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Human Reproduction

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Human Reproduction

Updates and New Horizons

Edited byHeide SchattenDepartment of Veterinary Pathobiology,University of Missouri, Columbia, Missouri, USA

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Copyright © 2017 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Names: Schatten, Heide, editor.Title: Human reproduction : updates and new horizons / edited by Heide

Schatten.Description: Hoboken, New Jersey : John Wiley & Sons Inc., [2017] | Includes

index.Identifiers: LCCN 2016038683| ISBN 9781118849583 (cloth) | ISBN 9781118849576

(epub)Subjects: LCSH: Human reproduction.Classification: LCC QP251 .H845 2017 | DDC 612.6–dc23 LC record available athttps://lccn.loc.gov/2016038683

Cover image: Getty Images/mikroman6

Set in 10/12pt WarnockPro by SPi Global, Chennai, India

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Contents

List of Contributors xv

1 Sperm Selection Techniques and their Relevance toART 1Luke Simon, Monis B. Shamsi, and Douglas T. Carrell

1.1 Introduction 11.2 Need of Sperm Selection in ART 21.3 Methodology of Sperm Selection 31.3.1 Intracytoplasmic Sperm Injection 31.3.1.1 Methodology 41.3.1.2 Advantages and Limitations 41.3.1.3 Conclusion 61.3.2 Intracytoplasmic Morphologically Selected Sperm

Injection 61.3.2.1 Methodology 71.3.2.2 Advantages and Limitations 71.3.2.3 Conclusion 81.3.3 Annexin V Labeling 81.3.3.1 Methodology 101.3.3.2 Advantages and Limitations 101.3.3.3 Conclusion 111.3.4 Microfluidics 111.3.4.1 Methodology 121.3.4.2 Advantages and Limitations 131.3.4.3 Conclusion 141.4 Electrophoretic Sperm Separation 141.4.1 Methodology 15

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1.4.2 Advantages and Limitations 161.4.3 Clinical Importance of Sperm Preparation by

Electrophoresis 171.4.4 Conclusion 171.5 Zeta Test 181.5.1 Methodology 181.5.2 Advantages and Limitations 191.5.3 Clinical Importance 201.5.4 Conclusion 201.6 Microelectrophoresis Sperm Selection 211.6.1 Methodology 211.6.2 Advantages and Limitations 231.6.3 Clinical Importance 231.6.4 Conclusion 241.7 Raman Spectroscopy 241.7.1 Methodology 241.7.2 Advantages and Limitations 261.7.3 Clinical Importance 261.7.4 Conclusion 271.8 Hyaluronic Acid Binding Assay 271.8.1 Methodology 281.8.2 Advantages and Limitations 281.8.3 Clinical Importance 281.8.4 Conclusion 291.9 Future Perspective 30

References 31

2 In Vitro Maturation of Human Oocytes: CurrentPractices and Future Promises 45Catherine M.H. Combelles

2.1 Introduction 452.2 Clinical Indications for IVM 462.2.1 Ovarian Hyperstimulation Syndrome (OHSS) 462.2.2 Polycystic Ovary Syndrome (PCOS) 462.2.3 Fertility Preservation 472.2.4 Other Indications: Poor Responders, Normo-Ovulatory

Patients, and Other Unique Cases 492.2.5 Patient Selection 50

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2.3 Ovarian Stimulation Approaches for the Retrieval ofImmature Oocytes 51

2.4 Maternal Conditions that may Influence IVM 542.5 Follicular Origins of Immature Oocytes for IVM 552.6 Clinical Safety of IVM 572.7 Concluding Remarks towards the Optimization of IVM 58

References 61

3 Molecular Biology of Endometriosis 71Jayasree Sengupta, G. Anupa, Muzaffer Ahmed Bhat, andDebabrata Ghosh

3.1 Introduction 713.2 Brief Background 713.2.1 Definition, Pathology, and Demography 713.2.2 Phenotypes and Classification 723.2.3 Theories of Endometriosis 733.2.4 Essential Cellular Pathology 743.3 Genetic Basis of Endometriosis 763.4 Molecular Mechanisms of Endometriosis 773.4.1 Estrogen Dependence and Progesterone Resistance 793.4.2 Inflammatory Dysfunction 813.4.3 Triad of Molecular Processes: Estrogen/Progesterone

Balance, Apoptosis, and Inflammatory Function 823.5 Molecular Etiopathological Basis of Endometriosis: Leads in

Genomics Era 833.5.1 Candidate Genes and Module-Based Studies 833.5.2 Large Scale Transcriptomic Studies 863.5.3 Genome-Wide Gene Expression Profiles 923.5.4 Genome-Wide Association Studies (GWAS) 963.6 Molecular Etiopathological Basis of Endometriosis: Leads in

the Post-Genomics Era 993.6.1 Endometrial Proteome of Endometriosis 993.6.2 Epigenetic Landscape in Endometriosis 1073.6.3 Biomarkers of Endometriosis 1123.7 Future Targets 113

Acknowledgments 115Conflicts of Interest 116References 116

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4 Novel Immunological Aspects for the Treatment ofAge-induced Ovarian and Testicular Infertility,Other Functional Diseases, and Early and AdvancedCancer Immunotherapy 143Antonin Bukovsky

4.1 Introduction 1434.2 Ovarian Infertility 1454.2.1 Rationale for Using In Vitro Developed Oocyte-like Cells

(OLCs) and Possible IVM/IVF Developments for ClinicalUse 146

4.2.2 Formal Terms Related to Ovarian Cellular Conditions andFunctions 147

4.2.3 Stem Cell Commitment by Embryonic Primordial GermCells 148

4.2.4 Role of the Immune System in Ovarian Function 1484.2.5 Steps Required for Follicular Renewal in Adult Human

Ovaries 1484.2.6 Origin and Development of Ovarian Germ Cells 1494.2.7 Ovarian Stem Cell Cultures 1594.2.8 Perspectives of Advanced Age of the Woman, Premature

Ovarian Failure, or Other Ovarian Infertility Etiologies byInVitro Developed Oocytes in Ovarian Stem CellCultures 161

4.2.9 Nuclear Transfer 1624.3 Novel In Vitro Proposals for Ovarian Infertility

Treatment 1634.3.1 Preliminary Strategies for In Vitro Approaches 1634.3.1.1 Formation of Germ Cells 1634.3.1.2 Formation of Granulosa Cells 1644.3.1.3 Separation of Donor Mononuclear Cells 1644.3.1.4 Collection of Ovarian Stem Cells for a Clinical

Approach 1644.3.2 Oocyte-like Cell Nuclear Transfer to Donor Oocyte 1654.3.3 Donor Oocyte Cytoplasmic Transfer to Oocyte-like

Cell 1654.3.4 Transfer of Granulosa Cells or Their Components to an

Established OSC Culture 1654.3.5 Transfer of Granulosa Cells or Their Components to Fresh

Secondary Ovarian Stem Cell Cultures During Early Stepsof Oocyte Reconstruction 166

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4.3.6 Fibroblasts in OSC Cultures Steal ZP3 Expression fromOLCs 169

4.3.7 What May Be Next in Ovarian Tissue Cultures? 1704.4 Novel In Vivo Proposal for Ovarian and Testicular Infertility

Treatment 1704.4.1 Systemic Treatment of Ovarian Infertility by Transfer of

Compatible Blood or Separated Mononuclear Cells fromYoung Fertile Donor Women 172

4.4.2 Systemic Treatment of Testicular Infertility by Transfer ofCompatible Blood or Separated Mononuclear Cells fromYoung Fertile Donor Men 174

4.5 Systemic Treatment of Other Functional Diseases by TissueRejuvenation 174

4.5.1 Utilization of Blood or Mononuclear Cell Transfusion fromYoung Individuals 174

4.5.2 Utilization of Sex Steroid Combinations for Altered Tissuesin Younger Individuals without Altered Stem CellNiche 175

4.6 Advantages of Local and Systemic Use of Honey BeePropolis and Cayenne Pepper 175

4.6.1 Propolis and Alopecia 1764.6.2 Propolis and Varicose Veins 1784.6.3 Propolis and Dental Calculus 1784.6.4 Systemic Use of Propolis Tincture 1784.6.5 Benefits of Cayenne Pepper Systemic Treatment 1804.7 The Promise of Pyramid Healing Systems 1804.8 Raw Shiitake Causes Early Neoplasia Regression and

Malignancy Recurrence Prevention 1804.9 Immune Modulation for the Treatment of an Advanced

Cancer 1824.10 Advanced Ovarian Cancer Regression Case Report 1844.10.1 Immune Modulation in Advanced Malignancy Should Be

Attempted in Novel, Not Yet Treated Cancer Cases 1844.10.2 Postoperative Development 1844.11 Discussion 1874.12 Conclusions 192

Abbreviations 193Competing Interests 193Author Contribution 193References 194

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5 Mitochondrial Manipulation for Infertility Treatmentand Disease Prevention 205Tetsuya Ishii

5.1 Introduction 2055.2 The Roles of Mitochondria in Fertilization, Embryonic

Development, and Disease 2065.3 The Genetics of Mitochondria and Mitochondrial

Diseases 2095.4 Ooplasmic Transfer to Treat Infertility 2105.5 Pronuclear Transfer to Achieve Pregnancy 2145.6 Germinal Vesicle Transfer to Restore the Viability of

Oocytes 2165.7 Mitochondrial Diseases and Prevention of their

Inheritance 2175.8 Mitochondrial Replacement by Transferring Pronuclei and

MII Spindle 2185.9 Discussion 220

Acknowledgments 222References 223

6 Novel Imaging Techniques to Assess Gametes andPreimplantation Embryos 231Jason E. Swain

6.1 Introduction 2316.2 Light and Impact on Mammalian Gametes and

Embryos 2326.3 Novel Imaging Approaches for Gametes and Embryos 2336.3.1 Polarized Light Microscopy 2336.3.1.1 Oocyte 2356.3.1.2 Sperm 2366.3.2 Multi-Photon Excitation Fluorescence Microscopy 2376.3.2.1 Oocytes and Embryos 2376.3.3 Harmonic Generation Microscopy 2396.3.3.1 Oocytes and Embryos 2396.3.4 Fourier Transformed Infrared 2416.3.4.1 Oocytes 2426.3.5 Raman Microspectroscopy 243

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6.3.5.1 Sperm 2436.3.5.2 Oocytes 2446.3.6 Coherent Anti-Stokes Raman 2466.3.6.1 Oocytes 2476.3.7 Optical Quadrature Microscopy 2476.3.7.1 Embryos 2486.3.8 Phase Subtraction 2486.3.8.1 Embryo 2496.3.9 Optical Coherence Tomography (OCT) 2506.3.9.1 Embryos 2506.3.10 Quantitative Orientation Independent Microscopy 2516.3.10.1 Sperm 2516.3.11 Biodynamic Imaging 2516.3.11.1 Oocytes 2536.3.11.2 Embryos 2546.3.12 Multi-Modal Microscopy 2546.3.12.1 Embryo 2546.4 Conclusion 255

References 256

7 Clinical Application of Methods to Select In VitroFertilized Embryos 267Kirstine Kirkegaard, Thomas F. Dyrlund, and Hans Jakob Ingerslev

7.1 Introduction 2677.2 Morphological Assessment 2687.2.1 Traditional Morphological Evaluation 2687.2.2 Time-Lapse Imaging 2707.3 Genomic and Transcriptomic Analysis 2797.3.1 Embryo Biopsy 2797.3.2 Pre-Implantation Screening (PGS) 2807.3.3 Gene Expression 2837.3.3.1 Cumulus Cells 2837.3.3.2 Embryos 2847.4 Analysis of Conditioned Culture Medium 2857.4.1 Metabolism 2867.4.2 Proteomics 2897.4.3 microRNA 293

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7.5 Summary 294References 295

8 New Horizons/Developments in Time-LapseMorphokinetic Analysis of MammalianEmbryos 313Munevver Serdarogullari, Necati Findikli, and Mustafa Bahceci

8.1 Introduction 3138.2 Utilization of Time-Lapse Morphokinetics in Mammalian

Embryos: A Historical Perspective 3148.3 What is TLM? 3158.4 What are the Benefits of TLM? 3168.5 Application of TLM in Human ART Practice 3188.6 The Possible Utilization of TLM Analysis in Aneuploidy

Detection 3248.7 Expected Contributions of TLM Technology in the Future of

Mammalian Embryology 327References 328

9 The Non-Human Primate Model for Early HumanDevelopment 339Stuart Meyers and Renee Riejo-Pera

9.1 Introduction 3399.2 Why Primate Models Are Critical to Understanding Human

Development and Subfertility 3409.3 NHP Model of Assisted Reproductive Technology

(ART) 3429.4 NHP Model of Early Embryo Development 3439.5 Research Perspective on NHP Embryo Development 3459.6 Summary 347

References 348

10 Cytoskeletal Functions, Defects, and DysfunctionsAffecting Human Fertilization and EmbryoDevelopment 355Heide Schatten and Qing-Yuan Sun

10.1 Introduction 35510.2 Components of the Cytoskeleton and their Important

Functions in Reproductive Biology 35610.3 The Role of the Cytoskeleton in Oocyte Maturation 360

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10.4 Maturation Failures and Oocyte Aging 36910.5 Fertilization and First Mitosis/Cell Division 37110.6 Cellular Differentiation/Polarization During

Pre-Implantation Embryo Development/CompactionStage 374

10.7 Perspectives and Future Directions 380References 381

Index 399

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List of Contributors

G. AnupaDepartment of PhysiologyAll India Institute of MedicalSciencesNew DelhiIndia

Mustafa BahceciBahceci Women’s Health GroupIstanbulTurkey

Muzaffer Ahmed BhatDepartment of PhysiologyAll India Institute of MedicalSciencesNew DelhiIndia

Antonin BukovskyThe Laboratory of ReproductiveBiology BIOCEV Institute ofBiotechnologyAcademy of Sciences of theCzech RepublicPragueCzech Republic

Douglas T. CarrellAndrology and IVF LaboratoryDepartment of Surgery(Urology)Department of Obstetrics andGynecologyand Department of HumanGeneticsUniversity of UtahSalt Lake CityUtahUSA

Catherine M.H. CombellesBiology DepartmentMiddlebury CollegeMiddleburyVermontUSA

Thomas F. DyrlundDepartment of MolecularBiology and GeneticsAarhus UniversityAarhusDenmark

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Necati FindikliBahceci Women’s Health GroupIstanbulTurkey

Debabrata GhoshDepartment of PhysiologyAll India Institute of MedicalSciencesNew DelhiIndia

Hans Jakob IngerslevThe Fertility ClinicAarhus University HospitalAarhusDenmark

Tetsuya IshiiOffice of Health and SafetyHokkaido UniversityHokkaidoJapan

Kirstine KirkegaardDepartment of MedicalBiochemistryAarhus University HospitalAarhusDenmark

Stuart MeyersDepartment of AnatomyPhysiologyand Cell BiologySchool of Veterinary MedicineUniversity of CaliforniaDavisCaliforniaUSA

Renee Riejo-PeraDepartment of Cell Biology andNeurosciencesand Department of Chemistryand BiochemistryMontana State UniversityBozemanMontanaUSA

Heide SchattenDepartment of VeterinaryPathobiologyUniversity of MissouriColumbiaMissouriUSA

Jayasree SenguptaDepartment of PhysiologyAll India Institute of MedicalSciencesNew DelhiIndia

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Munevver SerdarogullariBahceci Women’s Health GroupIstanbulTurkey

Monis B. ShamsiAndrology and IVF LaboratoryDepartment of Surgery(Urology)University of UtahSalt Lake CityUtahUSA

Luke SimonAndrology and IVF LaboratoryDepartment of Surgery(Urology)University of UtahSalt Lake CityUtahUSA

Qing-Yuan SunState Key Laboratory ofReproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijingChina

Jason E. SwainCenter for ReproductiveMedicineDepartment of Obstetrics &GynecologyUniversity of MichiganAnn ArborMichiganUSA

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Sperm Selection Techniques and their Relevanceto ARTLuke Simon1, Monis B. Shamsi1, and Douglas T. Carrell1,2,3

1Andrology and IVF Laboratory, Department of Surgery (Urology), University of UT, Salt LakeCity, UT, USA2Department of Obstetrics and Gynecology, University of UT, Salt Lake City, UT, USA3Department of Human Genetics, University of UT, Salt Lake City, UT, USA

1.1 Introduction

Fertilization is now possible using any available sperm throughintra-cytoplasmic sperm injection (ICSI) treatment (Palermo et al.,1992). As a result, andrological research has raised questions regard-ing the selection of suboptimal sperm used for assisted reproductivetechnology (ART) (Avendano and Oehninger, 2011). In recent years,the role of sperm in ART has been highlighted as the sperm providesone half of the genome to the developing embryo. The use of healthiersperm has showed to improved ART outcomes and subsequently,sperm selection has become an integral part of ART procedure (Saidand Land, 2011). Since the birth of first in vitro fertilization conceivedbaby in 1978, sperm selection for ART has been focused on selectingphysiologically motile and morphologically normal sperm (Bartoovet al., 2002). Despite success, it has become evident that physicalappearances of the sperm are inefficient to identify the most suitablesperm for ART success (Yetunde and Vasiliki, 2013). Hence, recentresearch is focused on developing novel sperm biomarkers to identifynon-apoptotic sperm with high DNA integrity for successful usein ART.

Our understanding of sperm physiology, as well as the technologyto select healthier sperm has progressively been improved. Initially,sperm selection was based on simple semen washing procedures and

Human Reproduction: Updates and New Horizons, First Edition. Edited by Heide Schatten.© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

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now more sophisticated sperm separation measures have evolved(Simon et al., 2015). The sperm is regarded unusable for the use inART, after being analyzed for its molecular parameters such as DNAintegrity, histone retention, protamine content, or ratio, and so on.Therefore, preserving the structural and functional integrity of thesperm is been the goal for recently introduced novel sperm selectionapproaches (Berkovitz et al., 2006a, 2006b). Some novel sperm selec-tion approaches aim to mimic the natural selection process, where thefemale reproductive tract is known to eliminate poor quality spermto enhance the chances of a successful fertilization (Holt and Fazeli,2015). Other methods have focused on sperm physiological changes inthe female reproductive tract, like capacitation, which are functionallyimportant for acrosome reaction (Bedford, 1963). Inclusion of suchnovel biomarkers along with standard sperm preparation procedureshas shown promises to enhanced fertilizing ability and improves ARTsuccess (Nasr-Esfahani et al., 2008a; Kheirollahi-Kouhestani et al.,2009; Polak de Fried and Denaday, 2010; Wilding et al., 2011).

1.2 Need of Sperm Selection in ART

Human semen is comprised of heterogeneous sperm population withvarying degrees of structural differentiation, maturity, fertilizing abil-ity, and functional quality (Huszar et al., 1993, 1998). During naturalconception the sperm from these subpopulations compete to traversethrough several anatomical and physiological barriers in the femaletract. The most competent and reproductively efficient sperm are ableto migrate through the cervical mucosa, uterus, uterine tube, cumuluscells, zona pellucida, and finally oolemma to participate in the fer-tilization (Suarez and Pacey, 2006). Further, selection takes place atthe level of sperm-oocyte interaction and out of a population of mil-lions, a single sperm is able to fertilize the oocyte and develop intoan offspring. These barriers for natural selection exclude the spermwith structural abnormalities as acrosomal absence, flagellar defor-mity, immature sperm, and sperm with aneuploidy or other chromatinabnormalities from participating in a successful fertilization (Suarezand Pacey, 2006). On the contrary, during ART, sperm are brought inproximity to oocyte, outside the female body, where no such anatomi-cal and physiological barriers exist. Depending upon the technique ofART, either the sperm fertilize the oocyte on their own as in IVF or

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the sperm are injected into oocyte for fertilization as in ICSI. DuringART, sperm does not have to overcome any anatomical and physio-logical barriers present in the female reproductive tract, natural spermselection are bypassed. Therefore, it is imperative to have an efficientartificial selection process that maximizes the probability of successfulpregnancy and birth of a healthy offspring. Further, the sperm selec-tion procedures also help to enrich the concentration of good qualitysperm that increases the chance of ART success.

Sperm contribute half of the genome to the offspring. Therefore,selection of sperm with intact chromatin and free of chromosomalabnormalities is important for ART success. Studies indicate that evenif the best quality sperm are used for ICSI, approximately, 55% of theselected sperm have normal DNA (Ramos et al., 2004). The primaryobjective of sperm selection approaches is to select good quality orhealthier sperm. In addition, sperm selection approaches are designedto reduce the physiological and oxidative damage induced to the spermduring the selection process. With these perspectives in sight, recentdevelopments in sperm selection approaches are focused on physio-logical properties or morphological characteristics or behavior in theelectric field or basis on their fluid kinetic properties. This chapter dis-cusses some of the novel sperm selection techniques that have beenthe focus of recent research and may have the ability to revolution-ize ART by improving the success rate, even in patients with severelycompromised sperm parameters.

1.3 Methodology of Sperm Selection

1.3.1 Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is a very useful gamete micro-manipulation technique for treating couples with severely compro-mised sperm parameters. Since its introduction in 1992, ICSI has rev-olutionized ART by providing hope to couples to achieve a pregnancy,who had few chances of a natural conception or by in vitro fertiliza-tion (IVF). The basic principle of ICSI is to manually select the bestsperm on the basis of motility and/or morphology and to inject it intoan oocyte. The premise for such gamete micromanipulation is that itenables a successful fertilization, when a sperm is unable to fertilizeon its own. During this procedure, initial events of fertilization like

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acrosome reaction are bypassed and now fertilization is possible withany available sperm.

1.3.1.1 MethodologyThe oocytes retrieved after ovarian hyper-stimulation is placed in apetri dish (specific for ART) in which they are fertilized with a sperm.The whole process is done with the help of a CCD attached micro-scope using a micromanipulator. The basic steps for ICSI manipula-tion are as follows: the oocytes retrieved after hyper-stimulation areheld by a specialized holding pipette in a micromanipulator. The mostvisually normal sperm by virtue of its motility and morphology arepicked by ICSI pipette. During this step, sperm are usually visualizedat 400× magnification to increase the chances of detecting and elimi-nating any sperm with morphological abnormalities. The pipette con-taining sperm is then carefully inserted through the membrane of theoocyte, into the cytoplasm. A sperm is injected into the cytoplasm andthe pipette is carefully removed. The oocytes are then incubated andchecked for pronuclear appearance to confirm fertilization after 24hours. After a successful fertilization, the embryos are cultured untilcleavage stage (Day 3 embryo transfer) or until blastocyst stage (Day 5embryo transfer) into the uterus.

1.3.1.2 Advantages and LimitationsICSI is the most widely used ART, accounting to 70–80% of thecycles performed (Palermo et al., 2009). ICSI has assisted millions ofinfertile couples to conceive, even with severely compromised spermparameters, as severe oligozoospermia, asthenozoospermia, or bothin the male partner. This technique has dropped down the numberof sperm required for fertilization from several thousand to a singleviable sperm. In men with obstructive or non-obstructive azoosper-mia, where there are no sperm in an ejaculate, testicular-epididymalsperm extraction (TESE) combined with ICSI has made it possible tosire a child (Vloeberghs et al., 2015).

Although ICSI has fulfilled the dreams of parenthood for millionsof infertile couples, but there are risks and concerns for the healthof mother and the child associated with this technique. ICSI bypassesnumerous physiological events of fertilization, which has always beena safety concern related to this technique. Many hazards are not spe-cific to ICSI they are common to most of the ART. Two specific demer-its of ICSI are: the injection of media into an oocyte along with the

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sperm and the bypass of natural selection process (Sánchez-Calabuiget al., 2014).

During natural conception, the sperm pass through differentbarriers with in the female reproductive tract, so that the mostcapable sperm, with normal morphology and vigorous motility canfertilize the oocyte (Barratt and Kirkman-Brown, 2006; Suarez andPacey, 2006). Three levels of barriers effectively hinder the reach ofan abnormal sperm to an oocyte; (1) the microenvironment of thefemale reproductive tract, (2) the sperm-oviduct interactions at thecaudal isthmus, and (3) the sperm-zona pellucida interaction (Suarezand Pacey, 2006). ICSI bypasses these steps of natural selection toselect the best sperm (observed by an embryologist), since it does notinvolve the sperm–oviduct interaction and other processes as zonapellucida binding-penetration. Further, the presence of acrosomalenzymes from the unreacted acrosome is introduced into the oocyteduring ICSI, which may lead to an increased risk of vacuole formation(Morozumi and Yanagimachi, 2005; Morozumi et al., 2006).

During ICSI, the selection of sperm is based on the embryologistexperience, which usually rely on the motility and morphology of thesperm. Since, these sperm parameters are not always reflective ofsperm DNA integrity, chances of selecting a poor DNA quality spermfor fertilization in ICSI is not ruled out (Celik-Ozenci et al., 2004).Therefore, in ICSI there is a realistic possibility that a sperm withhigh DNA fragmentation or a aneuploid sperm can be selected forfertilization, which may ultimately lead to adverse consequences fromfailed fertilization and retarded embryo development to increasedrates of miscarriage and diseases in the offspring.

In the post-natal life, children born after ART have been observedto have lower birth weights and higher peripheral fat, blood pressure,and fasting glucose concentrations than controls (Fauser et al.,2014). A meta-analysis of 19 publications selected by a qualityscore based on sample size and appropriateness of control groupobserved that major malformation rates ranged from 0 to 9.5% inIVF, 1.1 to 9.7% in ICSI, while 0 to 6.9% after natural conception,leading to a statistically significant overall odd ratio of 1.29 (Rimmet al., 2004). Further, it has been reported that 90–100% of the ARTchildren with Beckwith–Wiedemann had imprinting defects, ascompared to 40–50% of the spontaneously conceived children withBeckwith–Wiedemann (Manipalviratn et al., 2009). Similarly, 71%of the Angelman Syndrome cases in ART children were attributed

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to epigenetic defects as compared to 5% of the naturally conceivedchildren with Angelman Syndrome (Manipalviratn et al., 2009).

1.3.1.3 ConclusionICSI sperm selection is an option for couples who have failed the stan-dard IVF treatment and benefits men with severe male infertility. ICSIselected sperm is directly injected into the oocyte, which provides thebest chance of fertilization in couples with fewer available oocytes fortreatment. Despite advantages, the absence of natural sperm selectionprocess may lead to an increased risk of miscarriage due to injectionof any available sub optimal sperm, which subsequently increases therisk of health issues in ICSI born children. Research into the effectsof ICSI sperm selection method is still on going, as this technique isextensive in use for less than two decades. However, ICSI sperm selec-tion method does improve the odds of treating an infertile man, but itdoes by remove the key elements that often lead to male infertility.

1.3.2 Intracytoplasmic Morphologically Selected SpermInjection

The introduction of ICSI as a method of insemination revolutionizedthe treatment of male infertility. With the widespread use of ICSI,contradictory findings were reported in many studies with regard tosperm selection based on morphology. Some studies demonstrate thatsperm morphology according to strict criteria (Kruger et al., 1986,1988) has controversial prognostic value in ICSI outcomes (Svalanderet al., 1996; De Vos et al., 2003; French et al., 2010) and does notseem to influence embryo quality or development (De Vos et al.,2003; French et al., 2010). Therefore, need for more stringent spermselection procedures were recommended to effectively improve ARToutcome. As a major development in this direction introduced byBartoov et al. (1994, 2001, 2002), who devised a method of unstained,real-time, high magnification (6600×) examination of sperm called“motile sperm organelle morphology examination” (MSOME). Theintegration of MSOME with ICSI sperm selection was defined asintracytoplasmic morphologically selected sperm (IMSI) (Bartoovet al., 2003).

During IMSI, the motile sperm morphology that includes normalcyof the sperm nucleus (shape and chromatin content), acrosome size,presence and absence of vacuoles, are observed at high (6600×) mag-nification instead of around 400× used during conventional ICSI. The

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1 Sperm Selection Techniques: Relevance to ART 7

introduction of IMSI facilitated the observation of ultra-structuralmorphological details of live sperm, thereby assisting in selection ofhealthier sperm, to be used for ART.

1.3.2.1 MethodologyIMSI is a modification of ICSI, in which the sperm selection is done atmagnification many fold higher than ICSI. Its introduction in the fieldof ART facilitated the observation of live human sperm, particularlyby showing sperm vacuoles not necessarily seen at lower magnifica-tion. The sperm selection for IMSI relies on the evaluation criterion ofMSOME, which evaluates the presence, size, number, and location ofvacuoles. According to the MSOME criterion, if the sperm head con-tains one or more vacuoles (diameter of 0.78 ± 0.18 μm) occupyingmore than 4% of the normal nuclear area, it is considered abnormal foruse in ART (Bartoov et al. 1994, 2001, 2002). The MSOME criterionhas been modified to a scoring system, to simplify the sperm classifi-cation into different grades. Briefly, grade I sperm have normal spermhead and absence of vacuoles and they represent the optimal type.Grade II sperm are characterized by maximum two small vacuoles.Grade III sperm have either more than two small vacuoles or one largevacuole. The grade IV represents the poorest quality sperm, whichshow large vacuoles fully occupying the head, along with other mor-phological defects (Vanderzwalmen et al., 2008; Greco et al., 2013).Cassuto et al. (2009) introduced a similar protocol of sperm classifica-tion based on the detailed analysis of head, acrosome, vacuoles, baseof sperm head, and the presence of cytoplasmic droplet.

1.3.2.2 Advantages and LimitationsThe use of IMSI over ICSI or other sperm selection techniques hassignificantly improved ART success rate, since it involves the selec-tion of sperm with a strictly defined, morphologically normal nucleus.It has been particularly useful for couples with repeated ICSI failure(Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al., 2006; Anti-nori et al., 2008; Franco et al., 2008; Setti et al., 2010). It has beenreported that IMSI is associated with significantly higher implanta-tion and clinical pregnancy rates and a reduction in the abortion rates(Setti et al. 2010, 2011), where the pregnancy rate in IMSI has beenobserved to be 66% as compared to 30% in ICSI. The reported implan-tation rate in IMSI is 27.9% while it is 9.5% in ICSI (Bartoov et al.,2003; Berkovitz et al., 2005). In cases, where no sperm could qualify

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8 Human Reproduction

for selection in the IMSI procedure, an increase in abortion rate from10 to 57% has been reported (Berkovitz et al., 2005). Further, Cassutoet al. (2014) reported a lowering of congenital malformation in IMSIborn children to 1.3% as compared to 3.8% born after ICSI. In addi-tion, IMSI improved ART outcome in patients with severe degrees ofsperm DNA damage. It has also provided evidence for the associationof presence, size, and number of sperm nuclear vacuoles with embryoquality and development, and suggested that high number of vacuolesmay account for increased abortions (Figueira et al., 2011).

Though, IMSI has been documented to significantly improve theART outcomes, but the technique has its drawbacks. Undoubtedly,it is a time-consuming technique and selecting a normal sperm inaccordance with MSOME criterion may take 60–120 min (Antinoriet al., 2008). Further, the prolonged exposure to the microscope’sheated stage may itself cause damage to the sperm, as demonstratedby Peer et al. (2007) after 2 h on the microscope’s heated stage,sperm nucleus vacuolization significantly increases. Despite theseobservations, IMSI has proved itself as a valid tool for safe and anon-invasive method of sperm selection.

1.3.2.3 ConclusionThe IMSI sperm selection approach changed the perception of howa sperm suitable for insemination should appear. Sperm, which wasconsidered as normal when observed under a low magnificationmicroscope, is showed to contain ultra-structural defects that mayimpair ART outcomes. Recent studies have reported that IMSI is asso-ciated with improved ART outcomes; specifically, implantation andpregnancy rates, while a reduction in miscarriage rates was observedwhen compared to conventional ICSI insemination. Despite its advan-tages, clinical indications for IMSI procedure are still lacking andfurther prospective randomized clinical trials are required to identifypatient groups that are benefited by IMSI sperm selection approach.

1.3.3 Annexin V Labeling

Annexin V labeling is a well-recognized method to detectbio-molecules at the sperm membrane to identify apoptotic sperm.This method has been widely used to separate the apoptotic spermfrom non-apoptotic (healthier) sperm population. This methodis based on the affinity of protein coagulant, Annexin V, with a

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1 Sperm Selection Techniques: Relevance to ART 9

phospholipid, phosphatidylserine of sperm plasma membrane. Ina normal sperm, phosphatidylcholine and phosphatidylserine areasymmetrically distributed, with the former exposed to externalleaflet of membrane while the later located at the inner surface oflipid bilayer. However, this asymmetry is disrupted during apoptosis,when the phosphatidylserine is externalized to the outer side ofmembrane, which facilitates an apoptotic sperm to be recognizedby the macrophages and eliminated. A magnetic bead-conjugatedannexin V helps in the identification of an apoptotic sperm, in anexternal magnetic field, annexin-V conjugated to dead and apoptoticsperm by magnetic activated cell sorting (MACS; Figure 1.1).

Phosphatidylserine

Normal

sperm

Apoptic

sperm

Annexin V labeling

Annexin V labeled

apoptic sperm

Annexin V

magnetic spheres

External

magnets

Figure 1.1 Apoptotic sperm are labeled by annexin V magnetic beads. Amagnetic field separates the apoptotic sperm. (See color plate section for the colorrepresentation of this figure.)

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10 Human Reproduction

1.3.3.1 MethodologyAnnexin V is a phospholipid binding protein that has high affinityfor phosphatidylserine but lacks the ability to pass through an intactsperm membrane (van Heerde et al, 1995). Therefore, in sperm withcompromised membrane integrity, the annexin V binding takes placeat the phosphatidylserine exposed on the outer layer of membrane(Glander and Schaller, 1999). To separate the apoptotic sperm fromnon-apoptotic sperm, super-paramagnetic microbeads conjugatedwith annexin V are used to label sperm with externalized phos-phatidylserine. During this procedure of MACS, a mixture of sampleand conjugated annexin V is incubated, and loaded on a separationcolumn placed in the magnetic field. The attractive force between themagnetic field around the column attracts the magnetic beads conju-gated to annexin V-sperm complex, and hence the annexin V-positivefraction comprising of apoptotic sperm binds to the column, while theannexin V-negative fraction of non-apoptotic sperm elutes throughthe column. The column is removed from the magnetic field, andannexin V-positive fraction is eluted using the annexin V-bindingbuffer (Chan et al., 2006). Thus, this method yields two fractions:annexin-negative (intact membranes, non-apoptotic sperm) andannexin-positive (externalized phosphatidylserine, apoptotic sperm)(Grunewald et al., 2001; Glander et al, 2002).

1.3.3.2 Advantages and LimitationsAnnexin V labeling is a simple, convenient method for detectionand separation of apoptotic sperm. It provides optimal purity withreliable and consistent results. As opposed to other routine methodsof sperm separation, which rely on motility and sperm density, thistechnique acts at the molecular level. Combining this method, withother techniques such as density gradient centrifugation may yield asperm population with higher motility, viability, and lower number ofapoptotic sperm, though it makes the procedure for sperm isolationmore time and energy consuming (Said et al., 2006a). This techniquehas been reported to improve acrosome reaction and is associatedwith higher cleavage and pregnancy rates than spermatozoa selectedby density gradient centrifugation in oligoasthenozoospermic men(Dirician et al., 2008; Lee et al., 2010). Annexin V negative fractionhas low amounts of DNA damage, and higher oocyte penetrationcapacity than annexin V-positive sperm (Said et al., 2006). Although,sperm sorting with annexin V method was effective in the treatment