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The Heart and Soul of ART……is in the Laboratory The Inside Story
Prof Prakash Trivedi, Priti P Trivedi
SECTION 1: THE ART OF ART: PERSPECTIVES
CHAPTER 1:
ART: Embryologist's Viewpoint
INTRODUCTION
IN VITRO FERTILIZATION AND ITS ASSOCIATED TREATMENTS
In Vitro Fertilization
Metaphase II Stage Oocyte
Embryo Transfers
Embryo Polarity
Blastocyst Transfers
GAMETE INTRAFALLOPIAN TRANSFER (GIFT)
ZYGOTE INTRAFALLOPIAN TRANSFER (ZIFT) AND TUBAL EMBRYO TRANSFER (TET)
OOCYTE AND EMBRYO MANIPULATIONS
Intracytoplasmic Injection of a Single Spermatozoon into an Ooctye (ICSI)
EMBRYO CRYOPRESERVATION
ASSISTED ZONA HATCHING
PREIMPLANTATION GENETIC DIAGNOSIS (PGD)
ETHICS OF ASSISTED CONCEPTION: PAST AND PRESENT
SUMMARY
CHAPTER 2:
ART: Clinician's Viewpoint
INTRODUCTION
MILESTONES IN ART
WORLD'S FIRSTS IN ART PROCEDURES2
CLINICIAN'S POINT OF VIEW
BASIC REQUIREMENTS IN REGARD TO AN ART TEAM/LABORATORY/LEVELS OF SKILL
AN APPROACH TO INFERTILE COUPLE
PREPROCEDURE COUNSELING OF THE COUPLE
COMMON DILEMMAS IN ART PROGRAM
Poor Implantation Site: A Clinician's Despair
Troubleshooting in ART
Proper Laboratory Control
GG Hospital Statistics
CONCLUSION
CHAPTER 3:
Setting Up of an ART Lab as per Guidelines and Future Need
INTRODUCTION
LOCATION OF THE LAB
The Sterile Area
BASIC INFRASTRUCTURE
LABORATORY THEATER EQUIPMENT AND LAYOUT
Requirements of OPU/ET Operation Table
Semen Processing and Freezing Room
Culture Lab
Laminar Flow Hood (Clean air station) (Figure 3.6)
Stereozoom Microscope (Figure 3.7)
Compound Microscope
Inverted Microscope with Micromanipulator (Figure 3.8)
Incubators
Microscope Stage Warmer and Dry Baths
Freezing Unit (Figures 3.13 and 3.14)
Miscellaneous Instruments
Equipments
Consumables and Disposables
Culture Media and Other Chemicals for ART Lab
Standardization of Procedures
Proper Documentation and Data Maintenance
NEED OF ADVANCED ART CENTER
ART Team
CONCLUSION
CHAPTER 4:
An ART Research Unit for Future
INTRODUCTION
Towards Better Improvements in Down Regulation and Stimulation
TOWARDS SINGLE EMBRYO TRANSFER
Blastocyst Culture
Embryo Selection Based on Morphology
Oocyte Morphology as an Predictor of Embryo Morphology
Polar Body Morphology as an Indicator of Embryo Quality
Pronuclear Stage Scoring as an Indicator for Embryo Quality
Preimplantation Genetic Diagnosis (PGD)
IMPLANTATION
IMPROVEMENT IN OOCYTE MANIPULATION: CONCERNS ABOUT DAMAGING THE SPINDLE
USE OF LASER IN ART
IN VITRO MATURATION (IVM) OF GAMETES
IVM of Spermatocytes and Spermatids
IVM of Oocytes
CRYOPRESERVATION
MOLECULAR BIOLOGY APPLIED TO ART
GENETICS OF FEMALE AND MALE INFERTILITY
EMBRYONIC STEM CELLS
CLONING
SECTION 2: GAMETES AND FERTILIZATION: BASICS SIMPLIFIED
CHAPTER 5:
Fertilization and Development: Theory and Practice
INTRODUCTION
GAMETES (OOCYTE AND SPERM CELL)
Oocyte
Sperm
PROCESS OF FERTILIZATION
Capacitation of Sperm
Changes in the Oocyte
Cumulus Mass
Zona Pellucida
Acrosome Reaction
Cortical Reaction
Fusion of Sperm and Egg
Sperm Nucleus Decondensation
Zygote Scoring
0 pn
1 pn
3 pn, 4 pn etc
CHAPTER 6:
Understanding Gametogenesis and Fertilization
INTRODUCTION
DEVELOPMENT OF MORPHOLOGICALLY AND FUNCTIONALLY COMPETENT GAMETES
Gametes
Embryogenesis of Gonads and Gametes
Maturation and Fertilizing Competence of the Gametes
Oocyte Maturation
Follicle Formation
Spermatogenesis
Anatomy of Human Spermatozoa
Endocrine Regulation of Spermatogenesis
EVENTS LEADING TO RELEASE OF GAMETES, THEIR TRANSPORT, FUSION AND FERTILISATION
Egg Release and Transport
Sperm Release and Transport
Capacitation and Hyperactivation
Preparatory Changes in Gametes before Fertilization
Further Sperm Maturation (Acrosome Reaction)
Further Oocyte Maturation
Fertilization
Events in Sperm-egg Interaction
Pronucleus Formation: Syngamy-embryonic Cleavage
CHAPTER 7:
Understanding Male Factor in ART and Infertility
INTRODUCTION
INCIDENCE
SOME PHYSIOLOGICAL ASPECTS
GENETICS OF MALE INFERTILITY
AGE AND MALE FACTOR INFERTILITY
OBESITY AND MALE INFERTILITY
PRESERVING FERTILITY IN MEN UNDERGOING CHEMOTHERAPY/RADIOTHERAPY
MALE INFERTILITY AND ART
ART CURRENT LIMITATIONS
SPERM RETRIEVAL TECHNIQUES
FUTURE TRENDS
SECTION 3: SPERM PARAMETER: HOW TO UTILIZE CLINICALLY
CHAPTER 8:
Human Spermatozoa: Structure and Theory
INTRODUCTION
STRUCTURE OF THE HUMAN SPERM
ROLE AND CONTRIBUTION OF HUMAN SPERMATOZOON DURING FERTILIZATION
Role of Sperm Centriole in Fertilization
Pronuclei Formation
CHAPTER 9:
Can Semen Analysis Interpretation be Standardized to More Value for Results
INTRODUCTION
BASIC SEMEN ANALYSIS
Historical Review
Need of Standardization
Basic Difficulty in Standardization
PROCESS OF STANDARDIZATION
Background Data
Physical Parameter
Analysis of Quantitative and Qualitative Parameters
Number of Semen Analysis to be Performed
CONCLUSION
CHAPTER 10:
CASA and Makler…..Usefulness?
MAKLER CHAMBER
Makler Chamber and Semen Analysis
COMPUTER AIDED SEMEN ANALYSIS
Sources of Errors in CASA
Digitalization
Object Recognition Errors
Collisions
CASA for Semen Analysis
Sperm Kinematics
CASA Analysis of Hyperactivation
Morphology Assessment by CASA
CONCLUSION
CHAPTER 11:
Handling the Sperm with Respect for Fertilizing, Freezing and Beyond IUI and IVF
SWIM-UP TECHNIQUE
GRADIENT METHOD
CRYOPRESERVATION
Thawing of Semen Sample
CONCLUSION
SECTION 4: ERECTILE DYSFUNCTIONS AND SPERM RETRIEVED TECHNIQUES
CHAPTER 12:
Erectile and Ejaculatory Dysfunction in Male Infertility
INTRODUCTION
ERECTILE DYSFUNCTION
Evaluation
Treatment
EJACULATORY DISORDERS
Evaluation
Causes
Neuropathic Causes
Anatomic Causes
Psychogenic (Functional) Dysfunction
Treatment of Ejaculatory Dysfunction
Therapeutic Modalities Available
SUMMARY
CHAPTER 13:
Male Sexual Dysfunction and Infertility
INTRODUCTION
MSD CAUSING INFERTILITY
TREATMENT OF MSD
PREVENTING MSD DURING INFERTILITY TREATMENT
CONCLUSION
CHAPTER 14:
Practical Aspects of Operative Sperm Retrieval
INTRODUCTION
WHO NEEDS OPERATIVE SPERM RETRIEVAL?
FROM WHERE CAN SPERM BE RETRIEVED?
HOW SHOULD SPERM BE RETRIEVED?
CONCLUSION: WHICH PROCEDURE AND WHEN?
SECTION 5: THE OOCYTE STORY
CHAPTER 15:
Inheritance from the Oocyte
INTRODUCTION
INHERITANCE
Mandelian Inheritance
Criteria for Autosomal Dominant Inheritance
Criteria for Autosomal Recessive Inheritance
Criteria for X-linked recessive inheritance
Criteria for X-linked Dominant Inheritance
Non Mendialian Inheritance
Rules of Non-Mandelian Inheritance
FORMATION OF THE MATURE OOCYTE
STRUCTURE OF THE OOCYTE
Chromosomes
Oocyte Genome
OOCYTE ACTIVATION, FERTILIZATION AND GENETIC EXCHANGE
OOCYTE INHERITANCE
Nuclear Inheritance
Mitochondrial Inheritance
Clinical Implications of Mitochondrial Inheritance
ART and Oocyte Inheritance
Nuclear Transfer
Cytoplasmic Transfer
Formation of Pathogenic Stem Cells
Preimplantation Genetic Diagnosis
CHAPTER 16:
Communication between the Ovary and the Oocyte
INTRODUCTION
OOCYTE-GRANULOSA CELL COMMUNICATION
TRANSFORMING GROWTH FACTORS
STEROIDAL AUTOCRINE/PARACRINE REGULATION
INSULIN-LIKE GROWTH FACTORS
OTHERS
CHAPTER 17:
Oocyte: Theory, Growth and Development
INTRODUCTION
HISTORY
ORIGIN
OOGONIA
OOCYTES
FOLLICULAR GENESIS
FOLLICULAR UNIT: OOCYTE AND GRANULOSA CELLS
FOLLICLE RECRUITMENT AND GROWTH
OOCYTE RECRUITMENT, SELECTION AND DOMINANCE
DYNAMICS OF THE FOLLICULAR POPULATION: FROM BIRTH TO MENOPAUSE
CONCLUSION
CHAPTER 18:
Ovum Pick-up Techniques
INTRODUCTION
LAPAROSCOPIC OOCYTE RECOVERY
Preliminary Laparoscopy
Procedure
Ultrasound Guided Follicular Aspiration Techniques
Transabdominal-transvesical Ultrasound directed Oocyte Recovery
Procedure
Disadvantages
Transabdominal-transvaginal Ultrasound directed Oocyte Recovery
Transabdominal-perurethral Ultrasound directed Oocyte Recovery
Transvaginal Ultrasound directed Oocyte Recovery
INSTRUMENTATION
TECHNIQUE
FACTORS AFFECTING OOCYTE COLLECTION
PRECAUTIONS
ADVANTAGES
DISADVANTAGES
Complications
Difficulty
CONCLUSION
CHAPTER 19:
Oocytes: Handling, Grading and Maturity Evaluation
OBTAINING THE OOCYTES
HANDLING THE OOCYTES
OOCYTE GRADING AND MATURITY EVALUATION
CELLULAR ASSESSMENT
CHAPTER 20:
In Vitro Maturation of Oocytes: Indian Perspective
INTRODUCTION
CLINICAL BENEFITS LIKELY TO BE DERIVED FROM IVM
POSSIBLE ROLE OF IVM TECHNOLOGY IN CURRENTLY PRACTICED IVF/ICSI PROTOCOL
CLINICAL CRITERIA FOR RETRIEVAL OF IMMATURE OOCYTES
Current Status of Immature Oocytes Retrieval
Technical Aspects of Immature Oocyte Collection
‘Trouble-shooting’ Problems during and after IOC
Optimizing Culture Media
Molecular Basis of Oocyte Maturation
Nuclear Maturation
Cytoplasmic Maturation
Meiotic Competence
Regulators of Meiotic Competence
Grading and Culture of Immature Cumulus-oocyte-corona (COC) Complex
FERTILIZATION
Implantation and Pregnancy Rate of IVM Followed by ICSI
IVM IN INDIAN PERSPECTIVE
CHAPTER 21:
In Vitro Maturation of Oocytes
INTRODUCTION
HISTORY
PHYSIOLOGY
STRATEGIES TO MATURE OOCYTES IN VITRO
In Vitro Maturation from Antral Stage Follicles
IVM from Preantral Follicles
APPLICATIONS OF IVM
Oocyte Maturation after Controlled Ovarian Hyperstimulation (COH)
IVM in Patients with PCOS and in Patients at Risk of OHSS
Role of IVM of Oocyte in Cryopreservation of Ovarian Tissue
Role of IVM for Donors
STEPS INVOLVED AND FACTORS AFFECTING IVM
ADVANTAGES OF IVM
CONCLUSION
CHAPTER 22:
Control of Follicular Recruitment
INTRODUCTION
PHYSIOLOGY OF SPONTANEOUS CYCLE
CLOMIPHENE CITRATE (CC)
Selection of Patients for Ovulation Induction with Clomiphene
Guidelines
Monitoring of Clomiphene Citrate Induced Cycles
Results
Clomiphene Citrate in IVF Cycles
Side Effects
TAMOXIFEN
LETROZOLE IN COH
GONADOTROPINS IN FOLLICULAR RECRUITMENT
Gonadotropin Preparations
Evaluation of the Patient
Monitoring of Ovulation Induction in Non-IVF Patients
Adjuvants
Gonadotropins in IVF cycles
FSH THERAPY
Recombinant FSH
Advantages of rFSH
Disadvantage
Results of Gonadotropin Therapy
Problems Associated with Gonadotropin Therapy
GnRH AGONISTS
Types of GnRH Agonists
Mechanism of Action
Routes of Administration
Down Regulation Protocol (long)
Benefits of Long Protocol
Short Comings
Flare Protocol (Short Protocol)
Benefits
Drawbacks
Ultrashort Protocol (Sequential Protocol)
Newer Protocols
Depot Injections
Complications of GnRH Agonist in Superovulation Programs
Response Pattern to GnRH Agonist
Conclusion
GnRH ANTAGONISTS IN ASSISTED REPRODUCTION
Mechanism of Action
The Lubeck Protocol (Long Protocol)
The French Protocol
Advantages of the Use of GnRH Antagonist
RECOMBINANT LH
Is Recombinant LH Necessary?
CONCLUSION
SECTION 6: THE ART LAB: INSIDE STORY
CHAPTER 23:
IVF Lab Techniques Made Practical and Easy
INTRODUCTION
SEMEN COLLECTION
ROUTINE SEMEN ANALYSIS
Sperm Concentration
Motility
Grades of Motility
Vitality
Morphology
Head
Midpiece
Tail
INTRAUTERINE INSEMINATION
Methods for Preparation of Semen
Direct Layering Technique
Swim-up Technique
Gradient Method
Insemination for IUI
IN VITRO FERTILIZATION
Preparation of Plates
Preparation of Plates for IVF
Preparation of Plates for ICSI
Preparation of Hylase Plate
IVF Procedure
Ovum Pick-up (Figures 23.9 and 23.10)
Gradation of Oocyte
Insemination
Day 1 Procedure after 18 to 20 Hours
Day 2 Procedure after 24 to 48 Hours
Day 2 Procedure after 48 to 72 Hours
Day 4 Procedure—Blastocyst Culture
MICRODROPLET TECHNIQUE
Preparation of Dishes
Microdroplet Insemination
Grading and Scoring of the Embryos
Two Pronuclei Stage Scoring 18 to 20 Hours
Embryo Grading 48 to 72 Hours
Blastocyst Culture
ICSI PROCEDURE
Preparation of Plates and Cumulus Removal
Preparation of ICSI Dish or Plate
ICSI Setting Up the Pipettes
Injection of Sperm in the Oocyte
EMBRYO TRANSFER
Patient Preparation before Embryo Transfer
Loading of the Embryos
CRYOPRESERVATION OF SEMEN
Procedure
Thawing the Cryopreserved Sample
EMBRYO FREEZING
Introduction
Pronuclear Stage, Day 1 to Day 3 Embryo Freezing
Freezing Method
Thawing of Pronuclei and Cleavage Stage Embryo—Day 1–3
Method
Bench Marker—Expected Results
BLASTOCYST FREEZING
Blastocyst Thawing
EMBRYOLOGIST RESPONSIBILITIES
CHAPTER 24:
ICSI: From Mastery to Excellence
INTRODUCTION
MICROMANIPULATION AND SPERM-OOCYTE INTERACTION
ICSI: AN OVERVIEW
Instruments
Sperm
Oocyte
ICSI: AS A TECHNIQUE
ICSI: THE FUTURE
SECTION 7: ENHANCING RESULTS
CHAPTER 25:
History and Development of ART Culture Media
INTRODUCTION
HISTORICAL DEVELOPMENT OF CULTURE MEDIA FOR PREIMPLANTATION MAMMALIAN EMBRYOS
COMPONENTS OF ART MEDIA (CONDENSED AND MODIFIED FROM QUINN, 2003)
Sequential Media (Also known as two-step media)
Energy Sources
Ionic Composition
pH
Use of Colored pH indicators: Advantages and Disadvantages (from Quinn and Pool, 2004)
Amino Acids
Vitamins
Growth Factors
Osmolality
MEDIA FOR SPERM PREPARATION
CONDITIONS OF USE AND CULTURE STRATEGIES
CHAPTER 26:
Stage Specific Culture Media and Blastocyst
INTRODUCTION
CULTURE ENVIRONMENT
Acidity
Buffer System
Osmolarity
Mineral Oil
Protein Source
Energy Source
Salts
Water
CO2 Incubator
Gases
CULTURE OF EMBRYOS
DEVELOPMENT OF CULTURE MEDIUM FOR MAMMALIAN EMBRYOS
Basic Composition
Energy Source
Amino Acids
Suboptimal Culture Conditions
Factors which may Inhibit Embryonic Development
Glucose-Phosphate Interaction
Osmotic Stress
Hypoxanthine in Culture Media
Polypeptide Growth Factors
Remarks
EMBRYO DEVELOPMENT
Cell Division
Closing Remarks
FERTILIZATION, CLEAVAGE AND BLASTOCYST CULTURE PRACTICE
Transport and Flushing Medium
Fertilization Medium
Cleavage Medium
Blastocyst Medium
Embryo Transfer Medium
Freezing and Thawing Medium
CHAPTER 27:
Assisted Hatching: Benefits and Limitations
INTRODUCTION
CAUSES FOR IMPAIRED HATCHING
PLAUSIBLE MECHANISMS
PITFALLS OF ASSISTED HATCHING
Monozygotic Twinning
SELECTION FOR EMBRYO HATCHING
Indications
Contraindications
OVERVIEW OF TECHNIQUES
Mechanical: Partial Zona Dissection (PZD) on Day 2
Chemical: Zona Drilling with Acid Tyrode's Solution (pH 2.5)
Enzymatic: Zona Softening or Complete Removal with Pronase
Laser Assisted Drilling
Types of Laser System
Piezo Assisted Drilling
MEDICAL MANAGEMENT
CONCLUSION
CHAPTER 28:
Embryo Transfer: A Gentle Skill for Final Result
INTODUCTION
NUMBER OF EMBRYOS, DAY OF TRANSFER, SEQUENTIAL TRANSFER
ROLE OF MOCK TRANSFER
Pre-ET Preparation
Medication
Types of Catheters
Loading of the Catheter, Embryo Transfer
Ultrasound in Embryo Transfer
Types of Transfer
Post-ET Procedures
Embryo Transfer at AAKAR IVF Center
CONCLUSION
SECTION 8: THE COLD ZONE IN ART
CHAPTER 29:
Concepts of Cryofreezing, Cryoprotection, Freezing of Embryos: Myths, Facts, Results, Can it be Cost Effective?
INTRODUCTION
CELL CHANGES DURING FREEZING
CRYOPROTECTIVE PROCEDURES DURING EMBRYO FREEZING
EQUIPMENTS
FREEZING AND THAWING PROTOCOLS
For Cleavage Stage Embryos (Embryo Cryopreservation Vitrolife GISeries™ Manual 2.0.7, 2003)18
Example of Freezing Program for Cleavage Stage Embryos
Steps Involved
Thawing Program for Cleavage Stage Embryos (Embryo Cryopreservation Vitrolife GISeries™ Manual 2.0.7)
Steps Involved
Freezing Program for Blastocyst Stage Embryos (Embryo Cryopreservation Vitrolife GISeries™ Manual 2.0.7)
G-FreezeKit Blast™
Example of Freezing Program for Blastocyst Stage Embryos
Steps Involved
Thawing Program for Blastocyst Stage Embryos (Embryo Cryopreservation Vitrolife GISeries™ Manual 2.0.7)
G-ThawKit Blast™
Steps Involved
DISCUSSION
SECTION 9: FUTURE AND ADVANCES IN LAB
CHAPTER 30:
Preimplantation Genetic Diagnosis: Principles and Techniques
INTRODUCTION
TECHNICAL HISTORY OF PGD
WHY USE PGD?
SELECTION OF PGD TECHNIQUES
Blastomere Biopsy
Materials
Procedure
Polar Body Biopsy
Materials
Procedures
ANALYSIS OF BIOPSIES: FLUORESCENCE IN SITU HYBRIDIZATION
Use of FISH in PGD
Aneuploidy Analysis
Analysis of Structural Chromosome Abnormalities
Fish Protocols
Materials
Methods
ANALYSIS OF BIOPSIES: POLYMERASE CHAIN REACTION
Genetic Disease
PCR Technology
Apparatus Required
Materials Required
Procedures
CHAPTER 31:
Basics and Future of Stem Cell Research
INTRODUCTION
WHAT ARE STEM CELLS?
Embryonic Stem Cells
Fetal Stem Cells
Adult Stem Cells
PRINCIPLES OF STEM CELL-BASED TRANSPLANTATION THERAPY
SCOPE OF STEM CELL TRANSPLANTATION
SAFETY OF STEM CELL TRANSPLANT
OBSTACLES AND RISKS ASSOCIATED WITH THE USE OF STEM CELLS
STEM CELLS AND PHARMACOGENOMICS
STEM CELLS AND HUMAN DEVELOPMENT STUDIES
ETHICAL CONTROVERSY
CONCLUSION
SECTION 10: TROUBLESHOOTING AREAS: SOLUTIONS
CHAPTER 32:
Troubleshooting Areas in Lab: How to Overcome
INTRODUCTION
INFECTION CONTROL
SUPPLIERS AND SOURCES
RECORDS AND PROTOCOLS
MAINTENANCE CALIBRATION AND EQUIPMENT MONITORING
STAFFING REQUIREMENTS
AIR CONDITION SYSTEM
CRYOPRESERVATION
Storage Containers
Cryoprotectants
Freezing Machine
Cooling Program
Warming Rate
Cryoprotectant Removal
Final Caution
CONCLUSION
SECTION 11: ART: GUIDELINES FOR SAFE PRACTICE
CHAPTER 33:
ART and IUI Guidelines for Indian Scenario
INTRODUCTION
IMPORTANCE AND SIGNIFICANCE OF GUIDELINES
CATEGORIZATION OF INFERTILE COUPLES
GUIDELINES RELATING TO IUI
Sperm Donor
Requirement of Semen Bank
GUIDELINES FOR ART
Controversy on Minimum Requirements of ART Clinic Personnels
Clinical Gynecologist
Embryologists
Andrologists
Counselor
Other Controversial Issues
Gamete Donation
Oocyte Sharing
Surrogacy: General Consideration
Sourcing of Donor Oocytes and Surrogate Mothers
Right of a Child Born Through Various ART Technologies
ART in Unmarried Women
AIH or ART in an HIV Positive Woman
Ethical and Moral Responsibilities of ART Clinics
Institutional Ethics Committees
Possible Misuse of ART: Sale of Embryos and Stem Cell
Providing ART Services to the Economically Weaker Section of the Society
Offering Services at Cost Price or Exploring the Scope of Low-cost of IVF
Reduction of Drug Cost
Establishing a National Database for Human Infertility
CHAPTER 34:
Understanding Our Status as ART Specialists: A Legal Standpoint
CHALLENGES FACED BY ART SPECIALISTS
Human Fertilization and Embryology Authority
HFEA's Principal Tasks
HFEA's Other Statutory Functions
PRACTICE OF ART IN INDIA
CONCLUSION
SECTION 12: SPERMS TO STORE AND RECORD KEEPING
CHAPTER 35:
Making a Semen Bank of International Standards
INTRODUCTION
CRYOPRESERVATION
Process of Cryopreservation
VARIABLES OF SPERM CRYOPRESERVATION
Predicting Freezing Sensitivity
Other Factors Influencing Sperm Banking
Minimum Numbers of Motile Cells, Site of Insemination, Removal of Seminal Plasma Before Freezing
Assessment of Post-Thaw Fertility
Packaging, Leakage, Cross-contamination and Liquid Nitrogen Contamination
Long-term Stability of Frozen Specimens
Genetic Consequences
Consanguinity and Number of Pregnancies from a Donor
Cryoprotectant and Extenders
Freezing
Thawing
CLINICAL USE OF CRYOPRESERVED SPERMATOZOA
Artificial Insemination
Indication of AID
Evaluation of Couple
Factors Influencing AID Outcome
Sperm Cryopreservation as an Adjunct to Assisted Reproduction
Autoconservation
SEMEN BANK SET-UP (Figures 35.1 to 35.3)
Instruments, Plasticware, Packaging Material, Media and Cryoprotectant
Dilution of Semen with Cryoprotectant Media and Packaging
Neat Semen Freezing
Processed Semen Freezing
Record should be Maintained
CONCLUSION
SECTION 13: UNDERSTANDING HUMAN RESONURCES AND THE SILENT MALE/FEMALE ZONE
CHAPTER 36:
Human Resource Utilization in Infertility Practice
INTRODUCTION
CHAPTER 37:
Male Factor Infertility for the Gynecologist: Established Concepts and Myths
INTRODUCTION
HISTORY TAKING
PHYSICAL EXAMINATION
ENDOCRINE EVALUATION
GENETIC TESTING
ROUTINE SEMEN ANALYSIS
Interpretation of Semen Analysis
Antisperm Antibodies (ASA) in Semen
Abnormalities of Sperm Function
Reactive Oxygen Species
Computer Assisted Semen Analysis
Hypo-osmotic Swelling Test (HOS)
Postcoital Test
COMMON DISORDERS OF THE REPRODUCTIVE TRACT
Varicocele
Pathophysiology
Diagnosis
Treatment
Prognostic Factors Determining Success of Treatment
Y Chromosome and Infertility
Genetic Disorders Associated with Male Infertility
Pretesticular Effects
Testicular Effects
Current Practice
Genetic Conditions with Post-testicular Effects
Ejaculatory Dysfunction
Evaluation and Diagnosis
Treatment
Infections
Chlamydia Infection
Ureaplasma urealyticum (Mycoplasma hominis)
CURRENT TECHNOLOGY FOR GENETIC SCREENING
Karyotyping
Polymerase Chain Reaction
Fluorescence in Situ Hybridization Technique (FISH)
Comparative Genomic Hybridization
DNA Sequencing
CONCLUSION
CHAPTER 38:
Cervical Factor Infertility
INTRODUCTION
ANATOMY OF CERVIX
Types of Endocervical Cells
Nonciliated Secretory Cells
Ciliated Cells
Types of Cervical Mucus
Estrogenic Mucus
Gestagen Mucus
Cervical Mucus Proteins
PHYSIOLOGY OF CERVIX
Sperm Migration
Sperm Nutrition
Sperm Storage
Sperm Selection and Sperm Filtration
Fertility Related Function of Cervix and its Mucus
CLINICAL APPLICATION
In Vivo
Postcoital Test (PCT)
In Vitro Test
Sperm Penetration Test
Sperm Cervical Mucus Contact Test
INTERPRETATION
TREATMENT OF CERVICAL FACTOR INFERTILITY
Glossary
INDEX
TOC
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