Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives

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Textbook of Assisted Reproductive Techniques

Textbook of Assisted Reproductive Techniques Laboratory and Clinical Perspectives EDITED BY

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

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

MARTIN DUNITZ © 2001 Martin Dunitz Ltd, a member of the Taylor & Francis group First published in the United Kingdom in 2001 by Martin Dunitz Ltd, The Livery House, 7–9 Pratt Street, London NW1 0AE Tel: +44 (0) 20 7 482 2202 Fax: +44 (0) 20 7 267 0159 E-mail: [email protected] Website: http://www.dunitz.co.uk/ This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge's collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP. Although every effort has been made to ensure that drug doses and other information are presented accurately in this publication, the ultimate responsibility rests with the prescribing physician. Neither the publishers nor the authors can be held responsible for errors or for any consequences arising from the use of information contained herein. For detailed prescribing information or instructions on the use of any product or procedure discussed herein, please consult the prescribing information or instructional material issued by the manufacturer. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. A CIP record for this book is available from the British Library.

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Contents Preface

xii

Acknowledgments

xiii

List of contributors

xiv

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

1

LABORATORY PROCEDURES Setting up an ART Laboratory 1 2 3

4

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

25

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

38

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

49

Accreditation of IVF laboratories: the European perspective Cecelia Sjöblom

66

Handling of the Sperm 5 6

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

86 112

Handling of the Oocyte 7 8

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

129

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

142

9

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

156

Manipulation 10

11

12 13

14

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

214

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

230

Assisted hatching Anna Veiga, Irene Boiso

248

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

264

Human embryo biopsy for preimplantation genetic diagnosis Alan H Handyside

291

Handling of the Embryo and Embryo Transfer 15 16 17

Analysis of fertilization Lynette Scott

308

Embryo culture David K Gardner, Michelle Lane

324

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

359

Cryopreservation of Gametes and Embryos 18 19 20 21

Oocyte cryopreservation Eleonora Porcu

375

Cryopreservation of human embryos Jacqueline Mandelbaum, Yves JR Ménézo

392

Managing the cryopreserved embryo bank Phillip Matson, Neroli Darlington

416

Cryopreservation and storage of sperm Eileen A McLaughlin

423

Eileen A McLaughlin 22

23

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

443

Ovarian tissue cryopreservation and transplantation Kutluk H Oktay

450

Diagnosis of Genetic Disease in Preimplantation Embryos 24

25 26 27

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

459

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

481

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

518

Polar body biopsy Yury Verlinsky, Anver Kuliev

539

Implantation 28

29

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

551

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

572

Data Management and Interpretation 30

31

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

599

Evidence based medicine Salim Daya

623

CLINICAL APPLICATIONS AND PROCEDURES Patient Investigation and the Use of Drugs

32 33 34

35

36

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

643

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

657

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

677

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

698

Use of recombinant DNA technology in ART Colin M Howles

736

Stimulation Protocols 37 38

39 40 41 42 43 44 45

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

753

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

777

The use of GnRH agonists Roel Schats, Joop Schoemaker

792

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

807

Monitoring IVF cycles Matts Wikland, Torbjörn Hillensjö

821

Follicle aspiration Carl Wood

828

The luteal phase: luteal support protocols James P Toner

844

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

864

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

892

Different Technical Procedures 46 47 48 49 50

Ultrasound in ART Marinko M Biljan

922

Epididymal and testicular sperm extraction: clinical aspects Herman Tournaye

959

Gamete intrafallopian transfer (GIFT) Machelle M Seibel

977

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

992

Embryo transfer William B Schoolcraft

1017

Special Medical Conditions 51 52

Endometriosis Mark I Hunter, Alan H DeCherney

1023

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

1040

Complications of Treatment 53 54 55 56 57

Severe ovarian hyperstimulation syndrome Daniel Navot

1052

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

1069

Iatrogenic multiple pregnancy: the risk of ART Isaac Blickstein

1082

Reducing the incidence of multiple gestation David R Meldrum

1101

Multifetal pregnancy reduction and selective termination Shlomo Lipitz

1109

Egg Donation and Surrogate Motherhood 58 59

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

1126 1144

Peter R Brinsden The Support Team 60

Patient support in the ART program Sharon N Covington

1165

Ethics and Legislation 61 62

Worldwide legislation Jean Cohen, Howard W Jones, Jr

1182

Times of transition: modern ethical dilemmas Françoise Shenfield

1211

Index

1123

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Introduction The beginnings of human in vitro fertilization Robert G Edwards

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

INTRODUCTION So first of all, a few tributes must be expressed to my teachers. In fact, these include investigators from the far off days of earlier centuries when the fundamental facts of reproductive cycles, surgical techniques, endocrinology, and genetics were reported by many investigators. These fields really began to move in the twentieth century, and if one of these pioneers should be saluted, it must be Gregory Pincus. Famous for the contraceptive pill, he was a distinguished embryologist, and part of his work dealt with the maturation of mammalian oocytes in vitro. He was first to show how oocytes aspirated from their follicles would begin their maturation in vitro and a number would complete it by expelling a first polar body. I believe his major work was done in rabbits, where he found that the 10–11 hours timings of maturation in vitro accorded exactly with those occurring in vivo after an ovulatory stimulus to the female rabbit. Pincus et al also studied human oocytes.1 Extracting oocytes from excised ovaries, they identified chromosomes in a large number of oocytes and interpreted this fact as evidence of the completion of maturation in vitro. Many oocytes possessed chromosomes after 12 hours, the proportion remaining constant over the next 30 hours and longer. Twelve hours was taken as the period of maturation. Unfortunately, chromosomes were not classified for their meiotic stage. Maturing oocytes would be expected to display diakinesis or metaphase 1 chromosome pairs. Fully mature oocytes would display metaphase 2 chromosomes, and so be fully ripe and ready for fertilization.

Textbook of assisted reproductive techniques

2

Unfortunately, it is well known that oocytes can undergo atresia in the ovary involving the formation of metaphase 2 chromosomes in many of them. These oocytes complicated Pincus’s estimates, even in controls, and was the source of the error leading later workers to inseminate human oocytes 12 hours after collection and culture in vitro.2,3 Work on human fertilization in vitro, and indeed comparable studies in animals, then remained in abeyance for many years. Progress in animal IVF had also been slow. After many relatively unsuccessful attempts on several species in the 1950s and 1960s, a virtual dogma arose that spermatozoa had to spend several hours in the female reproductive tract before acquiring the potential to bind to the zona pellucida and achieve fertilization. Later, in 1969 Austin and Chang independently identified the need for sperm capacitation, identified by a delay in fertilization after spermatozoa had entered the female reproductive tract.4,5 This discovery was taken by many investigators as the reason for the failure to achieve fertilization in vitro, and why spermatozoa had to be exposed to secretions of the female reproductive tract. At the same time, Chang reported that rabbit eggs that had fully matured in vitro failed to produce normal blastocysts and none implanted normally.4

MODERN BEGINNINGS OF HUMAN IVF, PREIMPLANTATION GENETIC DIAGNOSIS, AND EMBRYO STEM CELLS When I started my PhD in Edinburgh in 1952, encouraged by Professor Conrad Waddington and supervised by Dr Alan Beatty, capacitation was gaining prominence. I decided to study the growth of mouse embryos with altered chromosome complements. It would be essential to expose mouse spermatozoa to x rays, ultraviolet light, and various chemicals in vitro to destroy their chromatin so they could fertilize eggs but make no genetic contribution to the embryo. These embryos would become gynogenetic haploid embryos. Later, I exposed eggs to colchicine, in order to destroy the second meiotic spindle in mouse eggs. All chromosomes are thus freed from spindle attachment and exit from the egg into tiny artificial polar bodies. A fertilizing spermatozoon would therefore enter an empty egg, to result in androgenetic haploid embryos with no genetic contribution from the maternal side. For three years, my work was concentrated in the mouse house, working at midnight to identify mouse females in estrus by vaginal smears, collecting epididymal spermatozoa from males, and practising artificial insemination with samples of treated spermatozoa. My research was successful, and mouse embryos were identified with haploid, triploid, tetraploid, and aneuploid chromosomes. Moreover, the wide scientific talent in the institute was a perfect place for fresh collaborative studies. Julio Sirlin and I applied the use of radioactive

Introduction

3

DNA and RNA precursors to the study of spermatogenesis, spermiogenesis, fertilization, and embryogenesis and gained knowledge unavailable elsewhere. An even greater fortune beckoned. Allen Gates, newly arrived from the United States, brought commercial samples of Organon’s pregnant mares’ serum (PMS) rich in follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) with strong luteinizing hormone (LH) activity to induce oestrus and ovulation in immature female mice. Working with Mervyn Runner,5 they had used low doses of each hormone at an interval of 48 hours to induce oocyte maturation, mating and ovulation in immature mouse females. He now wished to measure the viability of 3-day embryos from immature mice by transferring them to an adult host to grow to term.6 I was more interested in stimulating adult mice with these gonadotrophins to induce oestrus and ovulation, by now weary of taking vaginal smears. My future wife, Ruth Fowler, and I teamed up to test superovulating adult mice. HCG has stayed with me from that moment, even until today. Opinion in those days was that exogenous hormones such as PMS and HCG would stimulate follicle growth and ovulation in immature female mammals, but not in adults because they would interact badly with the adult’s reproductive cycles. The hormone preparations really worked. Doses of 1–3 IU PMS induced the growth of numerous follicles, and similar doses of HCG 42 hours later invoked oestrus and ovulation a further six hours later in almost all of them. Often, 70 or more ovulated oocytes crowded the ampulla, most of them being fertilized and developing to blastocysts. Multiple implantations occurred just as similar treatments in anovulatory women were to do some years later.7 Oocyte maturation, ovulation, mating and fertilization were each closely timed in all adults, another highly unusual aspect of stimulation.8 Diakinesis was identified as the germinal vesicle regressed, with metaphase 1 a little later and metaphase 2, expulsion of the first polar body, and ovulation at 11.5–12 hours after HCG. Years afterwards, germinal vesicle breakdown and diakinesis were to prove equally decisive in identifying meiosis and ovulation in human oocytes in vivo and in vitro. Even as these results were gained, Ruth and I departed in 1957 from Edinburgh to the California Institute of Technology, where I switched into immunology and reproduction, a topic that was to dominate my life for five or six years on my return to UK. The Institute had given me an excellent basis in genetics, but equally in reproduction. I had gained considerable knowledge about the endocrine control of oestrus cycles, ovulation, spermatozoa, and the male reproductive tract artificial insemination and the stages of embryo growth in oviduct and uterus, superovulation and its consequences, and the use of radiolabelled compounds. All this knowledge was to prove of immense value in my later career. Much of it was cemented in California. After California, London beckoned first, to the National Institute for Medical Research working with Drs Alan Parkes and Colin (Bunny)

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Austin. After two intense years on immunology, my curiosity returned to maturating oocytes and fertilization in vitro. It should be easy to stimulate maturation in mouse oocytes in vitro by using gonadotrophins if they matured so easily in vivo. In fact, to my immense surprise, oocytes matured immediately in vast numbers in all groups, exactly on time with those matured in vivo in Edinburgh. Adding hormones made no difference. Rabbit, hamster, and rat oocytes also matured with 12 hours, each at their own species specific rates. Then, again to my surprise, oocytes from cows, sheep, and rhesus monkeys, and the occasional baboon, did not mature in vitro within 12 hours, their germinal vesicles persisting unmoved. How would human oocytes respond? A unique opportunity emerged to collect pieces of human ovary, and to aspirate human oocytes from their occasional follicles. I grasped it with alacrity.

MOVING TO HUMAN STUDIES Molly Rose was a local gynaecologist who delivered two of our daughters. She accepted to send slithers or wedges of ovaries such as those removed from patients with polycystic disease as recommended by Stein and Leventhal, or with myomata or other disorders demanding surgery. Stein-Leventhal wedges were the best source, with their numerous small Graafian follicles lined up in a continuous rim just below the ovarian surface. Though samples were rare, they provided enough oocytes to start with. These oocytes responded just as those oocytes from other adult mammals, as germinal vesicles persisted and diakinesis was absent. This was disappointing, especially as Tjio and Levan, and Ford, identified 46 diploid chromosomes in humans and teams in Edinburgh (Scotland), France, and elsewhere discovered monosomy or disomy in many men and women. XO and XXX women, and XY and XYY in men were identified, and trisomy 21 proved to be the most common cause of Down’s syndrome. The origins of these genetic disorders were hidden in the meiotic chromosomes in maturing oocytes, especially diakinesis, and all this new information reminded me of my chromosome studies on the Edinburgh mice. I had to obtain diakinesis and metaphase I in human oocytes, and continue to metaphase 2 when the oocytes would be fully mature ready for fertilization. Despite being disappointed at current failure with human oocytes, it was time to write my findings for Nature in 1962.9 There was so much to write about the animal work, and describing the new ideas then taking shape in my mind. I had heard institute lectures on infertility, and realised that fertilizing human oocytes in vitro and replacing embryos into the mother could help to alleviate this condition. It could also be possible to type embryos for genetic diseases when a familial disposition was identified. Pieces of tissue, or one or two blastomeres, would have to be excised from blastocysts or cleaving

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embryos, but this did not seem to be too difficult. There were few genetic markers available for this purpose in the early 1960s, but it might be possible to sex embryos by their XX or XY chromosome complement, in mitoses in the excised cells. Choosing female embryos for transfer would avert the birth of boys with various sex linked disorders such as haemophilia. I was becoming totally committed to human IVF and embryo transfer. While reading in the library for any newly published papers relevant for my proposed Nature manuscript, I discovered those earlier papers of Pincus and his colleagues described above. They had apparently succeeded 30 years earlier in maturing human oocytes cultured for 12 hours, where I had failed. My Nature paper9 became very different to that originally intended, even though it retained enough for publication. Those results of Pincus et al had to be repeated. After trying hard, I failed to repeat them. Intact ovaries were perfused in vitro with gonadotropin solutions, different culture media failed, joint cultures with mouse oocytes failed, and added hormones had no effect. It began to seem that menstrual cycles affected oocyte physiology. Finally, after two years of fruitless research on the precious few human oocytes available, an idea struck. Perhaps maturation timings differed widely between mice and rabbits compared with cows, baboons, and humans. Even as my days in London were ending, Molly Rose sent a slither of human ovary. The few oocytes were placed in culture. The germinal vesicle remained static for 12 hours and then 20 hours in vitro. Three oocytes remained, as I waited for 24 hours. The first contained a germinal vesicle. So did the second. There was one left and one only. I was overjoyed as I gazed down the microscope. Its chromosomes were in diakinesis and its germinal vesicle was regressing. The chromosomes were superb examples of human diakinesis with their classical chiasmata. This was the step I had waited for, a marker that Pincus had missed. He never checked for diakinesis and apparently confused atretic oocytes, which contained chromosomes, with maturing oocytes. Endless human studies were now opening. It was easy now to calculate the timing of the final stages of maturation as ending at about 36 hours, which would be the moment for insemination. All these gaps in knowledge had to be filled, but now we were on the way to human IVF. And at this wonderful moment, John Paul, an outstanding cell biologist, invited me to join him and Robin Cole in Glasgow University to study differentiation in early mammalian embryos. This was exciting, to work in biochemistry with a leading cell biologist. We wanted to grow stem cells from mammalian embryos and study them in vitro. Over 12 months, stem cells migrate out from cultures of rabbit blastocysts, forming muscle and blood islands in vitro.10 A line of embryonic stem cells characterized for biochemical markers was established. Thoughts were strengthened of growing stem cells from human embryos to repair defects in tissues of children and adults. Almost at my last moment in Glasgow, a piece of ovary yielded

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several oocytes and two had reached metaphase 2 and expelled a polar body at 37 hours. The whole pattern of oocyte maturation was increasingly clear even if still in outline. Resuming a mixture of immunology and reproduction remained my dominant themes in the Physiological Laboratory at Cambridge as I rejoined Alan Parkes and Bunny Austin. Thoughts of human oocytes and embryos were never far away. One possible model of the human situation was the cow, and large numbers of cow, pig, and sheep oocytes were available from ovaries given to me by the local slaughterhouse. Each species had its own timing, all of them longer than 12 hours.11 Human oocytes trickled in, improving my provisional timings of maturation. One or two spare oocytes were inseminated, without signs of fertilization. More oocytes were urgently needed to conclude the timings of oocyte meiosis. Surgeons in Johns Hopkins Hospital, Baltimore (United States), performed the Stein-Leventhal operation, so I could collect the oocytes and send the remaining tissues to pathology if necessary. Victor McKusick worked there and supported my request for a visit to work with the hospital gynaecologists for six weeks. He made laboratory space available and, a wonderful invitation, introduced me to Howard and Georgeanna Jones. This significant moment was equal to my meeting with Dr Rose. The Jones’ proved to be superb and unstinting in their support. Sufficient wedges and other ovarian fragments were available to complete the maturation programme in human oocytes. Within three weeks, every stage of meiosis was classified and timed.12 With Howard and Georgeanna Jones, preliminary studies were made on human fertilization in vitro, using different media or fragments of ampulla in the cultures, and even attempting fertilization in rhesus monkey oviducts. Two nuclei were found in some inseminated eggs, resembling pronuclei, but sperm tails were not identified so no claims could be made.13 During those six weeks, oocyte maturation was fully timed at 37 hours, permitting me to predict that women would ovulate at 37 hours approximately after an HCG injection. For clinical trials, a simple access to aspirate human ovarian follicles in vivo was needed to aspirate them 36 hours after HCG, just before follicular rupture. Who could provide this? And how about sperm capacitation? Only in hamsters had fertilization in vitro been achieved, using in vivo matured oocytes and epididymal spermatozoa.16 I met Victor Lewis, my third clinical colleague, and we noticed what seemed to be anaphase 2 in some inseminated eggs. Again, no sperm tails were seen within the eggs. An attempt to achieve human capacitation, in Chapel Hill, North Carolina, United States, working with Robert McGaughey and his colleagues, also failed.17 A small intrauterine chamber lined with porous membrane was filled with washed human spermatozoa, sealed, and inserted overnight into the uterus of human volunteers at mid cycle. Molecules entering it could react with the spermatozoa. No matured

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human eggs were fertilized. Later evidence indicated that the chamber contained inflammatory proteins, perhaps explaining the failure.

DECISIVE STEPS TO CLINICAL HUMAN IN VITRO FERTILIZATION Back in the United Kingdom, my intention to conceive human children in vitro was even stronger. So many medical advantages could flow from it. Human embryos had been very rare, flushed from a human oviduct or uterus after sexual intercourse. It was time to attain fertilization, and move close to working with infertile patients. Ethical issues and moral decisions would emerge, one after the other, in full public view. Matters such as cloning and sexing embryos, the risk of abnormalities in the children, the clinical use of embryo stem cells, the ethics of oocyte donation and surrogate pregnancy, and the right to initiate human embryonic life in vitro were never very far away. I accepted all these issues, remaining confident that a study of human conception was correct ethically, medically, and scientifically even to use the increasing knowledge of genetic and embryology to be applied in such human studies. Few human oocytes were available in the United Kingdom. Despite this scarcity, one or two of those matured and fertilized in vitro possessed two nuclei after insemination. But there were no obvious sperm tails. I devised a cow model for human fertilization, using in vitro matured oocytes and insemination in vitro with selected samples of highly active washed bull spermatozoa. It was a pleasure to see some fertilized bovine eggs, with sperm tails and characteristic pronuclei, especially using spermatozoa from one particular bull. Things were suddenly changing. A colleague had stressed that formalin fixatives were needed to detect sperm tails in eggs. Barry Bavister joined our team to study for his PhD and designed a medium of high pH, which gave excellent fertilization rates in hamsters. We decided to collaborate by using it for trials on human fertilization. Third, and by no means least, while browsing in the library of the Physiological Laboratory, a paper in the Lancet caught my attention. Written by Dr P C Steptoe of the Oldham and District General Hospital,18 it described laparoscopy, with its narrow telescope and instruments and the minute abdominal incisions. He could visualize the ampulla and place small amounts of medium there. This is exactly what I wanted because access to the ampulla was equivalent to gaining access to ovarian follicles. He had worked closely with two pioneers, Palmer in Paris19 and Fragenheim in Germany.20 He improved the pneumoperitoneum, to gain working space in the abdominal cavity, and used carbon fibers to pass cold light into the abdomen from an external source.21 Despite advice to the contrary from several medical colleagues, I telephoned him about

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collaboration and stressed the uncertainty in achieving fertilization in vitro. He responded most positively, just as Molly, Howard and Georgeanna, and Victor had done. We began our collaboration six months later in the Oldham and District General Hospital, almost 200 miles north of Cambridge.

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

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Molly Rose sent me a small piece of ovary to Cambridge. Its dozen or more oocytes were matured in vitro for 37 hours when Barry and I added washed spermatozoa suspended in his medium. We examined them a few hours later. To our delight, spermatozoa were pushing through the zona pellucida, into the eggs. Maternal and paternal pronuclei were forming beautifully. We saw polar bodies and sperm tails within the eggs. That evening in 1969, we watched in delight virtually all the stages of human fertilization in vitro (Fig 1). One fertilized egg had fragments as Chang had forecast, strengthening the need to abandon oocyte maturation in vitro and replace it with stimulation of maturation in vivo by means of exogenous hormones. Our paper in Nature surprised a world unaccustomed to the idea of human fertilization in vitro.22 Incredibly fruitful days followed in our Cambridge laboratory. Richard Gardner, another PhD candidate, and I excized small pieces of trophectoderm from rabbit blastocysts and sexed them by staining the sex chromatin body. Those classified as female were transferred into adult females and were all correctly sexed at term. This work transferred my theoretical ideas of a few years earlier into the practice of preimplantation diagnosis of inherited disease, in this case sex linked diseases.23 Three years later we tried to repeat this work in human blastocysts, but they failed to express either sex chromatin or the male Y body. Human preimplantation genetic diagnosis would have to wait a little longer. Alan Henderson, a cytogeneticist, and I analysed chiasmata during diakinesis in mouse and human eggs, and explained the high frequencies of Down’s syndrome in offspring older mothers as a consequence of meiotic errors arising in oocytes formed last in the fetal ovary which were then ovulated last at the later maternal ages.24 Dave Sharpe, a lawyer from Washington, joined forces to write an article in Nature25 on the ethics of in vitro fertilization, the first paper ever in the field. I followed this up with a detailed analysis of ethics and law in IVF covering scientific possibilities, oocyte donation, surrogacy by embryo transfer, and other matters.25 So the first ethical papers were written by scientists and lawyers and not by philosophers, ethicists, or politicians.

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

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women by Bruno Lunenfeld.26 It removed the need for PMS, so avoiding the use of non-human hormones. Doses to patients were kept low: to 2–3 vials (a total of 150–225 IU) given on days 3, 5, and 7. 5000 IU of HCG were given on day 10. Initially, oocyte maturation in vitro was confirmed, performing the laparoscopic collection at 28 hours after HCG to check the oocytes were in metaphase 1,27 and then at 36 hours to collect mature metaphase 2 oocytes for fertilization. Those beautiful oocytes were surrounded by masses of viscous cumulus cells and maturing exactly as predicted. We witnessed follicular rupture at 37 hours through the laparoscope. Follicles could be classified from their appearance as ovulatory or non-ovulatory, this diagnosis being confirmed later by assaying several steroids in the aspirated follicular fluids (Fig 2). It was a pleasure and a duty to meet the patients, searching for help to alleviate their infertility. We did our best driving from Cambridge to Oldham at noon to prepare the small laboratory there. Patrick had stimulated the patients with HMG and HCG, and he and his team led by Muriel Harris arrived to prepare for surgery. Patrick’s laparoscopy was superb. Ovarian stimulation, even though mild, produced five or six mature follicles per patient, and the ripe oocytes came in a steady stream, into my culture medium for insemination and overnight incubation. The next morning, the formation of two pronuclei and sperm tails indicated fertilization had occurred, even in simple media, now with a near neutral pH. Complex culture media, Ham’s F10 and others each with added serum or serum albumin, sustained early and later cleavages,28 and, even more fascinating, the gradual appearance of morulae and then light, translucent blastocysts (Fig 3).29 Here was my reward—growing embryos was now routine, and examinations of many of them convinced me the time had come to replace them into the mothers’ uteri. I had become highly familiar with the teratological principles of embryonic development and knew many teratologists. The only worry I had was the chance of chromosomal monosomy or trisomy, on the basis of our mouse studies, but these conditions could be detected later in gestation by amniocentesis. And our human studies had surpassed work on all animals, a point rubbed in even more when we grew blastocysts to day 9 after they had hatched from their zona pellucida (Fig 4).30 This beautifully expanded blastocyst a large embryonic disc was a potential source of embryonic stem cells.

Fig 2 Eight steroids were assayed in fluids extracted from human follicles aspirated 36–37 hours after HCG. The follicles had been classified as ovulating or nonovulating by laparoscopic examination in vivo. Data were analysed by cluster analysis, which groups follicles with similar features. The upper illustration shows data collected during the natural menstrual cycle. Note that two sharply separated groups of follicles were identified, each with very low levels of

within group variance. Attempting to combine the two groups resulted in a massive increase of within group variation, indicating that two sharply different groups had been identified. These different groups accorded exactly with the two groups identified by means of steroid assays. The lower figure shows the same analysis during stimulated cycles on fluids collected at 36–37 hours after HCG. With this form of stimulation, follicle growth displays considerable variation within groups. Attempts to combine all the groups, result in a moderately large increase in variation. This evidence suggests that follicles vary considerably in their state of development in simulated cycles using HMG and HCG.

Fig 3 Successive stages of human preimplantation development in vitro in a composite illustration made in Oldham in 1971. Upper left: 4 cell stage showing the crossed blastomeres typical of most mammals. Upper middle: 8 cell stage showing the even outline of blastomeres and a small piece of cumulus adherent to

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the zona pellucida. Upper right: a 16–32 cell stage, showing the onset of compaction of the outer blastomeres. Often, blastocoelic fluid can be seen accumulating between individual cells to give a “stripey” appearance to the embryo. Lower left and middle: two living blastocysts showing a distinct inner cell mass, single celled trophectoderm, blastocoelic cavity, and thinning zona pellucida. Lower right: a fixed preparation of a human blastocyst at five days, showing more than 100 even sized nuclei and many mitoses. There were very very few plaudits for us, criticism being mostly aimed at me, as usual when scientists bring new challenges to society. Criticism came not only from the Pope and archbishops, but also from scientists who should have known better, including James Watson (who testified to a US Senate Committee that many abnormal babies would be born) and Max Perutz, who supported him. These scientist critics knew virtually nothing about my field, so who advised them to make such ridiculous charges? Cloning football teams or intelligentsia was always raised by ethicists, which clearly dominated their thoughts rather than the intense hopes of our infertile patients. Yet one theologian, Gordon Dunstan, who became a close friend, knew all about IVF from us, and wrote an excellent book on its ethics. He was far ahead of almost every scientist in my field of study. Our patients gave us their staunch support. So too did the Oldham Ethical Committee and Bunny Austin back home in Cambridge. Growing embryos became routine, so we decided to transfer one of them to their mothers’ uteri. Here again we were in untested waters. Transferring embryos via the cervical canal, the obvious route was virtually a new and untested method. We would have to do our best with it. And from now on, we worked with patients who had seriously distorted tubes or none whatsoever. We had to do this because no one would have believed us if we had claimed a test tube baby in a woman with near normal tubes. This had to be a condition of our work, yet it led many people to believe we started IVF to bypass occluded oviducts, when we already knew that embryos could be obtained for men with oligozoospermia or antibodies to their gametes, and for women in various stages of endometriosis. One endocrinological problem did worry me. Stimulation with HMG and HCG shortened the succeeding luteal phase, to an impossibly short time for embryos to

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Fig 4 A hatched human blastocyst after nine days in culture. Notice the distinct embryonic disc and the possible bilaminar structure of the membrane. The blastocyst has expanded considerably, as shown by comparing its diameter with that of the shed zona pellucida. The zona contains dying and necrotic cells and its diameter provides an estimate of the original oocyte end embryo diameters. implant before the onset of menstruation. Levels of urinary pregnanediol also declined soon after oocyte collection. This condition was not a result of the aspiration of granulosa and cumulus cells, and luteal support would be needed, preferably progesterone. Csapo stressed how this hormone was produced by the ovaries for the first 8–10 weeks before the placenta took over this function.31 Injections of progesterone in oil given over that long period of time seemed unacceptable since it would be extremely uncomfortable to patients. While mulling over this problem, my attention turned to those earlier endocrinologists who believed that exogenous hormones would distort the reproductive cycle although I doubt they even knew anything about a deficient luteal phase. This is how we unknowingly made our biggest mistake in early IVF days. Our choice of Primulot depot, a progestagen, meant it should be given every five days to sustain pregnancies, since this progestagen supposedly saved threatened abortions. So we began transfers in stimulated cycles, giving this luteal phase support. Even though our work

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was slowed by having to wait to see if pregnancies arose in one group of patients before stimulating the next, enough patients had accumulated after three years. None of our patients was pregnant. Disaster loomed. Our critics were even more vociferous as the years passed, and mutual support between Patrick and I had to pull us through. Twenty or more different factors could have caused our failure, e.g. cervical embryo transfers, abnormal embryos, toxic culture dishes or catheters, inadequate luteal support, incompatibility between patients’ cycles and that imposed by HMG and HCG, inherent weakness in human implantation, and many others. We had to get every scrap of information from our failures. I knew Ken Bagshawe in London who was working with improved assay methods for gonadotrophic hormones. He offered to measure blood samples taken from our patients over the implantation period using his new HCGß assay. He telephoned: three or more patients previously undiagnosed had actually produced shortlived rises of HCGß over this period. Everything changed with this information. We had established pregnancies after all, but they had aborted very early. Today we call them biochemical pregnancies. It had taken us almost three years to identify the cause of our failure, and the finger of suspicion pointed straight at Primulot. I knew it was luteolytic, but it was apparently also an abortifacient, and our ethical decision to use it had caused much heartache, immense loss of work and time, and despair for some of our patients. The social pressures had been immense, with critics claiming our embryos were dud and our whole programme was a waste of time. But we had come through it and now knew exactly what to do next. We reduced levels of Primulot depot, and used HCG and progesterone as luteal aids. We at least now suspected that single embryo transfers could produce a 15–20% chance of establishing pregnancy. Almost instantly, our first clinical pregnancy arose after the transfer of a single blastocyst in a patient stimulated with HMG and HCG.32 Fantastic news— a human embryo fertilized and grown in vitro had produced a pregnancy. Everything seemed fine, even with ultrasound images. My culture protocols were satisfactory after all. Patrick rang: he feared the pregnancy was an ectopic and he had to remove it somewhere after 10 gestational weeks. Every new approach we tested seemed to be ending in disaster, yet we would not stop, since the work itself seemed highly ethical, and conceiving a child for our patients was perhaps the most wonderful thing anyone could do for them. And, in any case, ectopic pregnancies are now known to be a regular feature with assisted conception. I sensed we were entering the final phase of our Oldham work, seven years after it began. We had to speed up, partly because Patrick was close to retiring from the National Health Service. Four stimulation protocols were tested in an attempt to avoid problems with the luteal phase: HMG and HCG, clomiphene, HMG and HCG to gain a better luteal phase, bromocryptine, HMG and HCG because some patients had high prolactin concentrations, and HCG alone at mid cycle. We also tested what became

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Fig 5 The first attempts at gamete intrafallopian transfer (GIFT) were called oocyte recovery for tubal insemination (OR+TI). In this treatment cycle, using HMG and HCG, including additional injections of HCG for luteal support, a single preovulatory oocyte and 1.6 million sperm were transferred into the ampulla. to be known as gamete intrafallopian transfer (GIFT), calling it ORTI (oocyte recovery with tubal insemination, by transferring one or two eggs and spermatozoa to the ampulla) (Fig 5). Natural cycle IVF was introduced, based on collections of urine samples at regular intervals eight times daily, to measure exactly the onset of the LH surge using a modified Higonavis assay (Fig 6). Cryopreservation was also introduced, by freezing oocytes and embryos that looked to be in good condition when thawed. A recipient was given a donor egg fertilized by her husband’s spermatozoa, but pregnancy did not occur. Lesley and John Brown came

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as the second entrants for natural cycle IVF. Lesley had no oviducts. Her egg was aspirated in a few moments and inseminated simply and efficiently. The embryo grew beautifully. Their positive pregnancy test a few days after transfer was another milestone—surely nothing could now prevent their embryo developing to full term in a normal reproductive cycle, but those nine long months lasted a very long time. Three more pregnancies were established using natural cycle IVF as we abandoned the other approaches. A triploid embryo died in utero—more bad luck. A third pregnancy was lost through premature labour on a mountain walking holiday two weeks after the mother’s amniocentesis.32,33 It was a lovely well developed boy. Louise Brown’s birth, and then Alistair’s, proved to a waiting world that science and medicine had entered human conception. Our critics declared that the births were a fake, and advised against attending the presentation on the whole of the Oldham work at the Royal College of Obstetrics and Gynaecology.

IVF WORLDWIDE The Oldham period was over. Good facilities were now needed, with space for a large IVF clinic. Bourn Hall was an old Jacobean house in lovely grounds near Cambridge. Facilities on offer for IVF in Cambridge were far too small, so we purchased it mostly with venture capital. It was essential to conceive 100 or 1000 IVF babies to ensure it was safe and effective clinically. The immense delays in establishing Bourn Hall delayed our work by two years after Louise’s birth. Finally, on minimal finance, Bourn Hall opened in September 1980 on a shoestring, supported by our own cash and loans. The delay gave the rest of the world a chance to join in IVF. Alex Lopata delivered an IVF baby in Australia, and one or two others were born elsewhere. Natural cycle IVF was chosen in Bourn Hall since it had proved successful in Oldham, and we became experts in it. Pregnancies flowed, at 15% per cycle. An Australian team of Alan Trounson and Carl Wood announced the establishment of several IVF pregnancies after stimulation by clomiphene and HCG and replacing two or three embryos,34 so they had moved ahead of us during the delayed opening of Bourn Hall. Our own effort expanded prodigiously. Thousands of patients queued for IVF. Simon Fishel, Jacques Cohen, and Carol Fehilly joined the embryology team, and new clinicians joined Patrick and John Webster. Patients and pregnancies increased rapidly, and the world was left standing far behind. Howard and Georgeanna

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Fig 6 Recording the progress of the human natural menstrual cycle for IVF. Three patients are illustrated. All three displayed rising 24 hour urinary oestrogen concentrations during the follicular phase, and rising urinary pregnanediol concentrations in the luteal phase. LH levels were measured several times daily and the data clearly reveals the exact time of onset of the LH surge. Jones began in Norfolk using gonadotrophins for ovarian stimulation. Jean Cohen began in Paris, Wilfred Feichtinger and Peter Kemeter in Vienna, Klaus Diedrich and Hans van der Venn in Bonn, Lars Hamberger and Matts Wikland in Sweden, and Andre van Steirteghem and Paul Devroey in Brussels. IVF was now truly international. The opening of Bourn Hall had not deterred our critics. They put up a fierce rearguard action against IVF, alongside LIFE, SPUC (Society for the Unborn Child), individual gynaecologists and others. The low rates of pregnancy, the possibilities of oocyte and embryo donation, surrogate mothers, unmarried parents, one sex parents, embryo cryopreservation, cloning, and endless other objections were raised against the procedures. LIFE even issued a legal action against me for the abortion of an embryo grown for some days in vitro. It was rejected by the senior government lawyer since the laws of pregnancy began after implantation. We fully respected the intense ethical nature of our proceedings and the need to

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protect the embryo even as those not replaced had to be used for research under strict controls, and for open publishing and discussion of our work. Each year, 1000 rising to almost 2000 patients passed through Bourn Hall. Different stimulation regimens or new procedures could be tested in very little time. Clomiphene/HMG was reintroduced. Bourn babies increased: 20, 50, 100, to 1000 after 5–6 years. This was far more than half of the world’s entire IVF babies, including the first born in the United States, Germany, Italy, and many other countries. Detailed studies were performed on embryo culture, implantation, and abortion. We even tried aspirating epididymal spermatozoa for IVF, without achieving successful fertilization. Among the immense numbers of patients, people with astonishingly varied conditions of infertility emerged, including poor responders in whom immense amounts of endocrine priming were essential, women with a natural menstrual cycle that was not as it should have been, previous misdiagnoses which had laid the cause of infertility on the wife when the husband had never even been investigated, where men brought semen samples that we discovered had been obtained from a friend. The collaboration between nurses, clinicians, and scientists was remarkable. Yet trouble—ethical trouble—was never far away. I purchased a freezing machine to resume our Oldham work, but unknown to me, Patrick talked to BMA officers and for some reason agreed to delay embryo cryopreservation. Apparently, it would be an unwelcome social development. I did not approve: David Whittingham had shown how low temperature cryostorage was successful with mouse embryos, without causing genetic damage. “Freezing and cloning” became a term of intense approbation at this time. I unwillingly curtailed our cryopreservation programme. One weekend major trouble erupted as a result of this difference between Patrick and me. My duties in Bourn Hall prevented me from attending a conference in London. Trying to be helpful, I telephoned my lecture to London. Reception at the other end was apparently so poor as to lead to misinterpretations of my talk. Next morning, the press furore about my supposed practice of IVF was awful, so bad, that legal action had to be taken. Luckily, my lecture had been recorded, and listening to the tapes with a barrister revealed nothing contentious. I had said nothing improper in my lecture nor during answers to questions. That day, I issued seven libel actions against the cream of British society: the BMA and its secretary, the BBC, London Times, and other leading newspapers. Seven and another one later. If only one was lost, I could be ruined and disgraced. But they were all won even if it took several years with the BMA and its secretary. These legal actions inhibited our research, the cryopreservation programme being shut down for more than one year. And every single embryological note of mine from those days in Oldham and from Bourn Hall was examined in detail for my opponents by someone who was clearly an embryologist. Nothing was found to incriminate me.

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That wretched period passed. Numbers of babies kept on growing, embryo cryopreservation was resumed and Gerhard Zeilmaker in Holland beat the world to the first “ice” baby.35 Colin Howles and Mike Macnamee joined us in endocrinology, Mike Ashwood Smith and Peter Hollands in embryology as the old team faded away. Fascinating days had returned. Working with barristers, we designed consent forms which

Fig 7 A happy picture of Patrick and I, standing in our robes after being granted our Hon. D.Sc. by Hull University. were far in advance of those used elsewhere. Oocyte donation and surrogacy by embryo transfer were introduced. Work on preimplantation diagnosis of genetic disease resumed, and the first papers using new DNA technology were published. But embryo research faltered as all normal embryos were cryopreserved for their parents, so that almost none were available for study. Alan Handyside, one of our Cambridge PhDs, joined Hammersmith Hospital in London to make major steps in introducing preimplantation genetic diagnosis.36 As we reached 1000 pregnancies, our data showed the babies to be as normal as those conceived in vitro. Test tube babies, an awful term, were no longer unique and were accepted worldwide, exactly as Patrick and I had hoped. Our work was being recognized (Fig 7). Clinics sprang up everywhere. Ultrasound was introduced to detect and aspirate follicles by the Scandinavians,37 making laparoscopy for oocyte recovery largely redundant. Artificial cycles were

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introduced in Australia, intracytoplasmic sperm injection (ICSI) in Belgium,38 and gonadotropin releasing hormone (GnRH) agonists were used to inhibit the LH surge. Ian Craft in London showed how postmenopausal women aged 52 or more could establish pregnancies using oocyte donation and endocrine support. Women over 60 years of age conceived and delivered children. This breakthrough was especially welcome to me, since older women surely had the right to at ages still far almost the same as possible by a man. And ethics continues side by side with advancing science and medicine. The UK governmental Warnock report recommending permitting embryo research and proposed a Licensing Authority for IVF. A year or so later, the UK House of Lords, in all its finery, responded with a 3:1 vote in favour, a decisive support for all we had done in Mill Hill, Cambridge, and Oldham. What a wonderful day! The British House of Commons passed a liberal IVF law after intense debate. So did the Spanish government, although elsewhere things were not so liberal. Ten years after the birth of Louise Brown, the British Parliament had therefore accepted IVF, research on human embryos until day 14, and establishing research embryos. Cloning and embryo stem cells still bothered the politicians in 1988, to re-emerge in 1998, grey shadows of my earlier times in Glasgow. IVF is now fundamental to establish embryonic stem cells for organ repair, or cloning. During all this activity, tragedy struck all of us in Bourn Hall. Jean Purdy died in 1986 and Patrick Steptoe in 1988. They at least saw IVF come of age. A burgeoning medical science was digging deeper into endless aspects of human conception in vitro. The intracytoplasmic injection of a single spermatozoon into an oocyte to achieve fertilization, ICSI, was one of the greatest advances since IVF was introduced. It transformed the treatment of male infertility, enabling severely oligozoospermic men to father their own children. It did not stop there, since epididymal spermatozoa and even those aspirated from the testis could be used for ICSI. Spermatids have also been used. ICSI became so simple that many clinics reduced IVF to fewer and fewer cases. And new antagonists of GnRH introduced novel ways to control the cycle, enabling many oocytes to be stimulated by HMG and subsequently recombinant FSH. Treatment in the natural cycle could be improved, since these antagonists control LH levels and prevent premature LH surges. My own interests were returning to embryology, as the molecular biology revolution influenced our thinking. I am convinced that the oocyte and egg must be highly programmed, timewise, in embryonic polarities and integrating genetic systems that the tight systems place every new gene in its right place in the 1 cell egg and cleaving embryo. This must be right—there can surely be no other explanations for the fabulous modification in embryonic growth in the first week or two of embryonic life.

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IVF OUTLOOK In one sense, opening human conception in vitro was perhaps among the first examples of applied science in modern hi tech. Human IVF has since spread throughout the world, with apparently more than 500000 babies born worldwide by 2000—yet Louise Brown is only just 21 years of age. The need for IVF and its derivatives is greater than ever, since up to 10% of couples may suffer from some form of infertility. Major advances in genetic technologies now identify hundreds of genes in a single cell, and diagnosing genetic disease in embryos promises to help avoid desperate genetic diseases in newborn children. Indeed, the ethics of this field have now become even more serious since typing embryo genotypes provides detailed predictions of future life and health. IVF could well combine closely one day with genetics, to eliminate disease or disability genes or lengthen the lifespan. But most of all, practising IVF teaches a wider understanding of the desire and love for a child and a partner, the wonderful and ancient joys of parenthood, the pain of failure, the deep motivation needed in donating and receiving an urgently needed oocyte or a surrogate uterus. Parenthood is more responsible than ever before. Its complex choices are gathered before couples everywhere by the information revolution, placing family responsibilities on patients themselves, where it really matters. And IVF now reveals more and more about miracles preserved in embryogenesis from flies and frogs to humankind, over 600 million years of evolution. Even as I write this text, I stumbled across a paper in development. Sequencing of the entire mouse genome has enabled genes to be identified that are active during preimplantation stages of mouse embryo. A staggering array of genes operate then—apparently a huge percentage of those acting throughout our complete life. The same must apply to the human embryo. We are indeed enmeshed in some of the most fundamental evolutionary stages of our existence as we pass from oocyte to blastocyst and to implantation.

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

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10 Cole R, Edwards RG, Paul J. Cytodifferentiation and embryogenesis in cell colonies and tissue cultures derived from ova and blastocysts of the rabbit. Dev Biol (1966); 13:385–407. 11 Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (1965); 208:349–51. 12 Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet (1965); 2:926–9. 13 Edwards RG, Donahue R, Baramki T, Jones H Jr. Preliminary attempts to fertilize human oocytes matured in vivo. Am J Obstet Gynecol (1966); 96:192–200. 14 Austin CR. Adv Biosci (1969); 4:5. 15 Chang M. Adv Biosci (1969); 4:13. 16 Yanagimachi R, Chang MC. J Exp Zool (1964); 156:361–76. 17 Edwards RG, Talbert L, Israestam D, et al. Diffusion chamber for exposing spermatozoa to human uterine secretions. Am J Obstet Gynecol (1968); 102:388–96. 18 Steptoe PC. Laparoscopy and ovulation, Lancet (1968) ii: 913. 19 Palmer R. Acad Chir (1946); 72:363. 20 Fragenheim H. Geburts Frauenheilkd (1964); 24:740. 21 Steptoe PC. Laparoscopy in Gynaecology (1967). Edinburgh: Livingstone. 22 Edwards RG, Bavister BD, Steptoe PC. Early stages of fertilisation in vitro of human oocytes matured in vitro. Nature (1969); 221:632–5. 23 Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferred sexed blastocysts. Nature (1968); 218:346–8. 24 Henderson SA, Edwards RG. Chiasma frequency and maternal age in mammals. Nature (1968); 218:22–8. 25 Edwards RG, Sharpe DJ. Social values and research in human embryology. Nature (1971); 231:81–91. 26 Lunenfeld B. In: W Inguilla, RG Greenblatt, RB Thomas, eds. The ovary. CC Thomas: Springfield, 111. (1969). 27 Steptoe PC, Edwards RG. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet (1970); 1:683–9. 28 Edwards RG, Steptoe PC, Purdy JM. Fertilization and cleavage in vitro of preovulator human oocytes. Nature (1970) 227:1307–9. 29 Steptoe PC, Edwards RG, Purdy JM. Human blastocysts grown in culture. Nature (1971) 229:132–3. 30 Edwards RG, Surani MAH. The primate blastocyst and its environment. Uppsala J Med Sci (1978); 22:39–50. 31 Csapo AI, Pulkkinen MO, Kaihola HL. The relationship between the timing of luteectomy and the incidence of complete abortions. Am J Obstet Gycecol (1974); 118:985–9. 32 Steptoe PC, Edwards RG. Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet 1976; 1:880–2.

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33 Edwards RG, Steptoe PC, Purdy JM. Clinical aspects of pregnancies established with cleaving embryos grown in vivo. Br J Obstet Gynecol (1980); 87:757–68. 34 Trounson AO, Leeton JF, Wood C, et al. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science (1981); 212:681–2. 35 Zeilmaker GH, Alberda T, Gent I, et al. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril (1984); 42:293– 6. 36 Handyside A, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA application. Nature (1990); 344:768–70. 37 Wikland M, Enk L, Hamberger L. Transvesical and transvaginal approaches for the aspiration of follicles by use of ultrasound. Ann NY Acad Sci (1985); 442:182–94. 38 Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet (1992); 340:17–18.

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

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

PERSONNEL AND EXPERIENCE While the surroundings, housing, and equipment require special consideration in the design of an integrated gamete and embryo treatment facility, it is really the staff who will conduct the procedures and who are essential to its success. Successful clinical practice is almost entirely dependent on high levels of experience and qualifications among medical and laboratory personnel. Hospitals usually do best when their human resource departments select key personnel on the basis of personality and experience, rather than official qualifications and status. The same principle applies to assisted reproductive laboratories, as experience is the key to success and because there are very few standard teaching and

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examination systems in place for the assisted reproductive technologies (ART) environment. Few new programs directed by novices have had flying starts, and, although we cannot underestimate the potential of human creativity, inexperienced teams may have poor results and eventually fail altogether. This chapter aims to protect experienced practitioners from unexpected failure during the setup of a new laboratory, when they are essentially placing themselves in a new environment. Laboratory staff, directors, and embryologists must consider their experience within the context of what will be required of them. Even though humility is sometimes difficult for medical professionals, an adequate clinical outcome requires a cautious and rational reassessment of individual abilities and acceptance that much of the environmental effects on ART are unknown. Certain regulatory bodies such as the College of American Pathology and the British Human Fertilization and Embryology Act provide guidelines and licensing for embryologists, sometimes even for subspecialties such as the performance of ICSI, and for those who are capable of setting up and running IVF laboratories. So far, such licensing has done little more than provoke debate, because the abilities of such licensed personnel are largely unproved and the licenses are not interchangeable between countries. Tradition also plays its part, as in many Asian countries embryology directors are usually medical professionals. So qualifications are often seen to be less important than tradition and habit. What then qualifies someone to be a laboratory director and/or an embryologist? The answer is not a simple one. In general, peer evaluation sidesteps the problem by accepting any individual that qualified as a general pathology laboratory director or a reproductive specialist with an MD or PhD degree. However, pathologists do not necessarily have experience in gamete cell culture and some reproductive specialists, such as urologists and immunologists, may never have worked with gametes and embryos at all. Equally, it is perfectly possible for a medical practitioner to direct a laboratory, without ever having practiced gamete and embryo handling. So how can it have been decided that they were actually qualified to set up and run an IVF laboratory? “Eppur si muove” (“And yet it moves”), as Galileo said when condemned to life imprisonment for heresy.

EMPIRICAL AND STATISTICAL REQUIREMENTS FOR STAFF There is considerable disagreement about the required experience of embryologists. Hands on experience in all facets of clinical embryology is an absolute requirement when starting a new program; even highly experienced veterinarian or basic science embryologists must be individually supervised by experienced clinical personnel. The period during which detailed supervision must continue depends absolutely on

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the types of skills required, the daily case load, and total time. It can be appreciated that 100 cases over one year is a very different matter than the same amount during six weeks; the period of supervision should vary accordingly. The optimal ratio of laboratory staff to the contemplated number of procedures is debatable, and, naturally, economics is the enemy. The ratio should be high, because embryologists can then spend enough time on quality control, training, and procedural details to ensure the high standards required for success. In practice, however, the staff/procedure ratio is often very low to save money, as happens sometimes in commercially oriented clinics. Sometimes it is a consequence of national health systems that must provide a wide range of services on a minimal budget. Needless to say, patients do not always benefit from these economic constraints; this is most obvious when comparing outcomes between the various health service systems in Western countries. The job description for the embryologist ideally includes all possible tasks, excepting initial patient intakes and medical and surgical procedures. Embryologists are often involved in important tasks related to general patient management, such as follicular monitoring, genetic counseling, marketing, administration, and nurse management. But it should be realized that these tasks seriously detract from their true work. Firstly, the embryo legist’s duty is to perform gamete and embryo handling and culture procedures. Secondly, but equally importantly, the embryologist should maintain full awareness of quality control standards, both by performing routine checks and tests as well as by maintaining detailed logs of incidents, changes, unexpected changes, and countermeasures. Across all these duties, the following seven job positions can be clearly defined: director, supervisor, senior embryologist, embryologist, trainee, assistant, and technician; their actual numbers varying according to the number of annual procedures. There may also be positions for others to do preimplantation genetic diagnosis, research or secretarial work. Obviously, not all these functions will apply to smaller centers. Although at first sight a seemingly unimportant detail, one of the most useful functions that ever existed was that of a professional witness that was implemented during the first few years of Bourn Hall Clinic. It effectively preserved a high level of security for embryo handling, even when large numbers of patients were being treated simultaneously. It also ensured that embryologists performed only those procedures and techniques for which they were properly qualified. In general, embryologists should concentrate only on gamete and embryo handling, and any laboratory with a relatively high number of annual procedures should have additional embryology technicians and assistants that can order and maintain equipment, and properly record laboratory data. In short, skimping on staff can be seriously self defeating in the IVF laboratory.

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FACILITY, BUDGET, AND DESIGN Historically some clinics were built in remote areas, based on beliefs that environmental factors such as stress would affect the patient and thereby the outcome. Today’s laboratories are commonly placed in city centers and large metropolitan areas in order to service large populations. The recent development of better assays for scanning the content of air samples combined with the awareness that some buildings or building sites could be intrinsically harmful to cell tissue culture makes today’s choice of a laboratory site even more important for a new program.5,6 A laboratory design should be based on the anticipated case load and any subspecialty. Obviously, local building and practice permits must be assessed prior to engaging in a full fledged design. There are five basic types of design: (I) laboratories using only transport IVF, (II) laboratories adjacent to clinical outpatient facilities that are only used part of the time, (III) full time clinics with intrafacility egg transport using portable breeding chambers, (IV) fully integrated laboratories with clinical areas, and (V) portable, temporary laboratories. Before developing the basic design for a new laboratory, environmental factors must be considered. While the air quality in modern laboratories can be controlled to a degree, it can never be fully protected from the exterior environment and adjoining building spaces. Designers should first determine if the building or the surrounding site will undergo renovations, demolition or major changes of any kind, in the foreseeable future. City planning should also be reviewed. Activity related to any type of construction can have a significant negative impact on any proposed laboratory. Prevalent wind direction, industrial hazards, and general pollution reports such as ozone measurements should also be determined. Even when these factors are all deemed acceptable, basic air sampling and determination of volatile organic compounds (VOCs) should be made inside and outside the proposed building area. The outcome of these tests will determine which design requirements are needed to remove VOCs from the laboratory area. In most cases an overpressured laboratory (at least 0.10–0.20 inches of water) that uses a high number (7–15) of air changes (ACH) per hour is the best solution, because it also provides for proper medical hygiene. The laboratory walls and ceiling should have the absolute minimum number of penetrations. This generally requires a solid ceiling, sealed lighting and airtight utility connections. Doors will require seals and sweeps and should be lockable. Ducts and equipment must be laid out in such a way that routine and emergency maintenance and repair work can be performed outside the laboratory with minimal disruption. Air handling should not use an open plan design. In the ideal case, 100% outside air with chemical and physical filtration will be used with sealed supply and return ducts. Alternately, the air supply equipment may balance outside air with re-circulated air, with processing to control the known levels of VOCs. Some laboratories will require full time air

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recirculation, while others may actually find the outside air to be perfectly clean (imagine that!). Outside air is often erroneously judged to be polluted without proper chemical assessment; while inside air is usually considered “cleaner” based on the presumption that it “smells” better.5 In many laboratory locations, conditions are actually the reverse, and designers should not by any means “follow their instincts” in these matters. Humidity must also be completely controlled according to climate and seasonal variation. The system must be capable of supplying the space with air with a temperature as high as 30–35 degrees centrigrade, at less than 40% relative humidity. Air inlets and outlets should be carefully spaced to avoid drafts that can change local “spot” temperatures or expose certain items of equipment to relatively poor air or changes in air quality. A detailed layout and assessment of all laboratory furniture and equipment is therefore essential prior to construction and has many other benefits. The organization and flow of persons and things in a world class restaurant results in a special ambience where more than just the food is considered. In the same way, appropriate modular placement of groups of incubators, gamete handling areas (laminar flow unit or isolette) and micromanipulator stations will minimize distances that dishes and tubes need be moved. Ideally, an embryologist should be able to finish one complete procedure without moving more than three meters in any direction; not only is this efficient, but it also minimizes possible collisions in a busy laboratory. The number of modules can easily be determined by the expected number of cases and procedure types, the average number of eggs collected, and thereby the number of embryologists expected to work simultaneously. Each person should be provided with sufficient workspace to perform all procedures without delay. Additional areas can contain simple gamete handling stations or areas for concentrating incubators. Cryopreservation and storage facilities are often located in a separate space; these should always be adjacent to the main laboratory. Another separate laboratory can contain an area for culture medium preparation, sterilization and water treatment. Administration should be performed in separate offices. Last but not least, it is preferable to prepare semen in a separate laboratory altogether, adjacent to a collection room. The semen laboratory should have ample space for microscopes, freezing, and sterile zoning. Some thought should go into planning the semen collection area. This room should be at the end of a hallway with its own exit; it should be soundproofed and not too large, with a sink and clear instructions of how to collect semen in preparation for ART. The room should be adjacent to the semen preparation laboratory with a double door cupboard type pass through for samples between them. This pass through should have a signaling device so the patient can inform the embryologist that the sample is ready; it also permits male patients to leave the area without a sample container in their hand.

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EQUIPMENT AND STORAGE A detailed list of equipment should be prepared and checked against the planned location of each item; it can later be used as the basis of maintenance logs. It is important to consider the inclusion of extra crucial equipment and spare tools in the laboratory design, to allow for the event of sudden malfunction. It is particularly important to have redundant elements of the Cryopreservation system, including cryopreservation and storage equipment. Similarly, two or more spare incubators should not be seen as excessive; at least one spare suction device and micromanipulator for micromanipulation should also be included. There are many other items whose malfunction would jeopardize patient care, although some spares need not be kept on hand as manufacturers may always have them available; however, such details need to be repeatedly checked as suppliers stock continue to change. It may also be useful to team up with other programs or an embryology research laboratory so that a crucial piece of equipment can be exchanged in case of unexpected failure. Some serious thought is needed when contemplating the number of incubators and incubator spaces. The ratio of cases per incubator varies considerably from program to program, and assuredly affects clinical outcome, depending on the number, type and length of incubator door openings. In principal, the number should be kept to a minimum; we prefer a limit of four cases per incubator. Several other incubators are used for general purpose during micromanipulation and other generic uses in order to further limit the number of incubator openings. Strict guidelines must be implemented and adhered to when separating dishes or tubes from patients. Separate compartments may be helpful and can be supplied by certain manufacturers. Servicing and sterilizing of equipment such as incubators may have to occur when the laboratory is not performing procedures. Placement of incubators and other pieces of equipment on large castors may be helpful in programs where downtime is rare. Pieces of equipment can then be serviced outside the laboratory. When there are several options available to the laboratory designer, supply and evacuation routes should be planned in advance. One of the most susceptible aspects of ART is cryopreservation. In case of an emergency such as fire or power failure, it may be necessary to relocate the liquid nitrogen filled dewars without using an elevator or to relocate the frozen samples using a temporary container. This may seem an extreme consideration, especially in the larger laboratories that stockpile thousands of samples, but plans should be made. It may be possible to keep a separate storage closet or space near the building exit, where long term samples that usually provide the bulk of the storage can be kept, but this would require repeated check ups of a facility that is not part of the laboratory. Liquid nitrogen level alarms, with remote notification capability, should be contemplated for all dewars. The route of delivery of liquid nitrogen and other medical gas cylinders must be relatively easy,

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without stairways between the laboratory and the delivery truck, and should be sensibly planned in advance. Note that the flooring of this route is usually destroyed within months because of liquid nitrogen spills and wear caused by delivery containers, so the possibility of an alternative delivery corridor should be considered for these units. Liquid nitrogen containers and medical gas cylinders are preferentially placed immediately adjacent to the laboratory in a closet or small room with outside access. Pipes and tubes enter the laboratory from this room, and cylinders can be delivered and changed to this room without compromising the laboratory area in any way. This allows liquid nitrogen to be pumped into the cryopreservation laboratory using a manifold system and minimal lining. Lines should be properly installed and insulated to insure that they do not leak nor allow condensation and conserve energy. Medical gases can be directed into the laboratory using prewashed vinyl/Teflon lined tubing. Alternately, solid manifolds made from stainless steel with suitable compression fittings can be used. Avoid soldered or brazed copper lines used in domestic plumbing applications wherever possible: copper lining can be used but should be cleaned and purged for a prolonged period prior to laboratory use. Copper line connections should not be soldered as this could cause continuous contamination. This recommendation may conflict with existing building codes, but non-contaminating alternatives must be found. In any case, a number of spare lines hidden behind walls and ceilings should be installed in case of later renovation or facility expansion. Placement of bulky and difficult pieces of equipment should be considered when designing doorways and electrical panels. Architects should be fully informed of all equipment specifications to avoid that truly classic door width mistake. Emergency generators should always be installed, even where power supplies are usually reliable; the requirements can easily be determined by an electrical engineer. Thankfully, these units can be well removed from the laboratory, but must be placed, mind you, in wellventilated areas that are not prone to flooding. Additional battery “uninterruptible power systems” (UPS) may be considered as well but are of very limited ability. Buildings should also be checked for placement of the main power inlets and distribution centers, especially because sharing power lines with other departments or companies may not be advisable. Circuit breakers should be easily accessible to embryologists or building maintenance staff. General knowledge of mechanical and electrical engineering of the building and the laboratory specifically will always be advantageous. Ample storage spaces should always be planned for IVF laboratories. In the absence of dedicated storage space, laboratory space ends up being used instead, filling all cabinets and playing havoc with the original design. This storage area should contain all materials in sufficient quantity to maintain a steady supply. A further reason to include storage areas in laboratory design, sufficient itself to justify the space, is that new supplies

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including sterilized disposable items, release multiple compounds for prolonged periods. This “outgassing” has been determined to be a major cause of adverse air content in a number of laboratories in which supplies were stored. Separate storage space therefore provides the best chance of good air quality; especially when it is supplied by separate air handling equipment. It should be large enough to handle bulky items as well as mobile shelving for boxes. One should be careful to avoid the natural inclination to save extra trips by bringing too many items into the laboratory, or the gains made by careful design may be lost.

MICROSCOPES AND VISUALIZATION OF CELLS Though dissecting microscopes are crucial for the general handling of gametes and embryos, many people still consider inverted microscopes to be a luxury, even though they are in regular use with micromanipulation systems. Proper visualization of embryos is key to successful embryo selection for transfer or freezing; if the equipment is firstclass, the visualization can be done quickly and accurately.7 Even so, appropriately detailed assessment is still dependent on the use of an oil overlay system to prevent damage by prolonged exposure. Each workstation and microscope should be equipped with a still camera and/or video camera and monitor. Still photos can be placed in the patient file, and video footage permits speedy review of embryonic features with colleagues after the gametes are safely returned to the incubator, as well as helping train new embryologists, an ever present task. Interference optics, such as Hoffman and Nomarski, are preferable because they permit the best measure of detail and depth. Novel visualization of internal elements such as spindles using the Pol-Scope requires more complicated equipment, but is still largely compatible with the limitations of routine embryo assessment.8 Ideally, the captured photos should be digitally stored for recall at their appropriate location in the clinic’s medical database.

CONSTRUCTION, RENOVATION, AND BUILDING MATERIALS Construction and renovation can introduce a variety of compounds into the environment of the ART laboratory, either temporarily or permanently. Either can have significantly adverse effects on the outcome of operations.5,6,9,10 The impact of the exterior environment on IVF success has been demonstrated. Pollutants can have a significant adverse effect on reproductive success in an IVF laboratory. These effects can range from delayed or abnormal embryonic development, absence of fertilization to complete reproductive failure. Many of the damaging materials are organic chemicals that are released or outgassed by paint,

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adhesives from flooring, cabinets, and general building materials as well as from laboratory equipment and procedures. It is important to realize that the actual construction phase of the laboratory can cause permanent problems. Furthermore, any subsequent adjacent renovation can also cause similar, or even greater, problems. Neighboring tenants can be informed of the sensitivity of cultured in vitro gametes, and at least these nearby changes can be supervised to minimize potential damage to a greater or lesser extent. However, new construction immediately outside the building is considerably more problematic. City works such as street construction are very hard to predict and nearly impossible to control. A good relationship with the neighbors is not always an easy task, and working relationships should always be maintained with building owners and city planners, in the hopes that, at the very least, the IVF laboratory will be kept informed of upcoming changes. In spite of one’s best wishes, or the fervent assurances of building owners, changes of this sort inevitably take place, so here we present some guidelines, all of which apply to new laboratory construction as well as changes in adjacent areas. First of all, the area to be demolished and then constructed needs to be physically isolated from the IVF laboratory (if this is not the new IVF laboratory itself). The degree of isolation should be equivalent to an asbestos or lead abatement project. The isolation should be done by the following techniques. Physical barriers should be erected consisting of poly sheeting supported by studding where needed. Access to the construction area should be restricted by the use of an access passageway with two doors in series. All construction waste should be removed via an exterior opening or properly bagged before using an interior exit. The construction area should be under a negative air pressure, exhausting to the exterior; naturally, this exhaust should be far removed from the laboratory’s air intake, and properly located with regard to the prevailing winds and exterior air flow. Extra interior fans should be used during any painting or use of adhesives, to maximize removal of noxious fumes. Material safety data sheets (MSDS) for all of the paints, solvents, and adhesives should be obtained, logged, and used to evaluate any potential material and manage industrial hygiene concerns. Follow up investigations with manufacturers and their representatives may be helpful because specifications of these items are changed without notice. The negative pressurization of the laboratory space requires continuous visual indication, such as a ball and tube pressure indicator, or simply paper strips. Periodic sampling for particulates, aldehydes, and organics could be done outside the demolition and construction site, provided this can be budgeted. Alternately tracer gas studies can be used to verify containment. The general contractor of the demolition and construction should be briefed in detail on the need to protect the IVF facility and techniques to accomplish this. When possible, the actual members of the construction crew themselves should be selected and briefed in detail. Large filter units using filter pellets of carbon and permanganate can be placed strategically

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(GenX International, Madison, Conn, USA). Uptake of organics can be assayed, but the tempo of routine filter changes should be increased during periods of construction activity.

SELECTION OF BUILDING MATERIALS Many materials release significant amounts of volatile organics, and a typical list includes paints, adhesives, glues, sealants, and caulking release alkanes, aromatics, alcohols, aldehydes, ketones, and other classes of organic materials. This section will outline steps to be taken in an effort to reduce these outgassing chemicals. Any and all interior painting throughout the facility should only be done on prepared surfaces with water-based paint formulated for low VOC potential. During any painting auxiliary ventilation should be provided using large industrial construction fans, with exhaust vented to the exterior. Paints that can significantly influence air quality should be emission tested (some suppliers already have these tests available). MSDS are generally available for construction materials. Suppliers under these specifications are encouraged to conduct product testing for the emission potential. The variety of materials and applications greatly complicates this testing, but several procedures have been developed to identify and quantify the materials released by building materials and furnishings. The interior paints must be water based, low volatile paints with acrylic, vinyl acrylic, alkyd, or acrylic latex polymers. Paints meeting this specification can also contain certain inorganic materials. Paints with low volatiles may still contain low concentrations of certain organics. No interior paint should contain materials like formaldehyde, acetaldehyde, benzene, toluene, styrene, xylenes, and other volatile organics. Adhesive glues, sealants, and caulking materials present some of the same problems as paints, but water based materials are generally not available for these applications, although their composition varies widely. Silicone materials are preferred whenever possible, particularly for sealants and caulking work. No adhesive, glue, sealants, or caulking used in the interior should contain materials such as formaldehyde, benzaldehyde, and phenol (for a complete review of potentially toxic materials contact Alpha Environmental, New Jersey, USA).

“BURNING IN” OF THE FINISHED FACILITY New IVF laboratories and new facilities around existing laboratories have often been plagued by complaints of occupants who experience discomfort from the chemicals released by new construction and furnishings. The ambient levels of many of these materials can be reduced by “burning in” the facility. A typical burn in consists of increasing the

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temperature of the new area by 10–20 degrees centigrade and increasing the ventilation rate; even higher temperatures are acceptable. The combination of elevated temperature and higher air exchange aids in the removal of the volatile organics. Upon completion of the construction, the air handling system should be properly configured for the burn in of the newly constructed area. As previously stated, the system must be capable of supplying the space with air with a temperature of 30–35 degrees centigrade, at less than 40% relative humidity. The burn in period can range from 10 days to 28 days, and the IVF laboratory should be kept closed during this time. If these temperatures cannot be reached by the base system, use auxiliary electrical heating to reach the minimum temperature. During burn in, all lighting and some auxiliary equipment should be turned on and left running the whole time. Naturally, ventilation is critical if redistribution of irritants is to be avoided; the whole purpose is to repeatedly purge the air. Auxiliary equipment should of course be monitored during the burn in. The same burn in principle applies to newly purchased incubators. Removal of volatile organics is especially important in the critical microenvironment of the incubator. Whenever possible, it is advantageous to purchase incubators months in advance of their intended initial use, and to operate them at an elevated temperature in a clean, protected location. After the burn in is complete, a commissioning of the IVF suite should be conducted to verify the laboratory meets the design specifications. The ventilation and isolation of the laboratory should be verified by a series of tests using basic airflow measurements and tracer gas studies. The particulate levels should be determined to verify the HEPE system is functional. Particulate sampling can be done by using USA Federal Standard 209E. Microbial sampling for aerobic bacteria and fungi is often done in new facilities using an Andersen Sampler followed by microbiological culturing and identification. The levels of VOC contamination should be determined. Possible methods are included in the US EPA protocols using gas chromatography/mass spectroscopy (GC/MS) and high performance liquid chromatography sensitive at the microgram per cubic meter level.11–13

INSURANCE ISSUES ART has become common practice worldwide, and is regulated by any combination of legislation, regulations or committee based ethical standards. The rapid evolution and progress of ART techniques reveal new legal issues that require consideration. Even the patients themselves are changing, as it becomes more acceptable for single mothers and homosexual and lesbian couples to present themselves for treatment. Donation of genetic material, age limitation, selective fetal reduction, preimplantation genetic diagnosis, surrogacy, and cloning each present a

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legal quagmire; their very definitions vary from country to country, along with types of application, as well as regional social factors, religion and law. Furthermore, financial and emotional stresses often oppress patients seeking treatment in countries where social medicine does not cover infertility treatment; and more especially after a failed cycle. This translates into an increasing number of ART lawsuits, in spite of generally improved success rates. Laboratory personnel and the institution owning the laboratory should therefore obtain an insurance policy of sufficiently high level and quality commencing prior to the first day of operations. Litigation prone issues need special consideration, and include (a) cancellation of cycle prior to egg retrieval, (b) failure to become pregnant, (c) patient identification errors and (d) interrupted cryo-storage events; even when experienced practitioners consider themselves at low risk. Prior to engaging in ART activity, protocols can be established to identify these problem areas and establish counter measures.

CONCLUSION All in all, it is surprising how many professionals continue to pursue the establishment of new ART clinics at a time when competition is high, obvious financial benefits are small and existing ART services approach saturation in most areas. Regardless, this chapter can provide some guidance to those medical professionals aspiring to independence in the world of ART, although it cannot safeguard from disaster in all cases. It should serve to provide useful suggestions and concepts that have been learned from practical experience for the wide variety of problems and solutions that have been used over many years.

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

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5 Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception invitro. Hum Reprod (1997); 12:1742–9. 6 Cohen J, Gilligan A, Willadsen S. Culture and quality control of embryos. Hum Reprod (1998); 13(S3): 137–44. 7 Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril (1999); 7:836–42. 8 Liu L, Trimarchi JR, Oldenbourg R, Keefe DL. Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes. Biol Reprod (2000); 63:251–8. 9 Hall J, Gilligan A, Schimmel T, Cecchi M, Cohen J. The origin, effects and control of air pollution in laboratories used for human embryo culture. Hum Reprod (1998); 13(S4): 146–55. 10 Boone WR, Johnson JE, Locke AJ, Crane MM 4th, Price TM. Control of air quality in an assisted reproductive technology laboratory. Fertil Steril (1999); 71:150–4. 11 Seifert, B. Regulating indoor air. Proceedings of 5th international Conference on Indoor Air Quality and Climate, Toronto (1990); 5:35– 49. 12 Federal Standard 209 E (1992) General Services Administration USA Federal Government, Washington, DC. 13 Compendium of Methods for the Determination of Toxic Organic Compounds in Ambient Air, US EPA 600/4–84–041, April 1984/1988. Available from the US EPA through the Superintendent of Government Documents, Washington DC.

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

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

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

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

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

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

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

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

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

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

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

Week 3

Week 4

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

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

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

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

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REFERENCES 1 Schiewe MC, Schmidt PM, Bush M, Wildt DE. Toxicity potential of absorbed/retained ethylene oxide residues in culture dishes on embryo development in vitro. J Anim Sci (1985); 60:1610–8. 2 Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception in vitro. Hum Reprod (1997); 12:742–9. 3 Nagata Y, Shirakawa K. Setting standards for the levels of endotoxin in the embryo culture media of human in vitro fertilization and embryo transfers. Fertil Steril (1996); 65:614–9. 4 Ackerman SB, Swanson RJ, Stokes GK, Veeck L. Culture of mouse embryos as a quality control assay for human in vitro fertilization. Gamete Res (1984); 9:145–52. 5 Boone WR, Shapiro SS. Quality control in the in vitro fertilization laboratory. Theriogenology (1990); 33:23–50. 6 Gerrity M. Mouse embryo culture bioassay. In: Wolf DP, Bavister BD, Gerrity M, Kopf GS, eds. In vitro fertilization and embryo transfer: a manual of basic techniques. New York: Plenum Press (1988):57–76. 7 Fleethman JA, Pattinson HA, Mortimer D. The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization. Fertil Steril (1993); 59:192–6. 8 Bavister BD, Andrews JC. A rapid sperm motility bioassay procedure for quality-control testing of water and culture media, J In Vitro Fertil Embryo Transfer (1988); 5:67–75.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4 Accreditation of IVF laboratories: the European perspective Cecilia Sjöblom

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

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

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

EUROPEAN AND INTERNATIONAL PRINCIPLES FOR ACCREDITATION The European Union (EU) has a goal to establish harmonized principles for the assessment of laboratories, certification and inspection bodies. These principles needs to be accepted regardless if the activity they carry out is encompassed by the EU or national regulations for conformity assessments or merely by voluntary requirements, when industries or specific buyers require harmonized quality system standards. The European conformity assessment system is based on European standards in the EN 45000 series. These European standards are more or less identical to the ISO/IEC guides for conformity assessment. When the EU’s new principles for conformity assessment were developed, it was clear at an early stage that some sort of quality assurance between the bodies, preferably accreditation bodies, was needed to assess the competence of laboratories, certification bodies, and inspection bodies. The European Commission considered that it was very important to create a system that could safeguard the quality assurance of such bodies, active both within the voluntary sector and within the mandatory sector. The latter should also be based on relevant standards in the EN 45000 series of standards. The European Commission therefore

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took the initiative in bringing together the national accreditation bodies in order to formalize their cooperation. European organizations for cooperation between accreditation bodies and their predecessors have existed in Europe since the mid-1970s, and in November 1997 the European cooperation for accreditation of laboratories (EAL) and the European accreditation of certification (EAC) merged into EA, the European cooperation for accreditation. EA covers accreditation in all fields of conformity assessment activities.16

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

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The European Commission states that accreditation must be a transparent, independent, and non-commercial activity. If these requirements can be assured it would ensure that accreditation could be the last level of control of technical competence. It further states that accreditation should be considered as the most favored technical basis for assessment of the competence of technical actors, and that accreditation bodies should not engage in other conformity assessment activities so as to not compromise their independence and integrity.4 At present the EA members represent nationally recognized accreditation bodies from 24 countries in the EU and the European Free Trade Area (EFTA) countries (Fig 4.1). To make accreditation effective across borders all EA members must apply the same standard of assessment to the laboratories, certification and inspection bodies etc. To ensure that this is happening EA members can apply for peer group evaluation of their activities by the other members. EA works mainly within the framework of the two international cooperation organizations for accreditation bodies, the International Laboratory Accreditation Cooperation (ILAC), and the International Accreditation Forum (IAF). A comprehensive program of inter-laboratory comparisons supports mutual confidence in laboratory accreditation. Accreditation will therefore have a part to play in the mutual recognition agreements on conformity assessment. As a consequence of this, accreditation systems are established in countries throughout the world and these accreditation bodies cooperate in regional bodies, such as EA. On the global level, the regional bodies as well as the national accreditation bodies cooperate in the international organizations such as ILAC and IAF. This structure ensures harmonized procedures for conformity assessment activities all over the world. Within the framework of IAF, a worldwide multilateral agreement, in the area of certification of quality management systems (ISO 9000), has been signed by EA as a regional body and national accreditation bodies from all parts of the world.

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

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

THE QUALITY MANUAL AND BUILDING OF A QUALITY SYSTEM The main purpose of the quality manual is to outline the structure of the documentation used in the quality system.17 It shall also include or refer to the standard operating procedures (SOPs). There should be clear definitions of the management’s areas of responsibility including its responsibility for insuring compliance with the international standards on which the system is built. A simple overview of the quality system requirements and the position of the quality manual is shown in Fig 4.2. A good quality manual shall be precise and brief; it should be an easily navigable handbook for the whole quality system. The most important procedures are preferably included in the manual itself, but deeper descriptions should be referred to in underlying documentation. An easy way to start building the system is to make up a table of contents for the quality manual, and there are some crucial chapters and documents that should be found in a manual.

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Fig 4.2 Quality system requirements. The elements of the system are separated into different levels with the quality manual as the highest document. The quality manual and related quality documentation outlines the clinic policies and includes manuals for the standardized routines at all levels of work in the laboratory. IDENTITY This chapter clearly states the identity of the clinic or laboratory, its address and official registration and organization numbers. Since the quality manual usually is an official document shown to both visitors and clients, it is good to include a bit of company history and background in this brief chapter. QUALITY POLICY (ISO 17025 4.2.2) As discussed previously, short powerful and according to the standard. THE QUALITY SYSTEM (ISO 17025 4.2) This chapter should contain a description of the system structure, the different levels and on what standards the system is built in accordance with. An important feature is to outline the areas covered by the certification and accreditation or both. An accreditation according to EN 45001 or ISO 17025 is aimed towards accredited methods. Not all methods performed in the laboratory have to be accredited for the laboratory to be called accredited, and not all methods can be accredited owing to lack of

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references or methods for validation. However, in this part of the manual there should be a list of which methods in the laboratory are covered by the accreditation and an authority approved accreditation CV that states the percentiles of variations acceptable within the methods. The laws, regulations, and legislation under which the laboratory and clinic work should be listed with full references. All the documentation of these should be kept as underlying documentation and as controlled documents included in the system and kept accessible for all personnel. This chapter shall also include a full master list of all the documentation included and controlled in the quality system. The list shall clearly state the document name, issue, and current revision status, date of approval level, and physical location. DOCUMENT CONTROL (ISO 17025 4.3) According to EN 45001, ISO 17025, and the ISO 9000 standards the laboratory or clinic shall establish and maintain procedures to control all documents that form part of its quality documentation. This includes both internally generated documentation such as SOPs and protocol sheets and externally generated documentation such as law texts, standards, and instruction manuals for equipment. Document handling and control are an important part of the quality system and, if not designed properly, can become a heavy load for a smooth running quality system. Since it is the part that touches every part of the system it is important to sit down and think through how this system of paperwork shall be handled in your laboratory or clinic. Firstly, ensure that the system you choose covers the demands of the standards. A checklist for document control is found in appendix II. The identification of the document should be logical and it is a good suggestion to use numbers. The same identification number could then be used for the file name within the computerized version. The issue number in brackets or after a dash could follow this number. The pagination is important. If you choose not to use pagination you must clearly mark where the document starts and ends. The dates of issue together with information on who wrote the document and who approved it (signature) are usually included in the document header together with a company logo. AUDITS AND MANAGEMENT REVIEWS (ISO 17025 4.13 AND 4.14) Internal and external audits are tools for improving and keeping your system up to date with the standards. The quality manual shall include specific instructions covering both how and how often they shall be performed (ISO 17025 4.13). The management usually chooses internal auditors, and they should be familiar with both the standards and the

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

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

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

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

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

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

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The embryo culture environment is an important factor in a successful IVF-ET program. It is the responsibility of the IVF laboratory to establish and maintain a stable, non-toxic, pathogen free environment providing optimal conditions for fertilization and embryo development in vitro. The gametes and early embryos are extremely sensitive to minor changes in the milieu. Small variations in temperature, pH, and the physical properties of media such as pH, and osmolarity will inevitably effect the outcome of an IVF cycle. Embryo toxic substances in the media and in materials used for embryo and gamete handling should be screened for and identified before they are clinically used. There is a lot of debate around the issue of what quality control assay to use, and different assays and variations of them to get increased sensitivity have been suggested.20 Many clinics today are using the “human embryo assay.” Fluctuations in pregnancy rates despite a constant clinical profile of the patients being treated are the sole indicator of suboptimal culture conditions and indicates that there might be a problem in the laboratory. Apart from the ethical dilemma there are two main limitations to this, namely the time lag in culturing embryos and diagnosing pregnancy in a substantially large number of patients, and failure to identify the source of the problem. Culture of excess embryos to the blastocyst and hatched blastocyst stages is another way of human embryo assay frequently used. The problem with this assay is that these poor quality embryos might suffer from genetic abnormalities. Conclusions from these culture results therefore are hard to make, and again there is a problem in identifying the source of toxicity. The use of an objective quality control strategy that is independent of patient factors can prove valuable in assessing the media and lab ware that comes in contact with the patients gametes and embryos. At Fertilitetscentrum we have chosen to use the mouse embryo assay for the testing, since this method, if used properly, provides the best means of testing resembling the human embryo culture system. There are two major parts of our testing system: the assay itself to detect toxicity and our record batch numbers to be able to identify the source of problems. We, like many other smaller privately owned units, have neither the economical nor the laboratory resources to facilitate a standardized mouse embryo assay. Nevertheless, we do have strict limits for the acceptance of a test. The test shall be done using an appropriate strain of mice, and fully hatched shall be the end point of the test. Many manufacturers state in their production sheet that their products have been mouse embryo tested, but we rarely see a certificate from this test. It is important to demand a copy of this certificate before the release of a product into the laboratory. If the product fails to meet our testing demands we send them to a test laboratory for testing (Scandinavian QC laboratories, Göteborg, Sweden). This assures that we get our equipment tested in the way that we find proper, and we get a certificate from every test.

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

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

CONCLUDING REMARKS Accreditation is an efficient and effective tool to demonstrate technical competence. Laboratory accreditation is the formal recognition of a laboratory’s technical competence. EA mutual recognition agreements are based on the evidence or assumption of equal technical competence of laboratories across borders. Such evidence is generally provided by the results of a comprehensive program of interlaboratory comparisons in calibration, although there are fields, such as ART, where harmonization is still needed. In testing and inspection, full evidence of equal technical competence is still missing, and mutual recognition is based rather on the assumption of equivalence. Full equivalence can only be achieved by harmonization of measurement procedures and identical requirements on uncertainty determination and reporting of results. In February 2000 the European Society of Human Reproduction and Embryology special interest group for embryology issued Guidelines for good practice in IVF laboratories.21 These guidelines partly incorporate standards previously published by the UK Association of Clinical Embryologists.22 A step towards harmonization could be to get these guidelines approved as standards by the EA. Throughout completing the long and work intensive process of applying a QC system in our ART laboratory one might have asked what it has meant for the laboratory. There is no doubt that introducing a fully implemented QC system has standardized the methods and the way in which the embryologists perform their work in the laboratory. The troubleshooting, the maintenance of the equipment, and the milieu improved and were standardized. This guarantees an optimal handling of the couples’ blood samples, gametes, and pre-embryos. Thus, introducing and working according to an accredited QC system in an ART unit is a never ending project. It is a system that shall guarantee a constant improvement of the work. Introducing and fully implementing a quality control system in our laboratory has standardized the methods and the way that the embryologists perform their work in the laboratory. It has also optimized the environment in which the patient’s gametes and embryos are handled. The accreditation of more ART laboratories to the same standards will bring about alignment through a wide base for external controls. This

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could lead to an improvement of IVF results worldwide, and we would for the first time be able to compare the results between different laboratories.

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

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

APPENDIX II

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

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

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

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2 Haeckel R, Kindler M. Effect of current and forthcoming European legislation and standardization on the setting of quality specifications by laboratories. Scand J Clin Lab Invest (1999); 59:569–73. 3 Libeer JC. Total quality management for clinical laboratories: a need or a new fashion? Acta Clin Belg (1997); 52:226–32. 4 The European quality assurance standards EN ISO 9000 and EN 45000 (1997). European Commission Doc Certif 97.4. 5 Hazekamp JT. Current differences and consequences of legislation on practice of assisted reproductive technology in the Nordic countries. The Nordic Committee on Assisted Reproduction of the Scandinavian Federation of Societies of Obstetrics and Gynecology. Acta Obstet Gynecol Scand (1996); 75:198–200. 6 Clinical and laboratory guidelines for assisted reproductive technologies in the Nordic Countries: NFOG bulitinen supplement 1997:3 7 Dawson KJ. Quality control and quality assurance in IVF laboratories in the UK. Hum Reprod (1997); 12:2590–1. 8 Pool TB. Practices contributing to quality performance in the embryo laboratory and the status of laboratory regulation in the US. Hum Reprod (1997); 12:2591–3. 9 Lieberman BA, Matson PL, Hamer F. The UK Human Fertilisation and Embryology Act 1990—how well is it functioning? Hum Reprod (1994); 9:1779–82. 10 The Swedish national board of health and welfare act on assisted reproduction. SOSFS 1989:35 11 EN 45001. General criteria for the operation of testing laboratories. 1989. 12 EN ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories. 1999. 13 ISO 9001:2000. Quality management systems. 2000. 14 ISO/IEC Guide 25. General requirements for the competence of calibration and testing laboratories. Third edn, 1990. 15 SWEDAC document ME 46b. Handling, storing, culture and cryopreservation of fertilized eggs and embryos. 1999. 16 Ettarp L. An overview of international conformity assessment systems. WTO Technical Working Group, (1999). 17 Huisman W. Quality system in the medical laboratory: the role of a quality manual. Ann Biol Clin (Paris)(1994); 52:457–61. 18 ISO 10011. Guidelines for auditing quality systems. 1992. 19 Van Kooij JR, Peeters MF, te Velde ER. Twins of mixed races: consequences for Dutch IVF laboratories. Hum Reprod (1997); 12:2585–7. 20 Ackerman SB, Stokes GL, Swanson RJ, Taylor SP, Fenwick L. Toxicity testing for human in vitro fertilization programs. J In Vitro Fert Embryo Transf (1985); 2:132–7.

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

5 Evaluation of sperm Kaylen M Silverberg, Tom Turner

INTRODUCTION Abnormalities in sperm production or function, alone or in combination with other factors, account for 35–50% of all cases of infertility. Although a battery of tests and treatments has been described and continues to be used in the evaluation of female infertility, the male has been essentially neglected. Most programs offering advanced reproductive technologies (ART) in 2000 apparently employ an only cursory evaluation of the male—rarely extending beyond semen analysis (with or without strict morphology), and antisperm antibody detection. Several factors certainly account for this disparity. First, most practitioners of assisted reproductive technologies (ART) are gynecologists or gynecologic subspecialists who have little formal training in the evaluation of the infertile or subfertile male. Second, the urologists, who perhaps theoretically should have taken the lead in this area, have devoted little of their literature (and research budgets) to the evaluation of the infertile male. Third, and perhaps most important, is the inescapable fact that sperm function testing remains a very controversial area of research. Many tests have been described, yet few have been extensively evaluated in a proper scientific manner. Those that have continue to be weighed down by persistent criticisms of poor sensitivity or specificity, a lack of standardization of methods, suboptimal study design, problems with outcome assessment, and the lack of long term follow up. Although many of these same criticisms could also be leveled against most diagnostic algorithms for female infertility, in that arena the tests continue to prevail over their critics. Fourth, like female infertility, male infertility is certainly multifactorial. It is improbable that one sperm function test will prove to be a panacea, owing to the multiple steps involved in fertilization. In addition to arriving at the site of fertilization, sperm must undergo capacitation and the acrosome reaction; they must then penetrate through the cumulus, bind to the zona pellucida, penetrate through the zona, fuse with the oolemma, and then undergo nuclear decondensation. Finally, with the advent and rapid continued development of microassisted fertilization, sperm function testing has assumed a role of even lesser importance. As fertilization and pregnancy rates improve with

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procedures such as intracytoplasmic sperm injection (ICSI), more and more logical questions are being asked about the proper role for sperm function testing. This chapter will review the most commonly employed techniques for sperm evaluation, and examine the issues surrounding their utility in the modern ART program. PATIENT HISTORY

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A thorough history of the infertile couple at the time of the consultation will often reveal conditions that could affect semen quality. Some of the important factors to consider are: Reproductive history of the couple, including previous pregnancies with this and other partners. Sexual interaction of this couple, including frequency and timing of intercourse and duration of their attempt to become pregnant. Past medical and surgical history, Exposure to medication, drugs, and toxins including occupational and leisure activities, either in the past or in the present.1 SEMEN ANALYSIS The hallmark of the evaluation of the male remains the semen analysis. It is well known that the intrapatient variability of semen specimens from fertile men can vary significantly over time.1 This decreases the diagnostic information that can be obtained from a single analysis, often necessitating additional analyses. What is also apparent from literature analyzing samples from “infertile” patients is that the deficiencies revealed may not be sufficient to prevent pregnancy from occurring. Rather, they may simply lower the probability of pregnancy, resulting in so called subfertility. Clearly, the overall prognosis for a successful pregnancy is dependent on the complex combination of variables in semen quality coupled with the multiple factors inherent in the female partner. The commonly accepted standard for defining the normal semen analysis is the criteria defined by the World Health Organization (WHO). These parameters are listed in Table 5.1. COLLECTION OF THE SPECIMEN When the semen analysis is scheduled, instructions should be given to the couple to ensure the collection of an optimum semen sample. Written instructions are useful, especially if the patient is collecting the specimen outside the clinical setting. During the initial evaluation, a specimen should be obtained following a two to seven day abstinence from sexual activity.2 A shorter period of time may adversely affect the semen volume and sperm concentration. A longer abstinence may reduce the sperm motility. In light of the natural

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variability in semen quality that all men exhibit, the initial semen collection may not accurately reflect a typical ejaculate for that patient. A second collection a minimum of seven days later can eliminate tensions associated with the initial semen collection and give another count from which a typical set of semen variables can be determined. This second collection may also be used to appropriately adjust the abstinence period. Masturbation is the preferred method of collection. The use of lubricants is discouraged since most are spermicidal.1 However, some mineral oils may be acceptable. Since masturbation may present significant difficulty for some men, either in the clinic or at home, an alternative method of collection must be available. The use of certain silastic condoms (seminal collection devices) during intercourse may be an acceptable second

Table 5.1. WHO normal values for semen analysis. Parameter Normal values Liquefaction Complete within 60 minutes at room temperature Appearance Homogeneous, gray, and opaque Odor “Fresh” and characteristic Consistency Leaves a pipette as discrete droplets Volume >2ml pH 7.2–8.0 Concentration 20 million sperm/ml semen or greater Motility 50% or more with forward progression, or 25% or more with rapid progression within 60 minutes of collection Morphology 30% or more with normal forms Viability 75% or greater Leukocytes Less than 1 million/ml Immunobead Less than 20% with adherent particles test MAR Test Less than 10% with adherent particles choice. Interrupted intercourse should not be considered, as this method tends to lose the sperm rich initial few drops of semen while transferring many bacteria to the specimen container.2,3 CARE OF THE SPECIMEN Appropriate care of the ejaculate between collection and examination is important. Specimens should be collected only in approved, sterile, plastic, disposable cups. Washed containers may contain soap or residue of previous contents, which can kill or contaminate the sperm. Delivery of the semen to the lab should occur within sixty minutes of collection, and

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the specimen should be kept at room temperature during transport. These recommendations are designed to maintain optimal sperm motility through the time of analysis. CONTAINER LABELING The information recorded on the specimen container label should include the husband’s name as well as a unique identifying number. Typically, a social security number, birth date, or clinic assigned patient number is used. Other helpful information recorded on the label should include the date and time of collection and the number of days since the last ejaculation. When the specimen is received from the patient, it is important to confirm that the information provided on the label is complete and accurate. EXAMINATION OF THE SPECIMEN 1. LIQUEFACTION AND VISCOSITY When the semen sample arrives in the laboratory it is checked for liquefaction and viscosity. Although similar, these factors are distinct from each other.4,5 Liquefaction is a natural change in the consistency of semen from a semiliquid to a liquid. Before this process is completed, sperm are contained in a gel-like matrix that prevents their even distribution. Aliquots taken from this heterogeneous distribution of sperm for the purpose of determining concentration, motility, or morphology may therefore not be accurate. As liquefaction occurs over 15–30 minutes, sperm are released and distributed throughout the semen. Incomplete liquefaction may adversely affect the semen analysis in several ways. The coagulum that characterizes newly ejaculated semen results from secretions from the seminal vesicles. The liquefaction of this coagulum is the result of enzymatic secretions from the prostate. Watery semen, in the absence of a coagulum, may indicate the absence of the ejaculatory duct or seminal vesicles. Inadequate liquefaction, in the presence of a coagulum, may indicate a deficiency of prostatic enzymes.6,7 Viscosity refers to the liquefied specimen’s tendency to form drops from the tip of a pipette. If drops form and fall freely, the specimen has a normal viscosity. If drops will not form or the semen cannot be easily drawn up into a pipette, viscosity is high. This highly viscous semen prevents the homogenous distribution of sperm. Treatment with an enzyme, such as chymotrypsin,8 or aspiration through an 18-gauge needle may improve the distribution of sperm before an aliquot is removed for counting. Any addition of medium containing enzymes should occur only after the semen volume has been measured.

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

Sperm motility may be affected by many factors including the following: The patient’s age, health, and length of time since last ejaculation. The patient’s exposure to outside influences such as excessive heat or toxins. The method of collection. The length of time and adequacy of handling from collection to analysis. When the aliquot of semen is placed on the room temperature counting chamber, the count and motility should be determined immediately. This will prevent the influence of the heat from the microscope light source from influencing the results. If a chamber is used to count the sperm, the motility can be determined at the same time as the concentration by using

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a multiple click counter to tally motile and non-motile sperm and then totaling this number to arrive at the sperm concentration. The accuracy improves as more sperm are counted. If a wet mount slide is used to determine motility, more than one area of the slide should be used, and each count should include at least 100 sperm. Prior to examining the specimen for motility, the slide or counting chamber should be examined for signs of sperm clumping. Sperm clumping to other sperm, either head to head, head to tail, or tail to tail, may indicate the presence of sperm antibodies in the semen. This should not be confused with clumping of sperm to other cellular debris in the semen, which is not associated with antibodies.2,3 Motility is one of the most important prerequisites to achieving fertilization and pregnancy. The head of the sperm must be delivered a great distance in vivo through the barriers of the reproductive tract to the site of the egg. Sperm must have sufficient motility to penetrate the layers of coronal cells surrounding the egg as well as the zona pellucida and the egg’s cell membrane (oolemma). An exact threshold level of motility required to cause fertilization and pregnancy, however, has never been described.9 This may be due to the variables in equipment and technique used in assessing motility. 5. PROGRESSION

0 1 2 3 4

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

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

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

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

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

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As described by Kruger et al, sperm forms that are not clearly normal should be considered abnormal. Normal sperm morphology greater than 14% should be interpreted as a normal result. Normal morphology 4–14% should be considered to be borderline, and normal morphology less than 4% should be considered abnormal.11,12 Normal sperm morphology has been reported to be directly related to fertilization potential. This may be due to the abnormal sperm’s inability to deliver normal genetic material to the cytoplasm of the egg. From video recordings, it appears that abnormal sperm are more likely to have diminished or absent motility. This reduced motility may result from hydrodynamic inefficiency due to the head shape, abnormalities in the tail structure which prevent normal motion, and/or deficiencies in energy production necessary for motility.13,14 In addition to compromised motility, abnormal sperm do not appear to bind to the zona of the egg as well as normal sperm. This has been demonstrated in studies employing the hemizona binding assay.15 In vitro fertilization has helped further elucidate the part that normal sperm morphology plays in the fertilization process and in pregnancy. Both methods of determining normal sperm morphology, the WHO method and the Kruger strict method, have been used to predict a patient’s fertility. Several studies have concluded that the Kruger method of strict morphology determination shows the most consistent prediction of fertilization in vitro following conventional insemination.12,16,17 This method of assessing normal sperm morphology, because of its precise, non-subjective nature, establishes a threshold below which abnormal morphology becomes a contributing factor in infertility. COMPUTER ASSISTED SEMEN ANALYSIS Computer assisted semen analysis (CASA) was initially developed to improve the accuracy of the manual semen analysis. Its goal is to establish a standardized, objective, reproducible test for sperm concentration, motility, and morphology. The technique also attempts, for the first time, to actually characterize sperm movement. The automated sperm movement measurements— known as kinematics—include straight line velocity, curvilinear velocity and mean angular displacement (table 5.2).18 The use of CASA requires specialized equipment, including a phase contrast microscope, video camera, video recorder, video monitor, computer, and printer. To perform CASA, sperm are placed on either a Makler or a MicroCell chamber and then viewed under a microscope. The video camera records the moving images of the sperm cells and the computer digitizes them. The digitized images consist of pixels whose changing locations are recorded frame by frame. Thirty to 200 frames per minute are produced. The changing locations of each sperm are recorded and their trajectories are computed (fig 5.3).18 In this manner, hyperactive motion can also be

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detected and recorded. Hyperactive sperm exhibit a whiplike, thrashing movement, which is thought to be associated with sperm that are removed from seminal plasma and ready to fertilize the oocyte.18,19 Persistent questions about the validity and reproducibility of results have kept CASA from becoming standard equipment in the andrology laboratory. The accuracy of sperm concentration appears to be diminished in the presence of either severe oligospermia or excessive numbers of sperm. In oligospermia, counts may be overestimated owing to the machine counting debris as sperm. High concentrations of sperm may be underestimated, as individual sperm cannot be accurately counted in the presence of clumping. Sperm concentration also seems to be closely related to the type of counting chamber employed. Similarly, the process of dilution can interfere with accurate motility determination.19,20 Sperm motion parameters identified by CASA have been assessed by several investigators for their ability to predict fertilization potential. Certain types of motion have been determined to be important in achieving specific actions related to fertilization, such as cervical mucus penetration and zona binding. However, the overall value of CASA for predicting pregnancy is still the subject of much debate.

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

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harmonic MAG Magnitude of the fundamental harmonic

is computed using the Fourier transformation) Amplitude squared height of the HAR spectral peak (MAG is a measure of the peak to peak dispersion of the raw trajectory about its average path at the fundamental frequency) VOL Area of Area under the fundamental harmonic peak in the fundamental magnitude spectrum (VOL is a measure of the harmonic power-bandwidth of the signal) CON Specimen Concentration of sperm cells in a sample in millions concentration of sperm per milliliter of plasma or medium MOT Percent motility Percentage of sperm cells in a suspension that are motile (in manual analysis, motility is defined by a moving flagellum; in CASA, motility is defined by a minimum VSL for each sperm) Reproduced from reference 18.

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

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In summary, persistent questions about results and their interpretation continue to limit the routine use of CASA. As reproducibility improves over all range of sperm concentration, CASA may become the standard for semen analysis. In addition, as the kinematics of sperm motion become better understood, CASA may play an integral part in determining the optimal method of assisted reproductive technology that should be utilized for specific types of male factor patients. SPERM ANTIBODIES Because mature spermatozoa are formed after puberty, they can be recognized as foreign protein by a man’s immune system. In the testicle, the sperm are protected from circulating immunoglobulins by the tight junctions of the Sertoli cells. As long as the sperm are contained within the lumen of the male reproductive tract, they are sequestered from the immune system, and no antibodies form to their surface antigens. If there is a breach in this so called blood:testis barrier, an immune response may be initiated. The most common causes of a breach in the reproductive tract, which could initiate antibody formation, include vasectomy, varicocele repair, testicular biopsy, torsion, trauma, and infection.21,22 Antibodies are secreted into the fluids of the accessory glands, specifically the prostate and seminal vesicles. At the time of ejaculation, the fluids from these glands contribute to the seminal plasma. They then come into contact with the sperm and may cause them to clump. In women, the atraumatic introduction of sperm into the reproductive tract as a result of intercourse or artificial insemination does not seem to be a factor in the production of sperm antibodies. However, events that induce trauma, or introduce sperm to the mucous membranes outside of the reproductive tract, can induce antibody formation. Proposed examples of such events include trauma to the vaginal mucosa during intercourse or the deposition of sperm into the gastrointestinal tract by way of oral or anal intercourse.22 Several tests are currently employed for detecting the presence of sperm antibodies. The two most common are the following: (1) The mixed agglutination reaction (MAR) This test is performed by mixing semen, immunoglobulin G (IgG) or IgA coated beads or rbcs, and IgG or IgA antiserum on a microscope slide. The slides are incubated and observed at 400×. If antibodies are present, the sperm will form clumps with the coated latex beads or coated rbcs. If antibodies are absent, the sperm will swim freely. The level of antibody concentration considered to be clinically relevant must be established by each center conducting the test. The WHO considers a level of binding of 50% or greater to be clinically significant. This test is used only for detection of direct antibodies in men, and is not specific for location of bead attachment on the sperm.

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(2) The immunobead binding test This test is performed by combining IgG or IgA coated latex beads and washed sperm on a slide. After incubation, the slides are read at 200× or 400×. If antibodies are present, the small beads will attach directly to the sperm. This test provides potentially greater information, as results consider the number of sperm bound by beads, the type of antigen involved in binding, and the specific location where the bead is bound to the sperm. If antibodies are absent, the beads will not attach. This test can be used for the detection of direct antibodies in men. However, unlike the MAR test, it may also be used to detect antibodies produced in a woman’s serum, follicular fluid or cervical mucus by incubating these bodily fluids with washed sperm that have previously tested negative for antibodies. To perform an indirect test, known direct antibody negative sperm are washed and mixed on a slide for a one hour incubation with the bodily fluid to be tested and IgG or IgA-coated latex beads. The test is interpreted by noting the percentage and location of bead attachment. The WHO considers a level of binding of 20% or greater to represent a positive test. Clinical significance is commonly considered to be a level of binding of 50% or more.9,23 The clinical value of antisperm antibody testing is predicated on the observation that the presence of a significant concentration of antibodies may impair fertilization. It has been reported that antibody-positive sperm may have difficulty penetrating cervical mucus. Although, in these cases, intrauterine insemination (IUI) or IVF may improve the prognosis for fertilization, antibody levels exceeding 80% coupled with subpar concentration, motility or morphology may necessitate the addition of ICSI in order to truly make a difference.24 As suggested by the literature, andrology labs may benefit greatly in their preparation of sperm if they are aware of the presence of antibodies. In summary, antisperm antibodies have been demonstrated to be a contributing factor in infertility. While their presence alone may not be sufficient to prevent pregnancy, their detection should encourage the andrologist to pursue additional appropriate action. SPERM VIABILITY An intact plasma membrane is an integral component of, and possibly a biologic surrogate for sperm viability. The underlying principle is that viable sperm contain intact plasma membranes that prevent the passage of certain stains, whereas non-viable sperm have defects within their membranes that allow for staining of the sperm. Several so called vital stains have been employed for this purpose. They include eosin Y, trypan blue, and/or nigrosin.25 When viewed with either bright field or phase contrast microscopy, these stains allow for the differentiation of viable, non-motile sperm from dead sperm. This procedure may, therefore, play a significant part in selecting appropriate sperm to use for ICSI when only

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immotile sperm are present. Unfortunately, however, dyes such as eosin Y are specific DNA probes which may have toxic effects if they enter a viable sperm or oocyte. Flow cytometry has also been used for the determination of sperm viability. Like vital staining, flow cytometry is based on the principle that an intact plasma membrane will prevent the passage of nucleic acid specific stains. Some techniques employ dual staining, such as the one described by Noiles et al, which can differentiate between an intact membrane and a damaged membrane.26 There are no studies that prospectively evaluate sperm viability staining as a predictor of ART outcome. HYPO-OSMOTIC SWELLING TEST Another means of assessing the sperm plasma membrane is the hypoosmotic swelling test (HOST). This assay is predicated upon the observation that all living cells are permeable to water, although to different degrees. The human sperm membrane has one of the highest hydraulic conductivity coefficients (2.4um/min/atm at 22°C) of any mammalian cell.27 As originally described, the HOST involves placing a sperm specimen into hypotonic conditions of approximately 150mOsm.28 This environment, while not sufficiently hypotonic to cause cell lysis, will cause swelling of the sperm cells. As the tail swells, the fibers curl, and this change can be detected by phase contrast microscopy. The normal range for a positive test has been described as a score greater than or equal to 60%—60% of the cells demonstrate curling of the tails. A negative test is defined as less than 50% curling.29 This test generated a significant amount of initial interest, and several investigators compared it to the sperm penetration assay (SPA) as an in vitro surrogate for fertilization, reporting good correlation.30,31 More recently, the test has been employed as a predictor of ART outcome, with conflicting results. Although one group reported a favorable correlation, another found no predictive value for the test.32,33 It has also been suggested that, owing to sperm morphology changes in response to the test, the HOST may impair an embryologist’s ability to select sperm appropriate for injection. In summary, the HOST lacks sufficient critical evaluation to determine its true role in the evaluation of the infertile male. ASSAYS OF THE SPERM ACROSOME The acrosome is an intracellular organelle, similar to a lysosome, which forms a cap-like structure over the apical portion of the sperm nucleus.34 The acrosome contains multiple hydrolytic enzymes, including hyaluronidase, neuraminidase, proacrosin, phospholipase, and acid phosphatase, which, when released, are thought to facilitate sperm passage

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through the cumulus mass, and possibly the zona pellucida as well (fig 5.4). Once sperm undergo capacitation, they are capable of an acrosome reaction. This reaction is apparently triggered by fusion of the sperm plasma membrane with the outer acrosomal membrane at multiple sites, leading to diffusion of the acrosomal enzymes into the extracellular space. This results in dissolution of the plasma membrane and acrosome, leaving the inner acrosomal membrane exposed over the head of the sperm (fig 5.5).

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

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Fig 5.5 Acrosome reacted sperm IA=inner acrosomal membrane Reproduced from reference 58. Although electron microscopy has produced many elegant pictures of acrosome intact and acrosome reacted sperm, it is not always possible to know if sperm that fail to exhibit an acrosome have truly acrosome reacted, or could possibly be dead. In addition, electron microscopy is not a technique available to all andrologists. This has lead to the necessity for the development of biochemical markers for the acrosome reaction. Throughout the 1970s and 1980s, multiple biochemical tests were described using a variety of lectins, antibodies, and stains. Although they apparently correlated well with electron microscopy, the tests were still time consuming and difficult to perform.35,36 Contemporary assays for acrosomal status determination employ fluorescent plant lectins or monoclonal antibodies, which can then be detected much more easily with fluorescence microscopy.37,38 These assays may prove to be of value if they can truly identify males who manifest deficiencies in their ability to undergo the acrosome reaction. Hypothetically, such patients may need to have their sperm specially

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preincubated—such as with follicular fluid or calcium ionophore—prior to insemination if they fail to acrosome react on their own. Conversely, this test may help identify a small sub-population of males who prematurely acrosome react. Several studies have reported an association between ejaculated sperm with low percentages of acrosome-intact sperm and poor subsequent fertilization.39 These areas certainly await additional study. OTHER BIOCHEMICAL TESTS As noted above, one of the predominant enzymes present in the acrosome is proacrosin. This enzymatic action of acrosin is not necessarily correlated to the presence of an intact acrosome, therefore assays for the presence of acrosin have been described.40 Acrosin activity has been reported to be greater in fertile males than in infertile males,41 however, there are no prospective evaluations correlating acrosin activity to fertilization rates in ART patients. Like all other tissues that require energy synthesis and transport, spermatozoa contain measurable levels of creatinine phosphokinase (CPK). Two isomers, CK-M and CK-B have been described, and differences have been noted in these levels in semen specimens from fertile and infertile males. Specifically, CK-M levels exceed CK-B levels in normospermic males, while CK-B levels are greater in spermatozoa from oligospermic males.42 In this same study, researchers found that semen samples in which CK-M ratios exceeded 10% exhibited higher fertilization rates in IVF than specimens with lower ratios. Few other studies have addressed this topic. SPERM PENETRATION ASSAY The sperm penetration assay or hamster egg penetration assay (HEPA) was initially described by Yanagimachi et al. in 1976.43 Oocytes from the golden hamster were treated in order to remove the zona pellucida. One of the functions of the zona is to confer species specificity, therefore its presence would preclude performance of this test. Human sperm were then incubated for 48 hours with the hamster oocytes, and the number of penetrations with nuclear decondensation were calculated. As originally described, it was hoped that the test would correlate with the ability of human sperm to fertilize human oocytes in vitro. Although the test was designed in order to assess the ability of sperm to fuse with the oolemma, it also indirectly assesses sperm capacitation, the acrosome reaction, and the ability of the sperm to be incorporated into the ooplasm. Unfortunately, however, intrinsic in the design of the test is the inability to assess the sperm’s ability to bind to—and penetrate through—the zona pellucida. This factor continues to be one of the major criticisms that plague this test.

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Throughout the 1980s, multiple modifications of the SPA were published. These included modifications of the techniques for sperm preparation prior to the performance of the assay such as inducing the acrosome reaction or incubation with TEST yolk buffer, changes in the protocol methodology itself, and modifications of the scoring system.44,45 Published reports demonstrated widely varying conclusions, such as the finding that the SPA could identify 0–78% of men whose sperm would fail to fertilize oocytes in ART procedures.46 Most criticisms of the SPA literature center on poor standardization of the assay, poor reproducibility of the test, and lack of a standard normal range. Although some reports suggest a correlation between the SPA and fertility, neither a large literature review46 nor a prospective long term (five-year) follow-up study demonstrate such a correlation.47 In light of these considerations, support for this test has waned over the past several years.

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

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HEMI-ZONA ASSAY Over the past several years, a growing body of research has demonstrated a significant correlation between tests of sperm:zona pellucida binding and subsequent fertilization in ART. This led the European Society of Human Reproduction and Embryology (ESHRE) Andrology Special Interest Group to recommend inclusion of such tests in the advanced evaluation of the male.48 Like the SPA, the hemi-zona assay (HZA) employs sperm and non-viable oocytes in an in vitro assessment of fertilization.49 In this test, however, both gametes are human in origin. Classically, oocytes that failed to fertilize during an ART procedure are bisected, and then sperm from a proven fertile donor (500,000/ml) are added to one hemi-zona, while sperm from the subject male are added to the other hemi-zona. Following a four hour incubation, each hemizona is removed and pipetted in order to dislodge loosely attached sperm. A comparison or hemi-zona index (HZI) is then calculated by dividing the number of test sperm tightly bound to the hemizona by the number of control (fertile) sperm bound to the other hemizona. HZI= # test sperm bound/# control sperm bound ×100 This test assesses the ability of sperm to bind to the zona itself. Although expensive, labor intensive, and difficult to perform, there are some data which suggest that the HZA may help identify individuals with a poor prognosis for success with ART (fig 5.6).50,51 A more recent prospective study employing receiver operating characteristic (ROC) curve analysis has also suggested that HZA results may be used to predict subsequent fertilization in ART procedures with both high sensitivity and specificity.52 MANNOSE BINDING ASSAY Another test has been recently developed in order to assess the ability of sperm to bind to the zona. This in vitro procedure is based on a series of observations which suggest that sperm:oocyte interaction involves the recognition by a sperm surface receptor of a specific complimentary receptor on the surface of the zona pellucida. This zona receptor appears to be a glycoprotein, the predominant sugar moiety of which is mannose.53 In an elegant series of experiments, Mori et al determined that sperm:zona binding could be curtailed by the addition of a series of sugars to the incubating media. Although many sugars impaired binding, the addition of mannose totally inhibited sperm:oocyte interaction.54 In vitro assays in which labeled probes of mannose conjugated to albumin are coincubated with semen specimens allow for the differential staining of sperm (fig 5.7). Those that bind the probe are thought to

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

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

CONCLUSION In summary, there have been many recent advances in the diagnostic evaluation of sperm and sperm function. Although many tests of sperm function have been described, there remains a lack of consensus as to both the role of testing and the identification of the appropriate test(s) to perform. Owing to the complicated nature of sperm function, it is improbable that a single test will emerge with sufficient sensitivity, specificity, and positive and negative predictive values required of a first line diagnostic tool for all affected males. A more likely scenario will be like that in female infertility, where a battery of tests—each evaluating a specific function—is employed as needed. In the light of profound recent advances in gamete micromanipulation, a more germane issue might be the overall relevance of sperm function testing in the contemporary andrology laboratory. Although this issue is

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quite controversial, it is likely that sperm function testing will continue to play a role in the evaluation of the infertile male. Just as ART is not the treatment of choice for all infertile women, it is not likely that micromanipulation will become standard treatment for all infertile men. The gold standard of sperm function remains the ability to fertilize an oocyte in vitro. Therefore, in order to continue to address the above questions, it is incumbent upon investigators to design appropriate prospective trials in order to thoroughly assess these tests. Those tests that demonstrate a significant correlation with fertilization in vitro must then undergo additional evaluation in order to assess clinical significance if we hope to develop an appropriate diagnostic algorithm.

REFERENCES 1 Gangi CR, Nagler HM. Clinical evaluation of the subfertile man. In: Diamond MP, DeCherney AH, Overstreet JW, eds. Infertility and reproductive medicine. Clinics of North America. Philadelphia: WB Saunders (1992); 3:299–318. 2 World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. New York: Cambridge University Press (1992):3–27. 3 Alexander NJ. Male evaluation and semen analysis, Clin Obstet Gynecol (1982); 25:463–82. 4 Overstreet JW, Katz DF, Hanson FW, Foseca JR, A simple inexpensive method for objective assessment of human sperm movement characteristics. Fertil Steril (1979); 31:162–72. 5 Overstreet JW, Davis RO, Katz DF, Overstreet JW, eds. Infertility and reproductive medicine. Clinics of North America. Philadelphia: WB Saunders (1992): 329–40. 6 Koren E, Lukac J. Mechanism of liquefaction of the human ejaculate: I. Changes of the ejaculate proteins. J Reprod Fertil (1979); 56:493–500. 7 Lukac J, Koren E. Mechanism of liquefaction of the human ejaculate: II. Role of collagenase like peptidase and seminal proteinase. J Reprod Fertil (1979); 56:501–10. 8 Cohen J, Aafjes JH. Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration. Life Sciences (1982); 30:899–904. 9 Van Voorhis BJ, Sparks A. Semen analysis: What tests are clinically useful? Clin Obstet Gynecol (1999); 42:957–71. 10 Zuckerman Z, Rodriquez-Rigau IJ, Smith KD, Steinberger E. Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril (1977); 28:1310–3. 11 Kruger TF, Menkveld R, Stander FS, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril (1986); 46:1118–23.

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12 Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF; Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril (1988); 49:112–17. 13 Katz DF, Overstreet JW. Sperm motility assessment by videomicrography. Fertil Steril (1981); 35:188–93. 14 Katz DF, Diel L, Overstreet JW. Differences in the movements of morphologically normal and abnormal human seminal spermatozoa. Biol Reprod (1982); 26:566–70. 15 Franken Dr, Oehninger S, Burkman LJ, et al. The hemizona assay (HZA): a prediction of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J In Vitro Fert Embryo Transfer (1989); 6:44–50. 16 Coetzee K, Kruger TF, Lombard CJ. Predictive value of normal sperm morphology: A structured literature review. Hum Reprod Update (1988); 4:73–82. 17 Enginsu MF, Pieters MGEC, Dumoulin JCM, Evers JLH, Geruedts JPM. Male factor as determinant of in vitro fertilization outcome. Hum Reprod (1992); 7:1136–40. 18 Davis R. The promise and pitfalls of computer aided sperm analysis. In Diamond MP, DeCherney AH, Overstreet JW, eds. Infertility and reproductive medicine: clinics of North America (1992); 93:341–52. 19 Irvine DS. The computer assisted semen analysis systems: sperm motility assessment. Hum Reprod (1995); 10 (suppl 1):53–9. 20 Krause W. Computer assisted semen analysis systems: Comparison with routine evaluation and prognostic value in male fertility and assisted reproduction. Hum Reprod (1995); 10 (suppl 4):60–6. 21 Marshburn PB, Kuttch WH. The role of antisperm antibodies in infertility. Fertil Steril (1994); 61:799–811. 22 Golumb J, Vardinon N, Hommonnai ZT, et al. Demonstration of antispermotozoal antibodies in varicocele-related infertility with an enzyme-linked immunosorbent assay (ELISA). Fertil Steril (1986); 45:397–405. 23 Helmerhost FM, Finken MJJ, Erwich JJ. Detection assays for antisperm antibodies: What do they test? Hum Reprod (1999); 14:1669–71. 24 Bronson R. Detection of antisperm antibodies: an argument against therapeutic nihilism. Hum Reprod (1999); 14:1671–73. 25 World Health Organization. Manual for examination of human semen and semen-cervical mucus. Cambridge: Cambridge University Press (1987): 1–12. 26 Noiles EE, Ruffing NA, Kleinhans FW, et al. Critical tonicity determination of sperm using dual fluorescent staining and flow cytometry. In Johnson LA, Rath D, eds. Reproduction in domestic animals. (Suppl 1.) Boar Semen Preservation II. Beltsville, MD: Proceedings of the Second International Conference on Boar Semen Preservation (1991):359–64.

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27 Noiles EE, Mazur P, Kleinhans FW, et al. Determination of the water permeability coefficient and its activation energy for human spermatozoa. Biol Reprod (1993); 48:99–109. 28 Jeyendran RS, Van der Ven JJ, Perez-Pelaez M. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil (1984); 70:219–28. 29 Zaneveld LJD, Jeyendran RS. Modern assessment of semen for diagnostic purposes. Semin Reprod Endocrinol (1988); 4:323–37. 30 Chan SYW, Fox EJ, Chan MMC. The relationship between the human sperm hypoosmotic swelling test, routine semen analysis, and the human sperm zona free hamster ovum penetration test. Fertil Steril (1985); 44:688–92. 31 Jeyendran RS, Zaneveld LJD. Human sperm hypoosmotic swelling test. Fertil Steril (1986); 46:151–4. 32 Mladenovic I, Micic S, Genbacev O, et al. The hypoosmotic swelling test for quality control of sperm prepared for assisted reproduction. Arch Androl (1995); 34:163–9. 33 Joshi N, Kodwany G, Balaiah D, et al. The importance of CASA and sperm function testing an in vitro fertilization program. Int J Fertil Menopausal Stud (1996); 41 (1):46–52. 34 Critser JK, Noiles EE. Bioassays of sperm function. Sem Reprod Endocrinol (1993); 11 (1):1–16. 35 Talbot P, Chacon RS. A triple stain technique for evaluating acrosome reaction of human sperm. J Exp Zool (1981); 215:201–8. 36 Wolf DP, Boldt J, Byrd W, et al. Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies. Biol Reprod (1985); 32:1157–62. 37 Cross NL, Morales P, Overstreet JW, et al. Two simple methods for detecting acrosome-reacted sperm. Gamete Res (1986); 15:213–6. 38 Holden CA, Hyne RV, Sathananthan AH, et al. Assessment of the human sperm acrosome reaction using Concanavalin A lectin. Mol Reprod Dev (1990); 25:247–57. 39 Chan PJ, Corselli JU, Jacobson JD, et al. Spermac stain analysis of human sperm acrosomes. Fertil Steril (1999); 72:124–8. 40 Kennedy WP, Kaminski JM, Van der Ven HH, et al. A simple clinical assay to evaluate the acrosin activity of human spermatozoa. J Androl (1989); 10:221–31. 41 Mohsenian M, Syner FN, Moghissi KS. A study of sperm acrosin in patients with unexplained infertility. Fertil Steril (1982); 37:223–9. 42 Huszar G, Vigue L, Morshedi M. Sperm creatinine phosphokinase Misoform ratios and fertilizing potential of men: A blinded study of 84 couples treated with in vitro fertilization. Fertil Steril (1992); 57:882– 8.

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43 Yanagimachi R, Yanagimachi H, Rogers BJ. The use of zona-free animal ova as a free system for the assessment of their fertilizing capacity of human spermatozoa. Biol Reprod (1976); 15:471–6. 44 Aitken RJ, Thatcher S, Glasier AF, et al. Relative ability of modified versions of the hamster oocyte penetration test, incorporating hyperosmotic medium of the ionophore A23187 to predict IVF outcome. Hum Reprod (1987); 2:227–31. 45 Jacobs BR, Caulfield J, Boldt J. Analysis of TEST (TES and tris) yolk buffer effects on human sperm. Fertil Steril (1995); 63:1064–70. 46 Mao C, Grimes DA. The sperm penetration assay: Can it discriminate between fertile and infertile men? Am J Obstet Gynecol (1988); 159:279–86. 47 O’Shea DL, Odem RR, Cholewa C, et al. Long-term follow-up of couples after hamster egg penetration testing. Fertil Steril (1993); 60:1040–5. 48 Consensus Workshop on Advanced Diagnostic Andrology Techniques. ESHRE Andrology Special Interest Group. Hum Reprod (1996); 11:1463–79. 49 Burkman LJ, Coddington CC, Franken DR, et al. The hemi-zona assay (HZA): Development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril (1988); 49:688–97. 50 Oehninger S, Acosta AA, Marshedi M, et al. Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil Steril (1989); 50:283–7. 51 Oehninger S, Toner J, Muasher S, et al. Prediction of fertilization in vitro with human gametes; Is there a litmus test? Am J Obstet Gynecol (1992b); 1760–7. 52 Coddington CC, Oehninger SC, Olive DL, et al. Hemizona index (HZI) demonstrates excellent predictability when evaluating sperm fertilizing capacity in in vitro fertilization patients. J Androl (1994); 15:250–4. 53 Mori K, Daitoh T, Irahara M, et al. Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization. Am J Obstet Gynecol (1989); 161:207–11. 54 Mori K, Daitoh T, Kamada M, et al. Blocking of human fertilization by carbohydrates. Hum Reprod (1993); 8: 1729–32. 55 Tesarik J, Mendoza C, Carreras R. Expression of D-mannose binding sites on human spermatozoa: Comparison of fertile donors and infertile patients. Fertil Steril (1991); 56:113–8. 56 Benoff S, Cooper GW, Hurley I, et al. Human sperm fertilizing potential in vitro is correlated with differential expression of a headspecific mannose ligand receptor. Fertil Steril (1993); 59:854–62. 57 Silverberg K, Dey T, Witz C, et al. D-Mannose binding provides a more objective assessment of male fertility than routine semen

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

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

OVERVIEW The aim of sperm preparation for ART is to maximize the chances of fertilization to provide as many normally fertilized oocytes as possible for transfer to the uterus or cryopreservation.1 With normal semen it is easy to obtain motile sperm by a variety of techniques. Abnormal semen, which will not yield adequate sperm for standard in vitro fertilization (IVF), needs to be recognized so that intracytoplasmic sperm injection (ICSI) can be used. Refinements of the preparation procedures are required to obtain spermatozoa or elongated spermatids with the highest potential for normal fertilization from grossly abnormal semen samples or from samples obtained directly from the male genital tract. Sperm characteristics important for fertilization with standard IVF include: normal morphology, normal intact acrosomes, straight line velocity (VSL) and linearity (LIN), and ability to bind to the zona pellucida, penetrate the zona pellucida, fuse with the oolemma, activate the oocyte, and form a male pronucleus.1 For ICSI live sperm with ability to activate the oocyte and form a pronucleus are necessary but morphology, motility, and acrosome status are generally not important.1,2,3,4,5 It is probably important to remove seminal plasma as it contains decapacitation factors and extraneous cells and degenerating sperm that may produce agents capable of damaging the sperm.6,7,8 For IVF or gamete intra-fallopian transfer (GIFT), the medium should contain protein and buffers that promote sperm capacitation.1 While serum or high molecular weight fractions from serum appear to be important for sperm motility, more recently comparatively pure preparations of human serum albumin, pasteurized to reduce the risk of transmitting infections, have been found to be adequate for sperm preparation for standard IVF and ICSI.9,10 The inclusion of protein in the culture medium is required to prevent sperm adhering to surfaces. Although the concentration of albumin in human periovulatory oviductal fluid is reported to be of the order of 30mg/ml, concentrations of around 4mg/ml will support normal sperm function in IVF. Bicarbonate ions are required for capacitation of sperm and are normally present at about 25mmol/l in the medium. Although glucose is utilized as a metabolic substrate by sperm it is not clear whether it is essential for normal function in vitro. It has been suggested that more recent media formulations, which do not contain

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glucose, may not be appropriate for fertilization stages of assisted reproductive technologies (ART) procedures. Damage to the sperm from dilution, temperature change, centrifugation, and exposure to potentially toxic material must be minimized. Dilution should be performed slowly, especially with cryopreserved sperm. Temperature changes should be gradual. Preparation of the insemination suspension should be performed at 37°C. Centrifugal force should be the lowest possible required to bring down the most motile sperm. Minimizing centrifugation, particularly in the absence of seminal plasma, and separating the live motile sperm from the dead sperm and debris early in the procedure should limit oxidative damage caused by free oxygen radicals released from leucocytes or abnormal sperm.6,7 Modifications of sperm preparation may be necessary for the various types of ART For example, for GIFT or intratubal insemination, suspensions of spermatozoa are to be introduced into the fallopian tubes, so debris and bacteria must be removed and no particulate material added that might damage the female genital tract. If cryopreserved donor sperm are to be used matching and extra care in preparation of the sample is usually required. If the semen is severely abnormal sperm are prepared for ICSI. Combinations of gradient centrifugation and swim up may produce higher yields of good quality sperm.11 However, in the era of ICSI the need for special preparation techniques has receded as simple procedures with swim up, washing, or allowing sperm to swim to the medium oil interface from a centrifuged pellet placed in droplets of medium under oil, produce fertilization and pregnancy results as good as those with sperm obtained by more careful and laborious preparation techniques.12 The optimal number of sperm for insemination is poorly defined, but several reviews of results of IVF suggest that there is an increase in fertilization rate with insemination of sperm at between 2000 and 500000 per ml.1 There may be some increase in risks of polyspermy with the higher sperm concentrations thus most groups inseminate oocytes with about 100000 sperm/ml for standard IVF or GIFT. This is more than surround the oocyte in vivo and, if better selection of high quality sperm could be achieved, insemination with lower numbers could be as or more successful. It has been suggested that reduced exposure of the oocyte to sperm may result in improvement in embryo quality and higher implantation rates.13,14 The total volume of sperm suspension added should be minimized to restrict dilution of the oocyte medium.

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

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COLLECTION OF SEMEN OR SPERM While semen for ART is usually collected by masturbation, sperm may be collected by a variety of methods from several sites in the male genital tract (Fig 6.1). The man should collect semen, in a room adjacent to the IVF laboratory, into a sterile disposable plastic jar. The sperm should be prepared soon after liquefaction of the seminal plasma. If liquefaction is delayed or the specimen is particularly viscous, syringing the sample through a 21 gauge needle or mixing the specimen 1:1 with medium followed by vigorous shaking may help. If the semen sample is unexpectedly poor, a second sample

Fig 6.1 Possible sites of collection of sperm or elongated spermatids from the male genital tract for ART. may provide sufficient sperm. Cryopreserved sperm can also be used, for example, as backup for ICSI for patients with motile sperm present in the semen only intermittently.

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The timing of semen collection and preparation does not appear to be critical, especially with good semen samples. In general the oocytes are inseminated four to six hours after collection and the sperm can be prepared during this time. The semen should be placed in a sterile area of the laboratory or in a laminar flow hood. The sample must be mixed thoroughly because ejaculation does not result in a homogeneous suspension of sperm in the seminal plasma. The semen sample is examined, any particulate material allowed to settle and the supernatant transferred to another tube. Following mixing, a small portion (~10µl) of the sample is taken to check the sperm concentration and motility. With normal semen samples, usually 1 ml of sample is sufficient for preparation of adequate numbers of motile sperm. If the semen sample is mildly to moderately abnormal but judged adequate for standard IVF then the whole semen volume should be distributed to several tubes for preparation of as many sperm as possible. SPERM PREPARATION Initially, IVF involved repeated “washing” of the spermatozoa by dilution of the semen with culture medium supplemented with protein, followed by centrifugation and resuspension of the pellet. This technique has been criticized as it may result in oxidative damage of the sperm by free oxygen radicals.6,7,15,16 The swim up procedure is now the most commonly used technique (Fig 6.2). Sperm for ICSI may be harvested from the oil medium interface after sperm containing material is placed in a drop of culture medium under oil (Fig 6.3). Some prepare channels to outlying smaller droplets for this purpose. All plastic, glassware, and media should be checked for toxicity to sperm or embryos. Sperm may be immobilized by contact with rubber. A variety of media are suitable for sperm preparation for IVF. The medium chosen should be equilibrated with the gas mixture and the temperature maintained constant at 37°C. The protein source for the medium needs to be checked for sperm antibodies, and, if pools are used, the donors must be tested for viral illnesses including HIV infection and hepatitis. However, the use of pooled serum samples is to be discouraged because of the risk of transmitting both known and unknown diseases. Heat inactivation of the serum should not be relied upon to overcome the risk of transmitting infections. SWIM UP Several variations of the swim up procedure are possible. The seminal plasma can be overlaid directly with culture medium and the sperm allowed to swim from the seminal plasma into the culture medium. Following this the sperm suspension should be washed to ensure adequate removal of seminal plasma constituents. Alternatively the semen sample

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Fig 6.2 Methods of sperm preparation for ART. may be diluted and centrifuged and the pellet loosened and overlaid, or the semen sample may be centrifuged without prior dilution of the seminal plasma and the pellet loosened and overlaid with medium for the swim up procedure. The latter technique may be particularly useful for oligozoospermia as the sperm may be damaged by the dilution procedure.

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If cryopreserved semen is to be used, dilution of the semen sample should be slow with dropwise addition of culture medium to the thawed sample. If the thawed semen is overlaid directly, the need for slow dilution is eliminated. After centrifugation the supernatant is aspirated off the pellet and the pellet gently resuspended in a small volume of liquid. The overlay medium is then gently pipetted onto the surface of the pellet and the tube incubated for 45–60 minutes. Prolonged incubation times may result in a reduced yield of motile sperm from gravitational effects. The use of a conical tube for centrifugation may help maximize yield as the pellet is easier to see and less likely to be disturbed during manipulation. Some recommend that the tubes be placed in the incubator on an angle to increase the surface area of the interface. Following incubation, the upper half to two thirds of the overlay is aspirated, mixed, and the sperm concentration determined. DENSITY GRADIENTS Various gradient separation procedures have been introduced. The advantage is that the gradient separation techniques are rapid, requiring 20 minute centrifugation compared with an average of one hour incubation for swim up. They are also relatively simple to perform under sterile conditions (Fig 6.2). The most popular of these is colloidal silica density gradient (CSDG) centrifugation, but other agents have also been used.1,11,17 The colloidal silica particles are coated with polyvinylpyrollidone, for example, PercollTM (Pharmacia AB, Uppsula, Sweden). However, concerns regarding the levels of endotoxins have resulted in the withdrawal of Percoll from use in ART. Other media containing silane coated silica have become available for clinical use including Isolate (Irvine Scientific, Santa Ana, Ca, USA) and PureSperm (Nidacon Laboratories, AB, Gothenburg, Sweden).18 Discontinuous gradients of two or more steps are used. Sperm and other material form distinct bands at the interfaces on the CSDG. It has been claimed that abnormal sperm as

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Fig 6.3 Procedure for seminiferous tubules obtained by fine needle tissue aspiration or open biopsy. well as immotile sperm and debris are largely eliminated, and a rapid and efficient isolation of motile human sperm, free from contamination with other seminal constituents, is possible. Several studies have compared CSDG centrifugation with a swim up and occasionally other sperm preparation techniques. The end points of the studies have been recovery of motile sperm, morphology, chromatin structure assessed by the various techniques, and ultrastructure. Generally the recovery of motile sperm is

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greater with the gradient techniques, but the percentage of sperm with progressive motility is usually lower and the proportion of sperm with good morphology lower with gradient centrifugation than with swim up.1,8,11,18–20 Some studies suggest that the gradient materials may damage the sperm.21,22 SPERM PREPARATION FROM SURGICAL ASPIRATES OR TISSUE SAMPLES Spermatozoa or elongated spermatids may be obtained for ICSI from the male genital tract by microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular open biopsy, fine needle aspiration biopsy, or other techniques (Fig 6.2) and prepared by the methods outlined in appendices 7 and 8.1,2 SPERM SELECTION FROM IMMOTILE SAMPLES ICSI with immotile sperm is often associated with low fertilization rates thus every attempt should be made to ensure that live sperm are injected.1– 4,23 Various agents have been reported to enhance sperm motility.1 Pentoxifylline (POF) has been used for ART. The maximally effective dose of POF is between 0.3mmol/l and 0.6mmol/l and many groups use 3.6mmol/l (1mg/ml). POF has been reported to provide greater stimulation of motility and velocity than caffeine or 2-deoxyadenosine. Appendices 10 and 11 give methods for stimulating sperm motility with POF and demonstrating membrane integrity by hypoosmotic swelling.

RESULTS The normal fertilization and embryo utilization rates are compared for swim up and CSDG in Table 6.1. Apart from the improvement in the normal fertilization rate with CSDG for IVF with oligozoospermic samples, the results are similar. Results with sperm or elongated spermatids obtained from the genital tract cryopreserved samples, and following the use of hypo-osmotic swelling have been published.4,24,25

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

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Table 6.1. Comparison of results with swim up and colloidal silica density gradient (CSDG) preparation of sperm from semen for IVF or ICSI from men with normal semen (sperm concentration ≥20×106ml, progressive motility ≥40% and abnormal morphology ≤85%), abnormal semen (sperm concentration 1–19×104ml or progressive motility 1–39% or abnormal morphology 86–100%) or oligoasthenoteratozoospermia (sperm concentration 1– 19×106ml, progressive motility 1–39% and abnormal morphology 86–100%) from 1990–1999. Men with sperm autoimmunity were excluded. Embryo utilization is the sum of embryos transferred fresh and those frozen for later transfer. Percentages using oocytes collected as the denominator are shown in italics. Asterisks indicate significant differences between results for swim up and CSDG (P20×106/ml with moderate to good forward progressive motility, swim up can be performed. • Borderline samples may be better prepared by CSDG centrifugation. • If the sample is unexpectedly poor on the day (for example, concentration 5000 motile sperm/ml should have sufficient yield of live sperm post thaw for subsequent ICSI treatments. A method for cryopreservation of such samples is given in appendix 9. APPENDIX 8— TESTICULAR BIOPSY • Place tissue into a small Petri dish of warm HTF/HEPES/ALB.

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

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APPENDIX 9— FREEZING PROTOCOL FOR OLIGOZOOSPERMIA AND WASHED SPERM Semen containing only a few motile sperm and sperm suspensions obtained from the genital tract can be stored for subsequent ICSI. If necessary the semen is centrifuged to concentrate the sperm into a minimum volume of 0.4ml. MESA samples or other sperm suspensions are processed in the IVF laboratory by swim up, centrifugation on density gradients, or washing and resuspended in IVF medium. The remaining sperm suspension can be cryopreserved with glucose citrate glycine (GCG) cryoprotectant, glycerol and patient’s serum or 5% albumin solution. GCG cryoprotectant: Dissolve glucose (1.0g) and sodium citrate (1.0g) in 40ml of sterile deionized water and add glycine (1.0g). (pH ~7.5 and osmolality ~500mOsm/ kg). It is stored in 2ml volumes at −70°C. When a sample arrives, thaw a vial of GCG. Add 10ml glycerol. If the sample volume is >2.0ml and contains few motile sperm, centrifuge at 1800g for 5 minutes at room temperature and aspirate the supernatant to leave about 1.0ml and resuspend the sperm. Determine percentage total motile sperm (not just progressing)—if very few motile sperm are present, estimate number motile/coverslip—and record result. Add 0.5–1.0ml of 5% albumin solution to the sperm sample, mix well. Add GCG-glycerol preparation in 1:2 ratio gradually with mixing. Package in straws or cryovials and freeze.

APPENDIX 10— USE OF PENTOXIFYLLINE • Prepare a 10x concentrated solution of pentoxifylline (POF, Sigma) in protein free HTF/HEPES (POF MW=278.3; 10x concentrate=10 mg/ml). • Sterilize through a 0.2µm filter and store at 4°C. • Dilute 1:9 with sperm suspension to expose sperm to a final concentration of 1mg/ml POF (3.6mM). • Spread the treated sperm suspension adjacent to the holding drops in the injection plate. • Functional sperm should show motility within 10 minutes of exposure to the stimulant. • Move the motile sperm to clean, stimulant free medium.

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• Aim to collect the motile sperm quickly (23mm diameter.36–37 Studies from Nayudu

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et al34 and others38 found that most normal pregnancies after IVF came from follicles in the 2.5–6.5ml volume or 15–23mm diameter ranges. Studies were conducted by us to analyse the relation between fertilizability, embryo cleavage, and clinical pregnancy rates in GnRHa and gonadotropin stimulation cycles showing a superior developmental capacity from oocytes aspirated from large follicles with a diameter over 20mm (Smitz, unpublished personal observations). All IVF studies consistently show that follicles ≤2ml (volume) or ≤14mm (diameter) generate a very low proportion of clinical pregnancies. Most commonly, oocytes from small follicles do not cleave after fertilization, and even if they succeed to implant early abortion occurred.34,38 When oocytes were stripped from their densely packed surrounding granulosa cumulus, it was found that a higher proportion of immature (germinal vesicle, GV) oocytes were recovered from these small follicles.39 When immature COC (GV oocytes from ICSI cycles) are injected, fertilization fails, and when denuded oocytes are injected 24 hours after an in vitro maturation period most preimplantation embryos are of poor quality, have a high aneuploidy rate, and yield karyotype anomalies.40 Retrospective analysis on a large number of cycles from couples undergoing ICSI testified that after a GnRHa and gonadotropin stimulation about 80% of the follicles aspirated yielded a metaphase II oocyte (the remaining 20% of the cohort were either MI or GV). The cycles that yielded a higher proportion of immature oocytes had experienced a poor stimulation management with an aspiration of smaller follicle diameters (15mm diameter) obtained by superovulation in view of IVF, COC can be easily recognized floating in between the other parts of the mural granulosa cells. The COC is recognized as a mucified clump of cells under the stereomicroscope (Fig 9.9). Owing to the more traumatic procedure of aspirating small follicles, the search for the unexpanded COC is more difficult than in IVF. Careful inspection of the bloody aspirates under the stereomicroscope or the use of filters might enhance the recovery of the densely enclosed oocytes. The more time consuming aspiration procedure and the prolonged handling of the COC during the searching procedure necessitates strict control of temperature and pH conditions. The COC from large follicles are graded on the basis of the expansion of the surrounding cells from corona and cumulus. In routine IVF practice, grading of the nuclear maturation stage is approximated by the degree of expansion of the surrounding cumulus and corona cells.67 Early work of Testart et al established that in natural cycles there is a good synchrony between the development of the cumulus cells and the nuclear maturation stage.68 This synchrony in maturation is observed less often in COC from superovulation cycles for ART.69

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In the aspirates from smaller follicles (