Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction

Cover
Frontmatter
	Foreword
	Preface
	Editor Biographies
	Contents
	Contributors
Part I: Human Sperm Chromatin: Structure and Function
	1: Sperm Chromatin: An Overview
		Origins of Sperm Chromatin Research
		Spermatogenesis: A Special Form of Terminal Differentiation
		Variability in the Composition of Sperm Chromatin
		Spermatid Differentiation and Chromatin Remodeling
		Higher Ordered Organization of Chromatin in Mature Sperm
		Mammalian Protamines
		Structure of the DNA–Protamine Complex
		Chromosome Territories, Loop Domains, and Matrix Attachment Regions
		Reorganization of Sperm Chromatin Following Fertilization
		Consequences of Disrupting Sperm Chromatin Remodeling
		Future Research and Practical Applications
		References
	2: Spermatogenesis: An Overview
		Neurological Pathways
		Steroid Hormone Interaction and Neurological Axis
		Organization of the Testis
			Supporting Cells: Leydig Cells
			Seminiferous Tubules and Sertoli Cells
		Spermatogenesis
			Types of Spermatogonia
		Spermatocytogenesis
			Mitosis
			Meiosis
			Spermiogenesis
			Spermiation
			The Cycle or Wave of Seminiferous Epithelium
			Chromatin Remodeling/Alterations During Sperm Differentiation
			Histone and Basic Nuclear Protein Transitions in Spermatogenesis
			Role of Transition Proteins
			Protamines as Checkpoints of Spermatogenesis
			DNA Methylation During Spermatogenesis
			Sperm Nuclear DNA Strand Breaks
			Sperm Apoptosis
			Oxidative Stress in the Testis
			Spermiogenesis and Etiology of DNA Damage
			Efficiency of Spermatogenesis
			Postspermiation Events
		Spermatozoa
			Head
			Acrosome
			Neck
			Tail
			Endpiece
		Regulation of Spermatogenesis
			Intrinsic Regulation
			Extrinsic Influences
		Immune Status of the Testis
		Disturbances of Spermatogenesis
		Sperm Transport in the Epididymis, Storage, and Capacitation
			Epididymal Sperm Storage
			Sperm Entry into Cervical Mucus
			Capacitation and Acrosome Reaction
		Conclusion
		References
	3: Sperm Nucleoproteins
		Protamines: The Major Components of the Sperm Nucleus
		The Nucleohistone to Nucleoprotamine Transition
		Organisation of the DNA in the Mature Sperm Nucleus
		Protamine Anomalies in the Sperm Cells of Infertile Patients
		References
	4: The Relationship Between Chromatin Structure and DNA Damage in Mammalian Spermatozoa
		Sperm Chromatin Structure Overview
			Protamine Condensation of the Sperm DNA
			Histone-Bound Sperm Chromatin
			Organization of DNA Loop Domains
			Ability of Protamine Condensation to Protect Sperm DNA from Damage
			Active Sperm Chromatin
			Conclusions
		References
	5: Chromosome Positioning in Spermatozoa
		Overview of Chromosome Positioning in Interphase Cells
		Methods of Determining Chromosome Nuclear Localization
		Positioning of Chromosomes in Spermatozoa
			Sperm Chromocenter
			Longitude and Radial Positioning
		Chromosome Movement during Spermatogenesis
		Deviant Chromosome Positioning in Sperm of Subfertile Males
		Possible Significance of Sperm Chromosome Positioning for Fertilization and Early Development
			Does Positioning of Sex Chromosomes Have Functional Importance?
			Potential Role of Sperm Chromosome Positioning for Introcytoplasmic Sperm Injection
		Conclusion and Perspectives
		References
	6: Sperm Mitochondrial DNA
		What is mtDNA?
		Why is mtDNA Important?
		OXPHOS and Sperm Function
		The Mitochondrial Nucleoid
		mtDNA Replication
		POLG, mtDNA-Type Disease, and Sperm Function
		TFAM, mtDNA Disease, and Its Role During Spermatogenesis
		Sperm mtDNA Replication
		mtDNA Inheritance
		Mitochondrial DNA Variants and Their Effect on Sperm Function
		mtDNA Haplotype
		Conclusions
		References
	7: The Sperm Epigenome
		Epigenetics and the Roles of DNA Methylation
		Enzymes Involved in DNA Methylation
			Germ Cell Expression
		DNA Methylation Patterns in Germ Cells
			Erasure and Acquisition of Germ Cell Patterns
			Histone Modifications and Epigenetic Memory
		Consequences of an Altered Sperm Epigenome for Male Reproductive Function
			Animal Models
			Drug Targeting
			Human Infertility
		Conclusions and Future Directions
		References
	8: RNA Expression in Male Germ Cells During Spermatogenesis (Male Germ Cell Transcriptome)
		The Transcription Landscape in Male Germ Cell Development
			Expression Profiling of Male Germ Cell Transcriptome: Past, Present, and Future
				From cDNA Library to High- Throughput Sequencing
				Overview of Germ Cell Transcriptome Studies
				Key Biological Findings and Implications
			Revealing Transcription Complexity of Male Germ Cell Development by Serial Analysis of Gene Expression
				Alternative Splicing
				Antisense Transcription
				Noncoding RNA Transcription
			Germ Cell Transcriptome Informatics
				Germ Cell Transcriptome Resources
			Chromatin Remodeling and Spermatogenesis
				Background
				Chromatin-Related Transcriptional Regulations in Spermatogenesis
		Conclusions
		References
Part II: Laboratory Evaluation of Sperm Chromatin
	9: Sperm Chromatin Structure Assay (SCSA ®): 30 Years of Experience with the SCSA ®
		Pioneering the First Sperm DNA Fragmentation Test: SCSA ®
		Development of SCSA ®
		Power of the SCSA ® Test: Six Important Parameters
		SCSA ® Method Overview
		SCSA ® Data
			SCSA ® Raw and Computer Reoriented Data
			Characterization of Sperm Populations Identified in a SCSA ® Analysis
		Other Probes that Shed Light on SCSA ® Data
			Disulfide Bonding of Chromatin
			Chromomycin A3 (CMA3) Staining of HDS Sperm
		Comparison Between SCSA ® and TUNEL
		Validation of Flow Cytometry and AO Biochemistry on Sperm DNA Integrity
			Requirements for Validating a New DNA Fragmentation Test
			Examples of Repeatable High-Quality SCSA ® Data
				Genotoxicant Exposure
				Mouse
				Human
			Potential RNA Staining Artifacts for SCSA ®
			Repeatability of SCSA ® Data Over Time for Men
			Repeatability of %DFI Values of Human Sperm Samples from Two Commercial SCSA ® Laboratories
			Animal Fertility
				Bulls
				Boars
			Human Fertility
				Natural Conception
				SCSA ® Test and ART Clinics
				TESA for Failed ICSI Cycles with High %DFI
		SCSA ® Defined Etiologies of Increased DNA Fragmentation
			Age
			Genetics
			Varicocele
			Cancer
			Environmental Heat
			Fever
			Medications
			Diabetes and Insulin Resistance
		Conclusions
			Clinical Utility of the SCSA ®
		References
	10: Sperm Chromatin Dispersion Test: Technical Aspects and Clinical Applications
		Sperm DNA Fragmentation: Now and Then
		Technical Basis of the SCD Test
		Validation of the SCD Test
		Methodological Versatility
			Assessing DNA Damage Intensity
			Assessing Chromosomal Abnormalities
			Assessing Oxidative DNA Base Damage
			Assessing DNA Methylation
				Assessing Sperm Protein Matrix
			The SCD and Low Sperm Counts
		The SCD Test in the Assisted Reproductive Technology (ART) Laboratory
			Fertility Assessment
		The SCD Test in the Andrology Laboratory
			Varicocele
			Genitourinary Infections
			Sperm DNA Damage and Cancer
			Azoospermia
			Toxicogenetics
		Sperm DNA Fragmentation Dynamics
		Conclusion: Value of the SCD Test
		References
	11: Basic and Clinical Aspects of Sperm Chromomycin A3 Assay
		Fluorochromes as Indicators of Sperm Chromatin Compaction
		Interaction Between CMA3 and Sperm DNA
		CMA3 and DNA Damage
		References
	12: Cytochemical Tests for Sperm Chromatin Maturity
		Cytochemical Properties of Human Sperm Chromatin and Basis of its Testing by Planar Ionic Dyes
		Sperm Chromatin Structural Probes
			Acidic Aniline Blue
			Toluidine Blue Stain Assay
			Chromomycin A3 Assay
		Conclusion
		References
	13: Acridine Orange Test for Assessment of Human Sperm DNA Integrity
		Introduction
		History
		Principle
		Mechanism
		Acridine Orange Staining Technique
			Procedure
				Acridine Orange Test by Fluorescence Microscope
					Reagent Preparation
					Sperm Preparation and AO Staining
		Critical and Troubleshooting Points
			Acridine Orange Test by Flow Cytometry (Sperm Chromatin Structure Assay)
			Chromatin Anomalies and Clinical Significance of Acridine Orange Test
		References
	14: Laboratory Evaluation of Sperm Chromatin: TUNEL Assay
		Mechanisms of Sperm DNA Damage
		Measuring Sperm DNA Damage with TUNEL
		Measurement of DNA Damage in Spermatozoa by TUNEL Assay
			Protocol #3: In Situ Death Detection kit (Roche Diagnostics, Indianapolis, IN)
				Reagents and Equipment
				In Situ Death Detection Kit
				Assay Principle
				Sample Preparation
			Protocol #4: APO-DIRECT ™ kit (BD Pharmingen, Catalog # 556381)
				Principal
			Measurement of Sperm DNA Damage
				Flow Cytometry
				Fluorescence Microscopy
			Protocol for Shipping Semen Samples for TUNEL Test
				Reference Ranges of Sperm Damage
				Sensitivity and Specificity
			Factors Affecting TUNEL Assay Results
		Future of TUNEL Assay
		Conclusions
		References
	15: Basic and Clinical Aspects of Sperm Comet Assay
		The Need for Novel Diagnostic and Prognostic Tests
		Causes of Sperm DNA Damage
			Environmental and Lifestyle Hazards
		The Comet Assay: What Does It Measure?
		Strengths of Comet Assay
		Weaknesses of Comet Assay
		Need for Standardized Methodology for the Comet
		Clinical Significance of DNA Fragmentation Measured by the Comet Assay
		Further Uses of the Comet to Measure DNA Adducts
		Clinical Significance of Existing Strand Breaks Plus Adducts Measured by the Comet Assay
		The Risks of Using Sperm with Damaged DNA
		The Clinical Usefulness of the Comet Test
		Two People but Just One Prognostic Test
		Protection of DNA from Damage
		Conclusions and Future Recommendations
		References
	16: Assays Used in the Study of Sperm Nuclear Proteins
		Histones
		Protamines
		Assays
			Isolation Techniques
			Isolation and Purification of Protamines
			Acid Gel Preparation
			Isolation and Purification of Histones
			Protamine Quantification and Ratio Generation
				Quantification
				Generation of P1–P2 Ratio
			Generation of the Histone to Protamine Ratio
			Staining Techniques
				Chromomycin
				Aniline Blue
					Aniline Blue Staining Technique
			Immunocytochemistry
				Immunocytochemistry Preparation
		Conclusions
		References
	17: Sperm Epigenetic Profile
		Epigenetic Regulation of Gene Expression
			DNA Methylation
			Histone Modifications
		Genomic Imprinting Mechanism in Mammals
		Epigenetic Modifications in Sperm
			Establishment of Paternal Imprints in the Male Germ Line
				DNA Methylation Imprinting Marks in the Male Germ Line
				DNA Methyltransferases Expression in the Male Germ Line
			Chromatin Organization of the Sperm Nucleus
		Epigenetic Defects in Assisted Reproduction Techniques (ART)
			Imprinting Syndromes in ART Children
			Imprinting Errors in Male infertility
		Methods for Assessing Epigenetic Modifications in Sperm
		Clinical Importance of Sperm Epigenetic Profiling in ART
		Conclusion
		References
	18: Clinical Significance of Sperm RNA
		Presence of Various RNAs in Spermatozoa
			Multiple Origins of the Sperm Transcripts
				Transcripts from Genes Coexpressed in Somatic and Male Germ Cells
				Transcripts from Male Germ-Cell-Specific Homologous Genes
				Transcript Variants
				Transcripts from Testis-Specific Genes
				Spermiogenic RNA Retention
			Classes of Spermatozoal Transcripts
			Localization of the RNA Within Sperm Cells
			RNA Involvement in Paternal Genome Packaging
			Interfering RNA in Mature Spermatozoa
			Functional Significance of the RNA During Embryo Development
				Delivery of Sperm RNAs to the Ovocyte
				RNA-Mediated Epigenetic Effects on the Embryo
		Clinical Significance of the Sperm Transcriptome
			Transcripts of Aromatase and Estrogen Receptors
			Significance of Other Transcripts
		Concluding Remarks
		References
Part III: Biological and Clinical Determinants of Sperm Chromatin Damage
	19: Role of Oxidative Stress in the Etiology of Sperm DNA Damage
		The Chemistry of Oxidative Stress
			Reactive Oxygen Species
			Lipid Peroxidation
			Oxidative DNA Damage
			Antioxidant Protection
		Measurement of Oxidative Stress in Spermatozoa
			Assessment of Reactive Oxygen Species Generation
				Confounding Effect of Leukocyte Contamination
				Chemiluminescence
				DHE and Mitosox Red
			Measurement of DNA Damage in Spermatozoa
			Criteria for Diagnosing Oxidative DNA Damage in the Germ Line
		Origins of DNA Damage in the Germ Line
			Physiological DNA Strand Breaks
			Antioxidant Depletion
			Leukocytic Infiltration
			Apoptosis
			Impaired Spermiogenesis
		Conclusions and Future Recommendations
		References
	20: Abortive Apoptosis and Sperm Chromatin Damage
		Cellular Characteristics of Apoptosis vs. Necrosis
		Programmed Cell Death Cascade
			Testicular Germ Cells Apoptosis in Normal Spermatogenesis
		Regulators of Testicular Apoptosis
			Intrinsic Regulators
				Genes Regulating Germ Cell Apoptosis
				Genetic Regulators of DNA Repair
				Extrinsic Regulation (Hormonal Regulation)
			Testicular Germ Cells Apoptosis During Testicular Dysfunction Conditions
				Aging
				Varicocele
				Spermatogenesis Failure
				Obstructive Azoospermia
		References
	21: Spermiogenesis in Sperm Genetic Integrity
		Chromatin Remodeling in Spermatids
			Specific Histones and Histone Variants Present During Spermiogenesis
			Posttranslational Modifications and Their Contribution to the Remodeling Program
			Nuclear Proteins Transition
		Endogenous DNA Breaks as Part of the Normal Differentiation Program of Spermatids
			Detection and Characterization of DNA Breaks in Elongating Spermatids
			Possible Origins of DNA Breaks
			Type II Topoisomerases as Likely Candidates
			DNA Breaks and DNA Packaging: The Chicken or the Egg?
		DNA Damage Response and DNA Repair Processes in Spermatids
			DNA Damage Response
			Do Topoisomerases Trigger DNA Damage Response?
			DNA Repair Mechanisms in Spermatids
				Nonhomologous End joining
				DNA Repair by Homology in a Haploid Cell
			Highly Conserved Process Among Higher Eukaryotes
		Possible Consequences and Clinical Relevance
			Impairment of Genetic Integrity in the Male Gamete
			Impact of This Transient Window of Genetic Instability on Clinical Practices
			Potential Recovery by the Oocyte After Fertilization
		Summary
		References
	22: Male Subfertility and Sperm Chromatin Damage
		Male Infertility/Subfertility
		Diagnosis of Male Infertility/Subfertility
		Assessment of Sperm Chromatin Damage
		Genesis of Sperm DNA Damage
		Factors Reported to Impact Sperm Chromatin Integrity
		Male Infertility and Sperm Chromatin Damage
			The Association Between the Traditional Sperm Parameters and Sperm DNA Integrity
			Intraindividual Variation of Sperm Chromatin Parameters
			Impact of Sperm DNA Damage on Fecundity in General Population
			Impact of Sperm DNA Damage on Fecundity in Subfertile Men
		Future Perspectives
		Conclusions and Clinical Recommendations
		References
	23: Aging and Sperm DNA Damage
		Semen Analysis
		Fertility of Aging Men
			Genetic Risks of the Aging Male
			Numerical Chromosome Disorders
			Structural Chromosomal Anomalies
			Autosomal Dominant Diseases
		Sperm DNA Damage
			Oxidative Stress and Aging
		Conclusion
		References
	24: Cancer in Males: Implications for Sperm Quality, Fertility, and Progeny Outcome
		Epidemiology of Cancer in Boys and Young Men
			Fertility After Cancer Therapy
			Cancer Management Strategies
				Reproductive Health Before Cancer Treatment
				Impact of Surgical Management for Cancer on Male Reproductive Status
				Radiation Therapy
				Chemotherapy
			Impact of Chemotherapy on Male Reproductive Health
				Animal Studies
				Clinical Studies
			Male Fertility Preservation and Restoration Strategies
				Sperm Cryopreservation
				Pharmacological Strategies
				Fertility Restoration with Germ-Cell Transplantation
			Looking to the Future
		References
	25: Sperm Chromatin and Environmental Factors
		Biological and Clinical Relevance
		How Can Environment/Lifestyle Affect Sperm Chromatin?
		Epidemiological Indications of Environmentally Induced Changes in Sperm DNA
		Sperm Chromatin and Environment
			Animal Experience
			Human Data
				Tobacco and Other Lifestyle Factors
				Occupational Exposure
				Air Pollution
				Persistent Organohalogen Pollutants
				Phthalates
				Insecticides and Pesticides Other than DDT
			Gene–Environment Interaction and Sperm Chromatin
		Conclusions
		References
	26: Effects of Male Accessory Gland Infection on Sperm Parameters
		Male Accessory Gland Infection
		Effects of MAGI on Sperm Parameters
			Effects of Various Microorganisms and Viruses
				Escherichia coli
				Neisseria gonorrhoeae
				Chlamydia trachomatis
				Ureaplasma urealyticum
				Mycoplasma hominis and Others
				Candida albicans
				Trichomonas vaginalis
				Hepatitis B Virus and Hepatitis C Virus
				Human Immunodeficiency Virus Type 1
				Papillomavirus
			Effects of Oxidative Stress
			Effects of Proinflammatory Cytokines
				Interleukin 1
				Interleukin 6
				Interleukin 8
				Interferon Gamma
				Macrophage Migration Inhibitory Factor
				Tumor Necrosis Factor-
		Conclusions
		References
Part IV: Sperm Chromatin and Assisted Reproductive Technology Outcomes
	27: The Impact of Sperm Processing and Cryopreservation on Sperm DNA Integrity
		The Clinical Need for Sperm Processing
		The Hazards of Seminal Plasma Removal During DCG
		Antioxidants: Physiological and Therapeutic Uses
		The Heightened Vulnerability of Testicular Sperm
		The Clinical Need for Sperm Cryopreservation
			The Impact of Cryopreservation on Conventional Sperm Parameters
		The Impact of Cryopreservation on Human Sperm DNA
		The Greater Susceptibility of Infertile Mens’ Sperm to Cryoinjury
			Mechanisms of Cryoinjury
			Is Apoptosis a Cause of DNA Cryodamage?
		Is Sperm DNA Damage a Result of Oxidation?
			Why Freeze Neat Semen?
		Recent Advances in Cryopreservation
			The Efficacy of Different Cryoprotectants
			Effects of Repeated Freezing and Thawing
			The Benefits of Antioxidant Supplements to Cryoprotectant Media
			Recent Advances in Freeze–Thaw Protocols
				Freeze-Drying of Sperm
				Vitrification of Sperm
		Conclusions and Future Recommendations
		References
	28: Structure of Chromatin in Human Sperm Bound to Hyaluronic Acid: The Benefits of PICSI Dish Mediated Sperm Selection
		Sperm Chromatin Maturation and Its Importance
		Impact of Sperm Chromatin Maturation and Imprinting
			Relationship Between the Nuclear and Cytoplasmic Aspects of Sperm Mismaturation
		Sperm–Hyaluronic Acid (HA) Binding: Spermatozoa with Full Cellular Development Selectively Binds to Solid-State HA
			Does Sperm HA-Binding Test Predict DNA Chain Integrity in Bound Sperm?
			Sperm Chromatin and Sperm Cellular Development
		Conclusions and Overview
		References
	29: Electrophoretic Sperm Separation
		Principles of Electrophoresis
			Electrophoretic Properties of Spermatozoa
			Development of Electrophoretic Technology for Sperm Sorting
		Equipment Set-Up and Separation Parameters
			Separation Cartridges and Sample Handling
			Electrophoresis Buffers and Temperature Settings
			Current and Voltage Settings
			Cleaning of Equipment
		Method Validation
			Sample Recovery and Purity
			Sperm Vitality and Motility
			Sperm Morphology and DNA Integrity
		Clinical Applications
		Closing Remarks
		References
	30: Antioxidants and Sperm DNA Damage
		Etiology of Sperm DNA Damage
			Relationship Between Oxidative Stress and Sperm DNA Damage
			Seminal Antioxidant Capacity and Sperm DNA Damage
		Clinical Studies
			Effect of Dietary Antioxidants on Sperm DNA Damage
			Effect of In Vitro Antioxidants on Sperm DNA Damage
		Summary
		Expert Commentary
			Five-Year View
		References
	31: Sperm Chromatin and ART (IUI, IVF and ICSI) Pregnancy
		Assisted Reproductive Technologies
		Traditional Markers of ART Fertility Potential
			Sperm Chromatin Integrity Testing
			Sperm Chromatin Integrity Testing in ART
				Intrauterine Insemination
				In Vitro Fertilization and Intracytoplasmic Sperm Injection
				Sperm DNA Damage in Relation to Pregnancy Outcome
				Sperm DNA Damage in Relation to Fertilization
				Sperm DNA Damage in Relation to Pre-Embryo Development
			Raw vs. Prepared Semen
			Incubation of Sperm
			Testicular vs. Ejaculated Sperm in ART
			The Use of Cryopreserved Sperm in ART
			Intraindividual Variation of DFI in Relation to ART
		Future Perspectives
		Conclusions and Clinical Recommendations
		References
	32: Sperm DNA Damage and Pregnancy Loss After IVF/ICSI
		Pregnancy Loss
			Definitions
			Etiologies
		Sperm DNA Damage
			Human Sperm DNA and Chromatin Structure
			Etiology of Sperm DNA Damage
			Tests of Sperm DNA Damage
		Relationship Between Sperm DNA Damage and Pregnancy Loss After IVF and ICSI
		Conclusion
		References
	33: Postnatal Effects of Sperm Chromatin Damage
		Long-Term Effects on Progeny of Paternal Exposure to Harmful Environmental and Medical Agents That Affect Sperm Chromatin Structure in Animals
			Long-Term Effects on Progeny of Paternal Exposure to Radiation
			Long-Term Effects on Progeny of Paternal Exposure to Chemical and Environmental Factors
		Long-Term Consequences of Fertilizing Oocytes Using Spermatozoa with Different Extents and Types of Chromatin Damage Determined in Mouse Models
			Sperm Chromatin Damage Induced by Sperm Preincubation Conditions
			Sperm Chromatin Damage Produced by Freeze–Thawing in the Absence of Cryoprotectants
			Sperm Chromatin Damage Produced by Freeze-Drying
			Sperm Chromatin Damage Produced by Scrotal Heat Stress
			Sperm Chromatin Damage Produced by Whole-Body Exposure to g -Rays
		Transgenerational Consequences of the Use of Spermatozoa with Fragmented DNA
		Postnatal Consequences of Sperm Chromatin Damage in Humans
		Conclusions
		References
	34: Evaluation of Chromatin and DNA Integrity in Testicular Sperm
		Biological Significance of Testicular Sperm DNA Damage
		Clinical Significance of Testicular Sperm DNA Damage
		Future Directions in the Field
		References
Part V: Protocols and Integrity Tests
	35: Sperm Chromatin Structure Assay (SCSA®)
		Basic Protocol Steps
		Materials
		Staining Solutions and Buffers
		Major Equipment
			Flow Cytometer(s)
		Cell Preparation
			Collection and Handling
			Freezing
		Flow Cytometer Setup
			Workstation
			Flow Cytometer Alignment
			Reference Samples
			Sample Measurement
			Gating and Debris Exclusion
		Critical Parameters or Points
	36: Measurement of DNA Damage in Spermatozoa by TUNEL Assay
		Assay Principle
		Specimen Collection
		Equipment and Reagents
		Sample Preparation
		Staining Protocol
		References
	37: Clinical Utility of Sperm DNA Integrity Tests
		Clinical Utility of Sperm DNA Tests
			Screening Test for First Pregnancy Planners
			Couples with Mild Male-Factor Infertility: IUI Candidates
			Couples with Severe Male-Factor Infertility: IVF or ICSI Candidates
			Couples with Pregnancy Loss After IVF or IVF/ICSI
		Guidelines on Clinical Value of Sperm DNA Tests
		Summary
		References
Index