Sperm Chromatin

Armand Zini • Ashok Agarwal Editors

Sperm Chromatin

Biological and Clinical Applications in Male Infertility and Assisted Reproduction Editors Armand Zini, MD Ashok Agarwal, PhD, HCLD (ABB) Department of Surgery Center for Reproductive Medicine Division of Urology Glickman Urological and Kidney Institute McGill University OB-GYN and Women’s Health Institute St. Mary’s Hospital Center Cleveland Clinic, Cleveland, OH, USA Montreal, QC, Canada [email protected] [email protected]

ISBN 978-1-4419-1781-2 e-ISBN 978-1-4419-6857-9 DOI 10.1007/978-1-4419-6857-9 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011932679

© Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com) Foreword

The composition, stability, and integrity of sperm chromatin have major bearings on the fertilizing potential of male gametes and their capacity to support normal embryonic development. This assertion is supported by clinical data generated over the past 10–15 years, indicating the existence of significant correlations between DNA damage in spermatozoa and a variety of adverse reproductive outcomes including reduced conception rates, a high frequency of miscarriage and an escalation in the incidence of birth defects. Despite this wealth of correlative data, uncertainties remain with respect to such key questions as the precise nature of the DNA damage, the relative importance of genetic, epigenetic and environmental factors in its etiology, and the most effective method for detecting its presence. Answers to these questions are critical if we are to develop rational strategies for alleviating or, ultimately, preventing DNA damage in spermatozoa. Furthermore, answers to these questions are needed if clinicians are to provide patients with informed

v vi Foreword advice about the significance of their DNA damage results. In Sperm Chromatin: Biological and Clinical Application in Male Infertility and Assisted Reproduction Ashok Agarwal and Armand Zini have assembled contributions from a panel of world experts in this area and, in so doing, created an invalu- able text that brings us up-to-date with recent advances in this field. The remodeling of sperm chromatin during spermiogenesis is as remarkable as it is complex. It involves a dramatic morphological and biochemical trans- formation of chromatin structure through the coordinated movement of proteins in and out of the nucleus to generate a unique, highly compacted matrix. Furthermore, this extraordinary metamorphosis takes place in the absence of de novo gene transcription. Indeed, spermiogenesis is an object lesson in how biological processes can be controlled through the regulated translation of pre- existing mRNA species. If we could only replicate this process in vitro, research into the underlying control mechanisms would be greatly facilitated. Unfortunately, this is not possible at the present time and, as a result, progress in this area has been painfully slow. Nevertheless, as the pioneer of sperm chromatin research, Rod Balhorn, indicates in Chap. 1, we might anticipate more rapid progress in this area in the future because we now have an array of sophis- ticated tools to investigate the chromatin remodeling process. Thus, our new found ability to generate detailed inventories of proteins and mRNA species in selected cells and tissues is one of the driving forces behind the modern systems approach to biological research, and the analysis of sperm chromatin will be one of the major beneficiaries of these technical developments. In the first section of this book, we see beautifully illustrated reviews of spermatogenesis (Chap. 2) and chromatin organization (Chaps. 4 and 5) followed by a number of authoritative reviews on aspects of the genome (Chap. 6), epigenome (Chap. 7), transcriptome (Chap. 8), and proteome (Chap. 3) from a spermatozoon’s perspective. The detailed chemical analysis of sperm chromatin using the tools of the -omics revolution will certainly provide important clues as to the formation and ultimate function of this material and act as a major stimulus for increased understanding in this area. The impetus to study the composition and integrity of sperm chromatin from a clinical perspective can be traced back to the pioneering studies of Don Evenson, who not only initiated research in this area long before it became fashionable but also pioneered one of the major analytical techniques used in the assessment of sperm chromatin, the Sperm Chromatin Structure Assay (SCSA) (Chap. 9). This assay has now become the industry standard against all other techniques. In the second section of this book Agarwal and Zini have gathered together a very impressive list of authors, all of whom share an interest in the laboratory evaluation of sperm chromatin composition and integrity. The list of techniques is extensive and comprises, in addition to SCSA, meth- ods that have been borrowed from research in toxicology (Comet; Chap. 15) or somatic cell apoptosis (TUNEL; Chap. 14) as well as a range of protocols that are more specific to the male germ line including the Sperm Chromatin Dispersion assay (Chap. 10), the acridine orange test (Chap. 13), the isolation, purification and quantification of protamines (Chap. 16), the aniline blue test for histone retention (Chap. 12), gene expression profiling (Chap. 18), and analyses of epigenetic modifications to the haploid paternal genome (Chap. 17). Foreword vii

Additional probes such as toluidine blue and chromomycin 3 (CMA3) are also important because they provide indirect evidence of the efficiency of chromatin packaging (Chap. 11). In this context, Denny Sakkas deserves special men- tion for his work on the development of CMA3 as an extremely valuable diagnostic probe, which has consistently been found to generate significant information about sperm chromatin status in infertile men. The clinical and environmental factors that contribute to the etiology of DNA damage are reviewed in the third section of this book. The range of possible contributory factors is again extensive and includes oxidative stress (Chap. 19), apoptosis (Chap. 20), and defective repair of physiological DNA nicks (Chap. 21). The primary drivers for the oxidative stress, abortive apoptosis, and defective DNA repair include infertility (Chap. 22), age (Chap. 23), cancer (Chap. 24), environmental pollutants (Chap. 25), infection (Chap. 26), and cryopreservation (Chap. 27). If we understood more about the chemical nature of the DNA damage seen in human spermatozoa, we might be able to reduce this long list of potential causative factors down to the major culprits. However, at present, the only clue we have to the types of DNA damage present in human spermatozoa is the high prevalence of oxidative base lesions detected in the patient population. The mechanism by which such stress is generated is still something of an unresolved mystery. If we do not understand the etiology of DNA damage, then there is little we can do to treat this condition or prevent it from arising. However, we can try to develop strategies for limiting the impact of such damage on human embryos conceived in vitro including antioxidant therapy (Chap. 30), electrophoretic sperm isolation (Chap. 29), and the selective binding properties of hyaluronic acid polymers (Chap. 28), all of which help select non-DNA damaged spermatozoa for the insemination of oocytes. The importance of developing strategies for avoiding conception with DNA damaged spermatozoa is generally supported by the clinical data, although the results are not always as clean cut as one would like (Chaps. 31 and 32). A major problem with such clinical studies is that pregnancy is a very bad test of sperm function because it depends on so many confounding variables. By contrast, the animal data are incontrovertible in demonstrating that DNA damage in the male germ line has profound, lasting effects on the viability of pregnancy and the health of the offspring (Chap. 33). In light of such certainty, it is critical that the information available on this topic is assembled and presented for careful consideration. Agarwal and Zini have done a remarkable job of pulling together the protagonists in this field and creating a compendium of knowledge that will be of intense interest to clinicians and scientists who share an appreciation of the significant contributions made by the male gamete to a healthy start to life.

Callaghan, NSW R. John Aitken, PhD, ScD, FRSE

Preface

The evaluation of sperm DNA and chromatin abnormalities has gained significant importance in the past several years, largely as a result ofthe recent advances in assisted reproductive technologies (ARTs). In vitro fertil- ization (IVF) and intracytoplasmic sperm injection (ICSI) have revolution- ized the treatment of male-factor infertility. However, we have come to realize that in the context of these ARTs, the genetic integrity of the sperm is a key aspect of the paternal contribution to the offspring. With the growing concerns about the long-term safety of ARTs (especially ICSI), we have seen an increasing number of studies on the male genome’s influence on reproductive outcomes. These studies now shed some light on the influence of sperm chromatin and DNA abnormalities on reproductive outcomes. Along with these clinical studies, we also have made real advances in our understanding of the basic aspects of sperm chromatin and DNA integrity. We are now starting to better understand the unique organization of the sperm chromatin, as well as the nature and etiology of sperm DNA damage. We assembled this textbook with the idea of bringing together the key fundamental and practical elements of this rapidly evolving field. The 34 chapters in our book and four supplementary sections covering test protocols and guidelines are written by contributors from 16 countries. These authors were carefully selected based on their expertise and proven track record of high-quality research in the field. Our book is intended for researchers and clinicians alike and is meant to bridge the gap between our basic and clinical knowledge on sperm chromatin and DNA integrity. For the basic scientist, this textbook will serve as a sound foundation for any further studies in this field. For the clinician, this book will help guide clinical practice in this area. We would like to thank Richard Lansing, executive editor, for his support and advice and Margaret Burns, developmental editor, for her tireless efforts in reviewing and editing each of the manuscripts. Furthermore, we would like to thank all of the outstanding contributors for sharing their knowledge and for submitting their manuscripts on time. Finally, we are indebted to our families who have endured many long nights when we were working late on this book.

Montreal, QC Armand Zini Cleveland, OH Ashok Agarwal

Editor Biographies

Dr. Armand Zini is Associate Professor of Surgery and Director of the Andrology Fellowship program at McGill University. Dr. Zini received his Medical degree and completed his urologic training at McGill University in Montreal. He then completed a fellowship in Male Infertility at the New York Hospital-Cornell Medical Centre and The Population Council in New York, working with Drs. Marc Goldstein and Peter Schlegel. Dr. Zini’s main expertise is in clinical male infertility. Over the past 10 years, he has focused his research activity on the study of human sperm chromatin and DNA integrity, and, he has published numerous important papers on the influence of sperm DNA damage on reproductive outcomes. In 2005, he gave the John Collins lecture entitled “Sperm DNA damage and Male Infertility” at the annual meeting of the American Society for Reproductive Medicine. In 2006, he was invited to present on the “Tests of sperm DNA damage” at the Canadian Fertility and Andrology Society (CFAS) annual meeting and in 2008 was invited to present on the “Clinical importance of sperm DNA damage” at both the Canadian Fertility and Andrology Society (CFAS) and the American Society of Andrology (ASA) annual meetings. Dr. Zini has recently presented on the “Role of antioxidants and sperm DNA damage” (Sperm DNA Symposium in Rome, Italy, March 2009) and at the 2009 European Society for Human Reproduction and Embryology (ESHRE) consensus workshop on sperm DNA testing in Sweden. Dr. Zini is currently funded for studies on sperm physiology and the epigenetic effects of vitamin supplements.

Dr. Ashok Agarwal is the Director of Research at the Center for Reproductive Medicine at Cleveland Clinic Foundation and a Professor at the Lerner College of Medicine of Case Western Reserve University. His current research interests include studies on molecular markers of oxidative stress, DNA integrity, apoptosis in the pathophysiology of male and female reproduction, and effect of radio frequency radiation on fertility and fertility preservation in patients with cancer. Dr. Agarwal has published over 500 scientific articles and reviews in peer reviewed scientific journals, authored over 50 book chapters, and presented over 700 papers at scientific meetings. He is on the edito- rial board of over a dozen scientific journals. His laboratory has trained more than 100 basic scientists and clinical researchers from the United States and

xi xii Editor Biographies abroad. He is the Program Director of the highly successful Summer Internship Course in Reproductive Medicine. In the last 4 years, over 100 premed and medical students from across the United States and overseas have graduated from this highly competitive program. Dr. Agarwal has been invited as a guest speaker to over 20 countries for important international meetings. He has directed more than a dozen Andrology Laboratory and ART Workshops in recent years. Contents

Part I Human Sperm Chromatin: Structure and Function

1 Sperm Chromatin: An Overview...... 3 Rod Balhorn 2 Spermatogenesis: An Overview...... 19 Rakesh Sharma and Ashok Agarwal 3 Sperm Nucleoproteins...... 45 Rafael Oliva and Judit Castillo 4 The Relationship Between Chromatin Structure and DNA Damage in Mammalian Spermatozoa...... 61 Kenneth Dominguez, Chris D.R. Arca, and W. Steven Ward 5 Chromosome Positioning in Spermatozoa...... 69 Andrei Zalensky, Olga Mudrak, and Irina Zalenskaya 6 Sperm Mitochondrial DNA...... 81 Justin C. St. John and Bianca St. John 7 The Sperm Epigenome...... 95 Donovan Chan and Jacquetta Trasler 8 RNA Expression in Male Germ Cells During Spermatogenesis (Male Germ Cell Transcriptome)...... 107 Tin-Lap Lee, Albert Hoi-Hung Cheung, Owen M. Rennert, and Wai-Yee Chan

Part II Laboratory Evaluation of Sperm Chromatin

9 Sperm Chromatin Structure Assay (SCSA®): 30 Years of Experience with the SCSA®...... 125 Donald P. Evenson

xiii xiv Contents

10 Sperm Chromatin Dispersion Test: Technical Aspects and Clinical Applications...... 151 Jaime Gosálvez, Carmen López-Fernández, and José Luís Fernández 11 Basic and Clinical Aspects of Sperm Chromomycin A3 Assay...... 171 Gian carlo Manicardi, Davide Bizzaro, and Denny Sakkas 12 Cytochemical Tests for Sperm Chromatin Maturity...... 181 Igor Tsarev and Juris Erenpreiss 13 Acridine Orange Test for Assessment of Human Sperm DNA Integrity...... 189 Alex C. Varghese, C. Fischer-Hammadeh, and M.E. Hammadeh 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay...... 201 Rakesh Sharma and Ashok Agarwal 15 Basic and Clinical Aspects of Sperm Comet Assay...... 217 Luke Simon and Sheena E.M. Lewis 16 Assays Used in the Study of Sperm Nuclear Proteins...... 233 Timothy G. Jenkins, Benjamin R. Emery, and Douglas T. Carrell 17 Sperm Epigenetic Profile...... 243 Cristina Joana Marques, Alberto Barros, and Mário Sousa 18 Clinical Significance of Sperm RNA...... 259 Jean-Pierre Dadoune, Isabelle Galeraud-Denis, and Serge Carreau

Part III Biological and Clinical Determinants of Sperm Chromatin Damage

19 Role of Oxidative Stress in the Etiology of Sperm DNA Damage...... 277 R. John Aitken and Geoffry N. De Iuliis 20 Abortive Apoptosis and Sperm Chromatin Damage...... 295 Hasan M. El-Fakahany and Denny Sakkas 21 Spermiogenesis in Sperm Genetic Integrity...... 307 Marie-Chantal Grégoire, Frédéric Leduc, and Guylain Boissonneault 22 Male Subfertility and Sperm Chromatin Damage...... 321 Mona Bungum, Aleksander Giwercman, and Marcello Spanò 23 Aging and Sperm DNA Damage...... 337 Fábio F. Pasqualotto and Eleonora B. Pasqualotto Contents xv

24 Cancer in Males: Implications for Sperm Quality, Fertility, and Progeny Outcome...... 351 Peter Chan and Bernard Robaire 25 Sperm Chromatin and Environmental Factors...... 361 Aleksander Giwercman 26 Effects of Male Accessory Gland Infection on Sperm Parameters...... 375 Aldo E. Calogero, Sandro La Vignera, Rosita A. Condorelli, Rosario D’Agata, and Enzo Vicari

Part IV Sperm Chromatin and Assisted Reproductive Technology Outcomes

27 The Impact of Sperm Processing and Cryopreservation on Sperm DNA Integrity...... 397 Dan Yu, Luke Simon, and Sheena E.M. Lewis 28 Structure of Chromatin in Human Sperm Bound to Hyaluronic Acid: The Benefits of PICSI Dish Mediated Sperm Selection...... 411 Gabor Huszar and Leyla Sati 29 Electrophoretic Sperm Separation...... 423 Steven Fleming and R. John Aitken 30 Antioxidants and Sperm DNA Damage...... 431 Armand Zini and Maria San Gabriel 31 Sperm Chromatin and ART (IUI, IVF and ICSI) Pregnancy...... 441 Mona Bungum 32 Sperm DNA Damage and Pregnancy Loss After IVF/ICSI...... 457 Armand Zini and Jason Matthew Boman 33 Postnatal Effects of Sperm Chromatin Damage...... 465 Miriam Pérez-Crespo, Raúl Fernández-González, Miguel Ángel Ramírez, Eva Pericuesta, Alexandra Calle, and Alfonso Gutiérrez-Adán 34 Evaluation of Chromatin and DNA Integrity in Testicular Sperm...... 479 Armand Zini and Naif Al-Hathal

Part V Protocols and Integrity Tests

35 Sperm Chromatin Structure Assay (SCSA®)...... 487 Donald P. Evenson 36 Measurement of DNA Damage in Spermatozoa by TUNEL Assay...... 495 Rakesh Sharma and Ashok Agarwal xvi Contents

37 Clinical Utility of Sperm DNA Integrity Tests...... 499 Armand Zini Index...... 505 Contributors

Ashok Agarwal, PhD, HCLD (ABB) Center for Reproductive Medicine, Glickman Urological and Kidney Institute, OB-GYN and Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA R. John Aitken, PhD, ScD, FRSE Discipline of Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW, Australia ARC Centre of Excellence in Biotechnology and Development, Priority Research Centre in Reproductive Science, University of Newcastle, Callaghan, NSW, Australia Naif Al-Hathal, MD Department of Surgery, Division of Urology, Royal Victoria Hospital, McGill University, Montreal, QC, Canada Chris D.R. Arca, BS Institute for Biogenesis Research (IBR), John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA Rod Balhorn, PhD Department of Applied Science, University of California, Davis, CA, USA Alberto Barros, MD, PhD Centre for Reproductive Genetics, Porto, Portugal Davide Bizzaro, PhD Department of Biochemistry, Biology and Genetics, Polytechnic University of Marche, Ancona, Italy Guylain Boissonneault, PhD Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada Jason Matthew Boman, MD, FRCS(C) Department of Surgery, Division of Urology, Centre Hospitalier Régional du Suroît, Montreal, QC, Canada Mona Bungum, MSc, Med Dr, PhD Reproductive Medicine Centre, Skåne University Hospital Malmö, Lund University, Sodra Forstadsgatan, Malmö, Sweden

xvii xviii Contributors

Alexandra Calle, BS Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain Aldo E. Calogero, MD Section of Endocrinology, Andrology and Internal Medicine, and Master in Andrological, Human Reproduction and Biotech- nology Sciences, Department of Internal Medicine and Systemic Diseases, University of Catania, Policlinico “G. Rodolico”, Catania, Italy Serge Carreau, PhD Department of Biochemistry, University of Caen, Caen, France Douglas T. Carrell, PhD, HCLD Andrology and IVF Laboratories, Department of Surgery, University of Utah, Salt Lake City, UT, USA Judit Castillo, MS Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain Biochemistry and Molecular Genetics Service, Hospital Clínic i Provincial, Barcelona, Spain Donovan Chan, BSc Departments of Pharmacology and Therapeutics, Pediatrics, and Human Genetics, McGill University and Montreal Children’s Hospital of the McGill University Health Centre Montreal, QC, Canada Peter Chan, MD Department of Surgery, McGill University and the Research Institute of the MUHC, Montréal, QC, Canada Wai-Yee Chan, BSc, PhD Reproduction, Development and Endocrinology Program, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, SAR, China Albert Hoi-Hung Cheung, PhD Reproduction, Development and Endocri- nology Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, SAR, China Rosita A. Condorelli, MD Section of Endocrinology, Andrology and Internal Medicine, Human Reproduction and Biotechnology Sciences, Department of Biomedical Sciences, University of Catania, Catania, Italy Jean-Pierre Dadoune, MD, PhD Department of Histology, Biology of Reproduction, Hôpital Tenon, Paris, France Rosario D’Agata, MD Section of Endocrinology, Andrology and Internal Medicine, Human Reproduction and Biotechnology Sciences, Department of Biomedical Sciences, University of Catania, Catania, Italy Geoffry N. De Iuliis, PhD, BSc Department of Biological Sciences, ARC Centre of Excellence in Biotechnology and Development, Priority Research Centre in Reproductive Science, University of Newcastle, Newcastle, NSW, Australia Contributors xix

Kenneth Dominguez, MS Institute for Biogenesis Research (IBR), John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA Hasan M. El-Fakahany, MD Department of Dermatology, STD’s and Andrology, Al-Minya University, Al-Minya, Egypt Benjamin R. Emery, M Phil Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA Juris Erenpreiss, MD, PhD Andrology Laboratory, Riga Stradins University, Riga, Latvia Donald P. Evenson, PhD, HCLD SCSA Diagnostics, Po Box 107, Volga, SD, USA Emeritus, South Dakota State University, Brookings, SD, USA Department of Obstetrics and Gynecology, Sanford Medical School, University of South Dakota, Sioux Falls, SD, USA José Luís Fernández, MD, PhD Genetics Unit, INIBIC-A Coruña University Hospital, As Xubias, Coruña, Spain Molecular Genetics and Radiobiology Laboratory, Centro Oncológico de Galicia, Coruña, Spain Raúl Fernández-González, BS Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain C. Fischer-Hammadeh, MD Department of Obstetrics and Gynecology, Assisted Reproduction Technology Unit, University of Saarland, Homburg/Saar, Germany Steven Fleming, BSc (Hons), MSc, PhD Greenslopes Private Hospital, Greenslopes, QLD, Australia Isabelle Galeraud-Denis, PhD Section of Biology of Reproduction, Université de Caen Basse-Normandie, Caen, France Aleksander Giwercman, MD, PhD Reproductive Medicine Centre, Skåne University Hospital Malmö, Lund University, Malmö, Sweden Jaime Gosálvez, PhD, BSc School of Biological Sciences, Madrid Autonoma University, Cantoblanco, Madrid, Spain Marie-Chantal Grégoire, MSc Department of Biochemistry, University of Sherbrooke, Sherbrooke, QC, Canada Alfonso Gutiérrez-Adán, PhD Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain xx Contributors

M.E. Hammadeh, MD Department of Obstetrics and Gynecology, Assisted Reproduction Technology Unit, University of Saarland, Homburg/Saar, Germany Gabor Huszar, MD Director, Sperm Physiology Laboratory and Male Fertility Program, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University, School of Medicine, New Haven, CT, USA Timothy G. Jenkins, BS Andrology and IVF Laboratories, Department of Andrology Development, University of Utah School of Medicine, Salt Lake City, UT, USA Sandro La Vignera, MD Section of Endocrinology, Andrology and Internal Medicine, Human Reproduction and Biotechnology Sciences, Department of Biomedical Sciences, University of Catania, Catania, Italy Frédéric Leduc, MSc Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Canada Tin-Lap Lee, PhD Reproduction, Development and Endocrinology Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Laboratory of Clinical and Developmental Genomics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Sheena E.M. Lewis, PhD Centre for Public Health, Queen’s University of Belfast, Room 208, Institute of Clinical Science, Belfast, Northern Ireland, UK Carmen López-Fernández, PhD Genetics Unit, Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain Gian Carlo Manicardi, PhD Department of Agricultural and Food Science, University of Modena and Reggio Emilia, Reggio Emilia, Italy Cristina Joana Marques, PhD Genetics Department, Faculty of Medicine, University of Porto, Portugal, UK Olga Mudrak, PhD The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA, USA Rafael Oliva, MD, PhD Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain Biochemistry and Molecular Genetics Service, Hospital Clínic i Provincial, Barcelona, Spain Eleonora B. Pasqualotto, MD, PhD Department of Gynecology, University of Caxias do Sul, Caxias do Sul, RS, Brazil Fábio F. Pasqualotto, MD, PhD Department of Urology, University of Caxias do Sul, Rua Pinheiro Machado, Bairro São Pelegrino, Caxias do Sul, RS, Brazil Contributors xxi

Miriam Pérez-Crespo, PhD Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain Eva Pericuesta, PhD Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain Miguel Ángel Ramírez, PhD Department of Animal Reproduction and Animal Genetic Resources Conservation, National Research Institute for Agriculture and Food Technology (INIA), Madrid, Spain Owen M. Rennert, MD Laboratory of Clinical Genomics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Bernard Robaire, PhD Department Pharmacology and Therapeutics, Department of Obstetrics and Gynecology, McGill University, Montréal, QC, Canada Denny Sakkas, PhD Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA Maria San Gabriel, BS Division of Urology, Department of Surgery, Royal Victoria Hospital, McGill University, Montreal, QC, Canada Leyla Sati, MSc Department of Histology and Embryology, School of Medicine, Akdeniz University, Antalya, Turkey Rakesh Sharma, PhD Andrology Laboratory and Center for Reproductive Medicine, Glickman Urological and Kidney Institute, OB-GYN and Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA Luke Simon, MSc, PhD Centre for Public Health, Queens University Belfast, Institute of Clinical Sciences, Belfast, UK Mário Sousa, MD, PhD Departments of Laboratory Cell Biology and Microscopy, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal Marcello Spanò, PhD Laboratory of Toxicology, Unit of Radiation Biology and Human Health, Italian National Agency for New Technologies, Engery, and Sustainable Economic Development, Casaccia Research Centre, Casaccia, Rome, Italy Bianca St. John, MD The Mitochondrial Genetics Group, Centre for Re- production & Development, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia Justin C. St. John, MD The Mitochondrial Genetics Group, Centre for Reproduction & Development, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia xxii Contributors

Jacquetta Trasler, MD, PhD Departments of Pharmacology and Therapeutics, Pediatrics, and Human Genetics, McGill University and Montreal Children’s Hospital of the McGill University Health Centre, Montreal, QC, Canada Igor Tsarev, MD Andrology Laboratory, Riga Stradins University, Riga, Latvia Alex C. Varghese, PhD Fertility Clinic and IVF Division, AMRI Medical Centre (A Unit of AMRI Hospitals), Kolkata, India Enzo Vicari, MD Department of Internal Medicine and Systemic Diseases, University of Catania, Catania, Italy W. Steven Ward, PhD Institute for Biogenesis Research (IBR), John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA Dan Yu, MD Centre for Public Health, Queens University Belfast, Belfast, UK Irina Zalenskaya, PhD CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, USA Andrei Zalensky, PhD, DSci The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA, USA Armand Zini, MD Department of Surgery, Division of Urology, McGill University, St. Mary’s Hospital Center, Montreal, QC, Canada