, Dietrich K. The comparison of early follicular and midluteal administration of long-acting gonaclotropin-releasing hormone agonist. Fertil Steril 1990; 54:233–37. 23. Geber G, Sales L, Sampiano AC. Comparision between a single dose of goserelin (depot) and multiple daily doses of leuprolide acetate for pituitary suppression in IVF treatment: a clinical endocrinological study of the ovarian response. J Assist Reprod Genet 2002; 19(7):313–18. 24. Dodson WC, Hughes CL, Whitesides DB, Haney AF. The effect of leuprolide acetate on ovulation induction with human menopausal gonadotropins in polycystic ovary syndrome. J Clin Endocrinol Metab 1987; 65:95–100. 25. Louvet JP, Harman SM, Schrieber JR, Ross GT. Evidence of a role of androgens in follicular maturation. Endocrinology 1975; 97, 366–72. 26. Testart J, Lefevre B, Gougeon A. Effects of gonadotrophin-releasing hormone agonists (GnRHa) on follicle and oocyte quality. Hum Reprod 1993; 8:511–18. 27. Filicori M, Campaniello E, Michelacci L. Gonadotropin-releasing hormone (GnRH) analog suppression renders polycystic ovarian disease patients more susceptible to ovulation induction with pulsatile GnRH. J Clin Endocrinol Metab 1988; 66:327–33. 28. Cedars MI, Surey E, Hamilton F et al. Leuprolide acetate lowers circulating bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval. Fertil Steril 1990; 53:627–31. 29. Homburg R, Eshel A, Kilborn J. Combined luteinizing hormone releasing hormone analogue and exogenous gonadotrophins for the treatment of infertility associated with polycystic ovaries. Hum Reprod 1990; 5:32–35.

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157. Golan A, Ron-El R, Herman A, Soffer Y, Weinraub Z, Caspi E. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 1989; 44:430–440. 158. Gidley-Baird AA, O’Neill C, Sinosich MJ, Porter RN, Pike IL, Saunders DM. Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in human and mice. Fertil Steril 1986; 45:69–74. 159. Forman R, Fries N, TestartJ, Belaisch-AllartJ, HazoutA, Frydman R. Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. Fertil Steril 1988; 49:118–22. 160. Jewelewicz R, James SL, Finster M, Dyrenfurth I, Warren MP, Vande Wiele RL. Quintuplet gestation after ovulation induction with menopausal gonadotropins and pituitary luteinizing hormone. Obstet Gynecol 1972; 40:1–5. 161. Nakano R, Mizuno T, Kotsuji F, Katayama K, Washio M, Tojo S. “Triggering” of ovulation after infusion of synthetic luteinizing hormone releasing factor (LRF). Acta Obstet Gynecol Scand 1973; 52:269–72. 162. Keller PJ. Treatment of anovulation with synthetic luteinizing hormone-releasing hormone. Am J Obstet Gynecol. 1973; 116:698–705. 163. Breckwoldt M, Czygan PJ, Lehmann F, Bettendorf G. Synthetic LH-RH as a theapeutic agent. Acta Endocrinol (Copenh). 1974; 75:209–20. 164. Crosignani PG, Trojsi L, Attanasio A, Tonani E, Donini P. Hormonal profiles in anovulatory patients treated with gonadotropins and synthetic luteinizing hormone-releasing hormone. Obstet Gynecol 1975; 46:15–22. 165. Blumenfeld Z, Lang N, Amit A, Kahana L, Yoffe N. Native gonadotropin-releasing hormone for triggering follicular maturation in polycystic ovary syndrome patients undergoing human menopausal gonadotropin ovulation induction. Fertil Steril 1994; 62(3):456–60. 166. Casper RF, Sheehan KL, Yen SSC. Gonadotropin-estradiol responses to a superactive luteinizing hormone-releasing hormone agonist in women. J Clin Endocrinol metab. 1980; 50:179–81. 167. Dericks-Tan JSE, Hammer E, Taubert HD. The effect of D-Ser (TBU) 6-LH-RH-EA10 upon gonadotropin release in normally cyclic women. J Clin Endocrinol Metab. 1977; 45:597–600. 168. Lemay A, Faure N. Labrie F, Fazekas ATA. Gonadotroph and corpus luteum responses to two successive intranasal doses of a luteinizing hormone-releasing hormone agonist at different days after the midcycle luteinizing hormone surge. Fertil Steril. 1983; 39:661. 169. Monroe SE, Henzl MR, Martin MC et al. Ablation of folliculogenesis in women by a single dose of gonadotropin-releasing hormone agonistrsignificance of time in cycle. Fertil Steril. 1985; 43:361–68. 170. Itskovitz J, Boldes R, Barlev A, Erlik Y, Kahana L, Brandes JM. The induction of LH surge and oocyte maturation by GnRH analogue (buserelin) in women undergoing ovarian stimulation for in vitro fertilization. Gynecol Endocrinol 1988:2(suppl 2):165. 171. Itskovitz J, Boldes R, Levron J, Erlik Y, Kahana L, Brandes JM. Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist. Fertil Steril. 1991; 56;213–20. 172. Lanzone A, Fulghesu AM, Villa P, Guida C, Guido M, Nicoletti MC et al. Gonadotropinreleasing hormone agonist versus human chorionic gonadotropin as a trigger of ovulation in polycystic ovarian disease gonadotropin hyperstimulated cycles Fertil Steril 1994 Jul; 62(1):35– 41. 173. Allahbadia GN, Gandhi GN, Phadke A, Allahbadia G. Stimulation of Endogenous Surge of Luteinizing Hormone with Subcutaneous Leuprolide Acetate (Lupride) after ovarian stimulation for Intrauterine Insemination. Abstract Book, In Second World Congress of APART, Budapest, Hungary, September 14–17, 2000. 174. Hoff JD, Quigley ME, Yen SSC. Hormonal dynamics at midcycle: a reevaluation. J Clin Endocrinol Metab. 1983; 57:792–96.

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175. Seibel MM, Smith DM, Levesque L, Borten M, Taymor ML. The temporal relationship between the luteinizing hormone surge and human oocyte maturation. Am J Obstet Gyneco. 1982; 142:568–72. 176. Zelinski-Wooten MB, Lanzendorf SE, Wolf DP, Chandrasekher YA, Stouffer RL. Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles, I: oocyte maturation and corpus luteum function. J Clin Endocrinol Metab. 1991; 73:577–83. 177. Peluso JJ. Role of the amplitude of the gonadotropin surge in the rat. Fertil Steril. 1990; 53:150–54. 178. Danforth DR, Sinosich J, Anderson T, Cheng Y, Bardin CW, Hodgen GD. Identification (GnSIF) in follicular fluid and its differentiation from inhibin. Biol Reprod. 1987; 37:1075–82. 179. Danforth DR, Hodgen GD. The regulation of pituitary gonadotropin secretion by gonadotropin surge-inhibing factor (GnSIF) and inhibin. In Hodgen GD, Rosenwaks Z, Spieler JM (Eds). Nonsteroidal Gonadal Factors: Physiological Roles and Possibilities in Contraceptive Development. Norfolk: Jones Institute Press: 1988:221. 180. Balasch J, Fabregues F, Tur R, Creus M, Casamitjana R, Penarrubia J et al. Further characterization of the luteal phase inadequacy after gonadotrophin-releasing hormone agonistinduced ovulation in gonadotropin-stimulated cycles. Hum Reprod 1995; 10(6):1377–81. 181. Gerris J, De Vits A, Joostens M, Van Royen E. Triggering of ovulation in human menopausal gonadotrophin-stimulated cycles: comparison between intravenously administered gonadotrophin-releasing hormone (100 and 500 micrograms), GnRH agonist (buserelin, 500 micrograms) and human chorionic gonadotrophin (10,000 IU). Hum Reprod 1995; 10(1):56–62 182. Shalev E; Geslevich Y; Ben-Ami M. Induction of pre-ovulatory luteinizing hormone surge by gonadotrophin-releasing hormone agonist for women at risk for developing the ovarian hyperstimulation syndrome. Hum Reprod 1994; 9(3):417–9. 183. Romeu A, Monzo A, Peiro T, Diez E, Peinado JA, Quintero LA. Endogenous LH surge versus hCG as ovulation trigger after low-dose highly purified FSH in IUI: a comparison of 761 cycles. J Assist Reprod Genet 1997; 14(9):518–24. 184. Lewit N, Kol S, Manor D, Itskovitz-Eldor J. Comparison of gonadotrophin-releasing hormone analogues and human chorionic gonadotrophin for the induction of ovulation and prevention of ovarian hyperstimulation syndrome: a case-control study. Hum Reprod 1996; 11(7):1399–402. 185. Kol S, Lewit N, Itskovitz-Eldor J. Ovarian hyperstimulation: effects of GnRH analogues. Ovarian hyperstimulation syndrome after using gonadotrophin-releasing hormone analogue as a trigger of ovulation: causes and implications. Hum Reprod 1996; 11(6):1143–4. 186. Lanzone A, FulghesuAM, Apa R, Caruso A, Mancuso S. LH surge induction by GnRH agonist at the time of ovulation. Gynecol Endocrinol 1989; 3:213–20. 187. Gonen Y, Balakier H, Powell W, Casper RF. Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization. J Clin Endocrinol Metab 1990; 71:918– 22. 188. Imoedemhe DAG, Sigue AB, Pacpaco ELA, Olazo AB. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization. Fertil Steril 1991; 55:328–32. 189. Imoedemhe DAG, Chan RCW, Sigue AB Pacpaco ELA, Olazo AB. A new approach to the management of patients at risk of ovarian hyperstimulation in an in vitro fertilization programme. Hum Reprod 1991; 6:1088–91. 190. Tulchinsky D, Nash H, Brown K, Paoletti-Falcone V, Polcaro JA. pilot study of the use of gonadotropin-releasing hormone analog for triggering ovulation. Fertil Steril 1991; 55:644–46. 191. Emperaire JC, Ruffie A. Triggering ovulation with endogenous luteinizing hormone may prevent the ovarian hyperstimulation syndrome. Hum Reprod 1991; 6:506–10. 192. Emperaire JC, Ruffie A, Auderbert AJM, Declenchment de l’ovulation par la LH endogene liberee par l’ administration d’un agoniste de la LHRH apres stimulation folliculaire pour fecondation in vitro [in French]. J Gynecol Obstet Biol Reprod 1992; 21:489–94.

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193. Segal S, Casper RF. Gonadotropin-releasing hormone agonist versus human chorionic gonadotropin for triggering follicular maturation in in vitro fertilization. Fertil Steril 1992; 57:1254–58. 194. Scheele F, van der Meer M, Lambalk CB, Schoute E, Schoemaker J, Hompes PG. Exploring the recovery phase after treatment with a gonadotrophin-releasing hormone-agonist for ovulation induction in polycystic ovary syndrome: three pilot trials. Eur J Obstet Gynecol Reprod Biol 1995; 62(2):221–24. 195. Hutchison JS, Nelson PB, Zeleznik AJ. Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys. Emdocrinology 1986; 119:1964–71. 196. Mais V, Kazar RR, Cetel NS, Rivier J, Vale W, Yen SSC. The dependency of folliculogenesis and corpus luteum function of pulsatile gonadotropin secretion in cycling women using a gonadotropin-releasing hormone antagonist as a probe. J Clin Endocrinol Metab 1986; 62:1250– 55. 197. Penarrubia J, Balasch J, Fabregues F, Creus M, Casamitjana R, Ballesca JL et al. Human chorionic gonadotrophin luteal support overcomes luteal phase inadequacy after gonadotrophinreleasing hormone agonist-induced ovulation in gonadotrophin-stimulated cycles. Hum Reprod 1998; 13(12):3315–18. 198. Messinis IE, Milingos SD. Current and future status of ovulation induction in polycystic ovary syndrome. Hum Reprod Update 1997; 3(3):235–53. 199. Colombo PL, Dumoulin S, Saint-Martin F, Caron P, Bennet A, Louvet JP. Use of a gonadoliberin agonist with or without gonadoliberin pulses for ovulation induction in ovarian dystrophies. J Gynecol Obstet Biol Reprod (Paris) 1995; 24(4):362–67. 200. Schmidt-Sarosi C, Kaplan DR, Sarosi P, Essig MN, Licciardi FL, Keltz M et al. Ovulation triggering in clomiphene citratestimulated cycles: human chorionic gonadotropin versus a gonadotropin releasing hormone agonist. J Assist Reprod Genet 1995; 12(3):167–74. 201. Shalev E, Geslevich Y, Matilsky M, Ben-Ami. Induction of preovulatory gonadotrophin surge with gonadotrophin-releasing hormone agonist compared to pre-ovulatory injection of human chorionic gonadotrophins for ovulation induction in intrauterine insemination treatment cycles. Hum Reprod 1995 Sep; 10(9):2244–47. 202. Shanis BS, Check JH. Efficacy of gonadotropin-releasing hormone agonists to induce ovulation following low-dose human menopausal gonadotropin stimulation. Recent Prog Horm Res 1995; 50:483–86. 203. Moor RM, Osborn JC, Cran OG, Walters DE. Selective effect of gonadotropins on cell coupling, nuclear maturation oocytes. J Embryol Exp Morphol 1981; 61:347–65. 204. Olivennes F, Fanchin R, Bouchard P, Taieb J, Frydman R. Triggering of ovulation by a gonadotropin-releasing hormone (GnRH) agonist in patients pretreated with a GnRH antagonist. Fertil Steril 1996; 66(1):151–53. 205. Wada I, Matson PL, Troup SA, Hughes S, Buck P, Lieberman BA. Outcome of treatment subsequent to the elective cryopreservation of all embryos from women at risk of the ovarian hyperstimulation syndrome. Hum Reprod 1992; 7:962–66. 206. Abdalla HI, Ah-Moye M, Brinsden P, Howe DL, Okonofua F, Craft I. The effect of the dose of human chorionic gonadotropin and the type of gonadotropin stimulation on oocyte recovery rates in an in vitro fertilization program. Fertil Steril 1987; 48(6):958–63. 207. Scott RT, Bailey SA, Kost ER. Comparision of Leuprolide acetate and hCG for the induction of ovulation in clomiphene citrate stimulated cycles. Fertil Steril, 1994; 61:872–79. 208. Mansour RT, Aboulghar MA, Serour GI. Study of the optimum time for human chorionic gonadotropin-ovum pickup interval in in vitro fertilization. J Assist Reprod Genet 1994; 11(9):478–81. 209. Doody K. Ovulation Triggering with Recombinant Choriogonadotropin Alfa. Optimizing ART outcomes with tailored treatment strategies. In Symposium Abstract Handbook at the 58th annual meeting of the ASRM, Seattle, USA, 2002.

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210. The International Recombinant Human Chorionic Gonadotrophin Study Group. Induction of ovulation in WHO group If anovulatory women undergoing follicular stimulation with recombinant hFSH—a comparison of recombinant-human chorionic gonadotropin (r-hCG) and urinary-hCG. Fertil Steril (In Press). 211. The US Multicenter Study 7927 Investigator Group et al. Recombinant hCG in ART: Results of a clinical trial comparing two doses of r-hCG (Ovidrel) to urinary hCG (Profasi) for induction of final follicular maturation. Fertil Steril (In Press). 212. The European Recombinant Human Chorionic Gonadotrophin Study Group. Induction of final follicular maturation and early luteinization in women undergoing ovulation induction for assisted reproduction treatment-recombinant HCG versus urinary HCG. Hum Reprod 2000; 151446–51. 213. Schoolcraft W, Surrey E, Gardner D, Adams C, Stevens J. Recombinant hCG (Ovidrel(R)) for ovulation triggering in ART. Fertil Steril 2002; 77(Suppl 3):S20 214. Loumaye EP, Engrand Piazzi A et al. Use of recombinant human LH to reduce the risk of OHSS. In Abstract Book of Recombinant LH and hCG for the new millennium: new solutions for old problems at the 11th World Congress on in vitro Fertilisation and Human Reprod Genet 1999; 4–5. 215. Emperaire JC, Ruffie A. Triggering ovulation with endogenous hormone may prevent ovarian hyperstimulation syndrome. Hum Reprod 1991; 6:506–10. 216. Shoham Z, Schachter M, Loumaye E et al. The luteinizing hormone surge-the final stage in ovulation induction: modern aspects of ovulation triggering. Fertil Steril 1995; 64:237–51. 217. Imthurn B, PiazziA, Loumaye E. Recombinant human luteinizing hormone to mimic midcycle LH surge. Lancet 1996; 348:332–33. 218. Simon JA, Danforth DR, Hutchinson JS et al. Characterization of recombinant DNA derivedhuman LH in-vitro and in-vivo. Efficacy in ovulation induction and corpusluteum support. J Am Med Assoc 1988; 259:3290–95. 219. Marshall JC, Anderson DC, Russel-Fraser T et al. Human LH in man: studies of metabolism and biological action. J Endocrinol 1973; 56:431–39. 220. ChandrasekherYA, Hutchison JS, Zelinski-Wooten MB. Initiation of preovulatory events in primate follicles using recombinant and native human LH to mimic gonadotropin surge. J Clin Endocrinol Metab 1994; 79:298–306. 221. Romeu A, Molina I, Tresguerres JAF, et al. Effect of r-hLH versus hCG: effects on ovulation, embryo quality and transport, steroid balance and implantation in rabbits. Hum Reprod 1995; 10(5):1290–96. 222. Porchet HC, Le Cotonnec JY, Neuteboom S. Pharmacokinetics of recombinant human LH after IV, Im and SC administration in monkeys and comparison with IV administration of pituitary human LH. J Clin Endocrinol Metab 1995; 80:667–73. 223. Loumaye E, PiazziA, Warne Dl et al. Clinical use of recombinant human LH. In Abstract Handbook at the 13th Annual Meeting of the ESHRE, Edinburgh, 1997; 50 (Abstr: No: -104) 224. Albano C, Smitz J, Camus M. Comparision of different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67:917–22. 225. Itskovitz-Eldor J, Kol S, Mannaerts B, Coelingh Bennink H. Case Report: first establishepregnancy after COH with recFSH and GnRH antagonist ganirelix (Org 37462). Hum Reprod 1998; 13:294–95. 226. Chillik CF. Itskovitz J, Hahn DW. Characterising pituitary response to a GnRH antagonisin monkeys: tonic FSH/LH hormone secretion versus acute GnRH challenge tests before during, and after treatment. Fertil Steril 1987; 48:480–85. 227. Felderbaum RE, Reissmann T, Kupker W. Preserved pituitary response under ovarian stimulation with hMG and GnRH antagonists (Cetrorelix) in women with tubal infertility Eur J Obstet Gynecol Reprod Biol 1995; 61:151–55.

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228. Olivennes F, Fanchin R, Bouchard P, Taieb J, Frydman R. Triggering of ovulation by gonadotropin-releasing hormone (GnRH) agonist in patients pretreated with a GnRH antagonist. Fertil Steril 1996; 66(1):151–53. 229. Itskovitz Eldor J, Kol S, Mannaerts B. Use of a single bolus of GnRH agonist triptorelin to trigger ovulation after GnRH antagonist ganirelix treatment in women undergoing ovarian stimulation for assisted reproduction, with special reference to the prevention of OHSS: preliminary report. Hum Reprod 2000; 15(9):1965–68.

CHAPTER 15 Role of LH in Stimulation Protoco Is for ART Lars Grabow Westergaard, Claus Yding Andersen INTRODUCTION The success of assisted reproduction technologies during the last two decades is intimately connected with the use of exogenous gonadotrophins. Administration of surplus gonadotropins results in multi-follicular development and maximise the number of oocytes for fertilisation in vitro. Historically, follicular stimulation protocols attempted to mimic the normal physiology of folliculogenesis by including both FSH and LH. The classical, urine-derived hMG preparations contain equal amounts of FSH and LH-like activity (i.e. 75IU per ampoule) and were originally founded in the 2-cell twogonadotropin concept. In the late 1980’s it became clear, that increased tonic levels of LH during the follicular phase of the cycle was associated with reduced rates of fertilisation and implantation, but also increased miscarriage rate.1–4 These observations in combination with studies, which questioned the role and necessity of LH in folliculogenesis5 pawed the way for the introduction of ovarian stimulation regimes, which aimed at reducing the concentration of LH as much as possible. One important measure in this endeavour was the development of gonadotropin preparations with reduced or very low levels of LH (e.g. Normegon®, Organon, Metrodin®, Serono, Metrodin-HP®, Serono). The ultimate goal has been achieved with the introduction of recombinant FSH preparations, which are “pure” and without concomitant activity of LH (e.g. Puregon®, Organon; Gonal F®, Serono). This bouquet of gonadotropin preparations in combination with the recently developed recombinant preparations of LH and hCG (i.e. Luveris® and Ovitrelle® both Serono) allow us to control for the specific contribution of either FSH and LH during folliculogenesis and provide us with the unique opportunity to study the precise gonadotropin requirements of the developing follicle. Indeed, recent research has demonstrated, that LH cannot be neglected in connection with preovulatory follicular development and several studies indicate that very low levels of LH may reduce treatment outcome in connection with ART.6–9 It therefore appears, that LH should neither be too high nor too low. However, the level and range of LH required during the follicular phase in order to secure an optimal maturation of oocytes, fertilisation, pre-embryo development, and conception of a child is still an open question. The present review focuses on the importance of LH for normal folliculogenesis and evaluates the potential negative effects of a profound reduction of LH in connection with ovarian stimulation protocols.

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Measures Taken to Lower Circulating Levels of LH and Characteristics of Exogenous Administered Gonadotropins The introduction of gonadotrophin-releasing hormone agonists (GnRH-a) to suppress the endogenous gonadotrophin secretion during ovarian stimulation with exogenous gonadotrophins was of major significance in reducing circulating levels of LH during the follicular phase and to prevent an untimely mid-cycle surge of LH10 and the use of GnRH-a throughout the period of ovarian stimulation secures LH levels, which are substantially lower than those observed during normal menstrual cycles (Table 15.1). The use of gonadotropin preparations with reduced levels of LH also contributed to the markedly reduced levels of LH as observed in the now classical long GnRH-a protocol. However, the half-life of LH is relatively short of around 2 hours contrasting that of FSH, which has a terminal half-life of about 9–15 hours. Therefore, during COH women experience a sustained rise in the circulating levels of FSH, whereas exogenous LH, which may be detectable in circulation only for a few hours after injection, shows a progressive decline positively correlated to follicle development, but inversely correlated to increasing levels of oestradiol.11 The traditionally used hMG preparation also contain hCG (up to 25% of immunological LH activity,12 which show similar hormonal activity to LH but differ in the terminal half-life resembling that of FSH. Consequently, hCG may also be accumulated during COH although only in relative low levels.9,13 The use of GnRH-a and exogenous preparations with reduced LH contents therefore both contribute to reduced levels of LH-activity during ovarian stimulation, although the GnRH-a is likely to be of utmost importance, since exogenous administered LH is present only a short period in the circulation.9

Table 15.1: Mid-follicular serum LH levels in womena) with natural cycle (cycle day 8) in womenb) pituitary down-regulated and gonadotrophin stimulated women (stimulation day 8) N

Se-LH(IU/L)

Natural cyclea 19 3.2±1.0 IN buserelin/hMGb 100 1.8±0.2 IN buserelin/FSHb 98 1.9±0.2 SC buserelin/hMGb 89 0.9±0.1 SC buserelin/FSHb 92 0.8±0.1 a. Data from Westergaard et al, Hum. Reprod. 13:2612–2619,1998. b. Data from Westergaard et al, Fertil Steril 76:543–549, 2001; including 379 normogonadotrophic women treated with IVF or ICSI and randomised to long protocol GnRH agonist down-regulation with either buserelin nasal spray (IN) 0.15 mg X 4/ day (Suprecur, Hoechst) or 0.5 mg by s.c.(SC) injectio X 1/day (Suprefact, Hoechst) and to ovarian stimulation with a standardized dose of 225 IU per day of either hMG (Menogon, Ferring) or recombinant FSH (Gonal-F, Serono) for 7 days. Serum LH was measured on stimulation day 8 before the injection of gonadotrophins of that day, using a method as previously published (Westergaard et al. Hum Reprod 2000; 15:1003–1008).

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Is the LH Content in hMG Preparations Detrimental to the Outcome of IVF? The relatively good results reported in a large multicentre study by using rec. FSH in combination with long protocol GnRH-a down-regulation in IVF seems to support this notion.14 In a meta-analysis of 8 prospective, randomised trials comparing COH with urinary FSH and hMG it was concluded, that in IVF cycles the use of FSH is associated with a significantly higher clinical pregnancy rate than hMG, and that this difference could be ascribed to a detrimental effect of the LH contained in hMG preparations.15–16 Since the publication of this meta-analysis, however, a considerable number of prospective, randomised trials comparing hMG and FSH in GnRH agonist downregulated women have appeared, which do not reach the same conclusions as in the metaanalysis. On the contrary, some studies found an improved IVF outcome in the HMG treated group indicating a possible negative effect of severely suppressed LH levels in the FSH treated group during the follicular phase.17–20 Taken together, available data question a harmful effect of exogenous LH in the doses normally administered for ovarian stimulation. As a matter of fact, mean mid-follicular serum LH levels as observed during the natural cycle in normogonadotrophic women are two to four times higher than those observed on stimulation day 8 in GnRH agonist down-regulated women whether HMG or FSH is used for ovarian stimulation (Table 15.1). The question, therefore, is rather whether the very low mid-follicular LH levels seen in a GnRH agonist/recombinant FSH treated normogonadotrophic women are as harmless as previously assumed).5 Negative Effects of Decreased Circulating Levels of LH During Ovarian Stimulation Studies in GnRH antagonist down-regulated primates indicate, that an intrafollicular environment depleted of LH and oestradiol exert negative effects on oocyte maturation, embryo development and the ability of the embryo to implant.21 Clinical studies in women with hypogonadal hypogonadism have shown, that while follicular development can be achieved by stimulation with pure FSH preparations, concentrations of circulating oestradiol and fertilization rates of retrieved oocytes are severely compromised compared to stimulation with preparations containing LH activity.22–23 Similarly, in normogonadotrophic women treated with GnRH analogues and highly purified FSH a lower yield of oocytes, lower fertilization rates and reduced embryo quality was found in those women (one third of all) who had low circulating levels of LH (<0.5 IU/L) and oestradiol in the mid-follicular phase as compared to the women with normal LH and oestradiol levels.6 In contrast to the above mentioned primate study, the ability of surplus embryos to form blastocyst in vitro was similar in the low and normal LH groups.6 However, a normal development to the blastocyst stage in vitro does not necessarily imply normal embryonic capacity for implantation and foetal development in vivo. Recent data from our clinic indicate, that detrimental effects of low LH levels in the midfollicular phase of ovarian stimulation may become manifest only after implantation.7 A total of 200 consecutive IVF cycles in normogonadotrophic women were analysed retrospectively. The standard long protocol was employed in all women including midluteal pituitary down-regulation with injection of Suprefact® (Hoechst, Denmark) 0.5 mg s.c. per day for 14 days, and thereafter reduced to 0.2 mg sc per day until the day of hCG

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injection. For ovarian stimulation recombinant FSH (Gonal-F, Serono Nordic) in a standard dose of 225 IU per day for 7 days was administered. Thereafter the dose could be individualised according to ovarian response. Blood samples for oestradiol, FSH and LH analysis were obtained on stimulation day 1 and 8. On stimulation day 8 (S8) serum LH levels were below 0.5 IU/L in 98 of the 200 patients and above 0.5 IU/L in the remaining 102 patients. The outcome of IVF (ICSI included) in the two groups of women withLHbelow and above 0.5 IU/L differed significantly. The women with LH<0.5 IU/ Lhad serum oestradiol levels on S8 that were significantly lower than those in women with LH>0.5 IU/L. Nevertheless, the mean number of oocytes retrieved, fertilization rates and mean number of transferable embryos were similar as well as the number of positive pregnancy tests obtained in the two groups. However, the rate of early pregnancy loss in percent of clinical pregnancies in the group with LH below 0.5 IU/L was significantly higher than in the group with LH above 0.5 IU/L (36% versus 3%), p <0.005. Consequently, the delivery rate per started cycle in the below 0.5 IU/L LH group (19%) was lower than in the above 0.5 IU/L LH group (26%).7 In a retrospective study, Balasch et al24 analysed follicular phase serum LH levels in 72 women, who became pregnant after long protocol GnRH agonist down-regulation, rFSH stimulation and IVF/ICSI, and matched them with 72 similarly treated women, who did not conceive in immediately following ART cycles. Applying receiver-operating characteristics (ROC) analysis these authors found that serum LH concentrations of 1.0, 0.7 or 0.5 IU/L on stimulation day 7 was unable to discriminate between conception and non-conception cycles. In addition, an overlap in median and range of LH was found between non-conception cycles (N=72), conception cycles (N=72), and between ongoing pregnancies (N=58) and early pregnancy losses (N=14). Clearly, these results do not confirm a harmful effect of profoundly suppressed LH levels as reported by others and us. The reason for this discrepancy may be ascribed to diff erences with regard to case selection criteria between the two studies. More importantly however, in the study by Balasch et al24 mid-follicular serum oestradiol levels between the normal and low LII groups were similar. This contrasts other findings that report, significant, positive correlation between mid-follicular LH and oestradiol levels.7,15 This might indicate, that although Balasch et al23 found reduced LH levels in some of their patients, these decrements were not large enough to exert biologically significant actions on ovarian steroidogenesis. Thus, only 7 percent and 15 percent of the patients in the Balasch et al24 study had mid-follicular LH below 0.5 IU/L and 0.7 IU/L, respectively, as compared to 49 percent and 32 percent in the studies by Westergaard et al7 and Fleming et al.25 In addition, applying the ROC analysis on our data from the above study7 we could confirm, that the LH threshold of 0.5 IU/L still discriminates significantly between a positive and negative reproductive outcome (unpublished data). Probably, these discrepancies can be ascribed to differences in dosage and mode of administration of GnRH agonists for down-regulation (see below), and also differences with regard to the methods of hormone assays may be important. In the retrospective study by Fleming et al25 mid-follicular eLH concentration of 0.7 IU/L was used to discriminate between normal and low LH in women, who were treated with GnRH agonist down-regulation and stimulated with urinary or recombinant FSH. No significant difference in frequency of early pregnancy loss between the two groups was found in that study. On the other hand, in a French study8 on 229 women undergoing 261 GnRH-a and rFSH/hCG cycles for IVF, mid-

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follicular LH concentrationbelow 2IU/L (36% of the patients) was associated with a significantly lower oestradiol concentration, lower number of oocytes, lower percentage of good embryos and significantly lower clinical and ongoing pregnancy rates than in the two thirds of the patients having mid-follicular LH concentration above 2IU/L. The reason for the discrepancies among the different studies is not obvious, but taken together the data give evidence to suggest, that a substantial proportion of normogonadotrophic women, who are subjected to GnRH-a down-regulation followed by ovarian stimulation with pure FSH, will experience follicular levels of circulating LH that are so low, that a negative impact on the reproductive outcome ensues. Our data indicate, that this lower threshold is around 0.5 IU/L of LH in the mid-follicular phase, and also that up to half of the normogonadotrophic women subjected to sc Buserelin down-regulation combined with ovarian stimulation with rec FSH experience mid-follicular LH below 0.5 IU/L. Data from the Ganirelix dose finding group26 on the use of GnRH antagonist in various doses (from 0.0625 to 2 mg per day) confirm the negative effects of profotmd suppression of LH during ovarian stimulation in IVF, both with regard to effect on ovarian oestradiol production and on reproductive outcome of IVF. Thus, significantly lower LH and E2 levels and significantly more cases of early pregnancy loss were found in the higher dose groups (2 mg, 1mg and 0.5 mg per day) as compared to the lower dose groups (0.25, 0.125 and 0.0625 mg per day).26 Whether or not the low oestradiol levels accompanying LH suppression mediate these negative effects on pregnancy outcome is presently unclear. Also, it is not certain whether modification of the pituitary downregulation regime and/or supplementation with exogenous LH as hMG or rec. LH during ovarian stimulation will restore treatment outcome to that seen in women with midfollicular LH levels above 0.5 IU/L. Supplementing urinary FSH with hCG (50 IU/day) during ovarian stimulation in pituitary down-regulated women has been reported to enhance FSH efficacy and improve ovulation induction outcome as compared to women treated with FSH without hCG supplementation.13 On the other hand, addition of recombinant LH (150 IU/day from day 6 of stimulation) to recombinant FSH treated pituitary down-regulated women with serum LH levels <1.5 IU at the start of stimulation had no effect on the outcome of IVF.27 The discrepancy between these results may be related to differences in the half-lives of LH and hCG, being substantially shorter for LH than hCG and, thus, that the dose of LH used is too low, or to the fact that endogenous serum levels of LH above 1.5 IU are sufficient for a normal follicular development and function. Only recently, a number of prospective, randomised studies comparing recombinant FSH with hMG in GnRH-a down-regulated normogonadotrophic women treated with IVF/ICSI have been published.28–31 These studies have shown either equality28–30 or superiority of hMG over rFSH with regard to clinical outcome, endocrinological and other parameters characterizing IVF/ICSI.9,29 The results from our clinic9 show, that both the type of pituitary down-regulation regime and the type of gonadotrophin preparation influence the outcome of IVF significantly. A total of 379 women were down regulated from the mid-luteal phase with buserelin either as intranasal spray (Suprecur®, Hoechst) 0.15 mg 4 times a day (IN group) or as sc injection (Suprefact®, Hoechst) 0.5 mg once a day (SC group) and, thereafter stimulated with a fixed dose (225 IU/day) of either hMG (Menogon®, Ferring) (hMG group) or recombinant FSH (Gonal-F®, Serono) (FSH group). Thus, four groups of women were analysed: the IN/hMG (N=100), the IN/FSH

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(N=98), the SC/hMG (N= 89) and the SC/FSH (N= 92) group. Serum levels of oestradiol, LH and hCG measured in the mid-follicular phase, i.e. on stimulation day 8. Serum oestradiol levels were significantly lower in the SC/FSH group as compared to the other groups. Levels of LH were significantly lower in the two SC groups than in the two IN groups, and levels of hCG significantly higher in the hMG treated women compared to those treated with FSH. Thus, it seemed that one daily sc injection of Buserelin (0.5 mg) depresses endogenous LH more effectively than intranasal administration (0.6 mg day). In the SC/hMG group LH levels were equally low, but probably due to the content of HCG in hMG the effect on oestradiol biosynthesis seem to be compensated. The clinical pregnancy rate per started cycle in these four groups of women was significantly correlated to the levels of LH-like activity (LH+hCG) in serum on stimulation day 8, being 29 percent in the SC/FSH group compared to 44 percent, 36 percent and 39 percent in women treated with IN/hMG. SChMG and IN/FSH groups, respectively (p <0.05). The trend towards an improved outcome related to increasing levels of mid-follicular LH activity becomes even more pronounced when only first IVF attempts are considered. Thus, in the IN/hMG group (N=75) number and percentage of positive pregnancy tests, clinical pregnancies, live births as well as implantation rate are all significantly higher than in the IN/FSH (N=66), SC/ hMG (N=66) and in the SC/FSH (N=65) groups. CONCLUSIONS It is now well recognised, that women with hypogonadotrophic hypogonadism stimulated with pure FSH show follicular development, oocytes can be retrieved and preembryo development in vitro can be achieved, but oestradiol levels remain low and only one clinical pregnancy which terminated in a spontaneous abortion at week 8 of pregnancy has so far been reported.32 Deliveries, however, in women with hypogonadotrophic hypogonadism without cotreatment of gonadotrophins with LH-like activity during ovarianstimulation have not yet been reported.33 The above information and the data presented in this article does not question FSH as the principal regulator of follicular growth, but raises the question as to whether there is a critical lower level of LH-like activity, which must be exceeded during the follicular phase in order to secure optimal chances for giving birth to a child as a result of treatment. Our data show, that addition of LH-like activity to a regimen employing pure FSH preparations during ovarian stimulation of normogonadotrophic women actually does affect endocrinological parameters, most clearly expressed in the augmented oestradiol secretion. In addition, our data show an improved clinical outcome in parallel with increased mid-follicular serum levels of LH-like activity, thereby suggesting the existence of a lower limit of LH-like activiiy, which needs to be exceeded during the follicular phase in order for treatment to be truly successful resulting in the birth of a child. These data have been obtained in a group of normogonadotrophic women indicating, that the results may apply in general, and do indeed suggest, that LH-like activity is needed for maturation of oocytes with optimal pregnancy potential. However, an absolute level of LH-like activity, which needs to be reached, is hard to define, partly because both LH and hCG undertake LH activity, and they differ profoundly in their terminal half-life in vivo. While LH is cleared rapidly from circulation, hCG has a half-

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life closer to that of FSH allowing it to be accumulated during ovarian stimulation. In connection with a profound suppression of an endogenous production of gonadotrophins, hCG may actually represent the most important LH-activity in vivo. The above reported data support this notion since pituitary down-regulation results in similar low levels of LH regardless of whether pure FSH or hMG is used as exogenous gonadotrophin (Table 15.1). These findings are therefore in agreement with previous reports on beneficial effects of supplementation with exogenous hCG during ovarian stimulation.13 On the other hand, supplementation with recombinant LH (75–150 IU per day) during ovarian stimulation with recombinant FSH has not shown any beneficial effects.27,34–35 Since the exact requirements of LH are unknown, it might well be, that the doses of recombinant LH applied have been too low to exert a sufficient effect, bearing in mind that doses of 225 IU of LH per day are not reflected in increasing serum levels (Table 15.1). Therefore, one way to circumvent the negative effects of very low levels of LH-like activity during the follicular phase may be to use the classical hMG preparations or use supplementation with hCG, urinary or, even better, recombinant hCG. One may consider hCG as a hormone supporting pregnancy and not a hormone which should be used to stimulate follicular development and oestradiol production. However, some studies have actually shown, that hCG in low concentration is present in the circulation of most women and is released in a pulsatile manner.36 In our opinion hCG may be an excellent choice for securing a sustained but sufficient LH-like activity during ovarian stimulation. From the data presented, another possibility is offered, since the mode of pituitary down-regulation influences the circulating levels of LH significantly Intranasal administration of buserelin four times a day results in serum levels of LH twice as high as when the same amount of buserelin is administered by sc injection once a day. This probably reflects that pituitary down-regulation is less prof ound with intranasal administration than with sc administration and indicates, that a less prof ound downregulation to a certain extend allows a higher endogenous production of LH. Furthermore, it may be speculated that an endogenous production of LH, during the course of down-regulation with intranasal administration, may be released in a pulsatile manner resembling normal in vivo conditions more closely. Whether a pulsatile release of endogenously produced LH is part of an explanation for a beneficial effect of LH is presently unknown. Alternatively, endogenously derived LH may be released in a more steady way, thereby securing a constant LH tonus and ovarian stimulation. The mechanism of action may also involve local, intrafollicular changes in steroidogenesis affecting maturation of the oocyte.37 Receptivity of the endometrium may represent yet another mechanism of action. However, in our opinion, mechanisms that govern the cytoplasmic maturation of the follicle-enclosed oocyte are likely to be of major importance to understand the beneficial effects of the combined action of both FSH and LH. The question of the importance of LH-like activity in connection with ovarian stimulation is likely to further be highlighted during the coming years, since GnRH antagonists, which have just recently been introduced, cause a rapid and profound suppression of endogenous LH release, and regimens using GnRH antagonists may benefit from including gonadotrophin preparations with LH-like activity

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REFERENCES 1. Stanger JD, Yovich JL. Reduced in vitro fertilisation of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol 1985; 92:385–93. 2. Howles CM, MacNamee MC, Edwards RG et al. Effect of high tonic levels of luteinizing hormone on outcome of in vitro fertilisation. Lancet. 1986; 2:521–22. 3. Conway GS, Honour JW, Jacobs HS. Heterogeneity of the polycystic ovary syndrome: clinical, endocrine and ultrasound features. Clin Endocrinol 1989; 30:459–70. 4. Regan L, Owen EJ, Jacobs HS. Hyper secretion of luteinizing hormone, infertility and miscarriage. Lancet. 1990; 336:1141–44. 5. Chappel SC, Howles C. Re-evaluation of the roles of luteinizing hormone and follicle stimulating hormone in the ovulatory process. Hum Reprod 1991; 6:1206–12. 6. Fleming R, Lloyd F, Herbert M et al. Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Hum Reprod 1998; 13:1788–92. 7. Westergaard LG, Laursen SB, Yding Andersen, C. Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction. Hum Reprod 2000; 15:1003–08. 8. Fanchin R, Schonauer LM, Jahjoub S et al. Residual LH concentration after GnRH agonist administration influence ovarian response to recombinant FSH, embryo quality, and IVFembryo transfer outcome. Hum Reprod 2001; 16:14–15. 9. Westergaard LG, Yding Andersen C. The impact of follicular phase levels of LH on IVF outcome in normogonadotrophic women. Middle East Fert Soc J 2001; 6:99–107. 10. Fleming R, Coutts JTR. Induction of multiple follicular growth in normally menstruating women after gonadotrophin-releasing hormone agonist suppression. Fertil Steril 1986; 45:226– 30. 11. Baird DT In. Gonadotrophins, Gonadotrophin-releasing Hormone Analogues and Growth Factors in Infertility: Future perspectives. Howles CM (Ed.). Medifax International. 1992; 43– 55. 12. Stokman PGW, Leeuw R, van den Wijngaard HAGW et al. Human chorionic gonadotrophin in commercial menopausal gonadotropin. Fertil Steril 1993; 60:175–78. 13. Filicori M, Cognigni GE, Taraborelli S et al. Luteinizing hormone activity supplementation enhances follicle-stimulating hormone efficacy and improves ovulation induction outcome. J Clin Endocrinol Metab 1999; 84:2659–63. 14. Out HJ, Mannaerts BMJL, Driessen SGAJ et al. A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle-stimulating hormone (Puregon versus Metrodin) in in vitro fertilization. Hum Reprod 1995; 10:2534–40. 15. Daya S, Gunby J, Hughes EG et al. Randomized controlled trial of follicle stimulating hormone versus human menopausal gonadotrophin in in vitro fertilization. Hum Reprod 1995; 10:392– 96. 16. Daya S. Follicle-stimulating hormone and human menopausal gonadotrophin for ovarian stimulation in assisted reproductive cycles. In: The Cochrane Library, Issue 2, 2000. Oxford: Update Software. 2000; 1–8. 17. Fleming R, Chung CC, Yates RWS et al. Purified urinary follicle stimulating hormone induces different hormone profiles compared with menotropins, dependent upon the route of administration and endogenous luteinizing hormone activity. Hum Reprod 1996; 11:1854–58. 18. Westergaard LG, Erb K, Laursen S et al. The effect of human menopausal gonadotrophin and highly purified, urine-derived follicle stimulating hormone on the outcome of in vitro fertilization in down-regulated normogonadotrophic women. Hum Reprod 1996; 11:1209–13.

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19. Söderström-Antilla V, Foudila T, Hovatta O et al. A randomized comparative study of highly purified FSH and hMG for ovarian hyperstimulation in an oocyte donation program. Hum Reprod 1996; 11:1864–70. 20. Bagratec JS, Lockwood G, Lopez Bernal A et al. Comparison of highly purified FSH (Metrodin-High Purity) with Pergonal for IVF superovulation. J Ass Reprod Gen 1998; 15:65– 69. 21. Weston AM, Zelinski-Wooten MB, Hutchison JS et al. Developmental potential of embryos produced by in vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone. Hum Reprod 1996; 11:608–13. 22. Shoham Z, Jacobs HS, Insler V. Luteinizing hormone: its role, mechanism of action and detrimental effects when hyper secreted during the follicular phase. Fertil Steril 1993; 59:1153– 61. 23. Balasch J, Miro F, Burzaco I et al. The role of luteinizing hormone in human follicle development and oocyte fertility: evidence from in vitro fertilisation in a woman with longstanding hypogonadotropic hypogonadism and using recombinant human follicle stimulating hormone. Hum Reprod 1995; 10:1678–83. 24. Balasch J, Vidal E, Penarrubia J et al. Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Hum Reprod 2001; 16:1636–43. 25. Fleming R, Rehka P, Deshpande N et al. Suppression of LH during ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant FSH. Hum Reprod 2000; 15:1440–45. 26. Ganirelix Dose-finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist Ganirelix (Org. 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon). Hum Reprod 1998; 13:3023–31. 27. Kelly EE, Nebiolo L. Recombinant FSH therapy alone (Gonal-F) versus combination therapy (Gonal-F+Lhadi, R-hLH) in patients down-regulated with low dose luteal GnRH agonist protocol. Preliminary results. 11th World Congress on In vitro Fertilization and Human Reproductive Genetics. Abstract S-003, 1999. 28. Ng EHY, Lau EYL, Yeung WSB et al. hMG is as good as recombinant human FSH in terms of oocyte and embryo quality: a prospective randomized trial. Hum Reprod 2001; 16:319–25. 29. Gordon UD, Harrison RF, Hennelly B et al. A randomized prospective assessor-blind evaluation of luteinizing hormone dosage and in vitro fertilization outcome. Fertil Steril 2001; 75:324–31. 30. Strehler E, Abt M, El-Danasouri I et al. Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome. Fertil Steril 2001; 75:332–36. 31. Westergaard LG, Erb K, Laursen SB et al. Human menopausal gonadotrophin versus recombinant follicle-stimulating hormone in normogonadotrophic women down-regulated with a gonadotrophin-releasing hormone agonist who were undergoing in vitro fertilization and intracytoplasmic sperm injection: a prospective randomized study. Fertil Steril 2001; 76:543– 49. 32. Battaglia C, Salvatori M, Regnani G et al. Successful induction of ovulation using highly purified follicle-stimulating hormone in a woman with Kallmann’s syndrome. Fertil Steril 2000; 73:284–86. 33. Gordon UD. Effects of different menotropin preparations for assisted reproduction in hypogonadotropic hypogonadism. In. The role of serum luteinizing in folliculogenesis and ovulation induction. M. Filicori (Ed), Bologna, Monduzzi Editore, 1999; 135–43.

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34. Scott Sills E, Levy DP, Moomjy M et al. A prospective, randomized comparison of ovulation induction using highly purified folliclestimulation hormone alone and with recombinant human luteinizing hormone in vitro fertilization. Hum Reprod 1999; 14:2230–35. 35. Howles CM. Role of LH and FSH in ovarian function. Mol Cell Endocrinol 2000; 161(1– 2):25–30. 36. Odell WD, Griffin J. Pulsatile Secretion of Chorionic Gonadotropin during the normal menstmal cycle. J Clin Endocrinol Metab 1989; 69:528–32. 37. Tesarik J, Mendoza C. Non-genomic effects of 17-estradiol on maturing human oocytes: relationship to oocyte developmental potential. J Clin Endocrinol Metab 1995; 80:1438–43.

CHAPTER 16 Role of hMG-HP in Stimulated Cycles for ART Gautam N Allahbadia, Kaushal Kadam, SPS Virk INTRODUCTION The first successful in vitro fertilization attempt and most treatment cycles for a while thereafter were conducted in spontaneous menstrual cycles. Nevertheless, realization that availability of a crop of mature oocytes markedly increased chances of success in this therapy, prompted most centers to adopt some form of Controlled Ovarian Hyperstimulation (COH). At the outset, clomiphene citrate alone or in combination with human menopausal gonadotropins (hMG) was used but eventually exogenous gonadotropins emerged as the sole stimulatory drug for COH. Exogenous gonadotropins, and specifically hMG products, have been used in the treatment of infertility since the 1960’s, when the first hMG product became available. Subsequently, over the last 20 years they have become the mainstay of fertility treatment worldwide. The gonadotropins are indicated in isolation as a treatment to induce ovulation, normally in cases where clomiphene has failed, or as a first-line treatment in specific cases of amenorrhea. They are also indicated for hypogonadotropic hypogonadism in men and women. The most widespread use of gonadotropins is for women undergoing superovulation within a medically assisted reproductive program, such as IVF. Superovulation is the stimulation of the ovaries to produce more than one follicle, which enables several embryos to be created. Human Menopausal Gonadotropins (hMG) were initially used for COH, but poor reproductive outcome was ascribed to high levels of circulating LH associated with hMG therapy Lunenfeld hypothesized “that a high concentration of LH through the follicular phase allows the developing oocyte to mature prematurely, producing at ovulation an oocyte that is physiologically aged. Such oocytes may have a decreased capacity to fertilize; if they fertilize, they are unlikely to implant; and if they implant, their survival rate is decreased, resulting in early abortion”1. The LH hypothesis changed worldwide thinking and the direction of stimulation protocols in ART. “Pure” FSH preparations with reduced LH content such as Purified FSH (PoFSH), Highly Purified FSH (PoFSHHP) and Recombinant FSH (recFSH) were introduced. Soon after the introduction of COH in ART it became evident that in the course of ovulation induction the midcycle LH surge could be unexpectedly triggered by rising ovarian steroid levels; as a result, premature ovulation or follicle luteinization could occur and cause cycle cancellations in upto 30 percent of cases. In order to identify patients on the verge of spontaneous ovulation, close monitoring of LH and progesterone (P) plasma levels with multiple daily determination used to be the rule in ART. Thus, in

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the mid-1980s GnRH agonist supplementation was tested in ovulation induction2 and then successfully applied in most cycles. The pituitary response to GnRH agonist administration is biphasic: an initial agonistic phase during which circulating levels of LH and FSH rise and this is followed by pituitary desensitization and down-regulation with shutdown of gonadotropin secretion. The major advantage in the use of GnRH agonist is the complete elimination of the preovulatory LH surge; in addition to preventing cycle cancellations due to premature ovulation, the use of GnRH agonist has permitted to dramatically reduce endocrine monitoring and its related costs. Very recently GnRH antagonists were introduced in stimulation protocols for ART. GnRH antagonists offer a new and powerful tool for ovarian stimulation. Administered at pharmacological dosages, they allow an immediate yet completely reversible competitive blockade of the GnRH receptors at the level of the pituitary. The use of GnRH analogs in conjunction with gonadotropins for COH has afforded better control of the cycle, and has provided versatility to tailor specific COH protocols to specific groups of Patients.3–6 Currently, there are four broad categories of gonadotropins available: hMG products, which are derived from human post-menopausal urine. These products contain both FSH and LH in a one-to-one ratio, highly-purified hMG products, such as Menopur, which also contain both FSH and LH, in a one-to-one ratio, purified urinary products with FSH activity only and genetically engineered recombinant products, containing FSH only or LH only Each of these gonadotropin products has been shown to be effective in inducing follicular growth and maturation. In addition, dominant clinical outcomes, such as pregnancy rates per embryo transfer or started cycle, have been similar between hMG and urinary FSHonly products in all comparative studies.7 This has been further confirmed by the results from the recent meta-analysis by Agrawal et al.8 Consequently, there remains considerable debate over the value of FSH-only preparations and whether they bring any significant clinical advantages to patients-particularly in the light of the significantly higher cost of recombinant products. The Rationale of Introducing Highly Purified hMG For much of modern infertility treatment’s history, naturally derived hMG therapy, which retains the essential hormones responsible for stimulation of ovulation (FSH and LH), has made a valuable contribution to infertility treatment. With the development of biotechnology, genetically engineered mono-component FSH preparations became available during the 1990s. While containing recFSH-only these preparations offered practitioners, products with the highest purity at the time. hMG-HP (Menopur, Ferring Pharmaceuticals A/S, Denmark) is a naturally-derived gonadotropin preparation with levels of purity comparable to recombinant mono-preparations. The idea for its development was based on the insight that natural reproduction depends on the coordinated action of FSH and LH, and probably the belief that patients would be best served with a modality that is close to nature’s own two-cell model.19 The high purity of hMG-HP confers two significant benefits: Firstly hMG-HP can be given by subcutaneous injection. This tends to be easier and less painful to administer than an intramuscular injection and allows for self-administration, which together mean more convenience for the patient. The subcutaneous route offers several potential advantages. Intramuscular injections often require the patient to visit a clinic or a nurse,

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while subcutaneous administration can be easily carried out by the patient herselfor her partner. Subcutaneously selfadministered gonadotropin treatment provides greater convenience for the patient during the stimulation period, which tends to be regarded as a difficult period in any case, both emotionally and physically. Secondly, hMGHP contains a much lower level of inactive ingredients than regular hMG, which eases the concerns of patients and staff regarding injection site reactions. This means that hMG-HP offers the patient the clinical features of a product containing both FSH and LH, together with the convenience and the practical benefits associated with subcutaneously administered high purity products. The purity level of hMG-HP is the same as that claimed for recombinant products.10 The production of hMG-HP is initially identical to that of regular hMG.10 Further hMGHP is subject to the same rigorous screening, viral testing, sterilization and chromatography purification procedures as other hMG products, to eliminate the risk of cross contamination and to remove contaminants. hMG-HP is then subjected to additional chromatography purification steps at the end of the manufacturing process, that result in less inactive ingredients and a step-wise increase in purity over the regular hMG preparation by a factor of up to 50 times, as determined by the activity/mg of substance.10 The significant increase in purity permits subcutaneous self-administration of hMG-HP, which is much more convenient for the patient. In addition, the purified product itself is tested to confirm the absence of HIV and Hepatitis B and C viruses using sensitive PCR techniques.10 The purification steps involved in the manufacturing of hMG-HP are very likely to remove or inactivate prions.10 The high standards of purity in hMGHP can be demonstrated by SDSPAGE analysis, as compared with regular hMG and recFSH (Fig. 16.1). Intramuscular injections of non-purified urinary gonadotropins carry a rare but recognized risk of injection site reactions of an anaphylactoid type, probably due to inactive proteins. The greatly increased purity level of hMG-HP and of high-purity and recombinant FSH preparations removes the risk of adverse events.

Fig.16.1: SDS-PAGE analyais of hMG-HP followed by silver staining reveals high purity in lane 3 compared

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to a conventional hMG product (lane 1) which is comparable to recFSH (lane2) A recent large-scale, randomized, comparative study compared the efficacy and safety of hMG-HP to that of recombinant FSH in female patients in an IVF/ICSI programlO. The objective was to show that highly purified menotropin (Menopur) is at least as efficientin terms of on-going pregnancy rates-and safe as recombinant FSH, in the treatment of females undergoing IVF or ICSI. A total of 693 patients were included in the primary analysis population: 357 received Menopur and 336 received recombinant FSH (as follitropin alfa). Both products were administered subcutaneously. The gonadotropins were administered following a long pituitary down-regulation protocol, achieved by either depot or daily injections of a GnRH analogue. Both products were given as a fixed dose of 225 IU (3 ampoules/vials) for 5 days. Subsequent dosage was adjusted, depending on ultrasound and/or estradiol levels) until there were 3 or more follicles with a diameter of a 16 mm and hCG was administered. Biochemical, clinical and on-going pregnancy rates were compared for both products. The primary endpoint was the ongoing pregnancy rate after one IVF/ICSI cycle. In this study, a higher on-going pregnancy rate was observed in the group receiving hMG-HP. Although this diff erence did not reach statistical significance, these data conclusively demonstrate that the efficacy of hMG-HP is at least equivalent to that of follitropin alfa.10 There were no significant differences between the two preparations in terms of their adverse event profile. Local tolerance, as assessed by application site adverse events, was assessed as good for both treatments. There was no significant difference between hMG-HP and follitropin alfa in terms of frequency of abortion and miscarriage, frequency of OHSS II and III and frequency of multiple births.10 The Role of Luteinizing Hormone (LH) in Ovulation Induction For Assisted ReproductionTechniques (ART) The dominant focus of this debate concerns the relevance and implications of LH levels in hMG products, such as Menopur, and its absence from recombinant and urinary FSHonly preparations. This becomes increasingly meaningful in the context of the pituitary down-regulation protocols that are so commonplace in assisted reproduction techniques throughout the world. The roles of LH in estrogen synthesis and in triggering ovulation have been understood for some time.11 LH is essential for the synthesis of estrogen within the ovaries. LH stimulates theca cells to produce androgens, such as testosterone. These androgens are the main substrate for the synthesis of estrogen in the granulosa cells, via the action of the aromatase system. Aromatase activity is stimulated initially by FSH. The involvement of two cell types and both FSH and LH is the principle that underpins the “two-cell” theory of estrogen synthesis within the follicle, as described previously.9 Phelps et al found that addition of exogenous LH significantly increased estradiol secretion in poor responders with low midfollicular endogenous LH concentrations.12 An average change in the slope of the estradiol patterns from 27.54 to 85.49 was seen after the addition of exogenous LH, which emphasized the importance of the synergistic action of the two-cell, two-gonadotropin system.12 Estrogen serves a variety of extragonadal

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functions during the reproductive cycle, including: priming the pituitary for the mid-cycle LH surge, which is the trigger for ovulation, stimulation of endometrial growth, to prepare for implantation and also for stimulation of adequate cervical mucus production. At the ovarian level, the role of estrogen in follicular development and oocyte maturation is less clear. The local actions of estrogen are subject to renewed debate, particularly since the identification of a second estrogen receptor gene (ERb), that is expressed in the human granulosa cells13 Their role remains unclear, but estrogen appears to exert its action on the cell’s cytoplasm and cell membrane. Estrogens appear to protect the follicle from atresia that may result from an excess of androgens in the environment surrounding the follicle.14 This is suspected because when follicular atresia is seen, the fluid within the follicles is low in estradiol and there is a lower estradiol: androgens ratio.14 The mid-cycle LH surge is the trigger for ovulation. At its peak, the LH level may be six times that seen at the start of the cycle. Ovulation itself tends to occur about 18 hours after the peak LH levels are seen.15 Interactions between the follicle and LH seem to disrupt contacts between the granulosa cells and to induce maturation of the oocyte, which together cause rupture of the follicle and release of the oocyte.15 In addition, to these two wellestablished roles for LH, evidence is constantly emerging for other actions of LH-either directly or via the action of the sex steroids-elsewhere within the female reproductive cycle.16–18 Several clinical situations, where LH is either absent or completely inactive, provide important clues to our understanding of the roles of LH in follicular development. In Kallmann’s syndrome, women are profoundly hypogonadotropic, and follicle development may be induced by the exogenous administration of gonadotropins. Treatment of these patients with purified or recombinant FSH alone allows multiple follicle development, but produces inadequate estradiol concentrations. In some studies, fewer pre-ovulatory follicles developed compared with patients treated with combination of FSH and LH,19 while others have observed no differences.20 Administration of FSH without LH to hypogonadotropic hypogonadal patients results in lower serum and follicular fluid estradiol concentrations, normal inhibin concentrations, decreased endometrial thickness, reduced occurrence of ovulation, reduced oocyte fertilization rates, and lower embryo cryosurvival rates, when compared with hMG treatment.19–22 More importantly, no pregnancies were observed in these women when they received FSH alone for ovarian stimulation, despite estradiol replacement.20,23 In the same way women with primary amenorrhea and infertility attributable to a homozygous inactivating mutation in the LH receptor gene, exhibit low concentrations of estradiol, although ovarian histology reveals all stages of follicular development up to large antral follicles.24–25 FSH is undoubtedly the principle regulator of follicle development, but it is becoming increasingly evident that LH activity also has a direct effect on the developing follicle. In addition, LH had an indirect influence through the action of estrogen on the follicle. It was originally thought that the appearance of LH receptors in the granulosa cells was essential only to mediate the changes necessary for ovulation. LH receptors are known to exist on granulosa cells in the later stages of follicular development, so that these cells become responsive to LH stimulation in the mid- to late follicular phase of the menstrual cycle (after day 6, approximately).26

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Studies in rhesus monkeys that have received chronic treatment with a GnRH antagonist have led ZelinskiWooten et al to speculate that “LH exposure during the follicular phase may facilitate zygote viability and preimplantation development.27” These studies have illustrated that fertilized eggs from FSH-only treated animals are capable of establishing viable pregnancies.27,28 However, important differences have been noticed between the embryos from animals treated with recombinant FSH only and those treated with recombinant FSH plus recombinant LH.28 In particular, embryos from the FSH only treated animals showed a reduced viability after thawing and a slower rate of development from the morula to the hatched blastocyst stage during in-vitro culture.28 The reasons remain unclear and lead the authors to the view that “the contribution of LH during antral follicle development to subsequent embryogenesis warrants further study”28 An important emphasis in ovulation induction must be to achieve quality of resulting oocytes-and not just quantity. Analysis of data from clinical situations where the action of LH is absent has prompted further speculation regarding the possible involvement of LH in follicular development.29,30 Treatment of profoundly hypogonadotropic women with FSH alone results in inadequate estradiol secretion and, although multiple follicular development may be seen, several studies have found that the number of preovulatory follicles and their rate of development can be impaired.31 In 1998, a study in patients with profound hypogonadotropic hypogonadism, who are deprived of endogenous FSH and LH activity, demonstrated that LH administration is necessary to achieve proper follicle development and estrogen secretion. Patients who were treated with exogenous FSH-only had a reduced number of preovulatory follicles and lower ovulatory and pregnancy rates.32 Westergaard et al33 showed the mean fertilization rate to be significantly higher with hMG (56%) compared to PoFSH (50%) in their randomized trial of down-regulated normogonadotropic women. Soderstrom-Antilla et al34 compared PoFSH with hMG in their oocyte donation program and showed higher fertilization with hMG (36% versus 48% respectively). In a woman with isolated congenital gonadotropin deficiency, oocytes obtained in a LH depleted environment showed considerably lower fertilization rates with recFSH or PoFSH (27–28%) than with hMG (93%).35 Fleming et al36 have identified a subgroup of patients on GnRH agonists and PoFSH treatment who show profound suppression of endogenous LH. These cases, with mean serum follicular phase LH levels <0.5 IU/l, showed significantly lower E2 levels in the circulation and lower oocyte fertilization rates.37 Concern has also been raised about a higher incidence of failed fertilization in IVF cycles with low LH. Westergaard et al, in their randomized trial comparing PoFSH-HP with hMG, reported a significantly higher percentage of complete failure of fertilization with PoFSH-HP (16% vs. 6% respectively).33 Results with regard to the role of LH in embryo development vary. A few clinical studies examined the effects of different urinary gonadotropin preparations on embryo cleavage rates following IVF, and reported no significant differences.38,42 In contrast, others reported better cleavage rates43 and more transferable embryos44 with hMG treatment compared to recFSH. Embryo viability and pre-implantation development were also shown to be optimal in GnRH-antagonist treated macaques when folliculogenesis occurred in the presence of exogenous LH.28 A significant difference in embryo cryosurvival (56% after recFSH versus 78% af ter recFSH +recLH) and developmental rate to the hatched blastocyst stage (12 days versus 10 days respectively) was noted. The

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rescue of the corpus luteum also occurred later (16 days versus 12–13 days respectively).28,45 Fleming et al36 showed that while the degree of LH suppression in the follicular phase influences the number of embryos available, due to a lower oocyte yield and fertilization rates, no impact was noted upon the fertilized embryos to undergo cleavage and expanded blastocyst formation. In 1999 Filicori et al, in Bologna, Italy explored the role of LH in down regulated, normo-ovulatory women.31 All women were given a high purity FSH-only product (Metrodin HP), but half were also given LH stimulation (as small daily doses of hCG). On an average, development of medium and large follicles occurred 4 days earlier in those patients who received LH stimulation than in those patients who received FSH only.31 In December 1999 the same unit published a case report on a woman with hypogonadotropic hypogonadism.46 The patient underwent several IVF treatment cycles: in three cycles, ovulation induction involved pulsatile GnRH therapy and in one cycle, highly purified FSH (Metrodin HP) was used. All cycles were unsuccessful. In May 1998, a new cycle was started and this time the highly purified FSH was supplemented with daily subcutaneous administration of hCG. In this cycle, gonadotropin administration lasted 9 days and 17 ampoules of highly-purified FSH were used before ovulation induction. In the previous cycle, where no daily hCG was given, gonadotropin treatment lasted for 16 days and 45 ampoules of FSH were used. As a result of the FSH plus hCG regime, she gave birth to twins in January 1999,46 The authors state that the addition of the hCG “can hasten follicle development and reduce the dose and duration of treatment with highly-purif ied FSH without causing follicle luteinization or excessive theca cell stimulation.46” Some similar “experiments of nature” models, involving hypogonadotropic women, are less equivocal and folliculogenesis has been seen during FSH-only stimulation in low estrogen environments.47,48 This may be explained by the heterogeneity of hypogonadotropic hypogonadism syndromes-and the possible existence of very low level LH secretion in some cases. However, fertilization rates and embryo viability appear to be compromised.48,49Consequently, Filicori et al suggest that, although f olliculogenesis can progress during FSH-only stimulation in hypo-estrogenic conditions, “optimal oocyte cytoplasm and oolemma maturation require the presence of estrogens.50” Levy et at postulate that, while pre-ovulatory follicular development can occur in the absence of high estradiol and in the absence of significant follicular phase LH bioactivity, “profound LH suppression, and the consequently decreased steroid concentrations, may interfere with optimal oocyte maturation and/or endometrial development.51 GnRH antagonist treatment at high doses can mimic naturally occurring LH deficiencies52. In the European Ganirelix Multi-centre Dose-Finding Study the use of GnRH antagonist in the highest dose group (2 mg daily) during ovarian stimulation cycles with recFSH resulted in profound gonadotropin suppression, decreased estradiol concentrations, and shortening of the follicular phase. These observations were accompanied by low implantation (1.5%), and pregnancy rates (3.8%), and higher early miscarriage rates (13%).52 Interestingly, these disparate outcomes occurred even though the six different dose groups exhibited a similar number of antral follicles, oocytes recovered, fertilization rates, and numbers of transferable embryos.52

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The aim of a recent study53 was to investigate the effects of adding human menopausal gonadotropin (hMG) during controlled ovarian stimulation in normo ovulatory normogonadotropic patients showing an initial suboptimal response to a standardized long protocol therapy with recombinant FSH (recFSH) (300 IU/day). The data suggested that LH supplementation improves the ovarian outcome in patients characterized by an inadequate initial response to recFSH therapy in a long protocol.53 The need for LH in a normal reproductive cycle is well established and the diverse role of LH within the cycle is becoming increasingly understood. Consequently when administering exogenous gonadotropin treatments to women during infertility treatment, the issue is not whether LH is necessary or not-we know that it is. Rather, it is more a question of how much LH is necessary-and is it necessary to administer LH exogenously? Resting levels of LH seem to be adequate to occupy the very small number of LH receptors necessary to elicit maximum androgen synthesis within the theca cells. It has been demonstrated that less than 1 percent of LH receptors need to be occupied for maximum steroid synthesis to occur.54 Furthermore, inappropriately high levels of LH, as seen in patients with polycystic ovary syndrome (PCOS) have been linked with premature luteinization, reduced fertilization, and poor embryo quality. Consequently, it has been questioned whether use of exogenous LH is necessary for the majority of assisted reproduction treatment cycles-and whether it may in fact be detrimental. This particular question has been high on the fertility agenda for several years and remains the subject of much research and debate. In 1991, Chappel and Howles indicated that a high basal level of LH during the f ollicular phase compromised the number of oocytes recovered and the fertilization rates, in women who were treated with gonadotropins during assisted reproduction cycles.55 In 1995 Daya et al published a meta-analysis of 8 studies comparing FSH with hMG in IVF treatment cycles.56 Although no individual trial showed a significant difference between the treatments in clinical pregnancy rates, the meta-analysis showed that the use of FSH was associated with a significantly higher clinical pregnancy rate than hMG.56 The authors questioned the value of hMG in ovarian stimulation protocols in patients with no endogenous deficiency of LH. It is very important, however, that any analysis of the impact of LH within an assisted reproductive cycle takes into account the pituitary down-regulation protocols used and the level of pituitary desensitization achieved. The meta-analysis by Daya et at has been criticized for taking “no account of the differing protocols of pituitary desensitization that are commonly used in IVF treatment.57” According to the authors, “hormone profiles during these protocols vary so that conclusions made on an amalgam of all types of pretreatment are of uncertain value.57” Another author noted that the meta-analysis included trials both with and without GnRH agonist protocols and that studies included those with either short flare-up or long suppression protocols.58 Consequently, this “makes interpretation of the results of the meta-analysis controversial, as endogenous LH concentrations probably differed markedly from one study to another.58 Agrawal et at conducted a metaanalysis on the results of using FSH and hMG during IVF treatment cycles, taking into account the different pituitary desensitization protocols that were used.8 The combined analysis of the 3 studies with no pituitary desensitization (an extremely infrequent treatment strategy) showed that the FSH-only was significantly more effective in terms of clinical pregnancies per IVF cycle. The single study with a short GnRH agonist protocol showed no significant difference between FSH and hMG. In the 11 studies where the

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long GnRH agonist protocol used, results were in f avor of hMG in 8 of them; the metaanalysis showed no significant difference between FSH and hMG. The authors concluded that “although the results demonstrate that in all except 3 of the trials, the point estimate was in favor of hMG, the overall results were not statistically significant.8” In addition, “the results suggested that in the long and short GnRH agonist protocol of IVF; FSH and hMG were equally effective in achieving ovarian stimulation, and there were no differences in the clinical pregnancy rates per cycle of IVF. However, in protocols where no pituitary desensitization was used, FSH alone was more efficacious.8 Despite concerns regarding the detrimental effects of excessive serum LH, to date no single comparative study has demonstrated a significant clinical benefit for FSH-only versus hMG, in terms of pregnancy and birth rates. This confirms that it is essential to keep the physiologically important roles of LH distinct from the negative effects related to excessive and highly abnormal secretion of LH. Treatment cycles involving the ultra-short GnRH agonist regimens or no down-regulation at all are most likely to be associated with elevated serum LH levels and the unwanted effects linked to them. However, in the majority of circumstances the long GnRH agonist protocol is the pref erred mode of down-regulation (when compared to the short and ultra-short regimens) and is used much more frequently in assisted reproduction cycles. This protocol induces much lower follicular LH levels. Furthermore, there is an increasing use of GnRH antagonists for pre-treatment in assisted reproductive cycles. These drugs cause a rapid and profound LH and FSH suppression, which has heightened the awareness of the possible need to provide exogenous LH when administering gonadotropins. There are concerns that if GnRH antagonists are used, then FSH-only gonado tropins may be insufficient.59 According to Prof Paul Devroey, “an investigation has shown that neither follicular growth nor estradiol production takes place in the presence of this combined treatment (i.e. when GnRH antagonists are used in conjunction with recombinant FSH)”.60 Is it possible that the estradiol level could fall so low as to prevent pregnancy?60” The initial dose-finding studies for the GnRH antagonists reflect this concern. Serum LH levels have been shown to be inversely related to the dosage of Ganirelix,52 so that the highest dosage of Ganirelix results in the lowest serum LH levels (and vice versa). In this study, the lower dosages (0.25 mg/day and lower) were associated with a significantly greater pregnancy rate than those above 0.5 mg per day.52 These dosages resulted in pregnancy rates of 14 percent or lower (down to 0 percent for a dosage of 2 mg/day). Five patients in this study experienced zero or minimal follicular growth after treatment with the highest dosages of Ganirelix-and their treatment cycles were successfully rescued after switching from recombinant FSH to hMG. This study confirms the need for a minimum level of LH: “In view of the current findings, it needs to be assessed whether in women undergoing ovarian stimulation with recombinant FSH (instead of hMG), a single dose protocol with a relatively high dose of GnRH antagonist would be feasible.52 The concerns regarding the LH component of hMG have been shown to be unfounded in an increasing number of clinical studies.61–64 Furthermore, several of these studies have f uelled speculation that hMG may be preferable to FSH-only treatments, particularly when a more prof ound LH suppression has been achieved using the longer GnRH agonist regimens.63,64 In 1996, Check compared pregnancy and abortion rates in patients with luteal phase defects related to releasing eggs prior to complete follicular maturation

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in 164 patients.65 Patients were randomized to receive an ultra-low dose treatment with hMG or pure FSH. Pregnancy and abortion rates were similar in both groups, as were quantities of study drugs used. Check concluded that “these results show no advantage of choosing a preparation devoid of LH.65” Clinical Studies In 1997, Gordon et al compared the results from 128 cycles using recombinant FSH and different combinations of FSH and LH.66 A long protocol down-regulation was used and each patient was randomized to one of 4 different gonadotropin preparations: Group A received 75 IU FSH: O IU LH, Group B received 75 IU FSH: 1 IU LH, Group C received 75 IU FSH: 25 IU LH and Group D 75 IU FSH: 75 IU LH. Median numbers of follicles recruited, oocytes retrieved and embryos transferred were similar for all four groups. There was no significant difference between the groups in the number of cycles cancelled due to poor ovarian response. Clinical pregnancy rates were: Group A 28 percent, Group B 13 percent, Group C 27 percent, Group D 38 percent. Although the small number of cycles meant that the differences in pregnancy rates did not reach statistical significance, the authors concluded “Group D patients, with an FSH/LH ratio of 75:75 required a significantly lower number of ampoules for follicular development. No detrimental effects of the additional LH were noted on pregnancy and miscarriage rates66” and that the data indicates that “additional LH at a dose of 75 IU/ampoule is supportive of follicular growth 66.” In a separate analysis of the same treatment cycles, Gordon et al investigated the effects of differing concentrations of LH on oocyte maturation and fertilization.6 Group A (75 IU FSH: O IU LH) showed a significantly lower percentage of mature and fertilized oocytes and a correspondingly higher percentage of immature and unfertilized oocytes. The authors concluded “a dose-related trend in total fertilization failure was observed, with Group A, containing no LH, showing the highest rate of total fertilization failure” (6 cases failing to fertilize, versus 0 in Group D).66 In 1996, Westergaard et al investigated the hypothesis that the LH activity of hMG preparations may have adverse effects on the reproductive outcome.67 114 women received hMG (as Pergonal) and 104 women received FSH (as Metrodin). All women were subject to 14 days of pituitary down-regulation with a GnRH analogue, prior to gonadotropin therapy In this study, significantly more cycles in the FSH group were cancelled, the mean fertilization rate was significantly higher in the hMG group, significantly more transferable embryos were obtained in the hMG group. The clinical outcome was similar in both groups. The authors concluded “no detrimental effect of the LH activity of hMG on the clinical outcome of IVF in GnRH analogue down-regulated normogonadotrophic women was found.67” To the contrary, some beneficial effects of hMG on fertilization rates and embryo development as compared with FSH were demonstrated: They speculated that “a substantial subgroup of normogonadotrophic women require more than resting concentrations of LH for optimal oocyte maturation, and thus do less well on pure FSH stimulation.67” A further study examined the impact of circulating LH concentrations in the mid-follicular phase of ovarian stimulation on the outcome of IVF and ICSI procedures in normogonadotrophic women subjected to pituitary suppression with a GnRH analogue, followed by ovarian stimulation with recombinant FSH.68 The study analyzed 200 cycles in 200 women; all cycles involved pituitary down-regulation

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for 14 days, with a GnRH analogue, prior to gonadotropin therapy with recombinant FSH (Gonal-F). Serum LH concentrations were assessed on stimulation days 1 and 8. A serum LH concentration of 0.5 IU/l was defined as the threshold to discriminate between those women with a low LH level (less than 0.5 IU/l) and a high LH level (greater than 0.5 IU/l). Consequently, the study group was divided into 2 groups, based on their LH levels on stimulation day 8: those with low LH concentration <0.5 IU/l and those with normal LH concentration >0.5 IU/l. Serum estradiol concentrations on stimulation day 8 were significantly higher in the group with the normal LH concentration (> 0.5 IU/I). Both groups had a similar number of positive pregnancy tests-however, the group with a low LH concentration had a five-fold higher risk of early pregnancy loss, which was statistically significant.68 The authors concluded that women with low LH concentrations “had a significantly increased risk of an early termination of pregnancy and consequently a poorer chance of delivery.68” The study findings suggest that “low concentrations of LH have a detrimental effect on the outcome of IVF and that circulating LH concentrations above a certain critical level are required for optimal oocyte maturation.68” In addition, “although the present study does not offer an explanation for the detrimental effects of low LH concentrations, the results clearly raise the question of whether women with low mid-follicular LH concentrations would benefit from supplementation with exogenous LH-like activity during ovarian stimulation.68” Further confirmation of the potential benefit of exogenous LH in assisted reproduction comes from a large-scale randomized study that compared the impact of hMG and recombinant FSH on treatment outcomes in IVF and ICSI cycles.69 A total of 578 patients were recruited (hMG 282; recombinant FSH 296) and all received a short downregulation protocol prior to gonadotropin treatment. The clinical pregnancy rate per embryo transfer was 32.3 percent in patients receiving hMG and 30.1 percent in the group receiving recombinant FSH. In terms of secondary variables, there was no difference in terms of embryo quality and duration of stimulation between treatments. Significantly more oocytes were retrieved from patients receiving recombinant FSH; however, no difference was seen in the number of mature oocytes retrieved. The mean cumulative amount of gonadotropins needed to reach the criterion for hCG administration was significantly higher in the group receiving recombinant FSH (mean total dose 28.69 ampoules) than in the hMG group (mean total dose 20.16) ampoules. The authors comment that these findings may be explained by the results from Filicori et al**, discussed previously, in terms of the potential of the LH component of hMG to bring added economy and efficiency to assisted reproduction cycles. Data from these studies support the value of exogenous LH in hMG preparations when used in assisted reproductive cycles**. They indicate that the degree of pituitary desensitization should be considered when selecting the gonadotropin preparation during an IVF cycle. In addition, they endorse certain arguments in favor of exogenous LH and hMG, in particular, that the possible negative effects of excess LH are only likely to be seen when no desensitization protocol is used, or when the ultra-short protocol is used the perception of increased risk when giving additional LH (in hMG preparations) is likely to be misplaced, particularly when the much more commonplace longer protocols are used. That when the short and long protocols are used, FSH-only confers no additional benefit to hMG and that there may be additional benefits from using hMG during assisted reproductive cycles on a

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variety of parameters, including cost, fertilization rates, embryo transfer and pregnancy loss.69 CONCLUSION In conclusion, there is no evidence-based clinical argument that the LH content of the available gonadotropin preparations negatively affects the outcome of IVF treatments. It is possible that a substantial number of normogonadotrophic women are profoundly downregulated by standard GnRH agonist suppression and could benefit from the addition of LH to their stimulation protocol. It is difficult to detect which women will need additional LH administration in a reliable or cost-effective way, so it seems practical to systematically include LH in ovarian stimulation protocols. REFERENCES 1. Lunenfeld B, Lunenfeld E. Gonadotropic Preparations-Lessons Learnt. Fertil Steril 1997; 67(5):812–14. 2. Dodson WC, Hughes CL, Whitesides DB et al. The effect of leuprolide acetate on ovulation induction with human menopausal gonadotropins in polycystic ovary syndrome. J Clin Endocrinol Metab 1987; 65:95–100. 3. Meldrum D. GnRH agonists as adjuncts for in vitro fertilization Obstet Gynecol Surv1989; 44:314–17. 4. Meldrum DR, Wisot A, Hamilton F et al. Routine pituitary suppression with leuprolide before ovarian stimulation for oocyte retrieval Fertil Steril 1989; 1:455–59. 5. Hughes FG, Fedorkow DM, Daya S et al. The routine use of gonadotropin releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian tube of randomized controlled. Fertil Steril 1992; 58:888–90. 6. Cedars MI, Surey E, Hamilton E et al. Leuprolide acetate lowers circulating, bioactive luteinizing hormone and testosterone concentrations during ovarian stimulation for oocyte retrieval. Fertil Steril 1990; 53:627–31. 7. Soderstrom-Anttila V. Clinical outcome of ovulation induction: highly-purified FSH versus hMG. Ovulation induction: update ‘98 proceedings from the 2nd World Conference on Ovulation induction held in Bologna, Italy 1997. 1998; 193–200. 8. Agrawal R, Holmes M, Jacobs H. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis Fertil Steril 2000; 73:338–43. 9. Gore-Langton RE, Armstrong DT. Follicular steroidogenesis and its control. In Knobil E, NeilC (Eds): The Physiology of Reproduction. New York: Raven Press: 1994; 571–627. 10. Menopur Product Monograph, Ferring Pharmaceuticals A/S, Copenhagen, Denmark, 2002; 1– 39. 11. Hillier SG. Current concepts of the roles of follicle stimulating hormone and Luteinizing hormone in folliculogenesis. Hum Reprod 1994; 9:188–91. 12. Phelps I, Figueira-Armada L, Levine A, Vlahos N, Roshanfekr D, Zacur H et al. Exogenous luteinizing hormone (LH) increases oestradiol response patterns in poor responders with low serum LH concentrations. J Assist Reprod Genet 1988; 16:363–68. 13. Enmark E, Pelto-Huiko M, Nilsson S. Human oestrogen receptor- gene structure, chromosomal localisation and expression pattern). Clin Endocrinol Metab 1997; 82:4258–65.

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33. Westergaard LG, K Erb, S Laursen. The effect of human menopausal gonadotropin and highly purified, urine-derived follicle stimulating hormone on the outcome of in-vitro fertilization in down-regulated normogonadotropic women. Hum. Reprod. 1996; 11:1209–13. 34. V Soderstrom-Anttila, T Foudila, O Hovatta. A randomized comparative study of highly purified follicle stimulating hormone and human menopausal gonadotrophin for ovarian hyperstimulation in an oocyte donation programme. Hum Reprod 1996; 1:1864–70. 35. Balasch J, Vidal E, Penarrubia J. Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in downregulated women stimulated with recombinant FSH. Hum Reprod 2001; 16(8):1636–43. 36. Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T, Murdoch A. Effects of profound suppression of LH during controlled ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified FSH. Hum Reprod 1988; 13(9):1788–92. 37. Stouffer R, Dahl K, Hibbert M, WestonA, Wolf D, Zelinski-Wooten M. Relative roles of gonadotropins and intraovarian steroids on the outcome of ovulation induction. Ovulation induction: update ‘98. proceedings from the 2nd World Conference on Ovulation Induction held in Bologna, Italy 1997, 1–10, 1998. 38. Rabinovici C, Blankstein 1, Goldman B, Rudak E, DorY, Pariente C. In vitro fertilisation and primary embryonic cleavage are possible in 17 alpha-hydroxylase deficiency despite extremely low intrafollicular 17 beta-oestradiol. J Clin Endocrinol Metab 1989; 68:693–97. 39. McNatty KP, Smith MD, Makris A et al. The microenvironment of the human antral follicle: interrelationship among the steroid levels in antral fluid, and population of granulose cells, and the status of the oocyte in vivo. J Clin Endocrinol Metab 1979; 49:851–60. 40. Meirow D, Schenker JG, Rosler A. Ovarian hyperstimulation syndrome with low oestradiol in non-classical 17alphahydroxylase 17, 20-lyase deficiency: what is the role of estrogens? Hum. Reprod., 1996; 11:2119–21. 41. Meldrum DR. Blastocyst transfer—a natural evolution. Fertil Steril 1999; 72:216–17. 42. Catt KJ, Dufau ML. Spare receptors in rat testes. Nature 1977; 244:219. 43. Tanbo T, Hhaug E, Dale PO et al. Stimulation with human menopausal gonadotropin versus follicle stimulating hormone after pituitary suppression in polycystic ovarian syndrome. Fertil Steril 1990; 53(5):798–803. 44. Imthurn B, E Macas, M Rosselli et al. Nuclear maturity and oocyte morphology after stimulation with highly purified follicle stimulating hormone compared to human menopausal gonadotrophin. Hum Reprod 1995; 11:2387–91. 45. ChandrasekherYA, Hutchison JS, Zelinski-Wooten MB. Initiation of preovulatory events in primate f ollicles using recombinant and native human LH to mimic gonadotropin surge. J Clin Endocrinol Metab 1994; 79:298–306. 46. Filicori M, Cognigni GE, Taraborrelli S et al. Low-dose human chorionic gonadotropin therapy can improve sensitivity to exogenous follicle-stimulating hormone in patients with secondary amenorrhea. Fertil Steril 1999; 72(6):1118–20. 47. Couzinet B, Lestrat N, Brailly S et al. Stimulation of ovarian follicle-stimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab 1988; 66:552–56. 48. Shoham Z, Balen A, Patel A et al. Results of ovulation induction using human menopausal gonadotropin or purified folliclestimulating hormone in hypogonadotropic hypogonadal patients. Fertil Steril 1991; 56:1048–53. 49. Schoot DC, Harlin J, Shoham Z et al. 1994 Recombinant human follicle stimulating hormone and ovarian response in gonadotropin-deficient women. Hum Reprod 9:1237–42. 50. Filicori M. The role of luteinizing hormone in folliculogenesis and ovulation induction. Fertil Steril 1999; 71(3):405–14. 51. Levy D, Navarro G, Schattman G, Davis 0, Rosenwaks Z. The role of LH in ovarian stimulation. Exogenous LH: let’s design the future Hum. Reprod 2000; 15:2258–65. 52. The ganirelix dose finding study group. A double-blind, randomised, dose-finding study to assess the efficacy of the gonadotropin-releasing hormone antagonist ganirelix to prevent

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premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone Hum Reprod 1998; 13:3023–31. 53. De Placido G, Mollo A, Alviggi C et al. Rescue of IVF cycles by HMG in pituitary downregulated normogonadotrophic young women characterized by a poor initial response to recombinant FSH. Hum Reprod 2001; 16(9):1875–79. 54. Uilenbroek JT, Kramer P, Karels B et al. Significance of oestradiol for follicular development in hypogonadotropic immature rats treated with FSH and hCG. J Reprod Fertil 1997; 110(2):231–36. 55. Chappel SC, Howles C. Re-evaluation of the roles of LH and FSH in the ovulatory process. Hum Reprod 1991; 6:1206–12. 56. Daya S, Gunby D, Hughes E, Collins I, Sagle M. Follicle-stimulating hormone versus human menopausal gonadotropin for in vitro fertilization cycles: a meta-analysis Fertil Steril 1995; 64:347–54. 57. Filicori M. The role of luteinizing hormone in folliculogenesis and ovulation induction Fertil Steril 1999; 71:405–14. 58. Gordon U, Gordon A, Bonnar D, Harrison R. A randomised prospective study of the effect of LH on follicular growth and development Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997; 52:1997. 59. Vandervorst M, Devroey P. Recombinant FSH: results in assisted reproduction Ovulation induction: update ‘98: proceedings from the 2nd World Conference on Ovulation Induction held in Bologna, Italy 1997. 1998; 137–46. 60. Devroey P. The Role of Gonadotrophins in Assisted Reproduction-Present and Future; edited proceedings of Consensus Development Conference, Canada, 1998; 12. 61. Rekha P, Mowat L, Jamieson ME et al. Effect of profound suppression of luteinising hormone during treatment with gonadotoropin releasing hormone analogue and purified follicle stimulating hormone upon development of cryopreserved embryos. Hum Reprod 1998; 13(3):696–98. 62. Jacob S, Drudy L, Conroy R et al. Outcome from consecutive invitro fertilization/intracytoplasmic sperm injection attempts in the final group treated with urinary gonadotrophins and the first group treated with recombinant follicle stimulating hormone. Hum Reprod 1998; 13(7):1783–78. 63. Jansen CA, van Os HC, Out HJ et al. A prospective randomized clinical trial comparing recombinant f ollicle stimulating hormone (Puregon) and human menopausal gonadotrophins (Humegon) in non-down-regulated in vitro fertilization patients. Hum Reprod 1998; 13(11):2995–99. 64. Gordon UD, Gordon AC, Bonnar J et al. Chronically Administered hMG or FSH in PCOS Leads To A Progressive Decline of Endogenous LH With Reduction Of Peripheral LH Concentrations. Hum Reprod 1997; 12(Suppl 1):53–57. 65. Check JH. Low-dose gonadotropin stimulation for luteal phase defects-does absence of LH help pregnancy rates? Del Med Art 1996; 68 223–26. 66. Gordon U, Gordon A, Bonnar D, Harrison R. Effect of differing doses of LH on oocyte maturation and fertilisation. Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997; 53–54. 67. Westergaard L, Erb K, Laursen S, Rasmussen P, Rex S. The effect of human menopausal gonadotropin and highly purified, urine derived follicle stimulating hormone on the outcome of in vitro fertilisation in down-regulated normogonadotrophic women Hum Reprod 1996; 11:1209–13. 68. Westergaard L, Laursen S, Andersen. Increased risk of early pregnancy loss by prof ound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction Hum Reprod 2000; 15 1003–08. 69. Strehler E, Abt M, EI-Danasouri I, De Santo M, Sterzik K. Impact of recombinant FSH and hMG on in vitro fertilisation outcome Fertil Steril 2001; 75:332–36.

CHAPTER 17 Luteal Phase Support Valery N Zaporozhan, Ruslan V Sobolev INTRODUCTION Luteal support is necessary in ovarian stimulation protocols, such as those usually prescribed for the in vitro fertilization (IVF) and embryo transfer (ET),1 as well as for the cycles with Intracytoplasmic Sperm Injection (ICSI) and in cryopreserved embryo replacement cycles.2 Exogenous progesterone supplementation has been well established in the treatment of ovulatory dysfunction as well as in standard infertility treatment protocols. Furthermore, exogenous progesterone is mandatory as part of hormonal replacement therapy in agonadal women undergoing oocyte donation program followed by IVF-ET. Physiology and Morphology of the Endometrium and its Correlation in Different Phases of the Menstrual Cycle Physiological and morphological changes of the endometrium are triggered by estrogens and progesterone secreted cyclically by the ovaries during the menstrual cycle of a women. The cyclic changes in the ovarian secretion is under control of the hypothalamohypophyseal hormones’ system and so endometrium morphology may be claimed as indicator of the proper activity of this functioning structure. During one cycle endometrium undergoes following changes starting from menstruation or desquamation transiting onto the regeneration phase, proliferation and then onto the secretory transformation, in case of absence of fecundation, menstruation occurs again. Such cyclic modifications in morphological structure of the endometrium provide favorable conditions for the embryo’s nidation that in natural cycle usually occurs on the 5th-7th day af ter fertilization of the oocyte. Implantation is a complex process, which requires interaction of the blastocyst and subsequently the developing embryo with the endometrial tissues. An extremely coordinated order of events is essential to enable the endometrium to become receptive to embryo implantation during a short and exactly defined period in the menstrual cycle. Two sets of factors one of which is endometrial and the other, embryonic, are obligatory for the creation of proper interaction between the blastocyst and the endometrium. It includes the systemic signals, steroid hormones and their receptors along with local factors, that are present in the uterine cavity and necessary for the adequate correlation of all the participating agents. The role of each of these factors has been reviewed several lines in the literature.3,4 The luteal or secretory phase starts after ovulation and lasts from the 14th till the 28th day of the menstrual cycle, if its length comprises 28 days in general, and if the cycle is ovulatory. Progesterone induces secretory transformation of the endometrium and also has an anti-estrogenic effect expressed in inhibition of the oestrogen receptors’ synthesis and in disruption of the estrogen metabolism. Features of secretory transformation are

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most extensively expressed on the 21st day of the cycle and usually coincided with the implantation of the blastocyst in case of fertilization. This time period the “implantation window” represents a short period of time when the uterine environment is best suited for the implantation of the blastocyst. Adequate secretory transformation of the endometrium at this period is essential for satisfactory embryonic implantation during the ‘implantation window’. Ultrastructurally, pinopodes are evidenced during this period, indicating that the endometrial surface epithelium is receptive to the eventual implantation of a blastocyst.4,5 They are progesterone-dependent cellular organelles appearing for only 2 or 3 days between days 19 and 21 of the normal menstrual cycle.5 When the period of “implantation window” is over, the receptivity of the endometrium decreases tremendously coming down to the total refractory period and making nidation and implantation impossible. Another perspective on the endometrium’s readiness to accept the fertilized oocyte was attempted by the study of specific gene expression during this critical period. It was shown that the expression of a crystallin B mRNA was absent during the proliferative phase and progressively increased in the secretory endometrium.6,7 Immunoreactive protein appear on the surface epithelial cells and the underlying glandular epithelium during the implantation window, and the immunoreactivity intensifies in the glandular epithelium during mid-and late secretory phases.7 Later on, the role of progesterone in the regulation of α crystallin B chain in human endometrial epithelium was examined in the in-vivo experimental model. The results of this investigation showed that single administration of 5 mg medroxyprogesterone acetate (synthetic progesterone derivative) significantly enhances the expression of a crystallin B chain mRNA as early as day one and persists for a longer period of time.6 Although at this time no specific function can be ascribed to á crystallin, which is a member of the family of small heat shock proteins, its expression in the epithelium of secretory human endometrium suggest that this protein may have role in protecting the native forms of other protein(s) essential to implantation. The timing of the cycle described above, and time of nidation is not always true for stimulation protocols for IVF when oocyte retrival takes place on the 12–16th day of the stimulated cycle. Embryo transfer is assumed to be perform best at the blastocyst’s stage of the embryo’s development that corresponds to the 4th or 5th day af ter oocyte retrieval. So, in practice, it is not always that embryo transfer is performed on the 21st day of the cycle when the ‘implantation window’ period is expected. It is all in power of the clinician to determine the dates of the luteal phase support initiation, adequate dosage and the route of intake for affecting proper changes in the endometrium’s morphology along with sufficient corpus luteum function. Corpus Luteum and its Function Corpus luteum presents itself as a complex endocrine tissue, whose function is under the regulation of extrinsic gonadotrophic stimulae and intrinsic regulationby a large number of paracrine and autocrine factors. The corpus luteum of the ovary, under the influence of luteinizing hormone (LH) is responsible for the production of a number of hormones with progesterone being essential for the endometrial secretory transformation, promotion of blastocyst nidation, and establishment of early pregnancy. It was demonstrated that the corpus luteum is dependent on the LH at all stages of its development8, and even if it may

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recover to some degree after 3 days absence of LH influence, longer deprivation results in luteolysis. The corpus luteum is a highly relevant tissue with many events observed during its development cycle. Extensive angiogenesis occurs within it, such that, the mature structure possesses plenty of endothelial cells. Hormone-producing theca-cells control this proliferation through the production of the vascular endothelial grpwth factor which is likely to be under LH control.8 At the same time, in further development, corpus luteum presents a site of rapid cell death and tissue remodeling, so apoptosis becomes an attribute of its regression. And the purpose is that these processes should be slowed down in the infertility treatment cycle and the equilibrium between the factors promoting development of the corpus luteum and factors of its regression should be balanced in order to maintain progesterone production at a sufficient level for the luteal phase and establishment of pregnancy in the woman. Luteal insufficiency, mostly unrecognized by the generally applied diagnostic procedures is the primary cause of recurrent pregnancy loss especially in the first few weeks after nidation and implantation events. Incidence of luteal insufficiency among patients with recurrent pregnancy loss comprise about 93%.9,10 Poor luteinization plays an important role in patients experiencing first trimester habitual abortions or in patients with inability of nidation and implantation. Luteal insufficiency of varying degrees is the principal cause of different forms of adverse pregnancy outcomes including early pregnancy loss, habitual abortion, spontaneous abortion, unexplained infertility, etc. Moreover, it was proved that luteal phase defects are responsible for the first trimester habitual abortions but not for the repeated mid-trimester abortions.10 Assessment of the Endometrial Maturity Endometrial biopsy is obviously the best procedure for investigation of the endometrial tissue and determination of the endometrial receptivity according to the criteria elaborated by Noyes.11 Insufficient luteal phase is clearly reflected by the morphological features presented in the biopsy material. For better assessment, biopsy should be provided on the 22nd-24th day of the cycle or 4–5 days after the ovulation. The ovulation date must not be estimated according to the date of last menses, but by detection of the pre-ovulatory LH-peak because of the possibility of cycle variations due to the varying duration of the proliferative phase. Luteal phase insufficiency is expressed by the delay in the endometrial maturation which may be definitely revealed by the histologic examination. A diagnosis of endomentrial maturation retardation should be made only after two biopsies that were taken in different cycles. Retardation of 2 days is considered incompatible with viable implantation of an embryo. Another alternative, less precise, but non-invasive method of endometrium maturation grade evaluation is ultrasonographical examination. It allows the follow up of the endometrial development from the early regeneration stage up to the ovulation and secretory phase along with the corresponding changes in the ovaries. Detection of follicle development and timely maturation of endometrium is essential for ovulation induction and is carried out in every single treatment protocol even though there is no correlation between luteal phase deficiency and endometrial echo pattern in the ovulatory period detected.12 The mid-luteal phase patterns of the endometrial echo allow the detection and consequent correction of the signs of the second phase insufficiency.

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Role of integrated progesterone threshold value in defining histologic endometrial retardation was investigated for the purpose to assess its diagnostic value as a marker of luteal phase deficiency It was proved that integrated progesterone concentrations in women with diagnosed luteal phase deficiency were significantly lower than in women without luteal phase deficiency, receiving infertility treatment.13 Medications for the Luteal Phase Support Progesterone and human chorionic gonadotropine (HCG) are the two main medications ordinarly used for luteal phase support in infertility treatment protocols. It has been suggested that HCG might be superior to progesterone in gonadotrophin-releasing hormone (GnRH) agonist cycles for IVF,14 but at present, with a more deep understanding of progesterone’s influence onto the endometrium and its mechanism of action it is used in almost every treatment cycle. Progesterone acts on the epithelium and stroma of the endometrium via the specific receptors which are proteins located in the nucleus of the endometrial cells and have specific affinity for progesterone. Two receptor subtypes A and B have been discovered in the endometrium.15 Progesterone receptor synthesis is regulated by estrogens through the estrogen receptor that is a good marker for endometrial estrogen dependence. Progesterone receptor levels are the highest during the preovulatory and immediate postovulatory periods during which serum estrogen titers are the highest.16 That is why estrogen priming during the follicular phase is so essential to ensure response to progesterone during the luteal phase. Hormonal supplementation by vaginal estradiol followed by vaginal progesterone in the ovulation induction with clomiphene citrate corrects delayed endometrial development in 100% of women.17 This suggests that adding vaginal estradiol preceding progesterone support in clomiphene citrate ovulation induction allows the normalize action of endometrial morphology. Proliferative phase that is under estrogen’s control during which progesterone receptor synthesis takes place is therefore essential for good secretory differentiation. Progesterone itself is capable of inhibiting synthesis of estrogen receptor, consequently, number of both estrogen and progesterone (subtype A and B) receptors in the epithelium decreases considerably after ovulation, with estrogen receptors disappearing completely from the stroma.16 Since the subtype B antibody fails to stain stromal nuclei during the secretory phase,15 it obviously evidences that progesteroneinduced changes in the endometrium during the luteal phase appear to be mediated primarily via subtype Areceptors. It is notable that endometrial accumulation of progesterone was higher in the luteal phase than in the proliferative phase.18 This might be attributed to two causes: the higher concentration of progesterone receptors in the early luteal phase and the existence of different patterns of uterine contractions during the mentstrual cycle.19 Uterine contractions’ frequency increases from early follicular to periovulatory phase of the normal menstrual cycle and then decreases progressively during the luteal phase. Lately obtained results indicate that uterine contraction characteristics are influenced by plasma progesterone concentrations during the luteal phase and on the day of embryo transfer as well, and are refractory to the changes in plasma estradiol concentration on the day of HCG administration and embryo transfer. Moreover, on the day of embryo

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transfer in comparison with the day of HCG administration, only patients displaying high progesterone concentrations (>100 ng/ ml) experienced a reduction in uterine contractions frequency.21 The fact that uterine contractility may influence sperm transport, oocyte migraton through tubes, embryonic transport from tubes to the uterine cavity and possibly embryo implantation itself makes the importance of luteal phase support more obvious.22 This suggests that high tissue concentrations of progesterone are needed to alter uterine contractility during the early luteal phase of ovarian stimulation since intense contractile patterns during ovarian stimulation for IVF and at the time of embryo transfer are associated with lower implantation and pregnancy rates.23 All progesterone medications can be divided into two main groups: natural progesterone and derivatives, synthetic progesterone and its derivatives. They might also be conditionally divided into groups by the different routes of progesterone deliveryintranasal,24,25 sublingual,25,26 rectal,25,27 oral,25,28 intramuscular (i.m.)25,29 and vaginal.25,20,31 Oral, i.m. and vaginal routes of the progesterone administration have been mostly investigated and compared in significant number of prospective and retrospective trials for implantation and pregnancy rates af ter each way of administration. Natural progesterone is quickly inactivated when is taken orally because of its rapid metabolism in liver and intestines. Therefore, synthetic derivatives were developed to improve its bioavailability and to make it resistant to enzymatic degradation. Attempts to improve the bioavailability of natural progesterone were investigated because of the limited therapeutic value of synthetic progestins due to the number of undesirable effects, especially on lipids25,33 and psychological effects34 such as sedative and hypnotic effects of progesterone metabolites on the affinity of GABA-receptors in the central nervous system18 and possible teratogenic effects.18,35 In addition, synthetic progestogens, mainly those with androgenic properties, have been associated with an increased risk of fetal congenital malformations.18,36 Natural progesterone has no adverse effects on high density lipoproteins,33 no known teratogenicity37,38 and is more effective in inducing secretory endometrial features.25 Reduction of the particle size of progesterone by micronization enhances its bioavailability39 However, the micronized form of a single oral dose is inefficient to provide adequate concentrations throughout the day,40 though it was shown that absorption of micronized progesterone was enhanced two-fold in the presence of food.28 Nevertheless due to the convenience of orally administered progesterone, its use is somehow is associated with systematic adverse effects, such as drowsiness, flushing and nausea.41,42 After oral digestion, progesterone, is rapidly absorbed and metabolized from the intestines and during the first hepatic pass, cleared from the circulation.43 After ingestion of 200 mg of micronized progesterone, mean serum progesterone concentrations (within the range of luteal phase are attained in 2–4 hours and remain significantly elevated for the next 6–7 hours.42,44 Anyway oral progesterone was inappropriate for inducing the endometrial transformations normally seen in the luteal phase29 and providing adequate support during the luteal phase in IVF cycles.45 Intramuscular delivery of the progesterone is uncomfortable and associated with pain from daily injections, occasional sterile abscesses or allergic responses to the oil component. Especially, this is of a great concern for women undergoing oocyte donation program or patients with ovarian failure who are in need of a long-term treatment for pregnancy support. Progesterone is rapidly absorbed after i.m. administration and its high

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plasma concentrations are achieved within 2 hours and peak concentrations are reached within 8 hours.28 Serum concentrations equivalent to those seen during the luteal phase have been attained after the injection of 25 mg progesterone.25 Comparing oral micronized progesterone with i.m. progesterone administration revealed that relative bioavailability of oral progesterone was significantly lower than that observed after i.m. progesterone though there is a certain percent of patients presented a delayed rate of progesterone absorption after i.m. administration.25 So, a comparison of oral micronized progesterone to i.m. progesterone supported the use of the latter, as it resulted in significantly higher implantation rates.45 At present vaginal route of progesterone administration is considered a the priority choice for both clinicians and patients.30,31,32,46 Advantages of the vaginal delivery are the following: vaginal route has a uterine efficacy that far exceeds the serum progesterone levels achieved because of avoidance of first pass hepatic metabolism and relatively high bioavailability along with rapid absorption. It also prevents pain that is associated with the i.m. route, and undesirable side-effects of the oral synthetic progestines. The most important is the so-called uterine first pass effect47 which presents homogeneous distribution of progesterone throughout the endometrium that suggests proper local endometrial effect with secretory transformation. Four main mechanisms explaining routes of the hormone transition f rom vagina to the uterus were suggested: first is the direct diffusion through tissues,47 second is the intraluminal passage from the vagina to the uterus,48 third is the venous or lymphatic circulatory system,49 the fourth is the counter-current transfer between utero-vaginal veins or lymph vessels and arteries.50 Evidence of the existence of such a mechanism was proved by nearly 10-fold high endometrial concentrations of progesterone af ter the vaginal administration of 100mg micronized progesterone as compared to the same dose given i.m..51 Furthermore, full secretory transformation of the endometrium is produced when serum levels of 1–3 ng/ml of progesterone are produced by vaginal administration, but not when produced by i.m. or nasal delivery.24,47 After vaginal application of micronized progesterone, endometrial morphology closely matched that of the natural cycles, while the endometrium was heterogeneous or inadequate in both i.m. and oral groups.40 So, micronized progesterone vaginal capsules, sustained-release polycarbophil gel or vaginal ring releasing progesterone is probably the best clinical option for progesterone delivery Human chorionic gonadotrophin (HCG) is the other medication used for the luteal phase support in IVF treatment cycles. Luteal support by HCG administration overcomes luteal phase inadequacy after GnRH agonist induced ovulation in gonadotrophinstimulated cycles.52 Therefore, a potential advantage of HCG over progesterone is that HCG administration may be more physiological than progesterone because it may assist in stimulating the secretion of other factors important to endometrial maturation. There are also some controversial studies of HCG usage showing no significant difference in pregnancy rates af ter administration of HCG for luteal support compared to protocols with progesterone luteal support.53 It is not effective in preventing first trimester abortion for patients with polycystic ovaries syndrome with elevated LH concentrations.54 Moreover, it is important from a perspective of the risk of ovarian hyperstimulation syndrome development. When the estradiol concentration is >2700 pg/ml54 or the number of follicles is >10,55 luteal support with HCG should be avoided.

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Luteal support by HCG significantly improved clinical and ongoing pregnancy rates as compared with oral micronized progesterone 400mg, especially in short protocols with GnRH agonist and to a lesser degree in long ones.55 HCG supplementation was found to be superior to i.m. progesterone for the ultrashort protocol using GnRH agonist/HMG57 and in patients with firsttrimester habitual abortions due to luteal phase insufficiency.58 Protocols of HCG administration vary individually for each patient, though approximate scheme for its prescriptions is 3000 to 5000 IU of HCG on the day of embryo transfer and then 1500 to 3000 IU HCG 3 and 8 days af ter the embryo transfer. Luteal phase support by both progesterone and HCG considered to be beneficial.18,25,52 Duration of the luteal phase support by either progesterone or HCG was the matter of several investigations. If the pregnancy test is found to be positive, progesterone support should be continued up to 30 days after the embryo transfer,56 until fetal heart activity is seen 58 or until the 12th week of gestation.59 On the other hand, lately conducted investigation studied rationality for continued administration of progesterone in case of pregnancy after IVF and stated that progesterone supplementation beyond the 2 first weeks of pregnancy has no effect on the ongoing pregnancy rate.60 Luteal phase support and endometrial maturation may be provided and controlled by the small dosages (5mg once a day) of prednisolone and aspirin (75–100 mg per day). Both of them are non-specific medications, which have some direct influence on reproductive processes, though prednisolone possesses significant anti-inflammatory, immunosuppressive activity. The immunosuppressive activity of prednisolone is especially important in couples with a history of immunologic incompatibility in view of the fact that every embryo is genetically and immunologically foreign to mother’s organism. Hormonal activity primarilly relies on its ability to cessate hypophyseal ACTH secretion, leading to decrease in the corticosteroids’ and androgens’ production that has an indirect influence on the general hormonal balance. It also considerably influences the metabolism, improving protein catabolism, increasing sodium reabsorption, potassium and hydrogen excretion. Prescription of such medication should be made very cautiously because of multiple contradictions and limitations for its usage. Aspirin along with antipyretic, analgesic and antiinflammatory properties also decreases thrombocyte aggregation. It is capable of cyclooxygenase inactivation that results in prostaglandins, prostacycline and tromboxane synthesis disruption As the result of prostaglandin synthesis reduction, the sensitizing activity of prostaglandin decreases Aspirin also leads to irreversible disruption of the thromboxane A synthesis in thromobocytes, stipulating antiaggregant property. Acetylsalicylic acid also blocks cyclooxygenase in the epithelial cells, here prostacycline is synthesized. Having such considerable influence on rheologic properties and prostaglandin synthesis, it should be considered as an assistant drug for inclusion in the infertility treatment protocols. Separate administration of small doses of either prednisolone or aspirin increase rates of nidation and implantation in women undergoing IVF programs but combined administration of both of them leads to reduction of those rates.61 Vitamins and minerals should be obligatory components for promotion of the satisfactory oxidation/ reduction processes, preventing possible insufficiency of vitamin intake with diet.

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CONCLUSIONS Luteal phase support is an obligatory component in the infertility treatment protocol. It should be performed by administration of progesterone medications such as oral, i.m. or vaginal forms. Dosages and routes for its administration should be the decision of the doctor along with patient’s preference. Increaseing evidence in the literature suggesting that vaginal progesterone might be superior to other routes, mainly due to the described first uterine pass effect, which results in a better local progesterone bioavailability in the uterus. Luteal support may be also provided by HCG administration on the day of embryo transfer and 3 and 6 days after it on the background of progesterone supply. Either prednisolone or aspirin may be components facilitating endometrial maturation and embryo nidation and implantation. REFERENCES 1. Belaisch-Allart J, De Mouzon J, Lapousterle C et al. The effect of HCG supplementation after combined GnRH-agonist/HMG treatment in an IVF program. Hum. Reprod., 1990; 5:163–166. 2. Miller KF, Fry KL, Arcaiga RL et al. Use of vaginal progesterone gel in cryopreserved embryo replacement cycles results in decreased ongoing pregnancy rate. Abstr. Of the 16th Ann. Meet. of the ESHRE., 2000; 131. 3. Beier HM, Beier-Hellwig K. Molecular and cellular aspects of endometrial receptivity. Hum. Reprod. Update, 1998; 4:448–458. 4. Carson DD, De Sourza MM, Kardon R et al. Mucin expression and function in the female reproductive tract. Hum. Reprod. Update, 1998; 4:458–464. 5. Nikas G, Psychoyos A. Uterine pinopodes in peri-implantation human endometrium. Clinical relevnce. Ann. N. Y Acad. Sci., 1997; 816:129–142. 6. Satyaswaroop PG, Tabibzadeh B. Progestin regulation of human endometrial function. Hum. Reprod. 2000; 15(Suppl. 1):74–80. 7. Gruidl M, Buyuksal A, Babakina A et al. The progressive rise in the expression of α-crystallin B chain in human endometrium is initiated during the implantation window: Modulation of gene expressionby steroid hormones. Mol. Hum. Reprod., 1997;3:333–342. 8. Fraser HM, Duncan WC, Illingworth PJ et al. Endocrine control of the corpus luteum. Abst. of the 13th Ann. Meet. of the ESHRE, 1997; 28. 9. Siklosi Gm, Gimes G, Bakos L et al. Primary role of luteal insufficiency in recurrent pregnancy loss. Abst. of the 13th Ann. Meet. of the ESHRE, 1997; 331. 10. Sallam HN, Agamia AF Ezzedin F, Sallam AN. Luteal phase defects in patients with first trimester habitual abortions. Abstr. of the 13th Ann. Meet. of the ESHRE, 1997; 194. 11. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fetil. Steril., 1950; 1:3–25. 12. Hassa H, Yildirim A, Sener T, Tarmergen E et al. The place of ultrasonographical endometrial echo evaluation in the detection of luteal phase deficiency, Abst. of the 13th Ann. Meet. of the ESHRE, 1997; 46. 13. Yildirim A, Hassa H, Tekin B et al. The integrated progesterone threshold value in the assessment of the luteal phase deficiency. Abst. of the 13th Ann. Meet. of the ESHRE, 1997; 126. 14. Soliman S, Daya S, Collins J, Hughes E. The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil. Steril., 1994; 61:1068–1076. 15. Wang H, Critchley HOD, Kelly RW et al. Progesterone receptor subtype B is differentially regulated in human molecular stroma. Mol. Hum. Reprod., 1998; 4:407–412.

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16. Bergeron C. Morphological changes and protein secretion induced by progesterone in the endometrium during the luteal phase in preparation for nidation. Hum. Reprod., 2000; 15(Suppl. 1):119–128. 17. Elkind-Hirsch K, Philips K, Keller MG, Bello S et al. Hormone supplementation (vaginal oestradiol and Crinone 8%) corrects delayed endometrial development during the late luteal phase. Abtr. of the 16th Ann. Meet. of the ESHRE, 2000; 174. 18. Posaci C, Smitz J, Camus M et al. Progesterone for the luteal support of assisted reproductive technologies: clinical options. Hum. Reprod. 2000; 5(Suppl. 1):129–148. 19. Buletti C, Prefetto RA, Bazzocchi G et al. Electromechanical activities of human uteri during extra-corporal perfusion with ovarian steroids. Hum. Reprod., 1993; 8:1558–1163. 20. Buletti C, Ziegler D, Polli V et al. Uterine contractility during the mentstrual cycle. Hum. Reprod., 2000; 15 (suppl. 1):81–89. 21. Fanchin R, Ayoubi J-M, Olivennes F et al. Hormonal influences on the uterine contractility during ovarian stimulation. Hum. Reprod., 2000; 15(Suppl. 1):90–100. 22. Buletti C, De Ziegler D, Rossi S et al. Abnormal uterine contractility in non-pregnant women. Ann. N. Y. Acad. Sci, 1997; 828:223–229. 23. Fanchin R, Righini C, Olivennes F et al. Uterine contractions at the time of embryo transfer after pregnancy rates in in vitro fertilization. Hum. Reprod., 1998; 13:1968–1874. 24. Cicinelli E, Nahoull K, Petruzzi D et al. Nasal spray administration of unmodifed progesterone: evaluation of progesterone serum levels with three different radioimmunoassay techniques. Maturitas, 1994; 19:43–52. 25. Tavaniotou A, Smitz J, Bourgain C, Devroey P. Comparison between different routs of progesterone administration as luteal phase support in infertility treatments. Hum. Reprod. Update, 2000; 6(2):139–148. 26. Stovall D, Van Voorhis B, Mattingly K et al. The effectiveness of sublingual progesterone administration during cryopreserved embryo transfer cycles: results of a matched follow-up study Fertil. Steril, 1996:65:986–991. 27. Chakmakjan ZH, Zacharian NY Bioavailability of progesterone with different modes of administration. J. Reprod. Med., 1987; 32:443–448. 28. Simon J, Robinson D, Andrews M et al. The absorption of oral micronized progesterone: the effect of food, dose proportionality and comparison with intramuscular progesterone. Fertil. SteriL, 1993; 60:26–33. 29. Bourgain C, Devroey P, Van Waesberghe L et al. Effects of natural progesterone on the morphology of the endometrium in patients with primary ovarian failure. Hum. Reprod., 1990; 5:537–543. 30. Alper MM, Penzias AS. Crinone offers excellent implantation rates in patients undergoing IVF. Abstr. of the 16th Ann. Meet. of the ESHRE, 2000; 123. 31. Schoolcraft WB, Hesla JS, Gee MJ. Experience with progesterone gel for luteal support in a highly successful IVF programme. Hum. Reprod.2000; 15(6):1284–1288. 32. Zegers-Hochschild E, Balmaceda JP, Fabres C et al. Prospective randomized trial to evaluate the efficacy of a vaginal ring releasing progesterone for IVF and oocyte donation. Hum. Reprod. 2000; 15(10):2093–2097. 33. Ottosson UB, Johansson BG, von Schoultz B. Subfractions of high-density lipoprotein cholesterol during estrogen replacement thrapy: a comparison between progestogens and natural progesterone. Am. J. Obstetr. Gynecol, 1985; 151:746–750. 34. Sherwin J, Gelfand M. Aprospective one-year study of estrogen and progestin in postmentopausal women: effects on clinical symptoms and lipoprotein lipids. Obstet. Bynecol., 1989; 73:759–766. 35. Hendrickx AG, Korte R, Leuscner F et al. Embryotoxicity of sex steroidal hormone combinations in nonhuman primates: I Norethisterone caproate+ethinylestradion and progesterone+ estradiol benzoyate (Macaca mulatta, Macaca fascicularis, and Papio cynocephalus). Teratology, 1987; 35:119–127.

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36. Aarskog D. Maternal progestines as a possible cause of hypospadias. N. Engl. J. Med.,1979; 3000:75–78. 37. Rock J, Colston Wentz A, Cole K et al. Fetal malformations following progesterone therapy during pregnancy: a preliminary report. Fertil. Steril., 1985; 44:17–19. 38. Check JH, Rankin A, Teichman M. The risk of fetal anomalies as a result of progesterone therapy during pregnancy. Fertil Steril., 1986; 45:575–577. 39. Hargrove JT, Maxon WA, Wentz AC. Absorption of oral progesterone is influenced by vehicle and particle size. Hum. Reprod., 1989; 8:1372–14375. 40. Devroey R, Palermo G, Bourgain C et al. Progesterone administration in patients with absent ovaries. Int. J. Retil. 1989; 34:188–193. 41. Mazon W, Hargrove J. Bioavailability of oral micronized progesterone. Fertil. Steril.,1985; 44:622–626. 42. Pouly JL, Bassil S, Frydman R et al. Luteal support after in vitro fertilization: Crinone 8%, a sustained release vaginal progesterone gel, versus Utrogestan, an oral micronized progesterone. Hum. Reprod., 1996; 11:2085–2089. 43. Nahoul K, Dehennin L, Jondt M et al. Profiles of plasma estrogens, progesterone and their metabolites after oral or vaginal administration of estradiol or progesterone. Maturitas, 1993; 16:185–202. 44. Norman T, Morse C, Jennerstein L. Comparative bioavailability of orally and vaginally administered progesterone. Fertil. Steril, 1991; 56:1034–1039. 45. Liccardi FL, Kwiatkowski A, Noyes NL et al. Oral versus intramuscular progesterone for in vitro fertilization: a prospective randomized study Fertil. SteriL, 1999; 71:614–618. 46. Cicinelli E, De Ziegler D, Matteo MG. Vaginal administration maximizes delivery of progesterone to the uterus through a local vagina-to-uterus functional “portal” system. Abstr. of the 16th Ann. Meet. of the ESHRE, 2000; 89. 47. Bulletti C, De Ziegler D, Flamigni C et al. Targeted drug delivery in gynaecoligy: the first uterine pass effect. Hu. Reprod., 1997; 12:1073–1079. 48. Wildt L, Kissler Sl, Licht R, Beckler W. Sperm transport in the human female genital tract and its modulation by oxytocin as assessed by hysterosalpingoscintigraphy, hysterotonography, electrohysterography and Doppler sonography. Hum. Reprod. Update, 1998; 4:655–666. 49. Magness RP, Ford SP. Estrone, estradiol-17 beta and progesterone concentrations in uterine lymph and systemic blood throughout the porcine estrous cycle. J. Anim. Sci., 1983; 57:449– 455. 50. Einer-Jensen N, Kotwica J, Krzymovski T et al. Rapid absorption and local redistribution of progesterone after vaginal application in gilts. Acta Vet. Scand., 1993; 34:1–7. 51. Miles R, Paulson R, Lobo R et al. Pharmacokinetics and endometrial tissue levels of progesterone after administration by i.m. or vaginal routes: a comparative study. Fertil. Steril., 1994; 62:485–490. 52. Penrubia J, Balasch J, Fabregues F et al. Human chorionic gonadotrophin luteal support overcomes luteal phase inadequacy after gonadotrophin-releasing hormone agonist-induced ovulation in gonadotrophin-stimulated cycles. Hum. Reprod., 1998; 13:3315–3318. 53. Ludwig M, FinasA, Bals-Pratsch M et al. Prospective randomized study to evaluate the pregnancy rate using HCG vaginal progesterone (Utrogest) or a combination of both for luteal phase support: preliminary results. Abstr. Of the 15th Ann. Meet. Of the ESHRE, 1999; 3. 54. Yergok YZ, Ergur AR, Ertekin A et al. Efficacy of the luteal phase supplementation with HCG in gonadotrophin-induced cycles. Abstr. of the 13th Ann. Meet. of the ESHRE, 1997; 323. 55. Buvat J, Marcolin G, Guittard C et al. Luteal support after luteinizing homone-releasing hormone agonist for in vitro fertilization: superiority of human chorionic gonadotrophin over oral Progesterone. Fertil. Steril., 1990; 53:490–494. 56. Araujo E Jr, Bernardini L, Frederick JL et al. Prospective randomized comparison of human chorionic gonadotrophin versus intramuscular progesterone for luteal phase support in assisted reproduction. J. Assist. Reprod. Genet., 1994; 11:74–78.

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57. Golan A, Herman A, Soffer Y et al. Human chorionic gonadotrophin is a better luteal support than progesterone in ultrashort gonadotrophin-releasing hormone agonist/menotropin in vitro fertilization cycles. Hum. Reprod., 1993; 8:1372–1375. 58. Claman P, Komingo M, Leader A. Luteal phase support in vitro fertilization using gonadotrophin releasing hormone analogue before ovarian stimulation: a prospective randomized study of human chorionic gonadotrophin versus i.m. progesterone. Hum. Reprod., 1992; 7:487–489. 59. Smitz J, Devroey P, Faguer B et al. A prospective randomized comparison of intramuscular or intravaginal progesterone as a lluteal phase and early pregnancy supplement. Hum. Reprod., 1992; 7:168–175. 60. Bredkjaer HE, Schmidt KT, Popovic B et al. Progesterone supplementation during early gestation after in vitro fertilization has no effect on the ongoing pregnancy rate. Abstr. of the 15th Ann. Meet. of the ESHRE, 1999; 37. 61. Lewin A. Endometrial evaluation amd manipulation in reproduction. Abstr. of 1st World Congess of W. A. R. M., 2002; 11.

CHAPTER 18 Seυere OHSS: A Critical Care Physician’s Point of View Sanjay Wagle INTRODUCTION Ovarian hyperstimulation syndrome (OHSS) is a potentially life threatening complication of assisted conception. Gynecologists as well as critical care physicians should be aware of the diagnosis and management of this disorder of unknown pathogenesis. We will discuss only the severe form of OHSS and its management in this chapter. Incidence Mild OHSS has an incidence of 8–23 percent; moderate 1–7 percent and severe 1–10 percent. The clinical and laboratory features determine the severity of OHSS. Massive ascites, generalized anasarca, breathing difficulty oliguria, hemoconcentration, coagulation abnormalities and azotemia are some of the characteristic features of severe OHSS (Tables18.1 and 18.2).

Table 18.1: Golan classification of severity of OHSS1 Grade Mild

Moderate

Severe

Abdominal distension and pain. Plus nausea, vomiting and/or diarrhoea. Ovaries are enlarged to 5–12 cm. Plus US evidence of ascites. Clinical evidence of ascites and/or hydrothorax or dyspnoea. Blood volume changes, increased blood viscosity, coagulation abnormalities and diminished renal perfusion and function.

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Table 18.2: Distinction between severe and critical OHSS by Navot et al2 Severe OHSS Critical OHSS Variably enlarged ovaries Variably enlarged ovaries Massive ascites+/−hydrothorax Tense ascites +/−hydrothorax Hct>45% Hct>55% WBC 15,000/cmm WBC 25,000/cmm Oliguria Oliguria Creatinine 1.0–1.5 mg% Creatinine>1.5 mg% Creatinine clearance>50 ml/min Creatinine clearance<50 ml/min Liver dysfunction Renal failure Anasarca Thromboembolic phenomenon ARDS

Pathogenesls The main pathological features of OHSS are due to increased capillary permeability leading to fluid sequestration into the extravascular space. This is triggered by the release of a vasoactive substance secreted by the stimulated ovaries. In the past histamine, serotonin, prostaglandins and prolactin have been implicated in this process. Currently, ovarian rennin-angiotensin system, inflammatory cytokines (endothelin-1, IL-1, IL-6, IL8, TNF-α, ICAM-1 and VEGF) and angiogenic growth factors are implicated in increased vascular permeability.3 The activities of these mediators lead to extravasation of plasma, followed by hemoconcentration and hypovolemia. OHSS also predisposes to thromboembolism. The hyper-estrogenic state and changes it induces in the coagulation and fibrinolytic factors are responsible for this. Clinical Features of Severe OHSS Severe OHSS is associated with nausea and vomiting, abdominal distension, pain and dyspnoea. On examination the patient is anxious with a rapid thready pulse. Orthostatic hypotension is a regular feature. This can worsen to supine hypotension in severe cases. The uncomplicated severe OHSS leads to hypovolemia, hemoconcentration, pre-renal azotemia and oliguria. The abdomen is tense because of ascites and this contributes to dyspnoea. Isolated hydrothorax can be a manifestation of severe OHSS but is usually associated with ascites.4 This leads to collapse of underlying lung, ventilation perfusion mismatch and resultant hypoxia and dyspnoea. Electrolyte disturbances, particularly hyponatremia, are a fairly common manifestation. The ovaries are grossly enlarged and tender. They may even reach beyond the level of umbilicus. Complicated severe OHSS has additional features of generalized anasarca, vulvar oedema and severe weight gain. Pre-renal azotemia may worsen to acute tubular necrosis and acute renal failure. Tense ascites, pleural effusion and fluid extravasation in the alveolar compartment can lead to Acute Respiratory Distress Syndrome (ARDS).5 Patient presents with tachypnoea and refractory hypoxia. The fluid can also accumulate in the pericardial cavity leading to tamponade; however this is a very rare manifestation. Thromboembolic events have been well documented in severe OHSS. Commonest sites

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described are internal jugular veins.6 Patient presents with sudden onset neck pain, corticovenous thrombosis,7 pulmonary embolism, subclavian vein thrombosis have also been mentioned in the literature. Arterial embolism is, however, very rare. The enlarged ovaries can undergo torsion and hemorrhage and present as acute abdomen.8 Severe OHSS is usually associated with mild asymptomatic elevation of liver transaminases. This can worsen and lead to hepatorenal failure. In most severe cases multiple organ failure ensues leading to death. The disorder is often self-limiting. Regression is observed in about a fortnight and complete recovery is expected at the onset of next menstrual cycle. In the event of pregnancy, it may persist longer and be more severe.9 Principles of Management Because the pathogenesis of this disorder is not clear, treatment is essentially supportive. Hospitalization is strongly recommended for severe OHSS. Those patients with hematocrit >45 percent, tense ascites and respiratory rate >30/min should be admitted to ICU. Careful clinical examination should be done to detect presence of complications. Blood should be sent for CBC, electrolytes, creatinine, liver function test, serum estradiol and coagulation screen. Ultrasonography of the abdomen should be done to demonstrate the size of ovaries and of the pleural cavity to detect pleural effusions and document their size. High estradiol levels and raised hematocrit along with ultrasonographic evidence of massive enlargement of ovaries and accumulation of ascites are good indicators of severity of OHSS. After admission, strict intake/output chart, daily weight measurement should be maintained. Abdominal girth should be monitored. Central venous pressure monitoring also aids in rationalizing fluid therapy and is strongly recommended in severe OHSS. Achieving euvolemia with colloids or crystalloids so as to achieve adequate urine output is the mainstay of therapy Initially the hypovolemia should be corrected with isotonic crystalloids. Albumin can also be used to correct hypovolemia and there is evidence in literature that use of albumin can reduce the severity of OHSS.10 However this is a very expensive proposition. Hydroxyethyl starch (a synthetic colloid) has also been used for this purpose with encouraging results.11 Patient should be encouraged to take protein rich fluids orally Even though oliguria is a presenting feature, diuretics should be avoided till hypovolemia is corrected. Patients with ascites benefit from large volume.3–5 It paracentesis. This relieves the distension and dyspnoea. The paracentesis should be done under ultrasound guidance and may be done by abdominal or transvaginal route.12 It is essential to infuse albumin at the time of large volume paracentesis to avoid sudden shifts of fluid from the intravascular compartment. There have been reports in the literature of reinfusing the drained ascitic fluid intravenously after filtration with good results.13 This avoids the expensive albumin infusions. The author does not have any experience of this technique. The hydrothorax should be treated by repeated thoracocentesis. Pericardiocentesis may also be required to relieve tamponade.

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The use of subcutaneous heparin (5000 u 12 hrly) should be considered in severe OHSS to prevent thrombosis. In the event of documented thrombosis patient should be anticoagulated with heparin. Patients with renal failure may require dialysis. Refractory hypoxia, despite adequate drainage of ascitic and pleural fluid may require mechanical ventilation. Termination of pregnancy should be definitely considered in the event of impending multiple organ failure. Needless to say that this is the last resort. Patients with ovarian torsion and cyst rupture will require exploratory laparotomy. CONCLUSION OHSS is an iatrogenic complication of ART. In its most severe form it is potentially fatal. The major clinical components are marked ovarian enlargement and increased capillary permeability leading to ascites, hydrothorax and pericardial effusion. Severe cases are associated with thromboembolic phenomena, respiratory distress and renal failure. The definitive pathophysiology is unknown. The available evidence would support a central role for inflammatory cytokines and angiogenic growth factors. Ultrasound examination and serum oestradiol values are currently used to predict patients at risk. The ideal treatment is prevention, but there has been only limited success. The main aims of treatment are to correct fluid imbalance, maintain renal perfusion and support the patient until the condition resolves. REFERENCES 1. Golan A, Ron-EI R, Herman A et al. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 1989; 44:430–40. 2. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril1992; 58:249–261. 3. Elchalal U, Schenker JG. The pathophysiology of ovarian hyperstimulation syndrome—views and ideas. Hum Reprod 1997; 12:1129–1137. 4. Rabinerson D, Shalev J, Royburt M, Ben-Rafael Z, DekelA. Severe unilateral hydrothorax as the only manifestation of the ovarian hyperstimulation syndrome. Gynecol Obstet Invest 2000; 49:140–42. 5. Shigematsu T, Kubota E, Aman M. Acute respiratory distress syndrome as a manifestation of ovarian hyperstimulation syndrome. Int J Gynaecol Obstet 2000; 69:169–70. 6. Schanzer A, Rockman CB, Jacobowitz GR, Riles TS. Internal jugular vein thrombosis in association with the ovarian hyperstimulation syndrome. J Vasc Surg 2000; 31:815–8. 7. Tang OS, Ng EH, Wai Cheng P, Chung Ho P. Cortical vein thrombosis misinterpreted as intracranial haemorrhage in severe ovarian hyperstimulation syndrome: case report. Hum Reprod 2000; 15:1913–6. 8. Beerdonk CCM, van Dop PA, Braat DDM, Merkus JMWM Ovarian hyperstimulation syndrome: facts and fallacies. Obstet Gynecol Surv 1998; 53:439–49. 9. Brinsden PR, Wada I, Tan SL, Balen A, Jacobs HS Diagnosis, Prevention and Management of ovarian hyperstimulation syndrome. Br J Obstet Gynecol 1995; 102:767–72. 10. Aboulghar M, Evers JH, Al-Inany H. Intra-venous albumin for preventing severe ovarian hyperstimulation syndrome. Cochrane Database Syst Rev2000; CD001302.

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11. Abramov Y, Fatum M, Abrahamov D, Schenker JG. Hydroxyethylstarch versus human albumin for the treatment of severe ovarian hyperstimulation syndrome: a preliminary report. Fertil Steril 2001; 75:1228–30. 12. Raziel A, Friedler S, Schachter M, Strassburger D, Bukovsky I, Ron-El R. Transvaginal drainage of ascites as an alternative to abdominal paracentesis in patients with severe ovarian hyperstimulation syndrome, obesity, and generalized edema. Fertil Steril 1998;69:780–3. 13. Koike T, Araki S, Minakami H, Ogawa S, Sayama M, Shibahara H et al. Clinical efficacy of peritoneovenous shunting for the treatment of severe ovarian hyperstimulation syndrome. Hum Reprod 2000; 15:113–7.

CHAPTER 19 Ovulation Induction: Surgical Approach Attila Vereczkey, Otto Kabdebo, Istυán Szabó INTRODUCTION Polycystic ovarian disease is still a relatively poorly understood and controversial entity, 60 years after its description by Stein and Leventhal.1 They described enlarged polycystic ovaries in conjunction with the clinical manifestations of menstrual irregularity, obesity and hyperandrogenism, which eventually became the polycystic ovarian syndrome (PCOS). Problems related to PCOS continue to present themselves with regularity in the practice of most gynecologists. Many treatment schemes have been proposed and implemented in an effort to circumvent the intrinsic block to ovulation and thus restore infertility. Although the polycystic ovary was described in textbooks from the early part of this century,2,3 the condition was considered untreatable and left alone. Treatment in America developed only in the third and forth decade of this century and initially consisted of laparotomy with bilateral ovarian wedge resection (BOWR). As ovulation- inducing drugs become available and concerns regarding postoperative adhesion formation surfaced, however, the medical induction of ovulation become the dominant form of treatment. In recent years, however, as a result of rapidly expanding field of operative laparoscopy, surgical treatment has received renewed interest. This chapter reviews literature and attempts to evaluate critically the utility of surgical approach in the restoration of ovulation. BILATERAL OVARIANWEDGE RESECTION (BOWR): HISTORY OF AN OLDTECHNIQUE During the period of 1902 to 1935, Stein and Leventhal noted that a small group of women with secondary amenorrhea displayed bilateral polycystic ovaries at the time of laparotomy In an effort to determine the etiology of the amenorrhea wedge biopsies of the ovaries were performed. Although the biopsy specimens did not reveal the etiology of the amenorrhea, the wedge resection produced “astonishing good results.” Regular menses were established, and pregnancies occurred.4 In 1935, they published their observations with series of seven patients displaying a constellation of symptoms now known as PCOS. Cystic degeneration of the ovary was a well-known and documented conditionbefore that time,2,3 it’s only recognized associations being abnormal uterine bleeding and endometrial hyperplasia. Stein and

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Leventhal were the first to integrate the unique ovarian morphology of PCOS with the attendant key symptoms. Interestingly textbooks of the period did not reflect this technique in their discussions. Indeed, treatment of cystic disease of the ovaries was said to consist of suspension of the uterus and adnexa under the theory that this would improve circulation, thus resolving endocrine problems. Resection of ovarian cysts themselves was still viewed with skepticism,5 due in part to concerns over the potential for adhesion formation and other pathology after ovarian surgery In keeping with this conservatism, a discussion of BOWR as a treatment for PCOS did not appear in Te Linde’s textbook of gynecologic surgery until 1953 edition.6 This edition expressed skepticism as to the value of BOWR, stating that, even though conceptions had been reported after this technique, spontaneous pregnancies after as many as 15 years of infertility as well as spontaneous resumption of normal menstrual function had been reported. Skepticism regarding its value began to fade as more gynecologists were able to cite positive experience with the procedure. It is interesting to note, that in the 1962 edition of Te Linde’s textbook of gynecologic surgery7 the previously mentioned concerns about BOWR are reiterated. The relevant section closes, by stating: “increased personal experience with BOWR has shown it to produce the most gratifying results”, and “the satisfaction of seeing one of our treated cases request sterilization after her third cesarean section.” Mechanisms of Action: Theory Over the past years several historically interesting were proposed to explain the outcome of BOWR. First, reduction of the intraovarian tension produced by the thickened capsule was envisioned to result in the mechanical facilitation of ovulation.1 Second, reduction of ovarian bulk might decrease the hypersensitivity to pituitary gonadotropin stimulation.8 Third, surgery might remove any androgen producing or gonadotropininhibitory testicular remnants left behind from the process of gonadal differentiation.9 Fourth mechanism is described in the 1952 edition of the Novaks’ textbook.10 This theory presupposes a constant outflow of pituitary gonadotropins. When this outflow is concentrated upon a smaller amount of ovarian tissue, more effective ovulation may occur. A more modern theory is that the removal of a portion of the ovary brings about a sudden decrease in estrogen and perhaps inhibin, thereby allowing an increase in pituitary gonadotropin secretion and subsequent ovulation.11 Posterior Endocrine Alterations Monitoring hormonal changes in response to BOWR, Judd et al17 noted, a significant but transitory decrease in the circulating levels of androstendione followed by gradual recovery. A long-lasting decrease in circulating testosterone concentrations was also observed. In addition, note was made of a temporary decrease in the circulating levels of both estrone and estradiol followed by a definite subsequent midfollicular rise. Importantly no changes were noted in the circulating levels of f ollicle stimulating hormone (FSH) or luteinizing hormone (LH). Although Judd et al17 did not detect discrete fall in the circulating levels of LH, Katz et al18 did note an early decrease in the

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circulating levels of LH, a phenomenon unaccompanied by a concomitant fall in the circulating levels of FSH and thereby resulting in a significant lowering of the LH:FSH ratio. Tanaka et al,19 also noted a reduction in serum LH levels after BOWR, a decrease that persisted for the duration of the 7 to 14 days of postoperative follow-up. Similarly Manesh et al20 observed a decline in LH as well as FSH after BOWR. This decrease did not become statistically significant until more than 16 days postoperatively. Adverse latrogenic Events In 1975, Buttram and Vaquero21 reported a series of 59 patients who underwent laparoscopy or laparotomy within 1 year of BOWR. All patients were found to have adhesive disease. In the same year, Toaff et al22 reported 7 patients with continuing infertility after BOWR. All were found to display peritubal adhesions. One patient had bilateral ovarian atrophy, and two others displayed unilateral ovarian atrophy. Four of the 7 patients were deemed amenable to reconstructive surgery After surgical intervention, 3 of the 4 patients conceived. Cohen23 likewise reported endoscopic evidence of ovarian adhesions extensive enough to prevent ovulation in women who remained infertile in spite of regular menstrual cycles. Using life table analysis, and correcting patients with other infertility factors, such as luteal phase defects, Adashi et al13 predicted a 73 percent expected pregnancy rate in series of 90 patients, who underwent BOWR. They observed 48 percent conception rate only Beginning in the mid-1960s, the use of BOWR was further discouraged by an increasing experience with the ovulation inducing medication clomiphene citrate.24 Large proportion of PCOS subjects became ovulatory on this medication, and a substantial proportion became pregnant. To Minimize Adhesions In an effort to produce a more precise closure of the ovarian cortex, thereby presumably decreasing postoperative adhesion formation, microsurgical techniques have been applied to BOWR. Eddy et al25 compared adhesion formation in rhesus monkeys undergoing BOWR with either micro- or macrosurgical techniques. Five of ten ovaries (50%) subjected to macrosurgical techniques displayed adhesion formation. McLaughlin26 in turn, performed microlaser ovarian wedge resection at laparotomy and the performed second look laparoscopy in 25 consecutive patients. A third (36, 7%) of the ovaries also treated was involved with adhesions. A pregnancy rate of 60 percent was noted. These data were taken to mean that microsurgical techniques can decrease but do not eliminate, adhesion formation after ovarian surgery. TECHNIQUES OF THE LAPAROSCOPIC APPROACH: LAPAROSCOPIC OVARIAN DRILLING (LOD) Because of the multiplicity of laparoscopic instrumentation, several techniques of laparoscopic therapy have been described in the literature. The first reports (29–31) resorted to ovarian biopsy in an effort to decrease ovarian bulk. Briefly, the ovary is

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stabilized by grasping the uteroovarian ligament, and single or multiple biopsy specimens are obtained from the surface farthest from the tube and pelvic side wall, thereby minimizing the likelihood of adhesions. It is estimated that this technique removes a total of about 0.5 to 1.0 cm3 of tissue. If additional hemostasis is required, unipolar or bipolar cautery can be utilized. As the use of electrocautery at laparoscopy increased, it too was utilized to create thermal damage and necrosis of the excess ovarian stroma. Because no tissue is removed, less raw bleeding from the surface would be anticipated. The relevant original technique was described by Gjönnaess32 in his 1984 report and consisted of stabilizing the ovary by grasping the uteroovarian ligament and applying unipolar coagulating current until the capsule had been penetrated (usually 2 to 4 seconds of application). A total of 4 to 10 points on each ovary were thus treated. This is the classic laparoscopic method and the one for which the most data are available. With the acceptance of laser technology and the development of laparoscopic laser delivery systems, it was inevitable that this modality would also be applied. Daniell and Miller33 first described the use of carbon dioxide and KTP lasers. Other reports soon followed, wherein the neodymium- doped yttrium iron garnet (Nd: YAG) laser was used. When the KTP and carbon dioxide lasers are used, the technique is similar to that of electrocautery; namely, the ovarian cortex is vaporized over follicles. Because KTP and carbon dioxide lasers focus their energy more precisely than electrocautery, less peripheral thermal damage is inflicted. Consequently, it is recommended that the number of punctures per ovary be greater (about 30 or so) or that all visible follicles be drained. As is the case with ovarian biopsy or electro cautery, the laser-induced lesions are placed away from the tuboovarian interface to minimize adhesions in this area. With the Nd: YAG laser, the principle is somewhat different. Specifically, this laser displays much greater thermal diffusion in the noncontact mode than other lasers. Also, the beam diverges greatly once past the sapphire tip of the delivery system. As a result, coagulation, not vaporization, of tissue is the goal. The latter is achieved by slowly moving the def ocused beam across the ovarian surface at a distance of 5 to 10 mm.34 A wedge-shaped area of ovarian tissue is thus coagulated up to a depth of 4 to 10 mm without opening the ovarian cortex. The depth of coagulation can be controlled by observing the changing color of the surface. The Nd: YAG laser has also been in used in the contact mode so as to cut out a wedge-shaped portion of the ovary35 in a manner not unlike BOWR. We Tested the Effect of the Monopolar LOD on the Embryo Quality, on the Implantation Rate in PCOS Undergoing IVF Materials and Methods Between 1999 and 2001, 31 patients with PCOS aged 31.6±4.6 years, were selected for the study undergoing treatment with ART. Duration of cycles was 29.6±2.7 days. PCOS diagnosis was based on the typical US patterns described by Adams, anovulation, or oligomenorrhea with LH/FSH ratio higher than 3. (mean values: LH: 15.1 ±4.8 mIU/mL, FSH: 8.3±4.6 mIU/mL), and testosterone levels=0.8 ng/mL. The BMI was 22.7±3.6 kg/m2.

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LOD was performed with using monopolar needle (STORZ). Each ovary was repeatedly punctured and coagulated from different angles in average 4–8 times in each ovaries using 50 Watts (Erbotom ERBE), for 3 seconds or more. We used immediate cooling and washing with Ringer solution. As an adhesion prevention we left 500–1000 ml Ringer solution in the abdominal cavity. We performed preoperative and postoperative IVF-ETICSI cycles with using midluteal-long protocols (0.1 mg Triptoreline sc., or 0.1 mg Decapeptyl s.c., from D3 to D5:50–150 IE FSH, from D5:50–100 IE FSH or 75 IE HMG, for uvulation induction 5000 IE HCG was injected). For prevention of OHSS we used 1000 ml HAES (Fresenius), or Human Albumin infusion. We performed ET on D2D3 with giving 5000 IE HCG. For luteal phase support 3×200 mg Utrogestan was used intravaginally. From the D3, 0.4 mg Folic Acid was given to the patients. We measured embryo quality using the scoring system mentioned by Steer et al in 1992: 1. Less then 20 percent fragmentation, symmetrical cells 2. 20–40 percent fragmentation, minimal asymmetry 3. Over 50 percent fragmentation, maximal asymmetry. Results The 31 patients underwent LOD without any complication. Preoperative OPU was performed in 77 cases, with 70 ET in 31 cases. We made postoperative OPU in 37 cases, with 37 ET (Table 19.1). In the preoperative group we transferred 168 embryos (EQ A: 18%, EQ B: 44%; EQ C:38%), in then postoperative group 92 (EQ A: 35%, EQ B: 41%, EQ C: 24%). Preoperative group used 27 ampoules/cycle, the postoperative group 34 ampoules/ cycles.

Table 19.1: Result of monopolar LOD in 31 cases Preoperative Postoperative OPU 77 37 ET 70 37 Transferred embryos 168 92 Embryo quality EQA 18% 35% EQB 44% 41% EQC 38% 24% Ampoules/cycles 27 34 E2 on day HCG 3.146±807 pg/ml 2.125±610 pg/ml OHSS 16/77(21%) 4/36(11%) Pregnancy rate 0 13(35% pro ET)

On the day of HCG the E2 level in the preoperative/ postoperative group was: 3.146±807 pg/ml/2.125±610 pg/ml. The OHSS incidence in the preoperative/ postoperative group was: 16/77 (21%)/4/36 (11%). There were no pregnancy in the preoperative group, the pregnancy rate/embryo transfer for the postoperative group was: 13 (35%).

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The implantation rate/Embryo was: 0%/1 embryo, 32%/2 embryos, 21%/3 embryos. The average Implantation rate/Embryo was 25% (Table 19.2).

Table 19.2: Postoperative implantation rate/ET for 92 embryos n=92

Singles Twins Triplets Implantation Rate/ET

1 Embryo: 1× — 2 Embryos: 17× 5 3 Embryos: 19× 1 Together: 62 6

— 3 4 7×2

— — 1 1×3

0% 32% 21% 25%

Postoperative Endocrine Alterations A number of investigators have studied the hormonal sequelae of laparoscopic methods of ovulation induction. Most studies agree that, although LH levels are transiently increased during the 24 to 48 hours immediately after surgery, means immunoreactive LH levels are significantly decreased thereafter. LH bioactivity also appears to be decreased postoperatively.36 In those studies that addressed the issue, it also seems clear that it is the LH pulse amplitude rather than its frequency that is decreased.41 Interestingly, in a number of studies the patients who failed to ovulate postoperatively were the same patients who did not experience a postoperative fall in circulating LH levels.45 Accordingly, early resumption of the anovulatory state was associated with a return to the pretreatment hormonal milieu. In contrast, no such regression was noted for persistently ovulatory subjects. In addition, those patients with the highest perioperative LH levels appear to be the most likely subjects to ovulate spontaneously after the operation.46 Several other investigators also noted a postoperative increase in the circulating levels of FSH. Testosterone and androstenedione levels were noted to fall postoperatively in most studies. The fall in androstenedione, however, appeared to be more transitory Temporary decrease in inhibin concentrations have also been reported.40 Intraabdominal Adhesion Formation and Ovarian Atrophy A primary rationale behind the development of the laparoscopic approach was the assumption that adhesion formation would be minimal in comparison to that in BOWR. According to some reports no adhesions were noted in women undergoing cesarean section after laparoscopic treatment after PCOS.33,37 Keckstein et al34 in turn reported postoperative adhesions in three of seven patients treated with a carbon dioxide laser and none of four treated with the Nd: YAG laser. Kovacs et al40 noted no adhesions in one patient. The effect of minimal adhesions on infertility is unknown. GüZrgan et al42 in a second report performed a prospective, randomized controlled study in which pregnancy rates were among 19 patients who underwent second look laparoscopy and lysis of adhesions when present (13 of 19) and 20 patients who did not undergo second-look laparoscopy. No significant difference in pregnancy rates was present.

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Thus, although high rate of adhesion formation may be undeniable, lysis of adhesions may not improve subsequent pregnancy rates. Other concerns in addition to adhesion formation are associated with laparoscopic therapy. A case of unilateral ovarian atrophy has been reported after the procedure.47 Even questions regarding the potential for inducing epithelial ovarian cancers have been raised. Abortion Rates It is generally agreed that (for reasons that are poorly understood) the rate of spontaneous abortion in women with PCOS is elevated compared with that of non-PCOS subjects. Sagle et al48 noted a disproportionately high incidence of PCOS in a group of women who had experienced recurrent abortion (82%) compared with a control group of parous women (18%). Furthermore (again for reasons that are not clear), it has been observed that the rate of spontaneous abortion in conceptions achieved through the use of ovulation-inducing medical agents appears to be higher (25 to 40%) than in those conceptions occurring spontaneously (10 to 20%), even when controlled for surveillance bias.49 It is not surprising, then, that some reports of early pregnancy loss in PCOS patients requiring medical ovulation induction have described abortion rates approaching 50 percent.50 One proposed reason for the increased rate of early pregnancy loss in PCOS may be the elevated circulating levels of LH described in many (but not all) of these women. Numerous studies in the literature have noted the adverse effect of elevated LH levels on fertility51 Studies in patients undergoing in vitro fertilization have noted that oocyte quality and subsequent fertilization, cleavage, and clinical pregnancy rates are significantly lower for those patients with circulating LH values greater than 10 IU/L.52,54 Homburg et al55 noted that serum LH concentrations in PCOS patients experiencing spontaneous abortion were higher compared with those of patients whose pregnancies progressed to term. Johnson and Pearce,50 in a randomized prospective study, found the abortion rate to be much lowered for women down regulated with the gonadotropinreleasing hormone (GnRH) agonist buserelin followed by FSH stimulation (9%) compared with the rate for women treated with clomiphene citrate (48%). Similarly, Homburg et al56 found the abortion rate in HMG-stimulated cycles to be 39.4 percent without and 16.7 percent with the addition of a GnRH agonist. The addition of the GnRH agonist increased the live birth rate to 64 percent compared with the rate for HMG alone (26%). In this context, Regan et al53 prospectively studied the clinical outcomes of 193 women who had regular menstrual cycles and were planning pregnancy. A sample of blood was obtained from each woman during the follicular phase and was stored for 2 years, during which time clinical data were gathered. At the end of 2 years, the blood samples were assayed for LH content, and thee values were compared with clinical outcome. Of 147 women with LH concentrations of 10 IU/L or less, 138 (88%) conceived. There was a 12 percent rate of spontaneous early pregnancy loss in this group. In contrast, of 46 women with LH levels greater than 10 IU/L, only 31 (67%) conceived, and the miscarriage rate was 65 percent. There are several proposed mechanisms by which excess LH in the follicular phase might impair fertility. Although elevated LH levels are often accompanied by elevated

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levels of circulating androgens, there does not appear to be an increase in miscarriage rates unless LH is also elevated.54 It would seem, therefore, that the answer lies in the elevated LH levels themselves. A recent review by Shoham et al51 discusses the major mechanisms proposed by which hypersecretion of LH might impair fertility. Briefly, the excess LH affecting the cumulus-oocyte complex may allow premature resumption of meiosis. The oocyte would then be prematurely aged upon ovulation or retrieval. Such postmature oocytes are well known to have lower rates of fertilization, cleavage, and implantation. A second potential mechanism would be via changes in the antral steroidogenic environment leading to premature progesterone production. In the mouse, at least, oocytes prematurely exposed to progesterone undergo mitotic arrest and atresia more readily than controls.56 It would seem, therefore, that there are adequate data to infer an association between LH hypersecretion and poor reproductive outcomes. It remains to be seen whether a medical or surgical method of ovulation induction is the best approach in dealing with the problem. Thus far, one prospective study does address the issue. Abdel Gadir et al 28 carried out a prospective trial in which patients were randomized to receive ovulation induction with gonadotropins after either downregulation with a GnRH agonist or laparoscopic ovarian electrocautery. They found no differences in pregnancy rates or ovulation rates between the two groups. They found that the group receiving electrocautery, however, had fewer cycles with multiple dominant follicles, consistently lower luteal phase serum testosterone levels, and a lower rate of early pregnancy loss (14% compared with 50%). The study was not large enough for the differences in miscarriage rates to reach statistical significance, and three of the four miscarriages in the GnRH agonist group occurred early in the second trimester. Nevertheless, as the investigators put it, the trend was evident. Gjönnaes57 also reported early pregnancy loss in 13 of 89 patients (14.6%) who conceived after laparoscopic electrocautery. Although these results appear to support laparoscopic methods of ovulation induction, future largescale trials comparing these methods with GnRH agonist will be required to provide an authoritative answer. Indications Clomiphene Citrate Resistant PCOS After a complete infertility evaluation and an unsuccessful trial of CC therapy the patient should be presented with all the available options for ovulation induction (Fig. 19.1). Figure 19.2. shows a suggested algorithm for those patients who respond poorly to laparoscopic ovulation induction. In our opinion it can not be overemphasized, that complete inf ertility evaluation and an adequate effort at ovulation induction with CC must be carried out before surgical ovulation induction is considered (Table 19.3.)

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Fig.19.1: Suggested algorithm for treatment of chronic anovulation

Fig. 19.2: Suggested algorithm for postoperative anovulation following laparoscopic ovulation induction

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Table 19.3: Requirements for laparoscopic ovulation induction • Documented PCOS • MFO syndrome (Multi Follicular Ovaries ) • Polycystic ovaries detected by US • LH:FSH ratio • Obesity • Insulin resistance • Failed CC therapy • Normal prolactin level • Normal uterine cavity with tubal patency • Normal endometrial biopsy • Informed consent

It is the physicians responsibility to present in as unbiased a manner as possible, the pros and cons of each option, including the financial implications of each. Laparoscopic Treatment Failure The effects of laparoscopic methods of ovulation induction are, rarely permanent. Armar et Lachelin43 performed second laparoscopic procedure in four patients. 50 percent conceived after second operation. This would be appear that operation can be repeated if necessary, although certainly these patients should undergo a trial of CC therapy because other studies demonstrate (27.31) that 50 to 90 percent of patients unresponsive to CC preoperatively and who fail to ovulate spontaneously postoperatively will now be responsive to CC. Because most pregnancies that follow these procedures seem to occur within 6 ovulatory cycles38 failure to conceive within this time frame warrants further investigation, including second-look laparoscopy and lysis of any significant adhesions if present. If there are no impediments to fertility seen at laparoscopy the patient should be treated as for unexplained infertility Indications that are Not Recommended Treatment of Clomiphene-Responsive PCOS A question largery left unanswered by these studies is: What does one do with the clomiphene-responsive PCOS patient who fails to conceive after ovulatory cycles induced with clomiphene citrate? Data by Gysler et al68 suggesting that 85 percent of pregnancies will occur in the first 4 cycles of clomiphene citrate therapy appear to be in direct contradiction to the work of Hammond et al60 showing that conception rates with clomiphene citrate approach 100 percent after 10 cycles of therapy The issue of how many ovulatory cycles on clomiphene citrate constitute an adequate trial remains unresolved, but a popular number used by many physicians (despite the limited amount of solid evidence for or against it) is 6 ovulatory cycles. In the presence of 6 normal

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cycles induced by clomiphene citrate therapy, and in the absence of any other infertility factors (including possible clomiphene citrate-induced luteal phase defects or cervical mucus abnormalities), these patients arguably meet the criteria for unexplained infertility Several of the laparoscopic studies cited32,39,44 included patients who did not conceive after ovulatory cycles were induced by clomiphene citrate. These studies did not from provide a separate analysis of pregnancy rates in this subcategory of patients. Daniell and Miller,33 however, did provide this information and showed a pregnancy rate of 47 percent in preoperative clomiphene citrate responders compared with a 68 percent pregnancy rate in those who did not ovulate in response to clomiphene citrate preoperatively It would seem intuitive that a group of patients failing to conceive within six ovulatory cycles would have a lower pregnancy rate than an essentially untried population. It would be hazardous to take home any clinical message such limited data, however. Given the scarcity of information available, we see no compelling reasons for extending the indications of laparoscopic ovulation induction to include what in essence is unexplained infertility. It would appear fair to state that the infertility associated with PCOS (in the absence of other positive findings in the infertility workup) is due to chronic anovulation. Attempts to make a connection between existing data that seem to support a decreased incidence of spontaneous abortion for surgically induced ovulation and a justification for the employment of laparoscopic treatments for unexplained infertility are, at this time, tenuous at best. Similarly, the substitution of a surgical method of ovulation induction for an already successful medical ovulation induction is unwarranted in light of concerns regarding adhesion formation and the potential for postoperative ovarian atrophy. Barring contraindications to gonadotropin therapy, these patients would be better served by treatments for unexplained infertility for which more data are available (such as superovulation or intrauterine insemination). The importance of monitoring the normalcy of the clomiphene citrate-induced cycles cannot be overstated. Should clomiphene citrate induce a short luteal phase, luteal phase progesterone levels routinely less than 10 ng/dL, or cervical mucus abnormalities, these patients would be better served by ovulation induction with gonadotropins rather than by continued clomiphene citrate therapy Treatment of the Hirsute Patient Data regarding the endocrine consequences of the laparoscopic procedures document a decrease in circulating androgen concentrations, so that consideration has been given to treating hirsutism via laparoscopic surgery The available data addressing the effect of laparoscopic drilling on hirsutism reveal, however, that at present hirsutism should be treated by other methods. Gjönnaess38 included 40 patients with hirsutism in his series and reported inconsistent and variable responses to treatment. Daniell and Miller33 and Gjönnaess32 noted some improvement in acne, but again they could not recommend the procedure for indications other than infertility due to anovulation. Even the original BOWR procedures were unsuccessful in alleviating hirsutism in 29 of 3114 and in 23 of 2412 patients studied. Consequently, it must be stated that laparoscopic ovarian drilling should only be used for ovulation induction in women unresponsive to clomiphene citrate who are currently attempting to achieve pregnancy

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A NEW SURGICAL TREATMENT: TRANSVAGINAL OVARIAN DRILLING (TVOD) Patients with PCOS respond differently to controlled ovarian hyperstimulation compared with women with normal ovaries, and they are more likely to have cancelled cycles because of the high risk of OHSS, or because of a lack of E2 increase, despite the growth of several small follicles. In addition, the general consensus from published studies is that patients with PCOS yield a greater number of oocytes per retrieval but have a lower proportion of fertilized eggs and lower percentage of morphologically normal embryos compared women with tubal infertility. When normal cleaving embryos available for transfer, the pregnancy rate and the life-birth rates are comparable to those for other infertile couples. An intrinsic egg-factor seems to be responsible for the limited performance of infertility treatments in some patients with PCOS, but when this ovarian factor is overcome, then implantation rate of the developed embryos seems to be similar to normovulatory women undergoing ART. A subgroup of patients with PCOS repeatedly produces poor results in IVF treatment and requires special care. Since LOD has replaced BOWR, this surgical treatment has been offered to CCresistant patients with PCOS even to increase the spontaneous ovulation rate and to decrease complications from gonadotropin treatment in simple ovulation induction or in ART cycles. In addition, a significantly better ongoing pregnancy rate af ter the IVFET procedure in women pretreated by means of LOD has been reported.70 We tested then efficiency of a new surgical approach, which consists of TVOD in difficult to treat patients with PCOS undergoing IVF. Materials and Methods Between December 2000 and July 2001, 15 patients with PCOS aged 33±6 years, were selected for the study undergoing treatment with ART. Duration of infertility was 6±2 years. PCOS diagnosis was based on the typical US patterns described by Adams, anovulation, or oligomenorrhea with LH/FSH ratio higher than 3 (mean values: LH: 15.1±4.8 mIU/mL, FSH: 8.3±4.6 mIU/mL), and testosterone levels=0.8 ng/mL. The BMI was 25.6± 4.1 kg/m.2 TVOD was performed with using 17-gauge, 35 cm long needle connected to vacuum pressure pump. Each ovary was repeatedly punctured from different angles, and all the small follicles visible by US were aspirated and scraped. Patients were discharged after 3 hours and followed up with by US, hematocrit, and hemoglobin concentration. Results The 15 patients underwent TVOD without any complication. The surgical procedure lasted 28±12 minutes, no bleeding was observed after the puncture, no abdominal pain was observed, in 3 cases minimal pelvic pain was mentioned, which has disappeared for the second day. No temperature rise was reported. Patients begun a new IVF cycle 2–6 months af ter TVOD, in these cycles all patients reached the egg retrieval stage and a mean number of 11.4±3.2 oocytes were collected. The fertilization rate was 64 percent.

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Significantly higher dosage of FSH was required by patients to develop adequate follicular growth and higher number of oocytes was collected. The fertilization and cleavage rates were signif icantly higher af ter TVOD, and normal cleaving embryos were available for transfer. The pregnancy and implantation rates were similar after TVOD in the PCOS group comparing to normovulatory patients undergoing IVF. Conclusion We showed that even the new TVOD technique can act at the ovarian levels to improve the subsequent follicular maturation, and theref ore the oocyte competence under COH for IVF. No cycle cancellation and significantly higher fertilization and cleavage rates, which suggest better intrafollicular oocyte maturation. The requirement of higher doses of FSH to develop equal number of preovulatory follicles after TVOD suggests modified ovarian responsiveness to FSH, more similar to normovulatory patients. However, TVOD did not completely prevent to occurrence of OHSS. Compared with LOD, TVOD seems to have the same efficacy, with the advantage of being less invasive and less expensive. In addition, TVOD leads to the aspiration of all small follicles present in the deeper ovarian structure visible by US and may reduce the risk of adhesion formation that can result from LOD. If the result is confirmed in a larger group of patients, the single surgical approach can evenbe evaluated for restoring spontaneous ovulatory cycles, as suggested by Mio et al.69 COMPARISONS WITH MEDICAL TREATMENT OPTIONS FOR PCOS Clomiphene citrate (CC) is relatively inexpensive and produces ovulatory cycles in a high proportion of chronically anovulatory patients. It can be used safely with minimal monitoring requirements. The risk of highorder multiple gestation and ovarian hyperstimulation are low. The clinician can expect 60 to 80 percent of patients to ovulate on standard CC regimens and 40 to 50 percent of patients will conceive.58,59 Hammond et al60 showing that, when life table analysis is employed, the most significant contribution to decreased conception rates is patient discontinuation of therapy. In our opinion

Table 19.4: Gonadotropin compared with laparoscopic ovulation induction

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CC must remain the first line of treatment for the chronic anovulation that accompanies PCOS. For patients who present CC failure, the choice of treatment is therefore between medical ovulation induction using gonadotropins or the use of laparoscopic surgical method (Table 19.4). Conventional Gonadotropin Therapy The use of gonadotropins in the anovulatory PCOS patient who has failed to ovulate on CC is well established. The combination of HMG and human chorionic gonadotropin stimulates ovarian follicular maturation and results in a high rate of ovulation. Using a life table analysis, Dor et al62 noted 91 percent cumulative pregnancy rate after six cycles of therapy in hypoestrogenic subjects. They noted only a 50 percent cumulative pregnancy rate after 12 cycles of therapy euestrogenic subjects who had failed CC therapy. Although one could argue that today’s readily available monitoring technology (ultrasonography and serum estradiol measurements) makes gonadotropin therapy safer in terms of risk of multiple gestations and OHSS. The ability to predict those situations for which the risk of multiple gestation or OHSS is increased and the recognition that the PCOS patient is particularly susceptible to these complications require a high rate of cycle cancellation. Spontaneous LH surges and premature luteinization can also be problems with the use of HMG and appear to occur more frequently in the PCOS patient than in subjects with other causes of chronic anovulation. In summary, although gonadotropin therapy is effective in the hypogonadtotropic hypoestrogenic subject, it is much less effective in the PCOS subject and has significant drawbacks, including its high cost, intensive monitoring requirements and potentially serious medical complications. Pulsatile GnRH Therapy Results with pulsatile GnRH in PCOS patients have been generally disappointing.63,64 Ovulation rates are the same or lower than with HMG or FSH. Eshel et al65 obtained ovulation in only 52 of 108 cycles. The conception rate after 6 cycles was 60 percent (23 of 52 patients). Low-dose GnRH Agonist-Gonadotropin Therapy 66

Brown et al in 1969, first suggested the use of low dose starting dose of gonadotropins with small, stepwise increases to encourage monofollicular development. Polson et al67 were able to induce ovulation successfully in all of 10 patients to whom low dose pure FSH was administered. Ovulatory cycles occurred in 72 percent of patients, and 78 percent of these cycles were monofollicular. The 6 months cumulative pregnancy rate was 55 percent. It has been demonstrated that the low-dose regimen works equally well with pure FSH or HMG. At present, the low-dose gonadotropin regimen, with its lower risk of multiple gestation and OHSS, appears to be the most promising of the medical treatments now available.

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FUTURE DEVELOPMENTS With the production of an iatrogenic tubal factor for infertility regarded as the main drawback to the laparoscopic treatment, modifications of current laparoscopic methods are already taking place. The laser is now regarded by some to produce excessive ovarian surface trauma (thought by some to be of little importance in producing ovulation but the primary factor in adhesion formation) in relation to the amount of deeper stromal damage (thought to be the critical element in producing ovulation). As a result, attention is now shifting toward the use of unipolar needle electrodes that are insulated where they contact the ovarian surface. We have heard reports of the development of a steerable needle that can be used to cauterize extensive areas of stroma through a single entry in the ovarian surface. We look forward to regarding the mechanisms responsible for the clinical effects of these procedures, however, we would urge caution in the widespread use of extensive stromal destruction, particularly because it has not been shown that stromal destruction is the factor responsible for the clinical effects. In fact, several methods that would appear to have minimal effects on the stroma seem to produce the same clinical results.16,33,61,69 The question as to whether even less invasive methods of surgical ovulation induction are possible has already been partially answered. As early as 1938, Zondek15 reported 40 cases of multiple follicular puncture of polycystic ovaries through the cul-de-sac. More recently, Mio et al69 described the use of transvaginal ultrasoundguided follicular aspiration to produce ovulation in the PCOS setting. Using a technique similar to that employed by in vitro fertilization programs, all follicles persisting after three anovulatory cycles were punctured and thoroughly aspirated. Successful ovulation was observed in 7 of 8 (87.5%) patients and in 20 of 38 (52.6%) cycles. Four of the 8 patients (50%) conceived. Given 10 patients with sonographic evidence of polycystic ovaries but without hormonal alterations, ovulation occurred in all 10 (100%) of the patients and in 50 of 79 (63.3%) cycles. Five of the 10 (50%) patients in this category conceived. CONCLUSIONS The precise mechanism through which surgical therapy improves the ovarian function in patients with PCOS has not yet been defined. It has been postulated that ovarian trauma may impair local androgen synthesis with associate reduction of intraovarian androgen levels and a diminished androgen inhibitory effect on follicular maturation. Decreased androgen levels may result a lowered peripheral conversion of androgen to estrogen and hence in a decreased positive feed-back on LH secretion. It is likely that other factors, such as inhibin and other local ovarian substances, may also be involved. The removal of these inhibitor factors through the drainage of ovarian microcysts or the destruction of ovarian tissue can permit the recruitment of a new cohort of follicles either spontaneously or under exogenous FSH stimulation. This review of the available literature strongly suggests some real benefits of laparoscopic treatment for certain carefully selected patients who fail clomiphene citrate therapy. While cautiously endorsing a place for this procedure in the armamentarium of the clinician, we would like to state emphatically that this procedure is not without significant risks to the fertility status of women. The notion that this procedure is not

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associated with adhesion formation has been demonstrated to be a false one, and the reports of ovarian atrophy, although rare, are also of serious concern. These issues, as well as the cost and risk associated with any operative procedure, underscore the importance of performing this operation only when all other available options have been exhausted. It is a great disservice to the patient to perform laparoscopic equivalents of ovarian wedge resection after only a cursory infertility evaluation and a brief attempt at clomiphene citrate ovulation induction. When one is faced with apparent clomiphene citrate failure, consideration must be given to extending the duration of therapy (beyond 5 days) or to adding glucocorticoid replacement (if appropriate). Thus when the standard regimen of clomiphene citrate fails to produce ovulation and the clinician lacks experience in other medical options for ovulation induction, the patient should be referred to a subspecialist for possible gonadotropin therapy or for further evaluation before undergoing a laparoscopic procedure. It is envisioned that in the not too distant future we may be able to produce ovarian reduction by medical means. Current methods under investigation include the prolonged (up to 6 months) use of oral contraceptives or GnRH agonists in the hope of producing an endocrine environment similar to that produced by surgical methods. The hope is that, when these medications are discontinued, the patients will ovulate spontaneously or with clomiphene citrate. We also anticipate that in the future the pathophysiologic basis of PCOS will be sufficiently understood the render surgery for the purpose of ovulation induction a rare event. REFERENCES 1. Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1935; 29:181–91. 2. Ashton WE. A textbook on the practice of gynecology for practitioners and students (2nd edn). Philadelphia: Saunders; 1906. 3. Crosson HS. Diseases ofwomen (5th edn). St. Louis: Mosby; 1922. 4. Stein IF. Ultimate results of bilateral ovarian wedge resection: twenty-five years follow-up. Int J Fertil 1956; 1:333–44. 5. Curtis AH. A textbook of gynecology (4th edn). Philadelphia: Saunders; 1942. 6. Te Linde RW. Operative gynecology (2nd edn). Philadelphia: Lippincott; 1953. 7. Te Linde RW. Operative gynecology (3rd edn). Philadelphia: Lippincott; 1962. 8. Parsons L, Sommers S. Gynecology. Philadelphia: Saunders; 1962. 9. Allen WM, Woolf RB. Medullary resection of the ovaries in the Stein-Leventhal syndrome. Am J Obstet Gynecol 1959; 77:826–34. 10. Novak E, Novak ER. Textbook of gynecology (4th edn). Baltimore: Williams and Wilkins; 1952. 11. Merrill JA. Lesions of the cervix, corpus, tubes and ovaries. In Danforth DN (Ed): Textbook of obstetrics and gynecology New York: Harper and Row; 1966:867–937. 12. Buxton CL, Van de Wiele R. Wedge resection for polycystic ovaries: a critical analysis of 40 operations. N Engl J Med 1954; 251:293–97. 13. Adashi EY, Rock JA, Guzick D, Wentz AC, Jones HW. Fertility following bilateral ovarian wedge resection: a critical analysis of 90 consecutive cases of the polycystic ovary syndrome. Fertil Steril 1981; 5:320–25. 14. Hjortrup A, Kehlet H, Lockwood K, Hasner E. Long-term clinical effects of ovarian wedge resection in polycystic ovarian syndrome. Acta Obstet Gynaecol Scand 1983; 62:55–57.

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15. Zondek B. Polyhormonal amenorrhoea and polyhormonal haemorrhage. Harefuah 1938; 14:12– 13. 16. Paldi F, Timor-Tritsch I, Brandes JM, Peretz A, Abramovici H, Fuchs K. Operative culdoscopy as treatment for the polycystic ovary. Int J Fertil 1972; 17:109–10. 17. Judd HL, Rigg LA, Anderson DC, Yen SSC. The effects of ovarian wedge resection on circulating gonadotropin and ovarian steroid levels in patients with polycystic ovary syndrome. J Clin Endocrinol Metah 1976; 43:347–55. 18. Katz M, Carr PJ, Cohen BM, Millar RP. Hormonal effects ofwedge resection of polycystic ovaries. Obstet Gynecol 1978; 51:437–44. 19. Tanaka T, Fujimoto S, Kutsuzawa T. The effect of ovarian wedge resection and incision on circulating gonadotropin in patients with polycystic ovarian disease. Int J Fertil 1978; 23:93–99. 20. Mahesh VB, Toledo SPA, Mattar E. Hormone levels following wedge resection in polycystic ovary syndrome. Obstet Gynecol 1978; 51:64s–69s. 21. Buttram VC, Vaquero C. Post-ovarian wedge resection adhesive disease. Fertil Steril 1975; 26:874–76. 22. Toaff R, Toaff ME, Peyser MR. Infertility following wedge resection of the ovaries. Am J Obstet Gynecol 1976; 124:92–96. 23. Cohen BM. Surgical management of infertility in the polycystic ovary syndrome. In: Givens JR, Andersen RN, Cohen BM, Wentz AC, eds. The infertile female. Chicago: Year Book Medical; 1979:273–92. 24. Kistner RW. Further observations on the effects of clomiphene citrate in anovulatory females. Am J Obstet Gynecol 1965; 92:380–411. 25. Eddy CA, Asch RH, Balmaceda JP. Pelvic adhesions following microsurgical and macrosurgical wedge resection of the ovaries. Fertil Steril1980; 33:557–61. 26. McLaughlin D. Evaluation of adhesion reformation by early secondlook laparoscopy following microlaser ovarian wedge resection. Fertil Steril 1984; 42:531–37. 27. Abdel Gadir A, Mowafi RS, Alnaser HMI, Alrashid AH, Alonezi OM, Shaw RW. Ovarian electrocautery versus human menopausal gonadotrophins and pure follicle stimulating hormone therapy in the treatment of patients with polycystic ovarian disease. Clin Endocrinol 1990; 33:585–92. 28. Abdel Gadir A, Alnaser HMI, Mowafi RS, Shaw RW. The response of patients with polycystic ovarian disease to human menopausal gonadotropin therapy after ovarian electrocautery or a luteinizing hormone-releasing hormone agonist. Fertil Steril 1992; 57:309–13. 29. Palmer R, de Brux J. Resultats histologiques, biochemiches et therapeutiques obtenus chez les femmes dont les ovaries avaient ete diagnostiques Stein-Leventhal a la coelioscopie. Bull Fed Gynec Obstet Fr1967; 19:405–12. 30. Neuwirth RS. A method of bilateral ovarian biopsy at laparoscopy in infertility and chronic anovulation. Fertil Steril 1972; 23:361–66. 31. Campo S, Garcea N, Caruso A, Siccardi P Effect of celioscopic ovarian resection in patients with polycystic ovaries. Gynecol Obstet Invest 1983; 15:213–22. 32. Gjonnaess H. Polycystic ovarian syndrome treated by ovarian electrocautery through the laparoscope. Fertil Steril 1984; 41:20–25. 33. Daniell JF, Miller W. Polycystic ovaries treated by laparoscopic laser vaporization. Fertil Steril 1989; 51:232–36. 34. Keckstein G, Rossmanith W, Spatzier K, Schneider V, Borchers K, Steiner R. The effect of laparoscopic treatment of polycystic ovarian disease by CO2-laser or Nd: YAG laser. Surg Endosc 1990;4:103–107. 35. Kojima E, Yanagibori A, Otaka K, Hirakawa S. Ovarian wedge resection with contact Nd: YAG laser irradiation used laparoscopically J Reprod Med 1989; 34:444–46. 36. Sumioki H, Utsunomyiya T, Matsuoka K, Korenaga M, Kadota T. The effect of laparoscopic multiple punch resection of the ovary on hypothalamo-pituitary axis in polycystic ovary syndrome. Fertil Steril 1988; 50:567–72.

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37. Armar NA, McGarrigle HHG, Honour J, Holownia P, Jacobs HS, Lachelin GCL. Laparoscopic ovarian diathermy in the management of anovulatory infertility in women with polycystic ovaries: endocrine changes and clinical outcome. Fertil Steril 1990; 53:45–49. 38. Gjonnaess H. A simple treatment for polycystic ovarian syndrome. World Health Forum 1990; 11:214–17. 39. Utsunomyiya T, Sumioki H, Taniguchi I. Hormonal and clinical effects of multifollicular puncture and resection on the ovaries of polycystic ovary syndrome. Horm Res 1990; 33(Suppl 2):35–39. 40. Kovacs G, Buckler H, Bangah M, Outch K, Burger H, Healy D et al. Treatment of anovulation due to polycystic ovarian syndrome by laparoscopic ovarian electrocautery Br J Obstet Gynaecol 1991; 98:30–35. 41. Rossmanith WG, Keckstein J, Spatzier K, Lauritzen C. The impact of ovarian laser surgery on the gonadotropin secretion in women with polycystic ovarian disease. Clin Endocrinol 1991; 34:223–30. 42. Gürgan T, Urman B, Aksu T, Yarali H, Develioglu O, Kisnisci H. The effect of short-interval laparoscopic lysis of adhesions on pregnancy rates following Nd-YAG laser photocoagulation of polycystic ovaries. Obstet Gynecol 1992; 80:45–47. 43. Armar NA, Lachelin GC. Laparoscopic ovarian diathermy: an effective treatment for antioestrogen resistant anovulatory infertility in women with the polycystic ovary syndrome. Br J Obstet Gynaecol 1993; 100:161–64. 44. Tiitien A, Tenhunen A, Seppälä M. Ovarian electrocauterization causes LH-ragulated but not insulin-regulated endocrine changes. Clin Endocrinol 1993; 39:181–84. 45. Abdel GadirA, Khatim MS, Mowafi RS, Alnaser HMI, Shaw RW. Endocrine changes following ovarian electrocautery in patients with polycystic ovarian syndrome. Ad Reprod Endocrinol 1991; 3:135–47. 46. Abdel GadirA, Khatim MS, Alnaser HMI, Mowafi RS, Shaw RW. Ovarian electrocautery: responders versus nonresponders. Gynecol Endocrinol 1993; 7:43–48. 47. Dabirashrafi H. Complications of laparoscopic ovarian cauterization. Fertil Steril 1989; 52:878– 79. 48. Sagle M, Bishop K, Ridley N, Alexander FM, Michel M, Bonney RC et al. Recurrent early miscarriage and polycystic ovaries. Br Med J 1988; 297:1027–28. 49. Ransom MX, Bohrer M, Blotner MB, Kemmann E. The difference in miscarriage rates between menotropin-induced and natural cycle pregnancies is not survellaince related. Fertil Steril 1993; 59:567–70. 50. Johnson P, Pearce JM. Recurrent spontaneous abortion polycystic ovarian disease: comparison of two regimens to induce ovulation. Br Med J 1990; 300:154–56. 51. Shoham Z, Jacobs HS, Insler V. Luteinizing hormone: its role, mechanism of action, and detrimental effects when hypersecreted during the follicular phase. Fertil Steril 1993; 59:1153– 61. 52. Stanger JD, Yovich JL. Reduced in-vitro fertilization of human oocytes from patients with raised basal luteinising hormone levels during the follicular phase. Br J Obstet Gynaecol 1985; 92:385–93. 53. Regan L, Owen EJ, Jacobs HS. Hypersecretion of luteinising hormone, infertility, and miscarriage. Lancet 1990; 336:1141–44. 54. Pearce JM. Hypersecretion of luteinising hormone: cause or marker of subfertility and miscarriage? Br J Hosp Med 1993; 49:726–63. 55. Homburg R, Armar NA, Eshel A, Adams J, Jacobs HS. Influence of serum luteinising hormone concentrations on ovulation, conception, and early pregnancy loss in polycystic ovary syndrome. Br Med J 1988; 297:1024–26. 56. Homburg R, Levy T, Berkovitz D, Farchi J, Feldberg D, Ashkenazi J et al. Gonadotropinreleasing hormone agonist reduces the miscarriage rate for pregnancies achieved in women with polycystic ovarian syndrome. Fertil Steril 1993; 59:527–31.

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57. Gjonnaess H. The course and outcome of pregnancy after ovarian electrocautery in women with polycystic ovarian syndrome: the influence of body-weight. Br J Obstet Gynaecol 1989; 96:714–19. 58. Garcia J, Jones GS, WentzAC. The use of clomiphene citrate. Fertil Steril 1977; 28:707–17. 59. Nunley WC, Bateman BG, Kitchen JD. Reproductive performance of patients treated with clomiphene citrate. South Med J 1985; 78:31–33. 60. Hammond MG, Halme JK, Talbert LM. Factors affecting the pregnancy rate in clomiphene citrate induction of ovulation. Obstet Gynecol 1983; 62:196–202. 61. Huber J, Hosmann J, Spona J. Polycystic ovarian syndrome treated by laser through the laparoscope. Lancet 1988; 2:215. 62. Dor J, Itzkowic DJ, Mashiach S, Linenfeld B, Serr DM. Cumulative conception rates following gonadotropin therapy. Am J Obstet Gynecol 1980; 136:102–05. 63. Franks S, Adams J, Mason H, Polson E. Ovulatory disorders in women with polycystic ovary syndrome. Clin Obstet Gynecol 1985; 12:605–32. 64. Kelly AC, Jewelewicz R. Alternate regimens for ovulation induction in polycystic ovarian disease. Fertil Steril 1990; 54:195–202. 65. Eshel A, Abdulwahid NA, Armar NA, Adams JM, Jacobs HS. Pulsatile luteinizing hormonereleasing hormone therapy in women with polycystic ovarian syndrome. Fertil Steril 1988; 49:956–60. 66. Brown JB, Evans JH, Adey FD, Taft HP, Towsend L. Factors involved in the induction of fertile ovulation with human gonadotropins. J Obstet Gynaecol Br Commw 1969; 76:289–307. 67. Polson DW, Mason HD, Saldahna MBY, Franks S. Ovulation of a single dominant follicle during treatment with low-dose pulsatile follicle stimulating hormone in women with polycystic ovary syndrome. Clin Endocrinol 1987; 26:205–12. 68. GyslerM, Jarch CM, Mischell DR, Bailey EJ. Adecade’sexperience with an individualized clomiphene treatment regimen including its effect on the postcoital test. Fertil Steril 1982; 37:161–67. 69. Mio Y, Toda T, Tanikawa M, Terado H, Harada T, TerakaWa N. Transvaginal ultrasoundguided follicular aspiration in the management of anovulatory infertility associated with polycystic ovaries. Fertil Steril 1991; 56:1060–65. 70. Cohen J. Laparoscopic surgical treatment of infertility related to polycystic ovary syndrome, In Kovacs GT (Ed). Polycystic ovary syndrome. Cambridge: Cambridge University Press 2000; 144–58.

SECTION 3 Polycystic Oυary Syndrome (PCOS)

CHAPTER 20 Polycystic Oυary Syndrome (PCOS): An Update Jane M Nani INTRODUCTION Polycystic ovary syndrome (“PCOS”) is the most common endocrinopathy of premenopausal women. PCOS is a major cause of infertility, menstrual disturbances and hirsutism. The syndrome is also associated with a unique insulin metabolism disorder, resulting in a substantially increased risk of noninsulin-dependent diabetes mellitus (“NIDDM”). Over the past fifteen years, the work of numerous clinical investigators has significantly increased our understanding of PCOS. Specifically, PCOS is now regarded not only as a reproductive and cosmetic disorder, but also as a metabolic disorder, similar to NIDDM. HISTORICAL BACKGROUND The classification of excess facial hair in women as an abnormality was first addressed by Stein and Leventhal in 1935.1 They described the clinical phenotype of PCOS, and the classic triad of amenorrhea, obesity, and hirsutism. Dr. Stein subsequently described PCOS women who experienced reversal to normal menstrual cyclicity after undergoing ovarian wedge resection (1964).2 The effectiveness of clomiphene citrate for inducing ovulation in PCOS women was shortly thereafter revealed (1965).3,4 In the two decades following the pioneering work of Stein and Leventhal, functional abnormalities at all levels of the hypothalamic-pituitary-ovarian axis were identified. In 1970, Yen et al reported altered LH/FSH ratios associated with PCOS women.5 According to Yen, such altered biochemical ratios are a product of the theca cell component hyperfunction, concomitant with granulosa cell component hypofunction. Altered acyclic estradiol production and chronic anovulation were similarly characterized. In the early 1980s, important advances were made correlating hyperandrogenism with hyper insulinemia PCOS women.6–8 Knowledge of this relationship was later expanded in the 1990s as the paracarmine role of IGF-1 and insulin at the ovary level was established.9,10 Throughout the 1980s and 1990s, advances in fertility science as a whole and assisted reproductive technologies (“ART”) in particular enhanced the practical effectiveness of treatment for PCOS women.11,12 The unique susceptibility of the PCOS ovary to hyperstimulation and the heightened potential for multiple birth under the influence of

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gonadotropin therapy was subsequently verified.13,14 Consequently, IVF treatment for PCOS women is suggested, in order to reduce the increased risk of such high order multiples (1995).15 Most recently, various therapeutic options have been tailored to address the inherent abnormalities characterizing PCOS. A predisposition to insulin resistance and even diabetes has been discovered, suggesting specific strategies for treatment. In 1997, Metformin, which was introduced as a new treatment for NIIDM, was reported to be effective for ovulation induction in clomipheneresistant PCOS women.16 New treatment options for hirsutism and infertility have also been implemented as the understanding of PCOS as a phenotypically heterogeneous disorder has expanded over the last half century. PCOS PHENOTYPE According to the 1990 National Institute of Health Consensus Conference,18 a clear-cut definition of PCOS could not be reached; however, the majority of participants agreed that PCOS should be generally defined as: • Ovulatory dysfunction • Clinical evidence of hyperandrogenism • Exclusion of related disorders such as hyperprolactinemia, thyroid disorders and nonclassical congenital adrenal hyperplasia (CAH). No definition of ovulatory dysfunction, hirsutism or hyperandrogenism was supplied.

Table 20.1: Polycystic ovary syndrome research diagnostic criteria (NIH, April, 1990, n=58)17 Definite or probable

Possible

Hyperandrogenism 64% Insulin resistance 69% Exclusion of other etiologies 60% Perimenarchal onset 62% Exclusion of CAH 59% Elevated LH/FSH ratio 55% Menstrual dysfunction 52% PCOS by ultrasound 52% Clinical hyperandrogenism 48% Clinical hyperandrogenism 52%

PCOS Prevalence Undoubtedly, PCOS is the most common cause of chronic anovulation and hyperandrogenemia, affecting approximately 5 percent of the general population of reproductive-aged women. • Most reports of PCOS prevalence have used populations of women seeking medical care. – If PCOS is defined histopathologically, 1.4 to 3.5 percent of unselected women and 0.6 to 4.3 percent of infertile women are affected.18

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– If PCOS is defined by ultrasound appearance, 20 to 25 percent of unselected women are affected.19–21 • In a recent study,22 369 consecutive women were evaluated: – 174 White and 195 Black women, ages 18 to 45 – 277 consented to H & P and hormonal evaluation – NIH “consensus” definition used – Prevalence of history of oligomenorrhea was 11.9 percent – Prevalence of PCOS was 4 percent (4.7 whites, 3.4 blacks). PCOS: Pathophysiology Although polycystic ovary syndrome involves highly complex and little understood hormonal dysfunction, the following symptoms provide a fairly comprehenshfe pathophysiology: • increased pulse frequency of GnRH pulse generator • increase pituitary response to GnRH • increased levels of LH • theca cell hyperplasia • increased ovarian androgen production • decreased SHBG • increased Free Testosterone • increased peripheral aromatization of androgens • relative hyper-estrogenemia (El) • decreased serum FSH, increased LH/FSH ratio • deficient follicular development • resultant anovulation. PCOS: Clinical Complex Sixty five percent ovary syndrome, a premenopausal endocrinopathy, may produce complex hormonal imbalances which directly and indirectly culminate in numerous symptoms. The three principal, nonexclusive expressions of PCOS include cosmetic, reproductive and metabolic disorders. The following sections describe these respective expressions of PCOS, and suggest the latest advances in therapy and prognosis. PCOS: Cosmetic Disorders Sixty-five to 85 percent of all women with androgen excess are diagnosed as having PCOS. Polycystic ovaries are probably the result of increased androgen levels, which inhibit follicular development, leading to the formation of multiple small subcapsular atretic follicles. The increased androgen levels also result in excess body hair in undesirable locations, tending toward midline predominant hair growth. Androgen excess in women may not only present itself as hirsutism, but also as acne and/or oily skin.

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Unfortunately, no universally accepted techniques for assessing hirsutism and treatment response currently exist. Hirsutism scales are often quoted, but remain problematic when applied to both research and clinical practice. The most commonly used grading system is the modified Ferriman-Gallwey scoring system.24 The system is highly subjective, based on visual inspection, and heavily influenced by peripheral hair on the extremities. Other assessments of hirsutism have included measurement of hair shaft diameter, hair follicle density, growth rate, and weight of shaved hair removed from a given area. Some authors have piloted prostate specific antigens as a serum marker of therapy response, but results have been mixed. Most medical methods, while improving hirsutism,25 do not produce the dramatic results wished for by patients. In general, combination therapies appear to produce better results than single agent approaches. PCOS Cosmetic Disorder Therapies The following therapies, in the form of hormonal, physical, surgical and medical interventions, are prescribed as necessary to treat the various cosmetic disorders resulting from PCOS: Hormonal Interventions • Oral contraceptives (OCPs) such as estrogen-progestin preparations which suppress circulating LH and FSH, are the most popular treatment for hirsutism to a decrease in ovarian androgen production.26 In addition, estrogen in the birth control pill increases SHBG, decreasing free testosterone levels. • Anti-androgen therapy is critical to the successful treatment of significant hirsutism. Available agents include spironolactone, flutamide, and cyproterone acetate. Spironolactone is an aldosterone antagonist and a mild diuretic useful for hirsutism treatment. Principally, it competes with circulating androgens for the androgen receptor, and has a suppressive effect on various enzymes important in the androgen biosynthesis.27 Since spironolactone acts through mechanisms different from OCPs, combining these two therapies is often beneficial.28 Dosage is generally increased in a progressive fashion over two to three weeks, up to a dose of 100 to 200 mg daily. Side-effects are mild and include dyspepsia, polyuria, fatigue, and headaches. • Flutamide is used as an adjuvant treatment for prostate cancer, and is as effect in the treatment of hirsutism as spironolactone, albeit with fewer side effects.29 Hepatotoxicity, a rare complication mandates the close monitoring of liver functions. • Cyproterone acetate a strong progestin and anti-androgen, is usually administered in combination with ethynyl estradiol as an oral contraceptive and is very effective in the treatment of hirsutism. Cyproterone acetate decreases circulating testosterone and androstenedione levels through a decrease in circulating LH levels, and antagonizes the androgen effect at the peripheral level. Side-effects may include adrenal insufficiency and loss of libido. • 5-alpha reductase inhibitors include Finasteride, used for the treatment of benign prostatic hyperplasia. Although finasteride is an effective agent for hirsutism treatment,31 it also has a significant teratogenic potential and is thus, less commonly used in reproductively aged women.

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• Long-acting GnRH analogs may be required to suppress the hypothalamic-pituitaryovarian axis in severely androgenized patients who have failed to respond to conventional therapy. Side-effects include hot flushes, vaginal dryness, and bone loss. GnRH agonists should only be used for six months in order to avoid calcium depletion from the bone. However, estrogen/progestin “add back” therapy eliminates this problem. The cost, however, may prove prohibitive. • Insulin-sensitizing agents such as metformin and thiazolidinediones have been proposed for the treatment of hirsutism in PCOS.32,33 These agents have various potential advantages over traditional therapies, including: – correction of both metabolic and endocrinologic aberrations of PCOS, – permitting normal ovulatory function reduction; and, – possibly decreasing the long-term risk of NIDDM. However, note that conclusive data regarding outcome, risks, and complications is not yet available. Physical Interventions Mechanical depilation Mechanical hair removal (shaving, plucking, waxing, depilatory creams, electrolysis, and laser vaporization) can control hirsutism, and is of ten is the front line treatment used by PCOS women. Bleaching is also useful, particularly for mild hair growth. Mechanical depilation methods are also used in conjunction with hormonal methods, usually necessary, since hormonal therapy alone will produce thinning and pigmentation loss in terminal hair (long, dark hair most affected by androgens), but will not cause terminal hair to revert to vellus hairs (short, fine containing little pigment). Approximately 80 to 90 percent of patients may note a cessation in the progression of their hirsutism, and a majority of patients may also observe variable degrees of slowing in the rate of hair growth. In order to completely resolve hirsutism, patients will most often require mechanical depilation as well as medical therapy. Surgical Intervention Surgical therapy has changed with the new technologies, as ovarian wedge resection is rarely done now. Either laser or laparoscopic stromal reduction of ovaries has been suggested for refractory treatment through ovulation induction with clomiphene citrate.35 Surgical procedures have greater utility for restoration of ovulation than for hirsutism improvement, but may lead to postoperative adhesions. Medical Interventions Ketoconazole An antifungal agent that also inhibits various steroidogenic steps, suppresses both adrenal and ovarian androgen biosynthesis in hyperandrogenemia.34 Unfortunately, the risk of adrenal crisis makes this drug rarely uSed one for the chronic inhibition of androgen production in PCOS women. Ornithine decarboxylase inhibitors can be used, since ornithine decarboxylase is necessary for the production of polyamines and is also a sensitive and specific marker of androgen action in the prostate.36 Inhibition of this enzyme limits cell division and

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function. Recently, a potent inhibitor of this enzyme, eflornithine, has been tested and found to be effective as a facial creme against hirsutism. The creme consists of 13.9 percent eflornithine hydrochloride, and is applied to affected areas twice daily for a minimum of four hours each. In clinical trials, 32 percent of patients showed marked improvement after 24 weeks, compared to 8 percent of placebo treated patients; benefit was first noted at 8 weeks. Pregnancy category C was observed, and appeared to be well tolerated. A variety of adverse skin conditions occurred in 1 percent of subjects. PCOS: Reproductive Disorders Approximately one-third of women who undergo an evaluation for infertility discover that they do not ovulate regularly. The vast majority of these women are ultimately diagnosed with PCOS. Not surprisingly, women find out for the first time in their lives that they have PCOS when they have difficulty achieving pregnancy on their own. While medical science fails to fully understand and explain much about PCOS, infertility treatment of women with PCOS is widely understood. The actual appearance of the PCOS ovary is only one feature of the typical syndrome. In fact, a PCOS diagnosis can be made for the vast majority of confirmed cases without ever having an ultrasound picture of the ovaries: Striking clinical symptoms make such a PCOS diagnosis possible. These include hirsutism (male-pattern hair growth), acne, weight gain, irregular periods, and infertility. Such symptom patterns can essentially be attributed to increased androgen production by PCOS ovaries.37 In fact, each symptom is a direct result of increased androgens in relation to estrogens. Such high ovarian androgen levels prevent normal ovulation from occurring, thus leading to infertility.38 A PCOS diagnosis is usually made based on clinical symptoms, as well as blood tests for the various hormones which characterize PCOS.

Table 20.2: Laboratory testing for chronic anovulation23 Test

Normal Value

Indication

B-hCG <5mIU/mL Rule out pregnancy TSH 0.5 to 4.5 mIU/mL Rule out thyroid disease Prolactin 3 to 24 ng/mL R/O hyperprolactinemia 17-OH-progesterone 15 to 300 ng/dL R/O late onset CAH Total Testosterone 10 to 55 ng/dL R/O ovarian tumor (>200) DHEA-S 60 to 255 mg/dL R/O adrenal tumor (>700) LH/FSH ratio <2:1 R/O PCOS (>2:1) Fasting insulin 2 to 20 mU/mL R/O insulin resistance (>20) Fasting glucose <126 mg/dL R/O NIDDM (>126) Fasting glc/insulin ratio >4.5 R/o insulin resistance (<4.5)

Any infertility treatment in women with PCOS must focus on inducing ovulation and/or lowering androgen levels. If fertility is not as issue, women with PCOS are usually treated for menstrual cycle regulation.

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While many women seek medical attention due to infertility or irregular periods, almost all PCOS women are unaware of a more serious medical concern: associated insulin resistance, which inevitably accompanies chronic hyperandrogenemia.39 Women with PCOS, while perhaps not frankly diabetic, tend to have higher insulin circulating levels as compared to women without PCOS. Interestingly, insulin seems to enhance the androgenproducing capability of the PCOS ovary.40,41 One approach toward treating PCOS, therefore, involves medication more commonly used to treat NIDDM. These medicines are known as insulin sensitizing agents, and their use, lowers insulin circulating levels. In women with PCOS, these medicines have been shown to simultaneously lower androgen circulating levels, leading to spontaneous ovulation in some women.42 Since women with PCOS are ultimately at long-term risk of developing NIDDM later in life, periodic diabetes screening is recommended (see PCOS: AMetabolic Disorder). PCOS Reproductive Disorder Therapies The following therapies, in the forms of physiological and hormal interventions, are prescribed as necessary to treat the various reproductive disorders resulting from PCOS: Physiological Interventions • Numerous medical approaches exist for infertility treatment associated with PCOS. Certainly first line therapy ought to include weight loss, nutritionally counseling, and increased exercise. Studies have shown that as little as a 5 percent loss of total body weight is of ten associated with an increased number of ovulatory cycles.37 Hormonal Interventions • If weight loss is unsuccessful, or if the patient is lean, most clinicians use clomiphene citrate to induce ovulation. Alarge case series review indicates that although approximately 80 percent of women with PCOS will ovulate with clomiphene, only 40 percent become pregnant. For women who do not become pregnant with clomiphene treatment, gonadotropins or ovarian surgery is commonly recommended. • Similarly, exogenous gonadotropins are very effective in inducing ovulation in PCOS women, but are associated with a high incidence of ovarian hyperstimulation and multiple pregnancy.43 Low dose and longer stimulation protocols appear to reduce these complications.44–46 The combination of GnRH agonists (given with a long regimen) and gonadotropins improves response in some patients, but may also result in greater ovarian hyperstimulation/multiple pregnancy risk due to enhanced follicular development. One recent trend designed to reduce multiple pregnancy risk is to use gonadotropin therapy only for PCOS patients in IVF cycles. Hormonal Interventions for IVF Patients IVF patients with PCOS present a distinct therapeutic challenge. PCOS ovary response to gonadotropin stimulation often differs significantly from the norm, frequently exhibiting

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an initial slow response, followed by an “explosive” development of large numbers of follicles, coincident with markedly elevated estradiol concentrations. IVF stimulation protocols have been developed to specifically address PCOS ovary concerns. Such protocols usually begin with a signif icant suppression phase utilizing OCP administration, subsequently overlapped with subcutaneous GnRH agonist therapy. Low dose recombinant FSH therapy is then started on the third day of withdrawal bleeding, when GnRH dose agonist is lowered. Gonadotropin stimulation is then undertaken using a step-down approach, with incremental decreases in dosage as follicular recruitment progresses.47 Additional strategies might include early thresholds for “coasting,” as well as cryopreservation of all embryos in selected cases. Regardless of the ovulation induction regimen, PCOS patients are exposed to a greater spontaneous abortion risk than other anovulatory patients. The pathogenic mechanism of this complication remains controversial. PCOS: Metabolic Disorders Considerable evidence suggests that intrinsic abnormalities in ovarian steroidogenesis are present in polycystic ovaries. Insulin resistance also appears to be a genetic defect in PCOS. In at least 50 percent of PCOS women, constitutively increased serine phosphorylation of the insulin receptor appears to inhibit normal signal transduction via the receptor tyrosine phosphorylation.48 A factor extrinsic to the insulin receptor (a serinethreonine kinase) appears to be responsible for the abnormal phosphorylation pattern. Insulin, acting through its own receptor, can stimulate ovarian steroidogenesis. Despite resistance to insulin action on glucose metabolism, insulin can act in synergy with LH and FSH to augment steroidogenesis in PCOS. Lowering insulin levels pharmacologically results in decreased ovarian and adrenal steroid production, as well as decreased LH release. PCOS Metabolic Disorder Therapies The following therapies, in the form of hormal interventions, are prescribed as necessary to treat the various metabolic disorders resulting from PCOS. Hormonal Interventions • Insulin sensitizing agents have become widely used in NIDDM treatment. When administered to insulin resistant patients, these compounds act by increasing target tissue responsiveness to insulin, thereby reducing the need for compensatory hyperinsulinemia.49 Current insulin sensitizing agents include biguanides and thiazolidinediones. Metformin, a second generation biguanide, was introduced into the US market in 1995. The drug works by activating glucose transporters which allow glucose passage into hepatic and muscle cells.50–51 Peripheral insulin resistance is decreased and serum glucose levels are lowered. Metformin does not stimulate insulin release and, when given alone, does not cause hypoglycemia. Metformin benefits in glucose utilization are seldom seen with doses of <1500 mg/ day An additional therapeutic benefit of Metformin is significant serum androgen level lowering in

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PCOS women. In hyperandrogenic PCOS women, metformin use has been associated with the resumption of normal menstrual cyclicity. Nestler et al reported improved ovulation rates in PCOS women treated with metformin in combination with Clomid.32 Metformin was administered in a dose of 500 mg tid for 35 days prior to clomid treatment initiation. Even more recently De Leo et al reported improved stimulation cycles when patients were pretreated with metformin prior to gonadotropin therapy.52 • Side-effects of metformin include gastrointestinal symptoms which are dose-related and tend to resolve after several seeks. Since metformin exclusively undergoes renal metabolism (no hepatic metabolism), the drug should not be used in patients with renal insufficiency Lactic acidosis is a rare adverse effect of metformin therapy • Thiazolidinediones, the second type of new antidiabetic agents, are under active investigation for PCOS treatment. To date, available drugs in the market include rosiglitazone (Avandia) and pioglitazone (Actos). Thiazolidinediones apparently work by binding to peroxisome proliferation activator receptor gamma, known to decrease peripheral insulin resistance.53 Short-term treatment with these compounds has resulted in a decrease in androgen levels, associated with an insulin resistance attenuation and insulin secretion reduction in obese patients with PCOS.33,54 In some women, ovulation also occurred during drug therapy periods. • The primary concern with thiazolidenediones has been liver toxicity. Such concern was particularly urgent for troglitazone (Rezulin), which has since been withdrawn from the market. Both rosiglitazone and pioglitazone are approved for use. Patients receiving these drugs should be monitored at regular intervals. Serum alanine aminotransferase (ALT) levels should be measured every two months for the first year, and periodically thereafter. Thiazolidenediones should not be initiated in patients with any evidence of liver disease, or in patients with elevated ALT levels. More clinical studies are necessary to determine the effects of these agents on hyperandrogenic women with insulin resistance. Based on clinical evidence to date, novel insulin sensitizer use such as biguanides and thiazolidenediones promise new treatment options for PCOS patients for both fertility52,55 treatment and long-term disease prevention. Long-term follow-up of women with PCOS has revealed increased NIDDM prevalence when compared with controls, with up to 40 percent developing impaired glucose tolerance or frank diabetes by the age of 40 years.56 Patients with PCOS should be screened annually for insulin resistance and/or frank diabetes. Such screening is best achieved by fasting serum glucose, and 75 gm two-hour glucose challenge test.

Table 20.3: Testing for diabetes57 Stage

Fasting Glucose

Casual Glucose

Oral GTT (75g)

Diabetes

> or =126 mg/dL

> or =200 mg/dL

2-h glc >or=200

Impaired glucose > or =110 and <126 tolerance mg/dL Normal <110 mg/dL *Fasting: No caloric intake for at least 8 hours.

2-h glc >or= 140 and <200 mg/dL 2-h glucose <140

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** Casual: Any time of day without regard to time since last meal.

Evidence also suggests that patients with PCOS have an increased risk of hypertension and cardiovascular disease. CONCLUSION Data accumulated since Stein and Leventhal first described the clinical syndrome of PCOS suggests a heterogeneous and complex disorder. Current medical understanding requires that PCOS no longer be considered simply a cosmetic disorder or reproductive disorder. Groundbreaking data since these first descriptions suggests that PCOS is a metabolic disturbance, similar to NIDDM. And undoubtedly, like NIDDM, PCOS women are susceptible to long-term morbidities such as eventual diabetes, dyslipidemia, and cardiovascular disease. REFERENCES 1. Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1935; 29:181,. 2. Stein IF. Duration of infertility following ovarian wedge resection. West J Surg 1964; 72:237. 3. Kistner RW. Induction of ovulation with clomiphene citrate (Clomid). Obstet Gynecol Surv 1965; 20:873. 4. Lamb EJ, Guderian AM. Clinical effects of clomiphene in anovulation. Obstet Gynecol 1966; 28:505. 5. Yen SSC, Vela P, Rankin J. Inappropriate secretion of folliclestimulating hormone and luteinizing hormone in polycystic ovarian disease. J Clin Endocrinol Metab 1970; 30:435. 6. Burghen GA, Givens JR, Kitabchi AE. Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease. J Clin Endocrinol Metab 1980; 50:113. 7. Chang RJ, Nakamura RM, Judd HL et al. Insulin resistance in nonobese patients with polycystic ovarian disease. J Clin Endocrinol Metab 1983; 57:356. 8. Dunaif A, Segal KR, Futterweit W et al. Profound peripheral insulin resistance, independent of obesity, in polycystic ovarian syndrome. Diabetes 1989; 38:1165. 9. Nestler JE, Powers LP, Matt DW et al. A direct effect of hyperinsulinemia on serum sex hormone-binding globulin in obese women with polycystic ovarian syndrome. J Clin Endocrinol Metab 1991; 72:83. 10. Conover CA, Lee PDK, Kanaley JA et al. Insulin regulation of insulin-like growth factor binding protein-1 in obese and nonobese humans. J Clin Endocrinol Metab 1992; 74:1355. 11. Hughes E, Collins J, Vandekerckove P. Ovulation induction with urinary follicle stimulating hormone versus human menopausal gonadotropin for clomiphene-resistant polycystic ovary syndrome. Cochrane Database Syst Rev 2000; 2:CD000087. 12. Wang CF, Gemzell C. The use of human gonadotropins for induction of ovulation in women with polycystic ovarian disease. Fertil Steril 1980; 33:479. 13. Filicori M, Flamigni C, Campaniello E et al. The abnormal response of polycystic ovarian disease patients to exogenous pulsatile gonadotropin-releasing hormone: Characterization and management. J Clin Endocrinol Metab 1989; 69:825.

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14. Forman RG, Frydman R, Egan D et al. Severe ovarian hyperstimulation syndrome using agonists of gonadotropin-releasing hormone for in υitro fertilization: a European series and a proposal for prevention. Fertil Steril 1990; 53:502. 15. MacDougal MJ, Tan Sl, Balen AH et al. A controlled study comparing patients with and without polycystic ovaries undergoing in υitro fertilization. Hum Reprod 1993; 8:223. 16. Velazquez E, Acosta A, Mendoza S. Menstrual cyclicity after metformin therapy in polycystic ovary syndrome. Obstet gynecol 1997; 90:392. 17. Zawadzki JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: Towards a rational approach. In: Dunair A, Givens JR, Haseltine F, Merriam GR (Eds). Polycystic ovary syndrome. Boston: Blackwell 1992; 377–384,. 18. Goldzieher JW. Polycystic ovarian disease. Fertil Steil 1981; 35:371. 19. Farquhar CM, Birdsall M, Manning P et al. The prevalence of polycystic ovaries on ultrasound scanning in a population of randomly selected women. Aust NZ Obstet Gynaecol 1994; 34:67. 20. Clayton RN, Ogden V, Hodgkinson J et al. How common are plolycystic ovaries in normal women and what is their significance for the fertility of the population. Clin Endocrinol 1992; 37:127. 21. Polson DW, Wadsworth J, Adams J et al. Polycystic ovaries—a common finding in normal women. Lancet 1988; 1:870. 22. Knochenhauer ES. Prevalence of the polycystic syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endo Metab 1998; 83:3078. 23. Kutteh WH. Treating PCOS-Related Infertility with Insulinsensitizing agents. OBG Manage, 2000. 24. Hatch R, Rosenfield RL, Kim MH et al. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981; 140:815. 25. Negri C, Tosi F, Dorizzi R et al. Antiandrogen drugs lower serum prostate-specific antigen (psa) levels in hirsute subjects: evidence that serum psa is a marker of androgen action in women. J Clin Endocinol Metab 2000; 85:81. 26. Azziz R, Gay F. The treatment of hyperandrogenism with oral contraceptives. Sem Reprod Endo 1989; 7:246. 27. Cummings DC, Yang JC, Rebar RW et al. The treatment of hirsutism with spironolactone. JAMA 1982; 247:1295. 28. Pittaway DE, Maxson WS, Wentz AC. Spironolactone in combination drug therapy for unresponsive hirsutism. Fertil Steril 1985; 43:878. 29. Cusan L, Dupont A, Gomez JL et al. Comparison of flutamide and spironolactone in the treatment of hirsutism: A randomized controlled trial. Fertil Steril 1994; 61:281. 30. Belisle S, Love EJ. Clinical efficacy and safety of cyproterone acetate in severe hirsutism: results of a multicenter Canadian study. Fertil Steril 1986; 46:1015. 31. Rittmaster RS. Finasteride. N Engl J Med 1994; 330:120. 32. Nestler JE, Jakubowicz DJ, Evans WS, Pasquali R. Effects of metformin on spontaneous and clomiphene-induced ovulation in the polycystic ovary syndrome. N Engl J Med 1998; 338:1876. 33. Dunaif A, Scott D, Finegood D et al. The insulin sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovarian syndrome. J Clin Endocrinol Metab 1996; 81:3299. 34. Vidal-Puig AJ, Munos-torres M, Joder-Gimeno E et al. Ketoconazole therapy: hormonal and clinical effects in non-tumoral hyperandrogenism. Euro J Endocrinol 1994; 130:333. 35. Muenstermann U, Kleinstein J. Long-term GnRH analogue treatment is equivalent to laparoscopic laser diathermy in polycystic ovarian syndrome patients with severe ovarian dysfunction. Hum Reprod 2000; 15:2526. 36. McCann PP, Pegg AE. Ornithine decarboxylase as an enzyme target for therapy Pharmacol Thera 1992; 54:195.

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37. Pasquali R, Antenucci D, Casimirri F et al. Clinical and hormonal characteristics of obese amenorrheic hyperandrogenic women before and after weight loss. J Clin Endocrinol Metab 1989; 68:173. 38. Goldzieher JW, Axelrod LR. Clinical and biochemical features of polycystic ovarian disease. Fertil Steril 1963; 14:631. 39. Rosenfield RL. Current concepts of polycystic ovary syndrome. Balilliere’s Clin Obstet Gynaecol 1997; 11:307. 40. Morales AJ, Laughlin GA, Butzow T et al. Insulin, somatotrophic, and LH axes in lean and obese women with polycystic ovary syndrome: Common and distinct features. J Clin Endocrinol Metab 1996; 81:2854. 41. Dunaif A, Segal KR, Shelley DR et al. Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes 1992; 41:1257. 42. Kolodziejczyk B, Duleba AJ, Spaczynski RZ et al. Metformin therapy decreases hyperandrogenism and hyperinsulinemia in women with polycystic ovary syndrome. Fertil Steril 2000; 73:1149. 43. Garcea N, Campo S, Pancetta V et al. Induction of ovulation with purified urinary folliclestimulating hormone in patients with polycystic ovarian syndrome. Am J Obstet Gynecol 1985; 151:635. 44. Homburg R, Howles CM. Low-dose FSH therapy for anovulatory infertility associated with polycystic ovary syndrome: rationale, results, reflections and refinements. Hum Reprod Update 1999; 5:493. 45. Balasch J, Fabregues F, Creus M et al Recombinant human folliclestimulating hormone for ovulation induction in polycystic ovary syndrome: a prospective, randomized trial of two starting doses in a chronic low-dose step-up protocol. J Assist Reprod Genet 2000; 17:561. 46. El-Sheikh MM, Hussein M, Fouad S et al. Limited ovarian sitmulation (LOS), prevents the recurrence of severe forms of ovarian hyperstimulation syndrome in polycystic ovarian disease. Eur J Obstet Gynecol Reprod Biol 2001; 94:245. 47. Neal GS, Sultan KM, Liu H-C et al. A successful approach to simtulation of the high responder patient using oral contraceptive pills, leuprolide acetate and menotropins. Presented at the 8th World Congress on In υitro Fertilization and Alternate Assisted Reproduction, Kyoto, Japan, 1993. 48. Dunaif A, Xia J, Book CB et al. Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest 1995; 96:801. 49. Antonucci T, Whitcomb R, McClain R et al. Impaired glucose tolerance is normalized by treatment with the thiazolidinedione troglitazone. Diabetes Care 1998; 20:188. 50. Ehrmann DA, Polonsky KS. Effects of metformin on insulin secretion, insulin action, and ovarian steroidogenesis in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997; 82:524. 51. Velazquez E, Acosta A, Mendoza SG. Menstrual cyclicity after metformin in polycystic ovary syndrome. Obstet Gynecol 1997; 90:392. 52. De Leo V, La Marca A, Ditto A et al. Effects of metformin on gonadotropin-induced ovulation in women with polycystic ovary syndrome. Fertil Steril 1999; 72:282. 53. Spiegelman BM. PPAR-gamma adipogenic regulator and thiazolidinedione receptor. Diabetes 1999; 47:507. 54. Ehrmann DA, Schneider DJ, Sobel BE et al. Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997; 82:2108. 55. Stadtmauer LA, Toma SK, Riehl RM et al. Metformin treatment of patients with polycystic oavary syndrome undergoing in υitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors. Fertil Steril 2001; 75:505.

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56. Ehrmann DA. Role of functional hyperandrogenism to noninsulin dependent diabetes mellitus. Baillere’s Clin Obstet Gynaecol 1997; 11:335. 57. Rayburn WF. Diagnosis and classification of diabetes mellitus: highlights from the American Diabetes Association. J Reprod Med 1997; 42:586.

CHAPTER 21 In vitro Oocyte Maturation Ved Prakash Singh Patients with PCOS are extremely sensitive to the administration of exogenous gonadotrophins during fertility treatment, and are consequently at increased risk of developing OHSS.1 For women with PCOS, it is thought that whilst the initial steps of folliculogenesis may be functional, the selection of a dominant follicle for ovulation does not always occur.2 In υitro maturation of immature oocytes is currently being developed as a potential method for the management of these patients. To date, successful pregnancies have been achieved using immature oocytes recovered from natural cycles,3 stimulated cycles4 and patients with PCOS.5 However, in terms of the number of mature oocytes obtained, the efficiency of the in υitro maturation process does not yet approach that for controlled ovarian stimulation for IVF. Much remains unknown about the molecular mechanisms governing oocyte maturation in the human species, but both nuclear and cytoplasmic factors play a role. Human oocytes recovered from follicles of less than 12 mm mean diameter have not been exposed to the growth and maturation conditions necessary to yield mature oocytes at metaphase II stage of development. The challenge with IVM is to mimic the conditions that serve to stimulate maturation and optimize developmental potential, by providing any signals that may ready the oocyte for embryo development. For conventional IVF, hCG is commonly administered to stimulate maturation of the oocytes when at least three follicles have reached a mean diameter of 18 mm, following gonadotrophin treatment. Although there is likely some flexibility with regard to the timing of hCG administration, it has been shown that when oocytes are recovered from follicles with a mean diameter of at least 23 mm, oocyte recovery cleavage and pregnancy rates may be reduced.6 Furthermore, for follicles of less than approximately 14 mm mean diameter, the oocytes recovered give rise to a very low percentage of observed clinical pregnancies with IVF.7 Generally, a higher proportion of immature oocytes (with a germinal vesicle, GV), are recovered from small follicles. Oocytes are obtained for IVM by aspirating from much smaller follicles than for conventional IVF. These follicles have not completed their final growth and maturation processes. To enable the retrieval of immature oocytes from small (2–12 mm) follicles, which is necessary for IVM, consideration of the problems associated with this is required. For instance, the use of a double lumen needle allows for flushing of the follicles under ultrasound guidance. However, it is important to ensure that the ultrasound has adequate resolution to detect smaller follicles. Furthermore, the inclusion of heparin in the flush medium may help avoid blockages from the increased amounts of blood in the follicular aspirates compared with IVF. Due to the complex nature of factors regulating the maturation of oocytes, optimal conditions for IVM are still to be defined. Several basal media formulations that have

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been developed for use with other cell culture systems have been used for IVM; including, TCM 199, alpha MEM, synthetic oviduct fluid (SOF), and Ham’s F-10.8–10 However, the exact benefits of various additives such as hormones, growth factors, energy substrates and protein sources are still to be better defined. Whilst the inclusion of certain additives may not be important for oocyte maturation, their effects on fertilization and embryo development potential may be significant. For instance, according to Durinzi et al,11 IVM in the presence of follicle stimulating hormone (FSH) does not improve the maturation ability of oocytes, but it does improve the fertilization rate and subsequent development of the resulting metaphase II oocytes.12 Other factors such as insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) may also be important. As with all procedures involving the manipulation of oocytes and embryos in the laboratory, the maintenance of physiological conditions with regard to temperature and pH is of utmost importance. Furthermore, the benefits of reduced oxygen tension for embryo development may extend to in υitro oocyte maturation,13 particularly if the cumulus cells facilitate lower oxygen tension in υiυo. For conventional IVF, oocytes are commonly inseminated between 4 to 6 hours after oocyte retrieval.14 Since cytoplasmic maturation is required to support fertilization and development until the embryonic genome is activated, this most probably allows time for this to occur. Likewise, for ICSI it is beneficial to culture the oocytes for a period of time before injection. However, the optimal time for injection of in υitro matured oocytes has not yet been identified. Fertilization rates for in υitro matured oocytes are considerably lower than for in υiυo matured oocytes inseminated using conventional IVF.15 It has been proposed that reduced fertilization rates may be the result of zona hardening due to extended culturing.16 Therefore, the use of ICSI may be advantageous even when male infertility is not a factor. The quality of oocytes has generally been poor following in υitro maturation, with embryos exhibiting slower development and cleavage arrest.17,18 This may be reflected in the lower pregnancy rates that have been obtained using in υitro matured oocytes. More recently however, results have shown improved pregnancy rates,19,20 success with PCOS patients by hCG priming,20 and pregnancies resulting from the transfer of blastocysts.21 Moreover, a live birth has been reported using cryopreserved embryos produced using in υitro matured oocytes derived from an unstimulated patient with PCOS.22 Looking to the future, the cryopreservation of immature oocytes, together with IVM, may offer opportunities to preserve fertility in cases when ovarian loss may be a factor. REFERENCES 1. Mac Dougall, MJ Tan SL, Balen A et al. A controlled study comparing patients with and without polycystic ovaries undergoing in vitro fertilisation. Hum Reprod 1993; 8:233–37. 2. Jakimiuk AJ, Jakowicki JA, Magoffin DA. Follicular development in polycystic ovary syndrome. Assis Reprod Reviews 1997; 7:54–57. 3. Russell JB, Knezevich KM, Fabian KF et al. Unstimulated immature oocyte retrieval: early versus midfollicular endometrial priming. Fertil Steril 1997; 67:616–20. 4. Nagy ZP, Cecile J, Liu J et al. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinalvesicle stage oocytes: case report. Fertil Steril 1996; 65:1047–50.

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5. Trounson A, Wood C, Kausche A. In vitro maturation and the fertilisation and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62:353–62. 6. Ben-Rafael Z, Kopf GS, Blasco L et al. Follicular maturation parameters of oocyte retrieval, fertilization and cleavage in vitro. Fertil Steril 1986; 45:51–57. 7. Haines CJ, Emes AL. The relationship between follicle diameter, ferilization rate, and microscopic embryo quality. Fertil Steril 1991; 55:205–07. 8. Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vitro maturation and fertilisation of oocyted from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril 2001; 76:936–42. 9. Cha KY, Han SY, Chung HM et al. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilisation and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril 2000; 73:978–83. 10. Smitz J, Nogueria D, Cortvindt R, de Matos DG. Oocyte in vitro maturation. Chapter 9. In Textbook of Assisted Reproductive Techniques. Gardner DK, Weissman A, Howles CM, Shoham Z(Eds), 2001. 11. Durinzi KL, Wentz AC, Saniga EM, Johnson DE, Lanzendorf SE. (Eds), 2001. Follicle stimulating hormone effects on immature human oocytes: in vitro maturation and hormone production. J Assist Reprod Genet 1997; 14(4):199–204. 12. Schroeder AC, Downs SM, Eppig JJ. Factors affecting the in vitro. Ann NY Acad Sci 1988; 541:197–204. developmental capacity of mouse oocytes undergoing maturation 13. Eppig JJ, Wigglesworth K. Factors affecting the developmental competence of mouse oocytes grown in vitro: oxygen concentration. Mol Reprod Dev 1995; 42:447–56. 14. Khan I, Staessen C, Van denAbbeel E et al. Time of insemination and its effect on in vitro fertilization, cleavage and pregnancy rates in GnRH agonist/HMG-stimulated cycles. Hum Reprod 1989; 4:921–26. 15. Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55:109–13. 16. De Vos A, Van Steirtegham A. Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction. Cells Tissues Organs 2002; 166:200–07. 17. Barnes FL, Kausche A, Tiglias J, Wood C, Wilton L, Trounson A. Production of embryos from in vitro-matured primary human oocytes. Fertil Steril 1996; 65:1151–56. 18. TrounsonA, BongsoA, SzellA. Maturation of human and bovine primary oocytes in vitro for fertilization and embryo production. Singapore J Obstet Gynaecol 1996; 27:78–84. 19. Mikkelsen AL, Smith SD, Lindenberg S. In vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum Reprod 1999; 14:1847–51. 20. Chian RC, Buckett WM, Tulandi T et al. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15:165–170. 21. Son WY, Yoon SH, Lee SW et al Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCSO after in-vivo HCG priming: Case report. Hum Reprod 2002; 17:134–36. 22. Chian RC, Gulekli B, Buckett WM, Tan SL. Pregnancy and delivery after cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum Reprod 2001; 16:1700–02.

CHAPTER 22 Polycystic Oυary Syndrome: Genetics and Health Consequences Sudip Basu, NajarAmso, Jaydip Bhaumik HISTORY AND BACKGROUND In 1721 Antonio Vallisneri,1 an Italian scientist, described for the first time the clinical and anatomo-pathological features of polycystic ovary syndrome. In 1935, Irving F Stein and Michael L. Leventhal2 described a complex symptom associated with anovulation. They described seven patients with amenorrhea, hirsutism and enlarged ovaries. They reported that all the patients resumed normal menses after bilateral wedge resection of ovaries. Out of these seven patients, two became pregnant. They concluded that wedge resection overcome the mechanical barrier produced by the thickened tunica which otherwise prevented follicles from reaching the ovarian surface and thus ovulating. The concept of wedge resection came from the observation that some of their patients resumed menstruation after ovarian biopsy. DEFINITION For many years the gynecologists debated over the very existence of the singular clinical entity. Surprisingly enough, there is still no agreement regarding the definition of the condition. While scientists from United States stress the importance of oligomenorrhea and hyperandrogenism, the European view is more towards morphological diagnosis based on ultrasound. In reality, polycystic ovary syndrome (PCOS) is a heterogeneous disorder varying from one extreme of classical triads of oligomenorrhea, hirsutism and obesity to the other extreme of complete absence of symptoms with only incidental findings of polycystic ovaries diagnosed on scan or during laparoscopy. EPIDEMIOLOGY The incidence of the syndrome varies greatly in different studies. It depends on which group of population has been included in the study Heterogeneity of the clinical and endocrine features has long been a confounding factor in the definition and investigation of the polycystic ovary syndrome. While assessing patients in a general endocrine or a reproduction-oriented clinic, the use of arbitrary definitions, based on specific endocrine or clinical criteria (e.g. raised serum LH concentrations or presence of menstrual disturbances), ensures inclusion of only extremes of a clinical spectrum resulting in a referral bias. In 1951, Vara and Niemineva,3 in a series of 12–160 unselected gynecological laparotomies, found polycystic ovaries in 1.4 percent of patients. Sommers and Wadman4 reported presence of polycystic ovaries in 3.5 percent of 740 autopsied

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women. Clayton,5 claimed that the finding of polycystic ovaries is more common, with 22 percent of the younger female population showing a polycystic pattern of ovaries on scan. Adams and colleagues,6 found polycystic ovaries in 26 percent of patient with amenorrhea, in 87 percent with oligomenorrhea, and in 92 percent of women with hirsutism by transabdominal ultrasound. Hull,7 calculated a possible incidence of PCOS in 90 percent of infertile oligomenorrheic, and in 37 percent of infertile amenorrheic patients. MORPHOLOGY Classical morphology can be defined as the detection of polycystic ovaries by ultrasound scan, enlarged ovaries with more than 10 cysts, 2–8 mm in diameter scattered either around or through an echodense thickened central stroma.8 Ovarian morphology appears to be the most sensitive marker for PCOS compared with the classical endocrine features of a raised serum LH and/or testosterone concentration which were found in only 39.8 percent and 47.8 percent of the patients respectively. So it is preferred to make the diagnosis of the polycystic ovary syndrome only when there are associated symptoms (menstrual irregularity, hyperandrogenism and obesity) or endocrine abnormalities (raised serum LH and testosterone concentrations) in addition to the ultrasound findings of polycystic ovaries. The polycystic ovary is usually detected by ultrasound, which correlates well with histopathology.9–10 The original diagnosis was provided by transabdominal ultrasound.11 It is now accepted that transvaginal ultrasound provides greater resolution and there is definite need to revise the radiological criteria to diagnose polycystic ovaries. One suggestion is the requirement of at least 15 cysts per ovary.12 Apart from the number of cysts, it is necessary to consider the stromal thickness or density—the latter being a subjective assessment and the ovarian volume, none of which has been clearly defined.13–15 Though it is fascinating to follow the evolution of the spectrum and pathogenesis of the disease, at the same time it is very frustrating not to reach an agreement regarding the definition. The importance of biochemical abnormality should not be underestimated. The clinical manifestations and long term health consequences depend on that and no discussion will be complete without due consideration. ENDOCRINE FINDINGS The prevalence of the well known increase in early to mid-follicular phase serum luteinizing hormone (LH) and testosterone concentrations depends on the diagnostic criteria used. In patients with persistent anovulation, the average daily production of estrogen and androgens is both increased and dependent on LH stimulation. This is reflected in higher circulating levels of testosterone, androstenedione, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulphate (DHAS), 17hydroxyprogesterone (17-OHP), and estrone. The testosterone, androstenedione and DHA are secreted directly by the ovary, whereas the DHAS, elevated in about 50 percent of anovulatory women with polycystic ovaries, is almost exclusively an adrenal

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contribution. The ovary does not secrete increased amounts of estrogen, and estradiol levels are equivalent to early follicular phase concentrations. Circulating estrone levels are slightly elevated. The increased total estrogen is due to peripheral conversion of the increased amounts of androstenedione to estrone. Both estrone and estradiol continue to be secreted from the ovaries but in low amounts. The patients with PCO have higher mean concentrations of LH but low or low normal levels of FSH. This is mainly due to the high estrogen component causing an increase in LH pulse amplitude and frequency while suppressing FSH. The gonadotropin pattern (high LH and low FSH) can also partly be due to increased frequency of GnRH pulsatile secretion. Though the increased pituitary and hypothalamic sensitivity can be attributed to the increased estrone levels, an important contributing factor is the impact of the decreased SHBG concentration. In anovulatory women with polycystic ovaries, there is an approximately 50 percent reduction in circulating levels of SHBG, a response to the increased testosterone, and in patients with hyperinsulinemia, due to a direct insulin effect on the liver. Despite no increase in estradiol secretion, free estradiol levels are increased because of the significant decrease in SHBG. The clinical consequences of uninterrupted estrogen stimulation (endometrial and breast cancer) as well as the high LH levels are the result of the two estrogenic influences, estrone and free estradiol. The lower FSH levels reflect the sensitivity of the FSH negative feedback system to the elevated estrogen, both free estradiol and the estrone formed from peripheral conversion of androstenedione. Because the FSH levels are not totally depressed, new follicular growth is continuously stimulated but does not proceed upto the point of maturation and ovulation. Follicular life-span may extend several months in the form of multiple cysts. These follicles are surrounded by hyperplastic theca cells often luteinized in response to the high LH levels. As various follicles undergo atresia, they are immediately replaced by new follicles of similar limited growth potential. The tissue derived from follicular atresia is also sustained by the steady state and now contributes to the stromal compartment of the ovary. OBESITY, HYPERINSULINAEMIA AND HYPERANDROGENISM Android obesity (fat deposited in the abdominal wall and visceral mesenteric locations) is very common in anovulatory women with hyperandrogenism. This fat distribution is associated with hyperinsulinemia, impaired glucose tolerance, diabetes mellitus, and an increase in androgen production rates resulting in decreased levels of sex hormonebinding globulin and increased levels of free testosterone and estradiol. Hyperinsulinemia and hyperandrogenism, however, are not confined to anovulatory women who are overweight. It is important to note that the combination of increased androgen secretion and insulin resistance has been reported in both obese and nonobese anovulatory women. The polycystic ovary is the result of a vicious cycle which can be initiated at any one of many entry points. The polycystic ovary is a consequence of the loss of ovulation and the achievement of the steady state of persistent anovulation. The characteristic morphology of the ovary reflects this dysfunctional state.

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INSULIN RESISTANCE, HYPERINSULINAEMIA AND HYPERANDROGENISM It is a well known observation that androgens are known to increase insulin resistance. Men exhibit greater insulin resistance than females. If the female to male trans sexuals receive prolonged testosterone treatment they exhibit increased insulin resistance. Despite this, when PCO women are treated with antiandrogens there is very little improvement in hyperinsulinaemia though there can be marked improvement in circulating androgen level. It would thus seem unlikely that the modest increase in androgens seen in women with polycystic ovary syndrome is directly responsible for the insulin resistance they exhibit. On the other hand hyperandrogenism could be the effect of hyperinsulinaemia. In women with syndromes of extreme insulin resistance e.g. Type A or B insulin resistance syndrome hyperandrogenism is a prominent feature. Although the insulin resistance is caused by variety of mechanisms, all patients exhibit marked hyperinsulinaemia along with hyperandrogenism. This supports the idea that hyperinandrogenism is rather the effect than the cause of hyperinsulinaemia. In fact insulin has been demonstrated to stimulate the ovarian androgen production in υiυo. Various theories have been proposed. Insulin may act directly or via insulin like growth factor 1 (IGF-1). Insulin and IGF-1 receptors are found in the human ovary and at high concentrations Insulin can mimic IGF-1 actions by binding with the IGF-1 receptors. Insulin can also act by binding with Insulin like growth factor binding proteins (IGFBPS) which regulate IGF-1 bioavailability. Insulin decreases hepatic production of IGFBP-1 and thus may increase the bioavailability of free IGF-1, augmenting its action. The influence of insulin on the ovary is preserved despite the increase in peripheral insulin resistance. That might be due to alternative pathway of C17, 20 lyase activity by which insulin stimulates the secretion of testosterone, estrogen and progesterone. Thus only the pathway regulating the carbohydrate metabolism is impaired in polycystic ovary syndrome. Further evidence for the causal association between hyperinsulinaemia and hyperandrogenism comes from studies in which insulin levels have been lowered in PCO patients with specific drug therapy with diazoxide, metformin and troglitazone. Improved insulin sensitivity resulting in a decrease in insulin levels, leads to a significant reductions in circulating androgens along with improvement in abnormalities of 17, 20-lyase activity, suggesting insulin mediated stimulation of this enzyme. Thus in polycystic ovary syndrome it seems that hyperinsulinaemia augments androgen production, that insulin acts via its own receptor and that its actions on the ovary are preserved, despite peripheral insulin resistance. GENETICS OF POLYCYSTIC OVARY SYNDROME Familial Studies For the last forty years, the possibility that polycystic ovary syndrome may be genetically inherited has been debated. As there was no historical consensus about the definition of polycystic ovaries, it was difficult to carry out genetic studies. Lack of consensus on the

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male phenotype of the disease was another problem. The problem regarding the agreed criteria used to assign first degree relatives was compounded. Different studies raised the possibility of the mode of inheritance of idiopathic PCOS. Consensus exists that one or more dominant genes can cause PCOS, but that this gene(s) probably does not account for all cases. One of the earlier studies assessed 18 patients with “Stein-Leventhal syndrome”16 Oligomenorrhea was present in four mothers of 13 subjects, but in none of 13 control mothers. Oligomenorrhea was much more common in sisters of cases (9/19) than in sisters of controls (1/18). Hirsutism was also more common in both male and female relatives. When subjected to culdoscopy, eight cases of “Stein-Leventhal syndrome” were detected among 12 sisters of the affected probands. Male relatives also showed an increased prevalence of pilosity and more widespread hair distribution, suggesting a male phenotype. The proposed mechanism of inheritance was autosomal dominant with decreased penetrance. In the 1970s Givens et al,17 published reports indicating that PCOS could be inherited in an X-linked dominant fashion. Diagnostic criteria consisted of hirsutism and either polycystic or bilaterally enlarged ovaries. They studied 18 kindreds showing affected women in several generationp and have examined some mails in detail. There was a high frequency of metabolic disorders, such as diabetes and hyperlipidemia, in both male and female pedigree members. Ferriman and Purdie,18 studied 707 patients using hirsutism and enlarged ovaries as a diagnostic criteria. Subjects were classified into those with or without hirsutism, as well as into those with or without enlarged ovaries. Familial tendencies were greatest among hirsute women with enlarged ovaries. Of 188 patients with hirsutism and enlarged ovaries, 38 first degree relatives had hirsutism. Of 96 patients with hirsutism but normal sized ovaries, 30 had oligomenorrhea and 19 infertility The numbers of first-degree relatives with the above traits were 73, 15 and 10 respectively. In 179 controls, numbers were 7, 8 and 8. Baldness was significantly increased in male relatives of hirsute women. Carey et al using polycystic ovaries on ultrasound as the female phenotype and premature male pattern baldness as the male phenotype, combined with an extensive biochemical evaluation, reported an autosomal dominant inheritance pattern in polycystic ovary syndrome families, perhaps caused by the same gene.19 An autosomal dominant mode of inheritance has also been suggested by Govind et al in an analysis of 29 polycystic ovary syndrome families.20 The results showed a 61 percent risk of PCOS in the women and 22 percent risk of early baldness in the men. In all, 39 of the 70 siblings of the PCOS probands were affected, with a segregation ratio of 55 percent, which is consistent with autosomal dominant inheritance with nearly complete penetrance. In United States, Legro21 and others studied 80 probands diagnosed on the basis of elevated testosterone associated with oligomenorrhea (< six menses/year) or amenorrhea. Nonclassical 21 hydroxylase deficiency was excluded. The sample consisted of “non Hispanic white” (87%), Hispanic (10%), and African-American (3%). Of 134 sisters, 115 agreed to allow body mass index measurements and a blood sample to be taken to determine serum testosterone (T), which was considered abnormal if greater than two standard deviations above the control mean value. Excluding 36 relatives there remained 81, of whom 46 were affected on the basis of either elevated testosterone alone or both elevated testosterone and oligomenorrhea. Concordantly affected twins have been observed. However, discordance has been seen even for monozygotic twins. In an Australian twin study, Jahanfar et al identified 19 MZ

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and 15 DZ twin pairs using ultrasound criteria; five MZ and six DZ pairs were discordant.22 Genetic Basis 23

In this context, Simpson suggested that PCOS should be treated as a quantitative trait disorder. This does not necessarily imply a truly polygenic aetiology because it would be possible to explain the variable phenotype on the basis of a small number of causative genes. Franks24 and his colleagues in St Mary’s Hospital used a linkage analysis program which makes no assumption about the mode of inheritance. Given the biochemical phenotype characteristic of women with polycystic ovaries they focused on genes coding for steroidogenic enzymes in the androgen biosynthetic pathway and those involved in the secretion and action of insulin. Genes Coding for Steroidogenic Enzymes The 17-hydroxylase/17, 20- lyase gene (CYP17) Linkage studies were performed in PCOS families using polymorphic markers close to the gene and on the basis of these, it was possible to exclude CYP17 as a major causative gene. Carey et al using RFLP screening of the—34 allele, suggested an association between the variant allele of CYP17 and PCOS.25 These findings were, however based on a relatively small population of subjects (71 patients and 33 controls) and subsequently other studies could not confirm this.26–28 None of the other studies found any relationship between the CYP17 variant and serum androgen levels. Cholesterol Side Chain Cleavage Gene, CYP11a It is known for years that PCO theca cells produce an excess of both androgens and progesterone.29,30 This prompted scientists to examine CYP11a (encoding P450 side chain cleavage) as a possible candidate gene for abnormal steroidogenesis. Franks et al examined 97 women with symptomatic PCOS, 51 subjects with polycystic ovaries and no symptoms, and 59 with normal ovaries. Using an informative, microsatellite marker in the promoter region of CYP11a, genotype analysis was performed after PCR amplification. The data from both association and linkage studies suggested that CYP11a is a major genetic susceptibility locus for PCOS.30 The Aromatase Gene There have been reports of hyperandrogenism occurring in rare patients with aromatase deficiency.31–32 In Immunohistochemical studies of polycystic ovaries, Takayama et al, were unable to detect aromatase in antral follicles of various sizes.33 On the contrary, Mason and others didn’t find any evidence of any intrinsic deficiency of aromatase in PCO women.34 Gharani et al, did not find any association of alleles of CYP19 with PCO and no evidence for excess allele sharing.35

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Genes Involved in Secretion and Action of Insulin Different studies have shown that PCOS patients are more insulin resistant than control subjects, even allowing for the effects of obesity The results of such studies raise the possibility that genes implicated in the secretion and action of insulin may have a role in the aetiology of PCOS. The Insulin Receptor Gene There have been sporadic reports of a PCOS-like phenotype occurring in patients with severe insulin resistance associated with defects of the insulin receptor gene,36 but Conway et al were unable to detect any abnormalities of the tyrosine kinase domain of the insulin receptor gene in a population of 22 hyperinsulinaemic women with PCOS.37 Talbot and others carried out molecular scanning of the entire coding region of the insulin receptor gene on DNA samples from 24 well characterized women with PCOS. Though common polymorphisms were detected, no missense or nonsense mutations were found. The authors concluded that mutations of the insulin receptor gene were rare in women with PCOS.38 Dunaif et al, tried to determine whether there is a genetic basis for the putative abnormality of serinethreonine phosphorylation (post receptor signaling).39 Since then, different studies have put forward the hypothesis hat a common perhaps genetically determined, biochemical abnormality could result in both insulin resistance and hyperandrogenism in patients with PCOS.40 The Insulin Gene As earlier mentioned, hyperinsulinaemia has been found in PCO patients, with and without a family history of NIDDM. Data from the Uppsala group have demonstrated that whereas insulin resistance was largely reversible by weight reduction (in obese PCOS subjects), abnormal insulin secretion still persisted suggesting a fundamental disorder in pancreatic beta cell function.41 Franks and others evaluated the insulin gene, VNTR (variable number tandem repeats) and PCO women.42 Calculation was done on the basis of odds ratio for insulin VNTR genotypes either by using a conventional case control approach or by the use of affected family based controls. In summary they uncovered that there was strong evidence for both linkage and association between alleles at the VNTR 5’ to the insulin gene and PCOS. They concluded that VNTR of the insulin gene was a major susceptibility locus for PCOS, particularly anovulatory PCOS, and may contribute to the mechanism of hyperinsulinaemia and to the high risk of NIDDM in women with PCOS. As far as the insulin gene VNTR is concerned, the number of subjects studied in each population was not large but the consistency of results suggested that this was likely to be a sustainable finding. In studies of the insulin gene VNTR, the results of linkage analysis were similar even if data from the men in these families were omitted. This indicated that the results did not very on the still controversial, assignment of premature balding as the male phenotype.

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Calpain-10 Genotype Ehrmann and others, studied the gene encoding the cysteine protease calpain-10 in association with pathogenesis of PCOS and diabetes. They studied 212 women with PCOS.43 Each subject was genotyped for 3 DNA polymorphisms in the calpain-10 gene for association with phenotypic traits related to PCOS (levels of total and free T, SHBG, and DHAS) and to type 2 diabetes. DNA polymorphisms in the calpain-10 gene were typed using PCR based methods. They didn’t find any evidence that calpain-10 gene has any effect on PCOS associated hormonal and metabolic measures. They also concluded that the findings must be interpreted with caution and must be confirmed in a series of patients examined prospectively Genes Involved in Folliculogenesis-Follistatin Gene Urbaneck et al, tested a carefully chosen collection of 37 candidate genes for linkage and association with PCOS or hyperandrogenemia in date from 150 families.44 The strongest evidence for linkage was with the follistatin gene, for which affected sisters showed increased identity by descent. In an affected sibling pair analysis, 72 percent of sisters were concordant for the follistatin genotype, and this remained significant after correction for multiple testing. Liao et al, could not identify a single mutation of follistatin gene either the activating or inhibiting type, using Polymerase chain reaction (PCR)-based singlestranded conformational polymorphism (SSCP) and DNA sequencing, in 64 Chinese patients.45 They concluded that mutations in the coding regions of the follistatin gene may not be a common cause of PCOS in the population studied. However it is possible that mutations may reside in the regulation region of the gene, which should be screened once its sequence is known. LONG-TERM HEALTH CONSEQUENCES AND IMPLICATIONS OF GENETICS While discussing the management of polycystic ovarian disease two issues should be separately addressed: Fertility related problems and long-term health issues like diabetes, and cardiovascular problems. Fertility Problems Women with polycystic ovary syndrome (PCOS) are frequently referred to the assisted reproduction unit either because of an intractable anovulatory problem, or failure to conceive after several treatment cycles with confirmed ovulation or for other associated factors. There is evidence to suggest that a high LH level in PCO patients is accompanied with reduced fertilization rates and increased miscarriage rates. Premature luteinization is often a problem in PCO women treated with gonadotrophin for ovulation induction though this problem can be tackled by long down regulation. It has been suggested that oocytes obtained following superovulation in PCOS patients are often immature, with resultant poor embryo quality. However, it is acknowledged that determination of oocyte

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quality is very subjective and is no prognostic indicator of subsequent pre-embryo potential. Ovarian hyperstimulation (OHSS) is a serious problem often encountered with PCO women undergoing infertility treatment. If the pathophysiology of PCO patients is better understood by genetics, then there is a possibility of modifying the treatment regime to obtain an optimum outcome. Non-fertility Related Problems Diabetes Insulin resistance and pancreatic beta cell dysfunction are both implicated in the pathophysiology of diabetes mellitus. As both are described in polycystic ovary syndrome one would expect to find increased rates of both diabetes and impaired glucose tolerance in patients with a history of this condition. Dunaif et al demonstrated that upto 40 percent of women with polycystic ovary syndrome have either impaired glucose tolerance or type 2 diabetes by the age of 40.46 On the basis of that Metformin has been tried as the medical treatment for polycystic ovary syndrome. Metformin treatment was associated with a reduction in circulating insulin along with a fall in total and free testosterone, a rise in sex hormone binding globulin, and a reduction in body mass index. Reproductive function also improved, with seven women resuming normal menstruation and a further three becoming pregnant. Though these findings have been supported by similar studies there is valid concern that it might not be beneficial for all PCO women. The importance of recognizing polycystic ovary syndrome as a significant risk for the development of diabetes should be recognized. Genetic studies will help us to understand the complex pathophysiology of this serious problem and hopefully better management as well. Further studies will show whether routine Glucose tolerance test screening will be necessary to identify PCO women at risk of developing diabetes in old age. Positive results mighthelp to determine which group of PCO women will get benefit from Metformin. Cardiovascular Problems Failure to diagnose and adequately treat diabetes allows the devastating micro vascular complication of this disease to develop. There is also mounting evidence that hyperinsulinaemia in nondiabetic women is independently associated with an increased risk of cardiovascular disease. Furthermore, it also seems to be a marker for a cluster of metabolic abnormalities that include hyperten sion, glucose intolerance, hyperlipidaemia and impaired fibrinolysis. Scientists have referred it as “Metabolic syndrome X”. Careful comparison of blood pressure levels in young women with polycystic ovary syndrome does, however, reveal an increase in both mean and systolic blood pressures on 24 h blood pressure recording. Furthermore, there is a reported three fold increased prevalence of hypertension in older postmenopausal women with a past history of polycystic ovary syndrome. Majority of the studies have shown that women with polycystic ovary syndrome have an atherogenic lipid profile, with increased levels of low-density lipoprotein cholesterol and triglyceride, and a reduced high-density lipoprotein

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cholesterol concentrations. It would sound logical that women with polycystic ovary syndrome, particularly if obese, have an atherogenic lipid profile, which would be expected to increase their risk of coronary heart disease. So if it becomes possible to identify women at risk of developing polycystic ovary syndrome by carrying out the genetic screening of the families of PCO patients, many diseases can be prevented by changing to a healthy life style and advising healthy diet. It will have profound effect on the community and health system. SUMMARY There is strong evidence that polycystic ovary syndrome follows a familial trend though the exact mode of penetration is yet to be universally agreed. Using a candidate gene approach, scientists have found evidence for the involvement of two key genes in the aetiology of PCOS. The steroid synthesis gene CYP11a and the insulin VNTR regulatory polymorphism are important factors in determining the genetic basis of PCOS and may go some way in explaining the heterogeneity of the syndrome. These findings remain to be confirmed in larger studies and in other populations. There is a possibility that PCOS is an oligogenic disorder although it is quite possible that, within a given family, there is indeed one major gene, which is dominantly inherited. The search for this gene is still going on. Thus PCOS represents a quantitative trait in which a relatively small number of key genes contribute, in conjunction with environmental (particularly nutritional) factors, to the observed clinical and biochemical heterogeneity Identif ication of susceptibility genes in the aetiology of PCOS is not simply an intellectual exercise, as illustrated by the data regarding the insulin gene VNTR. If these findings are confirmed by future studies, that will help in a better understanding of not only PCOS, but also of the future risk of NIDDM. REFERENCES 1. Vallisneri A. Storia della generazione deiruomo e deH’animale. Cited in Cooke ID, Lunenfeld B (Eds) Res Clin Forums 1989; 1721; 11:109–13. 2. Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries, Am J Obstet Gynecol 1935; 29:181. 3. Vara P, Niemineva K. Small cystic degeneration of ovaries as incidental finding in gynaecological laparotomies. Acta Obstet Gynecol Scand 1951; 31:94–9. 4. Sommers SC, Wadman PJ. Pathogenesis of polycystic ovaries. Am J Obstet Gynecol 1956; 29:181–87. 5. Clayton RN, Ogden V, Hodgkinson J, Worswich L, Rodin DA, Dyer S et al. How common are polycystic ovaries in normal women and what is their significance for the fertility of the population. Clin Endocrinol 1992; 37:127–34. 6. Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J 1986; 293:335–59. 7. Hull MGR. Epidemiology of infertility and polycystic ovarian disease: endocrinological and demographic studies. Gynecol Endocrino1987; 1:235–45. 8. Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J 1986; 293:335–49.

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9. Saxton DW, Farquhar CM, Rae T, Beard RW, Anderson MC, Wadsworth J. Accuracy of ultrasound measurement of female pelvic organs. Br J Obstet Gynaecol 1990; 97:695–99. 10. Takahashi K, Eda Y, Okada S, Abu-Musa A, Yoshino K, Kitao M. Morphological assessment of polycystic ovaries using transvaginal ultrasound. Human Reprod 1993; 6:844–49. 11. Swanson M, Sauerbrei EE, Cooperberg PL. Medical implications of ultrasonically detected polycystic ovaries. J Clin Ultrasound 1981; 9:219–22. 12. Fox R, Corrigan E, Thomas PA, Hull MGR. The diagnosis of polycystic ovaries in women with oligo-amenorrhoea: predictive power of endocrine tests. Clin Endocrinol 1991; 34:127–31. 13. Puzigaca Z, Prelevic GM, Stretenovic Z, Balintperic L. Ovarian enlargement as a possible marker of androgen activity in polycystic ovary syndrome. Gynecol Endocrinol 1991; 5:167–74. 14. Pache TD, de Jong FH, Hop WC, Fauser BCJM. Association between ovarian changes assessed by transvaginal sonography and clinical and endocrine signs of the polycystic ovary syndrome. Fertil Steril 1993; 59:544–49. 15. Dewailly D, Robert Y, Helin I et al. Ovarian stromal hypertrophy in hyperandrogenic women. Clin Endocrinol 1994; 41:557–62. 16. Cooper HE, Spellacy WN, Prem KA, Cohen WD. Hereditary factors in the Stein-Leventhal syndrome. Am J Obstet Gynecol 1968; 100:371–87. 17. Givens JR, Wiser WL, Coleman SA, Wilroy RS, Anderson RN, Fish SA. Familial ovarian hyperthecosis: A study of two families. Am J Obstet Gynecol 1971; 110:959–72. 18. Ferriman D, Purdie AW. The inheritance of polycystic ovarian disease and a possible relationship to premature balding. Clin Endocrinol 1979; 11:291–300. 19. Carey AH, Chan KL, Short F et al. Evidence for a single gene effect in polycystic ovaries and male pattern baldness. Clin Endocrinol 1993; 38:653–58. 20. Govind A, Obhrai MS, Clayton RN. Polycystic ovaries are inherited as an autosomal dominant trait: Analysis of 29 polycystic ovary syndrome and 10 control families. J Clin Endocrinol Metab 1999; 84:38–43. 21. Legro RS, Driscoll D, Strauss JF, Fox J, Dunaif A. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Natl Acad Sci USA 1998; 95:14956– 60. 22. Jahanfar S, Eden JA, Warren P, Seppala M, Nguyen TV. A twin study of polycystic ovary syndrome. Fertil Steril 1995; 63:478–86. 23. Simpson JL. Genetic factors in common disorders of female infertlity Reproductive Medicine Review 2000; 8:173–202. 24. Franks S, Gharani N, Waterworth D, Batty S, White D, Williamson R et al. The genetic basis of polycystic ovary syndrome. Hum Reprod 1997; 12:2641–48. 25. Carey AH, Waterworth D, Patel K et al. Polycystic ovaries and premature male pattern baldness are associated with one allele of the steroid metabolism gene CYP17. Hum Mol Genet 1994; 3:1873–76. 26. Franks S et al. The genetic basis of polycystic ovary syndrome. Hum Repro 1997; 12:2641–48. 27. Gharani N, Waterworth DM, Williamson R, Franks S. 5′ polymorphism of the CYP 17 gene is not associated with serum testosterone levels in women with polycystic ovaries (letter). J Clin Endocrinol Metab 1996; 81:4174. 28. Pugeat M, Nicholas MH, Cousin P et al. Polymorphism in the 5’ promoter of the human gene encoding P450c 17 and adrenal androgen secretion in hirsute women. Programme of 10th International Congress of Endocrinology, San Francisco, June 1996. Endocrine Society Press, Bethesda, 1996; 561. 29. Gilling-Smith C, Willis DS, Beard RW, Franks S. Hypersecretion of androstenedione by isolated theca cells from polycystic ovaries. J Clin Endocrinol Metab 1994; 79:1158–65. 30. Franks S, Willis D, Mason H, Gilling-Smith C. Comparative androgen production from theca cells of normal women and women with polycystic ovaries. In Chang RJ(Ed) Polycystic ovary syndrome. New York: Springer, 1996a; 154–64.

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31. Harada N, Ogawa H, Shozu M, Yamada K. Genetic studies to characterise the origin of the mutation in placental aromatase deficiency Am J Hum Genet 1992; 51:666–88. 32. Ito Y, Fisher CR, Conte FA et al. Molecular basis of aromatase defeciency in an adult female with sexual infantilism and polycystic ovaries. Proc Natl Acad Sci USA, 1993; 90:11673–677. 33. Takayama K, Takao T, Hironobu S, et al. Immunohistochemical study of steroidogenesis and cell proliferation in polycystic ovary syndrome. Hum Reprod 1996; 11:1387–92. 34. Mason HD, Willis DS, Beard RW et al. Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and to concentrations of sex steroids in follicular fluid. J Clin Endocrinol Metab 1994; 79:1355–60. 35. Gharani N, Waterworth DM, Batty S et al. Association of the steroid synthesis gene CYPlla with polycystic ovary syndrome and hyperandrogenism. Hum Mol Genet 1997; 6:397–402. 36. Moller DE, Flier JS. Detection of an alteration in the insulin receptor gene in a patient with insulin resistance, acanthosis nigricans and the polycystic ovary syndrome. N Engl J Med 1988; 319:1526–29. 37. Conway GS, Avey C, Rumsby G. The tyrosine kinase domain of the insulin receptor gene is normal in women with hyperinsulinaemia and polycystic ovary syndrome. Hum Reprod 1994; 9:1681–83. 38. Talbot JA, Bicknell EJ, Rajkhowa M et al. Molecular scanning of the insulin receptor gene in women with polycystic ovary syndrome. J Clin. Endocrinol Metab 1996; 81:1979–83. 39. Dunaif A, Xia J, Book CB et al. Excessive insulin receptor phosphorylation in cultured fibroblasts and in skeletal muscles. J Clin Invest 1995; 96:801–10. 40. Zhang LH, Rodriguez H, Ohno S, Miller WL. Serine phosphorylation of human p450c17 increases 17, 20-lyase activity: implications for adrenarche and the polycystic ovary syndrome: USA. Proc Natl Acad Sci 1995; 92:10619–623. 41. Holte J, Bergh T, Berne C et al. Restored insulin sensitivity but persistently increased early insulin secretion after weight loss in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 1995; 80:2586–93. 42. Franks S, Gharani N, Waterworth D, Batty S et al. The genetic basis of polycystic ovary syndrome. Hum Repro 1997; 12:2641–48. 43. Ehrmann D, Schwarz P, Hara M, Tang Xu et al. Relationship of Calpain-10 genotype to phenotypic features of polycystic ovary syndrome. J Clin Endocrinol Metab 2002; 87:1669–73. 44. Urbaneck M, Legro RS, Driscoll D, Azziz R, Thirty seven candidate genes for polycystic ovary syndrome: Strongest evidence for linkage is with Follistatin. Proc Natl Acad Sci 1999; 96:8573– 78. 45. Liao WX, Roy AC, Ng SC, Preliminary investigation of follistatin gene mutations in women with polycystic ovary syndrome. Mol Hum Repro 2000; 6:587–90. 46. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 1989; 38:1165–74.

SECTION 4 ART Procedures

CHAPTER 23 Vaginal Oocyte Retrieυal Gautam N Allahbadia, Goral N Gandhi, Kaushal Kadam “The υery essence of the creative is its noυelty, and hence we have no standard by which to judge it.” Carl R Rogers

INTRODUCTION Once oocyte maturation is achieved, patients receive 5000 to 10,000 IU of human chorionic gonadotropin in order to mimic an endogenous LH surge. Retrieval is performed 34 to 36 hours after human chorionic gonadotropin administration, at which time the oocyte resumes meiosis, approaching completion of its reduction division.1 Historically, laparotomy had been briefly used and abandoned because the morbidity associated with the procedure precluded its widespread use.1 Steptoe and his group2 achieved the first IVF success harvesting the oocytes laparoscopically. Laparoscopy became the standard procedure for oocyte retrieval and was universally employed by IVF/ET programs. As the peripheral follicles are aspirated, it is difficult to delineate and selectively aspirate follicles well within the stroma of the ovaries. Generally, these “internal” oocytes are aspirated blindly. Its major advantage is related to the clear view of the pelvic organs during the aspiration process. However, this is an invasive technique, requiring general anesthesia and it is also associated with significant postoperative patient discomfort, some morbidity and, rarely, mortality. An increased risk and mortality rate may be associated with repeated laparoscopies in patients with previous abdominal operations. Furthermore, in 5 to 10 percent of the patients admitted for IVF/ET, it is impossible to perform a laparoscopic guided oocyte retrieval because of severe pelvic adhesions.3 Although the effects of general anesthesia and CO2 pneumoperitoneum on the oocyte have not been fully investigated, it has been demonstrated that prolonged exposure of the oocyte to the anesthetic agents and the potential lowering of follicular pH associated with CO2 pneumoperitoneum, may have a deleterious effect.4 Laparoscopic oocyte retrieval is currently reserved only for gamete intrafallopian transfer (GIFT), but there are increasing attempts to carry out GIFT completely under ultrasonographic control, especially as newly developed transcervical-transuterine tubal cannulation techniques have become widely available.5 The ideal method of oocyte recovery should allow easy access to the largest number of available follicles and be simple to perform, and be associated with low morbidity and cost. In addition, the method of retrieval should not adversely affect the recovered oocytes.

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Lenz et al6 reported the first ultrasound-guided needle aspiration of preovulatory oocytes in 1981. It was performed using transabdominal ultrasound guiding a needle for a transvesical ovarian puncture. The retrieval rates were acceptable by the transvesical route, but the technique is cumbersome and uncomfortable. This method uses an abdominal sector scan transducer (3.5 MHz) with a needle guide attached to it. The needle is guided through a full bladder until it enters the ovary. This procedure was generally done with sedation and local anesthesia but a relatively high rate of complications like cystitis and significant hematuria was experienced. In 1983, Gleicher et al7 reported the first case of egg retrievalby culdocentesis or transvaginal puncture, sonographically controlled by an abdominal sector scanner. The use of ultrasound in the diagnosis and treatment of female infertility was introduced in a report by Kratochwil et al8 on visualization of pelvic organs. Later on, Hackloer9 correlated follicular size determined by ultrasound with 17-B-estradiol (E2) serum concentration. Since then, it is apparent that the transvaginal route is the superior method for egg collection. As endovaginal probes became available, transvaginal ovarian puncture was simplified by attaching a needle guide to the endovaginal transducer. As the probe is inserted into the vagina, the distance from the tip of the transducer to the follicle is only about 3 to 5 cm. All ovarian follicles can be easily accessed regardless of the presence of pelvic adhesive disease. Once the needle penetrates through the ovarian cortex, it will be continuously monitored during the procedure. Since the needle is fixed and stabilized against the probe, adequate control is maintained at all times. Once the transducer is rotated or moved, the path of the needle will adjust accordingly. Furthermore, in patients with displaced ovaries or severe pelvic adhesions, the transvaginal route was definitely proved to be superior. Presently, this less invasive technique is the preferred method of oocyte recovery in all IVF programs. Since these techniques offered the potential benefits of less trauma, morbidity and greater patient acceptance, laparoscopy was gradually replaced by ultrasound-directed techniques.10–14 Laparoscopy is now only employed for gamete intrafallopian transfers. TECHNIQUE Patients undergoing oocyte retrievals are placed in the lithotomy position. They receive short general anesthesia usually with the combination of Fentanyl and Propofol. Other institutions have used regional epidural anesthesia for these procedures with excellent results.15 Fertilization and pregnancy rates have not been affected by the method of analgesia or anesthesia used. Rosenblatt et al reviewed the anesthesia records of 106 egg donors for Propofol usage during the transvaginal needle aspiration of the ova.16 The medical records of the 117 patients who received these donated embryos were reviewed for cumulative embryo scores, clinical pregnancy rates, and implantation rates. The pregnancy rate among all patients who received ova from women who received Propofol (44 of 103=42.7%) was 14.1 percent greater than those whose ovum donors did not receive Propofol (4 of 14= 28.6). 78.6 percent of both Propofol and non Propofolexposed groups had cumulative embryo scores of greater than 50. Among patients who became pregnant, 52.3 percent of Propofol-exposed and 50 percent of non Propofol-exposed cases had greater than 20 percent implantation rates. They concluded that the

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administration of Propofol during the aspiration of ovarian follicles for oocyte donation had no negative impact on the oocytes as measured by cumulative embryo scores, probability of a clinical pregnancy, or implantation rate.16 A thorough sterile saline-only irrigation of the vagina is done using nearly 500 ml and a sponge on holder. At Rotunda—The Center For Human Reproduction, oocyte retrievals are done using a 5-MHz transvaginal probe (SonoSite, Philips-ATL, Chennai, India) or the Logic-Pro 200 5.0 MHz transvaginal probe (Wipro-GE, Bangalore, India) (Fig. 23.1). The probe is covered with a sterile powder free latex transvaginal probe cover (RS Sethi Associates, Chembur, Mumbai, India) in order to maintain sterility. Ultrasound jelly as a coupling media is placed between the probe head and the covering globe to obtain satisfactory sonographic images (Fig. 23.2). Care should be taken to remove any residual air bubbles between the interphase transducer cover to minimize distortion of the images and presence of acoustic shadows. All the pelvic organs should be identified. The ovaries can be easily located by directing the transducer to the right or left. Hyperstimulated ovaries are easily identified by the presence of many cystic structures representing follicles (Fig. 23.2). Lying immediately underneath the ovary, the iliac vessels are identified. Care should be taken not to puncture any vascular structure during the procedure. In our program, the needles (Wallace, UK, Hansraj Nayyar, Mumbai, India) used for the aspiration are 17 gauge, 33 cm long, and the tips of the needles are scored to enhance echogenicity and proper placement while the follicles are punctured throughout the procedure (Figs 23.3A and B). A suction trap system comprising of a 14 mL Falcon test tube connected to the needle is used in order to collect the follicular fluid (Fig. 23.4). The system is connected to a dual pressure foot pedal-controlled negative pressure suction pump (Gen-X, USA, Trivector Scientific, Mumbai, India) (Fig. 23.5). The negative pressure during the aspiration procedure is always maintained between 90–100 mm Hg. We have found this foot pedal-controlled aspiration system more practical and reliable than syringe aspiration, which provides uncontrolled pressure that is likely to result in damage of some of the oocytes.17 Once the needle penetrates into the ovarian substance, each one of the follicles will be aspirated separately. One milliliter of Flushing media (Medicult, Denmark; Trivector Scientific, Mumbai, India) is added to each of the tubes in order to facilitate dissection of the oocyte-corona cumulus complex in the laboratory. Follicles should be aspirated in a systematic fashion in order to avoid multiple punctures into the same follicle. Once maximal sonographic demonstration of the follicle is obtained by rotating the transducer, the follicle is placed under the path of the needle, which is advanced steadily until the tip penetrates the follicle (Fig. 23.5). As the follicular fluid is obtained, the follicle will be seen to collapse and disappear from the screen. Controversy exists regarding flushing of follicles with culture media after they are aspirated.18–21 At Rotunda no differences in retrieval rates were observed between the cycles that were flushed versus the non-flushed cycles. Once the procedure is completed, a speculum is replaced in the vagina and all the puncture sites are inspected for bleeding. Hemostasis is generally achieved with continued pressure with a gauze on a spongeholding forceps. Operative morbidity for the ultrasound transvaginal harvest is relatively low. Bleeding or infectious complications occur in less than 1 percent of cases.22–30 We give all our patients a single dose of a broad spectrum antibiotic

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Fig. 23.1: Transvaginal Probe with non-lubricated latex probe sheath

Fig. 23.2: Hyperstimulated Ovary

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Flg. 23.3A: Surface Serrations near the needle tip surface to enhance echogenecity

Fig. 23.3B: Image of the echogenic needle tip within a follicle

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Fig. 23.4: The Falcon Test-tube suction trap with heparinized flushing media

Fig. 23.5: The Dual Pressure foot operated Suction Unit intraoperatively. The vast majority of patients tolerate the vaginal follicular aspiration procedure without any difficulty. A large Canadian study that measured the intensity and

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quality of pain experienced during the procedure showed that the majority of patients reported manageable levels of discomfort.31 Transvaginal US-guided oocyte retrieval is a rather simple procedure. Ragni et al32 investigated the question of the ease with which the technique can be acquired and no difference was found between highly experienced physicians and a newly trained group in relation to follicles punctured, oocytes recovered, mean number of follicles aspirated or in duration of the procedure. Difficulties of a successful oocyte retrieval appear to lie in stimulation and endocrinological problems (e.g., empty follicle syndrome) rather than in technical difficulties.33–39 TIMING OF OOCYTE RETRIEVAL Ultrasound monitoring of follicular growth in stimulated cycles is today an indispensable investigation in assisted conception.40–41 Ultrasound provides more accurate information of follicular number and size than can be obtained by estrogen determinations alone40 and it provides information as to the side in which the follicles are located. Most assisted conception programs incorporate a baseline ultrasound scan before therapy is commenced. The scan is scheduled at the beginning of the menstrual cycle on day one of two of the patients menstrual cycle. This scan is to rule out any baseline ovarian cysts more than 15–16 mm in size. Their management however, remains controversial. It has been suggested that the presence of ovarian cysts reduces the success of controlled ovarian stimulation for IVF.36 However other studies suggest that small ovarian cysts identified at the baseline ultrasound scan do not have a negative impact on ovulation induction nor pregnancy rates in an IVF program and aspiration of a unilateral ovarian cyst prior to initiation of gonadotropin therapy does not improve folliculogenesis and oocyte recovery rates.37 The timing of hCG administration in protocols which include pituitary downregulation by gonadotropin releasing hormone analogs (GnRHa) is not as critical and some IVF programs have attempted to reduce the amount of ultrasound monitoring to a minimum.42 Furthermore, it has been shown that the entire monitoring of controlled ovarian hyperstimulation can be successfully performed by ultrasound only. Oocyte retrieval is performed approximately 34–35 hours after the hCG injection; which places the retrieval just prior to the time of ovulation.43–44 Precautions Failure of oocyte retrieval during in υitro fertilization (IVF) is considered as “empty follicle syndrome.” The empty follicle syndrome (EFS) is a frustrating condition in which no oocytes are retrieved in an IVF cycle. Although this is an infrequent event in IVF patients, the economic consequences as well as the emotional frustration of a cancelled cycle due to the inability to obtain oocytes are enormous. The mechanisms responsible for EFS remain obscure, though many hypotheses have been put forward ranging from dysfunctional folliculogenesis to a drug-related problem.33–34,45–46 Many theories have been postulated, some related to an underlying ovulatory disorder or premature oocyte atresia. EFS is a rare event (1.8% of oocyte retrievals) but with profound implications for

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counseling the couple about their future reproductive performance.46 The chances of recurrence of EFS increase with the age of the patient (24% recurrence rate for the 35–39 year age group, and 57 percent for those over 40 years).46 Some authors postulate that ovarian ageing, through altered folliculogenesis, may be implicated in the etiology of EFS and its recurrence. Others reiterate,45 often it is related to improper administration of human chorionic gonadotropin (hCG). A 40-year-old woman underwent IVF for a 10year history of unexplained secondary infertility Two ultrasound-guided oocyte retrievals were performed 34 hours apart due to improper hCG administration prior to the first procedure. Successful retrieval of 16 oocytes, all mature and fertilized, occurred subsequent to the second oocyte retrieval. No pregnancy was established with the fresh cycle. This case report supports the premise that an IVF cycle in which improper hCG administration occurs can be salvaged.45 After partial follicular aspiration, no ovulation or luteinization of the remaining follicles occurred because of continued suppression by the gonadotropin releasing hormone analog.47 It is critical to consider the possibility of improper hCG administration when facing failure of oocyte retrieval. The procedure should be terminated and hCG re-administered, and a second retrieval should be performed 34 hours later. Psychological disorders of infertile patients are traditionally thought to be chronic, to advance gradually, and to be long-term problems. A recent report described a patient in whom an acute psychiatric episode developed immediately after transvaginal ultrasoundguided oocyte retrieval.48 A 34 year old women without history of psychiatric disturbance or adverse reaction to drugs suffered an acute psychiatric episode immediately after oocyte retrieval. She exhibited tachycardia, tachypnoea, transient hypertension and limb rigidity, as well as alterations to stupor and posture. Her vital signs stabilized and she opened her eyes 6 hours later, but she persistently raised her head to the left and stared blankly without response to external stimuli. Nine hours later, she was able to look around but remained unresponsive to stimuli. Aphasia was noted in the next morning and a wishful thinking of having delivered a baby was noted in the afternoon. Memory loss was noted on the third day. The patient was diagnosed as having dissociative amnesia and was discharged after three courses of supportive psychotherapy. Assisted reproductive technology-related acute psychiatric episodes, which may initially mimic brainstem stroke, are rare; however, attention should be paid to high-risk patients, and they should be offered elective psychological counseling much before their elective procedure. A high lateral placement of the ovaries has been described in patients with mullerian agenesis undergoing oocyte retrieval for gestational carrier treatment.49 The high lateral ovarian position, in combination with a relatively inelastic neovagina, makes transvaginal ultrasound-guided oocyte retrieval difficult in a significant portion of these patients. Laparoscopic oocyte retrieval may therefore be frequently required in patients with mullerian agenesis.49 A few recent reports included abdominal ultrasound-guided oocyte retrievals (including two cases undertaken in women with mullerian agenesis), although the precise techniques used in these reports were not fully described.49,50 A26 year-old woman with a history of mullerian agenesis presented for consideration of gestational carrier treatment.51 She had been diagnosed by laparoscopy performed at age 12 years. She exhibited normal sexual development and was confirmed to have a normal karyotype (46, XX). At age 16 years, she had undergone an exploratory laparotomy and left oophorectomy for a 10 cm mucinous cystadenoma. At age 19 years, she had a primary

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Mclndoe vaginoplasty using a split-thickness skin graft harvested from the buttock. Postoperatively, she experienced vaginal stenosis, although this responded to an active dilatation program. She now had been married for 1 year and had experienced no coital difficulties. Her husband was 35 years of age and had fathered one child from a previous marriage and the surrogate mother, who was 44 years of age and had previously carried four pregnancies successfully to term, wished to be her carrier. The commissioning patient’s right ovary could not be adequately imaged transvaginally with a 4-MHz to 8MHz transvaginal ultrasound probe. The ovary was better visualized abdominally using the same probe and was found to be elongated (5.7 cm×1.6 cm×2.2 cm) and with stimulated follicles and was well visualized through the right lower quadrant abdominal wall. A 16-gauge, 35 cm long, double-lumen aspiration needle (Echo Tip Model KJOPSDX-163507; Cook OB/GYN, Spencer, IN) was then thrust briskly through the abdominal wall directly into the ovary using the transvaginal biopsy guide fitted onto the probe. All six follicles were aspirated through this single percutaneous puncture. Although a seemingly unconventional approach, transabdominal-transperitoneal ultrasound-guided oocyte retrieval conferred several advantages for this particular patient. It had been clear from her evaluation that a standard transvaginal sound-guided approach would not be feasible. In addition she had only one ovary, and her stimulation response—limited. Therefore, the follicular aspiration technique with the highest estimated efficiency was chosen. In this specific patient, a standard transvaginal probe was used for ovarian imaging through the anterior abdominal wall. This was feasible because of the patient’s thin body habitus as well as the fact that the abdominal wall could be depressed such that it immediately was in contact with the ovary, providing an adequate visualization window. A further advantage of the transvaginal probe included better near-field resolutionbecause of its higher intrinsic frequency (4 MHz to 8 MHz) than that of a typical abdominal probe (2 MHz to 5 MHz). In addition, most transvaginal probes are designed with an available fixed needle guide, whereas many transabdominal probes are not. Most IVF practitioners are quite accustomed to the transvaginal probefixed needle guide arrangement, although they would need to be prepared for the slight distortion in orientation that comes with this technique. This report excellently described an efficient follicular aspiration procedure in a patient with mullerian agenesis and ovarian malposition.51 Whereas this procedure may only be appropriate and feasible in a select number of cases, it may nevertheless provide certain clear advantages for these patients. Recently, a case of a patient with a history of heart conduction disease, symptom-free and without treatment in the last years, who experienced a severe cardiac complication associated during with vaginal oocyte retrieval (VOR) in υitro fertilization (IVF) was reported.52 Eighty-five minutes after the VOR a severe bradycardia and bradypnea occurred, requiring an emergency application of a pacemaker. Presumably the condition occurred because of a toxic effect of the 400 mg of mepivacaine administered paracervically. It was concluded that in the paracervical anesthesia in the IVF cycles the therapeutic range should be scrupulously followed in patients with heart condition.52

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COMPLICATIONS Although the technique is associated with few serious side effects, early recognition of potential pitfalls may help to prevent long-term sequelae.53–55 Aretrospective study of 7 years in the fertility clinic of an university hospital was undertaken to evaluate the different short-term complications after in vitro fertilization and embryo transfer.54 Shortterm medical complications were analyzed after 1500 transvaginal ultrasonographically guided oocyte retrievals. The authors concluded that short-term medical complications af ter oocyte retrievals are rare (2.8%) with intraperitoneal bleeding contributing to only 0.2 percent of the cases.54 Endovaginal sonography readily allows for identification of enlarged, multicystic ovaries, uterus, and bowel, but provides limited recognition of smaller structures such as small pelvic blood vessels, nerves, and ureters. The most common problem associated with transvaginal ultrasound guided follicle aspiration is undoubtedly minor vaginal bleeding, a result of trauma to vaginal wall vessels.23,24 Such bleeding is however of minimal significance since it invariably responds to application of temporary local pressure.23,24 Such pressure should be applied without using a speculum since stretching of the vaginal walls tends to prolong the bleeding time. Hemoperitoneum may occur from direct damage to pelvic organs (i.e. uterus, bladder and colon) or to pelvic blood vessels misinterpreted as ovarian follicles.55 Furthermore, during puncture of the ovary and follicles, hemoperitoneum may occur as a result of bleeding from a vessel at the time of flushing of the follicular bed with heparin containing solutions.56 Irrigation and aspiration of the follicle usually results in damage to the fine vascular network of blood vessels in the theca interna layer and tends to produce a heavily bloodstained aspirate followed by the accumulation of blood within the collapsed follicle within 2 to 5 minutes. Enhanced bleeding may consequently occur. The avoidance of overdistending of the aspirated follicles during follicular flushing reduces the risk of multiple follicular rupture, and aspiration of all follicles without withdrawing the needle helps to reduce the risk of hemorrhage. Given the ureter’s anatomical position immediately anterolateral to the upper fornices of the vagina, it is surprising that clinically recognizable ureteral injuries do not occur more often than reported. This seems especially true given the relatively high percentage of women presenting for oocyte retrieval who have distortion of pelvic anatomy secondary to adhesions or endometriosis. In a report.24 If 2670 transvaginal oocyte retrievals, not a single case of ureteral injury was encountered, in contrast to a background rate of 0.5–1 percent for all pelvic operations.57 The only case reports of ureteral injury known to the authors that, occurred at the time of oocyte retrieval involved presentation beyond the immediate post-operative period.58– 60 One patient developed symptoms seven days after oocyte retrieval,60 while the other presented for evaluation four months following her procedure.58 Ureteric damage leading to hydronephrosis, infection, and eventual nephrectomy has been described in one IVF patient.58 Another report was the case of a ureteral stricture secondary to ultrasoundguided follicular puncture for oocyte retrieval that was corrected by a laparoscopic approach.61 This approach can minimize postoperative pain, the length of hospitalization, and the period of convalescence and should be considered a minimally invasive option in the management of this rare complication of oocyte retrieval. To the best of our

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knowledge, the only report62 of acute ureteral injury presenting within 24 hours of the inciting event appeared in the year 2002. A 34 year old patient with primary infertility underwent transvaginal sonographic oocyte retrieval using a 6.5 MHz transducer. Patient comfort was maintained using IV. Fentanyl and Propof ol with continuously monitored anesthesia care. A total of 19 oocytes were retrieved with no technical difficulty encountered. Intraoperative blood loss was judged to be minimal, and the patient was transferred to the recovery area from which she was discharged home 45 min later after an uneventful postoperative course. Within 7 h she presented to the emergency department complaining of several hours of worsening right lower quadrant and right flank pain with nausea and emesis. Abdominal examination was notable for normal bowel sounds with moderate right lower quadrant tenderness and voluntary guarding but no evidence of peritonitis. Her right costovertebral area was also mildly tender. Laboratory analysis revealed normal hematocrit, leukocyte count, platelets and electrolytes with a large amount of blood on urinalysis. An endovaginal pelvic sonogram demonstrated a small amount of echogenic fluid in the pelvis with moderately enlarged, multicystic ovaries with normal Doppler blood flow. A renal sonogram revealed mild right hydronephrosis with debris in the right collecting system consistent with blood or pus. The following day, an abdominal/pelvic computerized tomography (CT) scan confirmed right hydronephrosis and mild hydroureter down to the level of the right adnexa, with density in the right collecting system consistent with blood. Subsequently, the patient underwent cystoscopy and right ureteroscopy with ureteral stent placement. On cystoscopy, the bladder mucosa appeared normal with normal urine efflux from the left orifice and none on the right. During right ureteroscopy, the scope could not be passed beyond a point 1 cm above the ureterovesical junction at which a thrombus with underlying mucosal disruption was detected. Stent placement was accomplished without complication. Postoperatively, the patient noted signifi-cant relief of her right lower quadrant pain. Three weeks after her initial cystoscopy, she underwent uncomplicated office cystoscopy and stent removal, followed by five days of fluoroquinolone prophylaxis. A IV pyelogram performed six weeks after stent removal was normal. The ureteral trauma discovered in this case undoubtedly occurred at the time of vaginal puncture as the needle traversed the tissues interposed between the vaginal wall and the ovary. This is the first case report of ureteral trauma in the medical literature in which the patient presented acutely within hours of her oocyte retrieval.62 In the report by Jones and co-workers, their patient had known endometriosis with pelvic adhesions, which, along with repeated ovarian punctures, predisposed her to ureteral injury58 The position of the obstruction at the level of the lower border of the left sacroiliac joint suggests that the injury occurred from passage of the needle through the ovary, as opposed to the case presented here of pre-ovarian needle trauma. For others who may face a similar postoperative patient presentation, the differential diagnosis should include ovarian pathology, such as intra-ovarian hemorrhage or torsion, but should also include an assessment of extraovarian structures; such as the urinary tract and pelvic blood vessels, looking for obstruction or hematoma formation, respectively. Inf ectious causes are not as likely to develop over such a short time course, but should not prevent the judicious use of empirical antibiotic therapy. Likewise, early consultation from a urologist may expedite resolution of symptoms and diminish the chance of more serious sequelae (e.g. fistula formation).

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The second most commonly described complication is pelvic infection. There are a number of theoretical routes for infection. Direct inoculation of vaginal organisms appears to be the route of infection in the majority of cases; reactivation of latent infection in patients with a history of PID22,32 and trauma to a loop of large bowel may be additional causative factors. The last possibility appears to be more theoretical than actual. Howe et al22 reported three cases complicated by pelvic infection out of 92 aspiration attempts: one case required hysterectomy and bilateral oophorectomy. Borlum and Maigaard,25 in a group of almost 400 transvaginal aspirations, reported on two cases of serious pelvic infections that proved to be ovarian abscesses, one of them required oophorectomy In their procedures they used only two vaginal douchings with sterile normal saline, and they also noted that minimizing the number of repeated vaginal penetrations may serve to lower the risk of infection. Curtis et al26 evaluated the risk of pelvic infection following transvaginal oocyte recovery by culturing peritoneal fluid from 25 women with unexplained infertility. The samples were collected laparoscopically at the time of zygote intraf allopian transfer, 24 to 48 hours af ter oocyte collection. The peritoneal cultures were negative in all but one patient. However, high vaginal swabs grew Candida albicans in three cases, and endocervical specimens were all negative. In this context, vaginal disinf ection with 1 percent solutions of Povidone-iodine and normal saline was compared.27 There was no increase in infection risk and no difference in fertilization and cleavages were found, but pregnancy rate was significantly higher in the normal saline group. Using only 10 percent Povidine-iodine vaginal preparations, Evers et al,23noted that no pelvic infection occurred in a group of 181 patients who underwent transvaginal follicular aspiration. Prophylactic antibiotic therapy is advised when vaginal route of ovum aspiration is used or when PID or other intra-abdominal infection has occurred in the past. Meldrum et al28 demonstrated 88 transvaginal retrievals with no pelvic infections, under the coverage of intravenous cefazolin and vaginal preparation with Povidine-iodine followed by saline irrigation. Completion of follicular aspiration, without reinserting the needle through the vaginal wall was recommended. Bennett et al24 reported a low incidence of pelvic infection in 2,670 consecutive transvaginal ultrasound—directed follicle aspirations and do not advocate routine antibiotic prophylaxis. Acute abdomen as a result of ruptured tuboovarian abscess, endometriomas or hemoperitoneum may be very severe complications of transvaginal oocyte retrieval that require accurate diagnosis and definitive and prompt intervention. Dicker et al29 reported on 14 out of 3,656 patients undergoing oocyte retrieval presenting with the clinical picture of acute abdomen. An Israeli group63 reported recently a case of vertebral osteomyelitis as a complication of vaginal oocyte retrieval. A41-year-old woman who underwent IVF-ET treatment with the use of transvaginal ultrasound guided needle aspiration for oocyte retrieval landed up with excruciating back pain hours after the procedure. Vertebral osteomyelitis was diagnosed and treated with antibiotics. The authors recommended that when severe low back pain occurs after ovum retrieval, vertebral osteomyelitis should be considered.63 Early diagnosis requires a high index of suspicion.

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CONCLUSIONS Transvaginal ultrasound-guided follicular puncture for oocyte retrieval is a highly efficient and minimally invasive method for assisted reproductive techniques and is definitely the worldwide gold standard today. The technique we use today has evolved over the past twenty years with volumes of literature written on all methods that were used in the journey towards the present day minimally invasive technique. This method has a very short training curve and is easily reproducible making it a relatively safe procedure even for fellows in training withbasic transvaginal ultrasound skills. Complications related to this procedure are rare and can be avoided. “Effort only fully releases its reward after a person refuses to quit.” Napoleon Hill

REFERENCES 1. Lopata A, Johnston IWH, Leeton JR. Collection human oocytes at laparoscopy and laparotomy. Fertil Steril 1974; 25:1030–34. 2. Steptoe P, Edwards RG. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries gonadotrophins. Lancet 1970; 1:683–84. 3. Daniell F, Pettway DE, Maxson WR. The role of lapararoscopic adhesiolysis in an in vitro fertilization program. Fertil Steril 1983; 40:49–52. 4. Edwards RG. Effects of gas phase on oocytes during difficult laparoscopies, in Human Conception In Vitro. Edwards RG, Purdy IM (Eds). London: Academic Press, 1982; 123–25. 5. Jansen RPS, Anderson JC. Transvaginal gamete and embryo transfer to the Fallopian tubes. In Capitanio GL, Asch RH, De Cocco L (Eds): GIFT: From basics to Clinics, Vol 63, New York: Raven Press, 1989; 383–89. 6. Lenz S, Lauritsen JG, Kjellow M. Collection of human oocytes for in vitro fertilization by ultrasonically guided follicular puncture. Lancet 1981; 1:1163–64. 7. Gleicher N, Friberg J, Fullan N et al. Egg retrieval for in vitro fertilizationby sonographically controlled vaginal culdocentesis. Lancet 1983; 1:508–09. 8. Kratochwil A, Urban GU, Friedrich E. Ultrasonic tomography of the ovaries. Ann Chir Gynecol 1972; 61:211–14. 9. Hackeloer BJ, Fleming R, Robinson HP, Adam AH, CourtJRT. Correlation of ultrasonic and endocrinologic assessment of human follicular development. Am J Obstet Gynecol 1979; 135:122–28. 10. Drugan A, Blumenfeld Z, Erlyk I et al. The use of transvaginal sonography in infertility: In: Timor-Tritsch IE, Rottem S (Eds). Transvaginal Sonography. NY: Elsevier Scientific Pub Co, 1988; 143–45. 11. Feichtinger W, Kemeter P. Laparoscopically or ultrasonically guided follicle aspiration for in vitro fertilization. J In Vitro Fertil Embryo Transfer 1984; 1:244–49. 12. Feichtinger W, Kemeter P. Transvaginal sector scan sonography for needle guided transvaginal follicle aspiration and other application in gynecologic routine and research. Fertil Steril 1986; 45:722–26. 13. Dellenbach P, Nisand I, Moureau L, Feger B, Plumere C, Brun B et al. Transvaginal sonographically controlled ovarian puncture for egg retrieval. Lancet 1984; 1:1467–68. 14. Parson J, Booker M, Goswamy R et al. Oocyte retrieval for in vitro fertilization by ultrasonically guided needle aspiration via the urethra. Lancet 1985; 1:1076–77.

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15. Kogosowski A, Lessing J, Amit A. Epidural block: A preferred method of anesthesia for ultrasonically guided oocyte retrieval. Fertil Steril 1987; 47:166–71. 16. Rosenblatt MA, Bradford CN, Bodian CA, Grunfeld L. The effect of a propofol-based sedation technique on cumulative embryo scores, clinical pregnancy rates, and implantation rates in patients undergoing embryo transfers with donor oocytes. J Clin Anesth 1997; 9(8):614–17. 17. Cohen J, Avery S, Campbell S. Follicular aspiration using a syringe suction system may damage the zona pellucida. J In Vitro Fert Embryo Transfer 1986; 3:224–27. 18. Scott R, Hofmann G, Muasher S, Acosta A, Kreiner D, Rozenwaks Z. A prospective randomized clinical comparison of single- and double lumen needles for transvaginal follicular aspiration. J in Vitro Fert Embryo Transf 1989; 6:98–102. 19. Haines C, EmesA, O’Shea R, Weiss T. Choice of needle for ovum pick up. J in Vitro Fert Embryo Transf 1989; 6:111–54. 20. Waterstone JJ, Parsons JH. A prospective study to investigate the value of flushing follicles during transvaginal ultrasound directed follicle aspiration. Fertil Steril 1992; 57:221–23. 21. Kingsland CR, Taylor CT, Aziz N, Bickerton N. Is follicular flushing necessary for oocyte retrieval? A randomized clinical trial. Hum Reprod 1991; 6:382–83. 22. Howe RS, Wheeler C, Mastroianni L Jr, Blasco L, Tureck R. Pelvic infection after transvaginal ultrasound guided ovum retrieval. Fertil Steril 1988; 49:726–28. 23. Evers HH, Larsen JF, Gnanny GG, Sieck UV. Complications and problems in transvaginal sector scan guided follicle aspiration. Fertil Steril 1988; 9:272–78. 24. Bennett SJ, Waterstone JJ, Cheng WC, Parsons J. Complications of transvaginal ultrasound directed follicle aspiration: A review of 2,670 consecutive procedures. J Assist Reprod Genet 1993; 10:72–77. 25. Borlum KG, Maigaard S. Transvaginal oocyte aspiration and pelvic infection [letter]. Lancet 1989; 2:53–54. 26. Curtis P, Amso N, Keith E, Bernard A, Shaw RW. Evaluation of the risk of pelvic infection following transvaginal oocyte recovery. Hum Reprod 1991; 6:1294–97. 27. Van Os HC, Roozenburg BJ, Janssen-Caspers HAB, Leerentveld RA, Scholtes MCW, Zeilmaker GH et al. Vaginal disinfection with povidine iodine and the outcome of in vitro fertilization. Hum Reprod 1992; 7:349–50. 28. Meldrum DR. Antibiotics for vaginal oocyte aspiration. J In Vitro Fert Embryo Transf 1989; 6:1–2. 29. Dicker D, Ashkenazi J, Feldberg D, Levy T, Dekel A, Ben Rafael Z. Severe abdominal complications after transvaginal ultrasonographically guided retrieval of oocytes for in vitro fertilization and embryo transfer. Fertil Steril 1993; 59:1313–15. 30. Barber R, Porter R, Picker R. Transvaginal ultrasound directed oocyte collection for in vitro fertilization: Success and complications. J Ultrasound Med 1988; 7:377–80. 31. Shatford LA, Brown SE, Yuzpe AA et al. Assessment of experienced pain associated with transvaginal ultrasonography guided oocyte recovery for IVF patients. Am J Obstet Gynecol 1989; 160:1002–05. 32. Ragni G, Lombroso GC, de Laurentis L. Echoguided transvaginal oocyte retrieval: effective and easy to learn technique. Acta Eur Fertil 1991; 22:89–90. 33. La Sala GB, Ghirardini G, Cantarelli M, Dotti C, Cavalieri S, Torelli MG. Recurrent empty follicle syndrome. Hum Reprod 1991; 6:651–52. 34. Ben-shlomo I, Schiff E, Levran D, Ben Rafael Z, Mashiach S, Dor J. Failure of oocyte retrieval during in vitro fertilization: a sporadic event rather than a syndrome. Fertil Steril 1991; 55:324– 27. 35. Haning RV, Austin CW, Kuzma DL, Shapiro SS, Zweibel WJ. Ultrasound evaluation of estrogen monitoring for induction of ovulation with menotrophins. Fertil Steril 1982; 37:627– 32. 36. Thatcher SS, Jones E, De Cherney AH. Ovarian cysts decrease the success of controlled ovarian hyperstimulation and in vitro fertilization. Fertil Steril 1989; 52:812–16.

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37. Karande VC, Scott RT, Jones GS, Muasher SJ. Non functional ovarian cysts do not affect ipsilateral or contralateral ovarian performance during in vitro fertilization. Hum Reprod 1990; 5:431–33. 38. Rizk B, Tan SL, Kingsland C, Steer C, Marson BA, Campbell S. Ovarian cyst aspiration and the outcome of in vitro fertilization. Fertil Steril 1990; 54:661–64. 39. Golan A, Herman A, Soffer Y, Bukovsky I, Ron-El R. Ultrasonic control without hormone determination for ovulation induction in in-vitro fertilization/embryo transfer with gonadotrophin-releasing hormone analog and human menopausal gonadotrophin. Human Reprod 1994; 9:1631–33. 40. Hodgen GD. Physiology of follicular maturation. In Jones HW et al (Eds): In vitro Fertilization. Norfolk: Williams and Wilkins, 1986; 21–25. 41. Keller D, Strickler R, warren J. Clinical Infertility. Norfolk, CT: Appleton-Century Crofts, 1984; 213–15. 42. De Ziegler D, Cedars MI, Randle D et al. Suppression of the ovary using a gonadotropin releasing hormone agonist prior to stimulation for oocyte retrieval. Fertil Steril 1987; 48:807– 10. 43. Cohen J, Debache C, Pez JP. Transvaginal sonographically controlled ovarian puncture for oocyte retrieval for in vitro fertilization. In Vitro Fert Embryo Transfer 1986; 3:309–11. 44. Mantzavinos T, Garcia J, Jones HW. Ultrasound measurement of ovarian follicle stimulated by human gonadotropins for oocyte recovery and in vitro fertilization. Fertil Steril 1983; 40:461– 63. 45. Esposito MA, Patrizio P. Partial follicular aspiration for salvaging an IVF cycle after improper hCG administration. A case report. J Reprod Med 2000; 45(6):511–14 46. Zreik TG, Garcia-Velasco JA, Vergara TM, Arici A, Olive D, Jones EE. Empty follicle syndrome: evidence for recurrence. Hum Reprod 2000; 15(5):999–1002. 47. Sandler B, Fox J, Garrisi J. Very low dose Lupron enhances follicular recruitement and prevents premature luteinization in controlled ovarian hyperstimulation. Scientific program, 36th Annual Meeting of the Society for Gynecological Investigation, San Diego, CA, 1989. 48. Hwang JL, Kuo MC, Hsieh BQChang CH, Jou LC, Chen WH, Wu GJ. An acute psychiatric episode following transvaginal oocyte retrieval. Hum Reprod 2002; 17(4):1124–26. 49. Wood EG, Batzer FR, Corson SL. Ovarian response to gonadotrophins, optimal method for oocyte retrieval and pregnancy outcome in patients with vaginal agenesis. Hum Reprod 1999; 14:1178–81. 50. Giacolone P-L, Laffargue F, B6nos P, Dechaud H, Hedon B. Successful in vitro fertilization surrogate pregnancy in a patient with ovarian transposition who had undergone chemotherapy and pelvic irradiation. Fertil Steril 2001; 76:388–9. 51. Damario MA. Transabdominal-transperitoneal ultrasoundguided oocyte retrieval in a patient with mullerian agenesis. Fertil Steril 2002; 78(1):189–91. 52. Ayestaran C, Matorras R, Gomez S, Arce D, Rodriguez-Escudero F. Severe bradycardia and bradypnea following vaginal oocyte retrieval: a possible toxic effect of paracervical mepivacaine. Eur J Obstet Gynecol Reprod Biol 2000; 91(1):71–73. 53. Bennett SJ, Waterstone JJ, Cheng WC, Parsons J. Complications of transvaginal ultrasounddirected follicle aspiration: A review of 2670 consecutive procedures. J Assist Reprod Genet 1999; 10,72–74. 54. Govaerts I, Devreker F, Delbaere A, Revelard P, Englert Y. Shortterm medical complications of 1500 oocyte retrievals for in vitro fertilization and embryo transfer. Eur J Obstet Gynecol Reprod Biol 1998; 77(2):239–43. 55. Azem F, Wolf Y, Botchan A, Amit A, Lessing JB, Kluger Y. Massive retroperitoneal bleeding: a complication of transvaginal ultrasonography-guided oocyte retrieval for in vitro fertilizationembryo transfer. Fertil Steril 2000; 74:405–6.

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56. Ashkenazi J, Ben-David M, Feldberg D, Shelef M, Dicker D, Goldman J. Abdominal complications following ultrasonically guided percutaneous transvesical collection of oocytes for in vitro fert. J In Vitro Fert Embryo Transf 1987; 4:316–18. 57. Daly J, Higgins KA. Injury to the ureter during gynecological surgical procedures. Surg Gynecol Obstet 1988; 167, 19–22. 58. Jones WR, Haines CJ, Matthews CD, Kirby CA. Traumatic ureteric obstruction secondary to oocyte recovery for in vitro fertilization: A case report. J in vitro Fert Embryo Transf 1985; 6:185–87. 59. Mourelle Fl, Hereter L. Urerteral lesion secondary to vaginal ultrasound puncture for oocyte recovery in invitro fertilization. Hum Reprod 1997; 12:948–50. 60. Coroleu B, Mourelle FL, Hereter L et al. Ureteral lesion secondary to vaginal ultrasound follicular puncture for oocyte recovery in in-vitro fertilization. Hum Reprod 1997; 12:948–50. 61. Fugita OE, Kavoussi L. Laparoscopic ureteral reimplantation for ureteral lesion secondary to transvaginal ultrasonography for oocyte retrieval. Urology 2001; 58(2):281. 62. Miller PB, Price T, Nichols JE Jr, Hill L. Acute ureteral obstruction following transvaginal oocyte retrieval for IVF. Hum Reprod 2002; 17(1):137–38. 63. Almog B, Rimon E, Yovel I, Bar-AmA, AmitA, Azem F. Vertebral osteomyelitis: a rare complication of transvaginal ultrasoundguided oocyte retrieval. Fertil Steril 2000; 73:1250–52.

CHAPTER 24 Gamete Intrafallopian Transfer (GIFT) Andrea Borini, Luca Dal Prato OVERVIEW Gamete intrafallopian transfer (GIFT) is a technique of reproductive assistance with a proven success rate in patients with patent tubes. In contrast to in vitro fertilization (IVF), in GIFT procedures oocytes and spermatozoa are transferred together into the fallopian tubes, where fertilization occurs immediately after eggs retrieval. Thus, this technique is simpler than IVF because it does not require sophisticated cellculture procedures. However, classically, the procedure is performed laparoscopically and needs general anaesthesia. For this reason, alternative approaches to laparoscopy have been explored. The first GIFT was carried out unsuccessfully in the early 1970’s, but the method was firstly described by Asch et al1 who reported the first pregnancy in 1984. INDICATIONS This procedure was suggested as an alternative technique to IVF for all the infertility causes, also associated with seminal pathology, but with maintained tubal function. However, there is clear evidence that in case of severe oligoasthenospermia or immunological infertility, results achieved with GIFT are poor. The most important indication for GIFT are: i. idiopathic infertility, ii. minimal or mild endometriosis (grade I and II of ASRM classification) with at least one normal fallopian tube. Other indications for which GIFT may be used are: 1. mild seminal factor, 2. failure of previous artificial insemination cycles 3. cervical factor 4. minimal pelvic adhesions.

TECHNIQUE The steps of the GIFT procedure are summarized in Table 24.1.

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Table 24.1: Steps of GIFT 1. Selection of the patients. 2. Controlled ovarian hyperstimulation with ultrasound and endocrine monitoring. 3. Oocyte retrieval (laparoscopy, transvaginal ultrasound guided). 4. Evaluation of oocyte maturity 5. Sperm preparation. 6. Loading of oocytes and spermatozoa into the transfer catheter 7. Tube cannulation (laparoscopy, minilaparotomy, transcervical) 8. Transfer of gametes into the tubal lumen.

Each couple should undergo a preliminary examination, in order to check at least if tubes are open (laparoscopy or hysterosalpingography) and to exclude severe oligoasthenozoospermia. Fully mature eggs and spermatozoa of good motility and morphology are important for GIFT. As in all ART procedures, to enhance the success rate a controlled ovarian hyperstimulation (COH) is needed. The most widely used protocol for COH, in women undergoing assisted reproduction, involves the use of Gonadotropin Releasing Hormone Agonists (GnRH-a), followed by the administration of gonadotropins. Ultrasound monitoring of follicular growth and serum 17β-estradiol assay is usually performed until one or more follicles reach a diameter >18–20 mm. Oocyte retrieval is then programmed 34–36 hours after the administration of 10000 IU of Human Chorionic Gonadotropin. In the original description, follicular aspiration was performed trans-laparoscopically, but it is invasive and requires general anaesthesia. More recently, transvaginal ultrasound guided aspiration has become the preferred method for oocyte retrieval;2 it is easier and allows a better visualization of ovarian follicles, even those localized deep in the ovary, therefore more oocytes are recovered. Moreover, the retrieval is feasible even when pelvic adhesions hamper a laparoscopic access to the ovaries. Finally, less manipulation of the pelvic viscera occurs, therefore general anaesthesia is not necessary and may be replaced by conscious sedation. After retrieval, oocytes are placed in culture medium to assess their maturity. The semen sample should be collected by the male partner two hours before oocyte retrieval. Following liquefaction, spermatozoa are prepared in the same way as for IVF, with the swim up method or by a three layer discontinuous gradient of a colloidal substance, depending on the degree of semen alteration. Spermatozoa and oocytes are then loaded in a transfer teflon catheter. Different kind of transfer catheter are available, none has been proven to be the best. In our Centre we use the Frydman and Belaisch-Allart Bi-set for GIFT (CCD, Paris, France). One to four mature oocytes are usually transferred, together with about 200000 motile sperms per oocyte in about 30 µ1 medium. The most frequently used method to replace gametes into the tubal ampulla is via laparoscopy, but minilaparotomy has been also used both for oocyte retrieval and gametes transfer.

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Laparoscopic GIFT The technique is the same as of a standard two-puncture operative laparoscopy, with a subumbilical incision for a 5 mm endoscope and a 3 mm operative way trocar inserted above the symphysis. The peritoneal cavity is inspected and the fimbriated end of the fallopian tube is exposed with a grasper and gently elevated. The GIFT catheter is inserted through the abdominal wall on the midline about half way between the two trocars and threaded about 3–4 cm into the ampulla. The contents of the catheter are gently expelled into the Fallopian tube and the catheter is returned to the laboratory and flushed to ensure that the gametes have been transferred. Gametes may be transferred in one or in both tubes. The decision on which tube to choose depends on their morphology and accessibility. No real advantage of bilateral on unilateral transfer has been documented.3–4 Transcervical GIFT Alternative transfer techniques have been suggested in order to avoid laparoscopy and general anaesthesia (Table 24.2). Jansen and Anderson in 1987 described transcervical ultrasound guided tubal cannulation with a coaxial system of catheters.5 The system is composed by a firm, but still flexible outer cannula, bearing a lateral curve for entering the uterine angle, with an obturator that straighten the cannula allowing transit of the cervix.

Table 24.2: Transcervical GIFT No of cycles Pregnancies (%) GEU Ultrasound guided Bustillo 1989 Hazot 1989 Jansen 1989 Lucena 1990 Anderson 1990 Blind Lisse 1990 Ferraiolo 1991 Hysteroscopic Wurfel 1990 Possati 1991 Seracchioli 1991 Seracchioli 1995 Falloposcopic Porcu 1997

17 18 10 7 44

1 (5.9) 1 (6.0) 1 (10) 3 (42.8) 8 (18.2)

0 0 0 1 1

44 26

10 (22.7) 7 (26.9)

0 1

21 27 49

4 (19) 7 (25.9) 13 (26.5)

0 0 0

25

7 (28)

0

Withdrawal of the obturator when the cannula is in the uterine cavity allows the tip to reach the uterotubal junction. Then the transfer catheter can be passed through the

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cannula into the tube. The results reported are variable (Table 24.2) and show a better outcome when replacing embryos than oocytes. Some authors have questioned about the need of the ultrasound guidance and performed a blind tubal cannulation with the Jansen Anderson kit. Bauer reported 4 pregnancies in 16 transcervical TET.6 Lisse and Sydow,7 and Ferraiolo et al,8 reported a 23 percent and 26 percent pregnancy rate respectively with GIFT. The low uniformity of the results and of the time needed for the tubal transfer (time between 3 and 40 minutes is reported by some authors) poses a question about the reproducibility of the techniques. The major problem in transcervical cannulation (both blind and ultrasound guided) is the impossibility to directly visualize the uterotubal ostium. To overcome this problem, hysteroscopy has been used to perform tubal transfer under direct view of the ostium. Wurfel et al,9 achieved the first pregnancy with this technique without general anaesthesia. In 1991 Possati et al, treated 26 women by putting a flexible transfer catheter through the operating channel of a chorionhysteroscope and reported a 25.9 percent pregnancy rate and no ectopic implantation.10 Later, the same group11 improved the technique using a flexible hysteroscope and a Jansen and Anderson kit, inserted side by side through the cervix. One hundred and thirty-three patients were treated. Pregnancy and implantation rates of hysteroscopic GIFT (28.9% and 9% respectively) were not significantly different from those obtained with laparoscopic GIFT (43.3% and 14%). Hysteroscopic GIFT can be an effective and simple alternative to traditional techniques in selected cases, especially in cases of pelvic adhesions where the tubes are out of reach. A further evolution of this studies led to the development of a falloposcopic approach. Intrafallopian transfer was performed by cervical cannulation of the tube using a linear everting catheter incorporating direct falloposcopic vision of the tubal lumen to confirm directly not only ostia visualization, but also placement in the best portion of the fallopian tube in a safe and atraumatic way. Twenty-five patients were treated; PR was 28 percent and delivery rate 20 percent. Neither ectopic pregnancies nor tubal damage or other complications during the procedure occurred.12 RESULTS A multicentric comparative trial on GIFT and IVF reported significantly higher pregnancy rate and delivery rate per transfer with GIFT than with IVF (27.5% and 20.2% vs. 16.4% and 11.9% respectively).13 The majority of reports of the same period agrees upon such results. The most recent data available come from the 1998 ASRM/SART registry.14 The clinical pregnancy rate was 29.1 percent per initiated cycle, 34.7 percent per retrieval and 35.2 percent per transfer. Ectopic pregnancies were 1.6 percent. The delivery rate was 23 percent per cycle and 27.8 percent per transfer; 30 percent were twins, 5.4 percent triplets and 0.3 percent more than triplets. Comparing these data with the previous annual registries, two major features may be observed (Table 24.3). Firstly the number of GIFT cycles progressively declines year by

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year, whereas the number of IVF/ICSI is increasing. Secondly, the outcome of GIFT has not significantly changed in the last 10 years, whereas IVF and ICSI have progressively improved.

Table 24.3: Result of GIFT and IVF/ICSI trough the years from the ASRM/SART registry No. of transfers 1991 1995 1996 1997 1998

4370 3269 2379 1640 1068

GIFT Pregnancy rate

Delivery rate

34.7% 34.9% 27.4% 35.1% 38.2% 35.2%

27.2% 29.3% 30.4% 27.8%

No. of transfers

IVF Pregnancy rate

18372 31794 35859 41270 47529

21.9% 30.7% 33.3% 36.5% 37.8%

Delivery rate 17.5% 25.0% 27.9% 29.8% 31%

Factors that may Influence the Results Quality and Number of Oocytes The quality and the number of oocytes seem to influence the results of GIFT. A report shows a 39.6 percent pregnancy rate with the transfer of 5 mature oocytes and only a 16.5 percent if immature oocytes are transferred, emphasizing the importance of oocyte quality for a positive outcome.15 The best number of oocytes to be replaced at GIFT is still a matter of debate, since it affects the expectation of pregnancy and the risk of high order multiple pregnancies. Several studies found a direct correlation between the number of oocytes transferred and the pregnancy rate.4,16–17 Corson reported a 30 percent pregnancy rate per cycle following the transfer 2 oocytes versus 52 percent with 3 oocytes.16 Another study17 performed on 399 cycles, found a three time increase in pregnancy rate in women in which four or more oocytes were transferred. An increase over 5 oocytes transferred, however did not change the pregnancy rate. On the other hand, as in IVF, an increase in the number of oocytes transferred increases the incidence of multiple pregnancies.18 Some authors suggest as a good practice not to transfer more than 4–5 oocytes,19 or even no more than 2–3.20 Oocytes in excess may be inseminated in υitro and the developed embryos frozen for a future use. This management allows a better outcome per oocyte retrieval, limiting the number of high order pregnancies. Age As in all ART treatments, the outcome of GIFT decreases with age. The decrease in results in women over 40 yeafs is dependent on the worsening in the quality of oocytes. This may justify a transfer of a higher number of oocytes, but some authors believe that an increase in the number of oocytes cannot compensate their decrease in quality20 Some authors suggested that GIFT may offer couples a higher probability of pregnancy than IVF, particularly in women aged between 30 and 40 years,21 but more

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recent data14 show a comparable decrease in pregnancy rate and increase in abortion rate with both procedures. Women over 40 years of age and no male factor inf ertility had a 6.8 delivery rate per initiated cycle and 9.1 percent delivery rate per transfer with GIFT versus 7.7 percent and 11.4 percent respectively with IVF. Cause of Infertility The cause of infertility is an important determinant of the outcome of GIFT. The best success rates have been achieved in unexplained infertility and mild endometriosis, the worst results in male factor and immunologic factor infertility. Rombauts et al21 reported that in patients aged >36 years with tubal and male factor infertility, the cumulative pregnancy rate was lower when compared to unexplained infertility and endometriosis. Semen parameters are a crucial factor for the outcome of GIFT. Several studies demonstrated a decrease in the success rate with GIFT in cases of low sperm motility and low normal morphology; the total number of spermatozoa seems to be a minor determinant. Guzick et al14 reported a 32 percent pregnancy rate with a sperm motility >30 percent and only 6.7 percent when the motility has <30 percent. With regard to concerning morphology, in the same study, a decrease in pregnancy rate from 36 percent to 11 percent has been described when spermatozoa with normal morphology were lower than 50 percent. We analyzed the influence of seminal characteristics on GIFT utilizing normozoospermic, oligoasthenospermic and donor spermatozoa. Pregnancy and implantation rates were significantly higher with donor spermatozoa than with the normozoospermic or oligoasthenozoospermic male partner, and couple with an oligoasthenospermic male partner had the worst outcome (Table 24.4). These data suggest that GIFT is not very effective for couples with male factor infertility23

Table 24.4: Influence of spermatozoa characteristics on GIFT. From Seracchioli et al, 1993, modified. Oligoasthenozoospermic Normozoospermic partner partner No. of transfers 80 110 Oocytes transferred 4.2±1.1 4.0±1.0 No. of sperms 6.4±5.7# 24.1±20.8# (106/ml) after treatment Motility after 74.7±9.6# 84.9±5.8# treatment Pregnancies (%) 14 (15.9%)* 38 (32.7%)* Miscarriages (%) 6 (42.8%) 8 (21%) Implantation rate 3.7%* 10.3%* # p<0.001 group 1 versus groups 2 and 3. * p<0.01 group 1 versus groups 2 and 3 and group 2 versus group 3.

Donor spermatozoa 78 3.8±1.3 18.4±12.7#

82.8±7.5# 44 (51.1%)* 10 (22.7) 17.1%*

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In another study,24 we investigated the role of GIFT and tubal embryo transfer (TET) in women with patent tubes, but with oligoasthenozoospermic partners. Women with normozoospermic partners had similar pregnancy rates either with GIFT or TET (about 38%). On the other hand women with oligoasthenozoospermic partners had a significantly lower pregnancy rate with GIFT than with TET (16.3% versus 38.2%). Some studies suggests that endometriosis at any stage adversely affects the outcome of GIFT, but there is no correlation with the severity of disease among endometriosis cases.15 Others37 showed that the number of oocytes retrieved is decreased in women with endometriosis, but, since the number of oocytes needed for GIFT is limited, the final outcome of GIFT is not affected. Among the factors affecting the outcome of GIFT, a recent report include the light source used for laparoscopy25 Pregnancy rate was 50 percent with a conventional halogen light source and 9 percent with a xenon light source. It was found that Xenon source emitted more ultraviolet light than halogen and therefore it could be more detrimental to oocytes. COMPLICATIONS Complications of oυarian stimulation: oυarian hyperstimulation syndrome. The incidence is the same in every ART program: moderate, 3–4 percent, severe 0.1–0.2 percent.26 Complications of ultrasound-guided oocyte retrieval. Infection is the most frequent complication. A0.3 percent incidence has been reported.27 Women at highest risk are those with endometriosis and a previous history of pelvic inflammatory disease. Prophylactic antibiotic treatment was recommended by some investigators.28 Other possible complications described are bleeding and injury of pelvic viscera. Complications of laparoscopy. Are related to the anaesthesia, pneumoperitoneum, or to visceral and vascular injuries (about 3/10000) caused by trocar insertion and other instruments used, and infections.26 Two cases of ovarian torsion after GIFT have been reported.29 Ectopic and heterotopic pregnancy The incidence of ectopic pregnancy is not increased when compared to other procedures. The 1998 ASRM/SART registry14 reported a rate of 1.6 percent per clinical pregnancy with GIFT and 2.1 percent with IVF. This low incidence, despite the fact that the gametes are placed directly into the tube is probably a consequence of a good selection of patients undergoing the procedure. This compared favorably to an estimated overall incidence of ectopic pregnancy in the USA of 2 percent.30 Heterotopic pregnancy is a rare event; its incidence after spontaneous conception was estimated to be about 1/2600.31 The incidence has increased with ART, after the transfer of multiple oocytes or embryos, but it is still very low: 1.4 percent per pregnancy,32 0.4 percent,33 0.8 percent.34

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FUTURE DIRECTIONS AND CONTROVERSIES Three major advantages have been assumed for GIFT over IVF:1) it does not require sophisticated cell-culture procedures and manipulation of gametes is minimal; 2) tubal environment may be better than a laboratory to enhance oocyte maturation and sperm function, resulting in improved fertilization and embryo development; 3) fertilization takes place in the ampulla, in a more “natural” manner; then the embryos, after their journey through the tube, reach the uterine cavity at a developmental stage which is synchronous with that of the endometrium. For this “natural” fertilization, GIFT procedures have been considered more acceptable than IVF by religious authorities, particularly the Catholic Church. On the other hand, a limitation of GIFT is that it does not provide any information on fertilization and embryo cleavage. Today, the improved systems of cell culture and the development of new media allow physicians to obtain embryos of better quality and also to transfer them at blastocyst stage,35 increasing the success rate of IVF procedures. Thus, some of the advantages of GIFT over IVF have been lost. Moreover, GIFT is a more expensive procedure, due to the higher costs of general anaesthesia and operative setting. Transcervical techniques have partially simplified the procedure, but they have provided a few worse results than the traditional technique, thus their fortune is decreased in the last years. Anyway, transcervical tubal transfer remains a useful alternative to laparoscopic transfer in selected cases. However, today the technique of choice for tubal transfer is still laparoscopy (associated to transvaginal ultrasound guided oocyte retrieval). For all these reasons, through the years, the number of GIFT treatments have decreased in contrast with the increasing number of IVF cycles. However, the success rate of GIFT is still good, therefore this procedure should not be abandoned, because it can still be a useful tool, in selected cases, provided that at least one Fallopian tube has normal function and no severe male factor is present. Today, GIFT remains indicated when a couple refuses IVF for ethical and religious reasons, and in situations in which an elaborate and adequate cell-culture laboratory is not available. The development of new minilaparoscopes allows the performance of outpatient GIFT in sedation and local anaesthesia. A recent study36 reports comparable pregnancy rates under conscious sedation (43.7%) and under general anaesthesia (45.7%). REFERENCES 1. Asch RH, Ellsworth LR, Balmaceda JP et al. Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 1984; 2:1034–35. 2. Dellenbach P, Nisand I, Moreau L et al. Transvaginal, sonographically controlled ovarian follicle puncture for egg retrieval. Lancet 1984; 1:1467. 3. Haines CJ, O’Shea RT. Unilateral gamete intrafallopian transfer: the preferred method? Fertil Steril 1989; 51:518–19. 4. Yee B, Rosen GF, Chacon RR et al. Gamete intrafallopian transfer: the effect of the number of eggs used and the depth of gamete placement on pregnancy initiation. Fertil Steril 1989; 52:639–44.

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5. Jansen RP, Anderson JC. Catheterisation of the fallopian tubes from the vagina. Lancet 1987; 2:309–10. 6. Bauer O, Diedrich K, Ven VDH. Transvaginal tubal embryo stage transfer. In “VI World Congress IVF and Alternate Assisted Reproduction”: Jerusalem. 1989; 31. 7. Lisse K, Sydow P. Transvaginal gamete intrafallopian transfer. Abstract of the II joint ESCO/ESHRE Meeting. Hum Reprod Suppl 1990; 5:99. 8. Ferraiolo A, Croce S, Anserini P et al. ‘Blind’ transcervical transfer of gametes in the fallopian tube: a preliminary study. Hum Reprod 1991; 6:537–40. 9. Wurfel W, Steck T, Spingler H et al. Hysteroscopy for gamete intrafallopian transfer. Acta Europaea Fertilitatis 1992; 21:133–37. 10. Possati G, Seracchioli R, Melega C et al. Gamete intrafallopian transfer by hysteroscopy as an alternative treatment for infertility. Fertil Steril 1991; 56:496–99. 11. Seracchioli R, Porcu E, Ciotti P et al. Gamete intrafallopian transfer: prospective randomized comparison between hysteroscopic and laparoscopic transfer techniques. Fertil Steril 1995; 64:355–59. 12. Porcu E, Dal Prato L, Seracchioli R et al. Births after transcervical gamete intrafallopian transfer with a falloposcopic delivery system. Fertil Steril 1991; 67:1175–77. 13. Testart J, Plachot M, Mandelbaum J et al. World collaborative report on IVF-ET and GIFT: 1989 results. Hum Reprod 1992; 7:362–69. 14. ASRM/SART. Assisted reproductive technology in the United States: 1998 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2002; 77:18–31. 15. Guzick DS, Yao YA, Berga SL et al. Endometriosis impairs the efficacy of gamete intrafallopian transfer: results of a case-control study Fertil Steril 1989; 62:1186–91. 16. Corson SL, Batzer F, Eisenberg E et al. Early experience with the GIFT procedure. J Reprod Med 1986; 31:219–23. 17. Penzias AS, Alper MM, Oskowitz SP et al. Gamete intrafallopian transfer: assessment of the optimal number of oocytes to transfer. Fertil Steril 1991; 55:311–13. 18. Nelson JR, Corson SL, Batzer FR et al. Predicting success of gamete intrafallopian transfer. Fertil Steril 1993; 60:116–22. 19. Weckstein LN, JacobsonA, Galen DI. The role of cryopreservation in GIFT and ZIFT. Assist Reprod Rev 1992; 2:2. 20. Wang XJ, Warnes GM, Norman RJ et al. Gamete intra-fallopian transfer: outcome following the elective or non- elective replacement of two, three or four oocytes. Hum Reprod 1993; 8:1231–34. 21. Rombauts L, Dear M, Breheny S et al. Cumulative pregnancy and live birth rates after gamete intra-Fallopian transfer. Hum Reprod 1997; 12:1338–42. 22. Guzick DS, Balmaceda JP, Ord T et al. The importance of egg and sperm factors in predicting the likelihood of pregnancy from gamete intrafallopian transfer. Fertil Steril 1989; 52:795–800. 23. Seracchioli R, Bafaro G, Bianchi L et al. Influence of spermatozoa characteristics on gamete intra-fallopian transfer procedures: analysis of results obtained utilizing normozoospermic, oligoasthenozoospermic and donor spermatozoa. Hum Reprod 1993a; 8:2098–01. 24. Seracchioli R, Maccolini A, Porcu E et al. The role of gamete intrafallopian transfer (GIFT) and tubal embryo transfer (TET) in the treatment of patients with patent tubes associated with male infertility factor. J Assist Reprod Genet 1993b; 10:266–70. 25. Evans J, Wells C, Hood K. A possible effect of different light sources on pregnancy rates following gamete intra-fallopian transfer. Hum Reprod 1999; 14:80–82. 26. Schenker JG, Ezra Y. Complications of assisted reproductive techniques. Fertil Steril 1994; 61:411–22. 27. Bergh T, Lundkvist O. Clinical complications during in vitro fertilization treatment. Hum Reprod 1992; 7:625–26.

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28. Curtis P, Amso N, Keith E et al. Evaluation of the risk of pelvic infection following transvaginal oocyte recovery. Hum Reprod 1991; 6:1294–97. 29. Guirgis RR, al Shawaf T, Craf I. Ovarian torsion: A complication of GIFT. A report on two cases and literature review. Hum Reprod 1992; 7:967–69. 30. Centers for Disease Control and Prevention. Ectopic pregnancyUnited States, 1990–1992. JAMA 1995; 273:533. 31. Richards SR, Stempel LE, Carlton BD. Heterotopic pregnancy: reappraisal of incidence. Am J Obstet Gynecol 1982; 142:928–30. 32. Molloy D, Deambrosis W, Keeping D et al. Multiple-sited (heterotopic) pregnancy after in vitro fertilization and gamete intrafallopian transfer. Fertil Steril 1990; 53:1068–71. 33. Porter JB. Statistics and results of assisted reproduction technologies. Assist Reprod Rev 1991; 1:28–37. 34. Li HP, Balmaceda JP, Zouves C et al. Heterotopic pregnancy associated with gamete intrafallopian transfer. Hum Reprod 1987; 7:131–135. 35. Gardner DK, Schoolcraft WB, Wagley L et al. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998; 13:3434–40. 36. Pellicano M, Zullo F, FiorentinoA et al. Conscious sedation versus general anaesthesia for minilaparoscopic gamete intra-Fallopian transfer: a prospective randomized study. Hum Reprod 2001; 16:2295–97. 37. Chang MY, Chiang CH, Hsieh TT et al. The influence of endometriosis on the success of gamete intrafallopian transfer (GIFT). J Assist Reprod Genet 1995; 14:76–82.

CHAPTER 25 Zygote Intmfallopian Tube Transfer (ZIFT): Patient Selection is the Key to Beneficial Utilization Daniel S Seidman INTRODUCTION Gamete intrafallopian transfer (GIFT) was one of the first techniques introduced in the late 1970s to assist infertile couples with underlying tubal and unexplained infertility. However, a disadvantage with GIFT was the inability to assess successful fertilization before transfer.1 Zygote intrafallopian transfer (ZIFT), a modification of GIFT, was subsequently developed as an alternative that allows assessment of fertilization. Devroey et al2 reported in 1986 the first pregnancy following ZIFT. Although the ZIFT technique was originally aimed specifically to treat couples with an underlying male factor and who had unsuccessful GIFT procedures, it was quickly expanded to include all infertility etiologies. The technique of embryo transfer into the fallopian tube has witnessed various modifications over the years.1 The terms pronuclear stage-embryo transfer (PROST), tubal pre-embryo transfer (TPET), tubal embryo-stage transfer (TEST), and tubal embryo transfer (TET) all refer to different stages of embryogenesis in which transfer to the fallopian tube occurs. Collectively, these methods are known as ZIFT.1 ZIFT has several hypothetical advantages (Table 25.1). Foremost, in comparison to GIFT, it allows confirmation of fertilization before transfer and exclusion of polypoid embryos.1 In addition, ZIFT also has several theoretical advantages over IVF-ET. Transfer of the zygote at the ampullary portion of the tube would allow further developmentbefore entry to the uterine cavity. The zygote could then enter the uterus at a more advanced stage of development or in greater synchronization with the uterus.3 The presence of numerous growth factors and cytokinis in the human tubal fluid may contribute to the development of the early embryo, all factors that may enhance implantation.4 This procedure alsovdoes away with the potentially traumatic insertion of a catheter into the uterus.

Table 25.1: Advantages of zygote intrafallopian transfer (ZIFT) • Fertilization is confirmed before transfer • Polypoid embryos are excluded • Shorter incubation of embryos in the lab • Further development of embryos possible before entry to the uterine cavity

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• Zygote enters uterus at a more advanced stage of development • Greater synchronization with the uterus • Tubal growth factors may contribute to development of early embryo • Cytokines in tubal fluid may enhance implantation • Avoidance of potentially traumatic insertion of a catheter into the uterus

The ZIFT procedure, however, is associated with several significant disadvantages including the need for general anesthesia, laparoscopy, and a longer postoperative recovery time (Table 25.2). The procedure entails greater medical risk and requires significant expense to the patient related to the laparoscopic procedure, as well as significant logistical difficulties for the center.1 There is also a potentially greater risk of ectopic tubal gestation.4 ZIFT is currently performed, according to the latest report of the Society for Assisted Reproductive Technology (SART) registry, by more than one fifth of the 360 programs participating in the SART registry.5 However, the proportion of ZIFT procedures performed in North America has decreased by about a third, from 1,200 (1.8%) of 65,863 ART cycles initiated in 19966 to only 1,054 (1.3%)

Table 25.2: Disadvantages of zygote intrafallopian transfer (ZIFT) • Proof that at least one tube is patent necessary • Need for laparoscopy • Operative risks, especially in mechanical factor patients • General anesthesia required • Longer postoperative recovery time • Added expense related to the laparoscopic procedure • Significant logistical difficulties for the ART center • Potentially greater risk of ectopic tubal pregnancy

of 81,899 ART cycles initiated in 1998.5 This suggests a growing sense that the added risks of the ZIFT procedure are not justified by the moderate increase, if any, in pregnancy rates achieved. We believe that a subgroup of patients does exist where ZIFT offers an important addition to our treatment options. This group of carefully selected patients mainly includes in our experience patients with repeated failures. THE TECHNIQUE The ovarian stimulation protocol, the criteria for hCG administration and the laboratory handling in the ZIFT treated patients are similar to those in the standard IVFET protocol. We perform ZIFT 24–26 hours after oocyte retrieval with the use of a three-puncture laparoscopy method. After introducing the umbilical trocar and optical equipment, the abdominal cavity is surveyed and, through a 5-mm midline suprapubic incision, we aspirate all peritoneal fluid and blood. In patients without tubal factor, the fallopian tube chosen for the transfer is the one with the more healthy-looking appearance. In patients

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with tubal factor, transfer is performed to the tube with proved patency according to data from laparoscopy or hysterosalpingography (HSG) done during infertility investigation. Four to six pronuclear-stage zygotes are loaded into the catheter (delivery catheter, 35 cm; Cook) and transferred through a third paraumbilical puncture deep into the fallopian tube to the ampular region. OUTCOME DATA The most recent data on ZIFT comes from the 1998 annual report of the SART.5 In the report published in January 2002 all data were collected electronically by using the SART Clinical Outcome Reporting System software that was submitted to the American Society for Reproductive Medicine/SART Registry.5 Overall ZIFT was performed by 75 (20.8%) of the 360 programs that submitted data on procedures performed in 1998. The 75 programs initiated 1,054 ZIFT cycles, with 943 retrievals, (10.5% cancellation rate) and 890 transfers (94.4% of retrievals). From this, 343 clinical pregnancies were established that resulted in 279 deliveries, for a delivery rate of 26.5 percent per initiated cycle, 29.6 percent per retrieval, and 31.3 percent per zygote transfer. Of these 1,054 initiated ZIFT cycles, 724 (68.7%) were performed with intracytoplasmic sperm injection (ICSI). The clinical pregnancy rate for ZIFT cycles using ICSI was 37.3 percent per retrieval, and 39.1 percent per embryo cycle, compared with 28.9 percent and 36.5 percent, respectively, in ZIFT cycles without ICSI. In comparison among the 58,937 cycles that involved IVF (with and without micromanipulation) the delivery rate per retrieval, during the same time period, was 29.1 percent.5 Twelve ectopic pregnancies were reported following ZIFT (3.5% per clinical pregnancy). Of the deliveries achieved through ZIFT, 62.4 percent were singletons, 31.2 percent were twins, 5.7 percent were triplets, and 0.7 percent were higher order deliveries. Two stillborn infants (5 per 1,000 neonates) were reported5 Most studies comparing the ZIFT procedure with regular IVF-ET demonstrate increased implantation and pregnancy rates for ZIFT compared with intrauterine transfers.7–11 However, these studies have been criticized by Habana and Palter1 as being retrospective, and not controlling for variables such as age or underlying cause of infertility. Sample size was also small, thus giving inadequate statistical power to compare both techniques. Habana and Palter1 consequently performed a metaanalysis of all articles published in English up to December 1998. They based their conclusions on 6 randomized controlled trials4,12,16 that included 548 cycles, 514 retrievals, and 388 transfers. The demographic, stimulation protocols used and the transfer details were found to be comparable between the groups. Implantation and pregnancy rates did not differ significantly. However, there was apparently a trend toward increased risk of ectopic pregnancy with ZIFT.1 They therefore concluded that based on their meta-analysis of randomized trials they could find no statistically significant difference in implantation or pregnancy rates among patients randomized to either ZIFT or IVF-ET, although there was a trend toward higher implantation and pregnancy rates after ZIFT.1

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PATIENTSELECTION Realizing that some authoritiescurrently consider ZIFT as a more expensive, inconvenient, and invasive technique1 we, at the IVF Unit of the Sheba Medical Center in Israel, currently use a defined set of criteria for patient selection for treatment with zygote intrafallopian transfer (ZIFT). These are based on the assumption that patients who undergo several IVF-ET treatment cycles and who fail to conceive remain a frustrating problem to the clinician. In such cases we believe the added medical risk and cost may be justified in order to provide the patient with the more physiologic specialized tubal environment. This may theoretically allow for better development of the embryos and consequently higher pregnancy rates. Our patient selection criteria include no evidence of bilateral tubal pathology, transfer of three to five normally cleaving embryos in at least four previous IVF-ET cycles, failure of implantation in all previous IVF-ET cycles, and a normal uterine cavity according to hysteroscopy (Table 25.3). The planned ZIFT procedure is undertaken only if the patient has a fertilization rate of >60 percent, produces four or more zygotes in the ZIFT cycle, and has an endometrial thickness of ≥7 mm on the day of hCG administration (Table 25.3).

Table 25.3: Criteria for patient selection for treatment with zygote intrafallopian transfer (ZIFT) according to the Sheba Medical Center protocol • No

evidence of bilateral tubal pathology

• Transfer of three to five normally cleaving embryos in at least four previous IVF-ET cycles • Failure of implantation in all previous IVF-ET cycles • Normal uterine cavity according to hysteroscopy • Fertilization rate of >60 percent • Four or more zygotes available in the ZIFT cycle • Endometrial thickness of 7 mm on the day of hCG administration.

Levran et al17 summarized the experience of our center with ZIFT in patients with repeated failure of implantation in IVF-ET cycles. Criteria for patient selection included male factor or unexplained infertility, normal uterine cavity, and at least three failures of implantation in IVF-ET cycles in which at least three embryos were placed per transfer. In a case-control study, 70 patients who underwent 92 ZIFT cycles were compared with a control group that consisted of patients with the same selection criteria who underwent an additional standard IVF-ET cycle during the same time period. The ovulation induction protocol consisted of down-regulation with GnRH analogue followed by ovarian stimulation with FSH and hMG. In all patients with male factor infertility, ICSI was performed on the oocytes. Zygotes were transferred by laparoscopy into the f allopian tube 24–26 hours after oocyte retrieval. The study results revealed that pregnancy and implantation rates were significantly higher in the ZIFT group than in the control group: 34.2 percent (24/70) and 8.7 percent (29/333) versus 17.1 percent (12/70) and 4.4 percent (13/289), respectively. The cumulative conception rate for two ZIFT cycles was 59.3

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percent. It was thus concluded that ZIFT should be considered a beneficial mode of treatment for patients with repeated failure of implantation in IVF and transcervical ET. Farhi et al18 in a more recent study extended the above initial observations regarding patients with repeated failure of implantation in IVF-ET. These authors showed that ZIFT could also be considered as a mode of treatment for and with tubal factor with proved patency of one tube. These investigators from the Wolf son Medical Center in Israel determined the implantation and pregnancy rates in 112 ZIFT cycles performed in 81 patients with repeated failure of implantation. They further stratified their data for patients with tubal factor (n=15) and patients without tubal factor (n=66). Their criteria for patient selection for ZIFT include at least four failures of implantation in IVF-ET cycles in which at least 3 embryos were replaced per transfer and a cause of infertility diagnosed as male, unexplained, or tubal factor with proof of at least one patent tube. They reported that the pregnancy and implantation rates for all ZIFT cycles were 35.1 percent and 11.1 percent, respectively. However, pregnancy and implantation rates per cycle in patients with tubal factor versus patients without tubal factor were 26.6 percent versus 37.1 percent and 9.4 percent versus 11.4 percent, respectively. It was concluded that ZIFT could be applied as a mode of treatment for patients with repeated failure of implantation in IVF-ET and with tubal factor with proved patency of one tube.18 CONCLUSION It has long been advocated that ZIFT is a superior ART method, since transfer of cleavage-stage embryos into the tube is more physiologic than replacement into the uterus and provides the most appropriate embryo culture conditions. It was therefore claimed that the specialized tubal environment might lead to better development and ultimately, higher pregnancy rates. However, ZIFT is linked with additional surgical risks to the patient, and is associated with significant logistical and financial burdens to the ART center. We at the Sheba Medical Center believe that a cautiously selected subgroup of patients, with repeated IVF-ET failures, are likely to benefit most from the ZIFT procedure. Careful selection of patients may theref ore hold the key f or optimal utilization of this long established procedure. REFERENCES 1. Habana AE, Palter SF. Is tubal embryo transfer of any value? A meta-analysis and comparison with the Society for Assisted Reproductive Technology database. Fertil Steril. 2001; 76:286–93. 2. Devroey P, Braeckmans P, Smitz J, Van Waesberghe L, Wisanto A, Van Steirteghem A, Heytens L, Camu F. Pregnancy after translaparoscopic zygote intraf allopian transfer in a patient with sperm antibodies (letter). Lancet 1986; 1(8493):1329. 3. Jansen R. Endocrine response in the fallopian tube. Endocr Rev 1984; 5:525–51. 4. Preutthipan S, Curtis P, Amso N, Shaw R. A prospective randomized crossover comparison of zygote intrafallopian transfer and in vitro fertilization-embryo transfer in unexplained infertility. J Med Assoc Thai 1994; 77:599–603. 5. Society for Assisted Reproductive Technology, the American Fertility Society. Assisted reproductive technology in the United States: 1998 results generated from the American Society

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for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2002; 77:18–31. 6. Society for Assisted Reproductive Technology, the American Fertility Society. Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 1999; 71:798–805. 7. Devroey P, Staessen C, Camus M, De Grauwe E, Wisanto A, Van Steirteghem A. Zygote intrafallopian transfer as a successful treatment for unexplained infertility. Fertil Steril 1989; 52:24–29. 8. Hammitt D, Syrop C, Hahn S, Walker D, Butkowski C, Donovan J. Comparison of concurrent pregnancy rates for in vitro fertilization-embryo transfer, pronuclear stage embryo transfer and gamete intrafallopian transfer. Hum Reprod 1990; 5:947–54. 9. Pool T, Ellsworth L, Garza J, Martin J, Miller S, Atiee S. Zygote intrafallopian transfer as a treatment for non-tubal infertility: a 2 year study. Fertil Steril 1990; 54:482–88. 10. Staessen C, Camus M, Khan I, Smitz J, Van Waesberghe L, Wisanto A et al. An 18 month survey of infertility treatment by in vitro fertilization, gamete and zygote intrafallopian transfer, and replacement of frozen-thawed embryos. JIVF ET 1989; 6:2–29. 11. Yovich J, Yovich J, Edirisinghe W. The relative chance of pregnancy following tubal or uterine transfer procedures. Fertil Steril 1988; 49:858–64. 12. Balmaceda J, Alam V, Roszjtein D, Ord T, Snell K, Asch R. Embryo implantation rates in oocyte donation: a prospective comparison of tubal versus uterine transfers. Fertil Steril 1992; 57:362–65. 13. Tanbo T, OlavDale P, Abyholm T. Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in vitro-fertilization, gamete intra-fallopian transfer and tubal embryo stage transfer. Hum Reprod 1990; 5:266–70. 14. Fluker M, Zouves, Bebbington C. A prospective randomized comparison of zygote intrafallopian transfer and in vitro fertilization embryo transfer for non-tubal factor infertility. Fertil Steril 1993; 60:5–9. 15. Tournaye H, Devroey P, Camus M, Valkenburg M, Bollen N, Van Steirteghem A. Zygote intrafallopian transfer or in vitro fertilization and embryo transfer for the treatment of male factor infertility: a prospective randomized trial. Fertil Steril 1992; 58:344–50. 16. Van Voorhis B, Syrop C, Vincent R, Chestnut D, Sparks A, Chapler F. Tubal versus uterine transfer of cryopreserved embryos: a prospective randomized trial. Fertil Steril 1995; 63:578– 83. 17. Levran D, Mashiach S, Dor J, Levron J, Farhi J. Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated failure of implantation. Fertil Steril 1998; 69:26–30. 18. Farhi J, WeissmanA, Nahum H, Levran D. Zygote intrafallopian transfer in patients with tubal factor infertility after repeated failure of implantation with in vitro fertilization-embryo transfer. Fertil Steril 2000; 74:390–93.

CHAPTER 26 Fallopian Tube Sperm Perfusion Gautam N Allahbadia, Swati G Allahbadia, Sonia Malik, Javaid Mugloo INTRODUCTION In a therapeutic insemination cycle, although the number of available oocytes can be increased by ovarian stimulation, the results are still not very promising, mainly because of low sperm count, suboptimal spermatozoa or total absence of spermatozoa at the site of fertilization. Mortimer and Templeton1 showed that there is a reduction in sperm number of five to six orders of magnitude along the length of the female reproductive tract. After ejaculation, even when all semen characteristics were normal, there was still only a 49 percent chance of spermatozoa being found in the peritoneal cavity. However, significantly greater numbers of peritoneal spermatozoa have been found after artificial insemination into the external os, as compared to intercourse.1 Ripps et al2 showed that the number of spermatozoa recovered in the peritoneal fluid at laparoscopy after IUI was very low; however, the number increased after uterotubal flushes. Uterotubal flushes required a certain intrauterine perfusion pressure for the achievement of spill into normal tubes or in tubes with minimal adhesions.3 Intrauterine insemination (IUI) with controlled ovarian stimulation (COH-IUI) has been a popular method in recent years for the treatment of unexplained infertility, immunologic infertility, and to a lesser extent for treatment of male infertility. The fecundity reported in the literature ranged from 5.7 to 17.7 percent per cycle.4– 7 Techniques for the preparation of spermatozoa used in vitro fertilization (IVF) have been applied in IUI treatment cycles. The cycle fecundity is positively correlated with the postswim-up concentration of spermatozoa and the total number of motile spermatozoa inseminated.8 The most common inseminated volume is 0.2–0.5 mL.9–10 In 1991, Kahn et al11 described a new method of assisted conception called fallopian tube sperm perfusion (FSP), combining controlled ovarian hyperstimulation (COH) and IUI with a large volume (4 mL) of sperm suspension. They used a Frydman catheter to perform FSP and an Allis clamp placed on the cervix to prevent sperm reflux.11– 15 However, clamping of the cervix produced some discomfort or even pain and occasionally bleeding from the cervix. Subsequently, this technique was used by other investigators16,17 and other techniques have also been proposed.18–22 Several randomized controlled studies comparing FSP and IUI have been performed, with conflicting results.15–23 In the first clinical experience using a blocking device (the FAST system®), Fanchin et al20 reported a very high cycle pregnancy rate (PR)(Fig. 26.1). They observed a clinical PR per cycle of 40 percent in patients treated with FSP compared with a clinical PR per cycle of 20 percent in patients treated with standard IUI. These results were not

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confirmed by studies using other FSP methods, and a long debate ensued.24–27 Two important criticisms were made of Fanchin’s study: 1. The original paper did not give a breakdown of patients by diagnosis, hence their results are not comparable with those of other studies 2. Why should a more costly device for FSP be used when the procedure can be done with a less expensive IUI catheter? Regrettably, no further studies on the FAST system® have been published since the first report. Another method was described by Li18 who used a Foley size 8 pediatric catheter with the balloon inflated in the uterine cavity to seal the uterocervical junction (Fig. 26.2). However, introducing the Foley catheter through the cervix presented some difficulties, mainly because of its flexibility, especially in nulliparous patients. The use of forceps was necessary to provide steady downward traction on the cervix which served to reduce the angle between the uterine cavity and the cervical canal. The discomfort and the possible endometrial injury caused by the compressive effect of the balloon are a disadvantage of this method.

Fig. 26.1: Fallopian tube sperm perfusion

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Fig. 26.2: Fallopian tube sperm perfusion The ZUI II catheter (Zinnanti Surgical Instruments, Inc., Chatsworth, CA, USA) that Trout and Kemmann22 used worked well, because the stopcock allowed for testing of the backflow pressure before removal of the cervical canal obstruction (a balloon in their case). The procedure is technically more demanding than IUI and takes a little longer to perform, but the patients apparently tolerated the procedure well. Our Technique of FSP Fallopian tube sperm perfusion is carried out roughly 24–28 hours after the hCG trigger in appropriately selected patients; mostly unexplained infertility, patients with some form of immunological infertility or those with mild endometriosis. Semen is collected by masturbation into a sterile container after 2–3 days of ejaculatory abstinence. After initial analysis, the semen preparation is done using the swim-up technique using Flushing Solution (Medicult, Denmark). Next the sperm suspension prepared for IUI was diluted with 3.5 mL of the same pre-warmed and gassed Flushing media. Four mL of the Flushing media containing capacitated sperms was now loaded into a five mL syringe attached to the Labotect IUI cannula. The FSPs are all performed using the Labotect IUI cannula. (Labotect GmbH, Goettingen, Germany). The basic principle in the technique is to ensure a good seal at the external os, followed by gentle infusion of the uterine cavity with 4mL of sperm suspension medium; as the pressure in the uterine cavity gradually built up, it opened up the tubal ostia and allowed the sperm suspension medium to enter into the Fallopian tubes. The procedure is carried out on an outpatient basis with the woman in a dorsal position. No analgesia was required. The infusion is carried out slowly over 1–2 minutes. As the pressure inside the uterine cavity builds up, the woman sometimes experiences mild and crampy abdominal discomfort. The patients rest for

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about 10–15 minutes after the procedure and are then sent home with the standard luteal support protocols. One of the most important criticisms of the FAST system® concerns its high cost.27 To reduce costs to a similar level as IUI, we have developed a similar method that, however, uses a less expensive commercial device for IUI. In our limited early experience, using the 5F Hysterosonography Elliptosphere Catheter set (Ackrad Laboratories, Inc, USA) (Fig. 26.3), the procedure is really simplified and patient tolerance is much improved with minimal reflux. Our clinical pregnancy rate was 13.3 percent per started cycle (Unpublished Observations-Paper submitted to Medical Research Center, Bombay Hospital Trust, Mumbai). A clinical pregnancy was defined as a gestational sac seen on ultrasonography. There were no ectopic pregnancies in our study. Other complications included one vaso-vagal episode with FSP. There was no clinical evidence of tubal infection, trauma or perforation in our cases.

Fig. 26.3 DISCUSSION Workers have been suggesting various means of ensuring a higher mean number of motile spermatozoa to be available closer to the point of fertilization in the Fallopian tube (FT) and increasing the number of oocytes available for fertilization with ovarian stimulation.28 Makler suggested a device for injecting and retaining a small volume of concentrated spermatozoa in the uterine cavity and cervical canal.29 He believes that an ideal insemination device must fulfill three main requirements. First, it should deposit the small volume without requiring a dead space in the injection system. Second, it should be semi-rigid and have a nontraumatic entry past the cervical canal into the uterine cavity.

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Thirdly, the injection system should be provided with a mechanism to prevent backflow of the injected contents, resulting from the tendency of the uterus to contract when irritated by any foreign medium. The 5F Hysterosonography Elliptosphere Catheter set (Ackrad Laboratories, Inc, USA) that we used recently for FSP satisfied all the above requirements for an ideal insemination device and in addition could be used efficiently for FSP. The technique was satisfactory but the results in term of pregnancy outcome were very disappointing in the limited number of cases in whom we tried the procedure (Unpublished Observations-Paper submitted to Medical Research Center, Bombay Hospital Trust, Mumbai). To date, a total of 14 studies11–24 have been published on FSP. In 9 of these studies, FSP was compared with IUI and different results were obtained: in 5 studies, the pregnancy rate was significantly higher than that for IUI,11–15 whereas the other 4 failed to find any statistically significant differences.16,17,19,23 In these studies, FSP was used to treat different types of infertility. However, it is only for unexplained infertility that comparable, numerically sufficient data are available, whereas for other types of infertility, such as male factor, endometriosis, tubal factor, ovulatory dysfunction, and cervical mucus inadequacy, the number of women treated in the various studies is very small; hence it is not possible to make valid statistical comparisons. A recent meta-analysis,22 which considered only women affected by unexplained infertility, has concluded that FSP, in patients with this type of infertility, is more effective than IUI. However, this meta-analysis brings together studies that are not directly comparable to each other because different stimulation protocols, study populations, and FSP techniques were used. The same technique was used only in two of the studies considered by this meta-analysis, and opposite conclusions were reached: in the first study,15 there was a significant difference in favor of FSP, whereas in the second study,16 IUI had a higher PR than FSP, although the difference was not statistically significant. Hence, it cannot be said that the superiority of FSP over IUI has been ascertained. Fanchin et al20 have reported highly interesting results using a new method of FSP with a blocking device, (the FAST system®) but this method has not yet been tested by other investigators. The PR observed by Fanchin et al20 with this technique of FSP is much higher (40%) than that in all other studies published, especially if we consider that it deals with a population with various types of infertility. Fanchin et al20 did not report PRs according to different infertility categories, hence comparison with other studies is difficult. Moreover, no other author has reported better results from FSP compared with IUI in an unselected infertile population. Some workers suggested Direct Intraperitoneal Insemination (DIPI) for infertile patients who havebeen unable to conceive despite COH and IUI before going on to IVF/GIFT.30 The processed sperm specimen is injected directly into the cul-de-sac into the pool of follicular fluid that accumulates after rupture of the follicle and extrusion of the oocyte. Gregoriou et al reported a randomized comparison of IUI and DIPI31. They achieved an overall PR of 28 percent in both groups with no significant difference between IUI and DIPI.31 In another study, DIPI gave an overall PR of 40.5 percent per patient and a cycle fecundity of 8.5 percent.32 The data suggest that it may be important to use a technique that allows the cervical canal to be obstructed to prevent backflow of the inseminate. The rationale of the

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procedure is that capacitated spermatozoa injected into the cul-de-sac will reach the fertilization area by their own motility and peritoneal movements. There they will be picked up with the oocyte. Recently Barros reported that the spontaneous abortion rate was significantly higher after IUI than after DIPI.33 He suggested that DIPI provides a more physiological gamete interaction than IUI and if a higher conception rate and greater embryo viability after DIPI are confirmed in some other series, the preferential or exclusive use of DIPI should be considered.33 Ajossa et al reporting on an open multicenter study comparing DIPI and IUI concluded that because IUI and DIPI allow us to obtain the same results and DIPI is more invasive than IUI, it should be only considered when IUI is difficult to perform, as in the presence of a tight cervical canal.34 Intrafollicular insemination (IFI) has recently been described as a new reproductive technique for the treatment of infertility.35 Not only is the follicular fluid (FF) environment a stable one, but a yet unidentified FF component appears to promote sperm function. Logically, IFI would seem to be superior to DIPI because during DIPI, the sperm concentration would be greatly diluted by peritoneal fluid. Both DIPI as well as IFI are essentially invasive procedure and when the results of FSP were first published,12 it looked to be the most promising outpatient therapeuticinsemination procedure devised as yet. Kahn et al results have not been reproduced consistently by any other groups including ours.36 In a recent randomized prospective trial19 comparing FSP and IUI, the overall PR per cycle (10.8% versus 10.8%) were similar for IUI and fallopian sperm perfusion, respectively. The PR was also similar when compared for ovulation induction with CC (6.8% versus 9.1%) and gonadotropins (13.2% versus 11.8%).19 The authors concluded that fallopian sperm perfusion offers no advantage over IUI and because the process of fallopian sperm perfusion is more time consuming and more costly (because of increased media usage), fallopian sperm perfusion did not seem indicated as a routine infertility therapy and should not replace IUI.19 In another prospective randomized study, the authors treated 60 couples with unexplained infertility with a combination of ovarian stimulation and either intrauterine insemination (IUI) or fallopian sperm perfusion (FSP).16 The pregnancy rate per cycle was 16.2 percentin the IUI group and 14.5 percent in the FSP group and the pregnancy rate per woman was 40 and 36.7 percent, respectively (not statistically different). Again this group concluded FSP has no advantages over IUI.16 Kahn et al12 have hypothesized that it is delivery of sperm into the fallopian tubes that increases the likelihood of pregnancy. However, this does not explain why patients with diagnoses other than unexplained inf ertility do not benefit from FSP. We hypothesize that the higher PRs are similar to the higher rates that have been reported after hysterosalpingography.29 It may be that the large volume of inseminate washes out tubal obstructions or some factor that is deleterious to fertilization. These obstructions or other factors may be some of the undetected causes that make up the category of unexplained infertility. This would explain why this group shows improved PRs with FSP. It remains unclear why patients with endometriosis tend to have a lower PR with FSP. It is of note that the other study22 that included patients with diagnoses other than unexplained infertility also showed a poorer PR in patients with endometriosis who underwent FSP (6% vs. 12% with IUI). However, they did specifically exclude patients with severe endometriosis from their study. Further data are needed to indicate whether this observation is significant.

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CONCLUSIONS Our study failed to demonstrate any benefit from the use of FSP suggesting that IUI is a well established effective method in alleviating infertility.36 However, different groups all over the world have been publishing papers suggesting higher pregnancy rates with minor modifications of the technique of FSP.21 Unless a standard technique which is easily reproducible, cost-effective and gives consistent results with the majority of working groups all over the world is established, FSP will not find a place in the established form of infertility therapeutics such as IUI. We believe that FSP must be appraised more critically in large-scale randomized studies and should be formally compared with other treatment modalities such as GIFT and IVF. We had read with interest the results of Kahn and his group which at the time had pioneered the technique of FSP with excellent results.11–15 We set out to assess the results of the procedure in our own setting and despite comparable preparation and perfusion techniques, our group got very disappointing PRs. We submitted our preliminary results28 to stimulate scientific research and discussions such as this, in that we hoped some more multi-centric studies would establish the efficacy of FSPbased on the principles of evidence based reproductive medicine.37 Hence, we compared the results of our first twenty cases of FSP to an already established IUI program.36 We have since followed up our FSP protocol from June 2000 to May 2002 to include perfusion using the 5F Hysterosonography Elliptosphere Catheter set (Ackrad Laboratories, Inc, USA) with disappointing results. The PR we achieved with fallopian tube sperm perfusion was surprisingly low. It could have resulted, for example, because of either the adverse effect on the endometrium caused by the pressure of the balloon or to the large volume of the sperm suspensions. The large volume of inseminate may flush the ova out of the tubes or induce myosalpingeal abnormal contractions, resulting in expulsion of the ova from the tubes.22,23 Substances may also be dissolved from the Ackrad catheter, which may possibly further disturb fertilization. Today there are studies16,17,19,23 from different parts of the globe that have been unable to emulate the initial flattering Pregnancy Rates (PRs) originating from selected centers11– 15,20 . We still maintain that the Ackrad catheter is an ideal catheter for performing FSP because of its design and is less traumatic than the Allis’ clamp method described by Kahn et al11–15 Following the ‘literature-hype’ of the technique by a handful of groups11– 15,20 there has been a commercial invasion of devices designed to deliver sperms including the cervical clamp double nut bivalve speculum21, the Prietl uterotubal insemination catheter (Cook IVF), Jeyenderan’s spermiator38 and the FAST system.20 Until we have evaluated the procedure of FSP using the principles of Evidence Based Reproductive Medicine,39 we should not designate FSP as one of the milestones of reproductive medicine as has been attempted by certain groups.

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REFERENCES 1. Mortimer D, Templeton AA. Sperm transport in the human female reproductive tract in relation to semen analysis characteristics and time of ovulation. J Reprod Fertil 1982; 64:401–08. 2. Ripps BA, Minhas BS, Carson SA, Buster JE. Intrauterine insemination in fertile women delivers larger numbers of sperm to the peritoneal fluid than intracervical insemination. Fertil Steril 1994; 56:984–86. 3. Baker VL, Adamson GD. Threshold intrauterine perfusion pressures for intraperitoneal spill during hydrotubation and correlation with tubal adhesive disease. Fertil Steril 1995; 64:1066– 69. 4. Allen NC, Herbert CM, Maxson WS. Intrauterine insemination: a critical review. Fertil Steril 1985; 44:569–75. 5. Chaffkin LM, Nulsen JC, Luciano AA, Metzger DA. A comparative analysis of the cycle fecundity rates associated with combined hMG and JUT versus either hMG or IUI alone. Fertil Steril 1991; 55:252–57. 6. Dodson WC, Haney AF. Controlled ovarian hyperstimulation ‘and IUI for treatment of infertility, Fertil Steril 1991; 55:457–67. 7. Balasch J, Ballesca JL, Pimental C. Late low-dose pure FSH for ovarian stimulation in IUI cycles. Hum Reptod 1994; 9:1863–66. 8. Horvath PM, Bohrer M, Shelden M, Kemman E. The relationship of sperm parameters in superovulated women undergoing IUI. Fertil Steril 1989; 52:288–90. 9. Patton PE, Burry K, Novy MJ, Wolf DP. Acomparative evaluation of intracervical and IUI routes in donor therapeutic inseminations. Hum Reprod 1990; 5:263–66. 10. Sunde A, Kahn JA. IUI with pretreated sperm. A collaborative report. Hum Reprod 1988; 3:69– 73. 11. Kahn JA, Sunde A, Von During V, Sordal T, Molne K. Intrauterine insemination. Ann NY Acad Sci 1991; 626:452–60. 12. Kahn JA, von During V, Sunde A, Sordal T, Molne K. Fallopian tube sperm perfusion: first clinical experience. Hum Reprod 1992; 7(Suppl)1:19–24.[Medline]. 13. Kahn JA, von During V, Sunde A, Molne K. Fallopian tube sperm perfusion used in a donor insemination programme. Hum Reprod 1992, 7:806–12.[Medline]. 14. Kahn JA, von During V, Sunde A, Sordal T, Molne K. Treatment of unexplained infertility. Fallopian tube sperm perfusion (FSP) Acta Obstet Gynecol Scand 1993; 72:193–99.[Medline] 15. Kahn J, Sunde A, Koskemies A, von During V, Sordal T, Christensen F et al. Fallopian tube sperm perfusion (FSP) versus intra-uterine insemination (IUI) in the treatment of unexplained infertility: a prospective randomized study. Hum Reprod 1993; 8:890–94.[Medline]. 16. Gregoriou O, Pyrgiotis E, Konidaris S, Papadias C, Zourlas PA. Fallopian tube sperm perfusion has no advantage over intrauterine insemination when used in combination with ovarian stimulation for the treatment of unexplained infertility. Gynecol Obstet Invest 1995; 39:226–28 [Medline]. 17. El Sadek MM, Amer MK, Abdel-Malak G. Questioning the efficacy of Fallopian tube sperm perfusion. Hum Reprod 1998; 13:3053–3056 [Medline]. 18. Li TC. A simple, non-invasive method of Fallopian tube sperm perfusion. Hum Reprod 1993; 8:1848–1850 [Medline]. 19. Karande VC, Rao R, Pratt DE, Balin M, Levrant S, Morris R et al. A randomized prospective comparison between intrauterine insemination and fallopian sperm perfusion for the treatment of infertility. Fertil Steril 1995; 64:638–40 [Medline].

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20. Fanchin R, Oliveness F, Righini C, HazoutA, Schwab B, Frydman R. A new system for fallopian tube sperm perfusion leads to pregnancy rates twice as high as standard intrauterine insemination. Fertil Steril 1995; 64:505–10 [Medline]. 21. Mamas L. Higher pregnancy rates with a simple method for fallopian tube sperm perfusion, using the cervical clamp double nut bivalve speculum in the treatment of unexplained infertility: a prospective randomized study. Hum Reprod 1996; 11:2618–22 [Medline]. 22. Trout SW, Kemmann E. Fallopian sperm perfusion versus intrauterine insemination: a randomized controlled trial and metaanalysis of the literature. Fertil Steril 1999; 71:881–85 [Medline]. 23. Nuojua-Huttunen S, Tuomivaara L, Juntunen K, Tomas C, Martikainen H. Comparison of fallopian tube sperm perfusion with intrauterine insemination in the treatment of infertility Fertil Steril 1997; 67:939–42 [Medline]. 24. Fanchin R, Oliveness F, Righini C, Frydman R. The efficacy of “tubal sperm perfusion”? Fertil Steril 1996; 66:169–70 [Medline]. 25. Karande VC, Rao R, Pratt DE, Balin M, Levrant S, Morris R, et al. The efficacy of “tubal sperm perfusion”? Fertil Steril 1996; 66:169–70 [Medline]. 26. Fanchin R, Oliveness F, Righini C, Frydman R. Reply to the reply on IUI. Fertil Steril 1997; 67:1178–1179 [Medline]. 27. Karande VC, Rao R, Pratt DE, Balin M, Levrant S, Morris R et al. Reply to the reply on IUI. Fertil Steril 1997; 67:1179 [Medline]. 28. Aboulgbar MA, Yehia A, Mansour RT. Ovarian superstimulation and IUI for treatment of unexplained infertility. Fertil Steril 1993; 60(2):303–6. 29. Makler A, DeChemey A, Naftolin F. A device for injecting and retaining a small volume of concentrated spermatozoa in the uterine cavity and cervical canal. Fertil Steril 1984; 42(2):306– 7. 30. Crosignani PG. Intraperitoneal insemination in the treatment of male and unexplained infertility. Fertil Steril 1991; 55:333–37. 31. Gregoriou O. A randomized comparison of intrauterine and intraperitoneal insemination in the treatment of infertility. Int J Gynecol Obstet 1993; 42(1):33–36. 32. Turban NO, Artini PG, Ambroggio GD. Studies on direct intraperitoneal insemination in the management of male factor, cervical factor, unexplained and immunological infertility. Hum Reprod 1992; 7(1):66–71. 33. Barros A. Spontaneous abortions after intraperitoneal or Intrauterine insemination. Lancet 1991; 337:02. 34. Ajossa S, Melis GB, Cianci A, Coccia ME, FulghesuAm, Giuffrida G et al. J Assist Reprod Genet 1997; 14:15–20. 35. Lucena E, Ruiz JA, Mandonza JC, Lucena A. Direct Intrafollicular Insemination. J Reprod Med 1991; 36:525–26. 36. Desai SK, Allahbadia GN, Kania PM. Fallopian Tube Sperm Perfusion versus IUI: a preliminary report from a University teaching hospital. Middle East Fertility Soc J 1998; 3(3):267–71. 37. Evers JLH. Evidence based Reproductive medicine are we chasing storks? (Editorial). Obstet Gynaecol Communications 1999; 2(1):11–12. 38. Allahbadia GN, Allahbadia SG. Intrauterine Insemination Techniques. In Allahbadia GN (Ed). Mumbai: Rotunda Medical Technologies (P) Ltd, 1998; 180–95. 39. Karlberg J. Consequences of Evidence-Based Medicine. (Editorial). Obstet Gynaecol Communications 1999; 2(1):13–14.

CHAPTER 27 Use of Lasers in ART: Clinical Applications and Potential Research Tools Yona Tadir INTRODUCTION Developments in ART (assisted reproductive technologies) are based on scientific data and require artistic skills. Once a new development is well established, and accepted by the scientific and the clinical communities as a proven modality, it usually becomes a simple procedure that has very little to do with the magic of creation in which nature is still The Major Player. However, the more options we have in the AKTbasket, the more artistic skills are needed in order to select the optimal path along the complicated road of procreation. During the summer of 1988 a team of scientists from various continents have gathered at the Laser Medical Microbream Program (LAMMP)/Beckman Laser Institute and Medical Clinic, in the University of California, in order to evaluate the potential use of laser microbeams in the fast moving area of ART. Prior to this time, the only clinical experience with lasers in reproduction was iri tubal reconstructive surgery, where laser beams were delivered through operative microscopes and laparoscopes in order to improve fertility potential, and the effective beam spot size was 300–800 µm.1 At this time, conventional gamete micromanipulation using glass pipettes was also at its very early stage of development, and clinical applications were not defined. The availability of a large variety of laser tools at the LAMMP, with an optional delivery through inverted microscopes to sub-micron spot sizes (Fig 27.1) enabled testing its potential on various cell types, including gametes and embryos.2,3 The main applications of laser beams tested in ART during the past fourteen years were: a. Sperm manipulations with optical tweezers to improve fertilization in vitro and to study basic sperm physiology of motility force b. Drilling of oocytes and embryos to improve fertilization potential, assist hatching, remove blastomeres for pre-embryo genetic diagnosis (PGD), and assess zona pellucida (ZP) properties. The purpose of this article is to review the progress and evaluate the current status and potential use of laser microbeams, vis-a-vis other technologies available for gamete micromanipulations, in the ART labo ratory

LASERS AND DELIVERY SYSTEMS AVAILABLE FOR MICROMANIPULATIONS Lasers, (Light Amplification by Stimulated Emission of Radiation) are electromagnetic waves with unique properties. The beam is collimated, monochromatic and coherent. Lasers differ from each other by the wavelengths (WL), and they are usually in the visible range (red, green or blue), or invisible in the ultra-violet (UV) or infrared (IR)

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range. Effects on gametes may also vary as a result of the different parameters used. Some heat may be generated in the micromanipulated object if exposure time is long enough. Conversely, heat formation may be minimized if short exposure time is used in the order of micro (10−6), nano (10−9), or pico-seconds (10−12). Laser beams for gamete manipulation are typically reduced to a spot size of 1–5 µm.3,4 In principle, lasers can be delivered to the target as a free beam (non contact mode), or via flexible quartz fibers (contact mode). This

Fig. 27.1: Laser beams spot sizes delivered through various systems for clinical use is mainly dependent on the wavelength (WL) and the degree of absorption by the nurturing liquid medium, which is relatively low, ranging between 200–2000 nm.5 As such, wavelengths that are shorter or longer than this range require fiber delivery as will be discussed later. Light absorption in proteins and DNA is also WL dependent, and this should be considered as an important factor when selecting the optimal laser for gamete manipulations (Fig. 27.2). The potential advantage of using a light beam as a cutting tool is the ability to

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Fig. 27.2: Relative light absorption in DNA, Protein and Water at various wavelengths depicted on the electromagnetic spectrum eliminate the need for disposable fine glass pipettes, or expensive quartz fibers. Moreover, effects at pre-set (fixed) parameters are accurate and easily reproducible. As such, among various laser tools, the non-contact free beam delivered through the microscope objective is the preferred approach. Availability of solid-state compact diode lasers inserted into the body of the microscopes makes this combination very practical for non-contact cell manipulations6 (Fig. 27.3).

Fig. 27.3: Inverted microscope with a multiple laser beams delivered through the objective for gamete

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manipulations. (Courtesy of ZILOS, HamiltonThorne Research) SPERM MANIPULATIONS The ability to trap and immobilize cells with optical tweezers, initially described by Ashkin/has opened new possibilities for manipulating sperm.8 The principles of cell trapping are based on mechanical force, which is exerted on a microscopic particle by light. A single beam gradient force trap consists of a laser beam with a Gaussian intensity profile, focused to a spot smaller than the particle being trapped. This trap confines the particle to a location just below the focal point of the laser beam in the axial direction and centered in the beam in the transverse direction. The magnitude and direction of the net force on the particle is determined by the scattering of the laser light through the object. The force generated by the light is greater than all other forces acting on the particle and as such creates a trapping/manipulating effect. Several studies have demonstrated how a single motile sperm could be trapped, and subsequently released, by reducing the trapping power.8–10 Aselected single sperm can be guided from one location to another without any mechanical tool. Although sperm can be optically trapped and guided through a hole in the ZP to fertilize an egg11 it is probably not the preferred approach to improve the fertilization potential in modern ART, since other technologies such as the intra-cytoplasmic sperm injection (ICSI) have already been proven to be more successful. Using these principles, a laser generated optical trap was applied to manipulate sperm in two4 and three dimensions.10 Initially, the continuous wave (CW)—Neodymium: Yttrium-Aluminum-Garnet laser (Nd: YAG) operating at 1064 nm was used to determine relative force generated by single sperm.8 The results demonstrated that zig-zag motile sperm swam with more force when compared to straight swimming sperm. Other experiments revealed that similar effects could be achieved with a tunable CW Titanium Sapphire laser (700–800 nm wavelength).12,13 Several studies were performed to explore relative and absolute sperm force under various physiologic conditions. This demonstrated a significant increase in swimming force following interaction with the cumulus mass14 and a significant force increase following exposure to pentoxyphlline, a motility-enhancing agent.15 Relative force of human sperm before and after cryopreservation demonstrated that there was no significant difference when a yolk buffer freezing media was used as cryoprotectant.13 In another study, relative escape force of human epididymal sperm (aspirated microsurgically for IVF) was tested and compared to normal sperm. Data suggested that the relative swimming force of the epidydimal sperm was significantly lower (60%) than ejaculated sperm12 ATP-driven motility forces were calculated from calibrated trapping forces generated during the interaction of an 800 nm laser beam with single sperm cells.16 Sperm heads were obtained by microsurgically removing the flagellum with a pulsed laser beam (“laser scissors”). A trapping efficiency of 0.12+/− 0.02 and a mean intrinsic motility force of 44+/−20 pN (Pico Newton) were determined for motile spermatozoa from healthy donors.

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Oocyte Manipulations LaserZona Drilling (LZD) Early days of fertilization improvement in IVF were focused on zona cutting techniques (partial zona dissection-PZD). Cutting the zona with glass pipettes while holding it with a vacuum pipette was the common practice, and mircroinjection of acid Tyrode was a common alternative. The laser offered an attractive alternative in various modes. The non-contact mode Laser-zona interaction and the fate of oocytes exposed to light beams delivered through the microscope objective has been studied by several groups. Initially, a tunable dye laser at various wavelengths (266–532 nm) was tested on mouse, hamster and discarded human oocytes.3,4 The beam was delivered through the microscope objective and the depth of incision was observed on a television monitor, and adjusted by a joystick activated motorized stage. This method is simple and accurate when compared to conventional micromanipulations. Subsequently, other groups used a krypton fluoride laser (operating at 248 nm)17, nitrogen laser (337nm),11,18 and a nitrogen-pumped dye laser (440 nm)19 to test simplicity, accuracy and local effects. The investigators concluded that from the technical point of view, these lasers could offer an elegant alternative for accurate zona incisions. In order to further elucidate the laser effect on the ZP we designed a set of experiments with various laser parameters. Oocytes were exposed to two different XeCl excimer laser systems (both operating at 308 nm) that offer a large variety of parameters such as pulse duration or pulse repetition rate. High quality images were video recorded, and analyzed, by computerized image processing, and the oocytes were further processed for scanning electron microscopy (SEM).20,21 Ablation holes smaller than 1 µm were obtained in a reproducible fashion without causing any apparent damage to neighboring cells. Pulse energy and the beam focal plane position were shown to be the most critical parameters in defining the ablated spot diameters. It was concluded that excimer lasers of 308 nm operating in a short pulse duration (15 ns to 250 ns) are effective microsurgical tools for achieving “clean” ZP removal, in a non-contact mode. At this particular wavelength, the optical absorption is strong enough to cause selective interaction with the zona pellucida, yet weak enough to induce generate heat or explosive ablation. In addition, the 308 nm radiation can be delivered through glass slides, microscope objectives, and liquid medium or oil. It can facilitate easy, accurate and highly reproducible material removal without the need for handling and maintaining a contact delivery system. However, the known potential damage of UV irradiation still raises some concerns, and the sensitivity of gamete’s genetic material deserve extra caution. Ng et al22 have studied the potential use of nitrogen laser (337 nm) delivered through an inverted microscope to provide a spot of less than 1 µm in the non-contact mode. The laser was used at 2.5 µJ/pulse, with a repetition rate of 10 pulses/sec. A10 p.m opening was made in each ZP of mouse oocytes. The drilled oocytes were then inseminated iri micro-droplets with murine sperm at 2× 105 sperm/ml. There was a significant improvement in fertilization and blastocyst formation at day 5 following LZD (89 out of 158 [65.2%] compared to 46/127 [36.2%] p<0.001). Lasers used in these studies were in the UV or the visible range. Amore advanced system that can selectively disrupt the ZP and be delivered as a “free beam” is the

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compact diode laser operating in the near IR range (1480 nm). This option will be discussed in the laser assisted hatching and the blastomere biopsy sections where it has more clinical relevance. Contact mode A different approach to zona drilling that uses a glass pipette or laser quartz fibers in a contact mode has been suggested by several investigators. In these studies, the argon fluoride excimer laser (ArFl) at 193 nm,21 Nd: YAG laser (1640 nm),23 Holmium: YAG laser (2100 nm)24 and Erbium: YAG (2940 nm)25,26 were applied to oocytes. The 193 nm short WL was delivered to mouse oocyte ZP22 through a series of mirrors and a long focal length lens connected to an alumina silicate pipette. The glass pipette was pulled from capillaries with a 1 mm outer diameter to a tip of about 3–5 µm and filled with positive air pressure. Insemination at low sperm densities led to fertilization and further development to the blastocyst stage.27 Successful fertilization and pregnancy in humans followinig Er: YAG—LZD indicated feasibility of the technique.27 Laufer et al28 examined the safety and efficacy of the 193 nm laser by drilling the ZP of mouse oocytes to improve fertilization rate. The LZD significantly enhanced fertilization rate over controls, and the hatching rate was also enhanced. Normal litters were born following the transfer of the embryos into the uteri of pseudopregnant recipients. This issue has been cleared by now, since the improved fertilization rate following ICSI, and the ability to use sperm of very poor quality in ART suggests that zona drilling for fertilization improvement is obsolete, and no “artistic” skills are needed for this decision any longer. Laser Assisted Hatching (LAH) Assisted hatching (AH) was introduced into the clinical practice in 1990 by Cohen et al29,30 to improve implantation rate in patients with thick ZP (>15µm) or in patients over the age of 38. Twelve years later, the place of AH following IVF is still controversial, and it is being offered to selective groups of patients as a standard procedure in some institutions. Literature search reveals conflicting data regarding the clinical use of assisted hatching. Some articles provided statistical data suggesting that AH improved pregnancy rates in patients following previous IVF failures, and over the age of 39.27,32–38 Liu et al have demonstrated that Assisted hatching facilitates earlier implantation,39 and Tucker et al have shown that partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates,40 However, in another study the same investigator find that chemical removal of the zona pellucida of day 3 human embryos has no impact on implantation rate.41 Two recent studies have shown that laser zona thinning provided significant advantages over hatching by drilling holes,42 and that the clinical pregnancy rates arising from quarter LAH were higher in comparison with partial and total LAH.43 In a recent study, Heish et al57 have concluded that laser-assisted hatching of embryos is more effective than the chemical method in enhancing the pregnancy rate of women with advanced age. This team referred also to other practical aspects and concluded that the laser system allows an easier, faster, and safer micromanipulation of the zona pellucida, which provided a better method in zona drilling. Micromanipulation techniques used in these studies were: mechanical slitting, microinjection of acid Tyrode solution, chemical zona thinning and laser drilling and

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thinning. More information is still needed to define the common denominators for patients and embryos that may benefit from assisted hatching. However, from the technical point of view, it appears that the laser is not only the most accurate technique, but also the easiest mode of operation. The procedure is well controlled, easily monitored, and accurately repeated in multiple locations, in the same embryo or in multiple embryos (Fig. 27.4). Moreover, a computer controlled IVF workstation

Fig. 27.4: The laser procedure can be controlled and accurately repeated in multiple locations can pre define the size and location of the crater in the ZP, document various parameters, and automatically transfer the data to a spread sheet or to the patient’s records. Wavelengths tested for LAH range from the UV at 308 nm to the IR operating at 2940 nm and in the contact or free beam modes. 308 nm XeCl laser: Several investigators20,44 studied topical effects of this UV laser on mouse blastomeres using a commercially available system coupled to an inverted microscope. Effects were determined by microinjection of a vital fluorescence dye (fluoresein isothiocyanate [FITC] dextran) into the cell immediately adjacent to the site of zona photoablation. This dye is only passed onto daughter blastomeres and therefore permits study of specific cell lines. Embryonic growth was assessed following cell separation at the morula and blastocyst stage. Four cell stage embryos treated with this laser had significantly fewer cells 12 hours after zona photoablation than control embryos. This information suggests that the 308 nm UV excimer laser has some detrimental effect on pre-compacted mouse embryos. However, a different set of laser parameters at the same WL may eliminate this problem.

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337 nm nitrogen laser: Antinori et al36 in a randomized trial evaluated pregnancy and implantation rate in three groups of women with repeated IVF failures. One hundred and seven patients received mixed embryos (with or without LAH), 72 patients received only laser treated embryos, and a control group of 98 patients were treated by regular IVF. The resulting clinical pregnancies were 39 (36.4%) in the mixed embryos group, 32 (44.4%) in LAH group and 19 (19.3%) in the IVF controls. The implantation rates per embryo were 9.3%, 16%, and 5.1% in the three groups respectively. In total, 17 normal babies have been delivered (10 in mixed group and seven in the pure LAH group). These results demonstrate that LAH increased the pregnancy and implantation rates. The increase was slight but significant in mixed embryos group (P <0.01 and P<0.02) it was even higher in the LAH group (P <0.05). The laser parameters used did not cause any visible damage to the embryos as assessed immediately after birth. 2100 nm Ho: YAG and Ho: YSGG lasers: Light absorption by fluid at this infra red WL (Ho: YAG or Holmium: Yttrium Scandium Gallium Garnet—Ho: YSGG) is significant and several factors may affect the amount of energy deposited in the ZP: (a) quality and thickness of the petri dish, (b) protein content of the culture medium; or (c) distance that the beam has to pass in fluid before it hits the ZP. For this reason some investigators used this WL in the contact mode and some used as a free beam. It is not the intent of this review to discuss technical details such as pulse duration, energy, or pulse repetition rate, however, it is important to realize that such details will further determine the laser effects.46 a. 2100 nm in the contact mode Reshef et al25 applied the Holmium: (Ho: YAG) laser delivered via fibers on the ZP of two to eight cell stage mouse embryos to assist hatching. The rate of development to blastocyst stage and the rate of hatching between the laser-treated and control embryos were compared. Further development was assessed 72 hr. post-lasing. Thirty three out of 49 laser-drilled embryos (67%) progressed to hatching blastocyst as compared to 36 of 82(44%) untreated controls (p<0.01). b. 2100 nm in non-contact mode. Our group45,46 used the Ho: YSGG (2100 nm) as a free beam delivered through an inverted microscope and quartz glass dish to perform LAH in two cell mouse embryos. Control embryos were treated with human tubal fluid (HTF) culture with or without serum (HTF-s, HTF-o), or with late serum supplementation (HTF-o/s). Fewer (P<0.05) embryos developed to the blastocyst stage in the HTF-s group (81%) in contrast to the LAH (90%), HTF-o (94%) and HTF-o/s (92%) treatments. The level of hatching was significantly increased (P<0.01) in the LAH treatment (57%) compared to HTF-o/s (32%), HTF-s (18%) or HTF-o (5%). Implantation rates were not impaired following the LAH treatment (21%). This data suggested that LAH using the Ho: YSGG laser is accurate and effective, however, in view of technical limitations of light delivery through fluids near the 2000 nm range there might be better wavelengths to perform LAH in the non-contact mode. 2940 nm Er: YAG laser This infrared WL has a high absorption peak in water and can be delivered to the ZP only via fibers in a contact mode. Embryos must be kept stable with a holding pipette during the procedure. Strummer and Feichtinger47 tested it in mouse embryos, and subsequently in human embryos. Groups of 10–15 mouse embryos were placed under oil on two slides. A control slide was maintained on a warming stage

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while embryos on the other slide were subjected to the laser to produce 20–30 pm holes in the ZP. Subsequently, embryos were assessed up to the blastocyst stage. There was no difference between the laser-treated mouse embryos and the untreated controls on day 1 and 2 of culture. On day 3 however, complete hatching was significantly enhanced in the laser-treated group (44/55 [80%] -laser, 17/58 [29.3%] for controls, p=0.0001). 1480 nm diode laser Near infrared solid-state lasers are small and can emit light at power levels sufficient to cause selective damage to the ZP. The laser module that contains the diode, the electronic board, and the collimated lens are all small and can be inserted into the inverted microscope. The 1480 nm WL is ideal since it is just minimally absorbed by water, but is highly absorbed by ZP glycoproteins (Fig. 27.2). This system may serve as an optimal cutting tool for the IVF laboratory as suggested by Rink et al.6 In these studies, the beam was delivered through a 45× objective of an inverted microscope (2–4 µm spot diameter) to produce zona dissection in mouse and human oocytes and zygotes. One laser exposure was sufficient to drill openings in the ZP ranging from 5–20 µm depending on laser power and exposure time. The same group48 demonstrated that the energy needed to drill a hole of a given diameter is greater for mouse and human zygotes than for oocytes. This confirmed a previous observation with the XeCl laser, that ethanol induced zona hardening can be verified and quantified with a non-contact laser.49 The issue of zona hardening with regard to assisted hatching is summarized by De Vos and Van Stertegham.50 The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 µm diode laser has been investigated by determining the ability of mouse oocytes to develop in υivo.51 Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocytecumulus complexes. Newborns derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. Another group of mouse zygotes were microdrilled by exposing their ZP to a short pulse of the same 1.4 µm diode laser and allowed to develop in υitro.52 Various sharpedged holes could be generated and sizes varied by changing irradiation time (3–100 ms) or laser power (22–55 mW). Drilled zygotes presented no signs of thermal damage under light and scanning electron microscopy. Embryos allowed to develop in vitro and showed no sign of abnormality. In a crossed-beam experiment a HeNe laser probe was used to detect the temperature-induced change in the refractive index of an aqueous solution, and estimate local thermal gradient. The authors find that the 1480 nm laser beam produces superheated water approaching 200 degrees C on the beam axis. Thermal histories during and following the laser pulse are given for regions in the neighborhood of the beam. They concluded that an optimum regime exists with pulse duration
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BLASTOMERE BIOPSY AND PREIMPLANTATION GENETIC DIAGNOSIS Preimplantation genetic diagnosis (PGD) is offered worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode’s solution). In several articles54,55 it is stated that the introduction of laser technology has facilitated this step enormously. CONCLUSIONS Competitive technologies should be tested by nonprejudicial investigators in clinical studies to avoid bias regarding the role and outcome of any the new technique. This is especially true when expensive tools are introduced into clinical practice. Advances in gamete manipulations has changed indications for ART and opened new avenues for research and clinical applications. Though laser MM may not be beneficial to the process of fertilization, it can be combined with ICSI for sperm immobilization,56 and may play a role as a simple preferred approach in assisted hatching57 and other delicate manipulations such as polar body or blastomere biopsy.58 The inverted microscope with the diode laser built-in may be the ultimate approach for non-contact gamete manipulations (Fig. 27.5) since no disposable tools are needed. The computerized workstation offers significant benefits since no extra handling is needed to assess various parameters, and data can automatically be stored and loaded in patient’s records. As more and more computer memory becomes available, this workstation can instantly store a large amount of visual information on oocytes, embryos and blastomeres for prospective and retrospective analysis. This data may further assist in the art of ART. Other delicate procedures such as blastomere or polar body biopsy can be assisted by the non-contact effects laser method. Systems that combine mechanical tools for gamete manipulations with diode lasers, or even systems that provide more than one laser beam delivered through the same optical system may be useful for cutting and trapping. This may be used in sperm tail cutting prior to ICSI, or for targeting subcellular organelles that should be removed or inactivated during the IVF.

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Fig. 27.5: Non-contact laser manipulation of the zona pellucida. The laser beam is delivered through the microscope objective. LB—Laser Beam, EM -Embryo, OL-Optic Lens. (Courtesy ZILOS, HamiltonThorn Research) REFERENCES 1. Y Tadir, J Ovadia, R Margara, RML. Winston Intraperitoneal adhesiolysis by CO2 laser microsurgery. In Atsumi K, Nimsakul N (Eds): Laser: Publisher Inter. Group Corp, 1981; 13– 27. 2. Berns MW, Aist J, Edwards J, Stras K, Girton J, McNeil P, Rattner JB et al. Laser microsurgery in cell and development biology. Science 1981; 213:505–213. 3. Tadir Y, Wright WH, Berns MW. Cell Micromanipulation with laser beam. In Capitanio GL, Asch RH, Cecco L De, Croce S et al (Eds): GIFT: From Basics to Clinics, Raven Press: New York. 1989; 359–68. 4. Tadir Y, Wright WH, Vafa O, Liaw LH, Asch R, Berns MW. Micromanipulation of Gametes using Laser Microbeams. Hum Reprod 1991; 6:1011–16. 5. Tadir Y, Neev J, Ho P, Berns MW. Lasers for Gamete Micromanipulation: Basic concepts. J. Assist Reprod Genetics 1993; 10:121–25. 6. Rink K, Delacretaz G, Salathe RP, Senn A, Nocera D, Germond M et al. 1.5 µm Diode laser microdissection of the zona pellucida of mouse oocytes. Biomedical Optics. The international Society for Optical Engineering 1994; 2134A–53. 7. Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S. Observation of a single beam gradient force optical trap for dielectric particles. Optics Letters 1986; 11:288–90.

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8. Tadir Y, Wright WH, Vafa O, Ord T, Asch R, Berns MW. Micromanipulation of Sperm by a Laser Generated Optical Trap. Fertil Steril 1989; 52:870–73. 9. Tadir Y, WH. Wright O, Vafa T, Ord R. Asch, MW Berns. Force Generated by Human Sperm Correlated to Velocity and Determined Using a Laser Generated Optical Trap Fertil Steril 1990; 53:944–46. 10. Colon JM, Sarosi P, McGovern PG, Ashkin A, Dziedzic JM, Skurnick J et al. Controlled micromanipulation of human spermatozoa in three dimensions with an infrared laser optical trap: effect on sperm velocity. Fertil Steril 1992; 57:695–98. 11. Schutze K, Clemeny-Sengewald A, Berg FD. Laser zona drilling and sperm transfer into the perivitelline space. Hum Reprod 1993; 8:390. 12. Araujo E, Tadir Y, Patrizio P, Ord T, Silber S, Berns MW et al. Relative force of human epididymal sperm correlated to the fertilizing capacity in vitro. Fertil Steril 1994; 62;585–90. 13. Zoentania, ND, Araujo E, Tadir Y, MW. Berns, Schell MW, Stone SC. Effect of freezing on the relative escape force of sperm as measured by laser optical trap. Fertil Steril 1995; 63:185–88. 14. L Westphal, El-Danasouri IE, Shimizu S, Tadir Y, Berns MW. Exposure of human sperm to the cumulus oophorus results in increased relative force as measured by a 760 nm laser optical tram. Hum Reprod 1993; 8:1083–86. 15. Patrizio P, Liu Y, Sonek JG, Berns WM, Tadir Y. Effect of pentoxifylline on the intrinsic swimming forces of human sperm assessed by optical tweezers. Int J Androl 2000; 21(5):753–6. 16. Konig K, Tadir Y, Patrizio P, Berns M, Tromberg B. Effects of ultraviolet exposure and near infrared laser tweezers on human spermatozoa. Hum Reprod 1996; 11:2161–64. 17. Blanchet BB, Russel JB, Fincher CR, Portman M. Laser micromanipulation in the mouse embryo: a novel approach to zona drilling. Fertil Steril 1992; 57:1337–47. 18. Schutze K, Clement-SengewaldA. Catch and move—cut or fuse. Nature 1994; 14, 368(6472):667–69. 19. Godke RA, Beetem DD, Burleigh DW. A method for zona pellucida drilling using a compact nitrogen laser. Presented at the VII World congress on human reproduction. June 26–July 1, 1990; Abs No 258. 20. Neev J, Y Tadir, P Ho, RH Asch, T Ord, MW Berns. Microscopedelivered UV laser zona dissection: principles and practices. J. Assist. Reprod. and Genetics 1992; 9:513–23. 21. Li L, Munne S, Licciardi F, Neev X Tadir Y, Berns MW, Godke R et al. Microinjection of FITC-Dextran into mouse blastomeres to assess topical effects of zona penetration. Zygote 1993; 1:43–48. 22. Ng SC, Liow SL, Schutze K, Vasuthevan S, Bongso A, Ratnam SS. The use of ultra- violet microbeam laser zona dissection in the mouse. In VIII World Congress of In Vitro Fertilization and Assisted Reproductive Technologies. Japan, Sept. 12–15, 1993. J Assisted Rerprod Prog Suppl. 1993; Abstract no 273. 23. Palanker D., Ohad S, Lewis A, Simon A, Shenkar J, Penchas S, et al. Technique for cellular microsurgery using the 193 nm Excimer laser. Laser in Surg and Med 1991; 11:580–86. 24. Coddington CC, Veeck LL, Swanson RJ, Kaufman RA, Lin J, Simonetti S et al. The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes. J Assist Reprod Genet 1992; 9:557–63. 25. Reshef E, Haaksma CJ, Bettinger TL, Haas GG, Schafer SA, Zavy MT: Gamete and embryo micromanipulation using the Holmiumr YAG laser. In 49th American Fertility Society, Montreal, Canada. Oct. 11–14, 1993. Fertil Steril Program 1993; (Suppl P-016, S88). 26. Feichtinger W, Strohmer H, Fuhrberg P, Radivojevic K, Antoniori S, Pepe G et al. Photoablation of oocyte zona pellucida by erbium: YAG laser for in-vitro fertilization in severe male infertility. Lancet 1992; 339:811. 27. Antinori S, Versaci C, Fuhrberg P, Panci C, Caffa B, Hossein Gholami G. Seventeen births after the use of an erbium: YAG laser in the treatment of male factor infertility. Hum Reprod 1994; 9:1891–96.

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28. Laufer N, Palanker D, Shufaro Y, Safran A, Simon A, Lewis A: The efficacy and safety of zona pellucida drilling by a 193-nm excimer laser. Fertil Steril 1993; 59:889–95. 29. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod; 5:7–13. 30. Cohen J. Assisted hatching of human embryos. J in Vitro Fertilization and Embryo Transfer 1991; 8:179–90. 31. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992; 7(5):685–91. 32. Obruca A, Strohmer H, Sakkas D, Menezo Y, Kogosowski A, Barak Y et al. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994, 9:1723–26. 33. Schoolcraft WB, Schlenker T, Jones GS, Jones HW Jr. In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genet 1995; 12(9):581–84. 34. Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J et al. Assisted hatching by partial zona dissection of human preembryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 1995; 63(4):838–41. 35. Antinori S, Panci C, Selman HA, Caffa B, Dani G, Versaci C. Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum Repro, 1996; 11590–94. 36. Antinori S, Selman HA, Caffa B, Panci C, Dani GL, Versaci C. Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy. Hum Reprod 1996; 11:2488–92. 37. Parikh FR, Kamat SA, Nadkarni S, Arawandekar D, Parikh RM. Assisted hatching in an in vitro fertilization programme. J Reprod Fertil 1996; 50(Supplement):121–25. 38. Hellebaut S, De Sutter P, Dozortsev D, OnghenaA, Qian C, Dhont M. Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? Aprospective randomized study. J Assist Reprod Genet 1996; 13:19–22. 39. Liu HC, Cohen J, Alikani M, Noyes N, Rosenwaks Z. Assisted hatching facilitates earlier implantation. Fertil Steril 1993; 60(5):871–75. 40. Tucker MJ, Cohen J, Massey JB, Mayer MP, Wicker SR, Wright G. partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 1991; 165:341–44. 41. Tucker MJ, Luecke NM, Wicker SR, Wright G. Chemical removal of the zona pellucida of day 3 human embryo has no impact on implantation rate. J Assist Reprod Genet 1993; 10;187–91. 42. Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinning—an alternative approach to assisted hatching. Hum Reprod 2001; 16:1959–64. 43. Montag M, van der Ven K, Delacretaz G, Rink K, van der Ven H Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy. Fertil Steril 1998; 69:539– 42. 44. Neev J, Gonzales A, Licciardi F, Alikani M, Tadir Y, Berns MW et al. A contact-free microscope delivered laser ablation system for assisted hatching of the mouse embryo without the use of a micromanipulator. Human Reprod 1993; 8;939–44. 45. Neev Y, Schiewe MC, Sung WV, Kang D, Berns MW, Tadir Y. Assisted hatching in mouse embryos using a non-contact Ho: YSSG laser system. J Assisted Reprod Genetics 1995; 12:228–93. 46. Schiewe MC, Neev J, Hazeleger NL, Balmaceda JP, Berns MW, TadirY. Developmental competence of mouse embryos following zona drilling using a non-contact holmium: yttrium scandian gallium garnet (Ho: YSGG) laser system. Hum Reprod 1995; 10:1821–24. 47. Strohmer H, Feichtinger W. Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil Steril 1992; 58:212–14.

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48. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995; 64:604–11. 49. Tadir Y, Neev Y, Schiewe M, Balmaceda JP, Ord T, Asch RH et al. Spontaneous and induced zona pellucida hardness: measurements using enzyme assay and a non-contact laser micromanipulation. Pacific Coast Fertility Society, Palm Springs, CA. Fertil Steril Prog 1993; (Suppl O-21):14–16. 50. De Vos A, Van Steirteghem A. Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction. Cells Tissues Organs 2000; 166:220–27 51. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Pedrazzini T et al. Improved fertilization and implantation rates after nontouch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam. Hum Reprod 1996; 11:1043–48. 52. Rink K, Delacretaz G, Salathe RP, Senn A, Nocera D, Germond M et al. Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser. Lasers in Surgery and Medicine 1996; 18:52–62. 53. Germond M, Senn A, Rink K, Delacretaz G, De Grandi P.. Is assisted hatching of frozenthawed embryos enhancing pregnancy outcome in patients who has several previous nidation failures? Presented at the Three country Fertility and Sterility meeting: Innsbruck, Austria, Oct 12–14, 1995. Abstract in J Fur Fertilitat and Reproduction 1995; 3:41. 54. Licciardi F, Gonzalez A, Tang YX, Grifo J, Cohen J, Neev Y. Laser ablation of the mouse zona pellucida for blastomere biopsy. Journal of Assisted Reproduction and Genetics 1995; 12:462– 66. 55. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993; 8:1055–60. 56. Ebner T, Moser M, Yaman C, Sommergruber M, Tews G. Successful birth after laser assisted immobilization of spermatozoa before intracytoplasmic injection. Fertil Steril 2002; 78:417–18 57. Hsieh YY, Huang CC, Cheng TC, Chang CC, Tsai HD, Lee MS. Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age. Fertil Steril 2002; 78:179–82. 58. Eroglu A, Nahum RT, Isaacson K, Toth TL. Laser-assisted intracytoplasmic sperm injection in human oocytes. J Reprod Med 2002, 47:199–203.

CHAPTER 28 Blastocyst Transfer: One Step Further in the Questfor the Magic Bullet Jacob Levron, Micha Baum, Jehoshua Dor, Daniel S Seidman INTRODUCTION The recent development of complex sequential culture systems has been attributed to the growing understanding of the intricate physiology of early human pre-implantation embryos.1,2 The availability of these new extended embryo cultures has led to a flurry of enthusiasm regarding the potential benefits of replacing embryos at the blastocyst stage as an alternative for transfer of earlier cleavage stages embryos during the early days of human IVF practice.3 The methods for blastocyst production and transfer were to a large extent adopted for humans based on initial experience with large domestic animals, where replacement of a single blastocyst usually results in high pregnancy rates. Overall, however, the outcome of extended human embryo culture before the era of sequential culture media was disappointing even when elaborate co-culture systems were used.4 The current ability to support in υitro development of human embryos to advanced cleavage stages has led to a significant improvement in the results with the use of complex sequential culture media. Consequently, these innovative techniques have invoked a debate regarding the efficacy of extended culture versus the traditional IVF-ET management.5–8 Blastocyst production and transfer appariently has potential benefits. However, it remains unclear whether extension of embryo culture towards blastocyst production is a good option for all of the patients or even for all embryos within the same cohort. Furthermore, accumulating evidence suggests that the adoption of a more selective approach is needed for the optimal utilization of blastocyst transfer. Blastocyst stage embryo transfer has rapidly gained many enthusiasts and has created a “blastocyst fever” over recent years, despite the lack of sufficient data from prospective randomized studies. Our daily preliminary experience with blastocyst culture did not confirm the high expectations generated by initial reports. The fact that assisted reproductive medicine and research often operates under unique circumstances whereas new treatment modalities, such as blastocyst production and transfer, are implemented on an empirical basis before they are tested using randomized clinical studies has long been subject for concern. In order to adequately implement new treatment options it is therefore essential to select the patients for whom the novel technique is most likely to succeed.

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OUR EXPERIENCE We undertook a study in order to compare the outcome of IVF after day-3 and day-5 embryo transfer in a prospective randomized manner.10 We randomly allocated 90 consenting patients for day-3 or blastocyst stage embryo transfer. Patients were recruited according to the following inclusion criteria: 1. Maternal age-less than 38 years. 2. Less than five previous IVF attempts. 3. A cohort of more than five zygotes on day-1. Ovarian stimulation and oocyte retrieval were conducted according to our conventional IVF protocols using midluteal GnRH agonist administration followed by gonadotrophin ovarian stimulation. All the gamete and embryo handling procedures were done using a commercial sequential IVF media (Cook, Eight-miles plains, Queensland, Australia). The oocytes were collected into IVF oocyte wash buffer. The cumulus of each oocyte was dissected out by hypodermic needles and the oocytes were cultured under standard conditions in 80 µL droplets of IVF fertilization medium under oil. Oocytes were inseminated 40 hours after hCG administration either by conventional insemination or by ICSI according to the regular medical indications. Zygotes were transferred into IVF cleavage medium for further culture up to day-3. Extended embryo culture was done by grouping day-3 embryos into 80 µL droplets of blastocyst medium under oil. Embryos were assessed on day five and six. Selection of blastocysts for replacement and cryopreservation was done according to the blastocyst morphology. This morphological assessment was based on the presence of inner cell mass, the estimated number of the trophectodermal cells and the extent of the cavity expansion. The number of embryos for replacement in each patient was based on our replacement policy considering the number of the available embryos, embryo morphology, maternal age, past IVF history and number of previous implantation failures. Accordingly, for most of the patients up to three embryos were replaced either on day-3 or day-5. Embryo transfer procedures were performed using a standard Soft Pass™ transfer catheter (Cook). The mean ages, number of oocytes and fertilization rates were not different in both groups. The mean±SD number of embryos replaced was 3.1±0.6 and 2.3±0.8 on day-3 or day-5, respectively (P<0.001) (Table 28.1). In the day-5 transfer group 3 out of 46 patients (6.5%) did not have blastocysts for replacement. 141 out of 412 (34.2%) zygotes in the day-5 group reached the blastocyst stage and were suitable either for replacement or cryopreservation. Freezing of spare embryos was done in 25 cycles in day-3 group and only in 12 cycles in day-5 group (P=0.006). The implantation rates were 38.7% and 20.2%, respectively (P=0.002). The clinical pregnancy rates were 45.5% and 18.6%, respectively (P=0.007). The multiple pregnancy rates were not significantly different; 40% (8/20); five sets of twins, and three sets of triplets and 50% (4/8); three sets of twins, and one set of triplets, on day-3 or day-5, respectively. We therefore concluded that the pregnancy and implantation rates were significantly higher in the day-3 transfer group than in the extended culture group. It appears that there is about a 50 per cent decrease in implantation and pregnancy rates after extended culture. Moreover, it is evident that transferring a mean number of 2.3 blastocysts per

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transfer has not eliminated the risk of multiple pregnancies significantly. Any further attempt to restrict the average number of blastocysts transferred below 2.3 would have considerably lowered the pregnancy rate in this group. Based on our prospectively

Table 28.1: Comparison of the patient variables and IVF outcome between day-3 and day-5 transfer groups.* Variable

Day-3 transfer group Blastocyst transfer group P-υalue

Group size Patient with ET Mean age (y) Mean number of oocytes Mean number of zygotes Patients with ICSI (%) Patients with freezing Mean # of embryos per ET Implantation rate (%) Pregnancy rate (%) Multiple pregnancy rate (%) Twins and Triplets *Revised from reference 10.

44 44 31.5±7.4 16.3±6.4 9.9±4.1 26/44(59.1) 25 3.1±0.6 53/137(38.7) 20/44(45.5) 8/20(40) 5 and 3

46 43 30.9±4.0 15.2±5.5 9.7±4.0 22/43(52.1) 12 2.3±0.8 20/99(20.2) 8/43(18.6) 4/8(50) 3 and 1

NS NS NS NS 0.006 0.0001 0.002 0.007 NS

randomized data, we therefore currently recommend our patients to adhere to day-3 embryo transfer and for our good prognosis patients, to restrict the number of replaced embryos up to two top grade embryos.10 THE PROS AND CONS OF BLASTOCYST TRANSFER Numerous recent reports about in-house and commercially produced media have indicated a favorable outcome after extended embryo culture with better implantation and pregnancy rates.10–14 The review of the literature published over the past few years indicates an emerging consensus among the authors about the potential advantages of blastocyst production and transfer. These advantages include: i) allowing selection of more competent embryos for replacement, ii) minimizing the risk of multiple pregnancies, iii) providing more time for genetic diagnostic procedures, and iv) optimizing the synchronization between the embryo and the uterus. It is surprising to find, however, that less than handful reports were properly designed in a prospective random manner (Table 28.2).15–17

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Table 28.2: Advantages of extended embryo culture and replacement of embryos at the blastocyst stage • Opporturrity to select better embryos for transfer • Reduction in the number of embryos to be transferred, thus potentially reducing the incidence of high order multiple gestation • Temporal synchronization of embryo and endometrium at the time of embryo replacement • Potentially higher implantation rate per blastocyst • Improved potential to perform preimplantation genetic diagnosis (PGD) in conjunction with ART • Possibility to study in υitro the development of the early embryo.

Blastocyst production and transfer also entails several disadvantages. One risk associated with attempting blastocyst transfer is the possibility that no embryos will be available for replacement (Table 28.3). It has been postulated that one strategy to prevent this from occurring is to establish rigorous criteria for ovarian responsiveness during controlled ovarian hyperstimulation in order to produce a robust cohort of oocytes with good developmental potential.9,15 Another possibility is to establish defined developmental criteria for cleavage-stage (e.g. day 3) embryos, such as blastomere number and presence/extent of fragmentation, before patients can continue as candidates for blastocyst transfer.18 If these criteria are not met, the embryos may be transferred without additional culture. Both of these strategies may prove successful in reducing the incidence of cancellation due to failure to form blastocysts.

Table 28.3: Disadvantages of extended embryo culture and replacement of embryos at the blastocyst stage • Increased costs due to need for sequential media and prolonged culture of embryos in the lab • Possible negative effect on in υitro embryo development and implantation potential • Higher cancellation rate due to the possibility that no embryos will be available for replacement • Higher incidence of monozygotic twins • Potential reduction in the number of embryos available for cryopreservation • Possibly reduced success rate with frozen-thawed blastocysts

Additional potential disadvantages of blastocyst transfer include a suggestion from preliminary evaluations that the sex ratio of offspring produced from blastocyst transfer may be skewed to more males than females. Clearly, this awaits rigorous statistical scrutiny. Secondly, a recent multicenter analysis of 200 pregnancies resulting from blastocyst transfer revealed that 10 (5%) were monozygotic twins, an incidence 10-fold higher than that seen in nature.19 This possible association between monozygotic twins and blastocyst transfer has also been suggested by other studies.20,21 A final concern regarding blastocyst culture is the potential reduction in the number of embryos available for cryopreservation, resulting in a possible reduction in the cumulative pregnancy rate per retrieval following the transfer of both fresh and frozen blastocysts.

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Coskun et al,22 Huisman et al,23 as well as Plachot et al,17 could not demonstrate differences in implantation, pregnancy and twinning rates between day-3 and extended culture embryo replacement groups. Our present study is also in agreement with these findings. In a prospective study comparing day-3 transfer using Ham’s F-10 medium versus blastocyst transfer using sequential G-1/G-2 media (1) Gardener et al1 also did not find differences in pregnancy rates although a higher implantation rate in the second group allowed them to reduce the number of blastocysts transferred to an average of 2.2. Nonetheless, one may argue that the use of sequential G-1/G-2 media for the former group might have blunted this difference as well. Racowsky et al24 have shown in a retrospective study that the outcome of extended culture is significantly affected by the presence of 8-cell embryos on day-3 of culture. In the absence of such embryos on day-3 there was a pregnancy rate of 33.4% after day-3 ET, however, there were no implantations in cases where day-5 ET was attempted. These authors have concluded therefore that: “…whenever there are no 8 cell embryos on day-3 a day-3 transfer is warranted”. It appears, therefore, that patients with compromised embryos are not good candidates for blastocyst culture. A buy and large proportion of our unselected patient population are as such patients. In a well-designed prospective non-randomized study Alikani et al25 have demonstrated that cleavage anomalies affect the outcome of extended culture. In this study the implantation and the pregnancy rates in their selected group of extended culture patients were comparable with the corresponding rates of the day-3 transfer group. They have shown, however, that embryos with low average number of cells on day-3 or increased rate of fragmentation have significantly lower chances of cavitation and blastocyst formation. Moreover, the proportion of blastocysts with poor morphology significantly affected the IVF outcome. They have concluded that the presently available sequential extended culture systems do not support appropriate development of compromised embryos in υitro. Therefore, a fair proportion of embryos with implantation potential are lost during extended culture. Karaki et al26 in a randomized prospective study found that the implantation rate for embryos transferred at the blastocyst stage was significantly higher than that for embryos transferred on day 3 (26% vs. 13%). The viable pregnancy rate was similar inboth groups (29% vs.26%) and significantly fewer embryos were required for transfer at the blastocyst stage compared with day 3 embryo transfer (2.0+/−0.1 vs. 3.5+/−0.63). The highorder multiple gestation rate was significantly less with the blastocyst transfer than with the day 3 embryo transfer (4% vs. 19%). They therefore concluded that with the use of blastocyst culture, a few embryos can be transferred without decreasing the overall pregnancy rate. This may reduce multiple gestations and improve human IVF outcome. The patient population profile is an important detrimental factor in the overall IVF success rate of any given clinic. During the time frame of the present study 39% of our patients were over 36 years old. Twenty seven per cent have had six or more failed IVF attempts and 33% had less than 6 oocytes retrieved. Most of the embryos produced by this type of patients are compromised and therefore they are not ideal candidates for blastocyst replacement. Although usually over 40% of the fertilized oocytes reach the blastocyst stage there is considerable variability in the ability to produce blastocysts between patients. In our

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selected study group about 6.5% of the patients did not have embryos for replacement. This risk may be even higher in cases with less favorable prognosis. Beside the differences in implantation and pregnancy rates between our two study groups, patients with blastocyst transfer had fewer embryos for cryopreservation. This outcome further limits their overall IVF success rate. CONCLUSION Our experience suggests that day-3 ET is more beneficial that blastocyst transfer.10 Extended culture may have under certain laboratory conditions a negative effect on in υitro embryo development and implantation potential. The delayed replacement policy has failed in our hands to eliminate the problem of high order pregnancies. It is most likely that any further attempt to restrict the number of the replaced blastocysts would have had more negative effect on the outcome. In the light of these findings we currently recommend that our patients to adhere to day-3 embryo transfer method and in good prognosis patients, restrict the number of the replaced embryos up to two top grade embryos. We believe that such an approach will help in maintaining a reasonable outcome on one side and provide an effective tool in fighting the problem of high order pregnancies on the other side. The search for the “magic bullet”, that would ensure successful implantation of the embryo continues. Blastocyst production does offer a significant clinical benefit by allowing an improved ability to select in υitro the best embryos for transfer. Yet, at present it is clear that blastocyst transfer does not offer the long sought solution for patients with poor prognosis such as advanced maternal age, decreased ovarian reserve, or a history of repeated implantation failures and poor embryo or oocyte quality. REFERENCES 1. Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update 1997; 3:367–82. 2. Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod 1998; 13(Suppl 3):148–59. 3. Cohen J, Simons RF, Fehilly C, Fishel SB, Edwards RG, Hewitt et al. Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage. Lancet 1985; 1:647. 4. Wiemer KE, Cohen J, Tucker MJ, Godke RA. The application of co-culture in assisted reproduction: 10 years of experience with human embryos. Hum Reprod 1998; 13:226–38. 5. Bavister BD, Boatman DE. The neglected human blastocyst revisited. Hum Reprod 1997; 12:1507–09. 6. Gardner DKand Schoolcraft WB. No longer neglected: the human blastocyst. Hum Reprod 1998; 13:3289–92. 7. Menezo Y, Hamamah S, Hazout A, Dale B. Time to switch from co-culture to sequential defined media for transfer at the blastocyst stage. Hum Reprod 1998; 13:2043–44. 8. Alper MM, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question. Hum Reprod 2001; 16:617–19.

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9. Gardner DK, Vella P, Lane M, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998, 69:84–8. 10. Levron J, Shulman A, Bider D, Seidman DS, Levin T, Dor J. A prospective randomized study comparing day-3 versus blastocyst stage embryo transfer. Fertil Steril 2002; (in press). 11. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 2000; 74:482–6. 12. Langley MT, Marek DM, Gardner DK, Doody KM, Doody KJ. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001; 16:902–8. 13. Alves da Motta EL, Alegretti JR, Baracat EC, Olive D, Serafini PC. High implantation and pregnancy rates with transfer of human blastocysts developed in preimplantation stage one and blastocyst media. Fertil Steril 1998; 70:659–63. 14. Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil Steril 2000; 73:126–9. 15. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trail of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998, 13:3434–40. 16. Scholtes MC, Zeilmaker GH. Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not on age. Fertil Steril 1998; 69:78–83. 17. Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Serkine AM, Tesquier L. Blastocyst stage transfer: the real benefits compared with early embryo transfer. Hum Reprod 2000; 15: 24–33. 18. Behr B. Blastocyst culture without co-culture: role of embryo metabolism. J Asst Reprod Genet 1997; 14:13S. 19. Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000; 17:349–51. 20. Peramo B, Ricciarelli E, Cuadros-Fernandez JM, Huguet E, Hernandez ER. Blastocyst transfer and monozygotic twinning. Fertil Steril 1999; 72:1116–7. 21. Sheiner E, Kivilevitch Z, Levitas E, Sonin Y, Albotiano S, Har-Vardi I. Monozygotic twins following blastocyst transfer: a report of two cases. Eur J Obstet Gynecol Reprod Biol. 2001; 98:135–8. 22. Coskun S, Hollanders J, Al-Hassan S. Day-5 versus day-3 embryo transfer: a controlled randomized trail. Hum Reprod 2000; 15:1947–52. 23. Huisman GJ, Fauser BC, Eijkemans MJ, Pieters MH. Implantation rates after in υitro fertilization and transf er of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril 2000; 73:117–22. 24. Racowsky C, Jackson KV, Cekleniak NA. The number of eight-cell embryos is the key determinant for selecting day-3 or day-5 transfer. Fertil Steril 1999, 73:558–64. 25. Alikani M, Calderon G, Tomkin G, Garrisi J, Kokot M, Cohen J. Cleavage anomalies in early human embryos and survival after prolonged culture in υitro. Hum Reprod 2000; 15:2634–43. 26. Karaki RZ, Samarraie SS, Younis NA, Lahloub TM, Ibrahim MH. Blastocyst culture and transfer: a step toward improved in υitro fertilization outcome. Fertil Steril 2002; 77:114–8.

SECTION 5 Laboratory Issues

CHAPTER 29 Semen Analysis for Clinical Interpretation Elizabeth Puscheck, RS Jeyendran INTRODUCTION The essential goal of semen analysis is the assessment of male fertilization potential. Should that potential be discovered as compromised, semen analysis should then aid in the diagnosis of any underlying etiology. Inherent complexities between various sperm factors, however, necessitate a logical conceptual framework and rigorous laboratory methodology. Most importantly, the proper interpretation of these analysis results cannot only provide vital diagnostic information, but further recommend a battery of specialized semen analysis assays for a more refined and comprehensive fertility evaluation, along with direct therapeutic interventions. Inherent Ambiguities While the result of a single, successful fertilization is obvious, the contingencies culminating in chronic infertility can prove astoundingly complex. Evaluation of male fertility, being simpler, less invasive and more cost effective than testing of the female, is the acknowledged first step in trying to discover any problems. Since natural fertilization involves direct sperm-oocyte union and chromosomal fusion between male and female, sperm incubation with oocytes under natural reproductive or controlled laboratory conditions could directly test male fertilization potential. Such a procedure, however, is completely unrealistic and impractical; and even if performed, it could never evaluate sperm parameters relevant to transport within the female reproductive tract. As a viable substitute and improvement on fertilization evaluation through actual gamete union, numerous semen assays have been designed to clinically test various sperm parameters. These tests then produce quantified results which must, in turn, be compared to prescribed ranges (based on values obtained from a larger number of fertile samples), deemed “normal” or “abnormal.” Unfortunately, a precise boundary between such “normal” and “abnormal” values remains elusive. After all, the spermatozoon itself is a highly complex chromosomal delivery vehicle. Each sperm parameter describes but one of many diverse, interconnected physical aspects of the spermatozoa. Not only can uncertainties in measurement cloud results, but a particular sperm parameter might test conclusively within the “abnormal” range, yet have no causal connection to fertility. Similarly, several

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other parameters might display relatively minor deviations, yet cumulatively debilitate sperm capacity to the point of chronic infertility To allow results to better reflect these inherent uncertainties and variabilities, an intermediate or “equivocal” range of values can be assigned whenever a measured value does not fall in a definitive or comfortably “normal” or “abnormal” range. In such a manner, an allowance is made not only for the naturally broad range of otherwise perfectly functional sperm parameters, but also for the many inherent ambiguities when considering the very concept “male fertilization potential.” Further Testing Should any one or several sperm parameters test comfortably within the prescribed “abnormal” range, an underlying etiology might be suggested. Such a diagnosis can be further refined and perhaps confirmed through the recommendation of additional, more specialized assays. A proper clinical diagnosis can then be made by comparing these results to a sound understanding of exactly how that particular parameter affects the physical interaction between the sperm and female reproductive system. Repeat Testing The evaluation of sperm parameters can vary tremendously between two consecutive samples taken from the same individual, obtained but a few days apart. Similarly, other variables such as abstinence duration, collection method and environment, and the intensity of stimulation can all influence semen quality to greatly varying degrees. Since so many diverse biological, psychological and environmental factors can so profoundly influence results, routine semen analysis should be conducted at least twice, preferably with even greater frequency. If the results of these analyses are markedly different, then additional samples should be analyzed until a consistent overall semen quality is determined. Should a specific etiology be suggested by a specialized test, then a repeat test is highly recommended to confirm the final clinical diagnosis. Logical Framework Semen analysis can be divided into two distinct types: Routine and Specialized analysis. Routine semen analysis involves numerous macro- and microscopic measurements of sperm. These tests are relatively inexpensive and practical, and, by definition, are conducted on a regular basis. Specialized semen analysis, in contrast, includes a host of more refined assays, less frequently performed and typically recommended by the results of an initial, routine evaluation. Proper clinical interpretation of the results from both routine and specialized analyses is linked to an understanding of direct sperm interaction with the female reproductive system. Each sperm parameter, in other words, is measured and the result interpreted in the light of how that particular sperm activity either facilitates or inhibits potential spermoocyte union. Similarly, the significant deviation of a specific sperm parameter might indirectly suggest an underlying etiology, thereby providing other approaches to treatment and, hopefully the restoration of natural fertilization potential.

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Interpretation through Question and Answer With so many diverse semen analysis assays currently available, choosing the specific regimen appropriate for each patient can prove complex and confusing. By answering the following basic questions concerning sperm progress within the female reproductive system, the relevant semen tests that appropriately facilitate clinical interpretation can be suggested: Can Sperm Get to the Fertilization Site? A routine semen anolysis and a satisfactory postcoital test or sperm mucus penetration assay are able to determine how efficiently a sufficient number of sperm can transport themselves through the cervical mucus and the female reproductive tract. A routine semen analyses should be performed at least twice. Inconsistent results require repeat analysis until consistency is attained. If these results suggest “normar” or “questionable” sperm qualities, and if the spouse is also tested, then testing specifically for sperm fertilization capacity is recommended. Can the Sperm Fertilize the Egg? Hypoosmotic swelling assay, acrosome reaction assay, zona binding assay and sperm penetration assay all test for various sperm processes necessary for oocyte fertilization. If semen tests definitively as “abnormal,” then appropriate specialized semen analysis assays should be performed, based on the specific type of abnormality. Are Immunological Factors Suspected? Anti-sperm antibodies have been implicated in 10 to 20 percent of unexplained infertility cases. In such instances, sperm agglutination, sperm immobilization and sperm surface binding immunoglobulins should be performed. Do Chemical Components Need to be Assessed? If an azoospermic ejaculate volume is less than 1 ml, or if semen volume is less than 0.5 ml, then seminal fluid components and pH leυel should be assessed. Based on these analysis results, in conjunction with overall medical history and the partner’s clinical findings, even more sophisticated, specialized tests may need to be performed: These advanced tests include nuclear integrity, reactive oxygen species, acrosin, creatine phosphokinase, and sperm ultrastructural eυaluations. Although these assays are not routinely performed or often even available, they remain extremely promising and may become standard in the near future. Routine Semen Analysis Including both macroscopic and microscopic semen factor measurement, routine semen analysis is typically prescribed for general fertility assessment, and before and after such procedures as vasectomy and cryopreservation. Such a routine semen analysis should

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follow a strict set of directions to ensure accurate interpretation: For example, sexual abstinence of 2 to 3 days, the avoidance of lubrication during masturbation, and precautions against spillage during collection. Macroscopic Analysis The macroscopic components of routine semen analysis consist of testing for semen appearance, coagulation, color, odor, viscosity and volume. Appearance Semen is characteristically turbid, but can vary significantly in appearance, even from one consecutive sample to the next. Unlike other species, such as the bovine, the relative turbidity or overall appearance of human semen has absolutely no bearing on fertilization potential. Coagulation and Liquefaction Following ejaculation, semen coagulates into a gelatinous mass and then gradually liquefies. Exact liquefaction time is of no diagnostic importance unless more than two hours elapse without a discernible state change. Semen unable to liquefy cannot sufficiently release sperm into the cervical mucus, thereby precluding sperm movement toward the fertilization sites deeper within the female reproductive tract. Fertilization potential is then compromised, since sperm have difficulty reaching the oocyte. From a male reproductive standpoint, inability of the semen to coagulate may aid in diagnosis of ejaculatory duct obstruction or the congenital absence of the seminal vesicles (where coagulating proteins originate), especially if semen volume is extremely low. Prolonged liquefaction time or complete liquefaction absence is also most likely due to poor prostatic secretion (since liquefying enzymes are derived from the prostate gland). Further testing is then recommended. Color and Odor Semen color and odor have no significance for sperm evaluation, and do not seem to in any way compromise fertilization potential. Abnormal color may indicate accessory sexual gland or other, nonreproductive system pathologies. To cite a few examples, a pink or reddish color can be due to a proliferation of red blood cells, known as “hematospermia”; yellowish semen is suggestive of jaundice; while urine contamination and the presence of drugs like methylene blue and pyridium may also color the semen. Viscosity Semen viscosity is the measure of friction between various seminal fluid components as they slide by one another. The relationship between viscosity and fertility is inconclusive, although high viscosity, combined with poor sperm motility, can lead to a marked decrease in fertilization capacity due to problems with sperm transport and delivery.

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Specifically, the semen is too “thick” to allow smooth sperm migration through the cervical mucus, a condition analogous to problems with liquef action. Volume Semen volume is measured in milliliters. Abnormally low semen volume can physically complicate sperm-oocyte union. Such low volume, in conjunction with low pH and fructose presence, can also suggest an ejaculatory duct obstruction or congenital absence of the vas deferens and seminal vesicles. In such an instance, further specialized tests should be conducted to localize any etiology Microscopic Analysis The microscopic components of routine semen analysis include sperm agglutination, nonsperm cellular compo nent detection, sperm concentration, motility viability and morphology. Sperm Agglutination “Sperm agglutination” is the clumping of sperm into aggregates, as assessed in wet microscopic smears. Such agglutination can occur in two basic types: Nonspecific agglutination, where sperm adhere to various seminal debris, leukocytes or mucus threads, and various other non-sperm cellular elements (typically making sperm nonmotile as a result); and, Site-specific agglutination, where sperm adhere to each other in a site-specific manner, such as head-tohead, head-to-tail, tail-to-tail, or any such combination. Diagnostically, the sperm agglutination level becomes relevant only when motility is compromised to the point of abnormalcy In such an instance, sperm are physically hindered in their capacity to reach the fertilization sites, thereby indirectly inhibiting fertilization potential. Site-specific sperm agglutination might suggest an immunological cause, and should be noted. However, a few clusters of immotile, agglutinated sperm clinging to debris or mucus is of no clinical significance. Non-Sperm Cellular Elements These include cellular elements such as leukocytes, erythrocytes, epithelial cells, microorganisms, and sperm precursors which may be present in seminal fluid: Leukocytes Leukocytes (white blood cells), predominantly neutrophils, possibly originate within the prostate, and may be present in seminal fluid. Leukocytes are typically about 10 to 14 µm in size, with a lobed nucleus and granular cytoplasm. Occasionally, macrophages and polymorphs are also visible, phagocytizing the sperm.

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Leucocyte detection, if observed in conjunction with seminal debris and poor sperm motility may aid in the diagnosis of a possible accessory sex gland infection. If leucocyte count is exceptionally high, the sample may appear yellowish-opaque, a condition sometimes known as pyospermia. Leukocytes may also play a part in sperm agglutination, and may produce oxidative stress (generation of free oxygen radicals) and cytotoxic cytokinins secretions (lymphokines and monokines), all contributing indirectly to varying degrees of subfertility. Erythrocytes Erythrocytes (red blood cells) are typically not present in a healthy ejaculate. Their presence is usually due to a reproductive tract pathology. Fertilization potential, however, is unaffected, unless that etiology interferes, directly or indirectly, with sperm production or transport. Even large numbers of erythrocytes in the ejaculate, however, do not appear to influence sperm fertilization potential. Epithelial Cells Epithelial cells, possibly originating from the urethra or meatus and glans penis, are seminal fluid contaminates typically resulting from samples obtained through masturbation. Excessive numbers of epithelial cells may suggest that collection was conducted via coitus interruptus, or orally. These collection methods may sometimes compromise sperm quantity and quality; a repeat collection via more conventional, recommended means may prove necessary should other problems arise. Microorganisms Microorganisms, mainly bacteria, may be found in the ejaculate. Large numbers of bacteria usually suggest a reproductive tract infection, almost exclusively stemming from the prostate. A bacterial culture with antibiotic sensitivity determination is recommended to identify microorganism type and any potential etiology. Such a test is very important if sperm are used for intrauterine insemination or any other assisted reproductive technology procedures, since the probability of infection is greatly increased. Cells of Spermatogenic Origin Physically, spermatids and spermatocytes are immature spermatozoa of irregular in size, with two or more discrete nuclei. If present in extremely high numbers, they may suggest acute distress situations such as fever, intoxications, exposure to radiation or cytotoxic drugs. A high number of precursor cells is usually associated with below normal sperm count and abnormal sperm morphology, and may result in an overall reduction of fertilization potential. A testicular biopsy may reveal any underlying etiology.

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Sperm Concentration and Sperm Count Sperm concentration is measured as the number of sperm per milliliter of seminal fluid. Sperm count is the total number of sperm within the ejaculate. Sperm concentration alone, although a potentially determining factor, is not an accurate indicator of fertilization potential. Fundamentally, the probability of fertilization can be increased with a greater number of sperm deposited within the female reproductive tract; theoretically, however, only one sperm is necessary for pregnancy to occur. Azoospermia can nonetheless be considered an absolute indicator, and is confirmed when a pellet centrifuged at 1000 g for 10 minutes reveals the complete absence of spermatozoa. Sperm Motility Motility is spontaneous sperm movement. Sperm motility is susceptible to temperature variation, and should be performed under controlled thermal conditions. Abnormal sperm (except for headless or “pin-point” sperm) never show good motility. As a general rule, good motility is an inherent characteristic of good sperm. However, motility is only one of many variables influencing fertility. If sperm concentration is in the acceptable range, sperm morphology is evaluated as “normal”, and no leukocytes or other abnormalities are present, then relatively low motility values remain insignificant. Consequently, sperm motility must be extremely low to actually be the sole cause of infertility. Sperm Viability Viability is simply defined as the number of “live” sperm. The bearing on fertility is obvious, as dead sperm are, by definition, infertile. Sperm Morphology The shape, size and surface appearance of sperm Although more than 65 percent abnormal sperm morphology indicates a testicular or epididymal impairment, fertility potential need not be compromised, even if such an otherwise extreme condition is confirmed: Morphological abnormalities must be extremely high for overall fertilization capacity to be adversely affected. Punctilious analysis of stained sperm can identify many minor deviations from an otherwise “perfect” sperm cell. If such detailed analysis reveals fewer than 4 percent “normal” sperm, then the sample can confidently be classified as “abnormal.” This sample nonetheless remains fertile in the clinical sense, since intracytoplasmic sperm injection (“ICSI”) with in vitro fertilization (“IVF”) can still result in pregnancy. Sperm Movement Through The Cervical Mucus Sperm may appear normal but prove functionally deficient, indirectly inhibiting overall fertilization potential. An inability to penetrate and migrate through the cervical mucus, for example, will interfere with sperm being able to reach the fertilization site. If a

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postcoital cervical mucus examination reveals the complete absence of sperm or an inadequate sperm number, then a migratory deficiency might be causing the fertility problem. Note that appropriate cycle-timing with the female partner is critical to ensure the correct interpretation of the postcoital test. Any abnormal sperm migratory capacity should then be tested in vitro with the following test: Sperm Mucus Penetration Test To reach the fertilization site, spermatozoa must penetrate and migrate through the cervix and cervical mucus. Failure of in vitro penetration is believed to imply analogous failure in υiυo. Diagnostic capacity is limited, since relevance depends on a negative or questionable postcoital cervical mucus test result. Infertility can be confirmed, however, if sperm penetration and migration into the cervical mucus are revealed as abnormal. Should in vitro migration prove to be within a normal range, then the female should also be tested, since an inadequate quality or quantity of cervical mucus might, in and of itself, prove detrimental to the overall fertilization process. Healthy cervical mucus typically allows only motile sperm with normal forms and size to smoothly penetrate and migrate. Such a natural sperm filter can therefore provide an indirect measurement of sperm morphology. Specialized Semen Analysis As previously recommended, routine semen analyses should be performed at least twice. If consistent findings suggest “normal” or “equivocal” sperm qualities, and the partner is also found to test normal, yet the couple remains infertile, specidized semen analysis may then be required. Many diverse and vital sperm factors remain outside the realm of routine semen analysis. Capacitation, for example, taking place when sperm are already within the female reproductive tract, primarily involves changes in the sperm membrane system. Since the sperm membrane must be intact and functional for these events to occur, an osmotic stress test, or hypo-osmotic swelling assay (“HOS” test) should be performed to determine membrane status. To determine whether capacitation can occur, an acrosome reaction should be artificially induced and evaluated by an acrosome reaction assay. Alternatively, since only acrosome-reacted sperm can successfully bind to the zona pellucida, capacitated sperm can be added to isolated human zona to determine binding extent with a zona binding assay. If sperm are capacitated and acrosome-reacted, they should also be able to penetrate zona free hamster oocytes, as measured by a sperm penetration assay. The sperm nucleus should then be able to naturally decondense. These and the other specialized and optional tests listed further are not routinely performed, and reference values have yet to be firmly established. Many are also timeconsuming and technically demanding, their interpretation complex and often highly subjective. Regardless, these tests, when properly conducted, can provide valuable and otherwise unobtainable diagnostic information for certain select fertility cases.

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Important note Routine semen analysis, with consistent results obtained, should always be conducted prior to any of these more specialized tests. Hypoosmotic Swelling Assay Membrane integrity is defined as the ability of the outer sperm membrane to maintain a equilibrium between the sperm and its environment. Factors essential for sperm viability, such as sperm motility, capacitation, the acrosome reaction, and sperm-to-oocyte surface binding can all be compromised should membrane integrity prove questionable or in any way inadequate. Consequently, sperm membrane integrity and functionality is significant, and membrane functional assessment may prove important in assessing fertility. Of these specialized tests, the HOS test is relatively simple, reliable and easy to perform. Since sperm membrane integrity/functionality is always a significant sperm variable, HOS can even be performed routinely, if so desired. Sperm Acrosome Reaction Assay The “acrosome reaction” is an exocytotic event that occurs immediately prior to fertilization. The outer acrosomal membrane fuses with the surrounding plasma membrane, culminating in acrosomal content release and dispersal. Although the exact relevance and practicality of the acrosome test remain contested, sperm testing negative with this assay can be conclusively diagnosed as infertile. Sperm utilized for ICSI with IVF need not undergo the acrosome reaction, making this test completely redundant for such procedures. Zona Binding Assay Tests for sperm ability to bind to the zona pellucida outer surface, a prerequisite prior to oocyte vitelline membrane binding and penetration. Negative test results can help identify patients who might benefit from ICSI with IVF. Also, more than 90 percent of the tightly zona-bound sperm undergo the acrosome reaction, so this test can be used to similarly evaluate sperm for capacitation and the acrosome reaction. Few laboratories presently perform the hemizona or zona binding assay, since human zonae acquisition remains a major problem. The test is also labor intensive, often lacks sufficient quality control, and requires experienced and well-trained personnel. Sperm Penetration Assay Determines sperm ability to bind and penetrate the oocyte vitelline membrane, a prerequisite for male gamete fusion with oocyte contents. The assay’s ability to assess whether the sperm nucleus can decondense may assist in ICSI.. If the assay is conducted following a failed IVF procedure, results may aid in determining etiology. A positive result represents potential fertility. A negative result can identify patients who may subsequently benefit from IVF.

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Implementation of the sperm penetration assay as a routine laboratory test is hindered by various difficulties, including technical complexities, relatively poor repeatability, and lack of standardization between laboratories. The test is also relatively labor intensive, lacks good quality control, and requires experienced and well-trained personnel. The presence of leukocytes can also adversely affect outcome. Leukocytes in semen may similarly decrease fertilization potential. Consequently, this assay is currently limited in its practical application and general efficacy. Spedalized tests—when routine test results are “abnormal” These specialized tests should be performed based on the specific type of abnormality in an effort to localize be specific etiology. These assays include the Anti-Sperm Antibody test, and various tests for semen chemistry. Anti-Sperm AntibodyTest Sperm are not only antigenic, but considered as foreign tissue, even by their own host, since sperm never come into direct contact with the bloodstream. When an individual’s blood is directly exposed to sperm (due to an injury or surgery), immunoglobulin cells within the circulatory system respond by producing antibodies against these sperm, or socalled “anti-sperm antibodies.” These anti-sperm antibodies primarily cause sperm agglutination, immobilization or become surface bound. Site-specific sperm agglutination and somewhat erratic or “jerky” progressive sperm motility suggest that immunoglobulins should be analyzed to look for antisperm antibody effects. Both asthenozoospermia and necrozoospermia may be caused by anti-sperm effects, and may necessitate similar testing. “Indirect Tests” check for anti-sperm antibodies within body fluids, while “Direct Tests” seek antibodies physically located on sperm cells. Anti-Sperm Antibody Assays These assays help to diagnose immunological causes of infertility. In fact, anti-sperm antibodies have been implicated in 10 to 20 percent of unexplained infertility cases. Anti-sperm antibody levels inboth partners’ blood and reproductive fluids should be assessed, particularly when other etiologies have been exhausted or ruled out. Surprisingly, most men who actually possess antisperm antibodies in seminal plasma may still test “negative” with the Indirect Test. Direct testing is therefore highly recommended whenever Indirect Tests are performed. A wide variation exists between different anti-sperm antibody tests. Various test combinations may be successfully utilized to reduce the inherent uncertainties and ambiguities of individual tests. Such a combinational approach may allow for more reliable interpretation and management of infertile patients whose fertility may be compromised by anti-sperm antibodies. A direct correlation between anti-sperm antibodies and infertility does not yet exist, adding confusion and ambiguity to test results. Typically, excessive antibody presence indicates some sort of genital tract disturbance; the antibodies themselves are usually a symptom to that underlying etiology, necessitating further testing. In other cases, infection, injury, vasectomy or idiopathic factors may produce antibodies against sperm:

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Should such antisperm antibody levels rise too high, overall fertility might become severely compromised. Semen Chemistry Chemical analysis of seminal plasma should be considered when semen tests reveal azoospermia, and sample volume is less than 1.0 ml. If semen volume is consistently 0.5 or less (regardless of sperm concentration), then chemical semen analysis is automatically recommended. Each accessory sex gland, including the seminal vesicles, prostate and epididymis, independently produces distinctive components. Analyzing the presence of these components assists in assessing respective accessory sex gland status, and can assist in the overall diagnosis. Throughout such analysis, however, the total quantity of such secretions within any given ejaculate can vary significantly from sample-to-sample, even when the sample is collected from the same individual. Determination of the relative contribution of each accessory sex gland component within semen is therefore sufficient. However, when the concentration of one particular component is markedly low, then further testing of other components is no longer considered relevant. Prostatic Contribution Analysis Comprised of the chemical analysis of seminal plasma for specific prostatic components. This assay is diagnostically very limited. However, the absence of prostatic component may aid in confirming that the loss of initial, sperm rich fraction occurred during semen collection. Abnormal prostatic function may also result in prolonged liquefaction time or the complete absence of liquefaction. Seminal Vesicular Contribution Analysis Involves chemical analysis of the seminal plasma for specific seminal vesicular components. Such an assay can diagnose an obstruction at the level of both ampulla, or congenital absence of seminal vesicles and vas deferens. The low concentration or complete absence of fructose within the seminal plasma may suggest some type of male reproductive tract obstruction. In such cases, further testing, or consultation with a urologist, is highly recommended Epididymal Contribution Analysis Involves chemical analysis of the seminal plasma for specific epididymal components. Absence of neutral—glucosidase activity may diagnose an obstruction at the epididymal level. In such cases, further testing, or consultation with a urologist, is highly recommended.

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Seminal Plasma pH Analysis Tests for hydrogen ion concentration (expressed as the reciprocal logarithm of concentration). Obstruction existence and even location might be inferred from pH values. Note that semen pH has little direct significance to sperm fertility potential, unless levels are excessively abnormal. However, if semen volume is less than 1 ml, pH becomes a vitally important parameter. CONCLUSION The proper assessment of male fertilization potential is surprisingly difficult, owing to the subtle relationship between numerous sperm parameters and the overall fertilization process. A successful semen analysis for clinical interpretation must determine sperm factors relevant to sperm transport and fertilization potential. Various tests have been constructed to evaluate these particular sperm parameters. Based on the results of these tests, additional, more specialized tests are in turn available to further refine and localize any potential etiology. By effectively coordinating these sperm tests with a parallel evaluation of the female partner, most male fertility problems can be properly diagnosed and hopefully resolved. ACKNOWLEDGMENT Special thanks to Michael Spitz for excellent editorial assistance. BIBLIOGRAPHY 1. Keel BA, Webster BW (Eds). The Handbook of Laboratory Diagnosis and Treatment of Infertility. Boca Raton, Florida: CRC Press, 1990. 2. Zaneveld LJD, Jeyendran RS. Sperm function tests. In Overstreet JW (Ed). Infertility Reproductive Medicine Clinics of North America. Philadelphia, Pennsylvania: WB Saunders Company, 1992. 3. Zaneveld LJD, Jeyendran RS, Vermeiden JPW, Lens JW. Sperm enzymes for diagnostic purposes. In AAAcosta, TF Kruger (Eds): Human Spermatozoa in Assisted Reproduction. The Pathenon Publisher Group, New York 1996. 4. Jeyendran RS. Semen Analysis: method and interpretation. In JJ Sciarra JP (Ed): Gynecology and Obstetrics. Philadelphia, Pennsylvania: Lippincott Company, 1998. 5. Insler V, Lunenfeld B. Infertility Male and Female. Edinburgh, United Kingdom: Churchill Livingstone, 1993 6. Rowe PJ, Comhaire FH, Hargreave TB, Mellows HJ. WHO manual for the standarized investigation and diagnosis of the infertile couple. Cambridge, Great Britain: Cambridge University Press, 1993. 7. Mortimer D. Practical LaboratoryAndrology. New York: Oxford University Press, 1994. 8. Lipshultz LI, Howards SS: Infertility in the Male (3rd edn). Mosby-Year Book Inc., St. Louis, Missouri 1997.

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9. Handbook of Andrology, The American Society of Andrology. Lawrence, Kansas: Allen Press Inc., 1998. 10. WHO. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. World Health Organization. Cambridge, Great Britain: Cambridge University Press, 1999. 11. Jeyendran RS: Interpretation of Semen Analysis Results: A Practical Guide. Cambridge, Great Britain: Cambridge University Press, 2000.

CHAPTER 30 Fundamentals of Sperm Processing Techniques RS Jeyendran, Vida Acosta, Milica Ivanovic INTRODUCTION Sperm processing involves various laboratory techniques designed to enhance the fertilization potential of semen. Since the female reproductive system naturally optimizes the conditions necessary for fertilization, laboratory duplication and augmentation of these physiological processes is the best approach to sperm processing. These sperm processing techniques include the separation of sperm from the seminal fluid, and several additional techniques to enrich and enhance the sperm sample. NATURE’S OWN SPERM PROCESSING CLINIC During coitus, semen is deposited within the female reproductive system following ejaculation. However, only a fraction of the total ejaculate comes into contact with the periovulatory cervical mucus. The cervical mucus acts as a selective filter by providing a natural barrier against various infectious agents that may be present in the semen. Other potentially deleterious factors and agents within the seminal fluid, such as decapitation factors and prostaglandins, are also inhibited from entering. Only progressively motile sperm with normal shape and size are able to steadily and methodically negotiate their way through narrow channels within the cervical mucus, and eventually migrate through the cervix. The cervical mucus also functions as sperm reservoir. However, only a small proportion of all the viable sperm initially present in the ejaculate are able to reach the fertilization site within the female reproductive system. Mimicry and Improvement Laboratory sperm processing techniques have been developed which mimic the cervical mucus. These in vitro techniques can actually improve nature’s own processes, since rigorous laboratory methods can be applied thoroughly to an entire ejaculate, as opposed to the relatively minuscule semen percentage capable of reaching the cervix in vivo. Consequently, much greater sperm quantities, exhibiting a marked increase in quality, is made available for insemination procedures. To successfully accomplish these sperm processing techniques, laboratory technicians must first remove all seminal fluids from the ejaculate. All progressively motile sperm with normal shape and size are then concentrated. The enriched samples resulting from

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these laboratory methods are essential for intrauterine insemination (“IUI”) and in vitro fertilization (“IVF”) procedures. Sperm processing for IUI and IVF procedures are often similar, although the underlying rationale varies tremendously. The goal for IUI is to obtain as many viable sperm as possible from a relatively small semen volume. However, IVF requires relatively few viable sperm (only approximately one hundred thousand) processed from any given sample. In vitro fertilization, by its very nature, necessitates the artificial inducement of sperm capacitation. Evolution of Laboratory Sperm Processing Throughout the late 1970s and early 1980s, the principal modus operandi of such sperm processing procedures was seminal plasma removal through centrifugation. Unfortunately, such a procedure often proved damaging to sperm viability, with the potentially deleterious side effect of inadvertently concentrating leucocytes and nonmotile, even abnormal, sperm. Today, these centrifugation side effects are minimized by new sperm processing methods whose functions have evolved well beyond the mere removal of seminal plasma: Increased concentrations of non-motile sperm and miscellaneous cellular debris can now be removed from sperm through a host of practical laboratory procedures. SPERM PROCESSING Sperm processing procedures that mimic the sperm separation abilities of preovulatory mucus can favorably influence reproductive outcome. These methods analogously and effectively select progressively motile and morphologically normal sperm from the overall sample population. The resultant sample contains an enriched sperm population with higher fertilizing potential. The first step in most processing procedures is sperm washing, designed to separate sperm from the surrounding seminal plasma. Such separation is necessary to remove decapacitation factors deleterious to the fertilization process, and along with prostaglandins. Various blood components, such as leukocytes and erythrocytes, might also have to be removed. Sperm washing also concentrates sperm, which further facilitates various sperm selection procedures. Sperm washing, common to both IUI and ART, specifically involves dilution and centrifugation. Centrifugation Centrifugation, or the mechanical induction of centrifugal force, is fundamental to sperm processing procedures. Most attempts to effectively remove seminal plasma from the ejaculate will involve some form of centrifugation. Centrifugation can be described through the behavior of particles in suspension. Particles respond to gravitational force, and when suspended in a fluid, will naturally move towards the container bottom. Such a filtration method is influenced by particle

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size and shape, density differential between particle and suspension media, media viscosity, relative centrifugal field (“rcf” or “g”) strength and exposure time to that “rcf.” Of these variables, only the last three (viscosity, intensity and duration) can be tweaked in the laboratory to obtain maximum yield. Improper intensity and duration, however, may compromise sperm quality or quantity: High centrifugal fields can exert too much force, damaging cell structures and thereby proving detrimental to sperm quality, while low centrifugal fields might exert insufficient force, resulting in samples of relatively low quantity. Optimized sperm processing through adroit centrifugation therefore requires a balanced compromise between these two contradictory tendencies. Such a balance can be obtained in numerous ways, including seminal plasma viscosity reduction through media dilution and by applying the inversely proportional mathematical relationship between intensity and duration. Innovative new techniques in centrifugation, such as “oscillating” and “pulse” centrifugation, can also help increase yield. Viscosity Reduction Sperm washing for most semen samples begins with an initial 4-to-5 fold media dilution of the original seminal plasma, especially for those samples containing high sperm concentration or extremely high viscosity. If a semen sample contains low sperm concentration in relatively high volume, then a relatively low dilution rate is necessary. Media not requiring a CO2 incubator, and generally available for sperm dilution and processing, include: • Tyrode’s Salt Solution (Sigma Chemical Co., St. Louis, MO 63178; Irvine Scientific, Santa Ana, CA 92705). • Modified Ham’s F-10 medium with HEPES (Irvine Scientific, Santa Ana, CA 92705). • Dulbecco’s Phosphate Buffered Saline solution (Irvine Scientific, Santa Ana, CA 92705; Sigma Chemical Co., St. Louis, MO 63178). Protein supplementation is essential during centrifugation and for the general maintenance of sperm viability. Such protein supplementation appears to stabilize sperm membrane integrity, and may also absorb free oxygen radicals already present or additionally generated during centrifugation. Protein supplements generally available for sperm dilution and processing include: • Bovine Serum Albumin, 0.2 to 0.5 gm percent (Sigma Chemical Co., St. Louis, MO 63178; Irvine Scientific, Santa Ana, CA 92705); or, as an alternative to bovinederived products: • Dried Chicken Egg Yolk, 0.6 gm percent (Sigma Chemical Co., St. Louis, MO 63178) In essence, sperm washing can be optimized by applying combined dilution ratio with appropriate rcf strength, continuing for sufficient time exposure. An example of such a regime would be the following: • Use a dilution of 4-to-5 • Centrifuge with an rcf of 500 at 1000 g • Continue centrifugation for a duration of 4 to 10 minutes

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The goal should be a thorough concentration of motile sperm, and the recovery of most if not all viable spermatozoa. Such separation procedures should be reliable, repeatable and relatively simple to perform. Such currently available Sperm Separation Procedures include: • Sperm migration • Density gradient centrifugation • Column adherence method. Sperm Migration Method Naturally motile sperm will effortlessly and efficiently move from one medium to another, whereas non-motile or morphologically compromised sperm will have greater difficulty. The Sperm Migration Method inspires procedures which take advantage of healthy sperm’s natural tendency for migration. Within a controlled environment, viable sperm can differentiate themselves from other non-viable sperm, and superfluous or deleterious elements within the sample. These methods are regularly used to obtain sufficient populations of viable sperm for IUI and IVF, and include the “Swim-Up” (with and without centrifugation), and the “Swim-Down” procedures, respectively. Swim-Up Procedure In the so-called Sperm Swim-Up Procedure, the natural ability of motile sperm to migrate against gravity is used to artificially select a motile sperm population. A light, low viscosity media is layered over semen (without initial centrifugation), or over a sperm pellet (obtained by centrifugation): Sperm will then naturally migrate up against gravity, leaving all other non-dynamic factors behind in the sample. Results can be further improved if sperm are exposed to antigravitational rcf strength. Centrifuges capable of generating such an antigravitational field are currently available. Sperm Swim-Up Procedure (Without Centrifugation) Sperm recovery following the standard Swim-Up procedure can be rather poor, since a centrifuged, compact sperm pellet has a relatively low surface area in relation to the overall sperm concentration. Due to the relatively long incubation period during Swim-Up, equilibrium will eventually be reached between the sperm migrating out and the ones migrating back into the pellet. More precisely, equilibrium is established between the downward gravitational force, sperm buoyancy, and the relative viscosity gradient differential between pellet and Swim-Up media. During Swim-Up, sperm will migrate more readily into a higher viscosity gradient than into a low gradient. Whether such migration is caused by a low sperm return to the pellet, or some kind of natural selective preference, is not yet known. For example, if 0.5 ml french straws containing media with different concentrations of bovine serum albumin are placed vertically into semen, a higher concentration of sperm with increased albumin concentration will be found. Similarly, if sperm migrating into a

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Swim-Up media are either continuously removed or trapped from re-entering, then higher numbers of sperm result. Acouple of examples of commercially available Swim- Up (sans centrifugation) systems include: • The Sperm Select® System (Select Medical Systems, Williston VT05495) utilizes the former concept, while • ZSC™II (ZDL Inc., Lexington, KY40523) utilizes the latter. These products come as complete kits, which incorporate all necessary items for insemination. Ideally, such a Kit would eliminate the need for extensive laboratory facilities and personnel, quality control and assurance. Such a Kit may be especially attractive to practices performing a limited number of insemination procedures, or possessing insufficient preparation space. Swim-Down Procedure In the Swim-Down Procedure, the natural ability of motile sperm to gravitationally migrate is used to select a motile sperm population. This concept is implemented by layering semen or washed sperm over a heavier, more viscous media. Sperm then migrate into and towards the gravitational force (quite literally “swimming down”), thereby separating motile sperm from the remaining sample, left above. Density Gradient Centrifugation Method The Density Gradient Centrifugation Method is a procedure utilizing either a continuous or discontinuous density gradient, in conjunction with centrifugation. Such a procedure selectively separates sperm based on their size, motility and density differential. For the Density Gradient Centrifugation Method to be effective, the density gradient should have no or minimal osmotic effect when added to media. The gradient should also have very low viscosity, thereby having a minimal effect on sperm sedimentation rate. The gradient should also have high specific gravity to facilitate sperm sedimentation, and be inert so as to not interfere with sperm quality. Common density gradient substances such as sucrose and ficoll cannot be used, since they are either hypertonic or highly viscous. (They can, however, be used to separate sperm from seminal plasma for biochemical analysis). Examples of two types of acceptable Density Gradient materials include colloidal Silica Based Density Gradient, and lodixanol Based Density Gradients. The former has increased in popularity over the last decade due to effectiveness and reproducibility in recovering most motile sperm. Toxicity and teratogenicity levels of the silica materials have yet to be substantiated. Colloidal Silica Based Density Gradient When sperm counts or motility are suboptimal, Multiphase Gradients can be utilized. Progressive sperm motility is generally not observed in 80 percent colloidal silica preparation, probably due to its relative viscosity, or to silica particle adherence to the

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sperm membrane. Although sperm washing prior to use renders sperm progressively motile, evidence suggests that all colloidal silica is not thoroughly removed from the sample. Silanized colloidal silica does not appear to interfere with progressive sperm motility, so sperm washing is not necessary. Whether or not the sperm membrane retains a residual coating of silanized colloidal silica is unknown. Since many women complain of irritation following IUI supplied with sperm centrifuged through 80 percent silanized colloidal silica, routine sperm washing prior to IUI is, however, vital. Available density gradient products include: • Pure sperm (Nidacon International AB, Goteborg, Sweden) • Isolate (Irvine Scientific, Santa Ana, CA 92705) • Pure ception (Sage BioPharma Inc,, Hartford, CT 06150) • Sperm grad (IVF Science Scandinavia, Vero Beach, FL 32965) • Enhance S Plus (Conception Technologies, San Diego, CA 92121). Iodixanol-Based Density Gradient In contrast to colloidal silica density gradients, where sperm with normal forms and motility are sedimented into pellet form, Iodixanol-based density gradients select sperm according to the density differential between various ejaculate components. In Iodixanol-based density gradients, motile sperm with normal morphology band together at the interface between two density gradients, and consequently do not collect into dense pellet form. Iodixanol was principally designed for the in vitro isolation of cellular organelles. The compound has been approved by the US. Federal Drug Administration for in vitro fertilization and for the isolation of a wide range of other cell types. Iodixanol is nontoxic, inert, and an iodinated acidic hydrocarbon. A relatively large molecule, Iodixanol’s chemical formula is: {S, S′-[(2-hydroxyl-1-3-propanediyl)-bis(Acetylamino)]-bis-{N1 N’bis (2,3-dihydroxypropyl-2,4,6-triiodo-1,3-benzene-carboxamide]}. Iodixanol is currently available under the following trade names: • OptiPrep (Nycomed Pharma AS, Oslo, Norway) • Accudenz (Accurate Chemical & Scientific Corporation, Westbury, NY 11590). The Density Gradient Centrifugation Method as a whole is not appreciably proficient in the separation and selection of extremely low sperm count or highly viscous semen samples. Samples containing a large percentage of cellular debris also take poorly to the Density Gradient Method. Column Adherence Procedure In the early 70s, Dr Graham at the University of Minnesota experimented with various sperm filtration procedures. He eventually demonstrated that a column made of hydrated Sephadex G15, an inert polysaccharide, in conjunction with a glass wool barrier support, effectively separated motile and functionally intact bovine sperm within an ejaculate. He

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also reported that nonfunctional sperm adhered to the column, effectively inhibiting their migration, thereby filtering motile sperm capable of passing through the filter. Although Dr Graham attributed the sperm filtration characteristic to the Sephadex column, replacing the glass wool barrier support with either cotton wool or any other synthetic fibers always yielded poor motile sperm recovery. Also, such modified filter columns resulted in a relatively high number of non-motile and non-viable sperm within the sample. Based on these findings, two types of filtration columns were developed for functional sperm separation within an ejaculate: These are based on the fundamental concept that non-viable sperm are “sticky,” and therefore more likely to adhere to substances within the column than otherwise motile and functionally intact spermatozoa. Procedurally, semen samples are first washed using media and subsequently placed over an adherence column, where downward filtration, fuelled naturally by the gravitational force, takes place. Two types of adherence columns currently exist for sperm selection: The Sephadex Column Filtration Procedure and the Glass Column Filtration Procedure. Sephadex Column Filtration Procedure Note that Sephadex column effectiveness is compromised if polysaccharide hydration is insufficient. Also, sperm separation efficacy will be further compromised if any substance other than glass wool is used as a supporting barrier. The Sephadex column is commercially available as SpermPrep ™ I and II (ZDL Inc., Lexington, KY 40523) Glass Wool Column Filter Procedure (“GWCF”) Glass wool column filtration was initially developed as an attempt to decrease the viscosity of semen, and to increase sperm motility percentage. Apparently, tightly packed soda lime glass wool filtration yielded a particularly high recovery of motile spermatozoa, but also resulted in an increased number of sperm with short or broken tails. Subsequent analysis revealed that filtration efficacy was dependent on the type of glass wool used: Glass wool micro fibre, Code #112, appeared optimal. Filter column density also influences procedural outcomes. For example, if glass wool micro-fibers were too tightly packed, sperm would be unable to traverse the column, regardless of vigor. Conversely, if fibers were too loosely compressed, non-viable spermatozoa could penetrate the column. Glass wool filters are commercially available, including: Cook Sperm Filter (Cook Ob/Gyn, Spencer, IN 47460). CONCLUSION Periovulatory mucus has the natural ability to selectively filter a sperm population based on complex sperm functions, including motility, shape, size and many other composite and even unknown aspects.

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Sperm Migration Methods all rely on progressive sperm motility, whereas Densiiy Gradient Centrifugation Methods also rely on sperm shape, size, and density. The Sperm Adherence Method, on the other hand, mainly selects and removes sperm with broken or non-functional sperm membranes. Essentially, no laboratory technique developed thus far truly and comprehensively mimics the natural ability of the periovulatory mucus for sperm population selection. The latter two methods are nonetheless able to concentrate most viable sperm into volumes not only sufficient for IUI, but in concentrated proportions actually greater than those attainable by the periovulatory mucus itself. In summary, although clinical sperm processing procedures attempt to mimic the innate capacities of the female reproductive system, in reality these laboratory techniques are able, at their best, to select a more suitable sperm population based solely on particular sperm characteristics. Although these methods have been demonstrated to effectively recover viable sperm populations, none except the Sperm Adherence Method has concomitantly enhanced overall fertilization result or pregnancy outcome. The Sephadex column has proven very effective, but only when a glass wool barrier support is utilized and properly placed to prevent Sephadex particles from filtering out. ACKNOWLEDGMENT Special thanks to Michael Spitz for excellent editorial assistance. BIBLIOGRAPHY 1. Acosta A, Kruger T. Human Spermatozoa in Assisted Reproduction (2nd edn). New York: Parthenon Publishing Group, 1996. 2. Speroff L, Glass RH, Kase NG. Clinical Gynecologic Endocrinology and Infertility (6th edn). Philadelphia, Pennsylvania: Lippincott Company, 1999. 3. Trounson A, Gardner D. Handbook of In Vitro Fertilization (2nd edn). New York: CRC Press, 2000.

CHAPTER 31 Sperm Separation Techniques: Comparison and Evaluation of Gradient Products KE Tucker, CAM Jansen INTRODUCTION The human ejaculate is comprised of a mixture of seminal plasma, mature and immature spermatozoa, non-reproductive cells, various micro-organisms and non-specific debris. In preparation for intrauterine insemination (IUI) or in υitro fertilization (IVF), the motile, and hopefully, the most fertilizable population of sperm must be separated from the surrounding milieu. Many studies have been performed comparing direct semen processing procedures (i.e. simple wash, swim-up, etc.) with gradient separation techniques. Sperm separation using, a polyvinylpyrrolidone (PVP)-coated, silica-based density gradient (Percoll®), has been shown by numerous investigators to be an effective and relatively simple way of producing a viable, highly motile, morphologically normal and fertilizable population of sperm for use in both IUI and IVF.1 The demands on sperm separation techniques have increased with our expanding knowledge of sperm physiology and on their genetic contribution to the embryo. Because of this, there has been rising concern over the safety of any sperm separation procedure with respect to not only the viability of the sperm, but to the long-term effects on any resulting pregnancy. Although extremely effective in sperm separation and apparently safe for clinical use, Percoll® (Pharmacia; Sigma Pharmaceutical) has been made unavailable for therapeutic use in human infertility. It has, therefore, become necessary to evaluate and select an alternative product or procedure that will compare favorably in light of all the studies that have advocated the use of Percoll for sperm separation. To address the need for Percollsubstitutes, a new line of density gradient products have been manufactured, specifically, the silane-coated, colloidal silica particle-based density gradients (i.e. PureSperm®, Isolate®, Enhance S+®). This paper will attempt to compare the efficacy of these new products with each other and with what has become the first choice for sperm separation, Percoll. Separation Techniques: General There are several sperm separation methods available. These include simple washing, sperm migration into culture medium (swim-up), Sephadex and glass wool columns and density gradient centrifugation. All of these techniques are capable of effectively separating sperm from the seminal plasma, but to varying degrees. Recovery rates, motility, morphology and degree of DNA damage vary greatly between procedures.2–6

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Separation Techniques: Density Gradient Separation (Percoll) vs Swim-up The most commonly used, non-density gradient technique involves separation of motile sperm from seminal plasma based solely on the intrinsic activity of the sperm population in a particular ejaculate. The swim-up (SU) separation procedure has been performed using either a washed or unwashed (direct) semen sample. It is a simple method, but in the case of a sample with poor motility, can be very time consuming. With the density gradient centrifugation method for sperm separation, liquefied seminal plasma is gently pipetted over a single (90%) or double (40/80% or 45/ 90%) layered-column of the gradient depending on the quality of the original semen sample. Subsequent separation relies not only on the percent of actively swimming sperm in the ejaculate, but also on the added force of gentle centrifugation to isolate this motile population from other cell types. A single Percoll layer was reported to be the most effective separation method for frozen-thawed semen.7 The double column was found to be more efficient in separating motile sperm than the single for normal samples,8 but, on average, good results can be achieved with both techniques.9 Sperm membrane binding characteristics were also improved following Percoll separation.10 Various rates of recovery have been reported for both SU and Percoll. Some investigators have found no difference in the number of motile sperm following SU or Percoll separation.11–12 Ng and co-workers13 reported that although Percoll and miniPercoll columns resulted in greater yields of motile sperm from abnormal samples, the SU technique appeared to enhance the velocity and number of morphologically normal sperm from normozoospermic samples. Others have demonstrated that the use of the twolayer Percoll gradient resulted in a higher overall rate of recovery and a significantly better percentage of motile sperm than did the SU method.14 Morphology results (based on WHO criteria),40 however, were similar. Differences in other sperm parameters were found between SU and Percoll. Discontinuous (two-layer) Percoll gradients were shown to select sperm with better motion characteristics, increased hyperactivation and improved longevity compared with direct SU.15 In addition, discontinuous Percoll gradients resulted in a more morphologically normal final population of sperm than SU for normal semen samples overall16–19 and within individual semen samples.9 More recently, Ding and co-workers20 reported that two- and three-layered Percoll gradients were both equally more effective at improving sperm recoverery rate, motility and motion characteristics compared to SU or an albumin column. It was also reported that density gradient sperm separation was better than SU at increasing the average acrosome index (higher number sperm undergoing acrosome reaction) and the number of vital sperm (as demonstrated by hypoosmotic swelling test-HOS).21 Compared with the direct method, SU from a washed pellet involves extra centrifugation steps due to the first step of washing the semen. Because of the potential for generating high levels of reactive oxygen species (ROS), this technique was criticized because of the possible damage that may occur to sperm due to centrifuging the sample before separating motile sperm from other cells such as immature sperm or leukocytes. In lieu of this, Mortimer22 vehemently contended that performing SU from a washed pellet be abandoned and replaced by either direct SU or the Percoll density gradient. Twigg and co-workers23 also reported that the SU technique caused an increase in ROS and was

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associated with an increase in the level of DNA damage. In addition, both SU methods have been shown to cause DNA damage,24 even though one of these still involves extra centrifugation steps. They concluded that both forms of the technique could be damaging to sperm. Conversely, density gradient separation was shown to improve both the percentage of morphologically normal forms and sperm nuclear integrity.25–26 Sperm Separation Techniques: Percoll, Glass Wool and Sephadex Columns Few investigators have found other methods superior to Percoll for physically separating sperm. Johnson and co-workers4 reported that glass wool filtration yielded more functionally intact sperm than did a mini-Percoll column from oligozoospermic samples. Conversely, the use of Percoll resulted in a more morphologically normal sample of sperm than did sephadex or glass wool (SperPrep®) columns.27 Joshi and others also reported that for matched asthenozoospermic samples, those prepared with a mini-Percoll column were of significantly higher quality in terms of computer-assisted sperm analysis (CASA) of various motion parameters, motility, morphology, HOS and nuclear stability. When tested for the presence of endotoxin, it was reported that only Percoll did not have positive formation gel following the LAL test.29 These investigators further demonstrated that sperm remained motile in the presence of Percoll and that washes from SpermPrep columns were toxic to mouse embryo development. Sperm Separation Techniques: Optipref, Ixaprep and Accudenz Even before of the withdrawal of Percoll for clinical use, there was strong effort to a find a suitable safe, effective and economically viable alternative. One density gradient proposed for use with human sperm was Accudenz® [5-(N-2,3dihydroxypropylacetamido)-2,4,6-tri-iodo-N,N’-bis(2,3-dihydroxypropyl) isophthalamide], which has been used extensively for preparing poultry sperm for insemination.30–31 When compared with Percoll, Accudenz was found to yield a higher concentration of motile sperm, with better motion characteristics and acrosome integrity from normospermic samples.32 The authors recommended this method for IUI. It is not known whether this product would be suitable for use in IVF. Another available product, Iodixanol (Optiprep®; Nycomed), is a non-ionic, polysucrose-based gradient and initial studies have demonstrated that a discontinuous gradient of 25/40 percent Optiprep yielded a sperm recovery of 32 percent with no evidence of toxicity as was found with the glass wool and sephadex columns.33 Smith and co-workers34 found no difference in sperm yield and survival and in the percent of motile or morphologically normal forms in samples processed with Optiprep or Percoll. Conversely, Anderson and Grinsted35 reported better overall motile sperm recovery with Optiprep compared to Percoll, but found no difference in the actual percentage of motile forms. The next version of Optiprep, Ixaprep® (Medicult), was compared with Percoll and one of the new-generation silica-based density gradient, Isolate. Isolate and Ixaprep had a higher recovery of motile sperm compared with Percoll; morphology and motion

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characteristics were similar.36 These authors recommended Optiprep and Ixaprep as suitable Percoll replacements. Sperm Separation Techniques: Silane-Coated Silica-Based Gradients Three new silica-based separation gradients have been proposed as Percoll replacements. These silane-coated products have been promoted as being safer than the PVP-coated product (Percoll) and studies have been performed to ascertain if these new gradients are superior to Percoll. Isolate® (Irvine Scientific) demonstrated higher recovery of motile sperm when compared to Percoll (Pharmacia)36 and both Isolate and Percoll were superior to SpermFertil® (EmbryoTech) in terms of sperm count, curvilinear velocity and percent of normal forms.14 Isolate has been approved for clinical use by the FDA, but is more expensive when compared to Percoll. Isolate is recommended as an excellent substitute for Percoll. Puresperm® (Nicadon International) is also composed of silane-coated silica particles and has been shown to be very effective with large volume loads in enriching the final sample with highly motile and morphologically normal sperm.37 Other investigators found higher sperm velocities and degrees of hyperactivation after separation with PureSperm compared to Percoll.5,38 Nicholson and co-workers39 also reported processing semen with PureSperm significantly reduced the level of bacterial contamination in the final preparation, even when non-aseptic techniques were used. All investigators agree that PureSperm is a superior alternative to Percoll. PureSperm is also FDA approved and high in cost. Enhance S+® (Conception Technologies) is also very effective in producing a more highly motile and morphologically normal population of sperm compared to Percoll9 (unpublished data). This silane-coated silica product is FDA approved, more costly when compared to Percoll preparations, but the cost can be reduced when ordered in large quantities. Enhance S+ is also an excellent Percoll alternative. SPERM SEPARATIONTECHNIQUES: SUMMARY AND CONCLUSIONS Several studies (published and unpublished) have shown density gradient centrifugation is the more effective method for separating motile, morphologically and genetically normal sperm. The “new generation” of density gradients has been shown to be a superior alternative to Percoll. Isolate, PureSperm and Enhance S+ are all bioassay and endotoxin-tested and easy to wash out and except for the cost, may produce a final sample of sperm for IUI or IVF that is at least as good as Percoll. More studies need to be performed to establish any possible long-term effects of these new products.

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REFERENCES 1. Moohan JM, Lindsay KS. Spermatozoa selected by a discontinuous percoll density gradient exhibit bettermotion characteristics, more hyperactivation, and longer survival than direct swimup. Fertil Steril 1995; 64:160–66. 2. Byrd W, Drobnis EZ, Kutteh WH, Marshburn P, Carr BR. Intrauterine insemination with frozen donor sperm: a prospective randomized trial comparing three different sperm preparation techniques. Fertil Steril 1994; 62:850–56. 3. Claasens OE, Kaskar K, Coetzee K, Lombard CJ, Franken DR, Kruger TF. Comparison of motility characteristics and normal sperm morphology of human semen samples separated by Percoll density gradient centrifugation. Arch Androl 1996; 36:127–32. 4. Johnson DE, Confino E, Jeyendran RS. Glass wool column filtration versus mini-Percoll gradient for processing poor quality semen samples. Fertil Steril 1996; 66:459–62. 5. Centola GM, Herko R, Andolina E, Weisensel S. Comparison of sperm separation methods: effect on recovery motility parameters, and hyperactivation. Fertil Steril 1998; 70:1173–75. 6. Adiga SK, Kumar P. Influence of swim-up method on the recovery of spermatozoa from different types of semen samples. J Assist Reprod Genet 2001; 18:160–4. 7. Bongso A, Jarina AK, Ho J, Ng SC, Ratnam SS. Comparative evaluation of three sperm-washing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples. Arch Androl 1993; 31:223–30. 8. Sharma RK, Seifarth K, Agrawal A. Comparison of single- and two-layer Percoll separation for selection of motile spermatozoa. Int J Fertil Womens Med 1997; 42:412–17. 9. Tucker KE, Hurst BS, Cymecki C, Mendelsberg B, Guadagnoli S, Awoniyi CA et al. Discontinuous percoll optimizes sperm populations used for assisted reproductive technologies (ART). J Androl 1996; 192 (Abstract). 10. De Maistre E, Bene MC, Foliguet B, Touati F, Faure GC. Centrifugation on Percoll gradient enhances fluorescent lectin binding on human sperm: a flow cytometric analysis. Arch Andol 1996; 37:179–87. 11. Mushayandebvu TI, Santoro NF, Lipetz KJ, Colon JM. Comparison of Percoll and swim-up techniques for sperm recovery in patients with male factor infertility. In Monduzzi (Ed): IX World Congress on In Vitro Fertilization and Alternative Assisted Reproduction. Italy: Bologna 1995; 577–80. 12. Smith S, Hosid S, Scott L. Use of postseparation sperm parameters to determine the method of choice for sperm preparation for assisted reproductive technology. Fertil Steril 1995; 63:591– 97. 13. Ng FL, Liu DY, Baker HW. Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod 1992; 7:261–66. 14. Sharma RK, Seifarth K, Garlak D, Agarwal A. Comparison of three sperm preparation media. Int J Fertil Womens Med 1999; 44:163–67. 15. Saad A, Guerin JF. Movement characteristics of human spermatozoa collected from different layers of a discontinuous Percoll gradient. Andrologia 1992; 24:149–53. 16. Van der Zwalmen P, Bertin-Segal G, Geerts L, Debauche C, Schoysman R. Sperm morphology and IVF pregnancy rate: comparison between percoll gradient centrifugation and swimup procedures. Hum Reprod 1991; 6:581–88. 17. Perez SM, Chan PJ, Patton WC, King A. Silane-coated silica particle colloid processing of human sperm. J Assist Reprod Genet 1997; 14:388–93. 18. Gravance CG, Champion ZJ, Sax-Gravance SK, Casey PJ. Percentage of normal sperm heads is significantly increased by Percoll separation of semen. Int J Androl 1998; 21:116–19. 19. Prakash P, Leykin L, Chen Z, Toth T, Sayegh R, Schiff I et al. Preparation by differential gradient centrifugation is better than swim-up in selecting sperm with normal morphology (strict criteria). Fertil Steril 1998; 69:722–26.

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20. Ding DC, Liou SM, Huang LY, Liu JY, Wu GJ. Effects of four nethods of sperm preparation on motion characteristics and nitric oxide concentration in laboratory-prepared oligospermia. Zhonghua Yi Xue Za Zhi (Taipei) 2000; 63:822–7. 21. Erel CT, Senturk, LM, Irez T, Ercan L, Elter K, Colgar U, Ertungealp E. Sperm-preparation techrdques for men with normal and abnormal semen analysis. Acomparison. J Reprod Med 2000; 45:917–22. 22. Mortimer D. Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum Reprod 1991; 6:173–76. 23. Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998; 13:864– 71. 24. Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF. Sperm swim-up techniques and DNA fragmentation. Hum Reprod 2001; 16:1950–53. 25. Tomlinson MJ, Moffat O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001; 16:2160–65. 26. Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, et al. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod 2000; 15:1112–16. 27. Lopez O, Mata A, Antich M, Bassas L. Sperm selection by PD-10 sephadex columns: comparison with SpermPrep filtration and Percoll centrifugation. Hum Reprod 1993; 8:732–36. 28. Joshi NJ, Raj JP, Sundaram GS. Evaluation of quality of spermatozoa prepared by SpermPrep method as compared to those prepared my MiniPercoll. Andrologia 1998; 30:79–83. 29. Scott L, Smith S. Mouse in vitro fertilization, embryo development and viablility, and human sperm motility in substances used for human sperm preparation for assisted reproduction. Fertil Steril 1997; 67:372–81. 30. McLean DJ, Feltman AJ, Froman DP. Transfer of sperm into a chemically defined environment by centrifugation through 12 percent (wt/vol) Accudenz. Poult Sci 1998; 77:163–68. 31. Parkhurst AM, Korn N, Thurston RJ. The effects of methylzanthines on the mobility of stored turkey sperm. Poult Sci 2000; 79:1803–9. 32. Sbracia M, Sayme N, Grasso J, Vigue L, Huszar G. Sperm function and choice of preparation media: comparison of Percoll and Accudenz discontinuous density gradients. J Androl 1996; 17:61–67. 33. Harrison K. Iodixanol as a density gradient medium for the isolation of motile spermatozoa. J Assist Reprod Genet 1997; 14:385–87. 34. Smith TT, Byers M, Kaftani D, Whitford W. The use of iodixanol as a density gradient material for separating human sperm from semen. Arch Androl 1997; 38:223–30. 35. Anderson CY, Grinsted J. A new method for the purification of human motile spermatozoa applying density-gradient centrifugation: polysucrose media compared to Percoll media. J Assist Reprod Genet 1997; 14:624–8. 36. Makkar G, Ng HY, Yeung SB, Ho PC. Comparison of two colloidal silica-based sperm separation media with a non-silica-based medium. Fertil Steril 1999; 72:796–802. 37. Chen MJ, Bongso A. Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum Reprod 1999; 14:759–64. 38. Soderlund B, Lundin K. The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization. Hum Reprod 2000; 15:857–60. 39. Nicholson CM, Abramsson L, Holm SE, Bjurulf E. Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces. Hum Reprod 2000; 15:662–66. 40. World Health Organization (WHO) laboratory manual for examination of human semen and cervical mucus interaction (4th edn), Cambridge: University Press, 1999.

CHAPTER 32 Prediction of ART Outcome in Male Factor Infertility Patients by a New Semen Quality Score Ashok Agarwal, Rakesh K Sharma INTRODUCTION Semen analysis is arguably the most important clinical laboratory test available in the evaluation of male infertility. Measures of semen quality are used as surrogate measures of male fecundity in clinical andrology, reproductive toxicology, epidemiology, and risk assessment. However, the implications of even moderate alterations in semen quality are poorly understood, and only limited data are available for relating these measures to the likelihood of achieving pregnancy. In general, the number and/or concentration of motile spermatozoa is a significant factor affecting success. In particular, success rates are poor when low numbers of motile spermatozoa (approximately less than 1×106/mL) are used for insemination.1 Some studies have suggested that computer-assisted semen analyzer (CASA) estimates of concentration and movement characteristics of progressively motile spermatozoa are significantly related to the fertilization rate in-υitro and time-toconception.2–4 Sperm motion characteristics, specifically amplitude of lateral head displacement (ALH) and linearity, have been related to sperm fertilizing capacity.5–7 Despite the current high profile of morphology examination, few IUI studies provide details on sperm morphology. Burr and colleagues8 found that pregnancy success rates were poor (4.3 percent per cycle) when sperm normal morphology in raw semen was less than 10 percent.9 In contrast, the IUI outcome was not significantly influenced when morphology was assessed on raw semen samples according to Kruger’s strict criteria10 (Kruger et al, 1986). Whether individual semen parameters are effective in predicting pregnancy outcome in couples with male-factor infertility undergoing IUI is a controversial subject.11–13 After the introduction of CASA, the number of semen parameters examined increased to the extent that each semen evaluation quantifies more than 10 semen characteristics. Couples affected by long-term male infertility have an estimated spontaneous pregnancy rate of 2 percent per menstrual cycle.14 To help increase the chances of pregnancy, assisted reproduction techniques such as intrauterine insemination (IUI) and in-υitro fertilization (IVF) are used. In-υitro fertilization has a comparable success rate but it is more invasive and expensive than IUI. The success rate of IUI can be increased to near that of IVF by combining it with ovarian stimulation.15–16 However, that increases the cost of the procedure from about $500 to $1,800 per cycle.17 Ovarian stimulation also can lead to a wide range of health risks, including ovarian hyperstimulation sydrome and multiple pregnancy. Clearly, couples with male-factor infertility seeking assistance should be counseled to undergo IVF and not IUI only when there is a clear indication for

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it and when no safer or less expensive alternative exists. Therefore, it is important to be able to predict the chances for successful IUI-induced pregnancy in couples who are seeking counseling. Unfortunately, we have no way of doing so that provides consistently reliable and accurate results. DEVELOPING AN OVERALL SEMEN SCORE Although semen parameters are unique measures of sperm quality, they are not independent of one another in the sense that patients with low motility tend to have low concentration and vice versa. Therefore, semen parameters are positively correlated with each other. In biological systems, when there are several correlated variables, principal component analysis can be used to reduce the variables to 1 or 2 variables, which are the linear functions of the original variables.18 This appears to be applicable to semen parameters, where several variables are recorded that have a generally strong correlation. Since many of these parameters are interrelated, we believed that an overall semen score could be developed using an appropriate statistical model. Developing an overall semen score by applying principal component analysis to the original semen variables obtained by CASA and also by the conventional or manual semen analysis is important. By employing a statistical model, overall semen scores that account for most of variability observed among the battery of interrelated semen variables can be calculated. These scores can be used to predict pregnancy in couples undergoing IUI for male-factor infertility. Secondly, determining if these 2 scores can be combined to produce an IUI-semen pregnancy score (IUI-SPS) using prewash measures is important. This can serve as a quick and reliable method for directing couples with male-factor infertility to either IUI (high IUI-SPS score) or IVF (low IUI-SPS score). Patient Selection in the Evaluation of Semen Scores In a retrospective study, we examined nine semen characteristics from 452 men (25 healthy donors, 250 men undergoing semen analysis as part of a fertility evaluation and 177 men with various clinical diagnoses of infertility). Of the 177 patients presenting with different male infertility clinical diagnoses the breakdown was prostatitis with infection (n=46), varicocele (n=77), varicocele with infection (n=11), and vasectomy reversal (n=43). In a separate set, the prewash and postwash semen analyses was examined in those couples where the male partner (n=93) was diagnosed with male-f actor infertility. Couples were excluded from participation if the woman had cycle disorders, untreated endometriosis (grade 1–4)19 or bilateral occluded tubes or if a semen sample yielded less than 1 million progressively motile spermatozoa. Investigation of infertility included basal body-temperature chart, late luteal-phase endometrial biopsy, postcoital test, hysterosalpingogram, diagnostic laparoscopy, and at least 2 semen analyses.

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Assessment of Semen Variables Sperm samples were analyzed using a computerized system (Cell-Trak, Model VP 110, Santa Rosa, CA). For each measurement, a 5-µL sample aliquot from either a donor or patient was loaded on a MicroCell slide (Conception Technologies, La Jolla, CA). The sperm motion kinetics measured by CASA were: sperm concentration (X 106/mL), percent motility, curvilinear velocity (VCL, µm/s), time-average velocity of a sperm head along its actual curvilinear path, as perceived in 2 dimensions in the microscope, straightline velocity (VSL; µm/s), time-average velocity of a sperm head along the straight line between its first detected position and its last, average path velocity (VAP; µm/s), and time-average velocity of a sperm head along its average path. This path is computed by smoothening the actual path according to algorithms in the CASA instrument. These algorithms vary between instruments. We also measured linearity (LIN; %), the linearity of a curvilinear path (VSL/VCL), amplitude of lateral head displacements (ALH; µm), and magnitude of lateral displacement of a sperm head about its average path, which can be expressed as a maximum or an average of such displacement. In addition to the computerized results, manual results were calculated for sperm concentration and motility. Measurement of Sperm Morphology For morphological evaluation, seminal smears were stained with Giemsa stain (DiffQuik, Baxter Scientific Products, McGaw Park, IL). Sperm morphology was assessed with World Health Organization (WHO) guidelines and Kruger’s strict criteria.10,20 STATISTICAL MODEL FOR CALCULATING THE SEMEN SCORES Nine semen parameters were included to compute semen quality scores: concentration, motility, VCL, VSL, VAP, ALH, linearity, and morphology by WHO guidelines and by Kruger’s strict criteria. Log transformation (base—10 logarithms after adding a constant of ‘1’ to each semen characteristic) was performed to reduce the effect of outliers and to scale the variables. Principal component analysis was applied to the covariance matrix of the 9 log-transformed semen parameters. This produced 9 new components each with “eigenvectors” (which were the weights assigned to the original variables). Only those components that accounted for at least 10 percent of the overall variability of the 9 semen parameters were used. The resultant principal component scores were converted to semen scores with a mean of 100 and a standard deviation (SD) of 10 based on the semen scores from the donor group. This involved both addition and multiplication of the values by a number of constants to derive the standardized scale. A mean of 100 was chosen simply because it provided a level of recognition. For example, a score of 100 or greater indicates a sperm quality better than the average male and vice versa for scores less than 100. This is similar to the intelligence quotient (IQ) scores, which are also standardized to a mean of 100. A SD of 10 was chosen because it provided an easily computable difference from 100 or any other value of interest. For example, in general, 95 percent of a population of

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normal individuals falls into a range of ±2 SD from the mean; therefore, the semen score would generally be expected to fall within a range of 80 to 120 for a group of donors. The steps involved in the calculation of the scores are illustrated in Figure 32.1. The same procedure was undertaken with the 4 non-CASA parameters: concentration, motility, and the two morphology measures. Pearson product-moment correlation between the 9parameter and 4-parameter semen scores were calculated. Calculation of the IUI-Semen Pregnancy Score The semen scores were combined to provide an IUI-Semen Pregnancy Score (IUI-SPS) using the regression coefficient estimates of both the pre-wash SQ and RQ score as weights. The regression coefficient of the RQ score was approximately twice as much as the SQ score, so the combined IUI-SPS score was calculated as: IUI-SPS=RQ Score+(SQ score/2) The sample size was sufficient to detect, with 90 percent power, whether a 10-point increase in IUI-SPS resulted in an increase in the odds of pregnancy of 1.5 times versus the null hypothesis of an odds ratio of 1.0 for such a IUI-SPS change. We attempted to predict IUI-induced pregnancy from male and female characteristics using multivariate logistic regression and generalized estimating equations (GEE). These equations used compound symmetry correlation structure to account for the fact that couples may attempt more than 1 IUI cycle. We also used multivariate GEE logistic regression analysis to predict IUI-induced pregnancy from the SQ and RQ scores based on 9 semen parameters taken from the semen analyses. Female factor infertility characteristics (ovarian stimulation, non-stimulation, and infertility duration) were also evaluated. If significant in univariate analyses, they were included in the logistic regression analyses. To summarize the overall sensitivity and specificity of the scores in predicting pregnancy, we calculated receiver operating characteristic (ROC) curves. The area under the curve (AUC) can range from 50 percent (no predictive value) to 100 percent (perfect prediction accuracy). Summary statistics are reported as median and inter-quartile range (IQR). Significance was assessed with p<0.05. Two-tailed tests were used, and all computations were performed with SAS version 8.1 (SAS Institute Inc., Cary, NC).

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Fig. 32.1: Steps involved in computing the SQ (Semen Quality) and RQ (Relative Quality) score Semen Quality (SQ) and Relative Quality (RQ) as Measures of Semen Score The correlation among the semen samples were generally positive (Table 32.1). Among the kinetic variables, a strong correlation was seen between VAP and VSL (r=0.98), between VAP and VCL (r=0.96), and between VSL and VCL (r=0.91). The weakest correlation was observed between linearity and Kruger’s morphology (r=0.01). Also, a weak negative correlation was observed between linearity and both VCL (r=−0.07) and ALH (r=−0.03). Given that most of the correlations were positive, it was anticipated that most of the variability would be explained by a weighted sum of the variables that would be related to the overall semen quality.

Table 32.1: Summary statistics and correlation among nine semen characteristics Variable Original υariable Mean ± Conc. (X 106/ml) Motility (%) VCL (mm/s) VSL (mm/s)

Log Correlation among log-transformed variable (r) transformed Mean ± Conc. Moti VCL VSL VAP Line ALH WHO Strict lity (µm/s) (µm/s) (µm/s) arity (µ/s) crit (%) (µm/s) eria

53.93±57.08 1.48±0.56

1.00

0.55 0.41

0.47

0.46

0.07

0.32

0.52 0.48

58.01±22.72 1.72±0.23

1.00 0.52

0.63

0.59

0.35

0.26

0.43 0.45

41.01±13.38 1.60±0.15

1.00

22.98±9.35 1.34±0.18

0.91

0.32 1.00

0.32

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VAP 27.74±10.17 1.43±0.17 0.96 0.98 1.00 0.32 (mm/s) Linearity 52.70±7.20 1.73±0.06 −.07 0.30 0.14 1.00 −.03 0.03 0.01 (%) ALH 4.10±1.28 0.69±0.16 1.00 (mm) WHO (%) 31.84±13.74 1.47±0.24 0.45 0.46 0.46 0.31 1.00 Strict 9.14±5.12 0.93±0.29 0.45 0.47 0.47 0.32 0.85 1.00 criteria (%) log10 transformation after adding ‘1’ to the original value; Conc.=concentration (X 106/ml); VCL=curvilinear velocity; VSL=straightline velocity; VAP=average path velocity; ALH=amplitude of lateral head displacement; WHO=World Health Organization

Principal component analysis indicated that 2 semen scores or components accounted for 80.3 percent of the variability observed among the 9 semen parameters. The first principal component was termed the “SQ” score to represent “overall semen quality.” This was a weighted sum of all semen characteristics and accounted for 64.8 percent or almost two-thirds of the overall variability observed among the 9 variables. The SQ score weighs each semen parameter based on the values listed in Table 32.2. For example, the variable with the greatest weight was sperm concentration (+0.81), and all the other scores had positive weights. Therefore, the SQ score sums up all the semen parameters with varying weights given to each score. The second component was termed the “RQ” score to represent “relative quality.” It assigns a negative weight for concentration and positive weights for all other variables. Therefore, an individual with a relatively low concentration but above average values for the other parameters would have a high RQ score compared with what would be expected based on other parameters. In contrast, individuals with low scores on this scale will have poor sperm motility poor morphology, or low scores on other parameters relative to their concentration. Therefore, the 2 new scores together accounted for more than 80 percent of the variability observed among the 9 semen parameters. Because these 2 scores are produced by principal component analysis, SQ and RQ are uncorrelated. Subsequently, the SQ and RQ scores were applied to samples from the 25 normal, healthy donors. Examples of the semen characteristics that produced the 5 highest and lowest SQ and RQ score are shown in Table 32.3. The 5 lowest SQ scores represent poor semen quality. The highest and lowest SQ scores are more obvious in interpretation, in that, individuals with high SQ scores

Table 32.2: Weights to calculate semen quality and relative quality scores in 250 patients*

Variable

CASA-derived Semen Score Weights SQ score RQ score

Non-CASA Semen Score Weights SQ score RQ score

Concentration (X 106/mL) Motility (%)

0.81 0.25

0.86 0.24

−0.57 0.15

−0.47 0.11

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VCL (mm/s) 0.15 0.18 — — VSL (mm/s) 0.19 0.20 — — VAP (mm/s) 0.18 0.19 Linearity (%) 0.009 0.001 — — ALH (mm) 0.09 0.07 — — Sperm morphology WHO (%) 0.27 0.42 0.27 0.51 Strict criteria (%) 0.33 0.61 0.34 0.71 Percentage of total variability 64.8% 15.6% 72.9% 17.6% accounted for by each score *Scores computed by multiplying each log-transformed variable by the semen score weights; VCL=curvilinear velocity; VSL=straightline velocity; VAP=average path velocity; ALH=amplitude of lateral head displacement; WHO=World Health Organization; CASA=computer assisted semen analysis; SQ=overall semen quality score; RQ=relative quality score

Table 32.3: Semen parameters of the 5 highest and 5 lowest scores observed for SQ and RQ among the samples of 250 patients Concentration Motility VCL VSL VAP Linearity ALH WHO Strict SQ RQ (X 106/mL) (%) (µm/s) (µm/s) (µm/s) (%) (%) (%) criteria score score (µm) (%) 1.02 0.4 0.36 0.48 0.96

6 32 22 42 29

9.1 7.1 7.9 18.3 9.7

198 230.8 389.4 193.7 414

91 89 97 80 95

45.8 37.3 34.8 62.9 44

24.3 9.6 21.3 7 77.1

36 27 49 36 87

18.4 13.5 16.7 17.4 35

3 0.2 2.04

39 50 65

22.1 28.3 38.2

Five lowest values of SQ score (Overall semen quality) 18 7.2 45 0 11 0 25.08 81.07 11.7 6 71 0 5 2 27.23 97.11 14.4 4.9 46 0 15 2 29.70 106.39 43.5 12.8 50 0 6 0 31.10 95.48 15.8 8.2 54 2.2 10 1 35.14 98.07 Five highest values of SQ score (Overall semen quality) 54 42.3 69 2.9 54 17 124.12 106.96 52 31.7 53 4.5 50 16 124.19 101.30 52.6 28.7 53 4.3 34 7 125.05 79.03 88.2 54.9 57 5.8 62 19 127.69 115.31 59.7 37.6 56 4.2 40 12 130.38 89.58 Five lowest values of RQ score (Relative semen quality) 27 15.6 55 3.3 1 0 61.20 45.17 21.3 10.8 55 5.3 3 0 50.74 59.63 24 12.4 53 3.5 8 0 65.95 61.16 27.8 13.7 51 4.9 2 0 48.94 64.68 49.5 28.3 54 3.9 10 2 95.31 74.27 Five highest values of RQ score (Relative semen quality) 32.5 18 55 3.7 39 13 65.96 141.18 47.3 14.2 36 2.3 26 7 48.19 147.24 60.3 33.4 55 5.6 32 10 67.46 149.72

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5.8 56 44.1 61.1 40.7 56 5.1 65 14 81.82 151.12 0.56 39 26.3 45.4 19.5 37 7.2 48 21 59.73 165.94 VCL=curvilinear velocity; VSL=straightline velocity; VAP=average path velocity; ALH=amplitude of lateral head displacement; WHO=World Health Organization; SQ=overall semen quality score; RQ=relative quality score

have semen profiles that are better than normal, whereas those with low SQ have semen profiles that are below normal ranges. The sample with the highest SQ score had a concentration of 414×106/mL, a motility of 95 percent, and normal morphology (WHO guidelines) of 40 percent, whereas the sample with the lowest SQ score had a concentration of 1.02×106/mL, a motility of 6 percent, and WHO morphology of 11 percent. In addition, the 5 highest values for the RQ scores showed that the concentration was lower than expected based on the other above average semen parameters. The RQ score is not as easily interpretable as the SQ score because it is based on the relationship of 8 parameters compared with their concentration. For instance, the highest RQ score was a sample with a concentration of only 0.56×106/mL but a motility of 39 and 48 percent normal sperm morphology. In contrast, the individual with the lowest RQ score had concentration of 24.3×106/mLwith a motility of 36 percent and 1 percent normal sperm morphology. Among the entire group of 250 patient samples, the mean SQ score was 89.9 ±20.9 and the mean RQ score was 106.1±16.4. (The distribution of the 250 patients and 25 donors for both the SQ and RQ scores is illustrated in Fig. 32.2.)

Fig. 32.2: Distribution of semen scores of 250 patients assessed for male infertility (à) and 25 donors (Æ). The circle encompasses two standard

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deviations from 100, where most donors’ scores should fall Patients are often classified as oligozoospermic, asthenozoospermic, or teratozoospermic based on concentration, motility, and morphology or a combination of these factors. (To illustrate how these classifications are related to the semen scores, the 250 patients in our study were classified into 8 groups (Fig. 32.3).) Patients without any abnormal semen parameters had a mean SQ and RQ score of greater than 100. Patients who were either asthenozoospermic or teratozoospermic had a mean SQ score of 90. Since concentration is the parameter that is assigned the maximum weight in defining the SQ score, patients who were oligozoospermic had a mean SQ score greater than 80, which was slightly similar to the score in asthenoteratozoospermic patients. When patients with oligozoospermia were combined with those patients who were either asthenozoospermic or teratozoospermic, SQ scores had a mean value between 70 and 80. Patients with all 3 combinations (oligoasthenoteratozoospermic) had a mean SQ score of less than 60. Therefore, SQ scores decreased as the number of abnormal parameter increased. This plot also helps in interpreting the less intuitive RQ score, which also is a measure of asthenoteratozoospermia because it examines the quality of the non-concentration parameters. Patients identified as having asthenoteratozoospermia had a mean RQ score of less than 90 compared with the oligozoospermic patients who had a mean RQ score of 130. The latter RQ score is high in the sense that the sperm concentration is poor, but motility and morphology were in the normal range. Like the healthy patients, the RQ score in oligoasthenoteratozoospermic patients was close to 100 because their motility and morphology was as expected based on their poor sperm concentration.

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Fig. 32.3: Distribution of mean and standard deviations of the 20 men classified as either normal, asthenozoospermic (A), teratozoospermic (T), oligozoospermic (O), oligoasthenozoospermic (OA), oligoteratozoospermic (OT), asthenoteratozoospermic (AT), and oligoasthenoteratozoospermic (OAT) The two semen quality scores were applied to patients with various clinical diagnoses and also compared with the semen scores in the healthy donors. All 4 clinical diagnoses were associated with significantly lower SQ scores than that of the donors (p<0.03) (Table 32.4). These

Table 32.4: Semen scores in 177 men with clinical diagnoses and 19 healthy donors (controls) Diagnosis

SQ (Mean±SD) Pa

RQ (Mean±SD) Pb

Control (n=19) 100.0±10.0 — 100.0±10.0 Prostatitis with infection (n=46) 83.3±18.0 0.001 102.4±19.9 Varicocele (n=77) 78.6±17.7 <.0001 104.0±15.7 Varicocele with infection (n=11) 84.8±20.6 0.03 109.4±15.4 Vasectomy Reversal (n=43) 78.2±16.8 <.0001 98.7±18.0 a Pairwise comparisons for SQ between control and other groups. b Pairwise comparisons for RQ between control and other groups. SQ=overall semen quality score; RQ=relative quality score

— 0.63 0.41 0.17 0.80

groups had mean SQ values that were 15 to 22 points below that of the donors. Using multivariate logistic regression analysis to determine the ability of the semen scores to discriminate between the donors and patients, the SQ score had an odds ratio of 2.53 (95% confidence interval [CI] 1.36 to 4.72; p<0.004) and the RQ score had an odds ratio of 0.86 (95% CI, 0.51 to 1.46; p<0.58). Therefore, as a diagnostic tool to classify patients regarding their potential for natural conception, SQ appears to be more important than RQ. We next calculated the conventional semen score. In this score, only conventional or manual semen parameters (i.e., sperm concentration, % motility, and %normal forms according to both WHO morphology and Kruger’s strict criteria) were used. The principal component results for these 4 variables are shown in Table 32.2. A highly significant positive correlation was seen between the CASA-SQ score and the conventional-SQ score (r=0.99). Similarly, a very strong correlation was seen between the CASA-RQ score and the conventional-RQ score (4 variables) (r=0.96).

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RELATIONSHIP OF SQ AND RQ SCORES WITH IUI PREGNANCY RATES A total of 192 IUI cycles were evaluated and of these, 14 percent (27/192) resulted in pregnancy. Both the SQ and RQ prewash scores were significantly associated with IUIinduced pregnancy (Table 32.5). Of the postwash scores, only the RQ score (p<0.001) was related to pregnancy outcome. Of the 32 cycles in which the post-wash RQ score was greater than 125, 40 percent (13/32) resulted in pregnancy. Only 9 percent (14/160) resulted in pregnancy when the SQ score was less than 125. The significance of the prewash SQ score is illustrated by the following data. When the postwash RQ score was greater than 125 and the prewash SQ score greater than 70, 53 percent of 17 cycles resulted in pregnancy When the postwash RQ score was greater than 125 but the prewash SQ score less than 70, 25 percent of 16 cycles were successful. When both the postwash RQ and SQ scores were less than 125 and the prewash SQ score was less than 70, none of the 15 cycles resulted in pregnancies. The remaining 144 cycles with prewash SQ scores of

Table 32.5: Relationship between prewash and postwash semen scores and IUI pregnancy Variable

Non-pregnant cycles Pregnant cycles (n=165) (n=27)

Odds Ratio* (95% CI)

P-υalue* AUC

Prewash SQ score 86.5(73.7, 95.8) 81.7(76.6, 95.2) 1.6(1.1–2.5) 0.02 80% RQ score 102.2(92.3, 114.7) 117.3(109.2, 129.5) 2.5(1.6–3.8) <0.001 Postwash SQ score 90.5(77.4, 100.9) 83.3(76.3, 94.6) 1.2(0.8–1.6) 0.34 78% RQ score 107.6(97.1, 118.7) 124.7(112.6, 128.2) 2.0(1.4–2.8) <0.001 IUI-SPS Prewash 147.5(136.2, 156.5) 159.2(152.4, 166.2) 2.4(1.6–3.7) <0.001 81% Postwash 153.3(141.0, 164.9) 164.7(160.0, 173.9) 2.0(1.3–2.9) <0.001 78% Results are reported as median and interquartile range; *P-value indicates relationship with IUI pregnancy from logistic regression with GEE methods to account for multiple attempts of couples; Odds ratio is increased odds of pregnancy for each 10-point increase in SQ, RQ, or IUI-SPS; SQ=Semen quality; RQ=Relative quality; IUI-SPS=Intrauterine insemination semen pregnancy score; AUC=Area under the receiver-operating characteristic curves (ROC), which represents a summary statistic of sensitivity and specificity of the association with IUI pregnancy.

greater than 70 and postwash RQ scores of less than 125 resulted in a 10 percent pregnancy rate. (The distribution of SQ and RQ scores in cycles with and without pregnancies is illustrated in Figure 32.4.)

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Fig. 32.4: Distribution of prewash SQ and RQ scores in pregnant (+) and non-pregnant (•) IUI cycles Relationship of IUI-SPS with IUI Pregnancy Rates When using the combined IUI score (IUI-SPS) as calculated by regression coefficient, it was f ound that the prewash IUI-SPS was significantly related to IUI pregnancy rate with an AUC of 81 percent. This score had a greater ability to predict the pregnancy rate as measured by the AUC of pregnancy than the postwash semen scores (Table 32.5). Sperm morphology examined by Kruger’s strict criteria was the only semen parameter that approached the predictive ability of the IUI-SPS with an AUC of 79 percent. The fact that several prewash parameters are associated with IUI-induced pregnancy illustrates that combining the parameters can provide an improvement in prediction of pregnancy. (The relationship between the composite IUI-SPS and individual semen parameters is illustrated in Fig. 32.5). Logistic regression analysis indicated that IUI pregnancies could be predicted using the IUI-SPS (Fig. 32.6). Of the 196 cycles, more than half (56%) had an IUI-SPS of less than 150. Patients with an IUI-SPS greater than 150 had a pregnancy rate of 28 percent (24 pregnancies out of 87 cycles) compared with 3 percent (3 pregnancies out of 109 cycles) in patients with IUI-SPS less than 150. No pregnancies occurred in the 48 cycles where the IUI-SPS was less than 137. Measures of predictive ability using several cutoff values of the IUI-SPS are illustrated in Table 32.6. At a cutoff value of 130, the IUI-SPS had a sensitivity and negative predictive value of 100 percent. However, the specificity and the positive predictive values were low (18% and 16%, respectively)

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Fig. 32.5: Illustration of the IUI-SPS of the pregnant and non-pregnant IUI cylces, and the prediction equation of pregnancy from logistic regression of the observed IUI-SPS

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Fig. 32.6: Relationship between individual prewash semen parameters and the composite IUI-SPS . The specificity of IUI-SPS was as high as 81 percent at a cutoff value of 160. Negative predictive values were always less than 90 percent so long as the IUI-SPS was greater than 130. In a multivariate logistic GEE regression, ovarian stimulation (p=0.19), nonstimulation (p=0.07), and infertility duration (p= 0.15) were not related to pregnancy after adjusting for semen scores, whereas the prewash IUI-SPS was statistically significantly related after adjusting for these factors (p<0.001).

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Table 32.6: Measures of accuracy to predict IUI pregnancy with different cutoffs of the prewash IUI-SPS IUI-SPS Cutoff

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

130 100 18 16 100 140 93 36 19 97 150 89 63 28 97 160 48 81 29 91 IUI-SPS=Intrauterine insemination semen pregnancy score; PPV=Positive predicitve value NPV=Negative predicitive value

Relationship between Female Characteristics and Pregnancy Rate In a univariate analyses of female characteristics, infertility duration was significantly (p=0.046) related to pregnancy rate. On evaluating the effect of ovarian of stimulation, it was found that mild ovarian stimulation (either with clomphine citrate or low dose gonadotrophine) in the IUI cycles was as significant as non-stimulation (p=0.01) with respect to the per cycle rate. However, cumulative pregnancy rate was non-significant for both groups (using logistic regression with GEE) (Table 32.7). However, after adjusting for semen scores, ovarian stimulation (p=0.19), non-stimulation (p=0.07) and infertility duration (p=0.15) were not significant in multivariate logistic GEE regression, whereas the IUI semen pregnancy score maintained its significance after adjusting for these factors (p<0.001).

Table 32.7: Relationship between ovarian stimulation and per cycle fecundity and cumulative pregnancy rates. Group

Per cycle pregnancy rate P-value*

Per couple pregnancy rate P-value*

Overall

14% (n=192) 24% (n=93) No ovarian stimulation Total 7% (n=77) 0.01 14% (n=36) 0.08 Ovarian stimulation Total 19% (n=115) 0.01 29% (n=57) 0.08 *P-values compare each subgroup to the remainder of the attempts; P-values indicates per cycle rates from logistic regression with GEE methods to account for multiple attempts of couples, and per couple P-values based on Chi-square or Fisher’s exact tests.

SIGNIFICANCE OF SQ AND RQ AS NOVEL SEMEN SCORES In this study, we used a statistical model-principal component analysis to narrow down the elaborate measures of semen analysis to 2 main components or scores. The results demonstrate that the SQ and RQ scores accounted for the majority of the variability of 9 individual semen parameters. A majority of the semen characteristic variables positively

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correlated with these 2 scores. In addition, we were able to measure the SQ and RQ scores using different subsets of infertile patients with various clinical diagnoses. The SQ scores decreased as the number of abnormal parameter increased. A low SQ score reflected low concentration and poor motility and morphology whereas a high SQ score represented high concentration and relatively good motility and morphology. The men with varicocele and patients who had undergone vasectomy reversal had significantly lower SQ scores than the 25 healthy donors. Therefore, semen quality was significantly poor in all patients with clinical diagnoses but it did not differ among the patients themselves. The RQ score was not significantly different between the donors and the clinical diagnostic groups, indicating that their concentration levels were not significantly different than expected based on their other parameters. From our results, we can conclude that the SQ score can significantly discriminate donors from patients whereas RQ was not a good discriminator of semen quality in all these patient groups. This also confirms our univariate findings. Computer Assisted Semen Analyzer Derived Versus Conventional Semen Scores Our next objective was to examine how the two scores could effectively be utilized in those programs where CASA is not available, and, secondly how much information is really provided by the sperm kinematics associated with computerized semen analyzers. As seen from our findings, very little information would be lost if the CASA parameters are excluded and only conventional or manual scores are used. Therefore, programs that are not equipped with more sophisticated computer analysis systems for semen analysis can still have an effective means of interpreting the results of semen analysis. However, it is imperative that these programs have high standards of quality control and efficient and well-trained technical staff to minimize intra-laboratory variations. Information Provided by SQ and RQ Scores The semen score can provide important information on the semen quality and the likelihood of establishing a pregnancy. Second, semen score provides more meaningful information than the individual semen parameters. What are the clinical applications of our findings? The utility of the SQ and RQ scores for clinical practice may differ based on the clinical situation. Because all semen parameters were added and included, the SQ score may have greater utility in assessing the male fertility status for natural conception. On the other hand, the RQ score is derived from the measurement of morphology, motility, and motion parameters after adjusting for concentration. Therefore, the RQ score may be more helpful in predicting the outcome of artificial reproduction methods where concentration may not be as essential as the other parameters. Our SQ-RQ scoring system is more advantageous than many of the other systems being used, which lack corresponding information for accurate characterization of their relationship with fertility

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CONTROVERSY OVER SEMEN QUALITY AND ART OUTCOME The “normal” values provided by the WHO manuals for the basic semen parameters (i.e., volume and qualitative and quantitative motility and morphology) were obtained mostly through studies on fertile populations.21–22 This may be a reason why the clinical value of traditional semen parameters in the assessment of male infertility is a subject of considerable debate.23 Although reports on the association between pregnancy outcome and individual semen parameters vary widely and some have concluded that individual parameters are not effective in predicting pregnancy outcome, they have been used extensively to predict IUI success in patients with male-factor infertility. Sperm motion characteristics (CASA values) were reported to be related to the outcome of IUI and IVF.3,24 A nonlinear relationship between motile sperm count and pregnancy has been reported.25–26 In addition, total motile sperm count (TMSC) has been shown to have a positive correlation with pregnancy rate.27–29 With similar processing techniques, pregnancy rates of 11 percent have been documented.29–30 In an exhaustive retrospective study, it was reported that the average sperm motility of inseminates of patients who did not conceive af ter IUI was attempted was statistically lower (p<0.002) than values for couples who conceived, indicating a possible direct association between postwash sperm motility and the likelihood of pregnancy.29–31 Poor postwash motility combined with either age or pelvic surgery was associated with a 0 percent success rate. According to one study, sperm parameters obtained by CASA measurements can be used to predict fertility potential inboth infertile and healthy men.32Oehninger et al33 did a meta-analysis to examine the predictive values of different sperm function assays for fertilization outcome in IVF therapy and found that there is a real need for standardization and further investigation of the potential clinical utility of CASA systems. Lindheim et al34 found that sperm morphology was a predictor of IUI pregnancy, while others did not Burr et al.8 Although the effect of sperm morphology on the success of invitro fertilization has been well studied, the utility of strict morphology has not been studied extensively in couples undergoing controlled ovarian hyperstimulation and IUI. It was also reported that strict morphology is not a useful prognostic factor in IUI performed because of male infertility.35 While advanced reproductive techniques have been able to overcome the disadvantage of morphologically abnormal sperm, assisted reproduction utilizing in υiυo conditions with IUI does not appear to be able to circumvent inherent abnormalities in sperm. The Role of SQ and RQ Scores and the IUI-Semen Pregnancy Score Because there is no reliable method of predicting the chances for successful IUI-induced pregnancy, we examined the variability of semen parameters among couples with male infertility undergoing IUI and based on that variability calculated SQ and RQ scores and the IUI-SPS. Furthermore, the efficacy of these scores was calculated using cycle fecundity and cumulative pregnancy rates as an end point. The cycle fecundity rate was

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found to be 14 percent (27/192), which was higher than the rate of 8.7 percent reported by Goverde et al36 and comparable to a previous report of 14.4 percent.37 A similar pregnancy rate was reported with or without ovarian stimulationby other investigators as well.38–46 Both the prewash SQ score (p=0.02) and RQ score (p< 0.001) were positively associated with the increased likelihood of pregnancy. On the other hand, only the postwash RQ score (p<0.001) was related to a successful outcome. Of the cycles in which post-wash RQ score was greater than 125, 40 percent resulted in pregnancy compared with 9 percent of cycles when the postwash SQ score was less than 125. When the prewash SQ score was greater than 70 and the postwash RQ score was less than 125, the success rate was low (10%), which illustrates the feasibility of using the scores in predicting pregnancy outcome. The advantages of the SQ and RQ scores are that they enable clinicians to quickly compare semen quality and provide an easy method of identifying patients with abnormal semen quality, thereby facilitating improved assessment of male fertility for clinicians. These scores can also provide information about the fertilizing potential and predicting pregnancy in an IUI setting. We anticipated that SQ may be more important in assessing the chances of natural conception, and RQ more important in ART procedures, because in these controlled situations, the importance of concentration is reduced, and RQ measures quality after adjusting for concentration. Information Provided by IUI-SPS and Relationship with Pregnancy By combining the SQ and RQ scores, the IUI-SPS was derived, which was significantly related to pregnancy rate (p<0.001) with an area under the curve (AUC) of 81 percent. When the predictive ability of the IUI-SPS as measured by the AUC was compared with that of the individual semen parameters, IUI-SPS was f ound to have greater predictive ability than the individual semen parameters and that of postwash semen scores. Only Kruger’s strict morphology approached the predictive ability of the IUI-SPS with an AUC of 79 percent. However, the presence of other significant prewash parameters such as WHO morphology, VCL, VSL, VAP, and ALH illustrates that combining all parameters can improve the prediction of pregnancy. On examining the female factors, duration of infertility and induction of ovulation were significantly related to the chances of pregnancy, which is in agreement with previous reports.39,41–42 In another report, pregnancies occurred in 7.3 percent of patients with ovulatory dysfunction.29 None of the 3 female factors were related to pregnancy after adjusting for semen scores and using GEE multivariate logistic regression. When the semen scores in our study were stratified, these factors became non-significant and the scores retained their significant relation to both cycle fecundity and pregnancy rates. The reported success of IUI in the treatment of male-factor infertility varies widely from 0 to 57 percent per patient,15 although the number of subjects in most published studies were low. In larger studies, pregnancy rates determined for couples with male-factor infertility were between 8 and 10 percent.29,43Patients with idiopathic infertility had a 13.2 percent conception rate per cycle.15,29 These results emphasize the importance of etiology in the counseling of couples for IUI. The lack of significance of female characteristics in this group is probably related to the fact that this study was restricted to

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male factor patients, and female factors would probably be significant if a wider range of IUI attempts were included. When we measured the predictive accuracy of IUI-SPS at different cutoff values, it produced satisf actory sensitivity and negative predictive values. However, both specificity and positive predictive values were less than sensitivity and negative predictive values. At a cutoff of 130, IUI-SPS had 100 percent sensitivity and negative predictive values, which denotes that those patients with an IUI-SPS of less than 130 have a very low chance of pregnancy with IUI. Those patients should be directly counseled to undergo IVF without trying IUI, thereby avoiding unnecessary IUI cycles. When we compared sperm quality necessary for successful IUI with WHO threshold values for normal sperm, we found that the sperm quality necessary for successful IUI was lower than the WHO threshold values. It was therefore concluded that IUI is effective therapy for male-factor infertility when the total motile sperm count is 5×106 or greater and the initial sperm motility is 30 percent or greater.44 When initial values were lower, IUI was associated with a lower chance of success. In one study, the likelihood of pregnancy was maximized when motile sperm numbers were 5×106 or greater and sperm motility was 60 percent or greater.29 However, these authors did not provide the relationship between cycle fecundity rate and the motion parameters as measured by CASA and morphology characteristics measured by WHO guidelines or Kruger’s strict criteria. Our study gave the appropriate weight to the semen quality parameters, notably morphology and sperm motion characteristics. Using baseline semen analysis and the 24 hour sperm survival, it was found that the number of motile sperm available for insemination and especially their 24-hour survival are highly predictive of IUI success.45 CONCLUSION We have demonstrated that semen characteristics can be reduced to 2 semen quality scores, which account for more than 80 percent of the variability expressed by all of the semen characteristics individually. We believe that reducing the 9 semen characteristics to 2 scores will be more efficient by allowing quick comparisons of semen quality. In addition, the semen scores may provide improved assessment of male fertility. Patient scores under 80 are below the expected normal range of donors. Similar information is obtained using either CASA or conventional or manual semen analysis variables. Prewash SQ and RQ scores are positively related to pregnancy in couples undergoing IUI because of male-factor infertility. The postwash RQ score appear to be more important in predicting pregnancy in the IUI setting, particularly when it is greater than 125. The IUISPS score based on prewash semen parameters in those patients could be used to counsel them about their chances of success with IUI. Up to half of the failed IUI attempts in patients with male factor infertility could be avoided based on their IUI-SPS. Patients with an IUI-SPS below 150 may be advised to seek IVF whereas IUI be recommended for couples with an IUI-SPS above this cutoff value. Moreover, the prewash IUI-SPS can potentially be used to screen individuals for the feasibility of IUI attempts among couples with male-factor infertility. These novel scores provide quick, simple, and reliable tool to predict pregnancy in patients undergoing IUI for male factor infertility.

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SUMMARY The purpose of this study was to determine: 1. If the 9 semen characteristics measured by computer assisted semen analysis (CASA) could be reduced to 1 or 2 measures, thereby providing a more efficient way of predicting the outcome of natural conception and assisted reproduction techniques. 2. If these 2 scores can predict pregnancy in patients undergoing intrauterine insemination (IUI) for male-factor infertility. 3. If they can be combined to produce an overall score than can be used to determine whether couples should undergo IUI or in-vitro fertilization (IVF). Nine semen characteristics from 452 men (25 healthy donors, 250 men undergoing semen analysis as part of a fertility evaluation and 177 men with various clinical diagnoses of infertility) were measured: concentration, motility, curvilinear velocity, straightline velocity, average path velocity, linearity, amplitude of lateral head movement, and sperm morphology (by WHO and Kruger’s strict criteria), which was evaluated manually. After the scores were log transformed (base 10), we used principal component analysis to reduce the measures into few components. In addition, in couples undergoing intrauterine insemination, pre and postwash semen analyses from 93 men diagnosed with male-factor infertility were evaluated. The prewash SQ and RQ scores were combined to produce an IUI-Semen Pregnancy Score (IUI-SPS). The semen characteristics could be effectively summarized as 2 semen scores, which accounted for 80.3 percent of all the variability among the original semen charaeteristics. The first principal component (a weighted sum of all the semen characteristics accounting for 64.7 percent of the overall variability) was named the “SQ” score (semen quality). The second component (a combination of 8 of the characteristics after adjusting for concentration) was considered a measure of relative quality and was named the “RQ” score. Of a total of 192 IUI cycles, 14 percent (27) resulted in pregnancy. Both the SQ and RQ prewash scores were statistically significantly associated with IUI-induced pregnancy (p=0.02 and p<0.001, respectively) as was the postwash RQ score (p<0.001). Of the IUI cycles in which the postwash RQ score was greater than 125, 40 percent (13/32) resulted in pregnancy compared with 9 percent of cycles (14/160) when the postwash SQ score was less than 125. Moreover, the prewash IUI-SPS score was significantly related to IUI-induced pregnancy (p<0.001) and had an area under the ROC curve of 81 percent. An SQ or RQ score greater than 80 would be within the normal range for healthy males. Similar information is obtained using either CASA or conventional or manual semen analysis variables. The prewash SQ and RQ scores can predict pregnancy in patients undergoing IUI. The postwash RQ score may also help predict pregnancy, particularly when it is greater than 125. Patients with an IUI-SPS of less than 150 may be advised to seek IVF whereas those with an IUI-SPS of greater than 150 may be advised to seek IUI.

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REFERENCES 1. Campana A, Sakkas D, Stalberg A, Bianchi PG. Comte I Pache T et al. Intrauterine insemination: evaluation of the results according to the woman’s age, sperm quality, total sperm count per insemination and life table analysis. Hum Reprod 1996; 11:732–36. 2. Liu DY, Clark GN, Baker HW. Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in-vitro. J Androl 1991; 12:231–9. 3. Marshburn PM, Mclntire D, Carr BR, Byrd W. Spermatozoal charcteristics from fresh and frozen donor semen and their correlation with fertility outcome after insemination. Fertil Steril 1992; 58:179–86. 4. Macleod IC, Irvine DS. The predictive value of computer assisted semen analysis in the context of a donor insemination programme. Hum Reprod 1995; 10:580–86. 5. Aitken RJ. Motility parameters and fertility. In Gagnon C, (Ed): Control of sperm motility: biological and clinical aspects. Boston: CRC Press, 1990; 285–302. 6. Irvine DS. Computer assisted semen analysis systems: sperm motility assessment. Hum Reprod 1995; 10:53–59. 7. Mortimer D, Aitken RJ, Mortimer ST, PaceyAA. Workshop report: clinical CASA-the quest for consensus. Reprod Fertil Dev 1995; 7:951–59. 8. Burr RW, Siegberg R, Flaherty SP, Wang XJ, Mathews CD. The influence of sperm morphology and the number of motile sperm inseminated on the outcome of intrauterine insemination combined with mild ovarian stimulation. Fertil Steril 1996; 65:127–32. 9. Karabinus DS, Gelety TJ. The impact of sperm morphology evaluated by strict criteria on intrauterine insemination success. Fertil Steril 1997; 67:536–41. 10. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA. Sperm morphologic features as a prognostic factor in in-vitro fertilization. Fertil Steril 1986; 46:1118– 23. 11. Crosignani PG, Rubin B. Guidelines to the prevalence, diagnosis, treatment and management of infertility. Hum Reprod 1996; 11:1775–807. 12. Hughes EG. The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: a meta-analysis. Hum Reprod 1997; 12:1865–72. 13. Cohlen BJ, Te Velde ER, Van Kooij RJ. Looman CW. Habbema JD. Controlled ovarian hyperstimulation and intrauterine insemination for treating male subfertility: a controlled study. Hum Reprod 1998; 13:1553–58. 14. Collins JA, Burrows EA, Wilan AR. The progosis for live birth among untreated infertile couples. Fertil Steril 1995; 64:22–28. 15. Byrd W, Ackerman GE, Carr BR, Edman CD, Guzick DS, McConnell JD. Treatment of refractory infertility by transcervical intrauterine insemination of washed spermatozoa. Fertil Steril 1987; 48:921–27. 16. Dodson WC, Hughes CL, Haney AF. Multiple pregnancies conceived with intrauterine insemination during superovulation: an evaluation of clinical characteristics and monitored parameters of conception cycle. Am J Obstet Gynecol 1988; 159:382–85. 17. Van Voorhis BJ, Barnett M, Sparks AE, Syrop CH, Rosenthal G, Dawson J. Effect of the total motile sperm count on the efficacy and cost-effectiveness of intrauterine insemination and invitro fertilization. Fertil Steril 2001; 75:661–68. 18. Seber GAF. Multivariate Observations, Wiley, New York, 1984. 19. American Fertility Society. Revised American Fertility classification of endometriosis. Fertil Steril 1985; 43:351–52. 20. World Health Organization. WHO Laboratory Manual for the Examination of Human Sperm and Semen-Cervical Mucus Interaction (4th edn), New York, Cambridge Press, 1999.

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21. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction (2nd edn), New York, Cambridge University Press, 1987. 22. World Health Organization. WHO Laboratory Manual for the Examination of Human Sperm and Semen-Cervical Mucus Interaction (3rd edn), New York, Cambridge Press, 1992. 23. Tomlinson MJ, Kessopoulou E, Barratt CLR. The value of diagnostic and prognostic value of traditional semen parameters. J Androl 1999; 20:588–93. 24. Chu GP, Yang YS, Chen SU, Ho HN, Chen HF, Lin HR et al. The correlation between in vitro fertilization and sperm motility assessed by computer assisted semen analyzer (CASA). J Reprod Infertil 1997; 2:10–17. 25. Kang BM, Wu TC. Effect of age on intrauterine insemination with frozen donor sperm. Obstet Gynecol 1996; 88:93–98. 26. Hughes EG, Collins JA, Gunby J. A randomized controlled trial of three low-dose gonadotrophin protocols for unexplained infertility. Hum Reprod 1998; 13:15270–31. 27. Ombelet W, Bosmans E, Janssen M, Cox A, Vlasselaer J, Gyselaers W et al. Semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing. Hum Reprod 1997; 12:987–93. 28. Pasqualotto EB, Falcone T, Goldberg JM, Petrauskis C, Nelson DR, Agarwal A. Risk factors for multiple gestation in women undergoing intrauterine insemination with ovarian stimulation. Fertil Steril 1999; 72:613–18. 29. Stone BA, Vargyas JM, Ringler GE, Stein AL, Marrs RP. Determinants of the outcome of intrauterine insemination: Analysis of outcomes of 9963 consecutive cycles. Am J Obstet Gynecol 1999; 180:1522–34. 30. Negri P, Grechi E, Tomasi A, Fabbri E, Capuzzo A. Effectiveness of pentoxifylline in semen preparation for intrauterine insemination. Hum Reprod 1996; 11:1236–39. 31. Hendin BN, Falcone T, Hallak J, Nelson DR, Vemulapalli S, Goldberg J. The effect of patient and semen characteristics on live birth rates following intrauterine insemination: A retrospective study. J Assist Reprod and Genetics 2000; 17:245–52. 32. Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH et al. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. The Danish First Pregnancy Planner Study Team. Hum Reprod 2000; 15:1562–67. 33. Oehninger S, Franken DR, Sayed E, Barroso G, Kolm P. Sperm function assays and their predictive value for fertilization outcome in IVF therapy: a meta-analysis. Hum Reprod Update 2000; 6:160–68. 34. Lindheim S, Barad DH, Zinger M, Witt B, Amin H, Cohen B et al. Abnormal sperm morphology is highly predictive of pregnancy outcome during controlled ovarian hyperstimulation and intrauterine insemination. J Assist Reprod Genetics 1996; 13:569–72. 35. Matorras R, Corcosteugui B, Perez C, Mandiola M, Mendoza R, Javier Rodriguez-Escudero J. Sperm morphology analysis (strict criteria) in male infertility is not a prognostic factor in intrauterine insemination with husband’s sperm. Fertil Steril 1995; 63:608–11. 36. Goverde AJ, McDonnell J, Vermeiden JP, Schats R, Rutten FF, Schoemaker J. Intrauterine insemination or in-vitro fertilisation in idiopathic subfertility and male subfertility: a randomised trial and cost-effectiveness analysis. Lancet 2000; 355:13–18. 37. Ecochard R, Mathieu C, Royere D, Blache G, Rabilloud M, Czyba JC. A randomized prospective study comparing pregnancy rates after clomiphene citrate and human menopausal gonadotropin before intrauterine insemination. Fertil Steril 2000; 73:90–93. 38. Karlstrom PO, Bergh T, Lundkvist O. A prospective randomized trial of artificial insemination versus intercourse in cycles stimulated with human menopausal gonadotropin or clomiphene citrate. Fertil Steril 1993; 59:554–59. 39. Balasch J, Ballesca JL, Pimentel C, Creus M, Fabregues F, Vanrell JA. Late low-dose pure follicle stimulating hormone for ovarian stimulation in intra-uterine insemination cycles. Hum Reprod 1994; 9:1863–66.

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40. Karanade VC, Rao R, Pratt DE, Balin M, Levrant S, Morris R et al. Arandomized prospective comparisonbetween intrauterine insemination and fallopian sperm perfusion for the treatment of infertility. Fertil Steril 1995; 64:638–40. 41. Guzick DS, Carson SA, Coutifaris C, Overstreet JW, Factor-Litvak P. Steinkampf MP et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network. N Eng J Med 1999; 340:177–83. 42. Kaplan PF, Austin DJ, Freund R. Subcutaneous human menopausal gonadotropin administration for controlled ovarian hyperstimulation with intrauterine insemination cycles. Am J Obstet Gynecol 2000; 182:1421–16. 43. Aafjes JH, Vijver JC, Schenck PE. The duration of infertility: an important datum for the fertility prognosis of men with semen abnormalities. Fertil Steril 1978; 30:423–25. 44. Dickey RP, Pyrzak R, Lu PY, Taylor SN, Rye PH. Comparison of the sperm quality necessary for successful intrauterine insemination with World Health Organizatiqn threshold values for normal sperm. Fertil Steril 1999; 71:684–89. 45. Branigan EF, Estes MA, Muller CH. Advanced semen analysis: a simple screening test to predict intrauterine insemination success. Fertil Steril 1999; 71:547–51.

CHAPTER 33 Why Should We Assess Oocyte and Embryo Morphology? KE Tucker, CAM Jansen INTRODUCTION The ultimate goal of in vitro fertilization (IVF) is the establishment of a viable pregnancy and the birth of a single healthy baby. In order to meet the pressures imposed by anxious patients and by individual centers to improve success rates, multiple embryos are transferred in the hopes that one will implant. This practice has lead to an increase in the incidence of higher order multiple pregnancies that present numerous complications to both the child and mother. Multiple gestations have been accepted as a natural consequence of IVF. In fact, the incidence of twins is considered to be an extra bonus by both patients and clinicians. Since twin pregnancies can also lead to severe obstetric and neonatal complications, more and more infertility programs are striving to optimize stimulation regimens, laboratory and culture conditions, transfer procedures and post-transfer endocrine support in order to increase implantation rates and significantly reduce the number of embryos transferred. Several centers have already begun to establish a policy of elective transfer of a single embryo in good prognosis or “twin-prone” patients.1 This policy, however, poses the somewhat daunting challenge of selecting and transferring one embryo without compromising pregnancy rates. Historically, the most reliable method of embryo selection has been morphology, generally just prior to transfer. Early observations revealed embryos that appeared to look and develop “normally” were more likely to result in clinical pregnancies.2–3 This paper will review those characteristics reported to contribute to the definition the “golden” embryo—the one most likely to result in a pregnancy. Morphology Assessment: How and When? The method of assessment of embryo or oocyte quality should not be detrimental to subsequent development. Selection methods should be not invasive or time-consuming, but still be predictive. Embryo selection is not a static description of a single point in time. Embryo morphology is based on a series of dynamic processes and influences that can change dramatically from moment to moment. Because of this, some researchers believe that embryo assessment should begin with the ovary and follicle.4–8 Others maintain that the secret lies with the oocyte or pronuclear stage embryo.9–10 This discussion will address several stages of embryo development, from the microenvironment of the follicle and the

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morphology of the oocyte to the pronuclear-, cleavage- and blastocyst-stage embryo.12–24 Efforts are constantly being made to further describe the morphological characteristic of oocytes and embryos that will predict enhanced pregnancy and implantation rates. Morphology Assessment The Follicle Adequate vascularization has been associated with good follicle health in several species. In large domestic animals, such as the horse, ovulatory follicles demonstrate a dramatic increase in thecal blood flow, which has been correlated with high intrafollicular and circulating levels of estradiol, highly metabolic follicle cells and viable and healthy oocytes.15 Recently, it has been noted that perifollicular blood flow, as assessed by power color Doppler ultrasound, may be a good predictor not only of follicle health, but also of oocyte competence.6,16,17 It has also been shown that follicular hypoxia may negatively affect spindle organization and mitochondrial and chromosomal segregation in the human oocyte, leading to abnormalities in the embryos that may result from it.5,18 Good oocyte quality has also reported to be directly related to an increase in intrafollicular oxygenation. Other investigators have further demonstrated that follicular blood flow and the content of dissolved oxygen was positively related to the ability of the oocyte-embryo to develop successfully, compared with these from avascular follicles which did not result in any pregnancies.19 Similar results were reported by Bhal and others.8 These investigators found that significantly more pregnancies occurred when embryos from highly vasculairzed follicles were transferred (34.7%), compared to those from follicles with a low Doppler vascularity score (18%). Interestingly, no obvious differences in Day 3 embryo morphology were observed between the two groups. Huey and co-workers16 also found a significant correlation between oxygen content and oocyte-embryo health, but were not able to correlate this with differences in Doppler patterns. This discrepancy with other published reports may possibly be due to differences in equipment or technical methods and expertise. The Oocyte Ovarian stimulation protocols make numerous oocytes available per cycle and it is has been shown that a large variability exists within a single cohort.5 Several studies have been performed to precisely define the morphology of these oocytes and correlate that with a higher incidence of fertilization, embryo development and pregnancy rates. A large and detailed analysis of over 400 IVF cycles (>4,000 oocytes) demonstrated that pregnancy rates increased when more oocytes were retrieved within an individual IVF cycle. Although the presence of fractured zonae resulted in lower fertilization rates, no morpho logical characteristic was significantly associated with pregnancy rates.46 Others have reported that better fertilization and embryo development rates occurred with “good quality” oocytes; those with a small perivitelline space and a regular and distinct first polar body (PB).20 Additional reports have indicated that the morphology of the first PB can be a reliable indicator of oocyte age and that the presence of a wellshaped, non-fragmented PB was associated with increased pregnancy rates.9

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Why is PB morphology an important indicator of oocyte health? Battaglia and coworkers maintained that an irregular PB may be indicative of abnormalities associated with the mitotic spindle. The spindle, composed of microtubules, is necessary for normal alignment and separation of the maternal chromosomes during Meiosis I and II. As with the PB, healthy, intact spindles were found most often in oocytes from young women. Disruption or absence of the spindle has been shown to result in aneuploid embryos.21 These investigators also observed that the presence of a bifringent spindle, viewed with polarized optics (Polscope®) was highly predictive of better fertilization and pregnancy rates. The cytoplasm of the oocyte is also thought to be predictive of treatment success in IVF. Kahraman and others22 believed that an increase in cytoplasmic granularity was associated with low success rates after intracytoplasmic sperm injection (ICSI). They reported that centrally located cytoplasmic granulation (CLCG) in oocytes was associated with a lower pregnancy rate, due primarily to an extremely high abortion rate (54.5%) and therefore, urged that patients with a high incidence of CLCG be counseled with regard to a possible negative outcome. The Pronuclear Embryo (Zygote) Some investigators agree that the pronuclear (PN) stage of embryo development can be a very powerful predictor of subsequent embryo development, blastulation rate, the incidence of genetic abnormalities and resulting pregnancy rates.11,23–27 The shape, position and number of PNs in the embryo have been correlated with embryo health and overall success. Normally appearing zygotes contain two distinct PNs, but it is not unusual to see zygotes with a single pronucleus. Although a fairly large percentage of these embryos turned out to be diploid, based on fluorescent in situ hybridization (FISH), they still were not always karotypically normal. Interestingly, even seemingly normal, bipronuclear embryos displayed abnormal or delayed cleavage, primarily in older women, and despite being diploid, were often chaotic in their chromosomal constitution.28 The relative size of the PNs has also been shown to be informative. Manor and co-workers11 reported that chromosome abnormalities were present in 88.5 percent of ICSI and in 50 percent of IVF zygotes exhibiting unequally sized PNs. Other studies have shown that PN size is not the only predictive parameter readily observed in the zygote. Several groups have concluded that the distribution pattern of nucleolar precursor bodies (NPB) within the PN may be the most reliable characteristic in determining embryo quality and pregnancy rate.23,24,26,27,29 Several patterns of NPB distribution have been documented and, based on this feature, Tesarik and Greco23 devised an initial classification system. Zygotes were divided into 6 different categories, one normal (Pattern 0) and 5 abnormal (Patterns 1–5). Pattern 0 zygotes had two PNs at the same stage of development; that is, NPB should be polarized at the time of evaluation. Several researchers in this field agree that Pattern 0 (see below) is the most predictive of good clinical outcome and has been reported to lead to a 72 percent blastocyst rate.24,26,29 Higher implantation and pregnancy rates were obtained when at least 1 blastocyst derived from a 0 Pattern zygote was transferred. No relationship has been observed between Pattern 0 and female age or cause of infertility.19,46

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The formation of the PNs and extrusion of the second PB have been extensively documented using time-lapse video. Payne and co-workers30 have described the appearance of periodic waves of granulation in the ooplasm. This “halo” of granulation is in contrast with the static granulation previously reported to occur in sub-optimal oocytes. Scott and co-workers27 have suggested that good quality embryos result from PN embryos exhibiting a peripheral “halo” and centrally located granulation and in an earlier study by this group, impressive pregnancy rates were achieved when 2 zygotes were transferred that displayed both the “halo” and polarized PNs (55%). In contrast, a study by Hurst and others31 demonstrated that selection of “good” zygotes for a Day 1 transfer resulted in severely reduced pregnancy rates when compared to Day 2 transfer results (15% vs. 67%, respectively). A later study also found that, although cleavage-stage morphology was better in embryos derived from “good” quality zygotes (“halo” and polarized PNs), no differences in pregnancy rates were seen when compared to those from sub-standard zygotes (one or both PNs with scattered NPB).32 In all cases, the best cleavage-stage embryos were transferred. The Cleaving/Compacting Embryo Most embryologists and clinicians are well aware of the general morphological characteristics that have been correlated with a good quality embryo and excellent atlases are available that describe and beautifully illustrate embryos of all stages and qualities.33– 34 In view of this, a detailed description of embryo quality will not be included in this review. In general, “good” quality cleaving embryos display stage-specific cell division, have blastomeres of fairly equal size with few to no cytoplasmic fragments. Recent studies continue to add new information to what has been reported regarding the appearance of a good quality embryo and the processes it undergoes before implantation. The phenomenon of uneven blastomere cleavage and how it relates to embryo health is not fully understood. Hardarson and co-workers35 demonstrated that embryos with unequal cell division had a lower developmental capacity which was attributed to the higher degree of genetic abnormalities detected by fluorescent in situ hybridization (FISH) (i.e. aneuploidy or multinucleated blastomeres). When otherwise good quality embryos were transferred, these investigators found that implantation (IR) and pregnancy rates (PR) were significantly lower when those with uneven-sized blastomeres were replaced [IR: 23.9 (uneven) vs 36.4 percent (even); PR: 29.4 (uneven) vs 36.4 percent (even)]. A fairly recent study by Desai and co-workers36 observed that cytoplasmic “pitting” occurs in some embryos, which is thought to be physically different from excessive granulation in the cytoplasm. These researchers further ascertained that a high degree of pitting in Day 3 embryos could be used as an additional morphological marker for poor prognosis embryos.

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A more familiar indication of embryo health and developmental potential is the degree of fragmentation. The actual cause(s) and mechanisms of fragmentation are still not well known. It has been proposed that generation of high levels of reactive oxygen species (ROS) throughout the culture procedure contributes to excess fragment formation.37 The presence of some fragmentation is not always indicative of poor embryo health and has been observed in embryos produced in υiυo in a number of species.38 Most groups will agree, however, that a high degree of fragmentation significantly reduces an embryo’s implantation rate and may be associated with an elevated percentage of apoptotic features and chromosomal disorders.39 Patients should, therefore, be advised of any potential risks if only highly fragmented embryos are available for transfer. Additional research in this area of embryo quality assessment has demonstrated that several types and patterns of fragmentation exist, leading to different IVF outcomes. Specifically, five different types of fragmentation have been described.38 Types I and II are associated with the lysis of an individual blastomere. Type III, where fragments are scattered throughout the embryo, is the most common. Types I, II and III are usually seen in embryos from younger women (<38 years). Types IV and V describe embryos with more than 50 percent fragmentation (based on total volume) and are the patterns least likely to result in a pregnancy, primarily because the remaining blastomeres are grainy and necrotic. Some investigators have suggested that removal of fragments may offer some hope for highly fragmented embryos. Clinical observations suggest that removal of fragments may not only improve the cosmetic appearance of the embryo, but may, in fact, enhance subsequent cell division and compaction, and may contribute to increasing implantation rates for these embryos.38 Fragmentation removal, however, is a very invasive procedure and any benefits derived from it are highly controversial. The presence of fragments is not an all-or-nothing condition. Van Blerkom and coworkers7 performed an extensive time-lapse investigation of fragment dynamics in human embryos. Based on his observations, he concluded that not all forms of spontaneous fragmentation were necessarily lethal. In certain cases, more “transient” fragments were observed that later disappeared either by re-absorption or lysis. Even when considering all that we know about cleavage stage embryos morphology, it is still difficult to identify those embryos that will continue to develop normally, even in culture. Rijnders and Jansen12 demonstrated that even good quality embryos on Day 3 do not always develop into blastocysts on Day 5 (Grade 1–50% blastocyst rate) and that a surprisingly high number of lesser grade embryos do (Grade 3–25%). Other studies have reported similar results, with only 48 percent of the embryos chosen for transfer on Day 3 becoming blastocysts.40 The morula and compaction stage embryo may be considered to be more of a transitional structure and more difficult to evaluate. Pregnancies have occurred when morulae were transferred on Day 5,12 but to a much lesser extent than when stage-specific embryos (blastocysts) were transferred. It is unclear whether this is due to entirely to developmental stage or in part to a reduced ability to select the appropriate embryo for transfer. Good quality morulae are uniform in appearance (individual cells are difficult to distinguish) and early cavitation on Day 4 is a good predictor for normal blastocyst development. The formation of cell-cell junctions on Day 3 can also be considered a good predictor for blastocyst development in υitro and subsequent pregnancies.

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In addition to cell number, degree of fragmentation, blastomere size and cytoplasmic pitting, one of the most diagnostic characteristics of the health and developmental potential of an individual embryo is the number of nuclei per blastomere.13,24,41 Several investigators believe that the multinuclear blastomere (MNB) index is highly indicative of embryos that will NOT lead to a pregnancy. Studies in Belgium have reported that embryos resulting from ICSI without MNBs, were chosen for transfer on Day 3, had a higher chance of implanting and leading to a viable pregnancy compared with control transfers that included at least one embryo with MNB (11.3 vs. 6.0% and 28.7 vs. 16.9%, respectively).41 In 1999, Van Royen and others13 reported that the presence or absence of MNB in embryos from high-responder patients may be THE pivotal characteristic in determining the implantation potential of an otherwise good quality embryo. When two “top” embryos (no MNB) were transferred, pregnancy and implantation rates were 63 percent and 49 percent, respectively, with 57 percent incidence of twins. Conversely, the transfer of two non-top embryos (good quality Day 3 embryos, both with MNB) resulted in significantly lower success rates (PR—23%, IR-12%). When a mixed group of embryos were transferred (1 top + 1 non-top) results were more intermediate (PR—58%, 21% twins; IR—35%), suggesting that better outcomes depend on at least one embryo being transferred without any MNBs.13,24 These authors went so far as to recommend that embryos with MNB NOT be transferred. The Blastocyst The transfer of as few as two blastocysts has been shown, on average, to be effective in reducing the number of multiple gestations in patients with good ovarian response14,42,43 and capable of establishing pregnancies in women with previous IVF failures.12 Generally, a good quality blastocyst contains a well-expanded blastocoelic cavity, homogenous trophoblast with multiple cell-cell contacts and distinct nuclei, and an inner cell mass that is clearly visible and intact. A large number of studies exist in the literature attesting to excellent success rates following blastocyst transfer, with implantation rates of 67 percent.43 Gardner and co-workers14 also concluded that if at least one high scoring, good quality blastocyst is available for transfer, pregnancy rates as high as 60 percent overall could be achieved. Selection of the “golden” blastocyst is also not well understood. Some investigators strongly believe that extending the culture of embryos to blastocysts represents a type of “natural selection” of those embryos that will be genetically normal and viable in utero. Despite all our current advances, there is still very little “natural” about the procedures involved in IVF and embryo culture. Studies have shown that blastocyst morphology alone cannot rule out the incidence of chromosomal abnormalities (mosaicisms) in these embryos44 and that even trisomic embryos become blastocysts at a very respectable rate (37%).45

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MORPHOLOGY ASSESSMENT: SUMMARY AND CONCLUSIONS Oocyte maturation, fertilization and embryo development are complex and dynamic events and it is difficult to pin-point a single moment in development that will identify the embryo destined for implantation. We have seen that unique characteristics are present at virtually every stage of development that may be predictive of an embryo’s implantation potential. Even the rates of cell division and differentiation themselves can influence embryo selection. It is important to remember, therefore, that morphology assessment cannot be accomplished by a one-time observation. The visible features that help us select the “golden” oocyte or embryo may appear gradually, starting with increased follicular size and vascularity, continuing with a healthy, mature oocyte that fertilizes normally, resulting in an embryo that divides regularly and in a timely fashion and has intact blastomeres that are homogeneous with one distinct nucleus. Depending on the day of transfer, the chance of success can be further influenced with selection of the most competent blastocyst and an by uneventful and atraumatic embryo transfer. The “artificial” selection IVF programs exert, is necessary for minimizing multiple gestations without compromising pregnancy rate and outcome. We are already faced with the reality of single embryo transfer for many of our patients; certainly those more likely to develop twin or triplet pregnancies. Selection of that “golden” embryo relies on the additive influence of distinct, yet related variables that will hopefully add up to the birth of a single healthy baby. REFERENCES 1. ESHRE Campus Course Report. Prevention of twin pregnancies after IVF/ICSI by single embryo transfer. Hum Reprod 2001; 16:790–800. 2. Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM et al. Factors influencing the success of invitro fertilization for alleviating human infertility. J in vitro Fert Embryo Transf 1984; 1:3–23. 3. Cummins JM, Breen TM, Harrison KL, Shaw JM, Wilson LM, Hennessey JF. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J in vitro Fert Embryo Transf 1986; 3:284–95. 4. Van Blerkom J. Epigenetic influences on oocyte development competence: perifollicular vascularity and intrafollicular oxygen. J Assist Reprod Genet 1998; 15:226–34. 5. Van Blerkom J. Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function. Hum Reprod 2000; 15(Suppl 2):173–88. 6. Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod 1997; 12:1047– 55. 7. Van Blerkom J, Davis P, Alexander S. A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos. Hum Reprod 2001; 16:719–29.

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8. Bhal PS, Pugh ND, Chui DK, Gregory L, Walker SM, Shaw RW. The use of transvaginal power Doppler ultrasonography to evaluate the relationship between perifollicular vasctdarity and outcome in in-vitro fertilization treatment cycles. Hum Reprod 1999; 14:939–45. 9. Ebner T, Moser M, Yaman C, Feichtinger O, Hartl J, Tews G. Elective transfer of embryos selected on the basis of first polar morphology is associated with increased rates of implantation and pregnancy. Fertil Steril 1999; 72:599–603. 10. Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryos quality in intracytoplasmic sperm injection. Hum Reprod 2000; 15:427–30. 11. Manor D, Drugean A, Stein D, Pillar M, Itskovitz-Eldor J. Unequal Pronuclear size—A powerful predictor of embryonic chromosomal anomalies. J Assist Reprod Genet 1999; 16:385– 9. 12. Rijnders PM, Jansen CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregftancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1998; 13:2869–73. 13. Van Royen E, Mangelschots K, De Neubourg D, Valkenburg M, Van de Meerssche M, Ryckaert G, Eestermans W, Gerris J. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999; 14:2345–9. 14. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000; 73:1155–58. 15. Tucker KE, Henderson KA, Duby RT. In vitro steroidogenesis by granulosa cells from equine preovulatory follicles. J Reprod Fert 1991; 44 (Suppl): 45–55. 16. Huey S, Abuhamad A, Barroso G, Hsu M, Kolm P, Mayer J et al. Perifollicular blood flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization. Fertil Steril 1999; 72:707–13. 17. Borini A, Maccolini A, Tallarini A, Bonu MA, Sciajno R, Flamigni C. Perifollicular vascularity and its relationship with oocyte maturity and IVF outcome. Ann NY Acad Sci 2001; 943:64–67. 18. Wilding M, Dale B, Marino M, di Matteo L, Alviggi C, Pisaturo ML et al. Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 2001; 16:909–17. 19. Chui DK, Pugh ND, Walker SM, Gregory L, Shaw RW. Follicular vascularity-the predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study. Hum Reprod 1997; 12:191–96. 20. Suppinyopong S, Choavaratana R, Karavakul C. Correlation of oocyte morphology with fertilization rate and embryo quality after intracytoplasmic sperm injection. J Med Assoc Thai 2000; 83:627–32. 21. Wang WH, Meng L, Hackett RJ, Keefe DL. Developmental ability of human oocytes with or without birefringent spingles imaged by Polscope before insemination. Hum Reprod 16:2001; 1464–68. 22. Kahraman S, Yakin K, D”nmez, Samh H, Bahje, Cengiz G et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum Reprod 2000; 15:2390–3. 23. Tesarik J, Greco E. The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum Reprod 1999; 14:1318–23. 24. Gerris J, Van Royen E. Avoiding multiple pregnancies in ART: a plea for single embryo transfer. Hum Reprod 2000; 15:1884–88. 25. Wittemer C, Bettahar-Lebugle K, Ohl J, RongiŠres C, Nisand I, Gerlinger P. Zygote evaluation: an efficient tool for embryo selection. Hum Reprod 2000; 15:2591–97. 26. Balaban B, Urman B, Isiklaf A, Alatas C, Aksoy S, Mercan R et al. The effect of Pronuclear morphology on embryo quality parameters and blastocyst transfer outcome. Hum Reprod 2001; 16:2357–61.

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27. Scott L, Alvero R, Leondires M, Miller B. The morphology of human Pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod 2000; 15:2394– 2403. 28. Lim AS, Goh VH, Su CL, Yu SL. Microscopic assessment of pronuclear embryos is not definitive. Hum Genet 2000; 107:62–8. 29. Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998; 13:1003–13. 30. Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod 1997; 12:532–41. 31. Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Pronuclear uterine transfer lowers in vitro fertilization success. J Assist Reprod Genet 1998; 15:575–77. 32. Salumets A, Hyd, n-Granskog C, Suikkari A, Tiitinen A, Tuuri T. The predictive value of pronuclear morphology of zygotes in the assessment of human quality. Hum Reprod 2001; 16:2177–81. 33. Veek LL. An Atlas of Human Gametes and Conceptuses, New York: Pathenon Publishing 1999. 34. Menezo YJ, Kauffanan R, Veiga A, Servy EJ. A mini-atlas of the human blastocyst in vitro. Zygote 1999; 7:61–5. 35. Hardarson T, Hanson C, Sjogren A, Lundin K. Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation. Hum Reprod 2001; 16:313–18. 36. Desai NN, Goldstein J, Rowland DY, Goldfarb JM. Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study Hum Reprod 2000; 15:2190–96. 37. Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh Ks. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod 1998; 13:998–1002. 38. Alikani M. Cytoplasmic fragmentation in human embryos in vitro: implications and the relevance of fragment removal. In: Gardner D, Howles CM, Weissman A, Shohan Z (Eds). Textbook of Assisted Reproductive Techniques. Martin Dunitz Publishers, 1999; 169–83. 39. Ebner T, Yaman C, Moser M, Sommergruber M, P” lz, Tews G. Embryo fragmentation in vitro and its impact on treatment and pregnancy outcome. Fertil Steril 2001; 76:281–85. 40. Graham J, Han T, Peroter R, Levy M, Stillman R, Tucker MJ. Day 3 morphology is a poor predictor of blastocyst quality in extended culture. Fertil Steril 2000; 74:495–97. 41. Pelinck MJ, De Vos M, Dekens M, Van der Elst J, De Sutter P, Dhont M. Embryos cultured in vitro with multinucleated blastomeres have poor implantation potential in human in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1998; 13:960–63. 42. Toledo AA, Wright G, Jones AE, Smith SS, Johnson-Ward J, Brockman WW et al. Blastocyst transfer: a useful tool for reduction of high-order multiple gestations in a human assisted reproduction program. Am J Obstet Gynecol 2000; 183:377–79. 43. Langely DT, Marek DM, Gardner DK, Doody KM, Doody KJ. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001; 16:902–8. 44. Evisikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 1998; 13:3151–5. 45. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munn, S. Developmental ability of chromosomally abnormal human embryos to devlop to the blastocyst stage. Hum Reprod 2001; 16:1954–58. 46. Wittemer C, Ohl J, Bettahar-Lebugle K, Viville S, Nisand I. A quantitative and morphological analysis of oocytes collected during 438 cycles. J Assist Reprod Genet 2000; 17:44–51.

CHAPTER 34 Benefits and Drawbacks of Extended Embryo Culture CAM Jansen, PM Rijnders, KE Tucker OVERVIEW Embryo transfer in in vitro Fertilization (IVF) was initially in general performed two or three days after follicle aspiration. However as the embryonic genome still has not come to full expression, genetic or chromosomal damage, as well as disturbances in the mitotic or meiotic division might go undetected. As the full expression of the embryonic genome only takes place after the 4 to 8 cell stage, there are theoretical advantages to delay transfer until the blastocyst stage. However extending the culture may lead to different requirements and culture conditions that are more critical. In addition delay has no advantage in case it is not possible to select embryos from the cohort. This means that if blastocyst culture is performed for all patients, the benefit for some may counter-balance the negative effects for others. HISTORY OF BLASTOCYSTTRANSFER Blastocyst transfer was first performed in 1981 in an oocyte donation program, initiated by John Buster, based on an idea of Richard Seed, and published in 1983.1 With this technique, in total four ongoing pregnancies occurred.2 However, these blastocysts were formed in vivo by insemination of sperm of the prospective father after ovarian hyperstimulation of the donor. After approximately 100 hours the embryos were flushed from the uterine cavity and transferred to the receptor. This technique has not had a long life span because of a number of inherent serious complications: in many cases the embryos remained either in the uterus of the donor, necessitating a pregnancy interruption, or were even flushed back into the fallopian tube, necessitating emergency laparotomies for an ectopic pregnancy. However, it has given a hitherto unknown insight into the development potential of the embryo in natural conditions. The embryo recovery rate was only 47 percent, but from the embryos that had fertilized and cleaved all stages were present, from 2-cell stage to blastocyst stage; the blastocyst formation rate after 100 hours was 20 percent. In addition all zonae were intact which does not argue in favor of those who claimed that the zona pellucida completely dissolves during the path to the uterine cavity, and that the process of ‘hatching’ would be an artificial phenomenon. Of course the possibility exists that embryos that had already lost the zona were less likely to be flushed out.

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Fig. 34.1: Development of flushed embryos approximately 100 hours after fertilization in υiυo. Data taken and converted from ref The first pregnancy from transfer of blastocysts after in vitro fertilization was published in 1985.3 This was also a cryopreserved embryo, frozen and thawed in the blastocyst stage. Since then, after a lingering phase, there has been a steady increase to almost 100 articles per year entirely dealing with transfer of blastocysts in human clinical practice.4 There was some heightened interest in the early nineties by Bolton et al,1 using a serum enriched Earle’s balanced salt solution as culture media, but as pregnancy rates as well as live birth rates per transfer were less, this interest waned.5 However, lesser embryos were replaced in the blastocyst group (2, 7 versus 2, 0) and ongoing implantation rates were similar, even slightly higher in the blastocyst group (6 and 7percent respectively; NS). One group has cultured blastocysts in co-cultures with Vero cells, but this practice has never become widespread due to the fears of inherent risks of co-culture.6 Renewed interest arose with the use of sequential media, in an attempt to meet the different needs of the developing embryos.7,8 RATIONALE OF BLASTOCYST CULTURE In theory culture to the blastocyst stage has several advantages. It has been shown that the full expression of the embryonic genome in the human only takes place after the four to eight cell stage.9 Thus chromosomal or genetic damage to the embryonic genome may only manifest itself afterwards. As a result many four to eight cell embryos arrest in growth and even degenerate, and it is conceivable that these would have had no implantation potential. However the extra-corporeal stay has also risks, resulting in damage to the embryos in a later stage and hence a diminished implantation potential. These risks are inherent to less optimal culture conditions than the natural situation in the uterine tract, which generally is perceived to be optimal. An analysis with Fluorescent In Situ Hybridization (FISH) of 9 chromosomes in Preimplantation Genetic Diagnosis on one or two blastomeres (PGD) (1, 13, 15, 16, 18,

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21, 22, X and Y) revealed that as much as 63 percent of all 8-cell embryos were abnormal, versus 48 percent of blastocysts.10 In this study the blastocyst formation rate was 66 percent for chromosomal ‘normal’ embryos, versus 34 percent for ‘abnormal’ embryos. Sandalinas et al obtained blastocyst formation rates in relation to the chromosomal constitution of embryos and showed that there were considerable differences.11 Cohen obtained similar figures earlier from the same group12 (Fig. 34.2). This study had some constraints: it only dealt with part of the chromosomes, i.e. those known to play a prominent role in miscarriage, and only detected aneuploidies. In addition, it is known that the outcome of PGD on one or two blastomeres is diminished due to a considerable incidence of mozaicism, leading to the possibility of erroneously classifying an embryo as normal.13 It is known that morphology as well a cleavage rate of day three embryos is of limited predictive value for blastocyst formation.14,15 In our own study, only about 47

Fig. 34.2: Blastocyst formation rate in relation to chromosomal constltution in embryos See ref 10 percent of all class one and two embryos-(the best; VUB classification) developed to blastocysts, whilst still 20 percent of all class three and four developed to blastocysts. The main differences between both groups were the percentage of arrested embryos. Only 57 percent of the embryos chosen on day three for transfer had developed into blastocysts. Methods and Results The renewed interest in blastocyst culture arose after reports of very high pregnancy and implantation rates after sequential culture. However these treatments were only performed for a very stringent selection of patients, i.e. those that were anticipated to have a large number to choose from. With an average blastocyst formation rate of 40 percent, this means that when a patient has few embryos, chances are that she will end up with no transfer at all, a risk that many clinicians are not prepared to take. Many studies that show an improvement in implantation rate deal with a highly selected group as said

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before. However these are not randomized controlled studies, and the selected patients also would have better pregnancy and implantation rates on day two or three than the average patient. A number of retrospective studies have suggested better pregnancy as well as implantation rates.16,17,18,19 Others have found similar pregnancy rates but higher implantation rates as there were large differences in the transfer policy: considerably less embryos were replaced on day five in an attempt to decrease the multiple gestation rate, prevalent in all of these studies,20,21,22,23,24 Nevertheless the multiple pregnancy rate in these studies is still considerable. Over all many authors claim that only a limited number of patients might benefit from transfer of blastocysts. In one center, Cornell, only 8.5 percent of all patients actually had blastocyst transfer; in addition, many that were scheduled for blastocyst transfer had their transfer on day three because of fear of failure to transfer on day five.25 There are very few prospective randomized trials in a non-selected populatiori as well as in a selected population. In addition it is not clear which selection criteria are the best for blastocyst transfer. No increase in pregnancy rate in a non-selected population was found by Scholtes et al26 and Huisman et al,27 in a selected population by Coskun,28 in prospective randomized trials, by Toledo AA,29 in a retrospective analysis with matched controls, also in a selected population. The methodology used in these studies is too different to draw any firm conclusions. In addition, culture conditions as well as culture media may determine the remaining viability of an embryo. One can never turn a bad embryo into a good one, but one can make a good embryo bad by suboptimal culture conditions. It may well be that differences can only be found when the culture conditions are optimal, and that under suboptimal conditions no differences will be found. Complications One still persisting problem is the very high multiple pregnancy rate. The only way to prevent this is the voluntary reduction of the number of embryos to be replaced; however this will undoubtedly have a negative impact in the pregnancy rate. One authoradmittedly not in blastocysts found no difference in ongoing pregnancy rates30 when either two or three embryos were replaced, but this can solely be attributed to the ‘self fulfilling prophecy’ as a result of the design of the study. The author retrospectively took the data from the large database of the HFEA from a time span that each center had determined their own replacement policy. For instance with a difference of an over-all implantation rate from 15 to 20 percent, the pregnancy rate will be the same for the center with the low implantation rate when three embryos are replaced, as the center with the higher implantation rate when two embryos are replaced. When in the latter center three embryos are replaced as a default, the number of triplets per hundred pregnancies will be double from that in the former center. Centers with a high implantation rate will thus be inclined to reduce the number of embryos to two whilst the centers with a low implantation rate will be inclined to stay with the policy of replacing three embryos.

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Monozygotic Twinning One undesirable side effect is the increased incidence in monozygotic twinning as a result of blastocyst transfer.

Fig. 34.3: incidence of monozygotic twinning in relation to the day of transfer, per pregnancy and per gestational sac We were the first to report on this feature in 1998 when we found a six fold increase in incidence over day 2 or 3 transfer31, and this finding has been confirmed by a number of others since.32–34 Our first hypothesis was that this might be due to the formation of a ‘figure eight’ blastocyst during the process of hatching from the zona pellucida, but this seems less likely as the Australian center of Alan Trounson, that completely dissolves the zona by chemical means before replacement, also found the same rate. Another possibility is that even small triggers may lead to the formation of a double inner cell mass, as has been experimentally induced in the mouse by Chida.35 It is known that monozygotic twins have a much worse perinatal outcome than dizygotic twins. It is clear that the patient should be informed in advance of this unwanted side effect. Blastocyst Registry It is clear that many aspects concerning the transfer of blastocysts are not yet fully known and understood. Nevertheless many centers all over the world perform day 5 transfer on a wide scale. Therefore it is imperative that there is a registry that documents the outcome of our efforts, where for instance the number of congenital abnormalities and that of the MZ twinning rate is registered. This registry can be found on the Web-site www.blastocyst.net as well as on the ESHRE Web site www.eshre.com. So far more than 60 centers, in total responsible for the birth of more than 3000 babies have agreed to

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participate. We encourage everyone who performs transfer of blastocysts to participate in the registry. CONCLUSION Blastocyst culture and transfer may be an important adjunct in our therapeutic armamentarium. However there is still no consensus which patients might benefit, and how to make the selection, and there are serious drawbacks such as a 6-fold increase in the monozygotic twinning rate. Therefore a number of issues need to be resolved before it can be advised as a general therapy. REFERENCES 1. Buster JE, Bustillo M, Thorneycroft I, Simon JA, Boyers SP, Marshall JR et al. Non-surgical transfer of an in-vivo fertilised donated ovum to an infertility patient. Lancet 1983 (I); 8328:816–7 (letter to the editor). 2. Buster JE, Bustillo M, Rodi IA, Cohen SW, Hamilton M, Simon JA et al. Biologic and morphologic development of donated human ova recovered by nonsurgical uterine lavage. Am J Obstet Gynecol 1985; 153:211–17. 3. Cohen J, Simons RF, Fehilly CB, Fishel SB, Edwards RG, Hewitt J et al. Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage. Lancet 1985 (I); 8429:647. 4. Pub Med search: Blastocyst culture human IVF, limited by year of appearance. 5. Bolton VN, Wren ME, Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 1991; 55:830–32 6. Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Coculture of embryos on Vero cells and transfer of blastocysts in humans. Hum Reprod 1992; 71:101–06 7. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69:84–88. 8. Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod 1998; 13:169–77. 9. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eightcell stages of preimplantation development. Nature 1988 31; 332(6163):459–61. 10. Kadam A Obasaju M, Munne S, Biancardi T, Fateh M, Sultan K. Blastocyst formation rate in chromosomally normal versus abnormal embryos. Fertil Steril 2001; 76 3S 83. 11. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne S. Developmental ability of chromosomally abnormal human embryos to develop fo the blastocyst stage. Hum Reprod 2001; 16:1954–58. 12. Cohen J. Fertil Steril ASRM meeting 2000. 13. Wells D, Delhanty JDA. Comprehensive chromosomal analysis of human Preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod, 2000:6:1055–62. 14. Rijnders PM, Jansen CAM. The predictive value of day 3 embryomorphology for blastocyst formation and implantation rate at day 5 in IVF. Hum Reprod 1998; 13:2869–73. 15. Boonstafar R, Jain JK, Slater CS, Tourgeman DE, Francis MM, Paulson RJ. The prognostic significance of day 3 embryos cleavage stage on subsequent blastocyst development in a sequential culture system. J Ass Reprod Gen 2001; 18:548–50.

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16. Marek D, Langley M, Gardner DK, Confer N, Doody KM, Doody KJ. Introduction of blastocyst culture and transfer for patients in an in vitro—fertilization program. Fertil Steril 1999; 72:1035–40. 17. Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72:218–20. 18. Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer. Fertil Steril 1999; 72:225–8. 19. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 2000; 74:482–86. 20. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998; 13:3434–40. 21. Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Serkine AM, Tesquier L. Blastocyst stage transfer: the real benefits compared with early embryo transfer. Hum Reprod 2000; 15(6):24–30. 22. Hsieh YY, Tsai HD, Chang FC. Routine blastocyst culture and transfer: 201 patients’ experience. J Assist Reprod Genet 2000; 17:405–8. 23. Vidaeff AC, Racowsky C, Rayburn WF. Blastocyst transfer in human in vitro fertilization. A solution to the multiple pregnancy epidemic. J Reprod Med 2000; 45:529–39. 24. Racowsky C, Jackson KV, Cekleniak NA, Fox JH, Hornstein MD, Ginsburg ES. The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil Steril 2000; 73:558–64. 25. Veeck LL. Blastocyst transfer or day 3 transfer. MEFS session at the 57th annual meeting ASRM, 2001. 26. Scholtes MC, Zeilmaker GH. A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil Steril 1996; 65:1245–48. 27. Huisman GJ, Fauser BC, Eijkemans MJ, Pieters MH. Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril 2000; 73:117–22. 28. Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: a controlled randomized trial. Hum Reprod 2000; 15:1947–52. 29. Toledo AA, Wright G, Jones AE, Smith SS, Johnson-Ward J, Brockman WW et al. Blastocyst transfer: A useful tool for reduction of high-order multiple gestations in a human assisted reproduction program. Am J Obstet Gynecol 2000; 183:377–79. 30. Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998; 339:573–77. 31. PM Rijnders, HC Van Os, CAM Jansen. Increased incidence of monozygotic twinning following the transfer of blastocysts in human IVF/ICSI. Fertil. Steril. 1998; 70: Suppl 15–16 32. Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000; 17:349–51. 33. da Costa ALE, Abdelmassih, de Oliveira FG, Abdelmassih V, Abdelmassih R, Nagy ZP et al. Monozygotic twins and transfer at the blastocyst stage after ICSI. Hum Reprod 2001; 16:333– 36. 34. Boonstafar R, Jain JK, Slater C, Tourgeman DE, Francis M, Paulson RJ. High order multiple gestations as a result of monozygotic twinning associated with advanced embryo culture and blastocyst transfer. J Soc Gynecol Investig 2001; 8S:86A 35. Chida S. Monozygous double inner cell masses in mouse blastocysts following fertilization in vitro and in vivo. J In vitro Fert Embryo Transf 1990; 7:177–80.

CHAPTER 35 Really, Just How Important is the Level of Room Lighting in the IVF Laboratory on Embryo Development? KE Tucker, CAM Jansen INTRODUCTION We all are instinctively aware of the many benefits of light, both natural and artificial. Simply, natural light (specifically sunlight) is vital to most plant and animal life on the planet, but the role of light in the in vitro Fertilization (IVF) laboratory is not as lifesupporting. When one considers the in situ environment of oocytes and embryos, the effects of any light may be prof ound as these structures are never exposed to light throughout their development. In nature, there are mechanisms in place that can protect the cell or organism from exposure to unnatural or detrimental environmental conditions and this resilience helps to ensure that development continues. When it comes to the amount of “safe” light exposure for oocytes and embryos in the IVF, how much is too much and under what conditions is the most damage, if any inflicted? Effect of Light: Early Studies One of the earliest studies examining the role of light on embryo development was by Hirao and Yanagimachi1 which revealed that short wavelength visible light (<480 nm), emitted from ordinary sources was detrimental to hamster oocytes. They hypothesized that light disturbs the completion of meiosis after fertilization and that fluorescent light is more harmful than light from incandescent lamps. They recommended minimal exposure to any light and the use of filters (red). Ten years later, other investigators reported a potential risk to rabbit pre-implantation embryos of light.2–3 They found that when early cleavage stage rabbit embryos (Day 1 and 3; morulae) were exposed to standard room light (1600 lux), cell proliferation was significantly impaired. No gross morphological differences were seen. Additional studies included incrementally increasing the exposure time of embryos to room lighting, along with a reduction in room culture temperature. Cleavage stage (Day 1) embryos were the most susceptible to light damage, and although both Day 1 and 3 embryos displayed more tolerance to a drop in temperature, the combined effects of light and temperature amplified the detrimental effects of light, alone;3 cleavage stage rabbit embryos demonstrating an 8-fold lower tolerance to light than to decreased temperature. Further work in this area involved exposing Day 1 rabbit embryos to standard light for 24 hours of culture and then comparing the development of these embryos to controls cultured normally (in the dark) inside the incubator. The results from this study demonstrated that light-exposed embryos exhibited extensive cell degeneration and death. The type of

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damage was different depending on the embryo type. Early cleavage embryos (Day 1) showed cytoplasmic degeneration and nuclear lobulation and Day 3 (morulae) were apoptotic.4–5 Later work by this group of investigators could not demonstrate a significant increase in DNA aneuploidies in control versus light-exposed post-compaction rabbit embryos.6 They did, however, report a significant reduction in cell number in treated embryos. THe Effect of Light: Lessons from Cell Culture The mechanism(s) Whereby light exerts a detrimental effect has been further elucidated using various cell lines. Parshad and co-workers7 demonstrated that exposing mouse lung cells to light during a 24-hour culture period produced chromatid breaks. This effect was enhanced by increasing the concentration of oxygen in the gas phase of the culture environment. Studies using Syrian hamster embryo and human mammary carcinoma cell lines demonstrated that fluorescent light can cause oxidative damage to nuclear DNA within one hour of exposure.8 Other investigators attempted to discern if all light was detrimental to cell replication and division by exposing pig kidney cells to varying wavelengths of light. They found that cells were the least sensitive to green light (wavelength >500 nm), slightly more sensitive to blue light (wavelength 450–490 nm) and highly sensitive to UV light (wavelength 360 nm) with respect to normal chromosome replication, separation and cell division.9 It was hypothesized that the mechanisms surrounding the effect of light seen on these cells involved the formation of intra-cellular free radicals and/or peroxide, perturbing the onset of anaphase. The Effect of Light: Recent Studies Newer studies have confirmed the early findings of Yanaigimachi and co-workers. When early (4–8 cell) hamster embryos were cultured in 1600 lux of light, they demonstrated significantly lower developmental rates than those maintained at 70 lux (darkness)10 (Fig. 35.1). This same study also demonstrated that light irradiation of 1-cell embryos could arrest development even with the use of a yellow filter and reduced light wavelengths. Suppression of development was seen in all embryos, regardless of initial stage at manipulation. Conversely, studies using mouse oocytes reported that exposure to highintensity visible light (4000 lux) for as much as 4 hours did not appear to have any effect on embryonic development, implantation and birth of normal pups.11 These results indicated that the oocyte, at least in the mouse, may be more resilient to the effects of ambient laboratory lighting.

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Fig. 35.1: Development of 1-cell hamster embryos collected and cultured under dark (70 lux) vs. light (1600 lux) conditions (10 min. exposure time); adapted from Umaoka et al, 1992. The precise effect of light on embryonic development is still unclear. It has long been thought that light may cause some degree of oxidative damage, which also occurs when oxygen levels in the culture micro-environment are elevated. Investigators believed that both exposure to light and higher oxygen concentrations combine to contribute to the 2cell block often seen with in vitro cultured rodent embryos.12–14 Spare human embryos cultured under low oxygen conditions (5%) and low illumination (20 lux room light and 100 lux from the microscope) had a blastulation rate of 58.5 percent, which was more than that of embryos exposed to higher oxygen concentration and light intensities.15 Dumoulin and co-workers16 reported similar results in human embryos cultured in 5 versus 20 percent oxygen and also found that more blastocysts in the high-oxygen group had an abnormally low cell number. When one-cell hamster embryos were exposed to 40, 20 or 5 percent oxygen, an inverse felationship could be demonstrated between oxygen concentration and embryonic development.17 These authors concluded that oxidative stress was more detrimental than a decrease in temperature or an increase in pH. This hypothesis was further tested using the comet assay, which measures the degree of DNA damage. This assay was used to confirm the reports that fluorescent light damages the DNA of an embryo via an elevated production of reactive oxygen species (ROS).18 The effect of increased exposure times on 1-cell hamster embryos to fluorescent light as measured by the comet assay is illustrated in Figure 35.2. DNA damage (changes in migration length across an electrically charged field) was significantly increased in a dose-dependent manner with increasing exposure time

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Fig. 35.2: Effect of increasing exposure of 1-cell hamster embryos to fluorescent light. Control embryos were maintained in the dark under the exact conditions as the treated embryos. (Control: n=25, 5 min: n=24, 15 min: n=27, 30 min: n=24). Adapted from Umaoka et al, 1993. and was higher overall when compared with control embryos cultured in the dark. The Effect of Light: Toxic Effect of Oxygen The role of oxygen in embryonic metabolism is crucial and requires the balance between useful and harmful effects. The concentration gradient of oxygen is regulated by its consumption during oxidative phosphorylation, by the availability of substrates and by the integrity of the mitochondrial membrane. The generation of ROS occurs if more oxygen is available, generating an excess of highenergy electrons, thereby damaging the mitochondria in the cells of the embryos.19,20 A schematic representation of the mechanism of cellular damage caused by ROS is depicted in Figure 35.3.

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Fig. 35.3: Proposed pathway leading to cellular damage by ROS, based on Catt and Henman, 200019 Investigators have shown that there is an overall increase in DNA damage in embryos developed in vitro rather than in vivo, possibly due to higher levels of ROS generated under normal culture conditions.14 Others have further reported that the higher concentration of ROS found in fragmented human embryos was associated with an increased level of apoptosis.21 A study by Saka and Schultz verified earlier findings, using the comet assay with UV light as a positive control. They showed that the DNA of 1-cell hamster embryos was damaged in a similar fashion by both UV light and peroxide. They also reported that even a very short exposure to UV light (<1 min.) had a negative effect on embryo development, even if cultured in the dark. In addition, these investigators were able to demonstrate that visible light also caused DNA damage, but that infrared light did not. Protection against ROS The evidence in the literature indicates that oxidative stress is directly involved in the etiology of defective embryo development and that light is not the only culprit in stimulating the production of ROS. Reactive oxygen species may originate from embryo metabolism or ctdture environment. The embryo is equipped with a fair number of mechanisms to cope with oxidative stress, such as antioxidant enzymes (e.g. superoxide dismutase, taurine, hypotaurine and ascorbic acid).20 The effects of ROS may, therefore, be reduced by altering the culture environment. In addition to minimizing exposure to high-intensity light, the oxygen tension in the incubator can be lowered. Certain incubators (MINC incubator; Cook) allow the internal environment to be set by the

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operator. The use of a gas pre-mix, made up of 5 percent carbon dioxide, 5 percent oxygen and 90 percent nitrogen is also a viable alternative. Other research has indicated that adding free radical scavengers to the culture medium (i.e. ethylenediaminetetraacetic acid—EDTA, superoxide dismutase—SOD, catalase, vitamins E and C, amino acids) might be beneficial to healthy embryo development in response to increased oxygen content in the microenvironment.22–23 High O2 concentration (20%) was shown to have a deleterious effect on mouse embryo development, but when cultured in the presence of anti-oxidants, these embryos displayed a significantly improved blastocyst formation rate, especially in the presence of EDTA. Although EDTA leads to higher numbers of blastocysts and increased hatching rates, these embryos, themselves, contained fewer cells. Reducing O2 content to 5 percent was as effective as EDTA in enhancing embryo development, but did not negatively affect cell numbers.23 THE EFFECT OF LIGHT: SUMMARY AND CONCLUSIONS Several investigators have demonstrated that room lighting, either incandescent or fluorescent, can be detrimental at some level to embryo development in vitro. The effects seen are similar to those seen with increased microenvironmental oxygen and are thought to be mediated via radical oxygen species. The application of the comet assay further demonstrated that light can directly cause nuclear damage in early or cleavage stage embryos. Adding free radical scavengers to culture media and reducing the O2 content in the incubator can help attenuate some of the damage caused by the production of ROS due in part to excessive exposure to light. Care should still be taken to minimize both the duration of embryo handling in lighted environments and the intensity of the light itself. The use of filters for microscopes and room lighting should be employed. If the appropriate filters are not available for overhead lights, then a simpler alternative would be to turn off all unnecessary lights when oocytes and embryos are not in the incubator. The effects of light do not appear to be absolute or entirely irreversible. Some room light is not only acceptable, but is also necessary for the safe handling of gametes and embryos. Working in the dark is hazardous for laboratory personnel and can pose a risk for embryo/oocyte identification if labels are not easily readable. The laboratory does not have to be a dungeon to provide an embryo-friendly environment. Judicious use of weak or indirect lighting, for short periods of time, may be enough. It is important to keep in mind that maintaining healthy embryos in culture is a multi-faceted undertaking, and minimizing ambient room lighting is only one of the many steps that we must take to ensure healthy embryos for our patients. REFERENCES 1. Hirao Y, Yanaigimachi R. Detrimental effect of visible light on meiosis of mammalian eggs in vitro. J Exp Zool 1978; 206:365–69. 2. Fischer B, Schumacher A, Hegele-Hartung C, Beier HM. Potential risk of light and room temperature exposure to preimplantation embryos. Fertil Steril 1988; 50:938–44.

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3. Schumacher A, Fischer B. Inluence of visible light and room temperature on cell proliferation in preimplantation rabbit embryos. J Reprod Fertil 1988; 84:197–204. 4. Hegele-Hartung C, Schumacher A, Fischer B. Ultrastructure of preimplantation rabbit embryos exposed to visible light and room temperature. Anat Embryol (Berl) 1988; 178:229–41. 5. Hegele-Hartung C, Schumacher A, Fischer B. Effects of visible light and room temperature on the ultrastructure of preimplantation rabbit embryos: a time course study. Anat Embryol (Berl) 1991; 183:559–71. 6. Schumacher A, Armin, Kesdogan, Jadigar, Fischer B, Bernd. DNA ploidy abnormalities in rabbit preimplantation embryos are not increase by conditions associated with in vitro culture. Mol Reprod Dev 1998; 50:30–34. 7. Parshad R, Sanford KK, Jones GM, Tarone RE. Fluorescent light-induced chromosome damage and its prevention in mouse cells in culture. Proc Natl Acad Sci USA 1978; 75:1830–33. 8. Mauthe RJ, Cook VM, Coffing SL, Baird WM. Exposure of mammalian cell cultures to benzo [a] pyrene and light results in oxidative DNA damage as measured by 8-hydroxyguanosine formation. Carcinogenesis 1995; 16:133–9. 9. Gorgidze LA, Oshemkova SA, Vorobjev IA. Blue light inhibits mitosis in tissue culture cells. Biosci Rep 1998; 18:215–24. 10. Umaoka Y, Noda Y, Nakayama T, Narimoto K. Effect of visual light on In vitro embryonic development in the hamster. Theriogenology 1992; 3:1043. 11. Barlow P, Puissant F, van der Zwalmen P, Vandromme J, Trigaux P, Leroy F. In vitro fertilization, development and implantation after exposure of mature mouse oocytes to visible light. Mol Reprod Dev 1992; 33:297–302. 12. Goto Y, Noda Y, Narimoto K, Umaoka Y, Mori T. Oxidative stress on mouse embryo development in vitro. Free Radic Biol Med 1992; 13:47–53. 13. Nakayama T, Noda Y, Unoaka Y, Narimoto K. Development to the four-cell stage of hamster embryos fertilized in vitro. Theriogenology 1993; 3:1221–6. 14. Johnson MH, Nasr-Esfahani MH. Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? Bioessays 1994; 6:31–8. 15. Noda Y, Goto Y, Umaoka Y, Shiotani M, Nakayama T, Mri T. Culture of human embryos in alpha modification of Eagle’s medium under low oxygen tension and low illumination. Fertil Steril 1994; 62:1022–27. 16. Dumoulin JC, Meijers CJ, Bras M, Coonen E, Geraedts JP, Evers JL. Effect of oxygen concentration on human in-vitro fertilization and embryos culture. Hum Reprod 1999; 14:465– 69. 17. Umaoka Y, Noda Y, Nakayama T, Narimoto K, Mori T, Iritani A. Development of hamster one-cell embryos recovered under different conditions to the blastocyst stage. Theriogenology 1993; 3(2):485. 18. Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano A. Assessment of DNA damage in individual hamster embryos by comet assay. Mol Reprod Dev 1999; 54:1–7. 19. Catt JW, Henman M. Toxic effects of oxygen on human embryo development. Hum Reprod 2001; 15:199–206. 20. Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 2001; 7:175– 89. 21. Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod 1998; 13:998–1002. 22. Bavister BD. Interactions between embryos and the culture milieu. Theriogenology 2000; 53:619–26. 23. Orsi NM, Leese HJ. Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, Oxygen tension, catalse, superoxide dismutase and pyruvate. Mol Reprod Dev 2001; 59:44–53.

CHAPTER 36 The Mouse Embryo Bioassay: Is It the “Gold Standard” for Quality Control Testing in the IVF Laboratory? KE Tucker, CAM Jansen INTRODUCTION In any diagnostic laboratory, the design and application of appropriate and conscientious internal quality control (QC) procedures and external quality assurance (QA) programs, is critical for establishing and maintaining the highest level of patient care. An excellent quality management program revolves around the creed of continuously striving to improve all aspects of every laboratory procedure; providing services that are safe and error-free. The role of QC procedures in the in υitro Fertilization (IVF) laboratory, is to fine-tune existing protocols in order to more effectively help infertile patients in their quest to have a healthy baby. Laboratory quality management consists of three main components, quality control (QC), Quality assurance (QA) and quality improvement (QI). Quality control can be defined as a systematic group of activities designed to recognize errors, to further ensure that results are correct (internal controls) and to establish thresholds for all aspects of any testing procedure. Quality assurance is based on the retrospective surveillance of all quality issues (results from QC testing and analyses) as they apply to clinical outcomes (i.e. fertilization, embryo development, pregnancy and implantation rates). Quality assurance encompasses those modifications made in current laboratory and clinical procedures, testing or policy that will ensure the highest standard of treatment and level of patient care. Since its introduction for use in the IVF laboratory, the mouse embryo bioassay has been instrumental for QC, QA and QI by establishing basic guidelines for and improving human embryo growth in vitro.1–4 Many laboratories have implemented this test to such a degree that it forms the very backbone of in-house troubleshooting. There are others, however, that maintain that the mouse embryo bioassay is insensitive; preferring other methods of bioassay (i.e. human or rodent sperm, cell lines, DNA analyses). The purpose of this paper is to reinvestigate the strength of the mouse embryo bioassay in monitoring the culture environment in the human IVF laboratory and to ascertain if this test is still the “gold standard” for QC testing. Why do Bioassay Testing? BecauseWe Have to! Shortly after the “conception” of IVF, the need for testing of all contact materials was realized.5 Recently, scientific societies and regulating bodies have strongly recommended that rigorous evaluation of the culture environment be mandatory and routine. The European Society for Human Reproduction and Embryology (ESHRE) and the

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Association of Clinical Embryologists (ACE) guidelines state that “laboratory protocols should include in-house quality control procedures using an appropriate bioassay system.” The laboratory accreditation inspection checklist issued by the College of American Pathologists (CAP) states specifically that “media must be evaluated using a bioassay system such as the one or two-cell mouse embryo or a sperm motility assay” and that all contact materials not previously tested by the manufacturer or supplier, be evaluated before use by a bioassay. Testing is particularly important when culture media are made in-house. Most laboratories do not require legislation to see the benefit of implementing some form of bioassay in their quality management program. Debates exist, however, as to which is the definitive test and the one to employ. Types of Bioassays There are several assay methods and cell-types that are useful for testing supplies, procedures and environmental influences in the IVF laboratory. In addition to the mouse embryo, which will be discussed in greater detail later, there are two other fairly widely used tests; the sperm motility assay and the use of cell lines. Sperm Motility Assay This test can be performed either with human or hamster sperm and is considered by its proponents to be a superior method of detecting embryotoxic or embryo-“unfriendly” substances in culture media. Several investigators have shown that the sperm bioassay meets the criteria for a reliable bioassay6 Bavister and Andrews6 reported that the Hamster Sperm Motility Assay (HSMA) is simple and sensitive. Rinehart and coworkers7 further demonstrated that the HSMA to be more sensitive in discriminating types of water used in preparing culture media and a slightly later study by Gorill and others8 found this test to be very repeatable. Other investigators found that mouse sperm more effective at detecting low levels of endotoxin in a short period of time (4–6 hours) compared to mouse embryos and to hamster and human sperm.9 Conversely, it was shown that human sperm could, in fact, detect similar levels of toxicity in surgical gloves.10 Later, Morimoto and others11 also reported that the human sperm survival test could reliably detect endotoxin in all contact supplies and culture media. A recent study further demonstrated that human sperm could detect toxins in serum albumin12 and that results from the sperm assay were comparable to those of the mouse embryo bioassay. Cell Lines ®

Hybritest (Medicult) is a commercially available cell line designed for testing culture media used for IVF. It is derived from the rapidly growing mouse hybridoma cell line (1E6) in a defined serum-free culture medium. The use of serum-free conditions has been shown to greatly increase the sensitivity of these cells to toxic substances, presumably due to the absence of binding proteins. The authors claim this test is simple and sensitive,13 but the largest drawback is that this cell line may not be useful when testing

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complete culture media, including those that may contain serum or serum components. There is little else published regarding other cell types used for quality control in IVF. The Mouse Embryo The mouse embryo bioassay is probably the most commonly used evaluative test in the IVF laboratory. The opponents of this assay maintain, however, that it is cumbersome and not sensitive enough to detect substances that may affect IVF outcome.14 This paper will attempt to identify methods of utilizing the mouse embryo assay in order to maximize its repeatability, sensitivity and predictive value in the IVF laboratory. The Two-Cell Embryo Mouse embryos can be used at different stages of development. Two-cell embryos can be harvested fresh or purchased frozen and have been used extensively in developing a competent bioassay for the IVF laboratory. In 1984, Ackerman and co-workers5 evaluated the use of the two-cell mouse embryo in testing contact materials. They found that when fresh two-cell B6CBAF1 mouse embryos were used to compare two different media (Ham’s F10+15 percent fetal cord serum vs. Kreb’s medium), no initial difference was observed in embryo development between the two. When exposed to several types of Vacutainer blood collection tubes, however, the development of the embryos cultured in Kreb’s medium was significantly impaired compared to those in the Ham’s F10. Other early work with the two-cell mouse embryo was able to detect a toxic effect of powdered or un-powdered latex gloves, in an exposure duration-dependent manner.15–18 In addition, Naaktgeboren17 reported a detrimental effect of a temporary drop in pH on the development of mouse embryos. Vijayakumar and others18 reported that, based on the predictive value of the two-cell mouse embryo to detect nontoxic culture media, this bioassay was routinely implemented to test all batches of new media. More recent studies have shown that when compared to the one-cell, the two-cell mouse embryo was on average less sensitive, but when a difference in test materials was detected, this test was, in fact, predictive of human embryo quality19 and instrumental in increasing their take-home baby rate. The One-Cell Embryo Most investigators agree that the one-cell mouse embryo is more sensitive than the twocell in responding to toxins in the culture environment Scott and others20 compared one and two-cell CD-1 embryos in their ability to test water quality, contaminants in the culture system and fluctuations in environmental conditions (using a culture medium optimized for this strain of mouse). They found that both embryo stages were unable to distinguish between human embryo culture media and was attributed to mouse strain. Even though both embryo stages responded to changes in water quality, pH, temperature, incubator conditions and contaminants when grown in “their” medium (medium optimized for this strain of mouse), the one-cell embryos were more sensitive overall. Studies have shown that one-cell embryos from CD-1 mice were even more sensitive to suboptimal conditions when the zona pellucida was removed.21 Conversely, other

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investigators reported that embryos from CD-1 mice were still able to develop in compromised culture conditions even with the zona removed. They did find, however, that embryos from a different strain of mouse (B6CBA/F1J), when cultured under identical conditions, were more sensitive. The one-cell was more sensitive that the twocell stage and this sensitivity was further increased with zona removal.22 Recently, results from the mouse one-cell assay were compared with those from the hamster oocyte comet assay, which measures the degree of DNA fragmentation. Both methods were used in a proficiency test, with matched results.23 Mouse Embryo Bioassay: Directions for the Future The reports in the literature give a vague impression of the definitive use of the mouse embryo bioassay. The effects of certain toxic substances have been reported, but it would appear that when compared with other bioassays, the sensitivity of the mouse embryo is less. How can we change that? There have been several proposals made in the last few years, but the most important fact that routinely eludes us, it that the embryo bioassay has settled into a position of providing laboratories with subjective impressions as opposed to objective facts. We have to remember that the mouse embryo assay is still an “assay” and should be treated as such. How do we go about doing this? Firstly, there must be both positive and negative controls whenever possible. Dubin and co-workers24 reported a huge variability in effects of endotoxin with the same reported potency on mouse embryo development. They concluded that their most important finding was that a well-defined positive control be included when performing every embryo assay Basically, individual laboratories must establish a mouse embryo assay that will fail under the appropriate conditions. Repeatability of the assay is also critical, but difficult to achleve in a constantly fluctuating environment. Marc van den Bergh19 writes that it is important to remember that it is not only the culture media that vary, but also the sum of the changes in the physical environment that occur during the culture procedure, such as incubator performance, lab temperature and, most importantly, in the technical ability of the laboratory personnel. In 1995, Roussev and coworkers25 had already published their quidelines for significantly decreasing the variability in the mouse embryo bioassay. When used in the detection of endotoxin, they were able to perform this test with a 9 percent coefficient of variation and an inter- and intraassay operator variability of as little as 4 percent. Validation of the assay or confirmation of results can be established. Additional tests can also performed in conjunction with select or all mouse embryo assays. Claassens and others12 have suggested that the use of the human sperm motility assay may have more relevance on human embryo development if run along with the mouse embryo bioassay. Perhaps this will not be necessary for all supplies and conditions; each laboratory must define its own criteria to optimize testing. As mentioned above, the type and quality of the embryos themselves may affect the outcome of the assay and must be closely controlled. Not every strain of mouse embryo will be appropriate for use in a bioassay.22,26 It is important for each laboratory to set individual limits and thresholds of response. This includes selecting the appropriate embryo stage and morphology. If a zona-free one-cell mouse embryo appears to be the

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most sensitive and gives the most reliable and repeatable results for your laboratory environment and culture conditions, then that is what should be used. Each laboratory is different and the mouse embryo bioassay should be validated for each new environment. SUMMARY During the infancy of IVF, the use of the mouse embryo to decipher optimal culture conditions for human embryos was considered important in establishing good quality control in the laboratory and clinic. Other investigators challenged the sensitivity of the mouse embryo in detecting toxic substances or conditions and introduced the sperm motility assay. Still others felt that the use of commercially produced cell lines may be more useful for QC in the IVF laboratory. Although possibly more sensitive to certain substances and definitely more time-efficient, the question remains as to whether a sperm cell (or cell line) can reliably respond to culture components and conditions in the same fashion as an embryo? The answer is, probably not, which is why the mouse embryo should not be disregarded. If the mouse embryo bioassay is not the “Gold Standard” for quality assessment in the IVF laboratory, then it is because of our own complacency when imposing strict assay guidelines to this test. Ask an endocrinologist and you’ll find out that the results of any hormone assay performed without defined standards, appropriate controls, low coefficients of variability and documented levels of sensitivity are considered essentially worthless. It has been documented that we can optimize the sensitivity of this bioassay with the appropriate stage, strain and morphology of the mouse embryo. We can validate embryo response with sperm motility and with the hamster oocyte comet assays, but ultimately, the best model to predict the development of one kind of embryo is another embryo. REFERENCES 1. Fleming TP, Pratt HP, Braude PR. The use of mouse preimplantation embryos for quality control of culture reagents in human in vitro fertilization. Fertil Steril 1987; 47:858–60. 2. McDowell JS, Swanson RJ, Maloney M, Veek L. Mouse embryo quality control for toxicity determination in the Norf olk in vitro fertilization program. J In Vitro Fert Embryo Transf 1988; 5:144–48. 3. Tucker KE, Hurst BS, Guadagnoli S, Dymecki C, Mendelsberg B, Awoniyi CA et al. Evaluation of synthietic serum substitute versus serum as protein for mouse and human embryos culture. J Assist Reprod Genet 1996; 13:32–37. 4. Quinn P, Horstman FC. In the mouse a good model for the human with respect to the development of thepreimplantation embryo in vitro? Hum Reprod 1998; 13(Suppl 4):173–83. 5. Ackerman SB, Taylor SP, Swanson RJ, Laurell LH. Mouse embryo culture for screening in human IVF. Arch Androl 1984; 12(Suppl):129–36. 6. Bavister BD, Andrews JC. A rapid sperm motility bioassay procedure for quality-control testing of water and culture. J In Vitro Fert Embryo Transf 1988; 5:67–75. 7. Rinehart JS, Bavister BD, Gerrity M. Quality control in the in vitro fertilization laboratory: comparison of bioassay systems for water quality. J in vitro Fert Embryo Transf 1988; 5:335– 42.

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8. Gorrill MJ, Rinehart JS, Tamhane AC, Gerrity M. Comparison of the hamster sperm motility assay to the mouse one-cell and two-cell embryo bioassays as quality control tests for in vitro fertilization. Fertil Steril 1991; 55:345–54. 9. Esterhuizen AD, Bosman E, Botes AD, Groeneveld OA, Giesteira MV, Labuschagne GP et al. A comparative study on the diagnostic sensitivity of rodent sperm and embryos in the detection of endotoxin in Earle’s balanced salt solution. J Assist Reprod Genet 1994; 11:38–42. 10. Critchlow JD, Matson PL, Newman MC, Horne G, Troup SA, Lieberman BA. Quality control in an in vitro fertilization laboratory: use of human sperm survival studies. 1989; 4:545–49. 11. Morimoto X Hayashi E, Ohno T, Kawata A, Horikoshi Y, Kanzaki H. Quality control of human IVF/ICSI program using endotoxin measurement and sperm survival test. Hum Cell 1997; 10:271–76. 12. Claassens OE, Wehr JB, Harrison KL. Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing. Hum Reprod 2000; 15:1586–91. 13. Bertheussen K, Holst N, Forsdahl F, Hoie KE. A new cell culture assay for quality control in IVF. Hum Reprod 1989; 4:531–35. 14. George MA, Braude PR, Johnson MH, Sweetnam DG. Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse embryos is unaffected by the quality of water used in culture media. Hum Reprod 1989; 4:826–31. 15. Kruger TF, Cronje HS, Stander FS, Menkveld R, conradie E. The effect of surgical glove powder on cleavage of two-cell mouse embryos in an in vitro fertilization programme. S Afr Med J 1985; 67:241–42. 16. Naz RK, Janousek JT, Moody T, Stillman RJ. Factors influencing murine embryo bioassay: effects of proteins, aging of medium and surgical glove coatings. Fertil Steril 1986; 46:914–19. 17. Naaktgeboren N. Quality control of culture media for in vitro fertilization. Ann Biol Clin 1987; 45:368–72. 18. Vijayakumar R, Simoni J, Ndubisi B, DeLeon F, Heine W. Mouse embryo growth in different culture media: selection of a medium for quality control cross-testing of human in vitro fertilizaton conditions. Arch Androl 1987; 19:149–58. 19. Van den Bergh M, Baszo I, Biramane J, Bertrand E, Devrecker F, Englert Y. Quality control in IVF with mouse bioassays: a four years’ experience. J Assist Reprod Genet 1996; 13:733–38. 20. Scott LF, Sundaram SG, Smith S. The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program. Fertil Steril 1993; 60:559–68. 21. Montoro L, Subias E, Young P, Baccaro M, Swanson J, Suedo C. Detection of endotoxin in human in vitro fertilization by the zonafree mouse mebryo assay. Fertil Steril 1990; 54:109–12. 22. Fleetham JA, Pattinson HA, Mortimer D. The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization. Fertil Steril 1993; 59:192–96. 23. Chan PJ, Calinisan JH, Corselli JU, Patton WC, King A. Updating quality control assays in the assisted reproductive technologies laboratory with a cryopreserved hamster oocyte DNA cytogenotoxic assay. J Assis Reprod Genet 2001; 18:129–34. 24. Dubin NH, Bornstein DR, Gong Y. Use of endotoxin as a positive (toxic) control in the mouse embryo assay. J Assist Reprod Genet 1995; 12:147–52. 25. Roussev RG, Stern JJ, Thorsell LP, Thamoson FJ, Coulam CB. Validation of an embryotoxicity assay. Am J Reprod immunol 1995; 33:171–75. 26. Kruger TF, Stander FS. A comparative study of two-cell embryos of CBA and F1 mice in a human in vitro fertilization program. S Afr Med J 1984; 65:209–10.

CHAPTER 37 Human Oocyte and Embryo Cryopreservation Michael J Tucker SUMMARY OF SYLLABUS • Brief overview of human egg and embryo freezing. • Discussion of merits of currently utilized cryopreservation protocols. • Practical issues of cryopreservation with reference to egg or embryo selection, and thaw replacement protocols. • Alternative cryopreservation technologies. Cryostorage of the Female Gamete The last few years have seen a significant resurgence of interest in the potential benefits of human egg freezing. Essentially, these benefits are: 1. Formation of donor “egg banks” to facilitate and lessen the cost of oocyte donation for women that are unable to produce their own oocytes. 2. Provision of egg cryostorage for women wishing to delay their reproductive choices. 3. Convenient cryopreservation of ovarian tissue taken from women about to undergo therapy deleterious to such tissue, which may threaten their reproductive health. The technology so far applied clinically has been based directly on traditional human embryo cryopreservation protocols, and has produced relatively few offspring. Fortunately to date, no abnormalities have been reported from these pregnancies, regardless of the persistent concerns that freezing and thawing of mature oocytes may disrupt the meiotic spindle and thus increase the potential for aneuploidy in the embryos arising from such eggs. With respect to cryostorage of donated oocytes there have been several reports that have shown some success with this approach.1–3 Six pregnancies have generated 10 babies from cryopreserved donor oocytes in these reports. Use of frozen donor oocytes post-thaw not for whole egg donation, but for ooplasmic transfer has been reported with a successful delivery of a twin following thawed ooplasmic donation.4 Cryostorage of women’s own oocytes was originally reported in the case of three births over a decade ago by two centers.5–6 More recently these successes have been reproduced by others,3,7,8 and giving9 rise to 10 babies. One other baby has arisen from a clinical circumstance that is not completely unfamiliar to IVF clinics: oocytes had been collected but no sperm were retrievable for insemination. In this case, the oocytes were frozen, and donor semen was selected for future use. Ultimately both sets of gametes were thawed and used in a subsequent IVF attempt, which achieved a healthy delivery (Moody and San Roman, personal communication). All of these pregnancies were from frozen-thawed mature oocytes, but for one notable exception, where a pregnancy arose from an immature germinal vesicle (GV) stage egg.8

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Interestingly, this stage of egg development might prove to be a more successful approach for cryopreservationbecause its oolemma is more permeable to cryoprotectant, and its chromatin is more conveniently and safely packaged in the nucleus.10 Such eggs, however, still have to undergo GV breakdown and maturation to the MII stage before fertilization, and therefore their developmental competency is not so clearly established as with fully mature oocytes that are frozen. Source of the GV eggs and whether they have been exposed to any exogenous gonadotropins may play a key role in the competency of these eggs.11 Whether mature or not, standard cryopreservation technologies appear to have their ultimate limitations in not only cryosurvival, but also more importantly in their lack of consistency. Fifty percent cryosurvival is an adequate overall outcome, but not if it is a statistic that is arrived at by 90–100 percent survival in one case, and 0–10 percent in the next. Consequently, radically different types of protocol may provide the answer to increased consistent success. One approach has been to replace sodium as the principal cation in the cryoprotectant with choline in an attempt to shut down ihe sodium ion pumps in the oocyte membrane during cryoprotectant exposure, thus minimizing potentially deleterious “solution effects” during cooling.12 This has provided significant improvements in murine egg freezing, though it has yet to be applied clinically in the human. Alternatively, traditional slow cooling/rapid thaw protocols might be replaced with vitrification. Which again has been successfully applied in the mouse,13 bovine,14 and very recently in the human.9 While the mouse can be a useful model, it must be remembered that the murine oocyte is only just over half the volume of a human oocyte; this can have a major impact on permeability and perfusion of the two types of egg.15 ICSI has become the accepted norm for insemination of oocytes post-thaw, to avoid any reduction in sperm penetration of the zona with premature cortical granule release.16 The most plentiful source of oocytes potentially is ovarian tissue itself, containing as it does many thousands of primordial follicles in healthy cortical tissue. Earlier successful work with cryopreservation of rodent ovarian tissue has led the way to successful cryostorage of both sheep and human tissue.16–17 Up to 80 percent survival of follicles has been reported, but the issue is how to handle this tissue following its thaw. Tissue that has been removed, for example, from a woman about to undergo cancer therapy may contain malignant cells, and therefore may not be safely used for auto-grafting to in such a woman if she were to survive. The tissue might be screened before or after thawing for the presence of malignant cells to enable some assessment of the safety of such an approach, or it may be grown in a host animal (e.g., SCID mouse) until such time as in vitro maturation could be undertaken more effectively. Extended culture of primordial follicles to full oocyte maturity, with subsequent embryonic development and birth has only been recorded in the mouse, and this was not from cryopreserved tissue.18 Early studies are being undertaken in the human44 with much to be done. Fertility has been restored in sheep, in a good model for the human ovary, following cryostorage of ovarian cortex and auto-grafting19 and this seems the most likely successful clinical model for restoration of fertility of women who are at risk of losing their ovarian function. This may include not only women about to undergo cancer therapy, but also women who have a family history of early menopause, and those with non-malignant diseases such as thalassemia or certain auto-immune conditions which may be treated by high-dose chemotherapy. Recently, it was reported that ovarian function was restored by such

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means in the human, in a 29 year old patient suffering from hypothalamic amenorrhea subsequent to removal of both her ovaries at age 17.20 The myriad routes for cryostorage of the female gamete makes for a confusing vision of where clinical applications may occur. However, different clinical needs may actually be met by differing technological approaches, whether they incorporate whole tissue freezing, separate follicle storage, or cryopreservation of mature oocytes themselves. For our current most consistent protocol for cryopreservation of oocytes retrieved following ovarian stimulation refer to Appendix 37.1. Cryopreservation of the Preimplantation Human Embryo While human embryo cryopreservation has become a well-established technology in assisted human reproduction, it has yet to become fully clear as to which stage preimplantation embryos are best cryostored. Indeed on the face of it, the superiority of blastocyst stage freezing over 1-cell pronucleate stage freezing in terms of implantation per thawed embryo transferred, is countered by the loss of embryos that lack the wherewithal to grow for five to six days in vitro.21 Countering the benefits of freezing cleavage stage embryos is the partial survival of multi-cellular embryos,22 where “partial” embryos may give rise to live births even from one surviving cell, but viability is reduced.23 Ultimately, there seems to remain some degree of clinic choice of philosophy of approach over when to freeze.23 If one were to assume, however, that the majority of in vitro culture of human embryos might eventually be carried out to the blastocyst stage, then it would seem redundant to freeze embryos at an earlier stage. Not to belabor the point, but selection is the central essence of extended culture, enabling poorer viability embryos to arrest in development so “selecting” themselves as non-candidates for fresh transfer or cryopreservation. Although to some this may seem wasteful of embryos, the net result is probably that chances of pregnancy are more clearly defined and potentially improved, whilst the risks of higher order multiple implantation is reduced. Additionally, fewer embryos are frozen as blastocysts, reducing storage requirements, and improving expectations of pregnancy from those embryos that are frozen. Therefore, overall efficiency will be increased. Nevertheless, given the consistently high rates of cryosurvival of cryopreserved early stage embryos, there will probably continue to be certain clinical circumstances where early stage freezing is justified. If a clinic wishes to move its cryopreservation program to blastocyst stage principally, a key question would be what to do with the early stage embryos already cryostored? One progressive approach may be to thaw all embryos at these earlier stages and grow them to blastocysts if possible. In this way, fewer embryos will be kept cryostored, and if an excess of embryos for transfer do reach the blastocyst stage, then they may be re-frozen for later use. The first successful reports of human blastocyst cryopreservation came from culture in a simple salt solution.24–25 More recently most cryopreserved blastocysts arose from extended culture of supernumerary embryos not transferred fresh on day-two or -three, usually using co-culture.26–27 However, with increasing confidence in growth stagesequenced culture media, blastocyst culture for fresh transfer has become increasingly common. More convenient cryopreservation protocols for blastocysts28 have also improved the ease with which this adjustment in a clinic’s protocols may be made.

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Consistently high cryosurvival rates (approximately 90%), and good post-thaw pregnancy rates (38%) are now being achieved by certain clinics with judicious selection of blastocysts for freezing (Marek and Langley, personal communication). The key is how to select potentially viable blastocysts. As culture is extended over a longer period, the rate of development becomes an increasingly important parameter for blastocyst seleo tion.29 However, a range of selection criteria need to be applied to optimize the choice of the blastocysts with the best potential for successful cryopreservation (see below). Selection Criteria forHuman Blastocysts for Cryopreservation • Expanded blastocyst growth rate: day-5> day-6> day-7. • Overall cell number ≥60 cells (depending on day of development). • Relative cell allocation to trophectoderm/inner cell mass. • Original quality of early stage embryo: PN formation, blastomere regularity, mononucleation, fragmentation. Issues such as how “early” a blastocyst can be frozen, or if blastocysts that are partially or totally hatched can be consistently cryopreserved, have yet to be adequately answered. Much data may exist from mouse and bovine models, for example, however cell number and levels of lipidation may have a profound differential impact thus minimizing the usefulness of such comparative studies. Hence, data will be collected, as has often been the case with human ART, prospectively and used to fine tune future protocols from clinical hindsight. Most embryo cryopreservation protocols currently use a slow freeze/ rapid thaw approach. Roughly speaking, slow freeze protocols utilize lower concentrations of cryprotectants (approx. 1.5 M) to avoid the toxicity of such agents during the initial exposure and slow cooling; higher concentrations of cryoprotectant (approx. 4.0 M) allow shorter exposure times to the cryoprotectant and “rapid freezing”. Vitrification, using concentrations as high as 6.0 M allow extremely high rates of cooling and thawing (>22,000°C/ min) without the formation of ice. However, these more convenient protocols of ultra-rapid freezing and vitrification, that eliminate the use of expensive controlled rate freezers, await cross over from use in other species, or validation from more extensive experimental study in humans.14,30,31 Regardless of the uncertainties of which protocols for cryopreservation will prevail, the future seems to point to increasing success and consistency with embryo cryopreservation. The preparation of the uterus into which the thawed embryos will ultimately be placed seems to be an area of study that is better resolved, with both natural and hormone replacement cycles providing comparable levels of receptivity in naturally cycling women, though differing levels of convenience.23 Indeed, artificially prepared cycles may even effectively dispense with the use of gonadotropin releasing hormone agonists to lessen cost and improve convenience without loss of success.32 Why Freeze Embryos at the Blastocyst Stage? The very first report of successful cryopreservation of the human embryo was in 198333 with a pregnancy arising from the freezing in DMSO, thawing and transfer of an eightcell embryo. Within a year or so appeared the first successes from the use of glycerol to cryopreserve human blastocysts.24–25 In the same year Lassalle et al34 published a simple

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but consistent protocol using propanediol plus sucrose (1985) that has become probably the most commonly used approach for freezing both early cleavage stage and pronucleate one-cell embryos. Attempts to improve on the consistency and convenience of cryopreserving blastocysts reappeared when, using Vero cell co-culture to enhance extended culture,35 explored the use of a combination of glycerol and sucrose as cryoprotectants to freeze “spare” expanded blastocysts. Essentially, all of the above protocols employed a “slow freeze/rapid thaw” approach, requiring the use of a programmable freezer for the controlled rate cooling to temperatures between minus 35°C to minus 150°C. Variants of these protocols remain the current standards for human embryo cryopreservation. With an increase in IVF-ET cycles being extended to incorporate the fresh transfer of blastocysts, blastocyst cryopreservation is no longer being considered as a last option for “left over” embryos that develop to this stage. Increasingly, it is being considered as the sole or at least principal stage at which to freeze. The reason for this is that if selection of blastocysts is to be optimized, then freezing embryos at an earlier stage would reduce the pool from which to choose fresh blastocysts for transfer. Concerns that embryos are in some way being “lost” due to extended culture, because fewer embryos are being used overall compared with previous approaches adopting day-two and three transfer, will be allayed by increasing consistency of extended culture. The central emphasis of blastocyst transfer in any event is to reduce the number of embryos at transfer while maintaining good pregnancy outcomes (see Table 37.1a).

Table 37.1 a: Blastocyst Cryopreservation outcomes: Center for Assisted Reproduction, Texas Del Marek et al Year

1998 1999

Thaws 110 Transfers 101 Del./ongoing pregnancy 24% Average # embryos ET 2.3

106 101 33% 2.1

Routine Freezing of Blastocysts Revisiting the original blastocyst cryopreservation protocol,28 modified the protocol such that it became extremely convenient and at least as successful as the earlier protocol (1992). Differing clinics, however, have struggled with inconsistent results with the latter protocol, and started research variants to improve on consistency. In fairness, much of this has probably been due to inexperience on the part of many embryologists, both with selecting blastocysts of sufficient quality to freeze, and also understanding the subtleties of cryopreservation and the impact that even the slightest variation, no matter how unintentional, might have on consistency. The most common practice to attempt improved consistency has been to reintroduce one or two glycerol concentration steps in the thaw, with one or two extra sucrose dilutions (for two examples of modified protocols, refer to Appendices 37.2 and 37.3). Not a major change, and not too great an increase in time commitment. A typical example of a shift in outcomes within a

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cryoprogram following a change to a modified protocol would be the results from Boston IVF, where Jeannine Witmyer reports that in their first 13 thaws using the “1997 Menezo/Veiga” protocol,28 they achieved one ongoing pregnancy. After the introduction of a modified thaw approach similar to that used at Shady Grove (Appendix 37.2), then they achieved six pregnancies from 19 thaws. Small numbers, but they changed nothing else in their approach. More complicated has been the experience at Shady Grove Reproductive Science Center, Rockville, MD, where initially we undertook eight thaws as per Menezo and Veiga28 and with only a 13.5 percent cryosurvival rate (7/ 52 blastocysts survived thawing), we did achieve one healthy pregnancy. Oddly many of the blastocysts appeared to survive initially, but upon subsequent culture for several hours the cells became increasingly degenerate. Subsequently, with no change other than to thaw into a protocol as per Appendix 37.2, we got 80 percent cryosurvival (35/44), with five ongoing pregnancies from 13 thaws. Many factors clearly have an impression on these experiences, not the least of which are the differences in the “holding media”, freezing in straws, vials, or ampoules and possibly even the type of programmable freezer. Subsequent to that time, we have experienced other fluctuations in outcome regardless of the quality of the blastocysts at the time of thaw, with respect to the hormone replacement protocol. Specifically, changes in the progesterone supplementation have seemed to have had a prof ound impact, such that with the use of Crinone we achieved only 3 ongoing pregnancies post-thaw in 25 cycles with an implantation rate of 6 percent (4/70). Moving to the use of intra-muscular progesterone, this returned results to a rate of 6 clinical pregnancies, with 4 ongoing/viable from 8 thaws, with an implantation rate of 25 percent (5/20). Again numbers here are small, and seemingly contradictory to some reports in the literature with the use of vaginal progesterone gels,36 or suppositories.37 This serves not to stimulate lack of credibility in other’s results, but to underscore the multi-factorial nature of assisted reproduction in general, and how the least variation in approaches clinic to clinic may have a significant effect on outcomes. Melanie Freeman (Table 37. 1b) reports that her clinic’s results in Nashville have become more successful with a shift away from the “multi-step” protocol35 to her own variant of the modified protocol for blastocyst cryopreservation (Appendix 37.3). Different freezing protocols can be thawed in the modified fashion as can be seen in the second column of Table 37.1b, with reasonable outcomes. This has been our experience also, suggesting that thawing, at least in these types of protocols, can seem to be more critical than the freezing.

Table 37.1 b: Blastocyst Cryopreservation outcomes: Nashville, Tennessee Period

1993–8 “Multi-step” “Multi-step Freeze/Mod. Thaw”*

“Mod. Freeze/ Thaw”*

Thaws Survived #/thaw #/ET Del. P. R. Emb. Imp.

720 embryos 533(74%) 4.2 3.1 38/173(22%) 67 sacs (12.6%)

54 46(85%) 3.0 2.6 9/18(50%) 18 sacs (39%)

73 59(81%) 2.9 2.4 7/25(28%) 14 sacs (23.7%)

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* 1999 onward, protocol used as in Appendix 2; more stringent selection criteria for cryopreservation including only mid- to fully expanded blastocysts on day-5/6. Also PZD Assisted Hatching used prior to transfer.

The impact of assisted hatching on thawed embryo implantation at Shady Grove RSC can be seen in Table 37.2. Numbers as yet are low. Interestingly, the cleavage/pronucleate thawed embryos appeared to gain no advantage from the assisted hatching procedure. Though due to the small numbers and lack of real discrimination of embryo quality at the earlier stages of embryo transfer, it is probable that it would take much higher numbers than with the thawed blastocysts to discern any real significance. It is logical that the hatching of the blastocysts should be beneficial for thawed blastocysts (Fig. 37.1), given that many of them have been frozen on day-six at which stage it appears that assisted hatching is beneficial for fresh blastocysts.38 Secondly, the zona pellucida is thought to undergo problematic hardening during the freeze/thaw procedure.39 In some cases zona fractures can be caused routinely22 depending on the means of cryopreservation. Embryos with holes already present in their zonae can successfully survive cryopreservation and give rise to pregancies.40

Fig. 37.1: Hatching Blastocyst PostThaw

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Table 37.2: Assisted Hatching and its impact on the cryopreservation program at Shady Grove Fertility RSC, Maryland Assisted Hatching No Intervention Post-Thaw Pregnancy Emb. Imp. Pregnancy Emb. Imp. Blastocyst (ET’s=33) 5/15(33%) 6/40(15%) 4/18(22%) Cleavage/ Pronucleate (ET’s=18) 3/9(33%) 3/27(11%) 4/9(44%)

4/50(8%) 5/29(17%)

Generally the cryopreservation protocols discussed above can be well-defined and controlled from the laboratory perspective, so if fluctuations in pregnancy outcomes continue regardless of good cryosurvival, then clinical management problems outside of the lab are probably at fault. An example of this is given above from the management of the “artificial” cycles with vaginal progesterone gel instead of intra-muscular progesterone during thawed blastocyst replacements. This was completely unanticipated. Many simple errors are possible, including, for example, calculation of the day of transfer. The easiest way to consider this is to calculate the “day of ovulation” (whether in a “natural” or “artificial” transfer) cycle then thaw and transfer all blastocysts on the fifth day of development, counting “ovulation” day as day-zero. This mirrors what would happen normally in an IVF cycle, but where some manipulation of timing may be needed for whatever practical reasons, then it is better to err on the “early” side when thawing the embryos. The Future of Egg and Embryo Cryopreservation Firstly, it is hoped that more clinics will become increasingly comfortable with blastocyst freezing as it currently exists. This will only be possible if extended culture is perceived to become sufficiently consistent. With production of good quality mid to fully expanded blastocysts with well-defined ICM and trophectoderm on day-five/six, it is possible to settle on consistently successful cryoprotocol for such embryos using the present technology. Even so, as Menezo and Veiga proved,28 protocols can always be made simpler and more convenient. To this end, it has to be noted that vitrification protocols are starting to enter the mainstream of human ART. Protocols successfully applied for bovine oocytes and embryos have been used initially with human oocytes,9 and initial trials been undertaken with human blastocysts.41 Vitrification is very simple, requires no expensive programmable freezing equipment, and relies especially on the placement of the embryo in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed cryostraws and vials. Whatever the approach to cryostorage, the aim of blastocyst cryopreservation will be to maximize the potential viability of each embryo thawed and replaced, such that the numbers of embryos thawed and transferred may be kept to a minimum. Oocyte cryopreservation will slowly enter the mainstream of techniques in ART, most likely in the area of oocyte donation. Here information, in terms of clinical success of protocols, is generated within months not years, as would be the case with freezing of eggs for single women concerned

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with their future reproductive choices. In accepting that cryopreservation of human eggs and embryos seems here to stay, if remains important that we research the consequences of these therapies carefully to ensure that we truly do no harm.42–43 APPENDIX 37.1 Human Egg Cryopreservation Freezing Human eggs is very similar in principle to early embryo freezing but with several key differences adopted to make outcomes more consistent. Freeze 1. After egg collection, maintain all eggs in culture for >5 hours before attempting cryopreservation. 2. Strip all eggs in hyaluronidase at some point in the 5 hrs pre-incubation period. 3. Place all eggs (GV through MII) to be frozen into warm modified HTF and then place them on to the bench at room temperature for 10 mins to cool down (Approx 22°C). 4. Expose to 1.5 M 1, 2-propanediol (propylene glycol) in modified HTF with 15 percent HSA for 20 mins. 5. Place into 1.5 M PROH+0.2 M sucrose for a further 10 mins. 6. Rinse cryovial with PROH+Sucrose medium and then fill with 0.3 ml of this medium ready to receive the oocytes. 7. Freeze in the following manner: −22°C down to −5.0°C at a rate of 2.0°C/min. Hold 15 mins @ −5.0°C, “seed” after Smins, ensure that the “seed” has been established afterwards (*this is a rather “high” temperature compared to −7.0°C for embryos). Cool further @ −0.3°C/min to −38°C then plunge into liquid nitrogen for storage. Thaw 1. Place cryovial at room temp for Imin, then place in 30°C water bath till ice crystals have gone. 2. Remove contents into room temp drop of 1.5 M PROH+0.3 M Sucrose in modified HTF with 10 percent HSA, then place into subsequent PROH dilutions for 8 mins each of 1.0 M, 0.75 M, 0.5 M, 0.25 M, 0.0 M all+0.3 M Sucrose. 3. Dilute slowly the final 0.3 M Sucrose drop with modified HTF+10 percent HSA, then wash eggs through 4–5 drops modified HTF, then 6–8 drops plain HTF+10 percent HSA, and place in the incubator. 4. Undertake ICSI on all mature thawed oocytes only after four hours in culture, after which any cytoskeletal damage that may have occurred during freezing will have had an opportunity to repair itself.mt. 10.19.99.

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APPENDIX 37.2 Blastocyst Cryopreservation: Shady Grove RSC Protocol Freezing 1. Holding Medium: modified HTF+10 percent HSA. 2. Freeze good expanded/hatched or hatching blastocysts on Day-5/6 (unless fertilization delayed, e.g. because of FICSI). 3. Embryos into modified HTF+HSA @ 37°C, then move onto cool bench (22°C), and wash through several droplets for about 1 to 2 min. 4. Move into 5 percent glycerol for 8 mins. 5. 10 percent glycerol+0.2M sucrose for Smins (including loading time). Load straws/cryovials. (SGRSC uses 1.2 ml Nunc cryovials containing 0.3 ml medium). 6. Cool @ −2°C/min to −7.0°C; hold for 15 min; “seed” after 5 min; −0.3°C/min to −38°C, then plunge into liquid nitrogen for storage. Thawing 1. Room temperature for 1 min. Waterbath @ 30°C till ice gone. 2. Locate blastocyst in 10 percent glycerol+0.4 M sucrose for 30–40 sec. 3. 5 percent glycerol+0.4 M Sucrose for 3 mins. 4. 2.5 percent glycerol+0.4 M Sucrose for 3 mins. 5. 0.4 M Sucrose alone for 2 mins. 6. 0.2 M Sucrose for 2 mins. 7. 0.1 M Sucrose for 1 min; move dish to warm scope/ bench. 8. Modified HTF+10 percent HSA @ 37°C for three washes, then into culture of HTF+10 percent HSA. 9. Undertake Assisted Hatching while blastocyst still collapsed post-thaw. 10. Culture for >4 hours, even overnight to observe reexpansion.

Blastocyst Freezing— Short Protocol Nashville Fertility Center, Tennessee. Reagents Glycerol (Sigma, G-2025) Sucrose (Sigma, S-1888) Dulbecco’s Phosphate Buffered Saline (PBS)(GIBCO) Human Serum Albumin (HSA) (IVC)

APPENDIX 37.3 Melanie R.Freeman, MSTS

Materiqls and equipment Nunc 4-well mnltidish 1.8 ml cryovials (COSTAR) Kryo-10, 1.7 cell freezer (T/S Scientific) Isolet (Hoffman IVF Chamber)

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Blastocyst Freeze/Thaw Media Make at least one day in advance to de-gas. • Cryo solutions: Filter solutions through 0.2 m filters and store in the refrigerator for 2 months. • Cryo solution #0: (PBS+5mg/ml HSA). Combine 5 ml of HSA with 100 ml of PBS. • Cryo solution #1: (PBS+5mg/ml HSA+5% glycerol). Add 2.0 ml Glycerol to 38.0 ml Cryo solution #1. • Cryo solution #2: (PBS+5mg/ml HSA +10% glycerol +0.2 M sucrose). Add 5 ml of Glycerol to 45 ml of Cryo solution #4. • Cryo solution #3: (PBS+5mg/ml HSA+5% glycerol+ 0.2 M sucrose). Add 6 ml of Cryo solution #2 to 6 ml of Cryo solution #4. • Cryo solution #4: (PBS+5mg/ml HSA+0.2 M sucrose). Add 3.425 g Sucrose to 50 ml of Cryo solution #1. Blastocyst Freezing Procedure 1. Prior to freezing, fill Nunc multi-dish wells #1 and #2 with 0.6 ml Cryo solution #0. Fill well #3 with 0.6 ml Cryo solution #1, and well #4 with 0.6 ml Cryo solution #2. Fill each cryovial with 0.3 ml Cryo solution #2. 2. Allow the dish and freezing vials to warm to 37°C for 10 minutes. 3. Freezing Program for Kryo-10: Start temp: ambient \\−2°C/min to −7°C\\−0.3°C/min to −37°C\\ Seeding: manual\\Seeding Temp: −7°C\\soaking (before seeding): 10 min\\Hold (after seeding) for 10 min\\End of program 4. MOVE DISH TO ROOM TEMPERATURE, place all embryos to be frozen in well #1 of the Nunc dish to wash out all culture media. Transfer to well #2 and incubate for 2 minutes. 5. Transfer the embryos to well #3 and incubate for 10 minutes. 6. Transfer the embryos to well #4 and set timer for 10 minutes. When embryos settle to bottom of dish (1–5 minutes) load into vials. 7. Once all vials are loaded, place in the Kryo-10 at the same time. Press the “Run” button when the timer rings (end of 10 minutes). The Kryo-10 will proceed to the seeding temp. Seed each vial carefully using ring-forceps dipped in LN2 8. At the end of the freezing program, fill the portable dewar with LN2. Quickly, remove freezer canes from the Kryo-10 and submerse in the LN2 in the portable dewar. Lift out and place each vial on a precooled storage cane and submerse storage cane in LN2 in portable dewar while the other vials are unloaded. Repeat for all other vials and place cane in LN2 storage dewar. Blastocyst Thawing Procedure 1. Allow at least 4 hours between thaw and embryo transfer. 2. Prior to thawing, fill Nunc multi-dish well #1 with 0.6 ml Cryo solution #2, well #2 with 0.6 ml Cryo solution #3, well #3 with 0.6 ml Cryo solution #4, and well #4 with 0.6 ml Cryo solution #0. Allow Nunc dish to warm to ROOM TEMP for 20 minutes.

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3. Prepare a 30? C waterbath. Remove freezing vial (s) from LN2 and leave at room temp approximately 1–2 minutes until surface frost appears, and seal is easily broken. Tighten vial top and immerse the bottom of the vial (s) in a 30C? water bath for 2 minutes until completely melted. Gently agitate the vials in the waterbath. After 2 minutes no crystals should remain. 4. At ROOM TEMP, transfer the contents of each vial to the center area of a Nunc 4well multidish and locate each embryo. Transfer them to well #1 as you find them. When all are in well #1, MOVE THE DISH TO 37°C (in isolet with no CO2). Allow the embryos to equilibrate for 30–45 seconds. Move the embryos to well #2 and incubate for 3 min. 5. Move the embryos to well #3 and let them remain for 2 minutes. Rinse in well #4, then rinse again several times with growth media (GM) and place in GM until Assisted Hatching (PZD) and transfer.

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40. Magli MC et al. Impact of blastomere biopsy and cryopreservation techniques on human embryo viability. Hum Reprod 1999; 14:770–73. 41. Lane M et al. Vitrification of mouse and human blastocysts using a novel cryoloop containerless technique. Fertil Steril 1999; 72:1073–78. 42. Wennerholm UB et al. Postnatal growth and health in children born after cryopreservation as embryos. Lancet 1998; 351:1085–90. 43. Dulioust E et al. Saftey of embryo cryopreservation: facts and artefacts. Hum Reprod 1999; 14:1141–45. 44. Abir R et al. Pilot study of isolated early human follicles cultured in collagen gels for 24 hours. Hum Reprod 1999; 14:1299–1301.

CHAPTER 38 In Vitro Maturation: Future Clinical Applications Jin Ho Lim, Weon Young Son, San Hyun Yoon OVERVIEW The in vitro maturation (IVM) of human oocytes retrieved from unstimulated ovaries is a reproductive technology of increasing interest. In comparison with controlled ovarian hyperstimulation (COH), the major benefits of IVM treatment include avoidance of the risk of ovarian hyperstimulation syndrome (OHSS), reduced cost, and less complicated treatment. Knowledge regarding the IVM of immature human oocytes and its clinical application has been accumulated during the past couple of years. Fertilization, embryo development and term pregnancies of IVM oocytes have been reported in stimulated cycles,1 natural cycles2 and PCO patients.3 Although some recent studies have shown improved pregnancy rates per embryo transfer,4,5 the pregnancy rate after IVM has in general been low. Likely reasons are suboptimal culture conditions during IVM or inadequate cytoplasmic maturation of the oocyte itself. To overcome these problems, some studies have focused on improved culture media; other studies have tried to optimize the quality of the oocyte by stimulation with oestradiol or gonadotropins. Chian et al5 reported that rates of oocyte maturation and pregnancy could be improved by hCG priming 36 h before immature oocytes retrieval in women with PCOS. In this article, we discuss our experience of several hundred cycles of unstimulated IVM for 2.5 years. Firstly we examined whether human IVM program can be applied clinically. Secondly, we reviewed the details of our experimental results on the IVM of oocytes and the clinical application of these programs in patients with high a risk of OHSS. Lastly, a new dimension of the IVM approach for the prevention of OHSS in COH cycles will be discussed. METHODS AND TECHNIQUES Patients In period I, we examined the possibility of clinical application of IVM program during October 1999 to March 2000. Immature oocytes were collected from unstimulated ovaries (143 cycles) of women (n=122) with various infertility factors. In period II, we examined the effect of hCG-priming in IVM cycles (n=103) in consented patients (n=84; mean age: 32.0±2.4 years) during April 2000 to October 2000. The patients had experienced OHSS or were prone to a high risk of OHSS. They were

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divided into two groups. Some patients (group I, n=50 cycles) received no treatment as control before their oocyte collection. Other patients (group II, n=53 cycles) were primed with 10,000 IU hCG about 36 hours before oocyte collection. The patients with anovulatory cycles received IM injection of P (Progestin; Samil Pharmacology, Seoul, Korea) for withdrawal bleeding. Based on the above result, we applied HCG-priming IVM cycles (n=303) in consented patients (n=174; mean age: 32.8±4.9 years) from November 2000 to April 2002. Out of total 303 cycles, embryos were transferred at the blastocyst stage in 67 cycles. During the same period, we applied the established IVM program for the prevention of OHSS in 17 patients undergoing COH for IVF or COH/IUI. Oocyte Recovery Follicle development was monitored by transvaginal ultrasonography (Aloka, Tokyo, Japan) beginning on cycle day 3–5, and immature oocytes were aspirated between cycle days 7 and 19 based on the patient’s cycle length and endometrium thickness. A transvaginal ultrasound machine with 19-gauge aspiration needle (Cook, Eight Mile Plains, Queensland, Australia) was used to aspirate follicles. A portable aspiration pump was used with a pressure between 80 to 100 mmHg. The aspirates were collected in tubes containing prewarmed heparinized Ham’s F-10 medium that contained bicarbonate and HEPES buffers supplemented with 0.3 percent BSA. Follicular aspirates were filtered (70-mm mesh size, Falcon 1060; Life Technologies) and washed with addition of medium to filtrate. The filtrate was further washed with medium by vigorous pipetting using 10 ml of serological pipette (Becton Dickinson and company, NJ, USA) to remove erythrocytes and small cellular debris. The retained cells then were resuspended in the medium. The oocytes were isolated under a stereomicroscope and washed twice in the same medium. In Vitro Maturation After oocyte collection, the maturity was evaluated and the immature oocytes were cultured in IVM medium, consisting of YS medium6,7,8 supplemented with 70 percent human follicular fluid during Period I6 or with 30 percent human follicular fluid (hFF), 1 IU/ml FSH, 10 IU/ ml hCG and 10 ng/ml rhEGF.9 After day 1 in culture, the oocytes were denuded of cumulus cells with hyaluronidase (IVF Science, Gothenburg, Sweden) and mechanical pipetting. Oocyte nuclear maturation was assessed by the presence of the first polar body under the dissecting microscope. Following examination, immature oocytes remaining at GV or MI stage were further cultured in the same medium and the meiotic status reexamined at day 2 and finally day 3 of culture. In vitro Fertilization (IVF), In vitro Development (IVD), and Embryo Transfer (ET) Matured oocytes were inseminated by ICSI using husband sperm. Fertilization was assessed 17–19 h after insemination for the presence of two distinct pronuclei and two polar bodies. The zygotes were co-cultured with cumulus cells in YS medium

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supplemented with 20 percent hFF.6,9 Embryos were transferred on day 4 or 6 after oocyte retrie val by the transcervical route in standard fashion. The date of embryo transfer was determined by the number of zygotes and the quality of day 3 embryos.7 Blastocyst stage transfer at day 6 was performed in patients who had more than seven zygotes and three good-quality embryos. The remaining patients were allotted to day 4 ET. After the embryo transfer, regardless of the embryo transfer date, surplus embryos were cultured until day 7, and the only embryos developed to the expanded blastocyst stage (diameter is more than 160 mm) were cryopreserved by vitrification on EMgrid.10,11 Endometrial Preparation For the preparation of the endometrium, 6 milligrams of E2 valerate (Progynova; Schering, Berlin, Germany) was administered daily from the day of oocyte collection. One hundred milligrams of Progest (progesterone) was administered daily, starting one day after oocyte collection. Both medications were continued until either a negative pregnancy or a positive fetal heartbeat was observed. Results IVM in Patients with Various Infertility Factors Table 38.1 summarizes the results from IVM cycles of women with various infertility factors.

Table 38.1: The rates of maturation, fertilization and pregnancy from IVM cycles of women with various infertility factors Variable

Value

No. of patients (mean age±SD) 122(34.8±4.0) No. of cycles 143 No. of oocytes collected (mean±SD) 1230(8.6±4.9) No. of MII oocytes (%) 918(74.6) No. of oocytes fertilized (%) 670(73.0) No. of embryos cleaved (%) 565(84.3) No. of cycles with ET 134 No. of mean embryos transferred 3.6 No. of clinical pregnancies (%) 22(16.4)

Follicles were aspirated on day 9.0±5.3 in 143 cycles from 122 patients (mean age=34.8±4.0 years). The mean number of oocytes retrieved was 8.6. The maturation, 2 PN and cleavage rates were 74.6 percent, 73.0 percent, and 84.3 percent, respectively. Nine cycles did not undergo ET by lack of maturation, fertilization failure, or poor embryo quality. The 134 cycles which underwent ET received a mean of 3.6 embryos. Aclinical pregnancy was observed in 22 cycles (16.4%, 22/34). The clinical pregnancy

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rate was increased with the increase in the number of oocytes collected per cycle (<6:7.3, 6–10:15.6%, 11–15:23.5%, >15:38.6%) (Table 38.2).

Table 38.2: The pregnancy rate according to the number of oocytes retrieved from IVM cycles No. of oocytes collected No. of cycles Pregnancy % <6 6–10 11–15 >15 Total

41 45 34 14 134

3 7 8 4 22

7.3 15.6 23.5 28.6 16.4

Effect of HCG-priming in IVM Cycles From the above result, we concluded that immature oocytes retrieved from women undergoing IVM cycles can undergo maturation in vitro, fertilize, cleave, and the resulting embryos can also establish pregnancies. However, although the number of embryos transferred (mean=3.6) in uteri was comparable to regular IVF/ICSI cycles, clinical pregnancy rate was only 16.4 percent. From the results that clinical pregnancy rate was increased with the increase in the number of oocytes collected per cycle, we suggest that an important factor for success of IVM program is the number of oocytes retrieved from a patient. Another factor could be the quality of the embryo derived from IVM. Therefore, we have attempted three strategies for improving IVM success; patient selection, HGG priming, and IVM medium. Firstly, the patients undergoing IVM were only with a high risk of OHSS. Secondly, we evaluated the effect of hCG-priming in human the IVM. Lastly, we have improved IVM system. We supplemented gonadotropins into IVM medium to a 1:10 ratio of rFSH: HCG. In addition, EGF, a growth factor that demonstrated increasing maturation of immature oocytes, was added to the IVM medium. To examine the effect of hCG-priming in IVM cycles of patients with a high risk of OHSS, the IVM cycles were divided into two groups; control group (group I) and HCGpriming group (group II). The mean number of the collected oocytes was comparablein these two groups (24 ±4.6 and 21.9±5.8). Oocytes that were classified as having a dispersed, compacted, and sparse CC appearance were used for culture (Fig. 38.1). Figure 38.2 shows the percentage of oocytes classified according to their cumulus appearance at the time of oocyte retrieval in two groups. As shown in Figure 38.2, oocytes with dispersed cumulus cells were only appeared in hCG-primed group compared with control group. Comparison of the time course of maturation to MII in two groups are shown in Figure 38.3. Eleven percent of oocytes collected at OPU were matured in hCG-primed group (group II), while there was no oocyte matured at that time in non-primed group (group I). Also, the oocyte maturation (56.9%) 1 day after culture in hCG-primed group (group II) was significantly hastened than that (40.8%) of control group (group I) (P<0.001). The embryos with good quality on day 4 in group II (23.7%) were significantly more than those in group I (13.6%) (P<0.01). In addition, the clinical pregnancy rate in group II (43.4%) was significantly higher than that in group I (28.0%) (P<0.05) (Table 38.3).

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The results indicate that the maturation time of oocytes collected from women with high risk of OHSS was hastened by HCG priming compared with non-priming. In addition, HCG-priming

Fig. 38.1: Human immature oocytes retrieved at the time of oocyte collection. (A) A GV-stage oocyte with dispersed cumulus cells appearance just after oocyte retrieval. (B) A GV-stage oocyte with

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compacted cumulus cells appearance just after oocyte retrieval. (C) A GVstage oocyte with sparse cumulus cells appearance.(Original magnification×200). before collection of immature oocytes may improve the developmental competence.

Table 38.3: Effect of HCG-priming on maturation, fertilization and pregnancy in women with high risk of OHSS Variable

HCG (−)

HCG (+)

No. of patients (mean age±SD) 41(32.2±2.9) 43(31.9±2.5) Infertility duration (mean±SD) 3.8±0.4 3.9±0.6 No. of cycles 50 53 No. of oocytes collected (mean±SD) 1200(24±4.6) 1161 (21.9±5.8) No. of oocytes matured (%) 883(73.6) 861(74.2) No. of oocytes fertilized (%) 671(76.0) 691(80.3) No. of oocytes cleaved (%) 599(89.3) 653(94.5) No. of good-quality embryos (%) 82(13.6) 155(23.7) * No. of embryos transferred (mean) 255(5.1) 265(5.0) No. of clinical pregnancies (%) 14(28.0) 23(43.4) ** *: P<0.01; **: P<0.05

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Fig. 38.2: The incidence of oocytes with cumulus cell appearances of elther dispersed ( ) compacted ( ), sparse ( ), or degenerated ( ) in nonprimed group (group I) and HCGprimed group (group II).

Fig. 38.3: Time course of human oocyte maturation during culture in vitro In non-primed (group I, ), and HCG-primed (group II, ). * Significant difference between two groups at the time point (P<0.001). The Experience of HCG-primed IVM Cycles Based on the above results, we performed 303 HCG-priming IVM cycles for 1.5 years in women with a high risk of OHSS. Among them, 106 were pregnant clinically (35.0 %) and an implantation rates of 12 percent was achieved (Table 38.4). Out of 303 cycles, ET was performed at the blastocyst-stage after IVM in 67 cycles. An implantation rate of

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27.5 percent (55/200) and high clinical pregnancy of 55.2 percent (37/67) were achieved from transfer (mean=2.9) at the blastocyst-stage.12 In addition, using the vitrification method which has been established in our hospital,10 we achieved three clinical pregnancies after vitrification of blastocysts produced by in vitro matured oocytes retrieved from patients undergoing IVM cycles.11

Table 38.4: Maturation, fertilization and pregnancy rate in hCG-priming IVM cycles Variable

Value

No. of patients (mean age±SD) 174 (32.8±4.9) No. of cycles 303 No. of oocytes collected (mean±SD) 5160 (17.0±4.9) No. of oocytes matured (%) 3861 (74.8) No. of oocytes fertilized (%) 3065 (79.4) No. of embryos transferred (mean) 1384 (4.6) No. of implantation (%) 166 (12.0) No. of clinical pregnancies (%) 106 (35.0)

Application of IVM Program for the Prevention of OHSS in COH Cycles Seventeen patients with PCOS were stimulated using FSH or HMG initially. Several ultrasound examinations were performed in all patients, to monitor the size and number of follicles. For the prevention of OHSS, gonadotropins withholding was offered to them, a full dose of HCG (10,000 IU) was administered when the leading follicle reached a mean diameter of 12–14 mm, and oocytes were retrieved 36 h later. The mean duration of COH was 10.5 days, and the mean dose of gonadotropins used during COH was 1465 IU. Among 233 oocytes (13.7 oocytes per patient) retrieved, 27 oocytes (11.6%) were in the MII stage at retrieval. The rates of maturation, fertilization and cleavage were 75.9 percent, 84.8 percent and 81.1 percent, respectively. The clinical pregnancy rate was 47.1 percent (8/17). Blastocysts were transferred in 4 cases, all of them were pregnant. There was no OHSS case in 17 patients. This preliminary study suggests that IVM program may be a solution to the management of PCOS patients before appearance of features of OHSS at ultrasound examination in stimulated cycles for IVF or COH/IUI. COMPLICATIONS The complications in the IVM program have not been reported, and we have also not found any complications in patients undergoing IVM cycles until now. Future Directions and Controversies Despite increasing interest and efforts, the number of births from IVM is still relatively low. The experiences of the IVM program for 2.5 years in our hospital are helping us to draw conclusions that, in near future, in vitro maturation could possibly replace standard

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stimulated IVF in selected patients. The selected group may include patients with a high risk of OHSS. PCOS patients are especially susceptible to OHSS,13 and recovery of immature oocytes and their maturation in vitro is a very attractive treatment strategy for pregnancy. It is beneficial to recover oocytes from small non-dominant follicles in patients with high risk of OHSS given HCG 36 hours before oocyte retrieval. Oocytes from these follicles appear to be developmentally competent and may contribute to improve the successful pregnancy. As shown in the results of period II, the oocytes with dispersed cumulus cells were only appeared in HCG-primed group compared with control group. Therefore, we wanted to compare the developmental competence between oocytes with different CC appearances in IVM-blastocyst transfer cycles. Collected cumulus-oocyte complexes (COCs) were classified into three groups in accordance with their CC appearances (Fig. 38.1): group I, oocytes with dispersed CC; group II, oocytes with compacted CC; and group III, oocytes with sparse CC. The total maturation rate (93.3%) in group I was significantly higher than those of group II (64.0%) and group III (73.0 %) (P<0.001) (Fig. 38.4).14 Comparisons of the time course of maturation to MII in three groups are shown in Figure 38.4. Thirty-five percent of oocytes with dispersed CC collected were matured at day of OPU, while there was no oocyte matured in oocytes with compacted or sparse CC. Also, the time course of oocyte maturation was hastened in the oocytes with dispersed (P<0.001) (Fig. 38.4). In addition, the development rate to the blastocyst stage in oocytes with dispersed CC (64.0%) was significantly higher than those in oocytes with compacted (39.6 %) or sparse CCs (46.6 %) (P<0.01). These results suggest that faster maturation and higher developmental competence of oocytes retrieved after HCGpriming in IVM cycles may be due to collection of oocytes with dispersed CC. Eventhough there is no evidence when LH receptors (LH-R) in folliculogenesis appear, some follicles at the time of HCG-priming in patients with high risk of OHSS might have LH receptors, resulting in retrieval of oocytes with dispersed cumulus cell appearance. Compact cumulus cells around oocytes following the administration of large doses of HCG (1000 IU), may be due to the presence of insufficient LH receptors to induce the cumulus cell response in vivo. To confirm these hypotheses, we analyzed the expression of LH-R between dispersed CC and compacted CC.15 The mRNA expressions of FSH-R and EGF-R in dispersed CC were comparable to those in compacted CC. However, compared to compacted CC, much abundant expression of LH-R mRNA was detected in the dispersed CC (Fig. 38.5). Of course, further studies are necessary to elucidate whether the expression of LH-R causes CC dispersion or whether the dispersion of CCs influences LH-R expression, but this result indicates that the expression of the LHR in CCs may be correlated with the dispersed CC pattern of oocyte retrieved.

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Fig. 38.4: Time course of the completion of maturation to MII (extrusion of first polar body) of oocytes cultured according to CC appearance. Oocytes with dispersed CC (□), oocytes with compacted CC (■), and oocytes with sparse CC (▲). *Significant difference among three groups at the time point (P<0.001). Barns et al16 suggested that the developmental competence of oocytes obtained from women with natural cycles is correlated with their follicle size. Therefore, we speculated that follicle sizes at the time of HCG priming are so different, that it is possible that oocytes retrieved with dispersed cumulus cells at OPU could be from some

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Fig. 38.5: Reverse transcriptionpolymerase chain reaction experiments showing the expression of FSH receptor (FSHR), LH receptor (LHR), EGF receptor (EGFR) genes in dispersed CC (A) and compacted CC (B). GAPDH is an internal control. The amplicons was confirmed by sequencing. large follicles. However, a well-defined further study is needed to ascertain this hypothesis. Our results suggest that CC appearances at the time of aspiration jplays a predictive role in the maturation and development of oocytes recovered in HCG-stimulated IVM cycles, and may be a relevant parameter in the development of a technology with successful higher oocyte maturation and blastocyst development in vitro. Generally, large numbers of unselected pronuclear or early cleavage stage embryos in IVM cycles have been transferred to patients for improving the pregnancy rates.17 However, the selection of the more developmentally competent embryos by extended culture would reduce the number of embryos transferred with a higher acceptable pregnancy rate. Therefore, improvements can be made to the maturation conditions to increase the developmental competence of immature oocytes and this aim should continue to be a priority for research on oocyte maturation. The recovery of oocytes from patients who have been treated with gonadotropins for IVF and with early administrated HCG, is a valuable method. This option could be adopted in clinical IVF to prevent the occurrence of OHSS with a reasonable pregnancy rate. The potential of this novel approach opens a new dimension in the management of patients with a high risk of OHSS during COH. In order to make the use of immature oocytes more efficient, further work is needed to define thebest conditions for both clinical and laboratory procedure. With further advances in these techniques, the IVM program will hopefully be extended to women with various infertility factors CONCLUSIONS In conclusion, in patients with a high risk of OHSS, an acceptable pregnancy could be achieved through the IVM program. HCG-priming about 36 h before oocytes collection in this program should increase the pregnancy rate especially. In addition, the IVM program may be a solution to the management of PCOS patients before the appearance of features of OHSS at ultrasound examination in stimulated cycles for IVF or COH/IUI. The results in our study suggest that recovery of immature oocytes followed by in-vitro maturation is a promising treatment for women with a high risk of OHSS.

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REFERENCES 1. Nagy ZP, Cecile J, Liu J, Loccufier A, Devroey P, Van Steirteghem A. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil Steril 1996; 65:1047–50. 2. Russell JB, Knezevich KM, Fabian KF, Dickson JA. Unstimulated immature oocyte retrieval: early vs. mid follicular endometrial priming. Fertil Steril 1997; 67:616–20. 3. Trounson A, Wood C, Kausche A. In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62:353–62. 4. Mikkelsen AL, Smith SD, Lindenberg S. In vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum Reprod 1999; 14:1847–51. 5. Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15:165–70. 6. Yoon HG., Yoon SH, Son WY, Lee SW, Park SP, IM KS et al. Pregnancies resulting from in vitro matured oocytes collected from women with regular menstrual cycle. J Assist Reprod Genet 2001; 18:249–53. 7. Yoon HG, Yoon SH, Son WY, Kim JG, Im KS, Lim JH. Alternative embryo transfer of day 3 or day 5 for reducing the risk of multiple gestations. J Assist Reprod Genet 2001; 18:188–93. 8. Yoon HG, Yoon SH, Son WY, Im KS, Lim JH. High implantation and pregnancy rate with transfer of human hatching day 6 blastocysts. Fertil Steril 2001; 75:832–33. 9. Son WY, Yoon SH, Lee SW, Ko Y, Yoon HG., Lim JH. Blastocyst development and pregnancies after in vitro fertilization of mature oocytes retrieved from unstimulated patients with PCOS after in vivo HCG priming. Hum Reprod 2002; 15:134–36. 10. Cho HJ, Son WY, Yoon SH, Lee SW, Lim JH. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts will be published in the September 2002 of Hum Reprod. 11. Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome will be published in the October 2002 of Hum Reprod. 12. Son WY, Yoon SH, Park SJ, Hyun CS, Lee WD, Lim JH. Blastocyst development and pregnancy from oocytes of unstimulated women with regular menstrual cycle after IVM approach (will be published in the November 2002 of J Assist Reprod Genet). 13. Cobo AC, Requena A, Neuspiller F, Aragon s M, Mercader A, Navarro J et al. Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size. Hum Reprod 1999; 14:1864–68. 14. Yoon SH, Son WY, Park SJ, Lee SW, Lee WD, Lim JH. In vitro maturation and the development to the blastocyst stage of immature oocytes collected from IVM/F-cycles. Will be presented at the 2002 American Society for Reproductive Medicine (ASRM), October 2002, Seattle. 15. Yang SH, Son WY, Lee SW, Yoon SH, Ko Y, Lim JH. Expression of Luteinizing Hormone Receptor (LH-R), Follicle Stimulating Hormone Receptor (FSH-R) and Epidermal Growth Factor Receptor (EGF-R) in Cumulus Cells of the Oocytes Collected from PCOS Patients in hCG-Priming IVM/F-ET Program. Presented at the 2001 ASRM, October 2001, Florida. 16. Barnes FL, Kausche A, Tiglias J, Wood C, Wilton L, Trounson A. Production of embryos from in vitromatured primary human oocytes. Fertil Steril 1996; 65:1151–56. 17. Cha KY, Chian RC. Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update 1998; 4:103–20.

CHAPTER 39 Oxidative Stress and DNA Damage in Human Sperm: The Cleυeland Clinic Story Ahok Agarwal, Ramadan Abdou Saleh INTRODUCTION Defective sperm function is the most prevalent cause of male infertility and is a difficult condition to treat. The mechanisms underlying abnormal sperm function are still poorly understood. This may be due to a lack of basic knowledge about biochemical and physiological processes involved in spermatogenesis. Over recent years, the generation of reactive oxygen species (ROS) in male reproductive tract has become a real concern because of their potential toxic effects, at high levels, on sperm quality and function. ROS are highly reactive oxidizing agents belonging to the class of free radicals. A free radical is defined as “any atom or molecule that possesses one or more unpaired electrons. Recent reports have indicated that high levels of ROS are detected in semen samples of 25 to 40 percent of infertile men. However, a strong body of evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities. Spermatozoa, like all cells living under aerobic conditions, constantly face the oxygen (O2) paradox, i.e. O2 is required to support life, but its metabolites such as ROS can modify cell functions, endanger cell survival or both. Hence, ROS must be continuously inactivated to keep only a small amount necessary to maintain normal cell function. It is not surprising that a battery of different types of antioxidants provide protection against oxidants. An antioxidant is defined as “any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidative damage to that substrate”. The term “oxidizable substrate” includes almost every molecule found in living cells including proteins, lipids, carbohydrates, and deoxyribonucleic acid (DNA). Spermatozoa are particularly susceptible to the damage induced by excessive ROS because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA) and their cytoplasm contains low concentrations of scavenging enzymes. In addition, the intracellular antioxidant enzymes cannot protect the plasma membrane that surround the acrosome and the tail, forcing spermatozoa to supplement their limited intrinsic antioxidant defenses by depending on the protection afforded by the seminal plasma, which bathes these cells. Excessive generation of ROS in the reproductive tract not only attacks the fluidity of the sperm plasma membrane but also the integrity of DNA in the sperm nucleus. DNAbases are susceptible to oxidative damage resulting in base modification, strand breaks and chromatin cross-linking. There is strong evidence that DNA fragmentation commonly observed in spermatozoa of infertile men is mediated by high levels of ROS.

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ROS AND SPERM PHYSIOLOGY Until recently, ROS were exclusively considered as toxic to human spermatozoa. The idea that limited amounts of ROS can intervene in a physiological manner in the regulation of some sperm functions was first evoked in a study by Aitken et al. It was observed that ROS, at low levels, enhanced the ability of human spermatozoa to bind zonae pellucida, an effect which was reversed by the addition of vitamin E. As a general rule, the incubation of spermatozoa with low concentrations of hydrogen peroxide (H2O2) stimulates the process of sperm capacitation, hyperactivation, and the ability of the spermatozoa to undergo acrosome reaction and oocyte fusion. Reactive oxygen species other than H2O2 such as nitric oxide and superoxide anion (O2), have also been shown to promote sperm capacitation and acrosome reaction. CELLULAR ORIGIN OF ROS IN HUMAN SEMEN A variety of semen components, including morphologically abnormal spermatozoa, precursor germ cells and leukocytes are capable of generating ROS in semen. However, seminal leukocytes and morphologically abnormal spermatozoa are the main sources of ROS in human ejaculates. ROS Production by Spermatozoa Clear evidence suggests that human spermatozoa produce oxidants. Levels of ROS production by pure sperm populations are negatively correlated with quality of sperm in the original semen. The link between poor semen quality and increased ROS generation lies in the presence of excess residual cytoplasm (cytoplasmic droplet). When spermatogenesis is impaired, the cytoplasmic extrusion mechanisms are defective, and spermatozoa are released from the germinal epithelium carrying surplus residual cytoplasm (Fig. 39.1). Under these circumstances, spermatozoa released during spermiation are thought to be immature and functionally defective. Retention of residual cytoplasm by spermato. zoa is positively correlated with ROS generation via mechanisms that may be mediated by the cytostolic enzyme glucose-6-phosphate-dehydrogenase (G6PD). This enzyme controls the rate of glucose flux through hexose monophosphate shunt, which in turn controls the intracellular availability of nicotinamide adenine dinucleotide phosphate (NADPH). The later is used as a source of electrons by spermatozoa to fuel the generation of ROS by an enzyme system known as NADPHoxidase. Spermatozoa may generate ROS in two ways: 1) NADPH-oxidase system at the level of the sperm plasma membrane, and 2) NADH-dependent oxidoreductase (diphorase) at the level of mitochondria. The mitochondrial system is the major source of ROS in spermatozoa from infertile men (Fig. 39.1). The primary ROS generated in human spermatozoa is the O2•. This one-electron reduction product of oxygen secondarily reacts with itself in a dismutation reaction, which is greatly accelerated by superoxide dismutase (SOD), to generate H2O2. In addition to H2O2 and O2•, a variety of secondary cytotoxic radicals and oxidants are

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generated by human spermatozoa. In the presence of transition metals such as iron and copper, H2O2 and O•2 can interact in a Haber-Weiss reaction to generate the

Fig. 39.1: Mechanism of increased production of reactive oxygen species (ROS) by abnormal spermatozoa extremely pernicious hydroxyl radical (OH•) as in Equation 1. Equation 1: O2•+H2O2→OH•+OH•+O2 Alternatively, the hydroxyl radical can be produced from hydrogen peroxide by Fenton reaction, which requires a reducing agent, such as ascorbate or ferrous ions (Equation 2). Equation 2: H2O2+Fe2+→ Fe3++OH•+OH• The hydroxyl radical is thought to be an extremely powerful initiator of the lipid peroxidation (LPO) cascade and can precipitate a loss of sperm functions. ROS Production by Leukocytes With respect to all non-sperm cells, the majority of the socalled ‘round cells’ consist of immature germ cells with less than 5 percent leukocytes under normal conditions. Peroxidase-positive leukocytes are the major source of ROS in semen. Peroxidasepositive leukocytes include polymorphonuclear (PMN) leukocytes which represent 50 to 60 percent of all seminal leukocytes, and macrophages, which represent the remaining 20

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to 30 percent of all seminal leukocytes. Peroxidase-positive leukocytes in semen are contributed largely by the prostate and the seminal vesicles. Sperm damage from ROS produced by leukocytes occurs if seminal leukocyte concentrations are abnormally high, i.e. leukocytospermia, if the patient has epididymitis, or if seminal plasma was removed during sperm preparation for assisted reproduction. Seminal plasma contains large amounts of ROS scavengers but confers a very variable (10–100%) protection against ROS generated by leukocytes. Activated leukocytes are capable of producing 100-fold higher amounts of ROS than non-activated leukocytes. Leukocytes may be activated in response to a variety of stimuli including inflammation and infection. Activated leukocytes increase the NADPH production via the hexose monophosphate shunt. The myeloperoxidase system of both PMN leukocytes and macrophages is also activated leading to respiratory burst with production of high levels of ROS. Such an oxidative burst is an early and effective defense mechanism in cases of infection for killing the microbes. ROS SCAVENGING STRATEGIES Interestingly, seminal plasma is well endowed with an array of antioxidant defense mechanisms to protect spermatozoa against oxidative insult. These mechanisms compensate for the deficiency in cytoplasmic enzymes in sperm. Seminal plasma contains enzymatic antioxidants such as SOD, glutathione peroxidase/glutathione reductase (GPX/GRD) system, and catalase, as well as non-enzymatic antioxidants such as ascorbate, urate, α-tocopherol, pyruvate, glutathione, taurine, and hypotaurine. Seminal plasma from fertile men have a higher total antioxidant capacity than that of infertile men. However, pathological levels of ROS detected in semen from infertile men are more likely due to increased ROS production rather than reduced antioxidant capacity of the seminal plasma. Antioxidant defense mechanisms include three levels of protection: prevention, interception, and repair. Prevention Prevention of ROS formation is the first line of defense against oxidative damage. An example is the binding of metal ions, iron and copper ions in particular, which prevents them from initiating a chain reaction. Chelation of transition metals is a major means of controlling LPO and DNA damage. When transition metals become loosely bound to biological molecules such as oxygen reduction products, they can produce secondary and more reactive oxidants, particularly OH•. Interception Free radicals have a tendency towards chain reaction, i.e. a compound carrying an unpaired electron will react with another compound to generate an unpaired electron, “radical begets radical”. Hence, the basic problem is to intercept a damaging species from further activity which is the process of deactivation leading to the formation of non-

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radical end products. Alpha-tocopherol (vitamin E), a chainbreaking antioxidant, inhibits LPO in membranes by scavenging peroxyl (RO•) and alkoxyl (ROO•) radicals thereby breaking the chain reaction. The ability of α-tocopherol to maintain a steady-state rate of peroxyl radical reduction in the plasma membrane depends on the recycling of αtocopherol by external reducing agents such as ascorbate or thiols. In this way αtocopherol is able to function again as a free radical chain-breaking antioxidant, even though its concentration is low. A prerequisite for efficient interception is a relatively long half-life of the radical to be intercepted. The peroxyl radicals are major reaction partners because their half-life extends into the range of seconds (7 seconds). In contrast, the hydroxyl radical, with its high reactivity and extremely short half-life (10–9 seconds), can not be intercepted with reasonable efficiency Repair Protection from the effects of oxidants can also occur by repairing the damage once it has occurred. Unfortunately, spermatozoa are unable to repair the damage induced by ROS because they lack the cytoplasmic enzyme systems that are required to accomplish this repair. This is one of the features that make spermatozoa unique in their susceptibility to oxidative insult. THE CONCEPT OF OXIDATIVE STRESS (OS) Oxidative stress (OS) is the term applied when oxidants outnumber antioxidants, peroxidation products develop, and when these phenomena cause pathological effects. Oxidative stress has been implicated in numerous disease states such as cancer, arthritis, connective tissue disorders, aging, toxin exposure, physical injury, infection, inflammation, acquired immunodeficiency syndrome, and male infertility. In the context of human reproduction, a balance is present between ROS generation and scavenging in the male reproductive tract. As a result of suchbalance, only a minimal amount of ROS remains, which is needed to regulate normal sperm functions such as sperm capacitation, acrosome reaction and sperm-oocyte fusion. Excessive ROS production, which is related to abnormalities of the male reproductive tract, can overwhelm all antioxidant defense strategies of spermatozoa and seminal plasma when exceeds critical levels, causing an OS status. MECHANISMS OF ROSTOXICITY Virtually every human ejaculate is contaminated with potential sources of OS such as peroxidase-positive leukocytes and morphologically abnormal spermatozoa. It follows that some of the sperm cells will incur oxidative damage and a concomitant loss of function in every ejaculate. Thus, the impact of OS on male fertility is a question of degree rather than the presence or absence of the pathology. All cellular components, lipids, proteins, nucleic acids and sugars are potential targets for ROS. The extent of damage caused by ROS depend not only on nature and the amount of ROS involved but

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also on the moment and duration of ROS exposure and of the extra-cellular factors such as temperature, oxygen tension and composition of the surrounding environment including ions, proteins and ROS scavengers. Lipid Peroxidation (LPO) Lipid peroxidation can be broadly defined as “oxidative deterioration of PUFA”, i.e. fatty acids that contain more than two carbon-carbon double bonds. The LPO cascade occurs in two fundamental stages: initiation and propagation. Initiation Stage •

The hydroxyl radical (OH ) is a powerful initiator of LPO. Most membrane PUFA have un-conjugated double bonds that are separated by methylene groups. The presence of a double bond adjacent to a methylene group makes the methylene C-H bonds weaker and therefore hydrogen is more susceptible to abstraction. Once this abstraction has occurred, the radical produced is stabilized by the rearrangement of the double bonds, which forms a conjugated diene radical that can thenbe oxidized. This means that lipids, which contain many methylene-interrupted double bonds, are particularly susceptible to peroxidation. Conjugated dienes rapidly react with O2 to form a lipid peroxyl radical (ROO), which abstracts hydrogen atoms from other lipid molecules to form lipid hydroperoxides (ROOH). Thus, the chain reaction of LPO is continued. Propagation Stage Lipid hydroperoxides are stable under physiological conditions until they contact transition metals such as iron or copper salts. These metals or their complexes cause lipid hydroperoxides to generate alkoxyl and peroxyl radicals, which then continue the chain reaction within the membrane and propagate the damage throughout the cell. Lipid peroxidation propagation will depend upon the antioxidant strategies employed by spermatozoa. One of the by-products of lipid peroxide decomposition is malondialdehyde (MDA). The later has been used as an end-product in biochemical assays to monitor the degree of peroxidative damage sustained by spermatozoa. The results of such an assay exhibit an excellent correlation with the degree to which sperm function is impaired in terms of motility and the capacity for sperm-oocyte fusion. Impairment of Sperm Motility The increased formation of ROS has been correlated with a reduction of sperm motility. The link between ROS and reduced motility may be due to a cascade of events that result in a decrease in axonemal protein phosphorylation and sperm immobilization, both of which are associated with a reduction in membrane fluidity that is necessary for spermoocyte fusion. Another hypothesis is that H2O2 can diffuse across the membranes into the cells and inhibit the activity of some enzymes such as G6PDH, leading to a decrease in the availability of NADPH and a concomitant accumulation of oxidized glutathione and

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reduced glutathione. This can cause a decrease in the antioxidant defenses of the spermatozoa, which ultimately leads to the peroxidation of membrane phospholipids. Sperm Nuclear DNA Damage Sperm DNA is organized in a specific manner to keep the chromatin in the nucleus compact and stable. In 1991, Ward and Coffey proposed four levels of organization for DNA packaging in the spermatozoon: 1) chromosomal anchoring, which refers to the attachment of the DNA to the nuclear annulus; 2) formation of DNA loop domains as the DNA attaches to the newly added nuclear matrix; 3) replacement of histones by protamines, which condense the DNA into compact doughnuts; and 4) chromosomal positioning. Chromosomes become organized, with their centromeres located in the center of the nucleus and the telomeres at the nuclear periphery with active genes being localized to the nuclear center and the inactive genes to the periphery (Fig. 39.2).

Fig. 39.2: DNA packaging in human spermatozoa This DNA organization not only permits the very tightly packaged genetic information to be transformed to the egg but also ensures that the DNA is delivered in a physical and chemical form that allows the developing embryo to access the genetic information. ORIGIN OF DNA DAMAGE IN SPERMATOZOA

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Defective Chromatin Packaging Endogenous nicks in DNA occur most frequently during the transition from round to elongated spermatids in the testis and occur before the completion of protamination in rat and mouse spermatozoa. Protamination for chromatin packaging might require the formation and ligation of nicks through endogenous nuclease activity. They proposed that the endogenous nuclease, topoisomerase II (topo II), might play a role in both the creation and ligation of nicks during spermiogenesis. These nicks are thought to relieve stress due to torsion and to aid chromatin re-arrangement as histones are replaced by protamines. Therefore, the presence of endogenous nicks in ejaculated spermatozoa indicates incomplete maturation during spermiogenesis. This hypothesis is supported by observations that the presence of DNA damage in mature spermatozoa is correlated with poor chromatin packaging due to under-protamination. Apoptosis Spermatogenesis is a dynamic process of germ cell proliferation and differentiation from stem spermatogonia to mature spermatozoa through a complex series of mitotic and meiotic divisions. Apoptosis, also described as programmed cell death, is a physiological phenomenon characterized by cellular morphological and biochemical alterations leading the cell to suicide. Apoptosis is genetically determined and takes place at specific moments during normal embryonic life to allow definitive forms of tissues to develop and in the adult life to discard cells which have an altered or no function at all. In the context of male reproductive function, apoptosis may be responsible for controlling overproduction of male gametes. Testicular germ cell apoptosis occurs normally and continuously throughout life. One factor postulated to be implicated in sperm apoptosis is the cell surface protein Fas. Fas is a type I membrane protein that belongs to the tumor necrosis factor-nerve growth factor receptor family, and mediates apoptosis. Binding of Fas-ligand (Fas-L) or agonistic anti-Fas antibody to Fas kills cells by apoptosis. In men with abnormal semen parameters, the percentage of Fas-positive spermatozoa can be as high as 50 percent. Samples with low sperm concentration are more likely to have a high proportion of Faspositive spermatozoa. This evidence suggests that, in sub-fertile men, the correct clearance of spermatozoa via apoptosis is not occurring. The presence of spermatozoa that possess apoptotic markers, such as positive Fas and DNA damage, indicates that in men with abnormal semen parameters such as abnormal morphology, abnormal biochemical functions, and nuclear DNA damage, an ‘abortive apoptosis’ has taken place. Failure to clear Fas-positive spermatozoa may be due to dysfunction at one or more levels. First, apoptosis limits any excess in the number of developing germ cells so that the supportive capacity of Sertoli cells is not overloaded. Because Sertoli cells can limit their proliferation by producing Fas-L, the production of spermatozoa may not be enough to trigger apoptosis in cases with hypospermatogenesis. In these men, Fas-positive spermatogonia may escape the signal to undergo apoptosis. Second, Fas-positive spermatozoa may also exist because of problems in activating Fas-mediated apoptosis. These problems could be inherent to a particular patient or may be due to lack of synchronization between apoptosis and spermatogenesis. In the latter case, the

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spermatozoa will go through spermiogenesis and fail to complete apoptosis even though apoptosis has been initiated. This hypothesis may explain why patients with abnormal semen characteristics also possess a high percentage of spermatozoa containing DNA damage and abnormal spermatozoa that display markers of apoptosis. Oxidative Stress-induced DNA Damage Two factors protect spermatozoal DNA from oxidative insult: the characteristic tight packaging of the sperm DNAand the antioxidants present in the seminal plasma. Exposing the sperm to artificially produced ROS caused DNA damage in the form of modification of all bases, production of base-free sites, deletions, frame shifts, DNA cross-links, and chromosomal re-arrangements. Oxidative stress is also associated with high frequencies of single and double DNA strand breaks. This information has important clinical implications, particularly in the context of assisted reproductive techniques (ART). Spermatozoa selected for ART most likely originate from an environment experiencing OS, and a high percentage of these sperm may have damaged DNA. There is a substantial risk that spermatozoa carrying damaged DNA are being used clinically in this form of therapy. When intrauterine insemination (IUI) or in vitro fertilization (IVF) is used, such damage may not be a cause of concern because the collateral peroxidative damage to the sperm plasma membrane ensures that fertilization cannot occur with a DNA-damaged sperm. However, when intracytoplasmic sperm injection (ICSI) is used, this natural selectionbarrier is bypassed and a spermatozoon with damaged DNA is directly injected into the oocyte. Whether DNA-damaged spermatozoa used in ICSI can impair the process of fertilization and embryo development is not clear. On one hand, a recent study has indicated that spermatozoa with significantly damaged DNA still retain a residual capacity for fertilization. On the other hand, the percentage of sperm with DNA damage has been negatively correlated to the fertilization rate. In addition, a recent study has linked sperm DNA damage to increased rates of early embryo death. This is also supported by the results of Sanchez and colleagues in 1996, who reported that the miscarriage rates after ICSI were higher than that af ter conventional IVF. Sperm preparation techniques involving repeated centrifugation may lead to high ROS production in sperm suspensions processed for ART. This may be important because exposure of spermatozoa to high levels of ROS may increase the DNA fragmentation rate, which can have adverse consequences if they are used for ICSI. In 2000, Zini and coworkers reported that the improvement in sperm motility af ter Percoll processing is not associated with a similar improvement in sperm DNA integrity. The authors recommended that the current sperm preparation techniques be re-examined with the goal of minimizing sperm DNA damage. This can be accomplished by using more gentle sperm preparation methods such as the swim-up technique, which allows for good sperm recovery with minimal sperm dysfunction. In a recent study in our center, we have demonstrated that recovery of sperm with intact nuclear DNA is significantly higher after the swim-up technique than the isolate gradient technique (unpublished data).

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COMPLICATIONS OF SPERM NUCLEAR DNA DAMAGE Failure of Fertilization Spermatozoa with DNA damage selected for ICSI may impede the initiation or completion of decondensation, leading to failure of fertilization. Lopes have shown that men with a sperm population containing more than 25 percent of sperm with DNA damage are more likely to experience a fertilization rate less than 20 percent after ICSI. However, Twigg et al found that the genetically damaged spermatozoa are able to achieve normal fertilization following ICSI. Embryo Death Several studies have indicated that damage to sperm DNA may be linked to an increase in early embryo death. Childhood Cancer Sperm nuclear DNA damage may have consequences for the health of the offspring, who show a particularly high incidence of childhood cancer. In a study from China, paternal smoking was associated with a four-fold overall increased risk of developing childhood cancer. Smoking may induce a state of OS that is associated with free radical-mediated damage to sperm DNA. Furthermore, an independent epidemiological study in the United Kingdom concluded that 14 percent of all childhood cancers could be attributed directly to paternal smoking. Infertility Another possible consequence of free radical-mediated DNA damage in the male germ line is infertility in the offspring. This possibility relates specifically to forms of male infertility involving deletions on the long arm (q) of the Y chromosome. In this nonrecombining area of the Y chromosome (NRY), three regions have been identified that contain genes of importance to spermatogenesis; these loci have been designated AZF (azoospermia factor) a, b, and c. Deletions in each of these areas produce a particular testicular phenotype. Deletions in AZFa produce Sertoli cell only syndrome. Deletions in AZFb are associated with germ cell arrest at the pachytene stage and deletions in AZFc generate arrest at the spermatid stage of development. These deletions are not observed in fertile men or in majority of fathers of affected patients. Therefore, the Y chromosome deletions leading to male infertility must arise de novo in the germ line of the patient’s fathers. Y chromosome deletions are found in approximately 15 percent of patients with azoospermia or severe oligozoospermia and in 10 percent of men with idiopathic infertility. Although these are not particularly high frequencies, it should be recognized that more than 90 percent of the human genome is non-coding and would not produce a phenotypic change on deletion. Moreover, for most of the genome, homologous recombination could provide a theoretical mechanism for repairing double-stranded DNA

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deletions on autosomes or on the X chromosome. However, since the Y chromosome does not possess a homologue, this repair mechanism cannot be invoked, and deletions on the non-recombining region of this chromosome will persist. Thus, for Y chromosomal deletions to occur at the frequency observed, there must be an extremely high spontaneous rate of DNA fragmentation in the male germ line, most of which is either undetected or is repaired. However, deletions on the AZF on the NRY cannot be repaired and produce an extremely obvious phenotype. CONTRIBUTIONS OFTHE CLEVELAND CLINIC The role of OS in male infertility has been the main focus of our research in the Cleveland Clinic Foundation during the last decade. Our research team has identified the critical role of OS in male infertility. The main objective of our research was to transfer this important knowledge from the research bench to clinical practice. This objective was stated in a review article by Sharma and Agarwal which described specific plans and strategies for future research in the area of OS. We designed studies with the aims of: 1) understanding the exact mechanisms by which OS develops in semen; 2) establishing assays for accurate assessment of OS status and running the quality control studies for this purpose; and 3) identifying the clinical significance of seminal OS assessment in male infertility practice. Mechanism of Seminal OS We investigated the cellular origins of ROS in semen to track the source of OS and, accordingly, to create strategies to overcome the problem. Role of Seminal Leukocytes Shekarriz et al reported that peroxidase-positive leukocytes are the main source of ROS in semen and found that positive peroxidase staining is an accurate indicator of excessive ROS generation in semen. Recently, Sharma et al observed that seminal leukocytes may cause OS even at concentrations below the WHO cutoff value for leukocytospermia (concentrations greater than 1×106 peroxidase positive leukocytes/mL semen). Levels of ROS production by pure sperm suspensions were found to be significantly higher in infertile men with leukocytospermia than in infertile men without leukocytospermia and showed strong correlation with seminal leukocyte concentrations (Table 39.1). This new finding led us to postulate a potential role for seminal leukocytes in enhancing sperm capacity for excessive ROS production either by direct sperm-leukocyte contact or by soluble products released by the leukocytes. This observation has significant implications for the fertility potential of sperm both in vivo and in vitro. Excessive production of ROS by sperm in the patients with leukocytospermia implies that both the free-radical generating sperm themselves and any normal sperm in the immediate vicinity are susceptible to oxidative damage. Furthermore, once the process of LPO is initiated, the self-propagating nature of this process ensures a progressive spread of the damage throughout the sperm population.

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Role of Abnormal Spermatozoa In addition to ROS production by seminal leukocytes, the production of ROS by human sperm was also the subject of extensive research by our group. Our data indicate that human sperm production of ROS was significantly increased by the repeated cycles of centrifugation involved in the conventional semen processing techniques (washing and re-suspension) for ART. In addition, we have demonstrated that the length of duration of centrifugation is more important than the force of centrifugation for inducing ROS formation in semen. Based on these findings, we recommended the use of more gentle techniques for sperm preparation, with shorter centrifugation periods, to minimize the risk of OS-induced injury to the sperm. Our group has also reported that ROS production by human sperm increases with the increase of sperm concentration, and decreases with time. In addition, we emphasized the importance of adjusting sperm concentration for ROS measurement when comparing ROS levels between different specimens. Results from our most recent studies indicate that there is significant variation in ROS production in subsets of human spermatozoa at different stages of maturation. Following isolate gradient fractionation of ejaculated sperm, ROS production was found to be highest in immature sperm with abnormal head morphology and cytoplasmic retention and lowest in mature sperm and immature germ cells. The relative

Table 39.1: Median (25% and 75% interquartile value) ROS levels in original cell suspension (basal), in leukocyte-free sperm suspension (pure sperm); and ROS-TAC score in normal donors, non-leukocytospermic and leukocytospermic patients Variable

Donors (n=13)

NonLeukocytospermic (n=32)

Leukocytospermic (n=16)

A

B

C

Basal ROS 0.4 (0.1, 2.7 (0.53, 12) 178 (32, 430) 0.06 0.0001 <0.0001 (X106 cpm) 2.5) 0.06 (0.01, 0.31 (0.09, 1.2) 3.3 (0.5, 7.4) 0.05 0.001 0.002 Pure sperm 0.2) ROS (X106 cpm) ROS-TAC 54.5 (52, 50.3 (42, 54.8) 27.8 (23.7, 35) 0.01 0.0003 0.0001 (Score) 60) A=P value of donors versus non-leukocytospermic; B=P value of donors versus leukocytospermic; and C=P value of non-leukocytospermic versus leukocytospermic. Wilcoxon rank-sum test was used for comparison and statistical significance was assessed at P<0.05 level

proportion of ROS-producing immature sperm was directly correlated with nuclear DNA damage values in mature sperm and inversely correlated with the recovery of motile, mature sperm. These interesting findings led to the hypothesis that oxidative damage of mature sperm by ROS-producing immature sperm during their comigration from seminiferous tubules to the epididymis may be an important cause of male

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infertility. This suggests that perhaps interventions directed to (i) increase antioxidant levels in immature germ cell membranes during spermatogenesis and (ii) isolate spermatozoa with intact DNA by in vitro separation techniques should be of particular benefit to these patients in which a defect in the normal regulation of spermiogenesis and spermiation leads to an abnormal increase in the production of ROS-producing immature sperm. Assessment of OS Status Extensive research in the field of male infertility has been conducted to develop adequate indices of OS that would help determine, with accuracy, if OS is a significant contributor in male infertility. Levels of OS vary greatly in infertile men. Because OS is an imbalance between levels of ROS production and antioxidant protection in semen, it is conceivable that assessment of OS will rely on the measurement of ROS as well as total antioxidant capacity (TAC) of semen. Recently, a statistical formula described as ROSTAC score has been introduced for assessment of OS using principal component analysis. Measurement of ROS (Fig. 39.3) Levels of seminal ROS can be measured by a chemiluminescence assay. Liquefied semen is centrifuged at 300 g for 7 minutes, and the seminal plasma is separated and stored at 80°C for measurement of TAC. The pellet is washed with phosphate-buffered saline (PBS) and resuspended in the same media at a concentration of 20× 106 sperm/mL. Levels of ROS are measured by a chemiluminescence assay using luminol (5-amino-2,3,dihydro-1,4-phthalazinedione; Sigma Chemical Co., St. Louis, MO) as a probe. Four hundred microliter-aliquots of the resulting cell suspensions (containing sperm and leukocytes) are used for assessment of basal ROS levels. Eight microliters of horseradish peroxidase (HRP) (12.4 U of HRP Type VI, 310 U/mg: Sigma Chemical Co., St. Louis, MO) are added to sensitize the assay so that it could measure extra-cellular hydrogen peroxide. Ten microliters of luminol, prepared as 5-mM stock in dimethyl sulfoxide (DMSO), were added to the mixture. Anegative control was prepared by adding 10 mL of 5-mM luminol to 400 mL of PBS. Levels of ROS are assessed by measuring the luminoldependant chemiluminescence with a lumino- meter (model: LKB 953, Wallac Inc., Gaithersburg, MD) in the integrated mode for 15 minutes. The results are expressed as X 104 counted photons per minute (cpm) per 20×106 sperm. Normal ROS levels in washed semen range from 10 to 100×104 counted photons per minute (cpm) per 20×106 sperm.

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Fig. 39.3: Measurement of reactive oxygen species (ROS) in washed semen by chemiluminescence assay Measurement of TAC (Fig. 39.4) Total antioxidant capacity in the seminal plasma can be measured with an enhanced chemiluminescent assay Frozen samples of seminal plasma are thawed at room temperature and immediately assessed for TAC. Seminal plasma is diluted 1:20 with deionized water (dH2O) and filtered through a 0.20-m filter (Allegiance Healthcare Corporation, McGaw Park, IL). Signal reagent is prepared by adding 30 mL H2O2 (8.8 molar/L), 10 mL paraiodophenol stock solution (41.72 mM), and 110 mL of luminol stock solution (3.1 mM) to 10 mL of Tris Buffer (0.1 M, pH 8.0). Horseradish peroxidase working solution is prepared from the HRP stock solution by making a dilution of 1:1 of dH2O to give a chemiluminescence output of 3×107 cpm. Trolox (6-hydroxyl-2,5,7,8tetramethylchroman-2-carboxylic acid), a water-soluble tocopherol analogue, is prepared as a standard solution (25, 50 and 75 mM) for TAC calibration. With the luminometer in the kinetic mode, 100 mL of signal reagent and 100 mL of HRP working solution are added to 700 mL of dH2O and mixed. The mixture is equilibrated to the desired level of chemiluminescent output (between 2.8 and 3.2×107 cpm) for 100 seconds. One hundred microliters of the prepared seminal plasma is immediately added to the mixture, and the chemiluminescence is measured. Suppression of luminescence and the time from the

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addition of seminal plasma to 10 percent recovery of the initial chemiluminescence are recorded. The same steps are repeated after the Trolox solutions are replaced with 100 mL aliquots of the prepared seminal plasma. The assay is conducted in a dark room as light affects the chemiluminescence. Plotting the three concentrations of Trolox solution versus 10 percent recovery time results in a linear equation (Fig.39.4A,B).

Fig. 39.4A: Measurement of total antioxidant capacity (TAC) in seminal plasma by enhanced chemiluminescence assay TAC calculation Seminal TAC levels are calculated using the following equation: y=(Mx±C)×d In this equation, M refers to the slope increase in the value of Trolox equivalent for onesecond increase of the recovery time, while C accounts for the daily background variability. The results were multiplied by the dilution (d) factor and expressed as molar Trolox equivalents. ROS-TAC Score: A New Development in Field of OS The fact that neither ROS alone nor TAC alone can adequately quantify seminal oxidative stress led us to the logical conclusion that combining these two variables may be a better index for diagnosis of the overall OS affecting spermatozoa. This conclusion was behind our landmark paper in which we introduced the ROS-TAC score as a new method for accurate assessment of OS status in infertile men. The new ROS-TAC score is derived from levels of ROS in washed semen and TAC in seminal plasma. The

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resulting score minimizes the variability present in the individual parameters of OS (ROS alone or

Fig. 39.4B: Linear relationship between the concentrations of standard Trolox solution (mM) and 10 percent recovery time (sec) TAC alone). The ROS-TAC score was calculated from a group of normal healthy fertile men who had very low levels of ROS. The composite ROS-TAC score calculated for these men was representative of the fertile population, and any scores sigrdficantly below levels in the fertile population were indicative of infertility. We found that individuals with ROS-TAC scores below ‘30’, the lower limits of normal range, are at particular risk for prolonged inability to initiate pregnancies. Quality Control of OS indices (ROS andTAC) It was of utmost importance to standardize the measures that we used as indices for OS including measurement of ROS in washed semen and TAC in seminal plasma. We have demonstrated that the luminol-dependent chemiluminescence assay for ROS measurement in washed semen is both accurate and reliable when the sperm concentration is greater than 1×106/mL, and the samples are analyzed within one hour after collection. Our results have also indicated that the enhanced chemiluminescence assay is both accurate and reliable for assessment of TAC in seminal plasma.

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Clinical Significance of Assessment of Seminal OS It was also of special interest to us to determine levels of seminal OS in different clinical settings (Table 39.2). We found a significant increase in levels of ROS in men with spinal cord injury, which was also associated with poor sperm motility and morphology. We also found elevated levels of ROS in infertile men with varicoceles. In a recent study, we demonstrated that varicocelectomy resulted in a significant increase in pregnancy and live birth rates for couples who underwent IUI, although standard semen parameters were not improved in all patients. We hypothesized that the improvement in pregnancy rates

Table 39.2: Mean and standard deviation between ROS, TAC, and ROS-TAC score in subgroups of infertility patients and controls Diagnosis

ROS Log P-value vs. TAC (Trolox (ROS+1) controls* Equivalent)

P-value vs. ROS-TAC P-value vs controls* controls*

Control (n=24) 1.39±0.73 1650.93±532.22 50.00±10.00 Varicocele 2.10±1.21 0.02 1100.11±410.30 0.0002 34.87±13.54 0.0001 (n=55) Varicocele 3.25±0.89 0.0002 1061.42±425.11 0.03 22.39±13.48 0.0001 with prostatitis (n=8) Vasectomy 2.65±1.01 0.0004 1389.89±723.92 0.30 33.22±15.24 0.0002 reversal (infertile; n=23) Vasectomy 1.76±0.86 0.80 1876.93±750.82 0.62 49.35±12.25 1.00 reversal (fertile; n=12) Idiopathic 2.29±1.20 0.01 1051.98±380.88 0.0003 32.25±14.40 0.0001 infertility (n=28) *Pairwise P-values from Student’s t-test adjusted using Dunnett’s method. TAC=total antioxidant capacity ROS=reactive oxygen species

following varicocelectomy may be due to a functional factor not tested during standard semen analysis such as seminal OS or sperm DNA damage. Currently, studies are underway in our center to investigate this hypothesis. Patients with varicocele also had low levels of TAC in their seminal plasma. We speculated that these patients might benefit from antioxidant supplementation. A study on rats has indicated that free radical scavengers such as SOD can prevent free radical-mediated testicular damage. Across all clinical diagnoses, the ROS-TAC score was a superior discriminator between fertile and infertile men than either ROS or TAC alone. Furthermore, analyses of patients with a diagnosis of male-factor infertility indicated that those with subsequent successful pregnancies had an average ROS-TAC score in the normal range compared with significantly lower ROC-TAC scores in those without subsequent pregnancies. In addition, we found that the average RQS-TAC score for the fertile vasectomy reversal

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group was nearly identical to that of controls. Infertile men with male-factor or idiopathic diagnoses had significantly lower ROS-TAC scores than the controls, and that men with male-factor diagnoses that eventually were able to initiate a successful pregnancy had significantly higher ROS-TAC scores than those who failed. Also, infertile men with chronic prostatitis or prostatodynia have been shown to have lower ROS-TAC score than controls, and this was irrespective of their leukocytospermia status. In addition, male partners of couples who achieved pregnancy did not have significantly different ROSTAC scores than controls. Therefore, the new ROS-TAC score may serve as an important measure in identifying those patients with a clinical diagnosis of male infertility who are likely to achieve a pregnancy over a period of time. ROS in Neat (Raw) Semen: An Accurate and ReliableTest for OS More recently, our group has introduced an additional test of OS by measurement of ROS levels directly in neat (raw) semen. The maximum ROS level observed in neat semen from normal healthy donors, with normal genital examination and normal standard semen parameters, was 1.5×104 cpm/20 million sperm/mL. The test was subjected to all quality control studies and proved to be an accurate measure for seminal OS status. At a cutoff value of 1.5×104 cpm/20 million sperm/mL, infertile men were reliably classified into OS-positive (>1.5×104 cpm/20 million sperm/mL) or OS-negative (1.5×104 cpm/20 million sperm/mL), irrespective of their clinical diagnosis or results of standard semen analysis. We also found that assessment of ROS directly in neat semen has diagnostic and prognostic capabilities identical to those obtained from ROS-TAC score (Table 39.3). Levels of ROS in neat semen were strongly correlated with levels of ROS in washed semen and with ROS-TAC score (Fig. 39.5). A strong positive correlation was seen between ROS levels in neat semen and the extent of sperm chromatin damage. However, the difference in the extent of sperm DNA damage between OS-negative and OS-positive patients was not statistically significant, an indication that OS is associated with and/or contributing to DNA damage in some but not all infertility patients.

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Fig. 39.5: Correlation of reactive oxygen species (ROS) levels in neat semen with: A) levels of reactive oxygen species in washed semen (r=

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0.87, P<0.001), and B) reactive oxygen species—total antioxidant capacity (TAC) score (r =−0.75, P<0.001) STRATEGIESTO REDUCE SEMINAL OS Long-term strateeies must determine the cause of the enhanced generation of ROS by spermatozoa of infertile men. Reduced levels of OS will be beneficial in ART such as IUI and IVF. An insight into the molecular basis of these defects is vital in order to identify the underlying cause of the etiology of sperm pathologies. Such an understanding will help researchers develop appropriate therapeutic strategies in the treatment for male infertility. Determining the level and origin of ROS production in the ejaculate and precise evaluation of the scavenger system may be useful in treating patients. If the error in spermatogenesis that leads to such atypical activity (excessive ROS production) could be defined, it would male infertility, and a rationale basis for the design of effective therapies could be prepared. The seminal leukocyte population should also be considered potentially detrimental and must be carefully monitored. It is important to minimize the interaction between ROS-producing cells in semen, e.g. PMN leukocytes, and spermatozoa that have a potential to fertilize. Differentiating between spermatozoa and leukocyte sources of ROS is important clinically, because this has a bearing on the strategies used to reduce OS on spermatozoa during the course of IVF therapy. It is important for the clinician also to know that sperm preparation techniques used for ART may induce damage to the spermatozoa by removing the seminal plasma with its powerful antioxidants and by inducing ROS generation by spermatozoa. FMTIIRF DIRECTIONS Future efforts should be directed to elucidate why spermatozoa of some patients become over-reactive in the generation of ROS. It is also important to determine the period of sperm differentiation at which this self-destructive activity first appears. Further efforts are also required to identify sperm population at risk of collateral

Table 39.3: Median (25th and 75th percentile) values of reactive oxygen species (ROS) in neat semen, ROS in washed semen, total antioxidant capacity (TAC) in seminal plasma, and ROS-TAC score, in donors, oxidative stress (OS)-negative and OS-positive patients Variable

Donors (n=9)

OS-negative (n=11)

ROS-neat semen (X 104 0.3(0.2, 0.9) 0.3(0.2, 1)

OS-positive (n=23)

A

B

C

19(6, 143)

0.9

0.0001 0.0001

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cpm) 79(13, 177) 1468(514, 11496) 0.03 0.0003 0.0008 ROS-washed semen (X 10(4, 17) 104 cpm) TAC (Trolox 908(736, 797(575, 966) 739(627, 1047) 0.31 0.24 0.94 Equivalents) 1129) ROS-TAC score 53(51, 55) 49(47, 52) 36(32, 44) 0.4 0.001 0.001 A: Donors vs. OS-negative patients; B: Donors vs. OS-positive patients; C: OS-negative patients vs. OS-positive patients. Results were analyzed by Wilcoxon rank-sum test, P<0.05 was significant

peroxidative damage to the sperm membrane. An interesting area of future research is to investigate precisely the oxidative damage to sperm DNA and its implication on male fertility potential and the outcome of assisted reproductive programs. SUMMARY The controlled generation of very low amounts of reactive oxygen species (ROS) appears to regulate normal sperm functions, while high levels of ROS endanger sperm viability and function. Oxidative stress develops as a consequence of excessive production of ROS and/or impaired antioxidant defense systems. It is proposed that such OS precipitates a range of pathologies currently thought to afflict male reproductive function. ROSmediated peroxidative damage to the sperm plasma membrane may account for defective sperm function observed in a high proportion of infertility patients. Excessive generation of ROS may also attack integrity of DNA in the sperm nucleus. DNA bases are susceptible to OS, and peroxidation of these structures can cause base modification, DNA strand breaks and chromatin cross-linking. DNA damage induced by excessive ROS may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility, and may explain the apparent deterioration of semen quality observed during the past four to five decades. Over almost one decade, our research team in the Cleveland Clinic Foundation has identified the critical role of OS in male infertility. The main objective of our research was to transfer this important knowledge from the research bench to clinical practice. We designed studies with the aims of: 1. Understanding the exact mechanisms by which OS develops in semen, which we thought will help setup strategies to overcome the problem 2. Establishing assays for accurate assessment of OS status and running the quality control studies for this purpose 3. Testing the correlation between OS and sperm nuclear DNA damage 4. Identifying the clinical significance of seminal OS assessment in male infertility practice.

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CHAPTER 40 Quality Management in an Assisted Reproductive Therapy Environment Malcolm Clarke INTRODUCTION The Assisted Reproductive Therapy (ART) providers are now more than ever conscious of the importance of Quality Management Systems (QMS) within their business operations. In Australia alone, three practices have embraced a QMS based upon the new ISO 9001:2000 Standard in the past 12 months. Practices are finding that they have been able to measure benefits to their businesses directly related to their QMS. To maximize potential gains within any business operation, it is imperative that practitioners understand the real value of a QMS. Many people believe that a QMS will ensure a product or service that meets the needs of the provider and hopefully the needs of the client. There is some wisdom in this viewpoint, however, I believe that a QMS alone does not necessarily achieve this outcome. Let us make the following assumption: “Product quality comes from having a good idea which is translated into a good product by the inherent or learned skills oftalented people”. If we accept for the time being that this assumption is correct, one has to ask “what contribution a QMS makes to the final product or service?” I believe that the contributions are two-fold: • It allows the manufacturer or service provider to control reproducibility within their process; and • It provides a baseline risk assessment for the operations of the business, which can be used to enhance or modify as necessary, the product or service provided. Initial product design or service definition are the driving forces behind the accuracy related to the product, that is, its ability to meet the need for which it was created. The QMS plays a significant part in ensuring the on-going reproducibility of the product or service because it can be crafted to create a template that can be used to control the manufacturing or service delivery process. My assertion, particularly with respect to ART providers, is that the greatest single benefit of a QMS is realized when the process is used to investigate, interrogate and review the operations of the business. It is the single most useful risk assessment tool available to the managers of the business.

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RISK ASSESSMENT WITHIN THE ART ENVIRONMENT The science of ART has long been part of the knowledge base available to clinicians treating infertile couples. However, in the past five years there has been a quiet revolution within that scientific base. Procedures such as direct insemination and embryo creation using historical in-vitro techniques (the media named “test-tube baby”) have been joined by procedures such as Gamete Intra-Fallopian Transfer (GIFT) and Intra Cytoplasmic Sperm Injection (ICSI). The knowledge gained in the development and implementation of these procedures as well as the peripheral advances in knowledge related to the causes of infertility have created an environment where successful pregnancy rates are significantly higher today than those realized even a few years ago. This increased success rate brings with it an increase in the potential risk to the businesses of ART providers related simply to volume of successful births. The risks associated with clinical management of the patient are covered by various legal and professional guidelines and requirements and as such, will not be discussed within this chapter. However, the risks associated with the delivery of the service by the service provider are very different and are discussed in some detail in the following pages. RISK CLASSIFICATION There are a number of activities that carry a defined risk within the ART business that could be considered generic in nature, that is, they are a function of the process itself rather than of the particular legal requirements existing in the country where the practice is based. Not all the risks have exactly the same potential to cause a difficulty for the business, therefore, the business should consider identification and classification of the risk as the first step in managing the issue. The process used to identify activities associated with any procedure or activity that has the potential to bring about a detrimental outcome is known commonly as risk assessment. There are well known risk assessment models used by any number of businesses to identify potential issues. The most common of these are the very simplistic SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis or the more sophisticated RBCA (Risk Based Corrective Action) model. However, all risk assessment models have one guiding and necessary requirement, the risk assessor must understand the nature of the business and the environment in which it operates. Without this detailed knowledge, the assessor may find that he/ she misses key issues because these issues do not necessarily present themselves with warning flags attached. Once the potential risk activities have been identified, the practice can assign levels of risk to each activity. A common definition uses the simple constants of frequency and severity of each event to define the associated risk potential. Finally a management plan can be designed to eliminate or minimize the risk associated with each discrete activity It is here that the QMS is most useful, as a tool to manage the efficient implementation and review of the defined remedial or preventative actions.

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This text is designed to be a practical tool and the following discussion relates to specific issues that should be considered by any ART practice as part of its QMS development. The list is not comprehensive and should not be relied upon as a definitive process. Each practice must assess and review its own internal operations and constraints when developing a QMS. Consent Form This document is the cornerstone of clinical liability management. As a minimum, it provides the prospective patient with an understanding of the procedures that the clinician intends to perform. As a maximum, it may also define known procedural risks, dependent upon the country of origin. In all cases, it requires the patient clearly acknowledge the intent and nature of the procedure has been explained to them, and they agree to submit themselves to the procedures described. In effect, this document is a simple contract between the service provider and the recipient or client. Generically in the ART business, the nature of the consent sought is fairly broad in that it asks the patient to agree to “common” or “general” investigations and/ or therapy The risk associated with many of the consent forms currently available is that at some time during the on-going therapy e.g. embryo implantation, the clinician may opt to use a very specific technique. This decision will be based upon the best possible outcome and general welfare of the patient. However, if the procedure chosen is specific it may actually be considered as “out of the main stream” of general therapy agreed to by the patient when they first signed the consent form. In effect, if one considers that the initial consent form is in fact a contractual agreement between two parties, then it is imperative that any variation to this contract be agreed in writing. To this end, it may well be wise to have the patient counselled by the clinician at the time of “contract variation” and the patient re-establish their consent to the procedure by noting such on the original consent form or on a supporting document. This whole process would take approximately five minutes but would ensure the patient understands the intended therapy and agrees with the procedure about to be undertaken. In effect, it allows the ART provider an opportunity to provide a higher service level to the patient and to demonstrate one of the basic requirements of the ISO 9001:2000 Standard, i.e. client satisfaction. Unique Patient Identification It is critical that the ART provider ensures that all patient sample matter is clearly identified at all stages of the process. The consequences of the product of conception created by the use of either own patient or externally donated sample matter not expressing the correct phenotype with respect to the genotypes are horrendous to say the least, and the ensuing legal proceedings have the potential to bankrupt the ART provider. To avoid such issues it is critical that data management is of a high standard. Laboratory information systems can benefit from the use of discrete function software. This software only allows the input of one set of patient details against a discrete identifier such as a patient number. Similar systems should be employed in the

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management of all documented patient data including their referral notes, laboratory results reports, unit history, clinical notes and associated paperwork. This ensures the clinician has the latest data at the time of patient review and the results etc. are in fact, those of the patient he/she is consulting with. Gene Pool Restriction One of the key issues, which will affect ART providers in the future, is the risk of decreasing the gene pool. Not all children conceived using ART are aware of this fact. Some parents choose not to tell their offspring of the circumstances of their birth for a number of cultural and other reasons. Additionally, the majority of ART providers are located in countries, which have strong privacy laws. These countries often restrict access to the register of patients and in particular children conceived with ART support. Statistically, the chances of two people who were conceived using the same donated genetic material creating a child increase with every success seen in an ART unit. The outcome of a legal case related to a genetic disorder created because of such circumstances is clearly unknown at this time. Society may or may not be sympathetic to a defence of “I just complied with the law”. ART providers may need to consider this issue in some detail and formulate a position that can be presented to the regulatory body in their country of origin. ICSI is of course, a procedure that alleviates the risk associated with recombination of similar genetic material. Any procedure that mimics natural conception between a couple has the potential to both maintain the gene pool and improve client satisfaction because it overcomes potential cultural and religious difficulties faced by some couples. This aspect of the QMS should be clearly explained in any documentation because of its potential to demonstrate management of potential risks for the clinical unit. NON-SPECIFIC REQUIREMENTS OF A QUALITY MANAGEMENT SYSTEM There are numerous requirements that need to be met by any clinic embarking on the development of a QMS. The majority of these relate to the development of appropriate controls for the business and the associated documentation. The ISO 9001:2000 Standard and its associated guidance text provide excellent advice related to the requirements of the International Standard. Some of the key features, which need to be considered by any ART clinic, are listed below: Policy It is important that the policy is a commitment to provision of excellent service and it should be used to define key goals and identify a framework to allow achievement of these goals.

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Training A well defined training matrix allows a business to define its current skill base as well as which skills selected staff need to acquire in the future. Legal Issues It is imperative that the business has a dedicated procedure, which allows it to ensure it is made aware of legal requirements and alterations to relevant laws. Some of the more common means of achieving this include, but are not limited to, in-house legal staff, liaison with regulatory bodies and review of legal databases. Internal Audit System It is imperative the clinic have a scheduled internal audit process. This process is necessary to demonstrate compliance with the requirements of the International Standard. More importantly, it provides a guide to the performance of the business, which can be used by management as a tool to “fine-tune” the operations of the business. It is important the internal audit procedure is based upon a risk assessment methodology. Not all functions of the business are of equal importance or carry equal risk. Utilization of a good risk assessment procedure allows the business to define appropriate time frames between audits for each identified activity. This leads to costeffective internal auditing and minimization of risk associated with specified activities. Management Review Regular review of all business functions is critical to maximization of profits and minimization of risk. Reviews should be scheduled in accordance with business needs. Customer Satisfaction The new ISO 9001:2000 Standard identifies customer satisfaction as a key requirement of a QMS. The clinic will need to consider how it can demonstrate this output of its activities. Customer surveys and patient interviews are common methods but thought should be given to other opportunities. Preventative and Corrective actions as well as Customer Complaint management obviously take on a new level of importance within the new Standard. DEVELOPMENT OF A QUALITY MANAGEMENT SYSTEM If you are managing a clinical unit contemplating Certif ication to an International Standards Organization QMS, you need to carefully consider the requirements of your unit. The QMS you install within your business must facilitate good management practices, but not direct them. It is important to seek advice from qualified professionals and to review their advice in light of your requirements for the clinical unit.

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The most critical stage in the development of a QMS is the starting point. Prior to any developmental work the clinical unit should consider consulting an experienced, professional management systems assessor who can demonstrate experience in the provision of third party audit services. Unfortunately, there are very few individuals with the necessary experience. The next stage requires the clinical unit to determine the nature and potential severity of the business risks it faces. Once this has been completed, a management plan can be developed which addresses the defined risks. The penultimate step is to develop documented procedures to cover the necessary requirements of both the ISO Standard and the clinical unit. Finally, the unit must implement, review and refine the system. Once this point has been reached, the unit can consider contacting a Certification Body to arrange for third party accreditation. For further information related to any point in this text, or to seek assistance in the field of risk assessment related to ART clinics, please feel free to contact the author.

SECTION 6 Contemporary Thoughts

CHAPTER 41 How to Improve Success Rates in IVF? Anjali Malpani, Aniruddha Malpani INTRODUCTION In the past 22 years, since the delivery of Louise Brown, the world’s first IVF baby, advances in the field of Assisted Reproductive Technologies have been remarkable. Innovations in the areas of ovulation induction and improvements in laboratory techniques have enabled many couples to achieve their dream of having a child. It is helpful to analyze the chances of success after IVF by dividing them into three groups of factors: patient factor, laboratory factor and physician factor. These include: Patient Factor 1. The cause of infertility 2. The age of the patient 3. The endometrial receptivity (thickness and texture) 4. The number of previous cycles attempted. 5. The number of eggs retrieved. Laboratory Factor 1. The quality of the embryos (mean cumulative embryo score). 2. The number of embryos transferred. 3. The embryo transfer technique. The Physician Factor 1. Ovulation induction skills. 2. Oocyte retrieval skills. 3. Embryo transfer technique. 4. Doctorpatientrelationship. In this paper, 3 areas will be focussed on: 1. how to produce good quality eggs using advances in ovulation induction technology 2. how to improve embryo quality using advances in lab technology

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3. how to use advanced fertilization techniques, such as ICSI, AH and PGD to optimize embryo implantation.

OVULATION INDUCTION Today most clinics in the world use a protocol for ovarian stimulation which involves a combination of GnRH agonist and Human Menopausal Gonadotropins. Down regulation with a GnRH agonist suppresses endogenous gonadotropin production, improves folliculogenesis and inhibits the spontaneous LH surge. This results in fewer cancelled cycles, higher number of oocytes and more pregnancies. GnRH agonists have simplified superovulation protocols and provided increased convenience for clinicians and their patients in terms of scheduling pickups, so that doctors no longer need to do egg retrievals on a Sunday The various protocols using GnRH and HMG are: 1. the long protocol where GnRH is initiated in the midtluteal luteal phase (D21) of the previous cycle. 2. the short protocol or the flare regime where GnRH is initiated in the early follicular phase (D1). 3. the microdose flare regime. Comparisons between the follicular and luteal phase regimen have shown that the luteal phase regime resulted in more rapid pituitary suppression and improved oocyte yield and pregnancy rates. However, this is not a panacea, and in poor responders, gonadotropin requirements are significantly increased in patients on a long protocol. The flare up protocol was therefore described as a treatment for poor responders. This approach eliminates the excessive ovarian suppression while taking advantage of the initial rise in endogenous gonadotropins after the initiation of GnRH agonist. Pure FSH HMG contains FSH, but because it contains significant amounts of LH, it was thought to lead to poor oocyte quality, reduced fertilization rates, poor embryonic viability and early pregnancy wastage. This is why pharmaceutical companies introduced purified FSH preparations (Urofollitropin, Fertines Metrodin, Metrodin HP) which contain significantly reduced or negligible quantities of LH. The purification of urinary FSH also allowed gonadotropins to be administered subcutaneously and gave lesser batch to batch variability However, the pregnancy rate with these was no better, and they are significantly more expensive. Recombinant Gonadotropins All urinary derived preparations have the drawback of requiring the collection of large quantities of urine from multiple donors, leading to variability in supply and batch to batch inconsistency. Developments in biotechnology have to led to the development of

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recombinant FSH preparations, (Follitropin Gonal-F, Follitropin, Puregon) which are devoid of any LH activity and extraneous human protein. This technology has three advantages. 1. FSH production is independent of urine collection. 2. A constant supply is ensured. 3. Batch to batch consistency is guaranteed. Several randomized clinical trials have compared r-FSH with urinary gonadotropins. While these studies show that r-FSH is as good as u-HMG, unfortunately, it is no better. Also, because it is significantly more expensive, uHMG is still far more cost effective (in terms of cost per baby delivered) than the newer r-FSH. GnRH Antagonists The other interesting advance in this field is the use GnRH antagonists which are competitive inhibitors of GnRH. They bind to the GnRH receptors and block the release of LH. This suppression is immediate and observed within hours of administration and may last 10 to 100 hours. Two different GnRH antagonists which have been used are cetrorelix and ganorelix. These antagonists need to be given daily from the 6th day of HMG until the day of HCG in the dose of 0.25 mg sc. Single dose studies showed that a single dose of 3 mg of GnRH antagonist administrated when the leading diameter of 14 mm was effective as a daily dose. The advantages of the use of GnRH antagonist include: 1. initiating gonadotropin stimulation at the beginning of the menstrual cycle, potentially reducing the dose and duration of gonadotropin treatment, 2. the ability to postpone or interrupt the LH surge, and 3. reducing The dose and duration of gonadotropin therapy. Several clinical trials have demonstrated the effectiveness of GnRH antagonists in IVF. Future studies need to evaluate the different protocols using the GnRH antagonist alone and in comparison with the agonist. TECHNIQUES TO IMPROVE EMBRYO QUALITY Despite all the advances in ovulation induction protocols, the implantation rates of cleavage stage embryos transferred on Day 2 or Day 3 have remained low-from 10 to 15 percent. Transfer of early cleavage stage embryos following IVF has been used traditionally based on the belief that in vitro lab culture conditions are sub optimal, and embryo viability is compromised if transfer is deferred to development at a later stage. Today however, development to the blastocyst stage can be achieved in vitro by culturing embryos in strict, quality controlled serum free, cell free, defined sequential culture media. These media are commercially available, and are embryo friendly, since their formulation is based on a better understanding of embryo physiology

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The percentage of embryos that develop to the blastocyst stage from the cleavage stage is around 46 to 60 percent and pregnancy rates have been reported to be as high as 70 percent in some centers. The high implantation rate is because of: A. improved selection of viable embryos and B. better synchronization between embryo and the endometrium at the time of transfer. The major advantage of blastocyst transfer is that the number of embryos transferred can be reduced without risking a decline in pregnancy rates, which helps to reduce the risk of a multiple pregnancy, and may allow us to achieve the goal of a single embryo transfer. In one study, the ability to transfer just one high scoring blastocyst led to a pregnancy rate of 60 percent. The 3-part scoring system was based on blastocyst expansion, inner cell mass development and trophectoderm development. One drawback of routinely offering blastocyst transfer is the risk that no embryos may reach the blastocyst stage, thus resulting in a failure to transfer embryos, and a pregnancy rate of zero for that patient. Micromanipulation A major advance in managing male factor infertility is the technique of intracytoplasmic sperm injection. With its high fertilization and pregnancy rate it has gradually replaced conventional IVF as first-line therapy in couples with severe male factor infertility. ICSI ensures high fertilization and pregnancy rates regardless of sperm concentration, motility, or morphology-even when epididymal or testicular sperm are used. Since fertilization, embryo cleavage and implantation rates are similar to rates with conventional IVF, it is tempting to use this technique for all cases requiring in vitro fertilizations. While using ICSI for all cases of IVF is a tempting proposition, in order to maximize the number of embryos and to reduce the risk of unexpected total fertilizations failure, the rational approach remains to use the least invasive and most cost effective technique to initiate pregnancy Assisted Hatching The other area that has created a great deal of interest is that of assisted hatching. This technique is based on the observation that embryos with a thin zona pellucida had better implantation rates during IVF cycles. It is believed that in some women (for example, those over 38 years old, women with elevated basal FSH, and embryos with thicker outer zona) the process of embryo hatching is impaired. It is also possible (but completely speculative) that the reason for repeated IVF failure in some patients may be because of failure of embryo implantation, because the embryos lack a sufficient amount of energy to complete the hatching process. The different techniques used for assisted hatching include: • PZD, partial zona dissection (mechanical) • Chemical (enzymatic) • Laser • Piezo Electric Technology.

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Preimplantation Genetic Diagnosis Preimplantation genetic diagnosis is a technique that permits diagnosis of the genotype of the embryo before implantation, enabling couples at risk for transmitting genetic abnormalities to have reproductive choices. This technique was made possible as a result of simultaneous advances in IVF and micromanipulation and in the field of genetics with the development of polymerase chain reaction and fluorescence in situ hybridization. One group has proposed the use of preimplantation genetic diagnosis to diagnose aneuploidy in embryos before transfer in an effort to improve implantation rates and decrease spontaneous abortion rates. This application would have particular importance in older women who are at greater risk for spontaneous miscarriage and implantation failure. The findings of that study demonstrated that preimplantation genetic diagnosis reduced embryo loss after implantation and increased the proportion of ongoing pregnancies and live-born infants yet did not improve implantation rates. With data suggesting an increased incidence of aneuploidy in the embryos from women of advanced maternal age, with recurrent miscarriage, or with unexplained infertility, one can expect more liberal uses of preimplantation genetic diagnosis in infertility treatment in an effort to reduce the incidence of aneuploid pregnancies that are conceived. The Role of the Endometrium Why does every embryo not become a baby? The endometrium obviously has a major role to play in successful implantation, and newer ways of improving endometrial receptivity by increasing the uterine blood flow include the use of low dose aspirin, nitroglycerine patches, and vaginal viagra. Nitroglycerine patches and viagra both act by releasing nitric oxide (NO) in smooth muscles, causing dilatation of uterine blood vessels, thereby improving uterine blood flow and delivery of estrogen to the uterine lining, and hence improving the growth of the endometrium. Defects in uterine receptivity are a major reason for the poor pregnancy rate after IVF, since this leads to implantation failure. Several endometrial proteins have been suggested as markers of uterine receptivity. Recent data have suggested that integrins subunits are useful markers of uterine receptivity, since their levels change in luminal and glandular epithelium at the approximate time that embryos attach. Embryo Transfer Technique Since embryo transfer is such a simple procedure, it has always been taken for granted, and little attention has been paid to the transfer technique in the past. Today, we know that the success of an IVF cycle depends to a large extent on how well the transfer is performed, and clinicians need to spend more time in mastering this deceptively simple procedure, so that it is as atraumatic as possible. The various factors that affect embryo transfer technique are: 1. The type of catheter used, and studies have shown that it is best to use the softest catheter possible. Ultrasound guidance has been very helpful to ensure accurate placement of embryos in the cavity, near the fundus.

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2. For difficult transfers, it may be necessary to use a tenaculum to straighten the uterine axis. Traumatic transfers which induce bleeding have been shown to reduce pregnancy rates, so that performing a “dummy” embryo transfer is a useful technique to ensure that the actual embryo transfer is performed atraumatically 3. Embryos should be transferred in as small a volume as possible, to minimize the risk of their expulsion, and all the cervical mucus should be carefully aspirated prior to transfer, to prevent entrapment of the embryos in cervical mucus. There is no doubt that significant progress has been made in the assisted reproductive techniques, and success rates have definitely improved over the years. However, it is important not to jump to conclusions too fast. A new tool does not necessarily mean it is the best and while it may be tempting to use the “newest technique”, unless a large number of patients are studied, and double blind prospective randomized trials performed, clinicians should be wary about changing standard clinical procedures. Published papers need to be read critically, and clinicians need to understand the basics of evidence-based medicine, so that they can sift the wheat from the chaff. Unfortunately, in order to inflate their pregnancy rates, many clinics play games with their numbers, and end up giving patients false hopes and unrealistic expectations of success. In reality, there is probably little that separates most competent ART centers, most of which follow standard protocols and procedures. No centre can guarantee a pregnancy, and no centre can accurately predict the chances of success. Each centre would obviously want to play this game of numbers to present a high success rate and indulge in what is called the “ART of embroidery” to present a rosy picture But it is now high time that we should downplay the business side of ART, and concentrate on individualized, cost effective patient care. In the final analysis, there is no substitute for a healthy positive doctor patient relationship, based on informed consent, and free, frank communication. REFERENCES 1. Albano C, Smitz J, Camus M et al. Comparison of different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 76:917–22 2. Bahee M, Cohen J, Munne S. Preimplantation genetic diagnosis of aneuploidy: Were we looking at the wrong chromosomes? J Assist Reprod Genet 1999; 16:176–81. 3. Droesch K, Muasher SJ, Brzyski RG et al. Value of suppression with a gonadotropin releasing hormone agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil Steril 1989; 51:292–97 4. Gardner DK, Vella P, Lane M et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfer. Fertil Steril 1998; 69:84– 88. 5. Gardner DK, Schoolcraft WB, Wagly L et al. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998; 13:3434–40. 6. Handyside AH, Kontogianni E, Hardy K et al. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification. Nature 1990; 344:768–70.

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7. Leondires MP, Escalpes M, Segars JH et al. Microdose follicular phase gonadotropin releasing hormone agonists (GnRH-a) compared with luteal phase GnRH-a for ovarian stimulation at in vitro fertilization. Fertil Steril 1999; 72:1018–23. 8. Manassiev NA, Tenekedjier KI, Collins J. Does the use of recombinant follicle stimulating hormone instead of urinary follicle stimulating hormone lead to higher pregnancy rates in in vitro fertilization-embryo transfer cycles? Journal of Assisted Reproduction 1999; 9:7–12. 9. Munne S, Magli C, Cohen J et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14:2191–99. 10. Out HJ, Mannaerts BMJL, Coelingh Bennink HJT, et al. for the European Puregon Collaborative IVF Study Group: A prospective, randomized, assessor blind, multicenter comparing recombinant and urinary follicle stimulation hormone (Puregon vs Metrodin) in in vitro fertilization. Hum Reprod 1995; 10:2536–40. 11. Van Steirteghem AC, Liu J, Joris H et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination: Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993; 8:1055–60.

CHAPTER 42 Current Immunological Assays: Are they Enough to Uncover the Supposed Immune Causes for Assisted Reproduction Failure? Aygül Demirol, Erdal Aktan, Timur Gürgan INTRODUCTION The contribution of the immune system to the fine-tuning of the implantation process of the embryo and the continuation of pregnancy until the birth of a healthy baby has long been of interest to reproductive biologists and clinicians. It was considered that modulation in the function of immune system has to be during pregnancy for a successful outcome. The inefficiency in this modulation and an unusual type of modulation in the function of immune system were also considered as probable causal factors particularly for an unsuccessful outcome in the therapy of unexplained infertility Many descriptive studies imply a role for immunology in reproductive success and failure, although the precise mechanisms of such a role remains insufficiently defined. Unlike the consensus on the use of tests to uncover the immunological causes for recurrent pregnancy loss, the use of the same tests has not been justified for the same purpose in couples undergoing IVF, yet. Antiphospholipid Antibodies Antiphospholipid antibodies (APAs) are the most known immune etiologic factors proposed for the unsuccessful outcome in IVF. APAs are the part of autoimmune aspect of the immunological problem and are antibodies IgG, IgM, and/or IgA, acquired against negatively charged phospholipids associated with slow progressive thrombosis and infarction in the placenta.1 Commonly tested APAs are lupus anticoagulant and IgG and IgM anticardiolipin antibodies. An increased prevalence of APAs has been defined in infertile women undergoing ovulation induction2, but the association of this finding with the clinical outcome has not been proved, yet. Gleicher et al3 Birdsall et al4 and Balasch et al5 have all failed to find a relationship between APAs and pregnancy outcome in IVF cycles. In a larger investigation published by Denis et al6 APA testing was performed using a commercial battery of 21 APA tests but they reported no differences in implantation and pregnancy rates for any of the 21 antibodies evaluated. In contrast, Sher et al7 reported that APAs were clinically important factors determining outcome in IVF cycles. They also reported increased pregnancy rates for patients with APAs who were treated with aspirin and heparin. In 1999, The ASRM Practice Committee decided to publish a report entitled “Antiphospholipid Antibodies Do Not Affect IVF Success.”8 Following an evidence

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based literature search, data from the published studies revealed that the clinical pregnancy and live birth rates were 57 percent and 46 percent in APA-positive patients and 49.2 percent and 42.9 percent in APA-negative patients, respectively, so the report concluded that APA testing is not warranted in IVF patients and treatment is not indicated in seropositive patients for successful IVF outcome. Hornstein et al9 also reported the same conclusion based on a meta-analysis of APA and IVF success. Following the publication of the ASRM Practice Committee report, a rebuttal by the Antiphospholipid Antibody Committee (AAC) was also published in 2000.10 AAC criticized the conclusions mainly for the inclusion of the heterogeneous studies in the meta-analysis. They concluded that the wide variation in outcomes of APA-positive patients among the studies precluded the conclusion reached in the report, but Hornstein11 stated that the heterogeneity among the studies was not statistically significant and clinical heterogeneity also seemed unlikely, because the study designs were markedly similar and the individual study odds ratios conformed to a normal distribution around 1.0 or no difference. Hornstein also refused the AAC statement that APA measurements were useful in some centers but not in others, since a test should not vary so widely in its usefulness if it has universal applicability in normal clinical practice. In 1999, Egbase et al,12 concluded in their study that although the high prevalence of APA in general IVF patients could not be shown to justify the routine evaluation to predict the outcome, the occurrence of two consecutive miscarriages (rather than three or more pregnancy losses) after repeat IVF or ICSI suggests a subset of women in whom routine APA screening prior to further assisted reproductive treatment might be advised. As a conclusion, since APA testing and treatment for patients undergoing assisted reproduction still remains controversial, any APA evaluation in IVF patients should be considered only for experimental studies, unless there is concomitant history of recurrent pregnancy loss.13 Antithyroid Antibodies Antithyroid antibodies are accepted as the markers of autoimmune thyroid disorders. Thyroglobulin is synthesized by thyroid cells and functions in the synthesis and storage of thyroid hormones. Thyroid peroxidase is an enzyme responsible for the iodination of thyrosine residues and coupling of iodinated residues to form thyroid hormones.13 Autoimmune antibodies against these antigens have been accused for recurrent pregnancy loss. Sher et al,14 reported their experience with antithyroid antibody screening and treatment using aspirin/heparin/ IVIG in IVF patients and concluded that IVIG had positive effect on the outcome in seropositive patients. But, this study was criticized by Hill and Scott15 for not being appropriately randomized and controlled. Also, Kutteh et al,16 in a larger multicenter study reported that there was no correlation between between the presence of antithyroid antibodies and pregnancy rates or delivery rates in patients undergoing IVF-ET. So, assessment of antithyroid antibodies and treatment of seropositive patients undergoing assisted reproduction may not be clinically useful.

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Reproductive Peripheral Blood Immunophenotyping Pregnant women have decreased natural killer (NK) activity compared to non-pregnant women,17 but in spontaneous abortions NK cell activity was not found to be decreased.18 Determining the NK cell number or percentage in peripheral circulation may also be considered in predicting the outcome in assisted reproduction. However, although the distribution of peripheral lymphocyte population was reported to be predictive for the pregnancy outcome, Hill and Scott15 reported no relationship between immunophenotype results and either pregnancy rates or outcomes including CD 56 NK cells, CD 4 (T helper/cytotoxic cells), CD 8 (T suppressor/ inducer cells), CD 4: CD 8 ratio, or gamma/delta cells. Thus, they reported that the assessment of reproductive immunophenotype was not clinically useful in women undergoing IVF-ET. On the other hand, recently Gallinelli et al19 reported that a prolonged condition of stress, which caused a decreased ability to adapt a transitory anxious state, was associated with high amounts of activated T-cells in the peripheral blood, and also this condition was associated with a reduced implantation rate in women undergoing IVF-ET. Antinuclear Antibodies and Antiovarian Antibodies Antinuclear antibodies are (ANA) autoantibodies against some nuclear components (DNA, histones, Sm, SS-A, SS-B, RNP). Antiovarian antibodies (AOA) are also a group of antibodies against ooplasm, zona pellucida, membrane granulosa, theca folliculi interna and lutein cells.13 However, the studies about the predictive value of these antibodies for the outcome of pregnancy or assisted reproduction are currently not enough to drive any clinically useful conclusions about their role in assisted reproduction failure.13 Human Serum Embryo-toxicity In women with reproductive failure, suggested embryotoxic unknown factors in serum have been investigated using mouse embryo culture system. Embryotoxicity of the serum from women with reproductive problems was proposed but this suggestion was also criticized since, the serum collection errors such as the contact of serum and the rubber stopper of the tube was one of the causes for the embryotoxicity. Also, heterotopic antibodies against rodent antigens in human serum should be considered in human serum embryotxicity tests using rodent embryo culture systems. So, evidence justifying the determination of human serum embryotoxicity currently is not available and is not recommended in patients undergoing IVF or other infertility therapies.20 Antisperm Antibodies Sperm have some antigens foreign to immune system of both men and women. When sperm are exposed to the immune system in certain situations, antisperm antibodies (ASA) are produced in serum of male or female, in seminal plasma or in cervical mucus. ASA may interfere with fertilization (i.e. zona pellucida binding, penetration of zona pellucida, zona reaction, gamete fusion, cleavage and development of embryo). It was reported, that after washing, sperm could recover the ability to penetrate zona.21,22 Since

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sperm antigens are found on the surface of fertilized oocytes,23,24 antisperm antibodies were also shown to be cytotoxic to fertilized oocytes in animal settings.23,25 On the other hand, contrary to the expressed concern that antisperm antibodies inhibit nidation or fetal growth, high implantation (23.5%) and pregnancy (37.1%) rates were reported upon IVFET treatment of immunologic infertile women.26 Similar results were also reported by Shibara et al,27 but they concluded that the observed high pregnancy rate in the antisperm antibody positive group was probably due to fewer associated infertility problems. Contrary to the retrospective analyses performed in the cycles with poor fertilization indicating deleterious effects of sperm-bound ASA in oocyte fertilization rates, embryo quality, or implantation; prospective controlled studies28,29,30 failed to demonstrate any adverse effect of ASA on IVF outcome. As a conclusion, currently available methods to test the dysfunctions of the immune system could not unequivocally demonstrate any immune system dysfunction associated with the poor outcome in assisted reproduction so, immunologic tests to predict poor outcome in patients undergoing IVF as well immune therapy in seropositive IVF patients are not justified. Future studies are needed to determine the effects of functional variations of immune system on the outcome of assisted reproduction. REFERENCES 1. Harris EN. Syndrome of the black swan. Br J Rheumatol 1986; 26:324–6. 2. Fisch B, Rikover Y, Shohat L, Zurgil N, Tadir Y, Ovadia J et al. The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid antibodies. Fertil Steril 1991; 56:718–24. 3. Gleicher N, Liu H, Dudkiewicz A, Rozenwaks Z, Kaberlein G, Pratt D et al. Autoantibody profiles and immunoglobulin levels as predictors of in vitro fertilization success. Am J Obstet Gynecol 1994; 170:1145–9. 4. Birdsall MA, Lockwood GM, Ledger WL, Johnson PM, Chamley LW. Antiphospholipid antibodies in women having in vitro fertilization. Hum Reprod 1996; 11:1185–9. 5. Balasch J, Creus M, Fabregues F, Reverter J, Carmona F, Tassies DJ, et al. Antiphospholipid antibodies and human reproductive failure. Hum Reprod 1996; 11:2310–5. 6. Denis AL, Guido M, Adler RD, Bergh PA, Brenner C, Scott RT Jr. Antiphospholipid antibodies and pregnancy rates and outcome in IVF patients. Fertil Steril 1997; 67:1084–90. 7. Sher G, Feinman M, Zouves C, Kuttner G, Maassarani G, Salem RW et al. High fecundity rates following in vitro fertilization and embryo transfer in antiphospholipid seropositive women treated with heparin and aspirin. Hum Reprod 1994; 9:2278–83. 8. American Society for Reproductive Medicine Practice Committee Report. Antiphospholipid antibodies do not affect IVF success. ASRM Practice Committee, 1999. 9. Hornstein MD, Davis OK, Massey JB, Paulson RJ, Collins JA. Antiphospholipid antibodies and in vitro fertilization success: a meta analysis. Fertil Steril 2000; 73:330–3. 10. American Society for Reproductive Immunology Antiphospholipid Antibody Committee. Arational basis for antiphospholipid antibody testing and selective immunotherapy in assisted reproduction: a rebuttal to American Society for Reproductive Medicine Practice Committee Opinion. Fertil Steril 2000; 74:631–4. 11. Hornstein MD. Antiphospholipid antibodies in patients undergoing IVF: the data do not support testing. Fertil Steril 2000; 74:635–6.

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12. Egbase PE, Al Sharhan M, Diejomaoh M, Grudzinskas JG. Antiphospholipid antibodies in infertile couples with two consecutive miscarriages after in vitro fertilization and embryo transfer. Hum Reprod 1999; 14:1483–6. 13. Ghazeeri GS, Kutteh WH. Autoimmunity and assisted reproduction. Infertility and Reproductive Clinics of North America 2002; 13(1):183–201. 14. Sher G, Maassariani G, Zouves C, Feinman M, Sohn S, Matzner W et al. The use of combined heparin/aspirin and immunoglobulin G therapy in the treatment of in vitro fertilization in patients with antithyroid antibodies. Am J Reprod Immunol 1998; 39:223–5. 15. Hill JA, Scott RT Jr. Immunologic tests and IVF: Please, enough already. Fertil Steril 2000; 74:439–42. 16. Kutteh WH, Schoolcraft WB, Scott RT Jr. Antithyroid antibodies do not affect pregnancy outcome in women undergoing assisted reproduction. Hum Reprod 1999; 14:2886–90. 17. Hill JA, Hsia S, Doran DM, Bryans CI Jr. Natural killer and antibody dependent cell medicated cytotoxicity in preeclampsia. J Reprod Immunol 1986; 9:205–12. 18. Aoki K, Kajiura S, MatsomotoY, Ogasawara M, Okada S, Yagami Y et al. Preconceptual natural killer cell activity as a predictor of miscarriage. Lancet 1995; 345:1340–2. 19. Gallinelli A, Roncaglia R, Matteo LM, Ciaccio I, Volpe A, Facchinetti F. Immunological changes and stress are associated with different implantation rates in patients undergoing in vitro fertilization-embryo transfer. Fertil Steril 2001; 76:85–91. 20. Haimovici F, Hill JA, Anderson DJ. Variables affecting toxicity of human sera in mouse embryo cultures. J In vitro Embryo Transfer 1988; 5:202–6. 21. Daioth T, Kamada M, Mori T. Studies on biological actions of sperm immobilizing antibody on human fertilization in vitro. Acta Obstet Gynecol Jpn 1986; 38:1057–65. 22. Kamada M, Maegawa M, Yamamoto S, Takikawa M, Kunimi K, Yoshikawa S. Physiology and pathology of antisperm immunity in pregnancy and infertility. Adv Reprod 1998; 1:241–50. 23. Koyoma K, Hasegawa A, Isojima S. Effect of antisperm antibody on the in vitro development of rat embryos. Gamete Res 1984; 10:143–52. 24. Yoshiki T, Yang YY, Lee Y, Lee CY. Generation and characterization of monoclonal antibodies specific to surface antigens of human trophoblast cell. Am J Reprod Immunol 1995; 34:148–55. 25. Menge AC. Effect of isoimmunization and isoantisera against seminal antigens on fertility process in female rabbits. Biol Reprod 1971; 4:137–44. 26. Daitoh T, Kamada M, Yamano S, Murayama S, Kobayashi T, Maegawa M et al. High implantation rate and consequently high pregnancy rate by in vitro fertilization -embryo transfer treatment in infertile women with antisperm antibody. Fertil Steril 1995; 63:87–91. 27. Shibahara H, Mitsuo M, Ikeda Y, Shigeta M, Koyama K. Effect of sperm immobilizing antibodies on pregnancy outcome in infertile women treated with IVF-ET. Am J Reprod Immunol 1996; 36: 96–100. 28. Culligan PJ, Crane MM, Boone WR, Allen TC, Price TM, Blauer KL. Validity and cost effectiveness of antisperm antibody testing before in vitro fertilization. Fertil Steril 1998; 69:894–98. 29. Mandelbaum SL, Diamond MP, DeCherney AH. Relationship of antisperm antibodies to oocyte fertilization in in vitro fertilization-embryo transfer. Fertil Steril 1987; 47:644–51. 30. Sucharoen N, Keith J. The effect of the antisperm auto-antibody-bound sperm on in υitro fertilization outcome. Andrologia 1995; 27:281–89.

CHAPTER 43 Endometriosis and ART Ved Prakash Singh, Angela Beaten INTRODUCTION Endometriosis is one of the most enigmatic conditions in gynaecology. Since the publication of the most accepted theory of pathogenesis of endometriosis,1 there have been over seven thousand publications on this subject. Indeed, endometriosis is one of the most frequently investigated disorders in gynaecology. Despite that, there remains a lack of consensus on various issues related to its pathophysiology and treatment. One of the most perplexing issues with endometriosis is the link between endometriosis and infertility. In fact, no clear-cut cause and effect relationship has been established between endometriosis and infertility especially in the early stages of the disease. Most of the older reports on endometriosis had major flaws in design and were often retrospective or descriptive. Recently, there have been a number of good scientific reports based on sound laboratory and clinical research which have helped advance our understanding of mechanisms involved in the endometriosis disease process. This chapter first outlines the relationship between endometriosis and infertility and then focuses of the evidence of the effect of endometriosis on the various assisted reproductive techniques. Endometriosis and Infertility As stated earlier, the relationship between early endometriosis and infertility is unclear. There is a body of indirect evidence of the association between infertility and endometriosis. In prospective studies, normal fertile women have much lower incidence of endometriosis than those who are infertile.2–3 Lower fecundity has been demonstrated in animal models with surgically induced endometriosis.4–6 Women entering a donor insemination programme with minimal endometriosis have significantly reduced cycle fecundity.7 Endometriosis and ART The link between endometriosis and assisted reproductive technologies (ART) is still more controversial. Several observational studies have suggested a lower success rate of ART in women with endometriosis.8–10 However there are other studies which do not support this view.11 It is difficult to interpret studies involving ART as usage of high dose GnRh analogues and gonadotrophins with consequent morphologic abnormalities in endometrium, extended luteal phase support and transfer of more than one embryo make direct comparison between stimulated and natural cycles extremely difficult.12

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GnRH-analogue Therapy in ART Cycles in Women with Endometriosis If endometriosis does indeed diminish pregnancy rates in ART cycles, then it is reasonable to assume that treatment of endometriosis immediately before the cycle may improve the pregnancy rate. Women with moderate and severe endometriosis have been found to have a significantly lower pregnancy rate in ART cycles than women with tubal disease and an improvement in the rate is noted with the use of a GnRh analogue.13–14 Two recent studies demonstrated the benefits of prolonged down regulation with GnRH analogues before initiation of IVF in patients with endometriosis.15–16 Folliculogenesis and Ovulation in Endometriosis The use of IVF as a therapeutic tool in women with endometriosis has enabled us to gain clinical knowledge of factors implicated in endometriosis-associated infertility. Anovulation, or luteinised unruptured follicle (LUF) syndrome may occur when, despite ovulatory changes such as an LH surge and a rise in progesterone concentration, the egg is not released from the follicle. LUF syndrome has been noted in some women with endometriosis,17 but it is also known to occur in normal fertile women.18 It is not clear whether LUF syndrome is a consistent change or sporadic event during ovulatory function, or whether it is associated with endometriosis. The significance of this syndrome in terms of ovulatory dysfunction and unexplained fertility is under debate. Impaired fertilisation is thought to be an adverse effect of endometriosis. Although some studies have reported that fertilisation rates were reduced in women with endometriosis compared to those with tubal or unexplained infertility,19–20 other studies have shown comparable fertilisation rates.21–22 It is possible that lower fertilisation rates in oocytes derived from women with endometriosis are a consequence of altered folliculogenesis. Follicular fluid from women with endometriosis has been shown to stimulate the proliferation of endometrial stromal cells in culture.23 In addition, reduced oestradiol concentrations during the pre-ovulatory phase, reduced oestradiol and progesterone concentrations in the early luteal phase, impaired follicular growth24 and altered LH surge profiles have also been noted.25 Somewhat inconsistent findings also suggest that preovulatory granulosa cell steroidogenesis may be reduced in vitro,26 in cells derived from women with mild endometriosis. In addition to gonadotrophins and ovarian steroids, growth factors and other chemical messengers are important for folliculogenesis and oocyte maturation. In particular, the interleukins IL-1b and IL-6, and vascular endothelial growth factor (VEGF) mediate a number of effects in the ovary. Interestingly the serum concentration of IL-6 was found to be significantly increased during natural, but not stimulated cycles of women with endometriosis.27 Additionally, IL-6 secretion was significantly increased, and VEGF accumulation significantly decreased, in follicular fluid and granulosa cell cultures derived from patients with endometriosis. Together, these findings contribute to a somewhat altered endocrine environment in women affected by endometriosis, which may result in alterations within the oocyte that lead to lower quality embryos.

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Embryo Qualityand Uterine Receptivity in Endomethosis For successful implantation, complex interactions between embryonic and uterine cells must occur. There remains some controversy whether endometriosis affects implantation and pregnancy rates during IVF cycles.28–32 Earlier studies focused on oocyte quality have noted a decreased pregnancy rate in endometriosis patients using either their own oocytes or donated oocytes from women with endometriosis.33 No decrease in implantation rate or on-going pregnancy rate was seen when these patients received oocytes from women without endometriosis. These observations suggest that a decreased implantation and pregnancy rate in women with endometriosis may be due to poor oocyte quality and not to a defect in the endometrium. In an attempt to further clarify whether oocytes derived from the ovaries of women with endometriosis have a reduced ability to implant, the quality of embryos derived from women with endometriosis has been investigated. In a retrospective study, the number of blastomeres and degree of fragmentation of embryos derived from women with endometriosis were analysed, and compared with data from embryos derived from women with tubal infertility.34 After 72 hours in culture, there was a significant decrease in the number of blastomeres, and a significant increase in the percentage of embryos that arrested in the endometriosis group. This data suggests that poor embryo quality may be the main factor contributing to lower implantation rates in women with endometriosis. However, it is possible that poor quality embryos with a decreased ability to implant, and alterations in the endometrium which result in a hostile endometrial environment, are both contributing factors in endometriosis-related infertility. Some research has focussed on the role of integrins and uterine receptivity Integrins are a family of molecules that act as receptors for the extracellular matrix, mediating interactions between cells and the matrix during cellular proliferation, differentiation and survival. The use of integrins as biochemical markers of uterine receptivity has been investigated. There is conflicting data, but aberrant expression of integrins may alter endometrial receptivity.35–38 Whether changes in the expression of some integrin genes occur before the development of endometriosis, or as a result of endometriosis, remains to be elucidated. While the exact mechanism by which the integrin genes are regulated is unknown, cytokines, growth factors and sex steroids, the levels of which are altered in endometriosis, may modulate their expression.39 Temporal changes in integrin gene expression may provide clues to the underlying molecular events that take place in the endometrium during natural cycles, and in conditions such as endometriosis. Finally, it appears that functional changes in several immunological components in the peritoneal fluid are associated with endometriosis. For instance, peritoneal fluid has been shown to contain increased levels of macrophages, which secrete cytokines and growth factors, in response to endometriosis-associated inflammation.40 Cytokines are involved during endothelial cell implantation, the proliferation and formation of endometriotic lesions, and play a critical role in decreased immunological surveillance, recognition and destruction of ectopic endometrial cells. Increased cytokine production is one of the similarities endometriosis shares with autoimmune diseases such as rheumatoid arthritis and Crohn’s disease.41 Other similarities include decreased cell apoptosis, and T-and Bcell abnormalities. It is generally accepted that women with endometriosis have autoimmunity to endometrial and ovarian targets.42 When autoantibodies are directed

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against ectopic endometrium, they may also affect normal uterine endometrium and lead to early implantation failure and increased early pregnancy loss. CONCLUSION IVF would seem to be an effective treatment for many of the mechanical alterations in reproductive events that are associated with endometriosis-related infertility. Ovarian hyperstimulation and luteal phase support may correct some effects of endometriosis on ovulation and the endocrine environment. Furthermore, oocyte aspiration and embryo transfer to the uterus may overcome problems involving ovum capture or transport associated with endometriosis, as well as effectively remove the oocyte, sperm, and early embryo from a potentially toxic environment. Decreased fertilisation, implantation and pregnancy rates in women with endometriosis may be a reflection of reduced oocyte quality, which leads to a reduction in embryo quality and the ability of these embryos to implant in an altered endometrial environment. Although there is conflicting evidence with regard to the effect of endometriosis on oocyte/embryo quality and uterine receptivity, the research in this area seems to lend support to the conclusion that these processes are altered in women affected by this disease. REFERENCES 1. Sampson J. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue in to peritoneal cavity. Am J Obstet Gynecol 1927; 14:422–469. 2. Verkauf BS. Incidence, symptoms, and signs of endometriosis in fertile and infertile women. J Fla Med Assoc 1987; 74:671–75. 3. Strathy JH, Molgaard CA, Coulam CB, Melton LJ. Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 1982; 38:667–75. 4. Schenken RS, Asch RH. Surgically induced endometriosis in the rabbit: effects on fertility and concentration of peritoneal fluid prostaglandins. Fertil Stril 1980; 34:581–87. 5. Vernon MW, Wilson EA. Studies on surgical induction of endometriosis in the rat. Fertil Steril 1985; 44:684–94. 6. Barragan JC, Brotons J, Ruiz JA, Acien P. Experimentally induced endometriosis in rats: effect on fertility and the effects of pregnancy and lactation on the ectopic endometrial tissue. Fertil Steril 1992; 58:1215–19. 7. Jansen RP. Minimal endometriosis and reduced fecundability: Prospective evidence from an artificial insemination by donor programme. Fertil Steril 1986; 46:141–43. 8. Matson PL, Yocich JL. The treatment of infertility associated with endometriosis by in vitro fertilization. Fertil Steril 1986; 46:432–34. 9. Chillik CF, Acosta AA, Garcia JE et al. The role of in vitro fertilization in infertile patients with endometriosis. Fertil Steril 1985; 44:56–61. 10. Guzick DS, yao YAS, Berga SL, Krasnow JS, Stovall DW, Hubik CJs et al. Endometriosis impairs the efficacy of gamete intrafallopian transfenresulta of a case control study. Fertil Steril 1994; 62:1186–91. 11. Olivennes F, Feldberg D, Liu HC, Cohen J, Moy F, Rosenwaks Z. Endometriosis: A stage by stage analysis-the role of in vitro fertilization. Fertl Setril 1995: 64:392–98. 12. Lessey BA. Embryo quality and endometrial receptivity: Lessons learned from the ART experience. J Assist Repod Genet 1997; 15:173–76.

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13. Oehninger S, Acosta A, Kreiner D et al. In vitro fertilization and embryo transfer(IVF/ET): An established and successful therapy for endometriosis. J in vitro Fertil Embryo Transfer 1988; 5:249–56. 14. DickerD, Goldman GA, Ashkenazi J, Feldberg D, Voliovitz I, Goldman JA. The value of pretreatment with gonadotrophin releasing hormone(GnRH) analogue in IVF-ET therapy of severe endometriosis. Hum Reprod 1990; 5:418–20. 15. Damario MA, Moy F, Moomjy M, Davis OK, Tortoriello D, Rosenwaks Z. Delay in gonadotropin stimulation in patients receiving GnRh-agonist therapy permits increased clinic efficiency and may enhance IVF pregnancy rates. Fertil Steril 1997; 68:1004–10. 16. Kim CH, Cho YK, Mok JE. Simplified ultralong protocol of gonadotropin-releasing hormone agonist for ovulation induction with intrauterine insemination in patients with endometriosis. Hum Reprod 1996; 11:398–402. 17. Mio Y, Toda T, Harada T et al. Luteinized unruptured follicle in the early stages of endometriosis as a cause of unexplained fertility. Am J Obstet Gynecol 1992; 167:271–73. 18. Kerin J, Kirby C, Morris D et al. Incidence of the luteinized unruptured phenomenon in cycling women. Fertil Steril 1983; 40:620–26. 19. Wardle PG, Foster PA, Mitchell JD et al. Endometriosis and IVF: Effect of prior therapy. Lancet 1986; 1:276–77. 20. Hull MGR, Williams JA, Ray B et al. The contribution of subtle oocyte or sperm dysfunction affecting fertilization in endometriosis-associated or unexplained infertility: a controlled comparison with tubal infertility and use of donor spermatozoa. Hum Reprod 1998; 13:1825– 30. 21. Geber S, Paraschos T, Atkinson G et al. Results of IVF in patients with endometriosis: the severity of the disease does not affect outcome, or the incidence of miscarriage. Hum Reprod 1995; 10:1507–11. 22. Arici A, Oral E, Bukulmez O et al. The effect of endometriosis on implantation: results from the Yale University in vitro fertilization and embryo transfer program. Fertil Steril 1996; 65:603–07. 23. Bahtiyar MO, Seli E, Oral E et al. Follicular fluid from women with endometriosis stimulates the proliferation of endometrial stromal cells. Hum Reprod 1998; 13:3492–95. 24. Tummon IS, Maclin VM, Radwanska E et al. Occult ovulatory dysfunction in women with minimal endometriosis or unexplained infertility. Fertil Steril 1988; 50:716–20. 25. Cahill DJ, Hull MG. Pituitary-ovarian dysfunction and endometriosis. Hum Reprod Update 1988; 6(1):56–66. 26. Harlow CR, Cahill DJ, Maile LA et al. Reduced pre-ovulatory granulosa cell steroidogenesis in women with endometriosis. J Clin Endocrinol Metab 1996; 81:426–29. 27. Pellicer A, Albert C, Garrido N, Navarro J, Remohi J, Simon C. The pathophysiology of endometriosis-associated infertility: follicular environment and embryo quality. J Reprod Fertil Suppl 2000; 55:109–19. 28. Garcia-Velasco JA, Arici A. Is the oocyte/embryo affected in endometriosis? Human Reprod Update 1999; 14(2):77–89. 29. Dmowski W, Rana N, Michalowska J et al. The effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates. Fertil Steril 1995; 63:555–62. 30. Tanbo T, Omland A, Dale P et al. In vitro fertilization/embryo transfer in unexplained fertility and minimal peritoneal endometriosis. Acta Obstst Gynecol Scand 1995; 74:539–43. 31. Arici A, Oral E, Bukulmez O et al. The effect of endometriosis on implantation: results from the Yale University in vitro fertilization and embryo transfer program. Fertil Steril 1996; 65:603–07. 32. Simon C, Gutierrez A, Vidal A et al. Outcome of patients with endometriosis in assisted reproduction: results from in vitro fertilization and embryo transfer success rates. Hum Reprod 1994; 9:725–29.

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33. Pellicer A, Oliverira N, Ruiz A et al. Implantation in endometriosis: Lessons learned from IVF and oocyte donation. In Spinola P, Coutinho EM (Eds): Progress in Endometriosis. Carnforth, UK: Parthenon Publishing Group, 1994; 177–83. 34. Pellicer A, Oliverira N, Ruiz A et al. Exploring the mechanism(s) of endometriosis-related infertility: an analysis of embryo development and implantation in assisted reproduction. Hum Reprod 1995; 10:91–97. 35. Lessey B, Castelbaum A, Sawin S et al. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 1994a; 62:497–506. 36. Lessey B, Castelbaum A, Sawin S et al. Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab 1994b; 79:643–49. 37. Bridges J, Prentice A, Roche W et al. Expression of integrin adhesion molecules in endometrium and endometriosis. Br J Obstet Gynaecol 1994b; 101:696–700. 38. Hii L, Rogers P. Endometrial vascular and glandular expression of integrin alpha (v) beta3 in women with and without endometriosis. Hum Reprod 1998; 13:1030–35. 39. Lessey B, Yeh I, Castelbaum A et al. Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation. Fertil Steril 1998; 65:477–83. 40. Harada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil Steril 1998; 76(1):1–10. 41. Nothnick WB. Treating endometriosis as an autoimmune disease. Fertil Steril 1998; 76:1–10. 42. Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001; 75:1–10.

CHAPTER 44 Infertility: Is ihere Success afterForty? Daniel B Williams, Anil B Pinto INTRODUCTION The issues of age and declining fertility rates have been well studied in general populations showing a decline at age 35 with a marked further decrease at age 40.1 The age of the woman has an inverse relationship to and is the primary determining factor in the successful outcome of fertility treatment. This decrease in female fecundity with age takes a marked down turn at about age 37 to 38, coincident with an accelerated loss of oocytes, and reaches almost zero by 45 years.2 This naturally occurring phenomenon has had a significant impact on physicians who treat patients for infertility as patients are presenting for intervention at later ages. This is in part due to increasing female age at marriage, as well as avoidance of pregnancy during the early years of marriage. Controlled ovarian hyperstimulation (COH) in this age group of patients may result in cycle cancellation, insufficient response with poor follicle recruitment in number or size, slow increase or drop in serum estradiol (E2) or low pregnancy rates. The purpose of this article is to briefly review tests to assess ovarian reserve, to address treatment options and success rates, and finally, to give general recommendations on patient management. OVARIAN RESERVETESTING Ovarian reserve decline has been clearly related to age; however, the prognostic value of age alone has not been established. It is well known that the success of ART decreases progressively after the age of 30 years and declines markedly after the age of 35 years. Lass et al3 recently evaluated 1087IVF cycles initiated in 471 women ≥40 years of age. They reported that the pregnancy rate was significantly lower in these women than in a control group of women <40 years of age (11.3% vs 28.2%). The pregnancy rate decreased sharply in women >42 years of age, and no woman >45 years achieved a live birth. Ovarian reserve refers to the ability of a woman to conceive in the absence of specific pathophysiologic changes in her reproductive system. This ovarian reserve, or reproductive potential, declines with age. This age-related decline is thought to occur secondary to follicle depletion and diminished quality of remaining oocytes. Some tests for diminished ovarian reserve have also demonstrated the ability to identify patients with a decreased reproductive potential that is independent of age.

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Basal FSH/Estradiol (E2) The use of basal FSH has been used to detect patients with diminished ovarian reserve. This is based on the premise that diminished oocyte quality is accompanied by a decline in granulosa cell function, which results in lower inhibin levels that are eventually unable to normally suppress FSH levels in the early to mid follicular phase. Batista et al4 looked at LH, FSH, E2, inhibin, progesterone, PP-14, and endometrial biopsies (EMB) in a group of patients with normal menstrual cycles. They found that in patients >40 years, FSH levels were increased and inhibin levels were decreased compared with younger patients, with no differences in EMB’s or progesterone levels. Klein5 and co-workers found that patients 40 to 45 years of age had a shortened follicular phase as well as increased urinary FSH levels in both phases of the menstrual cycle compared to subjects 20–25 years of age. These data would support the idea that diminished ovarian reserve does increase with age. But are these markers predictive of pregnancy outcome? Pearlstone et al6 looked at 402 ovulation induction cycles in women over 40 years. They found that no woman over the age of 43 conceived (demonstrating the effect of age), and that no woman 40–43 years of age conceived when the FSH was >25 mIU/ml (suggesting an independent but synergistic effect of diminished ovarian reserve). Toner et al7 reported in a series of 1478 consecutive IVF cycles that basal day 3 FSH levels screening showed that the cancellation rates for patients with levels of <15 mIU/ml, 20 mIU/ml, 25 mIU/ml and >30 mIU/ml were 5 percent, 10 percent, 20 percent and 40 percent respectively. Scott (5) and coworkers evaluated 785 IVF cycles and found that pregnancy rates were highest when FSH levels were <15mIU/ml, while pregnancy rates fell to <5 percent when FSH levels were >25 mIU/ml. Interestingly in this study age did not appear to play a predominant role as patients in the low FSH and high FSH groups had no significant difference in mean age (approximately 35 years). The issue of the significance of intercycle variability of FSH levels has been addressed. Scott8) and coworkers found that the intercycle variability in patients with normal basal FSH levels (<15 mIU/ml) was low, with a mean value of 2.6±0.2 mIU/ml. However, those patients with elevated basal FSH levels (>15 mIU/ml) had a higher degree of variability with a mean value of 7.9±0.9 mIU/ml. This might suggest that it would be reasonable to repeat basal FSH measurements in subsequent cycles, in those patients who have had an abnormal value, with the hope of proceeding with treatment, should levels normalize.9 Martin et al10 looked at this question and found that pregnancy rates were 15 percent when no abnormal values were seen. This was in contrast to pregnancy rates in patients with one previous abnormal value (5%) or multiple previous abnormal values (0%). More recently, Lass et al11 compared the IVF results in poor responder IVF patients with one previous abnormal FSH value, compared to those with no abnormal values. They found no difference in pregnancy or delivery rates per embryo transfer. Therefore, patients with an initial abnormal FSH value, that subsequently normalizes, should still have a reasonable chance of conception with their own eggs. However, those patients with multiple abnormal values will likely have a significantly decreased chance of conception using their own eggs. It is now recommended that an E2 level be obtained simultaneously with a basal FSH level. This is primarily to ensure that patients do not have elevated E2 levels on day 3, which might indicate that the patient is further along in her cycle than was apparent

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clinically. In addition, it is possible that an elevated E2 level could suppress an abnormal FSH level into the normal range. Basal FSH and E2 should be obtained between cycle days 2–4, as E2 levels have been shown to significantly rise by cycle day 5. Therefore, findings of an elevated basal FSH are highly predictive of decreased ovarian reserve, and can help to identify a subgroup of patients that might not be detected using age alone. It is inexpensive, and correlates well with pregnancy outcome. Clomiphene Citrate ChallengeTest (CCCT) The CCCT is a dynamic test, which has been used to identify patients with decreased ovarian reserve. Navot et al12 proposed the CCCT as a means of assessing ovarian reserve in women ≥35 years of age. This test consists of the administration of clomiphene citrate at a dose of 100 mg daily from cycle days 5–9. Serum FSH levels are obtained on cycle days 3 and 10. The test is based upon the premise that a patient with normal ovarian reserve should be able to overcome the central effect of clomiphene citrate, and suppress FSH levels back into the normal range. An abnormal test would consist of either an elevated day 3 FSH, or an elevated day 10 value. Scott et al13 assessed the usefulness of this screening test in a general infertility population. They found that the incidence of abnormal tests rose with age, with 10 percent abnormal testing at age 35–39 years, and 26 percent abnormal testing in patients >39 years. Pregnancy rates were significantly decreased in those patients with abnormal testing. In a subsequent study the same group found that this decrease in pregnancy rate with an abnormal CCCT was poor regardless of age. Interestingly only 7 of 23 patients with an abnormal test had an elevated day 3 FSH, suggesting that the CCCT may be more sensitive thanbasal FSH levels alone. OTHERTESTS FOR OVARIAN RESERVE There are other tests that have been proposed to assess ovarian reserve. These include basal inhibin B levels, GnRH-a stimulation testing, follicle stimulating hormone reserve testing, pre-treatment ovarian volume, and pre-treatment basal follicle counts. Further studies are needed to establish the clinical utility of these tests. Regardless of the test used, screening for ovarian reserve in older patients can allow one to define a population with a poor prognosis.13,14 Gonadotropin Releasing Hormone Agonist StimulationTest The Norfolk group15 proposed the gonadotropin releasing hormone agonist (GnRH-a) stimulation test, which evaluates the change in E2 level from cycle day 2 to 3 after administration of 1 mg of leuprolide acetate. Patients showing at least a doubling of their base line E2 level on day 3 compared to the day 2 value, had a higher number of oocytes retrieved and higher pregnancy rates than patients who experienced less than a doubling of their E2 level on day 3.

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ULTRASOUND EVALUATION OF OVARIAN RESERVE Zaidi et al16 investigated the relationship between ovarian stromal blood flow and subsequent follicular response. They found that the mean ovarian stromal peak systolic blood flow velocity was significantly lower in the low response group and that the odds of a poor response decreased significantly as peak systolic blood flow velocity increased. Chang et al17 reported a clear correlation between the number of antral (2–5 mm) follicles under basal conditions and the outcome of ART. In their study the number of antral follicles correlated significantly with patient age, day 3 serum FSH level, dosage of menotropin ampoules used, serum E2 levels, number of oocytes retrieved, and the number of embryos transferred. The group of patients who had a lower basal antral follicle count (≤3) also had a significantly higher rate of cycle cancellation compared with other groups who had a higher basal antral follicle count. Data at the present time suggests that ultrasound may be a useful noninvasive tool in the prediction of ovarian reserve and ovarian response to stimulating drugs. However, future standardization of the criteria used is necessary in order to achieve routine clinical applicability TREATMENT OF THE OLDER PATIENT As has been previously stated, pregnancy potential is diminished with increasing age. This effect is seen even with fertility treatment due to a decreased follicular response to gonadotropins, decreased oocyte quality and implantation rates, as well as an increase in the spontaneous abortion rate. These effects are even more pronounced in patients who are 40 or older. Typical treatment for patients with no specific treatable cause for their infertility (“empiric therapy”) is based on two principles: 1) increasing the number of eggs and 2) placing sperm and eggs closer together. These 2 objectives can be met by using clomiphene citrate plus intrauterine insemination (CC-IUI), injectable gonadotropin therapy combined with intrauterine insemination (INJ-IUI), or by moving to assisted reproductive technologies (ART) such as in vitro fertilization (IVF) or gamete intrafallopian transfer (GIFT). However, the optimal treatment approach for this age group has yet to be determined.14,18 Clomiphene Citrate with Intrauterine Insemination (CC-IUI) Pearlstone and co-workers6 evaluated 402 cycles in 85 women over the age of 40 who underwent ovulation induction with most of the cycles analyzed involving the use of CC. They found that the pregnancy rates in patients 40–44 were 3.5 percent per cycle with a delivery rate of only 1.2 percent per cycle. Additionally they found no difference in pregnancy outcome, regardless of type of ovulation induction agent used. Agarwal et al19 evaluated 664 cycles in 290 women undergoing treatment with CC-IUI. The pregnancy rate was 19 percent per cycle in patients under 30, compared to a pregnancy rate of 5 percent per cycle in patients 40 and older.

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Gonadotropins with IUI (INJ-IUI) A number of studies have looked at the effects of gonadotropin treatment in this patient population. Brzechffa and Buyalos20 retrospectively analyzed 363 cycles and found that the pregnancy rate per cycle was significantly lower in patients >40 years (3.6%) compared with patients 35–39 years (13.6%) and patients <35 years (19.4%). Frederick et al21 analyzed 210 treatment cycles utilizing CC-IUI, CC/INJ-IUI, or INJ-IUI in patients 40 and over. They found a pregnancy rate of only 5 percent per cycle in this age group. Corsan et al22 also looked at patients 40 and older undergoing ovulation induction cycles with a variety of agents and found that the pregnancy rates per cycle were only 5.22 percent. They had no viable pregnancies in women 43 years of age. ASSISTED REPRODUCTIVE TECHNOLOGIES (IVF, GIFT) The use of ART has been shown to significantly increase pregnancy rates compared with other treatments in patients <40 years. However, the most recent published data from the national registry from 1998 revealed that the pregnancy rate was 13 percent and the delivery rate was 8 percent per retrieval in women aged >40 years undergoing IVF.23 Indeed, the spontaneous abortion rate in the 40 and older age group can be as high as 50 percent –75 percent overall following ART. Lim et al24 looked at 158 cycles of patients undergoing IVF cycles with or without intracytoplasmic sperm injection (ICSI). They found a pregnancy rate of 43.2 percent in women <35 years versus 14.3 percent in women >40 years. Type of Stimulation 25

In women over 40 years, Dor et al suggest that despite more oocytes retrieved, fertilized and cleaved after GnRHa/hMG stimulation, the clinical pregnancy rate was the highest with clomiphene citrate and hMG. These findings were conf irmed separately by Pellicier et al26 and Ben-Rafael et al.27 The beneficial effect from adding clomiphene citrate (CC) to the stimulation protocol may be related to an enhanced embryo quality as suggested by a higher cleavage rate and a greater implantation rate per embryo. Ben-Rafael et al27 suggest that in elderly poor responders, CC/hMG is cost-effective and should be the firstline attempt despite the high cancellation rate and the possible non-desirable effects of CC on the reproductive tract when undergoing stimulation for an IVF cycle. The short or “flare-up” GnRH protocol has been proposed as a better stimulation protocol for the low responders and the elderly patient undergoing IVF.28 The flare protocol takes advantage of the initial stimulatory effect of GnRHa action on the pituitary hormone levels. An important drawback is that the increase in gonadotropins may also induce ovarian androgen release, corpus luteum rescue, and a secondary decline in oocyte quality and ongoing pregnancy rates. It has also been suggested that the microdose GnRHa protocol for the elderly patient results in significantly decreased cycle cancellation rates as well as increased clinical and ongoing pregnancy rates.29 Overall, the use of gonadotropins would be recommended over clomiphene citrate for ART cycles, regardless of female age.

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Assisted Hatching Assisted hatching is another mechanism that has been used to attempt to increase pregnancy rates in ART patients. Assisted hatching is a process whereby defects are made in the zona pellucida prior to embryo transfer to facilitate “hatching” of the embryo to allow implantation to occur. The rationale for its use in the older population, is the theory that the zona becomes thicker and harder with age. The protocol for assisted hatching by acid drilling of the zona pellucida using acid tyrode solution was first described by Cohen.30 Successful assisted hatching has also been carried with the use of an 1.48 öm diode laser (Fretilase, MTG Germany); a few milliseconds of laser irradiation instantly makes an opening in the zona pellucida, and apertures ranging from 3 to 25 öm can be obtained. The procedure has been shown to be safe, simple and rapid.31 Schoolcraft et al32 looked at IVF cycles in women 40 and older, and compared results with similar subjects who also underwent assisted hatching. They found that the delivery rate increased from 11 percent, to 47 percent per cycle with the addition of the assisted hatching procedure in this age group. Stein et al33 evaluated assisted hatching and demonstrated a significant increase in pregnancy rates in patients aged >38 years (23.9%) compared with control patients (nonhatched) in the sane age group (7%). In another study Tucker et al34 found the clinical pregnancy rates to be significantly higher following hatching in patients aged >35 years (45.2%) compared with control patients (16.7%). However, the ongoing pregnancy and implantation rates were not significantly differentbetween the two groups. In our program we routinely perform assisted hatching on patients 38 and older. However, the beneficial effects have not yet been proven to be effective in randomized, controlled trials. Oocyte Donation It is well known that oocyte donation can easily improve pregnancy rates to that seen in younger women, thus confirming that the age related changes in fertility are primarily due to decreased oocyte quality35,36 Keefe et al37 have suggested that oocytes from older women are more likely to contain deleted mitochondrial DNA than oocytes from younger women, and that these alterations could have adverse cellular effects by disrupting the normal electron and energy transport chain, leading to high levels of intracellular reactive oxygen species and cellular dysfunction. To overcome this, Cohen et al38 reported the use of ooplasm transfer in seven couples with multiple implantation failures by using either electrofusion of an ooplasmic donor fragmented into each patient’s egg or direct injection of ooplasm from a donor egg into each patient. However, currently these treatments are currently experimental in nature and their efficacy is unclear. In addition they raise a number of ethical issues, such as the introduction of a completely new set of mitochondrial DNA from the donor to the recipient oocyte, which may lead to the creation of a three-parent individual.39 WHAT IS THE OPTIMAL TREATMENT In order to determine the optimal treatment paradigm for this patient population, our group at Washington University School of Medicine performed a retrospective study

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looking at various fertility treatments in women 3 40, seen at our center from January ‘92 through June’ 98.40 The treatment groups were IUI alone (NC-IUI), CC-IUI, INJ-IUI, and ART (IVF or GIFT). We studied 401 cycles in 152 patients. We found that the pregnancy rates per cycle were 5.54 percent in all combined IUI treatment groups. This was significantly less than the pregnancy rate of 18.96 percent per cycle seen in patients treated with ART. There was not a significant difference in pregnancy rates between the IUI treatment groups.12 Based on this data, we would conclude that ART would offer these patients their best chances for conception. Furthermore, we do not see an advantage for INJ-IUI cycles in this patient population. Certainly randomized, prospective trials are needed to definitively answer this question. CONCLUSION It is readily apparent that the potential for female fertility decreases with age, with a significant drop at age 40. This is seen, both in the natural population, as well as in the infertility population. Testing for diminished ovarian reserve can help to identify a subgroup of patients who may have a significantly poorer prognosis. Patients should be routinely tested for ovarian reserve between the ages of 35 and 40 years. We would recommend testing for ovarian reserve starting at age 35. In our practice, we have favored basal FSH/E2 testing, which is highly predictive of patients with diminished reserve, and is very simple to perform. The CCCT is also a reasonable alternative, and may be more sensitive than basal FSH testing. However, it is a provocative test, which is not as simple and must be interpreted appropriately. It is also important to know that FSH threshold υalues can υaryfrom lab to lab, so that it is important to know how measurements from your laboratory assay compare to those assay υalues cited in the published literature. Because of the extremely low pregnancy/delivery rates in this age group, it is imperative that physicians educate their patients regarding treatment options and their potential success rates. Based on our experience, we feel that it is reasonable to proceed directly to ART in the 40 and older patient. However, if ovulation induction is desired, then a trial of clomiphene citrate is reasonable, due to it’s relatively low cost. At this time, there does not appear to be any advantage in using injectable gonadotropins with IUI in this age group. Based on the data currently available in literature, it is possible to recommend guidelines for the management of the patient over 40 years undergoing ART. It is important to bear in mind that the stimulation protocols should be tailored to individual needs keeping in mind the costs, duration of treatment and the attendant risks of the treatment or procedure to the patient. Clearly we need to continually re-evaluate our current practices and try to incorporate newer techniques in order to improve the chances of conception in the elderly patient, as they become available.

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REFERENCES 1. Rosenwaks Z, Davis OK, Damario MA. The role of maternal age in assisted reproduction. Hum Reprod 1995; (10 Suppl):165–73. 2. Wallach EE Pitfalls in evaluating ovarian reserve. Fertil Steril 1995; 63:12–14. 3. Lass A, Croucher C, Duffy S, Dawson K, Margara R, Winston RM. One thousand initiated cycles of in vitro fertilization in women>or=40 years of age. Fertil Steril 1998; 70:1030–34. 4. Batista MC, Cartledge TP, Zallmer AW et al. Effects of Aging on menstrual cycle hormones and endometrial maturation. Fertil Steril 1995; 64:492–99. 5. Klein NA, Battaglia DE, Fujimoto VY et al. Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women. J Clin Endocrinol Metab 1996; 81:1038–45. 6. Pearlsone AC, Fournet N, Gambone JC et al. Ovulation induction in women age 40 and older: the importance of basal follicle-stimulating hormone levels and chronologic age. Fertil Steril 1992; 58:674–9. 7. Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle stimulating hormone levels is a better predictor of in vitro fertilization performance than age Fertil Steril 1991; 55:784–91. 8. Scott RJ, Leonardi MR, Hoffman GE et al. A prospective evaluation of clomiphene citrate challenge test screening of the general infertility population. Obstet Gynecol 1993; 82:539–44. 9. Hansen LM, Batzer FR, Gutmann JN et al. Evaluating ovarian reserve: follicle stimulating hormone and oestradiol variability during cycle days 2–5. Hum Reprod 1996; 11:486–89. 10. Martin JSB, Nisker JA, Tummon IS, Daniel SAJ, Auckland JL, Feyles V. Future in vitro fertilization pregnancy potential of women with variably elevated day 3 follicle-stimulating hormone levels. Fertil Steril 1996; 65:1238–40. 11. Lass A, Gerrard A, Abusheikha N, Akagbosu F, Brinsden P. IVF performance of women who have fluctuating early follicular FSH levels. J Asst Reprod Genetics 2000; 17(10); 566–73. 12. Navot D, Rosenwaks Z, Margalioth EJ. Prognostic assessment of female fecundity. Lancet 1989; 2:645–47. 13. Scott RJ, Leonardi MR, Hoffman GE et al. A prospective evaluation of clomiphene citrate challenge test screening of the general infertility population. Obstet Gynecol 1993; 82:539–44. 14. AuyeungA, Klein ME, Ratts VS, Odem RR, Williams DB. Fertility treatment in the forty and older women. J Assist Reprod Genet (in press). 15. Winslow KL, Toner JP, Brzyski RG, Oehninger SC, Acosta AA, Muasher SJ. The gonadotropin-releasing hormcme agonist stimulation test. A sensitive predictor of performance in the flare-up in vitro fertilization cycle. Fertil Steril 1991; 56:711–17. 16. Zaidi J, Barber J, Kyei-mensah A, Bekir J, Campbell S, Tan SL. Relationship of ovarian stromal blood flow at the base line ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 1996; 88:779–84. 17. Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. Use of the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril 1998; 69:505–10. 18. Muasher SJ. Controversies in assisted reproduction: treatment of low responders. J of Assist Reprod Genet 1993; 10:112–14. 19. Argawal SK, Buyalos RP. Clomiphene citrate with IUI: is it effective therapy in women above the age of 35 years? Fertil Steril 1996; 65:759–63. 20. Brzechffa PR, Buyalos RP. Female and male partner age and menotropin requirements influence pregnancy rates with human menopausal gonadotropin therapy in combination with intrauterine insemination. Hum Reprod 1997; 12:29–33. 21. Frederick JL, Denker MS, Rojas A et al. Is there a role for ovarian stimulation and intrauterine insemination after age 40? Hum Reprod 1994; 9:2284–86. 22. Corsan G, Trias A, Trout S, et al. Ovulation induction combined with intrauterine insemination in women 40 years of age and older: is it worthwhile? Hum Reprod 1996; 11:1109–12.

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23. Society for Assisted Reproductive Technology, American Society for Reproductive Medicine. Assisted reproductive technology in the United States and Canada: 1998: results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. 24. Lim AS, Tsakok MFH. Age-related decline in fertility: a link to degenerative oocytes? Fertil Steril 1997; 68:265–71. 25. Dor J, Ben-Shlomo I, Levran D, Rudak E, Yunish M, Mashiach S. The relative success of gonadotropin-releasing hormone analogue, clomiphene citrate, and gonadotropin in 1099 cycles of in vitro fertilization. Fertil Steril 1992; 58:986–90. 26. PellicerA, LightmanA, Diamond MP, Russel JB, DeCherneyAH. Outcome of in vitri fertilization in women with low response to ovarian stimulation. Fertil Steril 1987; 47:812–15. 27. Ben-Rafael Z, Feldberg D. The poor responder patient in an in vitro fertilization-embryo transfer program. J Assist Reprod Genet 1993; 10:118–20. 28. Tasdemir M, Tasdemir I, Kodama H, Fukuda J, Tanaka T. Short protocol of gonadotropin releasing hormone agonist administration gave better results in long protocol low responders in IVF-ET. J Obstet Gynaecol Res 1996; 22:73–77. 29. Scott RT, Navot D. Enhancement of ovarian responsiveness with microdoses of gonadotropinreleasing hormone agonist during ovulation induction for in vitro fertilization. Fertil Steril 1994; 61:880–85. 30. Cohen J. Assisted hatching of human embryos. J IVF-ET. 1991; 8(4); 179–89. 31. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48 diode laser beam: efficiency and safety of the procedure. Fertil Steril 1995; 5:604–11. 32. Schoolcraft WB, Shlenker T, Jones GS, et al. In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genetics 1995; 12:581–84. 33. Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J et al. Assisted hatching by partial zona dissection of human preembryos in patients with recurrent implantation failures after in vitro fertilization. Fertil Steril 1995; 63:838–41. 34. Tucker MJ, Morton PC, Wright G, Ingargiola PE, Sweitzer CI, Elsner CW et al. Enhancement of outcome from intracytoplasmic sperm injection. Does coculture or assisted hatching improve implantation rates? Hum Reprod 1996; 11:2434–37. 35. Cano F, Simon C, Remohi J, Pellecier A. Effect of aging on the female reproductive system: Evidence for a role of uterine senescence in the decline in female fecundity Fertil Steril1995; 64:584–89. 36. Morris RS, Sauer MV. Oocyte donation in 1990s and beyond. Assit Reprod Rev 1993; 3:211– 17. 37. Keef DL, Niven-Fairchild T, Poell S, Burdangunta S. Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women. Fertil Steril 1995; 64:577–83. 38. Cohen J, Scot R, Alikani M, Schimmel T, Munne S, Levron J et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 1998; 4:269–80. 39. Fasouliotis SJ, Simon A, Laufer N. Evaluation and treatment of low responders in assisted reproductive technology: a challenge to meet. J Assisted Reprod Genetics 2000; 17:357–73. 40. AuyeungA, Klein ME, Ratts VS, Odem RR, Williams DB. Fertility treatment in the forty and older woman. J Assisted Reprod Genetics.

CHAPTER 45 Inheritance of Infertility Mirudhubashini Govindarajan, MS Lakshmi INTRODUCTION The problem of infertility is not uncommon; affecting 10–15 percent of the general population. A small percentage of these can be attributed to direct genetic causes of gonadal failure. However, with the recent advances in molecular biology, a clearer picture has emerged. Genomic control of the reproductive development and processes is better understood now. Numerous common female and male factor abnormalities have also been proven to have genetic basis. With this current level of Assisted Reproductive Technology, procreation has become possible in many of these situations. Therefore understanding the basis of such problems under realistic assessment of the possibility of transmission of the defect to a potential offspring has become an important issue. There is a genuine concern for possible risk of increasing the pool of genetic disorders in human population. The genetic causes of inf ertility may involve either the sex-chromosomes or the autosomes. The type of abnormalities may vary. • Numerical abnormalities — Usually the result of non-disjunction at anaphase of either mitosis or meiosis resulting in abnormalities such as aneuploidy, polyploidy or mosaicism. • Structural abnormalities — Due to chromosomal breaks induced by radiation, drugs or viruses. • Polygenic inheritance — Abnormalities transmitted through several different genes each one of them having lesser individual effect. • Multifactorial inheritance — Interaction between genetic and environmental factors. • Single gene defects — Mutation in specific genes transmitted according to Mendelian modes of inheritance, autosomal dominant or recessive and X-linked dominant or recessive. — Level of penetrance of a single mutant gene may vary from non-penetrant to mildly expressive to full blown clinical expression.

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Genetic Abnormalities in Female Infertile Population X-Chromosome Abnormalities Ninety-nine percent of conceptuses with a single X-chromosome abort. Remaining 1 percent account for an incidence of Turner’s syndrome between 1 in 2500 to 1 in 5000 live born girls. X-chromosome abnormalities accelerate germ cell loss in the intrauterine life. These individuals have a normal migration and mitosis of germ cells in utero but the oogonia do not undergo meiosis. Rapid loss of oocytes leaves the gonad without follicles by birth and it appears as a fibrous steak. Sixty percent of these patients have a total loss of one X-chromosome while the remainder have a partial loss. Normal ovarian development seems to require two loci, one in the short arm and the other in the long arm of the X-chromosome. Loss of either results in the gonadal failure. Loss of material from the short arm leads to short stature and other physical stigmata of Turner’s syndrome. Long arm deletions near the centromere are associated with primary amenorrhoea only Simpson JL.1 Distal long arm deletions are associated with normal growth and usually secondary amenorrhoea. Because of the high incidence of associated abnormalities these patients should also be evaluated with an IVP, renal ultrasound, ECHO cardiograph, audiometry, annual evaluation of thyroid function studies, lipid profile, glucose metabolism and antibody testing. XX-gonadal Dysgenesis Patients with this chromosomal disorder have normal external genitalia with streak gonads. Simpson JL, et al2 in 1971 proved that it is inherited in an autosomal recessive allele with varied expressions Gluchi,3 and Boczkoushi,4 have described various families with the above features. Malkora5 et al reported of one family in which one member had streak gonads and the other siblings either had ovarian hypoplasia or increased gonadotropins. Familial Premature Ovarian Failure (POF) It has long been suspected that the FSH receptor (FSH-r) gene was a candidate for mutations in women with premature ovarian failure. Aittomaki, et al6 finally demonstrated the presence of such an inactivating mutation in 1995. These investigators, led by Albert De La Chapelle, studied a homogenous population of Finnish women with premature ovarian failure (POF), and using linkage analysis, found that markers on chromosome 2p were informative. This region has been shown by in situ hybridization to be the location of both the human FSH-r and the human LH-r genes. After isolating the FSH-r gene, these investigators identified a point mutation in exon 7 that altered an amino acid. The FSH-r mutation identified was transmitted in an autosomal recessive fashion. It was identified in women with primary (80%) and secondary amenorrhoea (20%). All subjects with an FSH-r mutation in these multiplex families had the same exon 7 mutations.

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Recently, precise deletion mapping studies of many X-autosomal translocations by a group in Milan, Italy has resulted in the isolation of a gene that is homogenous to the Drosophila gene diaphanous (DIA). The human DIA protein is similar to the Drosophila homologue. Mutations in DIA produce sterile fruitflies. In man there are two diaphanous (DIA) genes. One maps to an autosome (5q and appears to be involved in hearing loss, where as the other maps to the distal part of the long arm of the X-chromosome and may be involved in ovarian development. It is interesting to note that neuro-auditory defects have been seen in affected sisters with ovarian failure, suggesting a single autosomal gene involving hearing that may be closely linked to, or to interact with the ovarycontrolling gene. Only time will tell us as to whether genes that clearly affect germ cell migration will be identified and provide a clearer picture of premature gonadal failure inbothsexes. Smith et al7 reported premature ovarian failure in three Greek siblings (one male, two female) of consanguineous parents. Coulam et al8 observed the same phenomenon in two affected siblings who had an affected mother and aunt. Metison et al9 reported five families in which relatives in more than one generation manifested ovarian failure before age 40. The authors believed their data were best explained on the basis of a mutant autosomal or X-linked dominant gene. Mullerian Aplasia Mullerian aplasia when associated with renal and vertebral disorders is termed as Rokitansky-Kustner Hauser syndrome. It is seen in 46XX individuals. Familial aggregates of Mullerian aplasia are documented stating it could be autosomal recessive. Other studies by Lischke et al10 and Shokeir11 have concluded that inheritance is polygenic or multifactorial, as it either affects a single organ system or single embryologically related organ system. Another explanation is genetic heterogenecity, e.g. Mullerian aplasia may exist with Klippel-Feil and middle ear anomalies. True duplication of the Mullerian ducts or incomplete Mullerian fusion in familial aggregation affecting siblings as well as mother and daughter has been reported. Hand Foot Genital Syndrome Skeletal malformation and either incomplete Mullerian fusion in women or hypospadiasis in men characterize it. It is an autosomal dominant disorder reported in four families by Verp MS et al.12 Leiomyomata This pathology is more common in black population suggesting racial prevalence. Winkler and Hoffemen13 conducted a study in 365 patients with the same number as controls. 62 relatives of the study group patients were affected when compared to 9 in control group (4 times in near relatives and twice in distant relatives), suggesting polygenic mode of inheritance.

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Endometriosis Endometriosis accounts for 35 percent of female infertiliiy. Various studies by Ranney14 and Simpson15 proved incidence of endometriosis in mother, siblings or daughters to be 7 percent when compared to 1 percent in the control group suggesting polygenic or multifactorial mode of inheritance. Some of these severe lesions may be asymptomatic Individuals with family history of endometriosis should have screening and be advised for early child bearing or oral contraceptives. Polycystic Ovarian Syndrome PCOS shows strong familial aggregation suggesting an underlying genetic causative factor. The first large study was carried out in 1968 by Cooper et al16 in which firstdegree relatives of affected patients had a higher prevalence of oligomenorrhea than the control population. Ferrimen17 in a 1979 in a study group of 700 patients concluded that there was a high prevalence of symptoms among the first-degree relatives, i.e. hirsutism, and oligomenorrhea in female, and premature balding in male relatives suggesting a dominant mode of inheritance. Govind, et al18 in 1999 studied 29 families who had polycystic ovarian morphology and symptoms of PCOS. They found that 66 percent of the first-degree female relatives were affected as were 22 percent of first-degree male relatives (premature balding). Of the total of 71 first-degree siblings of PCOS proband, 39 were affected giving a segregation ratio of 55 percent suggesting an autosomal dominant mode of inheritance. Environmental factors may also influence the manifestations of PCOS.19 Two key genes have been associated with the pathophysiology of PCOS. • Cholesterol side chain cleavage gene, which encodes cytochromic 450-side chain cleavage. The P450 SCC is the catalyst in the steroidogenesis of cholesterol to pregnenolone. The deficiency of this gene leads to androgen production. • The Variable Number Tandem Repeats (VNTR) of the insulin gene regulates insulin expression.20 Waterworth et al21 found that class III alleles were associated with anovulatory PCOS. Subjects who are homozygous or heterozygotus for class III alleles at the insulin gene VNTR focus are likely to have disturbances of insulin secretion and action. This gene type is causative factor for menstrual irregularity and Type II Diabetes mellitus. Genetic Abnormalities in Male Infertile Population Severe male subfertility is assumed to be the reason for infertility in upto 50 percent of childless couples. In about 30 percent of these cases, genetic defects seem to be the basis. Severe oligozoospermia or azoospermia are the predominant symptoms of these genetic disorders. In humans, genes on the Y-chromosome control male sex determination. The presence of Y-chromosome determines a male phenotype. This has been attributed to the presence of a dominant “Testis determining factor” or TDF the testis produces two major hormonal effectors, testosterone and Mullerian Inhibiting Hormone (MAH), allowing sexual differentiation to take place. Testosterone is responsible for the development of the epididymis, the vas deferens, the seminal vesicles, and via 5α-dihydrotestosterone, the

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penis and the scrotum. MIH causes the regression of the Mullerian ducts, which, in females develop into fallopian tubes, uterus and cervix. In 1990, the gene responsible for sex-determination, SRY (Sex-determining region, Y-chromosome) encoding TDF was identified. Several genes playing a role in sex determination were subsequently identified. Sexual dimorphism can hence be viewed as the result of functional and developmental integration of a number of different genes. Genes Involved in Sex Determination Sex-determining region, Y-chromosome has been mapped to the distal region of the short arm of the human Y-chromosome. SRY has shown to be expressed in the genital ridge just before testis differentiation. The role of SRY in testis development was confirmed by transgenic mouse experiments. These experiments demonstrated that SRY is the gene that initiates the cascade of male development and it encodes TDF. This gene encodes a high mobility group (HMG) box transcription factor. HMG box binds to specific target sequences in DNA and causes a bend in the chromatin. DNA bending subsequently triggers transcription of genes downstream in a cascade of gene regulation leading to maleness. These genes are the SOX genes (SRY-related HMG box). One SOX gene of particular relevance for sex determination is SOX9. Point mutations in SOX9 are associated with campomelic dysplasia, a congenital skeletal malformation syndrome, in which a majority of XY patients are XY females with pure gonadal dysgenesis. SOX9 therefore is involved in both chondrogenesis and gonad development. The SOX gene family includes more than 20 representatives that have important development functions in both sexes. SOX3 is a highly conserved and closely related sequence on the X-chromosome. SRY and SOX3 interact to regulate SOX9 action as a testis-determining trigger (Graves, ’98). SOX3 inhibits SOX9 function in females. In males, SRY inhibits SOX3 and permits SOX9 to enact its testis-determining role. This could be by the interaction of SRY and SOX3 in the transcriptional regulation of SOX9. DSS: X-Linked Switch Between Female and Male Development In 1994, a new region was reported named DSS for dosage sensitive sex reversal. XY individuals who have duplication of this Xp region develop as phenotypic females. The Congenital Adrenal Hypoplasia (CAH) an inherited disorder of the adrenal cortex development, is contained within this region. The gene for CAH was named DAX1 (DSSAHC on the X chromosome, gene 1). DAX1 is a candidate for a role in sex determination. It is expressed in the genital ridge during sex determination. Testicular histology of patients with DAX1 mutations revealed an absence of germ cells and an immaturity of sertoli cells. This suggests the role of DAX1 in sertoli cell function. Autosomal Gene Involved in Sex Determination The Williams’ tumour gene, WT1, was shown to be involved in early gonadal development. The WT1 gene has been expressed in male and female genital ridges. Mutations in WT1 cause kidney and gonadal agenesis. The absence of gonads is caused

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by gonadal degeneration during embryogenesis, suggesting a role of WT1 in the maintenance of gonadal development. Mutations in WT1 were identified in patients with Denys-Drash syndrome and Frasier syndrome, who have renal failure. Additional occurrence of Wilms tumor is seen in those with Denys-Drash syndrome. These observations suggest a role of WT1 in gonadal development. Steroidogenic factor (SF1) is expressed in the adrenal cortex, testis, ovary, hypothalamus and pituitary In gonads, SF1 is expressed at the early stage, later turned off in females, but continues to be expressed in male sertoli cells. The fact that SF1 expression declines in female gonads, suggests its major role in the control of Mullerian duct regression. Disruption of SF1 results in gonadal and adrenal agenesis, resulting in postnatal death by adrenal insufficiency. Deletions of Y-Chromosome Tiepolo and Zuffardi22 first suggested a role for the human Y chromosome in spermatogenesis. The existence of a spermatogenesis gene or azoospermia factor (AZF) on Yq was suggested. Cytologically deletions on the distal part of the Yq were also associated with infertility Voget et al,23 claimed the existence of three genetically active regions AZFa, AZFb and AZFc. These three regions on the Y-chromosome correspond to different histological patterns of spermatogenic failure. AZFa deletions display a complete absence of germ cells; men with AZFb deletions exhibit a maturation arrest of spermatogenesis before or at meiosis. AZFc deletions do not seem to be associated with a specific interruption phase of spermatogenesis. It can result either in azoospermia or in oligozoospermia with few mature spermatozoa in the ejaculate. Genes in the AZFa, b and c Families Deletions responsible for male infertility occur in the AZFc region more frequently than in AZFb and AZFa. DAZ (Deleted in Azoospermia) gene family is reported for the AZFc phenotype. A number of studies have reported a high incidence of deletion of this gene in azoospermic and severely oligozoospermic patients. Furthermore, DAZ is specifically expressed in the germ cells. Similarly the germ-cell-specific RBMY (RNA-binding motif, Y chromosome) gene family is associated with the AZFb region. It has not been clearly demonstrated that the loss of RBMY actually causes the testiculopathy in AZFb deleted patients. The lack of such a demonstration is probably due to the multicopy nature of this gene. It has several copies dispersed across the short and long arms of the Ychromosome. AZFa phenotype seems to be caused by the loss of one or more genes. The first identified gene was USP9Y Sun et al24 (Ubiquitin-specific protease 9, Y chromosome), previously known as DFFRY (Drosophila fat facets related), it has been shown to be absent in a fraction of infertile patients. However, USP9Y occupies only a small part of the AZFa interval while the majority infertile males carrying AZFa deletions show the absence of this entire interval. These findings suggest that other genes in this region may also be responsible for the spermatogenic disruption observed in AZFa deleted patients. Subsequently two other genes DBY (DEAD/H box polypeptide, Y chromosome) and

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UTY (ubiquitously transcribed tetracopeptide repeat gene, Y chromosome) were mapped in the AZFa interval by Ferlin, et al25 1999. These genes were suggested to have a role in human spermatogenesis. The absence of USP9Y is associated with an oligozoospermic phenotype, while a more severe testiculopathy (SCOS) may reflect the additional loss of DBY. Genes Involved in Spermatogenic Disruption The two genesRBM (RNA Binding Motif-formerly called YRRM) and DAZ (Deleted in Azoospermia) are those which when deleted cause spermatogenic disruption. DAZ Gene Family An autosomal homologue, the human DAZ gene, called DAZLA/DAZh (DAZ homologue), has been cloned on chromosome 3. The role of DAZLA in spermatogenesis is supported by its testis specific expression. Male infertility caused by DAZLA gene, is likely to be inherited in an autosomal recessive fashion. The fraction of azoospermic/ severe oligozoospermie males who have a defective DAZLA gene remains to be determined. RBM Gene Family RBM genes belong to a multigene family with a majority of genes on the long arm of the Y-chromosome. This gene is expressed only in the spermatogenic cells and primary spermatocytes. The manner in which the RBM gene family affects spermatogenesis remains unknown. There is some data suggesting that other genes may be involved in causing infertility associated with Y-chromosome deletions.26 This emerges from the fact that DAZ and RBM families remain intact in a proportion of patients having spermatogenic defects. The nature of these genes remains to be identified. Cystic Fibrosis: (CFTR Gene) Congenital bilateral absence of vas deferens (CBAVD) is found in 98 percent of men with cystic fibrosis. CBAVD occurs in 2 percent of healthy inf ertile men. Cystic fibrosis trans-membrane conductance regulator gene (CFTR) is located on the long arm of chromosome 7 (7q31). Mutations of the cystic fibrosis locus occurs with a frequency of about 1:25 (4%) i.e. every 25th personbeing heterozygous for the mutation.27 The autosomal recessive disease manifests as a dysfunction of the pancreas and results in an increased proneness to infections of the respiratory tract. The disease is caused by mutations in the structure of the CFTR gene Peter N Schlegel et al.28 The malformation of the vas deferens is caused by a complex interplay between different mutations and/or intronic polymorphisms in the CFTR gene. The most common mutations found among the CBAVD patients are; DF 508, R117H, R1017W and the 5T variant. CBAVD has recently become amenable to symptomatic treatment with micro-surgical sperm aspiration from the epidydymis or testes and subsequent ICSI. Men, who are affected by CBAVD, but not by CF, have a remarkable increase (60%) of heterozygous

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mutations in the CFTR gene. Therefore every couple, in which the male partner suffers from cystic fibrosis, should be counseled and both partners should be tested for the presence of mutation in the CFTR gene before offering them ICSI. The majority of patients who have Y-chromosome deletions have azoospermia or severe oligozoospermia. It is unlikely that pregnancies would have arisen naturally in such cases. However, the use of ICSI to achieve fertilization and pregnancy highlights the possibility that these Y-chromosome deletions may be transmitted to the next generation. Careful counseling of patients is mandatory, especially on the risk of simultaneously inheritance of several deletions in the offspring. An increasing awareness of these genetic systems in the human male stresses the need for care and follow-up during the application of new methods for the alleviation of male infertility. REFERENCES 1. Simpson JL. Ovarian dysgenesis and related genetic disorders in Gynecologic Endocrinology (4th edn). In Edward E Wallach (Ed): Genes and Chromosomes that cause Infertility, Modern Trends in Infertility and Conception Control. 1992; 4:15, 224. 2. Simpson JL, Christakos AC, Horwith M, Silverman FS. In Edwards E Wallach (Ed): Gonadal sysgenesis in individuals with apparently normal chromosomal complements: tabulation of cases and compilation of genetic data. Birth defects 1971; 7(6):215. 3. Guisti G, Borghi A, Salti M, Bigozzi U. In Edwards E Wallach (Ed): “Disgenesia gonadica pura,” con cariotipo 44 A+XX in sorelle figlie dicugini from Genes and Chromosomes that caiuse Female infertility, Modern Trends in Infertility and Conception Control. 1992; 4:15, 225. 4. Boczkowski K. Pure gonadal dysgenesis and ovarian dysplasia in sisters. Am J Obstet Gynecol 1970; 106:626. In Edwards E Wallach: Genes and Chromosomes that cause Female, Infertility, Modern Trends in Infertility and Conception Control. 1992; 4:15, 225. 5. Malkova J, Chrz R, Motik K, Starka L, Kobikova J. Slinkova-Malkova E: 46, XX gonadal dysgenesis and ovarian hypoplasia. Humangenetik 23:205, 1974. In Edwards E Wallach (Ed): Genes and Chromosomes that cause female infertility, Modern Trends in Infertility and Conception Control 1992; 4:15, 225. 6. Aittomaki K, Lucerna JLD, Pakarinen P et al. Mutation in the follicle stimulating hormone receptor gene causes hereditary hyper gonadrotrophic ovarian failure. Cell 82, 959–968 from Cystic fibrosis in infertility: screening bef ore assisted reproduction from Human Reproduction 15: number 11 November 2000 by DI Lewis-Jones1, MR gazvani2,3 R Mountford1, 1995. 7. Smith A, Fraser IS Noel ML. Three siblings with premature gonadal failure. Fertil Steril 32:528, 1979 from Genes and Chromosomes that cause Female infertility, Modern Trends in Infertility and Conception Control by Edwards E Wallach 1992; 4:15, 225. 8. Coulam CB. Stringfellow S, Hoefnagel D. Evidence for a genetic factor in the etiology of premature ovarian failure. Fertil Steril 1983; 40:693, from Genes and Chromosomes that cause Femaleinfertility, Modern Trends in Infertility and Conception. Controls by Edwards E Wallach 1992; 4:15, 225. 9. Mattison DR, Evans MI, Schwimmer WB, et al. Familial premature ovarian failure. Am J Hum Genet 1984; 36:1341–48. Cystic fibrosis in infertility: screening before assisted reproduction from Human Reproduction Volume 15 number 11 November 2000 by DI, Lewis-Jones 1, MR Gazvani 2, 3 R Mountford1. 10. Lischke JH, Curtis CH Lamb EJ. Discordance of vaginal agensis in monozygotic twins. Obstet Gynecol 1973; 41:920. from Genes an Chromosomes that cause Female infertility, Modern Trends in Infertility and Conception Control by Edwards E Wallach 1992; 4:15:227.

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11. Shokeir MHK. Aplasia of the Mullerian septum: evidence for probabale sex-limited autosomal dominant inheritance. Birth Defects 1978; 14(6c):147 from Genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards E Wallach 1992; 4:15, 227. 12. Verp MS, Simpson JL, Elias S, Carson SA, Sarto GE, Feingold M. Heritable aspects of ulterine anomalies. I. Three familial aggregates with Mullerian fusion anomalies. Fertil Steril 40:80, 1983 from genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards 1992; 4:15, 225. 13. Winkler H, Hoffman W. Zur Frage der Veriebbarket des uterusmyom. Deutsch Med Wochenschr 1938; 64:253 from Genes and Chromosomes that cause Female infertility Modern trends in infertility and Conception control Edwards E Wallach 1992; 4:15, 229. 14. Ranney B. Endometriosis. IV> Hereditary tendency. Obstet Gynecol 1971; 34:734, from Genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards W Wallach 1992; 4:15, 229. 15. Simpson JL, Malinal LR, Elias S. Carson SA, Radvany RA. HLA associations in endometriosis. Am J Obstet Gynecol 1984; 148:395 from Genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards E Wallach 1992; 4:15, 229. 16. Cooper HE, Spellacy WN, Prem KA, Cohen WD. Hereditary factors in the stein-Leventhal syndrome. Am J Obstet Gynecol 1968; 100:371, from Genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards E Wallach 1992; 4:15, 229. 17. Ferriman D, Purdie AW. The inheritance of polycystic ovarian disease and a possible relationship to premature balding. Clin Endocrinol (Oxf) 1979; 11:291, from Genes and Chromosomes that cause Female infertility, Modern trends in infertility and Conception control Edwards E Wallach 1992; 4:15, 229. 18. Govind Obhrai, Clayton. Polycystic ovaries are inherited as an autosomal dominant trait analysis of 29 polycystic ovary synsrome and 10 control families. J Clini Endocrinol Metab 1999; 84(1):38–43. 19. Jahanfar S, Eden JA, Warren PW, Seppala M, Nguyen TV. A twin study of polycystic ovary syndrome. Fertil Steril 1995; 63(3)478–86. 20. Benett ST, LucassenAM, Gough SCL et al. Susceptibility to human type 1 diabeted at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nature Genetics 1995; 9:284–92. 21. Waterworth DM, Bennet ST, Gharani N et al. Linkage and association of the insulin gene VNTR regulatory polymorphism with polycystic ovary sysndrom. Lancet 1977; 349, 986–89. 22. Tiepolo and Zuffardi O Localization of factors controlling spermatogenesis in the nonfluorescent portion of the Y chromosome long arm. Hum Genet 1976; 34:119–24 in transmission of de nova mutation of the detected in azoospermia genes from a severe oligozoospermic male to a son via intracytoplasmic sperm injection from Fertility and Sterility 1997, 71:1029. 23. Voget et al Human Y chromocome azoospermic factors (AZF) mapped to genetics 1996; 5:933–43 in different sub regions inYq11. Human Molecular Genetics 1996; 5:933:43. 24. Sun C et al. An azoospermia man with a point de novo point mutation in the Y chromosomal USP94 Nature Genet 1999; 23,429–35 in prognostic value of Y deletion analysis. Hum Reprod 16:3:400. 25. Ferlin A et al. Human male infertility and Y chromosomes deletions-role of the AZF-candidate genes DAZ, RBM and DFERY. Hum Reprod 1999; 14:1710–16. 26. Mallidis et al. The role of Y chromosome deletions in male infertility. Eur J Endocrinol 2000; 142,418–30. 27. Lewis Jones MR, Gazvani and R. Mountford from Cystic Fibrosis in infertility: Screening before assisted reprodution. Hum Reprod 2000; 15(11):2415.

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28. Peter N Schlegel, Jacques Cohen, Marc Goldstein, Mina Alikani, Alexis Adler, Bruce R Gilbert et al. Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micro manipulation for men with bilateral congenital absence of vas deferens. Fertil Steril 1995; 64(2):421.

CHAPTER 46 Sperm Sepamtion Mandakini Parihar INTRODUCTION The current interest in the possibility of pre-selecting or rather controlling the sex of human progeny is only a recent manifestation of an age old phenomenon. The sex of the off spring remains important historically, culturally and economically. The fascination one has with the possibility of exercising control over the sex of one’s offspring is thus understandable. There are numerous descriptions in history at attempts to influence the sex outcome as placing a hammer under the heel for a boy or placing an egg or scissors under the bed for a girl. The early Greeks (about 500 BC) believed that male determining sperm were derived from right testis and so removing the left testicle would guarantee a male offspring.1 There are various suggestions offered today as, “at home”, methods of sex preselection techniques, and include timing of intercourse, sexual positions, timing of female orgasm, acid or base techniques and other methods which have been described in various “How to” books available for enhancing the chance of achieving the desired sex. Dietary methods have also been suggested to alter the sex ratio.2 The desire to pre-select the sex of the progeny differs according to socioeconomic conditions, culture and race. However, in nearly all cases, if only one child is the option, it is the son who is preferred. Once this is achieved, then a daughter becomes the next choice. Such a control over the gender of offspring has sufficient economic value in agriculture where different livestock production systems favour a particular progeny In humans it is of special interest in the prevention of X-linked diseases in “at risk” families. There are approximately 6000 heritable defects in humans, and about 500 out of these are X-linked diseases, including Hemophilia, Duschenne’s Muscular Dystrophy, Fragile X Syndrome and many more. Here, sons born from carriers are affected while daughters are unaffected. The ability to selectively separate X and Y chromosome bearing sperm and use the X spermatozoa to preferentially produce female off spring reduces or may even eliminate, the probability of conceiving an affected male. This represents a powerful approach to disease prevention. Spermatozoa determine human gender and there are many methods of separation of X and Y sperms like gradient, electrophoresis, immunologic techniques, electronic charge, staining, protein sedimentation etc. It is necessary that any technique selected must fulfil certain criteria and these are: 1. Complete separation of X and Y bearing sperm. 2. Sufficient number of separated sperm should be available for insemination. 3. Sperm should be viable after separation. 4. Capable of fertilization.

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During the present century, interest has focused mainly on three methods of sperm separation 1. In vivo clinical manipulations of timing of intercourse, ovulation and insemination and dietary methods. 2. In vitro sperm separation techniques designed to enrich either X and Y bearing sperm. 3. Preimplantation genetic diagnosis. In Vivo Methods It has been speculated that genetics of particular ethnic groups, diet and environment are factors that may determine changes of sex ratio. The techniques range from folk methods like reciting chants during intercourse, timing of coitus in relation to ovulation, wind direction, rainfall, phases of the moon to certain advocated dietary changes. Confusion and uncertainty exist regarding these in-vivo methods, because of lack of well designed prospective controlled trials. The very first reports in this century centered largely on effects of timing of intercourse in relation to ovulation. The unproven theory is that since Y-bearing sperms swim faster, intercourse just after ovulation would result in higher chances of male offspring. Whereas, the X bearing sperm survive longer and hence intercourse remote from ovulation resulted in higher number of females. In 1954 Kleegman3 reported a higher percentage of males, when insemination was done just after ovulation. Shettles4 suggested ovulation time, preceded by alkaline douche and female orgasm before the husband’s orgasm with abstinence till the day of ovulation would increase the likelihood of male offspring though many studies have suggested a positive co-relationship,4,16–17 an equal number of studies have disproved or failed to confirm these findings.6–10 A metaanalysis of data from couples practicing natural family planning methods has hypothesized that the pre-selection of females was probably related to maternal mid cycle LH peak and that the hormonal milieu of genital tract may be responsible for this result.11–19 This was first reviewed by Zarutskie15 where he found that different ovulation induction protocols had a significant skew in the sex ratio, mostly towards female predominance. However, the same study suggests that it was of little practical importance, because its influence on sex ratio though suggestive, was slight, and hence not significant. Dietary Methods Animal studies have shown that a change in diet leads to a change in sex ratio. Those women desirous of a male offspring should have low calcium and magnesium with high sodium and potassium in diet. However, research study which reported a 77.6 percent success rate20 lacked a control group of females to verify the recommended ionic concentrations and it is at best still unconvincing.

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IN VITRO SEPARATION OF X ANDY BEARING SPERMS Attempts to influence the gender of human offspring by pre-conceptional in υitro techniques date as far back as 1942. However, since the 1970’s attempts at sex selection focus on the separation of X and Y bearing spermatozoa and can be classified into four main groups: 1. Separation by sperm density or mobility. This takes into account differences in swimming ability of X and Y sperms. 2. Sperm separation by cell sorting. 3. Sperm separation by immunofluorescence. 4. Sperm separation by electrophoresis. Since the latter two methods are ineffective for clinical use, the purpose of this chapter is to elaborate the first two methods of sperm separation for X and Y spermatozoa. The role of PGD and its implications in use for non-medical purposes is outside the purview of this chapter. Separation by Density or Motility There exists subtle differences in plasma membrane surface charges, density, swimming velocity, motility, chromosome and DNA content between X and Y sperm. These differences are used in the various gradient techniques described for separation of X and Y spermatozoa. These are: a. Ericsson’s method using discontinuous albumin gradients. b. Sephadex column c. Discontinuous Percoll gradient d. Swim up procedure. Ericsson was the first to report approximately 85 percent enrichment of Ybearing spermatozoa population using an albumin gradient.23 Many investigators have been able to duplicate and confirm Ericsson’s results24–28 whereas others have failed to do so.29–30 Beernink et al31 published data from 65 clinics which are franchised by Gametrics Ltd., who hold the patent for Ericsson’s method of albumin separation. They report a 72% positive result for couples wanting a male child and 69% result for girl child. The pregnancy rates however, were between 10–16 percent. Ericsson’s method has been a subject of extra controversy from the beginning. This is mainly because the degree of separation has shown considerable variation in individual samples. Qunilan(26) in 1982 reported an increase in Y chromosome from 52 to 74%, and X chromosome from 60 to 74 percent. But their results too could notbe confirmed. Brandriff32 also used this technique and compared results using unprocessed sperm as control. Surprisingly his report indicates a decrease in Y count after albumin gradient density and hence could not confirm Y enrichment techniques. Sephadex columns and discontinuous 12-layer Percoll columns have also been quoted in different studies.33,34 However, the yield of Y enriched spermatozoa is in the range of 72 percent. Engelman et al34 reported that nearly all sperm preparation techniques are supposed to increase Y spermatozoa. They tested from different types of separation

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techniques and could demonstrate Y enrichment in all. Wang et al33 reported 55:44 enrichment in favor of X chromosome but concluded that the degree of enrichment was insufficient for clinical use in preconceptional sex selection. The most frequent sperm preparation technique used is swim-up procedure. Check et al35–37 hypothesize that swimup procedure damages the X spermatozoa and hence had 81% incidence of male offspring. They also state that albumin gradient separation favors female gender selection, which is contrary to Engelman’s study.34 Many contradictory reports in literature stress the need of a controlled prospective clinical trial for validating the clinical efficacy of gradients and columns for sex preselection of sperm through density and motility. There is a consensus that none of these techniques are really successful in separation of X and Y spermatozoa.33–38 Houssain et al39 have suggested certain reasons for failure and they cited lack of ability to visualize the separation process as well as size of the column as another reason. They have suggested that if a convenient low number of sperm is utilized and visualized during the entire separation process, then the procedure may be effective. In order to do this, they have included a microscope based column. This new horizontal column technique in its present form seems to be ideal for isolating Y sperm. The principle is basically a race between X and Y sperm which is managed in several gradients of albumin, staged on a Petri dish overlaid with mineral oil and can be monitored under an inverted microscope. Since, the Y sperm is a f aster swimming sperm, as soon as few sperm reach the end of column, they are trapped by breaking the column and the hand picked sperm, can be used for ICSI. The obvious disadvantage is that it necessarily needs IVF with ICSI as method of conception and it is not necessary that all the Y sperm will be faster than all the X sperm and hence it is difficult to predict the percentage. The authors have not cited the percentage of Y sperms separated by this method in their review. Sperm Separation by Cell Sorting Cell sorting is a method of sex pre-selection based on differences in sperm DNA content. It has been used extensively in animal husbandry sections to obtain livestock of a particular gender since 1989. It uses a flow cytometer for measurements of sperm DNA content and then separates the X and Y chromosome bearing sperms populations. The sexed sperm are then used with differing delivery routes like Intrauterine insemination, IVF with Embryo transfer or ICSI. The first human child born after using cell sorting technology was born in 1995, to a family that carried X-linked hydrocephalus. Subsequently, the clinical application of this technology in humans has resulted in more than 300 births40–42 under the name of ‘Microsort R’ for human sperm. This technology has a patent as ‘Microsort R’which is by Genetics and IVF Institute, Fairfax, USA. Principle of Cell Sorting One of the first attempts at sex pre-selection was described by Lush.43 1925 in animal studies on basis of centrifugation. However, this wasn’t successful. Flow cytometry was available for clinical studies from late 1960’s and was mainly for cancer diagnosis. In

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1979, Moruzzi44 showed a variety of differences between X and Y sperm for different species by measuring the DNAcontent as there are differences in chromosomal length. He pointed out that this difference in DNA content can be used as potential marker for sex pre-selection. It is this basis which has successfully shown to be the only effective marker for separating viable X and Y sperm.45 Development of Sperm SortingTechnology After the development of more advanced data acquisition equipment, the interest in using cell sorter for sorting sperm nuclei was acquired. A cell sorter was modified to sort sperm nuclei in 1982 by Johnson et al.40 This system formed the basis for the current technology as various protocols were developed that led to sorting of several mammalian sperm nuclei before using it on human sperm.46 Method as described by Johnson et al47 This consists of: 1. Preparation of Sperm nuclei 2. Fluorescent staining of spermatozoa 3. Flow CytometricAnalysis and Sorted 4. Collection of sorted sperm. Preparation of Sperm Nuclei The spermatozoa were obtained from healthy donors and frozen by Fairf ax Cryobank which is a division of Genetics and IVF Institute. The frozen samples were then thawed at room temperature and washed two times by centrifugation in 10 µm of Phosphate Buffered Saline (PBS) and resuspended at 10×106 sperm/ml. The sperm heads (nuclei) were prepared by sonication to break the tail from the head. These nuclei were then centrifuged for 20 seconds and resuspended in 1 ml of PBS. Pre-sort processing of human semen removes majority of dead, immotile and abnormal sperm. The presort method may be by washing, density gradient centrifugation or glass wool filtration techniques. Fluorescent Staining of Spermatozoa The processed sperm sample is stained with a 9 um solution of the vital fluorochrome bisbenzimide dye (Hoechst 33342 for 1 hour at 35°C.) Flow Cytometric analysis and Sorting Spermatozoa were sorted using cell sorter (Epics 753 or Epics V, Coulter Corporation, USA) which was modified for sperm analysis using DNA content and their flow sorting. The modification consisted of replacing the forward angle light scatter detector by a fluorescent detector and the standard cylindrical sample injection tip was beveled to produce a ribbon-shaped sample core stream. These modifications are essential in order

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to control the sperm orientation towards the laser beam and help in accurately measuring the fluorescence from properly oriented spermatozoa, only then the small differences in DNA content between X and Y sperms was evident. The stained spermatozoa were exited with ultra violet lines (351, 364 nm) of a 5W 90– 5 Innova Argon-ion laser (Coherent, Inc., USA) operating at 175 mv and fluorescence detected through 418 nm long pass filters. A 76 um jet-in-air flow tip is used and data collected as 256 channel histograms. Sheath fluid is 10 mM PBS. Collection of Sorted Spermatozoa The spermatozoa are sorted into 0.5 ml microfuge tubes that are pretreated with 1% BSA and contain 50 ml of Test yolk to determine motility. In the early phases, the flow rate was 1000 cells/second, resulting in sort rates of 30–40 cells/second per population. A key factor in the development was use of concentrated buffer solution in which to catch the sperm. In the original method approximately 3,50,000 sperms could be sorted in one hour. Improving the efficiency of sperm orientation was the key factor since it would increase the sperm available for sorting. The increase in the speed of the sorter using standard bevealed needle can sort up to 2 million sperm per hour. Tubes used for collected can be of every size to fit the type of sort. A 0.6 ml microtube is used for short sort and 15 ml conical tube for long sorts. To reduce the loss of sperm through adhesion, tubes are pre-coated by filling them for one hour prior to use with 1 percent BSA solution. Then 0.05 to 0.5 ml of test yolk is added depending on size of tube. If the sorted sample is to be used for IVF then 2 percent test yolk is used and if for IUI, then 20 percent is used.40 Change to High Speed Sorting (HiSON) The difference from the standard sperm sorting is: 1. staining procedure 2. use of orienting nozzle 3. elliptical beam shaping optic. The staining solution used is Hoechst 33342 (5 mg/ml in water) and food coloring, FDC #40 (25 mg/ml).48,49 The food coloring penetrates the membrane of dead sperm, and thus eliminates them from viable sperm. After incubation with Hoechst 33342, 1 ml of FDC 40 was mixed and kept for 5 minutes. Samples are then filtered through a 30-u nylon mesh and then sorted. The high-speed sorter is equipped with an orienting nozzle. This helps in preventing loss of sperm orientation before it reaches the laser beam. This results on 2 to 3 times the number of oriented sperm that are available for sorting. (70% v/s 25%)/Another modification is addition of an elliptical beam shaping optic,50 which is fitted in the laser path just before intersection of laser beam and sample stream. The O0 fluorescent histogram displays the DNA content of the oriented sperm only and the sort windows are established according to the specific population desired. With flow rate of 14,000/sec with sperm that were 80 percent motile at the outset, the resultant purity of X and Y sperm was about 90 percent/Losses to the sort are due to the dead or mis-oriented sperm. The usual orientation rate is about 70 percent with sort window elimination of

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30%. This results in a final yield of 20–30 percent of total sperm with which one started and with high speed sorting the final result is about 6 million sperm per hour in each direction i.e. a total of 12 million/hour. Mammalian Studies The effectiveness of sexing sperm was first attempted on several species of livestock. The sperm sorting based on DNA difference between X and Y chromosome is inherent in all mammals. In animal studies, the sort purities ranged from 85 to 98 percent. The purity of sample was judged by FISH technology The ultimate test of sexing method was the sex of desired offspring produced. However, to cover all the details of animal studies are outside the purview of this chapter and can be referred to in any of these references.51–58 Human Applications Multiple molecular probing methods were employed to reconfirm that sorted samples were of highest purity. Each sample was subjected to probing using either a X or Y DNA probe. Sorted spermatozoa were on slides and then the X and Y centromere specific DNA probes were applied and validated using FISH (Fluorescent in situ Hybridization) Technology.47 The reason for this is that: 1. morphology of the human sperm head is angular rather than paddle shaped as in animals. 2. human spermatozoa are more polymorphic and have greater heterogeneity of chromatin composition than animals. 3. the difference in human sperm DNA is very small (2.8%) and the resolving capacity of the instrumentation is pushed to its inherent limits. After sorting the sperm membrane is monitored which are usually acrosome reacted and pre-capacitated. There is no difference with respect to swimming velocity between Y and X sperm as measured by CASA (Computer Assisted Motion Analysis). Of the 300 Microsort pregnancies, (as per their data in 2001) 61 resulted after IUI and 35 after IVF/ICSI. The pregnancy rate reported per cycle was 11.8 percent for IUI and 24.1 percent for IVF/ICSI. The average sperm-cell enrichment after X and Y sort as determined by FISH was 88 percent and 69 percent respectively. The number of pregnancies with know fetal or birth sex resulting in correct offspring was 94.4 percent for daughters and 73 percent for sons. The sorting of X sperm alone is the easiest due to the f act that it carries more DNA and hence is the brightest in response to excite laser beam. Hence, sorting of X sperm at higher speed results in a purer sample than can be obtained with Y sperm. This is due to the fact that slightly misanalyzed X sperm will fall in the Y sort. Methods used for reconfirmation of sorted samples are: 1. DNA probesusing FISH 2. Sort re-analysis

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In sort re-analysis, the sorted sample is subjected to the whole process of cell sorting again by adding it with more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double Gaussian curve to determine the x and y populations.54 ETHICALISSUES OF SEX SELECTION Though attempts have been made to restrict preconception gender selection for medical purposes, there are as yet no specific guidelines for its nonmedical use. The Ethics Committee60 has expressed concern which includes the “potential for inherent gender discrimination, inappropriate control over nonessential characteristics of children, unnecessary medical burdens and inappropriate and potentially unfair use of limited medical resources.” Many debates and discussions have been put forth by the proponents and opponents of preconception gender selection for non-medical reasons.61–63 These are summarized in Tables 46.1 and 46.2.64–69 Majority believes that sex control technology would have little effect on the sex ratio except for a temporary period.

Table 46.1: Arguments for preconception gender selection 1. It fulfils desire of couples who have strong gender preferences. 2. It prevents selective abortions or infanticide if the offspring is of undesired gender. 3. Parents with children of a particular sex might prefer a child of the opposite sex. 4. Child of desired gender will be wanted and loved (presumably). 5. As a relatively low cost procedure by IUI after mechanical sperm separation it is unlikely to drain resources from medical system. 6. Acts as population control measure.

Table 46.2: Arguments against preconception gender selection 1. It allows increase in gender discrimination by producing more males. 2. Welfare of children born as a result of gender selection, as they may be expected to act in a specific way and may disappoint parents if is fails. 3. A major concern is disruption of sex ratio balances as have occurred in many parts of India and China. 4. Emphasis is on child’s gender rather than his or her inherent worth. 5. In many societies women are discriminated against solely on basis of gender and this will only have adverse consequences.

Recommendations of Ethics Committee (2001)69 1. Until a method of separation X and Y bearing sperm is established as safe and effective in statistically valid, properly executed clinical trials, it should be labeled as experimental.

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2. If such trials show that these techniques are effective, they should be used for nonmedical purposes only for gender variety in the family, i.e. have child of opposite sex from the one present. 3. If flow cytometry or other methods prove effective for couples who seek gender variety in family they should be a. fully informed about risk of failure. b. affirm that they will accept child of opposite sex if the gender selection fails. c. counseled not to have unrealistic expectations from pref erred gender. 4. Practitioners offering assisted reproduction are under no legal or ethical obligation to provide non-medically indicated gender pre-selection.

CONCLUSION The never-ending story of choosing the gender of one’s offspring is soon becoming a reality. The long term global benefit of successful separation of X and Y sperms will help in prevention of sex chromosome linked diseases, balancing sex composition in the family, reducing the population growth of the world by lowering the birth rate, postconceptional abortions and selective gender infanticides. Additional applications of flow cytometry may include the identification and separation of chromosomally normal sperms, in men carrying certain translocations or those with high incidence of aneuploidy These can also be applied to selection of genetically normal sperm in men exposed to radiation or chemotherapy. Accurate sex pre-selection would improve therapeutic alternatives and in time will reduce or eliminate the use of selective abortion. Flow cytometric separation is a reliable procedure to be used with IUI and IVF/ICSI especially for X linked genetic diseases. Although the reported efficacy of selection process is high, it should be followed by prenatal or pre-implantation diagnosis to prevent birth of children affected by sex linked diseases, to eliminate the risk of birth of an affected child. Its use in non-medical use for gender selection still remains controversial. REFERENCES 1. Reubinoff B, Schenker J. New advances in sex preselection. Fertil Steril 1996; 66(3):343–50. 2. Stolkowski J, Choukroun J. Preconception selection of sex in man. Isr J Med Sci 1981; 17:1061– 67. 3. Kleegman SJ. Therapeutic donor inseminisation. Fertil Steril 1954; 5:7–30. 4. Sheetles LB. Factors influencing sex ratios. Int J Gynecol Obstet 1970; 8:643–47. 5. Bilings EL, Westmore A (Eds). Scientific research on the Bilings method. In The Bilings Method. New York: Penguin Books, 1982; 199:172–85. 6. France JT, Graham FM, Gosling L, Hair P, Knox BS. Characteristics of natural conceptual cycles occurring in a prospective study of sex preselection: fertility awareness systems, hormone levels, sperm survival and pregnancy outcome. Int J Fertil 1992; 37:244–55. 7. Guerrero R. Association of the type and time of inseminisation within the menstrual cycle with the human sex ratio at birth. N Engl J Med 1974; 291:1056–59.

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8. Harlap S. Gender of infants conceived on different days of the menstrual cycle. N Engl J Med 1979; 300:1445–48. 9. Gray RH. Natural family planning and sex selection: fact or ficition. Am J Obstet Gynecol 1991; 165:1982–84. 10. James WH. Time of fertilization and sex of infants. Lancet 1980; 1:1124–16. 11. James WH. Gonadotrpin and the human secondary sex ratio. Br Med J 1980; 281:711–12. 12. James WH. The sex ratio of infants born after hormonal induction of ovulation. Br J Med Obstet Gynecol. 1985;92:299–301. 13. Cholst I, Jewelewiez R, Dyrenfurth T, Vande Wiele L. Gonadotropins and the human sex ratio. Br Med J 1981; 283:1264–65. 14. Ben-Rafael Z, Matalon A, Blankstein J, Serr dM, Lunenfold B, Mashiach S. Male to female ratio after gonadotropins induced ovulation. Fertil Steril 1986; 45:36–40. 15. Zarutskie PW, Muller CH, Magone M, Soules MR. The clinical relevance of sex selection techniques. Fertil Steril 1989; 52:891–905. 16. France JT, FM Graham, L Gosling, P Hair, BS Knox. Characterisitcs of natural conceptual cycles occurring in a prospective study of sex preselection: fertility awarness symptoms, hormone levels, sperm survival, and pregnancy outcome. Int J Fertil 1992; 37(4):244–55. 17. Cohen MR. Differentiation of sex as determined by ovulation timing. Int J Fertil 1967; 12:28– 32. 18. Muehleis PM, SY Long. The effects of altering the pH of seminal fluid on the sex ratio of rabbit offspring. Fertil Steril 1976; 27(12):1438–45. 19. Simcock BW. Sons and daughters-a sex preselection study. Med J 1985; 142(10):541–42. 20. Papa F, R Henrion, G Breart. Preconceptional selection of sex using the ionic method. J Gynecol Obstet Biol Reprod 1983; 12(4):415–22. 21. Zarutskie PW, CH Muller, M Mangone, MR Soules. The clinical relevance of sex selection techniques. Fertil Steril 1989; 52(6):891–905. 22. Ali JI, FE Eldrige, GC Koo, BD Schanbacher. Enrichment ofbovine X- and Y-chromosomebearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter. Arch Androl 1990; 24(3):235–45. 23. Ericsson RJ, Langvein CN, Nishino M. Isolation of fraction rich in human Y sperm. Nature 1973; 246:421–24. 24. Ericsson SA, RJ Ericsson. Couples with exclusively female offspring have an increased probability of a male child after using male sex preselection. Hum-Reprod 1992; 7(3):372–73. 25. Ericsson SA, RJ Ericsson. Sex ratio of male sex preselected children born to couples with exclusively. 26. Quinlivan WLG, Preciado K, Long TL, Sullivan H. Separation of human X and Y spermatozoa by albumin gradients and sephadex chromatography. Fertil Steril 1982; 37:104–7. 27. Corson SL, FR Batzer, NJ Alexdander, S Schlaff, C Otis. Sex selection by sperm separation and insemination. Fertil Steril 1984; 42(5):56–760. 28. Jaffe SB, Jewelewicz R. A. Controlled study for gender selection? Fertil Steril 1991; 56(2):254–58. 29. Brandriff BF, Gordon LA, Haendel S, Singer S, Moore DH II, Gledhill BL. Sex chromosome ratios determined by Karyo-typic analysis in albumin isolated human sperm. Fertil Steril 1986; 46:678–85. 30. Ueda K, Yanagimachi R. Sperm chromosome analysis as a new system to test human X- and Ysperm separation. Gamete Res 1987; 17:221–28. 31. Beernink RC, WP Dmowski, RJ Ericsson. Sex preselection through albumin separation of sperm. Fertil Steril 1993; 59:382–86. 32. Brandriff BF, LA Gordon, S Haendel, S Singer, DH Moore, BL Gledhill. Sex chromosome ratios determined by Karyotypic analysis in albumin isolated human sperm. Fertil Steril 1989; 46(4):678–58.

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33. Wang HX, Flaherty SP, Swann NJ, Mattews CD. Discontinous Percoll gradientssitu hybridization. Hum Reprod 1994; 9:126 34. Engelmann U, EM Parsch, WB Schill. Modern techniques of sperm preparation-do they influence the sex of offspring? Andrology, 1989; 21(6):523–28. 35. Check JH, BS Shanis, SO Cooper, A Bollendrof. Male sex preselection: swim-up technique and insemination of women after ovulation induction. Arch Androl 1989; 23(2):165–66. 36. Check JH, D Katsoff. A prospective study to evluate the efficacy of modified swim-up preparation for male sex selection. Hum Reprod 1993; 8(2):211–14. 37. Check ML, Bollendrof A, Check JH. Separation of sperm through 12-layer percoll column decreases the percentage of sperm staining with quinacrine. Archives of Andrology 2000; 44:47; 50. 38. Cohen J. The future of international registries for ART. Fertil Steril 2001; 76(5):871–73. 39. Hossain AM, Barik S, Rizk B et al. Preconceptional sex selection: Past, Present and Future. Archiev Androl 1998; 40:3–14. 40. Johnson LA, Welch GR. Sex preselection: High-speed flow cytometric sorting of x and y sperm for maximum efficiency. 41. Fugger EF, Black SH, Keyvanfar K, Schulman JD. Births of normal daughters after Micro Sort sperm separation and intrauterine insemination, in vitro fertilization, or intracytoplasmic sperm injection. Human Reprod 1998; 13:2367–70. 42. Fugger EG. Clinical experience with flow cytometric separation of human X and Y chromosome bearing sperm. Theriogenology 1999; 52:1435–40. 43. Lush JL. The possibility of sex control by artificial insemination with centrifuged spermatozoa. J Ag Res 1925; 30:893–913. 44. Momzzi JF. Selecting a mammalian species for the separation of X and Y chromosome bearing spermatozoa. J Reprod Fertil 1979; 57:319–23. 45. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 41:199–203. 46. Johnson LA. Gender preselection in domestic animals using flow cyriometric sorted sperm. J Anim Sci 1992; 70(Suppl 2):8–18. 47. Lawrence A, Johnson, Glenn R. Welch. Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases. Human Reprod 1993; 8:10, 1733–39. 48. Johnson LA, Welch GR, Rens W, Dobrinsky JR. Enhanced flow of cytometric sorting of mammalian X and Y sperm: High speed sorting and orienting nozzle for artificial insemination. Theriogenology 1998; 49:361 (Abstract). 49. Johnson LA, Welch GR, Rens W. The Beltsville Sperm Sexing Technology: High-speed sperm sorting gives improved sperm output for in vitro fertilization and AI. J Anim Sci 1999; 77:213– 20. 50. Welch GR, Rens W, Johnson LA. High speed cell sorting: Modifications to a MoFlo for sorting X and Y chromosome bearing sperm based on DNA. Cytometry 1998; (Suppl 9):130 (Abstr). 51. Johnson LA. Sex preselection in swine: Altered sex ratios in offspring following surgical insemination of flow sorted X and Y bearing sperm. Reprod Dom Anim 1991; 26:309–14. 52. Johnson LA, Pinkel D. Modification of a laser-based flow cytometer for high resolution DNA analysis of mammalian spermatozoa. Cytometry 1986; 7:268–73. 53. Garner DL, Gledhill BL, Pinkel D, Lake S, Stephenson D, Van Dilla MA et al. Qualification of the X and Y chromosome-bearing spermatozoa of domestic animals by floe cytometry Biol Reprod 1983; 28:312–21. 54. Welch GR, Johnson LA. Sex preselection: Laboratory validation of the sperm sex ratio of flow sorted X and Y sperm by sort reanalysis for DNA. 55. Cran DG, Johnson LA, Polge C. Sex preselection in cattle: a field trial. Vet Rec 1995; 135:495– 96.

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56. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 41:199–203. 57. Welch GR, Waldbieser GC, Wall RJ, Johnson LA. Flow cytometric sperm sorting and PCR to confirm separation of X and Y chromosome bearingbovine sperm. Anim Biotechnol 1995; 6:131–39. 58. Seidel GE Jr, Allen CH, Johnson LA, Holland MD, Brink Z, Welch GR et al. Uterine horn onsemination of heifers with very low numbers of nonfrozwn and sexed spermatozoa. Theriogenology 1997; 48:1225–65. 59. Fugger EF. Clinical experience with flow cytometric separation of human X and Y chromosome bearing sperm. Theriogenology 1999; 52:1435–40. 60. Ethics Committee of the American Society of Reproductive Medicine. Preimplantation genetic diagnosis and sex selection. Fertil Steril 1999; 72:595–98. 61. Greenhalgh S, Li J. Engendering reproductive policy and practice in peasant China: for a feminist demography of reproduction. Signs 1995; 20:601–37. 62. Glover J. Comments on some ethical issues in sex selection. Contribution to the international Symposium on Ethics in Med and Reprod Biol. Paris France, July 1994. 63. Glover J. Ethics of new reproductive technologies: the Glover reports to the European Commission. Dekalb: Northern ILLINOIS university Press, 1989:141–44. 64. Liford RJ. Sex selection-etical issues. Hum Reprod 1995; 10:762–64. 65. Laland K, Kumm J, Feldman MW. Medical ethics and human reproduction: scientists predict unbalanced future with sex selection. BMJ 1994; 308:536. 66. Kumar TC. Gender pre-selection: prevention of perpetuation of female deaths. Hum Reprod 1995; 10:1319. 67. Dawson K, TrousonA. Ethics of sex selection for family balancing. Why balance families? Hum Reprod 1996; 11:2577–78. 68. Coale AJ, Banister J. Five decades of missing females in China. Demography 1994; 31:459–79. 69. Ethics Committee of ASRM. Preconception gender selection for non-medical reasons. Fertil Steril 2001; 75(5):861–64.

CHAPTER 47 Ovarian Tissue Cryopreservation in Cancerous Patients: State of the Art Zeev Blumenfeld INTRODUCTION As survival rates for young cancer patients continue to improve, protection against iatrogenic infertility caused by chemotherapy with or without radiotherapy assumes higher priority.1–4 Hodgkin’s, disease is the most common malignancy in the population aged 15 to 24 years.5 Prolonged survival of almost 90 percent of patients is now expected for a high proportion of young patients treated with cytotoxic chemotherapy for Hodgkin’s disease.1,5–7 This is due to the introduction of effective chemotherapy such as MOPP (mechlorethamine, vincristine, prednisone and procarbazine) and/or ABVD (adriamycin, bleomycin, vinblastine and decarbazine) and its variants.1,5,8–10 Similar rates of long-term survival have been reported about patients with non-Hodgkin’s lymphoma, as well as for patients with other types of tumors receiving chemotherapy1,2,5–8 Moreover, cytotoxic agents have also been used for chemotherapy for various autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and for the prevention of organ transplant rejection.1,6 Recent advances in the understanding of the molecular biology of lymphomas permit an additional dimension—the identification of molecular characteristics associated with a poor prognosis, such as patterns of gene expression that are associated with protection against apoptosis, chemotherapy resistance, or the stimulation of angiogenesis. If such attempts prove successful, the goal of 100 percent cure without serious late secondary effects, despite its apparent elusiveness a few decades ago, will have been positioned clearly on the horizon.7 Premature ovarian failure (POF) is a common long term consequence of chemotherapy1,6,8–12 and radiotherapy.6,13 Whereas the cytotoxic-induced damage is reversible in other tissues of rapidly dividing cells such as bone marrow, gastrointestinal tract and thymus,6,14 it appears to be progressive and irreversible in the ovary, where the number of germ cells is limited, fixed since the fetal life, and cannotbe regenerated.1,6 CHEMOTHERAPY-ASSOCIATED GONADOTOXICITY Breast cancer, the most common malignancy in women, affects approximately 185,000 women/year in the US.15,16 Almost 25 percent of the cases occur premenopausally.15–19 Whereas adjuvant chemotherapy prolongs survival, it has been shown to cause POF.15,20– 25 Premature menopause associated with vasomotor, psychosocial, genitourinary, skeletal

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(osteoporosis) and cardiovascular problems is usually treated effectively with hormone replacement.15,26 However, hormone replacement therapy is contraindicated by most in breast cancer survivors;15,27 therefore, it is of utmost clinical importance to minimize the chemotherapy-associated POF in these women. The first study of systemic adjuvant chemotherapy for breast cancer showed cessation of menses in 40 percent of the patients treated with thiotepa as opposed to 3 percent of the control group.28 Various chemotherapeutic regimens have been in use since the late 1960s, but reports on the incidence of chemotherapy-associated amenorrhea (CRA) have been erratic. Recent reviews reported on 15 to 40 studies with information on the effects of adjuvant chemotherapy on ovarian function in premenopausal women.15,29 The reported incidence of POF ranges from 0 to 100 percent among different chemotherapy regimens. Most data were collected at 12 months after the beginning of treatment.15 The average percentage of CRAin regimens based on cyclophosphamide, methotrexate and fluorouracil (CMF) given for at least 3 months is 68 percent (95% confidence interval [CI], 66 to 70%), with a range of 20 to 100 percent.15 This wide range can be attributed to the following variations in experimental design: 1. Definition of amenorrhea 2. Definition of menopausal status 3. Age distribution 4. Therapeutic regimen (drug, dose, duration and route of administration) 5. Population characteristics. The risk of gonadal damage is usually directly related to the age of the patient. Patients older than 40 years experience a consistently higher rate of amenorrhea when compared with those ≤40 years.15 Rates varied from 21 to 71 percent in the younger age group and from 49 to 100 percent in the older group. The average percentage of POF for CMFbased treatments (for at least 3 months) was 40 percent (95% CI, 36 to 44%) and 76 percent (95% CI, 74 to 78%) for younger and older groups, respectively About two-thirds of premenopausal women experience POF after CMF chemotherapy combination for breast carcinoma (95% CI, 66 to 70%).15 The action of a given chemotherapeutic agent on the ovaries can occur through the impairment of follicular maturation and/or depletion of primordial follicles.15,30–34 Combination chemotherapy is used more often than single agents and it is difficult to evaluate the contribution of each individual drug. Most available information concerning the effects of chemotherapy on ovarian function has come from studies of women who received MOPP for Hodgkin disease.35 Since the first description of drug-induced amenorrhea, many other drugs have also been implicated.34,36,37 Alkylating agents are the most common chemotherapeutic agents associated with gonadal damage.31–35,38 These agents are not cell-cycle-specific and thus do not require cell proliferation for their cytotoxic action. It is believed that they act on undeveloped oocytes and possibly on the pregranulosa cells of primordial follicles.15,30 Most information is available on the effects of cyclophosphamide.30,35,38 Recent animal studies suggest that phosphoramide mustard is the main metabolite responsible for the ovarian toxicity of cyclophosphamide.15,39 The association of doxorubicin with infertility is debatable. In a study that compared MOPP with doxorubicin, bleomycin, vincristine and dacarbazine (ABVD) for the

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treatment of Hodgkin’s disease by Santoro et al40 reported less ovarian toxicity in the ABVD group. However, in animal studies, doxorubicin has been shown to cause testicular damage.41 Mitoxantrone is likely to produce amenorrhea.42 The antimetabolites cause little ovarian toxicity when used as adjuvant treatment for breast cancer. This could be explained by their cytotoxic action on dividing cells.15 Data available from treatment of women with choriocarcinoma have demonstrated no adverse effects on the ovary Schamberger et al43 treated seven women (aged 13 to 31) with highdose methotrexate for osteosarcoma and found no gonadal dysfunction. Koyama et al44 observed no ovarian toxicity among nine breast cancer patients treated with adjuvant fluorouracil. Comparing melphalan (L-PAM) and fluorouracil plus L-PAM, Fisher et al45 found no difference in severity of ovarian dysfunction, which suggests that L-PAM alone was the toxic agent. Cobleigh et al46 confirmed these findings and presented markedly lower rates of POF in patients treated with methotrexate to 5-fluorouracil compared with CMF. There is not enough information available regarding which chemotherapy regimen causes the highest rate of ovarian failure. Few reports analyzed POF rates among patients who underwent different combination treatments. Cobleigh et al41 compared adriamycin and cyclo phosphamide (AC) with CMF and reported a significantly lower rate of amenorrhea among those treated with the former (34% v. 69%, respectively). Effect of Dose-intensity, Cumulative Dose and Route of Administration on Incidence of POF It is difficult to analyze the impact of different treatment practices on the incidence of amenorrhea in view of the multitude of confounding factors. Cyclophosphamide is the agent most commonly implicated in POF15,30–35,38,47,48 and therefore most of the discussion will concentrate on this drug. The variables analyzed were cumulative dose, dose-intensity, duration and route of administration. Kay and Mattison37 showed that increasing doses of cyclophosphamide caused progressive destruction of oocytes and follicles in mice. Human studies confirm these results.1,2,15 Padmanabhan et al49 compared the percentage of cumulative dose received by patients who developed POF with those who continued to have regular periods. Although the percentage of the cumulative dose of cyclophosphamide (at 80 mg/m2 by mouth on days 1 to 14 for 12 months) received by those who developed POF was higher (60% v. 54%), the difference was not statistically significant. Higher cumulative doses of cyclophosphamide cause higher POF rates.15 Goldhirsch et al50 reported that patients treated with one preoperative cycle of CMF experienced POF rates of 10 percent and 33 percent in the younger and older age groups, respectively. The rates increased to 33 percent and 81 percent with six cycles (cumulative dose of cyclophosphamide, 8,400 mg/m2) and to 61 percent and 95 percent with 12 cycles of CMF (cumulative dose of cyclophosphamide, 16,800 mg/m2). Other investigators reported similar results.15 The only discordant observation came from Moliterni et al,51 who found similar POF rates regardless of cumulative dose. Tancini et al,52 Goldhirsch et al,50 Bianco et al,53 Moliterni et al51 and Reyno et al54,55 showed different POF rates while keeping dose-intensity constant. It is of note that

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treatment duration also varied, compromising the analysis of dose-intensity as an independent variable. Wood et al56 analyzed variation in dose-intensity while keeping cumulative dose constant, but they did not report POF rates. Brincker et al57 held duration of treatment constant and varied the cumulative dose and dose-intensity They showed that these variables were directly related to POF rates, but could not tell whether they were independent. Thus, the preponderance of the data, with one exception,51 support the concept of a direct correlation between dose intensity and POF. Duration of treatment and route of administration of cyclophosphamide as independent variables on POF rate remain to be determined.15 Infertility represents one of the main long term consequences of combination chemotherapy given for Hodgkin lymphoma, leukemia and other maglinancies in young women.1,2,5,15,58 The impairment of gonadal function after chemotherapy is much more frequent in men than in women, occurring in up to 90 percent of post pubertal males.1,2,59 Because dividing cells are known to be more sensitive to the cytotoxic effects of alkylating agents than are cells at rest, it has been suggested that inhibition of the pituitary-gonadal axis would reduce the rate of spermatogenesis and oogenesis and thereby render the germinal epithelium less susceptible to the effects of chemotherapy.1,2,6,59,60 However, several alternatives have been attempted for preservation of fertility in young women undergoing chemotherapy treatment. Other Alternatives for Fertility Preservation Although GnRH-a treatment in parallel with chemotherapy1 is a promising adjuvant therapy for young women, it is by no means the only option for fertility preservation. Moreover, this adjuvant treatment is not applicable to those young women undergoing very aggressive chemo-and radiotherapy for bone marrow transplantation (BMT), since POF is associated with 32 percent or more of those women undergoing BMT.61–66 For these patients additional options may be available: A. Cryopreservation of “mature” metaphase II oocytes, usually after hMG/hCG ovarian stimulation. Although successful in rodents,65,67,68 it is stillfar from being a clinical alternative in humans.69,70 Moreover, a concern of possible chromosomal aberration associated with the freezing of MII oocytes has been raised.65,71 A future possible alternative may be the retrieval of human immature oocytes for cryopreservation and in vitro maturation after thawing.65,72 B. A much more clinically available option, is the cryopreservation of fertilized ova, after IVF, before chemotherapy73 However this alternative is relevant to married women or those who have a partner, and almost inapplicable to the very young, single women. Moreover, the ovarian stimulation with hMG/hCG before IVF-egg retrieval needs to postpone the initiation of chemotherapy, often contraindicated by the hematologists and oncologists.1,65 Furthermore, the increase in estradiol concentrations, caused by hMG/ hCG ovarian stimulation may possibly aggravate the clinical situation of patients with breast carcinoma or other estrogen-sensitive tumors, or that of SLE patients, by inducing a flare-up of the autoimmune disease.1 Preliminary clinical results in our and other centers suggest that it may be possible to retrieve immature ova from developing antral follicles, mature them in-vitro for a few days, fertilize

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them by ICSI and subsequently cryopreserve the few generated embryos. This option may enable for an incomplete resolution, although limited, for preservation of future fertility, without postponing the initiation of chemotherapy and without exposure of these patients to ovarian stimulation and hyperestrogenism. C. An old suggestion74 which has recently been the focus of intense investigation is the transplantation of ovarian tissue.1–3,65,75–79 However, a concern of possible associated malignancy in the transplanted ovary has been raised.1,3,65,79 Therefore, future endeavors may concentrate on cryopreservation of ovarian fragments, before chemotherapy, and thawing these fragments, dispersion of the primordial/ primary follicles, in vitro maturation and fertilization. Although not clinically available yet, the enormous progress of ART in the last two decades, lends hope that in the next few years this option may indeed turn into a practical option.78 Until then, every effort should be made to offer all the possible options available at present for minimizing the gonadotoxic effect of chemotherapy A recent editorial in Fertility and Sterility by Dr Oehninger80 has challenged the pertinent question: “Will ovarian autotransplantation have a role in reproductive and gynecological medicine?” for preservation of ovarian function. This has been successfully accomplished in rats, sheep and other animals.74–79,81–82 Such therapy could provide a source of ovarian tissue that, when autotransplanted, would maintain an adequate estrogenic milieu that protects against heart disease and osteoporosis. It has been suggested that it may be best to restrict ovarian transplantation to women whose ovaries are disease free because cancer can be transmitted with ovarian tissue grafts in animals.79 A second role for this therapy could be in the form of “oocyte banking” as a strategy to preserve the reproductive potential of younger women or girls before cancer therapy.65,76,79,81–84 Cryopreservation of ovarian tissue before initiation of oncologic treatment, followed by autologous transplantation after remission, could provide a means of protecting fertility80,85 Obviously, an option for these cases involves oocyte retrieval (today probably better accomplished with gonadotropin stimulation, although the natural cycle will be preferable when improved oocyte in vitro maturation protocols become available), followed by IVF and embryo cryopreservation.78–80 Oocyte banking could have a role also in women of advancing age who elect to delay conception into the years of diminished ovarian reserve.80,82 The feasibility of this approach has been reported in the rat.86 The potential therapeutic use of ovarian auto-transplantation mandates continued efforts to achieve better results with ovarian tissue cryopreservation (i.e., freezing of the isolated germ cell at different stages of maturity, cumulus-encased oocytes, follicles and sliced tissue) and with in vitro oocyte maturation procedures. Ovarian tissue can be recovered at the time of laparotomy, or ovarian biopsy specimens can be taken by laparoscopy When autografting to an orthotopic site succeeds in restoring ovulatory menstrual cycles, hormonal replacement therapy and medical intervention in the process of conception may not be needed.76,78,80 Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −1327°C has been reported.75 Alternatively, oocytes could be recovered from an ectopically located graft and matured in vitro or, after gonadotropin stimulation, mature oocytes could be retrieved and used in assisted reproductive technologies.78

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FERTILITY CONSERVATION OPTIONS Cryopreservation of ovarian tissue has several potential advantages over both oocyte and embryo freezing. Hundreds of immature oocytes may thus be cryopreserved without the necessity of ovarian stimulation and delay in initiating cancer treatment.87 Cryopreservation of ovarian tissue is of great benefit since immature oocytes are relatively quiescent, smaller and lack zona pellucida and cortical granules.87 These properties make them far more tolerant to freezing and thawing injuries as compared to mature oocytes. Furthermore, it has been hypothesized that primordial follicles have a greater potential to repair sublethal damage to organelles and other intracellular structures during their prolonged growth phase.87 However, a significant follicular loss occurs with freezing, thawing, and grafting. It is unknown yet how well and how long will a given frozen thawed ovarian segment function after autotransplantation in human.87 There are three optional methods for utilizing of cryopreserved ovarian tissues: Autotransplantation, xenotransplantation, and in-vitro maturation. Autotransplantation Cryopreserved ovarian tissue can be autografted either orthotopically or heterotopically, but as yet, it is unknown which will turn to be more practical and effective. The expected relatively short life span of frozen thawed ovarian grafts is a concern.87 Where few follicles remain and early graft exhaustion is expected, it may be more reasonable to use the heterotopic approach.87 Bearing in mind the potential risk of transmission of microscopic metastatic disease, attempts to confirm the safety of ovarian tissue transplantation, based on the absence of malignant cells by light microscopy may not be reassuring enough.87,88 A recent study,88 suggested that it is impossible to exclude the risk of cancer transmission in hematogenous diseases (such as leukemia) or systemic neoplasms. Xenotransplantation Using this option, the possiblity of cancer cells transmission and relapse is eliminated since cancer cells do not penetrate the zona pellucida. Another advantage of this technique is the possibility of application of patients for whom hormonal treatment is contraindicated (such as in breast cancer). It has been recently demonstrated,88,89 that after subcutaneous transplantation of human ovarian cortex into mice, exogenous gonadotropin stimulation, generated follicular growth in 51% of the grafts.89 Revel et al90 have demonstrated follicle maturation and subsequent formation of corpus luteum in human ovarian tissue xenografted subcutaneously into mice. The advantages of subcutaneous xenografting are: Simplicity, convenient monitoring of follicular development, and direct access to follicle aspiration.87 However, there is a serious concern to possible transmission of xenoses and animal pathogens to human.

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In Vitro Maturation Developing an in vitro culture system contains multiple variables that may possibly affect oocytes integrity since the development of primordial follicles to the preovulatory stage in humans may take more than six month.91 The in vitro growth and maturation of human primordial follicles, followed by IVF, is a very attractive and desirable option, but it is technically challenging due to the prolonged growing phase of lack of knowledge of the optimal conditions for growth and maturation of human oocytes. Although preliminary encouraging experiments have been recently generated in animal models,87 the ability to completely grow and mature human primordial follicles in vitro, however, will not be available until the development of an optimal culture system, which depends on the acquisition of a full understanding of the signal and control mechanism of folligular growth.87 In summary, several urgent issues need to be resolved for the clinical application of ovarian transplantation and/ or in vitro maturation to be usccessful and clinically applicable. Furthermore, unequivocal information is needed on the optimal dehydration times, cooling and thawing rates, and identification of the most effective cryoprotectant.87 The most crucial factor for tissue survival is the degreeof ischemic-reperfusion injury after transplantation.87 Indeed it has been reported that more primordial follicles suffer demise because of ischemia than of freezing injury87 Indeed using a sheep autograft model,92 only 5% of the primordial have survival autografting. The optimal site for transplantation is also not yet precisely known.87 The main hurdle with ovarian transplantation is ischemia-reperfusion injury during revascularization.87 It is crucial, therefore, to find a way for minimalization of the hypoxic injury, which may significantly jeopardize the future success of ovarian transplantation. Ovarian tissue cryopreservation has yet to prove itself in terms of its main goal: a human pregnancy93 Indeed, many centers around the world have begun extracting ovarian tissue in clinical settings, wagering that, by the time their patients need this tissue the optimal manner for its use and the proper technology will be developed.93 Therefore, it appears, that at the present time we should be prudent and reserve ovarian tissue cryopreservation for patients who have nothing to lose, when the intensity of anticancer treatments (such as in bone marrow transplantation) will render them menopausal.93 The study of Corleta et al82 confirmed that ovarian grafts with sliced tissue resulted in a lower degree of ischemic or degenerative changes than intact, transplanted ovaries. In different animal models (rat, rabbit, monkey) it has been demonstrated that autotransplantation of ovaries can result in a prompt revascularization of the gland. The transplanted ovary is (or becomes after grafting) able to produce substances that promote and direct angiogenesis.86 Recent studies have shown that the increase in gonadotropin secretion after ovarian transplantation in the rat contributes to revascularization of the graft by up-regulating the gene expression of two major angiogenic factors, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1).86 More information is needed to determine whether the oocytes from autotransplanted ovarian tissue are competent to achieve full maturation, fertilization and embryo development. Interesting, perplexing and challenging questions arise:

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1. Could the addition of angiogenic and other growth factors (such as VEGF, TGF-β, or others) to the transplanted ovary enhance the anchorage and number of functionally competent cells? 2. What is the survival rate of the grafts? 3. Could the functional longevity be prolonged by addition of survival factors? 4. Is the developmental potential of the female gamete preserved after transplantation (with or without an intermediate cryopreservation step)? 5. Could gene expression (regulatory genes of growth, steroidgenesis, oocyte maturation) be manipulated before transplantation (or after) to achieve better results or to treat specific abnormalities of the germ cell line.80 All these questions and concerns about ovarian autotransplantation in the human need to be addressed in preclinical, animal studies. Better cryopreservation methods of human ovarian tissue and oocytes must be sought. Only then should clinical studies be considered. Appropriate discussions not only at the scientific but also at the ethical level are mandatory to establish whether these techniques will benefit reproductive and gynecological medicine patients.78,80 CHEMOTHERAPY and GnRH-a CO-TREATMENT The possibility of administering an adjuvant treatment that might limit the gonadal damage caused by an otherwise successful treatment program is attractive.4,5,94 Glode et al95 tested this hypothesis using a murine model and concluded that an agonistic analogue of GnRH appeared to protect male mice from the gonadal damage normally produced by cyclophosphamide. It may be that decreased secretion of the pituitary gonadotropin, by decreasing gonadal function, could protect against the sterilizing effects of chemotherapy. Although previous suggestions have been made96,97 claiming that primordial germ cells fare better than germ cells that are part of an active cell cycle, this hypothesis has not been seriously tested clinically, until recently.1,98–100 Whereas several investigators have demonstrated that GnRH-a inhibit chemotherapy-induced ovarian follicular depletion in the rat,95–98 uncertainty remains about human application.1,98–100 The human ovary has lower concentrations of ovarian GnRH-receptors and may not necessarily exhibit the same response as rats.1,98–99 Ataya et al98 have found that GnRH-a protected the ovary against cyclophosphamide-induced damage in Rhesus monkeys by significantly decreasing the total amount of follicle loss during the chemotherapeutic insult, and by decreasing the daily rate of follicular decline. Chapman et al10 have found that of their female patients treated for Hodgkin disease (HD), 69 percent developed POF if they were younger than 29, while 96 percent developed POF if their age was more than 30 years. Advances in the treatment of all stages of Hodgkin and non-Hodgkin lymphoma with chemotherapy and irradiation have led to a long-term survival 90 percent or even more in several groups of patients.1,15,16,100–102 The improved long-term survival of relatively young patients treated for lymphoma, focused attention to the gonadal toxicity of the combined chemo- and radiotherapy Whereas 86 percent of men had azoospermia after the COPP/ABVD regimen,101 only 48–77 percent of women receiving chemotherapy for lymphoma exhibited hypergonadotropic amenorrhea and ovarian failure.101–103 Moreover, a long-term follow-up of 240 children, 15 years of age or younger, treated by MOPP for

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Hodgkin disease showed azoospermia in 83 percent of the boys, whereas only 13 percent of the girls suffered ovarian failure.99–104 The chances of maintaining gonadal function following combined modality treatment are significantly greater among girls than boys.99– 104 At variance with the results reported in adults, the MOPP chemotherapy in girls with Hodgkin disease did not induce ovarian failure.105 Since ovarian function was preserved in most long-term survivors who were treated prepubertally for lymphoma,104–106 but only in a minority of similarly treated adult patients,101 it is clinically logical and therefore tempting to temporarily create a prepubertal milieu in women in the reproductive age before and during the chemotherapeutic insult.1,99,100,102 It has been reported that 64 percent of adult female patients undergoing cancer therapy experienced one or more of the symptoms of ovarian failure.15,107 Whereas previous studies15,59108 suggested profound gonadal toxicity in men after adjuvant chemotherapy in patients with and without GnRH-a protection, for either malignant lymphoma59 or germcell tumors,108 the situation in females may be completely different. Ataya et al98 have shown, in female Rhesus monkeys, that GnRH-a may protect the ovary from cyclophosphamide-induced gonadal damage. Administration of GnRH-a in parallel with cyclophosphamide has significantly decreased the daily rate of follicular decline and the total number of follicles lost during the chemotherapeutic insult, as compared to cyclophosphamide alone (without GnRH-a).98 This preliminary experience in Rhesus monkeys is in keeping with the clinical results whereby only two of the 47 evaluable surviving women in our study group became menopausal after the GnRHa/chemotherapy cotreatment (4.3%) as compared to a 56.5 percent (26/46) rate of POF in the chemotherapy alone (control) group1,99,102 Table 47.1).

Table 47.1: Comparison of clinical data and the rate of premature ovarian failure (POF) in the two groups of young women undergoing che motherapy with or without GnRH-agonist cotreatment GnRH/ chemotherapy Chemotherapy P υalue Patients (total) 55 Evaluable patients 50 Hodgkin disease 33/55(60%) Non-Hodgkin lymphoma 22/51(40%) Age 15–40 Radiotherapy 33/55(60%) Radiation dose (cGy) 2320±1521 Pregnancies 18 in 12 women Age of pregnant women at chemotherapy (years) 18–33 Cyclic ovarian function 47/50(94%) POF 3/50(6%)

55 50 33/55(60%) 22/55(40%) 14–40 32/55(58%) 1882±1993 13 in 8 women 16–24 22/50(44%) 28/50(56%)

NS NS NS NS NS NS NS NS NS* <0.01 <0.01

This preliminary experience is encouraging. Whereas most of the survivors of the chemotherapy (with or without radiotherapy) who received the GnRH-a co-treatment resumed ovulatory menses (>94%, 47/50), only 22 of the 50 women (44%) who were treated with chemotherapy with or without mantle irradiation (control group) had normal

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ovarian function and more than half of these women (28/50) had premature ovarian failure (POF) and hypergonadotropic amenorrhea.1,99 Buserelin, another GnRH-agonistic analogue, administered by others94 in a small group of young male patients in parallel to chemotherapy with or without irradiation, failed to protect from azoospermia associated with chemotherapy. As opposed to young girls, most prepubertal boys receiving chemoand radiotherapy suffered azoospermia, therefore there is little rationale to expect a significant benefit from the GnRH-a cotreatment in men.99,104–106,109 In keeping with this hypothesis, Johnson et al59 concluded that no improvement in post-treatment fertility could be demonstrated by GnRH-a cotreatment in six men receiving chemotherapy for advanced lymphomas. Contrary to the apparent protective effect of GnRH-a cotreatment with chemotherapy, no protection from ovarian damage caused by irradiation to rats could be provided by the GnRH agonist.110 Neither the age, nor the dosages of the various cytotoxic drugs were significantly different between the study and control groups.99,102 The only significant difference between the two groups was the incidence of POF and hypergonadotropic amenorrhea (56% vs. 6%; P<0.01, Table 47.1).1,2,99–102 However, one should be very cautious about drawing long-term conclusions from these promising but still preliminary data, since our study was neither randomized nor double blind, and the follow-up in the study group was only 8 years as compared to up to 11 years in some of the patients in the control group.1,99,102 This difference is attributed to the nature of the control group (retrospective historical control), whereas the study protocol was a prospective one.1,99,102 Although the follow-up in the control group is longer than in the study group, the observations within the first three years in both groups suggest that longer follow-up will not significantly affect these results.99,102 Future prospective, double blind, randomized studies will be needed to unequivocally resolve this problem, although some ethical problems may be raised upon initiation of such studies. Notwithstanding all the above, two recent studies in the New England Journal of Medicine111–112 report that observational studies give results similar to randomized controlled trials. They “found little evidence that estimates of treatment effects in observational studies reported after 1984 are either consistently larger than or quantitatively different from those obtained in randomized, controlled trials”.111 Moreover, “the results of well-designed observational studied, do not systematically overestimate the magnitude of the effects of treatment as compared with those in randomized, controlled trials on the same topics.112 Few of our young patients, two in the study group, and several in the control group, who later underwent high dose chemotherapy and autologous bone marrow transplantation due to recurrence of the disease, have turned prematurely menopausal. This is in keeping with recent experience that such intensif ication regimens carry a high risk of permanent infertility and POF 58,61,66,99,113 It has been well established that chemotherapy with total body irradiation followed by allogeneic or autologous bone marrow transplantation causes permanent elevation of gonadotropin levels, hypoestrogenism, and amenorrhea in 92 to 100 percent of female patients.1,58,61,63,92 Future endeavors are obviously needed to challenge the long term infertility problem of young women treated with chemotherapy

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Future endeavors may also use GnRH-antagonists instead of, or in combination with, agonists for achievement of a faster pituitary-ovarian desensitization, eliminating the waiting period of 7–14 days needed by the GnRH-a to achieve down-regulation.1,99,100,114 What are the Reasons for Discrepancy between Various Studies? While it is convenient to perform preliminary and mating studies in rodents, due to their availability and low cost rat oocytes may respond to the GnRH-a/chemotherapy cotreatment protocol differently than human or non-human primates.1,6 An oestrous cycle duration is 4 days in rats versus 28 days menstrual cycle in women or Rhesus monkeys. Moreover, rats have oestrous cycles without endometrial shedding whereas women and monkeys menstruate by shedding the endometrial lining.1,6 Lastly, rat ovaries have been shown to contain GnRH receptors whereas the existence and presence of these receptors in human ovaries is equivocal.1,6 Except for one report,108 most investigators conclude that GnRH-receptors are not present in the human ovary6,116,117 Similar to the human, but different from rodents, the Rhesus monkey ovaries also lack GnRH receptors.1,6,98 Another point of possible concern, is the different length of waiting period between starting the GnRH-a and the administration of the gonadotoxic chemotherapy. In the clinical perspective, the issue of the waiting period required to establish the pituitarygonadal suppression becomes critical in light of the obvious pressure to start chemotherapy as soon as possible after the diagnosis has been made. Indeed, before pituitary-ovarian desensitization, a “flare-up” period of one to two weeks occurs during which the stimulated ovaries may be more vulnerable to the gonadotoxic effect of chemotherapy possibly due to increased gonadotropic and follicular stimulation.1,6,98,102 In order to minimize this possible shortcomings of GnRH-a “flare-up”, future endeavors should prefer the use of GnRH-antagonists rather than agonists (or their combination) for similar purposes, at least for the starting period. The discrepancy between the results of different clinical studies appears to be influenced by the inadequate time interval between the GnRH-a administration and the intiation of chemotherapy, since a short interval may possibly render the gonad hypersensitive to the cytotoxic effect of chemotherapy.1,98,99,102 Another problem accounting for the divergence among the results of different studies, may be the inadequacy of the used agonist dosage, and possibly also interspecies differences.1,6,98,102 INHIBIN MEASUREMENTS We have reported1,100,102 that temporary increased FSH concentrations were experienced in about a quarter of the young women who ultimately resumed ovarian cyclic function suggesting a reversible ovarian damage in a proportion of women in addition to those experiencing POF. Inhibin is a dimer of two subunits designated a and p, of which the latter appears in several forms: βA and βB, (and most recently also βC, βD, and βE in non-human species).100,118,120 The dimers are thus termed inhibin A and B, respectively.118,119 The α-β dimers, in contrast to the free a subunit or β-β subunits, are biologically active in suppressing FSH secretion.118

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Intensive basic research has been undertaken to characterize the biological activities of the 30 to 32 KD inhibin A. In addition, a number of immunoassays have been developed, including two site methods to specifically measure the 30–32 KD inhibin A dimer in biological fluids.118–120 Dimeric inhibinAspecific assays were found to detect bioactive inhibin forms in human follicular fluid and in serum.118 Inhibin-A concentrations in the sera of these patients were, therefore, measured before, during, and following the gonadotoxic chemotherapy100 Inhibin-A immunoactivity was measured by commercial ELISA kits (Serotec) employing the method of Groome and O’Brien.121 Longitudinal individual follow-up measurements of inhibin-A immuno-concentrations in the study group patients have shown a decrease during chemotherapy and GnRH-a with a subsequent increase to normal levels, within 1–2 months after the completion of chemotherapy/ GnRH-a protocol, in all the treated patients, except in the older patient who developed POF. The mean±SE inhibin-A concentrations were 40.73±10 pg/mL before starting the GnRH-a/chemotherapy treatment, decreasing to 4.77±1.4 and 1.83±0.5 pg/mL at 1–3 and 4–6 months of the treatment protocol, respectively.100 The mean (±SE) inhibin-A concentrations increased to 26.5±20 pg/mL at 2 months after the protocol, 96.4±47.6 at 6 months, 69.4±17 at one year, and 177±134.7 pg/mL at 2 years after treatment.100 The high variation at 2 years was generated by two pregnant women in the study group.100 The mean inhibin-A concentrations in the patients who developed POF after chemotherapy was below 4 pg/mL, compared to 300±200 pg/mL in women undergoing hMG/hCG superovulation, and 170±05 in those patients who spontaneously conceived after the GnRH-a/chemotherapy cotreatment protocol.100 Inhibin is secreted by the ovarian granulosa cells in the female and by testicular Sertoli cells in the male. The fully processed form of inhibin has a molecular weight of approximately 32 KD. Higher molecular weight forms, with precursor forms of the a subunit, also occur in follicularfluid and serum.121–127 In addition, free a subunit forms, unassociated with a b subunit, and lacking inhibin bioactivity, are also present.125 Until the recent development of Serotec’s Inhibin-A Assay by Nigel Groome,124–128 it was not possible to accurately distinguish between circulating functional dimeric inhibin and free a subunit in the normal human menstrual cycle, as the previously widely used Monash RIA128–132 was unable to make this distinction.111–128 Immunoreactive inhibin was undetected during perimenopausal cycles in which FSH concentrations were elevated, yet it was within the normal range during cycles with normal FSH levels.132 Moreover, in cross-sectional studies of women approaching menopause, immuno-reactive (ir)-inhibin decreased progressively in both serum and follicular fluid.131–133 We thought, therefore, that measuring the immunoactive concentrations of inhibin-A and B, reflecting the function and reserve of ovarian granulosa cells, may possibly add to our attempts at predicting the probability for the return of cyclic ovarian function in young women after the chemotherapeutic gonadotoxic insult. Not unexpectedly, the levels of inhibin-A and -B were low, compatible with menopausal levels in those women who experienced hypergonadotropic amenorrhea, whereas those who resumed cyclic ovarian function had ir-inhibin-A and-B concentrations within normal levels.1,100 Immunoreactive-inhibin-A and -B concentrations decreased to menopausal levels during the chemotherapy/GnRH-a cotreatment, increasing thereafter in parallel with

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returning ovarian cyclic activity In those who spontaneously conceived, the ir-inhibin-A and -B levels were higher than in those who did not. However, due to the small number of the pregnancies (fifteen) and since most of those who did not conceive were young unmarried women who were not interested in fertility, it is premature to draw any conclusion, at this time, whether the level of ir-inhibin-A and -B after chemotherapy is predictive of future fertility Obviously, further experience is needed to resolve this clinically practical question. Whereas 15 pregnancies spontaneously occurred in nine patents in the treatment group (receiving GnRH-a in parallel to chemotherapy), 12 pregnancies also spontaneously occurred in seven women in the control group (treated by chemotherapy without GnRHa). However, whereas all the patients who conceived in the control group were younger than 21 (between 16–21 years at chemotherapy), those who spontaneously conceived in the treatment group were between 18 and 30 years old at the GnRH-a/chemotherapy cotreatment. Thus, we believe the GnRH-a adjuvant treatment may possibly extend the “ovarian rescue”, and fertile period by almost ten years, or possibly even more. Inhibin-A, and possibly, also -B, immunoactivity as measured by the recently developed ELISA may serve as an additional means for the evaluation of ovarian activity, besides estrogen, progesterone and gonadotropins. It is premature to conclude whether the inhibins (A and B) role as markers is unequivocally superior to the traditional markers of ovarian function (FSH, LH, and sex steroids). Moreover, the decrease of inhibins concentration during treatment may be attributed to either GnRH-a treatment, or to the gonadotoxicity of chemotherapy, or possibly to both. The exact mechanism for the GnRH-a/chemotherapy-associated inhibin decrease needs further investigation.1,102,129–131 Future endeavors are obviously needed for long term assessment of the positive and negative predictive value of these gonadal peptides (inhibins and activins) in young women undergoing gonadotoxic chemotherapy for various indications (lymphoma, leukemia, breast cancer, SLE, organ transplantation, etc.). Future Endeavors If the protective effect observed in our preliminary study of GnRH-a and chemotherapy on future ovarian function is confirmed in larger and prospective randomized studies, it may become mandatory to use this cotreatment protocol in every woman undergoing chemotherapy. Thus, ovarian protection may enable the preservation of future fertility in survivors and prevent the bone demineralization and osteoporosis associated with hypoestrogenism and ovarian failure.1,2,6,99,101,114,116 This GnRH-a cotreatment may be also applied to young women receiving cytotoxic chemotherapy for non-cancerous, benign diseases. Since almost one quarter of young women with systemic lupus erythematosus in the reproductive age may develop premature ovarian failure after cyclophosphamide pulse therapy,6,134,135 the GnRH-a cotreatment may possibly be offered to these young women in parallel with the cytotoxic treatment, as well as to any woman with an autoimmune disorder treated by gonadotoxic chemotherapy, mainly alkylating agents.1,2,135 Until now, women undergoing aggressive cytotoxic therapy such as that experienced in BMT, could preserve their future fertility potential only by undergoing egg-retrieval and IVF with embryo cryopreservation for future thawing and embryo transfer after

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several years of no evidence of the malignant disease.1,81,83 However this assisted reproductive technology (ART) is pertinent to female patients who have a partner (husband, or a spouse who is the selected father of their future children). Due to the high prevalence of lymphoma and leukemia in young ages, many of these young women may be single. Unfortunately, although the technology of sperm cryopreservation is widely utilized and successful, the technology for cryopreservation of unfertilized ova is not yet successful. It may be possible to freeze unfertilized eggs, but upon thawing the fertilizability is very low, and therefore unpractical for clinical use at present. Although intense investigational efforts are being conducted in several medical centers,1,77,78,81,83,87,93 it may take at least a few years before such technology is clinically available. Furthermore, retrieving an egg in a natural, unstimulated cycle, may generate only one or two metaphase II oocytes. To increase the yield of egg retrieval by follicular aspiration, controlled ovarian hyperstimulation (COH) by hMG/hCG, with or without GnRH-a cotreatment needs to be experienced, as usually practiced in IVF programs.136 This may further postpone the initiation of cytotoxic chemotherapy for another two weeks or more and may be relatively contraindicated in breast cancer or other sex hormone sensitive tumors. Due to these short-comings the possibility of ART by IVF combined with embryo cryopreservation for future embryo transfer, may not be applicable to all the young women with malignant diseases who have not consumed their fertility potential. Therefore, for all these young patients, the GnRH-a cotreatment, and possibly in the future antagonists as well, in parallel with the gonadotoxic chemotherapy may offer an increased chance of preserving their unconsumed fertility potential. This practical option may be widely experienced clinically, until the technology of cryopreservation of immature prophase I or unfertilized, metaphase II oocytes is available for young women undergoing gonadotoxic chemotherapy for various clinical indication.77,78,81,83 The use of GnRH antagonists instead of or in combination with GnRH-agonists awaits future clinical testing. Recently, Morita et al,137 have identified several molecules that are required for chemotherapy-induced oocyte apoptosis. While much of their work has relied on gene knockout mice, they have identified a small lipid antagonist of the pro-apoptotic second messenger ceramide, termed sphingosine-1-phosphate (S-1-P), as a potent protective molecule in vitro. They have also found that in vivo S-1-P pre-treatment, in mice, resulted in a dramatic dose-dependent protection of oocytes following radiotherapy.138 Whether the GnRH-a adjuvant cotreatment positive effect is direct or possibly associated with an intraovarian increase in S-1-P is a question of tremendous scientific interest and clinical impact. It obviously awaits further investigation. Since most of the methods involving ovarian or egg cryopreservation are not yet clinically established and successful, one should be very careful in providing these young patients with all the information concerning the various modalities to minimize gonadal damage and preserve ovarian activity and future fertility. Furthermore, combining the various modalities for a specific patient may increase the odds of preservation of future fertility. There is no contraindication to ovarian biopsy for cryopreservation combined with GnRH-a administration and follicular aspiration for IVF and embryo freezing where the patient has a spouse/partner. In cases where the chemotherapy has caused POF, as is frequently the case in bone marrow transplantation, the patient has cryopreserved

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primordial follicles, and/or frozen embryos, to fall back upon. However, in cases where conventional chemotherapy regimens such as commonly used for young lymphoma patients are applied, the GnRH-a cotreatment may preserve ovarian function without necessitating the usage of cryopreserved ova or embryos. Of course, we hope that in the future many of the unanswered questions may receive appropriate scientific answers. Until then, let us be very cautious in supplying our patients with all the possibly relevant information. Holding back the whole information may violate the ancient dictum “primum non nocere.” Acknowledgement The help of Prof JM Rowe, Dr I Avivi, and the staff of the hematology institute and that of Mrs. Ruth Blumenfeld, Batia Navar, Ruth Tal and Dr M Ritter, is thankfully acknowledged. REFERENCES

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38. Averette HE, Boyce GM, Girl MA. Effects of cancer chemotherapy on gonadal function and reproductive capacity. CA 1990; 40:199–209. 39. Plowchalk DR, Mattison DR. Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide. Toxicol Appl Pharmacol 1991; 107:472–81. 40. Santoro A, Bonadonna G, Valagussa P, et al. Long-term results of combined chemotherapyradiotherapy approach in Hodgkin’s disease: Superiority of ABVD plus radiotherapy versus MOPP plus radiotherapy. J Clin Oncol 1987; 5:27–37. 41. Cobleigh MA, Bines J, Harris D, et al. Amenorrhea following adjuvant chemotherapy for breast cancer. Proc Am Soc Clin Oncol 1995; 14:A158 (abstr). 42. Shenkenberg TD, Von Hoff DD. Possible mitoxantrone-induced amenorrhea. Cancer Treat Rep 1986; 70:659–61. 43. Shamberger RC, Rosenberg SA, Seipp CA, et al. Effects of high-dose methotrexate and vincristine on ovarian and testicular functions in patients undergoing postoperative adjuvant treatment of ostosarcoma. Cancer Treat Rep 1981; 65:739–46. 44. Koyama H, Wada T, Nishizawa Y, et al. Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer. Cancer 1977; 39:1403–9. 45. Fisher B, Sherman B, Rockette H, et al. L-phenylalanine mustard (L-PAM) in the management of premenopausal patients with primary breast cancer: lack of association of disease-free survival with depression of ovarian function. Cancer 1979; 44:847–57. 46. Cobleigh MA, Bines J, Lincoln ST, et al. Amenorrhea following adjuvant chemotherapy for breast cancer. Proc Am Soc Clin Oncol 1994; 13:A55 (abstr). 47. Miller III JJ, Williams GF, Leissring JC. Multiple late complications of therapy with cyclophosphamide, including ovarian destruction. Am J Med 1971; 50:530–5. 48. Damewood MD, Grochow LB. Prospects for fertility after chemotherapy or radiation for neoplastic disease. Fertil Steril 1986; 45:443–59. 49. Padmanabhan N, Wang DY, Moore JW, et al. Ovarian function and adjuvant chemotherapy for early breast cancer. Eur J Clin Oncol 1987; 23:745–8. 50. Goldhirsch A, Gelber RD, Castiglione M. The magnitude of endocrine of adjuvant chemotherapy for premenopausal breast cancer patients. Ann Oncol 1990; 1:183–88. 51. MoliterniA, Bonadonna G, Valagussa P, et al. Cyclophosphamide, methotrexate and fluorouracil with and without doborubicin in the adjuvant treatment of resectable breast cancer with one to three positive axillary nodes. J Clin Oncol 1991; 9:1124–30. 52. Tancini G, Valagussa P, Bajetta E, et al. Preliminary 3-year results of 12 versus 6 cycles of surgical adjuvant CMF in premenopausal breast cancer. Cancer Clin Trials 1979; 2:285–92. 53. BiancoAR, Del Mastro L, Gallo C, et al. Prognostic role of amenorrhea induced by adjuvant chemotherapy in premenopausal patients with early breast cancer. Br J Cancer 1991; 63:799– 803. 54. Reyno LM, Levine MN, Skingley P, et al. Chemotherapy induced amenorrhea in a randomized trial of adjuvant chemotherapy duration in breast cancer. Eur J Cancer 1993; 29A:21–23. 55. Levine MN, Gent M, Hryniuk WM. A randomized trial comparing 12 weeks versus 36 weeks of adjuvant chemotherapy in stage II breast cancer. J Clin Oncol 1990; 8:1217–25. 56. Wood WC, Budman DR, Korzun AH, et al. Dose and dose intensity of adjuvant chemotherapy for stage II, node-positive breast carcinoma. N Engl J Med 1994; 330:1253–59. 57. Brincker H, Rose C, Rank F, et al. Evidence of castration-mediated effect of adjuvant cytotoxic chemotherapy in premenopausal breast cancer. J Clin Oncol 1987; 5:1771–78. 58. Muller U, Stahel RA. Gonadal function after MACOP-B or VACOP-B with or without dose intensification and ABMT in young patients with aggressive non-Hodgkin lymphoma. Ann Oncol 1993; 4:399–402. 59. Johnson DH, Linde R, Hainsworth JD, et al. Effect of a luteinizing hormone releasing hormone agonist given during combination chemotherapy on post-therapy fertility in male patients with lymphoma: preliminary observations. Blood 1985; 65:832–6.

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60. Sutcliffe SB. Cytotoxic chemotherapy and gonadal function in patients with Hodgkin’s disease. JAMA 1979; 242:1898,1901. 61. Nademanee A, Schmidt GM, O’Donnell MR, et al. High dose chemoradiotherapy followed by autologous bone marrow transplantation as a consolidation therapy during first complete remission in adult patients with poor-risk aggressive lymphoma: Apilot study. Blood 1992; 80:1130–34. 62. Carey PJ, Proctor SJ, Hamilton PJ. Autologous bone marrow transplantation for high grade lymphoid malignancy using melphalan/irradiation conditioning without bone marrow purging or cryopreservation. Blood 1991; 77:1593–99. 63. Sanders J, Buckner CD, Leonard JM, et al. Late effects on gonadal function of cyclophosphamide, total-body irradiation, and marrow transplantation. Transplantation 1983; 36:252–55. 64. Keilholtz U, Korbling M, Fehrentz D, et al. Long-term endocrine toxicity of myeloablative treatment followed by autologous bone marrow/blood derived stem cell transplantation in patients with malignant lymphohematopoietic disorders. Cancer 1989; 64:641–45. 65. Meirow D, Schenker JG. Cryopreservation and transplantation of ovarian tissue: a mode of preserving female fertility. Harefuah 1998; 134:461–64. 66. Sanders JE, Buckner CD, Amos D, et al. Ovarian function following marrow transplantation for aplastic anaemia or leukaemia. J Clin Oncol 1988; 6:813–17. 67. Carroll J, Wood MJ, Whittingham DG. Normal fertilization and development of frozen thawed mouse oocytes: protective action of certain macromolecules. Biol Reprod 1993; 48:606–12. 68. Bos-Mikkich A, Wood MJ, Candy CJ, Whittingham DG. Cytogenetic analysis and developmental potential of vitrified mouse oocytes. Biol Reprod 1995; 53:780–85. 69. Parks JE, Ruffing NA. Factors affecting low temperature survival of mammalian oocytes. Technology 1992; 37:59–72. 70. Trounson AO, Bongso A. Fertilization and development in humans. Curr Topics Dev Biol 1996; 32:59–101. 71. Vincent C, Johnson MH. Cooling, cryoprotectants and the cytoskeleton of the mammalian oocyte. Oxford Rev Reprod Biol 1992; 14:72–100. 72. Barnes FL, KauscheA. Tiglias J, et al. Production of embryos from in vitro matured primary human oocytes. Fertil Steril 1996; 65:1151–56. 73. Apperly JF, Reddy N. Mechanism and management of gonadal failure in recipients of high dose chemoradiotherapy. Blood Rev 1995; 9:93–116. 74. Biskind GR, Kordan B, Biskind MS. Ovary transplanted to spleen in rats: the effect of unilateral castration, pregnancy and subsequent castration. Cancer Res 1950; 10:309–18. 75. Gosden RG, Baird DT, Wade JC, et al. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196°C. Hum Reprod 1994; 9:597–603. 76. Newton H, Aubard Y, Sharma V, et al. The low temperature storage and grafting of human ovarian tissue into SCID mice. Hum Reprod 1996; 11:1487–91. 77. Wade JC, Gosden RG. Assessment of oocyte survival in ovarian cortical grafts after frozen storage and xenografting. In: Schats R, Schoemaker J (Eds). Ovarian endocrinopathies (The 8th Reinier de Graaf Symposium). New York: Parthenon Press, 1994:67–70. 78. Gosden RG. Transplantation of ovaries and testes. In: Edwards RG, ed. Fetal tissue transplants in medicine. Cambridge: Cambridge University Press, 1992:253–79. 79. Shaw JM, Bowels J, Koopman P. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to the graft recipient. Hum Reprod 1996; 11:1668–73. 80. Oehninger S. Will ovarian autotransplantation have a role in reproductive and gynecological medicine? Fertil Steril 1998; 70:20–21. 81. Meirow D, Ben-Yehuda D, Prus D, Poliack A, Schenker JG, Rachmilewitz EA et al. Ovarian tissue banking in patients with Hodgkin’s disease: Is it safe? Fertil Steril 1998; 69:996–98. 82. von Eye Corleta H, Corleta O, Capp E, Edelweiss MI. Subcutaneous autologous ovarian transplantation in Wistar rats maintains hormone secretion. Fertil Steril 1998; 70:16–19.

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83. Moomjy M, Rosenwaks Z. Ovarian tissue cryopreservation: the time is now. Transplantation or in vitro maturation: the time awaits. Fertil Steril 1998; 69:999–1000. 84. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996; 11:1268–72. 85. Gunasena KR, Villines PM, Critser ES, Critser JK. Live births after autologous transplant of cryopreserved mouse ovaries. Hum Reprod 1997; 12:101–6. 86. Dissen GA, Lara HE, Fahenbach WH, Costa ME, Ojeda SR. Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression. Endocrinology 1994; 134:1146–54. 87. Kim SS, Battaglia DE, Soules MR. The future of ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001; 75:10490–56. 88. Kim SS, Gosden RG, Radford JA, Harris M, Jox A, Rutherford AJ. A model to test the safety of human ovarian tissue transplantation after cryopreservation: xenografts of ovarian tissues from cancer patients into NOD/LtSz-Scid mice (abstract #O-003). In: Program and abstracts of the ASRM/CFAS Conjoint Annual Meeting, Toronto, Ontario, Canada, 25–30, 1999. Fertil Steril 1999; 72(suppl):S1. 89. Weissman A, Gotlieb L, Colgan T, Jurisicova A, Grenblatt EM, Casper RF. Preliminary experience with subcutaneous human ovarian cortex transplantation in to the NOD-SCID mouse. Biol Reprod 1999; 60:1462–7. 90. Revel A, Raanani H, Leyland N, Casper R. Human oocytes retrieval from nude mice transplanted with human ovarian cortex. Human Reprod 2000; 15:13. 91. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results Hum Reprod 1986; 1:81–7. 92. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen thawed ovarian cortex in sheep. Hum Reprod 1999; 14:2149– 54. 93. Aubard Y, Poirot C, Piver P, Galinat S, Teissier MP Are there indications for ovarian tissue cryopreservation? Fertil Steril 2001; 76:414–15. 94. Waxman JH, Ahmed R, Smith D, et al. Failure to preserve fertility in patients with Hodgkin disease. Cancer Chemother Pharmacol 1987; 19:159–62. 95. Glode LM, Robinson J, Gould SF. Protection from cyclophosphamide induced testicular damage with an analogue of gonadotropin-releasing hormone. Lancet 1981; 1:1132–34. 96. Chapman RM, Sutcliffe S. The effects of chemotherapy and radiotherapy on fertility and their prevention. Recent Adv. Clin Oncol 1986; 2:239–51. 97. Krepart GV, Lotocki RJ. Chemotherapy during pregnancy. In: Allen HH, Nisker JA, eds. Cancer in pregnancy, therapeutic guidelines. Mount Kisco, NY: Futura Publishing, 1986:69–88. 98. Ataya K, Rao LV, Laurence E, Kimmel R. Luteinizing hormone-releasng hormone agonist inhibits cyclophosphamide induced ovarian follicular depletion in Rhesus monkeys. Biol Reprod 1995; 86–92. 99. Blumenfeld Z, Avivi I, Linn S, Epelbaum R, Ben-Shahar M, Haim N. Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotropinreleasing hormone agonist in parallel to chemotherapy. Human Reprod 1996; 11:1620–26. 100. Blumenfeld Z, Ritter M, Shariki K, Haim N. Inhibin-A concentrations in sera of young women during and following chemotherapy for lymphoma—correlation with ovarian toxicity. Presented at the 44th annual meeting of the Society for Gynecologic Investigation, San-Diego, CA, March 20–22, 1997. J Soc Gyn Investig (Suppl.). Am J Reprod Immunol 1998; 39:33–40. 101. Kreuser ED, Felsenberg D, Behles C, et al. Long-term gonadal dysfunction and its impact on bone mineralization in patients following COPP/ABVD chemotherapy for Hodgkin’s disease. Ann Oncol 1992; 3(Suppl 4):S105–10. 102. Longo DL. The use of chemotherapy in the treatment of Hodgkin’s disease. Semin Oncol 1990; 17:716–35.

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103. Ratcliffe MA, Lanham SA, Reld DM, Dawson AA. Bone mineral density (BMD) in patients with lymphoma: the effects of chemotherapy, intermittent corticosteroids and premature menopause. Hematol Oncol 1992; 10:181–87. 104. Ortin TT, Shoshlak CA, Donaldson SS. Gonadal status and reproductive function following treatment for Hodgkin’s disease in childhood: the Stanford experience. Int J Radiol Oncol Biol Phys 1990; 19:873–80. 105. Backshine H, Brauner R, Thibaud E, et al. Chemotherapy and ovarian function. Retrospective analysis in 17 girls treated for malignant tumor of hematologic disease. Arch Fr Pediatr 1986; 43:611–16. 106. Wallace WH, Shalet SM, Tellow LJ, Morrris-Jones PH. Ovarian function following the treatment of childhood acute lymphoblastic leukemia. Med Pediatr Oncol 1993; 21:333–39. 107. Kreuser ED, Hetzel WD, Billia DO, Thiel E. Gonadal toxicity following cancer therapy in adults: Significance, diagnosis, prevention and treatment. Cancer Treat Rev 1990; 17:169–75. 108. Krause W, Pfluger KH. Treatment with the gonadotropin-releasing hormone agonist buserelin to protect spermatogenesis against cytotoxic treatment in young men. Andrologia 1989; 21:265– 70. 109. Byrne J, Mulvihill JJ, Myers MH, Connelly RR, Naughton MD, Krauss MR, et al. Effects of treatment on fertility in long-term survivors of childhood or adolescent cancer. N Engl J Med 1987; 17:1315–21. 110. Jarrell JF, McMahon A, Barr RD, Young Lai EV. The agonist (d-leu-6, des-gly-10)— LHRH—ethylamide does not protect the fecundity of rats exposed to high dose unilateral ovarian irradiation. Reprod Toxicol 1991; 5:385–88. 111. Benson K, Hartz AJ. A comparison of observational studies and randomized, controlled trials. N Engl J Med 2000; 342:1878–86. 112. Concato J, Shah N, Horwitz RI. Randomized, controlled trials, observational studies and the hierarchy of research designs. N Engl J Med 2000; 342:1887–92. 113. Hinterberger-Fischer M, Kier P, Kalhs P, et al. Fertility, pregnancies and offspring complications after bone marrow transplantation. Bone Marrow Transplantation 1991; 7:5–9. 114. Linde R, Doelle GC, Alexander N, Kirchner F, Vale W, Rivier J, Rabin D. Reversible inhibition of testicular steroidogenesis and spermatogenesis by a potent gonadotropin-releasing hormone agonist in normal men. N Engl J Med 1981; 305:663–68. 115. Bramley T, Menzies G, Baird D. Specific binding of gonadotropin-releasing hormone and an agonist to human corpus luteum homogenate: Characterization properties and luteal phase levels. J Clin Endocrinol Metab 1985; 61:834–40. 116. McLachlan R, Healy D, Burger H. Clinical aspects of LHRH analogues in gynecology: A review. Brit J Obstet Gynecol 1986; 93:431–54. 117. Clayton R, Huhtaniemi I. Absence of gonadotropin releasing hormone receptors in human gonadal tissue. Nature 1982; 299:56–59. 118. Robertson D, Burger HG, Sullivan J, Cahir N, Groome N, Poncelet E, et al. Biological and immunological characterisation of inhibin forms in human plasma. J Clin Endocrinol Metab 1996; 81:601–76. 119. Burger HG. Inhibin. Reprod Med Rev 1992; 1:1–20. 120. Porcelet E, Franchimont P. Two site enzymo-immunoassays of inhibin. Ares-Serono Symposia Series—Frontiers in Endocrinology 1994; 3:45–54. 121. Groome NP, O’Brien M. Two-site immunoassays for inhibin and its subunits. Further applications of synthetic peptide approach. J Immunol Methods 1993; 165:167–76. 122. Groome NP, Illingworth PJ, O’Brien M, Cooke I, Ganesan TS, Baird DR, McNeilly A. Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassys. Clin Endocrinol 1994; 40:717–23. 123. Lambert-Messer; ian G, Hall JE, Sluss P, Taylor AL, Martin KA, Groome NP, Crowley WF, Schneyer A. Relatively low levels of dimeric inhibin circulate in men and women. J Clin Endocrinol Metab 1994; 79:45–50.

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124. Muttukrishna S, Fowler PA, Groome NP, Mitchell GC, Robertson WR, Knight PG. Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotropin. Human Reprod 1994; 9:1634–42. 125. Groome NP, Illingworth PJ, O’Brien M, Priddle J, Weaver K, McNeilly AS. Quantification of Inhibin Pro-aC-containing forms in human serum by a new ultra sensitive two-site enzymelinked immunosorbent assay. J Clin Endocrinol Metab 1995; 80:2926–32. 126. Groome NP, Lawrence M. Preparation of monoclonal antibodies reactive with the beta-A subunit human ovarian inhibin. Hybridoma 1991; 10:309–16. 127. Groome NP, et al. Monoclonal and polyclonal antibodies reactive with the 1–32 amino terminal peptide of 32kD human ovarian inhibin. Hybridoma 1990; 9:31–42. 128. Knight PG, Muttukrishna S, Groome NP. Development and application of a two-site enzyme immunoassay for the determination of ‘total’ activin-A concentrations in serum and follicular fluid. J Endocrinol 1996; 148:267–79. 129. McLachlan RI, Robertson DM, de Kretser DM, Burger HG. Advances in the physiology of inhibin and inhibin-related peptides. Clin Endocrinol 1988; 29:77–112. 130. McLachlan RI, Robertson DM, Healy DL, Burger HG, de Kretser DM. circulating immunoreactive inhibin level during the normal menstrual cycle. J Clin Endocrinol Metab 1987; 65:954–61. 131. Woodruff TK, Krummen L, Baly D, Wong WL, Garg S, Sadik M, Davis G, Soules MR, Mather JP. Inhibin and activin-a measured in human serum. In: Inhibin and inhibin-related proteins—frontiers in endocrinology, Vol. 3. Burger HG (Ed). Rome: Ares-Serono Symposia, 1994; 55–68. 132. Hee J, McNaughton J, Bangah M, Burger HG. Premenopausal pattern of gonadotropins, immunoreactive inhibin, estradiol, and progesterone. Maturitas 1993; 18:9–20. 133. Woodruff TK, Mather JP. Inhibin, activin, and the female reproductive axis. Ann Rev Physiol 1995; 57:219–44. 134. Langevitz P, Klein L, Pras M, Many A. The effect of cyclophosphamide pulses on fertility in patients with lupus nephritis. Am J Reprod Immunol 1992; 28:157–8. 135. Blumenfeld Z, Shapiro D, Shteinberg M, Avivi I, Nahir M. Preservation of fertility and ovarian function and minimizing gonadotoxicity in young women with systemic lupus erythematosus treated by chemotherapy. Lupus 2000; 9:1–5. 136. Blumenfeld Z, Barkey RJ, Youdim MBH, Brandes JM, Amit T. Growth hormone—binding protein regulation by estrogen, progesterone, and gonadotropins in human: The effect of ovulation induction with menopausal gonadotropins, GH and gestation. J Clin Endocrinol Metabol 1992; 75:1242–49. 137. Morita X, Paris F, Perez GI, Kolesnick RN, Tilly JL. Protection of the ovary from radiationinduced damage by small molecule therapy in vivo J Soc Gynecol Investig 2000; 7(supplement): abstr. 429, p164 A. 138. Morita Y, Perez GI, Paris F, et al. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat Med 2000; 6:1109–14.

CHAPTER 48 Molecular Biology Applied to ART Silvio Cuneo, Alexandra Bermúdez, Alfredo Góngora, Beatriz Xoconostle, Alfonso Nájar Gutiérrez BACKGROUND During the last years, we are living a revolution in the field of scientific investigation. Such changes have provided new modern tools with high impact for the different scientific disciplines. This is the case of the molecular biology, that lead us to understand complex biological systems, using the knowledge from different areas. In particular, Assisted Reproduction Techniques use genetics, microbiology, cellular biology molecular biology and biochemistry to diagnose and treat the health of the relatives and their future offspring. Historically, the discipline of molecular biology has already advanced and specially with regard to the development of DNA manipulation techniques, the recent human genome sequencing being an example of it biggest contribution to science. With all of these advantages, we are awaiting a new era in different medical fields and specially in reproductive medicine, that will permit the design of new treatments, drugs, and therapies to achieve a better quality of live. INTRODUCTION The Genetic Material The structure of genetic material was described by the Nobel winners Watson and Crick, who found that DNA was formed by a double linear strand constituted of deoxyribonucleotides called: Adenine, Thymine, Guanine and Cytosine. These nitrogen bases form very long polymer, in which are encoded the instructions for protein synthesis. The process of gene expression involves 2 steps: first the messenger RNA synthesis from the strand of DNA. This RNA is then translated to produce a protein. The process is complex and highly regulated. In general, we can say that 3 residues of RNA, denominating “a codon” specify an amino acid a number of which constitute proteins. The Human Genome Project Among an international initiative place the work of deciphering the human genome began. Due to the development of automatic sequencers, the information was obtained from two sources: one proceeding from Celeron Company and the other arose in a Project of many universities from all over the world. The last information is being interpreted and allocated in big data bases. To date, it has been identified that there are numerous

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genes in every chromosome that constitute our genetic heap. There are some examples of deciphered genomes of eukaryotes, including the human. As it is now known the human genome has 2693 millions of pairs of bases! A colossal number to imagine! By this, it is valid to ask: What are we going to learn from knowing the sequence of our genes, and how is it going to impact the medicine of the reproduction? The answer has diverse implications. It is necessary to consider that in our genetics a change in a single base is able to give us susceptibility or protection against diseases. Without trying to make polemics, this information will give us the molecular basis to understand the nature of diseases that affect us, to propose new strategies to its prediction, prevention, diagnosis and treatment. In the present, exist some examples that support the last affirmation. It was described that a mutation by substitution in the gene APOE, is observed to be associated to Alzheimer’s disease or senile insanity. Token of the same way, a deletion of a base in the gene CCR5 is able to confer resistance to the HIV virus, that causes AIDS. There is an interesting analysis of the hereditary diseases and the mutations that are involved by Jimenez and Cols, (2001) who analyzed 923 genetically transmitted diseases, eliminating from the study an inherited forms of cancer and DNA maternal mitochondrial transmitted diseases. The scientists grouped the diseases in base to their nature and to the age of comparison. This analysis

Table 48.1: List of mutations of a single gene detected by PGD Diseases

Techniques

Syndrome of fragile X PCR PKU PCR and digestion Deficiency of OTC PCR Myotonic dystrophy PCR and sequencing Fanconi’s anemia PCR and digestion Achondroplasia PCR and digestion Neurofibromatosis type I PCR and digestion Alzheimer (early phase) PCR and sequencing Cystic Fibrosis PCR and digestion Tay-Sachs PCR and sequencing Hemophilia A and B PCR Pigmented Retinitis PCR and sequencing Thalassemia PCR Alporte disease PCR and sequencing Gaucher’s disease PCR and digestion Deficiency Hidroaxiacil-CoA desH. PCR Phenylketonuria (PKA) PCR

provides a powerful base for the diagnosis of diseases, because it could be use for preimplantation genetic diagnostic (PGD) at an embryonic stage and avoid the transmission of such diseases to the offspring. Obviously that means that the number of the diseases suitable to this diagnosis will increase importantly according to the discovery

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or comparison of new mutations. Considering the number of genes and mutations related to diseases, we are non only simply admiring the tip of a iceberg! DNA based Systemic Diagnosis Of routine manner, it uses the DNA in the investigation of a group of methodologies for the diagnosis of genetic diseases, and in assisted reproductive techniques they have been adapted and modified to the preimplantation genetic diagnostic. This procedure, although routine, requires a special ability, because the quantity of cells available for the diagnosis is a limitation. In general, it is possible to obtain one or two blastomeres extracted from a viable embryo or polar bodies from oocytes. In any case, the DNA quantity available is very low, and also a challenge for the detection of a single gene disorder. When possible to collect more number of cells, as in the case of a biopsy, cells from amniotic fluid, or spermatozoas from ejaculate, the task is more easy, because it is possible to add more steps of purification of the genetic material and improve the subsequent detection of a gene. In the next pages we will describe the principal techniques that we use for molecular diagnosis, trying to focus it in ART when it is possible. DNA Hybridization (Southern) It is necessary to extract and purify the total DNA originating from at least of 500 mg of sample, (biopsy of tissue or 5 ml of amniotic fluid). Subsequently the DNA is digested with enzymes that recognize specific sequences and cut them. These enzymes are denominated as restriction enzymes, and will generate reproducible segments of DNA, that are separated by weight on a trough of a mesh of jelly (agarose) and transferred to a membrane. This membrane containing digested DNA is hybridized with control DNA, previously marked with radioactivity. The bands are observed in films of X-rays where the expected size is analyzed and any change interpreted as a modification on its sequence. Polymerase Chain Reaction (PCR) This technique has wide applications in diverse areas, and has permitted the identification of a single copy of genes present in very small samples, including single cells. The process starts with the warming of a small sample that contains the DNA. With the heat, the double helix of DNA denatures and exposes the nitrogen bases. In a second step, the temperature decreases, allowing the binding of the DNA starters or primers on the gene extreme that is desired to be amplified. The third step consists of the synthesis of the amplified complementary chain using the primers and the open DNA as a model. This cycle is repeated for about 20 to 30 times, in the DNA of a limited size and it is logarithmic synthesized by the primers. If the reaction of a single copy from the gene is optimum, after 30 cycles, it is possible to have a million of copies of the sequence that we want to study At the end of the reaction, the products synthesized are separated in a mesh of jelly submitted to an electrophoresis field and the negative DNA will migrate to the opposite

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pole and it can be visualized by means of the use of a fluorescent stain. Diverse laboratories in the world had utilized this technique with success, but is very important to maintain an elevated quality control to every reagent, to assure that human DNA is free of contamination. The Use of PCR for PGD in Embryos We analyze the status of various genetics markers of morphologically normal blastomeres of arrested human embryos by PCR using a method developed in our molecular biology laboratory. We studied a total of 47 arrested embryos from consent patients of our ART program. Morphologically normal blastomeres were used to amplify by PCR the following genes: ZFX (chromosome X and Y), SOD (chromosome 21) and DCK (chromosome 18). For cell lysis, a buffer containing nonionic detergent and Pronase P was used. Further amplification was performed using 24 mers designed to possess high annealing temperature. PCR

Fig. 48.1 products were obtained using a Robocycler (Stratagen) and then resolved in agarose-gels. A single sharp band of the expected size was obtained in each case. In order to assess gene identity automated DNA sequencing was performed.

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It was observed that morphologically normal blastomeres from arrested embryos had chromosomal alterations varying from 56 percent when 2 oligonucleotides were used to 73 percent when 4 oligonucleotides were tested. The most common chromosomic alteration detected were: aneuploids (41.5%), haploids (7%), polyploids (6%), mosaicism (32.5%). The X/Y relation was 1.28, being 23 male embryos (56.1%) and 18 (43.9%) females. Ahighly reproducible and cost-effective PCR method was established to amplify genes from single cell that permitted the detection of high chromosomic abnormalities in morphologically normal blastomeres of non viable human embryos. Currently we are developing new oligonucleotides for detection of others genetic markers. Fluorescent in-situ Hybridization (FISH) This is a very common technique used in molecular biology, and particularly in ART, is useful for PGD, because it allows the diagnosis of the chromosomal status of single blastomere in a short time. The method consists in denaturing the double strand of DNA to separate them, then hybridized with a probe marked with different color fluorochromes (complementary to be DNA sequence that is in study), and after that read under a fluorescent microscope to check for aneuploidies or translocations.

Fig. 48.2: Example of FISH The use of FISH for Genetic Screening in Spermatozoa There is a growing concern to find out the status of the sperm used for ART, and its implications in the outcome of the offspring. We decided to test the genetic status of sperms from samples of patients with severe altered masculine factor by FISH using our own laboratory synthesized probes. Ten seminal fluid samples, 3 epididymal aspirations (MESA) and 5 testicular biopsies (TESA) from patients undergoing infertility programs treatments were analyzed by FISH to determine chromosomal alterations. A control group of 10 normozoospermic ejaculates was used. In order to analyze human sperm cells by FISH, probes detecting X, Y, 21, and 18 chromosomes were sensitized by PCR using a new designed set of primers. The identity of amplified DNA band was confirmed by sequencing, the purified band was

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then used as template for further in vitro synthesis of fluorescent probes by means of PCR in the presence of dUTP-fluorescein or dUTP-rodamine and the corresponding primer. The label probe was hybridized to previously treated sperms cells using a modified protocol. Fluorescent signals associated with the corresponding gene were then analyzed using an immunofluorescent microscope (Zeisse, Mexico) and then interpreted with the Axiovision Software (Zeisse, Germany). We observed that in normozoospermic patients the aneuploidy index for sex chromosomes is near 2.6 percent and for diploidy is 0.25 percent. On the other hand, that index increases upto 12 and 3 percent respectively in cases of severe oligoasthenoteratozoospermia, in samples from ejaculates or from TESA or MESA. Ahigh aneuploidy and diploidy index were detected in patients with a severe altered masculine factor by FISH using our own laboratory synthesized probes, yielding adequate signals to screening the genetic status of human sperm cells. Currently we are developing new probes for detections of others genetic markers.

Fig. 48.3: FISH in spermatozoa DNA Sequencing This methodology permits us to know the position at which the bases are present in a gene. The DNA needs to be previously purified and after a PCR it can be sequenced. The DNA is denatured and divided into four test tubes. A different nucleotide (A, C, G or T) is added to each tube to start the synthesis of the complementary chain. These special nucleotides have two additional characteristics: are marked with fluorescence and when the nucleotide is incorporated, the synthesis of the chain stops. Finally, the products of synthesis are separate as a function of size and fluorescent bands are detected. Using this technique is possible also to build probes and oligonucleotides to determine genetic markers for PGD.

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Fig. 48.4: Microarrays (Microchips) This methodology has been described like a discovery as important as the PCR. It permits the massive analysis of gene expression and consists of an arrangement in scale of a big number of genes distributed in a well known position. This arrangement takes place directly on a solid support (microchip) and the simultaneous hybridization of RNA is affected from two sources: The first corresponds to every messenger RNA of cells with a normal genotype, stained with red fluorescence. The second source comes from cells whose genotype is desired to be analyze, and is stained with green. Subsequent to the simultaneous hybridization, the color and the intensity of the drops formed in the chip is analyzed. The colors tells us that: if the RNA of the cell to be analyzed is normal, then the color that is observed is the combination of equal quantities of red and green. That combination gives orange. Nevertheless, if a gene in the test cell is extinguished, the red color is predominant. On the contrary, if there is an abnormal gene over expression, we will observe only green points. For example, a trisomy 21 is observed as extra green lights. Adeletion is observed as the presence of red lights, and a translocation is expressed with chip zones as red and green lights. There are now different chips kits available for the diagnosis of more that 300 genetic and metabolic disorders only in one microchip.

Fig. 48.5: Image of a microchip

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Perspectives The techniques of molecular biology have been demonstrated to be rapid and very informative. With the elucidation of the information originating from the analysis the human genome, we will have new tools for the diagnosis of genetic diseases. Nevertheless, the advances in this field are not limited only to the diagnosis, but will also provide a molecular therapy to restore the normal integrity and function of the damaged (Gene Therapy). Experimentally, now these gene therapies are tested by the introduction of the correct gene into the target cells. It will be really exciting to know the new applications and the development of new tools! The only limit will be the creativity of the research team. BIBLIOGRAPHY 1. Bernardini L, Martini E, Geraedts JPM et al. Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol Hum Reprod 1997; 3:431–38. 2. Bickerstaff H, Flinter F, Yeong CT, Braude P. Clinical application of preimplantation genetic diagnosis. Hum Fertil (Camb) 2001; 4(1):24–30. 3. Egozcue J Blanco J, Vidal et al. Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization. Hum Reprod Update 1997; 3:441–52. 4. Fugger EF, Blanck SH, Keyvanfar K et al. Birth of normal daughters after Micro-Sort sperm separation and intrauterine insemination, in vitro fertilization or intracytoplasmic sperm injection. Human Reprod 1998; 13(9):2367–70. 5. Greene PJ, Poonian MS, Nussbaum AL, Tobias L, Garfin DE, Boyer HW et al. Restriction and modification of a cell complementary octanucleotide containing the EcoRi substrate J Mol Biol 1975; 99:237. 6. Guttenbach M, Martinez-Exptfsito MJ, Michelmann HV et al. Incidence of diploid sperm and disomic sperm nuclei in 45 infertile men. Hum Reprod 1997; 12:468–73. 7. Human Genome. http://www.ncbi.nlm.nih.gov/; 2001. 8. Hung MC, Wensink PC. Different restriction enzyme-generated sticky DNA ends can be joined in vitro. Nucleic Acid Res 1984; 12:1863. 9. In’t Veld PA, Brockmans F, de France H et al. Intracytoplasmic sperm injection (ICSI) and chromosomically abnormal spermatozoa. Hum Reprod 1997; 12:752–54. 10. Kogan SC, Doherty M, Gitschier J. An improved method for prenatal duagnosis of genetic diseases by analysis of amplified DNA sequences. Amplifcation to hemophilia AN Engl J Med 1987; 317:985. 11. Magli C, Jones G, Gras L y cols. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocyst in vitro. Hum Reprod 2000; 15:1781–86. 12. Maxam AM y Gilbert W. A new method for sequencing DNA Proc Natl Acad Sci 1977; 74:560. 13. Mullis KF, Faloona S, Scharf R, Saiki G, Horny H Erlich. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 1986; 51:263. 14. Mullis KF, Faloona S. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987; 155:335. 15. Munn S, Alikani M, Tomkin G y cols. Embryo morphology, developmental rates, and maternal age are correlated with chromosomal abnormalities. Fertil Steril 1995; 64:382–91.

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16. Munn S, Magli M, Bahce M y cols. Preimplantation diagnosis of the aneuploidies most commonly found tn spontaneous abortions and live births: X, Y, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn 1998b; 18:1459–66. 17. NicitoA, Kononen J, Kallioniemi OP, Sauter G. Tissue microarrays (TMAS) for highthroughput molecular pathology research. Int J Cancer 2001; 94(1):1–5. 18. Pellicr A, Rubio C, Vidal F y cols. In vitro fertilization plus preimplantation genetic diagnosis in patients with recurrent miscarriage: an analysis of chromosome abnormalities in human preimplantation embryos. Fertil Steril 1999; 71:1033–39. 19. Rubio C, Sim¢n C, Blanco J et al. Implications of sperm chromosome abnormalities in recurrent miscarriage. J ssit Reprod Genet 1999; 16:253–58. 20. Sambrook, J, Fritsch EF y Maniatis T. Molecular cloning. A laboratory manual 1989. Cold Spring Harbor Press. 21. Schrck E, du Manoir S, Veldman t y cols. Multicolor spectral karyotiping of human chromosomes. Science 1996; 273:494–97. 22. Southern, EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98:503. 23. Vandervors M, Staessen C, Sermon K, De Vos A, Van de Velde H, Van Assche E et al. The Brussels’ experience of more than 5 years of clinical preimplantation genetic diagnosis. Hum Reprod Update 2000; 6(4):364–73. 24. Verlinsky et al. Single-cell DNA analysis of polar bodies and blastomeres in: An Atlas of preimplantation genetic diagnosis. Paternon Publishing 2000. 25. Verlinsky Y, Kuliev A. Preimplantation diagnosis of common aneuploidies in fertile couples of advanced maternal age. Hum Reprod 1996; 11:2076–77. 26. Vidal F, Fugger EF, Blanco et al. Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X-or Y-chromosomes haplotyipes: a bind trial assessed by double and triple colour fluorescent in-situ hybridization. Hum Reprod 1998; 13:308–12. 27. http://www.geneclinics.org/, Gene Tests-Gene Clinics; 2001. 28. Xoconostle-C zares B, Lozoya-Gloria E, Herrera-Estrella L. Gene Cloning and Identification in: Plant Breeding, Principles and Prospects; LONDON: CHAPMAN and HALL, 1993; 107– 25. 29. Xoconostle-C zares B, Ruiz-Medrano R, Ortega L¢pez J. Manual para el segundo curso de entrenamiento para profesores del Sistema COSNET “Expresion y purificaci¢n de enzimas recombinantes de inter, s biotecnol¢ico, Cinvestav IPN. 17. Gianaroli L, Magli C, Ferraretti A y cols. Preimplantation diagnosis for aneupioidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72:837–44. 30. Vidal F, Blanco J, Fugger EF et al. Preliminary study of the incidence of disomy in sperm fractions after MocroSort flow cytometry. Hum Reprod 1999; 14:2987–90.

CHAPTER 49 Assisted Reproductive Technologies in Human Immunodeficiency Virus (HIV) Sero-discordant Couples: Practice, Prognosis and Future Prospects Richard A Ajayi, Nwora A Melie INTRODUCTION The human immunodeficiency virus (HIV) and its associated disease, the acquired immunodeficiency syndrome (AIDS) swept into recognition like a modern day plague about a quarter of a century ago. It came as a disease with a lot of the initial attention focused on people in “high risk” groups such as intravenous drug abusers and homosexuals. It soon became clear that there was a high prevalence of the infection amongst so called ‘low risk” people who had acquired the infection through heterosexual contact. This led to wide spread dissemination of the disease particularly in developing countries and the problem has now reached epidemic proportions. Fortunately, the spread of the disease has been matched by concerted international research effort to effectively control it. This has led to the development of effective antiviral drug regimens that have dramatically improved the prognosis of people suffering with HIV/AIDS. Although the disease continues to wreak havoc in developing countries, where poverty compounds its effects and limits access to effective antiviral therapy, the situation is much better in developed countries like Europe and North America. In these countries, the effective use of antiviral therapy has changed the course of the disease from a fatal disease to a chronic disease such that most infected people can now expect to lead normal lives.1 Many HIV infected people are now in a good state of general health, having gainful employment with plans for the future, such as having children. The universally fatal prognosis originally associated with HIV/AIDS meant that consideration of assisted reproduction was not an issue in the management of HIV/ AIDS but the improved prognosis has since changed these views. Indeed, HIV is considered a disability in the American disability act and so withholding health care in the form of assisted reproductive technologies (ART) from couples infected with HIV could be considered discriminatory.2 All medical practitioners have a duty of providing care for their patients and the changing nature of HIV/AIDS story means that they could require treatment in the form of assisted reproductive techniques. This article will discuss the current practices, prognosis and future prospects of ART in the management of HIV serodiscordant couples.

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HIV, Gametes and Fertility Before looking at the various ways in which serodiscordant couples may receive fertility treatment, it may be worth briefly considering the relationship between HIV and gametes, and fertility. Although there is no direct evidence linking adverse effects of HIV infection on oogenesis, some studies have shown reduced pregnancy rates in HIV infected women3 and this may in turn be related to underlying subfertility.4 In addition, the oocyte membrane is thought to lack receptors for HIV. HIV particles may however be found in follicular fluid and associated blood. HIV infection has been linked to testicular germ cell loss and atrophy5–6 and reduced sperm parameters.7–8 Although the presence of viral particles in semen is well documented, there is no definitive evidence that they may be borne on, or in sperm cells. Current assisted reproduction practices for treating serodiscordant couples have therefore focussed on extracting virus-free gametes from potentially contaminating associated fluids. Two broad groups of HIV sero-discordant couples are likely to be the concern of the ART practitioner. Those who are fertile and can achieve pregnancy by themselves but wish to prevent the transmission of the virus to the uninfected partner and those who are infertile and need ART to achieve pregnancy Assisted Reproductive Techniques for Serodiscordant Fertile Couples The risk of HIV transmission via vaginal intercourse from the man to the woman and from the woman to the man has been estimated to be, 0.15% and 0.09% respectively. This risk is controlled by various factors such as the viral load, the degree of virulence and stage of the disease.9 Therefore, serodiscordant fertile couples may seek ART as a means of ‘safe procreation’; thereby obviating the need for the risky (in these circumstances), natural method of unprotected sexual intercourse. Where the woman is infected and the man is HIV negative the issues to consider include the following: the risk of transmitting the infection to the man, the effect of pregnancy on the cause of the disease, the effect of the disease on the pregnancy and the risk of vertical transmission of the infection to the child. A multi-disciplinary approach is essential in counseling such couples to assist them make an informed choice. Although the woman could be monitored and intercourse timed to the period of ovulation to minimise the period of exposure, this will still carry a risk and the sensible option has to be artificial insemination with the husband’s sperm. In this case no special precautions are necessary in the handling of the sample as the semen is not inf ected and standard intrauterine insemination protocols will suffice. A decision to carry out insemination on a HIV positive woman must involve the assistance of a physician experienced in the management of HIV particularly in pregnancy that will certify the fitness of the woman to go through the pregnancy The physician will also be involved in the management of the pregnancy from the point of view of providing anti-viral therapy to minimise the risk of vertical transmission of the HIV. There is evidence that the mother-child transmission is linked to the viral-load in the mother’s blood not only at the time of delivery but also when the pregnancy is diagnosed,10 with the risk more significant when the viral-load is more than 500 copies of RNA/ml.11

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The value of caesarian section before labour where the membranes have not ruptured leads to a 50% reduction of transmission especially when combined with timed use of antiviral agents.12 Avoidance of breastfeeding further reduces the risk of vertical transmission. Where the female is HIV negative and the man is HIV positive the objective is to obtain a population of virus-free spermatozoa for the treatment of the woman. The often quoted pioneering work of Semprini et al,13 and subsequent updates, though criticized for lacking a “meticulous publication covering its methodology or follow-up”,14 provide the basis for separation of spermatozoa from seminal plasma, with undetectable levels of viral contamination. This is achieved by density gradient centrifugation followed by ‘swim up’. To enhance the efficacy of this technique, further polymerase chain reaction (PCR) on recovered spermatozoa for detection of viral nucleic acid can be done.15 This appears to be widely applicable in Europe.16–18 Since it is not possible to guarantee an absolutely virus-free sperm population following the ‘sperm washing procedures, Jouannet and colleagues9 worked out a theoretical risk of viral transmission in assisted reproductive procedures. This was based on the sensitivity of nucleic acid assay technique used, number of spermatozoa tested and used for the ART process. They calculated a maximum risk of transmission of ten viral particles when 1×106 sperms are inseminated into the uterus, and minimum risk of 2×10−5 following injection of a single spermatozoon into an oocyte as in intracytoplasmic sperm injection (ICSI). This was done using a detection threshold of 20 nucleic acid copies whilst testing 2×106 spermatozoa. The French health authorities building on the above, have proposed some guidelines to help assess treatment options for HIV-serodiscordant couples where the male is infected with HIV.9 These were based on the level of viral RNA particles in the fresh semen and the possibility of residual particles, post-preparation. It was therefore posited that the choice of technique for fertile serodiscordant couples was between IUI and ICSI.9 Though treatment-related factors are same for ICSI and conventional IVF, the latter involves the insemination of a higher number of potentially contaminated spermatozoa. Other factors such as qost, success rates, clinical and peculiar problems of the couples also ought to be considered before arriving at a final decision.

Table 49.1: Proposed guidelines by French Health authorities for ART to be adopted in serodiscordant couples with an infected male partner9 HIV RNA copies in seminal plasm (copies/ml)

Provirus DNA and HIV RNA in the selected population of spermatoza

ART to be adopted

<1000

undetectable

IUI or conv IVF or ICSI ICSI none

1000–10,000 undetectable >10,000 IUI-Intrauterine insemination con IVF-Conventional In vitro fertilisation ICSI-Intracytoplasmic sperm injection

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Where the serodiscordant couple with the man HIV positive do not want to run a risk of using the man’s gametes, donor insemination with quarantined HIV free sperm could also be used. In France, donor insemination has been progressively researched since the early nineties as a way of assisting HIV sero-discordant couples to have children.19 Assisted Reproduction for Infertile Serodiscordant Couples The issues involved in the management of infertile serodiscordant couples are quite different from those of fertile couples. Here the couples have been attempting a pregnancy by themselves at a risk of having infected each other with the virus. In some cases, the couples will not know that they are HIV positive and the infection could have been picked up during screening for ART procedures. There are some suggestions that HIV couples should only have access to limited investigations and in particular, that invasive investigation such as diagnostic laparoscopy should not be carried out. However, this is inappropriate and could be considered discriminatory especially where not all couples seeking help for infertility are screened for HIV. Therefore, once a decision has been made to investigate an inf ertile couple, they should have access to the full test available. The link between HIV infection and impaired spermatogenesis has been cited above. In addition, women with HIV infection would probably also have a history of other sexually transmitted infections and pelvic inflammatory diseases and may therefore be infertile. These two factors could increase the requirement for ART in serodiscordant couples. The screening techniques used for the ‘washed’ semen may not always be applicable, as sufficient sperms may not be obtained post-wash to accurately quantify viral RNA/DNA, using current methods. However, assessment of the number of viral nucleic acid copies in seminal plasma and non-sperm components post-wash and comparison with the French guidelines, may offer a useful alternative.12,9 This would be applicable if it is the male partner that is infected. For serodiscordant infertile couples in which the female is inf ected, the clinical status of the woman regarding both the HIV infection and her infertility would be more important considerations. Overall, the determining factors for ART in serodiscordant infertile couples will include the standard clinical factors for infertility management and the eventual quantity and quality of gametes. With the apparent effects of HIV on semen quality and the reduction in transmission risks with ICSI as discussed above, ICSI would probably be the best treatment option in most cases. Procedurefor Handling Gametes from HIV Serodiscordant Couples There is no reported incidence of HIV transmission via infected gametes or embryo manipulation in an IVF laboratory. Current concerns arose from reported hepatitis B cross contamination of bone marrow and blood stem cells, stored in liquid nitrogen tanks like gametes and embryos. Also, other microbes have been shown to survive in liquid nitrogen. These therefore suggest a potential risk of cross-contamination when infected material is stored with uninfected material. Clarke20 reviewed the potential risk of crosscontamination in relation to sperm cryopreservation.

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To minimise the risk of HIV transmission to other patients by treating HIV infected (including serodiscordant) couples, several groups suggest use of dedicated facilities.12,9 As much as is possible, single-use disposable consumables should be utilised and patients with HIV put at the end of lists for clinical procedures. Handling of material by laboratory staff should involve strict observance of universal safety precautions for handling pathologic material. These would include working in class III safety cabinets, use of appropriate clothing as well as centrifuges with screwcapped bucket covers. In addition, adequate decontamination procedures with 1% hypochlorite solution must routinely be undertaken as well as proper disposal of all clinical waste. ART, Pregnancy and HIV Understanding the relationship between HIV infection and pregnancy will be useful in assessing the prognosis for ART in a HIV-infected woman in a serodiscordant couple. CD4 cell counts may decline in pregnancy perhaps due to haemodilution, although the percentages may remain fairly constant in HIV-infected pregnant and non-pregnant women. In the course of the pregnancy, both counts and percentages are similar in both groups. Pregnancy does not appear to have any significant impact on the disease as regards manifestation/progression and outcome. Therefore, once the clinical picture of the infected woman is satisfactory, ART may be considered appropriate, combined with adequate monitoring if she gets pregnant. However, there appears to be an association between HIV-infection and adverse pregnancy outcomes in HIV-infected women. This maybe as a result of the disease itself, or related causes. It was initially thought that antiretroviral therapy employed in the management of HIV infection had adverse implications for pregnancy, but a recent metanalysis could not categorically support such claims.21 Adverse outcomes of intrauterine growth restriction, premature delivery, low birth weight and spontaneous abortion have been linked to HIV infection. The above will be useful in counselling the couple regarding their chances of achieving a live birth whilst encouraging them to continue the drug therapy for the HIV infection. Indeed, the therapy for the infection will also minimise the risk of vertical transmission to the unborn child. A large meta-analysis in Europe showed a reduction in vertical transmission rates to 2.0% when antiretroviral therapy was combined with elective cesarean section.12 Direct transmission of HIV infection from man to child through an uninfected mother is unknown. Therefore, theoretically, controlling infection of the woman by some of the measures outlined above would reduce the chances of the child being infected. It has been suggested that some men remain infectious throughout the course of their disease, with significant spermatogenic activity, but they are unidentifiable by age or CD4 cell counts.5 In addition, the blood plasma viral load and seminal plasma viral load do not always correlate in both treated and untreated HIV infected males. Absence of viraemia does not preclude viral particles from semen and thus should not be the sole basis for transmission risk assessment, although it may seem more convenient. Serodiscordant couples with an infected male partner should therefore be encouraged to maintain precautionary measures in their conjugal obligations regardless of the outcome of the ART.

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In summary, the prognosis for ART in HIV serodiscordant healthy couples will be similar to those for uninfected couples with similar clinical characteristics (fertility wise) once adequate precautions have been taken to reduce transmission risks during the procedures. Future Possibilities In general, HIV infection has come to be regarded as a form chronic infection. Infected people now have the same expectations as those with other chronic ailments. These include as outlined above, access to infertility treatment. The concern for serodiscordant couples is to minimise transmission between couples and/or resulting child. In addition, other concerns are for the safety of the personnel involved in managing these patients. There is no uniform policy for ART in HIV infected patients, including serodiscordant couples. The French authorities seem to have set the pace for an ART policy for HIV infected couples. The trend in management appears to rely on the health of the partner involved and his/her infectivity as judged by CD4 counts and viral load in blood. These give an idea of the clinical status in relation to the infection and aid decisions on whether or not to treat for infertility. As for the facilities, it appears that using dedicated equipment to prevent cross contamination will become increasingly popular in endemic areas and where patients can afford the new therapies. In conclusion, ART for serodiscordant couples should be a multidisciplinary approach, involving the clinician managing the disease and fertility respectively, counsellors and scientists. A broader and long-term view of all the issues needs to be applied in arriving at a uniform policy. REFERENCES 1. Minkoff PH, Santoro N. Ethical considerations in the treatment of infertility in women with human immunodeficiency virus infection. N Eng J Med 2002; 342:1748–52. 2. Lyerly AD, Anderson J. Human immunodeficiency virus and assisted reproduction: reconsidering evidence, reframing ethics. Fertil Steril 2001; 75(5):843–58. 3. Gray RH, Wawer MJ, Serwadda D et al. Population-based study of fertility in women with HIV1 infection in Uganda. Lancet 1998; 351(9096):98–103. 4. Ross A, Morgan D, Lubega R et al. Reduced fertility associated with HIV: contribution of preexisting subfertility. AIDS 1999; 13(15):2133–41. 5. Shevchuk MM, Pigato JB, Khalife G et al. Changing testicular histology in AIDS: its implications for sexual transmission of HIV. Urology 1999; 53:203–08. 6. Mhawech P, Onorato M, Uchida T, Borucki MJ. Testicular atrophy in 80 HIV-positive patients: a multivariate statistical analysis. Int J STD AIDS 2001; 12:221–224 7. Umapathy E, Simbini T, Chipata, Mbizvo M. Sperm characteristics and accessory sex gland functions in HIV-infected men. Arch Androl 2001; 46:153–58. 8. Dulioust E, DuAL, Costagliola D et al. Semen altercation in HIV-1 infected men. Hum Reprod 2002; 17:2112–18. 9. Jouannet P, de Almeida M, Dulioust E et al. Assisted reproduction for HIV and/or HCV-infected patients. In: Healy D, Kovacs GT, Mclachlan R. and Rodriguez-Armas O (Eds). Reproductive Medicine In The Twenty-first Century New York: The Parthenon Publishing Grp Inc USA 2002; 152–62.

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10. Connor E M, Sperling R S, Gelbert et al. Reduction of maternal infant transmission of HIV type 1 with zidovudine treatment. Paediatric AIDS clinical trial group. N Eng J Med 1994; 331:1173–80. 11. Mofenson LM, Lambert JS, Stiehm ER et al. Risk factors for perinatal transmission of human immunodeficiency virus type 1 in women treated with zidovudine. N Eng J Med 1999; 41:385– 93. 12. The International Perinatal HIV Group. The mode of delivery and the risk of vertical transmission of Human immunodeficiency virus type-1: Ameta-analysis of 15 prospective cohort studies. N Eng J Med 1999; 340:977–87. 13. Semprini A E, Levi-Setti P, Bozzo M et al. Insemination of HIV-negative women with processed semen of HIV-positive partners. Lancet 1992; 340:1317–19. 14. Englert Y, Van Vooren J, Place I et al. ART in HIV-discordant couple; Has the time come for a change in attitude. Hum Reprod 2001; 16:1309–15. 15. Marina S, Marina, Alcolea, et al. Human immunodeficiency virus type 1: serodiscordant couples can bear healthy children after undergoing intrauterine insemination. Fertil Steril 1998; 70:35–39. 16. Lasheeb A S, King J, Ball J et al. Semen characteristics in HIV-1 positive men and the effect of semen washing. Genitourin Med 1997; 73:303–05. 17. Coll O, Vidal R, Martinez de Tejada B et al. Management of HIV serodiscordant couples: The clinician’s point of view. Contracept Fertil Sex. 1999; 27:399–413). 18. Gilling-Smith C. HIV prevention: Assisted reproduction in HIV-discordant couples AIDS Read 2000; 10:581–87. 19. Jouannet P, Dulioust E, Kunstmann JM et al. Management of fertile and infertile HIV positive patients wanting to become parents. In: Kempers RD, Cohen J, Haney AF, Younger JB (Eds). Fertility and Reproductive Medicine. Amsterdam: Elsevier Science 1998; 487–95. 20. Clarke G. Sperm cryopreservation: is there a significant risk of cross-contamination? Hum Reprod 1999; 14:2941–43. 21. Tuomala RE, Shapiro DE, Mofenson LM et al. Antiretroviral therapy during pregnancy and the risk of an adverse outcome. N Eng J Med 2002; 346:1863–1970.

SECTION 7 Third Party Reproduction

CHAPTER 50 Gestational Surrogacy Anil B Pinto, Nona Morgan Swank INTRODUCTION In the English dictionary the surrogate means “substitute” as compared to the real thing. Surrogacy has been used to treat certain forms of childlessness for centuries. Many argue that surrogacy dates back to biblical times when Rachel asked her maidservant to bear a child for herself and her husband. However commercial surrogacy was not available until relatively recently. Surrogacy involves a contracting couple and another woman, known as the surrogate. Traditionally, a surrogate agrees to be inseminated with the contracting husband’s sperm and then carries the baby to delivery. The surrogate then relinquishes her maternal rights after the birth of the child, and the contracting couple adopts the baby For a small group of women, surrogacy may be the only option available and this would be the option for a woman: (1) Patients with functioning ovaries but without a uterus, either secondary to a congenital anomaly or due to a hysterectomy (2) Women with medical conditions that make pregnancy life-threateningbut who are otherwise healthy, (3) Women who have experienced repeated miscarriages and for whom the possibility of carrying a baby to term is quite remote, as well as for those who have been unable to conceive following repeated attempts at IVF. With the development of advanced reproductive technologies (ART’s) for infertility, the concept of a third person, the gestational carrier, was introduced. A gestational carrier works with the contracting couple to carry the couple’s own genetic material to delivery The contracting couple’s embryos are transferred to the gestational carrier’s uterus to be carried to term. The gestational carrier is thus genetically inert in this process. For the sake of clarity it is important to refer to the process in which a woman supplies both eggs and gestation to the contracting couple as surrogacy. It is thus important to bear in mind that a gestational carrier and surrogate are two distinct conditions and should not be described in an inter-changeable manner. Once it is decided to proceed with a surrogate, it is important to bear in mind that the treatment and management of the pregnancy does not differ from the conventional management of a pregnant patient. In this chapter we will discuss the legal issues, psychological aspects, ethical and counseling issues central to this process.

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LEGAL AND ETHICAL ISSSUES SURROUNDING SURROGACY The issue of surrogacy raises many questions. Is it proper for surrogates to have children to be turned over to single people or homosexual couples? Does the impregnation of the surrogate mother by a married man’s sperm amount to adultery? Does the impregnation of a woman with her brother-in-law’s sperm constitute a type of incest? What if the surrogate mother decides to have an abortion or to keep the baby? What if the adoptive parents die or get divorced before the birth or decide that they do not want the baby after all? What if the child is born with a severe handicap? Many have argued that there is little distinction between surrogacy and gestational carrier from a medical or legal point of view. The American College of Obstetrics and Gynecology (ACOG) committee stated that the genetic link between the contracting couple and the baby born from the gestational carrier was less weighty than the linkbetween the gestational carrier and the fetus she gestates and delivers. The logical extension of this argument is that a traditional surrogate has a stronger bond with her offspring than the contracting couple. Thus, both the surrogate and the carrier would have a greater claim to any child born. This position argues that motherhood is ascribed to the act of gestating because the uterine environment plays an important role in fetal development. Furthermore, the effect of bonding has a permanent and significant role for both the gestational mother and the child. The counter argument is that parenthood should be determined solely on the basis of genes. This view is based on the fact that the genes predominantly determine personality, intelligence and behavior. Basing parentage solely on genetics has great merit, but it does not account for the influence of environment on personality and development. All consenting parties in the surrogate process must have an opportunity to consider their reasons for choosing to be involved in the process. All parties should begin by clearly stating their intentions about their roles in this third party reproduction scenario. The legal arguments surrounding surrogacy should also consider the issues of the enforceability of the contract, the commodification of human life, and constitutional issues. One simple option is to ban all surrogate arrangements and condemn the process as demeaning and exploitive of women. If surrogacy programs are not to be dismissed with a flat ban then society may be faced with the choice of whether or not to regulate surrogacy It is important to predetermine who will be considered the legal mother. PSYCHODYNAMIC ISSUES It was Parker who first suggested a three-step framework to understand a surrogate mother’s motivation: (1) The perceived desire and need for money, (2) The perceived degree of enjoyment and desire to be pregnant (3) The perception that the advantages of relinquishment outweigh the disadvantages. First, women often express a strong wish to give the gift of a baby to a couple who want a child. Second, women often feel that surrogate motherhood will help them master unresolved feelings they have for a previous voluntary loss of fetus or baby through abortion or relinquishment. The desire of women who choose to be surrogates in their wish to help infertile couples has often been labelled “Altruistic.” It is possible that altruism can coexist with a

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decision to seek financial gain. Surrogates frame financial gain as compensation for the emotional and physical risk to themselves and their family as well as the work of going through pregnancy Critics of surrogacy frequently overlook the fact that surrogates are not compensated for the time spent in the pursuit of pregnancy and may receive nothing for their efforts if pregnancy does not occur. The degree of emotional risk a surrogate takes on has often been well documented. Participation as a surrogate carries varying degrees of stress during pregnancy, delivery, and the postpartum period. Maternal anxiety has an inverse relationship with positive pregnancy outcome. Literature also suggests that surrogates experience a strong prenatal attachment, a postpartum separation process, and a sense of loss after delivery While the surrogate is faced with the task of separating from her own biologic child there is no evidence to suggest an increased incidence of psychiatric illness such as postpartum depression or psychosis. Relatively few centers offer the option of anonymous surrogacy in which the surrogate has no identifying information about the contracting couple. It is imperative that both the surrogate and the contracting couple develop a relationship. Even before a pregnancy is established, a surrogate may have to interrupt her life and family to be available for office visits and further investigations such as ultrasound scans, blood work, etc. Then, as the surrogate experiences the many physiological changes and/ or complications of the pregnancy, the contracting couple must manage their feelings of powerlessness to alleviate the surrogate’s strain. The couple’s feelings of powerlessness run parallel to their feelings of being out of control during the whole fertility and pregnancy experience. Many couples express their fear that the surrogate may change her mind and not take proper precautions of herself during the pregnancy or will not surrender the baby at birth. The surrogate on the other hand must balance the need to care for the pregnancy with the need to remain emotionally separate from the pregnancy. The needs of the surrogate and the parents may conflict if the surrogate wants to engage the couple more fully and the couple wishes to withdraw from her because of any guilt feelings they may have. The opposite extreme may also occur when the contracting couple and surrogate attempt to become fully integrated into each other’s lives and cease to have boundaries between them. The couple may hope to have more control in this manner, but it may backfire if the surrogate feels intruded upon or feels she is not fully trusted. Ideally if all the participants are able to express their feelings and conflicts, they will be able to act constructively. Many surrogates fear that the contracting couple will not accept a baby that is less than perfect, and that they will be burdened with a child that they did not want. The surrogate may have fears that the couple will blame her for any untoward events that occur during the pregnancy Others express concerns that they could be sued should they fall, are involved in a car accident, or miscarry at any point in the pregnancy. Clearly the relationship between the surrogate and the contracting couple can be very tenuous and this calls for psychological screening of both parties.

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PSYCHOLOGICAL SCREENING Psychological screening must identify two major areas: (1) The diagnosis of any current or previous major psychopathology, and (2) the evaluation of the surrogate and the contracting parents to understand the issues and procedures involved in the process. In addition, evaluation of the surrogate calls for a thorough evaluation of personality and ego strength. Evaluation should also extend to guiding the participant through anticipated events and reactions. Factors that must be considered include the surrogate’s motivation, her concerns regarding medical procedures, her ability to separate emotionally post-birth from the child and religious attitudes regarding surrogacy, support from family and friends, comprehension and acceptance of medical risks and emotional maturity. The psychological screening of surrogate mothers is very similar for both traditional artificial insemination surrogates and IVF gestational surrogates. It is crucial that the candidate already have at least one child that she has given birth to and parented. If she has not had pregnancy and parenting experience, it would seem impossible for her to give any level of informed consent and it may be difficult for her to empathize with the parents and the child. Additionally, it seems risky for a doctor to endorse women without such obstetrical histories. It is crucial that the candidate obtain something for herself beyond financial remuneration. Traits such as low self-esteem, low intelligence and martyr patterns should be evaluated carefully. Psychological testing, clinical interviews, observations in a group setting, and feedback from others involved in the case are all important. Specifically it is important to eliminate sociopaths, depressed persons, borderline personalities, and those who have little ego strength. It is important to assess their coping mechanisms, defenses, and resiliency especially when under duress. Surrogates also need an intellectual ability to do think abstractly, conceptualize, and retain a lot of information. It is vital that she have the ability to think independently, as well as take care of herself so to prevent exploitation. Over the years evaluating a surrogate’s support system, resources, and immediate family has become increasingly important. Assessing the husband’s beliefs and thinking is most revealing. A surrogate with minimal to no resources or minimal ability to use resources is often indicative of a person with poor judgment who will need a lot of case management. Furthermore, her children are of utmost concern. Discovering how she plans to tell her children, and assessing how much life trauma the children have undergone are important considerations for surrogacy to proceed safely. If a candidate answers that she may not tell her children the truth or if her children have a history with much loss and/or trauma it is often best not to accept her. During the evaluation, the question of the loss of the “spotlight” for the surrogate must be analyzed. She will have to deal with loss of attention once the baby is born and returns home with the contracting couple. It is also important to explore the potential impact on the surrogate’s husband or partner and her children. A strong communication and flexibility of all the participants is most beneficial for the children. Prior to beginning treatment, specific issues need to be discussed, such as the contracting couple’s willingness to meet and work with the children, ability of the children to see the baby that is born and the postpartum relationship between both families. In some cases it may be wise to involve the services of a child psychologist.

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Husbands of surrogates may face community and peer reactions to their wife’s choice to be a surrogate in addition to their own choice to be involved. Issues may arise about the husband’s masculinity and leadership role in the house because he “allowed” his wife to be a surrogate. The husband or partner also may be required to take on additional responsibilities in the house as the surrogate goes through different stages in the pregnancy. The extended family (grandparents etc) will have to process their own feelings and reactions about a child that is genetically related to them being given up for adoption. Religion and ethnic backgrounds may also play an important role in how the families choose to cope with the pregnancy. SCREENINGTHE PROSPECTIVE PARENTS As with surrogates, the psychological screening of couples attempts to assess the general mental health. In order for surrogacy to be successful, it is important that the couples are empathic, flexible, and respond to new situations with resilience and ego-strength. Participants who need unrealistic amounts of control, who are narcissistic, depressed, or have notable personality disorders, put themselves, the surrogate, the practice, and the child at risk. Couples who are very mistrusting and/or do not understand the importance of the process and just focus on the end result are likely to sabotage treatment and be angry. Asking questions about how they make decisions, observing how they treat you and your staff, and a review of history and lifestyle of ten reveals personality traits that may put the clients at risk. Again, often the tool that is most revealing is a full open discussion of the issues surrounding surrogacy. It is important to assess how they have come to this choice. Questions should be addressed if one spouse is pursuing this option, if the choice is an informed one, if they have exhausted other options, and what they believe surrogacy can realistically provide for them. As with the surrogate population, why they are choosing surrogacy is very revealing. There are probably inappropriate reasons, i.e., one spouse refuses to consider any other option consequently perhaps forcing an uncomfortable choice on the other. Most couples pursue surrogacy because of a desire for a genetic connection, a desire to be participants in the pregnancy and birth process, a desire to know and feel comfortable with their child’s birth mother, a need to avoid fears about returning the inf ant to birth parents, or the lack of adoption opportunities in their state or country. Another important area of assessment is the couple’s perception of the surrogate and their desire for contact. The intense and complex process of achieving conception, the long poignant pregnancy, and the future years of reflection and wondering need to be based on the couple being comfortable with the surrogate. If a couple cannot envision themselves in some sort of open relationship with a surrogate of their choice, then they should not involve that surrogate and her young family in their lives. The evaluation of couples also includes their beliefs about the future, what they may tell the child, and what they will tell others. Obviously, this assessment needs to be culturally sensitive. But again, the answers can provide insight into the couple’s resolution and readiness to proceed. Often the assessment is spent helping couples sort

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through the conflicting and confusing feelings about openness, about who is the mother, and about the others’ responses. Additional assessment can include psychological testing. Follow-up interviews or a second interview by a colleague can be helpful. Of course, interviewing spouses separately, as well as together, can be enlightening. As with the surrogates, feedback from other professionals and reading their answers to various questionnaires does highlight patterns. Additionally, as a service to the clients and as part of their informed consent process, couples should be offered contact with other couples participating in surrogacy In most centers, standard procedures regarding sexually transmitted disease screening is mandatory For surrogacy, the semen of the husband from the genetic couple should be frozen for six or more months before treatment. A repeat negative HIV status of the husband will then make it possible to transfer “fresh” embryos to the host. SELECTING THE SURROGATE Only normal fit women less than 35 years of age with no contraindication to pregnancy are selected as the surrogate. It is our opinion that a surrogate needs to have had a successful pregnancy experience prior to agreeing to be a surrogate. Though the carrier may never have been a surrogate before, if she has had a pregnancy she at least knows what it entails. One never knows exactly how a particular pregnancy will go but a prior experience gives a beginning road map. Additionally, the need to give the first child she has borne to the contracting couple could be difficult emotionally, especially for a woman who had never given birth before. It has also been shown that the surrogate who has really enjoyed a prior pregnancy but who may not want another child is the best option. The ability to verbalize and to appreciate the difference in having your own child versus being a host womb is also essential. This understanding defines the kind of attachment that will develop during the pregnancy. The support of those closest to the surrogate is also important. They will be the ones most directly affected by the medical experience prior to conception, the pregnancy and postpartum. Seeking their input in the evaluation process assures that they too, know what to anticipate and are “on board”. The contracting couple’s ability to appreciate what the surrogate and their family are agreeing to do for them is vital. It is imperative that before a woman becomes a surrogate, she should undergo rigorous psychological screening by a licensed psychologist to identify any personality characteristics that would hinder her ability to fulfill a surrogacy contract. The contracting couple should be provided with a psychological profile of the surrogate, based upon her scores in standard psychological tests. When attempting to identify a potential surrogate working through a reputable program is at times the better option. This streamlines the process by eliminating much of the guesswork and legwork ensuring that the parents-to-be and the surrogate are a good match. This also helps to maintain a smooth understanding and relationship between the surrogate and the contracting couple. Surrogate program coordinators play a large role in helping set the tone of the working relationship between the two parties. Working with a surrogacy program gives a couple an already established cohort of candidates from whom

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they can make a selection if they so wish, the benefit of experience and expertise and a guide through the necessary legal procedures. The program coordinator also checks with the surrogate and the prospective parents periodically for updates, to answer questions and to help ensure that the relationship remains untroubled. The coordinator also can ensure that the surrogate attends regular counseling sessions, ideally with a group of other surrogates. The coordinator can also ensure that all the paperwork is in order to minimize any confusion as to the identity of the child’s legal parents and to streamline the legal process, according to the existing laws. A contract should then be drawn using the help of attorneys familiar with the unique issues that surround surrogacy It would seem prudent to take out life insurance policies for both of the natural parents, which names the child as a beneficiary, and for the surrogate. It is also advised that written wills be drawn and estate-planning issues dealt with. A court order specifying that the child belongs to the prospective parents and not the surrogate can be obtained prior to the birth of the child. THE TEAM APPROACH A team of professionals who are both supportive and knowledgeable are a must. There must be a physician who is willing to perform the procedures for conception to take place as well as one who is willing to care for the surrogate during pregnancy and delivery. Physicians have a belief system too, and it can be quite a task to find those who will assist in reaching this goal. An attorney who has written surrogate contracts and is a local specialist in surrogate law is also essential. In addition to the contract, an attorney may assist one in considering questions about care of the surrogate that one would not have examined. Arrangements between family members and close friends are often spoken with such love that there is hesitancy to seek legal advice. It is my opinion that there is even more complexity in these relationships and a legal contract is very important in spelling out possible ramifications and outcomes; therapists serve a role in both evaluation and clarification. A therapist should also be available during and after a pregnancy to provide needed support. Like so many other things in life, “timing is everything.” The couple needs to have a sense of completion of infertility treatment if that has been their experience. A feeling of readiness to move on and a willingness to venture into other possibilities is very important. However, when one moves into an arena that involves another person making a commitment to you, the readiness and desire need to be mutual. Without this mutuality, the uncommitted spouse can undermine the effort. It becomes clear in meetings that include physicians, attorneys, therapists and the surrogate if one partner is dragging his/her feet or just not ready. A surrogate wants to know that “we are in this together and each partner is ready and willing to pull their weight.” In addition, practical matters involving timing for the surrogate are important. Many surrogates have a specific time frame in mind, for instance, doing the surrogacy in a time when the surrogate plans to be staying at home or is between jobs or school. Fitting the experience into a time when the pregnancy would be least disruptive to them or their children can be extremely important. Therefore, the synchrony of the couple’s readiness and the surrogate’s timing needs to be evaluated.

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The couple and the surrogate need a stronger belief that what they are doing is good and right in order to handle the various reactions and comments of others. This belief will be called forth in interactions with everyone from relatives to casual acquaintances who make comments to the surrogate about the pregnancy or who run into the couple with a newborn never having seen them pregnant. PREGNANCY There is a strong sense of success in having achieved a goal that was set out for months or even years earlier. Some groups may temper their excitement with the sobering possibility of miscarriage. In the event that the surrogate has more than one fetus, excitement may be accompanied by anxiety about handling the physical strain of twins or higher order births. Surrogates must also decide if they are willing to undergo prenatal testing and therapeutic interventions if medically indicated. Once past the initial couple months of pregnancy, different tasks confront the surrogate. As the pregnancy progresses, her heretofore-private decision now becomes public, as she grows visibly pregnant. Thus the surrogate must open herself to opinions of those she might have otherwise wished to avoid and, at the very least, must expend the emotional energy to give repeated explanations about surrogacy The most demanding and central task is for the surrogate and the contracting couple to develop defense mechanisms and coping strategies for the changes taking place and the increasing presence of the baby. Social visits between the surrogate and couple may be planned to help the surrogate remain focused on the couple’s experience of the pregnancy rather than her own. Several institutions recommend that the surrogate must meet with a mental health professional or a support group to help work through any conflicting feelings that may arise. DELIVERY AND POSTPARTUM Depending on her individual situation, the surrogate may lose immediate contact with the baby, whereas others have a gradual separation process during the hospital stay. The surrogate now needs to rely on using the emotional and intellectual fulfillment of her choice to put closure on the event. Even in situations where there will be an ongoing relationship, the surrogate must have a sense of an end to her active participation. The surrogate must also learn to deal with the fact that now with the birth of the child the focus of attention will now shift to the newborn baby. The surrogate must be able to mobilize and draw on other resources in her life to continue the support she has been receiving and will need. On a practical level, even though the contracting parents may wish to continue to be as supportive as before the birth, they are now caring for a newborn baby. This may leave the surrogate susceptible to feelings of mild depression or agitation. The transition from a very busy schedule filled with prenatal visits and phone calls to the contracting couple she must now settle into often times a quieter daily routine. Depending on state laws, the surrogate also must go through termination of her parental rights and other legal requirements involved in placing the child for adoption. It is also

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important that the surrogate and her family change the relationship with the contracting couple. It is also important that the couple decide as to how they intend to pass over the information of the existence of a surrogate mother to the child. It is uncommon for a couple to choose not to disclose to the child the use of a surrogate, but telling the child may take several routes. The first route is to incorporate the surrogate into the child’s life through correspondence, telephone calls and even visits. The second route is to provide the child, at an age the couple feels appropriate, with all the information they have about the surrogate but not to involve visits. The last route is to acknowledge the participation of the surrogate but to treat the situation more like a closed adoption with very little information available. It is reasonable to assume that childrenborn of surrogates will have some of the same issues or needs surrounding the knowledge of their genetic background and reasons why their biologic mother chose to do so. COUNSELING Counseling provides an excellent opportunity for the surrogate and the contracting couple to explore and manage their experience throughout the entire process of choosing to become pregnant, the pregnancy, labor and the postpartum period. Education as well as counseling must include the potential physical and emotional risks involved in surrogacy, It requires that both the surrogate and the contracting couple fully explore all possible outcomes of their involvement. Counseling helps each individual make his or her bestinformed choice about becoming involved in this type of third-party reproduction. During pregnancy, counseling can provide the opportunity for the surrogate to take time to explore all the feelings she is experiencing related to the pregnancy. Counseling also offers the contracting parents the chance to explore their feelings about the pregnancy As the pregnancy advances the contacting couple feels more certain that they will become parents, and normal fears and anxieties can arise independent of the overriding fears of having another woman carry their child. This will allow the couple to look at the many issues surrounding this process and help them to cope with normal parenting fears. Postpartum counseling allows for the feelings that have occurred throughout the surrogacy experience to be explored and resolved. Under pressure from family, friends and the public to defend or reassure the surrogate about their choice, counseling may be the safest place to express the wide range of feelings. Throughout all phases of the experience, counseling can provide an excellent resource to all participants if communication should falter or fail. Although not a guarantee that all issues will be resolved, counseling provides the best foundation upon which all parties can work all disagreements or issues. The organization “Parenting Through Surrogacy”, a nonprofit support group available to surrogates and contracting couples, also can provide both support and information. Thus counseling before the commencement of a cycle will help both the surrogate and the contracting couple to voice their expectations and concerns. Several issues need to be addressed and resolved, for instance: Is everyone in agreement regarding selective reduction and therapeutic abortion? How many cycles will be attempted? How will the

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obstetrician be selected? Will the surrogate be expected to make life style changes (diet, exercise, alcohol consumption etc)? Who will be present at birth? These and several questions will need to be dealt with from time to time. Unresolved family issues may surface during these stressful times. After a failed pregnancy attempt, the surrogate may blame herself. The intended mother may hide her feelings, while trying to take care of others. Counseling can help address all these obstacles and more importantly prepare for those yet to surface. SUMMARY Despite its notoriety in the media, surrogacy has persisted as an alternative choice to infertile couples attempting to form a family The psychological and emotional issues surrounding surrogacy are complex and not well researched. Each case is different and both the surrogate and the contracting couple must evaluate the emotional risks as well as the medical risks (in case of the surrogate) this third-party reproductive process involves. In the absence of legislation in most states, the surrogate also must determine what type of relationship she needs and wishes to have with the contracting couple. Tremendous debate exists over the morality and ethics of surrogacy and even more over the legal disposition of any children born through surrogacy. Surrogacy can work well if the real issues involved are acknowledged and anticipated through appropriate legal, medical and psychological counseling. It appears that surrogacy can work for the majority of those women and men who choose to participate. Both parties to the surrogacy arrangement sometimes have unreasonably high expectations of success, in spite of frank information and counseling. Because the host is fit, young and known to be fertile, she and the contracting couple expect success and feel badly let down if they fail. The incidence of miscarriage varies from study to study and is the same or higher than the background rates of miscarriage. Full support counseling for both parties in these unfortunate circumstances is essential. At least half of the surrogates will undertake further treatment cycles after a failure or a miscarriage. The long-term follow-up of the surrogates suggests that they feel fulfilled and are glad to have helped an infertile couple realize their goal. Researchers who have followed up hosts have found that surrogacy was a positive experience, with strong feelings of fulfillment and altruism, even when payment was received. Being a surrogate is a life experience that allows some women real success in altering their emotional state in a direction they desire and fulfilling ideal images of themselves. A very significant aspect of that image is that of being a mother and, by extension, enabling others to enjoy the pleasures of parenthood that they themselves had. Because surrogacy involves an act of giving that is meaningful to the surrogate, and because what is being given is of unique value, being a surrogate mother has a potential to be a “mutative” event, an experience capable of altering and transforming identity, self-image, and existing psychic structure.

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BIBLIOGRAPHY 1. American College of Obstetricians and Gynecologists: Ethical issues in surrogate motherhood. Washington, DC, ACOG, committee opinion number 1990; 88. 2. Andrews L. Between Strangers, New York, Harper and Rowe, 1989. 3. Annas GJ. Fairy tales surrogate mothers bell. In Gostin L (Ed): Surrogate Motherhood: Politics and Privacy. Bloomington: Indiana University Press, 1990; 52. 4. Annas GJ. The shadow lands- secrets, lies, and assisted reproduction. N Engl J Med 19983; 39:935–39. 5. Bouchard TJ, Lykken DT et al. Source of human psychological differences: The Minnesota study of twins reared apart. Science 1990; 250. 6. Brinsden PR, Appleton TC, Murray E et al. Treatment by in vitro fertilization with surrogacy: experience of one British Center. 2000; 320(7239):924–29. 7. English ME, Mechanick-BravermanA, Corson SL: Semantics and science: the distinctionbetween gestational carrier and traditional surrogacy options. Womens Health Issues 1991; 1:3. 8. Erlen JA, Holzman IR. Evolving issues in surrogate motherhood. Health Care Women International 11:3,1990 9. Ethics committee of the American Fertility Society Ethical considerations in the New reproductive Technologies. Fertil Steril 1986; 46(suppl 1):62–68. 10. Hayley D, Stern R, Stocking C, Sachs G. The application of health care surrogate laws to older population: How good a match? J Am Geriatr Soc 1996; 44:185–88. 11. High DM. Surrogate decision making. Who will make decisions for me when I can’t? Clin Geriatr Med 1994; 10:445–62. 12. Jones HW (Jr). Commentary on ACOG committee opinion number 88, November 1990, Ethical issues in surrogate motherhood. Women’s Health Issues 1991; 1(3):138–39. 13. Kleinpeter CH, Hohman MM. Surrogate motherhood: personality traits and satisfaction with service providers. Psychological reports. 2000; 87(3):957–70. 14. Kronhaus A. Assisted reproduction: who is the mother? N Engl J Med 1999; 340(8),656–57. 15. Marrs RP, Ringler GE, Stein AL et al. The use of surrogate gestational carriers for assisted reproductive technologies. Am J Obstet Gynecol 1993; 168:858–63 16. Parker PJ. Motivation of surrogate mothers: Initial findings. Am J Psychiatry 1983; 140:1. 17. Reame NE, Parker PJ. Surrogate pregnancy: Clinical features of forty-four cases. Am J Obstet Gynecol 1990; 162:5. 18. Robertson JA. Procreative liberty and the state’s burden of proof in regulating noncoital reproduction. In Gostin L (Ed): Surrogate Motherhood: Politics and Privacy. Bloomington: Indiana University Press, 1990; 39. 19. Rogal MJ. A legal perspective of surrogacy and paternalism. J Bone and Joint Surg-Amer 2001; 83-A(4):623. 20. Rothenberg KH. Gestational surrogacy and the health care provider: Put part of the “IVF” genie back into the bottle. Law, Medicine and Health Care 1990; 18:4. 21. Roupie E, Santin A, Boulme R et al. Patient’s preferences concerning medicl information and surrogacy: results of a prospective study in a French emergency department. Intensive care Med 2000; 26:52–56. 22. Samuels A. The surrogate child: who is his or her mother and who is the father? Medicolegal J 2000; 68(2):65–67.

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23. Serafini P. Outcome and follow-up of children born after IVF-surrogacy. Hum Reprod Update 2001; 7(1):23–27. 24. Shuster E: Non-genetic surrogacy: No cure but problems for infertility? Hum Reprod 1991; 6:8. 25. Van Waart J, Kruger TF. Surrogate pregnancies in patients with Mayer-Rokitansky-KustnerHauser syndrome and severe teratozoospermia. Archiv Androl 2000; 45(2):95–97. 26. Van ZL, Van NA. Interpretations, perspectives and intentions in surrogate motherhood. J Med Etnics 2000; 26(5):404–9.

CHAPTER 51 Oocyte Donation Siya S Sharma, Sucheta Jindal INTRODUCTION Oocyte donation is being practiced for about two decades in human infertility treatment. In 1984, Lutjen et al1 reported the first pregnancy and delivery following in-vitro fertilisation and embryo transfer (IVF and ET) in a patient with primary ovarian failure. A woman who is unable to produce her own oocytes due to malfunctioning, nonfunctioning or absent ovaries receives the oocytes. Oocyte donation is an emotional, expensive and time-intensive experience, but it offers a realistic, successful, option for many couples who would otherwise have no way to have a child. It is important to remember that each patient has her own unique response to the medication she receives and that each assisted reproductive technique (ART) cycle is different. In clinical practice not only is an individual likely to respond differently from others, but also one may actually respond differently from one cycle to the next. For this reason, treatment and testing differs in each patient and possibly on each occasion. Donor and recipient menstrual cycles are synchronized so that embryos are transferred to a receptive endometrium. Despite advanced reproductive age, perinatal and obstetric outcomes are generally good. Newer advanced techniques (i.e. germinal vesicle transfer, donor ooplasm transfer and ovarian cryopreservation and transplantation techniques) may permit the recipient to provide some genetic contribution to the offspring and are currently under investigation.2 PRE-TREATMENT COUNSELLING Patient and her partner should be explained about the following: 1. Oocyte donation programme 2. Treatment procedure and its success 3. Time and duration of treatment in relation to the menstrual cycles if one is menstruating 4. Investigations to be performed before, during and after the treatment 5. Cost of the treatment 6. Informed written consent.

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RECIPIENTS OF OOCYTES—INDICATIONS Broad Categorisation Two groups of women are possible candidates for the oocyte donation programme: 1. those lacking ovarian function due to the menopause, premature ovarian failure, and ovarian surgery 2. those who, despite normal ovarian function, cannot use their own oocytes, either because of their low quality hereditary transmissable disorders, or a poor response to ovulation stimulation treatment. Detailed Categorisation All patients with any of the following indications can opt for the oocyte donation programme: 1. Women with ovarian failure or nonfunctioning ovaries: a. Primary ovarian failure: About 10 percent patients face this problem in an oocyte donation programme.3 • Gonadal dysgenesis—Turner’s syndrome, Swyer’s syndrome, pure gonadal dysgenesis. Ovarian tissue even if present such as streak ovaries, does not have primordial follicles and does not produce steroid hormones. • Savage’s or resistant ovary syndrome. b. Premature ovarian failure: There is inherent depletion in number of primordial follicles in ovaries leading to early cessation of ovarian function before the age of 40 years. Approximately 85 percent patients in oocyte donation programme and 1 percent of infertility population belong to this group.3 • Hereditary factors: fragile X syndrome carriers. • Enzymatic changes: galactosemia, 17-α-hydroxylase deficiency, a defect in gonadotrophin secretion. • Autoimmune disorders: multiple endocrine neoplasia (MEN) syndrome, Addison’s syndrome, diabetes mellitus, hypothyroidism, antiovarian antibodies. • Infectious factors: parotiditis, rubella. • Environmental: tobacco addiction. • Surgical: bilateral oophorectomy. This condition includes about 5 percent patients of oocyte donation programme. • Previous chemo or radiotherapy. c. Menopause: The reported success of oocyte donation in older women makes pregnancy feasible in virtually any woman with normal uterus, regardless of age or the absence of ovaries or ovarian function.4–6 Prospective parents and their treating physicians must carefully consider the

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specifics of each case before oocyte donation, including a woman’s health, medical and genetic risks, and the provision for child rearing. 2. Women with normal ovarian function or functioning ovaries: a. Genetic abnormalities that can be transmitted to offsprings. • Autosomal dominant: familial alopecia, epidermolysis bullosa. • Autosomal recessive: cystic fibrosis. • Sex-linked diseases: haemophilia. b. Chromosome abnormalities: mosaicism, translocation, fragile X syndrome carriers, chromosome inversions and deletion. c. Women with recurrent in-vitro fertilization failure: • Low responders who do not respond adequately to ovarian stimulation. • Poor oocyte quality. • Recurrent fertilization failure even with intracytoplasmic sperm injection. • Recurrent failure of embryos to implant. d. Ovaries inaccessible to oocyte retrival in cases of frozen pelvis and multiple adhesions especially with the bowel. e. Women over 40 years with a normal ovarian cycle, to reduce the risk of trisomies such as Down’s syndrome and miscarriage rate. Source of Oocyte Donors Donors can be anonymous or known to the recipients. 1. A sister, relative or a friend willing to be an oocyte donor make a great option. 2. Patients who are undergoing IVF treatment, can provide their surplus oocytes. This is the common source of oocytes in most of the oocyte donation programmes. 3. Volunteer donors who wish to provide their own oocytes to the infertile women. Ideal Oocyte Donors Oocyte donors must fulfil the following criteria or requirements to take part in the programme: 1. Age: 18–35 years. 2. History: a. Donors should be encouraged to provide as much other non-identifying biographical information about them as they wish to be made available to prospective parents and any resulting child. b. No family history of any genetically transmitted disease. c. No personal history of transmissible infection.

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d. Normal reproductive system with adequate response to ovarian stimulation treatment and or history of previous fertility. 3. Examination: Normal physical and mental health with good IQ. 4. Investigations: a. Normal karyotype b. Negative tests for Human Immunodeficiency Virus, Hepatitis B and C, Chlamydia, Herpes, Cytomegalovirus, Toxoplasma, Rubella, and Syphilis All donations must be voluntary and anonymous. The only information about the donors that can be given to the recipients is general information of interest for correct monitoring of the pregnancy as blood group and age of the donor. A careful clinical and social consideration should be given to the suitability of donors. The attitude of the donor towards the donation should be positive. Counselling of Unsuitable Donors 1. If someone is unsuitable as an oocyte donor, it should be explained to the donor and recipient as well. The explanation should be presented sensitively. 2. If donor is rejected due to the physical or psychological problems that require treatment or specialized counselling, the centre should provide all reasonable assistance in obtaining this. 3. When the centre becomes aware that a donor has a previously unsuspected genetic disease or is a carrier of a deleterious recessively inherited condition (for example through the birth of a baby with cystic fibrosis), the concerned people (donor and recipient) should be informed and treatment and counselling offered to them. Investigations forDonors 1. A thorough family history of a donor is essential. Genetic testing should be limited to the determination of carrier status of inherited recessive disorders in which an abnormal test result carries no significant direct health implications for the donor. Donors should be informed of the result of their test and offered post-test counselling. 2. In relation to cystic fibrosis, donors from the population groups known to have high frequencies of cystic fibrosis carrier should be screened. 3. Screening for Tay-Sachs, thalassaemia and sickle cell anaemia should be carried out in appropriate population groups. 4. HIV screening must be carried out in all donors. This entails the testing for HIV antibody at the time of donation and rechecked 3–6 months later. If donor oocytes are to be used immediately, there is slight risk that donor infection that will not be identified. Informed consent should be taken before screening. If test is positive, centre should offer to arrange HIV counselling. 5. All donors should be screened for cytomegalovirus antibodies. Ensure that oocytes from seropositive donors should be used only for seropositive recipients. It should be ascertained that these seropositives should be IgM negative indicating that they are unlikely to have an active infection, however they are IgG positive. Any donor who is

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initially seronegative and becomes seropositive during the treatment should notbe used. 6. It is essential that all recipients of donated oocytes should receive information explaining the limitations of the testing procedures used and any risk associated with the treatment. Benefits to the Donors 1. Monitory benefits may be made to the donors in exchange of oocyte donation in accordance with policy and the guidelines by the centre and appropriate authorities. 2. If an oocyte donor becomes ill as a direct result of making a donation, ART centre should reimburse any direct expenses that the donor incurs. Investigations and Prerequisites for Oocyte Recipients All couples wishing to undergo this treatment must provide following details: 1. Previous medical and gynaecological history. 2. Previous fertility treatment history 3. Up-to-date hepatitis B and C, syphilis and HIV serological tests for both partners. 4. Blood groups and Rh factors of both partners. 5. Completed and signed application form and informed consent forms for in vitro fertilization by oocyte donation. Medication for Recipients The treatment of the recipient is of great importance because the recipient and donor cycles should be synchronised in terms of endometrial response. When donor is ready for oocyte retrieval, the endometrium of recipient should be well prepared and be ready to receive the embryo. Preparation of endometrium: The exogenous hormones are administered to simulate the effects of the ovarian hormones on the endometrium. 1. In those patients who have normal ovarian function, embryo transfer could take place in a natural cycle; given the difficulty of synchronization with the donor, this option is rarely used. 2. In patients with preserved ovarian function, GnRH analogues are used from day 21 for 2 weeks or more (subcutaneous injection) so as to neutralize the action of the endogenous hormones that may interfere with the transfer cycle. With the onset of the following menstruation, recipient should have transvaginal ultrasound (TVS) to check endometrial and ovarian response. The hormone replacement should be started. 3. Patients without ovarian function would have withdrawal bleeding with a short course of progesterone hormone for 5–7 days, norethisterone acetate 10 mg daily Hormone replacement therapy should be started immediately in these patients. Hormone replacement or substitution treatment consists of increasing oral doses of oestradiol valerate starting with 2 mg from day 1 to day 8, 4 mg day 9 to day 11, and 6 mg from

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day 12 onwards. Oestradiol administered continuously without interruption. Oestrogens can also be given in form of transdermal patches or intramuscular route. 4. All patients have TVS to measure endometrial thickness and serum oestradiol estimation between days 14–16 after the onset of substitution therapy. Depending on the response the doses of oestradiol may be increased. The treatment with oestradiol can be continued for 100 days as long as there is no vaginal bleeding, if there is problem in obtaining oocytes from donors. 5. Oral contraceptive pills can be used in recipients to synchronise the cycles with donor if required, before starting oestrogen therapy. 6. Once endometrium is prepared in recipients, collected oocytes from donors are inseminated with semen from recipient’s partner or donor. 7. On the day of oocyte pick up, progesterone intramuscular 50–100 mg or vaginal 200– 800 mg is added along with oestradiol and both of these will be continued till about 12 weeks of pregnancy when placenta is sufficiently capable of producing these hormones. 8. Fertilisation should be checked after about 24 hours of oocyte pickup (18 hours after the insemination or ICSI). 9. Once the pre-embryo has reached to the 4 to 8 cell stage it is transferred to the uterus between 48 to 120 hours (2 days to 5 days). 10. Extra viable pre-embryos are cryopreserved for future use. Medication for Oocyte Donors 1. Down regulation with GnRH analogues from day 21 of the previous cycle for 10–14 days or as required. 2. On day 2–3 of withdrawal bleeding, TVS is performed to assess endometrial thickness and ovaries to rule out any persisting follicular cyst. Also basal levels of oestradiol, FSH, and LH are measured. 3. Controlled ovarian hyperstimulation (COH) or ovulation induction is started with gonadotropins, which may include recombinant or pure FSH, and human menopausal gonadotropins (hMG). 4. Appropriate monitoring of COH is done by TVS to assess ovaries and endometrium, and by blood levels of Oestradiol and LH. 5. Human chorionic gonadotropin (hCG) injection is given intramuscularly when appropriate 6. GnRH analogues should be continued till the day of human chorionic gonadotropin (hCG) injection. 7. Oocytes are then retrieved. Success of IVF with Oocyte Donation 7–10

In literature, the pregnancy rate per embryo transfer cycle is about 50 percent. Approximately 15–20 percent of these pregnancies will result in abortion and 20–25 percent will result in multiple births as twins, triplets and more. The ectopic pregnancy rate is 1–1.5 percent.

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There is a significantly higher clinical pregnancy rate for recipients who have a fresh embryo transfer compared with recipients whose embryo transfer consists of frozen and thawed embryos.11 With fresh embryo transfers, the clinical pregnancy and delivery rates per recipient are 50 to 60 percent and 45 to 50 percent, respectively. These rates with frozen embryo transfer are 45 to 50 percent and 39 to 45 percent respectively.7,8,12 Implantation rates are 32 percent and 26 percent for fresh and frozen embryo transfers, respectively.8 With combined fresh and frozen embryo transfers, cumulative pregnancy rate of oocyte donation and embryo transfer is about 50 to 57 percent.13 The status of ovarian function in recipients has an influence on pregnancy rates. The clinical pregnancy rates per transfer for fresh embryo transfers are 20 percent for recipients with ovarian function and 46 percent for recipients with ovarian failure (P<0.05). The pregnancy rates for frozen embryo transfers are 17 percent for recipients with ovarian function and 23 percent for recipients with ovarian failure (not significantly different).7 The most reliable predictive factors for pregnancy in oocyte donation cycles are the quality of the embryos transferred and the recipient’s mid-cycle endometrial thickness.9,10 The clinical pregnancy rate in cycles where the endometrial thickness is less than 8 mm is significantly lower when compared to cycles with an endometrial thickness of more than 8 mm. Cycles where optimal quality embryos are transferred have the better implantation (36%), clinical pregnancy (63%) and live birth (54%) rates and these rates are significantly higher than those of cycles where only poor quality embryos are available for transfer (10% implantation, 17% clinical pregnancy, and 8% live birth rates, respectively; P<.05).10 Low dose Aspirin (75 mg daily) with other hormones replacement, for oocyte recipients with a thin endometrium of less than 8 mm helps to improve implantation.14 CONCLUSION Oocyte donation is an excellent option for the patients with gonadal dysgenesis, premature ovarian failure, genetic disorders, and menopause. An appropriate selection of patients and comprehensive evaluation of the recipients and donors are important aspects for the success of oocyte donation programme. Medical, psychological, and ethical factors weigh heavily in the decision making to have a child at any age. Because of these and legal issues which are not well defined for the oocyte donation programmes, medical professionals must carefully consider the specifics of each case before oocyte donation, including a woman’s health, medical and genetic risks, and the provision for child rearing. Proper documentation of consent, investigations, procedures and treatment should be maintained to minimise legal complications and to safe guard the personnel involved in oocyte donation programme.

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REFERENCES 1. Lutjen P, Trouson A, Leeton J, Findlay J, Wood C. The establishment and maintenance of pregnancy using in-vitro fertilisation and embryo donation in patient with primary ovarian failure. Nature 1984; 307:174. 2. Klein J, Sauer MV. Oocyte donation. Best Pract Res Clin Obstet Gynaecol 2002; 16(3):277–91. 3. Lutjen PJ, Leeton JF, Findly JR. Oocyte and embryo donation in IVF programme. Clinics in Obstet and Gynaecol 1995; 12:(4),799–813. 4. Sauer MV, Paulson RJ, Lobo RA. A preliminary report on oocyte donation extending reproductive potential to women over 40. N Eng J Med 1990; 323:1157–60. 5. Sauer MV, Paulson RJ, Lobo RA. Pregnancy after 50: Application of oocyte donation to women after natural menopause. Lancet 1993; 341:321–45. 6. Sauer MV, Paulson RJ, Lobo RA. Pregnancy in women 50 or more years of age: outcome of 22 consecutively established pregnancies for oocyte donation. Fertility and Sterility 1995; 64:111– 15. 7. Check JH, O’Shaughnessy A, Lurie D, Fisher C, Adelson HG. Evaluation of the mechanism for higher pregnancy rates in donor oocyte recipients by comparison of fresh with frozen embryo transfer pregnancy rates in a shared oocyte programme. Hum Reprod 1995; 10(11):3022–27. 8. Moomjy M, Mangieri R, Beltramone F, Cholst I, Veeck L, Rosenwaks Z. Shared oocyte donation: society’s benefits. Fertil Steril 2000; 73(6):1165–69. 9. Check JH. The use of the donor oocyte program to evaluate embryo implantation. Ann N Y Acad Sci 1994; 30; (734):198–208. 10. Noyes N, Hampton BS, Berkeley A, Licciardi F, Grifo J, Krey L. Factors useful in predicting the success of oocyte donation: a 3-year retrospective analysis. Fertil Steril 2001; 76(1):92–7. 11. Check JH, Choe JK, Nazari A, Fox F, Swenson K. Fresh embryo transfer is more effective than frozen for donor oocyte recipients but not for donors. Hum Reprod 2001; 16(7):1403–8. 12. Lindheim SR, Sauer MV. Embryo donation: a programmed approach. Fertil Steril 1999; 72(5):940–1 13. Opsahl MS, Blauer KL, Black SH, Dorfmann A, Sherins RJ, Schulman JD. Pregnancy rates in sequential in-vitro fertilization cycles by oocyte donors. Obstet Gynecol 2001; 97(2):201–4. 14. Weckstein LN, Hampton K, Jacobson A, Janine H, Galen D. Low dose Aspirin for oocyte donation recipients with a thin endometrium: Prospective, Randomised study. Fertil steril 1997; 68; 927–30.

CHAPTER 52 Oocyte-Sharing Programs Gautam N Allahbadia, Goral N Gandhi, Prashant L Kharche, Shashank R Karekar, Avinash Phadnis INTRODUCTION For patients with ovarian failure, IVF is not a medical option. Nonetheless, these patients have a good chance of achieving pregnancy with oocyte donation. The known time, effort, commitment, discomfort, and minor risks derived from ovarian stimulation and oocyte retrieval continue to limit donor availability.1–3 Egg sharing is a form of egg donation where complete strangers can collaborate anonymously to overcome their involuntary childlessness. Since the Australian team of Trounson and Wood first reported a successful oocyte donation >I 5 years ago,4 the procedure has spread around the world, although in a limited way (small number of patients treated) when compared with sperm donation or with in-vitro fertilization (IVF) (2930 cycles of oocyte donation worldwide in comparison with 119,992 IVF cycles in 1991).5 This modest activity is probably due more to the difficulties of the procedure for the oocyte donor (IVF) than to the lack of medical indications, since it is estimated that >100000 women in the USA present with premature ovarian failure (before the normal age of menopause).6 From a purely rational point of view, oocyte donation is the mirror of sperm donation; it consists of introducing, in the couple, half of the genetic material from a third party donor (a male donor in the case of sperm donation, a female donor in the case of an oocyte donation). However, the similarity stops there; symbolically, sperm donation and oocyte donation are experienced very differently by couples. This was shown by a Californian study on the reaction of recipient couples to the possibility of recruiting the brother (or sister) of the sterile partner as the donor; whilst 86 percent of the women and 66 percent of their partners involved in oocyte donation stated that they would prefer the patient’s sister to an anonymous donor (moreover 80% had asked for it), only 9 percent of the women and 14 percent of the men involved in sperm donation expressed the same preference for the brother of the patient and no one had actually asked for it.7 This difference has its origins in the different perception of feminine and masculine sterility, both by couples and by society; a perception which frequently leads to hiding masculine sterility and to openly accepting feminine sterility.8 Some recipient candidates may also worry about the transmission of infectious or genetic disorders or, in case of non-anonymity, fear the ‘genetic’ donor ringing at the door some years later. However, a comparative examination of the differences and similarities between oocyte and sperm donation shows that this is not the case. In addition, maternity by oocyte donation repairs a double major wound in women not only confronted by the failure to become a mother, but also disturbed in their female identification (absence of a cycle) and even in their sexual identity (Turner’s syndrome, gonadal dysgenesis). All these reasons explain the massive denial observed in pregnant

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women or having given birth after an oocyte donation, which can go as far as ‘forgetting’ the distinctive character of their filiation ties.9,10 As far as the future of the children is concerned, the paucity of available data does not indicate real particularities11 in accordance with the studies carried out on children conceived by AID.12,13 Unlike sperm donation, oocyte retrieval is not without risk, either the risk related to ovarian stimulation or to oocyte retrieval, even to anesthetic risk in case of general anesthesia. An informed consent document is therefore all-important, and it must be preceded by the provision of meticulous information to the potential donor on the nonexceptional risks of treatment especially when it concerns non-anonymous donations where the medical risks often appear to be minimized by the potential donor.14 Since this consent is only of value if it is freely given, the discussion, in case of a related donor, must aim at detecting the candidates who may be pressurized by the recipient couple. In this case, it is necessary to act with tact to help the ‘non-candidate’ to get out of a commitment which has not been freely agreed to without compromising her relationship with the recipient couple, whether these relations are effective or professional ties. India, being a very conservative country which is just opening up to the western “liberated” ideas courtesy television is in its infancy as far as truly successful egg sharing programs are concerned. Over the past few years most of the egg donation cycles at our Center involved ‘siblings’ or close ‘family friends’. It is only in the last three years that true “egg sharing” as we understand it today has become popular and contributes to nearly 20 percent of our total cycles. This growth has been fuelled by the opening up of the world wide web and popular Indian cinema which has come of age in tackling subjects such as egg-sharing and surrogacy. At our Center, there are two types of eggsharing done; a non-patient egg donor whose complement of eggs can be shared by two recipients and more commonly the IVF patient who agrees to share her eggs with the recipient in lieu of her IVF fees being waived off. Unlike the West, we still do not come across professional egg donors who donate for a fee. These programs exist openly in the USA, the candidates being recruited by way of classified advertisements.15 It seems to be the difficulty of donor recruitment which plays a crucial role in all oocyte donation programs, leading to ethically questionable or unacceptable practices under the pressures arising from the shortage of donors. It is therefore essential to fully analyze this situation to find more efficient solutions (reduce the shortage) whilst fully respecting the dignity and rights of the oocyte donors. Our Center has been very progressive on this front with our home page having a link at http://www.iwannagetpregnant.com/egg.asp where we update the available anonymous egg sharer’s profiles including their photographs which help potential recipients check out the phenotypic matching and other characteristics before embarking on the cycle. This facility is especially useful where the recipients come from countries other than India. The Non-patient Egg Sharer between two Egg Recipients Donor recruitment is a difficult endeavour, taking into account that a considerable number of potential donors may have a positive finding on medical, genetic, or psychological testing that prohibits donation. Occasionally, we get young volunteer egg sharers who are ready to donate either for monetary reasons or purely altruistic reasons.

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Providing oocyte donation to two recipients from a single cohort of eggs obtained from a single donor has the theoretic advantage of permitting a greater amount of fresh Embryo Transfers. Eggs from non-patient volunteers may be allocated to anonymous multiple recipients with no dilution of the expected success rates.16 Historically, shared donation involved IVF patients willing to donate half of their oocytes to a recipient, of ten for reduction of treatment costs.1,2,17 Shared oocyte donation has emerged as a more efficient use of this precious resource of human oocytes. From a recent series, 90 percent of the time there was an adequate number of healthy mature oocytes available at the time of donation for two recipients.18 Because the mean number of oocytes received in the shared donation in this report was 8.3, all recipients received the recommended fresh ET of two or three embryos to have a 57 percent chance of a clinical pregnancy. This form of oocyte disposition permits less exposure of donors per families completed. Rarely, there are women who, without any ties to a recipient, donate oocytes anonymously either spontaneously or when undergoing surgical procedures unrelated to a sterility problem. This group consists mainly of patients coming for a tubal sterilization, which offers the advantage that they are of reproductive age and in good health, having proved their fertility and who undergo an operation (sterilization by laparoscopy) in the course of which the oocytes can be retrieved. Although this situation could appear ideal, experience has shown that this type of recruitment has been disappointing, the teams encountering a large number of refusals. Although the practical aspects are often put first and can in part explain these refusals (these women have children, an active life and the constraints of stimulation discourage them), it seems that more fundamental reservations of an unconscious rivalry of a woman who renounces her fertility for good vis-à-vis another woman who could take advantage of her sterilization to become a mother.19 Spontaneous initiatives from the general public are rare, and according to our experience, are very often taken by women with a particularly fragile personality and psychologically disturbed, looking for a recognition or a massive repair. For these reasons, they are generally not considered able to give free and informed consent and their use as donors involves great risks of disrupting an extremely unstable psychological balance. Related Donors are women recruited as donors by the couples themselves within their family circle or friends. Many oocyte donation programs in Western Europe operate in this way. The donors have a close relationship, often intra-family donations in the broadest sense. These donations, even if they come from very close relatives, are valued as much by the requesting couples7 as by the public at large, according to a Californian study,19 although with a little more reluctance from women (59% agree) than from men (74% agree). On the other hand, authorities occasionally oppose them invoking the fear of psychological consequences for the children involved in family relations which are too complicated.20 Given that only about 3 percent of all the eggs retrieved at IVF result in live births, most (>80%) of patients attending licensed 1VF Centers in the UK require repeat treatments.18 This raises concerns for the long-term well-being of non-patient donors who might repeatedly be exposed to unconfirmed risks of ovarian stimulation with gonadotrophins.21–24 Even if sufficient non-patient volunteers could be recruited there is increasing unease about medical aspects of their treatment which cannot be ignored. There are reports of an association between ovarian stimulation and cancer.22,25–27 These

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have quite different implications for a patient who requires IVF in order to have a child and opts to take the risks associated with ovarian stimulation compared to a known or anonymous donor who is likely already to be a mother of young children.28 Would she be willing to take the risk once she understood the issues unless there was undue family or peer pressures? Sister donors have shown a decreased ovarian response to stimulation even when large doses of drugs are used.29 There is also evidence of a high incidence of family turmoil and reproductive traumas amongst non-patient volunteers, including child abuse. Also, known donor situations can be an emotional minified of manipulation and abuse, e.g. donation to pay a debt.30–32 These findings should not be ignored. However, there is a group of potential donors which apparently has been very little explored, namely that of former IVF patients who have had their child by this technique. In our experience, without any intervention of the IVF team, we had only one volunteer from this group (in this case they were ‘related donors’: donors recruited by patients among their relations) and these women were especially relaxed and positive in their action. Nevertheless, shared ovum donation does result in an overall reduction of embryos available for cryopreservation. Another advantage of this approach is that having potential recipients per donor permits completion of stimulation and retrieval in the event that one recipient cancels her cycle for medical or personal reasons. There are concerns about increasing the possibility of inadvertent consanguinity brought about by the use of shared oocyte donation. Because some parents may choose not to disclose to their children their genetic origins when oocyte donation is used, it is remotely possible to have unknown consanguinity between individuals genetically related to same oocyte donor. The risk of potentially yunknown consanguinity is not absent in unshared oocyte donation because donors may have their own children. This raises responsibility issues for the programs, to limit the number of donor attempts, for donor safety and to avoid future couples of unknowing of genetic half-siblings. National guidelines by the Indian Council for Medical Research (ICMR) for prudent limitation of retrievals and pregnancies per donor are currently being debated. Patients Undergoing IVFTreatment and Agreeing to Share Oocytes This involves asking patients who are undergoing oocyte retrieval for their own needs to donate some oocytes to an anonymous recipient, provided they have ‘a sufficient number’. In our program, egg-sharing is practiced in conjunction with other forms of egg donation. Couples who are suitable for egg-sharing in this form of egg donation normally participate in it because of the mutual-help nature of the scheme. Many who are prepared to donate some of their eggs in return for less expensive fertility treatment regard it as recompense and an acceptable compromise: much more acceptable than being content with no treatment at all. Although the unquestionable advantage of this approach is that the donor does not have to suffer any additional medical aggression, it does involve some ethical objections, e.g. is a patient who depends on the medical team for her own treatment able to give her consent freely to a process which potentially reduces her chances of success? From what number of collected oocytes would it be legitimate to retrieve oocytes? Nevertheless. This source of oocytes has been historically important for the development of this technique33,34 and is still current.35,36 At our Center, we aim for

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14–16 oocytes being retrieved from the egg-sharer which are then randomly and equally divided between the sharer and the recipient. There is a fear that the recipients who pay- receive ‘better’ eggs or, conversely, there could be a bias towards the donor. Egg selection is a random process as, at egg collection, the egg quality cannot be determined very accurately. In egg-sharing, eggs are allocated randomly to donor and recipient which maximizes the benefit to both parties. Secondly, it is argued that the donors with unsuccessful outcomes might suffer psychologically if they feel that the recipients might have been successful. An unsuccessful IVF treatment cycle will always cause the patient distress. There is no previous evidence to indicate that it is more upsetting for the patient who has also been the donor. Indeed, many donors participating in egg-sharing have either donated more than once or are waiting to do so again even though they have no knowledge of the outcome of the recipient’s treatment. For some donors the distress of an unsuccessful treatment is eased with the thought that their recipient may have been successful. Furthermore, in the comparative data on IVF patients who voluntarily donate a proportion of their oocytes, there is no reported evidence of adverse psychological effects on donors, even though this form of donation has been available in many countries for many years.35,37–40 We believe that access to counseling and informed consent are pivotal and, provided these are available, it cannot be argued that women of a reproductive age and their partners are incapable of making rational and informed decisions about donation. We have a qualified psychiatrist who counsels all such couples before they are enrolled into our program. The main motivation for donation is acknowledged to be the desire to help others start a family: a feeling greatly accentuated in volunteers, including IVF patients, who are knowledgeable about infertility.10,30,39,41 The fact that some IVF patients donate eggs whilst receiving a lower cost of treatment themselves does not, in our experience, diminish that feeling. A study from the UK has shown that by each using half the available eggs, donors and their anonymous recipients do not compromise their probability of success.2 Similar conclusions were reached in at least two other independent recent studies on egg sharing.42,43 Virtually, all the successes were achieved when eggs were provided mostly by sharers whose primary cause of inf ertility was either blocked Fallopian tubes, poor spermatozoa requiring treatment with intracytoplasmic sperm injection (ICSI) or a combination of these. In this highly selected group of patients, each completed egg collection cycle resulted in the birth of at least three infants to two sharing patients, in contrast to the more usual three or more egg collection cycles required on average for the birth of a single infant.18 For the non-patient donor, however, there is no gain whereas for the patient (egg sharing) donor there is at least the potential for her to produce a baby.43 There are a few negative aspects to egg sharing, chief of which is that the donor has to part with half of her eggs. This could lead to her believing that she is forfeiting her best eggs and she may become disillusioned if repeated treatments have no positive results for herself. Patients who become disillusioned with repeated failures are unlikely to advocate egg sharing; they might instead increase anxiety if their own experience leads them to believe that they have given away their best eggs or reduced their chances by reducing the number of eggs.44 It must be said that “robbing Peter to pay Paul” is unlikely to assist egg sharing. Conversely younger women with potential as providers or sharers of better eggs could

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help establish egg sharing as a meaningful solution to a growing social dilemma, provided the intended benefits are not undermined by the act of sharing. A criticism that subsidized IVF treatment represents a financial reward for an egg sharer is often voiced.44 But the authors’ experience indicates that the financial or social status of the donors is seldom a major consideration for treatment in an egg sharing program. The argument that the poor are being preyed upon by the wealthy is inconsistent with the facts. In almost every case the donor egg sharer is attracted principally by the basic human desire for reciprocation but feels some recognition is entirely appropriate.45 Technical Minutiae The HFEA criteria for egg donation from UK are applied at our Center, which include an upper age limit (i.e. 35 years) and a satisfactory day 3 hormone profile. A negative screen for Koch, HIV, HCV, HbsAG and antichlamydial antibodies is imperative. Donors and recipients are matched for their physical characteristics. They are offered independent counseling prior to being accepted into the egg sharing program. Protocols for ovarian stimulation (donors) and hormone replacement therapy (recipients) and details of laboratory procedures have been described in previous publications.2 There was no difference between the ovarian stimulation protocols used for egg share and non-egg share treatment cycles. The rules for selection are in principle identical to those which prevail for sperm donors, with regard to both the psychological aspects and the genetic or infectious aspects; nevertheless some differences exist. If it is ‘biblically’ simple to donate spermatozoa, the same is not true for oocytes; it is necessary for the female donor to undergo a full procedure of ovarian stimulation and oocyte collection, which represents a significant effort in terms of availability and is not entirely without risk, even though complications are rare.3 This can explain the difficulty in recruiting oocyte donors which has been reported by all oocyte donation programs and which requires recourse to various strategies. On the other hand, the difficulty associated with the small number of oocytes that can be collected during oocyte retrieval leads to the problem of the limitation of number of children by donor (the risk of inbreeding ignored for the descendants) being nonexistent in the field of oocyte donation. Lastly, the present inability to store non-fertilized oocytes makes the use of quarantine as a prevention of risks of infection as in sperm donation extremely difficult. Moreover, it complicates the programs of anonymous donation since female donors and recipients must follow the treatment in parallel. The increase in genetic risk with increasing age also implies more restrictive age limits than for sperm donors. It is generally accepted that no special precautions are required when the donor is aged <35 years. After this, the genetic risk increases and the efficacy of the procedure diminishes.46 Finally, the exceptional aspect of oocyte donation makes the matching procedure of morphological characteristics to a recipient couple more random and most of the centers restrict this to racial matching in the West. In India, again possibly due to the ingrained ultraconservative mindset which is just accepting this medical “advancement”, phenotypic matching is a lengthy and a “very important” part of the egg sharing cycle. The egg-sharing cycle is initiated with the recipient’s induced withdrawal bleed. From day one of her menses, she starts estradiol valerate at a prescribed dose and continues this

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till the dose requires to be altered, if at all; usually after serial transvaginal sonography guided examinations of the endometrial thickness and morphology. Some programs do a serial hormonal profile as well. This hormonal replacement therapy (HRT) can be continued with or without the cover of a GnRH agonist. After the initiation of the HRT in the recipient, we begin the ovarian stimulation of the egg sharer using the standard long protocol. Parenteral Progesterone is initiated for the recipient one day before the Eggsharer’s Oocyte pickup. Both their embryo transfers are done on the same day at our Center. From a purely medical perspective, the method described above offers researchers a highly useful model to design studies aimed at explaining the interplay between the ovarian and uterine factors at implantation. During the life span of a woman there is a continuous but variable decline in ovarian follicle production.46 It is logical to expect that women with a comparatively large ovarian reserve will achieve greater success in an egg share process and it is this very principle that we apply at our Center and advocate egg sharing to be done by women under the age of 30 at our Center. If such women could be identified simply, one might realize high efficiency in allocating eggs to recipients with consequent reduction in the number of repeat treatments needed to achieve success. So far a better system has not been identified than a day 3 FSH of <6 IU/L, and a history of previous stimulation attempts with fertilization score from a previous IVF attempt. We have a pregnancy rate of nearly 66 percent per started cycle in these egg-sharing cycles. It is indeed plausible that the embryo implantation results attained here simply point to the use of donors younger than the recipients. Even so, if a ready mechanism for identifying a high quality egg source is accomplished, it could enable us to target a highly desirable reduction in the frequency of multiple births.47,48 CONCLUSIONS Egg sharing provides a platform for anonymous but sophisticated social interaction among infertile couples. Different healthcare systems have universally failed to fill the widening gap between the demand and supply of donor eggs. Neither the pursuit of absolute altruism (volunteer non-patient donors) nor possibly, the selfish pursuit of trade (paid nonpatient donors) are universally acceptable or effective. There is still no consensus concerning the association between ovarian stimulation and epithelial ovarian carcinoma,49–53 but it is clear that this theoretical risk is a concern. By means of shared oocyte donation, one can decrease the population of donors exposed to ovarian stimulation for a given population of recipients. Alternatively, a conservative limit on total donation cycles per donor can be set to similarly reduce exposure to ovarian stimulation. This is an issue of importance when one must decide on risk exposures. Cost reduction with this form of treatment is obvious. For anonymous oocyte donation, sharing of oocytes between two phenotypically matched recipients provides a good opportunity for the recipient to experience pregnancy, while keeping medical risks and discomforts to an absolute minimum for the donor group. Precious human oocytes are well utilized in this form of shared oocyte donation. As the numerous direct and indirect benefits of egg sharing become more widely known, this practice is likely to become the preferred ethical option for qualifying couples. The ultimate objective is for supply to meet the

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demand in a rational and ethical manner. In conclusion, a strong case for a carefully controlled shared egg scheme exists since there are two infertile groups of women who can be of mutual help to each other by means of a single egg collection. Since the scheme is not restricted to the disadvantaged, there is no justification not to encourage the development of egg-sharing which offers treatment and hope to many who would otherwise be helpless. REFERENCES 1. Ahuja KK, Mostyn BJ, Simons EG. Egg sharing and egg donor attitudes of British egg donors and recipients. Hum Reprod 1997: I,, 2845–52. 2. Ahuja KK, Simons EG, Fiamanya W, Dalton M, Armar NA, Kirkpatrick P. Egg-sharing inassisted conception: ethical and practical considerations. Hum Reprod 1996; 11:1126–31. 3. Englert Y, Govaerts I. Oocyte donation: particular technical and ethical aspects. Hum Reprod 1998; 13 (Supp 2):90–7. 4. Trounson A, Leeton J, Besanko M. Pregnancy established in an infertile patient after transfer of a donated embryo fertilized in vitro. Br Med J 1983; 286, 835–36. 5. Cohen J, de Mouzon J, Lancaster P (Eds). World Collaborative Report 1991. Presented in VIIth World Congress on in vitro Fertilization and Alternative Assisted Reproduction, Kyoto, 1993. 6. Rozenwaks ZO. Donor eggs—the applications in modern reproductive technology. Fertil Steril1987; 47:895–97. 7. Sauer MV, Rodi LA, Scrooc M. Survey of attitudes regarding the use of siblings for gamete donation. Fertil Steril 1988; 49:721–22. 8. David D, Soule M, Mayaux MJ. lAD: enquete psychologique sur 830 couples. J Gynecol Obstet Biol Reprod 1988; 17:47–74. 9. Weil E. L’abord psychologique des couples receveuses de dons d’ovocytes anonymes. Contracept Fertil Sex 1987; 7:690–91. 10. Raoul-Duval A, Letur-Konirsch H, Frydman R. Les enfants du don d’ovocytes anonyme personnalise. J Gynecol Obstet Biol Reprod 1991; 20:317–20. 11. Raoul-Duval A, Bertrand-Servais M, Letur-Konirsch H et al. Que sont ces enfants devenus: les enfants de procreations medi- calement assistees. Medecine/Science 1993; 9:747–51. 12. Manuel C, Czyba JC. Les Aspects Psychologiques de L’insemination Artificielle par Donneur. SIMEP, Villeurban, 1983. 13. Golombok S, Cook R, Bish A, Murray C. Families created by the new reproductive technologies: quality of parenting and social and emotional development of the children. Child Dev 1995; 66:285–98. 14. Weil E, Comet D, Sibony C. Psychological aspects in anonymous and non anonymous oocyte donation. Hum Reprod 1994; 9:1344–47. 15. Schover LR, Rothmann SA, Collins RL. The personality and motivation of semen donors: a comparison with oocytes donors. Hum Reprod 1992; 7:575–79. 16. Englert Y, Rodesch C, Van den Bergh M, Bertrand E. Oocyte shortage for donation may be overcome in a programme with anonymous permutation of related donors. Hum Reprod 1996; 11:2425–28. 17. Peskin BD, Austin C, Lisbona H, Goldfarb JM. Cost analysis of shared oocyte in vitro fertilization. Obstet Gynecol 1996; 88:428–30. 18. Ahuja KK, Simons EG, Rimington MR, Nair SA, Gill A, Evbuomwan I. One Hundred and three concurrent IVF successes for donors and recipients who shared eggs: ethical and practical benefits of egg sharing to society. RBM online 2000; 1(3):101–05.

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19. Lessor R, Reitz K, Balmaceda J, Ash R. A survey of public attitudes toward oocyte donationbetween sisters. Hum Reprod 1990; 5:889–92. 20. Robertson JA. Ethical and legal issues in human egg donation. Fertil Steril 1989; 52:353–63. 21. Ahuja KK, Simons EG. Anonymous egg donation and dignity. Human Reproduction 1996; 11:1151–54. 22. Ahuja KK, Simons EG. Cancer of the colon in an egg donor: policy repercussions for donor recruitment. Human Reproduction 1998; 12:2230–34. 23. Nieto JJ, Rolfe KJ, MacLean AB, Hardiman P. Ovarian cancer and infertility: a genetic link? Lancet 1999; 354, 649–51. 24. Rossing MA, Daling JR. Complexity of surveillance for cancer risk associated with in vitro fertilisation. Lancet 1999; 354:1573. 25. Banderra CA, Cramer DW, Friedman AJ, Sheets EE. Fertility therapy in the setting of a history of invasive epithelial ovarian cancer. Gynaecol Oncol 1995; 58:116–19. 26. Dor J, Lemer-Geva L, Rabinovici J. Cancer incidence in a cohort of infertile women treated with in vitro fertilization. Presented in 52nd Annual meeting of the American Society for Reproductive Medicine. Suppl, 1996; 147. 27. ShusanA, Paltiel O, Iscorich J. Human menopausal gonadotropin and the risk of epithelial ovarian cancer. Fertil Steril 1996; 65:13–18. 28. Ramogida C. The views of the patients. In Shenfield F, Sureau C. (Eds): Ethical Dilemmas inAssisted Reproduction. Carnforth, UK: Parthenon Publishing Group, 1997. 29. Sung L, Karstaedt A, Mukherjee T. Sisters of women with premature ovarian failure may not be ideal ovum donors. Fertil Steril 1997; 912–15. 30. Schover LR, Collins RL, Quigley MM. Psychological follow up of women evaluated as oocyte donors. Hum Reprod 1991; 6:1487–91. 31. Saunders DM, Garner F. Oocyte donation using ‘known’ donors: it may seem the convenient answer but who pays? Hum Reprod 1996; 11:2356–57. 32. Lockwood GM. Donating life: practical and ethical issues in gamete donation. In Shenfield F, Sureau C (Eds): Ethical Dilemmas in Assisted Reproduction. Camforth, UK: Parthenon Publishing Group, 1997; 23–30. 33. Kemeter P, Frichtinger W, Bernat E. The willingness of infertile women to donate eggs. In Frichtingen W and Kemeter P. (Eds), Future aspects in Human in Vitro fertilization. Berlin: Springer Verlag, 1987; 145–53. 34. Junca AM, Cohen J, Mandelbaum J. Anonymous and non-anonymous oocyte donation. Preliminary results. Hum Reprod 1988; 3:121–23. 35. Power H, Baber R, Abdalla H. A comparison of the attitudes of volunter donors and infertility patient donors on an ovum donation programme. Hum Reprod 1990; 5:353–55. 36. Oskarsson T, Dimitry ES, Mills MS. Attitudes towards gamete donation among coules undergoing in vitro fertilization. Br J Obstet Gynaecol 1991; 98:351–56. 37. Flamigni C, Boring, Violini DF. Oocyte donation: comparison between recipients from different groups. Hum. Reprod 1993; 8:2088–92. 38. Check JH, Askari HA, Fisher C, Vanamann, L. The use of a shared oocyte programme to evaluate the effect of uterine senescence. Fertil Steril 1994; 61, 252–55. 39. Yaron Y, Amit A, Mani A. Uterine preparation with oestrogen for oocyte donation assessing the effect of treatment duration in pregnancy rates. Fertil Steril 1995; 63:1284–90. 40. Yaron Y, Amit A, Brenner SM. In vitro fertilisation and oocyte donation in women 45 years of age and older. Fertil. Steril 1995; 63:71–74. 41. Sauer MV, Paulson RJ, Lobo RA. Pregnancy in women 50 or more years of age: outcome of 22 consecutively established pregnancies from oocyte donation. Fertil Steril 1995; 64:111–15. 42. Check JH, Choe JK, Ketsoff D, Summers-Chase D, Wilson C. Controlled ovarian hyperstimulation adversely affects implantation following in vitro fertilisation and embryo transfer. J Assist Reprod Genet 1999; 16:16–20.

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43. Van den Branden K, Evenepoel J, Tournaye H et al. Egg sharing does not jeopardise the pregnancy rates in oocyte donors. Annual Meeting of ESHRE, Tours, France. 1999; 15 (Abstract 087). 44. Johnson MH. The medical ethics of paid egg sharing in the UK. Human Reproduction 1999; 14:1912–18. 45. Ahuja KK. Egg sharing: ethics and practice. In Progress Educational Trust Symposium Recruiting gamete donors in the 21st Century: Principles and Practice. London: Royal Society 1997. 46. Faddy MJ, Gosden RG. Mathematical model for follicle dynamics in human ovaries. Hum. Reprod 1995; 10:770–75. 47. Fisk NM, Trew G. Two’s company, three’s a crowd for embryo transfer. Lancet 1999; 354:1572–73. 48. Royal College of Obstetricians and Gynaecologists The management of infertility in tertiary care. Evidence-based clinical guidelines No.6. RCOG, London, UK, 2000. 49. Rossing MA, Daling MR, Peretz T. Ovarian tumors in a cohort of infertile women. N Engl J Med 1994; 331:771–76. 50. Shushan A, Patliez S, Peretz T. HMG and the risk of epithelial ovarian cancer. Fertil Steril 1996; 65:73–78. 51. Mosgaard BJ, Lidegaard U, Kruger S. Infertility, Fertility Drugs and Invasive Ovarian Cancer. Fertil Steril 1997; 67:1005–11. 52. Parazzini P, Negri F, Vecchia A. Treatment for infertility and risk of ovarian cancer. Hum Reprod 1997; 12:159–61. 53. Mosgaard BJ, Lidegaard U, Andersen A. The impact of parity, infertility and treatment with fertility drugs on the risk of ovarian cancer. Acta Obstet Gynecol Scand 1997; 76:89–95.

CHAPTER 53 Germinal Stem Cells: Culture and Replication Jayant G Mehta, Thankam R Varma INTRODUCTION Development of embryonic stem cells (ES) evolved out of work on mouse teratocarcinoma tumors that arise in the gonads of a few inbred strains and consist of a remarkable array of somatic tissue, juxtaposed together in a disorganized fashion. Origins of teratocarcinomas from germ cells in mice provided the concept of a stem cell (the embryonal carcinoma or EC cell) that can give rise to the multiple types of tissues found in the tumors.1,2 Following the generation of chimaeric mice by blastocyst injection of EC cells, investigators began to realize the potential value of cultured cell lines from the tumors or models for mammalian development.3 Teratocarcinomas can also be developed spontaneously from primordial germ cells in some mouse strains, or following transplantation of primordial germ cells to ectopic sites.4 These EG cells have a developmental capacity very similar to that of embryonic stem cells, though they differ in their expression of some imprinted genes. Pluripotent stem (PSC) cell lines have been developed from testicular teratocarcinomas which occur spontaneously in humans.5 Cloned cell lines from human teratocarcinomas, have been reported to differentiate in υitro into neurons and other cell types.6,7 Pera et al,8 developed cell lines that could differentiate into tissues representative of all three embryonic germ layers—mesoderm, endoderm and ectoderm. These three germ layers are the embryonic source of all cells of the body (Table 53.1).9 Pluripotent stem cells have also been derived from two embryonic sources, embryonic stem cells being derived from the inner cell mass of preimplantation embryos10,11 and embryonic germ cells from Primordial germ cells (PGC).12,13 A new era in stem cell biology began in 199814,15 with the derivation of cells from human blastocysts and fetal tissue with the unique ability of differenting into cells of all tissues in the body, i.e. the cells are pluripotent.

Table 53.1: Embryonic germ layers from which differentiated tissues develop Enibryonic germ layer

Differentiated tissue

Endoderm

Thymus Thyroid, Parathyroid glands, larynx, trachea, lung, urinary bladder, vagina, urethra, Gastro-intestinal (GI) organs (liver, pancreas), lining of G1 tract and lining of the respiratory tract

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Mesoderm

Bone marrow (blood) Adrenal Cortex Lymphatic tissue Skeletal, smooth and cardiac muscle Urogenital systems Connective tissues (including bone cartilage) Heart and blood vessels (vascular system) Ectoderm Skin Neural tissue (neuroectoderm) Adrenal medulla Connective tissue of the head and face Pituitary gland Eyes, ears Source: Chardran KJ, Mezey E (2001)9

They can be grown in vitro and expanded in number indefinitely in the primitive undifferentiated state characteristic of the embryonic cells from which they are derived. They meet the generic criteria, in that they retain a normal karyotype during extensive cultivation in vitro and possess telomerase activity, indicative of them being immortal. However, embryonic germ cells have not been grown for such long periods. But there is no indication that their life span is finite. In the case of the germ cell derived cultures, no evidence has been presented regarding formation of teratomas in vivo, but reports of in vitro differentiation within embryoid bodies has been observed. Embryoid bodies (EBs) are differentiated cell aggregates first described as arising in human16 mesoderm, endoderm and ectoderm, and mouse teratomas and teratocarcinomas.17–19 These aggregates range from a cluster of pluripotent stem cell enclosed by a layer of endoderm to complex structures closely resembling an embryo during early development. EBs from mouse PSC grow on feeder layers or in suspension and may contain a variety of cell types. This property has been used as evidence of cell pluripotency20,21 and as a source of differentiating cells. Although little is known about the primordial germ cell maturation in man, this period encompasses those developmental stages in which primordial germ cells arrive in the gonads and proliferate, and overt sexual differentiation of the gonad takes place. In the human embryonic and foetal gonad, through this period, cells expressing markers characteristic of primordial germ cells are present.22 In order to support mouse primordial germ cell survival and mitogenesis different groups have supplemented the culture medium with basic fibroblast growth factor, Leukemia Inhibitory Factor (LIF) and Forskolin.22,23 In presence of proper growth and differentiation factors, it has been reported that mouse EG cultures can generate cells of hematopoietic lineage and Cardio myocytes.24,25 The most definitive in υiυo test of developmental potential would be a demonstration of contribution to all lineages in chimerical animals. However, this test is not practical in all species and cannot be done in human cells. In addition, both ES and EG cells share several morphological characteristics such as high levels of intracellular alkaline phosphatase (AP) and presentation of specific cell surface glycolipids26,27 and glycoproteins.28 The potential applications envisioned for human PSC with these properties is very profound; new approaches to the study of human embryonic development and disorders thereof, such as birth defects and embryonal tumors; access to hitherto-unexplained areas

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of human embryonic gene expression for modern genomic data; new tools for discovery of polypeptide growth and differentiation factors that might find application in tissue regeneration and repair; new means of creating human disease models in vitro for basic research, drug discovery and toxicology; a potential answer to the issue of the chronic shortage of tissue for transplantation in the treatment of degenerative diseases, and an end to the use of immuno suppressive therapy in transplantation, if cloning techniques can be used to derive stem cells from patient’s own tissue; new delivery system for gene therapy. TISSUE—CULTURE MATERIALS AND ROUTINE PROCEDURES All stem cell isolation and maintenance should ideally be carried out in an independent sterile embryonic cell culture laboratory. Standard, sterile tissue-culture procedures should be employed throughout. Although the majority of tissue culture is performed within the vertical—flow cabinet, isolation of EG cells requires the use of dissecting microscope. Care should be taken to keep the microbial contamination to a minimum. It is advisable that testing for mycoplasma—infected cells should be periodically undertaken. Equipment The essential equipments, required for the tissue culture laboratory are listed in Table 53.2.

Table 53.2: Essential equipment of tissue culture laboratory • A-vertical—flow tissue -culture cabinet • Humidified 5 % CO2 (in air) incubator set at 37°C • An inverted, phase contrast microscope (magnification: 40, 100 and 200X) • A binocular dissecting microscope with transmitted illumination (mag 12–100x) mounted in a separate laminal flow cabinet • Disposable plastic pipets, 1, 5, 10 and 25 ml • A vacuum pump, with two waste traps to aspirate spent medium through a tube connected to disposable, heat-sterilized, long—form glass Pasteur pipets • A bench top centrifuge

Tissue Culture Media and Solutions Medium All tissue culture procedures use Dulbecco’s Modified Eagle’s Medium (DMEM) without Sodium Pymiate but high in glucose (4500 mg/ml). This can be purchased either in liquid or as powder from tissue culture suppliers (Life Technologies Inc). DMEM is

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routinely supplemented with Pencillin and Streptomycin to control bacterial contamination. Amphotencin B (Sigma Chemicals Co) added to a final concentration of 2 µg/ml will control fungal contamination. Other solution needed is Trypsin EDTA 0.25 percent (w/v) trypsin in 0.04 percent (w/v) EDTA, for use in routine subculture of both fibroblasts and stem cell lines. Serum For the successful growth of most tissue culture cells both Fetal Calf Serum (FCS) and Newborn Calf Serum (NCS) are required. The serum quality is critical and should be selected after testing several samples provided by suppliers for their respective planting efficiencies with established ES or feeder-dependent embryonal carcinoma cell lines. The batch giving the highest plating efficiency, with no toxicity at 35 percent serum concentration should be purchased in bulk order. Preparation of Feeder Cells Historically, stem cells have been isolated and maintained on layers of mitotically inactivated, embryonic fibroblast “feeder cells.” If cultured in the absence of feeders, the pluripotent cells rapidly differentiate. The most commonly utilized fibroblast feeder layers have been those prepared from continuous STO cell lines.29 It has been shown that a factor known as DIA/LIF, which inhibits the differentiation of stem cells, is produced by these feeder cells30 as both, a diffusible protein and in an immo bilized form, associated with the extracellular matrix.31 Further more, pluripotent cells in a co-culture system secrete a heparin-binding growth factor responsible for the stimulation of DIA/LIF expression in the feeder cells 30. If recombinant DIA/LIF is added to the culture media, PSC lines retain their capacity for germ-line transmission without feeder cells.32,33 Other embryonic.29,30 fibroblast cell lines which have been shown to be capable of maintaining PSC include C3H 1071/2 cells and BAL B-373/ A31 cells.32 In order to prepare feeder cell layers for co-culture with embryos or pluripotent cells, the fibroblast cells must be mitotically inactivated. This is achieved by treatment with the drug mitomycin C29 or by exposure to irradiation.33 Derivation and Culture of Human Embryonic Germ Cells The Embryonic Germ (EG) cells are derived from Primodial Germ Cells (PGC).13,14 PGC were obtained from gonadal ridge and mesentery of 5 to 9 weeks gestation fetal tissue (PGC are responsible for giving rise to germ cells eggs and sperm in adults). PGC were mechanically and chemically desegrated in Dulbeco’s minimum essential medium (Life Science Inc.). The chemical desegregation involves incubation of the PGC for 5–10 minutes in 0.25 percent trypsin-EDTA (Sigma) at 37°C or incubation in DMEM supplemented with 0.01 percent Hyaluronidase type V (Sigma), 0.1 percent collagenase type IV (Sigma) and 0.002 percent D nase type I (Sigma) at 37°C for 2 hours.15 Segregated cells are subsequently cultured and passage on a mouse STO fibroblast feeder layer, mitotically inactivated with 5,000 rads (1 rad=0.01 Gy, r=radiation). Cultures were maintained at 37°C in 5 percent CO2 in air and 95 percent humidity and

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routinely passaged every 5 days after desegregation with 0.25 percent trypsin EDTA at 37°C for 5–10 minutes. The culture medium was supplemented with 15 percent human serm albumin (Immuno), 0.15 mM non-essential amino acids, 0.15 mMβmercaptoethanol (Sigma), 2 mM glutamine, 1 mM sodium pymvate, 100 units of Penicillin, 50 µg/ml of Streptomycin, 1000 units/ml of human recombinant leukemia inhibitory factor (hr LIF, Genzyme), 1.5 µg/ml of human recombinant basic fibroblast growth factor (hrb FGF, Genzme) and 10 µg/ml forskolin (Sigma). After two to four weeks, in vitro PGCs form dense multilayered colonies of cells that resemble mouse ES or EG cells (Fig. 53.1). A small variable percentage (1–20%) of PGC -derived cells which colonised spontaneously formed embryoid bodies. The growth medium for embryoid bodies lacked LIF, bFGF and forskolin. At this stage, embryoid bodies were collected from the culture and examined for the cell types they contained or were replated into single wells of a 4 well tissue culture plate for a further 14 days.

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Fig. 531: Panels showing growth of colony of stem cells after (a) day 3, (b) day 4, (c) day 5 of culture. Colony increases in size in the absence of overt differentiation. The constituent cells

576

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adhere tightly to one an- other making difficult to see individual cells. This growth pattern is characteristic of stem cells It has been reported that based on the appearance of the cells and surface markers, they expressed, the embryoid bodies, included derivatives of all three embryonic germ layersendoderm, mesoderm and ectoderm. These results suggested that PGC—derivative cells were pluripotent, however, it was not possible to demonstrate pluripotency in vivo by generating the formation of teratomas in mice.11 Expansion of Pluripotent Stem Cells into Permanent Cell Lines The first passage in culture is used to screen out all of the differentiated colonies and to select only those that display stable PSC morphology. The cultures are monitored daily and desegregated once PSC colonies attain a “suitable large size: If several PSC colonies are present in the first passage, the entire culture can be typsinized as follows: 1. Aspirate the old medium and wash cells in situ with PBS and 20 ml of Trypsin EDTA (TEDTA) solution. 2. Incubate for 5 to 7 mins at 37°C. 3. Using mouth controlled pipet dissociate colonies further into single cells. 4. The cell suspension is then transferred into a STO feeder well of a four well plate containing 1 ml of Embryo stem cell culture medium (ES).21 5. Observe on daily basis, and after 2 to 3 days. Colonies of PSC like cells may be visualized in some of these cultures. From the four well plates, the PSC like cells are subsequently expanded into 25 cm2 flasks containing a layer of STO feeder cells. 6. The flask is then cultured in the 5 percent CO2 in air incubators at 37°C Maintenance of Pluripotent Cell Lines After the 5th passage, PSC can be “weaned” from the STO feeder layer and the serum concentration in the medium reduced by half. PSC cells are subsequently maintained on gelatin-coated flasks in ES medium supplemented with DIA/LIF to prevent PSC. differentiation. PSC are generally grown in media containing 10 µg DIA/LIF to maintain stem cell morphology34,35 Subculture of PS Cells Passage PSC lines every 3 to 4 days changing medium every other day or whenever the media becomes acidic. 1. When confluent the cells are refed 2 to 3 hours before the passage to maximize subsequent cell survival. Media is aspirated and cells washed twice in PBS 2. Add 2 ml of TEDTA and incubate flask at 37°C for 3 min. Observe for the cells to dissociate.

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3. Add 3 ml of ES+DIA/LIF medium and mix suspension thoroughly. 4. Cells can now be sub-cultured by transferring a 1/ 10 aliquot of this suspension into a pregelatinized flask. Freezing and Thawing of Pluripotent Cells Freezing Cells 1. Cells collected after trypsinization are then pelleted by centrifugation @ 1200 RPM for 5 minutes 2. Resuspend the pellet in 0.5 ml of ES+DIA/LIF medium. 3. Equal volume of 20 percent (v/v) DMSO in ES+ DIA/LIF medium is gently added to the resuspended pellet. 4. Dispense 1 ml suspension into the cryotube labeled with the PSC line and plunged into liquid nitrogen, clipped onto a freezing cane. Thawing Cells 1. Remove the cryotube from liquid nitrogen tank and thaw quickly in a 37°C water bath until ice crystals have all melted. 2. Sterilize the cryotube by wiping with 70 percent alcohol. 3. Transfer the 1 ml cell suspension into 9 ml of ES+ DIA/LIF medium, pellet at 1200 rpm for 5 minutes. 4. Resuspend the pellets in 10 ml of ES+DIA/LIF medium and transfer to a pregelatinized, 25 cm2 tissue culture flask and culture in 5 percent CO2 in air incubator at 37°C. 5. Change the medium after 4 to 6 hours to remove any cell debris. 6. Observe for confluence within 1 to 2 days. In Vitro Differenttetion in Pluripotent Cells Pluripotent cells can be induced to differentiate along the pathways thought to be analogus to those early embryonic development, leading to the formation of embryoid bodies. The embryoid bodies have been reported to contain alpha-fetoprotein and transferrin and are thus analogous to the visceral yolk sac of the postimplantation stage mouse embryo. Formation of Simple Embryoid Bodies 1. Add 1.5 ml of 2 percent (w/v) agrose in PBS to 6 cm Sterile plastic dish to give an even layer and leave at room temperature to set. 2. Once set, add a further thin layer this time using 1 percent (w/v) agrose in PBS—allow it to set. 3. 5 ml of DMEM+0.1 ml β-mercaptoethanol is added to each dish and incubated at 37°C to allow equillibration.

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4. Lightly trypsinize Pluripotent cells for 1 to 2 minutes. Once large clumps of cells are detached quickly neutralize trypsin with DMEM medium and 0.1 mM mercaptaoethanol. 5. Using a wide bore pipette, and taking care not to break up cellular aggregates dispense 1/20 aliquot of aggregate suspension on to the prepared agrose dishes. 6. Feed the culture regularly, aspirating the old medium and adding fresh medium. Observe for embryoid bodies which will form within 2 to 4 days and they will become cystic after 7 to 10 days. This is seen as a dark layer separating the endoderm cells from the undifferentiated core cells. Both visceral and parietal endoderm cell types are formed. The parietal endoderm cells secrete a thick layer of basement membrane material termed Reichert’s membrane. Culture of these embryoid bodies will give rise to an array of cell phenotypes for example, nerve, muscle, cartilage etc. As the differentiation occurs within a multilayered culture, it is difficult to see these cells by visual inspection. 7. If these embryoid bodies are allowed to attach to a tissue culture surface, the resulting differentiation can be identified utilizing standard histology or specific cell markers. Stem Cell Markers Coating the surface of every cell in the body are specialized proteins called receptors that have the capability of selectively binding or adhering to other “Signaling” molecules. Many different types of receptors exist that differ in their structure and affinity for the signaling molecules. Cells carry our their proper functions in the body communicating with other cells through these receptors and molecules. Each cell type, for example a liver cell, has a certain combination of receptors on their surface that makes them distinguishable from other kinds of cells. Identification of Stem Cell Markers One of the more popular technique, is fluorescence-activated cell sorting (FACS).36–38 This technique utilities a suspension of tagged cells (i.e. bound to the cell surface markers are fluorescence tags) which are sent under pressure through a very narrow nozzle—so narrow that cells must pass through one at a time. Upon exiting the nozzle, cells then pass one-by-one through a light source, usually a laser and then through an electric field. The fluorescence cells become negatively charged, while non fluorescent cells become positively charged. The charge difference allows stem cells to be separated from other cells. Assessment of how stem cells appear in tissue, is carried out by preparing a thin slice of tissue, and the stem cell markers are tagged by the signaling molecule that has the fluorescent tag attached. The fluorescent tags are then activated either by special light energy or a chemical reaction. The stem cells will emit a fluorescent light that can easily be seen under the microscope. Most recently, genetic engineering approach that uses fluorescence has been reported. The importance of this new technique is that it allows the tracking of stem cells as they differentiate or become specialised. Green fluorescent protein GFP39 is inserted into a stem cell and is only activated when cells are undifferentiated and is turned off once they

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become specialized. Once activated, the genes directs the stem cells to produce a protein that fluoresces in a brilliant green color (Fig. 53.2). Coupling this reporting method with the FACS and microscope methods described earlier to sort cells, one is able to track them as they differentiate or become specialised.

Fig. 53.2: Microscopic image of fluorescent lable stem cell Some of the cell marker for Pluripotent stem cells are listed in Table 53.3. Karyotype Analysis Karyotype analysis is mandatory to ascertain the chromosome complement and sex chromosome constitution of PSC line. This should be done as soon as possible once it is established. Euploid chromosome complement has been shown to be retained by PSC, for in excess of 15 passages (approximately 70 cell generations) by demonstration that cells can form functional germ cells in Chimaeras.20,40 It is inevitable that cell populations will drift away from the normal genotype and over a period of continuous culture, subsets of aneuploid cells will be selected. To estimate the range of variability and to determine modal number simple chromosome count can be performed, complemented by Gbanding analysis to enable the exact chromosomal constitution to be established.

Table 53.3: Stem cell markers Marker name

Cell type

Alkaline phosphatase Embryonic stem (ES) Embryonic carcinoma (EC) Alpha-fetoprotein Endoderm (AFP) Bone morphogenetic Mesoderm protein 4 Brachury Mesoderm

Significance Elevated expression of this enzyme is associated with undifferentiated pluripotent stem cells (PSC) Protein expressed during development of primitive endoderm reflects endodermal differentiation Growth and differentiation factor expressed during early mesoderm formation and differentiation Transcription factor important in the earlier phases of mesoderm formation and differentiation: used as

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Oct-4

ES. EC

Stage-specific ES. EC embryonic antige-4 (SSEA-4) Cluster designation 30 ES. EC (CD 30) Cripto (TDGF-1) ES cardiomyocyte GCTM-2

ES. EC

Germ cell nuclear factor Nestin

ES. EC

Telomerase

581

the earliest indication of mesoderm formation Transcription factor unique to PSCs, essential for establishment and maintenance of undifferentiated PSCs Glycoprotein specifically expressed in early embryonic development and by undifferentiated PSCs Surface receptor molecule found specifically on PSC Gene for growth factor expressed by ES cells, primitive ectoderm and developing cardiomyocyte Antibody to a specific extracellular matrix molecule that is synthesised by undifferentiated PSCs Transcription factor expressed by PSCs

Ectoderm, neural and Intermediate filaments within cells, characteristic of pancreatic Progenitor primitive neuroectoderm formation ES. EC An enzyme uniquely associated with immortal cell lines: Useful for identifying undifferentiated PSCs

Collection of Mitotic Cells 1. Add colecmid to a final concentration of 0.02 µg/ml to an exponentially growing plate of cells. Place the plate in the incubator at 37°C for 1 hr. 2. Transfer for the suspension to a 10 ml conical centrifuge tube and pellet the cells at 12000 rpm for 5 minutes. 3. Aspirate the supernatant as much as possible and gently tap the tube to disrupt the pellet. Add 1 ml of hypotonic KCl solution (0.56% w/v) and resuspend the cells. Add further 6 mls (excess) and invert the tube to ensure thorough mixing. 4. Allow the cells to swell at room temperature (approximately 10 minutes) Pellet the cells by gentle centrifugation (350 rpm for 5 min) Aspirate the supernatant. Flick the tube vigorously and add ice cold fixative (3:1 volumes of absolute methanol to glacial acetic acid freshly prepared. Ensure that the fixative is added dropwise, while flicking the tube prevent formation of large clumps. 5. Repeat 4 above further 5 times, with 5 min incubation at room temperature in between the changes. 6. Suspend the PSC in a final volume of 1 ml. Preparation of Mitotic Spreads 1. Pre-clear the slides by wiping with fixative 2. Dispense a single drop of PSC suspension approximately 5 to 10 cm above the center of the slide. 3. Once the suspension has spread evenly over the entire slide, blow gently across the surface until all the fixative has evaporated. (Work in a fume hood as fixative vapour is toxic if inhaled).

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4. 10–15 slides should be prepared. Examine the individual slides carefully under phase contrast (x 200) to check the number and quality of the chromosa spreads. Count the chromosomes by staining the slide with a 3 percent (v/v) solution of Gum’s Gremsa (Sigma) stain in PBS for 13–15 minutes. Allow to air dry after three rinses in two distilled water. G-banding Analysis G-banding is one of the most reliable methods of various banding techniques. In this technique mitotic spreads are subjected to various denaturation treatments before staining.41 Karyotype should be prepared according to the convention of Nesbitt and Franke method42 (Fig. 53.3 and 53.4). Incubate the slides at 60°C in a bath of 2×standard saline citrate (SSC) for 1½ hr (0.03 M NaCl+0.03 M trisodium citrate). After rinsing the slides extensively in several charges of distilled water store them in a rack under water. Immerse each slide in trypsin (4°C) for 3–4 units and rinse in three charges of Gurr’s pH 6.8 Phosphate Buffer (Sigma) Immerse in staining solution for 7 minutes. Rinse in three changes of buffer followed by two changes of distilled water. Allow the slides to air dry.

Fig. 53.3: Karyograms constructed from photographs of G-banded metaphase spread. Female cell line showing two chromosome

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Fig. 53.4: Karyogram constructed from photographs of G-banded metaphase spread. Male cell line showing XY sex chromosome complements Photograph suitable metaphase spreads after examining the slides under a high-power objective (X 100 oil immersion) for correct number of chromosomes. DISCUSSION PSC derived and grown as described here have been continuously maintained for more than 25 passages. The highly compacted nature of colonies obtained and resistant to trypsin digestion suggest strong cell—cell adhesion. This strong adhesion makes it difficult to disegregation of colonies into single cells, and a lower plating efficiency. Just like mouse ES and EG cells, human pluripotent stem cells form EBs, and have the ability to differentiate in vitro into ectoderm, endoderm and mesodermal derivatives. Morphologically no difference was observed in human EBs and those derived from mouse ES and EG cultures. The potential application of stem cells in biology and medicine is based on the assumption, that it will be possible to grow human pluripotent stem cells on large scale, enabling the introduction of genetic modifications in them and direct their differentiation. Although there is an indication of progress to come in form of novel factors driving pluripotent stem cells growth, or elimination of inhibitory influence of differentiated

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cells, and expansion of cloning of human PSC, the present technology falls short of the goals. REFERENCES 1. Kleinsmith LJ, Pierce GB. Multipotentiality of single embryonal carcinoma cells. Cancer Res 1964; 24:1544–49. 2. Stevens LC. Testicular ovarian and embryo-derived teratomas. Cancer Survey 1980; 2:75–91. 3. Martin GR. Teratocarcinoma and mammalian embryogenesis. Science 1980; 209:768–76. 4. Matsui Y, Zsebo K, Hogan BLM. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70:841–47. 5. Andrews PW. Human teratocarcinomas. Biochim Biophys Acta 1988; 948:17–36. 6. Andrews PW, Damjanor L, Simon D, Banting GS, Carlin C, Dracopoli NC, Fogh J. Pluripotent embryonic carcinomas clones derived from the human teratocarcinoma cell line. Lab Invest 1984; 50:147–62. 7. Thompson S, Stevin PL, Webb M, Walsh ES, Engstrom W, Evans EP, Shi WK, Hopkins B, Graham CF. Cloned human teratoma cells differentiate into neuron-like cells and other cell types in retinoic acid. J Cell Sci 1984; 72:37–64. 8. Pera MF, Cooper S, Mills J, Parrington JM. Isolation and characterization of a multipotent clone of human embryonal carcinoma cells. Differentiation 1989; 42:10–23. 9. Chardrass KJ, Mezey E. Plasticity of adult bone marrow stem cells. Mattson MP, Van Zant G (Eds): (Greenwich CI JAI Press). 10. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature (London) 1981; 292:154–56. 11. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Nat Acad Sci (USA) 1981; 78:7634–38. 12. Matsui Y, Torsoz D, Nishikawa S, Nishikawa S, WilliamsA, Zsebo K, Hogan BL. Effect of steel factor and leukemia inhibitory factor on murine primordial germ cells in culture. Nature (London) 1991; 53:750–52. 13. Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long term proliferation of mouse primordial germ cells in culture. Nature (London) 1992; 359:550–51. 14. Thomson JA, Itskorizeldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall US, Jones JM. Embryonic stem cells lines derived from human blastocysts. Science 1998; 282:1145–47. 15. Shamblott MJ, Axelman J, Wang SP, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PA, Huggins GR, Gearhart JD. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Nat Acad Sci (USA) 1998; 95:13726–731. 16. Reubinoff BE, Pera MP, Fong CY, Trounson A, Bongoso A. Embryonic stem cell line from human blastocysts: Somatic differentiation in vitro. Nature Biotech 2000; 18:399–404. 17. Martin GR. Teratocarcinoma and mammalian embryogenesis. Science 1980; 209:768–76. 18. Stevens LC. Testicular ovarian and embryo derived teratomas. Cancer Survey 1980; 2:75–91. 19. Stevens LC. The development of transplantable teratocarcinomas from intratesticular grafts of pre and post implantation mouse embryos. Dev Biol 1970; 21:364–82. 20. Evans MJ, BradleyA, Robertson E. Ek cell contribution to chimeric mice: from tissue culture populations. Banbury Report 20. Genetic Manipulation of the Early mammalian Embryos. Jaenish R, and Constantini F (Eds): Cold Spring Harbor Laboratory Press (New York) 1985; 93–95. 21. Doetrschman TC, Eisteller H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac blood islands and myocardium. J Embryol Exp Morphol 1985; 87:27–45.

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22. Jorgensen NM, Meyts ER, Gracin N, Mullter J, GineremanA and Skakkeback NE. Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells. Lab Invest 1995; 72:223–31. 23. Denovan PJ. Growth factor regulation of mouse primordial germ cell development. Curr Top Dev Biol 1994; 29:189–225. 24. Klug M, Soonpaa M, Field L. Formation of germ line chimaeras from embryo derived teratocarcinoma cell lines. Am J Physiology 1995; 269; H1913–H1921. 25. Rohwedel J, Sehlmeyer U, Shan J, Meister A, WobusA. Primodial germ cell derived mouse embryonic germ (EG) cell in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution. Cell Biol Int 1996; 20:579–87. 26. Solter D, Knowler B. DNA synthesis and multinucleation in embryonic stem cell derived cardiomyocytes. Proc Natl Acad Sci (USA) 1978; 75:5565–69. 27. Kannagi R, Cochran N, Ishigami F, Hakomori S, Andrews P, Knowles B, Solter D. Stage specific embryonic antigens (WSEA-3&4) preepitopes of a unique globo series gangliosede isolated from human teratocarcinoma cells. EMBO J 1983; 2:2355–61. 28. Andrews P, Banting G, Damjanov I, Arhand D, Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma 1984; 3:347–61. 29. Martin GR, Evans MJ. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Natl Acad Sci (USA). 1975; 72:1441–45. 30. Rathjen PD, TothS, Willis A, Heath JK, Smith AG. Differentiation inhibiting activity is produced in matrix associated and diffusible forms that are generated by alternate promoter usage. Cell 1990; 62:1105–14. 31. Rathjen PD, Nicols J, Toth S, Edwards DR, Heath JK, Smith AG. Developmentally programmed induction of differentiation inhibiting activity and control of stem cell populations. Genes Dev 1990; 4:2308–18. 32. Nichols J, Evans EP, Smith AG. Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 1990; 110:1341–1348. 33. Pease S, Braghetta P, Gearing D, Grail D, Williams RL. Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factory (LIF) Dev Biol 1990; 141:344–52. 34. Smith AG, Heath JK, Donaldson DD, Wong GG, Morean J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypephides. Nature (London) 1998; 336:688–90. 35. Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factory maintains the developmental potential of embryonic stem cells. Nature (London) 1988; 336:614–87. 36. Bonner WA, Hulett HR, Sweat RG, Herzenberg LA. Fluorescence activated cell sorting. Rev Sci Instrum 1972; 43:404–09. 37. Herzenberg LA, De Rosa SC. Monoclonal antibodies and the FACS: Complementary tools for immunology and medicine. Immunol Today 2000; 21:383–90. 38. Andrews PW, Casper J, Damjanov I, Duggan-Keen M et al. Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumors. Int J Cancer 1996; 66:806–16. 39. Eiges R, Schuldiner M, Drukker M, Yanuka O, Hskovitz-Eldor J, Benvenisty N. Establishment of human embryonic stem cell transfected clones carrying a marker for undifferentiated cells. Curr Bio 2001; 11:514–18.

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40. Bradley A, Evans MJ, Kaufman MH, Robertson EJ. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature (London) 1984; 309:255–57. 41. Gallimore PH and Richardson CR. Tandem duplications of drosophila inter chromosomal effects of a heterozygous tandem duplication. Chromosoma Berl 1973; 41:259–64. 42. Nesbitt MN and Franke O. A system of nomenclature for band patterns of mouse chromosomes. Chromosoma Berl 1973; 41:145–51.

CHAPTER 54 Cytoplasmic and Nuclear Transfer: New Life for an Old Egg? Hugh C Hensleigh, Samuel S Thatcher INTRODUCTION Oocyte quality is perhaps the single most limiting factor in fertility. It is logical that alteration in oocyte cytoplasm and especially adverse changes in intermediary metabolism may be the source of some oocyte defects. Cytoplasmic transfer (CT) and nuclear transfer (NT) are techniques whereby an attempt is made to correct cytoplasmic abnormalities allowing a couple a chance to have a child without use of donor oocytes. However, CT and NT are much more than a new infertility treatment. They represent two new probes into the ethics and basic science of human reproduction. The desire to have one’s own child is an inestimable force. There is little doubt that virtually every couple would like to maintain the genetic integrity of their children. Many are willing to spend large amounts of money, even take significant risks to accomplish this goal. Three decades ago, many believed that human in vitro fertilization (IVF) was impossible and/or dangerous. With several thousand of babies born after IVF and those IVF babies now having babies, most believe that IVF is a relatively safe procedure. Fifteen years ago, the same could be said for intracytoplasmic sperm injection (ICSI) which, by micromanipulation, alters the gamete overcoming various blocks to fertilization. ICSI now routinely allows men to father children whereas before, they may have been relegated to donor sperm. On the balance and despite the documented association of ICSI with microdeletions of the Y chromosome and an increased risk in cystic fibrosis carriers, ICSI is accepted as a valuable therapy. The greatest barriers to fertility are grouped into the category of “poor egg quality.” Whether created by an inherited abnormality or an acquired defect, we have little mechanistic understanding of what makes a “good egg” or what makes a good egg go bad. Certainly some of these disorders must have their origins in the egg’s cytoplasm. A transfusion of healthy cytoplasm, or the removal and transfer of the oocyte nucleus into donor cell, would seem an obvious treatment. Clearly cytoplasmic transfer (CT) and nuclear transfer (NT) are on the cutting edge of our understanding of reproductive biology and offer great scientific excitement. At the same time CT and NT may pose greater risks and more ethical dilemma than has yet been seen in human reproductive technology An analysis of the biology of this process, its rationale, risks and success is the purpose of this chapter.

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SEXUAL REPRODUCTION The sperm and the ovum each bring to the conception an equal complement of chromosomes, but an unequal amount of cytoplasm and DNA. The spermatozoon contributes a pair of centrioles, one of which forms the centrosome guiding pronuclear development and formation of the first mitotic spindle and is passed to all the subsequent cells.1 The sperm may also contribute cytoplasmic factors such as ossillin, that cause a flux in membrane potential in the embryo,2 as well as enzymes and metabolites. In the process of activation and penetration, sperm generate considerable quantities of reactive oxygen species (ROS). The role of ROS in embryonic development is unclear. The 50 to 75 mitochondria wrapped around the mid-piece of the sperm are highly differentiated and are eliminated from the embryo by a ubiquitin-dependent response during the cleavage stages, in the mouse at the 4 to 8 cell stage.3 This may be a positive process to preserve the homoplasmy of maternal mitochondria. Safety Mechanism The zygote receives virtually all of its cytoplasm from the oocyte. This includes a long list of structural and functional organelles, proteins, RNA, and mitochondria. Maternal messenger RNA (mRNA) has been shown to be transferred during cloning and expressed in the embryo in pigs.4 During the first three cleavage cycles in the embryo, the embryonic genome is not expressed; only mRNA from the maternal genome that is stored in the ovum during oogenesis is expressed. Maternal mRNA in donor cytoplasm may be able to substitute for a deficiency in poor quality ovum. All the functional mitochondria in the zygote are of maternal origin. It is possible that the mitochondrion holds the key to success with CT/NT, but also the greatest problem areas. Mitochondria and Development Relatively undeveloped mitochondria are present in the primordial germ cells (PGC) as they migrate from the primitive yolk sac into the germinal ridge during the forth week of development. Mitochondria number are at a minimum at the time of PGC migration, with perhaps as few as 6 to 21 mitochondria per PGC, each with one copy of mtDNA. PGCs multiply by mitosis to about 7 million at mid-gestation, but then are reduced by more than half by birth when they have entered meiosis, becoming primary oocytes. About 300,000 oocytes remain at menarche with their number falling to several hundred to several thousand around age forty when the ovary is no longer able to support normal follicular development. Mitochondria in the primary oocyte are still comparatively primitive, but soon change as a part of cytoplasmic maturation that accompanied the final stages of oocyte maturation. In the primary oocyte, they are spherical in shape and have few cristae. As the oocyte matures, matrix density and cristae increase in the mitochondria.

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Mitochondrial DNA Mitochondria are unique among cellular organelles because they contain their own DNA. MtDNA consists of a 16.6 kilobase histone free circular “chromosome”, present in one or more copies in each mitochondrion. Human mtDNA also encodes 2 ribosomal RNAs and 22 transfer RNAs. A single mitochondrion may be the founder for all later mitochondria. MtDNA has been strongly conserved through all vertebrates and invertebrates.7,8 It is theorized that our present human strain of mitochondria developed about 30,000 years ago and all since have been progeny of the “mitochondrial Eve.” Mitochondria are thought to arise from phagocytosised osised bacteria, subsequently forming over a billion year old symbiotic relationship that is the genesis of the eukaryotic cell. Subsequently, mitochondria have lost their capacity to exist outside the cell and likewise, the eukaryotic cell has an absolute dependency on the ATP provided by the mitochondria. This high level of intracellular cooperation continues. Mitochondrial Genetics It is hypothesized that all mitochondria in mature ovum are derived from a single, or few, most genetically fit mitochondria. Mitochondria multiply by asexual clonal expansion, which has a mutation rate much higher than nuclear DNA. However, defective mitochondria are not reproduced and thus are not passed on to the developing embryo. This tight selection process has been referred to as the “bottleneck” theory Still, random alterations occur as mtDNA replicates. Mitochondria have base and nucleotide excision repair pathways, though not as efficient as those in the nucleus. DNA alterations accumulate over time and at some point a threshold is reached beyond which there can be no further compensation. The mtDNA code becomes senseless. The cell dies. Mitochondria geneticists used the term “Muller’s ratchet” after W.H. Muller who hypothesized that organisms reproducing asexually would eventually become extinct to explain this form of genetic selection. Mitochondria compensate by a type of polyploidy or heteroplasmy, the presence of more than one mitochondrial genome in a cell. Thus, a small amount of heteroplasmy may be tolerated, even essential for normal development, but beyond a threshold, cellular dysfunction will occur interfering with growth and differentiation of the embryo or reduce the life span of the organism.5 Disorders of Mitochondrial Metabolism There is a wide range of relatively rare mitochondrial based genetic diseases including premature aging, neurodegenerative diseases, myopathies, and diabetes. The tissues most often affected are those that have high rates of OXPHOS, including the nervous system, muscle, heart, pancreas, kidney, and liver. Some are very severe with multi-system compromise, while others are very mild and identified only by esoteric testing. They markedly vary depending on which organ(s) are involved, age at onset, and progression. Genetic counseling can be difficult at best and treatment options are usually limited.10–12 Nuclear driven multiple mtDNA abnormalities are possible, which illustrates the close communication, but also obscures cause and affect.13 There are several hundred mtDNA derangements that have been described. Some may be highly deleterious while others are of limited biological or clinical importance. Hsieh,

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et al found 13 different mtDNA deletions in human oocytes and embryos.14 The largescale 4977 base pair mtDNA deletion called the “common deletion” or “hot spot” is the most common deletion in human oocytes and embryos. It is relatively easily detected and has been used as an assay of mitochondrial damage. This deletion results in OXPHOS pathway abnormalities a frequent finding in individuals with progressive external ophthalmoplegia (Kearns-Sayre syndrome). Brenner, et al, found 33 percent of 74 oocytes and 8 percent of 137 embryos using a nested primer strategy while 47 of 181 oocytes and 20 percent of 104 embryos using a “long PCR-short PCR” method.15 Hsieh, et al, found 66 percent of 124 unfertilized oocytes, 35 percent of 98 arrested or fragmented embryos, and 21 percent of 45 3PN embryos contained this 4977-bp mtDNA deletion.14 Role of Mitochondria in Atresia Reproductive biologists have long pondered on the origins and mechanisms of atresia, an orderly loss of oocytes from the ovary except by ovulation. Krakauer and Mira hypothesize that death of germ cells is a selective solution to an accumulation of mitochondrial defects and that prenatal apoptosis effectively removes oocytes carrying defective mitochondria.16 Perez et al17 disagree with the hypothesis of Krakauer and Mira16 in that they believe that defective mtDNA make a relatively small contribution to the prenatal loss of oocytes. In their fascinating study, Perez, et al,17 tested whether mitochondria influence oocyte fate by microinjecting a relatively small number of donor mitochondria derived for preovulatory mouse granulosa cells into mouse oocytes of the FVB strain that are characterized by a high rate of apoptosis in vitro. The approximate 5000 mitochondria injected compare with the native mitochondria population of 100,000 resulting in a donation equal to 5 percent of the native mitochondria. Since it is commonly believed that much of postnatal atresia originates in the follicle cells rather than oocyte, Perez et al17 theorize that postnatal atresia may occur from alteration in mtDNA and apoptosis in granulosa cells. Since the egg is dependent on granulosa cells for its metabolic substrates, alteration of granulosa cell mitochondria by any mechanism may result in egg quality compromise. That healthy mitochondria from granulosa or other cells may be used specifically to improve egg quality is a fascinating and potentially very important advance. Mitochondria and Aging A dictum of reproductive biology that can be recited by virtually every woman is that there are a finite numbers of eggs and that there is no new production after birth. Most also understand the link between age and the risk of chromosome defects, most notably Downs syndrome. Infertility and pregnancy loss increase before ovulation stops suggesting altered egg quality as one cause for the decline in reproductive capacity with aging. Clearly nuclear aging is real, but whether there is also cytoplasmic aging and whether cytoplasmic aging alone, or cytoplasmic aging perhaps acting through substrate availability can alter meiosis remains an unanswered. A mitochondrial connection for nondysjunction has been suggested by Schon.18 Mitochondria aggregate around the mitotic spindle indicating a need for energy in the process of cell division. Defective

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spindles have fewer mitochondria. It is very possible that the same “bottleneck theory11 where mtDNA is refined and fixed from one generation to the next could be the operating principle. It is thought that all of an individuars mtDNA have the same sequence until age related mutations start to occur. Fetal tissue contains few if any mtDNA mutations while slow dividing cells, such as neurons, myocytes, and oocytes have more mtDNA mutations. This suggests increased risk with longer exposure time to DNA mutagens such as ROS. Thusfar, no supporting data has been presented for an age-related increase in mtDNA deletions in oocytes.14,15,17 It may be because the right probe has not been found. Whether an injection of cytoplasm can breathe new life into old eggs is very much an issue of debate. Clinical use of Cytoplasmic Transfer Cohen, et al. in 199719 reported the first human live birth following transfer of donor cytoplasm with a sperm at the time of ICSI. The patient was a 39 year old woman with poor egg quality as evidenced by four unsuccessful IVF cycles, each with different stimulation protocol and utilizing co-culture, assisted hatching, and fragment removal prior to transfer. Nine of 14 injected ova fertilized, eight cleaved and six were reported to have better morphology than the untreated embryos and those from previous cycles. Cytoplasm derived from an egg donor undergoing controlled ovarian stimulation was injected, an amount equal to about 5 percent of the recipient cytoplasm. Donor mitochondria were not detected in the amniocytes at 16 weeks of gestation. However, subsequent studies from this center, one of two children born after CT, had both donor and recipient mtDNA present in the blood cells at one year of age.20 A series of nine patients who had a previous total of 83 cycles of IVF with transfers and no pregnancies were treated in an IVF cycle with ICSI and CT using donor cytoplasm from triploid embryos.21 Four pregnancies resulted in five healthy births after 39 weeks of gestation. The age of the patients ranged from 33 to 42 and the pregnancies were in women 36, 38, 42, and 42 years of age. In this series, the CT was asynchronous, cytoplasm coming from triploid embryos and being transferred into MII ova at ICSI. In addition, mitochondria tend to aggregate around the pronuclei so that CT from a pronuclear stage embryo will transfer fewer mitochondria. Cryopreserved ova were used by Lanzendorf, et al, as cytoplasmic donors so cycle synchronization between donors and recipients was not needed.22 The cytoplasmic transfer was done in conjunction with ICSI. Cytoplasm from each donor ovum was used to inject two ova. A total of 37 ova had cytoplasmic transfer and ICSI with a normal fertilization rate of 70 percent. Three patients of advanced maternal age (43, 47, and 47 years) did not become pregnant after transfer of 3, 2, and 6 embryos. The embryos for these patients had no improvement in morphology or cleavage. A fourth patient, 35 years of age, with 6 failed IVF cycles with poor embryo quality, delivered healthy twins. The 6 embryos transferred were reported to have improved morphology when compared with those of the previous failed cycles. Similar findings using cryopreserved donor oocytes for CT in patients over 40 years of age (n=15) or patients with low ovarian reserve under 40 years of age (n=3) were reported by Opsahl, et al.23 CT was carried out in 25 cycles of IVF for 18 patients.

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Embryos were transferred in 20 cycles resulting in three biochemical losses and one aneuploid clinical loss. No improvement was noted in the quality of embryos after CT. Dale, et al, reported CT in a patient 32 years of age with 3 cycles of IVF with poor embryo cleavage rates and morphology24 On the fourth attempt, 8 of 12 ova were injected with cytoplasm at the time of ICSI; 4 of 12 ova were injected with sperm (ICSI) but without CT. The embryos with CT had lower levels of fragmentation and higher cell number than those without. Transfer of 4 of the treated embryos led to pregnancy and birth of healthy twin babies. Nuclear Transfer Nuclear transfer (NT) requires a fusion of an excised nucleus (karyoplast) of one cell with the enucleated membrane bound cytoplasm (cytoplast) of a different cell. A small amount of cytoplasm and associated organelles will invariably accompany the karyoplast. The number of “peri” nuclear mitochondria accompanying the transfer depends on the stage of the cell cycle. Like CT, NT usually results in heteroplasmy with the accompanying above concerns. The difference between CT and NT is, on one side, a matter of the amount of cytoplasm and on the other side, it is a different technology. NT was used to unite a cell nucleus from mammary tissue with an enucleated oocyte in the cloned sheep, Dolly.25 NT remains the primary method of animal cell cloning with all of its ramifications. Clone size is only limited by the availability of nuclei, which in the case of present discussion are oocyte nuclei from the infertile female. For domestic animals there is desire to clone a relatively large number of identical animals for superior economic traits.25–29 Nuclear transfer may also have tremendous implications for drug development and potential gene therapy Colman, et al,30 and Schnieke, et al,31 cloned sheep fibroblasts transfected with the human clotting factor IX (FIX) gene linked to ovine B-lactoglobulin gene promoter. This resulted in the birth of three female sheep that produced FIX in their milk. FIX could be easily extracted for use in the treatment of hemophilia. Cloned pigs were born after NT of boar fibroblast nuclei containing human transferase gene, obtained from a ear punch biopsy, were transferred into sow ova.32 A study was also performed using nuclear transfer from a transgenic goat cell line containing a gene for human antithrombin III into cytoplasts from goat ovaries from the local abattoir. Using abattoir-derived ova, 2 offspring resulted from transferring 72 embryos from 183 fused coup. Wolf et al, at the Oregon Regional Primate Center have cloned monkeys using cytoplasts from MII ova and karyoplasts from 8-cell embryos.33 They propose this as a model for gene therapy and basic research. However, NT has a low efficiency with only 2 live births from 53 embryos transferred. Across all species, the efficiency of producing liveborn from NT is poor with a high percentage of developmental anomalies.33 The technique of NT is considerably more complex than that of CT. A significant problem in the standard technique of electrofusion of nuclei into oocytes is its tendency to induce oocyte activation. Less aggressive techniques with improved success are waranted in the future. Tesarik, et al, report the use of mechanical and chemical methods of induction of fusion of human cytoplasts and karyoplasts.34 Electrofusion results in immediate activation of the ovum, while these approaches do not. This is important in use of the technique for GV transfer where a maturation interval is desired.

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There is a single report of NT in humans.36 Extraction of germinal vesicles (prophase I ova) from 48 oocytes from women over age 38 yielded 12 successful transfers into cytoplasts of immature oocytes discarded at ICSI. Maturation to meiosis II was successfully accomplished in 7/12 couplets. DISCUSSION In one of the first international IVF meetings where a paper was presented on the effects of light on IVF success, the presenter said that their IVF success rate had significantly improved after changing to red light in the laboratory. When a member of the audience asked a question about wavelength, the presenter replied “it has nothing to do with wavelength, it’s the color.” There is no doubt that we have the tools to affect CT/NT. At present, it is not known what specific defect is being treated by CT/NT and there are only vague hypotheses on why, even if, the treatment works. There is little doubt that CT/NT and cloning will be increasingly performed in humans. The excitement of discovery and the desire for recognition is each too great an impetus for it not to be. In the United States, The Federal Drug Administration effectively stopped CT/NT in humans in June 2001 by requiring Investigational New Drug (IND) exemption for patient treatment and public hearings before permission can be granted. The FDA surmises that since the mitochondria contain DNA and since mtDNA for donor cytoplasm has been found in children born after donor cytoplasmic transfer, the technique represents “human germ line modification and is expressly prohibited”.37 In his most recent report, Barritt, et al20,38 states that about 30 babies have been born worldwide from CT. In their center there have been 13 clinical pregnancies in 30 fertilization attempts for a rate higher than the expected pregnancy rate or this group with low fecundity Two pregnancies were reported as 45, XO (Turner’s syndrome). Heteroplasmy was shown in children born after CT clearly indicating, mtDNA is contained in CT injections, but does it matter? Heterogeneity of mtDNA may cause disease, a mismatch between the mtDNA and the nuclear DNA, dissonance between mitochondrial and nuclear genes, contraindicating CT. Mitochondrial heteroplasmy may account for the highly unpredictable results and low number of cloned embryos that implant and result in healthy liveborn.3 Hawes, et al, caution against use of cytoplasmic transfer due to the incompatibility between nuclear and mitochondrial genome.39 The incompatibility may have epigenetic effects, such as defects in gene expression and development and these problems are heritable and therefore may only become apparent in the next generation. Asynchrony between the mitochondria and nucleus may be the basis of the Big Calf syndrome in cloned cattle. Questions remain as to whether this new mtDNA will replace the native mtDNA, integrate with it (form a functional heteroplasmy or a disease state), or be eliminated. This may vary in each individual. There can be no doubt that there is a cytoplasmic origin of alteration in oocyte quality, nor can there be doubt of a contribution of nuclear factors. A third category of communication disorders between these compartments could also be at fault. Cytoplasmic transfer will include mRNA that will be translated in the recipient.4 These could be translated into proteins that may have beneficial or harmful effects.

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We are still stuck for an explanation of the mechanisms responsible for reproductive aging. From Lazendorft et al. it appears that the cytoplasmic transfer was not effective in treating the ova of older patients.22 Explanations could be that there was not enough cytoplasm injected to renew these ova or that there was another problem not addressed by transfer of cytoplasm, c.f. chromosomal abnormalities. CONCLUSIONS The purpose of a species is to elaborate the cellular vehicles of evolution, the gametes. With our primitive stage of understanding of cellular dysfunction as it relates to reproduction, we bundle probably hundreds of fertility altering defects of oocytes under a single heading of “oocyte quality” Synchronous with life is the hunger for energy Each follicle, each oocyte, may have a different intrafollicular oxygen saturation.40 Altered ATP utilization, whether a consequence of the stoichometry of folliculogenesis, aging, or intracellular def ect, may have an adverse effect on oocyte health. Since mitochondria are so intimately connected with oxidative metabolism as well as ion fluxes and apoptosis, it is not surprising that they have taken center stage. With CT, more productive mitochondria or other cytoplasmic constituents may be transfused with improved cellular function. A “let’s try that and see if it will work” philosophy could be adopted that will no doubt be grasped by desperate infertile couples! REFERENCES 1. Sutovsky P, Schatten G. Paternal contributions to the mammalian zygote: fertilization after sperm-egg fusion. Int Rev Cytol 2000; 195:1–65. 2. Swann K. Soluble sperm factors and Ca2+release in eggs at fertilization. Rev Reprod 1996; 1:33–39. 3. Cummins JM. Fertilization and elimination of the paternal mitochondrial genome. Hum Reprod 2000; 15:92–101. 4. Parry TW, Prather RS. Carry-over of mRNA during nuclear transfer in pigs. Reprod Nutr Dev 1995; 35:313–18. 5. Jansen, RPS. Origin and persistence of the mitochondrial genome. Hum Reprod 2000; 15:1–9. 6. Motta P, Nottola S, Makabe, S, Heyn R. Mitochondrial morphology in human fetal and adult female germ cells. Hum Reprod 2000; 15:129–44. 7. Jansen, RPS. Germline passage of mitochondria: quantitative considerations and possible embryological sequelae. Hum Reprod 2000; 15:112–28. 8. Howell N, Chinnery P, Ghosh S, Fahy E, Turnbull D. Transmission of the human mitochondrial genome. Hum Reprod 2000; 15:235–39. 9. St. John, J C. The transmission of mitochondrial DNA following assisted reproductive techniques. Theriogenology 2002; 57:109–23. 10. Christodoulou, J. Genetic defects causing mitochondrial respiratory chain disorders and disease. Hum Reprod 2000; 15:28–43. 11. Naviaux RK, McGowna KA. Organismal effects of mitochondrial dysfunction. Hum Reprod 2000; 15:44–56. 12. Johns DR. Mitochondrial DNA and disease. New Engl J Med 1995; 333:638–44.

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13. Lestienne P, Reynier P, Chretien M, Penisson-Besnier I, Malthiery Y, Rohmer V. Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements. Mol Human Reprod 1997; 9:811–14. 14. Hsieh R-H, Tsai N-M, Au H-K, Chang S-J, Wei Y-H, Tzeng C-R. Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes. Fertil Steril 2002; 775:1012–17. 15. Brenner CA, Wolny YM, Barritt JA, Matt DW, Munne S, Cohen, J. Mitochondrial DNA deletion in human oocytes and embryos. Mol Human Reprod 1998; 4:887–92. 16. Krakauer DC, Mira A. Mitochondria and germ-cell death. Nature 1999; 400:125–26. 17. Perez GI, Trbovich AM, Gosden RG, Tilly JI. Mitohondria and the death of oocytes. Nature 2000; 403:500–01. 18. Schon EA, Kim SH, Ferreira JC, Magalhaes P, Grace M, Warburton D, Gross SJ. Chromosomal non-disjunction in human oocytes: is there a mitochondrial connection? Hum Reprod 2000; 15:160–72. 19. Cohen J, Scott R, Schimmel T, Levron J, Willadsen S. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 1997; 350:186–87. 20. Barritt JA, Willadsen S, Brenner CA, Cohen J. Epigenetic and experimental modifications in early mammalian development—Cytoplasmic transfer in assisted reproduction. Hum Reprod Update 2001; 7:428–35. 21. Huang C-C, Cheng T-C, Chang H-H, Chang C-C, Chen C-I, Liu J et al. Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures. Fertil Steril 1999; 72:702– 06. 22. Lanzendorf SE, Mayer JF, Toner J, Oehninger S, Saffan DS, Muasher S. Pregnancy following transfer of ooplasm from cryopreserved-thawed donor oocytes into recipient oocytes. Fertil Steril 1999; 71:575–77. 23. Opsahl M, Thorsell L, Geltinger M, Iwaszko M, Blauer K, Sherins R. Donor oocyte cytoplasmic transfer did not enhance implantation of embryos of women with poor ovarian reserve. J Assist Reprod Genet 2002; 19:113–17. 24. Dale B, Wilding M, Botta G, Rasile M, Marino M, Di Matteo L et al. Pregnancy after cytoplasmic transfer in a couple suffering from idiopathic infertility: case report. Hum Reprod 2001; 16:1469–72. 25. Wilmut I, Young L, Campbell KH. Embryonic and somatic cell cloning. Reprod Fertil Dev 1998; 10:639–43. 26. Kikyo N, Wolffe AP. Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J Cell Sci 2000; 113:11–20. 27. Wolf DP, Meng L, Ouhibi N, Zelinski-Wooten M. Nuclear transfer in the rhesus monkey: practical and basic implications. Biol Reprod 1999; 60:199–204. 28. Reggio BC, James AN, Green HL, Gavin WG, Behboodi E, Echelard Y et al. Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: oocytes derived from both follicle stimulating hormone-stimulated and nonstimulated abattoir-derived ovaries. Biol Reprod 2001; 65:1528–33. 29. Zuccotti M, Garagna S, Redi CA. Nuclear transfer, genome reprogramming and novel opportunities in cell therapy. J Endocrinol Invest 2002; 3:623–29. 30. Colman A, Kind A. Therapeutic cloning: concepts and practicalities. Trends Biotechnol 2000; 18:192–96. 31. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997; 278(5346):2130–33. 32. Bondioli K, Ramsoondar J, Williams B, Costa C, Fodor W. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol Reprod Dev 2001; 60:189–95.

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33. Wolf DP, Mitalipov S, Norgren RB Jr. Nuclear transfer technology in mammalian cloning. Arch Med Res 2001; 32:609–13. 34. Tesarik J, Nagy ZP, Mendoza C, Greco E. Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes. Hum Reprod 2000; 15:1149–54. 35. Ivakhnenko V, Cieslak J, Verlinsky Y. A microsurgical technique for enucleation of multipronuclear human zygotes. Hum Reprod 2000; 15:911–16. 36. Zhang J, Wang CW, Krey L, Liu H, Meng L, Blaszczyk A et al. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil Steril 1999; 71:726–31. 37. Food and Drug Administration. BRMAC Briefing Document May 9, 2002. Ooplasm transfer as method to treat female infertility. http://www.fda.gov/ohrms/dockets/ac/02/briefing/%203855B1_01.pdf 38. Barritt JA, Brenner CA, Malter HE, Cohen J. Mitochondria in human offspring derived from ooplasmic transplantation. Hum Reprod 2001; 16:513–16. 39. Hawes S M, Sapienza C, Latham KE. Ooplamic donation in humans: the potential for epigenic modifications. Hum Reprod 2002; 17:850–52. 40. Van Blerkom J. The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte. J Soc Gynecol Investig 1996; 3:3–11.

SECTION 8 Implantation

CHAPTER 55 The Endometrium and Implantation Anil B Pinto, Daniel B Williams, Odem R Randall INTRODUCTION The human endometrium is not receptive to embryos throughout most of the menstrual cycle. The endometrium acquires the ability to permit the developing embryo to implant between days 5–10 after ovulation. This temporal window in the secretory phase of the menstrual cycle is called the “implantation window.” During this period the endometrium undergoes structural and functional changes induced by ovarian steroids namely estrogens and progesterone. These steroids alter the endometrium and prepare it to be receptive to invasion by the embryo. Following fertilization in the f allopian tube, the human embryo enters the uterine cavity 3–4 days following ovulation. The blastocyst remains free floating within the endometrial cavity for one day and then initiates the process of implantation on day 5–10 after ovulation.1,2,3 Implantation is a complex process requiring interaction of the blastocyst and subsequently the developing embryo and placenta with the endometrium. In natural cycles, the human endometrium, in anticipation of implantation, undergoes a series of controlled changes at the tissue, cellular and molecular levels following ovulation. These event make the endometrium ready for implantation of the embryo around the time of its arrival in the endometrial cavity. This time frame coincides with the so-called “implantation window.”4 Initially during this process, the blastocyst establishes contact with the surface epithelium of the endometrium subsequently, during a series of exquisitely controlled steps, involving both activation and repression of genes and gene networks, the blastocyst invades and implants within the underlying stroma. Formation of the placenta, the so-called “placentation” completes the implantation process and establishes a means of supporting the embryo to the end of the pregnancy period. Therefore, for successful implantation to occur the arrival of the blastocyst in the uterine cavity should occur at a time when the endometrium is appropriately primed and developmentally receptive to signals transmitted by the blastocyst. Thus, endometrial receptivity is a result of “cross talk” between the developing embryo and the endometrium. If implantation fails, the endometrium undergoes a series of changes that will prepare it for menstrual shedding. The transition from the non-receptive to the receptive endometrial state is presumably determined by the regulated statement of membrane-bound, soluble or secretory factors that permit trophoblast attachment and its subsequent migration. In the study of human endometrial receptivity only factors expressed during the implantation window can be considered as either markers or functional mediators of the receptive state. A number of structural, morphologic, cellular and molecular changes occur in the endometrium during the “implantationwindow.”

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Structural Short and cystic microvilli, so called “pinopodes” appear on the postovulatory days 5–6 in about 78 percent of cases and regress shortly thereafter.4 Molecular The molecular repertoire that makes the endometrium receptive to implantation includes steroid hormones, Muc1, and a diverse group of cytokines and growth factors, adhesion molecules, heat shock proteins, calcitonin, products of the Hox genes and a number of newly identified molecules such as trophinin and tastin. MOLECULAR MARKERS OF ENDOMETRIAL RECEPTIVITY Steroid Hormone Among the systemic signals, steroid hormones seem to play a prime role in preparing the endometrium for implantation. In the rodent model, induction of receptivity requires a minimum of 3-days of priming of the endometrium with progesterone and minute amounts of estrogen.4 Administration of progesterone to the rat leads to the formation of pinpodes in surface epithelium of the endometrium. These structures remain as long as the administration of progesterone continues. When progesterone is administered daily along with low doses of estrogen, the pinopodes appear on day 5 of treatment.5 RU486, a progesterone antagonist displaces the time of appearance of the pinopodes from day 5 to day 6 or 7 in rats.6 This indicates that the appearance of pinopodes is progesterone dependent. MUC 1 The receptive status of the endometrium is the balance between the activation of adhesive molecules and the presence of natural barrier that inhibits the implantation process. Mucins are a family of highly glycosylated, high molecular weight 250–500 kDa glycoproteins present on the surface of human epithelial cells. The most studied mucin glycoprotein is designated as MUC 1 and is expressed on the surface epithelium of endometrium in the rodent model.7–8 as well as in humans.9 This epithelial surface glycocalyx inhibits the process of implantation. Therefore, loss of these apically disposed glycoconjugate moieties may be required for the development of a receptive phenotype by the surface epithelial cells. In humans, the statement of mRNA and protein of MUC 1 is minimal during the implantation window.8 This observation has led investigators to propose that such down-regulation of a relatively large cell surface molecule allows the “unmasking” of smaller molecules such as integrins or cadherins, which may mediate the specific adhesion of the trophectoderm to the endometrial cell during the receptive phase of the cycle. Cytokines Various cytokines namely tumor necrosis factor (TNF), Leukemia inhibitory factor (LIF), Transforming growth factor (TGF-beta 1); Interleukin (IL-6), IL-8, Vascular endothelial growth factor (VEGF) and several others have been implicated in the process of

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implantation. Leukemia inhibitory factor (LIF) is one of the cytokines with a well established role in the mouse. Deletion of the LIF gene leads to implantation failures in the homozygous mutant mice.10 The peaked statement of LIF in human endometrium in the mid-secretory phase of the menstrual cycle is consistent with the hypothesis that LIF is implicated in human implantation.11 To gain an insight as to whether the statement of various cytokines differs between the receptive and the non-receptive periods, Tabibzadeth et al carried out ribo-nuclease protection assays and simultaneously examined the statement levels of cytokines including TNF, IL-1 beta, IL-6, IL-8, LIF, TGF-beta 1, and VEGF mRNAS in human endometrium.12 A low level of statement of these cytokine mRNAs was found during the proliferative and early secretory phase. Statement of these cytokine mRNAs increased during the mid-secretory phase and progressively rose to a peak in the late secretory phase. The level of cytokine mRNA statement during early gestation was similar to that observed during the mid-secretory phase. These findings suggest that there is no definitive pattern of statement of the various cytokines that would help one characterize the receptive from the non-receptive phase. Receptivity may be the outcome of certain critical concentrations and balance in the statement levels of the various cytokines. Consistent with this, individuals with habitual abortion have been found to have an abnormally low statement of IL-1 beta and IL-6 mRNA in the endometrium during the mid-secretory phase.12 This suggests that certain cytokines such as IL-1 beta and IL-6 may play an important role in helping to sustain a pregnancy. Cadherins Cadherins belong to a large super family of glycoprotein adhesion molecules ensuring cell-to-cell adhesion in a calcium dependent manner. Of these molecules, E-cadherin is the one which has been the best characterized. This molecule is responsible for the maintenance of epithelial cell polarity, and the intracellular domain has been shown to bind cytoskeleton molecules, such as B-catenin. E-cadherin statement coincides with the differentiated state of the epithelial cell and malignant transformation is characterized by a loss of E-cadherin statement. Rather than the absolute level of statement, it is the membrane distribution, and the stability of the cadherin catenin complexes that are affected by ovarian steroids. Adhesion Molecules A number of adhesion molecules have been implicated in the process of implantation Many basement membrane and extracellular matrix components such as fibronectin, laminin, entactin, collagen type IV, hyaluronic acid and heparan sulphate proteoglycan promote the attachment and outgrowth of a blastocyst in vitro.13 Integrins are involved in cell-to-cell and cell-to-matrix interactions, and have been recognized to contribute to diverse functions of cell migration, organization of cytoskeleton, and transduction of differentiation signals.14 The immuno-reactivity of two members of this family α4β1 and β3 was found in the endometrium during the implantation window.15,16 The clinical observation that β3 was lacking in patients with documented luteal phase deficiency, and

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in certain patients with minimal or mild endometriosis and infertility is consistent with this view point.16 Heat Shock Proteins Immunoreactivity of HSP27 is significantly increased in the endometrium, 2 days after ovulation.17 Both mRNA and protein statement of crystallin B chain is found to increase at around the time of the implantation window as well as during the rest of the mid and late secretory phase.18 This specific statement pattern suggests a role for these HSPs in the implantation process. Calcitonin Immuno-histochemical staining shows that calcitonin is present in the endometrial glands. Calcitonin statement is exclusively localized in the glandular epithelium, and strictly controlled by progesterone through its receptor. The anti- progestin drug, RU486, which is known to block implantation abolished, whereas progesterone significantly enhanced calcitonin mRNA and protein statement in the rodent model.19 It has also been found that calcitonin statement increased 10–20 fold in the uteri of pregnant rats reaching a peak on day 4 of the pregnancy In the human endometrium calcitonin statement is maximum between days 19–21 of the menstrual cycle. Interestingly it is undectable before day 16 of the cycle and then disappears after day 25. This suggests that calcitonin could possibly play an important role in the process of implantation. Hox Genes Hoxa-9, 10, 11 and 13 are transcription factors essential to embryonic development. These genes are also expressed in the mammalian female reproductive system in a coordinated manner and exhibit a unique spatial pattern of statement. It has been showed that both Hoxa 10 and Hoxa 11 are essential for implantation in the mouse and appear to play a similar role in women.20 Hoxa 10 predominates in the epithelial cells, while Hoxa 11 is mostly expressed in the stroma. Statement is low in the follicular and early luteal phase, but rises to a maximum during the mid-luteal phase until menstruation. Estradiol and progesterone stimulate the statement of both Hoxa 10 and 11. EMBRYONIC REGULATION OF ENDOMETRIAL RECEPTIVITY HUMANS Endometrial receptivity, in the strictest sense, is defined as the temporal window of endometrial maturation during which the trophectoderm of the blastocyst can attach to the endometrial epithelial cells and can subsequently proceed to invade the stroma. In addition, to the presence of receptive endometrial environment, the implantation process requires a functionally normal embryo at the blastocyst stage and a dialogue or crosscommunication between maternal endometrium and the developing blastocyst. Embryonic implantation is a complex process by which the human embryo orientates,

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attaches and finally invades the underlying maternal endometrial tissue. In the adhesion phase, which occurs 6–7 days after ovulation and within the implantation window, direct contact occurs between the endometrial epithelium and the trophectoderm. Thereafter, the embryonic trophoblast penetrates the basal membrane and reaches the endometrial stroma and the underlying uterine vessels. Changes in the morphology of the plasma membrane and the cytoskeleton of the luminal endometrial epithelia are necessary to alter the endometrial status from nonreceptive to that of receptive, thus allowing for implantation to occur. It is now evident that the human embryo plays a relevant role in the induction of these changes in the endometrium prior to implantation. During the apposition phase this dialogue must be mediated via soluble factors produced and received in a bidirectional fashion, whereas in the invasive phase this interaction requires a direct contact and is mediated by membrane bound molecules. EMBRYONIC REGULATION OF HUMAN ENDOMETRIAL EPITHELIAL CELL CYTOSKELETON During blastocyst implantation, the endometrial epithelium undergoes a series of morphological and biochemical changes, resulting in the disruption of the cortical actin cytoskeleton in response to external signals. The action cytoskeleton provides a structural framework that defines cell shape and polarity Cell adhesion molecules act as signalling, receptors, interacting either directly or indirectly with proteins responsible for the initiation of the signalling cascade. Several proteins namely ezrin/ radixin/moesin (ERM) are located just beneath the plasma membrane are involved in the association of actin filaments with the membrane. Up-regulation of the ERM proteins has been shown to occur in the presence of the human blastocyst. EMBRYONIC REGULATION OF CHEMOKINES IN HUMAN ENDOMETRIAL EPITHELIAL CELLS Chemokines are a family of small polypeptides, 8 to 10 kDa and are responsible for attracting leukocytes to an injury site. During implantation lymphoid cells of bone marrow origin infiltrate the decidua. The predominant lymphocyte subsets include approximately 60 percent of natural killer cells and 15 percent of cytotoxic T lymphocytes. Regulation of leukocyte recruitment in the uterus during the implantation is thought to be mediated by uterine epithelial and stromal cells, which release an array of chemokines in a precise temporal pattern, driven by ovarian steroids.21 The cyclical pattern of leukocyte presence is suggestive of steroid control. However, since leukocytes do not posses either estrogen or progesterone receptors, it implies that this regulation is exerted indirectly via chemokines.

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EMBRYONIC REGULATION ADHESION AND ANTI-ADHESION MOLECULES IN HUMAN ENDOMETRIAL AND EPITHELIAL CELLS Integrins are transmembrane heterodimers, which contain two subunits (α and β) which are non-covalently associated. Endometrial integrins are hormonally regulated. Integrins α4 and α1 are progesterone driven and they appear when progesterone production starts and endometrial progesterone receptors (PR) are at their peak.22 In contrast, β3 integrins appear when progesterone production is maximal and endometrial PR are lowest. In the presence of a developing blastocyst endometrial epithelial cell statement of β3 is maximal.23 and appears to be driven via embryonic IL-1.23 This reinforces the concept of paracrine cross talk between blastocyst and endometrial epithelium. It has been shown that MUC1 statement in the plasma membrane of the endometrial epithelial cells in the apposition phase is increased in the presence of the human embryo.23 In humans MUC1 mRNA increases from the proliferative to secretory phase in endometrial tissue, decreasing in the late secretory phase. THE BLASTOCYST REGULATES APOPTOSIS OF ENDOMETRIAL EPITHELIAL CELLS In humans, embryonic induction of the endometrial epithelial cells apoptosis is crucial for the embryo to breach the epithelial barrier.25 In the apposition phase, the presence of a blastocyst, rescues the endometrial epithelial cells from the apoptotic pathway, maintaining more endometrial epithelial cells alive in preparation for the initial contact. However, when the blastocyst adheres to the endometrial epithelial cell monolayer, it induces a paracrine apoptotic reaction that is maximal for those cells in close contact with blastocyst. This apoptotic event enables the embryo to invade the luminal epithelium. The immediate consequence is that the trophectoderm comes in direct contact with the basement membrane and stroma-l invasion follows. CLINICAL IMPLICATIONS It has been suggested that the endometrium of patients with unexplained infertility may become non-receptive if the statement of factors that are crucial to implantation fails to occur.26 For example it has been shown that infertility is associated with aberrant statement αβ3 which is normally present in endometrium during the “receptive phase” of the menstrual cycles.16 Such a defect may be due to lack of endometrial receptivity or alternatively due to dysregulated statement of the “premenstrual molecular repertoire” which leads to menstrual shedding and bleeding.26 It has been also demonstrated that the endometrium of infertile patients with endometriosis failed to show the expected midluteal rise in HOX gene statement as compared to controls. This aberrant HOX gene statement suggests that altered development of the endometrium at the molecular level may contribute to the etiology of infertility in patients with endometriosis.27

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Exogenous estradiol (E2) and progesterone (P) can successfully induce endometrial receptivity. To date no study has demonstrated that any other agent or agents can generate a better environment. Although other biochemical substances are involved in the actual process of implantation, their production is secondary to the steroid stimulation of the endometrial cells. When preparing the endometrium for transfer, all of the strategies and clinical protocols focus on one of the following parameters i) the delivery of “adequate” E2 and P to the endometrium, ii) the timing of the delivery of E2 and P to the endometrium, and iii) the determination of the adequacy of the endometrial preparation. Navot et al28 studied 8 women with ovarian failure utilizing a combination of oral E2 valerate and intramuscular P in oil. They reported 2 successful pregnancies in women with no ovarian tissue, again demonstrating the adequacy of the replacement regimen with serum levels. Li et al29 examined the influence of variable doses of E2 on the endometrial response in artificial cycles. The outcomes measured included ultrasound measurement of endometrial thickness and endometrial biopsy. The authors concluded that normal endometrial development required adequate priming of the endometrium by E2, which could be achieved with a variety of dosing regimens. Following adequate estrogen priming of the endometrium, it is P that prepares the endometrium for implantation. It is important to remember that P cannot act on an unprimed endometrium. A variety of routes of administration for P have been suggested. These include oral, transdermal, IM and transdermal IM and transvaginal. There are 2 reasons why P delivery is more complex than estrogen replacement: i) Much larger doses of P are needed to achieve luteinization, and ii) P is susceptible to metabolism by 5 αreductase in the skin, making transdermal delivery not a feasible option. Endometrial P concentrations were found to be greater following transvaginal application as compared to the IM route.30 What represents “adequate” P stimulation? There is little doubt that a luteinization threshold exists in the endometrium. Since the serum levels of P during the follicular phase of the natural cycle are typically below 0.5 ng/ml, these levels must correspond to levels below the luteinization threshold. Typically levels above 3 ng/ ml have been associated with secretory changes in the endometrium.31 Thus the threshold must lie somewhere between 0.5 and 3.0 bg/ml. It is suggested that P acts on P receptors to induce predictable histological changes in the endometrium and that these changes occur only when the luteinization threshold is exceeded, but cannot be accelerated by higher P stimulation. The “implantation window” occurs in the luteal phase, and is dependant on adequate P stimulation, which in turn, is dependent on adequate E2 endometrial priming. Therefore, the correct timing of E2 administration is such that adequate P receptors must be induced. It is imperative that the P dose be above the luteinization threshold. Clinical implantation studies32,33 have suggested that optimal implantation rates occur when cleaving embryos (2 to 12 cells) are transferred on the third or fourth day of P administration. This corresponds to commencing P replacement in the recipient on the day of follicle aspiration. Thus, day 3 embryos are transferred on the fourth day of P. This synchronization is also consistent with modern day observation of blastocyst development, which typically takes place on day 5 or 6 post-aspiration. Thus blastocyst would be transferred on the sixth or seventh day of P. This is consistent with other

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markers of the implantation window; notably pinopodes are noted in the seventh day of P administration in artificial cycles.34,35 Assessing adequacy of steroid replacement had been studied earlier by endometrial biopsy.36 However endometrial biopsy has since been replaced in most IVF programs with ultrasound. Gonen et al37 correlated pregnancy outcomes with endometrial thickness and have suggested that an endometrium ≥6 mm was associated with successful pregnancy At our institution, we monitor endometrial thickness and continue estrogen stimulation until the endometrial stripe is ≥8 mm. If the endometrial stripe is less than 8 mm, E2 therapy is continued with ultrasound assessment every 3–4 days until adequate thickness is achieved. In those patients who cannot achieve adequate endometrial thickness with standard oral E2 administration, use of vagina E2 has been suggested.38 It has now been established that the endometrium is well prepared for the process of implantation during a well defined period referred to as the “Implantation Window.” This temporal window of uterine receptivity can be inferred from what has been learned from transfer of embryos to uteri of women primed with exogenous estrogen and progesterone preparations. There is a distinct window for embryo transfer leading to implantation, which spans endometrial cycle days 16 to 20. For the 4–12 cell stage conceptus, the optimal period of transfer appears to be on days 17–19 of the artificial cycles with day 15 being the first day of administration of progesterone.39,40 In some IVF trials, pregnancies have been established when the conceptus was transferred on days 16– 19 but not days 20–24 of the artificial cycles.40–41 The timing of this “transfer window” seems to vary, depending on the developmental state of the transferred conceptus and the method of hormone treatment. The shorter duration of the embryo transfer in some artificially induced cycles may be attributable to the rapidity by which the endometrium is prepared. For example, the endometrium when sampled was found to be more advanced than expected after induction of ovulation by hMG/hCG regimens.5,42 SUMMARY Endometrial receptivity is the outcome of coordinated and balanced statement of number of factors. The human blastocyst triggers a cascade of molecular events that overlap the effects of hormonal regulation. The blastocyst modulates endometrial receptivity by the regulation of endometrial epithelial adhesion molecules such as the integrin β3 subunit, the mucin MUC1 and the cytoskeleton associated proteins of the ERM family members. During the adhesion and the invasion phases of implantation, the blastocyst plays an important role in the induction of apoptosis in the endometrial epithelial cell barrier thus helping in the process of implantation. The available evidence thus suggests that for successful implantation to occur, it is necessary to have a receptive endometrium and the presence of a competent embryo. Exogenous administration of E2 and P results in adequate preparation of the endometrium for implantation. A variety of replacement regimens have been suggested. It appears that E2 is necessary for adequate priming of the endometrium, and that the time, dose and route of administration do not play a major role in the attainment of this priming. Adequate E2 stimulation can be substantiated by the demonstration of endometrial thickness that is approximately ≥6–7 mm. Endometrial receptivity is

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ultimately induced by adequate stimulation by P, which can only act on an adequately primed endo metrium. Many of the molecules that have been discussed only appear during the implantation period and hence may be largely responsible for regulation of uterine receptivity. In the future, recognition of reliable biochemical markers may enable us to improve uterine receptivity and therefore increase implantation rates following IVF procedures and optimize clinical success of the procedures. Hence, the ideal marker for clinical practice would be a ubiquitous molecule, which is, secreted de novo in the uterine cavity. Identification of this molecule in uterine secretions could potentially enable us to identify an endometrium capable of allowing implantation of a competent embryo. The measurement should be sensitive and reproducible, to enable the immediate modification of the cycle based on the results. Finally, screening for the marker must be non-invasive so as not to disturb the endometrial architecture. REFERENCES 1. Hertig A, Rock J, Adams EC. A description of 34 human ova within the first 17 days of development. Am J Anat 1956; 98:435–93. 2. Rogers PAW, Murphy CR. Uterine receptivity for implantation. Human Studies. In Yoshiaga (Ed). Blastocyst. Implantation, Serono Symposia USA 1989; 231–38. 3. Navot DM, Scott Rt, Droesh K. Kreiner D, Veeck LL, LiuHC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vitro Fertil Steril 1991; 55:114–17. 4. Psychoyos A. The implanation window Basic and clinical aspects. Inperspectives on assisted reproduction: Mori T, Aono T, Tominaga T, Hiroi M (Eds). Ares Serono Symposia 4.1993; 57– 62. 5. Martel D, Frydamn R, Glissant M, Maggioni C, Roche D, Psychoyos A. Scanning electron microscopy of postovulatory human endometrium in spontaneous cycles stimulated by hormone treatment. J Endocrinol 1987; 114:319–24. 6. Sarantis L, Roche D, Psychoyos A. Displacement of receptivity for nidation in the rat by the progesterone antagonist RU486: a scanning electron microscopy study. Hum Reprod 1988; 32:251–55. 7. Valdizan MC, Julian J, Carson DD. WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells. J Cell Psyial 1992; 151:451–65. 8. Brag VM, Gendler SJ. Modulation of Muc-1 mucin statement in the mouse uterus during the estrous cycle, early pregnancy and placentation J Cell Science 1993; 105:397–05. 9. Hey NA, Graham RA, Seif MW, Alpin JD. The polymorphic epithelial mucin Muc-1 in human endometrium is regulated with maximal satement in the implantation phase. J Clin Endocrinol Metab 1994; 78:337–42. 10. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadil I, Kontgen F, Abbondanzo SJ. Blastocyst implantation depends on maternal statement of leukemia inhibitory factor. Natue 1992; 359:76– 79. 11. Arcici A, Engin O, Attar, O live DL. Modulation of leukemia inhibitory factor gene statement and protein biosynthesis in human endometrium. J Clin Endocrinol Metab 1995; 80:1908–15. 12. M von Wolff, Thaler CJ, Broome J, Strowitzki T, Tabibzdeh S. Regulated statement of cytokines in human endometrium throughout menstrual cycle: Dysregulation in habitual abortion Mol Hum Reprod. 13. Armant DR. Kaplan HA, Lennarz WJ. Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts. Dev Biol 1986; 116:519–23. 14. Ruoslahti E. Integrins. J Clin Invest 1991; 87; 15.

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15. Tabibzadeh S. Patterns of statement of integrin molecules in human endometrium throughout the menstrual cycle. Human Reprod 1992; 7:876–82. 16. Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW, Sun J. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 1994; 62:497– 06. 17. Tabibzadeh S, Kong QF, Babaknia A. Statement of the heat shock proteins in human endometrium during the menstrual cycle. Mol Hum Reprod 1996; 11:633–40. 18. Griudl M, Buyuksal A, Babaknia A, Fazelbas AT, Sivarajah S, Satyaswaroop PG et al. The progressive rise in the statement of a crystallin b chain in human endometrium is initiated during the implanation window; Modulation of gene statement by steroid hormones. Mol Hum Reprod 1997; 3:333–34. 19. DingY-X, Zhu LJ, Bagchi MK, Bagchi IC. Progesterone stimulates clacitonin gene statement in the uterus during implantation Endocrinology 1994; 135:2265–274. 20. Taylor HS, Vanden Heuvel Gb, Igarashi P. A conserved Hox axis in the mouse and human female reproductive system: late establishment and persitent adult statement of the Hox cluster genes. Biol Reprod 1997; 576:1338–45. 21. Robertson SA, Mayrhofer G, Seamark Rf. Ovarian steroid hormones regulate granulocytemacropahge colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biology of Reprod 1996; 54:265–77. 22. Lessey BA, Yeh I, Castelbaum AJ. Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation. Fertil Sterill 1996; 65:477–83. 23. Simon C, Gimeno MJ, Mercader A. Embryonic regulation of integrins β3, α4 and α1 in human endometrial epithelia cells in virto. JCEM 1997; 82; 2607–616. 24. Hey NA, Graham RA, Seif MW. The polymorphic epithelium mucin MUCl is regulated with maximal statement in the implanation phase JCEM 1994; 78:337–42. 25. Galan A, O’Connor JE, Valbuena D. The Human Blastocyst regulates endometrial epithelial apoptosis in embryonic implantation. Bio Reprod 2000. 26. SperofL, Glass RH, Kase NJ. Female infertility In: Clinical gynecologic endocrinology and infertility: Mitchell C (Ed) (5th edn), Chapter 26. 1994; 808–39. 27. Taylor HS, Bagot C, Kardana A, Olive A, Arici A, Hox gene statement is altered in the endometrium of women with endometriosis. Hum Reprod 1999; 145:1328–331. 28. Navot D, Laufer N, Kopolovic, J, Rabinowitz R, Birkenfeld A, Lewin A et al. Artifically induced endometrial cycles and establishment of pregnancies in the absence of ovaries. N Engl J Med 1986; 314:806–11. 29. LiTC, Cooke ID, Warren MA, Goolamallee M, Aplin JD. Endometrial responses in artificial cycles: a prospective study comparing four different oestrogen doses. Br J Gynecol 1992; 99; 751–56. 30. Miles RA, Paulson RJ, Lobo RA, Press MF, Dahmouth L, Sauer MV. Pharmacokinetics and endometrial tissuen levels of progesterone after administration by intramuscular and vaginal routes: a comparative study Fertil Steril 1994; 62; 485–90. 31. Israel R, Mishel DR Jr, Stone SC, Throneycroft IH, Moyer DL. Single Luteal phase serum progesterone assay as an indicator of ovulation. Am J Obstet Gynecol 1972; 112:1043–46. 32. Navot D, Scott Rt, Droesch K, Veeck LL HC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vito. Fertil Steril 1991; 55:114–18. 33. Navot D, Bergh PA, Williams M, Garrisis GJ, Guzman I, Sandler B et al. An insight into early reproductive processes through the in vivo model of ovum donation. J C in Endocrin Metab 1991; 72:408–14. 34. Martel D, Frydman R, Glissant M, Maggoni C, Roche P, Psychoyos A. Scanning electron microscopy of postovulatory human. endometrium in spontaneous cycles and cycles stimulated by hormone treatment. J Endocrinol 1987; 114:319–24.

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35. Kolb BA, Paulson RJ. The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol 1997; 176:1262–269. 36. Paulson RJ, Hatch IE, Lobo RA, Sauer MV, Cumulative conception and live birth rates after oocyte donation implications regarding endometrial receptively. Hum Reprod 1997; 12:835–39. 37. Gonen Y, Casper RF. Prediction of implantation by songraphic appearance of the endometrium during controlled ovarian stimulation for in vitro fertilization (IVF). J Vitro Fert Embryo Transfer 1990; 7:156–52. 38. Tourgeman DE, Coulam C, Stanczyk FZ, Paulson RJ. Vaginal administration of estradol in preparation for oocyte donation Fertil Steril 1999; 71(supp I1):1. 39. Navott RW, Scott RT, Doresch K, Veeck LL, Liu HC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril 1991; 1:114–18. 40. Rosenwaks Z. Donor eggs, their application in modern reproductive technologies. Fertil Steril 1987; 47:895–09. 41. Navot D, Laufer N, Kopolovic J, Rabinowitz R, Birkenfeld A, Lew in A Grant M et al. Artificially induced endometrial cycles and establishment of pregnancies in the absence of ovaries. New Engl J Med 1986; 314:806–11. 42. Garcia Je, Acosta AA, Hsiu JG, Jones Jr HW. Advanced endometrial maturation after ovulation induction with human menopausal gonadotrop in human chorionic gonadotrop in for in vitro fertilization. Fertil Steril 1984; 1:31–35.

CHAPTER 56 Modulators of Endometrial Receptivity: A Molecular Symphony Rafael C Haciski SUMMARY The embryo implantation process remains the limiting factor in attaining higher success rates in Assisted Reproductive Technologies—while we are constantly pushing the envelope technologically, ethically, and legally, ever reaching to higher levels, successful implantation has remained at relatively low. This complex set of inter-related events continues to awe, inspire, and humble all who are involved with reproduction. This paper will review the parameters involved in successful outcome, describe the morphological events involved in implantation, review some of the cellular and molecular constituents, and offer suggestions for improved outcomes. THE BROAD PERSPECTIVE Over last two decades considerable advances have been made in the success rates of Advanced Reproductive Technologies (ART) procedures: ovulation stimulation protocols yield good oocyte harvests, with most oocytes being successfully fertilized and going on to pre-embryo development. However, the transfer of those pre-embryos into the uterus for implantation remains a low yield procedure, with implantation rates of 5–30% per embryo being the norm.1–3 While we have some rudimentary information about the early morphological events of implantation, and a great deal of progress has been made in understanding protein secretions, growth factors, and adhesion molecules associated with endometrial and trophoblastic interaction and function, there remains remarkable little information about the exact nature of interactions at the cellular and subcellular levels. Indeed, the endometrial cavity remains a “black box” wherein the activities governing the implantation of the newly arrived pre-embryo remain as yet unknown. The end point of ART endeavors remains successful pregnancy. In order to analyze how we may maximize success rates, we need to break down the overall process into independent components, and evaluate each component individually. If we look at the probability of a pregnancy (P), we find that it is a function4 of the probability of implantation of an embryo (EI) and the number of embryos transferred (n): P=1−(1−EI)n

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The single unknown factor in this formula is the implantation quotient EI. In order to have a better grasp of what this probability is, we have to understand what are the factors that influence successful embryo implantation. Logically these determining factors are: • Embryo quality (EQ) • Transfer efficiency (TE) • Endometrial receptivity (ER) These three factors may be combined into one equation that expresses the chances of embryo implantation:4 EI=EQ×TE×ER Continuing our analysis by breaking down the overall process into its smaller components, we need to determine what are possible modulators of endometrial receptivity. Such modulators may fall into these general categories: • Physical/mechanical problems such as intrauterine devices (IUD), fibroids, toxins (environmental or infectious), may adversely affect proper development of the endometrial surface ultrastructure5–6 thus inhibiting or preventing the implantation process; muscular contractions (following oocyte retrieval, or induced during embryo transfer) may displace the blastocyst, or prevent its attachment;7 • Alterations in hormonal effects, consisting primarily of estrogen priming in the proliferative phase, and progesterone-effected luteinization in the secretory phase, may give rise to inhospitable endometrium;8–9 • Vascular changes involving angiogenesis and vascular permeability, if not executed properly may also interfere with implantation; • Protein and enzyme moieties exert effects at the cellular level, these effects must be accomplished in a precise fashion, otherwise, they will inhibit implantation; • Immunological factors are necessary for normal implantation, and if not properly expressed, may alter the process in deleterious fashion. There is now considerable evidence that the pivotal modulation of implantation is achieved via immunologic, protein, and enzymatic changes; indeed, mechanical and toxic problems, as well as hormones, affect those protein and enzyme changes. Setting the Stage—the Overture Allow me to paint a bigger (but admittedly speculative at this point) picture and then look in detail at the individual players involved. There are two distinct but interrelated cyclic sequences of events that are operative in the process of reproduction: the preparation of the oocyte and the preparation of the endometrium for implantation. Factors instrumental in the preparation of a receptive endometrium originate in the maternal hypothalamic-pituitary-gonadal axis. The cyclic function of the ovary under control of the hypothalamic and pituitary hormones not only serves to complete the preparation of the oocyte for fertilization and subsequent growth, but, through the cyclic release of ovarian hormones estrogen (E2) and progesterone (P4), interacts with, and controls the cyclic endometrial development. During each menstrual cycle, a series of coordinated, architectural, morphological, cytochemical, and molecular changes

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ultimately lead to the preparation of a receptive endometrium, with the receptivity being confined to a very specific and time-limited implantation window.10–13 In the past, the debate about the primary defects in implantation failure centered on either the embryo or the endometrium. Such arguments are increasingly futile in light of emerging studies demonstrating that in order for implantation to occur, a coordinated molecular dialog must be established between the primed and receptive apical surface of the luminal epithelium (LE) and the trophoblast of the hatched blastocyst.14 If, for any reason, this dialogue is not established or is disturbed, the process of implantation is interrupted, and the embryo is aborted. The natural fate of the receptive endometrium, in the absence of implantation, is the continued development of a set of changes that ultimately lead to menstruation. The overall control of implantation is ultimately maternal, as it is through the stimulation of follicular growth that there is both the maturation of the oocyte, and the preparation of the endometrium with E2 and P4 regulating the changes in the expression of adhesion molecules, cytokines, heat shock factors, matrix metalloproteases, and transcriptional factors, many of which have been implicated in mediating implantation. Because of the complexity and dynamic nature of implantation, the molecular changes are still poorly understood.15 One of the difficulties in studying the process of implantation is that human studies are fraught with ethical restrictions, while not always may we infer from animal studies that the same set of endometrial genes in humans are implicated in the endometrial receptivity and implantation. For example, despite the finding that mucin MUC-1 is downregulated in the mouse, it is upregulated in the human. On the other hand, while ßhCG is necessary in the human to maintain the corpus luteum function until placenta can take over, in the mouse it is unnecessary as the pituitary maintains ovarian function until placenta may take over. With that caveat in mind, looking at what information we have from studies of rodents and primates, as well as the pioneering work of Hertig12 on early human embryos, the process of implantation may be viewed as a coordinated sequence of separate but interacting events: • Apposition of the hatched blastocyst to the apical endometrium; • Adhesion of the blastocyst to the endometrium; • Penetration of the endometrium by the trophoblast; • Invasion into the endometrial stroma; • Breaching of the maternal blood vessels • Placental formation We will look at each of these events separately. Act I—Apposition 16

The observations of Hertig that early stage implantation sites were all in the upper half of the endometrial cavity, and away from the lateral margins of the lumen suggest a specific mechanism of blastocyst placement. While the egg and the blastocysts are presumed to have been transported through the fallopian tube borne on the currents created by the cilia lining the tubal luminal epithelium, few cilia have been noted on the luminal epithelial cells of the endometrium; they appear to be insufficient in number to

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affect the transport of the blastocyst to the desired location within the uterus. Indeed, that specific intrauterine placement of the blastocysts suggests muscular contractions possibly of the myometrium, but more likely of the endometrial layer. Recent sono-videographic studies19 of the endometrium demonstrate slow motion peristaltic movement of the endometrium, which may be directly involved in proper placement of the blastocyst, or in cases of irritated endometrium, a displacement of the blastocyst. Time-lapse sonovideography of the endometrium demonstrates dispersal and expulsion of minute droplets of echogenic fluid18 following traumatic mock embryo transfer; the fluid was effectively and rapidly cleared to the endocervical canal, or into the fallopian tube. Once properly placed within the cavity, the blastocyst comes in contact with the luminal endothelial surface. Indeed, the uterine cavity is only a potential cavity; in normal situation that cavity is collapsed, and the endometrial surfaces are in direct contact. Scanning electron microscopy of the rat uterine epithelium19 demonstrated peculiar projections of the luminal epithelial cells. Subsequent studies12 demonstrated that these projections were fluid imbibing structures (pinopodes) that appeared approximately 7 days after ovulation, and marked the opening of the implantation window, disappearing after <48 hours.20 While these projections are also seen in human endometrial epithelia, there is no direct evidence of fluid uptake, hence these structures should be considered simply apical projections, recently more appropriately named uterodomes.21 The speculative suggestion is that in order for apposition to occur, the endometrial lumen must be closed; furthermore, the more tightly the blastocyst is pressed against the epithelium, the less effort is necessary for satisfactory trophoblastic adhesion. Thus blastocyst turgor, and increased pressure from the stromal edema, as well as outreaching uterodomes all contribute to pressing the blastocyst against the endometrial surface, facilitating adhesion. This theoretical consideration is supported by in vitro studies of rabbit endothelium where it was necessary to make an endometrial epithelial sandwich rather than simply placing the trophoblast on the epithelial surface to achieve a high percentage of attachment and invasion.22 Not only is the position within the cavity important, but so is the orientation of the blastocyst in respect to the luminal endothelium. In studying the early human and primate implantation, it has been noted that the blastocyst orients itself with the inner cell mass towards the implantation site. This suggests that the trophoblast are not all the same—it is only the trophoblast near the inner cell mass that exhibit adhesive properties. Act II—Adhesion Once the blastocyst finds itself apposed to the luminal epithelium, the extraordinary process of actual implantation begins, becoming even more remarkable in that the subsequent adhesion occurs not only between apical surfaces of two distinctly different epithelia, but also genetically different epithelia. Possibly the closest known process that may involve similar mechanics is the adhesion of leukocytes to the endothelial epithelium of the blood vessels and their transmigration through that endothelium. Based on studies revealing extensive syncytial trophoblast formation in the earliest known post-epithelial penetration stages of human implantation, as well as the appearance of syncytium formation in primate blastocysts cultured beyond the time of implantation, there is suggestion that the formation of syncytiotrophoblast, which has a surface that includes

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molecules capable of adhering to the luminal epithelium, is what allows this unusual process to occur.23 While this process is not well understood, adhesive molecules such as integrins24 and osteopontin,25 adhesion blocking molecules such as MUC-1,26 agents possibly involved in control of adhesion such as calcitonin,27 as well as a host of other proteins such as amphiregulin, leukocyte inhibitory factor (LIF), epidermal growth factor (EGF), colony stimulating factor (CSF), interleukin-1ß (IL-1ß), cyclo-oxygenase (COX), and the homeobox genes (specifically HOX A 10) are being investigated. Involvement of MUC-1 is interesting in that there appears a species-specific distinction: while in mice the MUC-1 is downregulated on the luminal epithelium, in human it appears to be upregulated during the implantation suggesting a different mechanism of action.28 Act III—Penetration The penetration of the luminal epithelium by the trophoblast was first described by Enders in 1983 using electron microscopy.29 After apparent loosening of the tight junctions between the luminal epithelial cells, the syncytial trophoblast processes insert themselves between those cells, and then establish the tight junctions with the epithelial cells. The adhesion junctions are likewise interrupted, and following insertion of the syncytial processes, re-established, thus maintaining the structural integrity of the luminal epithelium. This maintenance of structural integrity is important, as this process is not destructive, however, there is speculation that some leakage may occur,30 possibly providing a chemotactic signal to the trophoblast. This trophoblastic expansion then continues to the basal lamina. These findings were later confirmed in other primates31–32 During this process, which may resemble the leukocyte transendothelial migration,30 different transmembrane molecules are sequentially encountered and altered sufficiently resulting in temporary opening of the junctions, with subsequent interaction with the syncytial membrane to reestablish those junctions. Act IV—Invasion Having penetrated the perimeter, the blastocyst now continues to invade by breaking through the basal membrane and spreading into the endometrial stroma. Presumably to facilitate such penetration and insertion, the blastocyst collapses its cavity, subsequently re-expanding it. While this collapse was initially thought to be an artifact of specimen preparation, studies in animals where the blastocyst establishes a vascular connection with the endometrium without becoming interstitial have demonstrated no such collapse.12 Therefore, this appears to be an intentional process to facilitate the insertion of the blastocyst into a subdermal position. At the margins of the penetration, there is a mass of cytoplasm containing small, clustered nuclei suggesting fusion of the luminal epithelial cells forming a symplasma. This tissue also demonstrates presence of few much larger nuclei, suggesting either invasion by the syncytial trophoblast, or possibly fusion with the trophoblast forming a heterokaryon. While initially the syncytial trophoblast has no free spaces within it, over time clefts begin to form. Some of these clefts appear to be lined by microvilli suggesting a

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differentiation of invading trophoblast into absorptive trophoblast.33 These clefts are interconnected and extend inwards, eventually filling the space between the outer periphery of the syncytium and the cytotrophoblast layer surrounding the blastocyst cavity Act V—Vascular Breaching Upon penetration of maternal venules and capillaries, maternal blood begins to fill the microvilli-lined clefts within the syncytium. This penetration is achieved in a manner similar to the penetration of the luminal epithelium—the syncytial trophoblast interpose themselves between the cells lining the venules, and establish tight junctions allowing confluences between the venules and the lacunae with no leakage.34 As the penetration of maternal vasculature increases, the clefts fill with blood and expand into lacunae, with eventual establishment of sluggish blood flow.35 While the microscopic sections of the early implanting blastocyst show it buried within the endometrium, with an overlying covering of endometrial epithelium, it appears that the covering is achieved not by an overgrowth of the endometrium, but rather by the lateral and inward expansion of the syncytial trophoblast under the endometrium. Having achieved such expansion, the blastocyst is now completely surrounded by unilaminar syncytium formation. The thicker portion of the syncytium beneath the blastocyst is now filled with lacunae, and these are interrupted by syncytial septae. The margin of implantation appears to be relatively smooth spherical shape in contact with endometrial stroma, interrupted only at the confluence withblood vessels. At this stage, approximately 8 days post ovulation and 4–5 days post initiation of implantation, the trophoblast has differentiated from an invasive to absorptive type, forming an ovoid chorionic vesicle with interconnected lacunae, and has formed an organized layer of cytotrophoblast around the exocelom. Act VI—Placental Formation The final stage of implantation is marked by a tremendous increase in cytotrophoblast proliferation. This proliferation is directed through the lacunae towards syncytial periphery, where the syncytium is breached and the cytotrophoblast spreads on the endometrial surface forming both the anchoring villi and the trophoblastic shell. Simultaneously, extra embryonic mesoderm begins to indent the inner cytotrophoblast cell mass, forming secondary villi, which eventually become lined with capillaries formed by endothelial cells. While at the initial stages of implantation, the trophoblast was invasive component, it is now the cytotrophoblast that becomes invasive, extending into the endometrial stroma to complete the placental formation and anchor the embryo within the uterus. The Players—at the Molecular Level Now that we have looked at the morphological events, which give a rough outline of the overall process of implantation, let us take a look in greater detail at some of the interaction at the cellular and molecular levels between the blastocyst and the

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endometrium. The literature is rife with descriptions and actions of diverse cytokines, proteins, enzymes, messenger RNA, and genes, but the full significance and interactions of these moieties vis-a-vis implantation is far frombeing understood. While it may be beyond the scope of this article to describe all the known players in this process, I would like to describe several of the important ones, and what we know of their actions: Calcitonin • Peptide hormone induced by progesterone • Seen in glandular epithelium in midsecretory phase of the cycle36 • Possibly involved in cellular calcium homeostasis. Chemokines • Small polypeptides specializing in attracting leukocytes • Generally produced in response to exogenous irritants or endogenous mediators • Released by epithelial and stromal cells in a precise temporal pattern controlled by estrogen and progesterone.37 Epidermal Growth Factor (EGF) • Large family composed of EGF, transforming growth factor (TGF-a), and heparinbinding EGF-like growth factor (HB-EGF) • All interact with EGF receptor • Potent cellular mitogens, stimulate cell proliferation, and possibly differentiation • Associated with increase in serum estradiol • Independent of progesterone secretion • Seen in stromal cells in proliferative phase, then decline • Seen in epithelial cells in secretory phase38 • EGF receptor appears in the embryo at the 4- to 8-cell stage. Ezrin, Radixin, and Moesin (ERM) • Ezrin, radixin, and moesin are proteins involved in association of actin filaments with cell membrane (the actin cytoskeleton provides structural framework that defines cell shape and polarity)39 • Expressed by endometrial epithelium • Upregulated by progesterone • Upregulated by the blastocyst.40 Integrins • This is a family of adhesion molecules, that are transmembrane heterodimers containing one a and one 6 subunit

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• Several extracellular matrix proteins are capable of binding to integrins (fibronectin, vitronectin, collagen type IV) • Integrins a4 and a1 are progesterone stimulated and appear early in secretory phase • Integrin ß3 is also progesterone stimulated and appears in mid secretory phase41 • Presence of blastocyst (possibly mediated through the IL-1 system) appears to lead to ß3 expression by endometrial epithelial cells.42 Interleukin-1 (IL-1) • modulates cell proliferation and differentiation • stimulates expression of LIF receptors43 • stimulates prostaglandin synthesis • required for synthesis of nitric oxide (vasodilator) • IL-1ß decreases expression of prolactin and insulin-like growth factor binding protein1, which are involved with decidualization.44 Leukemia Inhibitory Factor (LIF) • Probably one of the key elements involved in implantation;45 • Maximal expression of this protein is seen in the endometrial glands during “window of implantation;”46 • Also expressed by the blastocyst starting on day 4 post ovulation • Required for promotion of blastocyst attachment and uterine decidualization, absence prevents implantation; Mucins • Family of glycoproteins forming natural barriers to adhesion • MUC-1 is the most studied mucin—it is a transmembrane molecule with a relatively long tail resting in the extracellular space, and shorter tail residing inside the cell • In humans (expression is variable in other species) MUC-1 expression increases in early secretory phase, decreasing in late secretory phase47 • MUC-1 expression is downregulated by the blastocyst in the rabbit48 • In human, however, MUC-1 appears to be upregulated by the blastocyst, suggesting that it actually may be involved in the initial attachment, rather than preventing it as would be expected • Progesterone administration preferentially increases luminal MUC-1 as compared to glandular MUC-1.49 Prostaglandins (PGs) and COX Enzymes • PGs are involved in increasing vascular permeability50 • PG secretion is mediated by ovarian steroids • COX enzymes are the rate limiting molecules in biosynthesis of PGs • COX-1 is expressed mainly in glandular and luminal epithelium • There are two isoforms: COX-1 and COX-2

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• COX-2 is expressed in luminal epithelium and perivascular cells;51 • The two enzymes COX-1 and COX-2 are coded by two different genes • Expression of the COX-2 gene is induced by IL-1ß52 • COX-2 deficient mice demonstrate defective implantation (not COX-1) • hCG and LH increase expression of COX-2. Proteinases Invasion requires proteolytic enzymes that may breach the basement membrane, and degrade the extracellular matrix (ECM); some of these enzymes are: • serine proteases: – urokinase- and tissue-plasminogen activators catalyze conversion of plasminogen to plasmin and have a broad proteolytic action. • matrix metalloproteinases: – family of zinc dependent endopeptidases, active against ECM – comprised of collagenases, gelatinases, and stromelysins.53 As may be deduced from the preceding extensive but at the same time incomplete listing, the players involved in this symphony are numerous, and while there may be much information available about each individual player, the understanding of how these players interact with one another is minimal. What is safe to say, is that there is exquisite coordination between all the players, each one having a specific effect on another, and that effect may also vary with the intensity of interaction (same player may have a stimulatory effect at low levels, inhibitory at higher levels). Similarly to a large orchestra, when the players do not do their part, cacophony results, but if they act in concert, they contribute to a coordinated final outcome, which is a beautiful symphony. Improving the Outcome With the minimal knowledge that we have, how can we control this process and maximize the end result? Indeed, how do we maximize the success of ART? The answer lies in Paulson’s equation, mentioned at the beginning of this paper, EI=EQ×TE×ER which states that successful implantation depends on embryo quality, transfer technique, and endometrial receptivity. In those three parameters lies the success of ART. If we look at last of those parameters, endometrial receptivity, we find that we lack sufficient knowledge or technique at present to alter, modify, or improve that receptivity at the molecular and cellular level. Our ability to control the receptivity is limited to endometrial preparation through appropriate hormonal stimulation (ovulation induction) and to the presentation to the endometrium of an embryo of superior quality. That brings us to the first of the controlling parameters: embryo quality. This is ultimately dependent on oocyte quality, which in turn depends on gamete quality and the maturation of that gamete (ovulation induction). Unfortunately, gamete quality is totally

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beyond our control, and ovulation induction is covered elsewhere in this collection. This leaves us with the last place where we can control the embryo quality and that is the embryo culture process. Embryo Culture It is a fallacy to think that poor quality oocytes will be converted to good embryos through in-vitro culture techniques.54 In computer technospeak jargon—garbage in, garbage out (GIGO). So it has to be assumed that we have received good oocytes for fertilization and culture. Furthermore, it is important to realize that while many different culture conditions will support embryos in-vitro, only few of these culture conditions will lead to a viable blastocyst, one capable of appropriate communication with the endometrial epithelium and continued development into a normal fetus. The fact that an embryo will grow in many diverse cultures is a testament to its plasticity and resilience, and not to the quality of our culture conditions.55 Therefore the essence of any culture technique is to minimize any stresses (metabolic, homeostatic) on the embryo and provide for the necessary nutrients. The more obvious stresses are the physical ones—abrupt temperature changes, photonic stimulation, acidbase disturbances. Less obvious are the different nutritional requirements of the oocyte and cleavage-stage embryo as compared with the blastocyst. The early embryo is relatively quiescent with low levels of oxidations and biosynthesis. The blastocyst, on the other hand, is metabolically active, with high levels of biosynthesis and high energy demands.55 These differences lead to sequentially changing nutritional requirements at different stages of the developing embryo. If we look at the fluids and nutrients produced by the fallopian tube and by the endometrium as the oocyte/embryo progresses through the reproductive tract while progressing through its development, we find that they are different at each location.56 These differences correspond to the specific nutritional demands of the embryo at that particular stage of its journey.55 It is noteworthy that early stage (day 1–3) is not as nutritionally demanding, supported by the evidence that many different culture media lead to similar implantation rates with early transfer (=8 cell stage). However, subsequent culture to morula and blastocyst stage (day 4–6) places a considerably greater burden on the laboratory, to provide culture media that meet the more critical requirements of the embryo—if such media is not provided, appropriate development fails to proceed. What are the required differences in the culture media? From the standpoint of carbohydrate metabolism, the early embryo uses pyruvate or lactate as the primary source of energy, while glucose becomes the main energy source in the blastocyst stage. It is important to point out that glucose, in addition to being a source of energy for the blastocyst, is also an important anabolic precursor required for the synthesis of triacylglycerols and phospholipids at all embryo stages. It is also an important precursor for the synthesis of mucopolysaccharides and glycoproteins. Furthermore, glucose is required for nucleic acid synthesis, and NADPH is required for synthesis of lipids and is also needed for intracellular reduction of glutathione, which is an important antioxidant.57 It is noteworthy that while blastocysts may be cultured satisfactorily in absence glucose, suchblastocysts demonstrate subsequent impaired implantation when compared to blastocysts cultured with glucose.58

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On the protein side, it has been shown that alanine, aspartate, asparagine, glutamate, glutamine, glycine, serine, taurine, and proline have a beneficial effect on the early embryo. Indeed, these amino acids are found in relatively high levels in the fallopian tube fluid,59 and have been shown to act as regulators of energy metabolism, osmolytes, and buffers.55 Furthermore, essential amino acids have been shown to negate the beneficial effects of the nonessential amino acids during the first few cleavage divisions. As the embryo physiology accelerates, there is an increased need for more amino acids in the culture medium; the essential amino acids are necessary for the development of the inner cell mass, while the nonessential amino acids help in the development of the trophectoderm.60 Such dynamic requirements have led to the development of sequential media, one designed for the early embryo up to the 8-cell stage, another for the development from day 3 to blastocyst.61 Timing of the Embryo Transfer Notwithstanding the increased burden placed on the laboratory for more critical culture support, the higher implantation rate, higher pregnancy rate, and lower multiple pregnancy rate seem to more than justify the increased effort of blastocyst transfer.62 There may be many reasons for this increased implantation rate: • Physiologically, the blastocyst arrives in the uterus on day 4–5 post ovulation, thus premature placement of the embryo in the endometrial cavity places it in a potentially compromising environment, leading to nutritional stress. • It is known from animal and human studies63 that hyperstimulated endometrium is less ideal environment for the embryo, thus later transfer may allow for normalization of that endometrium64 • Not all fertilized oocytes are normal, indeed with increasing age of the patient there is an increase in chromosomal abnormalities (from~20% at younger age, to over 50% above 40 years of age)65 which lead to arrest of development; however, since the embryonic genome is just beginning to be transcribed at the 8-cell stage, such arrest only becomes evident with longer embryo culture • Paternal contribution to the development begins after the 8-cell stage,66 thus any problems with paternal genome do not come into evidence until longer culture is employed • Uterine contractions have been negatively associated with implantation, and have been found to be strongest at the day of oocyte retrieval, gradually subsiding over several days, and absent on day 5, thus blastocyst transfer may have lesser chance for contractile expulsion of the embryo.67 pH and Gas Phase While the pH of the surrounding medium (pHo) is usually 7.4, the pH inside the embryo (pHi) is 7.2; furthermore, the pH does not necessarily control pHi, which is controlled by lactic and amino acids.68 Because the embryo has to make an effort to maintain a lower pHi, it would seem prudent to lower the pHo to lessen the stress on the embryo. Since using bicarbonate-buffered medium allows the CO2 concentrations to have a direct impact on the pH, it would be advisable to use CO2 concentrations around 6–7 percent.

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Looking at oxygen concentrations, the levels in the fallopian tube may be around 6– 8%, while uterine oxygen concentration is much lower at 1.5%.69 Following this physiologic suggestion, it has been shown that human embryos produce blastocysts with more cells at 5% oxygen concentration than at higher ambient concentration of 20 percent.70 As the presence of more blastocyst cells is related to improved viability, the obvious suggestion is to culture the embryos in an atmosphere of low oxygen concentration. Dilution Factor As the embryo travels through the reproductive tract, it exists in a minuscule volume of surrounding fluid. This makes conceptual sense as autocrine factors produced and released by the growing embryo need to be kept in relative proximity to their target receptors. Culturing the embryo in a larger volume as is typically found in the culture dish (1 ml) is counterintuitive, as those autocrine factors released by the embryo become diluted, and the embryo loses its ability to communicate effectively. It has been suggested by some that the embryos should be cultured in small volumes of media (20–50 µl) and co-cultured in groups of 2–4 embryos.71 CONCLUSION Evolving research into endometrial receptivity has led to the realization that many different causes of infertility have an adverse impact on implantation and it is through correction of these causes that endometrial receptivity can be modulated. Improvement in implantation rates cannot be achieved by focusing on one factor. Rather, the path to success lies in appropriate patient selection and quality ovulation induction, which will produce healthy oocyte and endometrium, as well as appropriate culture techniques which will facilitate and promote healthy embryo development, and ultimately in an atraumatic and timely embryo transfer. While it would be desirable to control the endometrial receptivity directly at the cellular and molecular level, this is not yet available with current level of knowledge and technology. Undoubtedly, further research into this complex process will lead to more successful management of infertility. REFERENCES 1. Centers for Disease Control, USA 1998 Report. 2. Kooij RJ et al. Fertil Steril 1996; 66:769–75. 3. SART 1998 report analysis. 4. Paulson RJ. Am J Obstet Gynecol 1990; 163:2020–23. 5. Kolb BA. Am J Obstet Gyn 1997; 176:1262–67. 6. Kolb BA. Fertil Steril 1997; 67:625–30. 7. Bedaiwy MA et al. Video presentation VP-4; 2001 Amer Society for Reproductive Medicine annual meeting. 8. Navot TD. J Clin Endocrinol Metab 1989; 68:485 9. Navot TD. NEJM 1986; 314:806.

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10. Bergh PA, Navot TD. Fertil Steril 1992; 58:537–42. 11. Finn CA, Martin L. J Reprod Fertil 1974; 39:195–206. 12. Hertig AT et al. Am J Anat 1956; 98:435–93. 13. Navot TD et al. Fertil Steril 1991; 55:114–18. 14. Stewart CL, Cullinan EB. Dev Genet 1997; 21:91–101. 15. Cross J et al. Science 1994; 266,1508–51. 16. Hertig AT. JAMA 1989; 261:434–35. 17. Eytan O et al. Med Eng Phys 2001; 23:475–84. 18. Lesny P et al. Hum Reprod 1998; 13:1540–46. 19. Psychoyos A. J Reprod Fertil 1971; 26:137–38. 20. Nikas G. Human Reprod 1997; 14(Suppl 2):99–106. 21. Murphy CR. Human Reprod 2000; 15:2451–54. 22. Hoffman LH In Carson DD (Ed): Embryo Implantation: Molecular, Cellular, and Clinical Aspects. New York: Springer, 1999; 151–60. 23. Pope VZ et al. Placenta 1984; 5:403. 24. Lessey BA. Hum Reprod 1998; 13 (Suppl 3):247–58. 25. Coutifaris C, et al In Carson DD (Ed): Embryo Implantation: Molecular, Cellular, and Clinical Aspects. New York: Springer, 1999; 141–48. 26. Carson DD et al. Bioessays 1998; 20:577–83. 27. Bagchi IC In Carson DD (Ed): Embryo Implantation: Molecular, Cellular, and Clinical Aspects New York: Springer, 1999; 83–91. 28. Carson DD et al: Devel Biol 2000; 223:217–37. 29. Enders AC et al. Am J Anat 1983; 167:275–98. 30. Allport JR et al. J Cell Biol 2000; 148:203–16. 31. Enders AC et al, Am J Anat 1991; 192:329–46. 32. Smith CA et al. Anat Embryol 1987; 175:399–10. 33. Enders AC. Am J Anat 1989; 186:85–98. 34. Knoth M. Acta Obstet Gyn Scand 1972; 51:385–93. 35. Carter AM, Placenta 1997; 18:83–87. 36. Kumar S et al. J Clin Endocrinol Metab 1998; 83:4443–50. 37. Robertson SA et al. Biol Reprod 1996; 54:265–77. 38. Leach RE et al. J Clin Endocrinol Metab 1999; 84:3355–63. 39. Hall A. Science 1998; 279:509–14. 40. Martin JC et al. Embryonic regulation of ezrin. Ares-Serono Workshop on Human Implantation, Valencia, 1999; 30. 41. Lessey BA et al. Fertil Steril 1996; 65:477–83. 42. Simon C et al. J Clin Endocrinol Metab 1997; 82:2607–16. 43. Simon C et al. Fertil Steril 1998; 70:896–906. 44. Frank GR et al. Biol Reprod 1995; 52:184–91. 45. Cheng G et al. Proc Natl Acad Sci USA 2001; 98:8680–85. 46. Vogiagis D et al. J Reprod Fertil 1997; 109:279–88. 47. Hey NA et al. J Clin Endocrinol Metab 1994; 78:337–42. 48. Hoffman LH et al. Endocrinology 1998; 139:266–71. 49. Meseguer M et al. Hum Reprod 1998; 13:121–22. 50. Psychoyos A, Martel D. Embryo-endometrial interactions at implantation. In Edwards RG, Purdy JM, Steptoe PC (Eds): Implantation of the Human Embryo. London: Academic Press, 1985; 197–219. 51. Marions L, Danielsson KG. Mol Hum Reprod 1999; 5:961–65. 52. Huang JC et al. J Clin Endocrinol Metab 1998; 83:538–41. 53. Matrisian LM. Trends Genet: 1990; 6:121–25. 54. Gardner DK et al. J Assist Reprod Genet 1998; 15:455–58. 55. Gardner DK. Theriogenology 1998; 49:83–102.

Modulators of endometrial receptivity 56. Gardner DK, Lane M. Fertil Steril 1996; 65:349–53. 57. Rieger D: Theriogenology 1992; 37:75. 58. Gardner DK, Lane M. Human Reprod 1996; 11:2703–12. 59. Moses DF et al. Theriogenology 1997; 47:36. 60. Lane M, Gardner DK. J Reprod Fertil 1997; 109:153–64. 61. Behr B et al. Human Reprod 1999; 14:454–57. 62. Schoolcraft WB, Gardner DK. Fertil Steril 2000; 74:482–86. 63. Ertzeid G, Storeng R. J Reprod Fertil 1992; 96:649–55. 64. Pellicer A et al. Fertil Steril 1996; 65:1190–95. 65. Munne S et al. Fertil Steril 1995; 64:382–91. 66. Janny L, Menezo YJ. Mol Reprod Dev 1994; 38:36–42. 67. Lesny P et al. Hum Reprod Update 1998; 4:440–45. 68. Phillips KP et al. Hum Reprod 2000; 15:896–904. 69. Fischer B, Bavister BD. J Reprod Fertil 1993; 99:673–79. 70. Gardner DK et al. Proc Am Soc Reprod Med 1999; 72:30. 71. Lane M, Gardner DK: Hum Reprod 1992; 7:558–62.

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CHAPTER 57 Endometrial Preparation for Patients Undergoing Frozen-Thawed Embryo Transfer Cycles Raoul Orvieto, Benjamin Fisch, Doυ Feldberg INTRODUCTION Controlled ovarian hyperstimulation (COH) is the key factor for the success of in vitro fertilization-embryo transfer (IVF-ET) cycles. Its aim is the recruitment of multiple, fertilizable, healthy oocytes with the avoidance of severe ovarian hyperstimulation syndrome (OHSS). Only 1 to 3 of the embryos obtained are transferred into the uterus in each cycle, in order to reduce the risk of multiple gestation; the remainding are cryopreserved for future replacement. Cryopreservation of extra embryos, thus, provides further possibilities for conception after the initial fresh transfer, leading to a higher cumulative pregnancy rate per cycle. Cryopreservation of all embryos may be also helpful in patients at high risk of late severe OHSS1 and in patients in whom endometrial pathology (endometrial polyp, poor endometrial development, etc. is detected during COH. In these situations, transfer of the cryopreserved embryos can be conducted in a subsequent non-stimulated cycle or after the intrauterine abnormality is treated, respectively. Embryo cryopre-servation may also simplify donor oocyte programs by obviating the need for meticulous synchronization between donor and recipient.2 SYNCHRONIZATION Embryos can be frozen at different stages during their development: pronucleated egg (zygote) stage; cleavage stage, i.e. 4-cell stage at 2 days after oocyte pick-up (OPU) or 8cell stage at 3 days after OPU; or blastocyst stage. Cryopreservation allows the transfer of frozen-thawed embryos without any time relation to the stimulated cycle from which they were arisen. It, therefore, offers several options for the timing of embryo transfer corresponding to the method of endometrial preparation. There are four possible endometrial preparation protocols, as described below. The success of all of them requires that the embryonic stage at thawing be synchronized with the date of the endometrium within the endometrial preparation cycle.3 For this purpose, the age of the embryo is calculated from the day of OPU (day 0), which corresponds to the day of ovulation in the endometrial preparation cycle. Since plasma progesterone rises after the initiation of the luteinizing hormone (LH) surge or the administration of human chorionic gonadotropin (hCG) and prior to ovulation, in hormone replacement cycles (see below), the OPU day is considered the day after the initial rise in plasma progesterone level.

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Endometrial Preparation Protocols There are four currently employed replacement protocols for frozen-thawed embryo transfer: • Naturalcycle • Hormone replacement using only estrogen and progesterone2,4 • Hormone replacement using GnRHa for ovarian suppression followed by estrogen and progesterone5 • Ovulation induction by clomiphene citrate or gonadotropins. Patients with functioning ovaries may be offered a choice of all four; however, patients with quiescent ovaries (e.g., donor oocyte recipients with ovarian failure) may use only hormone replacement with estrogen and progesterone. Whatever protocol is used, the success of frozenthawed embryo transfer cycle rely on an optimal synchronization between embryonic stage at the time of transfer and the “age” of the endometrium undergoing the preparation. Natural Cycle Ovulating patients with regular menstrual cycles may undergo frozen embryo transfer in a natural cycle. Starting 3–5 days prior to the estimated ovulation day patients are monitored by serial ultrasound for endo metrial thickness, follicular development, and LH and progesterone levels. The day when LH value exceeds 180 percent of the baseline value, calculated as the mean of the three previous morning samples,6 corresponds to a day prior to OPU/ovulation. Alternatively, HCG 5000IU may be administered whenever follicle diameters of 18 mm, 17 mm, or 16 mm combined with E2 levels of 450–550 pmol/l, 600–700 pmol/l, or 800–900 pmol/l, respectively are observed. HCG administration corresponds to the HCG administration day of the source cycle. No luteal phase support is required in these cycles, as corpus luteum formation is not hampered by an inadequate LH secretion. Hormone Replacement using Estrogen and Progesterone Protocols of endometrial preparation are based on the understanding of the physiology of human endometrial development after exogenous administration of estrogen and progesterone. A detailed description of this issue is beyond the scope of this chapter, and for a comprehensive review, readers are referred to the article by Younis et al.7 However, several key-points should be emphasized. • An endometrium measuring >6 mm in thickness and showing a triple-line pattern on ultrasound scan is considered favorable for embryo transfer. • Endometrial development is unaffected by the length of the follicular phase; studies have shown no adverse effects of a reduction of the duration of exposure to estrogen to 6 days or an increase up to 35 days.8–10 However, receptivity is best preserved when the follicular phase is kept between 12 and 19 days. • The endometrium is affected by either incremental or fixed estrogen levels, even in the supraphysiologic range.

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• Estrogen may be delivered orally, transdermally or via a vaginal ring. The oral route seems to be most popular. • This hormone may be administered in the form of estradiol valerate (4–8 mg) or micronized E2 (0.2–0.4 mg); neither drug has an advantage over the other. • Progesterone may be delivered intramuscularly or transvaginally in micronized form; the dose ranges from 50 to 100 mg/d I.M or 100–300 mg tid transvaginally. • The effect of different modes of progesterone delivery or doses remains unclear. • Some researchers prefer the transvaginal approach because the absence of clearance during the hepatic first-pass effect leads to higher progesterone levels in the target organ, the enodmetrium. • The dose of progesterone may need to be increased in women over 40 years old.11 Endometrial preparation with estrogen and progesterone is modeled on the natural menstrual cycle. The initial follicular or estrogenic phase is maintained with either daily oral estradiol or estradiol valerate (4–8 mg) or transdermal estrogen (0.2–0.4 mg). After 10 days of estrogenic exposure the patients are asked to attend the clinic, and from this point they are monitored by serial ultrasound scanning for endometrial thickness and serum estradiol and progesterone levels. Progesterone supplementation is added whenever a triple-line pattern endometrium reaches 8 mm thickness concomitant with follicular level of plasma progesterone. The dose of progesterone is typically 100 mg IM daily or 100–300 mg tid transvaginally. The total length of the estrogenic exposure may vary widely, probably with no detrimental effect on endometrial receptivity. Indeed, Borini et al12 recently reported a decreased in miscarriage rate in patients receiving E2 for more than 40 days. So, if prolongation of estrogen exposure is necessary due to inadequate endometrial development, no adverse effect is expected, unless breakthroughbleeding occurs. Estrogen support is continued at a maintenance dose of 4 mg/d through the progesterone period and both are continued up to 8–10 weeks’gestation, when the placenta has established adequate steroidogenesis.13–14 At least one study has shown, however, that the discontinuation of estrogen does not have a deleterious effect.15 Sauer and Cohen16 suggested that for practical purposes, patients can be monitored weekly for serum progesterone concentrations starting at 10 weeks after ET and the exogenous steroids stopped when a level of >30 ng/ml is attained. Although the estrogen/progesterone protocol is preferred for women with nonfunctioning ovaries, the high estrogen doses alone usually prevent LH surge.10,17 In these cases, estrogen supplementation should be started on day 1 or 2 of the menstrual cycle and the patients followed as previously described. Hormone Replacement using GnRHa followed by Estrogen and Progesterone Prior to commencing estrogen and progesterone treatment for endometrial preparation, older women and women with irregular cycles may require pituitary down-regulation with GnRHa to prevent premature LH release and the consequent nonsynchronized secretory changes in the endometrium. Neuspiller et al,18 in a study of the direct effect of short-and long-acting leuprolide and triptorelin on implantation in ovum-donation models, found no differences in pregnancy rate per transfer, or implantation rate per

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embryo replaced between the different groups. They concluded that GnRHa may be administered by any route and in any form available. If daily administration is chosen, the dose may be halved after pituitary downregulation is confirmed, with the concomitant introduction of hormone replacement therapy. GnRHa should be continued until progesterone therapy is commenced. Estrogen and progesterone treatment then follows according to the protocol described in the previous section. Ovulation Induction Patients with functioning ovaries may undergo cycle stimulationby clomiphene citrate or gonadotropins prior to frozen-thawed embryo transfer. For purposes of synchronization, it should be emphasized that the day of hCG administration corresponds to the day of hCG administration of the source cycle in which the embryos were retrieved. Moreover, it was suggested that when sperm parameters are adequate, the addition of intrauterine insemination performed 36 hours after hCG administration to the frozen-thawed embryo transfer, may increase the chances of pregnancy.19 Anovulatory patients who were previously responsive to clomiphene citrate may be offered a cycle consisting of clomiphene citrate doses equal to their last ovulatory clomiphene citrate cycle. Clomiphene citrate is usually started on day 3 to 5 of menstruation or withdrawal bleeding and continued for 5 consecutive days. Thereafter, E2 (2 mg/d) is given until the day of hCG administration to overcome the detrimental effects of clomiphene on endometrial development. Repeated testing of serum progesterone levels combined with ovarian ultrasonographic monitoring is required to assess follicular development. In the presence of plasma progesterone within follicular level, with adequate follicular size (20–24 mm) and endometrial thickness (≥8 mm), 10000 UhCG is administered to trigger ovulation. Anovulatory patients resistant to clomiphene citrate may be offered a chronic lowdose gonadotropin regimen. In those who have already undergone this regimen, the starting dose of gonadotropin should be calculated according to the previous threshold dose attained (otherwise, the starting dose should consist of 37.5 to 75 IU). The initial dose should be continued for up to 14 days. If no dominant follicle is recruited, the dose is increased at increments of 37.5 IU every week, to a maximum dose of 225 lU/d. Treatment is monitored by ultrasound and measurements of E2 and progesterone levels. When a single dominant follicle of 10–12 mm diameter is seen on ultrasound, the gonadotropin dose is maintained until the follicle reaches maturity (16–18 mm), at which point hCG is administered. Patients with other causes of ovulatory infertility or unexplained infertility are given an individually adjusted step-up dose regimen of gonadotropin. Gonadotropins are usually started on day 3 to 5 of the menstruation or withdrawal bleeding, and patients return 3 to 4 days later for measurement of serum E2 and progesterone and ovarian ultrasonography. Thereafter, injections are given daily until adequate follicular growth (>16 mm diameter) is verified on ultrasound and serum E2 reaches the appropriate level for hCG administration. It is noteworthy that follicles are larger before ovulation during clomiphene citrate protocols (20–24 mm) than during gonadotropin or natural cycles (16–18 mm).

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CONCLUSIONS Most studies found no statistically significant difference in outcome by the method of endometrial preparation used. The choice depends on the individual woman’s ovarian function and convenience of the method, as well as on the experience gained with the method by the team. In our unit, hormonal replacement with only estrogen and progesterone is the most frequently used protocol in women with and without functioning ovaries. We usually use daily oral estradiol or estradiol valerate 4–8 mg for 10 days, after which the patient attends our unit for serial ultrasound scanning of endometrial thickness, and serum estradiol and progesterone levels. Progesterone supplementation with either IM progesterone (50 mg/d) or intravaginal micronized progesterone (300 mg in the evening) is added whenever a triple-line pattern endometrium of at least 8 mm in diameter is observed with concomitant follicular level of plasma progesterone. A day later, which correspond to the OPU day, the progesterone dose is increased to 100 mg or 300 mg tid, respectively, after the OPU day, and maintained until 10 gestational weeks. REFERENCES 1. Queenan jt, Veeck LL, Toner JP, Oehninger S, Muasher SJ. Cryopreservation of all prezyotes in patients at risk of severe ovarian hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozenthawed transfers. Hum Reprod 1997; 12:1573–76. 2. Salat-Baroux J, Tibi C, Cornet D et al. Pregnancies after replacement of frozen-thawed embryos in a donation program. Fertil Steril 1988; 49:817–21. 3. Navot D, Scott RT, Droesch K, Veeck LI, Liu HC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril 1991; 55:114–18. 4. Lelaidier C, de Ziegler D, Gaetano J, Hazout A, Fernandez H, Frydman R. Controlled preparation of the endometrium with exogenous oestradiol and progesterone: a novel regimen not using a gonadotrophin-releasing hormone agonist. Hum Reprod 1992; 7:1353–56. 5. Sathanantan M, Macnamee MC, Rainsbury P, Wick K, Brindsen P, Edwards RG. Replacement of frozen-thawed embryos in artificial and natural cycles. A prospective semi-randomized study. Hum Reprod 1991; 6:85–87. 6. Ransil BJ, Seibel MM, Taymor ML. Estimating the onset of the LH surge by cumulative summation. Infertility 1981; 4:295–9. 7. Younis JS, Simon A, Laufer N. Endometrial preparation: lessons from oocyte donation. Fertil Steril 1996; 66:873–84. 8. Serhal PF, Craft IL. Ovum donation- a simplified approach. Fertil Steril 1987; 48:265–69. 9. Younis JS, Mordel N, Ligovetzky G, Lewin A, Schenker JG, Laufer N. The effect of a prolonged artificial follicular phase on endometrial development in an oocyte donation program. J In Vitro Fert Embryo Trans 1991; 8:84–88. 10. Yaron Y, Amit A, ManiA et al. Uterine preparation with estrogen for oocyte donation: assessing the effect of treatment duration on pregnancy rates. Fertil Steril 1995; 63:1284–86. 11. Meldrum DR. Female reproductive aging—ovarian and uterine factors. Fertil Steril 1993; 59:1– 5. 12. Borini A, Dal Prato L, Bianchi L, Violoni F, Cattoli M, Flamigni C. Effect of duration of estradiol replacement on the outcome of oocyte donation. J Assist Reprod Genet 2001; 4:185– 90.

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13. Devroey P, Camus M, Palermo G et al. Placental production of estradiol and progesterone after oocyte donation in patients with primary ovarian failure. Am J Obstet Gynecol 1990; 162:66– 70. 14. Scott R, Navot D, Liu HC, Rosenwaks Z. A human in vivo model for the luteoplacental shift. Fertil Steril 1991; 56:481–84. 15. Lewin A, Benshushan A, Mezker E, Yanai N, Schenker J, Goshen R. The role of estrogen support during the luteal phase of in vitro fertilization-embryo transfer cycles: a comparative study between progesterone alone and estrogen and progesterone support. Fertil Steril 1994; 62:121–25. 16. Sauer MV, Cohen MA. Egg donation. In Gardner DK, Weissman A, Howles CM, Shoam Z (Eds): Textbook of assisted reproductive techniques-laboratory and clinical perspectives. London: Matin DunitzLtd, 2001; 691–701. 17. Simon A, Hurwitz A, Pharhat M, Revel A, Zentner BS, Laufer N. A flexible protocol for artificial preparation of the endometrium without prior GnRH-a suppression in women with functioning ovaries undergoing frozen-thawed embryo transfer cycles. Fertil Steril 1999; 71:609–13. 18. Neuspiller F, Levy M, Remohi J, Ruiz A, Simon C, Pellicer A. The use of long and short acting forms of gonadotrophin releasing hormone analogues in women undergoing oocyte donation. Hum Reprod 1998; 13:1148–51. 19. Geva E, Yovel I, Lerner-Geva L, Lessing JB, Azem F, Amit A. Intrauterine insemination before transfer of frozen-thawed embryos may improve the pregnancy rate for couples with unexplained infertility: preliminary results of a randomized prospective study. Fertil Steril 2000; 73:755–60.

CHAPTER 58 Pathophysiology of Implantation Failure in IVF Jayant G Mehta, Thankam R Varma INTRODUCTION Recent advances in our understanding of ovarian stimulation, the techniques of oocyte retrieval, the handling of gametes, the methods of assisted fertilisation and improved conditions of culture medium have steadily increased the fertilisation rate, but implantation failure following embryo transfer still remains a major disappointment. For successful implantation, synchronization of the embryo development and uterine prepation is necessary Embryo implantation is a highly controlled physiological process, involving complex interactions between the implanting embryo and the maternal endometrium.1 Overall, responsibility for co-ordination of implantation lies with the ovarian hormones oestrogen and progesterone. However, it is now clear that, locally acting soluble factors secreted by the endometrium can act on the embryos to influence it’s development. Developing embryos in turn have been shown to produce soluble factors that can act in an autocrine manner or on the endometrium to influence the receptivity. Histological examination of early human pregnancies have revealed distinct patterns of blastocyst attachment to the endometrial surface and underlying stoma.2–3 First, the embryo apposes, then adheres to the maternal endometrial epithelium (EE) and finally, during the receptive phase or “window” for embryo implantation, it invades the EE. This window in women is from days 20–24 of the cycle.4–5 In an IVF cycle, the operational definition (the term of pregnancy success after embryo transfer replacement) of the beginning of the receptive phase (clinical implantation window) is not as precise as the end, i.e. embryos replaced before day 20 may implant. While those replaced after day 24 will not.4 Cell-cell contact occurs via respective apical cell membranes when the trophoblasts attach to the EE.6 Apical plasma membranes have been shown to express unique cell adhesion molecules that mediate to initiate implantation process.7 Recent reports have shown that researchers are starting to believe that penetration of epithelium by the blastocyst without adhesion to the luminal surface is also possible.8 The invasive phase of implantation occurs in a non-decidualized endometrium and is dictated by the blastocyst induced apoptosis of the EE, which allows blastocyst to traverse the epithelial barrier invade the maternal vasculture deep into the stroma, and access nutrients for its continued and growth sustenance. Several cell types have been reported to be involved in the process of implantation. The molecular dialog mediated by lectins, integrins, matrix degrading enzymes and their

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inhibitors, variety of growth factors and cytokines, their receptors and modulatory proteins, occurs via cell-cell and cell-extracellular matrix interactions between the endometrial epithelium, stroma, fibroblast, vascular endothelium, smooth muscle cells of the lymphoid lineage and trophectoderm of the implanting conceptus.10–14 The cyclic changes under the control of ovarian oestrogens and progesterone follow a precisely regulated series of morophophysiological events characterized by proliferation, secretory differentiation of oestrogen—primed endometrium and in the absence of conception degeneration and regeneration.15 To understand the molecular mechanism involved in the pathophysiology of implantation failure in IVF, requires a very close examination of endometrial receptivity, the presence of a chromosomally and functionally competent human embryo and the impact of the implanting blastocyst on the endometrial epithelium.16–18 APPOSITION AND ATTACHMENT: ROLE OF ENDOMETRIUM Role of Pinopodes The apical plasma membrane of luminal epithelial cells has been most closely studied, since it is this surface to which blastocyst attachment occurs. There is evidence from many species that marked changes in membrane morphology occur during the period of uterine receptivity. Particular interest has focussed on pinopodes, first described in rodents, which are large cytoplasmic projections from the uterine epithelium. In humans,19–22 during the period of receptivity, pinopodes, structures involved in endocytosis and pinocytosis appear on the apical surfaces of endometrial lumen epithelium under control of progesterones. The temporal and spatial expression of pinopodes has been suggestive of the development of uterine receptivity for blastocyst implantation.23 Scanning Electron Microscopy Study20 on post ovulatory day 6 endometrial biopsies, has revealed, presence of 15 percent pinopodes in women undergoing clomiphene citrate or gonadotrophin stimulation when compared with 78 percent presence in normal cycling women. These observation support the concept of a distinct period of endometrial specialization that coincides with the window of implantation. Further more, hormonal treatment used for ovulation induction, can modify normal development of pre-medatory endometrium and may also have negative effect on embryonic implantation.20 Adhesion Molecules in the Uterine Epithelium The uterine epithelium produces secreted and membrane bound glycoproteins in readiness for implantation.24 Changes in luminal epithelial cells opening the window of implantation increases thickness of positive glycoproteins and a more fibrillar glycocalyx structure. Decrease in the amount of sialic acid which confers surface negative charge on the epithelium is associated with non-receptivity in some species,25 so that less negative is compatible with the acquisition of uterine receptivity to implant. In addition, during the window of implantation numerous cell adhesions molecules are expressed by the endometrial epithelium.24,26,27

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Mucins Other molecules that may modulate endometrial receptivity include MUC-1, a highly glycosylated mucin, believed to play a role in embryo attachment. It is expressed on the apical side of the endometrial epithelial cells.25 MUC-I expression is up-regulated in the pre-implantation period.29 It is possible that alteration in the carbohydrate structure of mucins may permit embryo attachment during the window of implantation or that the upregulation of MUC-I in human endometrium may have functional significance per se for embryo attachment and nidation. MAG (mouse ascites Golgi) appears on the luminal epithelial surface in human endometrium at the earliest phase of implantation.30 In one study, 60 percent of patients with unexplained infertility had abnormal expression of MAG, which appears on the surf ace of the endometrium only on cycle days 18–19. Some of these electronegative mucins may act as specific ligands for the embryo, making the initial point of attachment, leading to later interactions mediated by integrins. Integrins Integrins belong to a f amily of cell adhesion molecules of immunoglobulin super family, which are heterodimeric non-covalently bound α and β subunits, with an extra cellular domain that serves as a receptor for various extracellular matrix ligands including fibronectin, collagen and lamini.31 They mediate adhesion to extracellular matrix (ECM). Under the influence of steroid hormones, constitute integrins such as α2β1,α3β1 and α6 β4 and cycle dependent stromal integrins including α1 β1, α3β1 and α4βl, αvβ3 and αv β5 are expressed on endometrial epithelial cells.32–35 Lack of functional estrogen and progesterone receptors during the window of implantation36 is specifically associated with initiation of αv β3 expression. Loss of the progesterone receptor is delayed, as is the expression of αv β3 in women with histologically evident maturational delay and infertility.33 Expression of β3 integrins by the endometrium may be associated with the classical luteal phase defect—‘type 1 defect’ or alternatively with histologically-normal endometrium—‘type II defect’ The latter findings suggest an intrinsic defect in endometrial function and is largely found in women with minimal or mild endometriosis37 and in women with hydrosalpinges.38 Further lack of αvβ3 and the loss of α4 β1 have been associated with unexplained infertility.39 Role of Growth factors and Cytokines There exist a wealth of data regarding the expression of mRNAencoding growth factor receptor, and proteins in embryos and the effect of growth factors on embryo development in vitro. Cytokine mRNA and growth factor mRNA, have been detected inblastomeres and in pre-implantation embryos as well as in human endometrium throughout the menstrual cycle. Vascular Endothelial Growth Factor (VEGF) has been known to play an important role during the post implantation period of embryo development. Studies have reported activation of the VEGF gene as early as the eight-cell stage during pre-implantation embryo development in the human. VEGF appears to be among the first actively

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transcribed genes and there is suggestion that, interaction between small amounts of embryonically synthesized VEGF and endometrial receptors exist. Growth factors and cytokines mediate the effects of oestradiol and progesterone on the endometrium, during the secretory phase of the cycle and for most part their expression continues during early pregnancy, suggestive of a major contribution in the cross talk between the decidua and the conception.32,40,41 They bind to specific cell surface receptors, resulting in cellular mitosis or differentiation, by autocrine, paracrine, juxtacrine or endocrine mechanisms.32,40,41 Members of the epidermal growth factor (EGF) f amily, colony stimulating factor-1 (CSF-1), leukemia inhibiting factor (LIF), interleukin-1 β (IL-1 β), transforming growth factor-β (TGF-β) and insulin like growth factor binding protein-1 (IGFBP-1) are known to participate in the apposition (attachment phase of and the invasive phase ofimplantation) Growth factors also play an important role in signalling between blastocyst and endometriumbefore attachment. Psycloyos (1986)42 reported that blastocyst attachment in mice occur late on day 4 of pregnancy, and coincides with increased vascular permeability at this site. Local up regulation of the mRNA encoding heparin -binding epidermal growth factor (HB-EGF) occurs in the luminal epithelium of the mouse uterus some 6 hrs before attachment.43 This upregulation is induced by an activated blastocyst at the site of its apposition, even before zona dissolution, suggesting that a embryo derived soluble factors are responsible. In vitro, the effects of HB-EGF is to enhance blastocyst hatching and trophoblast outgrowth through phosphorylation of the EGF receptor on the blastocyst. Other effects may include the induction of adhesion molecules such as perlecan on the surface of the blastocyst, and occur at the time to correlate with the acquisition of attachment competence.44 The expression and role in implantation of HBEGF in the reproductive tract of the mouse and human await further investigation, as does a potential role for them in implantation failure in humans. Leukemia Inhibitory Factor LIF is a pleiotropic cytokine which has both proliferative and differentiative effects on a variety of cells in vitro, including cells of embryonic, hematopoietic, osteoblastic and endothelial origin, and prevents embryonic stem cells from differentiating.45 The actions of LIF are mediated by a high affinity subunit and a glycoprotein 130 (gp 130) subunits.46 Glycoprotein 130 is also involved in the signalling of other cy tokines, including interleukin-6, IL-1 and ciliary neurotropic factor (CNTF). The role of LIF in implantation has been shown conclusively in a mouse model lacking a functional LIF gene, achieved by gene targeting and homologous recombination. Although the mechanisms underlying maternal endometrial LIF expression, and its role in embryonic implantation await further investigation, it is likely that LIF plays an important role in embryonic attachment to the epithelium and perhaps intrusion through the epithelium. In humans, LIF is expressed in the endometrium and decidua.47–49 and has been shown to act on human cytotrophoblasts, diverting them to differentiate to the anchoring phenotype by increasing synthesis of fibronectin and decreasing the production of human chorionic gonadotropin.50 In addition, in vitro LIF regulates tropho blast differentiation

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along the invasive pathway.51 and thus may play a role in the invasive phase of implantation, as well. Interleukin-1 The IL-1 “family” is composed of IL-1α, IL-1β, IL-1 receptor antagonist (IL-Iva) and two IL-1 receptors, only one of which mediates signal transduction (IL-IR L1).52 The entire IL system is expressed in human endometrium. IL-IR L1 mRNA is expressed in epithelial cells and is maximally expressed in the luteal phase.53–54Immune reactive IL1α, IL-1β, IL-1ra and IL-IR L are present in human embryos, and embryos co-cultured with endometrial epithelium antagonist.55 Steroid hormones56 regulate the embryonic expression and apparent regulation by endometrial epithelial product(s), endometrial IL-1 α and β and IL-1ra. Further more, human blastocysts selectively up-regulate αv β3 integrins in human endometrial epithelial cells, compared to arrested human embryos.57 This embryonic up-regulation of endometrial β3 integrin is mediated, in part, by the embryonic IL-1 system.57 Placental Invasion: Role of the Endometrium The human cytotrophoblast must be capable of adhering to the extracellular matrix and rapidly invading into the endometrial strom during the invasive phase of implantation. Trophoblast invasion requires attachment to the extracellular matrix via cell adhesion molecules, local extracellular matrix proteolysis (by matrix metallo proteinases (MMPs) cellular migration and inhibition of these processes.58 It has been reported that during the transition to the invasive phenotype, first-trimester cytotrophoblasts undergo integrin ‘switching’ from α6 β4 to α5 β1 and α1 β1 which probably enable the intermediate cytotrophoblast to anchor and migrate in the maternal decidua.59,60 Of the family of MMPs, MMP-9, α 92 KD gelatinase is required for human cytotrophoblast invasiveness, in vitro,61–64 and the production and activation of MMP-9 in cytotrophoblasts coincide with maximal invasive behaviour in vivo.64–66 Autocrine action of IL-1β probably promote cytotrophoblast invasiveness in the first trimester. While the cytotrophoblast has long been considered the leader in the invasive process of implantation, the maternal endometrium also plays a major active role in liminiting this invasion. It probably does so by several mechanisms, including the production of inhibition of trophoblast differentiation into the invasive phenotype, inhibition of autocrine or paracrine stimulation of invasion, direct inhibition of invasion and inhibition of trophoblast-derived matrix-degrading enzyme. Broad-spectrum Protease Inhibitors Specialised inhibition (T1 MP-1, -2 and -3) of primary decidual and cytotrophoblast origin are probably important in limiting cytotrophoblast origin and are probably important in limiting cytotrophoblast invasion.67 Recent evidence has shown that IL-1β inhibits TIMP-3 expression in human decidualised endometrial stromal cells, suggesting that trophoblast promotes its own invasiveness by inhibiting a maternal restraint on invasion.

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Endometrial stromal cells decidualized in vitro, secrete laminin and fibrorectin68,69 and progestin stimulates fibronectin mRNA expression and protein synthesis in these cells.69 The extracellular matirx (ECM) is therefore likely to play a major role in trophoblast invasiveness, because it is the substratum that supports cellular adhesion and cell-cell interactions and because interao tions with the ECM results in changes in trophoblast invasiveness. Laminin production by human endometrial stroma is more abundant in secretory compared to proliferative endometrium and it increases with implantation and in early pregnancy.69 Laminin also causes a decrease in prolactin and insulin like growth factor binding protein-1 during in vitro decidualization of endometrial stromal cells,70 suggestive of a role in facilitating trophoblast invasiveness. In vitro, human first trimester trophoblasts preferentially attach to laminin coated surfaces.71 Although mechanisms underlying this inhibition are not well understood, fibronectin is likely to be one of the several participants in the ‘maternal restraint’ to promote trophoblast invasiveness at the maternal-fetal interface. Insulin-like Growth Factor Binding Protein-1 In humans IGF-1 and IGF-II are mitogenic to decidualized endometrial stromal and glandular cells. The IGF-1 gene is expressed primarily in late proliferative and early secretory endometrium and is believed to mediate, the mitotic actions of estradiol in this tissue. IGFII has been shown to stimulate trophoblast migration and its mediation may be through progesterone action.72–75 Recent studies have demonstrated that IGFBP-1, α member of a family of proteins, with high affinity for the IGF peptides, binds to human cytotrophoblasts, and to the α5β1 integrin in the cytotrophoblast membrane and inhibits human cytotrophoblast invasion into decidualized human endometrial stromal cultures.76 Whether an excess of IGFBP-1 at the maternal fetal interface is detrimental to cytotrophoblast invasion, resulting in shallow, implantation or early pregnancy loss, is not certain at this time. However, IGFBP-1 levels are elevated at the maternal—fetal interface and in the circulation of women with severe pre-eclampsia, a disorder of shallow implantation.77 Potential roles of IGF peptides and IGFBP-1 in occult endometrial defects and uterine non-receptivity await further investigation. Many proteins that are secreted uniquely in the secretory-phase endometrium and whose functions we are just begging to appreciate have been discussed so far. However there still remains a group of proteins, whose functions are still unknown, although their temporal expression would suggest a functional role in the process of implantation. Two such proteins, prolactin and progestin-associated endometrial protein/placental protein W(P6P/PP14) are discussed here due to their potential roles in water transport and immune modulation respectively. Prolactin a product of decidualized endometrium78–79 is a progesterone—dependent protein in this tissue.80 Although prolactin is known to modulate water transport across membrane, its precise function during the invasive phase of implantation, as a stromal product, is not well defined. While on the progestogen-associated endo metrial protein (PEP) or PP14, is the major secretory protein of the glandular epithelium of luteal phase endometrium.81 PEP is also known as glycodelin which inhibits the actions of natural killer cells which are abundant in decidualized endometrium during early pregnancy.82

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Besides this immuno suppressive effect, PP14 or PEP strongly inhibits binding of sperm to the zona pellucida.83 Whether it has any effect on an implanting embryo is yet to be determined. Role of Macrophages, T Cells and Natural Killer Cells in the Endometrium During early pregnancy 30 percent of stromal cell population comprise of white cells or leukocytes. The predominant cell population comprises of macrophages, T cells (CD8+) and granulated lymphocytes.84 Macrophages increase in the late secretory phase and are present in the decidua during pregnancy. They are responsible for producing cytokines, e.g. CSF-1 and granulocytes marcophage CSF (GM-CSF), which may regulate placental growth.85 Marcophages at the maternal -fetal interface may be immunosuppressants, facilitating tolerance of the new allograph. They may also have phagocytic roles, and/or be anti-inflammatory agents. The number and location of T cells present in the endometrium do not change with the menstrual cycle or in early pregnancy. Although the majority of endometrial T cells express CD8 suppressor/cytotoxic markers, 30 percent express CD4 helper markers86 and are a rich source of cytokines, which as discussed earlier have the potential to influence placental growth and invasion. Interesting enough, large granular lymphocytes wich have markers consistent with an early differentiation stage of natural killer cells are abundant in the pre-implantation period and throughout the first trimester after which their number declines markedly While their function is not well understood, they secrete an isoform of transforming growth factor-b which has immuno suppressive properties.87 Trophoblasts do not express major histocompatibility complex (MHC) Class II antigen, but some express unusual MHC Class I antigens88 which are discriminate for self versus non-self, and express HLA-G.89 HLA-G protects against killing by natural killer cells and because of limited polymorphism in trophoblast HLA-G, the protein encoded by paternally derived HLA-G genes is not recognised as foreignby the maternal immune system. This provides a mechanism whereby maternal tolerance of the new allocraft is achieved.89 From a clinical perspective, before embryo replacement takes place, there is a need to evaluate adequacy of the endometrium for implantation—for attachment and invasion— in women with endometriosis, unexplained infertility, sonographically, thin endometrium in the periovulatory period, and in women with habitual abortion not due to genetic causes or anti-phospholipid, antibody syndrome. There is also a need to evaluate the maternal-fetal interface in repetitive miscarriage for adequacy of the conceptus to invade the maternal endometrium and of the decidua to support the invasive process.90 More recently, functional evaluation of histologically normal endometrium has revealed abnormalities in αvβ3 integrin expression that are associated with unexplained infertility, hydrosalpinges and endometriosis. In order fo evaluate endometrial adequacy, two biopsies would have to be taken, in the same or a different cycle, and performed during the window of implantation (for attachment) and during the late secretory phase (for invasion). A ‘receptivity’ test or attachment test would probably screen morphologically for pinopodes and presence of mucins, αvβ3 integrins; trophinin/tastin, HBEGF, EGF, IL-1β, CSF-1 and LIF-1. An invasion test would ideally screen for TGF-β1. IGFBP-1, fibronectin and TIMPS. In an

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aborted conceptus, an ideal test would include the invasion test and markers of trophoblast invasiveness, including MMP-9, MMP-2, IL-1β, perhaps IGF-11 and the expression of α1β4, α5β1 α6β1 integrins and TIMPS. Effect of Ovarian Stimulation and Embryo Development on Implantation Exogenous administration of gonadotrophins as in IVF, results in higher concentration of circulating steroids which may also effect oocyte and/or embryo quality oviductal and/or uterine environment as well as the synchrony that normally exists between the embryo and the endometrium at the time of implantation. Hence, a relationship may exist between ovarian stimulation with gonadotrophins and the low implantation rate and the gestational complications observed. For obvious ethical reasons, only minimal directly derived scientific information exists on the impact of ovarian stimulation on embryo development, implantation and gestation in humans. Although in general it is difficult to extrapolate from animal models to humans, considerable similarities exists among many mammalian species in the early stages of development with respect to morphology and metabolism. The causes of the loss of developmental competence of embryos from superovulated donors remain unknown. Chromosomal abnormalities may account for reduced viability of embryos from superovulated females because superovulation has been found to increase the proportion of chromosomal abnormalities in murine embryos91 as well as in oocytes in rats. As ovarian stimulation produces a cascade of hormonal and physiological events, oocytes mature in an environment diff erent from that of naturally matured oocytes92 and variation in the time of ovulatoin may also occur,93 New evidence from molecular biology studies of mammalian oogenesis has implicated a role for gonadotrophins in the control of meiosis in mammalian oocytes.94 In mice as well as in humans, there is evidence for steroids being regulators of gene expression, and that embryo morphology and rate of development—both of which reflect embryo quality— have a genetic basis.95 Successful implantation depends on embryo quality, uterine receptivity and synchronization of embryo development and endometrial maturation. Exogenous administration of gonadotrophins, affecting the concentrations of circulating ovarian steroids may have changed the local expression of cytokines in the endometrium in superovulated patients and hence it’s receptivity. In IVF, ovarian stimulation with high oestradiol concentrations has been reported to be detrimental to implantation and pregnancy rates. While high serum oestradiol concentrations in fresh IVF cycles significantly reduce the implantation rate, the implantation and pregnancy rates in frozen -thawed cycles for surplus embryos were similar to normal IVF cycles with optimal levels of oestradiol. Hence, the impairment in implantation was attributed to hostile environment in the endometrium and it may be associated with high oestradiol circulating concentration.

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Significance of Embryo Replacement Technique Clinical approaches to increasing implantation rates have focused mainly on endometrial receptivity and blastocyst culture and transfer, while embryo transfer has been relatively under-rated by most programmes in terms of evaluating changes that might improve clinical pregnancy rates. Under transabdominal ultrasound and not touching the endometrium and the uterine fundus, replacement of embryos approximately 2 cm below the fundus in the lumen of the endometrial cavity is now considered to be the most important factor for successful embryo transfer by most IVF teams world wide.96 SUMMARY The apposition/attachment phase of implantation involves cell adhesion, changes in cell motility, cell surface charge and cellular differentiation. The invasive phase of implantation involves extensive paracrine signalling between the invading trophoblast and the decidual stroma, vasculature and lymphoid cells.97 The expression, regulation, modes of action and functions of pinopodes, glycoproteins, cytokines, growth factors and related proteins in the deci-dua during the process of implantation and sustained successful pregnancy are just beginning to be understood. Further, research into such areas as: i) regulation and dysregulation of endometrial molecules, endowing the epitheliam with ‘receptivity’, ii) molecular mechanism and participants underlying cross talk between the trophoblast and the decidua, iii) microenvironment effects on trophoblast invasiveness and iv) which clinical syndromes are associated with abnormalities in the endometrium and the decidua, that results in abnormal implantation or implantation failure, will greatly facilitate in achieving successful implantation for couples with infertility, an pregnant women with implantation disorders, and in inhibition of implantation for contraceptive purposes. REFERENCES 1. Simon C, Gimeno MJ, Mercader A et al. Cytokinis-adhesion molecules-invasive proteinases. The missing paracrine/autocrine link in embryonic implantation? Mole Hum Reprod 1996; 6:405–24. 2. Hertig A, Rock JA. A description of 34 human ova within the first 17 days of develop first twelve weeks of trophoblast differentiation. Am J Anat 1956; 98:434–94. 3. Shiotani M, Noda Y, Mori T. Embryo-dependent induction of uterine receptivity assessed by an in vitro model of implantation in mice. Biol Reprod 1993; 49:794–801. 4. Bergh PA, Navot D. The impact of embryonic development and endometrial maturity in the timing of implantation. Fertil Steril 1992; 58:537–42. 5. Aplin JD. The cell biology of human implantation. Placenta 1996; 17:269–75. 6. Denker HW. Endometrial receptivity cell biological aspects of an unusual epithelium. Ann Anat 1994; 176:53–60. 7. Kliman HJ, Coutifaris C, Feinberg RF et al. Implantation: In vitro human models utilising human tissues: In Yoshinaga K (Ed): Blastocyst Implantation. Boston Adams Publishing 1989; 83–91. 8. Lapata A. Blastocyst-endometrial interaction: an appraisal of some old and new ideas. Mol Hum Reprod 1996; 7, 519–25.

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55. Delos Santos MJ, MercaderA, FrancesA et al. Role of endometrial factors in regulating secretion of components of the immuno reactive human embryonic Interleukin-1 system during embryonic development. Biol Reprod 1996; 54:563–74. 56. Simon C, Mercader A, Frances A et al. Hormonal regulation of serum and endometrial IL alpha, IL beta and IL-Ira; IL-1 endometrial microenvironment of the human embryo at the apposition phase under physiological steroid level conditions. J Reprod Immunol 1996; 31:165– 84. 57. Simon C, Gimeno MJ, Mercader A et al. Embryonic regulation of Intergrins beta 3, alpha 4 and alpha 1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab 1997; 82:2607– 16. 58. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular metric during invasion and metastasis. Annur Rev Cell Biol 1993; 9:541–73. 59. Damsky CH, Fitzgerald ML, Fisher SJ. Distribution patterns of extracellular matrix components and B1 integrin receptors are intricately modulated during differentiation of human cytotrophoblast to an invasive phenotype in vivo. J Clin Invest 1992; 89:210–22. 60. Damsky CH, Librach C, Lim KH et al. Integrins switching regulates normal trophoblast invasion. Development 1994; 120:3657–66. 61. Bischof P, Friedli E, Marelli M et al. Expression of extracellular matrix degrading metalloproteinases by cultured human cytotrophoblast cells. Effects of cell adhesion and immunopurification. J Obstet Gynecol 1991; 165.1791–1801. 62. Fisher SJ, Damsky CH. Human cytotrophoblast invasion. Cell Biol 1993; 4:183–88. 63. Polette M, Nawrocki B, PintiauxA et al. Expression of gelatinases A & B and their tissue inhibitors by cells of early term human placenta and gestational endometrium. Lab Invest 1994; 71, 838–46. 64. Librach GL, Werb Z, Fitzgerald ML et al. 92 Kda type IV collagens mediates invasion of human cytrophoblasts. J Cell Biol 1991; 113:437–49. 65. Bass KE, Monish D, Roth I et al. Human cytrophoblast invasion is up-regulated by epidermal growth factor. Evidence that paracrine factors modify this process. Dev Biol 1994; 164:550–61. 66. Shimonovitz. BS, Hurwitz A, Dushnik M et al. Developmental regulation of the expression 72 and 92 kd type IV f olleaghases in human trophoblasts: A possible mechanism for control of trophoblast. Am J Obstet Gynecol 1994; 171.832–38. 67. Bass KE, Li H, Hawkes SP et al. Epidermal growth factor and cytotrophoblast invasion. Presented at the 34th Annual Meeting of the American Society for Cell Biology. San Francisco. CA. 1994; 353 (Abstract). 68. Irwin JC, Utian WH, Eckett RL. Sex steroids and growth factors differentially regulate the growth and differentiation of cultured human endometrial stromal cells. Endocrinology 1991; 129:2385–92. 69. Zhu HH, Huang JR, Mazella J et al. Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells. Hum Reprod 1992; 7:141–46. 70. Brar AK, Frank GR, Richards RG et al. Laminin decreases PRL & IGFB P1 expression during in vitro decidualization of human endometrial stromal cells. J Cell Physiol 1995; 163:30–37. 71. Loke YW, Gardner L, Burland K et al. Laminin in human trophoblast deciduo interaction. Hum Reprod 1989; 4:457–63. 72. Boetim KD, Diamar M, Gorodeski IG et al. Expression of the insulin like and platelet derived growth factor genes in human uterine tissues. Mol Reprod Dev 1990; 27:93–101. 73. Guidice LC, Dsupin BA, Jin LH et al. Differential expression of messenger nibonucleic acids encoding insulin like growth factors and their receptors in human uterine endometrium and decidua. J Clin Endocrinol Metab 1993; 76:1115–22. 74. Zhu J, Dsupin BA, Giudice LC et al. Insulin like growth factor system gene expression in human endometrium during the menstrual cycle. J Clin Endocrinol Metab 1994; 79:1723–34.

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75. Irving JA, Lala PK. Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGE, IGR-II and IGFBP-I Exp Cell Res1995; 217:419–27. 76. Irwin JQUtian WH, Eckert RL. Sex steroids and growth factor differentially regulate the growth and differentiation of cultured human endometral stromal cells. Giudice LC. Growth Horm. IGF Res1991; 21–31. 77. Guidice LC, Martina NA, De Las Fuenter L. Insulin like growth factor binding protein-1 at the maternal fetal interface and insulin like growth factor-II and insulin like growth factor binding protein-I in the circulation of women with severe preeclampsia. Am J Obstet Gynecol 1997; 176:751–58. 78. Maslar IA, Riddick DH. Prolactin production by human endometrium during the normal menstrual cycle. Am J Obstet Gynecol 1979; 35:751–54. 79. Healy DL, Hodgen GD. The endocrinology of human endometrium Obstet Gynecol Surv 1983; 38:509–30. 80. Huana JR, Tseng L, Bischoff P et al. Regulation of Prolactin production by progestrin, estrogen and releasing in human endometrium stromal cells. Endocrinol 1987; 121.2011–17. 81. Naaktgeboren N. Devroey P. Wisanto A et al. Endocrine profiles in early pregnancies with delayed implantation. Hum Reprod 1992; 1:9–14. 82. Okamoto N, Uchida A, Takakura K et al. Suppression by human placental protein 14 of antural killer call activity. Am J Reprod Immunol 19912; 6:137–42. 83. Oehninger S, Coddington CC, Hodgen GD et al. Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. Fertil Steril 1995; 64:377–83. 84. Bulmer JN, Sunderland CA. Immuno histological characterisation of lymphoid cell populations in the early human placental bed. Immunology 1984; 52:349–57. 85. Tsoukato D, Skavpelis G, Athanassakis I. Placentaspecific growth factor production by splenic cells during pregnancy Placenta 1994; 15:467–76. 86. Tabibzadeh S, Sun XZ, Kong QF et al. Induction of polarized micro-environment by human T cells and interferon gamma in three dimensions spheroid cultures of human endometrial epithelial cell. Human Reprod 1993; 8:181–92. 87. Clark DA, Vince G, Flanders KC et al. CD 56+ lymphoid cell in human first trimester pregnancy deciduo as a source of novel transforming growth factor-beta 2 related immuno suppressive factors. Hum Reprod 1994; 9:2270–77. 88. Yeh IT, Kurman RJ. Functional and morphologic expression of trophoblast. Lab Invest 1989; 61:1–4. 89. Fisher SJ, Cui T-Y, Zhang L et al. Adhesive and degradative properties of human placental cytrophoblast cells in vitro. J Cell Biol 1989; 109:891–902. 90. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3–25. 91. Elbling L, Colot M. Abnormal development and transport and increased sister chromatid exchange in pre-implantation embryos following superovulation in mice. Mutat Res 1985; 147:189–95. 92. Foote RH, Ellington JE. Is a superovulated oocyte normal? Theriogenology 1988; 29.111–23. 93. Allen J, McLaren A. Cleavage rate of mouse eggs from induced and spontaneous ovulation. J Reprod Fertil 1971; 27:137–40. 94. Picton H, Briggs D, Gosden R. The molecular basis of oocyte growth and development Mol Cell Endocrinol 1998; 145:27–37. 95. Warner CM, Cao W, Exley GE et al. Genetic regulation of egg and embryo survival. Human Reprod 1998; 13(Suppl 3):178–90. 96 Delhanty JAD Handyside AH. The origin of genetics defects in the human and their detection in the preimplantation embryo. Hum Reprod Update 1995; 1:201–15. 97. Coroleu B, Barri PN, Carreras O et al. The influence of the depth of embryo replacement into the uterine cavity on implantation rates after FVF: A controlled, ultrasound guided study. Human Reprod 2002; 17, 2:341–46.

CHAPTER 59 Recurrent Implantation Failures: The Preferred Therapeutic Approach Sonia Malik If we think we have all the answers, we haven’t asked all the questions! TOM WEGMANN The process of implantation is a finely orchestrated series of events involving two major players—the embryo and the maternal endometrium. Embryo maternal dialogue has been the topic for many studies1 as also the fascinating fact that the embryo being a semiallograft is not rejected.2,3,4,5 It was the theory of Medawar in 1950’s that first helped to understand the concept of immune modulation during the process of mammalian implantation. Ever since then there is a constant endeavour to unfold the mystery of this intriguing process. Although ART in humans has helped many a couple reproduce, overall pregnancy rates are still 26 percent6 and recurrent implantation failure is seen in 30–40 percent of patients undergoing IVF.7 In majority of these patients, no karyotypic abnormality is found.7 It is known that the developing embryo directs the endometrium towards receptivity so that rejection does not take place.1,8 However, there are certain situations where the embryo seems to be overridden and pregnancy fails to occur. In an ART programme the various factors responsible for implantation failure are: • the Embryo • the Endometrium • the laboratory quality control and technique of embryo transfer. In the past decade all efforts have been focused on developing methods to improve the embryo potential by improved methods of COH9,10 culture and co-culture techniques11,12 assisted hatching13,14 and blastocyst transfer.15 Similarly, techniques of embryo transfer have been modified by the use of different catheters16 and ultrasound guided transfer.17,18 Despite this effort, infertility specialists come across young patients who having produced adequate numbers of good embryos fail to conceive following repeated attempts at IVF-ET. It has been observed that in such patients with repeated implantation failures, the embryos become better with each attempt.19 This implies that it is the endometrium rather than the embryo which is responsible for a failing pregnancy. Within the endometrium there can be only two reasons that account for asynchrony: hormonal imbalance and infections.20 Hormonal imbalance has received lot of attention ever since the evolution of ART and ways and means to correct this have also evolved to a certain extent. Infections on the other hand have not been addressed adequately. This neglected factor maybe the major cause for implantation failure.21 Many organisms have been implicated in uterine infections and infertility,22 but due to the overwhelming data on

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infertility techniques and sophisticated tests, scant attention has been given to this. The actual stage where the drama actually takes place, that is the endometrium has been ignored. The simple procedure of endometrial sampling and cultures is excluded from many an infertility work up because of the availability of sophisticated hormonal assays and ultrasound techniques. The aim of this text is to draw attention towards the endometrium and describe a module to enhance endometrial receptivity based on prevailing evidence. Hormonal Control of Uterine Receptivity Ever since the studies of Hertig and Rock in 1956, it is well known that embryos require a receptive endometrium controlled by hormones for their implantation. This has been further highlighted by donor-recipient model in ART.23 The proposed actions of estrogen are proliferation of the endometrium, upregulation of progesterone receptors, a blood flow enhancing effect24 and immune modulation. Progesterone on the other hand seems to be playing a larger role in opening the window of implantation by immune modulation,25 and causing decidualization of the endometrium. Conditions where there is inadequate primingby estrogen, or there is excess of estrogen, or protocols that upset the E2/P4 ratio in the luteal phase, or a primarily progesterone deficient state are known to cause implantation failure. The Embryo Maternal Cross Talk Implantation is brought about by a series of developmental phases starting with blastocyst hatching, attachment to the endometrium and final invasion and formation of placenta. The three stages of implantation are-apposition, adhesion and invasion. In each stage there is an expression of different genes and proteins that serve as regulators of immunity and biomarkers of endometrial receptivity. However, it has been seen that any state of disordered receptivity-whether is it adhesion or invasion, results in an incompetent syncytiotrophoblast with poor decidualization of the endometrium.26 The vascularization is hampered, this leads to final cessation of growth within the embryos and pregnancy fails. The Th1-Th2 Paradigm (Wegman 1993)27 The T helper cells of the CD4+ clones can be divided into two subsets—Th1 and Th2 depending on the type of cytokines that they produce. The Th1 cells produce Interferon INFg, TNFa, NK cells and IL2 and the Th2 cells produce Interleukin IL4, IL-5, IL-6, IL13. The currently popular paradigm in reproductive immunology states that pregnancy involves a shift from the proinflammatory Th1 response to the Th2 anti—inflammatory response and it is theTh1/Th2 balance that decides the success or failure of a pregnancy28 This balance is upset in many conditions, e.g. autoimmune diseases, infections and metabolic conditions like diabetes.

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Anti Phospholipid Antibodies The incidence of antiphospholipid antibodies is found to be higher in patients of recurrent pregnancy loss.29 Each subset has been seen to have a different effect.30 Antiphospholipid antibodies may be markers of a separate underlying process (autoimmune disease) or may act directly at various sites. The direct targets of these antibodies are endothelial cells31, trophoblastic cells,31 and pre-embryos.32 On the endothelial cells there is thrombosis due to the inhibition of prostacycline generation and resultant thromboxane predominance. On the trophoblast, these antibodies bind directly to trophoblast and inhibit diff erenciation into syncitiotrophoblast and cytotrophoblast so that there is decreased production of hCG and failure of pregnancy. On the pre-embryo, the locus of action is not known but it is presumed to be again through the prothrombin pathway with ultimate deposition of fibrin. The Disordered Blood Flow All the biochemical and hormonal changes that have been described above, result in a disordered blood flow to the pelvis and endometrium. The color Doppler studies indicate that there is indeed increased vascular resistance to uterine blood flow in patients of recurrent implantation failure.33 Pregnancy rates are known to jump when interventions to enhance blood flow are used.34 High uterine artery impedence is thought to be predictive of a failure of implantation in an IVF cycle.35 This evidence seems to support the view that the endometrium does play an important role in implantation. Let us take specific examples of non-genetic failures. Polycystic Ovary Syndrome There is a disturbance of the endocrine milieu.36 LH is high, androgens are in excess, there is hyperinsulinemia and low progesterone levels. Within the endometrium, this leads to a disturbance in the E2/P4 ratio, NK cells are high and IGF, and IGF2 levels are disturbed resulting in activation of Prothombin activatior inhibitor I and II. This leads to platelet adhesion, microthrombosis and implantation failure.37 Endometriosis Endometrial receptivity has been found to be disturbed in this disease both by virtue of an altered immune response and the presence of an embryo toxic factor within the endometrium.38 In addition to this, there is a release of antiphospholipid antibodies due to scarring and fibrosis in the pelvis. These again compromise the vasculature of the syncitiotrophoblast due to deposition of fibrin and the pregnancy fails. Infections and Inflammations Although infections are said to account for merely 1 percent of the recurrent pregnancy losses, it has also been suggested that the immune modulation within the decidua during a pregnancy mimics that seen at infection. There is release of cytokines of the Th1 response like IL2, NK cells and TNF α.28 The final result depends on how strongly the

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embryo can bear this insult.28 More importantly, if the ratio of the Th1: Th2 cytokines is tilted towards Th1, implantation failure results and vice versa if it is a dominant Th2 response.28 In an infection or inflammation the endotoxin or the cytokines themselves are known to secreate procoagulase which again leads to disordered vascularization and resorption of embryos.38 Unexplained Most of the cases show a shift in the Th1→Th2 response, high levels of LAK, NK cells and TNFα levels indicating thats these cases are mediated through an altered immune response.39 A new prothrombotic factor fg12 has been identified in these cases40 thereby again indicating a vascular phenomenon. The inference therefore is that, in majority of cases of implantation failures, it is either a primary or a secondary (consequent to a cause) immune modulation within the endometrium that is leading to imperfect vascularization and implantation failures. It has been indicated that the processes causing resorption of embryos and implantation failures after the appearance of the heart beat are different7, but other studies32 have clearly implicated antiphospholipids like phosphoserine in the cause resorption of pro-embryos and embryos.32 Yet, another study suggests that the disturbed endometrial milieu because of a Th shift is responsible not only for implantation failures but also for the high teratogenicity as seen in diabetes.41 Based on all this data, the concensus that evolves is that a disordered endometrium plays a definite role in implantation failure. Evidence from these markers and the immunohistochemical studies have helped in reclassifying luteal phase defect (Table 59.1). This also suggests that in routine histopathology may not be enough to understand problems within the endometrium (Fig. 59.1 and 59.2). Some of these markers are commercially available and may help to further differenciate various endometrial patterns in different disorders.

Table 59.1: Luteal Phase Defects Type

Histology

αvβ3

Type I Out of phase Absent Type II In phase Absent Lessey et al. Jr of Cl Invest 1992; 90:188–95.

PROTOCOLFOR ENHANCING ENDOMETRIAL RECEPTIVITY The basic aim is to achieve an optimal endometrium by ensuring: • adequate hormonal support • adequate immune modulation.

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Fig. 59.1: Tubercular Endometritis: 1st IV Failure showing granulomas

Fig. 59.2: Endometrium showing NKcell activity: 3rd IVF failure

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In order to achieve this, the tests that are recommended are: 1. Complete endocrinel profile: • FSH • LH • E2 • Prolactin • Thyroid profile and thyroid antibodies • S. Insulin • F. Test. • SHBG • DHEAS • S. Progesterone 2. Endometrialbiopsy: • Histopathology for dating and endometritis • Immunohistochemistry for markers like NKcells • Cultures e.g. Koch’s, anaerobic etc. 3. Vaginal cultures: aerobic, anaerobic and fungal 4. TORCH profile 5. ELISA for Chlamydia, Mycoplasma, etc. 6. Reproductive Immunophenotype 7. CD4: CD8 RATIO 8. Antiphospholipid antibody panel 9. Lupus anticoagulant 10. Anticardiolipin antibody 11. APTT 12. Platelet Count 13. IgA 14. Antisperm antibody. Treatment Luteal Support Adequate luteal support must be added to all IVF cycles. There has been a lot of debate regarding the usefulness of hCG especially because of the fear of OHSS. However, it has been seen that hCG also acts locally within the endometrium and enhances certain implantation factors. Most cycles are now supported by both hCG and progesterone. The preferred progesterone is natural progesterone and the preferred route is vaginal because of the uterine first pass effect. Estrogen has also been used vaginally for luteal support with good results especially where the lining is compromised or there is a deficient uterine blood flow. In patients where the endometrium seems to be compromised, the following methods have been adopted to get better results:

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• Freezing embryos • Cancelling cycles. Immune Modulation Immune modulation is indicated in patients of recurrent IVF failures even if the tests mentioned above do not reveal any positives. This is because these interventions are known to give better results7 and presently we do not have complete knowledge of the changes that are taking place within the endometrium. Present day interventions aim at: • Enhancing the blood flow • Correctingthe Th1-Th2 imbalance • Downregulating antibodies and proinflammatory cytokines. The various medications available for this are: — Low dose aspirin — Steroids like prednisolone — Low molecular wt. heparin — IVIG — Lymphocyte immune therapy. Low dose aspirin Many studies have been conducted using low dose aspirin alone42 or with steroids or heparin43 which seems to be particularly helpful if the cause is antiphospholipid antibodies. It acts on thromboxane 2, decreasing its levels and thereby corrects the disturbed thromboxane—prostacycline ratio. The dose varies between 75–81 mg. Steroids It acts by decreasing antibody formation, proinflammatory cytokines and increases platelet formation in the bone marrow.43 The preferred steroid is prednisolone in doses ranging from 10–40 mg/day. However the side effects are many and the most dreadful being osteoporosis and avascular necrosis of the head of femur. Heparins Low molecular weight heparins act by decreasing thrombosis. The side effects are negligible but osteoporosis is known. There are many forms available and the dosage varies (Table 59.2).

Table 59.2: Low molecular weight heparin dosage schedule Product Dalteparin

Dose

Trade name

2500 IU/0.2 ml Fragmin 5000 IU/0.2ml Nadroparin 3075 IU/0.3ml Fraxiparine 4100 IU/0.4 ml Enaxaparin 20 mg/0.2 ml Clexane 40 mg/0.4 ml LMWH Sodium salt 3200 IU/0.3 ml Fluxum 6400 IU/0.6 ml

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Intravenous immunoglobulins IVIG prolong the clearing time of immune complexes by binding to macrophages. A dose of 0.5 mg/kg body wt.44 is given every three weeks. Side effects include hypotension, nausea, headache and life threatening anaphylaxsis in IgA deficient patients. The cost is however prohibitive. Lymphocyte immune therapy This can be given using both paternal and donor or mixed lymphocytes. This restores the Th1/Th2 imbalance and decreases the proinflammatory lymphocytes.45 The dose is 4 ml of mononuclear cells suspended in ringer lactate solution given intradermally. Side effects are those of serum sickness— fever, malaise, arthralgia and occasional anaphylaxis. The results of all these interventions although impressive have been nullified by the fact that in many studies of implantation failure results are as good without the use of any such methods. However, with the growing evidence and knowledge of reproductive immunology, it seems rational to use these interventions in special situations like recurrent implantation failures.46

Table 59.3: Success rate of various therapies Author Year No of patients

No of pregnancy

Spontaneous abortion(%)

Aspirin and Predinisolone Reece, 1991 18 18 3(17) Out, 1992 11 11 1(9) Branch, 1992 33 39 8(21) Aspirin and heparin Rosove, 1990 14 15 NA Cowchock, 8 8 NA 1992 Branch, 1992 17 19 1(5) Mod from Ref 45 Clinical Obstetrics and Gynec, 1994 Vol. 37 No. 9

Fetd death Live birtks (%) (%) 1(5) 3(27) 8(21)

14(78) 7(64) 23(59)

NA NA

14(93) 6(75)

2(11)

16(84)

Table 59.4: Success Rate of various therapies Treatment tested Author

No of cases (%) No of control (%)

Paternal cells

Mowbrav 25/737(67.6) 14/730(46.7) Ho 33/42(78.6) 2/49(65.3) Cauchi 13/20(65.0) 16/22(72.7) Uonorcell Gatenby 8/11(72.7) 12/28(60.0) Christiansen 27/40(67.5) 16/28(57.1) Scott 7/18(38.9) 5/12(41.71) Intravenous Mueller-Eckhardt 20/27(74.0) 20/27(74.0) Ref. 46 Clinical Obstetrics and Gynec., 1994 Vol. 37 No. 9

Over the past two years (1999–2000 and 2000–2001) we have been using an immune modulating protocol for our patients of recurrent IVF failures after adequate immune testing. This has given us a pregnancy rate of 53 percent. (See tables) as against 29 percent of previous years. If the patient shows evidence of an immune problem, immune modulation is undertaken prior to the IVF cycle. After completion of treatment, patient is

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retested and the cycle is not started until the results are normal. If the patient shows evidence of infection, she is treated aggressively—may be with intravenous antibiotics, she is then retested for infection and her immune status before the cycle is begun. Patients are kept on hormonal and immune support all through the luteal phase and after a positive pregnancy test, the treatment is continued till twelve weeks of pregnancy and in certain cases up to 24 weeks of pregnancy- till the time the second phase of trophoblastic invasion is over. Pregnancy is monitored by assessment of uterine blood flow on color Doppler. Following this regime, the carry home baby rate is 45.2 percent. Early pregnancy loss was 4.8 percent and there were 2(1.3%) mid trimester losses.

Table 59.5 Total No. of cases 146 No of pregnancies 78(53.4%) Implantation failures 3(2.05%) 1st trimester loss 7 (4.8%) 2nd trimester loss 2(1.3%) Take home baby rate 66(45.2%)

Table 59.6 Details of pregnancy 146(total) IVF pregnancy 65(44.5%) IUI pregnancy 8(5.5%) Spontaneous pregnancy 5(3.4%)

Table 59.7 No. of patients accordmg to diagnosis (Total No. 146) PCOD Endometriosis Infections including tuberculosis Unexplained Multi factorial

48 20 51 17 102

Table 59.8 No. of failed attempts at IVF in relation to immune problemsl infection IVF attempts Immune problems

1 69 52

2 50 38

3 17 11

4 7 4

5 1 1

6 2 2

CONCLUSION Every day somewhere in a corner of this earth a new thought or observation is emerging which is helping scientists answer more questions. In Vitro Fertilization and ART have laid bare the entire process of fertilization in our laboratories. As we learn more and more

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of this intriguing process, the challenge increases and the urge to better our results is more. The techniques of oocyte—embryo manipulation like assisted hatching, blastocyst transfer, co-culture are yet not available in every laboratory. Hence, the clinician is at a loss as to how to better the results. Immune modulation does seem to enhance endometrial receptivity and oocyte quality. Moreover, achieving natural pregnancies through immune modulation and treatment for infections suggests that there is certainly more to recurrent implantation failure than what meets the eye. Since a large number of patients show immune problems on immune testing, it is suggested that larger studies using these treatments may be undertaken to test their efficacy. Aknowledgements I thank Dr. Rieta Ghosh of Double Helix Laboratory and Dr. Savita Nagpal, Apex Lab. For their help in preparing this manuscript. REFERENCES 1. Pablo JL, Meseguer M, Cabellero-Campo P, Pellier A, Senior C. Embryonic regulation in the process of implantation. In Gardner, Weissman, Howles, Shoham (Eds): Text book of Assisted Reproductive Techniques. 2001; 28:341–52. 2. Chaouat G, Menu E, Mognetti B. Immunopathology of Early Pregnancy Infectious. Dis Obstet Gyncol 1997; 5:73–92. 3. Beaman KD Nidation. Tolerance and Immunotrophoism. Am J Reprod Immunol 1990; 23:54– 56. 4. Raghupathy R, Krishnan L. Immunosuppressive mechanisms in normal pregnancy. Central Eur J Immunol. 1996; 21:133–40. 5. Arck PC, Clark DA. Immunobiology of decidua. Curr Top Microbiol Immunol 1997; 222:45–66. 6. Society for Assisted Reproductive technology and American Society for reproductive medicine. Assisted reproductive technology in the United States: 1996 results generated from ASRM/Society for Assisted Reproductive Technology Registry. Fertil Steril 1999; 71:798–807. 7. Clark DA, Coulam CB, Daya S, Chaouat G. Unexplained Sporadic and Recurrent miscarriage in the new millennium: A critical analysis of immune mechanisms and treatments. Hum Reprod Update 2001; 7(5):501–11. 8. Garrido N, Navarro J, Garcia-Velasco J, Remoh J, Pellice A, Simon C. The endometrium versus embryonic quality in endometriosisrelated infertility. Hum. Reprod Update 2002; 8(1):95–103. 9. WHO Scientific Group Report. Agents Stimulating Gonadal Functions in the Human. WHO Techn Rep Ser 1973; 514:1–28. 10. Hill GA. The ovulatory factor and ovulation induction. In Wentz AC, Herbur IIICM, Hill GA (Eds): Gynaecologic Endocrynology and Infertility. Williams and Wilkins: Baltimore, 1998; 147–60. 11. Sakkas D, Jaquenoud N, Leppins G, Campanna A. Comparison of results after In vitro fertilized human embryos are cultured in routine medium and in co-culture on Vero Cells: Randomized Study. Fertil Steril 1994; 61521–25. 12. Quinn P, Margalit R. Beneficial effects of co-culture with cumulous cells on blastocyst formation in a prospective trial with supernumery uhman embryos. J Assist Reprod Genet 1996; 13:9–14. 13. Cohen J. Assisted hatching of human embryos. J in vitro Fertil Embryo Transfer 1989; 51:820– 27.

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14. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using Zona drilling of human embryos with poor prognosis. Hum Reprod 1996; 11:590–94. 15. Schoolcraft WB, Gardener DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: Analysis of results and parameters affecting outcome in two in-vitro fertilization programs. Fertil Steril 1998; 72:604–09. 16. Prapas Y, Prapas N, Hatziparasidou A, Vanderzwalmen P, Nijs M, Prapas S et al. How homogeneous are comparison groups in any study evaluating techniques of embryo transfer? Hum Reprod 2002; 17:1130–31. 17. Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001; 76: 863–70. 18. Tang OS, Ng EH, So WW, Ho PC. Ultrasound-guided embryo transfer: a prospective randomized controlled trial. Hum Reprod 2001; 16:2310–15. 19. Ogasawara M, Aoki K, Okada S, Suzumori K. Embryonic Karyotype of abortuses in relation to number of previous miscarriages. Fertil Steril 2000; 73:304. 20. Meyer WR, Castelbaum AJ, Somkuti S et al. Hydrosalpinges adversely aff ect markers of endometrial receptivity. Hum Reprod 1997; 12:1393–98. 21. Outcome of Subsequent Pregnancies Following Antibiotic Therapy After Primary or Multiple Spontaneous Abortions. Surg Gynecol Obstet 163:243–50. 22. Scarselli G, Gargiulo A, Branconi F, Di Tommaso M. Post conception failures in reproduction: infectious diseases and immunitary problems. Acta Eur Fertil 1984; 15:363–67. 23. Navot D, Berg PA, Williams M et al. An insight into early reproductive processes through the in-vivo model of ovum donation. J Clin Endocrynol Metab 1991; 72:408–14. 24. Brenner RM, West NB, McClellan MC. Eastrogen and Progestin receptors in the reproductive tract of the male and female primates. Biol Reprod 1990; 42:11–19. 25. Lessey BL, Yeh L, Castlebaum AJ, Fritz MA et al. Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation. Fertil Steril 1996; 65:477–83. 26. Van Le L, On ST, Anners JA, Rineheart CA, Holm J. Interleukin1 inhibits growth of normal human endometrial stromal cells. Obstet Gynaecol 1992; 80:405–9. 27. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bi-directional cytokyne interactions in the maternal-fetal relationship: Is successful pregnancy a Th2 phenomenon? Immunol Today 1993; 14:353–56. 28. Raghupathy R, Makhseed M, Azizieh F. Th1 and Th2 cytokine profiles in successful pregnancy and unexplained recurrent abortions. In Gupta SK (Ed): Reprod Immunol 1999; 14:149–57. 29. Reece EA, Garofalo J, Zheng XZ Assimakopoulos E. Pregnancy outcome. Influence of antiphospholipid antibody titer, prior pregnancy losses and treatment. J Reprod Med 1997; 42:49–55. 30. Ranboy RAS, Hoffmann M. From antiphospholipid syndrome to anti body mediated thrombosis. Lancet 1997; 350:1491–92. 31. Rote NS, Vogt E, DeVere G et al. The role of placental trophoblast in pathophysiology of the anitphospholipid antibody syndrome. Am J Reprod. Immunol 1998; 39:125–38. 32. Azein F, Geva E, Amit A et al. High levels of anticardiolipin anti bodies in patients with abnormal embryo morphology who attended an IVF programme. Am J Reprod Immunol 1998; 39:161–63. 33. Goswamy RK, Williams G, Steptoe P. Decreased uterine perfusion-Acause of infertility. Human Reprod 1988; 3;955–59. 34. Battaglia C, Larocca E, LanzaniA et al. Dopler Ultrasound studies of the uterine arteries in spontaneous and IVF stimulated ovarian cycles. Gynea Col Endocrinol 1990; 4:245–50. 35. Steer CV, Miller C, Tan SL et al. The use of trans vaginal colour flow imaging after IVF to identify optimum uterine conditions before embryo transfer. Fertil Steril 1992; 57:372–76. 36. Rai R, Backos M, Rushworth F, Regan L. Polycystic ovaries and recurrent miscarriage—a reappraisal. Hum Reprod 2000; 15:612–15.

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37. Clark DA, Chaouat G, Ding JW et al. Fgl2 in pregnancy: A novel prothrombinaze at the feto maternal interface may prevent bleeding and cause abortions. Am J Reprod Immunol 1999; 41:374. 38. Reece EA, Homko CJ, Wu YK. Multifactorial basis of the syndrome of diabetic embryopathy. Teratology 1996; 54:171–83. 39. Roussev RG, Kaider BD, Price DE, Coulam CB. Laboratory evaluation of women experiencing reproductive failure. Am J Reprod Immunol 1996; 35:418–20. 40. Gatenby PA, Cameron K, Shearman RP. Pregnancy loss with phospholipid antibodies: Improved outcome with asprin containing treatment. Aust NZ Obstet Gynaecol 1989; 29:294– 98. 41. Cocochock FS, Reece EA, Balaban D. Branch DW. Plouffe L. Repeated fetal loses associated with antiphospholipid antibodies: Acollaborative randomized trail comparing prednisolone to lowdose heparin treatment. Am J Obstet Gynaecol 1992; 166:1318–27. 42. Sher G, Feinman M, Zouves C et al. High fertility rates following antiphospholipid antibody seropositive women treated with heparin and aspirin. Human Reprod 1994; 9:2278–83. 43. Sher G, Matznee W, Feinman M et al. The selective use of heparin/ aspirin therapy, alone or in combination with IVIG in the management of antiphospholipid antibody—positive women undergoing in- vitro ferilization. Am J Reprod Immun 1998; 40:74–82. 44. Spinnato JA, Clark AL, Pierangali SS, Harris EN. The antiphospholipid syndrome in pregnancy: Immunoglobulin therapy. Am J Obstet Gynaecol 1994; 170:334. 45. Silver RM, Branch DW. Recurrent Miscarriage: Autoimmune considerations. Clinical Obst Gynae (Review) 1994; 37:745–60. 46. Scott JR, Branch DW. PotentialAutoimmune factors and Immune therapy in Recurrent Miscarriage. (Review). Cl Obset and Gynaecol 1994; 37:761–67.

CHAPTER 60 Repeated Pregnancy Loss (RPL): Is Investigation Important? Asha Baxi INTRODUCTION Investigation forms an integral part of management of any medical problem. Then why has question been raised for repeated pregnancy loss (RPL). This is because even if nothing is done, spontaneous resolution occurs almost in sixty percent of cases. Our recent abilities to document a probable cause in more than 50 percent of cases presenting with RPL justify the investigation (Table 60.1). Overall, RPL is a very distressing condition and couples wish to know a cause. Once they have been investigated, and the cause has been found, the treatment can improve the outcome. If a cause has not been found then they can be reassured of 80 percent chances of a successful outcome.

Table 60.1: Results of investigations from 300 couples presenting with recurrent miscarriage • Potential karyotype abnormality—12 (04%) Polycystic ovaries—170 (57%) • Serum LH >10 IU/L (n=170)–18(11%) • Raised u LH levels (n=125)–74(59%) • Antiphospholipid antibodies—58(19%)* • LA—19 • ACA—43 • *4 had both LA and ACA. Source: Clifford and Regan In Progress in Obs Gynae vol 11 edn 1994. Chart -1 (This is missing, and you will have to contact Dr Asha for this item) Source: Jenkins D.M. In Contemp Rev Obs Gynae edn 1992.

Another question is when to start the investigation. Traditional teaching has been to investigate after 3 repeated successive miscarriages. Can we really wait for that long? Some couples are doomed to recurrent miscarriage right from the beginning. The risk of further miscarriage in a primigravida is 5.6 percent, it doubles after first abortion, becomes six times after second abortion and 7 times after the third abortion. So why wait for three RPL. Even after the first miscarriage the risk is significant. So the investigation is fully justified after the second RPL and if the couple demands even after the first miscarriage then it should be considered. A detailed past medical history and previous obstetric history can help us to identify the etiology and direct our investigation more effectively. Uncontrolled diabetes mellitus,

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thyroid disease have all been associated with RPL. Similarly, a history of SLE, recurrent thromboembolism, intrauterine growth retardation, and preeclampsia would help us consider antiphospholipid antibody syndrome. Obesity has been well-documented in association with RPL in 21 percent of cases. Subfertile patients are also high-risk cases for RPL (Table 60.2).

Table 60.2: Medical history: findings from 300 couples • Diabetes mellitus 1 • Thyroid disease 7 • SLE 2 • Thromboembolism 6 • Obesity 64(21%) • (BMI >27 Kg/m2) • Subfertility 102(34%) Modified from Clifford and Regan In Progress in Obs Gynae vol 11 edn 1994.

Reviewing previous ultrasound data would be of help. If the intrauterine death has occurred, or contractions and hemorrhage existed there, then that excludes incompetent os as a cause of recurrent miscarriage. There are numerous of tests available in the literature, but it is important to use the tests that are more informative and cost effective, especially in a country like India (Table 60.3). Parental Karyotyping Balanced translocations are more common in couples with RPL, almost 3 5 percent1 as compared to 0.2 percent in

Table 60.3: Investigations of recurrent miscarriage All patients Parenteral karyotyping Pelvic ultrasound Serum LH LA and ACAs Selected patients HSG/hysteroscopy Urinary LH screening

Non-informative tests Glucose tolerance tests Thyroid function tests HLA typing APCA Infection In unexplained aetiology

Day 3 serum FSH and E2 Modified from Clifford and Regan In Progress in Obs and Gynae edn 1994.

general population.2 Such patients need genetic counseling and prenatal diagnosis must be performed during pregnancies and in cases undergoing assisted conception, preimplantation genetic diagnosis should be considered. Karyotyping of the products of

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conception in any future pregnancy loss can be undertaken, but its cost is largely the limiting factor in our country. Frequency of normal karyotype significantly increases with the number of previous abortions.3 Though aneuploidy is more common in sporadic miscarriages, some couples may be at risk for recurrent aneuploidy4 Pelvic Ultrasound A detailed pelvic ultrasound helps in assessing uterine morphology and in detecting polycystic ovaries. In case of septate and bicornuate uterus, transvaginal ultrasound permits us a sensitivity of 100 percent and a specificity of 80 percent.5 For fibroids, transvaginal ultrasound in association with sonohysterography can also help in mapping the fibroids. This helps in evaluation as well as in planning the treatment.6 In nearly 10 percent of cases of RPL, there may be associated uterine malformation. Endometrial polyps and recently endometrial calcification has also been reported to be the cause of RPL.7 Hypersecretion of Luteinizing Hormone There is now good evidence that the presence of polycystic ovaries is associated with both subfertility and early pregnancy failure. This could be due to hypersecretion of LH. In a prospective study of 193 women conceiving spontaneously, hypersecretion of LH was associated with a subsequent miscarriage rate of 65 percent compared to a rate of 12 percent in women with normal LH level.8 Antiphospholipid Antibodies (APL) Antiphospholipid antibodies (APL) are a diverse family of autoantibodies reactive against negatively charged phospholipid-protein complexes. The clinically significant members include lupus anticoagulant (LA) and anti-cardiolipin antibody, and more recently beta-2glycoprotein-1-dependent anticardiolipin antibody (b-2-GP1-dependent aCL).9 The pathophysiology of APA is uncertain. Possible mechanism of action includes inhibition of endometrial prostacyclin production, increased platelet activation leading to thromboxane release, reduced antithrombin III or protein C def iciency. The reported pregnancy loss is of the order of 80 percent in these women who have lupus anticoagulant or raised titres of anticardiolipin antibody. Prevalence rate of APA in women with RPL is almost 20 percent.10 Infection Many infections of the genital tract have been reported to be associated with sporadic pregnancy loss. The majority of the organisms implicated do not persist for sufficient period to produce repeated miscarriages. Therefore, maternal infection with Toxoplasma gondii, cytomegalo virus, rubella and listeria can cause sporadic pregnancy loss. Evidence that they cause RPL is lacking. Thus, routine ToRCH screening in cases of RPL is not justified.

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Glucose tolerance test and thyroid function test do not have much role in asymptomatic women. However, in our own experience in India, screening of infertility patients for hypothyroidism invariably picks-up 35 percent cases of hypothyroidism who are asymptomatic. May be in Indian population checking the thyroid status is important. Thyroid antibodies do not increase the future risk of pregnancy loss, unless they are in association with generalized autoimmune phenomenon.11 Autoimmune Cause Failure to mount appropriate maternal immune response leads to recurrent pregnancy loss. However, there is no consensus as to investigate RPL due to this cause. That an increased degree of human leukocyte antigen sharing between the parents could be responsible for the lack of maternal immune recognition is now strongly disputed.12 Similarly, absence of antipaternal cytotoxic antibodies has not been shown to be of much value. HSG and hysteroscopy may be required in selected cases to confirm the ultrasound findings and further evaluate the uterine cavity. Hysteroscopy offers an advantage over other tests by offering the therapeutic options at the same time. Role of diminished ovarian reserve has also been suggested as a cause of RPL. This can be detected by the Day-3 serum FSH and estradiol values. Overall, by careful investigation one can detect the cause of RPL in more than 50 percent cases. In some 70 percent patients there may be an overlap of etiology.13 Once the cause is found and treated the chances of successful outcome in future pregnancy would be around 80 percent (Table 60.4) which is better than spontaneous resolution rate of 60 percent. When the risk of RPL significantly increases even after the first abortion then why wait for three abortions before starting the investigation.

Table 60.4: Success rate in pregnancy Multiple aetiologies 81% Endocrine 82% Uterine body defect 85% Endometrial infection 77% Cervical incompetence 77% Systemic disorder 80% Source: Jenkins DM. In Contemp Rev Obs Gynae 1992 Vol. 4.

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REFERENCES 1. Stenius Aarniala B et al. A prospective study of 198 pregnancies. Thorax 1988; 12–18. 2. Lehere S et al. Association between pregnancy induced hypertension and asthma during pregnancy. Am J Obste Gynaecology 1993; 168:1463–66. 3. Ogasawara M et al. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril 2000; 73–300–04 4. Bone J et al. Chromosomal analyses of two consecutive abortions in each of 43 women. Human Genetik 1973; 19:275–80. 5. Pellerito JS et al. Diagnoses of uterine anomalies: relative accuracy of MRI, TVS and HSG. Radiology 1992; 183:795–800. 6. Cohen et al. Role of vaginal sonography and hysterosonography in endoscopic treatment of uterine leiomyoma. Fertil Steril 2000:73; 197–204. 7. Feley V et al. RPL associated with endometrial hypoechoic are as (endometril calcifications). Clin Exp Obstet Gynaecol 2000; 27(1):5–8. 8. Regan L et al. Hypersecretion of LH, Infertility and Miscarriage. Lancet 1990; ii:1141–44. 9. Aoki K et al. Antiphpspholipid antibody syndrome in adverse pregnancy-Rinsho Byori 2000; 48(4):323–27. 10. Kutlen WH. Antiphosphospholipid antibody and reproduction. J Reprod Immunolo 1997; 35:151–71. 11. Rushworth FH et al. Prospective pregnancy outcome in untreated recurrent miscarriers with thyroid autoantibodies. Human Reproduction 2000; 15:1637–39. 12. Adinolphi M et al. Recurrent habitual abortion HLA sharing and deliberate immunisation with partners cells. Human Reprod 1986; 1:45–48. 13. Tho PT et al. Etiology and subsequent reproductive performance of 100 couples with recurrent abortion. Fertility Sterility 1979; 32:389–95.

CHAPTER 61 Antiphospho lipid Antibodies in ART Gautam N Allahbadia, Sonia Malik, SPS Virk INTRODUCTION Accumulating evidence suggests that the fetal-placental semi allograft is afforded protection by local immunomodulating factors and that immunologic recurrent abortion may result from an imbalance or breakdown in the mechanisms responsible for immune homeostasis.1–4 The most compelling association between pregnancy loss and autoimmune phenomena has been with the presence of antiphospholipid antibodies— lupus anticoagulant and anticardiolipin antibody. These auto antibodies are also strongly associated with both venous and arterial thrombosis and thrombocytopenia.5 Thrombosis occurs in 25 to 33 percent of people with the lupus coagulant6 and in over 75 percent of patients with elevated anticardiolipin antibodies.7 Antiphospholipid antibodies (APA) are a group of organ non-specific auto antibodies that bind to negatively charged phospholipids. Their presence has been associated with reproductive failure; the most consistently reported phenomenon is the association between recurrent spontaneous abortion and the presence of immunoglobulin (IgG) anticardiolipin and lupus anticoagulant (LAC).8–10 At present, there is convincing evidence that abnormal autoimmune function is an etiological factor in approximately 10 percent of patients with recurrent pregnancy loss11–13 and assessment of Antiphospholipid antibodies, namely the Lupus anticoagulant (LA) and Anticardiolipin antibodies (aCL), has become routine in the evaluation of women having recurrent abortion.13–16 A particular subpopulation of anticardiolipin antibodies may be strongly represented in the male partners, the clinical significance of which remains to be established.17 First of all, it is important to note that Antiphospholipid antibodies are present in virtually every individual. They are so called ‘natural’ antibodies which can be found in females as well as males, though they appear at higher levels in females.18,19 Moreover, puberty and/or exposure to semen appears to affect at least the isotype of Antiphospholipid antibodies produced, if not their quantity.20 Pregnancy per se does not result in increased Antiphospholipid antibody titers. The sexes vary in antibody concentrations and production and auto-antibody concentrations increase with age.21 Increased autoantibody concentrations cannot, however, auto matically be equated with the presence of a disease state. In fact, it is probably reasonable to assume that a majority of women with raised Antiphospholipid antibody concentrations are perfectly healthy. A good example are relatives of patients with established autoimmune diseases. While first degree relatives of patients with autoimmune diseases have an increased incidence of elevated auto antibodies, and while they also experience an increased risk of autoimmune disease, a majority, even amongst those with elevated auto antibodies, will never develop an autoimmune disease.22 The mere presence of auto antibodies does, therefore, only

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denote a risk of disease and not necessarily the presence of disease itself. One therefore has to question the notion that the presence of auto antibodies per se causes disease and has to raise the possibility that the presence of abnormal autoantibody concentrations is only indicative of either abnormal B cell production or abnormal antibody clearance, while the truly pathognomonic effect, leading to disease, may be at a completely different level within a complex and intertwined immune system.23 The mere presence of abnormal auto antibodies does therefore not necessarily suggest that those auto antibodies cause concomitantly observed disease phenomena. Association is not necessarily causation, and abnormal auto antibodies may be nothing but an epiphenomenon in such a circumstance.23 It is equally curious that women with abnormally high peripheral values of Antiphospholipid antibodies concentrate these antibodies at incredibly high amounts in follicular fluid, while other immunoglobulins demonstrate standard clinical gradients between blood and follicle.24 In consideration of this observation, one has to wonder whether many more cases of unexplained inf ertility than are usually expected may not be due to an immunological cause. In fact, one can almost conclude that this is the case. A picture thus seems to emerge that suggests that the immune system can cause infertility. Maybe more importantly, however, if we outwit the immunological cause of infertility and succeed in nevertheless establishing a pregnancy, this pregnancy is at considerable excessive risk. This risk involves an increased chance of pregnancy loss,25,26 intrauterine growth retardation27–31 and increased perinatal morbidity as well as mortality.32–35 Antiphospholipid Syndrome The ‘antiphospholipid antibody syndrome’ has been described in women with a history of recurrent pregnancy loss or thrombosis with positive APA or lupus anticoagulant on two occasions.36 Although several treatments have been advocated, heparin and aspirin treatment is emerging as the treatment of choice for the APA syndrome associated with recurrent pregnancy loss.37–40 However, the significance of APA in a woman without a prior pregnancy or in the absence of prior thromboembolic phenomena is unclear.41 Antiphospholipid antibodies (APL) are associated clinically with thrombocytopenia, recurrent thrombosis, repeated pregnancy losses or a combination of these events.42 Patients whose pregnancies last beyond the middle of the second trimester may have a variety of collateral obstetric complications, such as early and severe pregnancyinduced hypertension and intrauterine growth retardation.36 Although the specific antibodies most commonly detected in these patients are against cardiolipin- and phosphatidylserine dependent antigens, current advances in the field suggest that phospholipid-binding plasma proteins, such as beta-2-glycoprotein I (beta2GPI), human prothrombin, proteins C and S and annexion V are involved in the binding of sera from patients with the antiphospholipid syndrome (APS) to anionic phospholipids.43 Previously, the ‘reproductive autoimmune failure syndrome’ was described in women with increased auto antibodies, endometriosis and infertility, leading to a recommendation for immunological testing of women with infertility and endometriosis.25 Recently, several investigators have advocated testing women undergoing in-vitro fertilization (IVF) for APA.44,45 The theoretical rationale for the role of APA and the potential benefits of anticoagulation therapy for women undergoing IVF

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is based on several observations. Firstly, phospholipids function as adhesion molecules during the formation of syncytiotrophoblasts.46 Secondly, the attachment of APA to surface phospholipids on trophoblasts may result in direct cellular injury Moreover, inhibition of syncytiotrophoblast formation may cause indirect damage via intravascular thrombosis.47 More recently, some investigators have recommended treatment of all APA-positive women with heparin and aspirin.48–49 Exogenous heparin has been shown to inhibit binding of APA with phospholipids,50 and endogenous heparin manufactured by trophoblasts should function in the same fashion. The antithromboxane effects of aspirin on inhibition of platelet aggregation are thought to work in concert with heparin to promote and enhance implantation.51,52 Pathophysiology in the Clinical Context A large body of evidence is emerging which suggests that a series of complex immune mechanisms modulates implantation. It has been demonstrated that increased concentrations of prostaglandins (PGE2 and PGF2alpha) at the site of embryo implantation increase vascular permeability prior to implantation and are critical to the process.53,54 Platelet activation factor (PAF), an etherlinked phospholipid, is produced by the blastocyst, by invading trophoblast and by adjacent decidua for a few days around the time of implantation.55–57 PAF facilitates implantation by increasing local consumption of thrombocytes and by promoting the release of PGE2.58,59 Phospholipids function as adhesion molecules in the formation of myoblasts and syncytiotrophoblasts.46,60 Exposure of surface phospholipids (especially phosphoserine and phosphoethanolamine in the hexagonal phase II form) creates an immunogenic state leading to delayed syncytialization of the trophoblast. This mechanism could play an important role in the pathogenesis of recurrent spontaneous abortion.47 PAF promotes local production of early pregnancy factor, an immunosuppressive glycoprotein.59 Conceivably, antibodies to surface phospholipids and to this glycoprotein could reduce the efficiency of implantation and promote autoimmune rejection of the conceptus. It has been postulated that in situations of local or systemic tissue damage, cellular surface phospholipids convert from a bilaminar configuration to a hexagonal phase II structure. In this isomeric form phospholipids combine with lipoproteins to become antigenic and lead to APA production.61 These antibodies have been identified in a number of autoimmune disorders (including but not limited to systemic lupus erythematosus, scleroderma and Hashimoto’s thyroiditis) that are known to be associated with a high incidence of pregnancy wastage.32 Infertility associated with pelvic inflammatory disease, endo metriosis and postsurgical pelvic adhesions is likewise associated with a high prevalence of APA seropositivity. This phenomenon could explain the reduced implantation rate per embryo as compared with implantation rates in women without these pathologies and following the transfer of embryos to a third party (IVF/ovum donation and/or IVF/surrogacy) who does not have pelvic pathology.62 APA have been shown to be transiently produced during ovarian stimulation and/or as a consequence of oocyte retrieval with subsequent disappearance within several weeks.63 This might explain the reduced implantation rate per embryo that occurs following embryo transfer in cases of ovarian stimulation, as well as explain the increased miscarriage rate that occurs in spontaneous pregnancies that immediately follow failed cycles of IVF/embryo transfer.64,65 When present, APA bind with surface

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phospholipids on the trophoblast and result in direct cellular injury, inhibit syncytia formation and cause indirect damage through intravascular thrombosis.47 Heparin, whether endogenous (manufactured by trophoblast) or exogenously administered inhibits binding of APA with phospholipids, protecting the trophoblast from injury.66 Aspirin on the other hand, exerts an anti-thromboxane effect and inhibits platelet aggregation.67 It is postulated that HeparinAspirin (H/A) therapy facilitates and promotes implantation through these mechanisms. While it is reasonable to link recurrent reproductive failure in APA seropositive women (in whom pregnancy has already been diagnosed) to failed implantation, it is difficult to attribute a failure to conceive following IVF/embryo transfer (where pregnancy has not yet been diagnosed) to a similar mechanism. The prevalence of APA seropositivity in the general population ranges from 5 to 17 percent, while in patients who experience recurrent spontaneous abortion it is as high as 59 percent.68 Some studies indicate that the prevalence of APA seropositivity in patients undergoing IVF/embryo transfer due to organic pelvic disease is similar to that seen in women who suffer from recurrent spontaneous abortion. In contrast, patients undergoing IVF/embryo transfer cycles in the absence of female pelvic pathology demonstrate similar APA seropositive rates to the general population (14%). Damage to pelvic organs from endometriosis, infection or iatrogenic trauma may induce the production of APA, and these antibodies may contribute to a woman’s inability to conceive naturally or via IVF. Fisch et al63 have shown that there is a transient rise in APA titers in women undergoing ovarian stimulation. Thus, it is possible that some IVF/embryo transfer failures in the pre-cycle APAseronegative women are indeed due to antibody induction and/or transient rises in titers previously below detection. However, the mechanism by which APL might cause recurrent miscarriages remains the subject of research. Fetal losses have been attributed to thrombosis of the uteroplacental vasculature and placental infarction.69,70 Although thrombosis is observed frequently in the decidua and placentas of aPL-positive patients, this observation is not universal, nor present in a sufficient degree to account for the pregnancy loss associated with this syndrome. An alternative hypothesis proposed that aPL have a detrimental effect on the trophoblastic layer of the human placenta.71 In line with the recent idea that several pathogenic mechanisms can be present simultaneously in the same patient, monoclonal aPL have been shown to prevent placental human chorionic gonadotropin (hCG) and human placental lactogen (HPL) secretion.72 Though recurrent pregnancy loss involves the loss of clinically recognized (postimplantation) pregnancies, it has been postulated that the same immunological dysfunction that may lead to some cases of recurrent pregnancy loss could also affect earlier unrecognized pregnancies as well, leading to heretofore unexplained infertility.13,73 Interestingly, a very recent report74 showed that antiphospholipid antibody was not predictive of IVF outcome but the rate of miscarriage was 2-f old higher in the IVF patients positive for antiphospholipid antibodies compared with antibody-negative women. The authors concluded that consideration whether to perform antiphospholipid antibody testing should be given to all patients whose IVF cycle results in a miscarriage after a clinical pregnancy has been documented.74 Another essential criterion that applies to antiphospholipid antibody testing in patients with pregnancy loss, is that only patients with recurrent miscarriage should be tested and considered for treatment.14,15,75 Since

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repeatedly unsuccessful embryo transfers after IVF are thought to be because of occult abortion10,13,76 a role for antiphospholipid antibodies in failure of nidation after repeated IVF—embryo transfer could be postulated. Favoring this concept is the fact that all the four studies in the literature investigating antiphospholipid antibodies in patients failing three or more IVF—embryo transfer attempts are in agreement that such patients have a higher incidence of antiphospholipid antibody seropositivity than women becoming pregnant with their first IVF attempt.12,77–79 Therefore, as with the ‘primary antiphospholipid syndrome’, antiphospholipid antibody testing in IVF patients should be applied according to strict laboratory criteria in those women having repeated failures of implantation/clinical abortion after embryo transfer but not in an infertile general population reaching an IVF program. Rationale of Heparin-Aspirin (H-A)Therapy Several regimens have been proposed for the treatment of APS, including aspirin alone, prednisolone and aspirin, heparin and aspirin and more recently IV immunoglobulin (Ig). Recent studies have suggested that aspirin plus heparin may be superior to prednisolone or aspirin taken alone for the treatment of aPL associated recurrent pregnancy loss.39,48 This combination of aspirin and heparin may promote successful embryonic implantation in the early stages of pregnancy and protect against thrombosis of the utero—placental vasculature after placentation.80 The rationale for prescribing aspirin in cases of recurrent reproductive failure associated with APA seropositivity is that aspirin may counter APAmediated hypercoagulability in the choriodecidual space, a situation which if left unaddressed would traumatize the trophoblast and compromise feto-maternal exchange. However, a haemochorial relationship is only established with placentation (i.e. after establishment of a clinical pregnancy) and accordingly, it is unlikely that aspirin therapy would influence early implantation. Rather, the possible benefit of aspirin therapy could lie in an ability to protect the trophoblast from damage after placentation has been established. Rai et al80 suggested that low-dose aspirin might improve pregnancy outcome in women with APS by blocking the action of cyclooxygenase in platelets, thereby inhibiting platelet thromboxane synthesis and preventing thrombosis of the placental vasculature. Heparin on the other hand, through preventing APA from interfering with syncytialization of the early cytotrophoblast and by countering APA interference with phospholipid-induced decidual reactions that are vital to early implantation, might potentially promote both early implantation and subsequent placentation. Furthermore, authors have not only shown a direct interference of heparin in the IgG binding to primary trophoblast cells, but also that heparin treatment is able to restore normal trophoblast invasiveness.81 In a previous study, the same authors identified a failure of placental cells to respond to GnRH after 72 hours of incubation in the presence of sera containing aPL.82Subsequent heparin treatment of cytotrophoblast cells restored the GnRHinduced secretion of HCG.82 They suggested that this failure might be due to reduced syncytium formation and that the morphology and differentiation state of the trophoblast differed between untreated and heparin treated cultures. In line with this hypothesis, they have now shown that heparin treatment of cytotrophoblast cells is able to restore regular differentiation.81 Different regimens have been proposed for the treatment

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of APS, including aspirin monotherapy, prednisolone and aspirin, or heparin and aspirin. The success of heparin treatment on pregnancy outcome in women with APS stimulated interest on the drug’s mechanism of action. Mcintyre et al66 suggested direct binding of heparin to aPL and, using an ELIS A, showed a decrease in aPL concentrations with increasing doses of heparin. This was not thought to be due to an electrostatic interaction, as chrondroitin sulphate—which has a negative charge similar to that of heparin—had no effect on aPL concentrations in the ELISA. In addition, LMWH appeared more effective at pharmacological doses and lower concentrations than did regular heparin,82 suggesting that steric hindrance was not a significant problem. The mechanism by which heparin might bind to aPL has still to be ascertained, though investigators have considered the possibility that heparin either binds to and interferes with recognition of either the aPL—protein complex, or binds directly with the aPL. The 54 kDa serum beta2-glycoprotein (beta2-GPI) appears to serve as cofactor in the recognition of the putative antigens by aPL.43 Either alone or in complex with anionic phospholipid, beta2-GPI may form an antigenic site for these antibodies.83 Findings indicate that beta2-GPI binds to heparin,84 which in turn might interfere with the aPL binding and thus eliminate the requirement for a cof actor in the binding reaction. The data of Kutteh and Ermel85indicated that heparin plus low-dose aspirin provided a significantly better pregnancy outcome than low-dose aspirin alone for aPLassociated recurrent pregnancy losses. Recently, Kutteh39 also reported that heparin combined with aspirin is as effective as heparin alone for the treatment of pregnancy losses associated with APS. Di Simone et al82 suggested that aPL could interfere with gonadotropin-releasing hormone (GnRH)-induced signal transduction. When GnRH was added to human trophoblast cells pre-incubated with normal serum, human chorionic gonadotropin (hCG) secretion increased significantly, while GnRH-induced hCG stimulation was abolished in the presence of aPL-containing sera. In vitro, heparin treatment of cytotrophoblast cells was able to restore the GnRH-induced hCG secretion, suggesting direct interference in the binding of aPL to cytotrophoblast—syncytiotrophoblast membranes.82 Furthermore, low molecular weight heparin seems to be more effective at pharmacological and lower concentrations than regular heparin. Dawes et al86 demonstrated that low molecular weight heparin may be more effective than unfractionated heparin, because it is more effectively absorbed after sc administration and has a longer half-life in the circulation. This represents an important role for low molecular weight heparin in the treatment of APS in pregnancy, because it causes less bleeding in both vaginal and abdominal deliveries.87 Antithrombotic therapy during pregnancy is used for the treatment and prophylaxis of venous thromboembolic disease, for the treatment and prevention of systemic embolism associated with valvular heart disease and/or mechanical heart valves, and for the prevention of fetal growth retardation and pregnancy loss in patients with antiphospholipid antibodies. Based on an equal efficacy and safety profile, low molecular weight heparins have replaced unfractionated heparin in the prophylaxis and treatment of patients with venous thromoembolism.88 Compared to unfractionated heparin, LMW Heparins have the advantage of an increased half life and improved bioavailability.89,90 These LMW Heparins can be administered once daily and have a substantially lower incidence of heparin induced thrombocytopenia

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and osteoporosis.91,92 As with unfractionated heparin, LMW heparins do not cross the placental barrier and are suitable for use during pregnancy93 DISCUSSION In 1996, a 38-year-old nulliparous woman died from complications of a cerebral hemorrhage.94 She was approximately 9 weeks’ pregnant with triplets at the time of her death. The patient had undergone in-vitro fertilization (IVF) and was being treated with anticoagulants (heparin and aspirin) and intravenous immunoglobulin at the time of her death. The patient had undergone 3 years of infertility therapy, including the use of clomiphene citrate with intrauterine insemination, before beginning IVF in 1995. She had no history of recurrent pregnancy loss at initiation of IVF. Her infertility workup included a normal hysterosalpingogram; her husband had a normal semen analysis. An autoantibody screen revealed positive antithyroid antibodies (antimicrosomal [76.0 mg/mL] and antithryroglobulin [19.9 mg/mL]; normal: <0.5 mg/mL for both assays). Antiphospholipid antibodies were negative. In 1985, she had a transphenoidal resection of a pituitary adenoma, with normal prolactin levels thereafter. She underwent three IVF cycles (ovulation induction, IVF, and embryo transfer). The first ended with a spontaneous abortion at 8 weeks in 1995; the second IVF cycle did not result in a pregnancy; and the third cycle resulted in a pregnancy with triplets in 1996. The patient was treated with estrogen and progesterone during each pregnancy. In addition, with each IVF cycle she received 5000 units heparin subcutaneously twice a day, 81 mg aspirin daily, and intravenous gamma globulin each month. Platelets and prothrombin time (PT) and partial thromboplastin time (PTT) were normal throughout her treatment. During her ninth week of pregnancy, the patient experienced an acute headache, anxiety, and nausea while visiting a clinic. She was transferred to a general hospital and lost consciousness en route. On admission to the hospital, she underwent immediate radiologic and neurosurgical evaluation. Her platelets and PT and PTT were normal. Neurosurgery identified a hemorrhagic arteriovenous malformation, which was surgically clipped. A postoperative computerized axial tomography (CAT) scan revealed no re-bleeding, but her condition worsened. Massive cerebral swelling could not be controlled, and her condition became critical. On her third day of hospitalization, she was pronounced braindead, and life support was discontinued the following day. Treatment of IVF patients with immunotherapy (anticoagulation or immunoglobulin) is aimed at preventing early pregnancy loss. Heparin and aspirin therapy substantially reduces the risk for recurrent spontaneous abortion (more than two pregnancy losses) for women with elevated antiphospholipid antibodies (APA). However, the woman described in this report had no antiphospholipid antibodies and no history of recurrent spontaneous abortion at the initiation of her infertility therapy.94 Anticoagulation therapy can increase the risk for fatal hemorrhagic stroke.95,96 The inhibition of platelet activity with aspirin doses lower than 81 mg daily are well documented.97 Although heparin decreases the risk for death from pulmonary embolism in surgical patients, it has been associated with increased postoperative bleeding.98 A meta-analysis of randomized clinical trials of lowdose heparin (5000 units/twice daily) to prevent thromboembolism demonstrated an increase in wound hematoma formation associated with heparin treatment.99 In surgical

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patients receiving heparin, the concomitant use of aspirin has been associated with increased risk for serious bleeding.100 In July 1997, a survey of medical practices that provide assisted reproductive technology services indicated that combination therapies of heparin and aspirin for infertility treatment were used at least once by 74 percent of respondents (Society for Assisted Reproductive Technology, unpublished data, 1997). Of those providing immunotherapy treatment, 94 percent reported that they considered women who had had recurrent spontaneous abortions as potential candidates for anticoagulation treatment. In addition, 49 percent considered women who previously had an unsuccessful IVF attempt as potential candidates for immunologic treatment, and 19 percent considered new IVF patients as potential candidates for therapy. This case is the first reported pregnancy-related death associated with the use of heparin and aspirin for infertility.94 The patient died from a cerebral hemorrhage associated with a congenital arteriovenous malformation. Although a causal relation between anticoagulation and hemorrhage from an arteriovenous malformation cannot be established, pregnant women have the risks for bleeding associated with anticoagulation therapy found in the general population (cerebrovascular accidents, gastric ulcers, and trauma) in addition to unique hemorrhagic risks such as ectopic pregnancy Both heparin and aspirin therapy have been associated with increased risks for and severity of bleeding. The patient in this report94 did not have recurrent spontaneous abortions or a history of antiphospholipid antibodies, widely accepted as indications for heparin and aspirin therapy. CONCLUSION Because the potential for bleeding exists with heparin and aspirin, the risks for and benefits of anticoagulation therapy to improve success rates in IVF patients require vigorous scientific investigationbefore being accepted as routine practice. REFERENCES 1. Edmonds DK, Lindsay KI, Miller JR Early embryonic mortality in women. Fertil Steril 1982; 38:447–53. 2. Alberman E. The epidemiology of repeated abortion. In Sharp F, Beard RW (Eds): Early Pregnancy Loss: Mechanisms and Treatment, New York: Springer-Verlag, 1988; 9–17. 3. Stray-Pederson B, Stray-Pederson S. Etiological factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984; 148:140–46. 4. Hill JA. Immunological mechanisms of pregnancy maintenance and failure: A critique of theories and therapy. Am J Reprod Immunol 1990; 22:1–12. 5. Mueh JR, Herbst KD, Rapaport SL Thrombosis in patients with the lupus anticoagulant. Ann Intern Med 1980; 92:156–59. 6. Gastineau DA, Keizmer FS, Nichols WL. Lupus anticoagulant: An analysis of the clinical and Laboratory features of 219 cases. Am J Hematol 1985; 19:265–75. 7. Harris EN, Chan JKH, Asherson RA. Thrombosis, recurrent fetal loss and thrombocytopenia: Predictive value of the anticardiolipin antibody test. Arch Intern Med 1986; 146:2153–59. 8. Cowchock FS. The role of antiphospholipid antibodies in obstetric medicine. In Lee RV (Ed): Current Obstetric Medicine. St Louis, MO: Mosby-Yearbook. 1991; 229–47.

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9. Lockwood C, Reece LA, Roniero LT, Hobbins JC. Antiphospholipid antibody and pregnancy wastage. Lancet 1986; ii:742–43. 10. Gleicher N, Pratt D, Dudkiewicz A. What do we really know about autoantibody abnormalities and reproductive failure: a critical review. Autoimmunity 1993; 16:115–40. 11. Rai RS, Regan L, Clifford K, Pickering W, Dave M, Mackie I. Antiphospholipid antibodies and ß-2 glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach. Hum Reprod 1995; 10:101–15. 12. Balasch J, Creus M, Fábregues F. Antiphospholipid antibodies and human reproductive failure. Hum Reprod 1996; 11:2310–15. 13. Hatasaka HH, Branch DW, Kutteh Wit, Scott JR. Autoantibody screening for infertility: explaining the unexplained? J Reprod Immunol 1997; 34:137–53. 14. Balasch J, Font J. Antiphospholipid antibody testing in patients with pregnancy loss. Lupus 1994; 3:429–31. 15. Balasch J. Immunological factors in pregnancy wastage: truth or fiction. In Asch R, Studd JWW (Eds): Progress in Reproductive Medicine, London, Parthenon 1995; (2):117–38. 16. Rai RS, Cohen H, Regan L. Non-pregnant women with a history of recurrent miscarriage are in a pro-thrombotic state. Hum Reprod 1996; 11:27–29. 17. Panton IA, Kilpatrick DC. Anti-cardiolipin antibodies in sexual partners of recurrent aborters. Hum Reprod 1997; 12:464–67. 18. El-Roeiy A, Dmowski WP, Gleicher N Danazol. But not gonadotrophin-releasing hormone agonists suppress autoantibodies in endometriosis. Fertil Steril 1988, 50, 864–71. 19. El-Roeiy A. and Gleicher N. Definition of normal autoantibody levels in an apparently healthy population. Obstet Gynecol 1988; 77:596–602. 20. Ober C, Kamson T, Harcowe L. Autoantibodies and pregnancy history in a healthy population. Am J Obstet Gynecol 1993; 169:143–47. 21. Moulias R, Proust J, Wanga A. Age related increase in autoantibodies. Lancet 1984; 1:178– 1129. 22. Shoenfeld Y, Segol G, Segol D. Detection of antibodies to total bistones and their subfraction in systemic lupus erythematosus patients and their asymptomatic relatives. Arthritis Rheumatica 1987; 30:169–75. 23. Gleicher N, Luih-C, Dudkiewicz A. Autoantibody profiles and immunoglobulin levels as predictors of in-vitro fertilization success. Am J Obstet Gynecol 1994; 170:1145–49. 24. El-Roeiy A, Gleicher N, Fribert T. Correlation between peripheral blood and follicular fluid autoantibodies and impact on in-vitro fertilization. Obstet Gynecol 1987; 70:163–70. 25. Gleicher N, El-RoeiyA, Carfino E, Fnberg J. Reproductive failure because of autoantibodies: unexplained infertility and pregnancy wastage. Am J Obstet Gynecol 1989; 160:1376–80. 26. Geva F, Yaron V, Lessing JB. Circulatory autoimmune antibodies may be responsible for implantation failure in in-vitro fertilization. Fertil Steril 1994; 62:802–06. 27. El-Roeiy A, Myers SA, Gleicher, N. The relationship between autoantibodies and intrauterine growth retardation in hypertensive disorders of pregnancy. Am J Obstet Gynecol 1991; 164:1253–61. 28. Kajino T. Polyclonal activation of IgM antibodies to phospholipids in patients with idiopathic growth retardation. Am I Reprod Immunol 1991; 28:231–34. 29. Polzin WJ, Kopelman JN, Robinson RD. The association of antiphospholipid antibodies with pregnancies complicated by fetal growth retardation. Obstet. Gynecol 1991; 78, 1108–1111. 30. Out HJ, Bruinse HW, Christiaens GCML. A prospective, controlled multicenter study on obstetric risk of pregnant women with antiphospholipid antibodies. Am I Obstet Gynecol 1992; 167:26–32. 31. Yasuda M, Takakuwa K, Tokunaga A, Tanaka K. Prospective studies of the association between anticardiolipin antibody and outcome of pregnancy. Obstet. Gynecol 1995; 86:555–59. 32. Lockshin RD, Druzin ML, Goei S. Antibody to cardiolipin as a predictor of fetal distress or death in pregnant patients with systemic lupus erythematosus. N Engl J Med 1985; 313, 152–56.

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33. Tan SL, Doyle P, Campbell S. Obstetric outcome of in-vitro fertilization pregnancies compared with normally conceived pregnancies. Am. I. Obstet. Gynecol 1992; 167, 773–84. 34. Nurat P, Olivennes F, de Muzon J. Task force report on the outcome of pregnancies and children conceived by in-vitro fertilization (France: 1987 to 1989). Fertil. Steril 1994; 61:324– 30. 35. Olivennes F, Kadhel P, Rufat P. Perinatal outcome of twin pregnancies obtained after in-vitro fertilization: comparison with twin pregnancies obtained spontaneously after ovarian stimulation. Fertil Steril 1996; 66:105–09. 36. Harris EN. Special report. The second international anticardiolipin standardization workshop/the Kingston Antiphospholipid Antibody Study (KAPS) Group. Am J Clin Pathol 1990; 94:476–84. 37. Lockshin MD. Antiphospholipid antibody syndrome. JAMA 1992; 268:451–1453. 38. Cowchock FS, Reece EA, Balaban D, Branch DW, Plouffe L. Repeated fetal losses associated with antiphospholipid antibodies: a collaborative randomized trial comparing prednisone with low-dose heparin treatment. Am J Obstet Gynecol 1992; 166:1318–23. 39. Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low dose aspirin is superior to low dose aspirin. Am J Obstet Gynecol 1996; 174:1584–89. 40. Raziel A, Herman A, Bukovsky I. Intravenous immunoglobulin treatment of pregnant patients with unexplained recurrent abortions. Hum. Reprod. 1996; 11:711–15. 41. Bronson R. Editorial: immunology and reproductive medicine. Hum. Reprod 1995; 10:755–57. 42. Branch DW, Silver RM, Blackwell LL. Outcome of treated pregnancies in women with antiphospholipid syndrome: an update of the Utah experience. Obstet. Gynecol 1992; 80:614– 20. 43. Roubey RA. Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other phospholipid antibodies. Blood 1994; 89:2854–67. 44. Gleicher N, Liu H, Dudkiewicz A. Autoantibody profiles and immunoglobulin levels as predictors of IVF success. Am J Obstet Gynecol 1994; 170:1145–49. 45. Birdsall MA, Lockwood GM, Ledger WL. Antiphospholipid antibodies in women having invitro fertilization. Hum. Reprod 1996; 11, 1185–89. 46. Sessions A, Horowitz AF. Differentiation-related difference in the plasma membrane phospholipid asymmetry of myogenic and firbrogenic cells. Biochim. Biophys. Acta 1982; 728:103–11. 47. Rote NS, Walter A, Lyden TW. Antiphospholipid antibodieslobsters or red herrings? Am J Reprod Immunol 1992; 28:31–37. 48. SherG, Feinman M, Zouves C. High fecundity rates following in-vitro fertilization and embryo transfer in anti-phospholipid antibody seropositive women treated with heparin and aspirin. Hum Reprod 1994; 9:2278–83. 49. Kowalik A, Vichnin M, Branch W, Berkeley A. Mid-follicular anticardiolipin and antiphosphatidylserine antibody titers do not correlate with IVF outcome. Presented in American Society of Reproductive Medicine Meeting, November, 1996, Boston, MA, USA. Abstract P-130. 50. Ermel LD, Marshbum PB, Kutteh WH. Interaction of heparin with antiphospholipid antibodies (APA) from serum of women with recurrent pregnancy loss (RPL). Am J Reprod Immunol 1995; 33:14–20. 51. Patrono C. Aspirin as an antiplatelet drug. N Eng J Med 1994; 330:1287–94. 52. Hauth JL. Low-dose aspirin: lack of association with an increase in abruptio placentae or perinatal mortality. Obstet Gynecol 1995; 85:1055–58. 53. Malathy PV, Cheng HC, Dey SE. Production of leukotrienes and prostaglandins in the rat uterus during preimplantation period. Prostaglandins 1986; 32:605–14. 54. Hoffman LH, Davenport GR, Brash AR. Endometrial prostaglandins and phospholipase activity related to implantation in rabbits: effects of dexamethasone. Biol. Reprod 1984; 38:544–55.

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55. Johnston JM, Bleasdale JE, Hoffman DR. Functions of PAF in reproduction and development: involvement of PAF in fetal lung maturation and parturition. In Synyer F (Ed): PlateletActivating Factor and Related Lipid Mediators. New York: Plenum Press, 1987; 375. 56. O’Neill C. Embryo-derived platelet activating factor: a preimplantation embryo mediator of maternal recognition of pregnancy. Domestic Anim. Endocrinol 1987; 4:69–85. 57. O’Neill C, Gidley-Baird AA, Pike JL, Porter RN, Sinosich MJ, Saunders DM. Maternal blood platelet physiology and luteal phase endocrinology as a means of monitoring pre and postimplantation embryo viability following in-vitro fertilization. J. In Vitro Fertil. Embryo Transfer 1985; 2:87–93. 58. Holmes PV, Sjogren A, Hamberger L. Prostaglandin-E2 released by preimplantation human conceptuses. J Reprod Immunol 1989; 17:79–86. 59. van der Welden RMF, Helmerhorst FM, Keirse MJNC. Influence of prostaglandins and platelet activating factor on implantation. Hum. Reprod 1991; 6:436–42. 60. Sessions A, Horowitz AF. Myoblast aminophospholipid asymmetry differs from that of fibroblasts. FEBS 1981; 34:75–78. 61. Rauch J, Janoff AS. Phospholipid in the hexagonal 11 phase is immunogenic: evidence for immunorecognition of nonbilayer lipid phases in-vivo. Proc Nazi Acad Sci: USA, 1990; 87:4112–14. 62. Asch RH. High pregnancy rates after oocyte and embryo donation. Hum Reprod 1993; 7:734. 63. Fisch B, Rikover Y, Shohat L, Zurgil N, Tadir Y, Ovadia J et al. The relationship between invitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid antibodies. Fertil Steril 1991; 56:718–24. 64. Schwartz M, Jewelewicz R. The use of gonadotrophins for induction of ovulation. Fertil Steril 1991; 35:3. 65. Scialli AR The reproductive toxicity of ovulation induction. Fertil. Steril 1986; 45:315. 66. Mclntyre JA, Taylor CG, Torry DS, Wagenknecht DR, Wilson J, Faulk WP. Heparin and pregnancy in women with a history of repeated miscarriages. Haemostasis 1993; 23(suppl 1):202–11. 67. Harris EN, Gharavi AE, Hughes GRV. Antiphospholipid antibodies. Clin Rheum Dis 1985; 11:591–609. 68. Matzner W, Chong F, Xu HF, Chung W. Characterization of’ antiphospholipid antibodies in women with recurrent spontaneous abortions. J. Reprod Med 1994; 39:27–30. 69. DC Wolf F, Carreras LO, Moennan P. Decidual vasculopathy and extensive placental infarction in a patient with repeated thromboembolic accidents, recurrent fetal loss, and a lupus anticoagulant. Am J Obstet Gynecol 1982; 142:829–34. 70. Out HJ, Kooijman CD, Bruinse HW, Derksen RH. Histopathological findings in placentae from patients with intrauterine fetal death and anti-phospholipid antibodies. Eur J Obstet Gynecol Reprod Biol 1991; 41:179–86. 71. Lyden TW, Vogt E, Ng AK. Monoclonal antiphospholipid antibody reactivity against human placental trophoblast. J Reprod Immunol 1992; 22:1–14. 72. Katsuragawa H, Kanzaki H, Inoue T. Monoclonal antibody against phosphatidylserine inhibits in-vitro human trophoblastic hormone production and invasion. Biol Reprod 1997; 56:50–58. 73. Tartakovsky B, Bermas BL, Sthoeger Z. Defective maternal—fetal interaction in a murine autoimmune model. Hum. Reprod. 1996; 11:2408–24. 74. Kowalik A, Viehnin M, Li HC, Branch W, Berkeley A. Mid—follicular anticardiolipin and antiphosphatidylserine antibody titers do not correlate with in-vitro fertilization outcome. Fertil Steril 1997; 68:298–304. 75. Cowchock S, Reece EA. For the Organizing Group of the Antiphospholipid Antibody Treatment Trial. Do low-risk pregnant women with antiphospholipid antibodies need to be treated? Am J Obstet Gynecol 1997; 176:1099–1100.

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76. Clark DA. Current concepts of immunoregulation of implantation. In Chapman M, Grudzinskas G, Chard T (Eds): Implantation: Biological and Clinical Aspects. London: SpringerVerlag, 1989; 163–75. 77. BirkenfeldA, Mukaida T, Minichiello L. Incidence of autoimmune antibodies in failed embryo transfer cycles. Am J Reprod Immunol 1994; 31:65–68. 78. Geva E, Amit A, Lemer-Geva L. Autoimmune disorders: another possible cause for in-vitro fertilization and embryo transfer failure. Hum. Reprod 1995; 10:2560–63. 79. Kaider B, Price DL, Rouses RG, Coulam CR. Antiphospholipid antibody prevalence in patients in an IVF program. J Reprod Immunol 1996; 35:388–93. 80. Rai R, Regan L. Antiphospholipid antibodies in women undergoing in-vitro fertilization. Hum Reprod 1997; 12:197–98. 81. Di Simone N, Caliandro D, Castellani R, Freazzani, Carolis S, Caruso A. Low molecular weight heparin restores in-vitro trophoblast invasiveness and differentiation in presence of immunoglobulin G fractions obtained from patients with antiphospholipid syndrome. Hum Reprod 1999; 14:489–95. 82. Di Simone N, Ferrazzani S, Castellani R. Heparin and low-dose aspirin restore placental human chorionic gonadotrophin secretion abolished by antiphospholipid antibody-containing sera. Hum Reprod 1997; 12:2061–65. 83. Rand JH, Wu XX, Andree HAM. Pregnancy loss in the antiphospholipid antibody syndrome: a possible thrombogenic mechanism. N Engl J Med 1997; 337, 154–60. 84. Mcintyre JA, Wagenknecht DR. Interaction of heparin with beta2glycoprotein I and antiphospholipid antibodies in-vitro. Thromb Res 1992; 68, 495–500. 85. Kutteh WH, Ermel LD. A clinical trial for the treatment of aPL associated RPL with lower dose heparin and aspirin. Am J Reprod Immunol 1996; 35:402–07. 86. Dawes J, Bara L, Billaud E. Relationship between biological activity and concentration of a low molecular-weight heparin (PK 10169) and unfractionated heparin after intravenous and subcutaneous administration. Haemostasis 1986; 16:116–22. 87. Dulitzki M, Pauzner R, Langevitz P. Low molecular weight heparin during pregnancy and delivery: preliminary experience with 41 pregnancies. Obstet Gynecol 1996; 87:380–83. 88. Ginsberg JS, Hirsch J. Use of Antithrombotic agents during pregnancy. Chest 1995; 108:305S– 11S. 89. Bara L, Billaud E, Garamond G, Kher H. Comparative pharmacokinetics of a low molecular weight heparin (PK10169) and unfractionated heparin after intravenous and subcutaneous administration. Thromb Res 1985; 39:631–36. 90. Bara L, Samana M. Pharmacokinetics of low molecular weight heparins. Acta Chir Scand Suppl 1988; 543:65–72. 91. Monreal M, Lafoz E, Olive A. Comparison of subcutaneous unfractionated heparin with a low molecular weight heparin (fragmin) in patients with venous thromboembolism and contraindications to Coumarin. Thromb Hemost 1994; 71:7–11. 92. Shefras J, Farquarson RG. Bone density studies in pregnant women receiving heparin. Eur J Obstet Gynecol Reprod Biol 1996; 65:171–74. 93. Forestier F, Daffos F, Rainaut M, Toulemonde F. Low molecular weight heparin (Cy216) does not cross the placenta during the third trimester of pregnancy. Thromb Hemost 1987; 57:234– 39. 94. Centers For Disease Control, USA. Pregnancy-related death associated with heparin and aspirin treatment for infertility, 1996. MMWR Morb Mortal Wkly Rep. 1998 May 15; 47(18):368–71. 95. The SALT Collaborative Group. SwedishAspirin Low-dose Trial (SALT) of 75 mg aspirin as secondary prophylaxis after cerebrovascular ischaemic events. Lancet 1991; 338:1345–49. 96. Steering Committee of the Physicians Health Study Research Group. Final report on the aspirin component of the ongoing Physicians’ Health Study. N Engl J Med 1989; 321;129–35. 97. Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest 1982; 69:1366–72.

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98. Collins R, Scrimgeour A, Yusuf S, Peto R. Reduction in fatal pulmonary embolism and venous thrombosis by perioperative administration of subcutaneous heparin: overview of results of randomized trials in general, orthopedic, and urologic surgery. N Engl J Med 1988; 318:1162– 73. 99. Clagett GP, Reisch JS. Prevention of venous thromboembolism in general surgical patients: results of a meta-analysis. Ann Surg 1988; 208:227–40. 100. WalkerAM, Jick H. Predictors of bleeding during heparin therapy. JAMA 1980; 244:1209–12.

SECTION 9 Cryopreservation

CHAPTER 62 Cryopreservation of Oocytes and Embryos Foad Azem, Ben Yosef Dalit, Joseph B Lessing INTRODUCTION Cryopreservation of spare or surplus human embryos produced by ART was actively pursued in the early 1980s.1 The methods used for cryopreservation of human embryos were based on those used for embryos of farm animals.2 The introduction of this technology enhanced the opportunities for pregnancy from a single stimulation cycle. Currently the outcome of freezing and thawing increase the ultimate pregnancy rate.3 However, legal, moral and religious problems related to the cryopreservation of human embryos have been raised. The use of such technology has been restricted and even forbidden in some countries such as Germany, Austria, Switzerland, Denmark and Sweden.4 Alternatively, human oocyte cryopreservation represents a solution to the moral, legal and religious problems associated with embryo storage.5 Furthermore, this method enables the preservation of the reproductive capacity inpatients treated by chemotherapy, radiotherapy or in women at risk of premature ovarian failure or pelvic surgery Despite early disappointing results regarding survival, fertilization and cleavage rates, which led to only sporadic pregnancies in more than ten years, the recent introduction of technical modification especially the use of ICSI has greatly improved the clinical efficacy, with the birth of several healthy children. HISTORY The history of cryopreservation goes back to 1776, when Spallanzani reported upon attempts to maintain spermatozoa and oocytes of various animals in a frozen state.6 More studies began in 1930s and 1940s, but it was not until the discovery of glycerol, that a practical application was established. In 1972, Whittingham, et al obtained live mice after the transf er of freeze-thawed morulae, with the use of a new cryoprotective compound-dimethyl sulfoxide (DMSO).7 In 1983, Trounson and Mohr reported the first pregnancy from a frozen embryo in humans.1 Since then the technology of embryo freezing has been simplified and optimized due to new cryoprotectants such as propandiol and sucrose.8 Today embryo cryopreservation has been established as a routine procedure in ART. In 1977, Whittingham reported the first births from transfer of mice morulae.9 Ten years later Chen reported the first successful cryopreservation of human oocytes.10

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However, contrary to embryo cryopreservation, results of mature oocyte freezing have generally shown poor cryosurvival, poor fertilization, and high incidence of triploidy and limited live births.5 Furthermore, exposure to cryoprotective compounds and the freezing process were claimed to have deleterious effects on the zona pellucida, cortical granules, cytoplasmic microfilaments and organelles.11 However, recent studies have documented the presence of normal karyotype in oocytes after thaw. Moreover, clinical studies have shown that the use of ICSI yields good fertilization rates, implantation rates and the birth of healthy children.12 INDICATIONS FOR EMBRYO AND OOCYTE CRYOPRESERVATION Embryo cryopreservation was originally developed in order to preserve the surplus oocytes and embryos, which result from ovarian stimulation. However, the use of this technology has been extended to cases of endometrial inadequacy, premature luteinization, or in cases in which the mother suffers from acute illness or unexpected complications of the ovum pick up.

Table 62.1: Indications for cryopreservation • Freezing of surplus embryos • In cases of severe ovarian hyperstimulation syndrome • Endometrial inadequacy • Fever, or acute illness

Biological and Technical Aspects When cells are cooled to temperatures between −5°C and 15°C, the aqueous solutions, surround the cell, undergo several physical and chemical changes: i. Decrease in the solubility of dissolved gases, might produce giant gas bubbles13 ii. Crystal formation and ice fronts may destroy the surrounding the cell membranes14 iii. Solution effects related to changes in the pH and the external concentrations of salts, may cause lipoprotein denaturation and induce constraints in the membranes iv. The increase in salt concentration may result in diffusion of water from the cell and consequent passive dehydration and cell shrinkage. Cells undergoing cryopreservation are, therefore, liable to damage both from the formation of intracellular ice and from the build up of salts in the cells as they dehydrate. The cryopreservation procedure follows five important stages: 1. Exposition to cryoprotectants 2. Freezing beyond 0°C 3. Storage 4. Thawing 5. Return to previous physiological conditions.

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The most critical steps affecting cellular survival are the initial phase of freezing and the final step of return to physiological environment. At the temperature of (−196°C) most of the chemical reactions are inhibited, and the cellular clock is arrested, and prolonged storage has no negative effect on the survival rate. In order to maximize the survival rate, a mathematical model has been suggested which represents the volume modifications as a function of the permeability, membrane area and temperature.15 It has been found that the optimal freezing rate depends on the cytosolic water content, the changing permeability constant of the membrane, the area of the membrane and the temperature. As the cooling rate increases, the probability that ice will form increases as well. The highest survival rates are obtained with a cooling speed of 0.3°C/minute for zygotes and embryos.16 In order to ensure optimal cell survival during cryopreservation there is a need to use chemical cryoprotectants. Cryoprotectants are substances, which present different chemical compositions, and share high water solubility associated with toxicity directly related to their concentration and temperature. They are water soluble, able to form hydrogen bonds with water. These cryo protectants penetrate the cell membranes and subsequently stabilize the intracellular proteins, reduce the temperature at which cells undergo lethal intracellular ice formation and moderate the impact of concentrated intra- and extra-cellular electrolytes. The four most commonly used cryoprotectants for oocyte and embryo cryopreservation are glycerol, ethylene glycerol, 1,2-propandiol (PROH) and dimethyl sulfoxide (DMSO). The cryoprotectants are divided into two groups according to their capacity to penetrate inside the cells: intra- and extra-cellular agents. Glycerol, DMSO and PROH are intracellular or penetrating cryoprotectants. Their molecular weight is relatively low (<100). Compounds which as a result of their size or polarity remain in extracellular solution include large molecules such as sucrose, proteins and lipoproteins.

Table 62.2: Molecular weight and Density of main cryoprotectants Cryoprotectant Molecular weight Density Glycerol DMSO 1,2-propandiol Ethyleneglycol

92.09 78.13 76.09 62.07

1.47 1.10 1.04 1.11

Cryoprotectants bond to water and they reduce the toxic effects of high concentrations of other compounds. Furthermore they protect cells during slow cooling when the cells are very dehydrated and are surrounded by concentrated salts. At high concentrations, cryoprotectants minimize the damage caused by ice formation, as they cause the water to form a glass rather than ice crystal. For slow cooling procedures, cryoprotectant concentration of 1.5 M are many times higher than that of any other components of the medium. The cryoprotectant enters the cell by osmosis. Cells placed in a cryoprotectant solution shrink as the water rapidly leaves the cells to dilute the high concentration of the extracellular solution. Equilibrium,

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however, is soon re-established and the cell will return to its original size. When a cell containing a cryoprotectant is placed in a medium with a lower concentration of cryoprotectant, water enters the cells to dilute the cryoprotectant more rapidly than the cryoprotectant can leave the cells, with swollen or burst cells resulting. In order to overcome this obstacle, cryoprotectants are removed in successive steps of progressively lower concentrations of the cryoprotectant. When cells return to their normal size, they can be removed to the next solution. After thawing, the cryoprotectants must be removed from the cells in order to avoid their deleterious effect on further embryo cleavage and development. Human gametes and embryos can be frozen at any developmental stage, from primordial follicles, immature oocytes, M-II oocytes, zygote, morula to blastocyst. The choice of particular cryoprotectants is depending in the developmental stage of the oocyte or pre-embryo. The slow cooling with propanediol is widely used for freezing zygotes and embryos at early stages of development. Embryos are equilibrated with propanediol and slow cooled in straws. They are thawed rapidly and are dehydrated in several steps. The cooling procedure takes about 2 to 4 hours whereas the thawing procedure can be done in 45 to 60 minutes. While embryo cryopreservation is widely used and currently considered as a well-established procedure, unfertilized mature (MII) stage oocytes are more difficult to cryopreserve.17 This is related to the oocyte’s surface to volume ratio, single membrane, temperature-sensitive metaphase spindle and zona and susceptibility to parthenogenic activation and chill-injury. The female gamete is one of the biggest human cells and this increases the likelihood of intracellular ice formation, which decreases the overall survival rates. Exposure of M-II oocytes has been reported to be associated with alterations in the cytoskeleton, microtubular structure, and spindle organization.18,19 Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. Embryo Survival The morphological integrity and the embryo ability to further cleave in vitro and in vivo determine the efficiency of embryo cryopreservation.20 Early cleaved embryos are considered to have survived when they can keep at least 50 percent of their initial blastomeres intact after thawing and dilution of the cryopotectants. General survival rate is defined as the percentage of surviving embryos among all frozen and thawed embryos. Pronucleate oocytes are considered to have survived the freeze-thaw process when they cleave in vitro during the next 24 h of culture, providing that they appear intact with clear cytoplasm and no zona pellucida infringement. Blastocyst survival is more difficult to evaluate, considering the number of cells and their specialization. Usually only morphologically normal blastocysts that have re-expanded after 3–4 h of recovery are transferred in utero. Transfer of thawed embryos is performed following synchronization of embryonic and endometrial receptivity is achieved. In natural cycle, this can be achieved with close monitoring of follicular size and serum estradiol (E2) and progesterone. Anovulatory women may be treated with Clomiphene citrate. Alternatively, endometrial maturation can be achieved with administration of estrogen and progesterone, similar to that used in egg donation protocols.

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Clinical Outcome Of the 10,181 frozen embryo procedures recorded in the 1997 U. S registry, the clinical pregnancy rate was 21.5 percent of thaw and 23.8 percent of transfer procedure, with 1,719 deliveries, for a delivery rate of 16.9 percent per thaw and 18.8 percent per transfer procedure was used in 1,584 thaw and 1,467 transfer procedures, resulting in 400 clinical pregnancies (clinical pregnancy rate 25.3% per thaw, 27.3% per transfer procedure) and 235 deliveries (delivery rate, 20.5% per thaw and 22.2% per transfer procedure). A total of 2,653 live-born infants resulted from 2,044 deliveries with known outcome from all cryopreserved embryo transfer, including donated oocytes. A total of 2,580 normal infants, 49 infants with structural or functional abnormalities (18 per 1000 neonates), and 24 neonatal deaths (9 per 1000 live births) were reported. According to this registry the incidence of neonates with structural or functional abnormalities and neonatal death did not differ for transfer of cryopreserved embryos compared with fresh embryos.21 Cryopreservation of oocytes, despite its impact on conservation of genetic resources, is a developing technology. The main promise of oocyte cryopreservation is that it offers an alternative when embryo freezing is not possible for technical, regulatory, or religious reasons. Oocyte freezing is more suitable for a single woman when the concern is age-related decline in fecundity.22 Unfertilized MII stage oocytes are more difficult to croypreserve. Many factors contribute to this including the oocyte’s surface to volume ratio, single membrane, temperature sensitive metaphase spindle and zona, and susceptibility to parthenogenetic activation and chillinjury. Oocytes should be frozen after being harvested, between 38 and 49 h after HCG.23 Older oocytes present a significantly reduced fertilization potential and an increase in anomalous fertilization and polypoidy.24 Cryopreservation of oocytes in prophase I have resulted in disappointing results. Mandelbaum, et al found low survival rate (37%) and low rate of in vitro maturation (20%), and they concluded that prophase I, is not the best stage in which to freeze oocytes.25 The survival rate of human oocytes at thawing, reported in literature varies considerably (25–76%).5 In 1986 Chen reported the first pregnancy from frozen thawed oocytes. One year later two additional pregnancies and births were reported. Until recently cryopreservation of oocytes was considered an ineffective technique, with poor survival, fertilization and cleavage rates. In 1997 Porcu, et al, reported the first birth of a healthy female conceived after intracytoplasmic sperm injection of cryopreserved human oocytes.26 Since the introduction of ICSI, the results in-terms of fertilization, and embryo implantation have become similar to those obtained with fresh oocytes. The concerns about the safety of the procedure were examined by Gook, et al, who demonstrated a normal genetic patrimony and the absence of aneuploidy induction.27 Currently, the low survival rate of the thawed oocytes appears to be the main obstacle of this technique. Our Experience Our experience involves freezing pronucleate (2PN) stage oocytes. This stage was chosen because the available literature demonstrated good survival and pregnancy results associated with the one-cell conceptus before syngamy. Intrauterine transfer of thawed

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embryos is scheduled only after viability is ascertained and a regular cleavage pattern established, usually within 24 hours of thaw. We chose to require cleavage as proof that embryo viability was not compromised. Embryos may be thawed on the day of ovulation in either natural or controlled cycles. Alternatively, embryos may be transferred into a controlled endometrium after treatment with exogenous steroids. In either case, transfer is scheduled for the day after thawing. Recently, we retrospectively analyzed the outcome of 290 frozen-thawed cycles (Fig. 62.2) performed between January 1997 and December 1999. Embryos were frozen at the 2PN stage in women who had at least 6 fertilized oocytes. In addition, supernumeracy cleaved embryos were cryopreserved on either day 2 or 3. Pertinent data on women, embryos and IVF outcome variables in each cycle were recorded. We studied the cycle involving 204 day 1 (group A) and 86 day 2 or 3 (group B) frozen embryos. The age of women, mean number of frozen embryos per cycle and number of ET were similar in both groups. The post-thaw survival rate and cleavage rate were significantly lower in-group B compared to group A (72.5% vs. 82.6%; p<0.05, and 45.6% vs. 92.0%; p<0.05, respectively). Implantation rate (IR; 4.4% vs. 9.2%) and take home baby rate (2.8% vs. 15.7%) were significantly lower (p<0.05) in group B compared to group A. Furthermore, when a selected group of cycles from group B in which only good quality embryos (i.e. normal cleavage rate and 50% grade I+II) were frozen, embryo cleavage post-thawing, PR, IR and take home baby, rates were significantly lower compared to group A.

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Fig. 62.2 Our data indicate higher survival and cleavage rates in 2PN embryos resulting in better implantation potential post-thawing. Furthermore, it seems that even the good quality embryos in group B suffer some kind of impairment during cryopreservation, which gives an advantage to a policy of embryo freezing on day 1. RELIGIOUS AND ETHICAL ISSUES Buddhist View28 Buddhism accepts cryopreservation of pre-embryo due to the following reasons: 1. It saves some of the human embryos, which otherwise would be lost, for future use. 2. The patient’s burden is reduced psychologically, financially as well as physically. 3. The chances of pregnancy are improved by increasing the opportunity of the number of embryos transferred to the uterus per ovum collection 4. The freezing of embryos should be accepted only during the period in which the ovum donor is in the reproductive age and should not accepted after the death of the parents or the loss of the reproductive ability of the oocytes donor. Islamic View The general consensus is that as long as fertilization remains within limits of morality and does not create any social problem it would be permissible i.e. fertilization of wife’s

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ovum by the husband’s sperms. According to Islamic law freezing of pre-embryo is acceptable.29 Jewish View According to Judaism, freezing of embryos is permitted only if the father’s identity will be preserved. The main function of the father is to fertilize of the oocyte. The period of freezing may cause breaking of the relationship between child and his father. However, with regard to the mother, the issue is not complicated, since the mother embryo relationship, will renew following embryo transfer.21 Roman Catholic View “The freezing of embryos, even when carried out in order to preserve the life of an embryo, constitutes an offence against the respect due to human beings, by exposing them to great risks of death or harm to their physical integrity and depriving them, at least temporarily, of maternal shelter and gestation, thus placing them in a situation in which further offences and manipulations are possible”.30 REFERENCES 1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983; 305:707–9. 2. Massip A. Cryopreservation of embryos of farm animals. Reprod Dom Anim 2001; 36:49–55. 3. Veck LL, Amundson CH, Brothman et al. significantly enhanced pregnancy rates through cryopreservation and thaw: a five-year clinical study. Fertil steril 1993; 59:1202–7. 4. Kazem R, Thompson LA, SrikantharajahA, Lang MA, Hamilton MPR, Templeton A. Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1995; 10:2650–4. 5. Porcub E. Freezing of oocytes. Curr Open Obstet Gynecol 1999; 11:297–300. 6. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos, frozen to −196°C and −289°C. Science 1972; 178:411–4. 6. Kuzan FB, Quinn P. Cryopreservation of mammalian embryos. In Wolf DP (Ed): In vitro fertilization and embryo transfer: A manual of basic techniques. New York; Plenum Press, 1988:301− 47. 7. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos, frozen to −196°C and −289°C. Science 1972; 178:411–4. 8. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1.2 propandiol. Fertil Steril 1985; 44:89–94. 9. Whittingham DG. Fertilization in-vitro and development to term of unfertilized mouse oocytes previously stored at −196°C. J Reprod Fertil 1977; 49:89–94. 10. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; I:884–6. 11. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and prospective. Hum Reprod 1996; 2:193–207. 12. Porcu E, Fabbri R, Savelli L, Petracchi S, Flamigni C. Cryopreservation of human oocytes: state of the art. In Kempers RD, Cohen J, Haney AF, Younger JB (Eds): Fertility and reproductive medicine. Amsterdam; Elsevier Press, 1998; 599–613. 13. Ashwood-Smith MJ, The cryopreservation of human embryos. Hum Reprod 1986; 5:319–32.

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14. Morris GJ, Watson PF. Cold shock injury comprehensive bibliography. Cryo-Letters, 1984; 5:352–72. 15. Mazur P. Limits to life at low temperature and reduced water activities. Orig Life 1980; 10:137 16. Mandelbaum J, MenezoY. Cryopreservation of human embryos. Textbook of ART. 17. Shaw JM, Oranratnachai A, TrounsonAO. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology 2000; 53:59–72. 18. Vincent C, Johnson MH. Cooling, cryoprotectants and the cytoskeleton of mammalian oocyte. Oxford Rev Reprod Biol1992; 14:72–100. 19. Pickering SJ, Braude PR, Johnson M, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54:102–8. 20. Mandelbaum J. Embryo and oocyte cryopreservation. Hum Reprod 2000; 15:43–47. 21. Society for assisted reproductive technology and American society for reproductive medicine. Assisted reproductive technology in the Unites States: 1997 results generated from the American Society for Reproductive Medicine/Society for assisted reproductive technology registry. Fertil Steril 2000; 74:641–54. 22. Oktay K, Kan MT, Rosenwaks Z. Recent progress in oocyte and ovarian tissue cryopreservation and transplantation. Curr Opin Obstet Gynecol 2001; 3:263–8. 23. Chen C. Pregnancy after human oocyte cryopreservation. Ann NY Acad Sci. 1987; 541:541–9. 24. Toth T, Baka S, Veek L, Jones H, Muasher S, Lanzendorf S. Fertilization and in-vitro development of cryopreserved human prophase I oocytes. Fertl Steril 1994; 61:891–4. 25. Mandelbaum J, Junca AM, Tibi C, Plachot M, Alnot MO, Rim H et al. Cryopreservation of immature and mature hamster and human oocytes. In-vitro Fertil Assist Reprod 1988; 541–550– 61. 26. Porcu E, Fabbri R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 4:724–6. 27. Gook D, Schiewe MC, Osborn S, Asch RH, Jansen RPS, Johnston WIH. Intacytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1, 2-propandiol. Hum Reprod 1995; 10:2637–41. 28. Shenkar JG. Ethical, religious and legal debate on IVF and alternate assisted reproduction. In S Mashiach (Ed): Advances in assisted reproductive technologies. New York: Plenum, 1990; 1041–52. 29. Raja IA, Chaudhry MR. Islam and medical ethics. Acta Neurochir 1999; 74:29–34. 30. Congregation for the Doctrine of Faith. Vatican, 1987.

CHAPTER 63 Cryopreservation of Human Spermatozoa Frank M Köhn, Wolf B Schill SUMMARY Assisted reproduction techniques have widened the indications for human sperm cryopreservation, because semen quality is no longer a limiting factor. Therefore, cryopreservation can now be used more of ten than in the past by cancer patients for the purpose of fertility protection. Since the fertilizing capacity of cryopreserved testicular and epididymal spermatozoa is comparable to that of ejaculated spermatozoa, the most evident advantage of cryopreservation techniques is that active spermatogenesis can be identified before the female partner undergoes any stimulation protocol. INTRODUCTION Cryopreservation of human ejaculates is a wellestablished medical procedure to maintain the fertilizing potential of spermatozoa during storage in liquid nitrogen (−196°C). Pregnancies have been achieved with semen samples cryopreserved for more than 15 years. Modern trends in assisted reproduction technologies (ART) have influenced the indications for human sperm cryobanking. In addition to cryopreservation of donor spermatozoa for artificial insemination or cryobanking of ejaculates from cancer patients, new indications are the storage of epididymal or testicular spermatozoa prior to intracytoplasmic sperm injection. The most important benefits of cryopreservation in combination with ART are: • Biological material can be stored and is available for more than one microinjection without repeated surgery. • Surgery to achieve testicular or epididymal spermatozoa and oocyte retrieval do not have to be performed at the same time. The different aspects of cryobanking of human semen have been described in detail by Sherman (1986),1 Schill and Bollmann (1986),2 Brotherton (1990),3 Quinn (1993)4 and Van der Elst et al (1997).5 Cryobanking of semen has received greatest promotion through the emergence of AIDS, since from this time on the use of fresh semen for donor insemination was no longer acceptable. It has been shown that transmission of human-Tcell lymphotrophic virus type 3 (HTLV-III) to recipients of donor semen in artificial insemination was possible.6 To avoid any transmission of HIV, donor semen should be held in quarantine for at least three months prior to testing donor’s blood sample negative for HIV antibodies. Thus, frozen semen in AID has to be used in order to meet the

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standards of cryobanking as established by the American Association of Tissue Banks (AATB). There is no doubt that pre-therapy storage of semen from cancer patients is an important medical task, all the more since the prognosis and life expectancy of Hodgkin’s disease and testicular tumors is very good today.7 Since the only requirement for the new reproductive technology of intracytoplasmic sperm injection is at least one living spermatozoon per oocyte, cut-off values of standard semen parameters prior to cryopreservation of semen samples are no longer necessary. Even ejaculates of poorest quality can now be stored in liquid nitrogen. Indications for Human Sperm Cryopreservation Clinical applications for cryopreservation can be summarized as follows: 1. Possibility of timed multiple inseminations of donor semen (AID) or husband semen (AIH), including microbiological testing of semen or blood prior to insemination to exclude sexual transmitted diseases. 2. Preservation of semen before surgical, chemical or radiological cancer therapy leading to subsequent sterility 3. Storage of semen in the temporary or permanent absence of the donor or husband 4. Storage of semen before vasectomy 5. Use of cryobanking in reproductive medicine (MESA, TESE) New indications may also include testicular cryopreservation inboys prior to chemotherapy or radiation. However, this technique still presents practical and ethical dilemmas.8 Sperm Preparation Techniques Before Cryopreservation In contrast to caffein and kallikrein which were used in the past to stimulate sperm motility and the fertilizing capacity of cryostored human spermatozoa,9 the phosphodiesterase inhibitor pentoxifylline has received considerable attention.10–13 Another possibility to increase the percentage of living spermatozoa after cryopresefvation was thought to be a separation of motile spermatozoa before freezing.14– 16

Therefore, a study was performed to compare the effect of different preparation techniques on sperm viability after cryopreservation.17 In conclusion, pre-freezing selection of motile spermatozoa by preparation techniques such as glass wool filtration, sedimentation migration or underlay does not improve the recovery rate after thawing. Treatment with pentoxifylline or concentration of spermatozoa by centrifugation result in slightly higher numbers of motile spermatozoa/µl after thawing. However, more samples do not show any motility. Freezing of low quality ejaculates without any preparation techniques seems to be the most effective procedure of cryopreservation. In cases of severe oligozoospermia or non-obstructive azoospermia only few ejaculated or testicular spermatozoa are available. Since it may be difficult to recover these spermatozoa after freezing and thawing, zonae pellucidae can be used as vehicles for their cryo preservation.18

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Effects of Freezing on Sperm Quality Cryopreservation of human semen has been associated with decreased acrosin activity,19– a post-freeze reduction of motility,22,23 decrease of native DNA content24 and impaired ability of spermatozoa to penetrate cervical mucus. On the other hand, fresh and frozenthawed human spermatozoa bind in a similar pattern to the zona pellucida in the hemizona assay.25 Nevertheless, swelling of the plasma membrane, acrosomal leakage and breakdown as well as lesions in the midpiece and the flagellum may lead to a substantial reduction of sperm function. Remarkably, programmed stage freezing using the cryopreservative glycerol is far superior to DMSO, yielding the best chances to penetrate zona-free hamster ova.26 There is no marker available to predict the freezability of individual semen samples.27 Studies using the hypoosmotic swelling test failed to show any correlation with the postthaw motility or the survival rate of spermatozoa after cryopreservation.28 However, the hypoosmotic swelling test has been found suitable to assess the viability of cryopreserved spermatozoa, which is of great importance for the identification of immotile, however, still viable spermatozoa for successful intracytoplasmic sperm injection.29 Clinical experience has demonstrated that spermatozoa of some individuals nearly have no loss of motility and vitality, while others show such a tremendous cryoinjury that the application of cryopreservation in these cases is more than questionable before the use of intracytoplasmic injection. In general, however, the survival rate one hour after thawing of frozen semen is between 50 and 60 percent; four hours after thawing the survival rate decreases further to 35–40 percent, whereas the motility loss of fresh semen within the same observation period is only 10–15 percent. This indicates that the vitality of cryopreserved spermatozoa compared to native semen is significantly reduced, corresponding to the phenomenon of latent cryoinjury described by Sherman (1967).30 To minimize cryoinjury in human spermatozoa, sufficient concentrations of cryoprotectants to control the rise in salt concentration and to increase the unfrozen volume during cooling are necessary.31,32 After initial studies with dimethyl sulfoxide (DMSO) as the cryoprotectant medium, glycerol at 5–7 percent per volume is now almost universally used.3,33 Often glycerol-egg-yolkcitrate medium is also applied or “Testcy” -a semen extender which contains no glycerol but uses a zwitter ionbuffer, citrate and egg yolk. To store semen, containers for deep freezing are screw-cap plastic ampoules and plastic straws (French: paillettes) containing 0.25 ml semen mixture. 21

Fertilization Rates with Cryopreserved Spermatozoa Using cryopreserved donor spermatozoa within in vitro fertilization programs, Trounson and Conti (1982)34 and Mahadevan and coworkers (1983)35 already demonstrated that these spermatozoa yielded fertilization rates comparable to those achieved with fresh semen. In general, the success rate of cryopreserved semen is about 10 to 25 percent poorer than that achieved with fresh semen in donor programs. The most predictive variable for pregnancy is the post-thaw motility.36 Using intracytoplasmic sperm injection, only a few vital spermatozoa are needed after freezing and thawing for successful fertilization of oocytes. Thus, in contrast to the past, no minimal criteria for cryostorage of semen samples exist nowadays.

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The efficacy of cryopreservation of ejaculated, testicular and epididymal spermatozoa for ICSI was reported by Rubio et al (1996).37 The authors showed that sperm cryopreservation did not affect ICSI results in terms of fertilization and pregnancy rates. There was no statistical difference between fresh and frozen spermatozoa from ejaculate, epididymis and testis, with fertilization rates between 44.1 and 68.7 percent and pregnancy rates between 14.3 and 30.8 percent. Cryobanking of Semen for Fertility Protection from Cytotoxic Treatment Testicular cancer is the most frequent cancer in men between 25 and 34 years. Other malignant diseases with high prevalence in younger age are Hodgkin’s disease and leukemia. Most of these patients have not fathered children at the time of diagnosis.7 Even though better surgical techniques and chemotherapeutic agents and regimens in the treatment of these diseases are available to maintain male fertility, it is currently not yet possible to predict accurately which of these men will regain spermatogenic function. Chemotherapeutic agents cause azoospermia in 90 to 100 percent of treated adult men. In 1/3, spermatogenesis will eventually be recovered. After completion of treatment for testicular cancer, recovery of spermatogenic function may take 2–3 years, sometimes more than 5 years. This is the reason why cryopreservation of semen from patients with malignant diseases before specific chemotherapy, radiation or surgical therapy is a realistic option to preserve fertility.38 In addition, for many young patients the availability of a sperm cryobank and the possibility to store semenbefore chemotherapy is a great psychological relief. Interestingly, only few patients will later call for their spermatozoa to be used either for intrauterine insemination or in vitro fertilization.39,40 For example, of 112 patients whose semen had been stored at the Munich spermbank during 1974 and 1986, only 15 called for their spermatozoa to be used either for IUI or IVF.17 Hallak et al (1998)41 reported about the reasons of cancer patients to stop storing their semen in a sperm bank program. Only a minority of patients did not plan to have children. Most of the reasons included death of patients or restored fertility. A major problem of cancer patients is that they may have a disease-intrinsic suppression of spermatogenesis (e.g., 50% of testicular cancer patients). There are no criteria available to predict the quality of cryopreserved spermatozoa from cancer patients.42,43,44 However, deterioration in sperm function after cryopreservation of semen among patients with different malignancies and normal donors appears to be similar, indicating that the type of cancer is not related to the cryopreservation results. Through the introduction of intracytoplasmic sperm injection, cryobanking of semen from cancer patients has to be completely reconsidered. In these cases, fertility should be protected and semen samples of any quality and even from testicular tissue have to be frozen and kept in liquid nitrogen (−196°C) to be later available for assisted reproductive techniques.

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Use of Cryopreserved Epididymal and Testicular Spermatozoa for Intracytoplasmic Sperm Injection Sometimes the ovarian stimulation protocol may provide plenty of metaphase-2 oocytes but no spermatozoa that can be harvested for microinjection. Therefore, the approach using either cryopreserved ejaculated, epididymal or testicular spermatozoa seems to overcome at least some of the logistic problems.45–47 As early as 1992, a pregnancy was reported after subzonal insertion of cryopreserved spermatozoa from a patient with testicular seminoma and severe oligoasthenozoospermia.48 Through the introduction of ICSI, the chance to conceive has been considerably enlarged using cryopreserved ejaculated or epididymal spermatozoa.49,50 Fertilization rates, embryo cleavage, implantation, clinical pregnancy per embryo transfer and delivery or ongoing pregnancy rate after ICSI do not differ significantly between fresh or cryopreserved epididymal spermatozoa.51 Further progress was achieved by the application of cryobanking for testicular tissue specimens.52,53 Freezing and thawing of testicular tissue has the advantage that a single testicular specimen can be used for histological examination, another for wet preparation to get immediate information about the occurrence of spermatozoa. The main bulk of material will be cryopreserved. Thanks to cryopreservation, microinjection procedures in the female can be planned independently of the male partner. This approch also allows transfer of cryopreserved material to another center where ICSI can be performed. A further advantage is that the tissue sample must not be shreddered in order to extract spermatozoa; instead, testicular biopsy specimens are dissolved in a collagenase solution after freezing and thawing.54 Fischer and coworkers (1996)55 reported the successful application of this new method of sperm extraction from a frozen-thawed testicular biopsy specimen within an established program of intracytoplasmic sperm injection. Recently, no significant differences of ICSI-outcome could be observed using fresh or frozen-thawed testicular spermatozoa.56 However, lower implantation rates with frozenthawed testicular spermatozoa have been demonstrated by other groups.57 The most evident advantage of cryopreservation techniques is that active spermatogenesis can be identified before the female partner undergoes any stimulation protocol. In addition, no further operation is required to collect testicular spermatozoa when subsequent cycles are needed until a pregnancy is achieved. In addition, if no spermatozoa can be identified in the testicular tissue by reliable andrological pre-treatment diagnosis (histology, test preparation for TESE), this saves the female partner a frustrating IVF cycle. REFERENCES 1. Sherman JK. Current status of clinical cryobanking of human semen. In Paulson JD, Negro-Vilar A, Lucena E, Martini L (Eds): Andrology, Male Fertility and Sterility. Orlando: Academic Press, 1986; 517. 2. Schill WB, Bollmann W. Semen preservation, insemination, in vitro fertilization (German). Munich, Vienna, Baltimore: Urban and Schwarzenberg, 1986. 3. Brotherton J. Cryopreservation of human semen. Arch Androl 1990; 25:181. 4. Quinn R Cryopreservation. In Marrs RP (Ed): Assisted Reproductive Technologies. Oxford, London, Edinburgh, Melbourne, Paris, Berlin, Vienna: Blackwell Scientific Publications, 1993; 89.

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5. Van der Elst J, Verheyen G, Van SteirteghemA. Cryopreservation: sperms and oocytes. In: Rabe T, Diedrich K, Runnebaum B (Eds): Manual on Assisted Reproduction. Berlin: Springer-Verlag 1997; 223. 6. Stewart GJ, Cunningham AL, Driscoll GL et al. Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet 1985; 2:581. 7. Kohn FM, Schill W-B. Kryospermabank MiinchenZwischenbilanz 1974–1986 Hautarzt 1988; 39:91. 8. Bahadur G, Chatterjee R, Ralph D. Testicular tissue cryopreservation in boys. Ethical and legal issues. Hum Reprod 2000; 15:1416. 9. Schill WB, Pritsch W, Preissler G. Effect of caffeine and kallikrein on cryo-preserved human spermatozoa. Int J Fertil 1979; 24:27. 10. Mbizvo MT, Johnston RC, Baker GHW. The effect of the motility stimulants, caffeine, pentoxifylline, and 2-deoxyadenosine on hyperactivation of cryopreserved human sperm. Fertil Steril 1993; 59:1112. 11. Bell M, Wang R, Hellstrom WJG, Sikka SC. Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J Androl 1993; 14:472. 12. Brennan AP, Holden CA. Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation. Hum Reprod 1995; 10:2308. 13. Sharma RK, Tolentino MV, ThomasAJ, AgarwalA. Optimal dose and duration of exposure to artificial stimulants in cryopreserved human spermatozoa. J Urol 1996; 155:568. 14. Kaneko S, Kobayashi T, Lee HK et al. Cryogenic preservation of low-quality human semen. Arch Androl 1990; 24:81. 15. Bongso A, Jarina AK, Ho J, Ng SC, Ratnam SS. Comparative evaluation of three spermwashing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples. Arch Androl 1993; 31:223. 16. Perez-Sanchez E, Cooper TG, Yeung CH, Nieschlag E. Improvement in quality of cryopreserved human spermatozuoa by swimup before freezing. Int J Androl 1994; 17:115. 17. Kohn FM, Volk R, Schill WB. Cryopreservation of semen samples from severely oligozoospermic men. Hum Reprod 1997; 12(Abstract Book 1):237. 18. Hsieh YY, Tsai HD, Chang CC, Lo HY. Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae. Fertil Steril 2000; 73:694. 19. Schill WB: Acrosin activity of cryopreserved human spermatozoa. Fertil Steril 1975; 26:711. 20. Mack SR, Zaneveld JD. Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res 1987; 18:375. 21. Cross NL, Hanks SE. Effects of cryopreservation on human sperm acrosomes. Hum Reprod 1991; 6:1279. 22. Schill WB, Topfer-Petersen E, Hoffmann R, Michalopoulos M, Rübekeil A. Untersuchungen zur Schadigung von Kryosperma. In Schill WB, Bollmann W (Eds): Spermakonservierung, Insemination, In-vitro-Fertilisation. Miinchen, Wien, Baltimore: Urban und Schwarzenberg, 1986; 35. 23. McLaughlin EA, Ford WCL, Hull MGR. Motility characteristics and membrane integrity of cryopreserved human spermatozoa. J Reprod Fertil 1992; 55:527. 24. Royere D, Hamamah S, Nicolle JC, Barthelemy C, Lansac J. Freezing and thawing alter chromatin stability of ejaculated human spermatozoa, fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies. Gamete Res 1988; 21:51. 25. Gamzu R, Yogev L, Yavetz H, Homonnai ZT, HissY, Paz G. Fresh and frozen-thawed human sperm bind in a similar pattern to the zona pellucida in the hemizona assay. Fertil Steril 1992; 58:1254. 26. Serafini PC, Hauser D, Moyer D, Marrs RP. Cryopreservation of human spermatozoa: Correlations of ultrastructural sperm head configuration with sperm motility and ability to penetrate zonafree hamster ova. Fertil Steril 1986; 46:691.

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27. Centola GM, Raubertas RF, Mattox JH. Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival. J Androl 1992; 13:283. 28. Chan SYW, Craft IL, Chan YM, Leong MKH, Leung CKM. The hypoosmotic swelling test and cryosurvival of human spermatozoa. Hum Reprod 1990; 5:715. 29. Esteves SC, Sharma RK, Thomas AJ, Agarwal A. Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm. Fertil Steril 1996; 66:798. 30. Sherman JK. Freeze-thaw-induced latent injury as a phenomenon in cryobiology. Cryobiology 1967; 3:407. 31. Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 1990; 11:73. 32. Gilmore JA, Liu J, Gao DY, Critser JK. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum Reprod 1997; 12:112. 33. Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil Steril 1988; 50:314. 34. Trounson AO, Conti A. Research in human in vitro fertilization and embryo transfer. Br Med J Clin Res Ed 1982; 285:244. 35. Mahadevan MM, Trounson AO, Leeton JF. Successful use of human semen cryobanking for in vitro fertilization. Fertil Steril 1983; 40:340. 36. Mayaux MJ, Schwartz E, Czyalik F, David G. Conception rate according to semen characteristics in a series of 15,364 insemination cycles: results of a multivariate analysis. Andrologia 1985; 17:9. 37. Rubio C, Minguez Y, RuisA, Amorocho B, Romero J, De los Santos MJ. Efficacy of sperm cryopreservation of ejaculated, testicular and epididymal spermatozoa for ICSI. Hum Reprod 1996; 11 (Abstract Book 1):89. 38. Khalifa E, Oehninger S, Acosta AA et al. Successful fertilization and pregnancy outcome in invitro fertilization uring cryopreserved/thawed spermatozoa from patients with malignant diseases. Hum Reprod 1992; 7:105. 39. Holland-Moritz H, Krause W. Use of cryopreservation by tumor patients (German). Hautarzt 1990; 41:204. 40. Keck C, Nieschlag E. Cryopreservation of spermatozoa and its importance in the management of malignant diseases (German). Fertilität 1993; 9:145. 41. Hallak J, Sharma RK, Thomas AJ, Agarwal A. Why cancer patients request disposal of cryopreserved semen specimens posttherapy: a retrospective study. Fertil Steril 1998; 69:889. 42. Krause W, Brake A. Utilization of cryopreserved semen in tumor patients. Urol Int 1994; 52:65. 43. Agarwal A, Shekarriz M, Sidhu RK, Thomas AJ. Value of clinical diagnosis in predicting the quality of cryopreserved sperm from cancer patients. J Urol 1996; 155:934. 44. Padron OF, Sharma RK, Thomas AJ, Jr, Agarwal A. Effects of cancer on spermatozoa quality after cryopreservation: a 12-year experience. Fertil Steril 1997; 67:326. 45. Devroey P, Silber S, Nagy Z et al. Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen-thawed epididymal spermatozoa. Hum Reprod 1995; 10:903. 46. Nagy Z, Liu J, Cecile J, Silber S, Devroey P, Van Steirteghem A. Using ejaculated, fresh, and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil Steril 1995; 63:808. 47. Romero J, Remohri J, Minguez Y, Rubio C, Pellicer A, Gil-Salom M. Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Fertil Steril 1996; 65:877. 48. Levron J, Lightman A, Stein DW, Brandes JM, Itskovitz-Eldor J. Pregnancy after subzonal insertion of cryopreserved spermatozoa from a patient with testicular seminoma. Fertil Steril 1992; 58:839.

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49. Oates RD, Lobel SM, Harris DH, Pang S, Burgess CM, Carson RS. Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa. Hum Reprod 1996; 11:133. 50. Holden CA, Fuscaldo GF, Jackson P et al. Frozen-thawed epididymal spermatozoa for intracytoplasmic sperm injection. Fertil Steril 1997; 67:81. 51. Friedler S, Raziel A, Soffer Y, Strassburger D, Komarovsky D, Ron-El R. The outcome of intracytoplasmic injection of fresh and cryopreserved epididymal spermatozoa from patients with obstructive azoospermia-a comparative study. Hum Reprod 1998; 13:1872. 52. Hovatta O, Fondila T, Siegberg R, Johanson K, von Smitten K, Reima I. Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen. Hum Reprod 1996; 11:2472. 53. Khalifeh FA, Sarraf M, Dabit ST. Full-term delivery following intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissue. Hum Reprod 1997; 12:87. 54. SalzbrunnA, Benson DM, HolsteinAF, Schulze W. Anew concept for the extraction of testicular spermatozoa as a tool for assisted fertilization (ICSI). Hum Reprod 1996; 11:752. 55. Fischer R, Baukloh V, Naether OGJ, Schulze W, Salzbrunn A, Benson DM. Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy. Hum Reprod 1996; 11:2197. 56. Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril 2000; 73:955. 57. De Croo I, Van der Elst J, Everaert K, De Sutter P, Dhont M. Fertilization, pregnancy and embryo implantation rates after ICSI with fresh or frozen-thawed testicular spermatozoa. Hum Reprod 1998; 13:1893.

SECTION 10 Endoscopy and ART

CHAPTER 64 Fertility Following Laparoscopic Surgery and Hysteroscopic Surgery B Ramesh, Nirmala Sadasivam INTRODUCTION Both laparoscopic and hysteroscopic surgeries play a vital role in enhancing the fertility of female partner. In fact infertility is the commonest indication for laparoscopic and hysteroscopic surgery and all the gynaec endoscopists are witness to this fact. Now it is established fact that laparoscopy is the gold standard in the surgical management of endometriosis, pelvic adhesions, ovarian cysts, PCOD and many other conditions. Hysteroscopic surgery is an indispensable tool for the management of intracavity problems and there is no alternative for hysteroscopy in certain conditions like uterine septum, submucus fibroid, Asherman’s, etc. ADVANTAGES OF ENDOSCOPIC SURGERY IN AN INFERTILE PATIENT Endoscopic approach is particularly attractive for a number of reasons. Endoscopic surgery embraces the principle of microsurgery like adequate exposure, magnification, minimal tissue handling, good hemostasis and steps to prevent adhesions, all adding to enhance the fertility. Most infertile women are working and prefer return to work after surgery, and they need to have sexual intercourse soon after surgery exposure for early conception. Endoscopic approach assists this. In addition, laparoscopic approach is associated with less risk of infection and less scarring. Laparoscopic Surgical Treatment of Infertility Related to PCOD Gjonnaess(19841) proposed the use of laproscopic multi-electrocauteriztion in PCOs. He reported the results concerning 252 women with P Cos treated with ovarian electrocauterization during the

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Fig. 64.1 years 1979–91,2 ovulation was obtained in 92 percent of the total series and pregnancy in 84 percent. The response was inf luenced by body weight, with an ovulation range of 96 to 97 percent for the slim and moderately obese, decreasing to 70 percent in the very obese ones. Laparoscopic electrocautery reduces the rate of spontaneous abortion and it also increases the ovarian sensitivity to clomiphene citrate and gonado trophins. Our Experience We do three puncture technique. The ovary is grasped at the ovarian ligament and all the surfaces of the ovary exposed and the number of punctures depend upon the ovarian volume. Generally, we do 10–15 punctures per ovary. One should use a fine high frequency needle to minimize the ovarian tissue damage and current setting on the diathermy should be as minimal as possible. Generally, we keep at 30 40 watts. First we place the needle just above the ovarian surface. First activate the electrode and then pierce the ovarian tissue, instead if we first touch the ovarian tissue and then activate the electrode, there will be more ovarian tissue damage. One should avoid piercing the meso ovarian vessels and hilum of the ovary. We do not prefer to do mechanical drilling with a needle. We always prefer electrocautery. At the end of drilling, we give a repeated thorough wash, to wash all the follicular fluid which has come out, to clean blood and clots and also to remove the carbon debris from the ovarian surface. Washing also has a cooling effect on the ovary and at the end we leave 200 to 300 ml of ringer lactate or saline in the pelvis immersing the ovaries.

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Patients with ovarian hyperthecosis with severe stromal hyperplasia without much follicular cysts do not respond favorably to medical induction of ovulation, these patients are particularly benefitted by ovarian drilling. Following drilling, we have observed pregnency rates varying from 50 to 80 percent, we have also observed that the abortion rate following drilling is much lesser than medical induction. Complications of Drilling Intraoperative bleeding is possible when the needle pierces the nearby vessels. Post operative periovarian adhesions result when there is blood oozing from the ovarian surface and these adhesions are filmy in nature and usually do not interfere with fertility. Gonadal atrophy, premature ovarian failure is a possibility, incidence not known. Following drilling, most of the patients respond favourably within one to one and half year, with or without ovulation induction. If they do not respond within reasonable period of time, these patients are resistant PCOs which are very difficult to treat. In these cases one can consider repeat drilling or ART. We have done repeat drilling in few patients and have found a favorable response in some of these patients. Laser Drilling All types of laser have been used for drilling, CO2 laser. Argon, Yag, Ktp. The fertility outcome following laser drilling is same as Electrocautery and we do not see any additional benefit with laser. CONCLUSIONS From our experience and other reports of clinical experience, the advantages of laparoscopic surgical method for PCOs are: • multiple ovulatory cycles from single treatment • high pregnancy rate • lower rate of spontaneous abortion • its usefulness for the diagnosis of unexplained infertility • elimination of intensive monitoring and high cost treatment with gonadotrophins • elimination of risk of ovarian hyperstimulation syndrome and multiple gestation.

Table 64.1: Spontaneous conceptions after laparoscopic ovarian drilling (stimulated cycles excluded) Year Total laparoscopies No of PCOD Conception rate (%) 1996 1997 1998 1999

120 135 216 242

40 43 72 75

12(33.3) 13(30.2) 20(27.7) 18(24.0)

Fertility following laparoscopic surgery and hysteroscopic surgery 2000 2001 2002

256 240 121

78 74 40

697

23(29.4) 20(27.0) 12(33.0)

LAPAROSCOPIC SURGERY FOR ENDOMETRIOSIS IN AN INFERTILE PATIENT The goals of conservative laparoscopic surgery are to remove all endometriotic implants, resect adhesions, relieve pain, reduce the risk of disease recurrence and post operative adhesion formation and restore involved organs to a normal anatomic and physiologic condition. For an infertile patient, restoration of normal tubo-ovarian relationship is essential to enhance fertility. Laparoscopy Versus Laparotomy Now it is very well established that laparoscopic surgery is the gold standard in the surgical management of endo metriosis in an infertile patient. Laparoscopic Surgery is more effective for adhesiolysis, causes fewer de novo adhesions3 and reduces impairement of tuboovarian function4 than laparotomy. It has been reported that pain relief and pregnancy rates after operative laparoscopy are comparable or better than those following laparotomy for endometriosis.5 Fayez and Collazo noted better pregnancy rates following laparoscopy (36%).6 From the above observation the pregnancy rate following laparoscopic surgery for endometriosis is 50–55 percent depending upon the severity of the disease. Conservative Laparoscopic Surgery Conservative surgery is indicated for women who desire pregnancy and whose disease is responsible for their symptoms of infertility or pain. Although seldom curative, surgery improves the likelihood of pregnancy and offers atleast temporary pain relief. Approximately 25 percent of patients undergoing conservative operations will require a subsequent operation because of recurrence

Table 64.2: Number of patients (N) and pregency rates (%) following laparoscopic surgery for endometriosis.7 Minimal Mild Moderate and severe Feste Martin Nezhat etal Olive etal Chong etal Fayez etal

N % N % 47 51.1 6 66.7 27 25.9 19 15.8 24 75.0 51 62.8 59 39 48 45.8 0 0 0 0 0 0 0 0

N 5 4 27 20 11 44

% 40 25.0 44.4 50.0 54.5 50.0

The art and science of assisted reproductive techniques (ART) Luciano etal 0 0 36 61 Adamson etal 0 0 0 0 Nezhat etal 39 71.8 86 69.8 Total 196 51 210 57.6

60 48 118 337

698

60.0 37.5 67.8 55.5

of endometriosis or progression of residual disease. The rate of repeat surgical intervention is related directly to the extent of the disease and the ability to conceive postoperatively. Of those who achieve pregnancy after the initial operation, only 10 percent require another operation8. After putting the laparoscope, the surgeon must first explore the pelvic cavity and assess the extent of disease. The operative procedure begins by lysing adhesions between the bowel and pelvic organs to adequately expose the pelvic cavity. Lysis of Bowel Adhesions Bowel adhesions vary in thickness, vascularity and cohesiveness. Some adhesions are stretched, excised with electrocautery, Hydrodissection, sharp and blunt dissections are used in combination for effective adhesiolysis. Peritoneal Implants The implants should be destroyed in the most effective and least traumatic manner. Superficial peritoneal endometriosis is vaporized with laser, coagulated with bipolar or monopolar current or excised. For lesions greater than 5 mm deep vaporization or excisional techniques are used.

Fig. 64.2 Oυarian Endometriosis

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The ovaries are a common site for endometriosis, Endometrial implants or endometriomas less than 2 cm in diameter are bipolar coagulated, laser ablated or excised. All visible endometriotic lesions and scars must be removed from the ovarian surface. Endometriomas more than 2 cm diameter must be resected thoroughly to prevent recurrence. Draining the endometrioma or partial resection of its wall is inadequate because the endometrial tissue lining the cyst is likely to remain functional and can cause symptoms to recur. However photocoagulation of the cyst wall has been equally therapeutic. Brosens and Puttemansi recommend performing ovarian cystoscopy and biopsy of the cyst wall before ablating the cyst. The cyst wall is ablated to a depth of 3 to 4 mm, using a laser or an electro coagulator. This procedure is analogous to endometrial ablation and has been reported to be successful with no recurrence. For bigger endometriomas, the cyst is punctured by piercing with a 5 mm trocar and aspirated with suction irrigation probe, using high pressure irrigation at 500 to 800 mm Hg, the cyst is irrigated, causing it to expand and aspirated several times. Now using two teeth graspers the plane is developed between the cyst wall and the ovarian cortex at the puncture site, using traction and counter traction the complete enucleation of the cyst wall is accomplished. Another method involves hydro dissection of the plane between the cyst wall and the ovarian stroma. If the entire cyst can not be seperated from the ovary, the adherent sections are ablated or coagulated. Although rare, some patients present with localized symptoms and severe involvement of one ovary with disease while the opposite ovary is normal, requiring unilateral salpingo opherectomy. By removing the diseased ovary the risk of disease recurrence is minimized and the fertility potential is improved by limiting ovulation to the healthy side.

Fig. 64.3 Restoration of Tubo-Ovarian Anatomy Once all lesions are resected or ablated and adnexa freed of adhesions, the anatomic relationship between the ovary and ipsilateral tube is evaluated and any distortion caused by adhesions is corrected. The mesosalpinx often adheres to the ovarian cortex along the ampullary segment of the tube. These adhesions cover a significant surface area of the

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ovarian cortex and may interface with the ovulatory process. Restoration of tubo-ovarian anatomy is one of the most important goal of the surgery in patients with endometriosis presenting with infertility. Adhesiolysis along the ovarian surface and mesosalpinx can be accomplished with sharp scissors, fine monopolar electrode or CO2 laser. The fimbriae are frequently agglutinated which can be released with microscissors after floating the fimbriae underwater.

Table 64.3: Incidence of endometriosis in our study Year Total laparoscopies for infertility No. of Endometriosis 1996 1997 1998 1999 2000 2001 2002

120 135 216 242 256 240 121

35(29.1%) 42(31.1%) 60(27.7%) 64(26.4%) 67(26.1%) 70(29.1%) 30(24.7%)

Fertility following Laparoscopic Surgery for Endometriosis Operative treatment of more extensive disease does offer a greater likelihood of conception than does expectant management, because of correction of mechanical factors such as adhesions. Pregnancy is most likely to occur during the first 36 months after surgery. The duration of infertility and patient age and actual stage of the disease will have great impact on the cumulative pregnancy rate. The cumulative pregnancy rates after laparoscopic surgery for endometriosis is approximately—52 percent. Laparoscopic Reconstructive Tubal Surgery The prefered route of access for adhesiolysis, fimbrio plasty and salpingostomy for many gynaecologists is laparoscopy On occasion one or more of these procedures requires laparotomy Overshadowed by the success of ART, tubal surgery in properly selected women remains a valuable technology. The contra indications for these tubal surgeries include pelvic tuberculosis, absence of ampullary segments of both the tubes, ovarian failure active PID and severe disease in which correction is unlikely. Laparoscopic Survey A thorough laparoscopic survey will identify any adhesions along with their extent and nature, reveal other abdominal and pelvic lesions and permit assessment of the uterus, tubes and ovaries. The combined information yielded by HSG, ultrasound studies and laparoscopic survey enables the surgeon to undertake reconstructive laparoscopic surgery to recommend surgery by laparotomy or to recommend the use of assisted reproductive technique.

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Laparoscopic surgery has the inherent advantage of microsurgical principles. The laparoscope provides magnification, excellent visibility and illumination, operating within a closed peritoneal cavity prevents

Table 64.4: Pregnancy rates after laparoscopic electrocoagulation of endometiosis10 Investigator

Number ofpregnancies/Number treated (%) Minimal Mild Moderate Severe Combined Length of followup (M)

Edward Hasson Sulewski Daniell & Pittaway Reich & Mcglynn Seiler etal Nowroozi et al Murphy et al Total

4/7(57) 0/1(0)

10/18(56) 20/42(48)

14/25(56) 13 2/2(100) 4/5(80) 6/8(75) 7 20/58(35) 40/100(40) 37 33/60(55)

15/23(65) 18 20/45(44) 20/45(44) 7 42/69(61) 42/69(61) 8 24/36(67) 18/36(50) 2/7(29) 0/3(0) 44/82 8 28/44(64) 110/210(52) 24/67(36) 4/8(50) 214/412(52)

desiccation of the peritoneal surfaces, eliminates the need to use packs and prevents the introduction of foreign materials. It is possible to perform intraoperative irrigation to keep tissues moistened and the pressure effect of pneumoperitoneum diminishes the venous oozing. Salpingoovariolysis Pelvic and periadnexal adhesions usually are the sequelae of PID. They are usually not too vascular and extend from one structure to another. In doing so they tend to leave a space or potential space between the involved structures, an aspect that facilitates adhesiolysis. Dense cohesive adhesions often result from prior surgery. The lysis of such post-operative adhesions is technically difficult and is associated with high percentage of recurrence. Periadnexal adhesions usually co exist with other types of tubal disease. Thus salpingoovariolysis is often an integral part of other reconstructive procedures. Even in the presence of patent tube adhesions may prevent the ovum pickup. The performance of effective and safe salpingoovariolysis requires clear identification of each adhesive layer. The adhesions are incised parallel to the tube and ovary and about 1mm away to prevent damaging its mesothelial envelop. Division is done electrosurgically with a microelectrode or sharp microscissors. Shallow adhesions are simply divided whereas broad adhesions are excised. At the end of the procedure thorough pelvic lavage is done with ringer lactate. Results of salpingoovariolysis The pregnancy rates following Laparoscopic salpingoovariolysis are smililar to those obtained by microsurgery. The reported

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intrauterine pregnancy rates ranges from 51 to 62 percent and the ectopic pregnancy rates range from 5 to 8 percent of the operated cases.12 Fimbrioplasty Fimbrioplasty is the reconstruction of the fimbriae or infundibulum in a tube that exhibits fimbrial agglutination or prefimbrial phimosis and results in partial distal occlusion. Often the tube and ovary are involved in adhesions in which case a salpingoovariolysis must precede the fimbrioplasty. Fimbrial phimosis results from agglutination of the fimbriae. A small opening is usually present at the distal end of the tube unless this opening is covered by fibrous tissue. The latter usually becomes evident when the tube is distended by chromopertubation. When the opening is covered by fibrous tissue, this tissue must be incised or excised to gain access to fimbriae. Agglutination of fimbriae can be corrected simply by introducing the closed

Fig. 64.4 jaws of fine atraumatic grasper through phimotic fimbrial opening. The jaws of the grasper are opened within the tubal lumen and grasper gently withdrawn with the jaws open. Deagglutination is achived by repeating this movement few times, varying the direction in which jaws of the grasper are opened. When the stenosis is located at the level of true tubal ostium which is located at the apex of the infundibulum the fimbriae may have a normal appearance. In this instance, it is necessary to place an incision on the antimesosalpingeal border of the tube, which commences at the infundibulum and continues past the stenotic area to the distal ampulla. The edges of the two flaps thus created are folded back either by securing them to

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adjacent ampullarly serosa with no 7–0 to 6–0 vicryl sutures or by dessicating, the serosal aspect of the flaps causing them to fold backwards. Results of fimbrioplasty The intractive pregnancy rate following fimbrioplasty varies from 30 to 63 percent depending upon the severity of tubal disease and the procedure done. The incidence of tubal ectopic is 5 to 8 percent. Laparoscopic Salpingostomy Salpingostomy is the creation of a new stoma in a tube with a completely occluded distal end. Distal tubal occlusion is usually associated with varying degrees of pelvic and periadnexal adhesions that must first be lyed and tube freed. The tube is distended by chromopertubation. The occluded terminal end of the tube is examined under magnification which permits recognition of the relatively avascular zones that radiate from central punctum. The tube is entered at this cental point with the use of microelectrode or microsurgical scissors and incision is extended towards the ovary over an avascular line. This incision fashions a new fimbria ovarica that maintains the tuboovarian relation. At this point in the procedure it becomes possible to view the tube from within when placing additional incisions along its circumference to complete the creation of new stoma. These additional incisions are made between endothelial folds over avascular areas. Once a satisfactory stoma is achieved, the flaps created in the process are everted either by securing them without tension to the ampullary seromuscularis with interrupted no.8–0 vicryl sutures or by desiccating their serosal surface which causes them to fold backward. Fertility following Salpingostomy The major determinants of the outcome of salpingostomy are the degree of pre existing tubal damage and the extent and nature of periadnexal adhesions. In general, salpingostomy yields lower success rates than adhesiolysis and fimbrioplasty The pregnancy rate following salpingostomy varies between 18 to 30 percent and ectopic pregnancy rate ranges from 5 to 18 percent.14 Laparoscopic Myometomy Fibroids can cause infertility, abortions, premature delivery, still birth and considerable complications during labour and postpartm period. Anumber of factors may be responsible for infertility in a patient with uterine leiomyomata. There may be interference with sperm transport caused by distortion in the uterine cavity, impingement of leiomyoma on the endocervical canal or fallopian tube. Endometrial changes, vascular alterations and enlargement of the uterine cavity may be present. The decision regarding surgical removal depends on their size and location. Usually subserous fibroids are not considered a factor in abortion and infertility. When leiomyoma are intramural or submucus and of significant size, they may well be factors causing infertility or abortion and myomectomy is justified in such cases.

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Technique of Laparoscopic Myomectomy Pedunculated leiomyomas are easiest to remove by coagulating and cutting the stalk. For removal of intramural fibroids, an incision is made over the fibroid using monopolar electrode. The incision is extended until it reaches the capsule. The myometrium retracts as the incision is made exposing the tumour. A myoma screw is inserted in to the fibroid to apply traction, while suction irrigator and grasping forceps are used for blunt dissection. By traction and counter traction myoma is enucleated, haemostasis is achieved by bipolar coagulation. If the defect is superficial, single layer closureis sufficient and if the defect is large and deep, the defect is to be closed in two to three layers. In such situation, if the laparoscopic suturing is difficult, the surgeon can do minilap, retrieve the myoma through minilap close the defect in two to three layers. The removal of fibroids from the peritoneal cavity can be done by various types of marcellators or through colpotomy. Fertility following Laparoscopic Myomectomy The impact of laparoscopic myomectomy on infertility is difficult to assess. Other factors besides leiomyoma may be present to a varying degree. The extent to which uterine cavity or fallopian tubes are distorted also varies. The conception rate f ollowing laparoscopic myomectomy or conventional myomectomy are almost similar. In patients with leiomyoma associated inf ertility without apparent cause, the conception rate is approximately 59 percent following myomectomy.16 Among patients who had additional treated infertility factors, 50 percent conceive after myomectomy. Most of the pregnancies occur within 2 years of operation. The conception rate among women older than age 35 may not be as good. The post myomectomy conception rate may be lower when the uterus is greater than 12 weeks gestational size and when more than four myome are removed. Then reduction in abortion rate after myomectomy from 41 to 19 percent suggests improvement in reproductive salvage.17 According to Verkauf review

Table 64.4: Results of laparoscopic salpingostomy15 Inυestigator

Year Patients Intrauterine pregnancies Live births Ectopics

Gomel 1977 Daniell and Herbert 1984 Dubuisson etal 1990 Canis etal 1991 McComb Paleologon 1991 Dubuisson etal 1994

9 22 34 55 22 90

4 4 10 13 5 29

4 3(13.6) 6 5(22.7%) 26(29.9%)

1 1 1 4

69.2 percent of women with previous recurent pregnancy wastage conceived after myomectomy and had a reduction in foetal loss.

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HYSTEROSCOPIC SURGERIES IN INFERTILE PATIENTS Infertility evaluation and treatment is incomplete without Hysteroscopy in many situations. Hysteroscopy has become a standard practice to confirm or exclude the uterine cavity pathology and to treat the pathology at the same time. In fact there is no alternative for hysteroscopic surgery for condition like submucus fibroids, uterine septum and intrauterine synichae. Hysteroscopic surgery is a highly specialized, minimally invasive, and highly effective in the management of uterine cavity problems. Commonly performed hysteroscopic procedures for infertility and repeated abortions. • Division of uterine septum • Polypectomy • Submucus fibroid resection • Division of synechiae • Canulation for proximal tubal occlusion Instrumentation A thorough knowledge of instruments is key to success inhysteroscopy. Diagnostic We use 4 mm 30 telescope and 4.5 mm examination sheath for diagnostic hysteroscopy. Now we have smaller telescopes of different sizes (e.g. 2 mm, 3 mm) which further makes hysteroscopy least traumatic. Operative Hysteroscopy Operative hy steroscopy can be mechanical using mechanical instruments or resectoscopic surgery using energy source like current or laser. For operative hysteroscopy, we commonly use 7 mm operating sheath with obturator which is inserted into the uterine cavity after cervical dilatation, now the telescope bridge with the operating channel and telescope are assembled, there are channels for the inflow and out flow of the distending medium making it a continuous flow operative hysteroscopy. The desired surgery is performed with 7 Fr size semirigid operating instruments like scissors, grasper etc. For canulation of the cornual blocks we pass catheter and guidewire through the operating channel. Resectoscope consists of rescetoscope sheath (Standrad size-26 fr) working element and different type of electordes(cutting loop, knife, roller ball, etc). Distension Medium For diagnostic and operartive hysteroscopy (mechanical) we use normal saline or ringer lactate as the distending medium. For resectoscopic surgery or electrosurgical procedures we use glycine 1.5 percent as the distending medium.

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Various irrigation pumps are available for maintaining the desired flow rate of the distending medium and also the desired intrauterine pressure giving optimal view of the cavity. We generally keep intrauterine pressure of 100–150 mm of Hg during operative procedures. It is very important to keep the glycine inflow and outflow chart to minimize the chances of glycine toxicity. Energy Sources Three basic energy sources are available for operative hysteroscopy, mechanical, electrical and laser. Mechanical energy sources are primarily biopsy forceps, grasper and scissors. Canulation for proximal tubal obstruction uses mechanical energy Electrical energy uses both monopolar and bipolar current for hysteroscopic surgery. Resection of Submucous Myomas and Polyps Submucous myomas can be graded by the degree to which they project in to the uterine cavity. Pedunculated myomas are type 0. Those that appear to have more than 50 percent of their volume intrauterine are type 1 and those with less than 50 percent are type 2. All types can be removed hysteroscopically. Mechanical energy can be used for polypoid lesions and small myomas. Scissors, electrical energy or laser can be used to transect the base of the polypoidal lesions and cut larger lesions into the pieces for removal. The most common and convenient method of removing myomas is with unipolar resectoscope using loop electrode. Fertility following resection of submucus fibroids definitely is favorable. Coexisting factors frequently limit fertillity. Although successful pregnancy outcome is improved it is only slightly greater than 50 percent.19 Transection of Uterine Septum All the energy sources can be used to incise the septum. The septum is incised at the beginning of the triangle and carried towards the fundus. The septum incision continues till adequate cavity space is created, both ostium are properly visualized. All subseptate uterus need not be resected. Transection of complete uterine septum resulting in a double cervical canal, is still controversial.

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Fig. 64.5

Fig. 64.6 Results of hysteroscopic metroplasty are excellent. In a review of 421 patients whose prior pregnancies were associated with poor outcome, 233 achieved term pregnancy, 19 delivered prematurely and 31 had spontaneous abortion20.

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Lysis of Uterine Adhesions Filmy intrauterine adhesions can be lysed with the tip of the hysteroscope. For other type of adhesions, one can use either scissors or resectoscope. We prefer to insert IUCD following significant adhesiolysis and prescribe oestrogens post-opertively. Adhesion extent and type are critical in determining the outcome. In a litertature review 44 percent pregnancy rate (range of 11 to 79%) were observed.21 Canulation of FallopianTube Hysteroscopic canualtion using various types of catheters and guidewires are useful in treating interstitial obstruction secondary to cellular debris, mucus plugs etc. We use turmo guidewire which is economical for tubal carnulation. Pregnancy rates range from 25 to 54 percent.

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REFERENCES 1. Gjonnaess H. Poly cystic ovarian syndrome treated by ovarian electro cautery through laparoscope. Fertil Steril 1984; 41:20–5. 2. Gjonnaess H. Ovarian electrocautery in the treatment of women with pcos. Acta obstetricia et and gynaecologica scandinavica 1984; 73:407–12. 3. Diamond mp, Daniell SF, Feste J et al. Adhesion reformation and denovo formation after reproductive pelvic surgery. Fertil steril 1987; 47:864. 4. Lendorffp, Hahlin M, Kallfelt B et al. Adhesion formation after laproscopic surgery in ectopic pregnancy. A randomized trial versus laprotomy fertil steril 1991; 55:91. 5. Adamson DG, Hard SJ, pasta DJ et al. Laparoscopic endometriosis treatment is it better. Fertil Steril 1993; 59:35. 6. Fayez JA, Collazo LM. Comparison between laparotomy and operative laparoscopy in the treatment of moderate and severe endometriosis. Int J Fertil 1990; 35:272. 7. Operative gynecologic laparoscopy principles and techniques. Nezhat 1995; 11:122. 8. Schenken SR, Malinak RC. Reoperation after initial treatment of endometriosis with conservative surgery. Arm J Obstet Gynecol 1978; 131:416. 9. Brosens I, Puttemansi P. Double Optic Laparoscopy, Ballieres Clin, Obstet Gynaecol 1989; 3:595. 10. Telinde’s Operative Gynaecology (8th edn), 27:613. 11. Telinade’s Operative Gynaecology (8th edn), 26:565. 12. Telinade’s Operative Gynaecology (8th edn), 26:565. 13. Telinade’s Operative Gynaecology (8th edn), 26:566. 14. Female Infertility Surgery. Robert B Hunt (3rd edn), 21:319. 15. Telinade’s Operative Gynaecology (8th edn), 26:569. 16. Verkauf B. Myomectomy for fertility enhancement and preservation. Fertil Steril 1992; 58:1. 17. Buttaram VC, Reiter RC. Uterine leiomyomata, etiology, symptomatoloty and managament. Fertil Steril 1981; 36:433. 18. Deblok S, Dijkman AB, Hemrika DJ. Transcen/ical resection of fibroids results related to hysteroscopic classification. Gynaecal Encose 1995; 4:243–24. 19. Goldenberg M et al. Outcome of hysteroscopic resection of submucus myomas for fertility. Fertil Stril 1995; 64:714–16. 20. Valle RF. Uterine septae In Biebes EJ, Loffer FD (Eds): Gyneacologic resectoscopy, Cambridge mass 1995 Blackwell Scientific. 21. Valle RF. Intrauterine Adhesions in Beiber EJ, Loffer FD (Eds). Gyneacologic Resectoscpy, Cambridge Mass 1995 Blackwell Scientific.

CHAPTER 65 The Role of Hysteroscopy in the Management of Infertility Carlo De Angelis, Monica Antinori INTRODUCTION Hysteroscopy is a relatively young procedure that experimented in rudimental way at the beginning of past century has been impressively implemented in the last 20 years thanks to Jacques Hamou, father of modern hysteroscopy At the beginning, this procedure was performed under narcosis. Later on, different techniques and procedures have transformed hysteroscopy in office examinations, characterised by a great precision and diagnostic accuracy, well tolerated by the patient. At present, hysteroscopy is considered as the gold standard for the study of the uterine cavity Nevertheless at present it not very clear what may be its role in the study of the infertile couple and in ART procedures, owing to the wide range of utility of the technique: • No role in the study of the morphology of the uterine cavity: according to many authors hysteroscopy is not necessary because the uterine cavity may be studied adequately through ultrasonography or hysterosalpingography, and therefore not through invasive techniques. • Other authors consider hysteroscopy a second or third degree exam and thus it can be performed only when there is a positive evidence after ultrasound and/or histerosalpingographicexaminations. • In many IVF Units hysteroscopy is performed after 2 or 3 failures of IVF attempts. • Some studies evidencing the considerable influence of endouterine pathology in the infertile woman, suggest that hysteroscopy he performed as a routine procedure in the study of the infertile couple, or at least before submitting the patient to an IVF treatment.1 We have been in search of some advise from the Official Scientific Societies, but we have found only the Guidelines of the British Royal College of Obstetricians and Gynecologists on “Diagnostic Approach and first treatment of the infertile couple” published in the last few years. These guidelines state that hysteroscopy should not be included in routine investigations of the couple up to the moment when there are evidences that the treatment of possible uterine anomalies can be correlated to the improvement of the reproductive function of the patient”.

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This statement creates some doubts both as to the contents, because has been widely demonstrated the negative effect on the reproductive function of the woman of endouterine anomalies (uterine septum, submucous fibroids, sinaechiae), as well as to the references on which the guidelines are based. They include only 7 articles published between 1978 and 1993. Thus the most recent work is 10 years old. Recently infertility related to uterine cavity abnormalities has been estimated to be the etiologic factor in as many as 10%–15% of couples seeking treatment. Abnormal uterine findings occur in approximately 34%–62% of infertile women. Because of the high prevalence of uterine abnormalities, evaluation of the uterine cavity should be routinely performed in the basic evaluation of infertile women.2 Among the procedures evaluating uterine cavity, we can consider hysterosalpingography, ultranosography sonohysterography, by introducing saline solution inside the uterine cavity associated to ultrasound by endovaginal probe. Outpatient hysteroscopy is considered as, the gold standard for diagnosing the endouterine pathology HYSTEROSALPINGOGRAPHY (HSG) Historically the hysterosalpingography has been the most widely used examination for screening the uterine factor and still today it is commonly adopted. In the last twenty years many papers have reported that when it is advisable to examine the uterine cavity for diagnosing and managing infertility, hysteroscopy reveals to be much more accurate.3–4 Kessler has reported that in two thirds of the cases the hysteroscopic finding was not related to the histerosalpingographic one: more than 50% of the intrauterine sinaechiae observed in HSG were not revealed in hysteroscopy (false positive). Wang et al4 has reported in a study comparing HSG and hysteroscopy performed in a group of infertile patients that 28, out of 79 women with normal HSG, showed abnormal findings in hysteroscopy, with 35.4% false negative. Inside the group of 135 women with pathological HSG, hysteroscopy has shown a normal uterine cavity in twenty-one of them, therefore with false positive rate of 15.6% (Fig. 65.1).

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Fig. 65.1 Golan in two different works of 1992 and 1996 reported similar results: HSG performed on 312 patients evidenced normal uterine cavity in 40 women, whereas the remaining 272 patients were affected by an endouterine pathology. Hysteroscopy confirmed such abnormalities only in 156 cases. Therefore HSG revealed a sensitivity of 97% and a specificity of 23%. The percentage of false positive was 44% and the one of false negative 10%.1 Generally speaking we can observe that in one third of the cases where HSG shows to be negative, this finding is not real and the test may supply a false reassurance. These women are therefore erroneously considered as having a normal uterine cavity when this is not true and consequently the cause of their infertility is misunderstood. On the basis of what is mentioned here, and having demonstrated the advantages of the hysteroscopic diagnostics as compared to the hysterosalpingographic one it is difficult to realize why 96% of all board-certified reproductive endocrinologists in the US still prefer the HSG as initial screening test of the uterine cavity. A major group will agree that the most beneficial advantage is that HSG supplies information on uterine cavity and tubal status. However, the low diagnostic reliability of HSG as screening test is so important that the further information on the quality of the tubes do not justify a compromising evaluation of the cavity.5 It is well know that small intrauterine lesions (synechiae, small septa or polyps) that may play a significant role in the reproductive failures are often undetectable during HSG and also during transvaginal ultrasound.6

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PELVIC ULTRASOUND When comparing pelvic ultrasound, particularly the one performed by vaginal route, and the outpatient hysteroscopy, the first procedure is advisable due to the limited invasiveness and it is well accepted by the patient, whereas, the second one is preferable because of the possibility to identify accurately the kind of intrauterine problem, thus enabling the performance of well accurate biopsies. The ideal procedures would be of course the one that enables the best opportunities of the two previous ones, and namely the great diagnostic accuracy associated to the minor invasiveness that as we will see later on, appears to be possible thanks to the use of hysteroscopic miniendoscopes. The ultrasound examinations show also in the best hands the limits of the procedure, as reported6 in a paper on 770 patients affected by menorrhagia, when ultrasound diagnostic was compared to the outpatient hysteroscopy. Ultrasound was normal in 300 patients (39%) abnormal in 417 (54.2%) and doubtful in 53 cases (6.8%); hysteroscopy was negative in 325 patients (42.2%) and abnormal in 445 (57.8%). When comparing the two procedures it is evidenced that the ultrasound had a sensitivity of 96% and a specificity of 86%, a positive predictive value of 91% and a negative predictive value of 94% in the diagnosis of intrauterine abnormalities in patients affected by menorrhagia (Fig. 65.2). This accordance decreases further and significantly in small focal pathology (small myomas or small polyps), in endometrial hyperplasia and mainly, in very small uterine septa, that are often missed by the ultrasound procedures. In the study of Vercellini the ultrasound did not diagnose small submucous myomas in 6 cases, small

Fig. 65.2

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polyps in 9 cases (we will consider later on the importance of small focal pathology on the success of ART procedures) and endometrial hyperplasia in 4 cases. This technique led to a wrong diagnosis of submucous myomas in 31 cases (intramural myomas without any distortion of the uterine cavity) and of polyps in 12 cases: in all these patients hysteroscopy had shown a normal uterine cavity with eutrophic endometrial lining. In the fifty-three doubtful cases after ultrasound, hysteroscopy had evidenced a normal uterine cavity in 18 cases and small polyps of endometrium in the remaining 35 patients. Furthermore in the diagnosis of uterine myomas, ultrasound compared to hysteroscopy had shown the need of the endoscopic intervention for only 118 out of the 148 myomas with intramural extension <50% and not operable hysteroscopically only 55 out of the 80 with intramural extensions >50%. The sensitivity, specificity Positive Predictive Value and Negative Predictive Value of the ultrasound in identifying the submucous myomas hysteroscopically operable were respectively, 80, 69, 83 and 65%. Therefore the accordance between ultrasound and hysteroscopy in the diagnosis of intramural extension of submucous myomas was very low (k index=0.48; when the maximum accordance is k=1) and this affects negatively the decision of the endoscopic surgical approach on the basis of the ultrasound alone. In other terms, the only ultrasound examinations may lead to a wrong choice of the surgical intervention to perform myomectomy. SONOHYSTEROGRAPHY (SHG) Better results may be attainable through sonohysterography (SHG), by introducing saline solution in the womb through catheters, during abdominal or vaginal ultra sound examination: this creates a contrast medium in the uterine cavity thus enabling a better investigation of the inner profile of the uterus. The procedure with saline solution to be adopted as ultrasonographic contrast medium associated to abdominal ultrasound was first described by Randolph et al. The data available at present encourage the wider adoption of such a method, despite its limitations compared to the hysteroscopic procedure, and reported by Darwish et al,7 on eighty-four infertile patients submitted to SHG the day before the intervention of hysteroscopy and diagnostic laparoscopy. Each one had been submitted to hysterosalpingography 6 months prior the above intervention. SHG proved to be unreliable in 8 patients (9.8%) due to failed sounding, intolerable pain and significant leakage of saline solution. Furthermore, no significant differences between hysteroscopy and SHG were evidenced in case of normal uterine cavity whereas the hysteroscopy confirmed to be clearly superior in detecting and diagnosing endouterine abnormalities, particularly adhesions, polyps and expanded lesions such as endometrial hyperplasia (Fig. 65.3)

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Fig. 65.3 In such cases the equivalence between SHG and hysteroscopy in evaluating the uterine cavity was quit low (72.2%, Cohen index k=31), lower as to the equivalence between HSG and hysteroscopy (75.6%, k=0.39). Furthermore, the evaluation of the pelvic pain through verbal pain scoring at the end of SHG had given the following results: intolerable in 4 cases (4.8%), barely tolerable in 9 cases (10.7%), tolerable in 45 (53.6%), acceptable discomfort in 19 cases (22.6%) and minimal discomfort in 7 cases (8.3%). Different results in terms of pelvic pain during sonohysterography are reported by Brown et al:2 the medium pain score (0–10) for sonohysterography was 2.7 as compared to the score of 5.8 and 5.3 respectively for hysterosalpingography and hysteroscopy (Fig. 65.4).

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Fig. 65.4 In the same study Brown et al2 report a substantial equivalence in diagnostic terms between HSG, SHG and HSC. But if we carefully consider the reported data and when assuming hysteroscopy as “gold standard” we observe that the equivalence shown among the three procedures is limited to 78% of the cases, whereas the equivalence between SHG and hysteroscopy is 90.5% of the cases (total amount of normal and abnormal cases in both procedures) this means that at least in 10% of the cases SHG does not properly diagnose abnormalities of the uterine cavity, particularly underestimating the presence of submucous myomas, endometrial polyps and endouterine adhesions. This discrepancy is much more evident if the diagnostic accuracy of outpatient procedures for the evaluation of the uterine cavity (HSG, SHG, HSC) is compared to the final results attained after operative hysteroscopy, with rates of correct diagnosis amounting to 54% and 72%, respectively for SHG and outpatient hysteroscopy. In our experience8 sonohysterography has proved to be a better and more effective procedure as compared to traditional transvaginal ultrasonography, particularly in the study of the focal pathology of endometrium, represented in such a case by endometrial polyps and mainly when associated to tridimensional vaginal ultrasonography: eighteen patients with endometrial polyps, 3-D sonohysterography equivalence of 100% to diagnostic hysteroscopy. However we have observed important limits to SHG procedure that can be summarised as follows: It is a miniinvasive technique, at present as the hysteroscopic miniendoscopes, which consists of introducing a catheter into the uterine cavity and a distension fluid, usually saline, in the same way as for the diagnostic hysteroscopy. Undoubtedly, the discomfort

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is strictly connected to the sizes of the catheter which is used: in some patients relevant pelvic pain and also vaso-vagal reactions can be observed. The examination can not be conclusive because of the flexibility of the catheter, that in some cases, cannot reach the uterine cavity or also because of the leakage of the saline solution. The expanded lesions represent the big limit of SHG, particularly the diagnostic of the hyperplastic pathology of endometrium. In the inflammatory and infective pathology of endometrium, SHG does not allow a diagnosis to be made: endometritis is visually diagnosed or through endometrial biopsy. In such cases, the ultrasonographic examination is totally unuseful. In the cases of submucous myomas with important intramural incidence, SHG may lead to underestimate the same submucous compound, thus resulting in a wrong choice of the surgical intervention to perform (hysteroscopy or laparoscopy/laparatomy). In fact, in presence of large myomas (4–5 cm) with intramural compound evaluated less or equal to 50 % during diagnostic hysteroscopy or transvaginal ultra-sound, (borderline cases for operative hysteroscopic), it may occur that even if the intrauterine pressure has slightly increased because of the introduction of the saline solution and of the catheter balloon, it may reduce temporarily and only for the duration of the SHG examination, the submucous portions of the myoma. Thus resulting in a wrong evaluation of the possibility of removing hysteroscopically the fibroid (Figs 65.5 to 65.8). HYSTEROSCOPY AND ASSISTED REPRODUCTIVETECHNIQUES The study of human reproductive physiology, in the last few decades, has been impressively implemented due to the wish to defeat infertility in all its aspects. The role of the physician has been thoroughly transformed. From a simple observer he has become a capable assistant to the reproductive act, during its various and delicate steps: the production of gametes, their meeting and fusion at the moment of fertilization, the development of a good quality embryo and at last its implantation into the uterine cavity.

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Fig. 65.5

Fig. 65.6 Each of these steps has been re-examined and corrected in vitro in order to search for that natural equilibrium to be restored.

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At present, still numberless are the dark areas which do not allow the techniques of assisted reproduction to offer the patients a reliable result and not only a possible one. The only condition existing in a IVF cycle that can guarantee the success of the ART procedure adopted is embryo implantation. All the efforts of the researchers aim at attaining this unique objective, whichever might be the strategy proposed or the technical support adopted. The phenomenon of implantation takes place at the end of a chain of events, including maturation and

Fig. 65.7

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Fig. 65.8 differentiation phases which affect, from one side the embryo and from the other side endometrium or better the uterus in its whole. An accurate evaluation of the infertile couple must ensure the production of qualitatively optimal embryos and at the same time the development of an endometrium able to receive them from both structural and functional viewpoints, inside a morphologically normal uterine cavity. In this section we will take into account exclusively the uterine factor, as a significant variable to consider seriously, from a point of view of the endometrial regularity and of the structural normality of the uterus. Infertility provoked by endometrial and structural uterine abnormalities amount to 5– 10% of the general population. Such abnormalities (congenital malformations, fibroids, endometrial polyps, intrauterine sinaechiae) may interfere during the reproductive process, thus jeopardising the meeting with the gametes or being the main cause of repeated miscarriages or pre-term deliveries. At present the investigation procedure, considered as the gold standard, in evaluating the uterine cavity is hysteroscopy, but its effective role in the management of ART is still debated and controversial. Someone would limit its use to the only cases presenting a clinical history of IVF failures, considering it as third degree test, somebody else would prefer to recommend it as an examination to carry out mandatorily in screening the infertile couple, due to the low reliability of non-invasive procedures (HSG, Ultrasound, hysterosonography). Frydman and Eibschitz,8 were able to experiment the limits of HSG, in perf orming diagnostic hysteroscopy in two groups of infertile patients, undergoing IVF, the first one

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recording a clinical history suggestive of uterine pathologies, the second one, not presenting any particular condition. The percentage of false positive (30.6%) and false negative (37.5%) attained after comparing HSG and HSC highlighted the advantages offered by the endoscopical procedure. First of all, the possibility of obtaining a direct visualization of the uterine cavity starting from the external uterine os, up to the bottom of the uterine cavity, by enabling an accurate diagnose of both focal lesions (polyps, myomas, sinaechiae) and expanded lesions (endometrites, hyperplasia), otherwise undetectable. Secondly it appears to be of outstanding importance the possibility of acquiring useful information about the routing to follow when transferring embryos so as to check whether the channel is more or less regular or whether obstacles to the progression of catheter are present, with a view to minimize the traumatism during such a procedure. All this means a very rigorous selection of the patients: • direct admit to IVF program; • exclusion for the presence of very severe pathologies; • exclusion or inclusion after surgical or pharmacological treatment. After the studies carried out by Hamou and Eibschitz, also Brinsden in 1990 investigated hysteroscopically 100 patients with at least two previous IVF failures. Thirty patients included in this group resulted negative hysteroscopically and were admitted to a new IVF attempt with a pregnancy rate of 23% per transfer. Forty-eight patients showed the presence of an endouterine pathology previously misunderstood. Thirty-seven women in this group underwent a treatment prior to a further IVF. The remaining 22 patients were particularly interesting because each of them revealed endometritis through endoscopic examination. In almost half of them, the serum titre for Chlamydia trachomatis was greater than 1/32. All these women were administered 200 mg daily Doxycycline for 21 days, prior to the IVF attempt. Three of them had a spontaneous pregnancy whereas the remaining women underwent IVF with a pregnancy rate of 28%. The same considerations were reported by Dicker and Goldman in 19909 who evaluated the validity of applying hysteroscopy by correlating it to the age of the patients eligible for IVF. The percentage of uterine abnormalities appear to be significantly higher in the group aged >40 years (Group I) as compared to the group including 30 year (Group II), presenting different distribution of the various pathologies observed. The uterine pathology prevails on the pathology of the cervical channel in both groups; particularly in the first Group (older women) it is possible to observe mainly polyps, submucous myomas and endometrial hyper-plasia whereas in the younger patients the more frequently evident uterine lesions are sinaechiae, and tubal occlusion. It is difficult to say if such anomalies represent the main cause of the infertility In any case they can interfere as secondary factors, by creating an unf avourable environment for the development and the implantation of the blastocysts, with a further reduction of the percentage of pregnancies which feature both the reproductive physiology of the older

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woman and the assisted reproduction techniques (15–30%). Also in such a case, satisfactory results were obtained following the applied strategies applied according to the hysteroscopic findings (Clinical Pregnancy: Group 1=8.9% Group II =19.9%). Hysteroscopy is a basic contribution for the management of infertile couple. It key role in evaluating the patients whose age is >40, where the possibility of observing uterine abnormalities is higher than in younger patients, is more than clear. This must be carefully considered because the number of older women who wishes for a child is increasing compared to the past (Fig. 65.9).

Fig. 65.9 Up to now we have underlined how the undetectable uterine abnormalities can directly or indirectly affect the success of an IVF cycle. Let us consider as starting point the IVF failures. These failures that physiologically are included in the clinical history of the infertile couple are not commonly considered as recurrent miscarriages unless there is an increase even if temporarily of the levels of ßHCG, that in absence of clinical or ultrasound evidence of pregnancy, enables us to speak of pre-clinical miscarriage. Dicker et al in 199610 wanted to highlight this possibility in order to identify, thanks to hysteroscopy, the correlation existing among pre-clinical miscarriages following treatments of assisted reproduction and intrauterine pathologies. Of the 144 patients under investigation, all of them presented an history of pre-clinical miscarriages after IVF; those where the uterine abnormalities were present (11.8%) showed an higher incidence of pre-clinical miscarriages (relation miscarriageabnormalities).

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Further, this significant data, we consider to be interesting to underline how 17 women out of 144 were previously admitted to a program of IVF, even if they showed uterine abnormalities susceptible of spontaneous abortion, mostly (12/17) uterus septum. This finding is also observed in a report by Sala et al11 on 100 patients with 2 IVF failures. They detected the presence of previous uterine abnormalities and mainly important pathologies such as partial uterine septum in 6 cases, intrauterine adhesions of medium degree in 6 cases, submucous myomas in 4 cases, endometrial polyps in one case and also one case of bone metaplasia, through diagnostic hysteroscopy Therefore the criticism on the use of hysteroscopy as a first level examination in ART procedures because of the cost is not understood, if we consider the cost of an IVF cycle which a priori is already a failure because of the absence of a correct diagnosis. The new achievements on the role of the small endouterine pathology affecting the results of IVF techniques deserve a special consideration, whereas no more doubts exist on the negative influence of the major pathology, such as partial or total uterine septa and submucous fibroids, on ART procedures. To this end in view, the meta-analyses published by E. Pritts12 is very remarkable, mainly as to the clinical evidences of fibroids and infertility: women with submucous fibroids demonstrated lower pregnancy rate (R. R. 0.30) and lower implantation rate (R. R. 0.28) than infertile controls. Therefore such data suggest that patients with submucous myomas or with intracavitary compound have a reduced reproductive prognosis, spontaneous or associated to ART procedures, and that hysteroscopic myomectomy is highly beneficial. The prospective controlled study of Hart et al13 is interesting as it cites the impact of intramurally developing fibroids on the outcome of assisted conception. The patients with intramural fibroids whose size is less than 5 cm (mean size 2.3) show pregnancy, implantation and signif icantly reduced ongoing pregnancy rate (23.3%, 11.9% e 15.1%) as compared to the control group including infertile patients without myomas and admitted to the same IVF program (34.1%, 20.2% and 28.3%). Logistic regression analyses shows that the presence of an intramural fibroid reduces by 50% the possibility of an ongoing pregnancy after IVF. If we go back to the initial consideration on the importance of the small endouterine pathology on repro duction, we discover very interesting data14 on to the possible negative effect of endometrial polyps of small sizes (polyps<2 cm). A comparison is done on two groups of patients to submitted to assisted reproduction with evidence of small endometrial polyps: 49 women (Group I) had standard IVFembryo transfer while in 34 women (Group II) hysteroscopy and polypectomy were performed immediately following oocyte retrieval, the suitable embryos were all frozen and the replacement cycle took place a few months later. In Group II, it has been possible to achieve a higher percentage of clinical pregnancies (33.3%), of implantation rate (16.7%) and lower percentage of spontaneous abortions (14.3%) as compared both to Group I (22.4% 9.3% and 27.3%) and to those of the overall frozen embryo cycles at Bourn Hall (22.3%, 12.4% and 12.1%).

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CONCLUSIONS All the data we have reported are in favour of a basic hysteroscopic investigation in the diagnostic evaluation of the infertile couple and also before starting an IVF treatment when considering the advantages, both in terms of number and of percentage, to achieve an absolutely normal pregnancy within an uterine cavity The last question we have to consider is whether hysteroscopy must be still considered a highly invasive procedure hardly bearable by the patient and thus reserved to particular and well selected cases. Our answer, after many years of outpatient endoscopic activity which has enabled us to observe thousands of cases is no! Hysteroscopy is no more an invasive procedure as it was up to some years ago. The rapid evolution of the endoscopic technology has realized hysteroscopic minioptics, equipped with Hopkins lenses, whose calibre is 2.7 mm (Circon; Wolf; Storz etc.) which fitted with diagnostic sheat, have a diameter of 3.3 mm (Fig. 65.10).

Fig. 65.10 In the last few years, we have made efforts in order to reduce the pelvic pain associated with the outpatient hysteroscopic examination, in order to transform hysteroscopy from a painful procedure to a “pain-free” procedure. We have experimented different procedures of local anaesthesia. Very interesting is the introduction of Mepivacaine 2%, 2–3 ml through the diagnostic sheath of hysteroscope immediately prior the procedure, mainly when fibrous synaechiae of the cervical channel and of the hystmus are present; but the global results are still controversial as also reported in the international literature. We have also utilised an

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equipment of transcutaneous electric stimulation (TENS) of the last generation regulated autonomously by the patient during hysteroscopy with results under publication. The real revolution has been the use of small calibre optics (3.3 mm, diagnostic sheat included) which permit a “pain-free” hysteroscopy without loosing the quality of the picture of the traditional optics of greater calibre in the last few years. We have carried out a randomised prospective study to compare outpatient hysteroscopy performed with a traditional optic by 5 mm (Hamou I, Storz) and minioptic by 3.3. mm (Wolf) whose data are under publication and where a drastic reduction of the pelvic pain during the procedure performed by aminiendoscope is evidenced. In an analogic visual scale of the pain 0–10 we achieve a medium score in the group of minioptic which is exactly half of the control group where the traditional optic was used (2.3 vs 4.6) (Fig. 65.11).

Fig. 65.11 The hysteroscopic procedure, carried out in such a way, in order to provoke a minor traumatism for the uterine tissues (good technique during the performance, low pressures of distension, short operative time, no use of tenaculum and dilatators) can create only a mild discomfort for the patient: 2.3 points to the visual analogue scale for pelvic pain is absolutely comparable to the introduction of the speculum or when an ultrasound is performed. It will be therefore possible to implement the use of hysteroscopy whose application allows a better evaluation of endometrium and of the uterine cavity for reproductive purposes.

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REFERENCES 1. Goland A, Ron-EI R, Herman A et al. Diagnostic hysteroscopy: its value in an in vitro fertilization/embryo transfer unit. Hum Repro 1992; 7(10):1433–34. 2. Brown Samuel E, Coddington Charles C, Schonorr John, Toner James P, Gibbons William, Oehninger Sergio. Evaluation of outpatient hysteroscopy, saline infusion hysterosonography, and hysterosalpingography in infertile women: a prospective, randomized study. Fertil and Steril 2000; 74:5. 3. Golan A, Eilat E, Ron-El R et al. Hysteroscopy is superior to hysterosalpingography in infertility investigation. Acta Obstet Gynecol Scand, 1996; 75:654–56. 4. Wang CW, Lee CL, Lai YM et al. Comparison of hysterosalpingography and hysteroscopy in female infertility. J Am Assoc Gynecol Laparosc 1996; 3:581–84. 5. ShushanAsher, Rojansky Nathan. Should hysteroscopy be a part of the basic infertility workup? Hum Repro 1999; 14(8): 1923–24. 6. Vercellini Paolo, Cortesi Ilenia, Oldani Sabina, Moschetta Marta, De Giorgio Olga, Crosignani Pier Giorgio. The Role of transvaginal ultrasonography and outpatient diagnostic hysteroscopy in the evaluation of patients with menorrhagia. Hum Repro 1997; 12(8):1768–71. 7. Darwish Atef M, Youssef Alaa A. Screening sonohysterography in infertility. Gyn Obst Inv 1999; 48:43–47. 8. La Torre R, De Angelis, Coacci F, Mastrone M, Cosmi EV. Trans-vaginal sonographic evaluation of endometrial polyps: a comparison with two dimensional and three dimensional contrast sonography Clin Exp Obst and Gyn 1999; 3–4. 8a. Frydman R, Eibschitz I, Fernandez H, Hamou J. Uterine evaluation by microhysteroscopy in IVF candidates. Hum Repro 1987; 2(6):481–485. 9. Dicker D, Goldman JA, Ashkenazi J, Feldberg D, Dekel A: The value of hysteroscopy in elderly women prior to in vitro fertilization-embryo transfer (IVF-ET): a comparative study J. in vitro Fert. Embryo Trans 1990; 7(5):267–70. 10. Dicker D, Ashkenazi J, Dekel A, Orvieto R, Feldberg D, Yeshaya A et al. The value of hysteroscopic evaluation in patients with preclinical in vitro fertilization abortions. Hum Repr 1996; 11(4): 730–1. 11. La Sala GB, Montanari R, Dessanti L, Cigarini C, Santori F. The role of diagnostic hysteroscopy and endometrial biopsy in assisted reproductive technologies. Fertil Steril 1998; 70(2): 378–80. 12. Pritts Elizabeth A. Fibroids and Infertility: A Systematic Review of the Evidence Obstetrical and Gynecological Survey. 2001; 56:8. 13. Hart Roger, Khalaf Yacoub, Yeong Cheng-Toh, Seed Paul, Taylor Alison et al. A prospective controlled study of the effect of intramural uterine fibroids on the outcome of assisted conception. Hum Repro 2001; 16(11):2411–17. 14. Lass A, Williams G, Abusheikha N, Brinsden P. The effect of endometrial polyps on outcomes of in vitro fertilization (IVF) cycles J. of Assis. Reprod. And Genet. 1999; 16(8):410–5. 15. Dov Dicker, Jack A. Goldman, Jacob Ashkenazi, Dov Feldberg, Aryeh Dekel. The value of hysteroscopy in elderly women prior to in vitro fertilization-embryo transfer (IVF-ET): A comparative study, in vitro J Fert Embr Trans 1990; 7:5. 16. Dov Dicker, Jacob Askenazi, Arie Dekel, Raoul Orvieto, Dov Feldberg, Arie Yeshaya et al. The value of hysteroscopic evaluation in patients with preclinical in vitro fertilization abortions Hum Repro 1996; 11(4):730–31. 14. Lass G, Williams N, Abusheikha, P.Brinsden. The effect of endometrial polyps on outcomes of in vitro fertilization (IVF) cycles. J of Assisted Reproduction and Genetics 1999; 16:8. 16. Reis Soares Sergio, Messala Batista Barbosa dos Reis, Aroldo Fernando Camargos. Diagnostic accuracy of sonohysterography, transvaginal sonography, and hysterosalpingography in patients with uterine cavity diseases. Fertil and Steril 2000; 73:2.

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17. Shalev Josef, Meizner Israel, Bar-Hava Itay, Dicker Dov, Mashiach Reuben, Zion Ben-Rafael. Predictive value of transvaginal sonography performed before routine diagnostic hysteroscopy for evaluation of infertility. Fertil and Steril 2000; 73:2.

CHAPTER 66 Modern Monagement of the Ischemic BlackBlue Tivisted Adenexa Roy Mashiach, Shlomo Mashiach, Daniel S Seidman INTRODUCTION Adnexal torsion, is a severe, though uncommon gynecological emergency, with a prevalence of 2.7–3 percent.1– 3 The present chapter reviews the options for management of the ischemic black-blue twisted adnexa, mainly detorsion versus extirpative therapy, i.e. removal of the ischemic organ (oophorectomy or adnexectomy). Extirpative Therapy Torsion commonly occurs in children and women of childbearing age.4 The grave cost of loosing precious ovarian tissue, especially in neonates, premenarcheal girls and women of fertility age, is not disregarded by physicians who support extirpative therapy of the ischemic black-blue twisted adnexa. Why, then, was the surgical dogma of removing twisted ovarian tissue so common in the past, and still prevalenttoday?5–10 The foundations for this are three strong and common beliefs: • First, that the ischemic tissue cannot recover. • Second, that the ischemic tissue, if left in place after detorsion, can cause severe local and systemic damage, mainly thromboembolic events, peritonitis, and sepsis, and • Third, that ovarian malignant neoplasm could be masked and left untreated with detorsion. Conservative Management The earliest published report on conservative management of adnexal torsion was presented by Way11 in 1946. He reported 15 cases in which the adnexal structures were untwisted Subsequently, a group from Israel, headed by Prof Shlomo Mashiach, has been repeatedly advocating, since 1989, in favor of untwisting the adnexa and conserving the ovary regardless of the morphologic appearance of the adnexa and without even extending the surgery duration in order to detect evidence of reperfusion.12–18 We will try to critically examine those beliefs which consist of the foundation for the extirpative treatment dogma, and see if they have a strong evidence base.

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Can the Ischemic Tissue Recover? Some authors believe that “the delay in establishing the correct diagnosis can lead to ischemia of the tortuous adnexal structure, with subsequent tissue necrosis, at which point the damage is irreversible and adnexal salvage is not possible”.19 However, the necessity to excise apparently looking necrotic adnexa remains a matter of debate. Many authors differentiate between infarcted and non-infarcted torted adnexa. “If the tissue is not infarcted, the adnexa may be untwisted and a cystectomy may be performed. If necrosis has occurred, an oophorectomy is mandatory”.20,21 We think that these guidelines are supported only by the authors belief that necrotic looking tissue can not regenerate and become viable again. Accordingly, Mage et al,22 studied 35 patients with untwisted adnexa and timed the interval for reperfusion. If reperfusion had not occurred in 10 minutes, excision was performed. Some argue that clinical inspection alone is unreliable in diff erentiating reversible tissue ischemia from irreversible tissue necrosis. Therefore, they embrace the intraoperative use of intravenous fluorescein dye, as suggested by McHutchinson et al.23 As stated earlier, Mashiach et al have long managed women with acute adnexal torsion by detorsion only, regardless of the morphologic appearance of the adnexa, and without even waiting for evidence of reperfusion. Between Jan 1984 and Aug 1999 they treated 112 patients with black-bluish ischemic adnexa by detorsion: 35 patients were treated by laparotomy, in the years 1984 through 1988, and 77 by laparoscopy in the following years. Follow-up of 0.5 to 15 years (median 4.5) was available in 102 patients. Vaginal ultrasound revealed normal sized ovaries with normal function in 92.1 percent (94/102) of the patients. In 8(7.9%) patients there was no evidence of ovarian function. In 94 of 102 patients they demonstrated ultrasonographic evidence of ovarian survival, manifested as follicular development postoperatively. Nine women consequently underwent a cesarean section and a normal appearing ovary was found. Six women underwent further IVF treatment and in all cases oocytes were obtained from the previously ischemic ovary and fertilized. Other groups have reported similar results. In a report by Shalev24 follicular development was evident in 49 of 52 (94.2%) women with normal-sized ovaries. Panski et al, report that in seven of eight girls (87%) the ovaries seemed viable through ultrasound examination.25 Similar results were reported in other series, where preservation of the adnexa was decided upon during operation.22,26 The authors consider this fact as a proof of their ability to prospectively estimate the adnexal viability, but we believe that the natural history of untwisted adnexa, even the ischemic looking ones, is to recover. Does Detorsion Increase the Risk of Thromboembolism? Torsion of the adnexa is essentially a vascular event, in which thrombi are bound to occur in proximity to the adnexa. Therefore the concern regarding possible spreading of thromboemboli, from the infarcted adnexa to the circulation, during or after detorsion procedure, expressed by Nichols et al27 can be regarded as logical. This concern has led to the classical teaching that the twisted adnexa should be resected and not untwisted,28 for fear of thromboembolic events. Some have even suggested palpation of the infundibulopelvic veins in order to rule out thrombi before detorsion.28,29

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This concern has often been emphasized. However, as previously noted by others,30,31,22 not a single case of pulmonary emboli, disseminated intravascular coagulation, sudden hypotension, or pulmonary collapse, which might occur with vascular release of vasoactive substances from ischemic tissues after untwisting adnexa have ever been reported in the English literature. McGovern et al,32 reviewed the English literature in order to compare the risk of thromboemboli in relation to the management of adnexal torsion. They excluded young (under 13) patients, cancer patients, patients in whom adnexal torsion was seen in association with ectopic pregnancy or cancer, and articles in which clinical follow-up was not reported. They found two proven cases of pulmonary embolism (PE) occurring in a population of 981 reported patients with adnexal torsion (incidence of 0.2 percent). Three hundred nine patients underwent “conservative” surgery, consisting primarily of detorsion, with no thromboembolic complications reported (0 percent), whereas 672 were treated with adnexal resection without detorsion, with two cases of PE reported (0.3 percent). There difference in the incidence of this complication between the two methods of management did not reach statistical significance (P=.47, Fisher’s exact test, onetailed). In the two cases of PE reported, PE was seen in patients in whom delay in diagnosis led to prolonged preoperative hospitalization (up to 3–4 days). This suggests, according to the reviewers, that immobilization may increase the propensity for thrombosis, regardless of surgical management. Thus, a delay in diagnosis, rather than operative management, may be the critical factor. Can Detorsion Postpone Treatment of Hidden Malignancy? When surgeons confront an edematous, dark, ischemic ovary caused by adnexal torsion, the question arises about missing a tumor if resection is not done. Several authors mentioned the possibility of hidden malignancy, especially concerning cases where young girls are involved.33,26,17,34,35 The risk of torsion is the most common complication of ovarian tumors in children, with an incidence that ranges from 3 to 16 percent.36–39 On the other hand, some studies suggest that ovarian tumors that undergo torsion are usually benign.10,36,40,41 One explanation for the lower incidence of torsion among malignant neoplasms is their tendency to cause inflammation, adhesions, local invasion, and, thus, increased adherence to the surrounding structures.40 One group even expressed that they “found it very difficult to differentiate which ovaries may be normal, and did not feel it was saf e to leave a necrotic mass which may harbor a tumor”. Thus, “based upon their experience”, they “were not comfortable recommending ovarian detorsion”.34 Although they also agree that “in select cases in which it is clear that the twisted ovary is normal, detorsion, may be warranted.” Argenta et al,42 examined pathologic results of 104 patients undergoing adnexal torsion. Cancer was diagnosed in fewer than 1 percent of cases. They therefore concluded that a more-conservative approach seems warranted in light of the low incidence of malignancy. Mashiach et al (personal communication) treated since 1984 over 150 women with torsion of the ovary, and encountered no case of malignancy.15

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Kokoska et al10 report that one of 51 children with adnexal torsion had a malignant tumor (dysgerminoma) with disease confined to the ovary. The child had high serum hCG levels. Panski et al25 reported eight pre-menarcheal girls with torsion. Only one had a cystadenoma that required further surgery Additional reports on this topic, involving a small number of patients, did not report any cases of ovarian malignancy. The risk of encountering a tumor should be much lower, in young women, especially those who develop torsion in early pregnancy achieved following ovulation induction,15 and when ultrasound studies do not reveal any suspicious ovarian finding. Therefore we agree with Dolgin et al 26 that, if there is no tumor seen on preoperative sonography and none evident at exploration, the ovary can be left in place. As stated earlier,17 repeating a sonogram 6 to 8 weeks postoperatively is sensible. We think the risk of needing further surgery is worth taking since it is low and as we believe that postponing treatment should not change the outcome considerably One must note that due to the small numbers of patients, evidence supporting this belief will probably not be soon available. The Risk of Retorsion The risk of retorsion in our group and as reported by others,18, 19 is five percent or less.35 We believe that oophoropexy should be considered in case of repeated adnexal torsion. However, since we strongly advocate minimal surgical handling of the edematous ischemic ovary immediately after detorsion, oophoropexy may be technically difficult and can be associated with injury to the fragile ovary. CONCLUSION Adnexal torsion, although rare, is a major complication requiring prompt surgical therapy. The dogmatic historic extirpative management, still practiced in many places, is based upon fears of local and systemic complications, and disbelief in the capability of the ischemic tissue to recover. In 1989, Mashiach et al, challenged this dogma, first using laparotomy, and then, laparoscopy.16 Others have reported confirmatory results, as reviewed here. In this review we have shown that: • The necrotic appearing ovary is, in most cases, viable. • Thromboembolic complications do not occur more after detorsion. • The risk of malignancy is very low We therefore plead for conservation of the ischemic black-blue twisted adnexa in all cases.

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REFERENCES 1. Taskin O, Birincioglu M, Aydin A, Buhur A, Burak F, Yilmaz I, et al. The effects of twisted ischaemic adnexa managed by detorsion on ovarian viability and histology: an ischaemiareperfusion rodent model. Hum Reprod 1998; 13:2823–27. 2. Hibbard LT. Adnexal torsion. Am J Obstet Gynecol 1985; 152:456–61. 3. Burnett LS. Gynecologic causes of the acute abdomen. Surg Clin North Am 1988; 68:385–98. 4. Haskins T, Shull BL. Adnexal torsion: a mind-twisting diagnosis. South Med J 1986; 79:576–77. 5. Valk N, Davis EW, Blackford JT. Ovarian torsion as a cause of colic in a neonatal foal. J Am Vet Med Assoc 1998; 213:1454–6. 6. Mizuno M, Kato T, Hebiguchi T, Yoshino H Surgical indications for neonatal ovarian cysts. Tohoku J Exp Med 1998; 186:27–32. 7. Lee EJ, Kwon HC, Joo HJ, Suh JH, Fleischer AC. Diagnosis of ovarian torsion with color Doppler sonography: depiction of twisted vascular pedicle. J Ultrasound Med 1998; 17:83–89. 8. Quint EH, Smith YR. Ovarian surgery in premenarchal girls. J Pediatr Adolesc Gynecol. 1999; 12:27–9. 9. Moore RD, Smith WG. Laparoscopic management of adnexal masses in pregnant women. J Reprod Med 1999; 44(2):97–100. 10. Kokoska ER, Keller MS, Weber TR. Acute Ovarian Torsion in Children. Am J Surg 2000; 180:462–65. 11. Way S. Ovarian cystectomy of twisted cysts. Lancet 1946; 2:47- 48. 12. Shalev J, Goldenberg M, Oelsner G et al. Treatment of twisted ischemic adnexa by simple detorsion. N Engl J Med 1989; 321:546. 13. Bider D, Ben-Rafael Z, Goldenberg M, Shalev J, Mashiach S. Pregnancy outcome after unwinding of twisted ischaemichaemorrhagic adnexa. Br J Obstet Gynaecol 1989; 96:428–30. 14. Ben-Rafael Z, Bider D, Mashiach S. Laparoscopic unwinding of twisted ischemic hemorrhagic adnexum after in vitro fertilization. Fertil Steril 1990; 53:569–71. 15. Mashiach S, Bider D, Moran O, Goldenberg M, Ben-Rafael Z. Adnexal torsion of hyperstimulated ovaries in pregnancies after gonadotropin therapy. Fertil Steril 1990; 53:76–80. 16. Bider D, Mashiach S, Dulitzky M, Kokia A, Lipitz S, Ben-Rafael Z. Clinical, surgical and pathologic findings of adnexal torsion in pregnant and nonpregnant women. Surg Gynecol Obstet 1991; 173:363–66. 17. Oelsner G, Bider D, Goldenberg M, Admon D, Mashiach S. Long-term follow-up of the twisted ischemic adnexa managed by detorsion. Fertil Steril 1993; 60:976–79. 18. Cohen SB, Oelsner G, Seidman DS, Admon D, Mashiach S, Goldenberg M. Laparoscopic detorsion allows sparing of the twisted ischemic adnexa. J Am Assoc Gynecol Laparosc 1999; 6:139–43. 19. Bayer AI, Wiskind AK. Adnexal torsion: can the adnexa be saved?Am J Obstet Gynecol 1994; 171:1506–10; 1510–11. 20. Rapkin AJ. Pelvic pain and dysmenorrheal. In Berek JS, Adashi EY, Hillard PA (Eds): Novak’s Gynecology. Baltimore: Williams & Willkins, 1998; 404. 21. Munro MG. Gynecologic endoscopy. In Berek JS, Adashi EY, Hillard PA (Eds): Novak’s Gynecology Baltimore: Williams & Willkins, 1998; 679. 22. Mage G, Canis M, Manhes H, Pouly JL, Bruhat MA. Laparoscopic management of adnexal torsion. J Reprod Med 1989; 34:5204. 23. McHutchinson LB, Koonings P, Ballard C d’Ablaing G III. Preservation of ovarian tissue in adnexal tissue with fluorescein. Am J Obstet Gynecol 1993; 168:1386–88. 24. Shalev E, Bustan M, Yarom I et al. Recovery of ovarian function after laparoscopic detorsion. Hum Reprod 1995; 10:2965–66. 25. Pansky M, Abargil A, Dreazen E, Golan A, Bukovsky I, Herman A. Conservative management of adnexal torsion in premenarchal girls. J Am Assoc Gynecol Laparosc 2000; 7:121–24.

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26. Dolgin SE, Lublin M, Shlasko E. Maximizing ovarian salvage when treating idiopathic adnexal torsion. J Pediatr Surg 2000; 35:624–26. 27. Nichols DH, Julian PJ. Torsion of the adnexa. Clin Obstet Gynecol 1985; 28:375–80. 28. DiSaia PJ. Ovarian neoplasms. In Scott JR, DiSaia PJ, Hammond CB (Eds): Danforth’s obstetrics and gynecology. Philadelphia. JB Lippincott 1994; 990–91. 29. Chambers JT, Thiagarajah S, Kitchin JD III. Torsion of the normal fallopian tube in pregnancy. Obstet Gynecol 1979; 54:487. 30. Wagaman R, Williams RS. Conservative therapy for adnexal torsion. A case report. J Reprod Med1990; 35:833–34. 31. Pinto AB, Ratts VS, Williams DB, Keller SL, Odem RR. Reduction of ovarian torsion 1 week after embryo transfer in a patient with bilateral hyperstimulated ovaries. Fertil Steril 2001; 76:403–6. 32. McGovern PG, Noah R, Koenigsberg R, LittleAB. Adnexal torsion and pulmonary embolism: case report and review of the literature. Obstet Gynecol Surv 1999; 54:601–8. 33. Templeman C, Hertweck SP, Fallat ME. The clinical course of unresected ovarian torsion. J Pediatr Surg 2000; 35:1385–87. 34. Kokoska ER, Keller MS, Weber T. Letters to the Editor. Am J Surg 2002; 183:95–100. 35. Dolgin SE. Acute ovarian torsion in children. Am J Surg 2002; 183:95–96. 36. Lee CH, Raman S, Sivanesaratnam V. Torsion of ovarian tumors: a clinicopathological study. Int J Gynecol Obstet 1989; 28;21–25. 37. Comerci JT Jr, Licciardi F, Bergh PA et al. Mature cystic teratoma: a clinicopathologic evaluation of 517 cases and review of the literature. Obstet Gynecol 1994; 84:22–28;22–28. 38. Brown MF, Hebra A, McGeehin K, Ross AJ III. Ovarian masses in children: a review of 91 cases of malignant and benign masses. J Pediatr Surg 1993; 28:930–32. 39. Piipo S, Mustaniemi L, Lenko H et al. Surgery for ovarian masses during childhood and adolescence: a report of 79 cases. J Pediatr Adolesc. Gynecol 1999; 12:223–27. 40. Sommerville M, Grimes DA, Koonings PP, Campbell K. Ovarian neoplasms and the risk of adnexal torsion. Am J Obstet Gynecol 1991; 164:577–78. 41. Peterson WF, Prevost EC, Edmunds FT et al. Benign cystic teratomas of the ovary. A clinicostatistical study of 1,007 cases with a review of the literature. Am J Obstet Gynecol 1955; 70:368–82. 42. Argenta PA, Yeagley Tf, Ott G. Torsion of the Uterine Adnexa: Pathologic Correlations and Current Management Trends. J Reprod Med 2000; 45:831–36.

CHAPTER 67 Fertiloscopy: A New Technique and an Alternative to Conventional Laparoscopy in Infertility Radha Syed OVERVIEW Fertiloscopy has been defined as the combination of transvaginal hydropelviscopy, dye test, salpingoscopy/ microsalpingoscopy, and hysteroscopy. This can be of ten perf ormed in an outpatient setting under local anesthesia and/or with IV sedation. In its current technique, fertiloscopy was first introduced by Dr. Antoine Watrelot of Lyon, France. The instrumentation used is distributed by Soprane S. A Surgical Technology, based in Lyon, France. Following the initial work of Gordts and Brosens in hyrdropelviscopy, Dr. Watrelot described the concept offertiloscopy in 1997 and published his first series in 1999 in “HumanReproduction”. To date, more than 500 cases have been presented in a continuous series performed in CRES® in Lyon. Salpingoscopy was described first by Henry Suchet and Cornier in France and Brosens and Putemans in Belgium. Marana in Italy has also investigated extensively in salpingoscopy. All these investigators have emphasized the need for salpingoscopy for therapeutic decision-making in treatment of infertility. It is now well established that there is lack of concordance between the outer aspect of the fallopian tube and its internal pathology. More recently, Marconi from Argentina has introduced the concept of microsalpingoscopy and has evaluated the internal epithelium of the salpinx with the nuclear dye staining, usingmethylene blue as a predictive test of tubal normalcy. The staging ranges from Stage I (normal) to Stage IV (severely abnormal). The new procedure of fertiloscopy allows this examination and tubal staging. The diagnosis of unexplained infertility is not easy. Hysterosalpingography (HSG) is very often practiced but even at its best, this examination is of value only when it shows complete tubal blockage. In other cases, the existence of a degree of patency or apparent normalcy are later proved to be false negatives as shown by laparoscopy. Swart et al (1995) in a meta-analysis found that HSG was not suitable for the evaluation of periadnexal adhesions. Up until now, laparoscopy has been the gold standard to explore tuboperitoneal infertility. Nevertheless, laparoscopy is very often performed without discoveringany significant pathology. Additionally, laparoscopic complications can be significant, as shown in the French register by Chapron et al, 1997. In contrast, fertiloscopy as a procedure is well accepted by the majority of patients, is free of significant complications and avoids unnecessary laparoscopy. Fertiloscopy is a diagnostic tool) a one-time

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procedure which gives complete information on the status of the uterus, tubes, ovaries, and peritoneum. New techniques are being developed for therapeutic options. INDICATIONS “The strategy for managing unexplained infertility has often involved laparoscopy or a move towards invitro fertilization without having visualized the genital tract at all, However, the last option fails to recognize unsuspected pelvic pathology. The aim at fertiloscopy is to establish a new approach in patients’ with a suspicion of unexplained infertility Fertiloscopy is now recommended as a primary investigation in the infertility workup. Other applications include: 1. unexplained pelvic pain (acute and chronic) 2. menstrual abnormalities 3. undefined pelvic mass 4. ectopic pregnancy 5. pelvic endometriosis 6. polycystic ovaries 7. pain mapping 8. pelvic inflammatory disease (PID) and 9. congenital anomalies of the pelvic organs. Future directions are leaning towards therapeutic indications for: 1. lysis of adnexal adhesions 2. ovarian drilling in polycystic ovaries and 3. biopsies of peritoneal pelvic pathology and 4. cytological sampling. Another indication is to select adequate therapeutic option prior to initiating treatment. Once pelvic pathology is detected, one of the available therapeutic options must be selected, e.g. surgery or ART (Artificial Reproductive Technology). In these cases, salpingoscopy and microsalpingoscopy are very useful to decide whether the infertility is unexplained or surgical treatment can be instituted for allowing spontaneous pregnancy. Figure 67.1 “Decision Tree”.

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Fig. 67.1: The female genital tract TECHNIQUE Pre-operative Workup and Patient Selection Patients with infertility history of at least one to two years are selected prior to referring them for IVF. A gyn exam, pap smear, and pelvic sonogram to rule out gross pelvic pathology are performed after extensive history taking. Evidence of satisf actory ovulation, either spontaneous or stimulated, should be obtained. Cervical mucus studies and semenalysis should be normal. Patient is kept NPO after midnight. A preoperative enema is given at six a.m. on the morning of surgery to increase the space between the rectum and vagina. Instrumentation (See Fig. 67.2) 1. Fertiloscopy introducers- Especially designed introducers are the key to performing fertiloscopy. These disposable introducers (one in a kit) are for the uterine cavity (FH 1–29 Soprane France) and the other is Douglas introducer (FTO 1–40 Soprane France).

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a. Uterine fertiloscope® (FHI-29 Soprane France) is fitted with a balloon in order to have a good seal during dye test. It has a smooth mandrel to allow for easy insertion into the uterine cavity. Once in place, the mandrel may be removed and the flexible introducer is fixed to the patients thigh with velcrow which is provided. b. Douglas Fertiloscope® (FTO 1–40 Soprane France) has three channels, (1) The central channel is fitted with a sharp trocar for insertion into the pouch of Douglas. Once removed, it allows for insertion of the telesceope. The second channel is for inflation of the balloon at the tip of the introducer (Fig. 67.3a to c). The balloon serves three purposes. 1. It prevents involuntary slipping of the introducer from the pelvic cavity 2. It allows better viewing of the pelvic cavity when the introducer is pulled. 3. It allows as a ball joint for moving the telescope around. The last channel is the operative channel through which five French instruments can be introduced and an outflow channel 2. Veres Needle is necessary to create hydroperitoneum. 3. Telescope Hamon II by Karl Storz, Germany is strongly recommended-it is 2.9 mm in diameter with a 30 degree lens and 180X magnification which is the only telescope capable of performing micro-salpingoscopy. 4. Operative Instruments: 5 FRbiopsy forceps, grasping forceps and scissors are used. Bipolar electrode or forceps are useful for ovarian drilling and other therapy. 5. Room Setup (See Fig. 67.2): Patient is placed in lithotomy without TrendelenBurg. Mobile videocart should be at the left of the patient. Saline for infusion should be on the right of the patient in a standard infusion cart. 6. Procedure: 1. Pelvic exam is performed prior to fertiloscopy to rule out obstruction of Pouch of Douglas by

Fig. 67.2: Basic set for fertiloscopy: (1) Adjustable speculum, (2) Veress needle, (3) Currette for endometrial biopsy, (4) Swab forceps for asepsis, (5) Pozzi tenaculum, (6) Irrigation tube, (7) HAMOU III examination and contact hysteroscope III (8)

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Examination sheath, (9) F 42K fertiloscopy® Kit

Fig. 67.3: (a) Transvaginal Douglas fertiloscope® FTO 1.40 with three channels; (b) Close-up view of the inflated balloon of the transvaginal Douglas fertiloscope® FTO 1.40; (c) FTO 1.40 introducer with the HAMOU hysteroscope III, syringe and grasping forceps pathology like myoma, endometriosis which is a contrmdication to the surgery. Prophylactic antiobiotics are adminstered 30 minutes prior to the procedure intravenously. 2. Anesthesia a. general anesthesia as for protocol b. local anesthesia (strict without sedation). Anesthesia gel is introduced into the fornix (Emla®) Ten minutes later, 4–5 ml of one person xylocaine is injected near the uteros ligaments. 3. Introduction of the uterine fertiloscope: Colin speculum is inserted deeply into the vagina to expose the posterior culdesac. This speculum can be removed with instruments still in the vagina. A Pozzi tenaculum is fixed at 8:00 clock position on

Fertiloscopy: a new technique and an alternative to conventional laparoscopy in infertility

739

the cervix. Then the uterine fertiloscope is inserted into the cervix; occasional dilatation may be required. The mandrel is removed and the balloon is inflated with 2–3 ml of air. Do not over inflate the balloon as it may cause pain. The introducer is attached to the patients thigh with velcro provided. 4. Creating a hydroperitoneum: A Veres needle is used to create a hydroperitoneum. The point of entry is located 5–10 ml below the cervix. A small nick on the vaginal mucuosa facilitates entry of the Veres needle without sliding. The axis of penetration has to be parallel to the inferior blade of the speculum in retroverted uterus and horizontal in case of anteverted uterus. Once in the right space, Veres tap is opened and preheated isotonic saline solution is allowed to freely flow into the Pouch of Douglas. About 200 cc of saline solution is inserted. 5. Introduction of the Douglas Fertiliscope: The Douglas Fertiliscope is then inserted into the same plane and axis as the Veres needle which is now removed. Some saline will flow out if the introducer is in the correct position. The balloon is now inflated with 4–5 cc of air. If no liquid appears, remove the mandrel and check position of the introducer into the scope. The telescope is introduced by unscrewing the valve located at the proximal end of the main channel and irrigation is continued through the sheath of the scope. Observation can now start. 6. Operative Channel: The red tap on the introducer closes the operative channel. When opened, it allows the passage of five French instruments. It is necessary to rotate the introducer until the red tap is located on the red side. By doing this, the operative channel will be above the main scopeand therefore the instruments can be seen through the 30 degree lens. The operative channel is also useful for saline outflow channel and if blood is present in the Pouch of Douglas, this allows to rinse the cavity and improve quality of vision. 7. Exploration of the Pelvis: (See Figs 67.4 to 67.6) A sytematic method is used. a. Hydropelviscopy: The first element is to find the posterior part of the uterus which forms the roof of the explored space. Then, one has to move alternatively from one side to the other and find the origins of the adnexae, the uterine ovarian ligament, and the tubal isthmus. In following the uterine ovarian ligament, the ovary can be reached and every part of the ovary must be examined. The upper part of the ovary can be seen with a 30 degree lens by entering the space between the ovary and fossa ovarica and rotating the scope on its axis. The tube can be followed from the isthmus to the ampulla and thefirnbria. If visualization is difficult, one must have to wait until more liquid is instilled. (See Fig. 67.7 to 67.9). b. The dye test: When all genital structures have been recognized, the dye test can be performed. The dye is instilled through the appropriate channel of the uterine introducer. A 20 cc syringe is connected and the dye pushed gently inorder to avoid tubal spasm. The dye is visualized at the fimbria and it is necessary to move from one side to the other to confirm bilateral patency. c. Salpinoscopy: Salpingoscopy is known to be a very useful means of investigation of the tube. Brosens has clearly demonstrated thepathology of intratubal adhesions and described a salpinoscopic score used to classify the findings. The technique is simple and consists of stabilizing the firnbria by means of a grasping forceps introduced into the operative channel Then by pushing gently with the telescope

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into the firnbria, it is possible to enter the ampulla and further reach the isthmicoampuUary junction. It is necessary to irrigate the tube through the sheath of the telescope. Avoid high pressure irrigation in the ampulla by adjusting the inflow. By rotating the telescope on its axis, each portion of the ampulla can be examined. Pathological findings like adhesions or flattened folds as well as methlyene blue-stained nuclei are of critical importance to decide whether surgical repair is legitimate or IVF is necessary. Microsalpinoscopy is of great value to examine the number of dyestained nuclei on the tubal epithelium. These cells

Fig. 67.4: Ovarian drilling; (a) Ovarian drilling, first step; (b) Ovarian drilling; (c) Ovarian drilling; (d) Ovarian drilling

Fertiloscopy: a new technique and an alternative to conventional laparoscopy in infertility

Fig. 67.5: Microsalpingoscopy—an overview

741

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Fig. 67.6: Exploration of the pelvis (a) Close-up view on the ovary; (b) Ovulation; (c); (d) are either intermediary or inflammatory cells in the middle of the tubal folds. Stage I is when no nuclei are dye-stained as in a normal tube to pathological (Stage IV) where a great number of cells appear to be dye-stained. Amicrobiopsy can be obtained to confirm the diagnosis. d. Hysteroscopy: This is the last step of the procedure and is practiced with the same scope. Endometrial biopsies are performed at this time. e. Operative Fertiloscopy: This is a new challenge and has limited application at the present time, as the operative channel allows only a small diameter instrument. Ovarian drilling and adhesiolysis are being performed on a limited basis. f. End of procedure: The telescope is removed and the liquid from the Pouch of Douglas is allowed to flow out freely. It is not important to evacuate all of the saline instilled as it will be reabsorbed in the following hours. No sutures are required on the vaginal scar. The patient can be discharge immediately if the procedure was performed

Fertiloscopy: a new technique and an alternative to conventional laparoscopy in infertility

Fig. 67.7a: The female genital tract

Fig. 67.7b: Insertion of the speculum

743

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Fig. 67.7c: Introduction of the transvaginal uterine fertiloscope® FH 1.29 under local anesthesia. If general anesthesia has been administered, the patient may be discharged on recovery. The only recommendation is to avoid the use of tampon and intercourse for a period of six days.

Fig. 67.8a: Insertion of the veress needle

Fertiloscopy: a new technique and an alternative to conventional laparoscopy in infertility

Fig. 67.8b: Introduction of the transvaginal douglas fertiloscope FTO 1.40 and the HAMOU III telescope

Fig. 67.8c: Extension of the Douglas pouch g. Contraindications to fertiloscopy: There is only one real contraindication. It is the obstruction of the Pouch of Douglas, either by a fixed retro-verted uterus or a myoma or endometriosis of the retrovaginal septum should be prepared for

745

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Fig. 67.9a: Dye test

Fig. 67.9b: Movement of the telescope

746

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747

Fig. 67.9c: Salpingoscopy and microsalpingoscopy further laparoscopy. It is easy to detect this pathology by ultrasound or by careful vaginal exam. In case of doubt aboid performing fertiloscopy

COMPLICATIONS The only serious complication is rectal perforation. Following a strict criteria of avoiding fertiloscopy in Pouch of Douglas obliteration will keep this complication under 1%. If rectal perforation does occur, the treatment is always conservative, using antibiotics for 5 days. Minor problems during fertiloscopy may be encountered a. No visualisation: if everything is black when the scope is introduced, there is something wrong with the equipment. It may be attributed to inadequate light supply. An accurate diagnosis cannot be made under these circumstanes. b. Partial visualisation: When only some structures are discovered and not the other, there are two possibilities: inadequate hyroperitoneum, solution is to wait awhile to fill up and secondly, remember to always follow an identified structure to discover the other genital structures. c. Bleeding: Blood is frequently encountered due to puncture of the Pouch of Douglas. Even a small amount of blood can blacken everything. Allow the outflow tract channel to evacuate the blood. d. Retroverted Uterus: Fertiloscopy is possible if the uterus is not fixed retroverted. Change the axis of introduction to be parallel to the inferior blade of the speculum. Improved skills can improve the success under these conditions. e. False route: If dissection is created between the peritoneum and the vaginal vault while inserting the Veres needle, then the flow of water is not regular and has to be stopped to limit the dissection. To avoid such a false route, it is important to insert the Veres needle with a firm movement.

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f. Pelvic pathology: If pelvic pathology is discovered, one should be prepared for further laparoscopy. Patient should be counseled prior to the procedure that in case of pathology, laparoscopy becomes mandatory.

FUTURE DIRECTIONS With renewed challenge of single procedure accomplishing all the tasks, there is a drive for operative fertiloscopy to emerge as a new technique. Several procedures for operative fertiloscopy are currently feasible such as ovarian drilling, limited adhesiolysis and biopsy. All of these procedures make use of five French operating channel and thus are limited with regard to the size of the instruments. Another limitation is the use of coaxial orientation without the advantages of a triangular approach as in laparoscopy. Nevertheless, increasing potentials of fertiloscopy procedures will soon become available. Future tubal ligation by this method may become possible as well as treatment of a wide range of pathologies limited to the pelvis. Study of Comparison between fertiloscopy and laparoscopy have been conducted by various investigators in Europe and V.S.A and the conclusions will be presented in Paris at a meeting in September, 2001 as well as the 30th AAGL annual meeting in San Francisco in November, 2001. Preliminary results indicate that the procedure of Fertiloscopy is effective in diagnosing all significant pelvic pathology in spite of its inherent difficulty in evaluating the anterior compartment of the pelvis. The laparoscopy performed concomitantly proves that there were no significant pathology in the anterior pelvis which was not present in the posterior Pouch of Douglas. Therefore, fertiloscopy appears well ensconced as the primary investigative tool for pelvic pathology. CONCLUSION There is a broad agreement that hysterosalpingography is the first line of examination for the detection of tubal pathology However, there is up to 40% false negative rate reported by the various authors. Additionally, the method fails to detect ovarian and periotoneal adhesions and endometriosis. Consequently, there is a strong need to have a more precise diagnostic tool. Fertiloscopy could easily replace diagnostic laparoscopy. Moreover, fertiloscopic examination is performed with a view to the physiological conditions of the individual patient. Example: Adhesions to the posterior part of the ovary are better documented by fertiloscopy because there is no need to mobilize the adnexa for the examination. Fertiloscopy is a minimally invasive and very safe technique compared to laparoscopy as there is no need for the use of carbon dioxide, Trendelenburg position, or risk of injury to large vessels. Most importantly, this simple technique can easily be carried out in a day surgery or outpatient setting after a short learning period without the inherent risks of laparoscopy The entire examination can be performed in a total time frame of 10–15 minutes with minimal recovery period for the patient.

CHAPTER 68 Hysteroscopic Assessment of Selective Tubal Pressures and Tubal Cannulation by Air Bubble Stents Atul Kumar, Alka Kumar HSG reveals only a mechanical patency of the tubes while selective tubal pressure (STP) measurement gives additional information about the physiological status also. STP can be easily assessed by hysteroscopy without cannulating the tubes with a plastic catheter. The steps of the procedure are as follows: The patient is taken in the usual lithotomy position, the diagnostic hysteroscope is introduced and both the tubal openings are visualized. By raising the head end of the patient by about 45 degrees the uterus becomes relatively more anteverted with respect to the ground and the fundus becomes the highest point of the uterine cavity. In such a situation any micro air bubble which is introduced any where inside the uterine cavity shall immediately rise and park itself in the fundus. It is essential to use a continuous flow uterine distending system, preferably of the pressure cuff or gravity type and not the peristaltic pump. It is also very essential that the outflow is not kept open but instead a continuous flow peristaltic pump is connected to it which removes liquid distending media from the uterine cavity at a constant rate of 20 ml per minute. This rate of 20 ml per minute is not a fixed entity and it is the surgeon’s and rsquo’s preference. We have observed that at this flow rate the debris is constantly being removed from inside the uterine cavity to give a clear visualization and at the same time the turbulence created inside the uterine cavity is so minimal that none of the microbubbles are dislodged even if the tip of the hysteroscope is kept 1 cm proximal to the cornuae. An approximately one meter long rubber tubing is shaped in the form of a coil. One end of this coil is connected to an ordinary needle type aneroid manometer which is routinely used for measuring blood pressure and the other end is connected any where in the inflow tubing by the help of a ‘ T’ shaped cannula. The liquid distending media enters inside the coiled tube and compresses the air column distal to the liquid column and the pressure registered by the manometer gives actual intrauterine pressure if the outflow rate of the peristaltic pump is kept low, e.g. 20 ml per minute. The inflow tubing of the continuous flow hysteroscope is punctured with an insulin syringe and the entire hysteroscope is rotated by 180 degrees so that the inflow channel of the hysteroscope comes to lie at the 6 and Isquo; O and rsquo; clock position instead of the usual 12 and Isquo; O and rsquo; clock position. In such a situation if 0.2 ml air is injected into the inflow tubing, after about 10 seconds, this air is seen entering the uterine cavity in the form of microbubbles which shall immediately get lodged at the fundus. If the STP of the left tube is to be assessed, the left side of the operation table, as seen from the perineal end of the patient, is tilted up by rotating the table in the anti clock wise direction along the long axis of the table, till the bubble comes in contact with the left tubal opening. The intrauterine pressure is now gradually increased and the pressure at

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which the bubble is seen entering retrogradely into the tube is the STP of the left tube. By raising the head end and by rotating the table, we are trying to make the tubal ostium the highest point inside the uterine cavity. In a similar manner if the STP of the right tube is to be assessed the table has to be rotated clockwise. The lower the STP the better is the physiological patency of that tube. One tube may have a STP of only 50 mm Hg (good) while the other tube may have a STP of more than 160 mm Hg (bad physiologic function). In our study the STP findings were found to collaborate with simultaneously performed laparoscopic CPT’s and past HSG findings. In cases of blocked tubes, the bubble stent can replace the routinely used plastic stents. (Note: Force exerted= Pressure×area of cross section). A solitary bubble of 5 mm diameter lodged at the cornuae can have a area of cross-section about 10 times more than that of the stents which are used for opening proximal tubal blocks. If the intrauterine pressure is raised to 160 mm Hg the cannulation force exerted by such a bubble shall be many times more in comparison to a simple plastic stent. Further, the possibility of trauma to the endosalpinx by bubble stents is much less when compared to plastic stents. Condusion Hysteroscopic assessment of the Selective Tubal Pressure gives an accurate idea about of the physiological patency of the tube and air bubble stent is a non invasive alternative to the plastic stent cannulation in the treatment of blocked tubes. HYSTEROSCOPIC MARKERS FOR AN OPTIMAL SEPTOPLASTY Enlarged septum is not an unusual cause for repeated pregnancy loss and primary infertility A septum is confirmed on ultrasound and a bi – cornuate uterus is ruled out. We cut the septum transversely by the collins knife using non modulated current. We have never felt the need of using a simultaneous control laparoscopy for assisting any hysteroscopic septoplasty The fibrous tissue of the septum does not bleed during resection but the underlying fundal myometrium does bleed, thus towards the completion of the septoplasty procedure, frequent pulses of low intrauterine distension pressure are given at regular intervals. The pressure in each of these pulses has to be much lower than mean arterial pressure. When minute superficial fundal myometrial bleeders are encountered it signifies that the septum has been accurately resected to the optimal depth and any further resection would amount to an over resection. During the course of septoplasty, the physiological patency of the tubes is also evaluated by recording the minimum intrauterine pressure required to push the micro bubbles retrogradely into each tubal opening.

Hysteroscopic assessment of selective tubal pressures and tubal cannulation by air bubble stents

HYSTEROSCOPIC FIBROID RESECTION: AN ENDOMETRIAL AND MYOMETRIAL CONSERVING APPROACH With hysteroscopy, it is possible to remove both intramural and submucous fibroids. Aprior ultrasonography is essential to map the exact location of the fibroid. The measurement of the intracavitary bulge of the fibroid and the thickness of the myometrium between the outer surface of the fibroid and the serosa are essential if a complete removal of the fibroid has been planned via the hysteroscopic route. A sagittal incision is made over the fibroid by the collins knife. As this incision is deepened the plane of cleavage between the pseudocapsule and the overlying circular muscle fibres of the myometrium becomes visible. The resectoscope loop is then insinuated in the plane between the pseudocapsule and the circular myometrial fibres and the resection of the fibroid is started. Thus the entire fibroid can be removed without sacrificing the overlying endometrium. It is important not to cause thermal injury to the myometrium because this may lead to adhesion formation later on. If a fibroid is resected along with its overlying endometrium, a very large amount of overlying endometrium is sacrificed, and this may impair the post operative fertility status. Thus the endometrial and myometrial conserving approach to myomectomy can be useful when resecting a fibroid in a case of fertility impairment. HYSTEROSCOPIC MARKERS FOR ENDOMETRITIS Hysteroscopic visualization of the endometrium is a subjective analysis because the interpretation may vary from observer to observer. But, with increasing experience of a surgeon more and more objectivity is ultimately induced into the procedure. Endometrial evaluation also has some practical problems. Either the endometrium appears healthy or it can look unhealthy. The color and the glandular architecture are important, e.g. a healthy endometrium in the early proliferative phase has smooth contours and a pink to red color. A closer inspection under video magnification shows the openings of the endometrial glands. But it is important to see whether these gland openings are surrounded by a zone of hyperemia or red congestion because this type of clinical picture may be associated with endometritis. Hysteroscopic visualization may help in raising a strong suspicion of endometrial tuberculosis. In the immediate post menstrual phase the endometrium may have a rough surface because the healing process has just started. But if the surface is rough even in the late proliferative phase, the color is whitish and the individual gland openings cannot be seen even under magnification, under such a situation a suspicion of endometrial tuberculosis may be raised. Such patients, if put on an empirical antitubercular therapy (ATT) often show an improvement in the menstrual flow which supports the hysteroscopic suspicion in favor of endometrial tuberculosis. The possibility of endometrial tuberculosis becomes even more if the endometrium appears thin, rough, whitish and shabby, with complete loss of glandular architecture even in the premenstrual phase. Interspersed islands of good looking endometrium may also be very often seen surrounded an overall whitish shabby looking endometrium. This may be a good

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prognostic indicator if an empirical ATT has to be started. All video films of the initial hysteroscopy should be compared with repeat hysteroscopies to look for any visible improvementin the endometrium after ATT.

CHAPTER 69 Intrauterine Adhesions Rakesh Sinha INTRODUCTION Intrauterine adhesions (synechiae), relatively uncommon in the developed countries, is increasingly witnessed in the developing countries. The diagnosis of this lesion is easier now with the increased usage of hysteroscopy. These adhesions most often occur secondarily to the uterine wall trauma by severe abrasion of the endometrium, particularly when the myometrium is soft and congested, as is the case with the gravid uterus. This condition can be responsible for disturbances of the menstrual cycle (amenorrhoea and hypomenorrhea), infertility, spontaneous abortion and placenta accreta. In 1948, Asherman described ‘amenorrhea traumatic’ as amenorrhoea secondary to intrauterine adhesions, following a curettage for incomplete or missed abortion and postpartum hemorrhage.1 The term ‘Ashermans Syndrome’ is used to describe this condition. Aetiology and Pathogenesis Only infection rarely causes adhesions, except in cases of tuberculous, endometritis (increased incidence in developing countries). Most frequently, more than 90 percent intrauterine adhesions develop after a curettage.2 The most important factor in the development of intra uterine adhesions is traumatic curettage or manipulation of the endometrium during the postpartum or post-abortal period. The denudation of the basalis layer and exposure of the muscularis layer produces adhesions by coaptation between the opposing uterine walls.3 Diagnosis Hysterosalpingography is the most accurate screening method in the diagnosis of intrauterine adhesions. Radiographic filling defects are suggestive of synechiae. Dilatation and curettage are not of diagnostic value for intrauterine adhesions. In a patient who complains of hypomenorrhea or amenorrhoea following history of postpartum or post abortal curettage, failure to elicit withdrawal bleeding after a progesterone challenge test may suggest the diagnosis. Hysteroscopy confirms the diagnosis and allows for treatment-lysis at the same sitting. The ideal time to perform hysteroscopy in these patients is post-menstrual, (when they

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have hypomenorrhea), unless they have amenorrhoea, in which case the hysteroscopy may be done earlier. Classification According to Hamou (1981), intrauterine adhesions are classified as endometrial, myofibrous or fibrous. Valle and Sciarra4 classified intrauterine adhesions as mild, moderate and severe, based on the degree of intrauterine involvement on HSG and the extent and type of adhesions found on hysteroscopy. Mild adhesions are filmy adhesions composed of basalis endometrial tissue, producing partial or complete uterine cavity occlusion. Moderate adhesions are fibromuscular characteristically thick and still covered with endometrium; severe adhesions are composed of connective tissue only.5 The American Fertility Society6 has proposed a classification of intrauterine adhesions based on the findings at HSG and Hysteroscopy and the correlation with menstrual patterns. J Donnez and M Nisolle use their own classification essentially based on the location of the Intrauterine adhesions7 Table 69.1. Technique Hysteroscopic adhesiolysis can be performed by breaking filmy, thin adhesions with help of the telescope itself. In some cases mere dilatation of the cervix results in the breaking of adhesions.

Table 69.1: Classification according to the location and the aspect of the adhesions7 Degree Location I.

II.

III.

Central adhesion (bridge-like) a. Thin or firmly adhesion (endometrial) b. Myofibrous or connective adhesions. Marginal adhesions (always myofibrous or connective) a. Ledge-like projections b. Obliteration of one horn Uterine cavity ‘absent’ on HSG a. Occlusion of the internal os (upper cavity normal psudoasherman’s syndrome) b. Extensive coaptation of the uterine walls (absence of uterine cavity)—true Asherman’s syndrome

True hysteroscopic adhesiolysis can be performed by using semi-flexible scissors, the bipolar electrode or laser fiber through the accessory port of a therapeutic continuous flow hysteroscope. Alternately the resectoscope can be used with the right angled electrode. The therapeutic hysteroscope is 21 French or 24 French and can be inserted by dilating the cervix to 7.5 mm to 8.5 mm Hegar dilatation. The medium for distension should be

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saline if the bipolar electrode is used. A 4 mm 30° fore oblique view telescope is ideal, however, never 3 mm telescope with small diameter sheath are now available. These offer operative hysteroscopy with lesser dilatation thereby facilitating surgery in nulliparous patients. The inflammation and tissue damage is lesser when adhesiolysis is performed with the help of scissors, bipolar electrode and Nd: YAG laser fiber. May be the postoperative recurrence of adhesions are less with these modalities in comparison to the monopolar current utilized with right angled electrode. The resectoscope is available as 24 French or 27 French and is an extremely versatile instrument. It utilizes monopolar current to achieve adhesiolysis. If used correctly with appropriate current 70 watts and unmodulated, the results obtained are good. The risk of perforation is high in grade III adhesions where the cavity has to be reformed. Extreme care is necessary and the lysis should proceed very gently so that a triangular cavity is achieved. The distension pressure of the fluid medium helps in identifying the cavity. Fluid overload may occur if 1.5 glycine is utilized and the procedure lasts for a long time. Hemorrhage is uncommon but may result if larger lateral wall vessels open up. Postoperative insertion of a pediatric Foley’s catheter, distended with 5 ml of fluid, for 4–6 hrs achieves good hemostasis. The end point of adhesiolysis would depend on the grade of adhesions. In mild to moderate adhesions, a good view of the entire cavity with ostial openings being visualized is a good indicator. It is extremely difficult to decide the end point in severe cases where there is total obliteration of the uterine cavity. It is not practical to perform hysteroscopic adhesiolysis under laparoscopic control. Theoretically the concept is good, but is extremely difficult to visualize all the walls of the uterus laparoscopically while the hysteroscopy is being performed. Postoperative Prophylactic antibiotics are given intraoperatively and for 4–5 days postoperatively. Several gynecologists perform a second look hysteroscopy but we find this should be reserved for women who continue to have amenorrhoea/ hypomenorrhea or those who are relieved of their symptoms but hypomenorrhea recurs. The subsequent insertion of an inert intrauterine device is being questioned by several workers, since the presence of a foreign body in contact with raw endometrial wall can itself precipitate adhesions. The postoperative hormonal treatment is recommended for patients of severe adhesions. Ideal treatment is conjugated estrogen from 3.75 mg to 5 mg daily in divided doses for 21 days. To this progesterone is added in the last 10 days. The doses are monitored depending on the menstrual response. A study by San Fillipo and lan S Fraser et al8 reported the use of hysteroscopy for lysis of intrauterine adhesions under ultrasound guidance. In their study the aim was to assess the value of simultaneous abdominal ultrasound scanning for the accuracy of hysteroscopic localization of endometrial or adenomyotic islands deep to synechiae in women with Asherman’s syndrome. The patients included 6 women with Asherman’s Syndrome. At hysteroscopy, under general anaesthesia, the ultrasound scans through

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adhesions to reach the island, treat the condition appropriately under direct vision. In women with Asherman’s syndrome the synechiae were successfully divided to restore normal cavity shape in all cases except one. Menses were restored or increased in volume in all cases and three women had successful pregnancies while two continued to have infertility. Fitzgerald, showed no significant difference between hormonal treatment and IUDC versus hormonal therapy alone. Success Rate Collectively success rates of 74–94 percent have been obtained (Table 69.2).6 It is incorrect to compare different series because the results have not been evaluated according to the degree of severity. In a recent review,6 the pregnancy rate was 60.5 percent and 80 percent of those pregnancies reached term.

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Fig. 69.1 A to F: Techniques of Hysteroscopic Versa Point Adhesiolysis

Fig. 69.2A: Right lateral wall adhesions

Fig. 69.2B: Left periostial adhesions

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Fig. 69.2C: Obliteration of right cavity

Fig. 69.2D: Anterior wall adhesions

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Fig. 69.2E: Left lateral wall adhesions

Fig. 69.2F: Fundal adhesions

Fig. 69.2G: Fundal adhesions

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Fig. 69.2H: Fundal adhesions

Fig. 69.2I: Obliteral of left side cavity Fig. 69.2A to I: Preoperative hysteroscopic view of uterine cavities

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Fig. 69.3A to I: Post-adhesiolysis hysteroscopic view of uterine cavities The reproductive outcome after hysteroscopic treatment of uterine synechiae at various centers is summarized in Table 69.2.

Table 69.2 No of cases Pregnancy % Livebirths % Sugimoto9 March & Israel10 Hamou et al11 Lancete Kessler12 Friedman et al13 Valle & Sciarra4

192 38 39 137 30 187

41.2 100 51.3 49 80 76.4

56.9 87.2 75 64.4 76.6 79.7

On the whole, after treatment hysteroscopy, one out of two women become pregnant and one out of three will have a live birth.

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CONCLUSION Hysteroscopic adhesiolysis is the gold standard since there is no other appropriate alternative treatment for intrauterine adhesions. The hysteroscopic treatment of intrauterine adhesions restored normal menstruation in more than 80 percent of treated patient.6 The results in terms of normal menses and pregnancy rates are excellent for those with adhesions of grades Ia,b; IIa,b. Operative hysteroscopy should be the first-choice treatment for any patient presenting with infertility caused by synechiae. The initial severity of lesion and quality of the rest of the endometrium are the important factors determining the prognosis. The results obtained for menstrual outcome and pregnancy seem encouraging. REFERENCES 1. Asherman JG. Amenorrhoea traumatic J Obtet Gynaecol Br Empire 1948; 55:23–30 2. Schenker JG, Margalioth EJ. Inrauterine adhesions; and update appraisal. Fertility Steril 1982; 37:593. 3. J Donnez, M Nisolle. Laser operative laproscopy and Hysteros-copy. Parthenon Publishing Group, 1994; 32:305. 4. Valle RF, Sciarra JJ. Intrauterine adhesions: Hysteroscopic diagnosis-classification treatment and reproductive outcome. Am Jr Obstet Gynecol 1988; 158:1459–70. 5. Valle R. Lysis of intra-uterine adhesions (Asherman’s Syndrome). In Sutton C, Diamond M (Eds): Endoscopic surgery for Gynaecologists. London: Saunders 1993; 38. 6. American Fertility Society. The American Fert Soc classification of adnexal adhesions, distal tubal occlusion, tubal occlusion secondary to tubal ligation, tubal pregnancies, mullerian anomalies and intra-uterine adhesion. Fertil Steril 1988; 49:944–55. 7. Donnez J, Nisolle M. Operative laser hysteroscopy in mullerian fusion defects and uterine adhesions. In Donnez J (Ed): Laser Operative Laparoscopy with Hyst. Leuren: Nauwelaerts Printing 989; 249–61. 8. lan S Fraser, Jing-Yu Song, Robert PS Jansen, Phillippa Ramsay, Tom Brogert. Gynaecological Endoscopy 1995; 4–35–40. 9. Sugimoto O. Diagnostic and therapeutic hysteroscopy in intrauterine adhesions. Am Jr of Obstetric and Gynaecologist 1978; 131:539–47. 10. March CM, Israel R. Gestational outcome following hysteroscopic lysis of adhesions Fertility and Sterility 1981; 36:455–59. 11. Hamou J, Salat-Baroux J, Sieglar AM. Diagnosis and treatment of Intra-uterine adhesions by microhysteroscopy. Fertil Steril 1983;3:39. 12. Lancet M, Kessler I. Traintement du syndrome d’ Asherman par Fhysteroscopie. Journal de Gyne’ cologie, Obstetrique et Biologie de la Reproduction 1986; 15:464. 13. Friedman A, Defazio J, De Cherney AM. Severe obstetric complication following hysteroscopic lysis of adesions. Obstet Gynaecol 986; 67:864.

SECTION 11 Ultrasonography

CHAPTER 70 Update on Ultrasound Guided Embryo Transfer Vishvanath C Karande INTRODUCTION Historically, embryo transfer (ET) technique is a topic that is less studied compared to other aspects of assisted reproduction. Over the past few years, this has changed and it is now generally accepted that paying a lot of attention to individual variations in transfer technique can positively impact success rates with IVF.1–2 Till recently there have been few randomized studies with large numbers evaluating variations in transfer technique. In this chapter, we will review some of the details of ET technique that impact outcomes. The role of ultrasound-guidance will be discussed in detail, and recommendations regarding the best transf er technique will be made. What Factors are Important for Successful ET? Kovacs3 reported on the results of a survey of the program directors of each IVF Unit in Australia and New Zealand. The questionnaire asked them their attitude with respect to 12 factors, which constitute the ET matrix. They were requested to rate each step on a scale of one to ten, where one was irrelevant and ten was very important Table 70.1 represents the cumulative experience of the 50 clinicians with >500 years of ‘hands on’ IVF practice. The most highly ranked factor was the removal of hydrosalpinges prior to starting a cycle. Number two on the list was the absence of bleeding/blood on the catheter. This probably is a strong indicator as to whether or not the transfer was traumatic. The use of ultrasound was almost at the bottom of the list. This was considered to be technically difficult when done transvaginally Surprisingly, the use of abdominal scanning was criticized as the bladder needed to be filled and the catheter tip was not easily visualized on the scan! ET techniques have changed significantly since Kovacs data were published and the use of ultrasound guidance is becoming routine. The impact of hydrosalpinx on success rates is beyond the scope of this chapter. We routinely remove hydrosalpinges that are visualized on ultrasound before proceeding with an IVF cycle.4–5 Before proceeding with the discussion on the use of ultrasound guidance, let us discuss some of the other factors that affect ET success rates.

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Table 70.1: The relative importance of each factor as rated by the total, mean score and SD. The maximum possible score for each variable was 500 Priority

Mean score SD Total score

1. Removal of hydrosalpinges 6.8 2.8 340 2. before treatment Absence of bleeding/blood on catheter 6.6 2.5 330 3. Type of catheter used 6.1 2.7 255* 4. Not touching the fundus 5.8 3.2 292 5. Avoiding the use of a tenaculum 5.7 2.9 283 6. Removal of all mucus from cervix 5.2 3.2 258 7. Ultrasound details of cavity before treatment 4.3 2.8 216 8. Leaving catheter in place for 4.2 3.1 211 9. at least 1 min 30 min rest after transfer 3.8 2.8 192 10. Dummy transfer before treatment 3.1 3.1 157 11. Ultrasonic monitoring of transfer 2.6 2.2 125 12. Antiprostaglandins to prevent contractions 1.9 1.5 93 *Only 42 clinicians responded to this question. Modified from Kovacs GT, Hum Reprod 1999; 14:3, 590–92.3

Absence of Bleeding/blood on Catheter The presence of blood inside the catheter tip after a transfer is associated with lower pregnancy rates.6 The blood is probably an indicator of trauma during transcervical passage of the catheter. Just having a difficult transfer does not necessarily result in lower pregnancy rates. Tur-Kaspa et al7 reported on a large series of patients where they categorized transfers as “easy” or “difficult” as assessed by the physician performing the transfer. They also evaluated the impact of cervical dilatation and the performance of multiple transfers due to retained embryos. The surprising finding was the similarity in pregnancy rates in patients with “easy” (23.3%), “difficult” (23.6%), cervical dilatation (23.8%), multiple transfers (29.6%) (Table 70.2). Groutz et al8 on the other hand, reported on 41 women that required cervical dilation at the time of oocyte retrieval and only 1 achieved an intrauterine pregnancy. Details on the presence or absence of blood at the catheter tip were not provided. Type of Catheter Used At our Center there was a definite increase in pregnancy rates when we switched to the Wallace catheter (Sims Portex, Ltd., UK).2 Over the past few years, there is a worldwide trend toward the use of “soft” catheters.9 The possible advantage of this type of catheter is its ability to follow the direction of the cervix and enter the cavity. Rigid catheters tend to penetrate the endometrial surf ace, and have the catheter tip plugged with mucus or endometrial tissue and cause bleeding. Wisanto et al10 studied three types of catheters in 400 patients retrospectively. The pregnancy rate with the Frydman catheter (32.3%) was better than the Wallace (19.2%), which did better than the TDT (19.4%).

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For difficult cervical insertions, we use the Wallace stylet. This consists of an outer sheath with a firm, coated rod in its lumen that provides stability to negotiate highly convoluted cervices. In rare instances, we use the Frydman catheter which has a wireguide which is used to negotiate the cervix. Details of the transfer technique are discussed later. Not Touching the Fundus The general consensus seems to be that the best area to deposit the embryos is 1 cm beyond the internal os and 1 cm below the fundus. This is usually 5–7 cm from the external os. Waterstone et al11 in a letter to the Lancet reported that the pregnancy rate between two clinicians differed based on the technique used. One clinician advanced the catheter until resistance was felt and then withdrew 5 mm before injection. His success rate (24%) was lower than that of another clinician (46%) who routinely deposited embryos at 5 cm past the cervical os. The pregnancy rate of the first physician increased to 46 percent when the low transfer technique was used. Rosenlund et al12 on the other hand could not correlate success rates with the site of deposition in a study using ultrasound measurement. The deposition of embryos close to the fundus may increase risk of an ectopic pregnancy.13 Avoiding the Use of a Tenaculum The use of a tenaculum or some kind of a “claw” instrument to hold the cervix and straighten out the canal to facilitate transfer should be avoided. Holding the cervix with a tenaculum stimulates uterine contractions. Lesny18 et al14 recently confirmed this. They performed mock ETs on 20 patients in the mid-luteal phase of the cycle. The patients were assessed with a transvaginal scan for 2 min to obtain baseline junctional zone activity and the images recorded on videotape. The cervix was then grasped with a Littlewood’s tissue forceps and the uterine position was corrected. The instrument was then released and the recording continued for a further 2 min. The images were then digitized into a computer and converted to 5 times normal speed to allow analysis of junctional zone contractions. The data clearly show an increase in the total number of contractions, the number of cervico-fundal, opposing and random contractions (Table 70.3). The mechanism for this increase in contractions remains elusive. Fanchin et al15 have shown that uterine contractions at the time of ET, negatively impact success rates. The same group assessed uterine contractility during the luteal phase of ovarian stimulation.16 They noted a slight, yet significant, decrease in uterine contraction frequency, observed from day of HCG (4.4±0.2 contractions/min)

Table 70.2: PR and outcomes for the different categories of ET Pregnancy outcome

Easy (%)

Difficult (%)

Ongoing or (delivered) Ectopic

118/171(69) 11/17(64.6) 3/5(60)

5/8(62.5)

137/201(68.2)

0

0

2/201(1)

2/17(11.8)

Cervical Dilatation Multiple (%) (%) 0

Total (%)

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3/8(37.5) 0 8/27(29.6)

768 33/201(16.4) 29/201(14.4) 201/854(23.5)

to HCG+4 (3.5±0.2 contractions/min), was followed by a more pronounced, additional decrease between HCG+ 4 and HCG+7 (1.5±0.2 contractions/min; P<0.001). They concluded that it is possible that such a uterine relaxation assists blastocyst implantation.

Table 70.3: Total number of junctional zone (JZ) contractions in a oup of 20 patients before and after the application of a tenaculum to the cervix Pattern of JZ Contractions Before application After application P-value* Cervicofundal 4 Fundo-cervical 0 Random 24 Opposing 35 Total 63 *Wilcoxan matched-pairs signed-rank test. Lesny P. et al Hum Reprod 1999; 14:2367–70.

34 9 45 58 146

0.005 0.067 0.001 0.007 0.0003

Removal of all Mucus from Cervix Poindexter et al17 showed the presence of embryos in the cervical mucus as well as the vagina and the speculum after transfer. The removal of cervical mucus seems to be important. Cervical mucus plugging the catheter tip may result in an increase in retained embryos, damage to embryos and improper placement. Cervical mucus adherent to the embryos may result in embryo expulsion after transfer (“sling-shot” effect). Mansour et al18 performed dummy transfers and showed that prior aspiration of cervical mucus led to methylene blue in the cervix on 23 percent of the transfers. Without aspiration, 57 percent of patients demonstrated methylene blue in the cervix. MacNamee recommends a vigorous cervical lavage with culture medium to remove all cervical mucus. A 10 cc syringe containing culture medium with the outer Wallace sheath is inserted 2 cm into the cervical canal. This can sometimes result in medium entering the uterine cavity. This does not seem to effect pregnancy rates. In addition to removing cervical mucus, cervical lavage may also reduce the amount of bacteria present in the cervical canal. The presence of bacteria at catheter tip has been shown to negatively impact pregnancy rates.19–20 A recent multicenter randomized study21 prospectively evaluated 253 patients. Surprisingly, the group with cervical irrigation showed a lower pregnancy rate (45% vs 57%, p=0.051). At our Center we carefully remove cervical mucus using plastic Q-tips. In patients with copious cervical mucus, we use a 10 cc syringe to aspirate the mucus. We do not irrigate the cervix.

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Ultrasound Details of Cavity Before Treatment Evaluation of the uterine cavity by hysterosonography, hysterosalpingography or hysteroscopy is routinely performed by most centers. This is done to rule out polyps, fibroids, adhesions and other intrauterine abnormalities. We routinely evaluate the cavity with one of these techniques. In experienced hands, all three techniques are adequate. Leaving Catheter in Place for at Least 1 min 22

Martinez et al investigated the influence that the time interval before withdrawal of the catheter after ultrasoundguided ET might have on pregnancy rates with IVF. They prospectively randomized 100 women about to undergo transfer of at least two optimal embryos to two groups. Fifty-one women had the catheter slowly withdrawn immediately after embryo deposit. In 49 women, there was a 30s delay before catheter withdrawal. The pregnancy rates for transfer in the two groups were 60.8 and 69.4 percent respectively, with no significant differences. The results indicate that either the waiting interval was insufficient to detect differences, or that the retention time before withdrawing the catheter is not a factor that influences pregnancy rate. Thirty min rest After Transfer In the early days of IVF, it was routine to keep the patient in bed rest often in a Trendelenberg position for 4 hours post transfer. The rest period seems to have been reduced by most programs to 30–60 min. Sharif et al23 showed no decrease in pregnancy rates with no bed rest after transfer. This was also confirmed in another study.24 With the use of transabdominal ultrasound guidance, we routinely require the patients to have a full bladder prior to ET. We allow patients to rest for a few minutes if they so desire. We have noticed no decrease in pregnancy rates in patients who get up immediately after transfer to void. Dummy Transfer Before Treatment Mansour et al25 ina large randomized study demonstrated the benefit of a dummy transfer. Dummy transfer reduced the incidence of difficult transfers from 29.8 percent to nil, and the pregnancy rates significantly increased from 13.1 to 22.8 percent. This can be carried out in a prior cycle or at the time of retrieval. The use of ultrasound guidance mandates a full bladder and this sometimes changes the direction of the cervical canal. If the dummy transfer is carried out with the bladder empty, one should be aware that the cervical direction might change. This is not a major problem as the catheter tip can usually be easily followed into the uterine cavity. A full bladder itself can sometimes make a difficult transfer easy by straightening out the cervix. Transmyometrial transfers have been recommended in patients where the cervix is completely inaccessible.26 Ultrasound Monitoring of Transfer Ultrasound guidance for ET was initially described by Strickler27 and then by Leong28 more than 15 years ago. Patients underwent transvaginal ET, which was monitored by

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transabdominal ultrasound. This necessitated the need for a full bladder at the time of transfer for proper visualization of the catheter tip. Initial publications showed marginal improvements in success rates. However, more recently there have been several publications, which showed statistically significant improvement in pregnancy rates with ultrasound guidance. This is also conformed by the fact that an increasing number of programs in the US now routinely use ultrasound-guidance. Kan et al29 randomly assigned 187 patients to ultrasound-guided or clinical touch transfers. There was a nonsignificant trend towards higher pregnancy rates with ultrasound guidance in the whole group (37.8 versus 28.9%) and in subsets of older women (38.1 versus 20.4%). Kan et al29 suggest that ultrasound-guidance should be used in clinically difficult transfers and in older women as it seemed to improve the pregnancy rate over clinical touch transfers (Table 70.4). Hurley et al30 showed some improvement in patients with single ETs. Lindheim et al31 showed improved pregnancy rates with ultrasound guidance in donor oocyte cycles. In patients with easy transfers, ultrasound guidance improved implantation (28.8% vs 18.4%) and pregnancy rates (63.1% vs 36.1%). Coroleu et al32 conducted a large prospective randomized study and compared 182 patients who had an ultrasoundguided ET with 180 patients who had clinical touch ET. There were no significant differences between the two groups in terms of age, cause of infertility or in the characteristics of the IVF cycle. The pregnancy rate, however, was significantly higher among the ultrasoundguided group (50%) compared with the clinical touch group (33.7%). Furthermore, there was also a significant improvement in the implantation rate: 25.3 percent in the ultrasound group compared with 18.1 percent in the clinical touch group. Wood et al9 also noted a statistically significant improvement in pregnancy rates when they switched to soft catheters and used ultrasound guidance. This study, however, was a retrospective analysis of data and lacked randomization.

Table 70.4: Ultrasound vs clinical: outcome “Ultrasound” group “Clinical” group Pregnancy rate 37/98(37.8) Implantation rate Pregnancy rate 53/260(20.4) One embryo transferred 1/5(20) Two embryos transf erred 5/24(20.8) Three embryos transferred Pregnancy rate 31/69(44.9) <37 years old 21/56(37.5) ≥37 years old Ease of transfer 16/42(38.1) ‘Easy’ and ‘difficult’ 13/26(50) ‘Difficult’ only 6/11(54.5) Values in parentheses are percentages. There were no significant differences between the groups. Kan AKS et al Hum Reprod 1999; 14:1259–61.

28/97(28.9) 41/253(16.2) 2/12(16.7) 4/14(28.6) 22/71(31) 17/43(39.5) 11/54(20.4) 6/22(27.3) 1/10(10)

Sub-endometrial transfers are associated with lower pregnancy rates than when the embryos are transferred in the cavity itself,33 With ultrasound visualization of the tip it is possible to avoid this from occurring. In cases with sub-endometrial transfer, the transf er-associated air bubble remains beneath the endometrium following withdrawal of the transfer catheter. These authors using a Jansen-Anderson K-JITS 2000 catheter set

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analyzed 121 embryo transfers. The transfers were performed using tactile assessment. Once the catheter was in a position the operator believed to be in the endometrial cavity, the speculum was removed and a transvaginal ultrasound probe was inserted to assess the position of the catheter. They concluded that tactile assessment of ET catheter placement is unreliable. In 17.4 percent of patients, the outer guiding catheter inadvertently abutted the fundal endometrium. The outer guiding cannula indented the endometrium in 24.8 percent and the transfer catheter embedded the endometrium in 33.1 percent. Unavoidable sub-endometrial transfers occurred in 22.3 percent of transfers. In a subsequent publication, Woolcott and Stanger34 tracked the movement of embryoassociated air bubbles on standing after transfer. Ninety-three patients undergoing 101 consecutive ETs were evaluated. After undergoing ultrasound-guided ET, the patients underwent a second scan in standing position immediately after transfer, allowing the movement of the embryo-associated air to be assessed. No movement occurred in 94.1 percent (95/101) of transfers, movement of <1 cm in 4 percent (4/101) of transfers and movement of 1–5 cm in 2 percent (2/101) transfers. In none of the patients did the embryo-associated air move out of the uterine cavity either into the cervix or the intramural portion of the Fallopian tube. They conclude that standing shortly after ET does not play a significant role in the final position of embryo-associated air and is unlikely to be a factor in determining the position of embryos transferred to the uterine cavity during ET. A recent development is the availability of a coaxial catheter system with an echodense tip for ultrasound guided ET (1999). The Cook Echo-Tip® catheter (Cook Ob/Gyn, Spencer, Indiana) (Fig. 70.1) is a modification of the universally used soft-tip Wallace catheter (SIMS Portex Ltd. Hythe, Kent, UK) and has an echogenic band at its tip. This is supposed to make visualization of the tip easier and facilitate ET. Since the tip is echo dense, it is seen easily in obese patients as well as in patients where the bladder is not optimally full. Letterie et al35 performed 20 transfers with a clinical pregnancy rate of 45 percent per cycle. They noted that the outer sheath of the system was well visualized during passage through the cervix and into the lower uterine segment due to thickness of the catheter. With the echo-dense tip, immediate recognition of the tip of the inner sheath was achieved in all patients. With small movements of the ultrasonographic transducer in the transverse plane, the echodense catheter tip could be easily tracked during passage through the entire uterine cavity into the fundal region during the first pass. The ability to identify the tip of the inner catheter by the movements of the transducer minimized the amount of to-and-fro motion necessary to identify the catheter tip. This is unlike conventional catheters where one has to often move the tip for proper identification. The authors commented that this would maximize the ability to transfer embryos atraumatically, which is extremely important in embryos that have undergone micromanipulation. We prospectively compared the Cook Echo-Tip catheter with the Wallace catheter in a randomized study36 (Fig. 70.2). In order to eliminate any variation in success rates due to “physician factor” a single physician performed all ETs.2 All ET were performed on Day3 after retrieval. A similar technique was used in all patients. Transabdominal ultrasound guidance took place using a 3.5 MHz linear array probe (RT-3200 Advantage I, General Electric, Milwaukee, Wisconsin) with the patients having a full bladder. The cervical mucus was carefully cleaned prior to transfer using plastic Q-tips. In patients with

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copious mucus, a 10 cc syringe was used to aspirate the mucus. Initially, ET was attempted with the loaded intact catheter connected to a tuberculin syringe. If the first pass failed to negotiate the cervix with ease, the catheter was gently withdrawn. A Wallace stylette (SIMS Portex Ltd. Hythe, Kent, UK) was then inserted under ultrasoundguidance till its tip reached the base of the endometrial stripe. The obturator was then removed and replaced by the floppy inner catheter, which was loaded with the embryos. In either case, the embryos were then gently expelled into the uterine cavity with the catheter tip positioned within 1–2 cm of the fundus. Care was taken to avoid white-knuckling the fingers while expelling the embryos. We were careful to avoid touching the uterine fundus. The catheter was then slowly withdrawn over 30s with a rotating motion. A tenaculum was not used in any of the cases. Care was also taken to avoid a subendometrial transfer and the embryos were placed within 1–2 cm from the fundus by monitoring the “embryo bubble” which is formed by the fluid and/or the air surrounding the embryos. The catheters were closely inspected under a stereomicroscope to confirm that all embryos had been expelled and for the presence of blood at the tip. Patients were encouraged to void and leave within a few minutes after the transfer.

Fig 70.1: The Cook Echo-Tip® Catheter sysetm with its echogenic tip

Figs 70.2A ahd B: Comparison of the appearance of the Wallace (A) and the Echo-Tip® Catheter (B) at the time of ET. In both cases, the “embryo

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bubble” is clearly seen. The echogenic band at the tip of the Cook Catheter is clearly seen whereas the Wallace tip can be discerned less clearly As expected, the catheter tip was always seen with the Echo-Tip, but not clearly visualized in 5 (9.4%) patients in the Wallace catheter group. The stylette was used in 14 (26%) patients in the Wallace group and 16 (34%) with the Echo-Tip catheter. The embryologists initially observed that the echogenic band of the Cook Echo-Tip® catheter interf ered with the loading of embryos but over a period of time adjusted to it. Pregnancy and implantation rates, however, were similar in both groups (Table 70.5). A recent publication surveyed ET practice in the UK.37 The factor that got the highest rating was the need for a standardized protocol for all unit staff regarding ET policy. The second critical factor was the presence of blood on the embryo catheter at the end of the ET process. Not touching the uterine fundus, type of catheter used and avoiding the use of tenaculum rounded off the top five. Ultrasound monitoring of ET was listed number 14 on the list after removal of all mucus from cervix, removal of hydrosalpinges before treatment, catheter rotation, ultrasound details of cavity before treatment, leaving catheter in place for 30s, full bladder for ET, dummy transfer before actual ET, and dummy transfer before treatment cycle. The least important factor was prolonged

Table 70.5: Comparison of the Cook Echo-Tip and the Wallace catheter for ultrasound guided ET (PR=pregnancy rates; IR=implantation rates). PR

Cook Echo-Tip® IR

PR

Wallace IR

Female Age <30 y 55% (6/11) 39% (9/23) 73% (8/11) 44% (12/27) 30–34 y 52% ( 12/23) 37% (17/46) 39% (7/18) 25% (11/43) 35–39 y 63% (5/8) 25% (6/24) 50% (8/16) 32% (12/38) ≥40 y 0% (0/5) 0% (0/12) 63% (5/8) 23% (6/26) No. of embryos 1 20% (1/5) 20% (1/5) 0% (0/4) 0% (0/4) 2 55% (17/31) 37% (23/62) 52% (13/25) 40% (20/50) 3 33% (2/6) 22% (4/18) 62% (10/16) 31% (15/48) 4 60% (3/5) 20% (4/20) 63% (5/8) 19% (6/32) Total 49% (23/47) 31% (32/105) 53% (28/53) 31% (41/134) Karande et al 2001.

bed rest following ET. This publication would be more interesting if they had compared the embryo transfer techniques between programs with high and low pregnancy rates.

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SUMMARY The ultimate goal of a successful ET is to deposit embryos without trauma into the uterine cavity. This apparently simple task is fraught with subtle variations that can significantly affect outcomes. Data from large programs have shown wide variation in success rates based upon the individual physician doing the transfer.2,38 Ultrasoundguidance during ET can be a valuable tool that can improve success rates. Direct observation of the catheter tip can be useful especially in patients with variations of normal anatomy e.g. long cervix, acutely anteverted or retroverted uteri. This will minimize the possibility of transf erring embryos in a blind passage in the cervix, detect when the catheter tip is f olding back on itself, and makes it possible to avoid subendometrial transfer of the embryos.33 Ultrasound-guided embryo transfer does require a certain degree of eye-hand coordination and like any other new technique has a learning curve.38 At our Center, we have been routinely using this technique and our current pregnancy rates are the highest they have ever been since the inception of the IVF program more than 15 years ago. REFERENCES 1. Naaktgeboren N, Broers FC, Hijnsbroek I et al. Hard to believe, hardly discussed, nevertheless very important for the IVF/ICSI results: embryo transfer technique can double or halve the pregnancy rate. Hum Reprod 1997; 12 (Suppl.) 149. 2. Karande V, Morris R, Chapman C, Rinehart J, Gleicher N. Impact of the “physician factor” on pregnancy rates in a large assisted reproductive technology program: do too many cooks spoil the broth? Fertil Steril 1999;71:1001–09. 3. Kovacs GT. What factors are important for successful embryo transfer after in-vitro fertilization? Hum Reprod 1999; 14:590–92. 4. Strandall A, Lindhard A, Waldenstrom U et al. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod 1999; 14:2762–69. 5. Strandell A, Lindhard A. Salpingectomy prior to IVF can be recommended to a well-defined subgroup of patients. Hum Reprod 2000; 15:2072–74. 6. Englert Y, Puissant F, Camus M, van Hoech J, Leroy F. clinical study on embryo transfer after human in vitro fertilization. J. In Vitro Fert Embryo Transfer 1986; 3:243–46. 7. Tur-Kaspa I, Yuval X Bider D, Levron J, ShulmanA, Dor J. Difficult or repeated sequential embryo transfers do not adversely affect in-vitro fertilization pregnancy rates or outcome. Hum Reprod 1998; 13:2452–55. 8. Groutz A, Lessing JB, Wolf X Yovel I, Azem F, Amit A. Cervical dilation during ovum pick-up in patients with cervical stenosis: effect on pregnancy outcome in an in-vitro fertilizationembryo transfer program. Fertil Steril 1997; 67:909–11. 9. Wood EG, Batzer FR, Go KJ, Gutmann JN, Corson SL. Ultrasound-guided soft catheter embryo transfers will improve pregnancy rates in in-vitro fertilization. Hum Reprod 1999; 14:107–12. 10. Wisanto A, Janssens R, Deschacht J, Camus M, Devroey P, Van Steirteghem A. Performance of different embryo transfer catheters in a human in-vitro fertilization program. Fertil Steril 1989; 52:79–84. 11. Waterstone J, Curson R, Parsons J. Embryo transfer to low uterine cavity. Lancet 1991; 337:1413.

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12. Rosenlund B, Sjoblom P, Hilensjo T. Pregnancy outcome related to site of embryo deposition in the uterus. J Assist Reprod Genet 1996; 13:511–13. 13. Yovich JL, Turner SR, Murphy AJ. Embryo transfer technique as a cause of ectopic pregnancies in in-vitro fertilization. Fertil Steril 1985; 44:318–21. 14. Lesny P, Killick SR, Robinson J, Raven G, Maguiness SD. Junctional zone contractions and embryo transfer; is it safe to use a tenaculum? Hum Reprod 1999; 14:2367–70. 15. Fanchin R, Righini FO, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998a; 13:1968– 74. 16. Fanchin R, Ayoubi JM, Righini C, Olivennes F, Schonauer LM, Frydman R. Uterine contractility decreases at the time of blastocyst transfers. Hum Reprod 2001; 16:1115–19. 17. Poindexter A, Thompson D, Gibbons W, Findley W, Dodson M, Young R. Residual embryos in failed embryo transfer. Fertil Steril 1986; 46:262–67. 18. Mansour RT, Aboulghar MA, Serous GI, Amin Y. Dummy embryo transfer using methylene blue dye. Hum Reprod 1994; 9:1257–59. 19. Egbase P, al-Sharhan M, Al-Othman S et al. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in vitro fertilization and embryo transfer. Hum Reprod 1996; 11:1687–89. 20. Fanchin R, Harmas A, Benaoudia F, Lundkvist U, Olivennes F, Frydman R. Microbial flora of the cervix assessed at the time of embryo transfer adversely affects in-vitro fertilization outcome. Fertil Steril 1998; 70:866–70. 21. Glass KB, Green CA, Fluker MR, Schoolcraft WB, McNamee PI, Meldrum DR. Multicenter randomized controlled trial of cervical irrigation at the time of embryo transfer. Fertil Steril 2000; 74(Supplement): S31 22. Martinez F, Coroleu B, Parriego M, Carreras O, Belil I, Parera N et al. Ultrasound-guided embryo transfer: immediate withdrawal of the catheter versus a 30 second wait. Hum Reprod 2001; 16:871–74. 23. Sharif K, Afnan M, Lashen H et al. Is bed rest following embryo transfer necessary? Fertil Steril 1998; 69:478–81. 24. Botta G, Grudzinskas G. Is a prolonged bed rest following embryo transfer useful? Hum Reprod 1997; 12:2489–92. 25. Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in-vitro fertilization. Fertil Steril 1990; 54:678–81. 26. Kato O, Takatsuka R, Asch R. Transvaginal-transmyometrial embryo transfer: the Towako method; experience of 104 cases. Fertil Steril 1993; 59:51–53. 27. Strickler RC, Christianson C, Crane JP et al. Ultrasound guidance for human embryo transfer. Fertil Steril 1985; 43:54–61. 28. Leong M, Leung C, Tucker M et al. Ultrasouind-assisted embryo transfer. J In Vitro Fert Embryo Tranf 1986; 3:383–85. 29. Kan AKS, Abdalla HI, Gafar AH, Nappi L, Ogunyemi BO, Thomas A et al. Embryo transfer: ultrasound-guided versus clinical touch. Hum Reprod 1999; 14:1259–61. 30. Hurley V, Osborn J, Leoni M, Leeton J. Ultrasound-guided embryo transfer: a controlled trial. Fertil Steril 1991; 55:559–62. 31. Lindheim SR, Cohen MA, Sauer MV. Ultrasound guided embryo transfer significantly improves pregnancy rates in women undergoing oocyte donation. Int J Gynaecol Obstet 1999; 66:281–84. 32. Coroleu B, correras O, Veiga A et al. Embryo transfer under ultrasound guidance improves pregnancy rates after in-vitro fertilization. Hum Reprod 2000; 15:616–20. 33. Woolcott R, Stanger J. Potentially important variables identified by transvaginal ultrasoundguided embryo transfer. Hum Reprod 1997; 12:963–66.

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34. Woolcott R, Stanger J. Ultrasound tracking of the movement of embryo-associated air bubbles on standing after transfer. Hum Reprod 1998; 13:2107–09. 35. Letterie GS, Marshall L, Angle M. A new coaxial system with an echodense tip for ultrasonographically guided embryo transfer. Fertil Steril 1999; 72:266–68. 36. Karande VC, Hazlett D, Gleicher’N. A prospective randomized comparison of the Wallace catheter and the Cook Echo-Tip catheter during ultrasound-guided embryo transfer. Oral Presentation at the Annual Meeting of the American Society for Reproductive Medicine, Orlando, Florida, October 2001. 37. Salha OH, Lamb VK, Balen AH. A Ipostal survey of embryo transfer practice in the UK. Hum Reprod 2001; 16:686–90. 38. Papageorgious TC, Hearns-Stokes RM, Leondires MP, Miller BT, Chakraborty P, Cruess D et al. Training of providers in embryo transfers: what is the minimum number of transfers required for proficiency? Hum Reprod 2001; 16:1415–19.

CHAPTER 71 Cervical Mucus Evaluation by Transvaginal Ultrasonography: A Novel Approach Ran Keidar, Ariel Jaffa, Igal Wolman INTRODUCTION The morphological configuration of the uterine cervix and the unique biochemical structure of the cervical mucus with its cyclical changes, accounts for the following roles of the cervix: 1. providing periovulatory receptive environment for spermatozoa, while in other parts of the cycle, this microenvironment is inhibitory in terms of sperm propagation. 2. acting as a reservoir for spermatozoa. 3. defending the spermatozoa from the hostile acidic vaginal secretions. 4. providing the energy demands of the spermatozoa. 5. refining the ejaculate of morphologically abnormal and immotile sperm. 6. providing terms for sperm capacitation. The chemical composition of the cervical mucus, its physical characteristics and the volume secreted, as well as the dimensions and structure of the cervix itself, show cyclical changes along the menstrual cycle. These changes are hormone-dependant: estrogen induces secretion of a large volume of clear acellular watery mucus, which is highly receptive to spermatozoa. Progesterone, on the other hand, inhibits the secretory activity of the cervical epithelium to produce low volume of thick, cellular mucus that can not be penetrated by the spermatozoa. The “open window” for fertilization (at least as far as the cervix is concerned) starts with the estrogen-peak just prior to ovulation which provides maximal stimulation to the cervical glands. It lasts till only 2–3 days after ovulation when progesterone levels increase. Based on this knowledge, examination of the cervical mucus (its quantity and physical characteristics) was used as a clinical marker for the timing of ovulation, as well as an indicator of the cervical reaction to estrogenic stimulation.2,5 The accepted parameters for cervical scoring were: 1. amount of mucus 2. mucus stretchability (Spinnbarkeit) 3. ferning pattern 4. mucus viscosity 5. mucus cellularity3,4 The cervical scoring systems based on such parameters were highly reliable as indicators for folliculogenesis and dating of the LH surge and ovulation.3,6–9

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In addition to the normal dynamics of ovulation and sufficient estrogen secretion, conception depends on adequate response of target organs to these stimuli. If the uterine cervix, as a target organ, fails to respond and create an optimal passage for spermatozoa, low sperm-receptivity leads to infertility.9 Cervical scoring, besides being an indirect parameter of folliculogenesis and normal estrogenic environment, is an indication for the cervical response and reflects the functional status of the genital tract, regarding sperm transport: poor cervical mucus at ovulation, as a consequence of poor hormonal milieu, prior morphological damage1 or due to pharmacological treatment like clomiphene citrate, should be diagnosed and treated either by an estrogenic boost or by intrauterine insemination.10 Although vaginal ultrsonography is part of the mainstay of follow-up during ovulation induction, no attention was paid till now to the application of this modality for cervical status assessment. The aim of this study was to evaluate ultrasonographic cervical parameters, and to correlate them with the common cervical mucus parameters in patients undergoing inf ertility workup or treatment. MATERIALS AND METHODS Our study group consisted of 101 patients evaluated and treated for infertility in our institute between 1998–1999. The patients were divided into three subgroups according to treatment protocol: 1. Natural cycle (during preliminary investigation), N=23. 2. Patients treated by clomiphene citrate for ovulation induction. N=56. 3. Patients treated by menotropins for ovulation induction, N=22. Each subject was fully informed about the study protocol and provided signed informed consent, which was approved by the institutional review board. ATL Ultramark 9 HDI. All patients were prospectively evaluated, through two phases: Phase I Vaginal ultrasonographic scanning was performed (ATL Ultramark 9 HDL 5–9 MHz vaginal probe). The following parameters were registered: 1. maximal cervical canal diameter (millimeters) 2. thickness of the cervical glandular layer (millimeters) 3. cervical canal length (centimeters) 4. number of follicles in each ovary 5. largest follicle diameter (millimeters)

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Phase II Immediately after the vaginal sonography, a second examiner who was blinded to the scanning results, performed cervical-mucus evaluation. Three parameters were recorded and graded as follows: Amount of Mucus scant: 1, medium: 2, abundant: 3 Quality of Mucus poor (thick, turbid): 1, good (clear, watery): 3 Spinnbarkeit <2 cm: 1, 2–5 cm: 2, >5 cm: 3. A cervical mucus-score was talculated for each patient. (range: 3–9) The ultrasonography data and the numerical cervical scores were then compared. The results were analyzed using the BMDP statistical software.13 The ultrasonography results were expressed as mean±SE. Dichotomous variables were analyzed by Pearson’s chi-square for ordered categories. Continuous data were analyzed by analysis of variance (ANOVA). Correlation by Pearson’s correlation test. P<0.05 was considered to indicate statistical significance. RESULTS The mean values of the investigated parameters are shown in Table 71.1.

Table 71.1: Mean values of parameters Mode

Parameter

Mean±SE (Range)

Cervical inspection

Amount of mucus 1.3±0.09 (0–3) Quality of mucus 1.3±0.04 (1–2) Spinnbarkeit (cm) 3.9±0.3 (0–10) Vaginal Cervical canal diameter (mm) 1.5±0.1 (0–4) Ultrasonography Cervical length (cm) 2.1±0.08 (1–4.5) Glandular layer thickness (mm) 10.1±0.3 (4.4–23) Number of follicles (both ovaries) 1.7±0.08 (0–6) Diameter of largest follicle (mm) 16.8±0.7 (10–32) Laboratory Estradiol level pg/ml 325±29.3 (24–1498)

Positive linear correlation was found between the number offollicles, the diameter of the dominant follicle and the serum estradiol level: r=0.506 (P<0.001) and r= 0.59 (P<0.001) respectively. No correlation was found between the serum estradiol levels and cervical

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mucus parameters on inspection, nor the cervical parameters on vaginal ultrasonography. Analysis of data after dividing the patients into two subgroups: I—natural-cycle patients and patients treated with menotropins, II-patients treated with clomiphene citrate, revealed significant difference in terms of cervical canal diameter and glandular layer thickness. (Table 71.2). Only the cervical canal diameter was in significant positive correlation (r=0.47, P=0.008) with the serum estradiol level in subgroup I. No correlation was fonnd between serum estradiol level and cervical ultrasonography parameters in subgroup II.

Table 71.2 Treatment P υalue HMG+No treatment Clomiphene citrate (mean± se) (mean± se) Cervical canal diameter 1.9±0.2 Cervical length 2.1±0.1 Glandular layer thickness 11±0.5

1.1±0.1 2.1±0.1 9.4±0.4

0.001 N. S. <0.01

The correlation between the mean cervical mucus parameters and cervical ultrasonography parameters is shown in Table 71.3.

Table 71.3 Amount of mucus P value Quality of mucus P υalue Scant Medium abundant poor good (Mean± se) (Mean± se) Cervical canal diameter 1.0±02 1.9±0.1 2.9±0.2 <0.001 0.8±0.1 1.8±0.1 <0.001 Cervical length 2.0±0.2 2.2±0.1 2.4±0.2 N. S. 2.0±0.2 2.2±0.1 N. S. Glandular layer thickness 9.3±0.4 9.5±0.5 12.8±1.0 <0.002 9.4±0.4 10.5±0.4 0.07

The cervical-mucus score values ranged between 3–, with a mean value of 5.6. The mean value (±se) for each treatment group was 6.1±0.5, 6.8±0.4, and 4.9±0.3, for natural cycle, HMG and clomiphen citrate patients respectively. (P<0.002). The correlation and calculated correlation-coefficient (R) between the cervical-mucus score and each of the cervical ultrasonography parameter is shown in Figure 71.1. Defining a cervical-mucus score of 5 as a cut-off value between favorable and hostile mucus, only the parameter of cervical canal diameter correlated significantly with the score, as shown in Table 71.4. By regressive analysis, a cervical-canal diameter of 0.97 mm correlated with a cervical-mucus score of 5 (Table 71.4).

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Table 71.4 Score=5 (Mean±se) Score >5 (Mean±se) Cervical canal diameter 0.9±0.1 Cervical length 2.0±0.2 Glandular layer thickness 9.6±0.3

2.1±0.1 2.2±0.09 10.6±0.5

P<0.001 N. S. N. S.

The distribution of patients according to the cervical canal diameter and cervical-mucus score is shown in Table 71.5. The sensitivity and specificity of cervical canal diameter measuring by transvaginal ultrasonography as a predictor of cervical mucus quality were found to be 83.7% and 80.8% respectively, with a PPV of 82.2% (Table 71.5).

Table 71.5 Low score (=5) High score (>5) Total P-υalue Canal diameter=1 mm* 42 (84%) 8 (16%) Canal diameter >1 mm* 10 (19.6%) 41 (80.4%) Total 52 49 * Approximation of the value –0.97 mm.

50 51 101

P<0.001

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Fig. 71.1: The correlation between the cervical score and the cervical parameters on transvaginal ultrasonography DISCUSSION Cervical mucus volume, its quality and stretchability (Spinnbarkeit) are well-accepted parameters of cervical receptivity under estrogenic influence. We prospectively studied vaginal ultrasonography as a method for indirect evaluation of the cervieal mucus. Primarily, as no correlation was found between the serum estradiol levels (the positive effector) and the cervical mucus parameters either by direct inspection or by sonography, we subdivided our patient population according to treatment protocol. Indeed, two ultrasonography cervical parameters-the cervical canal diameter and glandular layer width, were significantly different, comparing natural cycles and menotropins-treated patients with clomiphen citrate treated. Furthermore, the mean mucus cervical score, based on direct inspection, was significantly lower in the clomiphene-treated group. As clomiphene citrate might interfere with the estrogenic effect on the cervix,12 this emphasizes the importance of mucus evaluation in clomiphene-treated patients, in order to recognize those who will benefit from estrogenic boost or IUI. Correlating the cervical mucus parameters with the parameters of cervical ultrasonography, we were able to demonstrate a statistically significant positive correlation between the calculated cervical-mucus score and each of the cervical ultrasonography parameters. The cervical canal diameter was the single ultrasonographic parameter of which the correlation coefficient was the most significant. As this parameter’s correlation with the different inspected cervical parameters was consistently significant, we regressively evaluated its validity as a predictor of cervical mucus quality. At a cut-off value of 1 mm, the canal diameter had a good discriminative value between favorable and unf avorable cervical mucus (sensitivity=83.7%, specificity=80.8%, +PPV=82.2%). It can be concluded, from this study that transvaginal ultrasonography, which is routinely-used tool for follicular growth monitoring in patients undergoing ovulation induction, might also be implemented for evaluating the cervical mucus. With no additional effort or expanse, this data may help to optimize the treatment protocol. REFERENCES 1. Moghishi KS. Ovulation detection. Endocrinol Metabol Clin North Am 1992; 21:39–55. 2. Cardone A, Guida M, Lampariello C, Bruno P, Motemagno U. Objective and subjective data for fertile period diagnosis in women: comparison of method. Clin Exp Obstet Gynecol 1992; 19:15–24. 3. Insler V, Melamed H, Eichenbrenner I, Serr DM, Lunenfeld B. The cervical score. Int J Gynaecol Obstet 1972; 10:223. 4. Moghissi KS. The cervic in infertility. Clin Obstet Gynaecol 1979; 22:27–42.

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5. Depares J, Ryder RE, Walker SM, Scanlon MF, Norman CM. Ovarian ultrasonography highlights precision of symptoms of ovulation as markers of ovulation. Br Med J (Clin Res Ed) 1986; 292:1562. 6. Nulsen J, Wheeler C, Ausmanas M, Blasco L. Cervical mucus changes in relationship to urinary luteinizing hormone. Fertil Steril 1987; 48:783–86. 7. Kossoy LR, Hill GA, Parker RA, Rogers BJ, Dalglish CS, Herbert GM 3rd et al. Luteinizing hormone and ovulation timing in a therapeutic donor insemination program using donor semen. Am J Obstet Gynecol 1989; 160:1169–72. 8. Oelsner G, Pan SB, Barnea ER, Boyers SP, Tarlatzis BC DeCherney AH. The value of the cervical score in monitoring ovulation induction for in vitro fertilization: a prospective doubleblindstudy J Infert Embryo Transf 1986; 3:366–69. 9. Overstreet JW, Katz DF, Yudin AI. Cervical mucus and sperm transport in reproduction. Semin Perinatol 1991; 15:149–55. 10. Moghissi KS. Inflammatory and traumatic conditions of the cervix. In Gondos B, ed. Pathology of Infertility, New York: Thieme Medical Pub, 1987,1–10. 11. Coleman BG, Arger PH, Grumback K, Menard MK, Mintz MC et al. Transvaginal and transabdominal sonography: prospective comparison. Radiology 1988; 168:639–43. 12. Assad M, Abdulla U, Hipkin L. The effect of clomifene citrate treatment on cervical mucus and plasma estradiol and progesterone levels. Fertil Steril 1993; 59:539–43. 13. BMDP Statistical Software manual (1990). Chief editor: WJ Dixon. Berkeley CA: University of California Press.

CHAPTER 72 Multifetal Pregnancy Reduction Ashok Khurana, Reeti Sahni, Sonia Malik, Kuldeep Singh INTRODUCTION Ovulation induction for infertility treatment with or without assisted reproductive technologies such as insemination and in vitro fertilisation has resulted in a phenomenal increase in high order (three or more) multiple pregnancies.1,2,3,4 Perinatal mortality and morbidity are directly proportional to the number of fetuses5 and are also largely consequent to the high incidence of premature deliveries6 in multifetal pregnancies. Although earlier diagnosis, increased antenatal surveillance and improved neonatal care have improved pregnancy outcomes, the overall prognosis of multiple pregnancy with four or more fetuses is pathetic. Even in triplet pregnancies, the incidence of long term neurologic deficits and recurrent hospitalisation in inf ancy is high as is neonatal and inf ant mortality when compared to twin gestations. The aim of multifetal pregnancy reduction (MFPR) is to improve pregnancy outcome by striking a balance between the risks of ongoing multifetal pregnancy and the risks associated with the procedure. In recent years, a plethora of studies on multifetal pregnancy reduction has appeared in literature and this experience can be prudently drawn upon in obstetric decision making. RELIGIOUS, EMOTIONAL AND PRACTICAL CONSIDERATIONS Multifetal Pregnancy Reduction is an area of extreme stress and distress for the infertile patient. It is probably a straightforward option for the fertile patient who already has one living child and a simple option for the primigravida with a spontaneous conception of a multi-fetal pregnancy For the patients of Islamic and Roman Catholic faiths, it may not even be a considered an option. For the patient who has had long years of infertility and has conceived after extensive treatment, the diagnosis of a multif etal pregnancy itself, and its implications, can be emotionally devastating and the concept of multifetal pregnancy reduction can come as an even more rude shock. The route to a final decision leading to multifetal pregnancy reduction is always complicated and should be discussed with the patients extensively and an informed consent with a detailed, signed information sheet obtained prior to carrying out the procedure.

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HISTORICAL AND TECHNICAL CONSIDERATIONS Pioneering attempts in multifetal pregnancy reduction utilised a transcervical5–9 approach after cervical dilatation. Embryos were aspirated under ultrasound guidance. This route is now, however, less used because of the need for dilatation, frequent intraprocedural hemorrhage and the inability to be able to use it after 9–10 weeks of gestation because of large fetal size. The approach currently used is transvaginal needling or transabdominal needling and the choice depends on operator experience and period of gestation. The foremost consideration is operator expertise. The procedure shows a drastic learning curve with a high incidence of failed procedures and fetal losses in the initial cases whenever the operator is new to the procedure. Experienced operators are later often equally comfortable with either approach and have a considerably lower rate of complications. Operators experienced with only one type of approach tend to prefer the same approach. The transvaginal needling approach is used in early gestation and has the advantages of emotional convenience to the mother and a possibly lower rate of fetal loss. The transabdominal approach has the advantage of pre-assessing (a) early growth lag of any one fetus, (b) assessment of spontaneous fetal loss in early pregnancy and (c) ability to exclude abnormal nuchal translucency, thickness and abnormal fetal cranio-caudal differentiation, since it is performed at a later period of gestation. The continuing emotional stress to the mother is, however, prominently in evidence as the weeks go by. ULTRASOUND GUIDANCE Pre-operative ultrasound scans are necessary to assess the number of pregnancies, viability, size and location. It is also important to exclude any monochorionic twins as these will be almost always lost consequent to the procedure. The incidence is rare but not unknown. A crucial mapping of the embryos is important and it is wise to record photo-impressions or a map, particularly for later reference in case a needled embryo survives or revives. Intraoperative needle guidance is, of course, a prerequisite for the procedure and postprocedure monitoring is necessary The success of the procedure needs to be checked a few hours after the procedure and the following day. Medication Owing to the duration of the procedure, the frequent need of a multipuncture and maternal anxiety, general anaesthesia is often used. The procedure can, however, be performed under a local anaesthetic. Most centres outside India do not use an antibiotic or a tocolytic. Our personal experience shows a lower incidence of chorio-amnionitis, leukocytosis, fetal loss, spontaneous abortion and premature delivery if appropriate antibiotic cover is given for 7 days and tocolysis for a period of 72 hours after the procedure. The patient is advised bed rest for 48 hours with toilet f acility permitted.

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Needling For the actual procedure the vagina or the abdominal wall is prepared as per surgical norms and the urinary bladder is kept partially full (25–100 ml). This ensures avoiding a transvesical puncture, although inadvertent punctures have not been associated with any specific morbidity For the vaginal approach a 17–19 gauge, 25 cm needle is introduced through the needle guide and advanced through the myometrium into the nearest sac and then into the embryonic heart. Blood/fluid is aspirated and cardiac activity observed. Cessation is usually instantaneous. Some workers6,10,11,12 prefer infecting KCI into the heart. 01–02 ml of 2 meq/L are usually required. Most workers currently do not report any difference in success rates when KCI is used in early pregnancy, compared to when only needling and aspiration are used.13,14,15 When KCI is injected it is imperative to monitor the maternal ECG with care. The procedure is separated for each gestational sac. For transabdominal procedures, a 20 gauge spinal or equivalent needle is used in the same way. However, mechanical trauma, aspiration and saline injections are not always effective and KCI gives superior results. Fetal Loss and Maternal Morbidity Post multifetal pregnancy reduction, spontaneous demise in utero of surviving embryos/fetuses is rare. Spontaneous abortion of the entire pregnancy may occur in 9–15 percent of cases” and these are usually seen to occur 4–8 weeks postprocedure. However, this is to be viewed in the perspective of pregnancy losses before 24 weeks in triplet and higher order multifetal pregnancies not undergoing multifetal pregnancy reduction where the loss rate is about 10 percent. The risk of spontaneous abortion after MFPR is higher not only with an increase in the initial number of embryos but also with the number of fetuses reduced. “Miscarriage rates decrease with the increasing experience of the operator.” Clinical and hematological studies seem to rule out the risk of coagulation disorders following a MFPR. Prematurity Gestational age at birth after a MFPR is the major criterion to evaluate the efficacy of MFPR since prematurity is the major contributor towards pediatric morbidity in multiple pregnancies. Obstetrical follow up of large series has shown that among potentially viable deliveries (>27 weeks), mean gestational age at birth is 36 menstrual weeks. However, nearly half of the patients deliver after 37 weeks and over 85 percent deliver later than 33 weeks.11,16,18 Following MFPR to a number of three embryos, 89 percent of patients will deliver before 37 weeks and 33 percent before 32 weeks. With a final number of 2 embryos, 52 percent of the MFPR patients deliver before 37 weeks and 13 percent before 32 weeks. With one embryo, 30 percent of the MFPR patients deliver before 37 weeks and 10 percent before 32 weeks. This clearly shows a major decrease in the risk of premature delivery afterMFPR.

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CONCLUSION Multifetal pregnancy reduction reduces but does not eliminate the risk of prematurity in high order multif etal pregnancies. Patients with four or more fetuses should be encouraged to have a multifetal pregnancy reduction and patients with triplets should receive very extensive counselling to help them reach a decision. Infertility specialists need to be vigilant in using drugs and during IVF the number of embryos transferred should be limited. REFERENCES 1. Holcberg G, Biale Y, Lewenthal H, Insler V. Outcome of pregnancy in 31 triplet gestations. Obstet Gynecol 1982; 59:472–79. 2. Schenker JG, Yarkoni S, Granat M. Multiple pregnancies following induction ofovulation. Fertil Steril 1981; 35:105–23. 3. Dommergues M, Nisand I, Mandelbrot L, Isfer E, Radunovic N, Durnez Y. Embryo reduction in the management of multifetal pregnancies following infertility therapy: obstetrical risks and perinatal benefits are related to the operative strategy Fertil Steril 1991; 55:801–11. 4. Evans MI, Fletcher JC, Zador IE. Selective first trimester termination in octuplet and quadraplet pregnancies: Clinical and ethical issues. Obstet Gynecol 1988; 71:289–96. 5. Botting BH, McDonald Davies I, McFarlane AJ. Recent trends in the incidence of multiple births and associated mortality. Arch Dis Childh 1987; 62:941–50. 6. Berkowitz R, Lynch L, Chitkara U. Selective reduction of multifetal pregnancies in the first trimester. N Engl J Med 1988; 318:1043–47. 7. Salat-Baroux J, Aknin J, Antoine JM, Alamowitch R. The management of multiple pregnancies after induction for superovulation. Human Reprod 1988; 3:399–401. 8. Martene Duplan J, Aknin J, Alamowitch R. Aspiration embryonnaire partielle au cours de grossesses multiples. Contraception Fertilite Sexualite 1983; 11:745–48. 9. Dumex Y, Oury JF. Method for first trimester selective abortion in multiple pregnancy Contr Gynaecol Obst 1986; 15:50–53. 10. Bessis R, Milanese C, Frydman R. Partial termination of pregnancy. 2nd Congress on the Fetus as a Patient. Jerusalem, May 1985. 11. Evans MI, Dommergues M, Wapner RJ et al. Efficacy of transabdominal multifetal pregnancy reduction—collaborative experience among the world’s largest centers. Obstet Gynecol 1993; 82:61–66. 12. Jeny R, Leroy B. Reduction selective en cas de grossesses multiples. Ann Radiol 1983; 26:446. 13. Timor-Tritsch IE, Peisner DB, Monteagudo A, Lemer JP, Sharma S. Multifetal pregnancy reduction by transvaginal puncture—evaluation ofthe techniques use in 134 cases. Am J Obstet Gynecol 1993; 168:799–804. 14. Itskovitz J, Boldes R, Thaler I, Bronstein M, Eriik Y, Brandes J. Transvaginal ultrasonography guided aspiration of gestation sacs for selective abortion in multiple pregnancy. Am J Obstet Gynecol 1989; 160:215–17. 15. Itskovitz-Eldor J, Drugan A, Levron J, Thaler I, Brandes JM. Transvaginal embryo aspiration— a safe method for selective reduction in multiple pregnancies. Fertil Steril 1992; 58:351–55. 16. Evans M, Dommergues M, Timor-Tritch I et al. Transabdominal versus transcervical/vaginal multifetal pregnancy reduction: international collaborative study of more than 1000 cases. Am J Obstet Gynecol 1994; 170:902–09. 17. Pons JC, Le Moal S, Dephot N, Papernik E. Liquor amnii is normal, grossesse quadruple en France. In E Papernik (Ed): Les Grossesses Multiples, Paris Doin: 1991; 319–28.

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18. Evans M, Dommergues M, Wapner R et al. International collaborative experience of 1789 patients having multifetal pregnancy reduction: a plateauing of risks and outcome. J Soc Gynecol Invest 1996; 3:23–26.

SECTION 12 The Male Factor

CHAPTER 73 Non-obstructive Azoospermia: Predictive Criteria for Sperm Retrieval Vijay Kulkarni INTRODUCTION Azoospermia, the absence of sperm in ejaculated semen, is the most severe form of male factor infertility and is present in approximately 5 percent of all investigated infertile couples.1 It can be a result of obstruction to the passage of sperms where spermatogenesis is essentially normal, i.e. obstructive azoospermia or due to failure of the spermatogenic process without any obstruction, i.e. non-obstructive azoospermia. For this latter group, i.e. Non-obstructive azoospermia (NOA), Intracytoplasmic Sperm Injection (ICSI) with in vitro fertilization (IVF) has become an accepted mode of treatment. Retrieval of testicular spermatozoa from non-obstructive azoospermic patients for ICSI is a recent advance in the treatment of male infertility. Sperm retrieval by testicular sperm extraction (TESE) may not always be successful in patients with NOA, as only some patients have sperms, and those who do, only have minute foci of active spermatogenesis randomly scattered in the whole testis from which a tiny number of spermatozoa can be extracted (Fig. 73.1). Failure to recover sperms risks wastage of the cycle, of ovarian stimulation and necessitates use of donor sperms about which the couple may be undecided. Hence it has become important to prognosticate such failures whereby the couple can be suitably counseled for the alternatives. The predictive criteria for finding sperms or for failure to find the sperms during sperm retrieval procedures in non-obstructive azoospermia are yet to be conclusively established. Parameters Considered Testicular volume The testicular volume seemed the most obvious and the simplest of the indicators for the

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Fig. 73.1 presence of sperms within the testis—smaller the volume lesser the chance, larger the volume better the chance. One wished such simple logic ruled the world. While a smaller volume (<15 ml), denoting testicular atrophy, may indicate fewer chances of finding sperms, the vice versa was not true. Neither does the normal volume (>15 ml), assure the presence of sperms, nor does the small volume rule out its presence. However, the large volume testis does provide physically more tissue-mass for extraction during multiple biopsies which would contain more seminiferous tubules, therefore giving a better chance of findingminute foci of spermatogenesis. There are many studies which concluded that the testicular volume is not a reliable indicator.2,3,4 Serum follicle stimulating hormone (FSH) Raised levels of serum FSH were considered to be a negative indicator of spermatogenesis and hence, consequently a negative predictor for successful sperm retrieval. While the former is generally true, the latter belief is not substantiated by many studies.2,3,5 The normal or raised levels of serum FSH can neither predict success at sperm retrieval nor indicate pattern of spermatogenesis in NOA. Inhibin B This product of Sertoli cells was considered as a marker for spermatogenesis. Hence, the estimation of serum Inhibin B levels as a predictor of successful sperm retrieval in NOA, was studied. The studies revealed that estimation of Inhibin B, either alone or in conjunction with serum FSH levels, failed to prognosticate the success of sperm retrieval.5,6

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Study of the Ejaculate For spermatogenic cells Presence of round spermatid cells in the ejaculate may indicate a possibility of spermatogenic foci in the testes predicting the success of sperm retrieval. Efforts were made to corroborate the detection of these cells using May-GrunwaldGiemsa stain to stain the spermatids and carrelate successful sperm retrieval. There was a statistical positive correlation between the two.7 Biochemical examination The seminal plasma was subjected to assessment of the contents like fructose, lactate dehydrogenase, total acid phosphatase, Zn, K, Na, Cl, pH. They were used as parameters to correlate with successful sperm retrieval. No statistically significant correlation was found in both groups under study. So biochemical examination of the ejaculate cannot provide any prediction of sperm retrieval in nonobstructive azoospermia.8 For spermatozoa In one of the studies, a thorough microscopic search through many droplets of ejaculate sediment was made to detect the presence of sperms. When successful, it was in fact considered as an alternative to TESE.9 In a strict sense of the term, this should be cryptoazoospermia rather than azoospermia. Testicular histology This appears to be the most reliable of all the predictive criteria described so far.10 Whether the testicular tissue for histological examination was obtained by open biopsy or by f ine needle aspiration, its value for the prediction did not seem to alter. Based on the advanced pattern of spermatogenesis seen on histopathological examination, three categories are recognized; severe hypospermatogenesis, maturation arrest and Sertoli-cell-only syndrome (SCOS).11 In the series by Seo and Ko, spermatozoa were successfully recovered in 94 of 178 (52.8%) non-obstructive azoospermic men, in 13 of 80 (16.3%) men with Sertoli-cellonly syndrome, 15 of 24 (62.5%) men with maturation arrest, and 66 out of 74 (89.2%) men with severe hypospermatogenesis.2 Poor retrieval rates in Sertoli cell-only syndrome (SCOS) may be better predicted if the histological classification of the germinal aplasia is correctly done i.e. pure (congenital) or mixed (secondary) according to Anniballo R.12 Based upon the visualization of the spermatids per seminiferous tubule in histological examination, a good predictive criterion may be obtained. Silber et al13 showed in their study that men with non-obstructive azoospermia caused by germinal failure had a mean of 0–6 mature spermatids/seminiferous tubule seen in a diagnostic testicular biopsy, compared to 17–35 mature spermatids/ tubule in men with normal spermatogenesis and obstructive azoospermia. These findings were the same for all types of testicular failure whether Sertoli cell only, maturation arrest, cryptorchidism, or post-chemotherapy azoospermia. The study suggests that 4–6 mature spermatids/ tubule must be present in the testis biopsy for any spermatozoa to reach the ejaculate. More than half of azoospermic patients with germinal failure have minute foci of spermatogenesis which are insufficient to produce spermatozoa in the ejaculate. While this study provides an excellent indicator to differentiate between obstructive azoospermia and NOA, it does not take into account the distribution of the spermatogenic foci in NOA. While partial testicular failure in NOA may involve a sparse multifocal distribution of spermatogenesis throughout the entire testicle as often seen in severe hypospermatogenesis, it also may

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have a regional distribution. Thus testicular mapping becomes relevant while doing prior diagnostic testicular biopsies in NOA. In addition to the visualization of the spermatids, concurrently done Johnson Score14 may enhance the accuracy of the prediction. Ezeh et al in their study found 77 percent correct prediction rate with visualization of spermatids and 71 percent with Johnson score in an overall prediction of 87 percent.3,5 Genetic Considerations Genetic abnormalities, including partial deletions of the Y-chromosome, are commonly detectable in men with non-obstructive azoospermia. Among the AZFa, AZFb and AZFc deletions, the presence of an AZFb deletion appears to be a significantly adverse prognostic finding for a successful TESE. Men with AZFb deletions should be apprised of these possibilities before attempting TESE-ICSI.15 Karyotyping has its value while investigating a case of non-obstructive azoospermia, but has not proven its significance as a predictor for presence or absence of sperms.16 Predictability for Repeat Procedure There is a good degree of predictability for the second time sperm retrieval procedure if it has been successful the first time. However, there is no unanimity as to the time interval between the two procedures. Serial ultrasonographic images, histological analyses and evaluation of the success of the repeat TESE indicate the optimal period to be six months after the first procedure.17 Minimizing False Negative Sperm Retrieval Failure to retrieve sperms due to inadequacy of the procedure would be a false negative result. Procedures like open biopsy,18 multiple biopsies,19 and microdissection of the seminiferous tubules20 have been described in many studies to facilitate a better yield of spermatozoa. Hence, if percutaneous aspiration of epididymis or of testis (i.e. PESA or TESA) does not reveal sperms then, before accepting it as a “failure to retrieve”, the above mentioned procedures should be resorted to, since they have better chance of locating the spermatic foci and thus minimize the false negative results. Though discussion on these individual procedures is out of the scope of the present review, they certainly deserve a mention while discussing success or failure of sperm retrieval. The enzymatic digestion of testicular biopsy tissue has been attempted by Crabbe E to retrieve the spermatozoa with encouraging results.21 CONCLUSION It is now clear that even in men with azoospermia, due to absence of spermatogenesis or to a block in meiosis, there are of ten a few spermatozoa available in the testes which allow TESE with ICSI to be carried out. Open multiple testicular biopsies using magnification to minimize the trauma appears to be the best strategy to retrieve sperms.

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Predictive criteria for sperm retrieval are not yet precise. Testicular histology has a highest index for such prediction but involves prior diagnostic biopsy/biopsies. The most promising non-invasive criterion appears to be detection of micro-deletions on Ychromosome. In fact the most important arena for research into male infertility in the next decade will be to map out the deletions on the Y chromosome and some autosomal chromosomes that might result in defective spermatogenesis, and which probably cause most cases of non-obstructive male factor infertility.22 Finally, it is likely that some forms of severe male factor infertility are genetically transmitted and although ICSI offspring have been shown to be completely normal, it is possible that the sons of these infertile couples will also require ICSI when they grow up and wish to have a family.23 REFERENCES 1. Van Perperstraten AM, Proctor ML, Phillipson G, Johnson NP. Techniques for surgical retrieval of sperm prior to ICSI for azoospermia. Cochrane Database Syst Rev 2001; (4):CD002807 2. Seo JT, Ko WJ. Predictive factors of successful testicular sperm recovery in non-obstructive azoospermia patients. Int J Androl 2001; 24(5):306–10 3. Ezeh UI, Taub NA, Moore HD, Cooke ID. Establishment of predictive variables associated with testicular sperm retrieval in men with non-obstructive azoospermia. Hum Reprod 1999; 14(4):1005–12. 4. Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D, Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 1997; 12(7):1488–93 Comment in: Hum Reprod. 1998; 13 (4):1111–13. 5. Weiss DB, Gottschalk-Sabag S, Zukerman Z, Bar-On E, Kahana Z. Follicle-stimulating hormone in azoospermia in prediction of spermatogenic patterns [Article in Hebrew]. Harefuah 1998; 135(5–6):169–75, 256. 6. Vernaeve V, Tournaye H, Schiettecatte J, Verheyen G, Steirteghem AV, Devroey P. Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia. Hum Reprod 2002; 17(4):971–76 7. Amer M, Abd Elnasser T, El Haggar S, Mostafa T, Abdel-Malak G, Zohdy W. May-GrunwaldGiemsa stain for detection of spermatogenic cells in the ejaculate: a simple predictive parameter for successful testicular sperm retrieval. Hum Reprod 2001; 16 (7):1427–32. 8. Bartek J, Sobek A, Hrbkova K, Mucha Z, Zat’ura F, Psotova J. Biochemical findings in the ejaculate of men with non-obstructive azoospermia. Acta Univ Palacki Olomuc Fac Med 1998; 141:25–26. 9. Ron-El R, Strassburger D, Friedler S, Komarovski D, Bern O, Soffer Y et al. Extended sperm preparation: an alternative to testicular sperm extraction in non-obstructive azoospermia. Hum Reprod 1997; 12(6):1222–26 10. Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S et al. Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum Reprod 1997; 12(1):80–86. 11. Westlander G, Hamberger L, Hanson C, Lundin K, Nilsson L, Soderlund B et al. Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. Hum Reprod 1999; 14(1):118–22. 12. Anniballo R, Ubaldi F, Cobellis L, Sorrentino M, Rienzi L, Greco E et al. Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval. Hum Reprod 2000; 15(11):2269–7.

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13. Silber SJ, Nagy Z, Devroey P, Tournaye H, Van Steirteghem AC. Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum Reprod 1997; 12(11):2422–8 Erratum in: Hum Reprod 1998; 13(3):780 Comment in: Hum Reprod 1998; 13(7):2034–35. 14. Johnson SG. Testicular Biopsy Score for count: Method for Registration of Spermatogenesis in Human Testes: Normal Values and Results in 335 Hypogonadal Males. Hormone 1970; 1:2. 15. Brandell RA, MielnikA, Liotta D, Ye Z, Veeck LL, Palermo GD et al. AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum Reprod 1998; 13:2812–15. 16. Westlander G, Ekerhovd E, Granberg S, Hanson L, Hanson C, Bergh C. Testicular ultrasonography and extended chromosome analysis in men with nonmosaic Klinefelter syndrome: a prospective study of possible predictive factors for successful sperm recovery. Fertil Steril 2001; 75(6):1102–5. 17. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997; 12(8):1688–92 Comment in: Hum Reprod 1998; 13(2):505–6. 18. Ezeh UI, Moore HD, Cooke ID. A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 1998; 13(11):3075–80. 19. Hauser R, Botchan A, Amit A, Ben Yosef D, Gamzu R, Paz G et al. Multiple testicular sampling in non-obstructive azoospermia—is it necessary? Hum Reprod 1998; 13(11):3081–85. 20. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999; 14(1):131–35. 21. Crabbe E, Verheyen G, Silber S, Tournaye H, Van de Velde H, Goossens A et al. Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia. Hum Reprod 1998; 13:2791–96. 22. Silber SJ. A modern view of male infertility. Reprod Fertil Dev 1994; 6(1):93–103. 23. Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C et al. The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum Reprod 1995; 10(8):2031–43.

CHAPTER 74 Epididymal and Testicular Sperm Retrieval Rupin Shah INTRODUCTION The advent of Intracytoplasmic Sperm Injection (ICSI) has revolutionized the management of azoospermia. Previously, the only options for treating azoospermia were microsurgery (which could be performed only in some cases) or donor insemination. Now, ICSI enables most azoospermic men to become biological fathers, provided some sperm can be obtained from their epididymis or testis. Several studies have confirmed that the pregnancy rates with ICSI are the same whether ejaculated, epididymal or testicular sperm are used.1 Many methods of sperm retrieval have been described. In this review we will discuss each method, analyze its advantages and disadvantages, and highlight the author’s preferred methods. INDICATIONS ICSI using epididymal or testicular sperm is indicated in the following cases: 1. Obstructive Azoospermia when reconstruction is not possible e.g. vas aplasia. 2. Obstructive Azoospermia if reconstruction has failed (after vaso-epididymal or vasovasal anastomosis). 3. Obstructive Azoospermia if the couple chooses ICSI rather than reconstruction (since results with ICSI aremuchfaster). 4. Non-Obstructive Azoospermia, in those men who have some areas of spermatogenesis. 5. Failure to ejaculate during an ART cycle (if vibrator fails and electro-ejaculation is not available). 6. Total astheno-/necro-zoospermia. When all sperm are immotile they may be viable or nonviable. If a man has nonmotile but viable sperm, these can be identified by the hypo-osmotic swelling test2 and used for ICSI. If, instead, a man has all immotile sperm and tests reveal that most of these sperm are dead, then it is preferable to use sperm aspirated from the testis since these are more likely to be viable and may sometimes even be motile.

SPERM RETRIEVAL METHODS Sperm may be retrieved from the epididymis or the testis

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Epididymal Sperm Retrieval Open Procedures • MESA (Microsurgical Epididymal Sperm Aspiration) • OFNA (Open Fine Needle Aspiration) Percutaneous Procedures • PESA (Percutaneous Epididymal Sperm Aspiration) Testicular Sperm Retrieval Open Procedures • Conventional Open Biopsy • Microsurgical Open Biopsy • SST (Single Seminiferous Tubule) biopsy technique • Microsurgical selective biopsy technique Percutaneous Procedures • TESA (Testicular Sperm Aspiration) • NAB(Needle Aspiration Biopsy) • Needle Biopsy

OPEN EPIDIDYMAL SPERM RETRIEVAL MESA (Microsurgical Epididymal Sperm Aspiration) Technique Through a scrotal incision the epididymis is exposed. Under an operating microscope the epididymal tunica is incised and an epididymal ductule is mobilized. The ductule is opened and the spermatic fluid that flows out is aspirated. The ductule is then closed with microsutures.3,4 Advantages Microsurgical visualization of the epididymis allows for precise, bloodfree aspiration from multiple locations. A large number of motile sperm can be recovered and cryopreserved for future cycles of ICSI. Microsurgical handling of the ductule may preserve it for future repeat aspiration, if required. Disadvantages This is a time-consuming and demanding procedure that needs an operating microscope and a trained andrological microsurgeon. There is no evidence that closing the ductule microsurgically improves chances of future retrieval.

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OFNA (Open Fine Needle Aspiration) Technique The epididymis is exposed and a ductule is directly punctured through the tunica with a 26 G needle. Sperm-containing epididymal fluid is aspirated from the ductule. On withdrawing the needle, epididymal fluid continues to flow out of the punctured ductule and is aspirated from the epididymal surface. Advantages As in MESA, spermatic fluid can be aspirated under vision from different locations, thus obtaining the maximum number of sperm. As no microsurgical dissection or suturing is involved, the procedure is very quick, does not need special equipment or training, and can be performed under local anaesthesia in the operation theatre of the IVF unit. Disadvantages It is an open surgical procedure. MESA Versus OFNA Since OFNA offers the advantages of MESA withoutbeing cumbersome, it is the preferred technique when open epididymal aspiration is required. PERCUTANEOUS EPIDIDYMAL SPERM RETRIEVAL PESA (Percutaneous Epididymal Sperm Aspiration) Technique Under local anaesthesia, the epididymis is punctured directly through the scrotal skin using a 21G scalp vein needle. Suction is applied with a 20 ml syringe and the needle is moved back and forth in the epididymis, thus aspirating sperm-containing epididymal fluid.5 We have modified the technique by using an insulin syringe with a 26G needle which gives the same result while being less traumatic. Advantages The procedure is simple, quick,6 avoids open surgery and can be repeated.7 We have had several cases in whom we have repeated the procedure 2 to 4 times and obtained adequate sperm each time. Disadvantages Since the epididymis is not visualized the location of puncture is guided by palpation alone and cannot be precisely controlled. As a result, occasionally the sperm-containing ductule may be missed; we have had rare cases of vas aplasia where very little epididymis was present and was covered by fat: as a result PESA failed, but subsequent OFNA could retrieve motile sperm. Further, there is also the possibility of puncturing a blood vessel and contaminating the sample with red blood cells. Open Versus Closed Epididymal Sperm Retrieval Since PESA can obtain enough sperm for ICSI and for cryopreservation there is no reason to subject a patient to an open surgical procedure. Only if PESA fails should OFNA be performed, though even in such a situation an open surgical procedure can be avoided by doing TESA.

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PERCUTANEOUSTESTICULAR SPERM RETRIEVAL TESA (Testicular Sperm Aspiration) Technique This is like an aspiration cytology procedure. A 19G butterfly needle is jabbed around the testicular substance while applying suction with a 20 ml syringe. Sperm are recovered from the aspirate.8 TESAcanbe used for men with non-obstructive9 as well as obstructive azoospermia.10 Recently, color Doppler ultrasonography has been used to guide the aspiration so as to avoid blood vessels and reduce hematoma formation.11 Advantages This is a simple, nonsurgical procedure that can be performed without special training or equipment. Disadvantages Since this is a blind procedure, there is the risk of puncturing a tunical vessel and causing a hematocele. Multiple passages of the needle through the testicular tissue could disrupt a large number of seminiferous tubules or cause intra-testicular hemorrhage. The total amount of cellular material is scanty and in our experience, and that of others,12 TESA has often failed to recover sperm in men with non-obstructive azoospermia in whom sperm could be found when the testicular biopsy was examined. NAB (Needle Aspiration Biopsy) Technique An 18G scalp vein needle is introduced into the testis under local anaesthesia. Once the needle enters the tunica, suction is applied using a 10 ml syringe. The needle is pushed in up to its hub, then pulled partly out (staying within the tunica) and then pushed in again. The needle is rotated 180 degrees (to cut the tissue) and the out-and-in motion of the needle is repeated. The tubing of the scalp vein is then clamped and the needle is withdrawn slowly and carefully. As the needle emerges from the scrotal skin, a strand of testicular tissue is pulled out. This is grasped near the skin and pulled out till it snaps or a sufficient length of tubule is obtained (Fig. 74.1). An additional length of testicular tubule is often found in the needle and is flushed out gently.

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Fig. 74.1: Needle Aspiration Biopsy (NAB)—a loop of seminiferous tubule has been extracted by an 18G scalp vein needle and is being pulled out of the testis using a microforceps Advantages This is a simple, quick, nonsurgical procedure that can obtain tissue equivalent to a small open biopsy Technically, it is similar to TESA, but its cellular yield is many times greater. Disadvantages Since it is a blind procedure with a large needle it carries the risk of producing a hematocele or causing intra-testicular hemorrhage; hence, we prefer to limit the number of NAB biopsies to three per side. Further, since the testis is not visualized, multiple biopsies cannot be plotted as accurately as when doing open biopsies. TESA Versus NAB Both TESA and NAB can retrieve enough sperm in cases of obstructive azoospermia; however, NAB gives far more sperm than TESA and is therefore preferred, especially in cases where spermatogenesis is impaired. In our experience, NAB has often procured sperm when TESA has failed.

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Needle Biopsy Technique: A testicular biopsy can also be obtained using a tissue-cutting biopsy needle (e.g. Truecutä needle or Bioptyä gun.13 These dedicated biopsy needles are springactivated. When the needle is placed against the testis and released, it enters the stroma, cuts a sliver of tissue and withdraws it into a sheath. Advantages This is a simple method that is routinely being used to biopsy a variety of tissues. It is particularly useful in men with fibrosis following prior testicular surgery in whom NAB may fail to retrieve sufficient tissue. Disadvantages Unlike the NAB technique in which a tubule is unraveled out of the testis, the biopsy needle cuts through a number of tubules thus causing more trauma while retrieving less tissue. Also, these special needles represent an additional expense. OPENTESTICULAR SPERM RETRIEVAL Conventional Biopsy Technique Through a scrotal incision the testis is exposed, the tunica is incised, and a piece of protruding testicular tissue is excised. The tunica is sutured and the incision is closed. Advantage It is an easy method that can be performed by any surgeon. Disadvantage Apart from the fact that it is an open procedure, its main disadvantage is that it can damage the testis. As the procedure involves incision and closure of the tunica, sub-tunical vessels that cross the incision are permanently occluded. Further, during excision of the testicular tissue, intra-testicular vessels will be cut. Since testicular vessels are end-arteries, multiple open conventional biopsies in men with testicular failure can demonstrably impair testicular function.14,15 SST (Single SeminiferousTubule) Biopsy Technique The scrotum is opened and the testis is exposed. An avascular area of the tunica is punctured with a 26G needle. Microforceps are introduced into the puncture hole, dilating it till a loop of seminiferous tubule pops out. The seminiferous tubule is held with the microforceps and pulled out till sufficient tissue is obtained. The procedure is repeated at multiple sites. Deeper tissue can be obtained by inserting the microforceps into the depth of the testicular stroma. There is no need to close the tunica since the tunical opening is very small. Adυantage This technique allows procurement of a large biopsy though a tiny hole in the tunica. Since the tunica is not incised or sutured, no blood vessel is damaged; hence, multiple biopsies can be taken without affecting the testis. Disadυantage It is an open procedure. Occasionally, a small intra-testicular vessel may be cut when the seminiferous tubule is divided.

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Microsurgical Selective Biopsy Technique The testis is exposed and a long incision is made in the tunica. The seminiferous tubules are gently separated and examined under the microscope. Fibrous tubules can be distinguished from “healthy” tubules that are more likely to contain sperm.16 Only the healthy tubules are biopsied. Dissection and biopsy are continued till adequate sperm are retrieved. Advantages Since only promising tubules are biopsied, less tissue needs to be removed resulting in less testicular damage. The entire testicular tissue can be visually evaluated and biopsied, improving the chances of finding sperm in cases with focal spermatogenesis. Disadvantages Though only a small amount of tissue is removed, the large tunical incision and the dissection of the testicular tissue can cause considerable devascularization of the testis. Percutaneous Versus Open Biopsy In men with normal spermatogenesis, needle biopsy techniques can obtain adequate sperm without the need for an incision.10 Hence, in men with obstructive azoospermia NAB is preferred to open biopsy However, in men with testicular failure, percutaneous biopsies may fail to procure sperm and multiple openbiopsies are needed.12 Open Conventional Biopsy Versus Microsurgical Biopsy Techniques If a single biopsy is required the method of biopsy does not matter significantly. However, in men with testicular failure, often multiple biopsies have to be done before sperm can be found.17,18 Since microbiopsy techniques minimize testicular damage they are preferred to the conventional method of testicular biopsy in cases where multiple biopsies are required. CHOICE OF PROCEDURE In obstructiυe azoospermia, PESA is the first choice. If PESA fails then NAB is done. In non-obstructiυe azoospermia, initially NAB is tried. If 3 to 4 NAB biopsies from each side fail to procure adequate sperm, then multiple microbiopsies by the SST method are takenbilaterally. Epididymal sperm retrieval is not possible in these men. When operative sperm retrieval is required for men with ejaculatory dysfunction, a NAB biopsy will provide adequate number of sperm. PESA should not be done as it can cause an iatrogenic block of the epididymis. In men with total asthenozoospermia NAB is preferred to PESA since testicular sperm are more likely to be viable in these cases. CONCLUSION A variety of methods are available for retrieving epididymal and testicular sperm. Preference should be given to techniques that are non-invasive and simple.

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REFERENCES 1. Tarlatzis B, Bili H. Clinical outcome of ICSI: Results of the ESHRE Task Force. In: Filicori M, Flamigni C (Eds): Treatment of infertility: the new frontiers. New Jersey. Communications Media for Education 1998. 2. Verheyen G, Joris H, Crits K et al. Comparision of different hypoosmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update 1997; 3:195–203. 3. Silber SJ, P NZ, J L et al. Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod 1994; 9:1705–9. 4. Girardi SK, Schlegel P. MESA: Review of techniques, preoperative considerations and results. J Androl 1996; 17:5–9. 5. Shrivastav P, Nadkarni P, Wensvoort S, Craft I. Percutaneous epididymal sperm aspiration for obstructive azoospermia. Hum Reprod 1994; 9:2058–61. 6. Craft I, Tsirigotis M, Bennett V et al. Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril 1995; 63:1038–42. 7. Rosenlund B, Westlander G, Wood M et al. Sperm retrieval and fertilization in repeated percutaneous epididymal sperm aspiration. Hum Reprod 1998; 13:2805–7. 8. Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular sperm. Hum Reprod 1995; 10:1623–27. 9. Turek PJ, Givens CR, Schriock ED, Meng MV, Pedersen RA, Conaghan J. Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia. Fertil Steril 1999; 71:552–57. 10. Tournaye H, Clasen K, Aytoz A et al. Fine needle aspiration versus open biopsy for testicular sperm recovery: A controlled study in azoospermic men with normal spermatogenesis. Hum Reprod 1998; 13:901–4. 11. Belenky A, Avrech O, Bachar G et al. Ultrasound-guided testicular sperm aspiration in azoospermic patients: a new sperm retrieval 1 method for intracytoplasmic sperm injection. J Clin Ultrasound 2001; 29:339–43. 12. Friedler S, RazielA, Strassburger D et al. Testicular sperm retrieval by percutaneous fine needle aspiration compared with testicular sperm extraction by open biopsy in men with nonobstructive azoospermia. Hum Reprod 1997; 12:1488–93. 13. Morey AF, Deshon GEJ, Rosanski TA, Dresner ML. Technique of biopty gun testis needle biopsy. Urology 1993; 42:325–26. 14. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997; 12:1688–92. 15. Manning M, Junemann KP, Alken P. Decrease in testosterone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men (letter). Lancet 1998; 352:37. 16. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999; 14:131–35. 17. Tournaye H, Camus M, Goossens A et al. Recent concepts in the management of infertility because of non-obtructive azoospermia. Hum Reprod 1995; 10(Suppl 1):115–19. 18. Altay S, Hekimgil M, Cikili N, Turna B, Soydan S. Histopathological mapping of open testicular biopsies in patients with unobstructed azoospermia. Brit J Urol International 2001; 87:834–37.

CHAPTER 75 Microdeletions in the Y-Chromosome and Male Infertility Vida Acosta, Michael Spitz, RS Jeyendran INTRODUCTION Male factors have been attributed as a primary or contributory cause in 30 to 50 percent of all infertility cases. Given these statistics, successful measures for clinically circumventing or even curing male infertility would greatly benefit couples experiencing fertility problems. Possible causes of male infertility include immunological difficulties, infectious agents, chemical exposure, extreme physical trauma and genetic disorders. Numerous treatment procedures designed to overcome these problems through clinical and even surgical intervention have proven limited in their success. Uncertainties regarding the etiology of these male factors play a significant role, especially for non-obstructive azoospermia and oligozoospermia cases. The genetic basis for many human diseases has been substantiated and described in detail. Extrapolating this logic to the etiology of male infertility in terms of genetic defects has proven productive. Since the primary differentiating factor between genders is the sex chromosome, this research has focused on regions of the Y-chromosome presumed responsible for spermatogenesis. Chromosome karyotyping, for example, has revealed overt genetic etiologies such as XX males (with a Y- to -X translocation) and even 47XXY males (known as Klinefelter’s syndrome). In such patients the testis are usually devoid of germ cells, rendering these individuals sterile. Studies have subsequently revealed a strong correlation between micro-deletions in the Ychromosomes and azoospermic or oligozoospermic men. Tests capable of routinely and accurately detecting these deletions now provide invaluable tools for the diagnosis and treatment planning of genetically linked male infertility cases. BACKGROUND Genetic testing as a diagnostic and planning tool was first introduced when concerns were raised that male f ertility patients could transmit birth defects to their offspring through intracytoplasmic sperm injection (“ICSI”) procedures. Ironically enough, by revolutionizing fertility treatment and offering many sterile men the opportunity to procreate, ICSI also facilitated the transmission to their male children of those congenital anomalies first circumvented by the ICSI procedure itself.

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Most significantly, these transmissable abnormalities include not only those responsible for fertility problems, but also numerous diseases congenitally associated with male infertility factors. For example, approximately 70 percent of patients suff ering from congenital bilateral absence of the vas deferens (“CBVAD”) are either carriers or display vulnerability to cystic fibrosis and exocrine pancreatic insufficiency Similarly, men with spinal and bulbar muscular atrophy also suffer from oligozoospermia. As genetic testing became an accepted and vital component of many ART procedures, clinical research further expanded with the hope of discovering a genetic basis for male infertility. Research investigating the significance of micro-deletions of the Ychromosome (“MYC”) and specific male infertility problems has since proven particularly intriguing and productive. SIGNIFICANCE OF THEY-CHROMOSOME Comparative cytogenetic studies of azoospermic men and their fertile fathers led to the discovery that the long arm (Yq, region 11.23) of the Y-chromosome was indeed involved with spermatogenesis (Fig. 75.1). Additional research

Fig. 75.1: The AZF region of the Ychromosoem revealed that several Y-chromosome linked genes are apparently necessary to ensure normal sperm production. These studies have demonstrated a correlation between micro-deletions along several regions of the Y-chromosome and infertility. The apparent paucity of these same deletions in fertile men further reinforced this conclusion. Specifically, the development of a clinical protocol involved the screening of a fertile population; Y-chromosome regions found to be deleted in this fertile control population were excluded. Once “MYC” testing was standardized and conducted on a broad population of infertile males, statistics were compiled. These studies indicate that approximately 14 percent of azoospermic and roughly 10 percent of severely oligozoospermic men have micro-deletions in their Y-chromosome. Consequently, if 1 in 1,000 males is azoospermic and 12 to 15 percent have micro-deletions, then roughly 1 in 8,000 to 10,000 males are born with this birth defect of their sex chromosome.

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Further research has localized these micro-deletions to the Azoospermia Factor (“AZF”) locus on the Y-chromosome’s long arm. The AZF region is further subdivided into subregions, specified as AZFa, AZFb, AZFc and AZFd, respectively. Although the exact interrelationship between spermatogenesis and the coding sequence for these particular regions remains ambiguous, research has linked particular AZF deletions with observable changes in the spermatogenetic process (Table 75.1).

Table 75.1 Deleted Region (Genotype) Effect (Phenotype) AZFa AZFb AZFc AZFd

Oligozoospermia, or spermatogenic arrest Azoospermia or oligozoospermia Azoospermia or oligozoospermia Azoospermia to normal sperm counts with teratozoospermia

Region AZFa accounts for less than 10 percent of microdeletions, yet 75 percent of these patients display severe oligozoospermia. Region AZFb and AZFc micro-deletions may result in varied spermatogenetic arrest; as a general rule, long micro-deletions along multiple regions result in severely compromised sperm quality Some researchers classify AZFd as a distinct region, while many consider the region a mere extension of AZFc. Regardless, microdeletions within this little understood region can result in an extremely wide range of symptoms, from normal sperm counts with abnormal morphology to azoospermia. Additional genes affecting spermatogenesis both within the Y-and along other chromosomes is possible, further complicating the etiology. Phenotypes observed in patients with Y-deleted chromosomes are perhaps modified by genes outside the AZF region that might, in turn, influence the AZF deletions effect. For example, mammalian male germ cells experience an interconnected series of developmental steps as they mature and become capable of fertilization; in like manner, many additional genes and their secondary effects will probably soon be identified as responsible for somehow regulating this highly complex process. Since these outside regions currently remain unknown, definitively linking genotype to phenotype often proves problematic at best. Further complicating this etiology is the lack of correlation between the length of the deletion and the type of spermatogenic defect. Although an exact correlation between specific MYC regions and male infertility etiology and symptoms remains ambiguous, certain facts and general trends have nonetheless become apparent: • Deletions aff ecting spermatogenesis exist on numerous regions of the Y-chromosome. • Deletions can occur in one region or several, and can overlap between regions • These deletions display tremendous variability from patient-to-patient; a one-to-one correspondence between Y-chromosome genotype and phenotype as revealed by routine and even specialized semen analysis remains indeterminate • Large deletions can safely be associated with azoospermia • Small deletions, in contrast, should not necessarily be associated with proportionately less severe defects in spermatogenesis.

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METHODOLOGY AND TESTING As a general overview, patient DNA is typically extracted from a buccal swab sample or from peripheral blood. Microdeletions located along the Y-chromosome are then analyzed using polymerase chain reaction amplif ication of sequence tagged sites (“STS”) in the DNA extracted from male infertility patients. These STSs are relatively short, non-repetitive sequences of base pairs in the genetic code. Amplified sections of DNA are then observed through electro phoresis using ethidium bromide stain under an ultraviolet light source. These results are compared to a positive control containing amplified STSs which represent specific encoded sections from fertile patients. Absence of one or more of these amplified segments suggests, in turn, that a specific DNA section is absent in the sample. Unfortunately, such PCR tests can only evaluate sections specifically amplified, leaving other potentially deleted sections undetected. Similarly, although many sites along the Y-chromosome can have micro-deletions, testing all sites is not necessary since a particular microdeletion may not be related to sperm production or sperm quality at all. One should therefore concentrate on sites that are known to be directly responsible for spermatogenesis, and compare them to the intact strands of demonstrably fertile males. All patients with idiopathic azoospermia, severe oligozoospermia or teratozoospermia should be tested for the presence of micro-deletions in their Y-chromosomes. Since early embryonic cell differentiation is under male genomic influence, embryonic arrest may actually be due to a sperm defect cause by micro-deletions. Consequently, males whose partners suffer from repeat IVF failure due to early embryonic arrest should also be “MYC” tested. The “MYC” test is a very practical and efficient way to potentially determine the etiology of infertility. If the presence of Y-chromosomal microdeletions is indeed confirmed, then further evaluations or therapy are no longer necessary. And since microdeletions are transmissable from father-to-son, a “MYC” test can warn prospective ICSI patients that their male off spring might suffer from similar congenital defects. Genetic counseling is usually then recommended. TRANSFERENCE MECHANISM AND MUTATION ETIOLOGY Genetic screening of male infertility patients and their off spring demonstrated the heritability of micro-deletions in their Y-chromosomes. Further research has substantiated that all males with micro-deletions will either pass on those same microdeletions or even larger mutations to their male children. The origin of these micro-deletions are either a result of mutation de novo, or are transmitted directly from father to son. Several theories have been developed to help explain the etiology of these micro-deletions. For any given instance, one or several of these factors may be at work: • Abermnt crossover events Can result in relatively large sections of a chromosome’s genetic material being rendered incapable of transcription. Note also that various recombinations between similarly sequenced areas or homologous repeats on genes outside the specific pairing region could also give rise to microdeletions.

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• Abermnt or unbalanced sister chromatid exchange These can result from Ychromosome instability. Such instability might be due to an excessive occurrence of short and long interspersed tandem repeats, overcrowding the chromosome’s length. In such cases, micro-deletions increase in size when transmitted from male infertility patients to their soris. The exact transfer mechanism of these mutations f rom father to son can be extremely complicated. Mosaicism can further complicate the process, since some men might carry both intact and mutant chromosomes. To rule out mosaicism as a potential factor, multiple tissue sources from the same patient should be tested. Patients whose infertility is due to mosaicism have micro-deletions that occur as a de novo event from sister chromatid exchange early in development. In these instances, germ cell lineages contain both mutant and natural gene sequences. Consequently their male offspring can be either fertile or infertile. (Note that infertile offspring will necessarily pass their infertility on to all their sons.) In the more common non-mosaic fertility patient, the micro-deletion must have occurred in late germ cell development of the fathers. In these cases the patients’ fathers are or have once been fertile, with an intact Y-chromosome in their peripheral cell’s lineage; however, their Y-bearing sperm will nonetheless carry the microdeletion responsible for infertility to all their sons. CONCLUSION Genes vital for spermatogenesis are located in the Y-chromosome’s long arm. Any genetic material missing from this area can result in a marked or total deficiency in sperm production, leading to conditions such as azoospermia, severe oligozoospermia or teratozoospermia. Studies demonstrate that males with Y-chromosome micro-deletions will always transmit this defect to their male offspring through ICSI, resulting in their son’s infertility Testing for the presence of these micro-deletions can help patients decide whether or not to screen embryos for these birth defects. Gender selection techniques and donor sperm use can then provide viable alternatives to the 100 percent certainty of this kind of birth defect transmission. However, couples also have the option to simply allow whichever Y-chromosome mutations present to transfer from father to son. After all, these congenitalbirth defects are rarely critical. In the meantime, increasingly more sophisticated assisted reproductive techniques will no doubt make themselves available, further reducing the significance of these congenital infertility problems for future generations. Research into male infertility remains productive and ongoing. Subjects of study include identification of chromosomal regions involved with spermatogenesis and the association of genotype and phenotype. Investigations continue in the hope of determining the origin and transmission of mutations, while refinement of the epidemiological parameters related to infertility continues unabated. As gene therapy becomes more and more advanced, researchers should eventually be able to correct congenital birth defects. In fact, the very localized nature of abnormalities within the Y-chromosome suggest that this area of genetic research should prove most

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productive in the not-too-distant future. Although the exact etiology remains highly complex, the replacement of mutated strands of the Y-chromosome through advanced gene therapy may some day completely restore male fertilization potential at the genetic level. BIBLIOGRAPHY 1. Kent-First MG, Kol S, Muallem A, Ofir R, Manor D, Blazer S et al. The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Molecul Human Reprod 2, 1996; 12:943–50. 2. Pryor JL, Kent-First MG, Muallem A, Van bergen AH, Nolten WE, Meisner L, Roberts KP. Microdeletions in the Y-Chromosome of Infertile Men. New Engl J Med 1997; 336:534–39. 3. Kent-First MG, Ryan A, Schifreen R, Frackman S. The genetic bases of male infertility. IVD Technology, Canon Communications LLC, 1999. 4. Patrizio P. Mapping of the Y-chromosome and clinical consequences. In Barrat C, De Jonge C, Mortimer D, Parinaud J (Eds): Genetics of human male fertility. EDK: Paris, 1997.

CHAPTER 76 Isolated Teratozoospermia—ICSI or IUI Kemal Ozgur, Caner Sonmez INTRODUCTION The diagnosis of male factor infertility has been the corner stone of the work up for the infertile couple. It is a common practice to start the workup with semen analysis when the couples have their first visit to the specialist. In spite of the extensive research only a few tests have been used to diagnose male infertility. Basic semen analysis is perhaps the simplest but the most important one in clinical practice. However the lack of qualitative and quantitative calibration tools in basic semen analysis has made the test results vary from one lab to another. Several professional organizations including WHO have made an effort to standardize of basic semen analysis. Perhaps sperm morphology has been one of the most difficult to standardize in the last 15 years. The only parameter that has undergone a major change in the 5 years edition of the WHO lab manual has been the criteria for sperm morphology. The parameters of the basic semen analysis have been reviewed extensively by Coetze et al., and sperm morphology has been found to be the most valuable in predicting the outcome of in vitro fertilization.1 Therefore sperm morphology has been given special importance in deciding the prognosis of assisted conception of the couple. WHO Criteria for Normal Sperm In 1987, a normal spermatozoon was defined as described by the WHO, i.e. an oval shape with regular outline and acrosomal cap covering more than one third of the head surface.2 The head length was 3–5 µm and width ranged between 2 and 3 µm; the width was between one—half and two thirds of the length. The tail was slender, uncoiled and regular in outline and at least 45 µm in length. Spermatozoa were classified into % normal, % head defects (amorphous, small, large, pyriform, tapering), % midpiece defects (including cytoplasmic droplets) and % tail defects. Two counts should be performed on each occasion and if the difference was >10% than a repeat count should be performed and the mean value should be calculated. At least 200 spermatozoa should be examined in an attempt to reduce clinical variation. However following two editions of 1992 and 1999 both the description and the normal reference values of normal spermatozoon have gone through evolution.3,4 In table 76.1 normal sperm criteria and the comparison between WHO 1987, 1992 and 1999 methods have been demonstrated

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Table 76.1: Normal sperm criteria-comparison between WHO 1987, 1992 and 1999 methods WHO 1987

WHO 1992

WHO 1999

Head shape

Regular oval shaped

Oval in shape

Normal frequency Acrosome

50%

Oval; borderline forms abnormal; pinheads not counted 30%

>1/3 of head surface

Head size

3–5µm long; 2–3 µm wide Length/width 1.5–2.0 ratio Vacuoles No details Midpiece Slender, <1/3 width of head, straight and regular, aligned with longitudinal axis of head: 7–8 pm Tail

Slender, uncoiled, regular at least 45µm

Cytoplasmic No details droplets

Well defined 40–70% of head area 4.0–5.5 µm long; 2.5–3.5 µm 4.0–5.0 µm long; 2.5–3.5 µm wide wide 1.5–1.75 1.5–1.75 <20% of head area No dimensions; no description of normal midpiece (defects only given, e.g. insertion of tail >90% to head long axis is abnormal) No dimensions; no description of normal tail (defects only given) <1/3 normal head

>20% of the head area Slender, less than 1µm in width, about one and a half times the length of the head and attached to axially to the head. The tail should be straight, uniform, thinner than the midpiece, uncoiled and approximately 45 µm long Less than half size of the sperm head

Why Morphology has a Specific Role? Is morphology the crucial end point for the evaluation of human sperm? There are two possible explanations for the power of morphology to predict the fertility potential of the male. First, the concentration and the motility has little variability from one lab to another and the tools for these parameters are easier to standardize. However it has become difficult to have similar cut off values for the normal definitions on sperm morphology from one lab to another. Therefore different definitions of spermatozoa may have weakened the power of morphology in the past. Second, studies on using the basic semen analysis; sperm morphology has been found to be the most valuable parameter. Extensive review by Coetze et al have explored that sperm morphology according to strict criteria, has the most powerful impact in predicting fertilization in IVF.1 In keeping with the move to a more strict definition, the World Health Organization has redefined the normal spermatozoa and subsequently dropped to an empirical reference value from 50% to 30% in 1992.2–3 However the def inition of a normal spermatozoon as described by the WHO in 1987 and 1992 is different from that used by other authors.5,6 In contrast to the more strict scoring methods of sperm morphology adopted by Kruger and Liu, there appears no clinical and biological data to clarify the

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definition of normal spermatozoon in WHO (1992). Therefore, in 1999, WHO has changed the direction and redefined the criteria for normal spermatozoon to what Kruger et al5 and Liu et al6 defined previously However, for this edition no reference value has been given and multiple clinical studies have been expected to report a reference value for the normal frequency. The strict criteria are superior to the previous WHO criteria for the morphology in predicting fertilization in vitro. Because a biological end point has been used to aid the definition of normal spermatozoa, e.g. examination of morphological characteristics of spermatozoa recovered from cervical mucus and/or those binding to the zona pellucida. Menkveld et al has found increased proportion of normal spermatozoa in the swim-up fraction and zona pellucida bound spermatozoa. In this study the percentage of normal spermatozoa has increased from 21.5% to 27.5% in the swim-up fraction and to 44.8% in zona pellucida bound sperm.7 We have also found that the percentage of normal morphology positively correlates with the zona binding capacity of the sperm and these two parameters were the best predictors for the fertilization rate in in vitro fertilization.8 Is Isolated Teratozoospermia Another Entity Than Teratozoospermia with Oligoasthenospermia? During the 80’s and the early nineties most of the studies on the sperm morphology were aimed on the prediction of the fertilization failures in the field of IVF. Total fertilization failures have been the most scary part on the next day when checking the inseminated eggs in IVF. However with ICSI, fertilization failures have seldom occurred as the insertion of the sperm into the egg cytoplasm has almost eliminated the male factor problem. When there is a male factor case, which presents with oligoasthenoteratospermia, ICSI is commonly offered to solve the infertility problem. However, in cases with isolated teratospermia with a normal count and motility, there exists a dilemma as to whether they should be considered for ICSI. According to the WHO 1999 Lab manual, the count should be more than 20 million and the motility 50%.4 Although the cut off for normal morphology has not been reported, in clinical practice, 4% has been defined for teratozoospermia. In Table 76.2, we have shown the characteristics of the isolated teratozoospermia cases compared to the normal ones. We have found 6.0% incidence of isolated teratospermia in our patient population. Interestingly, there was a statistically significant increase in smoking in this group compared to the OAT cases. The incidence of male smoker in our patient population was 19.2%, which was significantly different from the isolated teratozoospermic cases. Wong WY et al have investigated the effect of cigarette smoking in male factor subfertility by correlating cotinine in seminal plasma and semen morphology.9 These authors have found a small but statistically significant correlation between cotinine concentrations in seminal plasma and the percentage of abnormal sperm morphology, but not for other semen parameters (r(s)=0.19). We believe that

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Table 76.2: Characteristics of teratozoospermic patients Oligoasthenoteratozoospermic cases Number of cases perinfertility 112/395 (28.3%) population Smoker (more than 5 19/112 (16.9%) cigarette/day)

Isolated teratozoospermia cases 24/395 (6.0%) 9/24 (37.5%)

the effect of smoking on sperm morphology needs further investigation. What Therapy should be offered to the Isolated Teratozoospermia Cases? Perhaps the question is to employ controlled ovarian hyperstimulation plus intrauterine insemination (COH +IUI) or ICSI for these cases. The answer lies on the pregnancy rates and cost of the treatment. However, there are no studies in the literature to compare the cost-effect of COH+IUI to ICSI in these cases at the moment. We believe that the answer to this question will result in better success rates for the clinicians in treating their patients. We have therefore designed a study to evaluate the role of sperm morphology on COH+IUI success in patients with normal motility and concentration. Materials and Method The study design was a prospective cohort type and inclusion criteria were as follows; female age less than 40, day 3 serum FSH <10 IU/L, at least one patent tube at hysterosalpingography or laparoscopy, sperm concentration of at least 20 million/ml and motility of at least 50%, presence of at least 10 million progressive motile sperm after swim-up. Human menopausal gonadotropins and/or pFSH were used for controlled ovarian hyperstimulation (COH). When the mean diameter of the leading follicle reached 18 mm, 5000/1000IU hCG was administered. One person performed sperm morphology assessment by Kruger’s strict criteria with the semen used for the insemination.5 Standard swim-up was used for sperm preparation and all women were inseminated with 10 million progressive motile spermatozoa 36 hrs after hCG administration. For luteal support 50 mg/day IM progesterone in oil was used and serumbeta-hCG 14 days after IUI was determined. Main outcome measure was the presence of pregnancy Receiver operating characteristic (ROC) curve was constructed for assessment of effectiveness of sperm morphology in predicting pregnancy and Student’s tor Mann-Whitney U and Chisquare or Fisher’s exact tests were used for statistical analysis where appropriate. Results One hundred and sixteen cycles were performed on 88 patients and 21 pregnancies achieved of which 5 were biochemical and 16 were clinical. There were 3 healthy

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deliveries, 4 abortions and 10 ongoing pregnancies. The pregnancy rate per cycle was 18.1% and 23.8% per patient. The patient characteristics are shown in Table 76.3.

Table 76.3: Patient characteristics of pregnant and no pregnant patients Pregnant Not pregnant P Age (years) 28.4 (±5.2) 29.6 (±4.4) Spouse’s age (years) 33.3 (±4.9) 34.0 (±4.3) Duration of infertility (months) 51.3 (±34.9) 67.8 (±50.4) Previous gravidity 0.45 (±0.68) 0.35 (±0.74) Smoking (%) 20.0 27.9 Smoking in spouse (%) 50.0 42.6

0.31 0.71 0.24 0.46 0.57 0.56

There was no difference in the semen characteristic of these patients. The concentration and the motility of the semen in pregnant and non-pregnant patients are shown in Table 76.4.

Table 76.4: Sperm concentration and motility before semen preparation. Pregnant Not pregnant P Sperm concentration (milyon/ml) 76.5 (±35.6) 80.7 (±41.6) Motile sperm percentage 59.8 (±14.2) 61.9 (±14.0)

0.69 0.59

We could find a statistically significant optimal cut-off value in sperm morphology but not in concentration and motility. The ROC curve of sperm morphology with IUI outcome is shown in Figure 76.1.

Fig. 76.1: ROC curve of sperm morphology with IUI outcome

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In Table 76.5 the relationship of sperm morphology at certain cut-off points is shown and 2 and 7% are found to be the thresholds to predict a pregnancy.

Table 76.5: Relationship of sperm morphology at certain cut-off points Morphology Pregnant Not pregnant Pregnancy rate (%) P* <2 1 25 3.8 0.01 2–7 20 56 26.3 >7 0 14 0.0 0.03 *When compared with the 2–7% normal morphology group

Discussion In this study we have aimed to explore the effect of sperm morphology on the success rate of COH+IUI in a specific group; isolated teratozoospermia cases. We have found two cut-off values to predict pregnancy. Less than 2% and more than 7% normal morphology were statistically significant in predicting a pregnancy. We have the highest pregnancy at the interval 2–7% normal morphology. The reason we have 2% as lower cut-off not 4% compared to the literature was controversial. There could be two explanations. First, the 2% may be due to our readings to be stricter. The second is that in the literature except one study by Lyndheim et al, all the studies were heterogeneous regarding to concentration and the motility.10 Therefore, the results were conflicting regarding the effect of morphology. Our study was a specific group performed only on the isolated teratozoospermic cases. We have found that regardless of the concentration and the motility, if the case has a poor morphology there is a very low chance to get pregnant in COH+IUI. The literature review has been shown in Table 76.6.

Table 76.6: The effect of sperm morphology on the success of COH+IUI Author

Criteria

Study design

Concentration and motility Result

Strict Prospective Low No effect Matorras et al (1995)11 Strict Retrospective Normal or low No effect Karabinus et al (1997)12 Ombelet et al (1997)13 Strict —do— Normal or low No effect Toner et al (1995)14 Strict —do— Normal or low Effect + Burr et al (1996)15 WHO —do— Normal or low Effect + Lindheim et al Strict —do— Normal Effect + (1996)10 —do— Normal or low Effect+ Hauser et al (2001)16 Strict Perhaps the treatment should be defined with the perspective of the cost analysis. Van Voorhis BJ et al. in a recent article have analyzed the cost of IUI and ICSI in subfertil cases.17 These authors have found ICSI to be more cost-effective than IUI if there is less than 5 million progressive motile sperm per ejaculate. Therefore we believe that the cost analysis for the isolated teratospermia cases may reveal ICSI to be more cost-effective than IUI.

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How is Sperm Function in Isolated Teratozoospermia? The prediction of the capacity of human sperm to achieve fertilization in υitro and in vivo has been a major goal within the assisted reproductive technology practice. Different laboratories have highlighted the diagnostic power of a variety of such assays, and the World Health Organization has incorporated some of these assays under the category of functional tests.4 Sperm-zona pellucida binding studies have been recommended because of the powerful evidence of their ability to predict fertilization success IVF failure.10 However, there are no studies to incorporate zona-binding function on the success of IUI. Therefore, we have designed a study to measure the zona binding function of isolated teratozoospermia cases. Ten cases with normal count and normal motility but poor morphology according to WHO criteria have been tested for Hemizona Assay. Salt-stored immature human oocytes were used in the experiments after desalting and microbisection into matching hemizonae following procedures extensively published elsewhere.18,19 A wash/swim-up separation of the motile sperm fraction was effected in Ham’s F-10 medium supplemented with 0.3% human serum albumin. Control (fertile donor) and test (patient) sperm droplets (100 ml of a final dilution of 0.5×106 motile sperm per ml) were incubated separately under heavy white mineral oil with a matching hemizona from the same pair for 4 hours at 37°C in 5% CO2 in air. After the incubation period, the hemizonae were washed to remove loosely attached sperm, using a finely drawn glass pipette and the sperm tightly bound to outer zona surface were counted under phase contrast microscopy (X200). The numbers of the tightly bound sperm are seen for each case in Fig. 76.2. The mean number of the sperm bound to each hemizona for the patient and the donor cases were 6±2.8 (mean±SD) and 22.7±7.5 (mean± SD), respectively.

Fig. 76.2: The mean number of the sperm bound to each hemizona for the patient and the donor

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These results are the first preliminary data on a selected population of isolated teratozoospermia. We have found that there is a considerable decrease in the number of the spermatozoa bound to zona pellucida if the spermatozoa is teratozoospermic. We believe that a major defect in sperm function may decrease the zona binding capacity revealing the decrease in the pregnancy rates in IUI. REFERENCES 1. Coetzee K, Kruger TF, Lombart CJ. Predictive value of normal sperm morphology: a structured literature review. Hum Reprod Update 1998; 4:73–82. 2. World Health Organization. WHO Laboratory Manuel for the Examination of Human Sperm and Semen-Cervical Mucus Interaction, (2nd edn), Cambridge: Cambridge University Press, 1987 3. World Health Organization. WHO Laboratory Manuel for the Examination of Human Sperm and Semen-Cervical Mucus Interaction, (3rd edn), Cambridge: Cambridge University Press, 1992 4. World Health Organization. WHO Laboratory Manuel for the Examination of Human Sperm and Semen-Cervical Mucus Interaction, (4th edn), Cambridge: Cambridge University Press, 1999 5. Kruger TF, Menkveld R, Stander FSH, Lombart CJ, Van der Merwe JP, van Zyl JA. Sperm morphologic features as prognostic factors in in vitro fertilization. Fertil Steril 1986; 46:1118– 23. 6. Liu DY, Baker HWG. The proportion of human sperm with poor morphology but normal intact acrosomes detected with Pisum sativum agglutinin correlates with fertilization in vitro. Fertil Steril 1988; 50:288–93. 7. Menkveld R, Stander FSH, Kotze TJvW, Kruger TF, van Zyl JA. T. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990; 5:586–92. 8. Oehninger S, Mahony M, Ozgur K, Kolm P, Kruger T, Franken D. Clinical significance of human sperm-zona pellucida binding. Fertil Steril 1997; 67(6):1121–7 9. Wong WY, Thomas CM, Merkus HM, Zielhuis GA, Doesburg WH, Steegers-Theunissen RP. Cigarette smoking and the risk of male factor subfertility: minor association between cotinine in seminal plasma and semen morphology Fertil Steril 2000; 74(5): 930–35 10. Lyndheim SR, Barad DH, Zinger M, Witt B, Amin H, Cohen B, et al. Abnormal sperm morphology is highly predictive of pregnancy outcome during controlled ovarian hyperstimulationand intrauterine insemination. J Assist Reprod Genet 1996; 13:569–72. 11. Matorras R, Cortostequi B, Perez C, Mandiola M, Mendoza M, Rodriguez-Escudero FJ. Sperm morphology analysis (strict criteria) in male infertility is not a prognostic factor in intrauterine insemination with husband’s sperm. Fertil Steril 1995; 63:608–11. 12. Karabinus DS, Gelety TJ. The impact of sperm morphology evaluated by strict criteria on intrauterine insemination success. Fertil Steril. 1997; 67:536–41. 13. Ombelet W, Van de Putte G, CoxA, Janssen M, Jacobs P, Bosmans E, et al. Intrauterine insemination after ovarian stimulation with clomiphene citrate: predictive potential of inseminating motile count and sperm morphology. Hum Reprod. 1997; 12(7):1458–63 14. Toner JP, Mossad H, Grow DR, Morshedi M, Swanson RJ, Oehninger S. Value of sperm morphology assessed by strict criteria for prediction of the outcome of artificial (intrauterine) insemination. Andrologia. 1995; 27(3): 143–8. 15. Burr RW, Sieberg R, Flaherty SP, Wang XJ, Matthews CD. The influence of sperm morphology and the number of motile sperm inseminated on the outcome of intrauterine insemination combined with mild ovarian stimulation. Fertil Steril. 1996; 65(1): 127–32. 16. Hauser R, Yogev L, Botchan A, Lessing JB, Paz G, Yavetz H. Intrauterine insemination in male factor subfertility: significance of sperm motility and morphology assessed by strict criteria. Andrologia 2001; 33(1):13–7

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17. Van Voorhis BJ, Barnett M, Sparks AE, Syrop CH, Rosenthal G, Dawson J. Effect of the total motile sperm count on the efficacy and cost-effectiveness of intrauterine insemination and in vitro fertilization. Fertil Steril 2001; 75(4):661–8. 18. Burkman LJ, Coddington CC, Franken DR, Kruger T, Rozenwaks Z, Hodgen GD. The hemizona assay: development of a diagnostic test for the binding of spermatozoa to the human zona pellucida to predict fertilization potential. Fertil Steril 1988; 49:688–93. 19. Oehninger S, Acosta AA, Veeck L, Bryzski R, Kruger TF, Muasher SJ, et al. Recurrent failure of in vitro fertilization: role of the hemizona assay in the sequential diagnosis of specific spermoocyte defects. Am J Obstet Gynecol 1991; 164:1210–5.

CHAPTER 77 Hormone Substitution in Male Infertility Frank M Köhn, Wolf B Schill SUMMARY Causal medical treatments in andrology include the treatment of endocrine insufficiency, sperm transport disturbances, and male adnexitis. Hormone therapy will mainly improve sperm concentration only in cases of male hypogonadotropic hypogonadism. In addition a variety of therapeutic procedures have been developed to modify sperm functions in vitro. Adjuvant male hormone therapy before or during IVF procedures has been suggested by several groups. For example, administration of exogenous testosterone or systemic treatment with FSH may improve the fertilizing capacity of spermatozoa. INTRODUCTION Therapeutic approaches to improve impaired male fertility are still limited.1 This is due to the fact that the cause of reduced fertility is unknown in most cases. Causal medical treatments in andrology include the treatment of endocrine insufficiency, sperm transport disturbances, and male adnexitis.2 Hormone therapy will mainly improve sperm concentration only in cases of male hypogonadotropic hypogonadism. In addition a variety of therapeutic procedures have been developed to modify sperm functions in vitro.3 Adjuvant male hormone therapy before or during IVF procedures has been suggested by several groups.4–8 For example, administration of exogenous testosterone or systemic treatment with FSH may improve the fertilizing capacity of spermatozoa. Ashkenazi et al7 and Ben-Rafael et al18 treated male partners of ICSIcouples with pure FSH or urinary FSH before oocyte retrieval and observed significantly higher implantation rates. Human Chorionic Gonadotropin (hCG) hCG is a polypeptide with the action of luteinizing hormone (LH) which is released from the pituitary gland under physiological conditions. LH simulates testicular Leydig cells and thus the testosterone production. hCG is approved for treatment of undescended testis and retarded puberty in children as well as therapy of hypogonadotropic hypogonadism in adults. The usual dosage for hypogonadotropic hypogonadism is 1000–2500IU hCG 2–3 times a week. During this therapy, serum testosterone concentrations return to normal and the testicular volume increases.9,10 In cases of hypogonadotropic hypogonadism and wish for a child, hCG substitution is combined with the injection of human menopausal gonadotropin (hMG) and usually continued for several months. A positive effect of

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hCG/hMG on the ejaculate quality in normogonadotropic men has not yet been demonstrated in controlled studies.11 However, therapy with hCG was recommended for patients with sustained Leydig cell dysfunction after varicocelectomy12 Human Menopausal Gonadotropin (hMG) hMG is obtained from the urine of menopausal women and possesses both FSH and LH activity. Follicle-stimulating hormone (FSH), physiologically released from the pituitary gland, stimulates spermatogenesis via Sertoli cells. In men, approval is restricted to male sterility and induction of spermatogenesis in cases of hypogonadotropic hypogonadism. The usual dosage is 75–150 IU hMG. In patients whose hypogonadotropic hypogonadism remained untreated for a longer time or who received testosterone substitution therapy, initial application of hCG is recommended to stimulate the endogenous testicular testosterone production. hMG is then additionally given after 1–3 months. Spermatogenesis is restored in up to more than 90 percent so that spermatozoa can be found in the ejaculate. Kliesch et al9 required average treatment periods of 6.7 months in men with hypogonadotropic hypogonadism after operation or trauma. In men with idiopathic hypogonadotropic hypogonadism or Kallmann syndrome, the first spermatozoa reappeared after an average of 9 months. The therapy was sometimes continued over more than 2 years until pregnancy occurred. The combination with hCG also increased the testicular volume significantly. Comparing this treatment with pulsatile GnRH therapy, Schopohl et al10 observed a more rapid restoration of spermatogenesis and higher increase in testicular volume during GnRH therapy, while Kliesch et al9 and Buchter et al13 did not record significant differences. A controlled study in normogonadotropic men failed to achieve improved ejaculate quality during combined hCG and hMG therapy.11 Pure and Recombinant FSH Highly purified FSH and recombinant FSH have higher specific activities than hMG with no LH activity. The amino acid sequence of recombinant FSH is identical to that of natural FSH. Highly purified urinary FSH and recombinant FSH have also been approved for therapy of male hypogonadism. The mechanism of action is identical to that of hMG. The response rates concerning initiation of spermatogenesis with recombinant and highly purified FSH appear to be consistent with those of hMG.14,15 Acosta et al4 reported on the use of pure FSH in 24 men who had failed to fertilize in an IVF program (group 1) and 26 men with reduced ejaculate quality (group 2). The patients received 150 IU pure FSH IM 3 times a week for at least 3 months. During this period, there were no significant changes in the ejaculate quality; however, the average fertilization rate in the FSH-treated group 1 increased from 2.2 to 54.4 percent, and in group 2 it was found to be 52.3 percent. These results implied effects of FSH therapy on sperm functions. In accordance with Acosta et al4 Glander and Kratzsch16 did not observe effects on sperm quality after 10 weeks therapy with pure FSH in 41 men with idiopathic infertility. On the other hand, a significant increase in sperm concentration and total motile sperm count was found in men who had shown lower FSH secretion after injection of GnRH. Like urinary FSH,

Hormone substitution in male infertility

recombinant FSH in combination hypogonadotropic men.17

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induces

spermatogenesis

in

Gonadotropin Releasing Hormone (GnRH) As a synthetic product GnRH is available as gonadorelin. Its structure is identical to that of native GnRH which is secreted from the hypothalamus. It stimulates the release of LH and FSH from the pituitary gland. Under physiological conditions, pulsatile secretion of GnRH occurs every 60–120 minutes. The substance is approved as solution for intranasal application in cases of unilateral or bilateral undescended testis, as injection solution for diagnostic application in patients with hypothalamic, pituitary or gonadal dysfunction as well as for treatment of retarded puberty and tertiary hypogonadotropic hypogonadism in men with testicular dysfunction. Of highest andrological-therapeutic significance currently is the pulsatile subcutaneous administration by means of a portable infusion pump. It is indicated in cases of tertiary hypogonadotropic hypogonadism (e.g. Kallman syndrome or idiopathic hypogonadotropic hypogonadism) and retarded puberty. For these indications, the usual dosage is 5–20 µg gonadorelin per pulse every 120 minutes over several months. During this therapy, serum testosterone levels return to normal and an increase in testicular volume is achieved.9,10 Complete normalization of the ejaculate quality can be expected only rarely. In men with Kallmann syndrome or idiopathic hypogonadotropic hypogonadism, the first spermatozoa have been redetected after an average of 9 months. GnRH is not effective in patients with disturbed spermatogenesis and a concomitant increase in FSH concentrations.18 In addition, pulsatile GnRH administration does not improve semen parameters in patients with idiopathic normogonadotropicoligozoospermia.19 Androgens Androgens are used for substitution therapy of male hypogonadism. Testosterone enanthate is an esterified form of endogenous testosterone, an anabolic steroid. Previously, the drug was used for release of a so-called “rebound effect” in oligozoospermic men; weekly injections resulted in azoospermia by suppression of gonadotropins. Thereafter, the injection therapy was interrupted until spermatogenesis was initiated. Today, this treatment must be considered as obsolete. Other techniques of testosterone substitution such as transdermal systems by films, tapes or gels will also not improve semen quality and are not indicated in cases of male infertility. Testosterone Undecanoate Testosterone undecanoate is a testosterone that is esterified with undecanoic acid at position 17*. Testosterone undecanoate is approved for substitution therapy of hypogonadism, treatment of male climacteric symptoms, impaired spermatogenesis due to androgen deficiency, and osteoporosis associated with androgen deficiency. For substitution therapy, 80–160 mg testosterone undecanoate are administered daily. The effect on the ejaculate quality of patients with a normal hormonal status is controversial.

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In a double-blind, randomized, placebo-controlled study in 60 men, Pusch20 observed significantly increased sperm concentrations and a higher number of normally shaped spermatozoa after 100 days therapy with 120 mg testosterone undecanoate daily. However, the pregnancy rates were not significantly different from those in the control group. Abdelmassih et al5 reported improved fertilization rates in an IVF program after the male partners had been treated with testosterone undecanoate. Later controlled studies, sometimes using higher concentrations (240 mg per day), failed to demonstrate positive therapeutic effects on sperm parameters and fertilizing capacity.6,21,22 For therapy of eugonadal infertile men, testosterone undecanoate is only indicated within the framework of clinical studies and for special indications (stimulation of epididmyis or seminal vesicles). Mesterolone Mesterolone is the 1*-methyl compound of 5*-dihydrotestosterone which acts on androgen-dependent tissues as a testosterone metabolite. The substance is approved for treatment of relative or absolute androgen deficiency and its sequelae, such as renal anemia, vegetative psychiatric disorders and decreased vitality in middleaged and aged men. Inhibition of spermatogenesis is less pronounced during mesterolone therapy because of lower central inhibition of gonadotropins. Mesterolone has also been used for treatment of male fertility disorders. However, a significant effect on the pregnancy rates could not be documented in controlled studies. In a doubleblind, placebo-controlled trial, Gems et al23 investigated the effects of mesterolone in 52 men with idiopathic oligozoospermia and/or teratozoospermia. The patients were given daily doses of 150 mg mesterolone for 12 months. While no improvement in sperm concentration was observed, the number of motile and morphologically normal spermatozoa increased in the group receiving mesterolone or placebo. The pregnancy rates were not significantly different. A more careful judgement resulted from a randomized, double-blind and placebo-controlled study previously performed by the WHO24. A total of 157 men were treated with daily doses of 75 or 150 mg mesterolone for 6 months. No significant differences in ejaculate quality were seen, but there was a tendency towards higher pregnancy rates in the mesterolonetreated group (control group 1:11 percent; mesterolone 75 mg:12 percent; mesterolone 150 mg:19%). REFERENCES 1. Schill WB. Survey of medical therapy in andrology. Int J Androl 1995; (Suppl 2):56. 2. Schill WB, Haidl G. Medical treatment of male infertility. In Insler V, Lunenfeld B (Eds): Infertility: Male and Female. Edinburgh: Churchill Livingstone, 1993:575. 3. Naz RK, Minhas BS. Enhancement of sperm function for treatment of male infertility J Androl 1995; 16:384. 4. Acosta AA, Khalifa E, Oehninger S. Pure human follicle stimulating hormone has a role in the treatment of severe male infertility by assisted reproduction: Norfolk’s total experience. Hum Reprod 1992; 7:1067. 5. Abdelmassih R, Dhont M, Comhaire F. Pilot study with 120 mg Andriol treatment for couples with a low f ertilization rate during in-vitro fertilization. Hum Reprod 1992; 7:267.

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6. Comhaire F, Schoonjans F, Abdelmassih R et al. Does treatment with testosterone undecanoate improve the in-vitro fertilization capacity of spermatozoa in patients with idiopathic testicular failure? (results of a double blind study). Hum Reprod 1995; 10:2600. 7. Ashkenazi J, Bar-Hava I, Farhi J et al. The role of purified follicle stimulating hormone therapy in the male partner before intracytoplasmic sperm injection. Fertil Steril 1999; 72:670. 8. Ben-Rafael Z, Farhi J, Feldberg D et al. Follicle-stimulating hormone treatment for men with idiopathic oligoteratoasthenozoospermia before in vitro fertilization: the impact on sperm microstructure and fertilization potential. Fertil Steril 2000; 73:30. 9. Kliesch S, Behre HM, Nieschlag E. High efficacy of gonadotropin or pulsatile gonadotropinreleasing hormone treatment in hypogonadotropic hypogonadal men. Eur J Endocrin 1994; 131:347–59. 10. Schopohl J, Mehltretter G, von Zumbusch R et al. Comparison of gonadotropin-releasing hormone and gonadotropin therapy in male patients with idiopathic hypothalamic hypogonadism. Fertil Steril 1991; 56:1143. 11. Knuth UA, Honigl W, Bals-Pratsch M et al. Treatment of severe oligospermia with human chorionic gonadotropin/human menopausal gonadotropin: a placebo-controlled, double blind trial. J Clin Endocrin Metab 1987; 65:1081. 12. Yamamoto M, Hibi H, Katsuno S et al. Human chorionic gonadotropin adjuvant therapy for patients with Leydig cell dysfunction after varicocelectomy. Arch Androl 1995; 35:49. 13. Buchter D, Behre HM, Kliesch S et al. Pulsatile GnRH or human chorionic gonadotropin/human menopausal gonadotropin as effective treatment for men with hypogonadotropic hypogonadism: a review of 42 cases. Eur J Endocrin 1998; 139:298. 14. European Metrodin HP Study Group. Efficacy and safety of highly purified urinary folliclestimulating hormone with human chorionic gonadotropin for treating men with isolated hypogonadotropic hypogonadism. Fertil Steril 1998; 70:256. 15. Liu PY, Turner L, Rushford D et al. Efficacy and safety of recombinant human f ollicle stimulating hormone (Gonal-F) with urinary human chorionic gonadotrophin for induction of spermatogenesis and fertility in gonadotrophin-deficient men. Hum Reprod 1999; 14:1540. 16. Glander HJ, Kratzsch J. Effects of pure human follicle-stimulating hormone (pFSH) on sperm quality correlate with the hypophyseal response to gonadotrophin-releasing hormone (GnRH). Andrologia 1997; 29:23. 17. Kliesch S, Behre HM, Nieschlag E. Recombinant human follicle-stimulating hormone and human chorionic gonadotropin for induction of spermatogenesis in a hypogonadotropic male. Fertil Steril 1995; 63:1326. 18. Bals-Pratsch M, Knuth UA, Honigl W et al. Pulsatile GnRH-therapy in oligozoospermic men does not improve seminal parameters despite decreased FSH levels. Clin Endocrin 1989; 30:549. 19. Crottaz B, Senn A, Reymond MJ et al. Follicle-stimulating hormone bioactivity in idiopathic normogonadotropic oligoasthenozoospermia: double-blind trial with gonadotropinreleasing hormone. Fertil Steril 1992; 57:1034. 20. Pusch HH. Oral treatment of oligozoospermia with testosteroneundecanoate: results of a double-blind-placebo-controlled trial. Andrologia 1989; 21:76. 21. Comhaire F. Treatment of idiopathic testicular failure with high-dose testosterone undecanoate: a double-blind pilot study. Fertil Steril 1990; 54:689. 22. Comhaire F, Milingos S, Liapi A et al. The effective cumulative pregnancy rate of different modes of treatment of male infertility. Andrologia 1995; 27:217. 23. Gerris J, Comhaire F, Hellemans P et al. Placebo-controlled trial of high-dose Mesterolone treatment of idiopathic male infertility. Fertil Steril 1991; 55:603. 24. WHO Task Force on the Diagnosis and Treatment of Infertility. Mesterolone and idiopathic male infertility: a double-blind study. Int J Androl 1989; 12:254.

SECTION 13 Preimplantation Genetic Diagnosis (PGD)

CHAPTER 78 Preimplantation Genetic Diagnosis in Cases with Abnormal Gamete Cell Morphology

Semra Kahraman OVERVIEW The results of assisted reproductive techniques (ART) are directly related to the nuclear and cytoplasmic maturation of the gamete cells. Nuclear or cytoplasmic defects may reflect certain morphological features in the gametes. However, to date, there have been few reports regarding the characteristics of embryos that were developed f rom morphologically abnormal oocyte or spermatozoa. Oocyte morphology has been thought to be insignificant in terms of fertilization, embryo quality and pregnancy rate.1–2 However, we have shown that cytoplasmic granulation of an oocyte may be a poor prognostic factor as the implantation and ongoing pregnancy rates were found to be unexpectedly low in this group of patients, despite normal fertilization rates, embryo quality and pregnancy rates.3 Further, in-vivo development of the embryo developed from a granular oocyte is thought to be compromised reflecting a poor implantation capacity or inability to go far beyond a viable pregnancy. Furthermore, it has been shown that patients with only megalo-pinhead spermatozoa in the ejaculate had low fertilization and pregnancy rates.4 This particular morphological abnormality results in defective acrosomal or nuclear maturation of the spermatozoa yielding poor capacity for conception.

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We had performed this study in order to evaluate the chromosomal complement of embryos that developed from two different forms of abnormal gametes; either from oocytes with centrally located cytoplasmic granulation or megalo-pinhead spermatozoa. Centrally Located Granular Oocytes Centrally located granular oocytes (CLGO) group was defined as the cycle in which all the oocytes retrieved following controlled ovarian hyperstimulation revealed central cytoplasmic granulation. Central granulation is of concern when the granulation is located centrally within the cytoplasm with a clear border, easily distinguishable with a darker appearance than normal cytoplasm.5 Cytoplasmic granulation was diagnosed as a larger, dark, spongy granular area, located centrally in the cytoplasm of the oocyte. In 23 cycles, a total of 322 oocytes were retrieved, all presenting central cytoplasmic granulation. Following intracytoplasmic sperm injection (ICSI), 73.2% of the oocytes were fertilized. On examination 64–68 hours after ICSI, 117 embryos containing more than 7 or 8 blastomeres were available for embryo biopsy A successful FISH analysis was carried out in 113 embryos and 52.3% were found to be abnormal. The majority of the abnormal embryos revealed aneuploidy (60.9%), followed by complex aneuploidy (19.6%). Atotal of 54 embryos were transferred in 21 cycles. Pregnancy was achieved in 7 cycles (36.6%). The on-going pregnancy (PR) and implantation rates (IR) were 28.5% (6/21) and 16.6% (9/54), respectively. METHODS OF TECHNIQUE The study group consisted of 35 ART cycles undertaken with the indication of severe male factor infertility. Third day embryos were biopsied in 23 cycles due to CLGO and in 12 cycles due to megalopinhead spermatozoa (MPHS). The couples were informed about the procedure and an informed consent was signed for all the couples

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Fig. 78.1: Oocytes with centrally located cytoplasmic granulation before blastomere biopsy. All couples were karyotyped and no numerical or structural abnormalities were detected. Megalo-pinhead Spermatozoa In 12 cases, absolute teratozoospermia was observed in the semen samples. For each patient, at least two consecutive semen samples showed 100% morphologically abnormal spermatozoa according to strict criteria.6 The predominant morphological anomaly was megalopinhead spermatozoa while the remainder was an abundance of free heads without tails or immature spermatids. Only motile spermatozoa with slightly abnormal heads were selected for use in ICSI. A total of 180 oocytes were injected with megalo-pinhead spermatozoa and 59.5% were fertilized. Blastomere biopsy was realized in 59 embryos. In MPHS sperm group, 59 embryos were biopsied for 12 cycles. A successful FISH analysis was carried out in 56 embryos and 60% were found to be abnormal. The majority of the abnormal embryos revealed aneuploidy (60%). Atotal of 23 embryos were transferred in 12 cycles (1.9 per ET). Pregnancy was achieved in 4 cycles (33.3%). The ongoing PR and IR were 25% (3/12) and 13% (3/23), respectively

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Blastomere Biopsy and FISH Analysis Only embryos with seven or more blastomeres having < 20% fragmentation were biopsied. Assisted hatching prior

Fig. 78.2: Megalo-pinhead spermatozoa with abundance of free heads without tails or immature spermatids

Fig. 78.3: Megalo-pinhead spermatozoa and FISH analysis of a megalohead sample

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to blastomere biopsy was performed either with three-dimensional partial zona dissection or by the help of non-contact infrared diode laser. Three dimensional partial zona dissection in the shape of a cross-hatching or V-shape was performed to facilitate biopsy procedure, as was originally introduced by Verlinsky’s group.7 A V-shaped

Fig. 78.4: Blastomere biopsy

Fig. 78.5: Ca+ −Mg+ free medium facilitates easy removal of the blastomeres

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assisted hatching was performed in most of the embryos to create a triangular flap opening with a diameter of 25–30 mm to allow the replacement of the blastomere biopsy pipette. For diode laser, 3 shots of 8 milliseconds were performed to create the same gap through the zona pellucida. A hand drawn holding pipette, microneedle and biopsy pipette (Cook, IVF, K-EBPH-3535, Australia) were used for the purpose of the biopsy. A double pipette holder (Narishige HD-21, Japan) was used for microneedle and biopsy pipette on the same side. The blastomere biopsies were performed as previously described25 in calcium and magnesium free medium (EB™−10 medium, Scandinavian IVF Science, Gothenburg, Sweden). A single blastomere was biopsied from each embryo. Biopsied embryos were cultured for a further 24 hours in order to observe their further development. Only cleaving embryos were transferred. Flourescence in-situ hybridization (FISH) procedure was undertaken by the protocol described by Munne et al.8 The only difference from Munne’s protocol was the 3 hours of hybridization. A Nikon E-600 microscope was used with filters recommended for the probes by Vysis. The scoring was performed according to criteria described by Munne and Weier.9 Five DNAprobes were used for simultaneous detection of chromosomes X, Y, 13, 18 and 21 in multicolor FISH analysis. The chromosomal content was classified as complex aneuploid when two or more chromosomes had abnormal count but were not completely polyploid or haploid. The patients were given 100 mg progesterone intramuscularly beginning from the next day of oocyte retrieval until the serum beta-hCG assay, 12 days after the embryo transfer. If pregnancy was achieved, the patients were then instructed to use micronized progesterone tablets vaginally; 200 mg three times a day. Clinical pregnancy

Fig. 78.6: FISH analysis presenting trisomy 13

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was defined as the presence of fetal heart beats 21 days following the beta-hCG assay. Results Centrally Located Granular Oocytes (CLGO) In CLGO group, 117 embryos were biopsied in 23 cycles. The mean age of the female partners was 29.8±5.8. In two blastomeres, nucleus could not be observed and in two blastomere, fixation procedure failed. A successful FISH analysis was carried out in 113 embryos and the results are presented in Table 78.1.

Table 78.1: Results of chromosomal analysis in the centrally lcated cytoplasmic granular oocytes (n=88) Results Normal Abnormal Aneuploidy Trisomy Monosomy Nullisomy Tetrazomy Complex aneuploidy Haploidy Polyploidy Triploidy Tetraploidy

Embryos analyzed n % 56 61 37 16 16 3 2 12 4 4 3 1

47.7 52.3 60.9 44.0 44.0 8.0 4.0 19.6 6.5 6.5 4.3 2.2

Following blastomere biopsy, 91 embryos (78.3%) revealed an increase in the blastomere number after 24 hours of cultivation. A total of 54 embryos were transferred in 21 cycles (2.57 embryos per cycle). In two cases, all three embryos biopsied revealed abnormal chromosomal constitution and embryo transfer could not be realized. Pregnancy was achieved in seven cycles (Pregnancy rate: 36.6%). The on-going pregnancy and implantation rates were 28.5% (6/21) and 16.6% (9/54) respectively. Megalo-pinhead Spermatozoa In megalo-pinhead sperm group, 59 embryos were biopsied in 12 cycles. The mean age of the female partners was 27.9±6.8. In two blastomeres, fixation procedure failed and in a single blastomere, nucleus could not be observed. A successful FISH analysis was carried out in 56 embryos and the results are presented in Table 78.2.

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Table 78.2. Results of chromosomal analysis in megalopinhead spermatozoa group (n=56) Results Normal Abnormal Aneuploidy Trisomy Monosomy Nullisomy Complex aneuploidy Triploidy Haploidy

Embryos analyzed n % 22 34 21 14 5 2 3 7 3

40.0 60.0 62.0 69.2 23.1 7.7 9.5 19.0 9.5

Following blastomere biopsy, 34 embryos (60.5%) revealed an increase in the blastomere number after 24 hours of cultivation. A total of 23 embryos were transferred in 12 cycles (1.9 embryos per cycle). In a single case, an embryo defined as monosomy 13 was transferred following the discussion with the couple regarding their request for the embryo transfer. Pregnancy was achieved in two cycles (Pregnancy rate: 33.3%). The ongoing pregnancy and implantation rates were 25% (3/12) and 13% (3/23) respectively. Future Directions and Controversies Megalo-pinhead spermatozoa frequently accompany spermatozoa with certain other morphological defects such as sperm heads without tails and immature spermatids either in the ejaculate or testicular samples. These features suggest an insult in the spermatogenesis leading to anomalies either in the head or tail. There are only studies indicating the relationship between the sperm structure and their chromosomal constitution. Controversy exists if sperm head structures are indicative of genotype abnormalities.10–12 However, low implantation and ongoing pregnancy rates were observed in cases with absolute teratospermia with 100% abnormal head morphologies.11,13 Abnormal morphological parameters in the spermatozoa from men with oligoasthenoteratozoospermia may be associated with an increased frequency of meiotic and cytogenetic chromosomal abnormalities.14 Using FISH analysis, Viville had found no increase in the aneuploidy rate in single case samples from shortened flagella syndrome, globozoospermia or abnormal acrosomes. However, in the case with macrocephalic sperm, the aneuploidy rate by FISH was found to be higher than the controls.15 Lee has studied the chromosome constitution of human spermatozoa after injecting individual spermatozoa into mouse oocytes. The incidence of structural chromosome aberrations was found to be four times higher in spermatozoa with amorphous, round and elongated heads. Authors suggest that some morphological abnormalities in the sperm heads are associated with their chromosomal defects. They found no increase in chromosome aberrations in spermatozoa with large heads however, their sample size was too small to draw a conclusion.16 Kishikawa has reported abnormal

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karyotypes to be significantly higher in mouse oocytes injected with severely amorphous spermheads.17 It is possible that abnormal sperm head morphology reflects an abnormality in spermatogenesis as well as abnormal chromosomal constitution. This high rate of abnormality may be manifested by the embryos with a low potential to establish a viable pregnancy. On the other hand, there are conflicting data concerning the relationship between the oocyte morphology and fertilization and embryo development. We have previously described a subgroup of patients who present with centrally located granulation in the cytoplasm.3 ICSI in CLG oocytes were shown to yield poor on-going pregnancy rates despite normal fertilization rates and embryo quality. Additionally, there is only little data regarding the relationship between oocyte morphology and their chromosomal content. Genetically functional paternal genome is an important factor for fertilization and embryo development. However, a normal nuclear material probably will not be enough for the development of a healthy embryo. Defects in the cytoplasmic activation may directly block gene activation, cleavage and cell determinations, which also take place in the oocyte cytoplasm. In humans, successful male pronuclear assembly and fertilization are largely determined by the quality of the oocyte cytoplasm.18 Sperm chromosome decondensation, release of protamines, DNA repair, chromosome remodeling, assembly of organelles and the nuclear envelope around the reprogrammed haploid chromosomes are all accomplished by specific proteins accumulated in the cytoplasm.19–20 The definite role of oocyte cytoplasm in the stages of early embryo development is still not clear. Ooplasmic factors may play important roles in the continued development of the zygote, particularly during early cleavage, when transcription of the embryonic genome is minimal.21–22 Some non-genetic anomalies that originate in the oocyte cytoplasm rather than in the embryonic genome may interfere with normal development and viability of the embryo. Like the oocytes of the older women, dysmorphic oocytes may also contain defective or deleted mitochondrial DNA, that may lead to adverse cellular effects by disrupting the normal electron and energy transport chain, producing high levels of reactive oxygen species and cellular dysfunction.23 One may suggest that the central granulation in the cytoplasm may reflect a subtle cytoplasmic defect that will lead to development of an abnormal embryo. The high frequency of aneuploidy in the oocytes retrieved after controlled ovarian hyperstimulation is well documented.24 The selective transfer of chromosomally normal embryos may help to increase the implantation and ongoing pregnancy rates in these particular cases. We have previously reported that the implantation rate was poor as 4.2% in cases with CLG oocytes and the ongoing pregnancy rate was only 12.8%.3 The results were found to be relatively better following the transfer of embryos after PGD. The implantation and ongoing pregnancy rates were 13.3% and 25% for the PGD group, respectively. However, the limited number of the cases will comply the comparative results of a statistical analysis.

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CONCLUSION Abnormal gamete cell morphology may give clues about an impending risk of aneuploidy in the resultant embryo. Use of megalo-pinhead spermatozoa with possible nuclear defects or oocytes with central granulation with possible cytoplasmic defects may be associated with an increased risk of chromosomal abnormalities. The number of cases in this series is too limited to define a conclusion. Further studies and reports are warranted for the clarification of some subgroups of patients in which the aneuploidy risk may be increased and preimplantation genetic diagnosis should be offered. REFERENCES 1. Alikani M, Palermo G, Adler A, et al. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote 1995; 3:283–88. 2. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997; 12:1750–55. 3. Kahraman S, Yakýn K, Dönmez E et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome f ollowing intracytoplasmic sperm injection. Hum Reprod 2000; 15:2390– 93. 4. Kahraman S, Akarsu C, Cengiz G, et al. Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection. Hum Reprod 1999; 14:726–30. 5. Serhal PF, Ranieri DM, KinisAet al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997; 12:1267–70. 6. Kruger TF, Menkveld R, Stander FSH et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1996; 46:1118–23. 7. Cieslak J, Ivakhnenko V, Wolf G, et al. Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. J Assist Reprod Genet 1999; 16:176– 81. 8. Munne S, Grifo J, Cohen J et al. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8:2185–91. 9. Munne S, Weier HUG. Simultaneous enumeration of chromosomes 13, 18, 21, X and Y in interphase cells for preimplantation genetic diagnosis. Cytogenet Cell Genet 1996; 75:263–70. 10. Nagy ZP, Liu J, Joris H et al. The result of intracytoplasmic sperm genetic diagnosis. injection is not related to any of the three basic sperm parameters. Hum Reprod 1995; 10:1123–29. 11. Engel W, Murphy D, Schmid M. Are there genetic risks associated with microassisted reproduction? Hum Reprod 1996; 11:2359–70. 12. Vegetti W, VanAssche E, Frias A et al. Correlation between sperm parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum Reprod, 2000; 15:351–65. 13. Tasdemir I, Tasdemir M, Tavukcuoðlu S ei al. Effect of abnormal sperm head morphology on the outcome of intracytoplasmic sperm injection. Hum Reprod 1997; 12:1214–17. 14. Pieters MH, Speed RM, de Boer P et al Evidence of disturbed meiosis in a man referred for intracytoplasmic sperm injection. Lancet, 1998; 28:351(9107):957. 15. Viville S, Mollard R, Bach ML, et al. Do morphological anomalies reflect chromosomal aneuploidies? Hum Reprod 2000; 15:2563–66. 16. Lee JD, Kamiguchi Y, Yanagimachi R. Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Hum Reprod 1996; 11:1942–46.

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17. Kishikawa H, Tateno H, Yanagimachi R. Chromosome analysis of BALB/c mouse spermatozoa with normal and abnormal head morphology. Biol Reprod, 1999; 61:809–12. 18. Banerjee S, Lamond S, McMahon A et al. Does blastocyst culture eliminate paternal chromosomal defects and select good embryos? Hum Reprod 2000; 15:2455–59. 19. Collas P. Cytoplasmic control of nuclear assembly. Reprod Fertil Dev 1998; 10:581–92. 20. Collas P. and Poccia D. Remodeling the sperm nucleus into a male pronucleus at fertilization. Theorigenology 1998; 49:67–81. 21. Cohen J, Scott R, Alikani M, et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 1998; 4:269–80. 22. Krey LC, Grifo JA. Poor embryo quality; the answer lies (mostly) in the egg. Fertil Steril 2001; 75:466–68. 23. Fasouliotis S, Simon A, Laufer N. Evaluation and treatment of low responders in assisted reproductive technology: Achallenge to meet. J Assist Reprod Genet 2000; 17:357–73. 24. Martini E, Flaherty SP, Swann NJ et al. Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes. Hum Reprod 1997; 12:2011–18. 25. Kahraman S, Bahçe M, Þamlý H, et al. Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure. Hum Reprod 2000; 15:2003–07.

CHAPTER 79 Preimplantation Genetic Diagnosis (PGD) for Sex Selection Aniruddha Malpani, Anjali Malpani, Alan Thornhill, Deepak Modi INTRODUCTION Preimplantation genetic diagnosis (PGD), is a new technique, which marries the recent spectacular advances in molecular genetics and assisted reproductive technology, and our clinic has achieved the first pregnancy in India using this technique. Preimplantation genetic diagnosis enables physicians to identify genetic diseases in the embryo, prior to implantation, before the pregnancy is established. This means that the newest patient for modern medicine is now the embryo. Sexing the embryo to avoid X linked disease remains the commonest reason for preimplantation diagnosis, and this is now optimally carried out by the molecular cytogenetic technique of FISH (fluorescent in situ hybridization) with DNAprobes derived from the X and Y chromosomes. In the present communication we describe our initial experience with use of FISH for PGD for sex selection, followed by blastocyst transfer. We used a novel strategy whereby the embryos were biopsied and analyzed on day 3, cultured in sequential media, selected on day 5 for signs of blastulation and then transferred. By adopting this strategy, PGD centres have the option of using the extra time available to confirm the diagnosis or introduce additional diagnostic tests for the same embryo. The selection of blastocysts for transfer should increase pregnancy rates and also reduce the risk of multiple gestations as a consequence of the expected higher implantation rate. MATERIALS AND METHODS 15 couples were enrolled for preconceptional sex selection. All had at least one female child, and they wished to have a boy to complete their family Many had previously terminated pregnancies after prenatal diagnosis biopsy had shown that the fetus was female. Since sex determination by amniocentesis or chorionic villus sampling has now become illegal in India, they wanted to explore the possibility of preimplantation sex selection. Patients were superovulated with Buserelin, 0.5 ml sc daily from Day 1; and 225 IU of HMG (Humegon, Organon, Netherlands) from Day 3. On Day 14, 36 hours after 10,000 IU of HCG (Pregnyl, Organon, Netherlands), oocytes were retrieved by vaginal ultrasound collection. Oocytes were fertilized by conventional in vitro means using a sperm concentration of 100,000 motile sperm per ml in organ culture dishes containing

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up to five oocyte cumulus complexes in IVF-50 medium (Scandinavian IVF Sciences AB). Motile sperm were separated on a 45 percent Percoll gradient in IVF-50. Commercial medium from Scandinavian IVF Sciences AB was used throughout and culture took place in 5 percent CO2 at 37°C On day 3, the cleaving embryos were biopsied as previously described1 using Narishige micromanipulators in combination with screw-actuated air syringes (Research Instruments, Penryn, Cornwall, UK) to obtain a single mononucleate blastomere f rom each embryo, which is considered to be sufficient for FISH diagnosis of sex.2 All biopsy procedures were carried out in a petri dish (Falcon 1006, Becton Dickinson, USA) containing 20 µl drops of Ca+/Mg+-free embryo biopsy medium (EB-1, Scandinavian IVF Sciences AB) under oil (Ovoil, Scandinavian IVF Sciences AB); the efficacy and safety of which has been established by Dumoulin et al3 The Narishige double pipette holder (HD-21) was used in conjunction with axial-drive micropipette holders (Research Instruments) modified for use with Narishige equipment. Micromanipulators were mounted on an Olympus IX-70 inverted microscope with Hoffman modulation contrast optics. During biopsy, the embryo was held in position using a PGD holding pipette (Research Instruments, UK), a hole drilled through the zona pellucida using a stream of acid salt solution pH 2.3 (ZD-1, Scandinavian IVF Science AB) delivered via a zona drilling micropipette (Research Instruments, UK) and a single blastomere removed by gentle suction, using a blastomere biopsy pipette with an inner diameter of 30µm (Research Instruments, UK). Followingbiopsy, embryos were cultured in 100µl drops of G2.2 medium for a further 2 days under Ovoil. Meanwhile each biopsied blastomere was fixed under visual control (using an Olympus SZHIO stereomicroscope) on a silanated slide (PGC Scientific Ltd., Bristol, UK) using a mixture of 0.1 percent Tween 20 and 0.01 NHCl as described by Coonen et al.4 A rapid FISH procedure was carried out according to the method described by Harper et al.5 Briefly, slides were air dried, rinsed in PBS and dehydrated through an ethanol series (70%, 90% and 100%). After air-drying, CEP X (spectrum green) and CEP Y (spectrum orange) chromosome-specific DNA probes (Vysis Inc. Downers Grove, IL) were applied to the slides and sealed with rubber cement under coverslips. Simultaneous denaturation of probe and target DNA was achieved by placing the slide on a prewarmed hotblock (Hybrite, Vysis, Inc.) at 75°C for 5 minutes, followed by hybridization at 37°C for 4 hours. A post-hybridization wash in 0.4X SSC at 70°C for 2 minutes was followed by a wash in 2XSSC/0.1 percent NP-40 at room temperature for 2 minutes. Slides were counterstained with DAPI in antifade (125 µg/ml), and the signals analyzed under an Olympus microscope (BX-40) fitted for fluorescence and equipped with a triple bandpass filter set (Vysis, Inc.) to detect spectrum orange, spectrum green and DAPI simultaneously. The selected XY embryos were transferred back into the uterus on Day 5, by which time the majority had formed hatching blastocysts. RESULTS The mean number of oocytes retrieved per couple was 12 (range: 8 to 28) and we were able to biopsy a mean of 8 embryos per couple (range: 6 to 14). We transferred a mean of 2.6 (range: 2 to 6) XY embryos to each patient. Of the 15 couples we treated, 5

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conceived. Of these, one had a miscarriage, 3 have delivered (one a set of twins), and 1 has an ongoing pregnancy. All the children have beenboys. DISCUSSION One significant problem with current PGD procedures is the limited time available for diagnosis. While current FISH protocols for sexing (such as is described here) require very little time, many other diagnostic tests require up to 24 hours. Dramatic improvements in culture media, including the advent of commercially available sequential media,6–7 have led to the possibility of higher implantation rates as a result of selection at day 5 or 6. An increasing number of clinics routinely transfer blastocysts as a result of this advance, but for routine IVF it is still not clear whether blastocyst transfer will replace cleavage stage transfer since a proportion of infertile couples will have no embryos which develop to the blastocyst stage and therefore no embryo transfer.8 However, PGD cases involving “fertile” couples, may benefit from such an approach.9 Moreover, the selection of blastocysts for transfer should reduce the risk of multiple gestations as a consequence of the expected higher implantation rate. While PGD represents the cutting edge of reproductive technology, and gives us an idea of what may be possible for the future, it also raises a number of worries and concerns, especially in India, where people are worried about its being used for sexselection. However, if we allow people to choose when to have babies; how many to have; and even to terminate pregnancies if they inadvertently get pregnant, then why should we not allow them to select the sex of their child, if it is possible? We should allow patients freedom to choose for themselves—medical technology should empower them with choices they can make for themselves! A common criticism against PGD for sex selection is that it will cause an unbalanced sex ratio. In reality, PGD will allow couples to balance the sex ratio in their families, rather than unbalance it! For example, take a couple with a baby girl, who want to have a second baby. If they leave things upto chance, half of them will have a second baby girl—causing unbalanced intrafamily sex ratios! PGD will allow them to make sure that they have a balanced sex ratio in their family, if they so desire. Seen in this light, PGD is perhaps the ultimate form of family planning there is. Preimplantation genetic diagnosis is the most reliable method available today for sex selection. A common criticism of PGD for sex selection for social reasons is that it will produce an unbalanced sex ratio. Such arguments have recently been debated in the literature.10 The expense, limited availability and comparative inefficiency of sexing by PGD makes the technique an unlikely source of a significant gender skew in any country. In societies where this is a major concern, one simple safeguard would be to restrict its use only for couples who have at least one child, and who desire a child of the opposite sex. Furthermore, the supernumerary embryos (of the “undesired” sex) could be frozen and donated to infertile couples. Preimplantation genetic diagnosis is now also being used in order to increase pregnancy rates for older infertile women. One of the reasons older women have a poorer pregnancy rate is because their embryos are often chromosomally abnormal, because of

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the fact they have older eggs (which may have genetic defects). PGD allows the doctor to select only the chromosomally normal embryos, so that only these can be transferred back into the uterus, resulting in a higher pregnancy rate. Preimplantation genetic diagnosis can also be useful for selected women with recurrent pregnancy loss, who have recurrent miscarriages because of aneuploidy or other chromosomal errors. Preimplantation genetic diagnosis allows the doctor to select the chromosomally normal embryos, thus reducing the risk of another miscarriage. The FISH method routinely employed for sexing requires only a few hours for completion following biopsy However, the time afforded by delaying transfer until day 5 could be extremely beneficial. Using this strategy, more time is available to perform the same PGD procedures (making it a more flexible clinical service). This extra time provides an opportunity to confirm the initial diagnosis or to institute additional analyses on the embryo. For example, following the diagnosis of sex, blastomeres could be reprobed with other chromosome specific probes in order to screen for common aneuploidies with the aim of improving the implantation rate and decreasing the miscarriage rate.11 By delaying transfer until day 5, a period of at least 24 hours is available for the confirmatory or additional diagnoses. REFERENCES 1. Handyside AH, Thornhill AR. Cleavage stage human embryo biopsy for preimplantation genetic diagnosis. In Kempers RD, Cohen J, HaneyAF, Younger JB (Eds). Fertility and Reproductive Medicine. Proceedings of the XVI World Congress on Fertility and Sterility. Elsevier Science BV Amsterdam, The Netherlands 1998; 223–29. 2. Kuo H-C, Mackie Ogilvie C, Handyside AH. Chromosomal mosaicism in cleavage-stage human embryos and the accuracy of single-cell genetic analysis. Journal of Assisted Reprod Genet 1998; 15(5):276–80. 3. Dumoulin JCM, Bras M, Coonen E, Dreesen J, Geraedts JPM, procedure for preimplantation genetic diagnosis and further Evers JLH. Effect of Ca2+/Mg2+-free medium on the biopsy development of human embryos. Human Reproduction 1998; 13(10):2880–83. 4. Coonen E, Dumoulin JCM, Ramaekers FCS, Hopman AHN. Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in situ hybridization. Hum Reprod 1994; 9:533–37. 5. Harper JC, Coonen E, Ramaekers FCS et al. Identification of the sex of human preimplantation embryos in 2 hours using an improved spreading method and fluorescent in situ hybridization (FISH) using directly-labelled probes. Hum Reprod 1994; 9:721–24. 6. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocyst increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69:84–88. 7. Jones GM, Trounson AO, Lolatgis N, Wood C. Factors affecting following in vitro fertilization and embryo transfer. Fertil Steril the success of human blastocyst development and pregnancy 1998; 70(6):1022–29.

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8. Tsirigotis M. Blastocyst stage transfer: pitfalls and benefits. Human Reprod 1998; 13:3285–89. 9. Edwards RG, Beard HK. Blastocyst stage transfer: pitfalls and benefits. Is the success of human IVF more matter of genetics and evolution than growing blastocysts? Human Reprod 1999; 14(1):1–6. 10. Malpani A. Preimplantation Genetic Diagnosis (PGD) of the Embryo for Preconceptional Sex Selection: Right or Wrong? Obstetrics and Gynaecology Today 1998; 3(8):486–90. 11. Gianaroli L, Magli MC, Ferraretti AP, Fiorentino A, Garrisi J, Munn, S. Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilisation by avoiding the transfer of chromosomally abnormal embryos. Fertil Steril 1997; 68:1128–31.

SECTION 14 Present and Future of Infertility

CHAPTER 80 Present and Future of Infertility Therapy Bruno Lunenfeld INTRODUCTION 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, novel challenges are arising in relation to scientific, ethical and humanitarian aspects in infertility management. How do we use current and evolving technologies to allow parents to achieve the joy of having children with dignity to make treatment available to all, secure universal reimbursement and to increase short and long term safety and decrease any adverse impact on the health of both mother and child. Medicine is racing from triumph to triumph. But somehow, the more medicine achieves, the less it satisfies. Medicine does not seem able to fulfil the ever—increasing expectations that have been raised by the promise of new breakthroughs. Until about the mid sixties, medical research was primarily driven by the desire to cure sick people, today the shift in research is focusing on the understanding of disease processes and the prevention of disease as well as in improving the quality of life. This is also true for reproductive health. According to a position paper by The World Health Organization (WHO),1 reproductive health is a state of complete physical, mental and social well being and not merely the absence of disease or infirmity, in all matters relating to the reproductive systems and its functions and processes. Reproductive health therefore, implies that people are able to have a satisfying and safe sex life and that they have the capability to reproduce and the freedom to decide if when and how of ten to do so (WHO 1994).1 In order to fulfill this task reproductive medicine must, on the one hand, make all possible efforts to enable conception to all couples desiring it and, on the other hand, to avoid maternal and neonatal complications such as the hyperstimulation syndrome, multiple pregnancy and premature delivery. These are formidable goals and in order to reach them, a constant battle for obtaining new detailed information regarding physiology of reproduction and devising new efficient treatment methods mustbe pitched. The ethical, sociopolitical and economic problems related to these goals must not be underestimated. They have to be discussed, taken into consideration and dealt with. In the near future, we should expect significant advances inbasic reproductive and applied research, diagnostic tools, drug development, and in the clinical management of infertility

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Post-infectious Infertility Post-infectious infertility is obviously a worldwide problem. It is estimated that 11 percent of patients will be infertile after one episode of PID, 23 percent after two episodes and 54 percent after three or more incidents.1 Upto 64 percent of African woman and 25 to 35 percent of patients in other areas of the world, had inf ertility that could be traced to prior infection. Specifically an infectious etiology for infertility could be directly related to a history of sexually transmitted disease, pelvic inflammatory disease (PID), male genital tract inf ections and pregnancy complication of an infectious nature. Clearly, the sequel of sexually transmitted diseases is a major cause of infertility throughout the world and prevention of pelvic infection should be a high priority both for the medical scientists and for governments. Chlamydia is probably the most common sexually transmitted disease today. WHO estimates that the minimal global incidence is 50 million per year.2 Therefore, new techniques for the rapid diagnosis of chlamydial infection are essential. There is now some promise that a rapid screening test to indicate the presence or absence of Chlamydiol Salpingitis can be developed. In addition, developing a vaccine a against the major outer membrane protein of chlamydia should be possible, and should be a high priority worldwide. This development alone would have a dramatic effect in every area of the world in reducing female infertility due to tubal disease. Basic Research In vitro Maturation of Gametes Priority areas for basic research should be: In vitro maturation of gametes, regulation of meiosis and the ability to control the production of the sperm head decondensation factor by the oocyte and of the oocyte activation factor by the male gametes. In vitro maturation of spermatocytes or spermatids have to date not been possible. Very few reports on children born following ICSI with spermatids have been reported2 and should be viewed with caution since it is difficult to differentiate between a late spermatid and early spermatozoon. It has been argued that cytoplasmic changes in spermiogenesis, mainly concern the Golgi apparatus (acrosome vesicle), centrosomal material (flagellum), mitochondria (peroxisomal ring) and cytoplasm volume (drastic reduction). These changes may not be necessary in case of ICSI and their absence could be without consequences on post fertilization development. Spermatid gene transductionbrings new proteins which are related to fertilization (acrosomal enzymes, flagellum proteins, protamines, etc.) and seem no longer useful after gamete fusion. There is no report of genomic imprinting occurring during spermiogenesis in mammals. Moreover, the important sperm changes during epididymal maturation (formation of disulfude bonds, acquisition of motility and of molecules for oocyte recognition, methylation of certain genes) were not found to be necessary for embryo development in case of ICSI with epididymal or testicular sperm.3 However, nuclear changes affect chromatin with the substitution of histones by protamines and other transitional proteins to ensure sperm DNA stability across male and

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female genital tracts and may also be important or even essential in the fertilization process. The in vitro maturation of oocyte will become reality and a routine procedure in the not too distant future. Successful pregnancies followed by life born children have been reported.4,5,6 If these procedures prove themselves and become a routine procedure they will simplify and significantly decrease the costs and risk of assisted reproductive procedures (ART) to the mother but may increase the risks of faulty pre or post-meiotic development of the gametes with profound consequences on fertilization, embryonic development and expression even in later generations due to gene deletions, mutation or amplification of mutations. Examples of such amplification of mutations are “the fragile X syndrome” “myotonic dystrophy”, “Huntington’s disease” Kennedy’s disease”, etc. All these syndromes show “trinucleotide repeats.” Thus, the potential risk of this procedure will have to be decreased by the use of genetic screening and improved preimplantation diagnosis. These diagnostic procedures have become even more important since the “Human Genome Project” has now been partially completed. Within the next 10 years the 60,000 to 100,000 genes in the human genome and their specific functions will have been fully deciphered. This will have an enormous impact on genetic testing and preimplantation diagnostics. It will however bring new and difficult ethical questions into light such as the question of whether pregnancy becomes a matter of choosing among embryos with different traits, and whether the prospective parents have the right to know or influence the genetic makeup of their future children. These are perplexing questions and we have hardly begun to consider them and their implications on society, but the advances of genetic research are already upon us. Early Follicular Development and Apoptosis Regulating the number of follicles that reach gonadotropin dependency should also be a high priority research area. Although today we are able to control, monitor and regulate gonadotropin-dependent follicular growth and development, we do not have the theoretical know-how nor the technical ability to increase the number of follicles prior to their gonadotropin dependency We can not control the processes leading to degeneration, atresia or apoptosis of follicles or oocytes. We are starting to understand their dependency on vascular components, neovasculogenesis and diverse growth factors. Designing a fine-tuned instrument enabling control of the whole span of follicular development from primordial to Graafian follicle, would obviously have a significant impact on the reproductive potential of the female. Drug Development Recombinant Gonadotropins New developments in the area of drug research will also create a significant impact on the future of reproductive medicine. With recombinant DNA technology and highly defined cell culture techniques genetically engineered gonadotropins are now being

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prepared on an industrial scale, and are already in the market in most developed countries.7,8 Besides increased safety and simpler use, comparison of the efficacy of recombinant FSH (rFSH) and urinary FSH (uFSH) in more than 2000 cycles demonstrated the rFSH yielded more oocytes, more embryos produced a higher pregnancy rate with lesser amounts and with lesser treatment days than urinary compounds.9 For couples who have cryopreserved embryos available, the potential for subsequent pregnancy is only slightly less than that obtained after the transfer of fresh embryos. This is a significant benefit for the couple, providing them with an additional boost in their pregnancy potential per stimulated cycle. In patients with hypogonadotropic hypogonadism, rFSH alone was sufficient to stimulate follicular growth but was inadequate to induce competent follicular function. However, rFSH combined with recombinant LH (rLH) was able to produce follicular development and following administration of hCG, ovulation and pregnancy The first birth following infertility treatment of a hypopituitary hypogonadotropic woman (WHO110) with rec-FSH, rec-LH to stimulate follicular growth and rechCG to induce ovulation was reported by Agrawal et al.11 This highly potent, safe and pure pharmaceutical grade gonadotropin with full batch to batch consistency and the additional convenience of S.C. self administration, will hopefully replace all urinary preparations in a short time. When such recombinant gonadotropin preparations will have reached all the world markets, hMG and urinary FSH will have served their purpose and become history. We are starting to understand the role of subpopulations of the microheterogenetic family of gonadotropin Isoforms, and carbohydrate complexity of FSH. Changing cell culture conditions and purification techniques, permits the selection of desired proportion of gonadotropin isoforms to produce tailor-made gonadotropins for specific phases of the cycle or for specific conditions. However, one should not exaggerate the importance of isoform profiles in gonadotrophic preparations until we know more about the fate and composition of these isoforms following injection. Moreover, r-DNA technology permits the design of potential therapeutically active gonadotropin agonists and antagonists by altering core proteins and carbohydrate moieties in the alpha and beta subunits of FSH and LH. Using site-directed mutagenesis and gene transfer techniques, it was possible to fuse the carboxyterminal extension of hCG beta (CTP) to the prime-3’ end of the FSH coding sequence. The FSH-CTP fusion protein retained the same biological activity as FSH but had a prolonged circulating halflife and, consequently a higher in vivo potency than native FSH. This is an obvious precursor for a future long acting FSH agonist. Alternatively, deglycosylated mutants of this chimera can be engineered, and together with deglycosylated alpha subunit, could serve as a model for production of various gonadotropin antagonists which would competitively bind to and desensitize gonadotropin receptors on ovarian cell membrane. A mutant FSH lacking the N-linked asparagine residue as Asn-52 on the oc-subunit exhibits 10 fold less bioactivity than the fully glycosylated wild type. Further, FSH devoid of all four linked carbohydrates on the oc and β-subunit is a potent antagonist of FSH action in vitro.12 However, a word of caution may be appropriate when we produce such compounds. Elaborate changes in the protein backbone or carbohydrate panel in any

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of the gonadotropin sub units might cause such preparations to provoke antibody formation. Functional Proteino-mimetic Molecules Better understanding of gonadotropin-receptor interaction combined with crystallography and sophisticated computer technique has permitted the design of proteinomimetic orally active gonadotropin agonists and antagonists. The current challenge inbiotechnology is to reduce the size of these proteins by developing small functional mimetic synthetic molecules that could be administered through the oral or trans dermal route. To achieve this objective for gonadotrophins it was necessary to develop: 1. a working model to explain how gonadotrophin activates its receptor. 2. develop a high throughput assay specific for each gonadotrophin and 3. create a large number of molecules with possible agonistic or antagonistic activity to be tested. Advances in the field of molecular reproductive endocrinology and the development of a number of molecular tools permitted to identify small molecular weight FSH agonistic molecules that could interact with a catalytic region of the FSH receptor that controls G protein coupling and adenylate cyclase activation. With better understanding of FSH receptor activation, it was possible to create small molecules predicted to induce gonadotrophic signal transduction without even the necessity to bind to the extracellular domains of the membrane protein. A number of such molecules are already being actively tested. Such molecules will ultimately be converted into high potency orally active therapeutic preparations to replace the dimeric glyco protein hormones or to act as antagonists. GnRH Analogs Third generation GnRH antagonist have appeared in the market. Due to their high affinity to the GnRH receptors, these compounds lead to a suppression of gonadotrophin secretion within hours. Acting through competitive binding, the duration of their effect is dose dependent. Their immediate inhibition of gonadotrophins, without the flare up effect has a profound influence on their use in ovulation induction and ART procedures. They can be administered either daily during several days in the late follicular phase (usually from day 6 of stimulation onwards) the “multiple dose protocol”13 or just at the moment when the LH-surge is expected (single dose protocol.14 Systematic structure-activity relationship studies of linear analogues are being performed. The development of mono-and di-cyclic analogs is being actively pursued. Anumber of such compounds are already available and are under active investigation. Functional Peptido-mimetic Molecules In an attempt to assess the functional relationship between GnRH and its receptor, reporter gene systems for signal transduction by the human GnRH receptor have been developed. This permits a better understanding of the GnRH receptor. Applying a high

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throughput screening allows the examination of thousands of potential candidates of peptidomimetic GnRH analogs. These throughput assays are performed for example by using mouse fibroblasts transfected with a luciferase reporter gene plasmid and a selected cell clone super transfected with the respective wild or mutant GnRH receptor expression plasmid. These methods also enabled the identification and characterization of diverse agonists and antagonists in wild type and site specific receptor mutants and permitted the development of a 3 dimensional model of receptor-ligand interaction. This permitted the identification and selection of potent peptidomimetic GnRH agonist and antagonist which could be orally active. Applied Clinical Research In applied clinical research, our aim should be to improve embryo viability, implantation and reduce the hyperstimulation syndrome and multiple pregnancy rate. The success rates for in vitro fertilization (IVF) and embryo transfer (ET) depend on two major factors: embryonic viability and uterine receptivity The nature and sequence of signals passing between the male and female pronucleus inside the freshly fertilized egg which result in the formation of a centromere and re-arrangement of male and female chromatin material must be studied indepth in order to redefine the fertilization process and to understand, and possibly to manipulate this decisive stage of embryo development. Implantation The most important bottleneck in obtaining a clinical pregnancy is implantation. The management of the implantation signal depends largely on our ability to understand and control neovascularisation, adhesion and invasion processes permitting the viable embryo to adhere to and invade the endometrium and to create proper vascularization necessary for its nourishment, growth and development. We must also learn more about the immune modulation system which prevents expulsion of the fetus by the mother. Since the fetus can be considered an allograft, maternal immune regulatory mechanisms are set in motion to prevent the rejection of the fetus and even aid in its growth and development. A defect in this maternal- fetal recognition of tolerance is thought to result in immunologically mediated damage to the fetus. The success of embryonic implantation relies upon a perfect dialogue between good quality embryos and a receptive endometrium. In response to endometrial factors the human embryo secretes the complete IL-1 system as well as other growth factors. The humanblastocyst regulates up the endometrial receptivity Any derangement in cross communication between embryo and endometrium can interfere with the implantation process. The sequel of pelvic inflammatory processes such as hydrosalpinx are known to reduce the pregnancy rate following ART. Recently, Sharara et al15 have demonstrated that in cases with hydrosalpinx, the window of implantation which is expressed by the appearance of αvβ3 endometrial integrins is impaired, and might be corrected by salpingectomies.

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Embryonic Development On the human embryo, a developmental block characterized by an arrest in cleavage generally occurs at the four to eight cell stage as first reported by Braude et al16 The fourcell block occurs at the point where the embryo is switching from the use of maternal ovum derived messenger RNA (mRNA) for protein synthesis to embryonic mRNA resulting from de-novo synthesis. If such a block occurs, it may lead to degeneration and or lack of implantation. Following “fertilization” with good and bad sperm, the resulting embryos showed equal development till the 4 cell stage, however at the 16 cell stage or at the blastocyst stage the difference of development with good sperm as compared to bad sperm became highly significant, demonstrating that after the maternal derived mRNA is switched off, paternal factors may influence further development. Besides the important scientific contribution of these findings, they can also be applied to differentiate between maternal and paternal causes in the arrest of embryonic development. Culture of embryos to the blastocyst stage may represent a significant advantage since it will select only those embryos with an implantation potential. It has become clear that nutrient requirements and metabolism of the zygote and blastocyst are completely different and that the oviduct and uterus provide diff erent nutritional support to the human embryo as its develops.17 The changing requirements of the embryo in this short but important period of growth and development are of utmost importance. In this period the embryo switches from the use of maternal, ovum derived, messenger RNA for synthesis of proteins, to embryonic mRNA resulting from de-novo synthesis. This is crucial for the normal development and differentiation of the inner cell mass and trophoectoderm. Sequential serum-free culture media were formulated for the development of the pronuclear and 8 cell embryo, respectively17–21 The benefits of providing embryologists with sufficient oocytes to enable selection among those embryos which develop into blastocysts with well differentiated trophoectoderm and inner cell mass lineage, with chromosomal normality or even metabolic capacity, could be substantial. It has been clearly demonstrated for blocked 4–8 cell human embryos that there is a lack of synthesis of a specific set of embryonic coded proteins, which are present in non blocked embryos.16 It is most probably that a redefinition of fertilization leading to embryo development will require the inclusion of the “ability of the embryo to produce its own messenger RNApermitting production of embryo specific proteins.” It is likely that inherent viability of the embryo is a natural selection process which may be compromised by suboptimal culture conditions at the time of embryonic gene activation. Re-implantation of accurately selected blastocysts would obviously increase the pregnancy rates on the one hand, and enable better control of multiple gestations by replacing only one or two blastocysts on the other hand. Gardner18–20 demonstrated that it was possible to obtain around 50 percent blastocyst development and a pregnancy rate of about 70 percent with the transfer of just two blastocysts. Therefore, in order to obtain a pregnancy rate of 50 percent one needs only transfer a single blastocyst. Importantly, suchblastocysts can be readily frozen and give rise to pregnancies post thaw. Gardner and Lane16 reported a 60 percent survival rate of frozen embryos, with a 50 percent ongoing pregnancy rate following the transfer of such thawed embryos. The application of novel viability markers will help to identify those blastocysts with the highest developmental potential

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before transfer, therefore increasing the overall success of blastocyst transfer in human. This will avoid unnecessary multiple births with increased risks and morbidity to mother and child, and the requests for fetal reduction. In addition, when indicated, the longer stay of fertilized eggs in the laboratory would permit genetic studies, thus avoiding transfer of genetically inadequate embryos on the one hand and further development of endometrium in preparation for nidation on the other hand. Recently Cohen22 demonstrated that removing cytoplasm with mitochondrial DNA from a younger woman’s ovum and injecting it into an ovum from an older women which contributes the nuclear DNA increases the chance of implantation. This new technology, a significant scientific advance, however raises new ethical and legal implications of a child inheriting genes from two mothers. Successful pregnancy also requires normal post implantation embryo growth and development. Aunderstanding of these complex events and interactions will lead to better care of the early embryo and fetus. Application of this knowledge would offer opportunities to treat the heretofore untreatable conditions of unexplained recurrent pregnancy loss. Cryopreservation To improve cryopreservation techniques some basic biophysical research is needed. This would be useful in better freezing of embryos, freezing of oocytes, of ovarian tissue, of testicular tissue and of individual spermatozoa or spermatids. Preliminary data show that similar results in terms of fertilization and clinical pregnancies can be obtained from fresh or cryopreserved testicular tissue. Such techniques could have a significant impact on the logistics, safety and expense of assisted reproductive technologies. The in vitro growth, development, and maturation of oocytes from cryopreserved ovarian tissue has become reality23 If these techniques are improved and become routine procedures, they will permit preservation of healthy oocytes prior to irradiation or chemotherapy or even for delaying conception for personal reasons. Freezing eggs, although reported with occasional success should become a reality within the next few years. This technique offers an ethically “soft” alternative to embryo freezing. The social implications of freezing ova can also be enormous. The nineties woman is financially independent and sexually liberated but she is not free of her biological clock if she wants to become a mother. The single woman in her 30S has become a burning social phenomenon of our times as she is caught between her body clock and her career, her desire for independence and her desire to have a child. Feminism has been unable to fulfill the twin desire of a modern women-career and children- because of the ticking biological clock in each woman. However, science has come to the rescue in a way that feminism never could, and promises to take the clock out of the equation. Now, it is possible for women like men to concentrate on their careers and put the issue of having children on hold. Human eggs can be stored for years and when the woman has broken all the glass ceilings, then the egg can be thawed and fertilized in vitro and implanted into the woman even after menopause. However, it is questionable if this technique will or should become routine.

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Reduction of Multiple Pregnancy Rate In the management of infertility our goal has to be to increase pregnancy rates and reduce abortion rates resulting in an increase in the birth rate of normal healthy children. Furthermore, we have to decrease the rate of the hyperstimulation syndrome as well as multiple pregnancy rate with a concomitant reduction of gestational and obstetric complications. The 1995 American Society for Reproductive Medicine/Society for Assisted Reproductive Technology, annual report revealed a 37 percent incidence of multiple deliveries with 7 percent being triplets or higher.24 Whereas premature delivery can be expected in 24 percent of singleton pregnancies it will rise to 67.5 percent in twin pregnancies and attain 93 percent in triplets. Whereas admissions to neonatal intensive care units are in about 15 percent following singleton deliveries, it will be about 48 percent following twin deliveries and 78 percent following triplet deliveries. The economic consequences of multiple gestations represent ‘hidden costs’ of infertility treatment Goldfarb et al25 estimated the cost of triplet or greater gestations in the USA at 340, 000 US$. The cost of twins has been estimated at 21000– 39000 US$. Even this figure does not consider the long term cost of long term care for children handicapped as a result of prematurity. In 40 percent of quadruplet pregnancies, significant developmental delay is present in at least one of the resulting children.26 Therefore, multiple pregnancies are not only a health risk to mother and child but they represent an enormous financial burden to society. How do we proceed to reach the goals mentioned above? This could be achieved by developing stimulation and suppression techniques enabling fine-tuned follicular development, precisely timed ovulation of healthy gametes, and appropriate control of the implantation processes. Obviously, a real progress in genuine improvement of the results of reproductive medicine will also be achieved with practical application of preimplantation diagnosis of genetic diseases. This would provide a blessed substitute for early or late abortion as a means for preventing the birth of disabled individuals. However, using such techniques to permit choice among healthy embryos with different traits is a much more complicated question which society will have to debate. ETHICAL, SOCIAL AND POLITICAL CONSIDERATIONS The pace at which advances are made often seems to exceed our ability to incorporate them in our life styles. Nearly every new advance brings a host of new ethical, social or even political changes with it. We must remember that society can not survive without biotechnology and at the same time, can not survive its unethical use. Therefore, the hopes of accumulating and applying the information needed to advance to the next phase of technological evolution in reproductive medicine must also be amalgamated with clear guarding directives: 1. Public policy to restrain population explosion appropriate as it may be, must not be confused with the basic human right to procreation. The agony of childless couples in overpopulated areas such as India is just as painful as that of couples in industrialized nations.

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2. Infertility is a reproductive health disorder and must therefore be considered in terms of the World Health Organization definition, a disease and the cost of its prevention and/or management should be covered by third party providers such as national or private health insurance. It is our task to persuade political decision makers that infertility is a medical condition and should be included in the health care benefit package of every individual. An audit of one of the major health care providers in the state of Massachusetts showed that the cost of infertility treatment accounts for only one tenth of a percent of a total family premium for insurance. In Germany, 0.2 percent of a mean family’s total health insurance package is used by inf ertility treatment. Moreover, in 1995 the entire expense of out patients care was 38.6 billion DM and of drugs 31.4 billion DM, in Germany. However, the entire cost of all ART cycles including drugs was only 0.3 percent of these sums. In many developed countries infertility accounts for a large and increasing number of childless couples. This has become a problem to society in the sense of an unfortunate demographic shift. It is our duty to provide the public and politicians with information on the consequences of delaying the moment of wanting the first child. It is also our duty to show that reproductive medicine offers a set of safe and effective solutions. 3. Since age is the single most important factor for infertility, it is our duty to educate the public and politicians on “planning a child.” We should encourage an environment that allows for combining successful career and motherhood. 4. The public as well as regulatory agencies should be informed that infertility per se, as well as its causes can be a serious and costly health risk involving hormone dependent cancers and cardiovascular diseases and that conceptions may reduce this risk. Furthermore, infertility can cause significant emotional stress. Psychological symptom scores were comparable to patients with cancer, cardiac problems, and chronic hypertension and all patients studied expressed a considerable loss of well being.27 5. The public as well as regulatory agencies should be informed that with the present and emerging sophisticated technologies for induction of ovulation and assisted medical procreation most infertile couples can expect the joy of parenthood today. 6. Attempts to prevent infertility by reducing sexually transmitted diseases, abortions and adolescent pregnancies should be a primary concern and should be given a high priority in educational, and public health programs. 7. Not only must short and long term safety of infertility therapy be of foremost awareness, but also the physical, mental and social welfare of each partner of the future parents as well as that of the planned child must be taken into consideration. These considerations are particularly important when we consider the use of donor eggs, donor sperm, or plan pregnancies in older women. 8. The patients must be informed on cost, effectiveness and short and long term safety of each procedure and if possible even for each specific center. 9. Sex selection for political, economic and for extraneous reasons must not be permitted. 10. Genetic selection for ethical purification, social or political reasons must be condemned. 11. Hundreds of thousands of embryos are steadily accumulating in tanks of liquid nitrogen in many countries. In the UK alone, 300000 surplus human embryos have

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been created. These spare embryos raise a host of ethical social and legal questions. These questions become even more complex when the people who provided the eggs or sperm divorce or die or simply loose contact with the center where the embryos are stored. 12. Ethical aspects must be of primary concern to the society and the medical profession. A line must be drawn between the theoretically possible and the practically acceptable and between the technically feasible and the socially reasonable. Research procedures must not be confused with good medical practice. 13. We who are near the peak of research and development in the area of reproductive medicine have to acknowledge that we also have the commitment to integrate new solid knowledge and new proven technical developments into our patients information package and into our daily practice. This should decrease the negative influence of “sensational” media reports, permit us to regain confidence and respect from our patients, and transform theoretical science into practical solutions to our patients problems. The mobile consumer society, constantly remodelled by mass media, and the modern explosively developing medicine must find appropriate means of communicating and developing mutual trust. To achieve this goal, the doctors must not only be scientifically competent and medically skillful, but also sensitive and honest human beings, aware of their duty to the society With these recommendations in mind we should be able to help mankind fulfill the first commandment in the bible in the spirit and intent as it was bestowed: “So God created man in His own image, in the image of God created He him; Male and female He created them. God blessed them, and God said unto them, be fruitful and multiply and replenish the Earth and subdue it” (Genesis 1:27–28). REFERENCES 1. World Health Organization. Health, Population and Development. WHO position paper for the International Conference on Population and Development, Cairo 1994 WHO/FHE/94 Geneva, WHO. 2. Westrom L. Impact of sexually transmitted diseases on human reproduction. Swedish studies of infertility and ectopic pregnancy in sexually transmitted diseases, Status Report NIAID Study Group. Washington DC NIH Publication 1980; 81(2213):43. 3. Testart J. De la spermatide au spermatozoide: quels changements necessaires au developpement?. Contracept. Fertil Sex 1996; 24:526–33. 4. Cha KY, Koo JJ, Choi DH. Pregnancy after in vitro fertilization of human follicular fluid oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55:109–18. 5. Russel JB, Knezevish KM, Fabian KF, Dickson JA. Unstimulated immature oocytes retrieval: Early versus mid follicular endometrial priming. Fertil Steril 1997; 67:616–20. 6. Trounson AO, Wood C, Kausche A. In vitro maturation and fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62:353–62. 27. Chappel S, Kelton C, Nugent N. Expression of human gonadotrophins by recombinant DNAmethods. In GenazziAR, Petraglia F (Eds): Proceedings of the 3rd World Congress on Gynecological Endocrinology. 1992; 179–84.

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8. Howles CM. Genetic engineering of human FSH (Gonal-F). Hum Reprod Update 1996; 2:172– 91. 9. Lunenfeld B, Lunenfeld E, Howles C. Development and use of recombinant Gonadotropins. Asian Journal of Endocrinology (In press), 1998. 10. Insler V, Melmed H, Mashiach S et al. Functional Classification of patients selected for gonadotropic therapy. Obstet Gynecol 1968; 32:620–28. 11. Agrawal R, West C, Conway GS, Page ML, Jacobs HS. Pregnancy after treatment with three recombinant gonadotropins. Lancet 1997; 349:29–30. 12. Keene J, Nishimori K, Boime I. Recombinant deglycosylated human FSH is an antagonist of human FSH action in cultured rat granulosa cells. Endocr J 1994; 2:175–80. 13. Diedrich K, Felberbaum R. Multiple dose protocol for the administration of GnRH antagonists in IVF: the “Luebeck protocol”. J Ass Reprod Genetics 1997; 14(Suppl):15S. 14. Olivennes F, Bouchard P, Frydman R. The use of a new GnRH antagonist (Cetrorelix) with a single dose protocol in IVF. J Ass Reprod Genetics 1997; 14(suppl):15S. 15. Sharara FI, Meyer WR, Lessey BA, Castelbaum AJ. Effects of hydrosalpinx on IVF outcome. Hum Reprod 1997; 12:2853–54. 16. Braude P, Boloton V, Moore S. Human gene expression first occurs between the four and eight cell stages of preimplantation development. Nature 1988; 332:459. 17. Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Rep Update 1997; 3:367–82. 18. Gardner DK, Vella P, Lane M. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfer. Fert Steril 1998; 69:84–88. 19. Lane M, Gardner DK. Selection of viable blastocysts prior to transfer using metabolic criteria. Hum Reprod 1996; 9:1975–78. 20. Gardner DK, Lane M. Culture of viable Human blasocysts in defined sequential serum free media. Hum Reprod 1998; 13(Suppll): 101–12. 21. Servy EJ, Kaufmann RA, Liu Z, Menezo Y, Keskintepe Human pregnancies after transfer of fresh (four- to eight-cell) versus frozen-thawed blastocysts resulting from intra cytoplasmic sperm injection. J Assist Reprod Genet Emb 1998; 15(7):422–26. 22. Cohen J, Scott R, Schimmel T, Levron J, Willadsen S. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 1997; 350(9072)186–87. 23. Edirisinghe WR, Junk SM, Matson PL, Yovich JL. Birth from cryopreserved embryos f ollowing in vitro maturation of oocytes and intra cytoplasmic sperm injection. Hum Reprod Emb 1997; 12(5):1056–58. 24. Bustillo M, Zarutskie P. Assisted Reproductive technology in the United States and Canada: 1995 results generated from the American Society for reproductive Medicine/Society for Assisted reproductive tewchnology registry. Fertil Steril 1998;69:389–398. 25. Goldfarb GM, Austin C, Lisbona H. Cost-effectiveness of in Vitro fertilization. Obstet Gynecol 1996; 87:18–21. 26. Evans MI, May M, Drugan A. Selective termination: Clinical experience and residual risks. Am J Obstet Gynecol 1990; 170:902–09. 27. Domar D, Zuttermeister PC, Friedman R. The psychological impact of infertility, a comparison with patients with other medical conditions. J Psychosom Obstet Gynecol 1993; 14:45–52.

CHAPTER 81 Patient Support in the ART Program Rubina Merchant OVERVIEW Infertility, treatment with reproductive technologies, and abortion are among the most emotionally weighty and philosophically contentious experiences in most patients’ lives involving the most intimate body parts and behaviors and the most heartfelt hopes and profound disappointments.1 Pregnancy and motherhood in women are developmental milestones that are highly emphasized by our culture and the experience of infertility can be devastating for the couple desiring a child.2 Its farreaching, multi-faceted effects engulf the couple in a vicious circle of events.

In other words infertility breeds infertility off-shooting a host of unpleasant emotional responses like shock, denial guilt, anger, depression, isolation, and loss of control, and hopelessness. These responses arise from an inability to accept the problem and may take a traumatic toll of the patient. Infertility plagues not only the more vulnerable female partner and her spouse, but also their respective families, the society, and ultimately the economy of the country The unyielding power of human innovation and the marvels of technology have unleashed promising therapeutic options in the field of Assisted Reproductive Technology rekindling dying hopes of the infertile and giving them one more reason to go on. However, as encouraging as these options may seem at the outset, the possibility of a failed attempt is still an unfortunate reality The emotional, psychological, and financial pressure that patients experience before, during and after treatment is an aspect that demands considerable thought and deep insight. Medical procedures that form a part

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of the infertility work-up may be more than mandatory, however, assessment would only be complete if the emotional and mental framework of the patient is simultaneously looked into. Patient support in an ART program is an integral function of an inf ertility setup that serves to oil the cog wheels of the life crisis of infertile couples. STRESS AND INFERTILITY About 15–20 percent of the infertile patients are expected to experience high levels of distress that interface with everyday activities at some point in their infertility experience.3 The very diagnosis of infertility is likely to cause stress. In addition, the many investigations and procedures may have compounded distress.4 Kee et al,5 have reported significant increases in trait anxiety and depressive symptoms in infertile women than the fertile women. Anxiety and depression in the in vitro fertilization (IVF)-failed women were significantly higher than the IVF-success women. However, contrary to the expectation, demographic factors such as religion and husband cooperation were not related to the experience of stress. There was a trend of a decreasing psychological stress with advanced infertility duration. On depression scales, the intermediate and final duration of infertility patients showed fewer symptoms than the first-stage patients. For some couples, the infertility crisis can be seen as a cumulative trauma, which indicates that these couples have a marked need for infertility counselling.6 OBJECTIVES OF COUNSELLING Counselling may be defined as a context for support advice and guidance rather than a vehicle for change.7 The main purpose of counselling is to: • give patients an opportunity to unravel and express their innermost feelings and to help alleviate their misery • resolve gender differences between them and help them view their main goal collectively • provide adequate medical information regarding the nature, course, outcome of treatment, success rates associated with the particular procedure and with that particular indication, and possible side effects of any medication used • accept and deal subjectively with the wide spectrum of emotions that might grip infertile patients • show them ways of coping with the stress associated with infertility before, during and after the treatment • help them address ways of tackling difficult situations such as unexpected results, treatment failure, pregnancy loss, financial loss, family differences, effectively • help them make decisions and consider alternate options of treatment • provide necessary information on the legal and ethical issues involved in Third Party Reproduction such as donor insemination, oocyte donation, embryo donation, surrogacy and adoption • inform them of the consequences of dealing with a biologically unrelated child • help them decide when to terminate treatment

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• help them accept childfree living in the event of continuous treatment failure, or when all available options may be unacceptable • help them deal with sexual problems that may be a cause or result of the infertility. Counselling may be considered a platform where individuals can be given the opportunity to explore, discover, and identify ways of living more resourcefully.8 INDICATIONS FOR COUNSELLING Counselling is indicated when there is an obvious deterioration in the mental stability or psychological state of the patient and a decrease in the overall quality of life. It is indicated in the following situations: • persistent feeling of guilt, anger, pessimism, loss of selfesteem, self-denial, worthlessness and suicidal feelings • highly distressed state culminating in persistent depression • increased anxiety prompted by an unfulfilled desire • withdrawal or isolation from routine activities or society • marital disharmony due to gender differences or threatened sexuality • dissatisfaction with life • disorientation and confusion leading to a difficulty in making sound decisions • disturbed sleep pattern • mood fluctuations.

FUNDAMENTAL AREAS OF CONCERN/SITUATIONS DEMANDING COUNSELLING INCLUDE Continued Treatment with Persistent Failure as is the Case in Patients Suffering from Idiopathic Infertility often leading to Advanced Maternal Age Infertile patients suffering this lengthy unpredictable phase in their lives have almost reached their lowest emotional, psychological and financial ebb and would require constant reassurance, support and help at every step during and after the course of treatment. Success is probably not far away but the very prospect of one more attempt and a seemingly endless ritual to satisfy and fulfil the yearning for a child could be gruelling. Infertility long after failed IVF treatment contributes to psychological dysfunction. It highlights the need to prepare women better for treatment failure and to ensure that appropriate counselling is available when further IVF treatment is no longer appropriate.9 The infertility team must be able to counsel this category of patients and advise them appropriately on future therapeutic options, their limitations and outcome and when to stop treatment. The age of the female partner, the ability of the couple to withstand the stress of treatment, the financial costs, and the reality of the situation must be given

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paramount importance to avoid any undesirable consequences. Pragmatism and insight must be used in helping the patients arrive at a decision. Third Party Reproduction (TPR) Third Party Reproduction is a means of alleviating rather than treating infertility as the problem persists despite the acquisition of a child. TPR has complex, social, ethical and legal aspects to it and couples who have been offered the option must be adequately informed about the fundamental issues that govern it, the implications and the consequences. Donor Insemination (DI) Donor insemination is offered in cases of severe male factor infertility where advanced treatment options like Intracytoplasmic sperm injection (ICSI) may be unacceptable owing to financial problems, evidence of a genetic basis for the infertility such as Ydeletions, hypospermatogenesis, germ cell arrest, failed fertilization following IVF/ICSI, evidence of HIV infection or a transmissible physiological disorder or repeated failure following IUI. Patients given the option of DI must be provided with substantial information on screening tests for anonymous donors, and the implications of accepting a partially biologically non-related child. The relation of the donor with the off spring must be clearly defined in the case of both anonymous and known donors and a written consent taken to this effect. Counsellors must address and help resolve the loss of male selfesteem a consequence of his inability to become a biological father. Possible anger experienced by the female partner against her spouse must be dealt with before proceeding with DI so that the welfare of the future offspring is protected at all costs. The expectations of all concerned parties namely the infertile couple, the semen provider, and the future offspring must be addressed during counselling. Confidentiality of the donor and of the procedure with regard to the couple, family, society, or the future off spring must be given careful consideration. Oocyte Donation (OD) Oocyte donors may be i) voluntary (known/anonymous to the recipient), ii) participants in an oocyte-sharing program in which they share their oocytes with recipients in an IVF cycle with mutual benefit. Oocyte-sharing programs may be ‘open’ or ‘closed’ depending on whether the recipient is known or anonymous to the donor respectively. Recipients in turn share or bear the entire cost of treatment of the donor. Oocyte donation may be indicated in: • advanced maternal age and/or poor quality oocytes • repeated failure with ART techniques including failed fertilization and/or cleavage where the semen quality is endorsed • premature ovarian failure (POF)

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• history of a sex-linked chromosomal disorder or a genetic disease confined to the ovary, ovarian carcinoma warranting chemotherapy, evidence of HIV infection where a risk of transmission of the disease to the offspring may be feared. Counselling may be especially warranted in the case of voluntary oocyte donors who may have a significant history of trauma/loss and donation may serve as a vent for overcoming or satisfying that loss. Such ‘occasional’ donors are psychologically fragile women looking for recognition or massive self-repair10 and their motives must be clearly understood before proceeding with the donation. The ability of such donors to cope with the stress of treatment must be assessed. The limitations of voluntary donors mustbe defined and the confidentiality of the therapy and its consequences must be evaluated in lieu of the family, society, and the offspring. Ethical issues must be given forethought and discussed with the donors and recipients in an oocyte-sharing program prior to the procedure with emphasis on possibilities that the recipient may get pregnant and the donor may not. The recipient must be prepared to bear the cost of the treatment and any adverse consequences in the event of a donor cycle cancellation due to inadequate eggs, or lesser than expected embryos, or a pregnancy loss. The legal, ethical and financial issues involved must be explicitly laid down in the form of a written consent. The motives of known oocyte donors, the expectations of the recipient and her partner from the donor, and of the off spring must be clearly perceived and a written approval taken. Embryo Donation The criteria for embryo donation include: severe combined factor infertility such as azoospermia and POF, repeated IVF failure, H/O of genetic disease. There may be two options for embryo donation: transfer of embryos developed in a fresh IVF cycle or transfer of cryopreserved embryos. Couple/sperm and oocyte donor may be known or anonymous to the recipient. Counselling in patients who have been offered the option of embryo donation must cover the following issues: • implications of accepting a biologically non-related child • secrecy/openness with regard to the family, society, or the off spring • financial costs involved especially in a fresh IVF cycle in which the expected number of embryos cannot be obtained due to failed fertilization and/or cleavage. Patients must be mentally prepared to accept failure at any point and bear the consequences • medical, legal and ethical issues that revolve around embryo donation • approval of donors of cryopreserved embryos and the consequences thereof which must be clearly documented as a written informed consent. The rights of such donors over the off spring that might be born as a result must be clearly defined • thorough screening of the couple/sperm and oocyte donor for HIV, Hepatitis, genetic diseases, social and ethical background • motives of all individuals/parties concerned and their relation to the future offspring. This must be documented in the form of an informed consent especially in the case of known donors.

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Surrogacy Surrogacy involves the use of a substitute birth mother to furnish a couple with a child owing to an inherent inability of the female partner of the latter to do so. It may be of two types: i. Gestational surrogacy and ii. Traditional surrogacy. Gestational surrogacy is surrogacy with donor gametes in which the embryo developed as a result of an ART procedure as IVF is not biologically related to the gestational carrier. Indications for gestational surrogacy include: • absent uterus due to hysterectomy or a uterine disease • malformed uterus which is anatomically or physiologically unfit to bear a pregnancy • repeated pregnancy loss due to a possible immunological factor • medical problems owing to which pregnancy may become a life-threatening situation such as cancer, surgery, ongoing chemotherapy, pelvic infection. Gamete donors may be known or anonymous. It is imperative that the intentions of all individuals/parties concerned such as the sperm donor, egg donor, surrogate her partner and offspring if any, and the commissioning couple, and the limitations of their relation with the future offspring be carefully considered, documented and a written approval taken before the procedure. The emotions of known surrogates such as family or friends towards the child being borne must be clarified in the best interests of the commissioning couple and the future offspring. The surrogate mother must be clear that the child she is bearing is wholly that of the commissioning couple or only partly so. The sperm donor, egg donor and the surrogate must be thoroughly screened for undesirable genetic traits, HIV, Hepatitis, STDs, cardiovascular diseases and high-risk obstetric complications prior to the medical procedure. The surrogate must be given adequate medical information and precautions regarding the pregnancy to be sustained, and the commissioning couple must be prepared to accept complications in the pregnancy of the surrogate that may ensue in abortion. Traditional surrogacy involves the insemination of the surrogate with sperm from the male partner of the commissioning couple. It may not necessarily involve an ART procedure like IVF and the offspring is partially related to the surrogate. Indications for traditional surrogacy include: • women without ovaries or ovaries removed f ollowing chemotherapy. • premature ovarian f ailure (POF) • advanced maternal age • medical problems that preclude pregnancy. Counselling issues in traditional surrogacy would essentially be the same as that in gestational surrogacy. However, emphasis must be placed on the fact that the surrogate would be biologically related to the offspring and any future claims of the surrogate and her partner on the offspring must be given careful consideration prior to undertaking the procedure. The financial cost to be incurred by the commissioning couple and the surrogate couple must be included in a written consent. The legal issues pertinent to the

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jurisdiction must be clarified to facilitate adoption of the child by the commissioning couple. The infertility team must be well acquainted with all the medical, legal and ethical procedures that govern surrogacy and its implications and must be able to provide followup counselling if necessary. Adoption In the eventuality of treatment failure the willingness of the infertile couple to consider adoption as an option must be explored. Prior to undertaking the child, the couple must be counselled on the implications of living with a biologically unrelated child whose background is often unknown. Adoption may be ‘open or ‘closed’. In open adoption, as is practised in most European countries, the natural parents retain the right to access the child. However, the limitations of access and the boundaries of the relationship must be clearly defined when the child is legally adopted so that the welfare of the child is secured at all points. In UK, adoption procedures allow all legal rights and inheritance to be transferred from the natural to the adoptive parents and access may only require a mere letter of permission. In ‘closed adoption’, the natural parents remain anonymous to the legal or adoptive parents and the latter have to be prepared to accept the child as an anonymous being. Adoptive parents must understand issues of difference, intended or unintended racism, and be prepared to tackle identity problems that may arise.11 A social attitude towards adoption has an important bearing on the intention of the infertile couple to adopt and this aspect must be considered when counselling infertile patients to take up adoption. Complications Associated with Pregnancy after Infertility such as Miscarriages, Multiple Pregnancies, Ectopics, and Genetic and Congenital Abnormalities Counselling may be especially indicated in complications associated with pregnancy after infertility such as miscarriages, repeated pregnancy loss (RPL), multiple pregnancies, ectopics, and genetic and congenital abnormalities. Reciprocal chromosome translocations (RCT) are the risk factor for the occurrence of reproduction disturbances, such as infertility spontaneous abortion, and congenital anomalies in the offspring.12 RPL can be a traumatic experience for patients suffering this consequence especially when the cause is unexplained. The apprehension of a future conception after an abortion and the fear of making the pregnancy last can take a heavy mental and emotional toll of the patients and perhaps deprive them of the pleasure of pregnancy Repeated pregnancy loss may often culminate in shock and denial and lead to persistent depression, the possibility of any future success seeming a distant reality. Guilt feelings, particularly connected with previous abortions, seem to be torturing to most women.13 The infertility team is endowed with an important responsibility of comforting these patients and helping them see a positive yet realistic attitude.

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Threatened Sexuality Sexual functiordng is a key aspect of individual experience and is particularly sensitive to the infertility crisis14,15 with various repercussions such as lack of infrequent intercourse, sexual desire, vaginismus, dyspareunia, and orgasmic dysfunction in 10–60 percent of the patients.16 Sexual dysfunction may predate the diagnosis of infertility, be reinforced by investigations and treatment or even cause infertility.17 There are probably a small number of patients in whom psychological factors may induce infertility. However, in the majority of patients, psychological factors may exacerbate infertility and influence the patient’s and partner’s responses affecting mental, sexual, marital and social adjustment.4 Tarlatzis et al,13 reported sexual dysfunction often associated with a degree of deterioration in marriage in infertile patients. The pressure associated with scheduled sex, the psychological presence of the medical team in the patients’ ‘intimacy’ and the fact that intercourse becomes goaloriented and a reminder of infertility itself14,15 may aggravate sexual problems. Few gender differences have been observed with regard to the impact of IVF on the couple’s sexual and marital relationship, although both men and women felt that there was a greater likelihood that IVF had decreased the female partner’s desire for sex.18 Bringing sexual problems to the forum of discussion is a delicate issue for both the patients and the counsellor, and the counsellor must create an ambience where patients can freely unfold and discuss their problem. Counselling patients for sexual disturbances requires professional communication and psychological skills, and a high degree of sensitivity, emotional quotient and perception on the part of the infertility team. Counsellors must be able to judge the real nature and history of the sexual problem, realize their limitations in the counselling task and direct patients to specialized personnel such as a sex therapist or psychiatrist whichever may be deemed necessary. Gender differences must be carefully considered and patients counselled professionally and tactfully without aggravating marital problems. Premarital counselling seems to be of primary importance in assuring the survival of marriages involving paraplegic men and should include the importance and advantages of artificial donor insemination as a solution to infertility.19 Gender Differences Couples undergoing infertility therapy have special needs and fears which may be both general and treatment specific. The stress of infertility has been identified in both sexesdepression, high defensive anxiety, numerous psychosomatic symptoms and more difficulties in social adjustment in women, and a higher tendency towards repressed anxiety and thus a greater risk of psychosomatic illness in men. However, irrespective of the one in whom the etiological problem was found both partners may have psychological problems.13 Findings from a study investigating gender differences on the General Health Questionnaire (GHQ) and questionnaire ratings of the impact of IVF, stressful aspects of treatment and reaction to a failed IVF attempt have also supported greater emotional distress for women than men in relation to infertility diagnosis and treatment, more stress than men at a number of stages of treatment, and a higher likelihood than men to endorse negative reactions in relation to IVF failure.20 Hence infertile couples undergoing

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different treatments may be candidates for psychological counselling and supportive psychotherapy Chronic H/O of lllness or Psychiatric Disease Medical personnel and members of the infertility team must make it a point to record any chronic history of illness or psychiatric disease during the infertility evaluation and history taking so that infertile patients are referred to licensed psychological counsellors to treat the specific problem prior to initiation of the medical therapy for infertility. It would take counsellors a deep psychological insight to perceive such problems because infertile patients may often be reluctant to admit the problem or in most cases not even be aware of an existing problem. TYPES OF COUNSELLING Infertility medical procedures recognize the following counselling tasks:21 • information gathering, analysis, and processing • therapeutic counselling • support counselling • implications and decision making counselling. Therapeutic Counselling Infertility involves suffering, anxiety and insecurity and it is the physician’s task to ensure that such distress is minimized during the diagnostic and treatment procedures.22 Therapeutic counselling mainly involves the physician or infertility specialist whom the patients are first introduced to. The primary care practitioner who is informed about the psychological impact of these experiences can play an essential role in interpreting medical information; helping patients think through their own values, resources, and options, facilitating communication between the couple and with their friends and family providing emotional support, and identifying and treating psychiatric disorders that sometimes occur before, during, or after these experiences.1 The first approach of the medical doctor plays an instrumental role in channelling the patients through the course of treatment and may have a significant impact on the patient. The physician must create an ambience where patients can freely discuss their personal issues without hesitation. He must be able to relate, reassure, comfort and perceive feelings during the first visit. Personal views or biases must not overpower the patient’s interests and personal experiences and feelings must be kept at bay when dealing with the patient. Dissemination of adequate medical information and helping patients make important decisions regarding their problem is an important responsibility that rests with the physician. He must be professionally skilled to prescribe the right tests and arrive at a sound diagnosis so that patients are not put through long rambling treatment protocols which may ultimately prove futile. He must give patients a comprehensive explanation of the therapy that is decided upon, the financial costs involved, the probability of success,

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any possible side effects of the therapy and complications in case of a pregnancy, and possible alternative options in case of failure so that patients are mentally prepared to cope with difficult circumstances at any point of the treatment. Physicians must be prepared to deal with the emotional and psychological issues provoked by the important role they play.23 Patients reactions thoughts and feelings may be determined by early childhood and past life experiences which have occurred outside the physician-patient relationship.24 Pre-existing psychological factors are independently related to treatment outcome in IVF/ICSI, and should therefore be taken into account in patient counselling.25 Hence physicians must be able to clearly discern such reactions from those that are solely the cause or result of inf ertility. More general therapeutic counselling can comprise supportive, coping-oriented and problem-solving strategies.26 The theoretical framework of the counselling models include psychodynamic psychotherapy, cognitive-behavioral techniques, solution focussed psychotherapy, crisis intervention and process-experiential grief counselling.7 Psychodynamic approaches examine low feelings of loss provoked by infertility, reawaken past losses while behavioral approaches focus on modifying maladaptive thinking pattern. Implications Counselling Implications counselling deals with the consequences of the course of treatment. Patients must be informed and educated at every step of the treatment protocol so that they are psychologically well prepared to accept situations and results that could otherwise be unnerving. The legal and ethical issues that revolve around the various forms of Third Party Reproduction must be clearly explained and detailed written informed consents that spell out the process, implications and consequences must be taken in the best interest of all parties involved. Support Counselling Support counselling aims at giving patients emotional, moral and psychological support during periods of distress, conflicts, decision making, accepting treatment failure or considering alternate options, coping with complications during and after treatment, and dealing with gender differences, sexual, marital or interpersonal problems. The infertility team must be equipped with adequate subject knowledge and special psychological skills, an important ingredient being compassion, in order to impart effective support counselling. They must be perceptive enough to grasp problems beyond those that are verbally expressed by the patient and must be able to amicably resolve gender differences regarding infertility or treatment choices. Inter-personnel biases between the staff must be set aside where the patient is involved so the patients can comfortably approach all members of the team without inhibitions.

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Counselling Approach Single Counselling Patients may be counselled individually in the following cases. • where one partner is more vulnerable and more adversely affected by infertility • in couples with sexual problems and consequent marital disharmony • where there are gender differences in — making decisions — coping with difficult situations — accepting treatment failure and alternate treatment options — facing the end of treatment — accepting a biologically non-related child as may be the case in third party reproduction —dealing with family or society pressures. Counselling patients singly may offer the advantage of coming to a rational conclusion based on the interpretation and coordination of individual views without affecting the self-esteem or emotions of either partner. Couple Counselling Patients may be counselled together where there is obvious need for orientation in decision making while accepting the reality of an unfulfilled desire. Fundamental issues involved in Third Party Reproduction may often require combined counselling. Group Counselling Group counselling is often necessary in open donor programs, surrogacy and adoption where • the motives and expectations (emotional, moral, and financial) of the various parties involved in the procedure must be clearly and unanimously understood for the welf are of the future child • the relationship of the donors/surrogates/biological parents with the future child must be defined and a detailed written consent taken to this effect • the legal and ethical issues within the particular jurisdiction must be explained to all parties concerned before proceeding with any of the mentioned programs. Group counselling may also be warranted in cases where the couple’s decisions are interrupted by family interference. In such cases the couple would have to be counselled with the respective family members, their views taken and a common judgement arrived at which best favors all parties concerned.

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Counselling Team Responsibilities A psychologist, or other mental health professional on the health care team, is essential to treatment of the biopsychosocial nature of infertility.2 In order to achieve the various counselling objectives and help infertile patients cope with their problems more subjectively it is imperative that the infertility team be equipped with specialized communication, professional medical and psychological skills. The medical, laboratory and nursing staff must be sensitive to the patients’ vulnerability and feelings regarding their wish for an unfulfilled desire, and must be able to reach out to them in an effective way The importance of the counselling role of the nurse, and the development and application of counselling services in ART centers, in lowering the anxiety and depression levels of couples and ensuring success of the treatment has been demonstrated.27 Place et al, 28 have also stressed on the need for better emotional preparation of couples through psychological counselling after the diagnosis of infertility, a constant demand of availability and empathy on behalf of the team and post-treatment counselling. An inherent quality that is central to counselling is compassion. The counsellor must be able the to allow the patients to express delicate issues like threatened sexuality which is of ten only brought to the forum of discussion after careful deliberation. Self-expression must be encouraged rather than forcing specific treatmentoriented answers which the personnel may deem fit. The concerned staff must be able to gauge and interpret problems that may be a sole result of the infertility from those that are driven by dif ferences within the family or society, and find effective solutions for the same. They must be tactful to settle differences within the relationship. While helping patients express and deal with their problems, they must be careful to avoid giving them the misconception of becoming an object of psychiatric dysfunction. Psychiatric illness must be diagnosed so that specific treatment can be given before initiating therapy for infertility. It would be the primary duty of the medical doctor to inform the patient of the limitations of the therapy to be used, and any possible complications and associated side effects that may be expected. A written informed consent explaining the entire treatment protocol, its limitations, legal and ethical issues and the financial considerations involved mustbe taken. A perfect coordination among the staff would be absolutely essential to achieve the objectives that are intended. Media for Patient Support In addition to personalized counselling, the most frequently provided adjunct services in infertility that may be used to enhance patient awareness and support are written information in the form of patient support booklets and telephone counselling. Findings from a study investigating perceived support and desire for support in couples referred for IVF have reported that both men and women felt that a routinely provided information booklet about the practical aspects of IVF would improve knowledge of and passage through an IVF cycle.20

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Patient Support Booklets Information covered in the patient support booklets must be lucid, comprehensive, and devoid of technical jargon that might be difficult to comprehend. It must include preparatory information that covers the psychosocial, emotional, social and ethical aspects of the treatment process. Medical information including side-effects of the treatment must be explained elaborately so that patients are well prepared to accept and face adverse results if any. Telephone Counselling Telephone counselling demands special communication skills to understand and interpret patients’ problems and guide them accordingly. Telephone counsellors must use their discretion when to invite a personal meeting with the clinic staff or a specialized counsellor. Self-Help Groups Self-help groups and patient organizations have been active since the 1970s’ in the area of assisted human reproduction.29 Self-help groups are independently functioning support groups organized by lay individuals experiencing infertility who aim to help infertile patients cope better with the crisis of infertility on both a cognitive and emotional level.30 Self-help groups may function through meetings in which infertile patients can share their woes and gather moral and emotional support from a similar group of patients who have met with success following infertility. Enabling interaction among patients on a personal level facilitates the exchange of experiences, feelings and coping mechanisms which may be mutually productive both mentally and psychologically. The inhibitions that patients often experience with mental health professionals may of ten be overcome in meetings with patients who can be related to at the same level. Patient Support Organizations Anetwork of several such groups may form a’ consumer organization’ that may function at a national and international level to disseminate information and increase the awareness of infertility. Resolve in the USA, Issue in the UK, Wunschkind in Germany and IFIPA (International Federation of Infertility Patient Associations) are examples of a few international organizations that have developed to offer patient support in infertility. These organizations aim to empower patients and increase their autonomy by increasing public awareness, influencing political decisions and actively seeking contact with relevant professions to facilitate cooperation.30 CONCLUSION Infertility often poses a kaleidoscope of problems that may grip infertile patients in ways and by measures that may prove self-destructive. A genuine treatable cause for the infertility may often be complicated with negative emotions arising from the inability to

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conceive naturally making the course of therapy and its aftermath a highly stressful ordeal for infertile couples. Psychotherapeutic counselling in infertile patients may have a mentally, psychologically, and emotionally healing effect by helping them through the decision making, educating them on the possible therapeutic options, understanding and helping self-expression, and showing them ways of coping with seemingly impossible situations. It may be morally reparative and constructive in helping infertile patients see a more positive yet realistic attitude towards childlessness and lead a more meaningful and resourceful life reminding them that Hope is a good thing and good things never die Shawshank Redemption

REFERENCES 1. Stotland NL. Psychiatric issues related to infertility, reproductive technologies, and abortion. Prim Care 2002; 29: 13–26. 2. Kainz K. The role of the psychologist in the evaluation and treatment of infertility. Womens Health Issues 2001; 11:481–85. 3. Leiblum SR, Williams E. Screening in or out of the new reproductive options: who decides and why. J Psychosom Obstet Gynaecol 1993; 14:1384–91. 4. Dennerstein L, Morse C. Psychological issues in IVF. Clin Obstet Gynaecol 1985; 12:835–46. 5. Kee BS, Jung BJ, Lee SH. A study on psychological strain in IVF patients. J Assist Reprod Genet 2000; 17:445–8. 6. Wischmann T, Stammer H, Scherg H, Gerhard I, Verres R. Psychosocial characteristics of infertile couples: a study by the ‘Heidelberg Fertility Consultation Service’. Hum Reprod 2001; 16:1753–61. 7. Applegarth LD. Individual counselling and psychothrapy. In Hammer Burns L, Covington SN (Eds): Infertility Counselling. Acomprehensive Handbook for Clinicians. Parthenon, London, UK, 1999; 85–102. 8. British Infertility CounsellingAssociation. J. Fertil. Counse. Spring and Summer; 1999. 9. Bryson CA, Sykes DH, Traub AI. In-vitro fertilization: a longterm follow-up after treatment failure. Hum Fertil (Camb) 2000; 3:214–20. 10. Englert Y. Ethics of oocyte donation are challenged by the health care system. Hum Reprod 1996; 11:2353–55. 11. Thoburn J, Norford L, Rashid J. Permanent Placement for Children of Minority Ethnic Origin. Jessica Kingsley, London, UK, 2000. 12. Midro AT. Genetic counseling in the case of carrier state with reciprocal chromosome translocations. Wiad Lek 1992; 45:775–80. 13. Tarlatzis I, Tarlatzis BC, Diakogiannis I, Bontis J, Lagos S, Gavriilidou D et al. Psychosocial impacts of infertility on Greek couples. Hum Reprod 1993; 8:396–401. 14. GreilAL, Porter KL, Leisco TA. Sex and intimacy among infertile couples. J Psychol Hum Sex 1989; 2:117–38. 15. Hammer Burns L. Sexual counselling and infertility. In Hammer Burns L, Covington SN N (Eds): Infertility Counselling. A Comprehensive Handbook for Clinicians. Parthenon, London and New York, 1999; 149–76. 16. Moller A, Fallstrom L. Psychological consequences of infertility: a longitudinal study. J Psychosom Obst Gynecol 1991; 12:27–45. 17. Joelle D. Sexuality. In ESHRE Monographs: Guidelines for Counselling in Infertility; 2002; 27–28.

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18. Laffont I, Edelmann RJ. Perceived support and counselling needs in relation to in-vitro fertilization. J Psychosom Obstet Gynaecol 1994; 15:183–88. 19. David A, Gur S, Rozin R. Survival in marriage in the paraplegic couple: psychological study. Paraplegia 1977; 15:198–201. 20. Laffont I, Edelmann RJ. Psychological aspects of in-vitro fertilization: a gender comparison. J Psychosom Obstet Gynaecol 1994; 15:85–92. 21. Human Fertilization and EmbryologyAuthority. Code of Practice. HFEA, London, UK; 1998. 22. Golombok S. Psychological functioning in infertility patients. Hum Reprod 1992; 7:208–12. 23. Lalos A. Breaking bad news concerning infertility. Hum Reprod 1999; 14:581–85. 24. Kentenich H. Emotional considerations within an infertility unit. In: Wijma, K. and von Schoultz, B. editors. Reproductive Life. Advances in Research in Psychosomatic Obstetrics and Gynaecology. Parthenon, Carnforth, UK; 1992; 551–56. 25. Smeenk JM, Verhaak CM, Eugster A, van Minnen A, Zielhuis GA, Braat DD. The effect of anxiety and depression on the outcome of in-vitro fertilization. Hum Reprod 2001; 16:1420–23. 26. Strauss B (Ed). Involuntary Childlessness. Psychological Assessment and Counselling. Hogrefe, Seattle, USA; 2002. 27. Terzioglu F. Investigation into effectiveness of counseling on assisted reproductive techniques in Turkey J Psychosom Obstet Gynaecol 2001; 22:133–41 28. Place I, Laruelle C, Kennof B, Revelard P, Englert Y. Gynecol Obstet Fertil 2002; 30:224–30. 29. Shapiro CH. Group counseling. In Hammer Burns L, Covington SN (Eds). Infertility Counseling. A Comprehensive Handbook for Clinicians. Parthenon, London and New York, 1999; 117–27. 30. Petra T. Self-Help Groups. In: ESHRE Monographs: Guidelines for Counselling in Infertility; 2002; 47–48.

CHAPTER 82 Politics, Partnerships and IVF Sandra K Dill OVERVIEW Governments worldwide have demonstrated a reluctance to acknowledge that infertility is a disability or medical condition. In most countries it is viewed as an elective procedure and therefore not worthy of reimbursement. The need to have access to health care is balanced against the need for governments to responsibly manage scarce resources and to distribute them justly and equitably for the good of the whole community. The challenge for consumers of infertility services is to persuade governments that inf ertility is a medical disability which causes suffering and as such is worthy of inclusion in their national health plan. The Australian experience has shown that before governments are willing to reimburse from the public purse, the medical community must demonstrate that quality standards of health care are met and that the practice of Assisted Reproductive Technology (ART) is effectively monitored. While some are proponents of restrictive legislation, others have argued that there is too much legislation for ART and cite existing legal choices for women in relation to human reproduction which respect individual autonomy In Australia, crucial to achieving good outcomes in the regulation and oversight of ART, has been the genuine involvement of consumers in all components of regulation, legislation, accreditation and policy The inclusion of a consumer representative on the Federal Council of the Fertility Society of Australia (FSA) and on the Reproductive Technology Accreditation Committee (RTAC), ensures that consumers have access to reliable information about treatment outcomes, possible drug side effects and the quality of service provided by individual clinics. A significant factor in the success of negotiations with government in relation to regulation and reimbursement issues, has been the commitment of consumers and providers to work in partnership to achieve common goals. This has proved to be a powerful tool in the political arena in Australia and has provided a model for similar representation in other countries. In the late 1980s this coalition of consumers and physicians successfully lobbied the Australian federal government for recognition of infertility as a medical condition and reimbursement for ART treatment in the national health plan. This has helped to provide equity of access to health care for infertile people in Australia. The continuing participation of consumers in public policy and the regulation of IVF clinics is a reassuring demonstration of openness by health ministers, physicians and bureaucrats in ensuring transparency and quality in the delivery of infertility services. It is also appropriate as it recognises that ultimately it is consumers who must live with the consequences of policy and treatment decisions.

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INTRODUCTION Most people take for granted their ability to have a child. Some choose not to but most of those who try to have a child have no difficulty in achieving that goal. However, for between 13 and 24 per cent of couples who would like to have a child but are not able to, it can be a very painful experience and one difficult to manage.1,2,3 Infertility is an extremely isolating experience. This is exacerbated because infertility and the death of a child are taboo subjects. As a society we have difficulty in dealing with these sad experiences. Infertile people need medical and social choices to help them deal with infertility Some pursue adoption and for over 30 years, Assisted Reproductive Technology (ART) has provided IVF and related treatments as another way of overcoming infertility and childlessness. The Limited Recognition of Infertility as a Disease or Medical Condition Governments worldwide have demonstrated a reluctance to acknowledge that infertility is a medical condition. In most countries it is viewed as an elective procedure and theref ore not worthy of reimbursement. For example, in Bangladesh, infertility is considered a curse that brings couples bad luck and the possibility of treatment is unknown to the majority.4 The need to have access to health care is balanced against the need for governments to responsibly managescarce resources and to distribute them justly and equitably for the good of the whole community. The challenge for consumers of infertility services is to persuade governments that inf ertility is a medical disability which causes suffering and as such is worthy of inclusion in their national health plan. This is one of the objectives of the Infertility Consumer Symposium, (ICSI), an international collaboration, whichbrings patient leaders together from more than 20 countries to discuss common interests and concerns. ACCESS Australia Infertility Network, a consumer based patient association, helped to found ICSI and is a partner and organiser of annual meetings held in conjunction with ESHRE.5 Australia is the only country in the world with public reimbursement for unlimited attempts of ART treatment. The government pays 85 per cent of the cost and the patient contributes the balance, so there is a shared responsibility. The following two sections provide a summary of how this was achieved. Australia does not have the benefit of the rich history of tradition enjoyed by some other nations. However, perhaps because we are such a young country, change has been easier to implement. Early History In 1987, the Human Embryo Experimentation Bill was introduced into the federal parliament. If passed it would have threatened the closure of all IVF clinics in Australia. Consumer groups formed a national alliance to lobby government about their concerns. The Fertility Society of Australia formed a sub committee of IVF clinic Directors to lobby government about political issues. Fortunately, the Bill didn’t get up in it’s original form.

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In 1988, the Department of Health released a discussion paper titled “Commonwealth Perspectives on IVF Funding”. As there was no coverage for IVF and related procedures in the national health plan, Medicare, the government had difficulty in determining the real costs and success rates of IVF treatment and they wanted this information. Clinicians and consumers suggested that introducing government reimbursement through Medicare would enable the government to track and collect this information, so negotiations commenced to discuss possible reimbursement for IVF and related procedures. From the outset, consumers accompanied clinicians to these negotiations. We represented our respective interest groups but met before each meeting to discuss any issues we thought would arise, to avoid being in conflict in front of Departmental officers. This co-operation seemed natural and appropriate to us, although it was unheard of in any other area of medicine. Two years later, the Health Minister took the unprecedented step of inviting consumers to his office in Canberra for two days, at taxpayers expense, so that he and his Department could meet with consumers on their own. The meeting began with representatives of the Financial Strategies Branch of the Department telling us that they had no extra money to spend. They told us that what IVF doctors wanted, was to make money out of us. So does the local supermarket, we said, but no-one seems concerned about that. When we asked why they didn’t want doctors present, they told us that they thought it would be helpful to get us away from their influence so that we could think more clearly about things. They said that they were concerned that our desire for a baby made it easy for the doctors to manipulate us into not opposing their point of view in the discussions. We assured him that their concern was misplaced as infertility affected our reproductive organs, not our minds, and that while we respected the doctors’ opinions, they were mistaken if they thought us as weak minded individuals, unable to consider the issues and make independent judgments about the matters being discussed. The Minister joined the meeting and put to us the government’s preferred option to fund IVF and asked us to support this. He seemed surprised and disappointed when we told him that we could not do that and explained, respectf ully, why we did not believe that this option would ensure quality and equity of access in delivering health care to infertile people. After the Minister left, his officers told us that we were wasting our time refusing to accept the government’s proposal and that we would not get what we wanted as with only 15 thousand women affected, we were ‘not electorally significant’. Some of my colleagues were very upset. But I encouraged them to be hopeful. The officer had told us what we needed to do in order to influence political opinion. And we realised finally that decisions about our medical treatment were going to be politically motivated and not based on rational discussion about our health needs. We assured him that it was not just the women undergoing treatment who would be concerned about the government’s attitude but their husbands, and their families and friends on both sides. We returned home and agreed with IVF doctors to launch a letter writing campaign. We asked patients to write to the federal Health Minister and their local Member of Parliament, with their concerns. We gave them the Minister’s fax number and asked them to fax their letter too. The Minister told us later that he had never received so many letters on a single issue as he had done in relation to IVF. And he was soon persuaded that infertile people were indeed ‘electorally significant’.

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After several more meetings with the Health Minister a compromise was reached and in 1990, the Prime Minister announced in his Budget speech that eight item numbers were to be introduced for IVF services, into the national health plan, Medicare. There was however, a restriction imposed for reimbursement of six cycles in a woman’s lifetime for a full, stimulated cycle. A condition of committing public money to reimburse ART services was the establishment of an effective accreditation process. The Reproductive Technology Accreditation Committee (RTAC), was established in 1987 by the FSA. In addition to clinicians there are representatives for Nurses, Counsellors and Scientists, each appointed by their respective professional associations. ACCESS appoints a consumer representative. These independently appointed representatives distinguish RTAC from other self regulation models. 1996 Campaign In 1996, the reimbursement for IVF item numbers was cut by 10 per cent in the first Federal Budget of a new government. This action caused distress for thousands of couples who were now being asked to contribute significantly more towards the cost of each treatment cycle. The IVF Directors’ Group met with ACCESS and with our combined resources we instituted another letter writing campaign, targeting Ministers and Members of Parliament (MPs) in marginal seats. IVF directors provided the resources we didn’t have to help us to provide literature to display in the clinics. This time we also provided MPs addresses, paper, pens, envelopes and suggested paragraphs to help people to write their letters while they were waiting to give their morning bloods. Then the clinics posted the letters for us. Clinicians also wrote to past patients with children telling them of this and suggesting they write to express their concern at the threat to their ability to return for another child, with government assistance. We tried unsuccessfully to meet with the Minister to discuss our concerns. Afew weeks later we were told that the Minister’s office was having difficulty in managing over 4,000 letters that had arrived protesting the cuts. One was complete with signatures of both parents of a six week old IVF child-and their daughter’s footprint. Then a business paper, the Sydney Morning Herald, published an article about a letter I had sent to the Prime Minister ‘s wife, seeking her support as a mother, and questioning the commitment of the government to the family. The same day one of the Minister’s senior advisors called me with the encouraging news that he was unhappy about the story. She said that we needed to understand that the government had limited resources and that we could not expect to have all our infertility health care paid for. I told her that all we were asking for was equity of access to appropriate health care services for the medical condition of infertility—and a 20 minute meeting with the Minister. She insisted that he was very busy and would not be able to see us for at least six months and f ollowed with the familiar, irritating warning to be wary of being manipulated in our campaign by IVF doctors. For an IVF doctor, I told her, inf ertility describes what he does but it defines who we are and I assured her that this provided all the motivation for consumers to do what ever was needed for however long it took, to have our concerns heard.

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Three days later I was invited to meet with the Minister but doctors were again excluded. In spite of this they provided expertise to brief me about the new Minister and government policy The new Minister spent twice the allotted time talking and listening sympathetically about issues that concerned patients. He also agreed to my request for a second meeting a few weeks later with doctors represented, to address the medical and technical issues. The Minister agreed to review the increased costs to consumers, in return for our undertaking to stop the letter writing campaign. He also agreed to consider removing the six cycle limit restriction and he kept his word and did this in November, 2000. Equity of Access to ART Legal access to ART services in Australia is both facilitated and restricted by legislation in some Australian states. The United Nations Declaration of Human Rights recognises that, “Men and women of full age, without any limitation due to race, nationality or religion, have the right to marry and found a family”.6 This is supported by the European Convention on Human Rights, which guarantees respect for family life and the right to found a family.7 It can be argued that these provisions create a positive right to access ART to achieve this goal, one taken for granted by fertile people in the community Because the limitless demand for health care can often not be met due to the scarcity of resources to service it, the need for micro allocation of health resources becomes apparent. While rationing is a necessity, it is important that the system used to decide who gets health care be one that promotes equity of access between people with health needs. Methods of rationing can use medical or social criteria. The use of social criteria is necessarily subjective and arguably immoral. However, it is difficult to see how those making decisions about rationing resources can avoid such judgments. Value judgments can be made based on an individuars past and potential contribution to society or in the case of ART, old fashioned prejudices masquerading as new ethical dilemmas.8 For example, there has been discussion about whether it is ethical to allow single women, lesbian or homosexual couples access to ART. Many believe that this is morally wrong, arguing that it is pref erable for a child to be raised within a stable, heterosexual relationship. Whatever our personal views, those who argue that the traditional concepts of family should be maintained, fail to recognise a different reality. An Australian government statistical report found that 69 per cent of households had no children, 32 per cent comprise two persons, 19 per cent had two or more children and that 13 per cent of households had one child. Marriage rates continue to fall, divorce occurs in more than 40 per cent of marriages and 27 per cent of births were to single women9. These figures demonstrate the diversity of family arrangements. The concept of family is different things to different people. Those who conceive naturally have assumed a genetic connection as normal. It is a matter of fact rather than a specific preference. The law in the state of New South Wales, through the Status of Children Act, plays a significant role in established rules where the social parents sometimes with no genetic link, are deemed to be the legal parents, such as in the case of donor insemination and donor oocyte procedures. Custom in some cultures also dictates what families mean to some people where there is a community approach to the raising children, rather than adherence to the notion of the nuclear family with which some of us

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are more familiar. The common thread in all of these arrangements is that people have been free to determine their own meaning of family and to live their lives accordingly. Where there is no evidence of detriment to the child, there appears to be no need for society to interfere in these arrangements. It is important to distinguish the question of public funding from that of legal access to services, where people without a medical dysfunction could pay for services needed because of social choices made. Decisions about who will access health care resources can be complex and difficult. The scarcity of resources available to meet the needs of everyone seeking them, compels health professionals and governments to make decisions about which individuals should have priority access to them and we are mindful that there is “very little distance between policy and politics”.10 Adherence to the “best interests of the child” principle while laudable can be difficult to apply in practice. It would be difficult to argue that it would be in the best interests of a child not to be born at all. In South Australia, the Reproductive Technology Act requires that a couple seeking assisted conception must demonstrate that they have no outstanding criminal charges or a history of an offence that was sexual or violent in nature. It also states that a couple must have no disease or disability, which could interfere with their capacity to parent a child. DeLacy argues that “while plausible, such requirements are extraordinary and unjust, and are likely to be both ineffective in protecting the welf are of children and harmful to individuals in the long term”. She identified the assumptions on which these requirements rest. Firstly, that “a parental history of a crime of violence will result in the child being exposed to violence.” Secondly, that parents who have had a child removed from their care have been proven to be abusive or neglectful which does not account for children removed from care for reasons other than poor parenting.11 Also, the requirement about a disability that could interf ere with the capacity to parent, offers no parameters with which to make that judgment. Given that reproductive medicine is called upon to intervene in situations of infertility caused by disease and disability, this presents a paradox for practitioners. This is supported by Douglas who argues that instituting a ‘fitness to parent’ code is “difficult enough to apply in cases concerning children who are in existence, let alone those who are only a twinkle in the doctors’ eye and it is open to many different assessments, depending on the person making the judgment.”12 Judgements are being made about a child who does not exist when patients who do exist and to whom the practitioner owes a fiduciary duty, are being refused treatment, which may not be in their best interests, leaving a practitioner vulnerable to an accusation that she may have acted in an ethically questionable manner. The Impact of Legislation on ARTTreatment While some are proponents of restrictive legislation, others have argued that there is too much legislation for ART. In Australia, the question is whether particular legislation will necessarily protect citizens from harm and where it is considered necessary, what degree of protection should be imposed by the law in a society where most citizens are free to make a multitude of choices about their lives or health care, including reproduction,

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without government interference. There is only intervention when the child is at risk as in adoption. In Australian States free from restrictive legislation, there has been no evidence that consumers or society have been disadvantaged. It can be argued that where genuine informed decision making occurs and there is a process for legitimate ethical review, restrictive laws make little sense and in some cases deny access to appropriate treatment for some couples who have no other means of forming their families. History has demonstrated that governments can often make ill inf ormed, politically expedient decisions, which are not necessarily in the best interests of their constituents. Furthermore, legislation is difficult to repeal. Even the most well intended legislation in a high-tech, rapidly evolving area such as ART, can quickly prove obsolete. Nationally, crucial to achieving good outcomes in the regulation and reimbursement of ART in Australia has been the genuine involvement of consumers in all components of regulation, legislation, accreditation, and policy development. This has been made possible largely because of the effective partnership that has developed between IVF clinicians and infertile patients over the last 14 years. It is a model unique in medicine in Australia and in ART worldwide. The inclusion of a consumer representative on the Federal Council of the Fertility Society of Australia (FSA), the Reproductive Technology Accreditation Committee (RTAC) and the IVF Directors’ Group, ensures that consumers have access to reliable information about treatment outcomes, possible drug side effects and the quality of service provided by individual clinics. Reproductive Technology Accreditation Committee (RTAC) It can be argued that the self regulation model is weak as the locus of control lies with doctors. However, a strength of the RTAC model in Australia is that consumers participate as equal partners. Any successful attempt to inappropriately manipulate the process would quickly destroy the credibility and effectiveness of RTAC. Despite the initial skepticism of the government, RTAC has demonstrated that self regulation can work. Access to government funded drugs used in treatment in Australia, is provided only to those clinics which have been accredited by RTAC. The availability of counselling is a requirement of accreditation, as is provision of detailed, written information on treatment, prior to its commencement. This is crucial to ensure that genuine, informed decision making occurs. Clinics must also demonstrate compliance with guidelines laid down by the National Health and Medical Research Council, the Australian Health Ethics Committee and a code of practice, together with relevant statutes in three States. To gain approval to conduct research or undertake new treatment with ethical considerations, individual clinics must apply to their local Institutional Ethics Committee. This ensures that the concerns of the community are addressed and that the interests of consumers are protected. Benefits of self regulation include its flexibility as it is more able to respond to emerging medical evidence and as Jansen argues, “enable delegation of the making of decisions to those who are to be the most personally affected by the consequences.13 “It

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also removes the need to rush to legislation every time a new procedure becomes available. In some countries, this has resulted in strange anomalies, such as: • allowing sperm donation but not in an IVF cycle (Norway and Sweden) or • allowing sperm donation but not oocyte donation (Denmark and Germany) or • recommending that use of her frozen embryo by a woman if her husband dies be disallowed but allowing that same woman to receive donor sperm (United Kingdom, France, Germany and Canada). Consumers of ART services seek politicians with integrity who have the courage to act fairly rather than expediently. In addition, almost 1 million IVF children have been born worldwide. Some of them have reached voting age and will show great interest in how their elected officials value their existence. Real Costs of Infertility: Societal, Social, Emotional Governments have argued that the costs of providing reimbursement for infertility treatment are too high but it can be argued that the financial costs are less significant than the real costs of infertility The Royal College of Obstetricians and Gynaecologists and the British Infertility Counselling Association found, based on papers by infertility specialists and interviews with medical, scientific and psychological experts, that infertility costs the nation in absenteeism, poor productivity and wasted resources.14 There are also social costs to consider such as marital relationships, taking time off work, refusing promotions, strained family relationships, and isolation from friends. The quality of life for some infertile people can become marginal when they have difficulty coping with a friend’s pregnancy, seeing babies and young children or watching television advertisements featuring babies. Events such as Christmas, Mother’s Day, and Father’s Day can be painful reminders of other people’s fertility and success and are times to be endured. Many couples do not participate in these family celebrations. The emotional costs can be the most signif icant. Nicol, in examining the impact of maternal loss, found that on average, ten per cent of women suffered some form of reproductive loss each year. Furthermore, she found that the death of a child had as significant emotional and physical impact on a woman as the death of a spouse and that with multiple losses, that the impact was exacerbated.15 It is easy to see the implications for women who have undergone successive attempts with assisted conception. In 1993, the London newspaper, the Daily Mail reported on the 15th birthday of “Bubbly Louise” (Brown), the world’s first baby born through IVF.16 A few pages away appeared a story headlined “Tragic teacher who longed for a baby”. Gillian Martin, a 34 year old primary school teacher from Southampton and her husband Michael, after trying to conceive for some years, had been told by their doctors the heartbreaking news that they would never have a child. Depressed and discouraged, she committed suicide.17 On the same day the joy of assisted parenthood and the desperation and despair of infertility were graphically contrasted. The question is not whether infertile people have a right to Infertility treatment reimbursement but rather, why they should be discriminated against in being denied affordable access to appropriate health care services.

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The profound impact which infertility and involuntary childlessness has had on millions of people worldwide, means that the global f amily of infertility associations will continue to lobby and represent the needs of our constituents. We will not rest until all those we represent are treated with the dignity enjoyed by others in the community. Infertile people, as citizens and taxpayers of their respective countries, seek rather to claim their right to equity of access, with fellow citizens, to affordable quality, health care and appropriate recognition of ART as a standard, proven treatment for infertility CONCLUSION This paradigm shift from consumers as passive participants to partners has been difficult for IVF clinicians in some countries but the political benefits for consumers and providers can be significant. These partnerships are also appropriate as they recognise that consumers of ART services must live with the consequences of policy and treatment decisions. The challenge for consumers is to ensure that all stakeholders have confidence in our integrity, professionalism and our ability to work effectively with the medical profession, government Ministers and senior public officials. This may not always be an easy task but may I suggest that the suffering of those who come to all of us for support, compels us to commit to nothing less.

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REFERENCES 1. Page H. Estimation of the prevalence and incidence of infertility in a population: Apilot study. Fertil and Steril 1989; 51(4): 571–77. 2. Greenhall E, Vessey M. The prevalence of subfertility. Fertil and Steril, 1990; 54(6):978–983. 3. Templeton A, Fraser C, Thompson B. The epidemiology of infertility in Aberdeen, Br Med J 1990; 301:148–152. 4. Personal conversation with Infertility Awareness support group 5. The European Society of Human Reproduction and Embryology 6. Universal Declaration of Human Rights, Article 16.1, United Nations, 1948 7. European Convention on Human Rights and its Five Protocols, Articles 8 and 12 8. Tizzard J. The deserving and the undeserving. Progress in Reproduction. London: Progress Educational Trust, 1996. 9. Tizzard J. The deserving and the undeserving, Progress in Reproduction. London: Progress Educational Trust, 1996. 10. Madden Richard. ‘Women’s Health’ Australian Bureau of Statistics Cat No 4365.0, Canberra. Australian Bureau of Statistics, 1994. ‘Marriages and Divorces’ Cat No 3310.0, Canberra, 1995. 11. Shenfield F. Justice and access to fertility treatments. In Shenfield F, Sureau C (Eds): Ethical Dilemmas in Assisted Reproduction. London: Parthenon Publishing Group, 1997. 12. De Lacey S. SA infertilty regulations labelled ‘unjust. FSA Newsletter, 1998; 39. 13. Douglas G. Law, fertility and reproduction (1991). In Kennedy and Grubb (Eds): Medical law (2nd edn). London: Butterworth 1994; 119–22. 14. Jansen R, Personal conversation 15. Kon A, Infertility: The Real Costs, ISSUE, CHILD for National Fertility Week, 1993 16. Nicol M. Loss of a Baby, Understanding Maternal Grief, Transworld, Moorebank, NSW, 1989 p. 4 17. The Daily Mail, 27/3/1993 p. 15 18. The Daily Mail, 27/3/1993 p. 9.

CHAPTER 83 IVF Success from a Clinician’s Viewpoint: How to Get it, How to Keep it? Sarah L Keller, Anil B Pinto, Daniel B Wllliams “In IVF, there are many opinions but very fewfacts” Anon.

INTRODUCTION There are many factors that could affect the success of an Assisted Reproductive Technology (ART) Program. Some of these factors include patient selection, the stimulation protocol utilized, as well as oocyte retrieval. The embryology laboratory is arguably the most crucial part of a successful ART program, as it is responsible for culture and insemination of oocytes, as well as monitoring embryo growth and development. Fertilized eggs are then cultured for an additional 1–5 days prior to embryo transfer. Micromanipulation procedures such as assisted hatching (AH) or ICSI (intracytoplasmic sperm injection) also require appropriate equipment and laboratory conditions in addition to skilled laboratory personnel. The embryologist then loads the catheter microscopically, the physician appropriately inserts the catheter into the uterine cavity and then gently expels the pre-embryos, although in some programs, the embryologist actually performs the transfer of pre-embryos. Thus, it takes a wellcoordinated team effort, as well as a good patient candidate, to make an ART cycle successful. As the importance of the embryology lab is well documented, this chapter will focus on some of the ways that clinical parameters can be adjusted so as to optimize success rates in an ART program. Patient Selection Patient selection is a very important factor that can influence success with IVF. After a careful history physical testing on both partners must be carried out in a pretreatment evaluation. In our program, both partners are tested serologically for HIV, Hepatitis B and C, and syphilis. Being positive for any of the above does not necessarily preclude treatment, although it does impact one’s ability to cryopreserve gametes or embryos. Evaluation of the uterine cavity is done prior to initiating treatment and can be done using sonohysterography, hysteroscopy, or a hysterosalpingogram. In our program, we favor sonohysterography, as this can easily be performed in the office, and is better tolerated. Correction of any abnormality that may affect a patient’s outcome should be carried out prior to cycle initiation. Additional testing should be offered as indicated. Patients should be evaluated regarding their immunity to rubella when their status is unknown. Depending on the

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ethnicity of the couple, screening for genetic disorders should be offered. In couples with known genetic disorders, genetic counseling should be off ered prior to treatment. When indicated, couples should be offered preconception counseling. The male partner should undergo a semen evaluation, which can determine the need for ICSI. When indicated, the patient should undergo a urologic evaluation. In partners with severe oligozoospermia or azoospermia, genetic testing should be offered for microdeletion of the Y chromosome and chromosomal analysis. Either partner should be offered screening for cystic fibrosis.1 Screening for ovarian reserve is an important part of the pretreatment evaluation for IVF in selected patients. The term “ovarian reserve” describes oocyte quality in the patient. It is accepted that older women have an overall diminished response to stimulation and a poorer prognosis for success in IVF. Studies have documented that ovulatory women may begin having subtle elevations in their FSH levels in their mid thirties.2 Scott et al, in a large retrospective study, demonstrated a decrease in pregnancy rates as basal FSH levels increased.3 Tests that can be used to screen for diminished ovarian reserve include basal FSH and estradiol, the clomiphene challenge test, ultrasonographic measurements of uterine volume and basal antral follicle counts, day 3 inhibin-B levels, and the GnRH-a stimulation test. While functional ovarian reserve can be assessed by all of these methods, the most widely used tests are the basal FSH and estradiol levels and the clomiphene challenge test. Our center primarily uses basal FSH and estradiol levels to assess a patient’s ovarian reserve, and screening is used for patients ≥35 years of age, as well as patients with a prior poor response. It is important to realize that different centers may use cut-off values to “screen-in” only the most optimal patients, in order to maximize success rates. This issue as well as more specific information about ovarian reserve testing is covered in more detail in another chapter in this book entitled, “Infertility: Is there success after forty?” Stimulation Protocol Determining which stimulation protocol to use in a particular patient depends upon multiple factors, including chronological age, previous response to ovarian stimulation, ovarian reserve testing, and diagnosis. An important factor for optimizing success inυolves standardization of protocols, particularly when there is more than one physician involved in a particular practice. The following are examples of some of the protocols that are used in our center. Normal Responders There are two protocols that we routinely employ to treat patients who are considered by clinical experience and/ or prior stimulation response as “normal responders”. For the most part they may be used interchangeably. Although we primarily use FSH for ovarian stimulation, we have not found overall response and pregnancy outcome to change with the use of HMG. The most widely used has been the Mid-Luteal Lupron Protocol (MLL). Leuprolide acetate (LA) is initiated in the mid-luteal phase at a dose of 1 mg per day and is given subcutaneously. Following the onset of the patient’s menses, a baseline estradiol

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and follicle scan is performed and FSH is initiated at a dose of 225IU subcutaneously At that time, the dose of LA is decreased to 0.5 mg daily After five days of medication, an estradiol level is checked. The FSH dose is adjusted based on this level. Estradiol levels and ultrasounds are then checked at appropriate intervals until the patient has a minimum of two follicles greater than or equal to 18 mm with an estradiol level greater than or equal to 500 pg/ml. Human chorionic gonadotropin (hCG) is given 36 hours prior to oocyte retrieval. This is given at a dose of 250mcg subcutaneously (recombinant hCG) or 5,000 to 10,000 units intramuscularly (urinary hCG) intramuscularly. The other protocol being used f or the normal responder is the GnRH antagonist protocol, which has the advantage of avoiding pre-stimulation treatment. FSH at a dose 225 IU is initiated on cycle day two or three following a normal baseline estradiol and follicle scan and is given for four days. An estradiol level and an ultrasound are then obtained. The dose of FSH is then adjusted based on the initial level. The antagonist is initiated at a dose of 250 mcg per day when the follicular size is 12–14 mm. In our program, we also give low-dose hCG 10 units per day at the time of antagonist initiation, to prevent a potential drop in estradiol, which can be seen with the use of an antagonist alone. Human menopausal gonadotropins (HMG) at a dose of 75 IU can be used in place of lowdose hCG. Testing is continued until the patient is ready for hCG administration prior to oocyte retrieval. The Low Dose Lupron Protocol is typically used in the following patients: 1) Those who do not respond well to the Mid-Luteal Lupron Protocol and 2) Patients who are 38 years of age and older. In this protocol LA is initiated at a dose of 0.5 mg in the midluteal phase of the cycle. With the ensuing menses, FSH is initiated at a dose of 300IU and the dose of LA is decreased to 0.25 mg. After five days of drug, the estradiol level is measured. The patient is monitored with serial ultrasounds and estradiol levels until the same parameters as outlined above are reached. Poor Responders The Lupron Flare Protocol is used f or the following patients: 1. Those who have failed to respond to the abovementioned protocols 2. Women 40 and over and 3. Those patients with a past history of abnormal ovarian reserve testing (basal FSH, E2 must be normal in the Flare cycle to proceed with treatment). Following normal basal FSH, E2 and follicle ultrasound, LA at a dose of Img per day as well as HMG 225IU in the a.m., and FSH 225IU in the p.m. is begun for 4 days. The dose of Lupron is then decreased to 0.25 mg per day and is continued at this dose until hCG is administered. An E2 level is drawn at that time and medication adjustments are made accordingly. Serial ultrasounds and estradiol levels are obtained serially until the patient is ready for hCG. Additional stimulation protocols that have been used for this difficult patient population are reviewed elsewhere in this book. While we have not found a definite advantage for using one protocol versus another, we routinely use the standard flare protocol in these patients based on our experience with this regimen.

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High Responders The Hyperresponder Protocol is used for: 1. Patients with a prior exaggerated response to ovarian stimulation 2. Patients <30 years of age 3. Patients with medical conditions that may predispose to a hyper-response (i.e. polycystic ovarian syndrome (PCOS)). These patients are at risk for Ovarian Hyperstimulation Syndrome (OHSS), especially if they become pregnant. Patients are given oral contraceptives for at least 21 days prior to starting LA, which is then started at a dose of 1 mg per day. During this time, they continue the oral contraceptives. Five days after starting the LA, the pill is discontinued. FSH is then started at a dose of 150 IU three to six days following withdrawal bleeding and the dose of leuprolide acetate is decreased to 0.5 mg. After four days of medications, an estradiol level is obtained and the patient is monitored serially with ultrasounds and estradiol levels as indicated. Human chorionic gonadotropin is given following the same guidelines as above. Oocyte Retrieval Oocyte retrievals are performed 36 hours after hCG administration. In our program, these are performed in a procedure area in our office that contains a procedure room with an anesthesia machine, an embryo transfer room, and four recovery rooms. The patient is placed in the dorsal lithotomy position. Once positioned, a nurse anesthetist who is in attendance for the entire procedure gives them intravenous sedation. The vagina and perineum are prepped with warm saline solution. We do not use betadine due to concerns about embryo toxicity Using a vaginal ultrasound probe with an attached needle-guide, follicles are aspirated under direct visualization using a sixteen-gauge double-lumen aspiration needle. Flushing of follicles is not routinely performed and is reserved for cases where there are very few follicles, or simply to clear the tubing. We routinely aspirate multiple follicles in one tube. It is important to use warmed tubes that are nonembryo toxic, and to also hand off aspirates to the embryologist expeditiously to minimize cooling of the tubes. Following oocyte retrieval, the patients are allowed to recover and are typically discharged home after 1–3 hours. Embryo Transfer Introduction Embryo transfer (ET) is one of the most important steps in an IVF cycle from a clinical standpoint. Indeed, meticulous detail to this part of the process will have a major impact on overall success rates. The following section will outline our approach to this procedure. Trial Embryo Transfer (TrET) The use of TrET prior to an IVF cycle is vitally important and serves two basic functions:

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1. To determine the length from the fundus to the external os, aiding in proper placement of the embryo transfer catheter 2. To identify patients who may have a potentially difficult embryo transfer that may require additional assistance such as ultrasound-guided transfer. If a patient is found to have cervical stenosis, this should be treated prior to beginning IVF treatment. We do trial transfers in the follicular phase and use either a tomcat catheter or a stiffer IUI catheter. One can also use actual ET catheters, but this would add significant cost. At the time of embryo transfer, the measurement from the trial transfer can be correlated with the ultrasound-derived measurement of the endometrial cavity length obtained during monitoring. A study by Mansour et al4 compared embryo transfers between groups in which patients were randomized into those having TrET trial versus those without TrET. In the group without TrET, 29.8% of the transfers were difficult and the overall pregnancy rate was 13.1%. In the group who underwent TrET, none of the actual transfers were considered difficult. This group had a 22.8% pregnancy rate. A number of other studies have also demonstrated a positive correlation between ease of embryo transfer and pregnancy rates.5,6 Variables that may negatively affect the success of successful ET can include the presence of blood or mucus at the catheter tip, effect of excessive uterine contractions, potential retention/ expulsion of embryos, bacterial contamination of the catheter tip, or “difficult ET”. Careful technique may help to eliminate these factors, and thus optimize overall pregnancy rates. Embryo Transfer (ET) The next step in the IVF process is the actual embryo transfer. At our Center, the patient is told to drink approximately 20–30 ounces of water approximately 1 hour prior to the procedure to ensure that the bladder is at least partially filled. This can serve to straighten the cervical canal of patients with an anteverted uterus, allowing easier ET catheter placement.7,8 If the patient becomes uncomfortable prior to the actual ET secondary to bladder filling, the patient can void into a 10 ounce cup to decrease any discomfort. The patient is placed in the lithotomy position and the cervix is gently washed using warm media. We tend to use Dulbecco’s Phosphate Buffered Sodium. A soft catheter attached to a tuberculin syringe is then used to gently aspirate mucus from the cervical canal. The presence of cervical mucus can potentially have a detrimental affect on transfer outcomes. Cervical mucus at the tip of the catheter has been associated with a higher rate of retained embryos.5 In addition, the presence of cervical mucus may interfere with embryo placement as it may increase the chances of embryo expulsion.4 Cervical mucus may also serve as a source of contamination adversely affecting both the endometrium as well as embryos resulting in a decrease in pregnancy rates.9 Attempts have been made to show that vigorous cervical lavage prior to transfer improves pregnancy rates but a large multi-center study could demonstrate no benefit for this technique.10 If a significant amount of mucus is obtained initially, the catheter is flushed and aspiration is repeated until no mucus is present. Using trial transfer measurements and correlating these with ultrasound measurements, the caiheter tip is υery slowly inserted through the cervical os to a distance of approximately 1.5 cm. from the fundus. Corolueu et al11 demonstrated under ultrasound guidance, that when embryos were placed approximately 1.5–2.0 cm from the fundus, both pregnancy and implantation rates

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were significantly higher than when embryos were placed approximately 1 cm from the fundus. If there is a discrepancy of >1 cmbetween the endometrial cavity length measurements by ultrasound and TrET, the last ultrasound measurement is utilized for ET catheter placement. Great care is taken to avoid contact with the uterine walls. In the case of non-ultrasound guided ET, the catheter and syringe is slightly disengaged from the outer sheath and rotated clockwise to ensure that the catheter did not kink within the cervical canal. The catheter is reengaged, and the embryos are then injected into the uterine cavity in a slow and steady manner. The ET catheter is rotated 90° and then very slowly withdrawn, again being careful to avoid contact with the walls of the uterus. Whether the practice of waiting for a certain period of time prior to catheter withdrawal is beneficial is unknown. However, a recent study demonstrated no difference in pregnancy outcome whether the ET catheter was withdrawn immediately following expulsion of embryos versus 30 seconds later.12 It is also important to remember to keep the plunger firmly depressed to minimize the risk of negative pressure that could draw the embryos back towards the cervix. After withdrawal, the embryologist inspects the catheter microscopically to verify that there are no retained embryos. While we do have patients maintain bed rest for thirty minutes following embryo transfer, there is no evidence that this has an impact on pregnancy outcome.13,14 A list of steps for ET is shown in Table 83.1. Particularly when there is more than one physician, it is important that everyone follows the same general guidelines for ET.

Table 83.1: Steps for Successful ET — Trial embryo transfer — Partially-filled to full bladder — Use of soft ET catheter — malleable stylet when indicated — SLOWLY insert loaded catheter through os to a distance of 1.5 cm from fundus — Slightly disengage ET catheter and syringe from outer sheath and rotate 360° clockwise — Reengage ET catheter and SLOWLY and STEADILY inject embryos — Rotate entire catheter 90° and VERY SLOWLY and CAREFULLY withdraw catheter — Inspection of catheter by embryologist for retained embryos

Other Issues Regarding ET Choice of ET Catheters The choice of an ET catheter is a very important one. Most authors distinguish between “hard” and “soft” ET catheters. Hard catheters would be stiffer, thereby making catheter placement theoretically easier. However, this could potentially result in trauma to the endocervical canal or uterine wall, as well as causing uterine contractions, compared to the use of softer catheters.15 It has been demonstrated that the frequency of uterine contractions increase with difficult mock transfer16 and a negative impact on pregnancy outcome has been associated with an increase in frequency of uterine contractions.17 Also, the presence of blood on the outside of the catheter may indicate a difficult or traumatic transfer and has been associated with lower pregnancy rates.18 A softer catheter would be flexible, malleable, with a smooth tip, which could serve to potentially minimize trauma during ET. Common examples of hard catheters would include:

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1. Tom Cat catheter (Sherwood Medical, St. Louis, MO, USA) 2. Tefcat catheter (Cook Ob-Gyn, Spencer, IN, USA) 3. Norfolk catheter (Cook Ob-Gyn).19 Examples of soft catheters would include: 1. Wallace catheter (Cooper Surgical, Shelton, CT, USA) 2. Cook Soft-Pass (Cook Ob-Gyn) 3. Frydman Catheter (Laboratoire CCD, Paris, France). A number of studies have demonstrated the benefit of using soft catheters versus hard catheters.20–22 Wood et al19 demonstrated that clinical pregnancy rates per transfer were significantly increased with the use of soft versus hard catheters (36% vs. 17%). The superiority of one soft catheter versus another has not been demonstrated. Urman and coworkers23 evaluated the use of Wallace versus Frydman catheters in 428IVF cycles and found no significant differences in pregnancy outcome. Another study by Karande et al24 evaluated pregnancy outcome in IVF patients undergoing ultrasound-guided ET, and compared the Cook Echo-Tip catheter (Cook ObGyn) with the Wallace Catheter. They found no differences in the ongoing pregnancy rates (49% vs 47%) or implantation rates (30% vs. 35%) between groups. Difficult ET’s In the best of circumstances, a potentially difficult ET would be predicted by prior history (i.e. prior difficult ET, cervical stenosis, prior cervical conization, etc) or on the basis of a difficult TrET. Routinely performing TrET on patients prior to an IVF cycle can significantly reduce the risk of unexpected difficult ET. Also, f ollowing some of the simple steps reviewed above under the section entitled “Embryo Transfer” will help to prevent unexpected problems. In the event of a difficult ET, there are a number of techniques that can be utilized to successfully enter the uterine cavity (See Table 83.2).

Table 83.2: Techniques for difficult ET — Routine use of Trial ET — Use of Stylet — Precycle Cervical Dilatation — mechanical, laminaria — GIFT — Transmyometrial Transfer — Hysteroscopic Shaving

A Malleable stylet can be inserted into the outer sheath of a soft catheter. The stylet can be angled to correspond to the curvature or angle of the cervical canal. It is important to make sure that the embryos have been placed back into an isolette or incubator until successful entry into the uterine cavity is achieved. Once you are just past the level of the internal os, the embryos can then be reloaded into the soft ET catheter. The stylet is removed, leaving the outer sheath in place. With the help of the embryologist, the ET transfer catheter is gently threaded through the outer sheath, and the ET is then performed as previously described. There does not appear to be any adverse effect on pregnancy outcome when the stylet is used.25,26

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Cervical dilatation has also been used in patients with cervical stenosis. Manual dilatation of the cervix within a few days of embryo transfer has been attempted with minimal success.5,27 This would suggest that recent trauma to the cervix/endometrium could adversely affect pregnancy outcome. However, the use of cervical dilatation used prior to the start of an IVF cycle has met with more success. Abuscheikkha et al28 performed cervical dilatation prior to initiation of the IVF cycle in patients with a history of cervical stenosis and found favorable results. Following hysteroscopy and cervical dilatation in patients with a prior history of difficult ET, IUI, or EMB, Yanushpolsky and coworkers29 placed a Malecot catheter (CR Bard Inc., Covington. GA, USA) for an average of 10 days. Thirty-two of 36 patients had significantly easier procedures following this treatment. In a case report, Glatstein et al30 demonstrated successful pregnancy outcome in two IVF patients with cervical stenosis and difficult prior ET following placement of laminaria tents approximately one month prior to the IVF procedure. Other techniques that have been suggested include transmyometrial ET31,32 and hysteroscopic shaving of the cervix33. It is also important to remember that in centers that have access to laparoscopy, GIFT is also an excellent way to treat the small number of patients with unexplained infertility who present with a history of severe cervical stenosis. Ultrasound-guided ET One of the more recent advances in embryo transfer technique from a clinical standpoint involves the use of ultrasound-guidance for ET catheter placement and transfer of embryos. This technique had initially been described by Strickler et al34 and would be potentially useful in cases where the cervical canal was difficult to negotiate. It would also allow one to visually confirm embryo placement, and could serve to direct the catheter along the contour of the uterus, potentially minimizing unwanted trauma to the endometrium. This technique can be performed using either transabdominal or transvaginal ultrasound. With transabdominal ultrasound, it is necessary that either an ultrasound technician or a trained assistant who can maintain a good view of the endometrial stripe is available. Transvaginal ultrasound relies on the skill of the physician to perform both ultrasound and ET simultaneously. A number of studies have demonstrated that ET with ultrasound guidance improves pregnancy outcome in terms of both implantation and clinical pregnancy rates compared to “blind” insertion.35,36,37,38,39 Prapas et al37 was able to demonstrate this benefit only with day 3 or 4 ET, but not with day 5 ET. Tang et al,40 in a prospective, randomized trial was able to demonstrate a significant improvement in implantation, but not clinical pregnancy rates. Other studies have demonstrated no improvement in pregnancy outcome with the use of ultrasound-guided ET.41,42 A recent retrospective study did demonstrate a benefit using ultrasound-guided ET in patients with a previous failed IVF cycle.43 Our current practice is to offer ultrasound-guided ET to: 1) patients with a difficult 2) TrET, history of cervical stenosis or difficult ET 3) patients with a history ≥2 failed IVF cycles. Some of these studies are summarized in Table 83.3. The use of a soft catheter with an echogenic tip may also make ultrasoundguided ET easier.24

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Table 83.3: Efficacy of ultrasound-guided ET Author

ET/+ Ultrasound ET/−Ultrasound Patient # IR CPR IR CPR

Kan 1999 prospective 187 20.4% 37.8% 16.2% 28.9% Lindheim 1999 retrospective 137 28.8%* 63.1%* 18.4% 36.1% Coroleu 2000 randomized 362 25.3%* 50%** 18.1% 33.7% Papras 2001 prospective 1069 23.3%** 47%* 15.8% 36% Tang 2001 randomized 800 15.3%* 26% 22.5% 12.0% Kojima 2001 retrospective 846 15.2%** 28.9%** 7% 13.1% Matorras 2002 randomized 515 11.1%* 26.3%* 7.5% 18.1% IPR—Implantation Rate; CPR—Clinical Pregnancy Rate; * p<0.05; **p<0.01

CONCLUSION Both the laboratory and clinical components of an IVF program are essential to the success of the program. While the IVF laboratory is the linchpin of any successful program, it must be complemented by excellence on the clinical side. Just as the laboratory has written protocols, the clinical side should also have standardized stimulation protocols, as well as a standardized technique f or embryo transfer. As previously mentioned, the procedure of embryo transf er is probably the most important aspect of the process from a clinical standpoint. Strict attention to detail in this area is absolutely essential to ensure the success of an IVF program. Finally if both the embryologists and clinicians follow the appropriate practices, consistency will be the key to ensuring good results. REFERENCES 1. ASRM/SREI Committee Opinion. Practical genetic evaluation and counseling for infertile couples. American Society for Reproductive Medicine/Society for Reproductive Endocrinology and Infertility. February 2002. 2. Sherman BM, Korenman SG. Hormonal characteristics of the human menstrual cycle throughout reproductive life. J Clin Invest 1975; 699–706. 3. Scott RT, Hoffman GE. Prognostic assessment of ovarian reserve. Fertil Steril 1995; 65:1–11. 4. Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization. Fertil Steril 1990; 54:678–81. 5. Visser DS, Fourie FL, Kruger HF. Multiple attempts at embryo transfer: effects on pregnancy outcome in an in vitro fertilization and embryo transfer program. J Assist Reprod Genetics 1993; 10:37–43. 6. Tomas C, Tapanainen J, Martikainen H. The difficulty of embryo transfer is an independent variable for predicting pregnancy in in vitro fertilization treatments. Fertil Steril 1998; 70:S433. 7. Sundstrom P, Wramby H, Persson PH et al. Filled bladder simplifies human embryo transfer. Br J Ob Gyn 1984; 91:506–7. 8. Lewin A, Schenker JG, Avrech O et al. The role of uterine straightening by passive bladder distention before embryo transfer in IVF cycles. J Assist Reprod Genetics 1997; 14:32–4.

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9. Egbase PE, al-Sharhan M, al-Othman S et al. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in vitro fertilization and embryo transfer. Hum Reprod 1996; 11:1687–9. 10. McNamee P, Huang T, Carwile A. Significant increase in pregnancy rates achieved by vigorous irrigation of endocervical mucus prior to embryo transfer with a Wallace catheter in an IVF-ET program. Fertil Steril 1998; 70:Suppl 1:228. 11. Coroleu B, Barri PN, Carreras O, Martinez F, Parriego M, Hereter L et al. The influence of the depth of embryo replacement into the uterine cavity on implantation rates after IVF: a controlled, ultrasound-guided study. Hum Reprod 2002; 17:341–6. 12. Martinez F, Coroleu B, Parriego M et al. Ultrasound-guided embryo transfer: immediate withdrawal of the catheter versus a 30 second wait. Hum Reprod 2001; 16:871–4. 13. Sharif K, Afnan M, Lenton W et al. Do patients need to remain in bed following embryo transfer: the Birmingham experience of 103 in vitro fertilization cycles with no bed rest following embryo transfer. Hum Reprod 1995; 10:142–30. 14. Botta G, Grudzinskas G. Is prolonged bed rest following embryo transfer useful? Hum Reprod 1997; 12:2489–92. 15. Lavie O, Margalioth EJ, Geva-Eldar T, Ben Chetrit A. Ultrasonographic endometrial changes after intrauterine insemination: a comparison of two catheters. Fertil Steril 1997; 52:79–84. 16. Fanchin R, Righini C, Oliveness F et al. Uterine contractions at time of embryo transfer alter pregnancy rates after in vitro fertilization. Hum Reprod 1998;13:1968–74. 17. Lesny P, Killick SR, Tetlow RL et al. Embryo transfer-can we learn anything from the observation of junctional zone contractions? Hum Reprod 1998; 1540–46. 18. Goudas VT, Hammit DG, Damario MA et al. Blood on the embryo transfer catheter is associated with decreased rates of embryo transfer on the outcome of IVF. Hum Reprod 1998; 70:878–82. 19. Wood EG, Batzer FR, Go KJ et al. Ultrasound-guided soft catheter embryo transfers will improve pregnancy rates in in vitro fertilization. Hum Reprod 2000; 15:107–12. 20. WisantoA, Janssens R, Deschacht J et al. Performance of different embryo transfer catheters in a human in vitro fertilization program. Fertil Steril 1989; 52:79–84. 21. Mansour RT, Aboulghar MA, Serour GI et al. Dummy embryo transfer using methylene blue dye. Hum Reprod 1994; 9:1257–59. 22. Ghawazzawi IM, Al-Hasani S, Karaki R et al. Transfer technique and catheter choice influence the incidence of transcervical embryo expulsion and the outcome of IVF. Hum Reprod 1999; 14:677–82. 23. Urman B, Aksoy S, Alatas C et al. Comparing two embryo transfer catheters. Use of a trial transfer to determine the catheter applied. J Reprod Med 2000; 45:135–8. 24. Karande V, Hazlett D, Vietzke M et al. A prospective randomized comparison of the Wallace catheter and the Cook Echo-Tip® catheter for ultrasound-guided embryo transfer. Fertil Steril 2002; 77:826–30. 25. Helsa J, Steven J, Schlenker T. Comparison of malleable stylet Wallace catheter to Tomcat catheter for difficult embryo transfers. Fertil Steril 1998; 70:Suppl 1:S222. 26. Nielsen IK, Lindhard A, Loft A et al. A Wallace malleable stylet for difficult embryo transfer in an in vitro fertilization program: a case-control study. Acta Obstet Gynecol Scand 2002; 81:133–7. 27. Groutz A, Lessing JB, Wolf Y et al. Cervical dilation during ovum pick-up in patients with cervical stenosis; effect on pregnancy outcome in an in vitro fertilization-embryo transfer program. Fertil Steril 1997; 67:909–11. 28. Abusheikkha N, Lass A, Akagbosu F et al. How useful is cervical dilation in patients with cervical stenosis who are participating in an in vitro fertilization-embryo transfer program? The Bourn Hall experience. Fertil Steril 1999; 72:610–12. 29. Yanushpolsky EH, Ginsburg ES, Fox JH et al. Transcervical placement of a Malecot catheter after hysteroscopic evaluation provides for easier entry into the endometrial cavity for women

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with histories of difficult intrauterine inseminations and/or embryo transfers: A prospective case series. Fertil Steril 2000; 73:402–5. 30. Glatstein IZ, Pang SC, McShane PM. Successful pregnancies with the use of laminaria tents before embryo transfer for refractory cervical stenosis. Fertil Steril 1997; 67:1172–74. 31. Kato O, Takatsuka R, Asch RH. Transvaginal-transmyometrial embryo transfer: the Towako method; experiences of 104 cases. Fertil Steril 1993; 59:51–53. 32. Groutz A, Lessing JB, Wolf Y et al. Comparison of transmyometrial and transcervical embryo transfer in patients with previously failed in vitro fertilization-embryo transfer cycles and/ or cervical stenosis. Fertil Steril 1997; 67:1073–6. 33. Noyes N, Licciardi F, Grifo J et al. In vitro fertilization outcome relative to embryo transfer difficulty: a novel approach to the forbidding cervix. Fertil Steril 1999; 72:261–65. 34. Strickler RC, Christianson C, Crane JP et al. Ultrasound guidance for human embryo transfer. Fertil Steril 1985; 43:54–61. 35. Lindheim SR, Cohen MA, Sauer MV. Ultrasound guided embryo transfer significantly improves pregnancy rates in women undergoing oocyte donation. Int J Gynaecol Obstet 1999; 66:281–84. 36. Coroleu B, Carrereas O, Veiga A et al. Embryo transfer under ultrasound guidance improves pregnancy rates after in vitro fertilization. Hum Reprod 2000; 15:616–20. 37. Prapas Y, Prapas N, Hatziparasidou A et al. Ultrasound-guided embryo transfer maximizes the IVF results on day 3 and day 4 embryo transfer but has no impact on day 5. Hum Reprod 2001; 16:1904–8. 38. Kojima K, Nomiyama M, Kumamoto T et al. Transvaginal ultrasound-guided embryo transfer improves pregnancy and implantation rates after IVF. Hum Reprod 2001; 16:2578–82. 39. Matorras R, Urquijo E, Mendoza R et al. Ultrasound-guided embryo transfer improves pregnancy rates and increases the frequency of easy transfers. Hum Reprod 2002; 17:1762–66. 40. Tang OS, Ng EH, So WW et al. Ultrasound-guided embryo transfer: a prospective randomized controlled trial. Hum Reprod 2001; 16:2310–15. 41. Al-Shawaf T, Dave R, Harper J et al. Transfer of embryos into the uterus: how much do technical factors affect pregnancy rates? J Assist Reprod Genet 1993; 10:31–36. 42. KanAK, Abdalla HI, GafarAH et al. Embryo transfer: ultrasoundguided versus clinical touch. Hum Reprod 1999; 14:1259–61. 43. Anderson RE, Nugent NL, Gregg AT et al. Transvaginal ultrasound-guided embryo transfer improves outcome in patients with previous failed in vitro fertilization cycles. Fertil Steril 2002; 77:769–75.

CHAPTER 84 Bandwagon IVF: All for One and One for All KE Tucker, CAM Jansen INTRODUCTION “Jumping on the bandwagon” by definition, refers to the complete and immediate support of an individual or a concept because of perceived popularity or initial success. How could this phrase be applied to the practice of In Vitro Fertilization (IVF)? Because of our strong sense of responsibility to our patients we are tempted to perform every new and promising technique in the hope of helping each of them achieve their goal of the birth of a healthy baby. Not every procedure is applied without carefully considering the ramifications, but sometimes our enthusiasm in offering what is initially thought of as a better treatment clouds our ability to objectively consider all outcomes; short- and long-term. BANDWAGONS IN IVF: WHAT ARETHEY? Three procedures that have been proposed at one time or another, to be performed for all IVF patients, assisted hatching (AH)—originally reported to improve implantation rates, intracytoplasmic sperm injection (ICSI)—wellestablished for severe male-factor cases, and extended embryo culture with blastocyst transfer—developed to facilitate embryo selection and thereby, increase implantation and pregnancy rates and reduce the incidence of multiple gestations. Initial results for all three of these procedures were very encouraging for selected groups of patients, but continued research revealed, however, that not all patients benefit from the “one for all and all for one” approach to these techniques. This, in conjunction with the possible risks imposed by each of these procedures requires that great care must be taken when performing any one of them. This paper will review the benefits and risks associated with AH, ICSI and blastocyst culture and transfer and attempt to establish if these procedures should be applied to all patients. Bandwagons in IVF: Assisted Hatching General Considerations The oocyte is surrounded by an acellular glycoprotein matrix called the zona pellucida (ZP) which is responsible for a variety of important functions. Specific sites exist on the surface of the ZP which enable sperm binding to occur prior to fertilization and then may

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subsequently, also contribute to the block to polyspermy. Throughout early development, the ZP provides protection and maintains the integrity of the embryo, but once in the uterus, however, the embryo must escape or “hatch” from the ZP in order to implant. It has been postulated that one of the reasons healthy-appearing embryos do not always implant after transfer is due to hatching failure.1,2 The inability of some in vitro—produced embryos to hatch has been attributed to perturbations of the culture environment that result in artificially hardening the zona,3,4 to cryopreservation or to in vivo zona thickening observed in some patients.5,6 Assisted hatching (AH) was developed in the IVF laboratory and is a microprocedure whereby an opening is made in the ZP, enabling a possibly compromised embryo to escape or “hatch” more easily and consequently improve the chance of implantation. There are three accepted methods of AH currently performed in ART, partial zona dissection, zona erosion and laserassisted zona drilling. The application of this procedure to all patients has been met with mixed results and recommendations. Partial Zona Dissection (PZD) The first report of AH in human embryos was by Cohen and co-workers,3 using a technique originally devised to assist fertilization, partial zona dissection (PZD). PZD is generally performed by inserting a microneedle through a small section of the ZP A tear or rift is made by rubbing the area of zona isolated by the needle against the holding pipette. Although these investigators reported higher overall implantation rates, other studies using this method of AH had mixed results. When PZD was performed for patients with elevated Day 3 FSH or for patients over 38 years of age, pregnancy rates were significantly elevated.7,8 A more recent, non-randomized study by Edirisinghe and others,9 however, could not show any benefit of AH by PZD for older patients (>38 years), for those with previous IVF failures or f or any patients with embryos displaying a thickened ZP. Arandomized study of the effects of PZD on patients with previous IVF failures reported some benefit, although not significant, of AH for IVF patients.10 Aprospective, randomized controlled study in 120 patients undergoing IVF/ICSI demonstrated no improvement of pregnancy and implantation rates with PZD.11 Zona Drilling using AcidTyrode’s Solution This widely used method of AH involves the application of an acidified medium (Acid Tyrode’s—AT; pH=2.5) in close apposition to the embryo in order to erode a hole in the ZP. This procedure is normally performed on Day 3 of embryo development, but it can also be successful with Day 2 embryos.12 Due to the increase in perivitelline space, it is believed that the use of AT to erode the ZP would be not only efficient, but also safe.2 It is still important to limit the exposure of the embryo to AT and care should be taken to perform the procedure quickly and rinse the embryo well in fresh culture medium to remove any excess AT. Discrepancies in results of AT for Ati were initially attributed to inconsistencies in the size of the hole created in the ZP. It has been reported that an opening of 30–40 µm is adequate for the embryo to hatch completely from the ZP and minimize the chance for

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monozygotic (MZ) twinning, the incidence of which has been reported to increase following the transfer of hatched embryos.13 Zona Thinning/Removal using AcidTyrode’s Medium Overall thinning of the ZP using AT has also been described for both mice and humans.5 This procedure was shown to be successful in mice, but had no significant effect on the implantation rates in humans. For patients undergoing intracytoplasmic sperm injection (ICSI), complete removal of the ZP of Day 3 human embryos was successful in significantly increasing pregnancy rates, but only for patients with poor prognosis (>40 years of age, previous failed IVF attempts). No differences were seen in younger women undergoing their first ICSI attempt.14 Zona Drilling using Laser Assisted Methods It has been suggested that one the main problem of the more traditional methods of AH is the requirement of extensive technical skill to consistently produce holes of the appropriate size using only microtools and conventional micromanipulation equipment.15 It was proposed that laser and other light delivery systems could be a viable alternative to chemical or mechanical AH methods. Two laser assisted AH methods exist. As the name implies, zona drilling using contact lasers involves a glass micro-fiber that comes in direct contact with the embryo. A criticism of this method is that the embryo is exposed to excess heat, possibly damaging the blastomere nearest the drilling site. Conversely non-contact lasers provide touch-free, objective-delivered accessibility to the ZP, with minimal absorption of heat by the embryo.6 Care must be taken, however, that the position of the objective has not shif ted between pulses, otherwise serious damage to the embryos can occur. Both methods can very precisely produce a single or series of pulses (result in a ridge-like opening in the ZP), but varying clinical results are seen depending on whether a single hole or ridge is produced. A study using excess human embryos demonstrated that contact laser ZP thinning increased in vitro hatching compared to non-manipulated controls.16 Mantoudis and coworkers6 found that non-contact, quarter laser AH, which creates a long ridge (essentially “thinning” about one-f ourth of the ZP) resulted in higher clinical pregnancy rates compared to total (single hole completely through the ZP) or partial (single hole not through the ZP) laser AH. Assisted Hatching: Who Benefits? Regardless of the AH methods used, very different results have been published over what patient population would benefit the most from this procedure. From 1992 to 1999, the results of 24 studies were compared and summarized.2 Overall, 12 of these studies reported a statistically significant improvement in pregnancy rates for all or some of the patients treated. One study reported significant improvements in success rates when AH was applied for all IVF/ICSI patients.

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Of the remaining 12 studies, no improvements were reported for either implantation or pregnancy rates for the same patient populations as the previously mentioned studies, including those couples undergoing frozen embryo transfer. Of the 14 RCT evaluated, 11 either found no significant improvement in pregnancy rates at all or an improvement only in a selected sub-population of patients.2 Assisted Hatching: Summary and Conclusions The majority of trials with AH have been performed on embryos from patients with poor or compromised IVF outcomes, including older patients (>38 years), those with elevated FSH or with poor quality embryos (Grades 3 and 4, thickened ZP). Results from these studies are contradictory. Approximately 50 percent indicated some improvement in implantation and pregnancy rates. Success rates for clinics advocating this procedure for all their patients, including those with good prognoses, are also conflicting. When considering routinely performed AH for any IVF patient, it is vital to remember that with any procedure or treatment, the benefits should outweigh the limitations or risks. This is especially true when considering hatching good quality embryos from good prognosis patients. One study investigating the effects of AH for embryos from good prognosis patients found not only no difference in pregnancy rates compared with untreated controls, but actually demonstrated a significant decrease in implantation rates in the study group.12 This is understandable considering the invasiveness of the procedure itself. Care must also be taken to avoid rupturing fragile embryos, either during the AH procedure itself or during embryo transf er. In any case, it is important to remember that this is an invasive procedure that exposes the embryo to various potentially destructive elements and should be used judiciously and cautiously. If implantation occurs, another risk of AH is a significant increase in the incidence in MZ twinning, based on population analysis of all IVF-ET cycles initiated in US clinics in 1996.13 Although AH may benefit a very select group of patients, the evidence is strong against applying this procedure to all IVF/ICSI patients. Bandwagons in IVF: Intracytoplasmic Sperm Injection (ICSI) General Considerations Conventional IVF methodologies have been expanded in an attempt to improve fertilization rates (FR) in cases of male factor infertility. High insemination concentration (HIC) has been proposed as a bridge procedure between IVF and intracytoplasmic sperm injection (ICSI) for oligozoospermic or teratozoospermic cases, but good results are sporadic.17 The introduction of ICSI has overshadowed these earlier modifications of the IVF procedure and has been shown to be particularly effective in patients with pervious fertilization failure 72% PR).18 Generally, the procedure involves the microinjection of a single sperm, collected either from the ejaculate, epididymis or testicle, into a mature, healthy oocyte. Specifically, a small sample of sperm is placed in a viscous medium, hindering forward progression of the sperm and exaggerating its motion characteristics. This makes it possible for a single cell to be selected for injection. It is subsequently immobilized with

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a microinjection pipette and injected into the egg without disrupting the mitotic spindle, generally thought to be associated with the first PB. The first pregnancies following sperm-injected oocytes were reported by Palermo and assoeiates in 1992.19 Since then, most IVF programs have included ICSI in their list of treatment options and high fertilization and pregnancy rates could now be possible regardless of low sperm concentration, reduced/abnormal motility or gamete source.20–22 Mansour and other14 reported that fertilization and pregnancy rates following ICSI were unaffected by most sperm parameters, as long as morphologically good, live cells were used for injection. When performed properly, ICSI can almost guarantee fairly high fertilization rates. This feature and the surprisingly high subsequent pregnancy rates have tempted some IVF programs to consider abandoning conventional in vitro insemination or any other method of assisted fertilization (PZD or sub-zonal sperm insertion—SUZI) for ICSI. ICSI: Who and When? Some investigators believe that ICSI should only be used when previous fertilization failure following IVF has occurred, or if the number and/or quality of the sperm population are not appropriate for IVF.23 Others maintain that the simplest (and cheapest) method, with the greatest long-term chance of healthy children, should be the f irst course of action.5 ICSI and IVF were compared in a prospective study, including non-male factor, infertile couples (n=96), no differences were observed in fertilization occurrence or failure, embryo quality, number of embryos available for transfer or pregnancy rates.24 A multi-center, controlled and randomized trial (RCT) involving MII sibling oocytes from 221 patients, demonstrated that although fertilization rates were higher after ICSI than conventional IVF (79 vs. 60%, respectively), in the absence of any male factor, no differences in embryo quality or cleavage rates were noted.25 Another RCT including 91 good prognosis couples also could not demonstrate any differences in study population variables such as patient age, BMI and endometrial thickness on outcome parameters, including fertilization rate, embryo score and grade at transfer.26 On average, ongoing PR (33 υs 23%) or IR (18 υs 11%) between IVF and ICSI, respectively, were also not statistically different, but were higher for the IVF group (except for the pathological PRthose that resulted in miscarriage, which was higher in the ICSI group). Although not statistically different, the data was compelling enough for these authors to still recommend that ICSI be applied only when conventional IVF fails. ICSI: Risks from the Technique Aspiration of a small amount of cytoplasm from the egg is considered necessary by those who regularly perf orm ICSI to confirm plasma membrane perforation and to initiate oocyte activation. It has been reported, however, that blastocyst formation was lower from oocytes that had >6 picoliters of cytoplasm aspirated into the ICSI injection pipette and was attributed to the damage of cytoskeletal structures. No effect of the volume of cytoplasm aspirated or of the position of the PB on chromosomal abnormalities was noted.27 Conversely, Jean and others28 reported that exposure of sperm to

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polyvinylpyrolidone (PVP) during selection and immobilization caused microscopic nuclear alterations. The effects on the oocyte, if any, were not obvious, but the use of alternatives was still recommended (i.e. Motilowâ—hyaluronic acid; Medicult). ICSI: Risks of Chromosomal Abnormalities A study comparing 3 PN embryos from IVF and ICSI cycles found that triple colored fluorescent in situ hybridization (FISH) indicated a significantly higher incidence of paternally-derived chromosomal abnormalities (7.4% from ICSI vs 1.5% from IVF) in those from ICSI.29 Another study by Calogero and others30 demonstrated that ICSI patients had significantly higher aneuploidy and diploidy rates for chromosomes 8, 12, 18, X and Y than normozoospermic controls. These authors also reported that the absence of a pregnancy tended to be associated with the transfer of aneuploid embryos, but if a pregnancy did occur, a significant number of these resulted from the transfer genetically abnormal embryos. Similarly, microdeletions on the Y chromosome have been detected in 3–15 percent of men with severe oligozoospermia and non-obstructive azoospermia31 and pregnancies still occurred in couples where the man had these genetic abnormalities. ICSI: Summary and Conclusion The 1998, ESHRE Task Force evaluated 23,932 ICSI cycles performed in 1995 and, not surprisingly, reported that overall success rates with this procedure were high. It was also reported that the incidence of de-novo chromosomal anomalies in ICSI children was higher than in the general population, but ultimately concluded that the procedure was generally safe.32,33 The ASRM released a Practice Committee Report in November 2000, maintaining that ICSI is compatible with normal child development and is no longer regarded as an experimental procedure. It was recommended that, although a safe and effective therapy, the application of ICSI should be performed carefully and only for select couples and even then, counseling should be offered to these patients, especially if a genetic def ect could be transmitted by this procedure. Since there does not appear to be any “natural selection” for abnormal ICSI embryos, either in-vitro or in-vivo, ICSI should not be used routinely f or all inf ertile couples. Bandwagons in IVF: Extended Embryo Culture/ Blastocyst Transfer General Considerations Attempts to improve ART outcome has drawn attention to developing culture environments that will support in vitro embryo growth to the blastocyst stage. The major benef its of blastocyst culture and transf er in human IVF include the possibility to further select a better embryo, which in turn will contribute to reducing the number of embryos transferred and consequently, the incidence of multiple gestations. Another rationale for transferring embryos at the blastocyst stage is that this is the most physiologically appropriate stage for intrauterine transfer. Initial results withblastocyst culture and transfer were quite encouraging and several different methods emerged designed to maximize the rate of blastocyst formation. These

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included the use of a single, enriched medium throughout culture,34 coculture with autologous cumulus cells,35,36 oviduct-endometrial cells37,38 and non-human (Vero) cells39 and most recently, the application of sequential media designed to more appropriately meet the needs of the embryo at different stages of development.40,42 Pregnancy and implantation rates in high responding, good prognosis patients were impressive when sequential media were used (70 and 48%, respectively:40 patients <35years, IR=54%:43). Results with other groups were somewhat less, but still very encouraging. Patients with previous multiple IVF failures could become pregnant following blastocyst transfer (53% clinical PR).44 Similar improvements with blastocyst transfer in selected populations of patients have been reported (those with at least 2 previously unexplained IVF attempts using a single medium: Rijnders and Jansen45—poor prognosis using Day 3 matched controls.46 Conversely, a RCT by Huisman and co-workers47 (2000) did not show a benefit of Day 5 (blastocyst) vs. Day 3 transfer in 1787 cycles using a single culture medium. In addition, these investigators also found no differences in pregnancy, implantation or multiple gestation rates. Another randomized trial using sequential media also could not demonstrate any differences in pregnancy, implantation or twinning rates.48 Extended Culture/BlastocystTransfer: Risks and Limitations On average, approximately 40 to 60 percent of f ertilized oocytes become blastocysts in culture, even with the newer sequential media, but variations among patients to produce blastocysts is significant. There is always the risk that no blastocysts will be available for transfer. Unfortunately, there is no reliable way to ascertain if a group of cleavage stage embryos will eventually become blastocysts. Morphology alone has been shown to be an inconsistent indicator of potential blastocyst development. Rijnders and Jansen34 demonstrated that not only did as little as 50 percent of all good quality Day 3 embryos (Grades 1 or 2) become blastocysts by Day 5, but so did as many as 25–30 percent of those of poorer grades (3 and 4). Morphology has also been shown to be a poor predictor of mosaicism in both cleavage stage embryos and blastocysts.49 Prolonged selection of embryos with culture to the blastocyst has been thought by many to eliminate those embryos with chromosomal abnormalities, especially those contributed by defective sperm. It is thought that the paternal genome was indispensable for blastocyst development.50 Trisomic embryos can reach the blastocyst stage a an alarming rate of 37 percent,51 but interestingly, only those monosomies associated with first trimester development in utero (mono X and 21) were detected at the blastocyst stage. It has also been shown that there is a significant increase in the number of MZ twins following transfer of blastocysts than with cleavage stage embryos.45,52 Rijnders and Jansen reported a 4.6-fold increase in the incidence of MZ when blastocysts were transferred compared to the results with Day 2 or 3 embryos. Behr and co-workers52 also found an incidence of MZ twinning as high as 5 percent, which represented an incidence 10-fold higher than seen in the general population. Since many pregnancies have resulted following frozen embryo transfers, the contribution of these cryopreserved embryos to the overall IVF success rates cannot be ignored. Blastocyst formation rate in most laboratories is generally <50 percent, leaving

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significantly fewer good quality blastocysts available for cryopreservation.53 Results following blastocyst thaw and transfer are still too limited to say that this procedure be applied to all IVF patients, providing any remaining embryos are available for freezing. Blastocyst Transfer: Summary and Conclusions Although a very effective means of reducing the incidence of high order multiple pregnancies, blastocyst culture and transfer is not without risks. It is important to note that 2–40 percent of patients will have their embryo transfer cancelled because no embryo continued to develop to blastocyst by Day 5.53 Some clinicians have maintained that this is a form of “natural selection”, and are prepared to accept this unfortunate outcome as the fate of abnormal embryos. Since the uterus may be a significantly better environment than even the newer sequential blastocyst culture media, we as embryologists and clinicians must be cautious in assuming that failure to reach the blastocyst stage indicates the embryo never had any potential for implantation. As we have seen, even genetically abnormal blastocysts can result in pregnancies and single blastocysts can lead to obstetrically complicated MZ twins. Extending embryo culture and transferring blastocysts can be an extremely effective treatment for selected patients, especially those prone to multiple gestations, but it is not the answer for all. For others, blastocyst culture may lead to cancelled transfers and in more extreme cases, to compromised and complicated pregnancies and births, and should only be offered to those couples who will most benefit from this therapy. BANDWAGONS IN IVF: CONCLUSIONS It is tempting to apply an initially successful procedure to all IVF cases. We all want to do the most for our patients and the vast majority of ART programs want their patients to leave with the promise of a single, healthy baby. There will always be new protocols or procedures that have good or encouraging results, but as with most things, everything comes at a price. Although it may seem an obvious point to mention, it is crucial to remember that not all patients are the same. Each will present different obstacles to overcome, and an individualized strategy should be devised for all couples. One treatment for all patients, whether AH, ICSI or extended embryo culture and blastocyst transfer, might be successful for some, prof oundly disappointing for others and even dangerous for a selected few. All treatments must be carefully evaluated and matched to the needs and wants of couples seeking treatment. In the field of Assisted Reproductive Technologies, one size does not fit all.

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19. Palermo G, Joris H, DeVroey P, Van SteirteghemAC. Pregnancies after intracytoplasmic injection of single spermatozoan into an oocyte. Lancet 1992; 340:17–18. 20. DeVroey P, Liu J, Nagy Z, Tournaye H, Silber SJ, Van Steirteghem, AC. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril 1994; 62:629–41. 21. Nagy ZP, Liu J, Joris H, Verheyen G, Tournaye H, Camus M et al. The results of intracytoplasmic sperm injection are not related to any of the three basic sperm parameters. Hum Reprod 1995; 10:1123–29. 22. Silber SJ. What forms of male infertility are there left to cure? Hum Reprod 1995; 10:503–4. 23. Baker HEG, Liu DY, Bourne H. Diagnosis of sperm defects in selecting patients for assisted fertilization. Hum Reprod 1995; 8:1779–80. 24. Moreno C, Ruiz, Simon C, PellicerA, Remohi J. Intracytoplasmic sperm injection as a routine indication in low responder patients. Hum Reprod 1998; 13(8):2126–29. 25. Fischel S, Aslam I, Lisi F, Rinaldi L, Timson J, Jacobson M, et al. Should ICSI be the treatment of choice for all cases of in-vitro conception? Hum Reprod 2000; 15:1278–83. 26. Poehl M, Holagschwandtner M, Bichler K, Krischker U, Jurgen S, Feichtinger W. IVF-patients with nonmale factor “to ICSI” or “not to ICSI” that is the question? J Assist Reprod Genet 2001; 18(4):205–7. 27. Dumoulin JCM, Coonen E, Bras M, Bergers-Janssen JM, IgnoulVanvuchelen RCM, van Wissen LCP et al. Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure. Hum Reprod 2001; 16:306–12. 28. Jean M, Mirallie S, Boudineau M, Tatin C, Barriere P. Intracytoplasmic sperm injection with polyvinylpyrrolidone: a potential risk. Fertil Steril 2000; 76:419–20. 29. Macas E, Imthurn B, Keller PJ. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI. Hum Reprod 20001; 16(1):115–20. 30. Calogero AE, De Palma A, Grazioso C, Nunziata B, Burrello N, Palermo I, et al. High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome. Hum Reprod 20001; 16:1433–39. 31. Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosome of infertile men. Hum Rerpod 1997; 12:1635–41. 32. Tarlatzis BC, Bili H. Survey on intracytoplasmic sperm injection: report from the ESHRE Task Force. Hum Reprod 1996; 13(Suppl 1):165–77. 33. Bonduelle M, Camus M, De Vos A, Staessen C, Tournaye H, Van Assche E, et al. Seven years of intracytoplasmic sperm injection and follow-up of 1087 subsequent children. Hum Reprod 1999; 14(Suppl 1):243–64. 34. Rijnders PM, Jansen CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1998a; 13:2869–73. 35. Planchot M, Antoine JM, Alvarez S. Granulosa cells improve human embryo development in vitro. Hum Reprod 1993; 8:2133–40. 36. Quinn P, Margolit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assist Reprod Genet 1996; 13:9– 14. 37. Bongso A, Fong CY, Ng SC, Ratnam S. Human embryonic behavior in a sequential human oviduct-endometrial coculture system. Fertil Steril 1994; 61:976–85. 38. Rubrio C, Simon C, Mercader A, Garcia-Velasco J, Remohi J, Pellicer A. Clinical experience employing co-culture of human embryos with autologous human endometrial epithelial cells. Hum Reprod 2000; 15(Suppl 6):31–38. 39. Menezo Y, Chouteau J, Veiga A. In vitro fertilization and blastocyst transfer for carriers of chromosomal translocation. Eur J Obstet Gynecol Reprod Biol 2001; 96(2):193–5.

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40. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increase implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 2000; 69:84–8. 41. Quinn P. Some arguments on the pro side. Hum Reprod 1998; 13:3292–95. 42. Mortimer D. Human blastocyst development media. Hum Reprod 2001; 16:2725–6. 43. Langely DT, Marek DM, Gardner DK, Doody KM, Doody KJ. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 20001; 16(5):902–8. 44. Sakkas D, Jaquenoud N, Leppens G, Alda Campana. Comparison of results after in vitro fertilized human embryos are cultures in routine medium and co-culture on Vero cells: a randomized study. Fertil Steril 1994; 61:521–25. 45. Rijnders PM, Jansen CAM. Increased incidence of monozygotic twinning following the transfer of blastocysts in human IVF/ ICSI. Fertil Steril 1998b70 (Suppl 1): 515–16. 46. Gutknecht DR, Lens JW, van Weering H, Schats R, Vermeiden JPW. Day 5 versus day 3 embryo transfers in patients with repeated implantation failure: A case-controlled study. Hum Reprod 2001; 16(Suppl 1):3. 47. Huisman GJ, Fauser BC, Eijkemans MJ. Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril 2000; 73:117–22. 48. Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: a controlled randomized trial. Hum Reprod 2000; 15:1947–52. 49. Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 1998; 11:3151–55. 50. Banerjee S, Lamond S, McMahon A, Campbell S, Margund G. Does blastocyst culture eliminate paternal chromosomal defects and select good embryos?: inheritance of an abnormal paternal genome following ICSI. Hum Reprod 2000; 15:2455–59. 51. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001; 16(9):1954–58. 52. Behr B, Fisch JD, Rakowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000; 17:349–51. 53. Alper M, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question. Hum Reprod 2001; 16:617–19.

Index

A A fresh IVF cycle 547 A serinethreonine kinase 149 Abdomen 128 Abdominal distension 127 Abdominal scanning 485 Abnormal embryo 531 Abnormal gamete cell morphology 531 Abnormal sperm 231 Abortion 84, 317, 350, 415, 422, 543 Abortion rate 84, 116, 236, 542 Accessory sex gland 207, 211 Accudenz 216, 219 Acetylsalicylic acid 124 Achondroplasia 334 Acid drilling 304 Acid phosphatase 504 Acid salt solution 533 Acid Tyrode’s medium 568 Acid Tyrode’s solution 568 Acne 24, 146 Acrosin 206 Acrosin activity 436 Acrosomal enzymes 538 Acrosome 316, 516 Acrosome index 219 Acrosome reaction 209, 210, 268 Acrosome reaction assay 206, 209 ACTH 10 ACTH secretion 124 Activin 11, 328 β-β activin dimer 12 Activin receptor II subtype 10 Activin-A 10 Activin-follistatin system 11 Acute respiratory distress syndrome (ARDS) 128 Addison’s syndrome 353 Adenine 333

Index

905

Adhesiolysis 445, 447, 450, 469, 471, 476, 477, 481 Adhesion 137, 139, 389, 409 Adhesion molecules 381, 382, 385, 388, 389, 392, 419 Adhesions 15, 132, 133, 184, 444, 445, 453, 461, 476, 477, 487 Adhesions are filmy adhesions 476 Adhesions or endometriosis 170 Adhesive disease 138 Adhesive molecules 382 Adipocytes 47 Adipose tissue fibroblasts 47 Adjuvant 29 Adjuvant treatment 322 Adnexa 24, 130, 446, 460, 461 Adnexal adhesions 465 Adnexal torsion 461 Adolescent pregnancies 543 Adoption 350, 546, 548 Adrenal cortex 47, 147, 309, 364 Adrenal insufficiency 147, 310 Adrenal medulla 364 Adrenal steroid 149 Adriamycin 320 Advanced reproductive technologies 345 Agarose-gels 335 Age 40, 301–303, 305 Agglutination 210 Agonist 89 Agonist/receptor complex 66 Alanine 394 Albumin 128, 315 Albumin gradient 314 Aldosterone antagonist 147 Alkaline phosphatase (AP) 365, 369 Alkoxyl and peroxyl radicals 270 Alkylating agents 321, 322, 328 Alleles 158 Allergic 66, 70 Allis’ clamp method 187 Allogeneic 326 Alpha-fetoprotein (AFP) 367, 369 5-alpha reductase inhibitors 147 Alpha-tocopherol (vitamin E) 269 Alporte disease 334 Alumina silicate 192 Alveolar 128 Alzheimer’s disease 5, 333 Amenorrhea, hirsutism 154 Amenorrhea, obesity, and hirsutism 145 Amenorrhoea 15, 63, 130, 157, 308, 321, 326, 476, 476 Amino acid 78, 247, 308, 333, 394 Amniocentesis 532 Amniotic fluid 334

Index

906

Amphiregulin 390 Amphotencin B 365 Amplitude of lateral head displacement (ALH) 222 Amplitude of lateral head movement 233 Ampoules 7, 41, 87 Ampoules of gonadotropins 40 Ampullary seromuscularis 448 An acellular glycoprotein 567 An anteverted uterus 563 Analgesia 166 Analgesic 124 Anaphase 246, 307 Anaphylactoid, local hypersensitivity 70 Anaphylaxis 411 Anasarca 127, 128 Anastrozole 49 Anatomy 491 Androgen 48, 81, 89, 112, 124, 148, 156, 409, 522 Androgen action 147 Androgen biosynthetic pathway 157 Androgen levels 146, 149 Androgen production 146 Androgen synthesis 10 Androgenic precursors 47 Andrology 222 Androstenedione 47, 131, 133, 147 Androstenedione to estrone 155 Aneroid manometer 473 Anesthesia 78, 165, 169, 174, 175, 180, 467 Aneuploid 236, 375 Aneuploid cells 368 Aneuploidies 242, 335 Aneuploidy 55, 237, 253, 291, 317, 416, 528, 529, 531, 570 Aneuploidy embryos 291 Angiogenesis 324, 389 Angiogenic factors 324 Angiogenic growth factors 127, 129 Angiography 41 Anionic phospholipid 417, 421 Annealing temperature 334 Annexion V 419 Anonymous donor 359 Anovulation 136, 137, 145, 146, 155 Anovulatory 31, 32, 91, 93, 149, 155, 158, 261, 431 Anovulatory infertility 32, 47 Anovulatory PCOS 158 Antagonist 402 Antagonistic analog 78 Antagonistic response 78 Antagonists 66, 540 Antiandrogen 147, 156 Anti-androgen therapy 147

Index

907

Antibiotic therapy 171 Antibiotics 171 Antibodies 293, 307, 419, 539 Anticardiolipin 418 Anticardiolipin antibodies (aCL) 411, 416, 418, 293, 418 Anti-chlamydial antibodies 360 Antiestrogen 29–32, 47, 48, 120 Antiestrogenic effect 31, 32 Anti-FSH antibodies 51 Antithromboxane 419 Antihypertensive 22 Anti-inflammatory 124 Anti-inflammatory agents 404 Antinuclear antibodies are (ANA) autoantibodies 294 Anti-oestrogens 32 Antiovarian antibodies (AOA) 294, 353 Antioxidant 247, 267, 269, 271, 274, 277 Antioxidant capacity 269, 275 Antioxidant defense mechanisms 269 Antioxidant defense systems 278 Antioxidant enzymes 267 Antipaternal cytotoxic antibodies 416 Antiphospholipid antibodies (APA) 293, 409, 411, 415, 416, 418–420, 422 Antiphospholipid antibodies—lupus anticoagulant 418 Antiphospholipid antibody syndrome 415, 419 Antiphospholipids 410 Anti-progestin drug, RU 486 383 Antiprostaglandins 485 Antipyretic 124 Anti-recombinant FSH antibodies 57 Antiretroviral therapy 341 Antisperm antibodies 206, 295, 411 Anti-sperm antibody test 210 Antithrombin III 416 Antithrombin III into cytoplasts 375 Anti-thromboxane effect 420 Antithyroglobulin 422 Antithyroid antibodies 294, 422 Antithyroid antibody screening 294 Antiviral therapy 338, 339 Antral follicle 561 Antral follicle count (AFC) 40, 77, 303 Antral follicle development 113 Antral follicles 10, 41, 113, 157, 322 Aphasia 10, 169, 247 Apoptosis 271, 299, 320, 328, 374, 384, 400, 538 Apoptotic 245 Apoptotic markers 271 Apoptotic pathway 384 Apposition 389, 405, 409 Arcuate nucleus 66

Index

908

Argon fluoride 192 Aromatase 47, 49 Aromatase activity 112 Aromatase expression 47 Aromatase gene 157 Aromatase inhibitors 47–49 Aromatase system 112 Aromatization 146 ART 191, 199 ART cycles 137 Arterial embolism 128 Arterio-venous malformation 423 Arthralgia 411 Artificial insemination 18, 174, 339 Ascites 95, 127, 128 Ascorbate 269 Ascorbic acid 247 Asherman’s syndrome 476, 477 Asparagine 394 Asparate 394 Aspiration 4 Aspirin 124, 401, 412, 419, 421 Aspirin for infertility 423 Aspirin monotherapy 421 Aspirin/heparin/IVIG 294 Assist fertilization 567 Assisted conception 6, 55, 127, 559 Assisted fertilisation 400, 569 Assisted hatching (AH) 192, 195, 257, 258, 291, 374, 528, 529, 560, 568, 567, 569 Assisted hatching, blastocyst transfer, co-culture 412 Assisted human reproduction 254 Assisted reproduction 62, 231, 268, 340 Assisted reproduction failure 294 Assisted reproductive procedures (ART) 81, 538 Assisted reproduction technique (ART) 8, 50, 55, 62, 85, 90, 104, 112, 145, 189, 233, 271, 289, 292, 297, 303, 307, 323, 328, 333, 338, 339, 352, 388, 435, 515, 527, 545, 554, 560, 571 Assisted reproductive therapy (ART) 282 Assistesd hatching 304 Asthenoteratozoospermic 227 Asthenozoospermia 210, 219, 227, 510 ATP 376 Atresia 112, 134, 374 Atretic follicles 146 Atrophy 339 Audiometry 307 Audit 284 Autoantibodies 299, 416, 418 Autocrine 10, 77, 121, 395, 403 Autocrine, paracrine, juxtacrine 402 Auto-grafting 254 Autografts 323 Autoimmune 418, 419

Index

909

Autoimmune antibodies 294 Autoimmune disease 320, 409, 418 Autoimmune disorder 328, 353 Autoimmune phenomenon 416 Autoimmune rejection 419 Autoimmune thyroid disorders 294 Autoimmunity 299 Autosomal dominant 156, 308, 309, 353 Autosomal dominant inheritance 157 Autosomal gene 308, 310 Autosomal recessive 308 Autosomal recessive 308, 311, 353 Autosomes 307 Autotransplantation 324, 322, 323 Average path velocity 223, 233 AZF (azoospermia factor) 272 AZF deletions 513 AZF region 513 Azoospermia 15, 17, 22, 23, 208, 211, 272, 309–311, 325, 437, 503, 504, 513, 514, 547, 560, 325 Azoospermia factor (AZF) 310, 513 Azoospermic 310, 512 Azoospermic men 512 Azotemia 127 B Bacteria 208, 487 Bacterial culture 208 Balanced translocations 415 Baldness 157 Basal body temperature 23, 31 Basal FSH 86 Basement membrane 384 Batch to batch consistency 94, 290, 539 Belaisch-Allart Bi-set 175 Benign 32, 461 Benign diseases 328 Beta-2-glycoprotein I 419 B-hCG 148 Bicornuate uterus, transvaginal ultrasound 416 Biguanides 149, 150 Bilateral oophorectomy 353 Bilateral salpingo-oophorectomy 49 Bioassay 250 Bioassay system 249 Biochemical 112 Biochemical analysis 215 Biochemical assays 270 Biochemical markers 298 Biochemistry 333 Biological parents 551 Biomarkers of endometrial receptivity 409

Index

910

Biopsy 195, 334, 375, 445, 471, 504, 510, 529, 533 Biosynthetic 94 Biotechnology 539 Bi-pronuclear embryos 236 Birth control pills 56, 77 Birthdefects 514 Birth rate 242, 317 B-lactoglobulin gene promoter 375 Bladder 485, 488, 490 Bladder mucosa 170 Bladder prior 487 Blastocoelic cavity 238 Blastocyst 53, 105, 120, 192, 198–201, 237, 239, 257, 258, 266, 381–385, 389–392, 394, 400, 402, 431, 540 Blastocyst attachment 392 Blastocyst cavity 391 Blastocyst cryopreservation 255, 257, 258 Blastocyst culture 198, 200, 241–243, 405, 567, 570, 571 Blastocyst development 238, 541, 571 Blastocyst expansion, inner cell mass 290 Blastocyst formation 241, 242, 570 Blastocyst formation rate 242 Blastocyst freeze/thaw media 259 Blastocyst hatching 192, 409 Blastocyst implantation 383, 401, 487 Blastocyst injection 364 Blastocyst morphology 199, 238 Blastocyst plays 385 Blastocyst rate 236 Blastocyst stage 105, 113, 178, 192, 193, 198–201, 241, 242, 254, 255, 265, 290, 383, 396, 570 Blastocyst stage transfer 262 Blastocyst survival 431 Blastocyst thawing procedure 259 Blastocyst tranfer 53, 121, 198–201, 238, 241, 243, 254–258, 264, 390, 394, 532, 533, 541, 567, 570, 571 Blastocysts cryopreservation 256 Blastocysts for 256 Blastocysts stage 264, 290 Blastocyst-stage embryo 235 Blastomere 189, 193, 195, 237, 239, 242, 298, 334, 335, 401, 431, 528, 529, 568 Blastomere biopsy 192, 194, 195, 528, 533 Blastomere cleavage 237 Blastomere nucleus 530 Blastomere number 529 Blastomere regularity 255 Blastomere size 238 Blastulation rate 236 Bleeding irregularities 63 Bleomycin 320, 321 Blocked tubes 5 Blood cells 375 Blood group 353

Index Blood plasma viral load 341 Blood vessel 364, 391, 508 Blood viscosity 127 Body mass index 21 Bone damage 5 Bone loss 147 Bone marrow 364, 411 Bone marrow transplantation 324, 326, 329 Bone metaplasia 457 Bone morphogenetic protein 4 369 Bovine 254 Bovine embryos 11 Bovine oocytes 257 Bovine serum albumin 214 Bowel 170 Bowel adhesions 445 Bradycardia 169 Bradypnea 169 Brainstem stroke 169 Breaching 389 Breast cancer 29, 30, 47, 155, 320, 321, 323, 328 Breast carcinoma 30, 322 Breastfeeding 339 Broken spinal cord 5 Buserelin 7, 51, 68, 81, 82, 85, 88, 93, 107, 108 Buserelin acetate 39 Buserelin down-regulation 106 Buserelin, nafarelin, triptorelin and leuprolide 73 Bybritest 250 C 2-cell two-gonadotropin concept 104 Cadherin 382 Calcitonin 381, 383, 390, 392 Calcium and magnesium 314 Calcium and magnesium free medium 529 Calcium dependent 382 Calcium homeostasis 392 Calcium-dependent 66 Calcitonin mRNA 383 Calpain-10 gene 158 cAMP response element binding protein (CREB) 47 Campomelic dysplasia 309 Cancer 320, 323, 333, 359, 461 Cancer cells 323 Cancer therapy 254 Cancer, arthritis, connective tissue 269 Candida albicans 171 Cannula 175 Cannulation for 449 Canulation 449

911

Index

912

Capacitation 209, 210, 267 Capillary permeability 127, 129 Carbohydrates 267 Cardiac activity 498 Cardiac muscle 364 Cardiomyocyte 369 Cardiovascular disease 159, 548 Cardiovascular disorders 5 Carrier 353 Carrier status 354 Catabolism 124 Catalase 247, 269 Catenin 382 Catheter 176, 181, 449, 485–487, 489, 563 Catheter tip 487 Cation in 254 CC resistant anovulation 31 CC therapy 31, 134, 136 CCCT 39 CD-1 mice 251 CD4 cell counts 340 CD4 counts 341 CD4:CD8 ratio 411 Cefazolin 171 Cell adhesion molecules 383 Cell culture 152 Cell division 237 Cell lines 250, 364 Cell proliferation 392 Cell sorter 315 Cell sorting 314, 315 Cell surface receptors 402 Cell survival 267 Cell type 368 Cell-cell junctions 238 Cell-to-cell adhesion 382 Cell-to-matrix interactions 382 Cellular 381 Cellular biology 333 Cellular dysfunction 304, 376, 531 Central nervous system 123 Centrally located cytoplasmic granulation (CLCG) 236 Centrally located granular oocytes (CLGO) 527, 529 Centrifugation 214 Centrioles 372 Centromere 270, 307 Centrosomal material 538 Centrosome 372 Cerebral hemorrhage 423 Cerebrovascular accidents 423 Cervical canal 186, 563 Cervical canal diameter 496

Index

913

Cervical canal length 494 Cervical conization 564 Cervical epithelium 493 Cervical glandular layer 494 Cervical incompetence 417 Cervical length 494 Cervical mucus 30, 48, 62, 186, 207, 209, 213, 292, 295, 465, 487, 489, 493–496, 517, 563 Cervical mucus parameters 496 Cervical mucus score 31, 494, 496 Cervical mucus volume 495 Cervical parameters 494 Cervical status 493 Cervical stenosis 564 Cervical ultrasonography 494 Cervix 23, 24, 175, 184, 209, 213, 309, 467, 476, 477, 485–487, 491, 493, 563 Cesarean section 131, 133 Cetrorelix 62, 68–70, 78, 79, 290 Cetrotide 78 Chelation transition 269 Chemical messengers 298 Chemiluminescence 274, 275 Chemiluminescence assay 274, 275 Chemokines 383, 392 Chemotherapeutic agents 321 Chemotherapeutic regimens 320 Chemotherapy 320–322, 324–328, 429, 437, 547, 548 Chemotherapy and smoking 77 Chemotherapy regimen 321 Chemotherapy-associated amenorrhea (CRA) 320 Childhood cancer 272 Chimaeras 368 Chinese hamster ovary cells 50 Chlamydia 353, 411, 537 Chlamydia infection 538 Chlamydia salpingitis 538 Cholesterol 309 Cholesterol side 157 Chondrogenesis 309 Chondroitin 421 Chorio-amnionitis 498 Chorionhysteroscope 175 Chorionic gonadotropin (hCG) 94 Chorionic villus sampling 532 Chromatid breaks 246 Chromatids 35 Chromatin 271, 540 Chromatin cross-linking 278 Chromatin packaging 271 Chromatography 111 Chromopertubation 447 Chromosoame deletions 311 Chromosomal aberration 322

Index

914

Chromosomal abnormalities 35, 376, 394, 404, 530, 570, 571 Chromosomal abnormalities (mosaicisms) 238 Chromosomal alterations 335 Chromosomal analysis 560 Chromosomal anomalies 84 Chromosomal breaks 307 Chromosomal constitution 529 Chromosomal defects 194, 530 Chromosomal disorder 307 Chromosomal fusion 205 Chromosomal length 315 Chromosomal or genetic damage 242 Chromosomal re-arrangements 271 Chromosomal segregation 235 Chromosome 7, 270, 333, 334, 368, 369, 372, 373, 512, 514, 529, 530 Chromosome 2p hybridization 308 Chromosome aberrations 530 Chromosome abnormalities 236, 353 Chromosome inversions 353 Chromosome specific DNA probes 533 Cimetidine 22 Cleavage 11, 134, 171, 264, 295, 547 Cleavage divisions 394 Cleavage rate 9, 89, 137, 262, 304, 429, 432 Cleavage stage 9, 198, 245 Cleavage stage embryos 238, 247, 254, 266, 290, 571 Cleavage stage rabbit embryos 245 Cleavage-stage embryo 237, 393 Cleaved embryos 431, 237 Climacteric 34 Clinical 112 Clinical pregnancies 52, 57, 86, 106, 107, 177, 262, 293, 355, 529, 564 Clinical pregnancy rate 38, 51, 75, 105, 115–117, 134, 166, 176, 181, 199, 263, 264, 303, 304, 355, 431, 568 Clinical touch transfers 488 Clomid 149 Clomiphene 148, 302 Clomiphene challenge test 561 Clomiphene citrate 5, 16, 18, 29, 32, 38, 47, 48, 55, 56, 59, 86, 122, 131, 134–137, 139, 147, 303– 305, 396, 398, 401, 422, 431, 443, 494, 496 Clomiphene citrate challenge test (CCCT) 24, 38, 56, 77, 302 Clomiphene citrate on peripheral tissues 48 Clomiphene citrate therapy 136 Clomiphene-resistance 8 Clomipheneresistant PCOS 145 Clomiphene-responsive PCOS 136 Clomphine citrate 230 Clone size 375 Cloned cattle 376 Cloned sheep 375 Cloning 370, 373 CO2 incubator 214

Index

915

Coagulation 127 Coagulation screen 128 Coasting 149 Co-culture 198, 242, 255, 366, 374, 408, 570 COH 38, 39, 69, 186 COH protocols 34 Cohort 198, 236 Coital frequency 21, 35 Coitus 313 Coitus interruptus 208 Colchicines 22 Collagen type IV 382 Collagenase 437 Collagenase type IV 366 Collagenases 393 Colloidal silica 216 Colloidal silica based density gradient 216 Colloids or crystalloids 128 Colony 366 Colony/stimulating factor (CSF) 390 Colony stimulating factor-1 (CSF-1) 402 Color Doppler 77, 412 Color Doppler ultrasonography 508 Colpomicrohysteroscope 23 Combined factor infertility 547 Comet assay 247 Commissioning couple 548 Compact cumulus cells 265 Compacted cumulus 263 Complex aneuploidy 529 Computer assisted semen analysis (CASA) 233 Computer assisted semen analyzer 230 Computer-aided analysis 41 Computerized axial tomography (CAT) 422 Conception 6, 12, 18, 47, 104, 182, 232, 249, 302, 304, 348, 400, 402, 448, 527, 543 Conceptus 385, 400, 404, 419 Congenital 207, 423 Congenital abnormalities 548 Congenital adrenal hyperplasia (CAH) 145, 309 Congenital anomalies 465, 549 Congenital anomaly 345 Congenital bilateral absence of the vas deferens (CBVD) 311, 512 Congenital birth defects 514 Congenital gonadotropin deficiency 113 Congenital infertility 515 Congenital malformations 123 Congenital skeletal malformation syndrome 309 Connective 364 Connective adhesions 477 Connective tissues 364 Consanguineous 308 Consanguinity 359

Index

916

Contact lasers 568 Contraceptive 4, 21 Contracting couple 346, 350 Controlled ovarian 59 Controlled ovarian hyperstimulation (COH) 18, 34, 59, 64, 110, 136, 174, 200, 231, 261, 301, 396, 518, 531 Controlled ovarian hyperstimulation (COH) or ovulation induction 355 Controlled ovarian stimulation 6, 31, 114, 168 Controlled ovarian stimulation for IVF 152 Cook Echo-tip 490 Cook soft-pass 563 Corneal opacities 32 Cornuae 474 Cornuate uterus 474 Coronary heart disease 159 Corpus lutem 15, 92, 63, 86, 92–94, 121, 304, 323, 389 Corpus luteum formation 84, 94 Corpus luteum luteinization of immature follicles 89 Cortex 310 Cortical granules 323 Corticosteroids 124 Corticovenous thrombosis 128 Cosmetic 145 Counseling 350, 546 Counsellors 547, 549 Couple counselling 551 COX enzymes 392 COX-2 expression 49 Cranio-caudal differentiation 497 Creatine kinase 268 Creatine phosphokinase 206 Creatinine 127, 128 Cristae 373 Critical OHSS 127 Crohn’s disease 22, 298 Cross-links 271 Cryo solutions 259 Cryobanking 436, 437 Cryopotectants 431 Cryopreservation 16, 69, 93, 149, 191, 199, 201, 206, 253–255, 257, 258, 322–324, 328, 329, 396, 429, 431–433, 435–437, 541, 567, 571 Cryopreservation oocytes 431 Cryopreservation protocols 253, 257 Cryopreservation techniques 438 Cryopreserved embryo replacement cycles 120 Cryopreserved embryos 51, 52, 69, 241, 547 Cryopreserved follicles 254 Cryopreserved semen 436 Cryopreserved tissue 254 Cryoprogram 256 Cryoprotectant 191, 254, 255, 324, 430, 431, 436 Cryoprotocol 257

Index

917

Cryostorage 253, 254 Cryosurvival 253, 254, 257, 429 Cryosurvival rate 255, 256 Cryotube 367 Cryptorchidism 504 Crystallin B 383 Crystallin B mRNA 121 Cul-de-sac 139, 186, 467 Culdocentesis 165 Culture 53, 238 Culture environment 249 Culture media 243 Culture media (ES) 175, 247, 250, 251, 255, 367 Cultures 408 Cumulative dose 322 Cumulative pregnancy rate 200, 230, 231, 355, 446 Cumulus 199, 263, 265 Cumulus cell 262, 263, 265 Cumulus oocyte complexes (COC) 9, 11, 69, 134, 265 Curvilinear velocity 223, 233 Cycle fecundity 6, 186, 231, 232 Cyclic AMP 47 Cyclooxygenase 124, 421 Cyclo-oxygenase type 2 (COX-2) 49 Cyclophosphamide 321, 322, 324, 325, 328 CYP19 gene 47 Cyproterone acetate 63, 147 Cystrupture 128 Cystwall 445 Cystadenoma 462 Cystectomy 460 Cysteine protease calpain-10 158 Cystic degeneration 130 Cystic fibrosis 311, 334, 353, 354, 372 Cystic fibrosis carrier 354 Cystic microvilli 381 Cystic tumours 63 Cystoscopy 171 Cytochromic 450 309 Cytokine 402 Cytokine mRNA 401 Cytokines 8, 129, 180, 298, 381, 382, 389, 391, 400–402, 404, 405, 409, 411 Cytokines (endothelin-1, IL-1, IL-6, IL-8, TNF-α, ICAM-1, VEGF) 127 Cytokines growth factors 405 Cytomegalovirus 353 Cytoplasm 208, 236, 375, 431, 530, 538, 570 Cytoplasmic 527 Cytoplasmic “pitting” 237 Cytoplasmic abnormalities 372 Cytoplasmic activation 530 Cytoplasmic defect 531 Cytoplasmic degeneration 245

Index Cytoplasmic droplet 268 Cytoplasmic factors 152, 372 Cytoplasmic fragments 237 Cytoplasmic granulation 527 Cytoplasmic maturation 10, 77, 108, 153, 261, 373 Cytoplasmic microfilaments 429 Cytoplasmic pitting 238 Cytoplasmic transfer 372, 374–376 Cytoplasts 375 Cytosine 333 Cytoskeleton 382, 383, 431 Cytotoxic 208, 320, 322 Cytotoxic cells 294 Cytotoxic markers 404 Cytotoxic T lymphocytes 384 Cytotrophoblast 391, 402, 403, 421 D Dacarbazine 321 Danazol 48 Daughters 317 DAZ (deleted in azoospermia) 310 DCK (Chromosome 18) 334 Death 93 Decapeptyl (D-triptorelin) 88 Decarbazine 320 Decidual stroma 405 Decidualization 392, 409 Decision making counselling 550 Decondensation 530 Defective sperm function 278 Deficiency Hidroaxiacil-CoA desH 334 Dehydroepiandrosterone sulfate 17 Deleted in azoospermia (DAZ) 310 Deletion 272, 336, 353 Deletions in AZFa 272 Deletions, frame shifts 271 Delivery 116, 349 Delivery rate 36, 176, 305, 355 Density gradient centrifugation 214, 215, 217, 218 Denys-Drash syndrome 310 Deoxyribonucleic acid (DNA) 267 Deoxyribonucleotides 333 Depot 58, 73 Depot GnRH 84 Depot leuprorelin 83 Desensitisation 40, 66, 67, 110 Desogestrel 64 Desquamation 120 Developing countries 338 Developing follicles 6

918

Index

919

Developmental 431 Developmental competence 265, 266 Developmental potential 238 Developmental rates 246 Developmental stages 9 DHEA-S 148 DHEAS 410 Diabetes 5, 21, 145, 148, 150, 156, 158, 159, 373, 409, 410 Diabetes mellitus 415, 353 Diagnostic laparoscopy 91 Dialysis 128 Diaphanous (DIA) 308 Diarrhoea 127 Diathermy 443 Diazoxide, metformin 156 Diethylstilbestrol (DES) 29 Diethylstilboestrol 32 Differentiated cells 368, 365 Differentiation factor 369 5α-dihydrotestosterone 309 Dilatation 467 Dimeric inhibin 11, 37, 327 Dimers 37 Dimethyl sulfoxide (DMSO) 429, 430, 436 Diode 195 Diploid 236 Diploidy 570 Direct intraperitoneal insemination (DIPI) 186 Discontinuous Percoll gradient 314 Dispersed CC 265 Distal long arm deletions 307 Disulfude bonds 538 Diuretics 128 DMSO 4, 255, 430 DNA 190, 246, 247, 270, 271, 333, 334, 513 DNA aneuploidies 245 DNA content 218, 219, 316, 436 DNA damage 218, 219, 246, 247, 269, 271, 278 DNA double helix 270 DNA fragmentation 35, 251, 272 DNA fragmentation rate 272 DNA hybridization (Southern) 334 DNA in 334 DNA loop 270 DNA manipulation 333 DNA packaging 270 DNA probe 316, 317, 532 DNA repair 530 DNA sequence 335 DNA sperm nucleus DNA 267 DNA stability 538 DNA strand breaks 278

Index

920

Dominant 309 Dominant follicle 7, 10, 12, 37, 38, 152, 265, 398 Donadotropins 266 Donor 352, 375, 547 Donor insemination 35, 340, 435, 546 Donor insemination anastomosis 507 Donor oocyte 253, 354, 372, 375, 396, 557 Donor oocyte cycles 35 Donor ooplasm 352 Donor semen 18, 253, 435 Donor sperm 372 Donor spermatozoa 177, 436 Donor, surrogate 548 Donors 191, 224, 276, 354, 359, 360, 551 Doppler 236 Doppler studies 409 Doppler ultrasonography 22 Dose of gonadotrophin 77 Dose-dependent 8, 9 Dose-intensity 322 Double helix 334 Double-lumen aspiration needle 169 Down regulation 7, 51, 55, 57, 62, 63, 75, 82, 85, 86, 92, 110, 115, 134, 158, 168, 182, 289, 355 Down’s syndrome 353, 374 Down regulation of receptors 78 Down-regulation protocol 117 Doxorubicin 321 Doxycycline 456 Drosophilia gene 308 Drug development 537 Dual pressure foot operated suction unit 167 Ductule 508 Dulbecco’s modified Eagl’s medium (DMEM) 365 Dulbecco’s phosphate buffered saline solution 214 Dulbecco’s phosphate buffered sodium 563 Dulbecco’s minimum essential medium 366 Duschenne’s muscular dystrophy 313 dUTP-fluorescein 335 dUTP-rodamine 335 Dysfunction 311 Dysfunctional bleeding 63 Dysmenorrhoea 63 Dyspareunia 21, 549 Dyspepsia, polyuria, fatigue 147 Dyspnoea 127, 128 Dysregulation 405 E E2 biosynthesis 49 E concentration 40, 67 E2 elevations 39

Index E2 levels 38, 67, 68 E2 values 42 balanced salt solution 242 Early abortion 110 Early cleavage stage 255 Early embryo 199, 394 Early embryonic loss 91 Early follicular phase 79, 82, 89, 155 Early gestation 382 Early luteal phase 298 Early luteinization 93, 95 Early miscarriage 84 Early pregnancy 84, 92, 106, 121, 498 Early pregnancy loss 85, 106, 116, 134, 403, 412 Early pregnancy wastage 290 Early stage embryo 255 Echodense 489 Echogenic needle tip 167 Echotip 489 Echotip catheter 490 Ectoderm 364 Ectopic endometrial tissue 48 Ectopic endometrium 63, 299 Ectopic pregnancy 176, 177, 241 Ectopic pregnancy rate 355 Ectopic tubal pregnancy 181 Ediology of 30 Eflornithine 147 Eflornithine hydrocloride 147 EGF receptor 265, 392 Egg 137, 191, 206, 253, 289, 302, 358, 570 Egg cryopreservation 258 Egg donation 357 Egg donation protocols 431 Egg donor 358, 548 Egg freezing 253 Egg recipients 358 Egg retrieval 322 Egg sharing 357, 361 Egg-sharing and surrogacy 358 Egg sharing program 360 Eight cell stage 242 Ejaculate 206, 208, 213, 218, 310, 334, 504 Ejaculate quality 522 Ejaculation 184, 207, 213 Ejaculatory duct 207 Ejaculatory duct obstruction 207 Ejaculatory dysfunction 510 Electrocauterization 443 Electrocautery 132, 444, 445 Electrofusion 375

921

Index

922

Electrolytes 128 Electrons 267 Electrophoresis 313, 314, 334 ELISA 411, 421 Embolism 422 Embryo 3, 4, 8, 105, 121, 124, 201, 235, 239, 241, 243, 282, 291, 304, 334, 354, 372, 373, 383– 385, 388, 389, 391, 394, 398, 400, 401, 403, 404, 408, 410, 420, 454, 487, 532, 540, 541, 567, 568 Embryo assessment 235 Embryo bioassay 249 Embryo bubble 489 Embryo cleavage 31, 178, 291, 437 Embryo cleavage rate 87, 90, 113, 375 Embryo cryopreservation 253, 254, 256, 328, 396, 429–431 Embryo cryosurvival rates 85, 113 Embryo culture 9, 182, 198, 238, 294, 393, 394, 571 Embryo culture media 251 Embryo death 272 Embryo development 9, 105, 116, 152, 177, 235, 236, 238, 247, 251, 272, 324, 400, 530 Embryo development potential 152 Embryo donation 547 Embryo donation surrogacy 546 Embryo freezing 253, 323, 329, 542 Embryo hatching 291 Embryo implantation 120, 122, 283, 289, 291, 361, 388, 400, 419 Embryo implantation and pregnancy rates 57 Embryo manipulation 340 Embryo maternal cross talk 409 Embryo maturation 51 Embryo morphology 9, 199, 235, 236 Embryo nidation 124 Embryo of 393 Embryo quality 7, 8, 59, 95, 105, 133, 159, 257, 262, 289, 295, 298, 299, 303, 375, 388, 393, 527, 530 Embryo quality uterine receptivity 405 Embryo secretes 235, 540 Embryo stem cell 367 Embryo storage 429 Embryo survival 431 Embryo transfer (ET) 3, 4, 51, 95, 117, 120, 121, 122, 124, 133, 180, 198–200, 239, 257, 261, 262, 289, 290, 291, 292, 302, 304, 315, 328, 352, 354, 355, 361, 385, 389, 390, 394, 397, 398, 400, 408, 405, 420, 485, 529, 540, 563, 562, 564, 568, 571 Embryo transfer in vitro fertilization (IVF) 241 Embryo transfer techniques 491 Embryo-endometrial 89 Embryogenesis 113, 310 Embryoid bodies (EBs) 364, 366, 367, 368 Embryology 6 Embryonal carcinoma 364, 365 Embryonic 120 Embryonic arrest 514 Embryonic capacity 105 Embryonic carcinoma (EC) 369

Index

923

Embryonic cells 364 Embryonic development 9, 35, 246, 541 Embryonic expression 402 Embryonic fibroblast 366 Embryonic genome 241, 242, 394, 531 Embryonic germ cells 364, 366 Embryonic germ layers 367 Embryonic germ layers—mesoderm 364 Embryonic growth 193 Embryonic heart 498 Embryonic implantation 383, 401, 402 Embryonic mesoderm 391 Embryonic metabolism 247 Embryonic mRNA 541 Embryonic stem (ES) 369 Embryonic stem cells 5, 364, 402 Embryonic viability 290 Embryonic-maternal dialogue 9 Embryos 4, 40, 51, 53, 55, 57, 69, 83, 84, 86, 87, 89, 93, 106, 110, 113, 114, 137, 149, 152, 166, 176, 177, 189, 192, 194, 198–201, 235–238, 241, 242, 245–248, 251, 253–257, 262–264, 289–291, 303, 329, 334, 335, 340, 353, 355, 374, 381, 396, 402, 408, 410, 429–433, 456, 486, 489, 491, 498, 527, 530, 533, 534, 547, 562–564, 567–570 Embryos cryopreservation 256 Embryos and pregnancy 409 Embryos development 247 Embryos group 193 Embryos loss 291 Embryos/fetuses 498 Embryo-toxic 250 Embryotoxicity 294 Embryotropic factors 9 Empty follicle syndrome 168 Emtryo transfer 51 Endocervical canal 23, 390, 563 Endocrine 84, 130 Endocrine tissue 121 Endocrine, paracrine 77 Endocrinology 6 Endocytosis 401 Endoderm 364, 367, 369, 370 Endogenous FSH 86, 93, 113 Endogenous GnRH 66 Endogenous gonadotrophin 68, 78, 82, 104, 289 Endogenous hormones 354 Endogenous LH 6, 51, 90, 107 Endogenous LH concentrations 112 Endogenous LH exists 57 Endogenous LH surge 83, 92, 94, 95, 165 Endogenous luteinizing hormone 91 Endogenous nuclease 271 Endogenous oestrogen 30 Endogenous pituitary gonadotropin 87

Index

924

Endogenous preovulatory LH surge 82, 83 Endogenous serum LH 69 Endometrial 120, 396 Endometrial atrophy 63 Endometrial biopsies (EMB) 301 Endometrial biopsy 121, 135, 223, 384, 385, 411, 466 Endometrial cancer 63 Endometrial carcinoma 32 Endometrial cavity 394 Endometrial cell 382 Endometrial development 48, 114 Endometrial epithelial cells 401 Endometrial epithelium 391, 400, Endometrial estrogen 122 Endometrial glands 383 Endometrial growth 57, 63, 112 Endometrial hyperplasia 32, 63, 453, 456 Endometrial implants 445 Endometrial integrins 540 Endometrial maturation 63, 121, 124, 383, 405 Endometrial myometrial 87 Endometrial polyps 454, 456, 457 Endometrial preparation 9 Endometrial receptivity 9, 121, 289, 381, 383, 385, 388, 395, 405, 409, 410, 412, 431 Endometrial sampling 408 Endometrial stroma 389 Endometrial stromal cells 298, 403 Endometrial thickness 63, 85, 113, 182, 355, 361, 384–386, 397, 398, 570 Endometrial thickness, follicular development 397 Endometrial tissue 120, 121, 383, 384, 476 Endometrial volumes 41 Endometrial-embryonic 87 Endometric implants 49 Endometriomas 445 Endometriosis 15, 17, 47–49, 63, 90, 174, 177, 185, 186, 297, 298, 308, 383, 384, 401, 404, 409, 412, 443–446, 467, 470, 444 Endometriotic 48 Endometriotic deposits 49 Endometriotic growth 63 Endometriotic lesions 445 Endometriotic tissue 48 Endometritis 21, 454, 474, 476 Endometrium 8, 9, 23, 24, 31, 32, 48, 62, 64, 69, 108, 120–123, 177, 199, 262, 290, 291, 297, 354, 355, 381–386, 389–391, 393–395, 397, 400–404, 408–411, 432, 454, 458, 474, 475, 488, 540, 563, 564 Endometrium maturation 122 Endometrium thickness 48, 261 Endosalpingeal 21, 87 Endoscopic surgery 443 Endothelial cells 409 Endothelium 390

Index

925

Endotoxin 219, 250, 251, 410 Endouterine abnormalities 453 Endouterine adhesions 454 EnhanceS plus 216 Enhance S+® 218, 220 Entactin 382 Enucleated oocyte 375 Environmental and genetic 77 Environmental factors 206, 309 Enzyme glucose-6-phosphate-dehydrogenase 268 Enzymes 267, 269, 294, 391 Epidemiology 222 Epidermal growth factor (EGF) 152, 390, 392, 402 Epidermal growth factor (HB-EGF) 402 Epidermolysis bullosa 353 Epididymal 437 Epididymal ductule 507 Epididymal fluid 508 Epididymal maturation 538 Epididymal sperm 191 Epididymal sperm retrieval 510 Epididymal spermatozoa 435 Epididymis 211, 274, 309, 311, 507, 508, 523 Epididymitis 22, 268 Epidural anesthesia 166 Epithelial 390 Epithelial barrier 384 Epithelial cell 124, 383–385, 207, 208, 383, 384, 392, 401 Epithelial ovarian cancers 133 Epithelial ovarian carcinoma 361 Epithelium 121, 383, 401, 464 Erbium: YAG 192 Ericsson’s method 314 Erythema 66 Erythrocytes 207, 208, 214 Estradiol 56, 113–115, 145, 155, 235, 403 Estradiol (2) 384 Estradiol (E2) 24, 34, 91 Estradiol concentrations, inhibin concentrations 85 Estradiol levels 87, 155, 494, 561 Estradiol synthesis 85 Estradiol valerate 361, 397, 398 Estradiol: androgens ratio 112 Estrogen 47, 77, 113, 131, 148, 328, 384, 389, 392, 397, 398, 411, 431 Estrogen levels 83 Estrogen metabolism 120 Estrogen receptor gene (ERb) 112 Estrogen receptors (ER) 30, 48, 120 Estrogen receptors (ER)-ERa ERb 30 Estrogen response 39

Index

926

Estrogen stimulation 385 Estrogen synthesis 112 Estrogene 155 Estrogenic 30 Estrogenic effect 31 Estrogen-progestin 146 Estrogens 62, 93, 120, 381 Estrogen-sensitive tumors 322 Estrone 155 ET catheter 563 Ethidium bromide 514 Ethylenediaminetetraacetic acid-EDTA 247 Ethyleneglycol 430 Ethynyl estradiol (EE) 62 Etiology 205 Eukaryotic cell 373 Euploid chromosome 368 Euvolemia 128 Evidence based reproductive medicine 187 Exocelom 391 Exogenous follicle stimulating hormone ovarian reserve test 56 Exogenous follicular stimulation 84 Exogenous FSH 139 Exogenous FSH ovarian reserve test (EFORT) 39 Exogenous gonadotrophin(s) 42, 50, 67, 81, 82, 84, 86, 104, 108, 110, 114, 148, 253 Exogenous gonadotropin stimulation 88, 323 Exogenous hCG 93, 94, 108 Exogenous hormones 354 Exogenous human chorionic gonadotropin (hCG) 81 Exogenous LH 74, 105, 106, 115, 117 Exogenous peptides 10 Exogenous progesterone 120 Exogenous rLH 57 Exogenous testosterone 521 Exon 7, 308 Expanded blastocysts 255 Extended culture 255, 256, 571 External genitalia 307 External os 184 Extra embryos 396 Extra ovarian sources 47 Extracellular 540 Extracellular domain 401 Extracellular matrix (ECM) 298, 366, 369, 382, 393, 400–403 Extracellular matrix ligands 401 Extracellular space 392 Extragonadal 10 Extragonadal functions 112 Extrahypothalamic GnRH 9 Extrapituitary 69, 87 Extrapituitary reproductive cells 8 Extravasation 127, 128

Index

927

Ezrin 392 Ezrin/radixin/moesin (ERM) 383 F Factor nerve growth factor receptor family 271 Failed fertilization 546 Failed fertilization in IVF cycles 113 Fallopian sperm perfusion 187 Fallopian tube (FT) 8, 9, 175, 178, 180, 186, 241, 381, 389, 390, 393, 448, 450, 489 Fallopian tube sperm perfusion (FSP) 184, 185 Fallopian tubes 23, 174, 187, 309, 360 Falloposcope 23 Falloposcopy 23 Familial aggregation 308 Familial alopecia 353 Familial premature ovarian failure (POF) 308 Family planning 314 Fanconi’s anemia 334 Fas antibody 271 FAS-L 271 Fas-ligand (Fas-L) 271 Fas-mediated apoptosis 271 FAST system 184 Fasting glc/insulin ratio 148 Fasting glucose 148 Fasting insulin 148 Fasting serum glucose 150 Fecundity 35, 184, 230, 297 Federal council of the Fertility Society of Australia (FSA) 558 Feedback mechanisms 84 Female fecundity 301 Female gamete 254 Female reproductive system 35, 206, 213, 383 Female reproductive tract 205, 207, 209 Fentanyl 166 Fertilisation 68, 299, 355 Fertilisation rate 69, 298, 400 Fertility 16, 21, 110, 134, 153, 189, 205, 208, 210, 223, 231, 254, 304, 305, 327, 328, 329, 338, 353, 354, 358, 359, 437, 443, 444, 449, 474 Fertility potential 278, 328 Fertility rates 35, 301 Fertility status 139 Fertilization 9–11, 57, 59, 79, 83, 85, 87–89, 94, 104, 114, 116, 120, 134, 137, 152, 153, 166, 171, 174, 177, 178, 180, 184, 186, 187, 189, 192, 194, 195, 205, 209, 211, 236, 238, 245, 253, 258, 262, 264, 272, 289, 291, 295, 311, 313, 322, 324, 361, 381, 429, 433, 454, 464, 493, 513, 527, 530, 538, 540, 541, 547, 567, 569, Fertilization capacity 208, 435 Fertilization failure 116, 262, 353, 353, 569, 517 Fertilization in 517 Fertilization oocytes 437 Fertilization potential 189, 191, 205–211, 213

Index

928

Fertilization rate 8, 35, 36, 57, 84–87, 105, 106, 113, 114, 116, 117, 137, 153, 158, 182, 192, 199, 236, 290, 295, 375, 429, 437, 522, 527, 569 Fertilization rate less 272 Fertilization site 207, 213 Fertilization, embryo development 261 Fertilization, embryo development, pregnancy 249 Fertilization-embryo transfer (IVF-ET) 396 Fertilizations failure 291 Fertilize 110 Fertilized egg 3, 113, 560 Fertilized oocytes 116 Fertilizing capacity 523 Fertilizing potential 214 Fertiloscope 467 Fertiloscopy 464–466, 470, 471, 472 Fetal calf serum (FCS) 365 Fetal cord serum 250 Fetal heart 124, 529 Fetal loss (s) 498, 420 Fetal-placental semi allograft 418 Feto-maternal exchange 421 Fetus 349, 497 Fibrinolytic 127 Fibroblast 47, 400 Fibroblast feeder layers 366 Fibroblast growth factor 365 Fibroblasts 375 Fibroid (s) 24, 388, 448, 456, 474, 487 Fibroids on 457 Fibronectin 382, 403 Fibronectin collagen and lamini 401 Fibronectin mRNA expression 403 Fibrorectin 403 Fibrous tissue 447 Fimbriae 447 Fimbrial phimosis 447 Fimbrioplasty 447 Finasteride 147 Fine needle aspiration 504 Fiollicular development 57 First degree relatives 309 First trimester 121, 412 First trimester habitual abortions 124 FISH (fluorescent in situ hybridization) technology 194, 236, 237, 242, 291, 316, 335, 529, 532, 570 FISH analysis 528 Fistula 171 Flagellum 191, 436, 538 Flare-up effect 63 Flare-up protocol 56, 83, 86 Flow cytometer 315 Flow cytometric analysis 315, 317

Index

929

Flow cytometry 315 Fluorescein dye 460 Fluorescein isothiocyanate (FITC) dextran 193 Fluorescence 316 Fluorescence-activated cell sorting (FACS) 368 Fluorescent light 245 Fluorescent probes 335 Fluorochrome bisbenzimide 315 Fluorochromes 335 Fluoroquinolone prophylaxis 171 Fluorouracil 321 Flutamide 147 Focal lesions (polyps, myomas, sinaechiae) 456 Foetal development 105 Foetal gonad 365 Foley’s catheter 477 Folic acid 132 Follicular stimulation 85 Follicle (theca cell, granulosa cell, cumulus cell, oocyte) 10 Follicle aspiration 74, 323 Follicle counts 302 Follicle development 104, 122, 261 Follicle growth 74, 75 Follicle loss 325 Follicle luteinization 110, 114 Follicle maturation 11, 12, 63, 323 Follicle numbers 83 Follicle puncture 75 Follicle recruitment 301 Follicle size 265 Follicle stimulating hormone (FSH) 50, 77, 91, 131, 152, 302, 504 Follicle storage 254 Follicle ultrasound 561 Follicle yield 82 Follicles 8, 23, 37, 38, 55, 68, 77, 86, 89, 112, 116, 124, 139, 152, 165, 166, 170, 186, 235, 236, 254, 262, 303, 307, 321, 323, 376, 494, 538 Follicles on ultrasound 74 Follicles undergo atresia 155 Follicles/oocytes 81 Follicular 94, 327 Follicular growth 62 Follicular and luteal phase regimen 289 Follicular aspiration 4, 11, 86, 168, 169, 174, 329 Follicular atresia 83, 112, 155 Follicular blood flow 41 Follicular cycle 122 Follicular cysts 355, 444 Follicular density 41 Follicular depletion 35, 325 Follicular development 6, 8, 40, 48, 51, 81, 94, 105, 107, 108, 112, 113, 146, 323, 373, 398, 461, 538, 539 Follicular diameter 78

Index

930

Follicular fluid 10, 11, 37, 89, 92, 166, 186, 298, 339 Follicular FSH 36 Follicular function 10, 88 Follicular growth 29, 30, 32, 48, 56, 62, 64, 66, 86, 89, 107, 155, 168, 298, 323, 389, 496 Follicular hypoxia 235 Follicular level 398 Follicular LH 115 Follicular luteinization 81, 82 Follicular maturation 6, 10, 62, 64, 67, 82, 94, 95, 115, 137, 139, 321 Follicular maturity 57 Follicular phase 12, 30, 37, 40, 41, 56, 78, 82, 83, 85–87, 104–107, 110, 114, 116, 122, 134, 289, 301, 562 Follicular phase LH 7 Follicular phase of ovarian stimulation 68 Follicular phase serum androgen 83 Follicular phase serum LH 106 Follicular recruitment 10, 55, 149 Follicular rupture 91, 92, 95 Follicular size 59, 165, 239, 398, 431 Follicular steroid production 6 Follicular stimulation 57, 84, 85 Follicular stimulation protocols 57 Follicular synchronization 90 Follicular wall 92 Follicular-luteal 92 Folliculogenesis 10, 12, 30, 42, 84, 85, 87, 89, 104, 113, 114, 152, 168, 265, 289, 297–298, 493 Folliculogenesis-follistatin gene 158 Follistatin 10, 11 Follistatin gene 158 Follistatin genotype 158 Follitropin alpha 50, 112 Follitropin beta 50 Follitropin Gonal-F, follitropin, puregon 290 For HIV, Hepatitis 547 Forskolin 366 Four cell stage embryos 193 Fragile X syndrome 313, 352, 353, 538 Fragmentation 200, 237, 238 Frasier syndrome 310 Free IFG-1 156 Free oxygen radicals 208 Free radical scavengers 247 Free radicals 267 Free testosterone 63, 146, 159 Free testosterone levels 146 Freeze/thaw procedure 257 Freeze-thaw cycles 69 Freezing 199 Freezing protocols 256 Freezing thawing of mature oocytes 253 Frozen blastocysts 200 Frozen embryo cycles 51

Index

931

Frozen embryo transfers 355 Frozen embryos 329 Frozen ET cycles 70 Frozen semen 436 Frozen-thawed blastocysts 200 Frozen-thawed embryo 48 Frozen-thawed embryo transfer 398 Frozen-thawed semen 219 Fructose 211, 504 Frydman 175 Frydman catheter 184, 486, 563 FSH 8, 10, 12, 17, 24, 30, 35, 38, 56, 62, 63, 66, 73, 85, 104, 111, 115, 137, 182, 290, 301, 327, 328, 521, 561 FSH agonist 539 FSH coding sequence 539 FSH concentration 36, 39, 327 FSH dose 8 FSH HP (high purity) 55 FSH levels 36, 39, 56, 302 FSH or hMG 108 FSH preparations 105, 110 FSH receptors (FSH-r) 11, 12, 265, 308 FSH secretion 37 FSH stimulation 64, 116 FSH surges 69 FSH therapy 149 FSH threshold values 305 FSH values 36, 37, 302 FSH, LH 42, 83 FSH/E2 testing 305 FSH: LH ratio 38, 55, 56, 74 FSH-r gene 308 FSH-r mutation 308 FSP 186 Functional cyst 63 Fundus 291, 473, 485, 486 G GABA-receptors 123 Galactorrhea 21 Galactosemia 352 Gamete 205, 210, 393 Gamete fusion 295 Gamete intrafallopian transfer (GIFT) 15, 18, 79, 165, 166, 180, 282 Gamete manipulations 190, 195 Gamete micromanipulations 189 Gametes 175, 177, 189, 248, 338–340, 400, 431, 527, 538, 542 Gametes transfer 175 Gamete intrafallopian transfer (GIFT) 174 Gamma/delta cells Ganirelix 62, 68, 70, 79, 84, 106, 114, 115, 290

Index

932

Ganirelix acetate 64 Ganirelix and cetrorelix 67 Gastric ulcers and trauma 423 Gastrointestinal organs (liver pancreas) 364 Gaucher’s disease 334 G-banded metaphase 370 G-banding 369 G-banding analysis 368, 369 Gelatinases 393 Gender 313, 317 Gender selection 317 Gene 157, 309, 310, 334, 369 Gene deletions 538 Gene expression 121, 324, 336, 404 Gene family 310 Gene networks 381 Gene pool 284 Gene regulation 309 Gene therapy 375 General anaesthesia 175, 178, 181, 477, 498 Generation 308 Genes 50, 157, 158, 270, 307, 310, 333, 334, 334, 381, 391 Genes distributed 336 Genes on 310 Genes predominantly 345 Genetic 548 Genetic abnormalities 237, 353, 504 Genetic chromosomal damage 241 Genetic disease 336, 353 Genetic disorders 307, 355, 357 Genetic makeup 538 Genetic markers 335, 336 Genetically engineered 111 Genetically engineered recombinant products 111 Genetics 333 Genital ridge 309 Genital tract 87, 210, 314, 470 Genital tuberculosis 21 Genitourinary skeletal (osteoporosis) 320 Genome 333 Genotype 283, 368, 513, 515 Germ cell 308, 546 Germ cell apoptosis 278 Germ cell arrest 272 Germ cell loss 339 Germ cell membranes 274 Germ cell nuclear factor 369 Germ cells 309, 310, 364, 368, 374 Germline 376 Germ-cell tumors 325 Germ-cells specific RBMY (RNA-binding motif, Y chromosome) 310 Germinal epithelium 268

Index

933

Germinal epithelium 322 Germinal failure 504 Germinal vesicle (GV) 10, 152, 253, 352 Germinal vesicle transfer 352 Germinal vesicle breakdown 92 Germ-line 366 Gestation 35, 124, 345, 404, 497 Gestational age 498 Gestational carrier 345 Gestational sac 75, 185 Gestational surrogacy 347, 548 GIFT 9, 176, 178 Glandular epithelium 121, 291 Glandular layer thickness 494 Glans penis 208 Glass column filtration procedure 216 Glass wool 219, 220 Globozoospermia 530 Glucocorticosteroids/hMG 12 Glucose 394 Glucose challenge test 150 Glucose metabolism 307 Glucose tolerance 150, 159 Glucose tolerance test 159, 416 Glucose transporters 149 Glutamate 394 Glutamine 366, 394 Glutathione 269, 270 Glycerol 255, 256, 258, 259, 429, 430, 436 Glycine 394 Glycocalyx 382, 401 Glycodelin 403 Glycolipids 365 Glycoprotein(s) 194, 365, 382, 392, 394, 401–402, 405, 419 Glycosylated mucin 401 GnRH 7, 9, 66, 146, 289, 522 GnRH a dose 90 GnRH agonists 8, 58, 69, 74, 78–79, 81–85, 87, 95, 105, 110, 123, 134, 198, 289, 326, 361 GnRH agonist down-regulation 106 GnRH agonist protocol(s) 115 GnRH agonist regimens 115 GnRH agonist stimulation test 77 GnRH agonist therapy 149 GnRH agonist/HMG 124 GnRH agonists 6, 48–50, 67, 73, 83, 113, 139, 147, 148 GnRH analogs 78, 84, 88, 116, 147, 182, 540 GnRH analogue protocols 56 GnRH analogues 16, 75, 297, 354, 355 GnRH antagonist 113, 114 GnRH antagonist (s) 8, 9, 16, 57, 59, 66–69, 75, 78, 79, 84, 93, 95, 108, 110, 115, 114, 290, 540 GnRH antagonist protocol 95, 561 GnRH challenge test 95

Index

934

GnRH depot 82 GnRH dose 73, 74 GnRH protocol 68 GnRH pulsatile 155 GnRH pulse generator 146 GnRH receptor 9, 64 GnRH receptor down-regulation 67 GnRH receptor mRNA 87 GnRH receptors 8, 58, 62, 66, 69, 87, 92, 110, 290, 326, 540 GnRH structure 66 GnRH suppression 74 GnRH-a 67, 78, 82, 86–89, 92, 105, 324–328, 396, 397 GnRH-a administration 7 GnRH-a chemotherapy cotreatment 327 GnRH-a dose 89 GnRH-a down-regulation 93, 105, 106 GnRH-a flare-up protocols 86 GnRH-a long protocol 68 GnRH-a microdose protocol 89 GnRH-a protocol 83, 88, 104, 327 GnRH-a protocols with 86 GnRH-a stimulation 302 GnRH-a stimulation test 561 GnRH-a stimulation test (GAST) 39, 86 GnRH-a suppression 7 GnRH-a therapy 93 GnRH-a treatment 322 GnRH-a/chemotherapy 326 GnRH-agonist 12, 58, 93, 325 GnRH-agonistic analogue 325 GnRH-agonists 62, 63, 328 GnRH-a-induced LH surge 93 GnRH-analogue therapy 297 GnRH-antagonist(s) 62, 64, 326 GnRH-receptors 326, 325 Golactorrhea 24 Golgi apparatus 538 Gonad development 309 Gonadal 10 Gonadal atrophy, premature ovarian failure 444 Gonadal damage 321, 329 Gonadal degeneration 310 Gonadal development 310 Gonadal dysfunction 321, 522 Gonadal dysgenesis 309, 352, 357 Gonadal failure 307 Gonadal peptides 328 Gonadal steroids 83 Gonadorelin 522 Gonadotoxic chemotherapy 326, 328 Gonadotoxic effect 322 Gonadotoxicity 320

Index

935

Gonadotrophic cells 57, 66 Gonadotrophin dose 37 Gonadotrophin FSH 47 Gonadotrophin preparations 3, 48 Gonadotrophin secretion 57, 352 Gonadotrophin stimulation 57 Gonadotrophin treatment 152 Gonadotrophin-releasing hormone (GnRH) 50, 59, 73 Gonadotrophin-releasing hormone agonist (GnRHa) stimulation test 36, 56, 302 Gonadotrophin-releasing hormone GnRH 122 Gonadotrophin (s) 4, 6–8, 16, 17, 29, 36, 48, 55–57, 59, 66, 67, 78, 82, 83, 85, 86, 88, 94, 104, 108, 110, 115, 117, 138, 148, 263, 290, 297, 298, 303, 308, 324, 326, 328, 355, 396, 398, 404, 405, 443, 444, 522, 539, 540 Gonadotropic hypogonadism syndromes 85 Gonadotropin dependency 538 Gonadotropin doses 40 Gonadotropin inhibin surge-inhibiting factor 92 Gonadotropin levels 85 Gonadotropin preparation 107, 117 Gonadotropin receptors 539 Gonadotropin regimens 81 Gonadotropin releasing 302 Gonadotropin releasing hormone agonists 255 Gonadotropin releasing hormone agonists (GnRH-a) 174 Gonadotropin releasing hormone analogs (GnRHa) 91, 168 Gonadotropin stimulation 7, 40, 42, 77, 149, 323 Gonadotropin suppression 114 Gonadotropin surge-attenuating factor (GnSAF) 42 Gonadotropin therapy 16, 18, 93, 116, 136, 139, 145, 149, 168, 290, 303 Gonadotropin-releasing hormone (GnRH) 62, 66, 85, 134, 421 Gonadotropin-releasing hormone agonists (GnRH-a) 81, 104 Gonadotropin-resistant ovary 11 Gonads 310 Gonal 104 (GOnal F)-Cetrorelix 79 Good quality embryos 569 Goserelin 82 Graafian follicle 10 Graft 323, 324 Granulocytes macrophage CSF 404 Granulosa cell(s) 12, 37, 38, 47, 88, 112, 113, 145, 298, 301, 374 Granulosa cell steroidogenesis 298 Granulosa layer 83 Granulosa tumours 32 Granulosa-cell estradiol synthesis 75 Granulosa-luteal cells 86 Green fluoresent protein 368 Group counselling 551 Growth factor 8, 180, 298, 369, 381, 388, 401, 402, 538 Growth hormone 56, 86 Growth media (GM) 259 Guanine 333

Index

936

GV breakdown 253 GV stage 262 GV stage oocyte 263 GV transfer 375 GV-breakdown (GVBD) 10 GV-stage oocyte 263 Gynecomastia 22 H Habitual abortions 121 Haemophilia 353 Hair shaft diameter, hair follicle density, growth rate 146 Half-dose LA depot 83 Half-life 78, 91, 104 Ham’s F 10 250 Ham’s F-10 medium 214, 262 HAMOU hysteroscope 466 HAMOU III telescope 470 Hamster 250 Hamster embryos 247 Hamster sperm motility assay (HSMA) 250 Haploids 335, 529 Hatching process 291 Hauser syndrome 308 Have cervical stenosis 562 HbsAG 360 hCG 10, 68, 74, 117, 124 hCG administration 31, 64, 67, 68, 82, 122 168 hCG administration gonadotropin dosage 83 hCG administration 82 hCG-Primed IVM cycles 264 hCG-priming 264, 265, 266 Head defects 516 Hearin 421 Heart 364 Heat shock factors 389 Heat shock proteins 121, 381, 383 Helper cells 409 Hematocrit 128, 137 Hematospermia 207 Hemizona 519 Hemizona or zona binding assay 210 Hemoconcentration 93, 127 Hemoglobin 137 Hemoperitoneum 170, 171 Hemophilia 313, 375 Hemophilia A and B 334 Hemorrhage 128, 170, 477 Hemostasis 132, 443, 477 Heparan sulphate proteoglycan 382 Heparin 128, 152, 170, 411, 412, 419, 420–423

Index

937

Heparin-aspirin (H-A) therapy 421 Heparin-binding 402 Heparin-binding EGF-like growth factor (HB-EGF) 392 Heparin-binding growth factor 366 Hepatitis 548 Hepatitis B 111, 340 Hepatitis B and C 353, 354 Hepatocellular carcinoma 32 Hepatorenal failure 128 Hepatotoxicity 147 HEPES buffers 262 Herniorrhaphy 22 Herpes 353 Heterokaryon 391 Heteroplasmy 373, 375, 376 Heterotopic pregnancy 177 Heterozygotus 309 Hexose monophosphate shunt 268 High density lipoproteins 123 High mobility group (HMG) 309 High speed sorting (HiSON) 316 High-density lipoprptein cholesterol 159 Highly purified FSH 522 Highly purified u-hFSH 52 High-risk obstetric complications 548 Hilum 444 Hirsutims 21, 24, 63, 136, 146–148, 156 Hirsutism oligomenorrhea 309 Histamine 70, 127 Histology 438 Histones 270, 271 Histopathology 411 HIV 111, 338 HIV antibodies 354, 435 HIV infection 546, 547 HIV negative 339 HIV positive 339, 340 HIV screening 354 HIV serological tests 354 HIV virus 333 HIV, hepatitis B and C 560 HIV-serodiscordant 339 hMG 7, 50, 67, 68, 73, 74, 86, 88, 107, 113, 115–117, 138, 182, 289, 561 hMG and triptorelin 69 hMG preparations 6, 105, 116 hMG treatment 16 hMG-HP 111, 112 Hodgkin lymphoma 322, 325 Hodgkin’s disease 325, 320, 321, 437 Hoffman modulation contrast optics 533 Holmium: YAG laser 192 Holmium: Yttrium Scandium Gallium Garnet 193

Index

938

Homeobox genes 390 Homosexual 338, 345 Homozygous 309 Homozygous mutantmice 382 Hormonal imbalance 408 Hormonal profile 91, 361 Hormonal replacement therapy (HRT) 323, 361 Hormone 9, 16, 24 Hormone administration 6 Hormone agonist (GnRH-a) stimulation test 302 Hormone assays 64 Hormone replacement 354, 396, 397, 320, 354, 360 Hormone therapy 521 Hormones 408 Hormones, growth factors 152 Horseradish peroxidase 274 Hoxgenes 381, 383 H-P-O axis 69 Human immunodeficiency virus (HIV) 338 Human blastocysts 364 Human chorionic gonadotrophin (hCG) 34, 78, 90, 91, 122, 123, 138, 165, 168, 174, 355, 396, 402, 420, 421, 521, 561, 562 Human chorionic gonadotropin (hCG) injection 355 Human clotting factor IX (FIX) 375 Human eggs 258 Human ejaculate 268, 269 Human ejaculates 435 Human embryo 4, 246, 247, 254, 381, 541, 567 Human follicle 11 Human follicle stimulating hormone (FSH) 94 Human follicular fluid 262 Human FSH 50 Human FSH-r 308 Human gametes 4, 333 Human genome 336 Human genome project 333 Human GnRH receptor 540 Human granulosa cells 112 Human immunodeficiency virus 353 Human mammary carcinoma cell lines 246 Human menopausal gonadotrophin 50 Human menopausal gonadotrophin (hMG) 4, 6, 55, 77, 81, 87, 88, 110, 114, 289, 355, 518, 521, 561 Humano ocyte 69, 254, 261 Human primates 326 Human prothrombin 419 Human recombinant leukemia inhibitory factor (hr LIF, Genzyme) 366 Human recombinant LH 90 Human reproduction 8 Human reproduction and embryology (ESHRE) 249 Humanserum 295 Human serum albumin 366, 519

Index

939

Human sperm 250 Human spermatozoa 270 Human tubal fluid (HTF) 193 Humans 568 Human-T-cell lymphotrophic virus type 3 (HTLV-III) 435 Humegon 532 Huntington’s disease 538 Husband’s sperms 262, 433 Hyaluronate migration test 22 Hyaluronic acid 382 Hyaluronidase 194, 258, 262 Hyaluronidase type V 366 Hybridization 336 Hydrodissection 445 Hydrogen 124 Hydrogen ion 211 Hydrogen peroxide 268, 274 Hydrogen peroxide (H2O2) 267 Hydronephrosis 170 Hydropelviscopy 467 Hydroperitoneum 467 Hydrosalpinges 401, 404, 485 Hydrosalpinx 540 Hydrothorax 127, 128, 129 17-α hydroxylase 10 21-hydroxylase deficiency 157 17-α hydroxylase deficiency 352 17-hydroxylase/17, 20 laysegene (CYP17) 157 Hydroxyl radical 268–270 Hydroxytamoxifen 29 Hyper stimulation syndrome 540 Hyperactivation 267 Hyperactivation assay 22 Hyperandrogenemia 21, 148, 158 Hyperandrogenic 150 Hyperandrogenic PCOS 149 Hyperandrogenism 130, 145, 146, 154, 155 Hyperandrogenism occurring 157 Hyperemia 474 Hyper-estrogenemia (E1) 146 Hyperestrogenism 127, 322 Hyperfertile 16 Hypergonadotropic amenorrhea 17, 18, 325–327 Hyperinsulinaemia 145, 149, 155–159, 409 Hyperlipidemia 156 Hyperplasia 456 Hyperprolactinemia 17, 22, 24 Hyperprolactinemia, thyroid 145 Hyper-responder 37 Hyperstimulated ovary 167 Hyperstimulation 138 Hyperstimulation 145

Index

940

Hyperstimulation 328 Hyperstimulation (COH) 59 Hyperstimulation syndrome 261, 537 Hypertension 168, 419, 543 Hypertension, glucose intolerance, hyperlipidaemia 159 Hyperthyroidism or hypothyroidism 24 Hypoandrogenism 22 Hypodermic 199 Hypoestrogenic 114, 138 Hypoestrogenic 78 Hypoestrogenism 21, 326, 328 Hypofertile 17 Hypoglycemia 149 Hypogonadal hypogonadism 105 Hypogonadism 522 Hypogonadism 78 Hypogonadotrophic hypogonadism 107 Hypogonadotropic 84, 113 Hypogonadotropic amenorrhea 17, 81 Hypogonadotropic hypoestrogenic 138 Hypogonadotropic hypogonadal 113 Hypogonadotropic hypogonadism 22, 110, 113, 114 Hypogonadotropic hypogonadism syndromes 114 Hypomenorrhea 476, 477 Hyponatremia 128 Hypo-osmotic swelling 22 Hypo-osmotic swelling assay 206 Hypo-osmotic swelling assay (HOS test) 209 Hypo-osmotic swelling test 507 Hypo-osmotic swelling test failed 436 Hypoosmotic swelling test-HOS 219 Hypophysectomy 66 Hypopituitary-hypogonadotrophic 57 Hypospadias 22 Hypospermatogenesis 271, 504, 546 Hypotaurine 269 Hypothalamic 9, 47, 48, 155, 389 Hypothalamic amenorrhea 38, 254 Hypothalamic anovulation 31 Hypothalamic decapeptide GnRH 78 Hypothalamic pituitary axis 7 Hypothalamic-hypophyseal-ovarian axis 85 Hypothalamic pituitary-gonadal axis 389 Hypothalamic-pituitary-ovarian 147 Hypothalamic-pituitary-ovarian axis 38, 145 Hypothalamohypophyseal hormones 120 Hypothalamus 30, 62, 66, 64, 310 Hypothyroidism 21, 353, 416 Hypovolemia 127, 128 Hypovolemic shock 93 Hypoxia 128 Hypoxic injury 324

Index

941

Hysterectomy 49, 345 Hysterosalpingo-contrast sonography 23 Hysterosalpingogram 91, 223, 422, 560 Hysterosalpingography 15, 17, 174, 187, 451 453, 454, 472, 476, 487 Hysterosalpingography (HSG) 181, 451, 464, 465 Hysteroscope 23, 466, 473, Hysteroscopy 15, 17, 23, 175, 182, 416, 443, 449, 451–454, 457, 458, 464, 469, 473–477, 481, 487, 560, 564, 477 Hysterosonography 487 Hysterosonography elliptosphere catheter set 186 I Iatrogenic infertility 320 ICMR 5 ICMR’s guidelines 5 ICSI 153, 192, 195, 199 ICSI (intracytoplasmic sperm injection) 560 ICSI embryos 570 ICSI injection 570 Idiopathic 15, 156 Idiopathic hypogonadotropic hypogonadism 522 Idiopathic infertility 174, 232, 272, 276, 522 Il-1 382 IL-1 beta 382 IL-6 298, 382 Iliac vessels 166 Immature 116 Immature follicles 10 Immature germ cell 268, 273 Immature oocytes 152, 176, 261–263, 265, 266, 376, 322, 323, 431 Immature sperm 219, 273, 274 Immature spermatids 528 Immature spermatozoa 208, 218 Immortal cell lines 369 Immune modulation 409–412 Immune modulation system 540 Immune response 410 Immune system 293, 295, 404, 418 Immunoassays 327 Immunofluorescence 314 Immunoglobulin 206, 210, 411, 422 Immunohistochemical 10, 194 Immunohistochemistry 411 Immunologic infertility 184 Immunologic techniques 313 Immunological infertility 174, 185 Immunological LH 105 Immunomodulating factors 418 Immunophenotype 294 Immunopurification techniques 50

Index

942

Immunoreactive (ir)-inhibin 10, 86, 327 Immunoreactive GnRH 9 Immunoreactive inhibin 10, 86, 327 Immunoreactive protein 121 Immunoreactivity 121 Immunosuppressive 124 Impaired fibrinolysis 159 Implantation 9, 48, 69, 87, 93, 104, 105, 112, 114, 120, 121–124, 134, 177, 180, 182, 192, 200, 201, 237, 242, 246, 254, 256, 264, 291, 295, 298, 299, 355, 361, 381–383, 385, 386, 389–392, 400– 405, 408, 409, 419, 421, 432, 437, 454, 455, 531, 534, 540, 541, 564, 569, 571 Implantation capacity 527 Implantation development 113 Implantation embryos 9 Implantation failure 91, 199, 201, 291, 299, 382, 400, 402, 405, 408–410, 412 Implantation is 384 Implantation lymphoid cells 384 Implantation of 389 Implantation on 381 Implantation potential 84, 200, 238, 242 Implantation rate 8, 9, 35, 38, 51, 57, 68, 75, 81, 83, 86, 88, 89, 95, 107, 133, 137, 166, 175, 182, 193, 194, 199, 235, 238, 242, 243, 249, 290, 291, 294, 298, 303, 304, 304, 355, 386, 388, 395, 398, 404, 405, 420, 429, 488, 490, 521, 530–532, 533, 534, 563, 564, 569, 570 Implantation rates in 298 Implantation window 120, 121, 381–383, 385 Implants 81 Implications counselling 550 In situ hybridization 10 In utero 498, 571 In utero exposure 32 In vitro 4 In vitro culture 5, 88, 324, 393 In vitro decidualization 403 In vitro development (IVD) 262 In vitro differentiation 367 In vitro embryo development 200 In vitro fertilization 3, 4, 6, 15, 16, 34, 50, 55, 66, 81, 84, 88, 110, 120 168, 174, 184, 209, 213, 218, 222, 233, 235, 245, 271, 303, 354, 357, 372, 412, 419, 437, 497, 503, 517, 540, 545, 567 In vitro fertilization (IVF) cycles 90 In vitro fertilization (IVF) laboratory 249, 262 In vitro fertilization embryo transfer 170 In vitro hatching 568 In vitro maturation 152, 153, 261, 262, 265, 266, 322–324, 538 In vitro oocyte maturation 152, 323 In vitro separation X and y bearing sperms 314 In vivo 108 Inducer cells 294 Infant abnormalities 84 Infanticide 317 Infanticides 317 Infarction 293 Infection 223, 228, 340, 341, 416 Infection, inflammation, acquired

Index

943

immunodeficiency syndrome 269 Infections 17, 409 Inferferon 409 Infertile couples 5 Infertile serodiscordant 340 Infertility 3, 15, 47, 49, 81, 89, 110, 113, 134, 136–138, 145, 148, 181, 186, 205, 208, 210, 218, 224, 232, 233, 237, 272, 282, 289, 291, 297, 301, 307, 310, 322, 326, 345, 352, 360, 384, 395, 401, 408, 416, 419, 444, 446, 448, 449, 452, 456, 465, 477, 493, 512, 515, 537, 543, 545, 546, 549, 550– 552, 554, 558, 559 Infertility therapy 549 Infertility and endometriosis 419 Infertility counselling 545 Infertility duration 263, 545 Infertility or unexplained infertility 398 Infertility treatment 122, 341, 77 Infertility treatment protocols 120, 124 Infertility, spontaneous abortion 549 Infertiloity 383 Inflammation 410, 298 Infundibulopelvic veins in 461 Infundibulum 447 Inheritance 309 Inheritance autosomal dominant 307 Inhibin 10, 11, 36, 38, 92, 113, 131, 327, 328 α-inhibin 12 α-β inhibin dimer 12 α-inhibin precursor 11, 12 Inhibin α-subunit 11, 12 Inhibin α-subunit mRNA 11 Inhibin α-subunit proteins 11 Inhibin A 37, 327 Inhibin A and B 327 Inhibin B 37–39, 56, 77, 504 Inhibin-A 328 Inhibin-A immunoactivity 327 Inhibin-B 10, 85, 86 Inner cell mass 199, 238, 243, 394, 541 Inositol phosphate, leukotrienes 66 Insection 432 Insemination 186, 199, 213, 219, 222, 241, 282, 313 Insemination concentration 569 Insulin 157, 410 Insulin expression 309 Insulingene 158, 309 Insulin like growth factor 1 (IGF-1) 156 Insulin like growth factor binding protein-1 (IGFBP-1) 402 Insulin like growth factor binding proteins (IGFBPS) 156 Insulin receptor 149 Insulin receptor gene 157, 158 Insulin resistance 135, 145, 146, 148, 149, 155–157 Insulin resistance syndrome hyperandrogenism 156

Index

944

Insulin sensitizer 150 Insulin sensitizing agents 148, 149 Insulin-like growth factor 1 (IGF-1) 152 Insulin-like growth factor binding protein-1 403 Insulin-sensitizing agents 147 Integrins 298, 382, 384, 390, 392, 400, 401, 402, 404 Integrins (fibronectin, vitronectin, collagen type IV) 392 Integrins; trophinin/tastin 404 Intercourse 313, 339, 549 Intercycle fluctuation 85 Interleukin 409 Interleukin (IL-6) 382 Interleukin-1 392, 402 Interleukin-1β (IL-1β) 402 Interleukin-1β (IL-1β) cyclo-ocygenase (COX) 390 Interleukins IL1b 298 Internal os 486 Intraabdominal adhesion 133 Intracellular 66 Intracellular free radicals 246 Intracytoplasmic 271 Intracytoplasmic sperm 432 Intracytoplasmic sperm injection 18, 51, 81, 120, 209, 236, 282, 303, 339, 353, 360, 372, 435–437, 503, 507, 512, 546, 567, 568, 569 Intrafallopian transfer (GIFT) 176, 303 Intrafollicular 105, 108 Intrafollicular insemination (IFI) 186 Intrafollicular oocyte maturation 137, 376 Intrafollicular oxygenation 235 Intraluminal passage 123 Intramural fibroids 448 Intramuscular 51 Intramuscular administration 7 Intramuscular progesteone 257 Intramuscular u-hFSH 51 Intraovarian 131 Intraovarian androgen levels 139 Intraovarian androgens 83 Intraovarian fluid 63 Intraovarian follicular phase 83 Intraovarian hemorrhage 171 Intraovarian pulsatility index (PI) 41 Intraperitoneal 63, 170 Intratesticular hemorrhage 509 Intrauterine abnormalities 452, 487 Intrauterine adhesions (synechiae) 476, 477, 481 Intrauterine devices 388 Intrauterine endometrium 48 Intrauterine growth retardation 419 Intrauterine insemination (IUI) 18, 31, 91, 184, 186, 187, 208, 213, 215, 218, 222, 233, 303, 315, 339, 422, 493, 518 Intrauterine insemination semen pregnancy score 228

Index

945

Intrauterine lesions (synechiae, septa, polyps) 452 Intrauterine perfusion 184 Intrauterine pressuer 473, 449 Intrauterine synechiae (Asherman’s syndrome) 23, 452, 456 Intrauterine transfer 4, 181, 432 Intravaginal 77 Intravascular 128 Intravascular coagulation 461 Intravascular thrombosis 420 Intravenous 412 Invasion 409 Invasion test 404 In-vitro culture 113 In-vivo ovulation induction 7 Iodixanol 219 Iodixanol based density gradients 215 Iodixanol is 216 Ion fluxes 376 Ipsilateral tube 446 Iron and copper 268 Irregular menses 35 Ischemic 460 Ischemic black-blue twisted adnexa 460 Ischemic tissue 460, 461 Ischemic tissue to 462 Isoforms 50 Isolate 216, 220, 218 Isotonic crystalloids 128 Issue in 552 IUI cannula 185 IUI catheter 562 IUI or IVF 220 IUI pregnancy 228 IUI pregnancy rates 228 IUI semen pregnancy score-prewash 229 IUI-semen pregnancy score (IUI-SPS) 224, 228, 231, 233 IVF 77, 95, 145, 168, 176, 215, 302 IVF cancellation rates 42 IVF cycle 36, 63, 39, 56, 77, 558 IVF failures 193 IVF fertilization medium 199 IVF outcome 37, 35, 42, 55 IVF pregnancy outcome 37 IVF protocols 198 IVF success 41 IVF success rate 201 IVF treatment cycles 115 IVF/ET 165 IVF/GIFT 186 IVF/ICSI cycle 112 IVF-ET 51, 85, 182, 198 IVF-ETcycles 182

Index

946

IVM medium 262 J Jugularveins 128 K Kallman’s syndrome 17, 85, 112, 522 Karyograms 370 Karyoplast 375 Karyotype 169, 353, 364, 369, 415, 530 Karyotype analysis 368 Karyotyping 505 Kennedy’s disease 538 Ketoconazole 147 Kidney cells 246 Klinefelter syndrome 17, 22, 512 Kreb’s medium 250 Kruger’s morphology 225 Kruger’s strict criteria 233, 518 L Laboratory 205 Lactate dehydrogenase 504 Lactic acid dehydrogenase 268 Lactic acidosis 149 Lacunae 391 Laminal flow 365 Laminaria 564 Laminin 382 Laparoscope 189, 445 Laparoscopic drilling 136 Laparoscopic electrocautery 134 Laparoscopic oocyte retrieval 169 Laparoscopic ovarian electrocautery 134 Laparoscopic ovulation induction 135 Laparoscopy 5, 15, 17, 23, 130–132, 136, 165, 166, 169, 174, 175, 177, 180, 181, 184, 223, 358, 443, 444, 446, 453, 461, 464, 471, 472 Laparoscopy/laparotomy 454 Laparotomy 128, 130, 131, 169, 323, 444, 461 Large follicles 7 Larynx 364 Laser 191, 192, 291, 444, 449, 568 Laser assisted hatching (LAH) 192 Laser beam 191, 317 Laser delivery systems 132 Laser drilling 444 Laser microbeams 189 Laser path 316 Laser pulse 194 Laser zona drilling (LZD) 191 Laser-assisted zona drilling 567

Index

947

Lasers, (Light Amplification by Stimulated Emission of Radiation) 189 Laser-zona interaction 191 Late follicular phase 41, 113 Late secretor phase 404 Late secretory phase 121, 382, 383, 384 Leading follicle 59, 67, 518 Lectins 400 Legal procedures 348 Leiomyoma 448 Leiomyomata 308, 448 Lesion 308, 446 Letrozole 48 Leucocytes 213 Leukemia 322, 323, 328, 437 Leukemia inhibitory factor (LIF) 365, 382, 392, 402 Leukocyte 170, 277, 384, 391 Leukocyte antigen 416 Leukocyte inhibitory factor (LIF) 390 Leukocytes 207, 208, 210, 214, 219, 268, 269, 383, 390, 498 Leukocytospermia 268, 273, 276 Leuprolide 51, 74, 86, 88 Leuprolide acetate 39, 68, 82, 85, 86, 88, 90, 91, 302 Leuprolide acetate (LA) 40 Leuprolide dosage, clomiphene citrate 86 Leuprorelin 82–84 Leydig cell dysfunction after varicocelectomy 521 LH 17, 23, 55, 62, 64, 66, 68, 73, 74, 85, 89, 104, 110, 111, 121, 328, 397 LH and hCG 104 LH bioactivity 133 LH concentration 83, 115, 116 LH hypersecretion 134 LH levels 7, 73, 74, 88, 105, 133, 134 LH peak 75, 314 LH pulse amplitude 155 LH receptor gene 113 LH receptors 113, 114, 265 LH stimulation 114 LH suppression 7, 82, 115 LH surge 23, 55, 64, 67, 70, 78, 92–94, 297, 397, 493, 540 LH threshold 106 LH/FSH ratio 135, 146, 148 LH-like activity 104, 107, 116 LH-like activity hMG preparations 108 LHRH pulse generator 30 Libido 147 Life-birth rates 137 Ligation 271 Light 190, 245, 248, 368 Lightbeam 190 Light irradiation 246 Light microscopy 323 Linearity of a curvilinear 223

Index

948

Linkage 158 Lipid hydroperoxides 270 Lipid peroxidation 268, 270 Lipid profile 159, 307 Lipids 267, 270 Lipoproteins 419 Liquefaction 207, 211 Liquefying enzymes 207 Listeria 416 Lithotomy 166, 465 Live birth 275, 341, 355, 374 Live birth rates 293, 355, 359 Liver 147, 150 Liver disease 32 Liver dysfunction 127 Liver function test 128 Liver transaminases 128 Local factors 120 Locus 311 LOD 137 Long GnRH agonist 83 Long GnRHa protocol 69 Long protocol 50, 58, 78, 83, 89, 90, 106, 114, 117, 361 Long regimens 83 Low dose aspirin 355 Low oestradiol 106 Low sperm concentration 569 Low-density lipoprotein 30 Low-density lipoprotein cholesterol 159 Low-dose gonadotropins 81 Low-dose step-up regimen 8 Lubeck protocol 57 Luminal endothelium 390 Luminal epithelial cells 401 Luminal epithelium 384, 389, 390, 392 Luminol-dependent chemiluminescence 274 Luminometer 274 Lung 128, 364 Lupride depot protocol 90 Lupron flare protocol 561 Lupus anticoagulant 293, 411, 416, 418 Lupus coagulant 418 Luteal cells 87 Luteal cysts 83, 93 Luteal E2 92 Luteal insufficiency 121 Luteal leuprolide protocol 40 Luteal phase 121, 122 Luteal phase 6, 9, 24, 56, 58, 67, 68, 78, 79, 83, 84, 88, 92, 95, 123, 385, 412 Luteal phase adequacy 92 Luteal phase defect 31, 136, 410 Luteal phase deficiency 68, 83, 91, 122, 383

Index

949

Luteal phase dysfunction 31, 91 Luteal phase insufficiency 121, 124 Luteal phase long protocol 90 Luteal phase support 91, 93, 122, 124, 132, 297 Luteal phases 59 Luteal support 120, 124, 411 Luteal support protocols 185 Luteinization 7, 23, 62, 74, 75, 95, 121, 385, 389 Luteinized unruptured follicle syndrome 91, 297 Luteinizing hormone (LH) 50, 56, 81, 91, 94, 112, 121, 131, 155, 396, 416, 521 Luteinizing hormone (LH) surges 55 Luteinizing hormone-releasing hormone (LHRH) analogues 55 Luteolysis 68 Luteotrophic 31, 91, 93 17, 20 lyase 156 17, 20 lyase steroidogenetic enzymes 10 Lymph vessels 123 Lymphatic tissue 364 Lymphocyte immune therapy 411 Lymphocytes 411 Lymphoid 400 Lymphokines 208 Lymphoma 320, 325, 326, 328 M Macrophages 208, 269 Macrosurgical techniques 131 Magnitude of lateral displacement 223 Major histocompatibility complex (MHC) 404 Male adnexitis 521 Male factor 90, 178, 180, 307 Male fecundity 222 Male fertility 205, 232, 270 Male fertilization potential 515 Male gametes 538 Male genital tract infections 537 Male germ 272 Male hypogonadism 522 Male infertility autosomal recessive 310 Male reproductive function 278 Male reproductive tract 211, 267, 269 Male-factor infertility 18, 177, 222, 223, 231, 233, 269, 273, 274, 276–278, 291, 310, 311, 503, 505, 512–514, 516, 522, 553, 569 Malformation 70, 311 Malignancy 320, 322, 461, 462 Malignant lymphoma 325 Malignant neoplasm 460 Malignant transformation 382 Malignant tumor (dysgerminoma) 462 Malondialdehyde (MDA) 270 Mammalian 404

Index

950

Mammary tissue 375 Marital disharmony 550 Marriage rates 557 Massive ascites 127 Masturbation 185, 208 Maternal age 35, 198, 201, 291, 548 Maternal endometrium 383 Maternal mRNA in donor cytoplasm 373 Maternal tolerance 404 Maternal-fetal interface 403 Maternal-fetal recognition 540 Matre oocytes 69 Matrix metallo proteinases (MMPs) 389, 402 Matrix-degrading enzyme 403 Maturation 10, 541 Maturation arrest 310, 504 Mature follicles 77 Mature oocytes 41, 83, 85, 86, 110, 254, 323, 358 Mature sperm 274 Mature spermatozoa 310 Mature unruptured follicles 32 May-Grunwald-Giemsa stain 504 Mean fertilization rate 90 Mechlorethamine 320 Medical ovulation induction 136 Medically assisted reproductive technologies 3 Medroxy-progesterone acetate 48, 121 Megalo-pinched spermatozoo 527, 528, 530 Megesterol acetate 49 Meiosis 10, 75, 134, 165, 245, 307, 310, 373, 404, 505, 538 Meiosis I 236 Meiosis II 236, 376 Meiosis luteinization 92 Meiotic division 92 Meiotic maturation 91 Meiotic spindle 253 Meiotic status 262 Membrane 209, 269, 339 Membrane fluidity 270 Membrane functional assessment 209 Mendelian modes 307 Menopausal 24, 34, 35, 50, 321, 327, 352, 353, 355, 357 Menopause 42 Menopur 111 Menorrhagia 63, 452 Menotropin 303, 494 Menstrual abnormalities 21, 464 Menstrual cycle 9, 24, 36, 56, 62, 66, 91, 92, 110, 120, 122, 128, 168, 301, 327, 352, 381–383, 397, 401, 404, 493 Menstrual dysfunction 146 Menstrual irregularity 130, 309 Menstrual irregularity, hyperandrogenism and obesity 154, 155

Index

951

Menstruation 86, 90, 120, 354, 383 Mental health professional 551 β-mercaptaoethanol 368 MESA (Microsurgical epididymal sperm aspiration) 335, 507 Mesoderm 364, 367, 369 Mesoovarian vessels 444 Mesosalpinx 446 Messenger RNA 391, 540, 541 Mesterolone 523 Metabolic syndrome X 159 Metaphase 10 Metaphase II oocytes 84, 92, 94, 322, 328 Metaphase II stage 10, 92, 152, 328, 437 Metaphase spindle 431 Metformin 145, 147, 149, 159 Methanol to glacial acetic acid 369 Methotrexate 321 Methylene blue 207, 464, 487 Metrodin HP 50, 104 Metroplasty 450 Meutation 311 MI stage 262 Mice 364, 568 Micro dose agonist 74 Micro dose GnRH 74 Micro vascular 159 Microbiology 333, 469 Microbiopsy techniques 510 Microchips 336 Micro-deletion 372, 512–514, 560 Micro-deletions in their Y-chromosome 505, 514 Microdissection 505 Microdose GnRH agonist 58 Microdose GnRH agonist stimulation protocol 40 Microdose GnRH protocols 73, 75 Microdose GnRH a protocol 304 Microdose lupride luteal phase long protocol 90 Microelectrode 447 Microinjection 191, 437 Microinjection pipette 569 Microlaser ovarian wedge resection 131 Micromanipulation 16, 181, 189, 192, 291, 372, 532, 568 Micronized progesterone 123, 398 Microorganisms 207, 208, 218 Microsalpingoscopy 464, 465, 467, 469, 471 Microscopes 189 Microsurgery 507 Microsurgical biopsy technique 510 Microsurgical open biopsy 507 Microsurgical techniques 131 Microsutures 498 Microthrombosis 409

Index

952

Microtubular structure 431 Mid follicular phase 301 Mid trimester 412 Mid-cycle 67 Midcycle FSH surge 93 Midcycle LH surge 69, 82, 91–93, 104, 112, 113 Mid-follicular phase 105, 107, 155 Mid-follicular serum 107 Mid-follicular serum oestradiol 106 Midluteal estradiol-peak 95 Midluteal long protocol 74 Mid-luteal lupron protocol (MLL) 561 Mid-luteal phase 32, 57, 82, 86, 88, 122, 383 Mid-secretory phase 382, 392 Mid-trimester abortions 121 M-II oocytes 431 M-II stage 253 M-II stage oocytes 431 Mild endometriosis 176 Minerals 124 Miniendoscope 452, 454, 458 Minilaparotomy 174, 175 Mini-percoll 219 Miscarriage 7, 31, 35, 85, 89, 242, 349, 351, 404, 415, 457, 533, 534 Miscarriage rate 6, 7, 38, 48, 74, 75, 88, 104, 116, 134, 158, 294, 345, 353, 397, 420, 548 Mised abortion 476, 486 Missense 158 Mitochondria 247, 268, 372–376, 538 Mitochondrial damage 374 Mitochondrial DNA 304, 531 Mitochondrial genome 373, 376 Mitochondrial heteroplasmy 376 Mitochondrial membrane 247 Mitochondrion 373 Mitosis 307, 373 Mitosis germ 307 Mitotic arrest 134 Mitotic spindle 236, 372, 374 Mitoxantrone 321 Mltiple conception 81 Moderate OHSS 59 Moesin 392 Molecular 381 Molecular biology 307, 333, 334 Molecular events 385 Molecular probing 316 Monkey 8, 92, 95, 324 Monkey ovaries 83 Monogenic diseases 194 Monokines 208 Mononucleation, fragmentation 255 Monopolar 132

Index Monosomies 529, 571 Monovulatory 138 Monozygotic (MZ) 568 Monozygotic twinning 243 Monozygotic twins 157, 200 Morbidity 165, 419, 497, 498, 541 Morphology 361 Mortality 75, 165, 419 Morulae 113, 238, 429, 431 Mosaicism 307, 335, 353 Mouse 251, 254, 383, 389 Mouse and bovine 255 Mouse embryo 219, 249, 251 Mouse embryo assay 251 Mouse embryo bioassay 249, 250 Mouse embryos 194, 250 Mouse hybridoma 250 Mouse oocytes 192, 194 Mouse spermatozoa 271 Mouse teratomas 364 Mouse uterus 402 Mozaicism 242 mRNA 9, 265, 541 mtDNA deletions 374 mtDNA mutations 374 MUC 1 382 Mucin 382, 389, 392, 401 Mucopolysaccharides 394 Mucus 485, 493, 563 Mullerian agenesis 169 Mullerian aplasia 308 Mullerian duct regression 310 Mullerian ducts 308, 309 Mullerian inhibiting hormone 309 Multi-cellular embryos 254 Multicystic ovaries, uterus 170 Multifactorial 15, 17, 18, 308 Multifactorial inheritance 307 Multifetal pregnancy 497 Multifetal pregnancy reduction (MFPR) 497 Multi-follicular development 78, 104 Multinuclear blastomere (MNB) 237, 238 Multiple 542 Multiple adhesions 353 Multiple biopsy 132, 505 Multiple births 361 Multiple corpora lutea 91 Multiple dominant follicles 134 Multiple dose protocol 540 Multiple dose regimen 62 Multiple embryos 192, 235 Multiple endocrine neoplasia (MEN) syndrome 353

953

Index

954

Multiple follicular development 31, 41, 51, 52, 85, 113 Multiple follicular growth 7, 8, 51 Multiple follicular stimulation 55, 56 Multiple folliculogenesis 81 Multiple genital tract abnormalities 32 Multiple gestation 137, 199, 396, 444 Multiple gestation rate 138, 200, 235, 239, 242, 541, 542, 567, 571 Multiple implantation failures 304 Multiple organ failure 128 Multiple ovarian follicles 4 Multiple ovulation 51 Multiple pregnancies 91, 93, 138, 148, 176, 199, 222, 290, 497, 537 Multiple pregnancy rate 199, 243, 542 Multiple tissue 514 Multiple-dose 67 Multiple-dose protocol 58 Murine 192 Murine embryos 404 Murine oocyte 254 Mutant autosomal 308 Mutant GnRH receptor expression plasmid 540 Mutation 158, 307, 311, 333, 334, 374, 515, 538 Mycoplasma 411 Myeloperoxidase system 269 Myocytes 365 Myoma 454, 467, 470 Myomectomy 448, 453, 457, 474, 476 Myometrium 9, 498 Myopathies 373 Myotonic dystrophy 334, 538 N NAB (Needle aspiration biopsy) 507–509 NADH-dependent oxido-reductase 268 NADPH-oxidase system 268 National health and medical research council 558 Native GnRH 16 Natural conception 284 Natural cycle 31, 66, 83, 92, 151, 261, 323, 354, 396–398, 494 Natural cycle IVF 56, 77 Natural killer cells 294, 384 Natural menstrual cycles 94 Natural progesterone 122, 123 Nausea 32, 127 Nd: YAG laser 132, 192 N-desmethyl-tamoxifen 29 Necrosis 132, 460 Necrozoospermia 210, 507 Needle biopsy 507 Negative feedback 47, 48, 155 Neonatal 181, 460, 537

Index

955

Neovagina 169 Neovascularisation 540 Neovasculogenesis 538 Nephrectomy 170 Nestin 369 Neural tissue 364 Neurodegenerative 373 Neurofibromatosis type I 334 Neurologic deficits 497 Neurons 364 Neurotrophic factor 402 Neutral—glucosidase 211 Neutrophils 207 Newborn calf serum (NCS) 365 Nicotinamide adenine dinucleotide phosphate (NADPH) 268 Nidation 120, 121, 401, 420 Nidation or fetal growth 295 Niradozole 22 Nitric oxide (NO) 291 Nitric oxide (vasodilator) 392 Nitrofurantoin 22 Nitrogen laser 193 Nitroglycerine patches 291 Non-disjunction 307 Nondysfunction 374 Non-essential amino acids 366 Non-Hodgkin lymphoma 320, 325 Noninsulin-dependent diabetes mellitus (NIDDM) 145 Non-obstructive azoospermia 17, 436, 503–505, 507, 508, 510, 512, 570 Non-obstructive male factor 505 Non-recombining area of the Y chromosome (NRY) 272 Nonsense mutations 158 Norethisterone 62 Norfolk catheter 563 Norgestrel-containing COCs 63 Normal estradiol 86 Normegone 104 Normogonadotrophic 30, 31, 57, 105, 107, 116 Normo-ovulatory 84 Normoovulatory normogonadotropic 114 Normoprolactinaemic 30, 31 Normozoospermic 177 Nuclear 10, 527 Nuclear annulus DNA 270 Nuclear chromatin decondensation 22 Nuclear damage 247 Nuclear DNA 272 Nuclear DNA damage 272 Nuclear dye staining 464 Nuclear genes 376 Nuclear integrity 206 Nuclear lobulation 245

Index

956

Nuclear matrix 270 Nuclear transfer (NT) 372, 375 Nuclease 271, 382 Nucleic acid copies 340 Nucleic acids 270 Nucleolar precursor bodies (NPB) 236 Nucleotide excision 373 Nucleus 270, 376 Nulliparous 422 Nullisomy 529 Numerical abnormalities 307 O OAT syndrome 17 Obese 159 Obesity 24, 63, 130, 135, 154, 157, 415 Obesity, hyperinsulinaemia 155 Obstetric complications 542 Obstructive azoospermia 23, 503, 507, 509, 510 Oestradiol 7, 50, 56, 57, 64, 105, 107, 108, 355, 402 Oestradiol concentration 8, 74, 77, 106 Oestradiol levels 6, 105 Oestradiol valerate 354 Oestrogen therapy 354 Oestrogenic potency 30 Oestrogens 56, 354, 400, 450 Oestrogens and progesterone 400 Oestrogens growth hormone 77 Oestrone 47 Oestrous cycles 326 Offspring 549 OFNA (Open find needle aspiration) 507, 508 OHSS 6, 59, 69, 95, 138 OHSS II 112 Oligoamenorrhea 24 Oligoasthenoteratozoospermia 227, 335, 517 Oligoasthernoteatozoospermia 530 Oligoasthenozoospermia 174, 177, 437 Oligomenorrhea 81, 137, 146, 156, 157, 308 Oligomenorrhea, hirsutism 154 Oligonucleotides 335 Oligo-ovulation 15 Oligo-terato-asthenospermia 15 Oligozoospermia 22, 219, 227, 309–311, 436, 512–514, 560, 570 Oligozoospermic 522, 523 Oliguria 93, 127, 128 One-cell embryos 251 Ongoing clinical pregnancy rates 52 Ongoing pregnancies 8, 39, 106, 256, 530 Ongoing pregnancy rate 51, 106, 112, 124, 298, 304, 530, 531, 564 Oocyte activation 538

Index

957

Oocyte banking 323 Oocyte competence 137, 235 Oocyte cryopreservation 258, 429 Oocyte cumulus complexes 194 Oocyte cytoplasm 114, 372, 530, 531 Oocyte development 11 Oocyte donation 56, 120, 123, 166, 253, 258, 304, 352–355, 357, 358–361, 546, 547, 558 Oocyte donation and embryo transfer 355 Oocyte donation programme 353, 355 Oocyte donor 353, 355, 357–359, 547 Oocyte embryo manipulation 412 Oocyte for fertilization 389 Oocyte function 87 Oocyte in vitro maturation 323 Oocyte maturation 6, 10, 11, 84, 91–95, 105, 112, 114, 116, 152, 165, 238, 254, 261, 263, 266, 298, 373 Oocyte morphology 527, 530 Oocyte nuclear maturation 262 Oocyte quality 31, 35, 87–89, 93, 201, 235, 290, 299, 303, 304, 353, 372, 376, 412 Oocyte recipients 354, 355 Oocyte recovery 95 Oocyte recovery rates 168 Oocyte retrieval 66, 69, 78, 121, 153, 165, 166, 168, 170, 171, 174, 175, 181, 198, 263, 289, 323, 353, 354, 357, 359, 360, 389, 394, 400, 420, 435, 457, 521, 562 Oocyte vitelline membrane 210 Oocyte yield 74, 289 Oocyte-cumulus complex 10, 93 Oocyte-embryo 236 Oocytequality 31 Oocytes 3, 8, 9, 36–40, 50–53, 55, 56, 59, 66, 69, 77, 79, 81, 83, 89, 92, 94, 104–106, 108, 110, 112, 114, 117, 120–122, 134, 152, 153, 165, 168, 174–177, 184, 186, 191, 192, 194, 198–201, 205, 207, 210, 235–237, 245, 246, 248, 253, 254, 261–266, 289, 295, 293, 301, 303, 304, 307, 321–324, 326, 334, 338, 339, 352–355, 358, 361, 374, 375, 376, 388, 389, 393, 395, 429, 431–433, 527, 531, 533, 538, 547, 567, 569 Oocyte donation 86 Oogenesis 322, 338, 373 Oogonia 35, 307 Oolemma maturation 114 Oophorectomy 10, 78, 169, 171 Oophoropexy 462 Ooplasm transfer 304 Ooplasm zona pellucida membrane granulosa, theca folliculi interna and lutein cells 294 Ooplasmic donor 304 Ooplasmic factors 530 Ooplasmic transfer 253 Openbiopsy 505, 507, 510 Operative adhesion 131 Ophthalmoplegia 374 Opposite sex 533 Optical tweezers 189 Optimal laser 190 Optipref 219

Index

958

Optiprep 216, 220 Optiprep, Ixaprep® 220 Oral contraceptive pill Oral contraceptives 48, 63, 64, 308 Oral contraceptives (COC) 48, 62–64, 146, 147, 308, 354 Organ transplantation 320, 328 Organelles 323, 373, 375 Organon 532 Organon metrodin 104 Orgasm 314 Ornithine decarboxylase 147 Ornithine decarboxylase inhibitors 147 Ossillin 372 Osteomyelitis 171 Osteopontin 390 Osteoporosis 323, 328, 411, 422, 522 Osteosarcoma 321 Ova 542 Ovarian stimulation protocols 34 Ovarian (hyper) stimulation (COH) 66 Ovarian abscess, endometriomas 171 Ovarian activity 329 Ovarian adhesions 78, 131, 146, 148, 156, 304 Ovarian androgen biosynthesis in hyperandrogenemia 147 Ovarian androgens 7, 48 Ovarian antral follicle count 56 Ovarian atrophy 133, 136 Ovarian biopsy 132, 154 Ovarian cell membrane 539 Ovarian cells 8 Ovarian cortex 34, 35, 132, 165, 254, 323, 445 Ovarian cryopreservation 324, 352 Ovarian cyst 32, 168 Ovarian cystoscopy 445 Ovarian cysts 24, 32, 63, 130, 168, 443 Ovarian damage 327 Ovarian drilling 465, 468, 469, 471 Ovarian endometriosis 445 Ovarian enlargement 93 Ovarian factor 42, 137 Ovarian failure 24, 308, 325, 328, 355, 357 Ovarian follicle production 361 Ovarian follicles 16, 83, 94, 165, 170 Ovarian follicular maturation 138 Ovarian folliculogenesis 88 Ovarian function 55, 79, 139, 254, 320, 321, 325, 327, 328, 352, 353, 355, 398 Ovarian grafts 324 Ovarian granulosa 87 Ovarian hormones 354 Ovarian hyperstimulation 6, 32, 81–84, 86, 137, 148, 168, 241 Ovarian hyperstimulation protocols 78

Index

959

Ovarian hyperstimulation syndrome (OHSS) 8, 37, 48, 51, 55, 57, 64, 67, 75, 90, 91, 94, 127, 159, 177, 222, 396, 429, 444, 562 Ovarian hyperstimulation syndrome development 124 Ovarian hypoplasia 308 Ovarian microcysts 139 Ovarian morphology 154 Ovarian pituitary axis 42 Ovarian reserve 35–39, 42, 77, 85, 201, 301–303, 305, 323, 561 Ovarian reserve screen 35 Ovarian reserves 79 Ovarian responder 34, 36, 39, 42 Ovarian response 5, 34, 35, 38, 40, 41, 55, 63, 64, 74, 77, 85, 86, 88–90, 116, 354 Ovarian response to gonadotropins 36 Ovarian rserve 301 Ovarian screening test 34 Ovarian steroid production 7 Ovarian steroidogenesis 88, 92, 106, 149 Ovarian steroids 66, 298, 381, 382, 405 Ovarian stimulation 31, 34, 37, 41, 51, 55–57, 63, 77, 79, 83, 84, 86, 87, 91, 93, 104–106, 108, 110, 113, 115, 116, 120, 184, 186, 187, 198, 222, 254, 322, 353, 357, 359, 360, 361, 400, 404, 420, 561 Ovarian stimulation cycle 64 Ovarian stimulation 122 Ovarian stimulation for in vitro fertilization 94 Ovarian stimulation protocols 79, 104, 236 Ovarian stimulation regimes 104 Ovarian stroma 132 Ovarian stroma blood flow 56, 77 Ovarian stromal flow index 41 Ovarian stromal peak 303 Ovarian structure 137 Ovarian suppression 83, 86, 396 Ovarian surgery 77, 131, 148, 352 Ovarian tissue 131, 139, 253, 254, 323, 324, 352, 384, 443, 444, 460, 541 Ovarian tissue cryopreservation 323, 324 Ovarian tissue grafts 323 Ovarian torsion 128, 177 Ovarian toxicity 321 Ovarian transplantation 324 Ovarian tumors 461 Ovarian volume 40–42, 56, 63, 74, 77, 95, 155, 302 Ovarian wedge resection 139 Ovarian wedge resection 145 Ovaries 3, 55, 74, 112, 127, 130, 166, 169, 174, 254, 326, 345, 352, 353, 355, 375, 398 Ovaries as 156 Ovaries with 461 Ovary 10, 40, 48, 89, 155, 169, 171, 310, 325, 444–446, 462, 467, 494 Oviduct-endometrial 9, 570 Ovulation 5, 7, 10–12, 15, 23, 30–32, 48, 49, 62, 63, 74, 75, 83, 85, 90–94, 120–122, 130, 131, 139, 148, 313, 381, 383, 385, 443, 469, 493, 542 Ovulation and pregnancy 539 Ovulation date 121 Ovulation in endometriosis 297

Index

960

Ovulation inducting agents 32 Ovulation induction 6, 7, 29–31, 55, 57, 81–83, 92, 93, 110, 112, 114, 122, 133, 135, 137, 138, 139, 158, 289, 289, 293, 303, 314, 393, 395, 398, 444, 462, 493, 494, 496, 497, 540 Ovulation induction agent 303 Ovulation induction by 396 Ovulation induction protocols 59, 67, 290 Ovulation induction regimen 149 Ovulation initiation 31 Ovulation rate 7, 137, 138 Ovulation stimulation 4 Ovulatory 131 Ovulatory cycle 6, 32, 94, 136, 137, 444 Ovulatory disorder 81, 168 Ovulatory dysfunction 23, 145, 186, 232, 297 Ovulatory follicles 235 Ovulatory status 23 Ovulatory stimulus 93 Ovum 372, 373, 541 Ovum donation 359 Oxidants 267, 269 Oxidative damage 246, 267, 269, 274 Oxidative insult 269 Oxidative phosphorylation 247 Oxidative stress 208, 246, 269, 271, 278 Oxidative stress-induced DNA damage 271 Oxidizing agents 267 Oxygen 152, 246, 247, 394 Oxygen (O2) paradox 267 Oxygen levels 246 Oxygen tension 270 P P level 92 P450 cytochrome 17a 10 Pacemaker 169 Pachytene stage 272 Pain 127 Pancreas 311 Pancreatic beta cell 158 Pancreatic beta cell dysfunction 159 Pap smear 24 Paracrine 10, 121, 384, 403, 405 Paracrine/autocrine role 87 Paraplegic 549 Parathyroid glands 364 Parenthood 351 Parkinsonism 5 Parotiditis 353 Parthenogenic activation 431 Partial zona dissection (PZD) 191, 567 Paternal genome 571

Index

961

Paternal smoking 272 Pathophysiology 400 Patient monitoring 6 Patient support 545, 552 Patient support booklets 552 Patient support organizations 552 PCO 261 PCO syndrome 63, 64 PCOD 17 PCOS 6–8, 59, 133, 134, 138, 148, 149, 150, 152, 158, 309 PCOS ovary 149 PCR techniques 111 PCR using 335 Peak estradiol 86, 87 Pedal-controlled aspiration 166 Pellucida, cortical granules 429 Pelvic 132, 165 Pelvic adhesions 165, 171, 174, 176, 443 Pelvic adhesive disease 165 Pelvic blood vessels 170 Pelvic computerized tomography (CT) 170 Pelvic endometriosis 465 Pelvic infection 171, 537, 548 Pelvic inflammatory disease 17, 340, 420, 465, 537 Pelvic mass 464 Pelvic organs 170 Pelvic organs 445, 465 Pelvic pain 464 Pelvic ultrasonography 31 Pelvic ultrasound 416, 452 Pelvic viscera 177 Pelvis 467 Pencillin 365, 366 Penile discharge 22 Penis 309 Pentoxyphlline 191 Peptide 9, 66 Peptidomimetic GnRH analogs 540 Peptidomimetic molecules 540 Percoll 218–220, 272, 314 Percoll density gradient 219 Percoll layer 219 Percutaneous aspiration of epididymis 505 Percutaneous testicular sperm retrieval 508 Perforation 185 Perfusion 127 Periadnexal adhesions 447 Periadnexal adhesions 447, 448 Pericardial cavity 128 Pericardial effusion 129 Pericardiocentesis 128 Perifollicular blood flow 41

Index Peri-implantation embryos 8 Perimenarchal 146 Perinatal mortality 497 Perioperative LH levels 133 Periovulatory 92, 404 Periovulatory mucus 217 Periovulatory phase 122 Peripheral hair 146 Peripheral insulin resistance 156 Peripheral LH 85 Peripheral receptors 9 Peristaltic pump 473 Peritoneal cavity 23 Peritoneal fluid 184,186 Peritoneal implants 445 Peritonitis 170 Peritubal adhesions 131 Perivascular cells 392 Peroxidase 247, 273 Peroxidase positive leukocytes 269, 273 Peroxidase-positive 268 Peroxidation 269, 270 Peroxidative damage 268, 278 Peroxide 246, 247 Peroxisomal ring 538 Peroxisome proliferation 149 Peroxyl radical 269, 270 PESA 17, 505, 507 pH 246, 250, 394 pH level 206 pH values 211 pH temperature 251 Pharmacokinetics and pharmacodynamics 85 Phase contrast microscope 365, 519 Phenotype 283, 310, 513, 515 Phenotypic matching 358, 361 Phenotypic traits 158 Phenylketonuria 334 Phimosis 22 Phosphate Buffer 370 Phosphate buffered saline (PBS) 274, 315 Phosphodiesterase inhibitor pentoxifylline 436 Phosphoethanolamine 419 Phospholipid antibodies 411, 422 Phospholipid-binding plasma proteins 419 Phospholipid-protein complexes 416 Phospholipids 270, 293, 394, 419, 420 Phosphoramide mustard 321 Phosphorylation 149, 402 Phosphoserine 410 Photocoagulation 445 Physiological range 84

962

Index

963

Physiology 198 Piezo electric technology 291 Pigmentation loss 147 Pigmented retinitis 334 Pinocytosis 401 Pinopodes 381, 382, 390, 401 Pioglitazone 149, 150 Pituitary 10, 16, 47, 48, 66, 68, 95, 110, 155, 310, 422 Pituitary desensitization 51, 78, 81, 84, 85, 88, 91, 115, 117 Pituitary down regulation 51, 78, 83, 86, 89, 90, 93, 105–108, 112, 398 Pituitary down-regulation protocols 112 Pituitary FSH 12 Pituitary gland 56, 62, 86, 91, 521, 522 Pituitary glant 364 Pituitary gonadotrophins 30, 131 Pituitary gonadotrophs 68 Pituitary gonadotropin stimulation 131 Pituitary hormone 304, 389 Pituitary LH 90, 92 Pituitary secretion 7 Pituitary suppression 41, 57, 68, 82, 90, 95 Pituitary-gonadal axis 322 Pituitary-gonadal suppression 326 Pituitary-ovarian desensitization 326 Placenta 293, 381, 389, 409, 420 Placental cells 87 Placental infarction 420 Placental vasculature 421 Plasma concentrations 48 Plasma FSH 6 Plasmah hCG 92 Plasma membrane 209 Plasma oestradiol 7 Plasma progesterone 398 Platelet activation factor (PAF) 419 Platelet adhesion 409 Platelet aggregation 419 Platelet count 411 Pleural effusion 128 Pleural fluid 128 Ploasminogen 393 Pluripotency 365 Pluripotent 364 Pluripotent cells 366, 367 Pluripotent stem 364, 370 Pluripotent stem cells 364, 367, 370 Plus low-dose aspirin 421 PMN leukocytes 277 Pneumoperitoneum 165, 177, 447 POF 12 Po-FSH 90, 113 Point mutations 309

Index

964

Polar 195 Polar bodies 10, 194, 262 Polar body (PB) 236 Polarized optics 236 Polarized PNs 237 Polscope® 236 Polyamines 147 Polycystic ovarian disease 47, 48, 81, 89, 130, 158 Polycystic ovarian disease (PCOD) 17 Polycystic ovarian disease resistant 30 Polycystic ovarian morphology 309 Polycystic ovarian syndrome 308 Polycystic ovarian syndrome (PCOS) 83, 130 Polycystic ovaries 24, 40, 130, 135, 139, 146, 149, 154, 155, 157, 416, 465 Polycystic ovaries and acne 63 Polycystic ovary 130, 155 Polycystic ovary syndrome 114, 145, 146, 154, 156–158, 159, 409 Polygenic 157, 308 Polygenic inheritance 307 Polygenic mode inheritance 308 Polygenic multifactorial 308 Polymerase chain reaction 158, 194, 291, 334, 339 Polymorphism 157–159, 311 Polymorphonuclear (PMN) leukocytes 268 Polymorphs 208 Polypectomy 449, 457 Polypeptid 335, 365, 373, 383, 392 Polyploidy 307, 529 Polypoid embryos 180 Polyps 449, 453, 456 Polyps or submucus fibroids 23 Polysaccharide 216 Polyspermy 567 Polyunsaturated fatty acids (PUFA) 267 Polyvinylpyrolidone (PVP) 218, 570 Poor egg quality 372 Poor ovarian responder 34 Poor ovarian responder 34 Poor ovarian response 34 Poor quality embryos 355 Poor responder 37 Poorer oocyte 7 Positive feed-back 49, 62, 139 Post coital test 15 Post menopausal endometriosis 49 Post multifetal pregnancy 498 Postcoital test 209, 223 Posterior colpotomy 4 Postimplantation stage mouse embryo 367 Postmature oocytes 134 Postmenopausal 7, 30, 47, 55, 56, 66 Postnatal death 310

Index

965

Postoperative 133 Postoperative adhesion 130, 133, 444 Post-ovulatory 122 Postpartum 346, 349, 350 Postpartum depression 346 Postpartum hemorrhage 476 Post-thaw pregnancy rates 255 Postwash 228 Postwash scores 228 Potassium 124, 314 Pouch of Douglas 467, 470–472 Povidine-iodine 171 Pozzi tenaculum 466 Precursor 394 Precursor germ cells 268 Prednisolone 124, 411, 412 Prednisone 320 Pre-eclampsia 403 Pre-embryo 388, 509, 431, 433, 560 Pre-embryo development 104, 107 Pre-embryo genetic diagnosis (PGD) 189 Pregnancy 412 Pregnancy 115, 116, 128, 137 Pregnancy 16 Pregnancy 175, 177 Pregnancy 18 Pregnancy 3, 32, 34, 39, 41, 53, 57, 69, 84, 85, 94, 182, 192, 200, 209, 218, 228, 231–233, 235, 237, 242, 261, 275, 282, 291, 293, 295, 311, 338, 339, 345–350, 353, 385, 402, 409, 417, 421, 422, 429, 444, 446, 450, 461, 490, 518, 530, 537, 539, 541, 549, 563, 571 Pregnancy rates 523 Pregnancy after 445 Pregnancy and motherhood 545 Pregnancy loss 106, 117, 299, 374, 418, 416, 419, 420 Pregnancy mimics 409 Pregnancy or assisted reproduction 294 Pregnancy outcome 52, 231, 301, 302, 561 Pregnancy outcomes 38, 121, 256, 257, 497, 564 Pregnancy potential 107 Pregnancy rates (PRs) 7, 18, 22–32, 24–43 51–52, 56, 73–75, 79, 82, 83, 86, 88, 93–94, 106–07, 114–15, 122–24, 133, 136–38, 152–53, 166, 168, 171, 176–77, 181,185, 187, 198, 199, 200, 235– 39, 266, 289–92, 298–99, 302–03, 314, 355, 361, 394, 405, 409, 429, 437, 444, 457, 485, 486–88, 491, 527, 541, 563–64, 568–69 Pregnant mare serum gonadotropins (PMSG) 90 Pregnenolone 309 Pregnyl 532 Pregranulosa cells 321 Pre-implantation 8, 404 development 57, 113 diagnosis 318, 538 embryo 198, 402, 198, 254, 364, 401 embryonic development 9 genetic diagnosis 194, 199, 242, 291, 313, 334, 531, 533, 534

Index

966

sex selection 532 Prematre LH surges 79 Premature 24, 55, 309, aging 373 balding 158 cortical granule release 254 delivery 498, 498 elevation of LH 88 endogenous LH surges 81 gonadal failure 308 LH 89 LH release 398, LH surges 57–58, 66–67, 75, 77–79, 84, 87 luteinisation 6, 7, 42, 73, 138, 158, 429 luteinization of follicles 81 menopause 320 oocyte atresia 168 ovarian failure 11, 21, 63, 83, 308, 320, 325, 325, 328, 352, 355, 429, 547, 548 ovulation 55, 56, 82 surge of LH 89 Premenopausal 30, 47, 145, 146, 321 Premenstrual phase 475 Prenatal 318, 346 Preoperative 133 Preovulatory 92, 94, 112, 324, 374 follicles 69, 85, 113 follicular development 104, 114 LH 91 LH surge 82, 110, 121 mucus 214 oocytes 92, 165 phase 298 surge 7, 94 Pre-renal azotemia 128 Pre-selection 315 Prewash 228 Prewash scores 228 Primary amenorrhea 21, 113, 307 oocyte 373 ovarian failure 352 spermatocytes 310 Primate ovary 10 Primer 334, 335 Primigravida 415 Primitive yolk sac 373 Primordial follicles 35, 55, 254, 321, 329, 352, 431 Primordial germ cells (PGC) 324, 364, 365, 366, 373 Pro-αC protein 11, 12 Pro-αC really 12 Pro-apoptotic second messenger ceramide 328 Procarbazine 320 Pro-embryos 410

Index

967

Progeny 313 Progesterone 7, 10, 11, 23, 30, 31, 32, 59, 69, 78, 79, 88, 95, 110, 120–24, 134, 157, 256, 262, 297, 301, 328, 354, 361, 381–85, 392, 396, 400, 402, 409, 411, 422, 431, 493 Progesterone (P) 91, 84, 384 Progesterone (P4) 389 Progesterone 397, 398 Progesterone antagonist 382 Progesterone bioavailability 124 Progesterone challenge test 16 Progesterone concentrations 122 Progesterone gels 256 Progesterone levels 38, 301, 136 Progesterone metabolites 123 Progesterone receptor 401 Progesterone receptor levels 122 Progesterone receptor synthesis 122 Progesterone receptors 384, 401, 409 Progesterone rise 68 Progesterone’s 122 Progesterones 401 Progesterone-to-E2 ratio 39 Progestin 64, 147, 403 Progestogen-associated endometrial protein (PEP) 403 Prognostic factor 527 Prognostic indicator 35 Prognostic tests 35 Programmable freezer 256 Programmed cell death 271 Prolactin 16, 17, 22, 30, 127, 135, 403, 410 Proliferation 120, 400 Proliferative 24 Proliferative endometrium 403 Proliferative phase 23, 121, 122 Proline 394 Promoter region 157 Pronuclear development 372 Pronuclear embryo (zygote) 236 Pronuclear stage embryo 375 Pronuclear stage-embryo transfer (PROST) 180 Pronuclear-stage zygotes 181 Pronucleate one-cell embryos 255 Pronucleated egg 396 Pronuclei 375 Pronucleus 540 Propanediol 431 Prophase I 328, 431 Propofol 166 Prostacycline 124, 409 Prostaglandin E2 49 Prostaglandin PGE 48 Prostaglandins 124, 127, 419, 48, 392 Prostate 207, 208

Index

968

Prostate cancer 147 Prostate specific antigens 146 Prostatic hyperplasia 147 Prostatitis 22, 223, 228 Prostatitis or prostatodynia 276 Protamines 270, 530 Protease inhibitors 403 Proteins 152, 190, 214, 267, 270, 333, 373, 383, 391 Protein kinase C 66 Protein phosphorylation 270 Protein sedimentation 313 Protein synthesis 540 Proteinases 393 Proteino-mimetic molecules 539 Proteolytic action 393 Prothombin activator 409 Prothrombin 409 Prothrombin time (PT) 422 Prothrombotic factor 410 Protocols 59 Pseudo Asherman’s syndrome 477 Pseudocapsule 474 Pseudopregnant 192 Psychiatric disease 549 Psychiatric episodes 169 Psychiatric illness 346 Psychodynamic 346 Psychological counselling 551, 549 Psychological screening 347 Psychopathology 346 Psychosis 346 Psychosocial 320 Psychosomatic symptoms 549 Psychotherapy 169 Pulmonary collapse 461 Pulmonary embolism 128, 422, 461 Pulsatile 66, 522 Pulsatile GnRH 81, 114, 138 Pulsatile gonadotropin-releasing hormone (GnRH) 81 Pulsatile injections 81 Pulsatile signalling 66 Pulse 194 Pulsed color Doppler 41 Pulsed Doppler 56 Pure ception 216 Pure FSH 355 Pure sperm 216 Puregon 104 Puresperm 218, 220 Purified FSH (Metrodin HP) 114 Purified urinary-derived-FSH 6 Pyospermia 208

Index

969

Pyridium 207 Pyruvate 269 PZD or sub-zonal sperm insertion—SUZI for ICSI569 PZD, partial zona dissection 291 Q Quality assuarance 249 Quality control 249, 250 Quality management system (QMS) 282, 284 Quartz 192 R Rabbit 324, 392 Rabbit endothelium 390 Rabbit pre-implantation embryos 245 Radiation 208 Radiation drugs or viruses 307 Radiotherapy 320, 325, 328, 353, 429 Radiotherapy for bone marrow transplantation 322 Radixin 392 Rats 10, 271, 323–24 RBM (RNA binding motif) 310 RBM gene family 310 Reactive oxygen species (ROS) 206, 267 Reactive oxygen species 22 Reactive oxygen species 219, 237, 246, 247, 268, 274, 277, 278, 304, 372, 531 Receiver operating characteristic (ROC) 85, 106, 224, 518 Receptor gamma 149 Receptors 120, 368 Recessive 307 Recessive allele 307 Recessive disorders 354 recFSH 111, 114 RecFSH 84, 85, 111 Recipient 361, 547 Recipient couple 357 Recipient menstrual cycles 352 Reciprocal chromosome translocations (RCT) 548 RecLH 95 Recombinant 51, 355, 522 Recombinant basic fibroblast growth factor 366 Recombinant choriogonadotropin 94 Recombinant DNA technology 50, 55, 56 94, 539 Recombinant FSH (r-FSH) 6, 16, 57, 73–75, 104, 105, 107, 108, 110–114, 116, 117, 522, 539 Recombinant gonadotrophins 85, 539 Recombinant hCG 561 Recombinant human FSH 56 Recombinant human LH 94, 95 Recombinant LH 57, 95

Index

970

Recombinant products 94 Recombinant rFSH 57 Recombinant technology 84 Reconstructive surgery 131, 189 Recruitment 38 Recto-vaginal 21 Recurrent abortion 133, 418 Recurrent failure 353 Recurrent implantation failure 408, 409, 411 Recurrent miscarriage 291, 415, 420 Recurrent pregnancy 409, 418 Recurrent pregnancy loss 38, 121, 293, 294, 409, 416, 419, 420 Recurrent spontaneous abortions 422, 423 Recurrent thromboembolism, intrauterine growth retardation 415 Refractory hypoxia 128 Refractory phase 87 Regeneration 120 Regimen 8, 78, 82, 83 Reichert’s membrane 368 Relative quality (RQ) 225, 228 Renal 127 Renal and vertebrla disorders 308 Renal failure 93, 128, 129 Renal perfusion 129 Rennin-angiotensin system 127 Repair mechanism 272 Repeated pregnancy loss 415 Repeated pregnancy loss (RPL) 548 Reproduction 94, 189 Reproductive 146 age 360 cycles 115 endocrinology 6, 29 failure 421, 452 function 451 health 537, 253 immunophenotype 411 outcome 106 potential 35, 77, 301 technology accreditation committee (RTAC) 556, 558 toxicology 222 tract 9, 304 tract infection 208 Resectoscope 449, 450, 477 Resistant ovary syndrome 55, 352 Resolvein 552 Respiratory burst 269 Respiratory distress 129 Respiratory rate 128 Retinopathy 32 Retrograde ejaculation 22 Retroverted uterus 471

Index r-FSH 290 rSH: HCG 263 Rh factors 354 Rhesus 10 Rhesus monkey 113, 325, 326 Rheumatoid arthritis 298, 320 r-hFSH 50, 51 r-hFSH (follitropin alpha) 50 Risk assessment 282–284 RNA 373 RNA synthesis 333 Rodent antigens 295 Rodent embryos 246 Rodent ovarian tissue 254 Rodents 322 Rokitansky-Kuster 308 Rokitansky-Kuster-Hauser syndrome 17 ROS generation lies 268 ROS scavengers 270 Rosiglitazone 149, 150 RQ score 228 Rubella 353, 416 S S. progesterone 410 Saline inj 498 Salpingography 465 Salpingo-oophorectomy 445 Salpingoovariolysis 447 Salpingoscopy 465, 467, 471 Salpingoscopy/microsalpingoscopy 464 Salpingostomy 446, 447, 448 Salpinx 464 Scanning electron microscopy 390, 401 Scanning electron microscopy (SEM) 191 Scavenging enzymes 267 Scavenging peroxyl (RO•) 269 SCID mouse 254 Scrotal thermography 22 Scrotum 309 SDSPAGE 111 Seborrhoeic acne 63 Secondary amenorrhoea 307 Second trimester 412 Secretory differentiation 122 Secretory endometrium 403 Secretory factors 381 Secretory phase 23, 24, 120, 122, 381, 389, 392, 404 Secretory transformation 120, 121, 123 Selective tubal pressure (STP) 473 Self-help groups 552

971

Index

972

Semen 5, 15, 207, 210, 213, 215, 216, 222, 268, 273–75, 315, 341, 435, 437 Semen analysis 5, 22, 91, 205, 207, 209, 211, 222, 230, 232, 233, 276, 513, 516–17 Semen characteristics 222, 232, 33, 271 Semen morphology 517 Semen parameters 222, 223, 225, 227, 228, 231, 275 Semen pregnancy score 223 Semen processing techniques 273 Semen quality (SQ) 222, 223, 225, 227–32, 268, 340, 522, 547 Semen quality scores 227 Semen score 223–25, 227–228, 230, 232 Semen variables 223 Semen viscosity 207 Semen volume 207 Semi-allograft 408 Seminal fluid 206–208, 213, 335 Seminal leukocytes 273 Seminal OS 277 Seminal plasma 210, 211, 213–215, 218, 268, 269, 271, 274, 275, 277, 339, 517 Seminal plasma viral load 341 Seminal smears 223 Seminal vesicles 207, 211, 309, 523 Seminiferous tubule 507, 509 Seminiferous tubule biopsy 504 Seminiferous tubules 23, 274, 505 Seminoma 437 Senile insanity 333 Separation 318 Sephadex 217, 218, 219, 314 Sephadex column 216 Sephadex column filtration procedure 216 Sephadex columns 220 Septoplasty 474 Sequence tagged sites (STS) 513 Sequential culture 198, 242 Sequential media 200, 394 Serine 394 Serine proteases 393 Serinethreonine phosphory 158 Serodiscordant 338, 339 Serodiscordant 341 Sero-discordant couples 339, 340 Serono 104 Serosa 474 Serotonin 127 Sertoli cell only syndrome 272 Sertoli cells 271, 309, 327, 521 Sertoli-cell-only syndrome (SCOS) 504 Serum 327 Serum alanine aminotransferase 150 Serum albumin 250 Serum androgen 149 Serum beta2-glycoprotein 421

Index

973

Serum E2 68, 69, 92 Serum E2 levels 39 Serum E2α 89 Serum estradiol 66, 75, 95, 128, 301, 397, 398, 494 Serum estrogen titers 122 Serum FSH 35, 36, 37, 146 Serum FSH level 303 Serum LH 68, 92, 115, 116, 154, 155 Serum markers 34, 37, 42 Serum oestradiol 30, 106, 107 Serum progesterone 83, 123 Serum prolactin 78 Serum quality 365 Serum renin 95 Serum testosterone 78, 522 Serum-free culture media 541 Severe endometriosis 297 Severe endometriosis pelvic inflammatory disease 77 Severe male subfertility 309 Severe OHSS 127, 128 Severe oligozoospermia 272 Severe PCO syndrome 63 Severe testiculopathy (SCOS) 310 Sex 313, 317 Sex chromosome 335, 368, 370, 512, 513 Sex chromosome linked disease 317 Sex determination 194, 309, 310, 532 Sex determining region 309 Sex hormone 328 Sex hormone binding globulin (SHBG) 63, 155, 159 Sex life 537 Sex of 316 Sex pre-selection 315, 317 Sex ratio 200, 313, 314, 533 Sex resulting 317 Sex reversal 309 Sex selection 314, 317, 532 Sex steroids 112, 298, 328 Sex-chromosomes 307 Sex-hormone-dependent disease 62 Sex-linked diseases 353 Sex-selection 533 Sexual desire 549 Sexual dimorphism 309 Sexual dysfunction 549, 549 Sexual functioning 549 Sexual intercourse 339 Sexual problems 546, 550 Sexually transmitted disease (STD) 24, 435, 543 Sexually transmitted infection 340 SHBG 146 Sheep 11, 323

Index

974

Short protocol 62, 89 Short-acting GnRH agonist 90 Shortened flagella syndrome 530 Sibling pair analysis 158 Signal transduction 149, 540 Silane-coated silica 220 Silica-based density gradient (Percoll) 215, 218, 220 Single counselling 550 Single dose protocol 115 Single follicle 8 Single gene defects 307 Single gene disorder 334 Single-stranded conformational polymorphism (SSCP) 158 Sister chromatid 514 Sister donors 359 Skeletal 364 Skeletal malformation 308 Skin 364 Skin fibroblasts 47, 48 Small follicles 152 Smoking 42, 272, 517, 518 Smoking status 40 Smooth 364 Smooth muscle cells 400 Smooth muscles 291 Sodium 314 Sodium pymiate 365, 366 Sodium reabsorption 124 Soft catheter 565 Somatic tissue 364 Sonographic assessment 42 Sonography 462 Sonohysterography 23, 416, 451, 453 454, 560 Sons 317 Spatial expression 401 Speculum 166, 170 Sperm 3, 18, 122, 184, 189, 195, 206–209, 213–220, 272–273, 345, 375, 493, 507, 508, 530, 532, 541 Sperm adherence method 217 Sperm agglutination 206, 207 Sperm antigens 295 Sperm aspiration 311 Sperm binding 567 Sperm bioassay 250 Sperm capacitation 493 Sperm capacitation acrosome reaction 269 Sperm cells 339 Sperm chromosome 530, 214, 215, 228, 523 Sperm count 18, 208, 278 Sperm counts with teratozoospermia 513 Sperm cryopreservation 340, 435 Sperm damage 268

Index

975

Sperm density 314 Sperm DNA 272, 278, 315 Sperm DNA damage 272 Sperm donation 357, 558 Sperm donor 548, 360 Sperm dysfunction 272, 205 Sperm Fertil® 220 Sperm fertilization capacity 206 Sperm fertilization potential 208 Sperm fertilizing capacity 222 Sperm filtration 216, 177 Sperm function 216, 267, 520, 522, 530 Sperm immobilization 206, 270 Sperm injection (ICSI) 272 Sperm membrane 217219, 278 Sperm migration 207, 214 Sperm morphology 208, 209, 222, 227, 229, 231, 233, 516, 517, 518 Sperm motility 177, 208, 216, 225, 231, 232, 249, 251, 270 Sperm motility assay 250 Sperm motion characteristics 231 Sperm mucus penetration assay 206 Sperm mucus penetration test 209 Sperm nuclear DNA damage 270, 278 Sperm nucleus 210, 270 Sperm parameters 205, 206, 339 Sperm penetration assay 206, 209, 210 Sperm plasma membrane 267, 268, 278 Sperm precursors 207 Sperm preparation 174, 518 Sperm preparation techniques 272 Sperm processing 213, 214 Sperm processing procedures 217 Sperm quality 206, 208, 214, 267, 436, 513, 219, 220 Sperm retrieval 503, 504, 504, 505 Sperm sedimentation rate 215 Sperm separation 218, 219, 314 Sperm separation techniques 219, 313 Sperm sorting 315 Sperm surface binding 206 Sperm surface lectin binding 22 Sperm tail 195 Sperm transport 207, 211, 521 Sperm ultrastructural 206 Sperm viability 213 Spermatic fluid 508 Spermatic veins 17 Spermatid 208, 504, 271, 538 Spermatocytes 208, 538 Spermatogenesis 22, 267, 268, 271, 274, 277, 310, 322, 437, 438, 503, 504, 505, 507, 509, 510, 512, 513, 514, 515, 521, 522 Spermatogenic cells 310, 504 Spermatogonia 271

Index

976

Spermatozoa, 174, 175, 177, 184, 186, 191, 208, 209, 217, 222, 223, 267, 268, 268, 269, 272, 277, 278, 313, 314, 316, 335, 339, 340, 360, 436, 437, 493, 493, 504, 505, 517, 518, 520, 522, 523, 527, 527, 528, 530, 530, 334 Spermatozoon 205, 268, 339, 516, 517, 538 Spermiation 268, 274 Sperm-injected oocytes 569 Spermiogenesis 271 Spermiogenesis 271, 538, 274 Sperm-leukocyte contact 273 Sperm-oocyte fusion 269, 270 Sperm-oocyte union 205, 206, 207 Sperms 317, 340, 503, 504 Sperm-to-oocyte surface binding 209 Sperm-zona 22 Sperm-zona pellucida 519 Spern in ejaculated semen 503 Sperniatozoa 9 Sphingosine-1-phosphate 328 Spididymis 569 Spindle 431, 235, 236 Spindle organization 431 Spinnbarkeit 493, 495, 494 Spironofactone 22 Spironolactone, flutamide 147 Sponge-holding forceps 166 Spontaneous abortion 107, 121, 133, 136, 138, 149, 294, 341, 418, 420, 476, 498 Spontaneous abortion rates 138, 291, 303 Spontaneous cycle 31, 56 Spontaneous LH surge 89, 138, 289 Spontaneous miscarriage 291 Spontaneous ovulation 110 Spontaneous pregnancy 412 Sporadic 416 SQ score 228 SRY sex-determination 309 SST (Single seminiferous tubule) biopsy 509 Stage-specific embryonic antige-4 (SSEA-4) 369 STDs 548 STDs (HIV, HPV infection) 21 Stein-Leventhal syndrome 156 Stem cell 5, 340, 364, 366 Stem cell markers 368 Stenosis 447 Step down 149 Step-up and step-down protocols 8 Stereomicroscope 262, 489 Sterility 42, 358 Sterilization 358 Steroid hormones 120, 381, 402 Steroid synthesis gene CYP11a 114, 159 Steroidogenesis 10, 57, 87, 92, 108, 157, 309, 397 Steroidogenesis in granulosa cells 79

Index

977

Steroidogenic enzymes 157 Steroidogenic factor 310 Steroidogenic factor-1 (SF-1) 47 Steroids 411, 404 Stillborn infants 181 Stimulation protocols 38 Stimulated cycle 4, 6, 152, 261 Stimulation 6, 55, 77, 87, 184 Stimulation cycle 67, 69, 149 Stimulation protocol 56, 75, 77, 84, 85, 95, 121, 148, 186, 303–305, 435, 438, 561 Stimulation regimen 74, 75 Stimulatory cycle 90 Stimuli 169 Straight-line velocity 233 Streak gonads 307, 308 Streak ovaries 352 Streptomycin 365, 366 Stress 545–546 Stroma 381, 383, 384, 400, 509 Stromal cell 384, 392, 404 Stromal edema 390 Stromal hyperplasia 444 Stromal reduction 147 Structural abnormalities 307 Structural chromosome aberrations 530 Stylet 564 Subcellular organelles 195 Subclavian vein thrombosis 128 Subcutaneous 82 Subcutaneous administration 62 Subcutaneous gonadotropins 7 Subcutaneous injections 52 Subcutaneous r-hFSH 51 Subfertility 415 Submucous fibroids 451, 457 Submucous myomas 449, 452, 453, 454, 456, 457 Submucus fibroid 449 Subseptate uterus 449 Subserous fibroids 448 Success rates 83, 235 Sucrose 256, 258, 259 Suction pump 166 Sudden hypotension 461 Sugars 270 Sulfasalazine 22 Supernumerary 533 Superovulation 16, 53, 88, 158, 327 Superovulation cycles 91 Superoxide anion 268 Superoxide dismutase (SOD) 247, 268 Support counseling 351, 550 Supraphysiologic 91

Index

978

Supraphysiological 66 Supraphysiological levels 7 Suprapubic incision 181 Surface receptor 369 Surgery 6, 447 Surgical ovulation induction 138 Surrogacy 345–348, 350, 547 Surrogate 169, 345, 346, 348–351, 548, 551 Survival rate 110, 324 Swim-down 215 Swim up 175, 184, 215, 218, 517 Swim up technique 185 Swyer’s syndrome 352 Synaechiae 449, 451, 481 Syncitiotrophoblast 390, 409 Syncytialization 421 Syncytiotrophoblast 390, 409 Syncytium 391, 421 Synergistic 16 Synthetic analogs 78 Synthetic peptides 78 Synthetic progestins 123 Syphilis 353, 354 Systemic allergic reactions 53 Systemic illnesses 77 Systemic lupus erythematosus (SLE) 320, 328 Systemic lupus erythematosus, scleroderma and Hashimoto’s thyroiditis 419, 420 Systemic neoplasms 323 Systolic blood flow 303 Systolic blood pressures 159 T T suppressor 294 Tachycardia 128, 168 Take home baby rate 250, 412 Tamoxifen 29, 30, 31, 32, 47 Tamoxifen-ER complex 30 Tamponade 128 Tastin 381 Taurine 269, 394 Taurine hypotarine 247 Tay-Sachs 334 TDF 309 Tefcat catheter 563 Telephone counselling 552 Telescope 465 Telomerase 364, 369, 270, 270 Temperature 270 Temperature decreases 334 Temporal window 383 Tenaculum 485, 486

Index

979

Teratocarcinoma tumors 364 Teratocarcinomas 364, 364, 364 Teratogenic 32, 123 Teratomas 364, 367 Teratomas or epithelial cysts 63 Teratozoospermia 514, 517, 518–519, 523 Teratozoospermic 227 TESA (Testicular sperm aspiration) 17, 335, 505, 507, 508 TESE 17 Test tube baby 5, 282 Test yolk 316 Testes 311 Testicle 569 Testicular atrophy 22 Testicular biopsies 23, 208, 335, 437, 438, 505, 509, 510 Testicular damage 321, 510 Testicular dysfunction 512, 522 Testicular feminization syndrome 17, 21 Testicular germ cell 271 Testicular histology 309 Testicular injury 22 Testicular mapping 504 Testicular sperm 291 Testicular sperm extraction (TESE) 503 Testicular spermatozoa 437 Testicular tissue 437, 509 Testicular tubule 509 Testicular tumors 435 Testicular volume 22, 503, 504, 522 Testiculopathy 310 Testis 9, 309, 310, 505, 507, 509, 510 Testis determining factor 309 Testis development 309 Testosterone 17, 22, 133, 137, 147, 148, 155, 155, 309, 309, 521 Testosterone, androstenedione, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulphate (DHAS), 17-hydroxy-progesterone (17-OHP) 155 Testosterone enanthate 522 Testosterone levels 134 Testosterone undecanoate 522, 523 Tetracopeptide repeat gene 310 Tetraploidy 529 Tetrazomy 529 TGF-beta 1 382 Thalassemia 334 Thawed blastocysts 257 Thawed embryo implantation 257 Thawed oocytes 258 The intra-cytoplasmic sperm injection (ICSI) 191 The low dose lupron protocol 561 Theca cell 10, 114, 121

Index

980

Theca cell hyperplasia 146 Theca cells to oestradiol 47 Therapeutic counselling 549, 550 Therapeutic regimen 321 Thermal 132 Thiazolidinediones 147, 149, 150 Third generation antagonists 78 Third party reproduction (TPR) 546, 550, 551 Thoracocentesis 128 Three-dimensional power Doppler imaging (3D-PDI) 23 Three-puncture laparoscopy 181 Threshold 8 Thrombocyte aggregation 124 Thrombocytes 124, 419 Thrombocytopenia 32, 422 Thromboembolic disease 32, 127, 128, 129, 460, 461 Thromboembolism 127, 415, 422 Thromboembolism adult respiratory distress syndrome 93 Thrombolic disease 63 Thromboplastin time (PTT) 422 Thrombosis 128, 293, 409, 411, 419, 420, 421 Thrombosis and thrombocytopenia 418 Thromboxane 2, 124, 409, 411 Thromboxane A 124 Thromboxane—prostacycline ratio 411 Thymine 333 Thyroglobulin 294 Thyroid 364 Thyroid antibodies 410 Thyroid cells 294 Thyroid function 307 Thyroid hormones 294 Thyroid peroxidase 294 Thyroid profile 410 Tissue 381 Tissue culture 365, 366 Tissue regeneration 365 Tissue-plasminogen 393 TMX 31 TMX administration 32 TNF 382 Tobacco 353 Tomcat catheter 562, 563 Topoisomerase II 271 TORCH 416 Total motile sperm count (TMSC) 231 Toxins 388 Toxoplasma 353 Toxoplasma gondii cytomegalo virus 416 Trachea 364 Traditional surrogacy 548 Transabdominal needling 497

Index

981

Transabdominal sonography 24 Transabdominal-transperitoneal ultrasound-guided oocyte retrieval 169 Transabdominal ultrasound 154, 405, 488, 489 Transcervical 175, 182, 497 Transcervical-transuterine tubal cannulation 165 Transcription 309, 369 Transcription factor 369 Transcriptional 389 Transcutaneous electric stimulation (TENS) 458 Transdermal skin 70 Transfer catheter 174 Transfer efficiency 388 Transferrin 367 Transforming growth factor (TGF-α) 392 Transforming growth factor (TGF-beta 1) 324, 382, 402 Transition metals 268 Translocation 317, 353, 335 Trans-membrane conductance regulator gene (CFTR) 311 Transmembrane heterodimers 384 Transmembrane receptors 66 Transmissable disorders 352 Transmission rates 341 Transplantation 323, 324, 352, 364, 365 Transplantation of ovarian tissue 322 Transvaginal biopsy 169 Transvaginal Douglas fertiloscope FTO 470 Transvaginal Douglas fertiloscopy 466 Transvaginal follicular aspiration 171 Transvaginal hydropelviscopy 464 Transvaginal needle aspiration 166 Transvaginal oocyte recovery 171 Transvaginal oocyte retrieval 171 Transvaginal ovarian drilling (TVOD) 136 Transvaginal ovarian puncture 165 Transvaginal probe 169 Transvaginal probe cover 166 Transvaginal route 128 Transvaginal sonography 23, 41 Transvaginal ultrasonography 32, 261 Transvaginal ultrasonography 40, 41, 454, 495, 496 Transvaginal ultrasound 41, 87, 488 Transvaginal ultrasound guided needle aspiration 171, 178 Transvaginal ultrasound probe 169 Transvaginal ultrasound-guided follicular aspiration 139 Transvaginal ultrasound-guided oocyte retrieval 169 Transvesical ovarian puncture 165 Trauma 138, 185 Trauma, morbidity 166 Treatment cycle 359 Treatment regimens 8 Tridimensional vaginal ultrasonography 454

Index

982

Triglyceride 159 Trinucleotide repeats 538 Triphenylethylene 30 Triple-line 397 Triplet 542 Triplet pregnancies 497 Triplets 133, 176, 181, 199, 355 Triploid 375 Triploid embryos 375 Triploidy 529 Triptorelin 84, 88, 95 Trisomic embryos 571 Trisomy 529 Troglitazone 150, 156 Trophectoderm 255, 257, 290, 382–384, 400 Trophinin 381 Trophoblast 400, 404, 419, 238, 381, 383, 389, 390, 391, 402, 403, 405, 409, 419–421 Trophoblast cells 421 Trophoblastic cells 409 Trophoblast-syncytiotrophoblast 421 Trophoectoderm 541, 541 Trypsin 370 Trypsin EDTA 365, 367 TSH 148 Tubal ampulla 175 Tubal cannulation 175 Tubal embryo transfer (TET) 177, 180 Tubal embryo-stage transfer (TEST) 180 Tubal factor 23, 90, 138, 182, 186 Tubal hydrosalpinx 24 Tubal infection 185 Tubal isthmus 467 Tubal occlusion 449 Tubal patency 135 Tubal pathology 182 Tubal pre-embryo transfer (TPET) 180 Tubal sterilization 4, 18, 358 Tubal surgery 446 Tubal transfer 175 Tube cannulation 174 Tubercular orchitis 22 Tuberculosis 474–476 Tuboovarian 21, 132, 444 Tubo-ovarian anatomy 446 Tubo-ovarian relation 448 Tumor necrosis factor (TNF) 271, 382 Tumors 24, 328, 364 Tumour gene 310 Tunica 509 Turner’s syndrome 21, 352, 357, 376 TVOD 137 Twin gestations 497

Index

983

Twinning 568 Twinning rates 200, 571 Twins 133, 176, 199, 238, 349, 355, 375, 542 Two cell mouse embryos 193, 250 Two-cell embryos 250 Type II diabetes mellitus 309 Tyrode solution 304 Tyrode’s salt solution 214 Tyrosine kinase 157 Tyrosine phosphorylation 149 Tyrosine receptor 42 U Ubiquitin-dependent response 372 Ubiquitin-specific protease 9 310 u-hFSH 51 u-hFSH HP 51 u-HMG 290 Ulcerative colitis 22 Ultra-low dose 116 Ultra-short protocol 83, 117 Ultra-short regimens 115 Ultrasonographic guidance 4 Ultrasonographic images 505 Ultrasonography 31, 128, 138, 185, 451, 474, 494 Ultrasonography or hysterosalpingography 451 Ultrasound 41, 90, 112, 128, 148, 156, 168, 264, 354, 416, 452, 458, 561 Ultrasound guidance 152, 291, 485 Ultrasound guidance for ET 488, 78, 565 Ultrasound guided follicle aspiration 170 Ultrasound measurement 56 Ultrasound scan 64, 154, 397, 497 Ultrasound, ECHO cardiograph 307 Ultrasound-guided follicular puncture 170 Ultrasound-guided oocyte retrieval 177, 168 Ultrasound-guided transfer 562 Umbilical trocar 181 Umbilicus 128 Undescended testes 22 Undifferentiated stem cells 368 Unexplained infertility 16 18, 24, 39, 90, 121, 136, 176, 180, 182, 184–187, 206, 210, 293, 298, 401, 404, 444 Unfertilized oocytes 35, 116 Unruptured follicles 63 Unruptured luteinized follicle: LUF 23 Unstimulated cycle 6, 55, 91 Urate α-tocopherol 269 Ureteroscopy 170 Urethra 208, 364 Urethral discharge 22

Index

984

Urinal FSH 521 Urinary bladder 364 Urinary extracts 56 Urinary FSH (u-hFSH) 50, 107, 539 Urinary gonadotropin preparations 113 Urinary gonadotropins 16, 52, 53, 111, 290 Urinary hCG 561 Urinary hMG 6, 90 Urinary LH 31 Urinary products 85 Urine-derived hMG 104 Urokinase 393 Uterine adhesions 450 Uterine anomalies 451 Uterine blood flow 291 Uterine blood vessels 291 Uterine cavity 23, 120, 122, 135, 177, 180, 182, 185, 186, 381, 451, 452, 454, 458, 473, 489, 560, 564 Uterine cells 298 Uterine cervix 493 Uterine contour 23 Uterine contractility 122 Uterine contractions 122, 394, 563 Uterine endometrium 299 Uterine epithelium 401 Uterine fibroids 17, 30 Uterine fundus 405 Uterine lining 291 Uterine malformation 416 Uterine ovarian ligament 467 Uterine preparation 400 Uterine receptivity 291, 298, 385, 386, 401, 408 Uterine secretions 386 Uterine septum 449 Uterine septum, submucus fibroid 443 Uterine vessels 383 Uterine wall 563 Uterocervical junction 184 Uteroovarian ligament 132 Uteroplacental 420 Uteroplacental vasculature 421 Uterotubal insemination catheter 187 Uterotubal ostium 175 Uterovaginal veins 123 Uterus 4, 23, 24, 123, 130, 180, 182, 186, 199, 241, 299, 309, 345, 353, 391, 448, 476, 571 Uterus, bladder 170 Utrogestan 132 UV laser 193 UV light 247 V

Index

985

Vacularization 410 Vacutainer 250 Vacuum 191 Vacuum pressure 137 Vagina 123, 166, 170, 364, 385 Vaginal progesterone 124 Vaginal apex 49 Vaginal cuff 49 Vaginal cultures 24, 411 Vaginal estradiol 122 Vaginal mucosa 467 Vaginal oocyte retrievals 78 Vaginal progesterone 122, 257 Vaginal route 452 Vaginal secretions 493 Vaginal sonography 494 Vaginal stenosis 169 Vaginal swabs 171 Vaginal vault 471 Vaginal wall vessels 170 Vaginismus 21, 549 Vaginoplasty 169 Vamins 124 Variable number tandem repeats (VNTR) 309 Varicocelectomy 275 Varicoceles 17, 22, 223, 228, 275, 276 Vas aplasia 508 Vas deferens 207, 211, 309, 311 Vascular endothelial growth factor (VEGF) 42, 298, 324, 382, 402 Vascular endothelium 400 Vascular follicles 236 Vascular permeability 93, 127, 389, 392, 402, 419 Vascularization 409 Vasculature lymphoid cells 405 Vasectomy 22, 206, 210, 223, 436 Vasectomy reversal 228, 276 Vasoactive 127 Vaso-epididymal anastomosis 507 Vasomotor 320 Vaso-vasal anastomosis 507 Ventilation perfusion 128 Veres needle 465, 467, 471 Vertebral osteomyelitis 171 Vertebrates 373 Vertical transmission 339 Viability 208 Viagra 291 Vinblastine 320 Vincristine 320, 321 Viraemia 341 Viral 111 Viral load 341, 339

Index

986

Virtual organ computer-aided analysis (VOCAL) 41 Viscosity 207 Vitamin E 267 Vitamin E and C 247 Vitrification 255, 257, 264 Vitrification medium 258 Volumetric 41 Vomiting 127 Vulvar oedema 128 W Wallace catheter 486, 489, 563 Water 251 Wavelength 245 Wedge biopsies 130 Wedge resection (BOWR) 130, 154 WHO threshold values 232 Window 383 Window of implantation 392, 401 Withdrawal bleeding 64, 354 Womb 3, 4 World Health Organization (WHO) 223, 516, 517, 537 Wunschkind 552 X X and Y bearing sperm 313 X and Y chromosome 313, 532 X and Y sperm 315 X chromosome 272, 307, 308, 314 X sperm 317 X spermatozoa 314 Xaprep 219 X-autosomal translocations 308 X-chromosome abnormalities 307 Xenotransplantation 323 X-linked 318 X-linked disease 532, 313 X-linked dominant 156, 307 X-linked dominant gene 308 X-linked hydrocephalus 315 XX males 512 Y Y enrichment 314 Y sperm 315, 317 Y spermatozoa 314 Y-chromosome 272, 309, 310, 372, 504, 505, 512–515, 560 Y-chromosome deletions 311 Y-chromosome micro-deletions 514 Y-chromosome mutations 514 Y-deletions 546

Index

987

Yolk buffer 191 Yolk sac 367 Z ZIFT 9, 182 Zona 153, 191 Zona binding assay 206, 209 Zona binding capacity 520 Zona dissection 194, 567 Zona drilling 192, 568 Zona erosion 567 Zona pellucida (ZP) 189, 192, 194, 210, 241, 243, 251, 257, 291, 295, 304, 323, 403, 431, 517, 520, 567 Zona photoablation 193 Zona reaction 295 Zona thinning 568 Zonae pellucidae 436 Zona-free hamster egg 22 ZP 191 Zygote 180, 237, 373 Zygote and blastocyst 541 Zygote intrafallopian transfer (ZIFT) 15, 171, 180, 182 Zygote viability 113 Zygotes 9, 182, 194, 198, 199, 236, 237, 430, 431

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