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IVF Techniques for the Beginners
Pankaj Talwar, Kuldeep Jain
SECTION 1: IVF SIMPLIFIED
1:
Ovum Pick-up
EGG RETRIEVAL PROCESS
Stimulating Follicular Development
Monitoring Follicular Development
Size of Follicles for Egg Retrieval
Egg Collection Procedure (Fig. 1.2)
After Egg Retrieval—Egg Retrieval Recovery
WHAT HAPPENS IN THE LAB AFTER THE EGGS ARE ASPIRATED?
Oocyte Scoring at OPU Time
Sperm Collection
Egg Insemination
Fertilization Check
Embryo Culture
Embryo Transfer
2:
Embryo Transfer
SUMMARY
INTRODUCTION
CLINICAL DISCUSSION
Factors Affecting the ET Outcome
Ultrasound Guidance Versus Clinical Touch
Depth of Embryo Transfer
Routine Uterine Cavity Evaluation
Hysteroscopic Revision of the Cervical Canal
Uterine Position
Catheter Type
Use of a Stylet
Difficulty of Transfer
Experience of the Provider
Trial Transfer
Fluid Volume
Embryo After Loading
Injection Speed
Removal of Cervical Mucus Prior to ET
Catheter Removal
Blood on the Catheter
Retained Embryos
Bacterial Contamination
Intrauterine Injection of Human Chorionic Gonadotropin (hCG)
Air in the Transfer Catheter
Air Bubble Position
Antibiotics Prior to ET
Embryo Quality
RECENT ADVANCES
CONCLUSION
3:
Media and Quality Control in IVF Laboratory
INTRODUCTION
QUALITY CONTROL
QUALITY ASSURANCE
QUALITY IMPROVEMENT (QUALITY AUDIT)
INTERNAL QUALITY CONTROL
QUALITY MANAGEMENT SYSTEM
ART LABORATORY
Equipment
How Important is Air Quality within the ART Laboratory?
Can we Protect in Vitro Culture Cells from VOCs?
Incubators
How Critical is Culture Temperature?
What should be the Optimal Incubator Temperature Setting?
Measurement of Temperature
Relative Humidity Inside the Incubator
Measurement of CO2 Concentration in the Incubator Chamber
Why is Measurement of CO2 Critical?
Importance of Measuring O2
How to Prevent VOCs Inside the Incubator?
Exposure to Light
Media
Use of Mineral Oil
CONCLUSION
4:
Sperm Preparation for In Vitro Fertilization and Intracytoplasmic Sperm Injection
METHOD
Pelleting and Swim-up Method (Fig. 4.1)
Disadvantage
Discontinuous Buoyant Density Gradient Centrifugation
Two-step Gradient (80/40) (Fig. 4.2)
Clinical Tip
Special Situations
Centrifugation and Washing
Swim-out Under Oil
Obstructive Azoospermia: Epididymal and Testicular Sperm5
Retrograde Ejaculation
Learning Points
5:
Sperm Retrieval Techniques (PESA, TESA, MESA, Micro-TESE, TESE)
INTRODUCTION
ETIOPATHOGENESIS OF AZOOSPERMIA
DIAGNOSIS
MANAGEMENT
Percutaneous Sperm Retrieval Methods
Percutaneous Epididymal Sperm Aspiration
Testicular Sperm Aspiration
Microsurgical Sperm Retrieval techniques
Microsurgical Epididymal Sperm Aspiration
Microsurgical Testicular Sperm Extraction (micro-TESE)
Conventional Testicular Sperm Extraction
CONCLUSION
6:
Surgically Retrieved Sperm Samples for ICSI
BACKGROUND
RETRIEVAL PROCEDURES
PATIENTS INDICATED FOR MESA/TESE/PESA/TESA
Choice of Method
Fresh or Frozen
SURGICAL RETRIEVAL OF SPERM
CRYOPRESERVATION OF SURGICALLY RETRIEVED SPERM
Cryoprotectant
Preparation of Sperm Samples for Cryopreservation
PESA/MESA Samples
TESE/TESA Samples
Adding Cryoprotectant
Freezing
Maintenance of Sperm Bank
Post-thaw Testing
SPERM PREPARATION ON THE DAY OF ICSI
SAFETY OF SURGICALLY RETRIVED SPERM
7:
Intracytoplasmic Morphologically Selected Sperm Injection
INTRODUCTION
SELECTION OF SPERMS—NATURAL CONCEPTION AND ART
SPERM MORPHOLOGY AND PREGNANCY OUTCOME
VACUOLES—DNA FRAGMENTATION AND ABNORMAL CHROMATIN PACKAGING
ABNORMAL DNA PACKAGING AND ITS IMPACT ON FERTILIZATION, EMBryONIC DEVELOPMENT AND PREGNANCY
SELECTION OF SPERMS FOR ICSI
Increasing Magnification of Sperm Without Compromising Resolution
STEPS FOR IMSI
PREPARATION OF IMSI DISH
Assessment and Scoring of Spermatozoa
Impact of IVF-IMSI on the Outcome of the ART Procedure
CONCLUSION
8:
Vitrification
INTRODUCTION
PRINCIPLE OF VITRIFICATION
VARIOUS FACTORS DETERMINE ULTIMATE SURVIVAL
Cooling and Warming Rates
CRYOPROTECTANTS
VITRIFICATION CARRIER DEVICES
VITRIFICATION—CLOSED SYSTEMS
ELEMENTS OF A TYPICAL VITRIFICATION PROCEDURE
CONCLUSION
9:
Ovarian Cortex Freezing
INTRODUCTION
Vulnerability of the Reproductive System to Cancer Treatment
Mode of Action
Radiotherapy-induced Gonadal Damage
Chemotherapy-induced Gonadal Damage
INDICATIONS FOR OVARIAN CRYOPRESERVATION
Patients Receiving Chemo- and/or Radiotherapy for Cancer
Breast Cancer
Cervical Cancer
Childhood and Juvenile Cancers
Patients Undergoing Bone Marrow Transplantation
Endometriosis
Autoimmune Diseases
Benign Ovarian Tumors
ASSESSMENT OF OVARIAN RESERVE
CURRENT TECHNIQUES OF FERTILITY PRESERVATION
OVARIAN CORTEX CRYOPRESERVATION
Ovarian Tissue Cryopreservation in Humans—Background
LIMITATIONS FOR OVARIAN TISSUE CRYOPRESERVATION
COLLECTION OF OVARIAN TISSUE
CRYOPROTECTANT PREPARATION
TISSUE PREPARATION (FIGS 9.5 TO 9.9)
HISTOLOGICAL ANALYSIS
STEPS OF OVARIAN CORTEX FREEZING (FIGS 9.11 TO 9.20) (TABLES 9.6 AND 9.7)
PECULIARITIES OF OVARIAN TISSUE FREEZING
METHODS OF THAWING (Table 9.8), (FIGS 9.21 TO 9.23)
TRANSPLANT (Table 9.9)
ETHICAL CONCERNS IN OVARIAN TISSUE CRYOPRESERVATION
10:
Preimplantation Genetic Diagnosis: An Emergent Technology
INTRODUCTION
INDICATIONS FOR PREIMPLANTATION GENETIC DIAGNOSIS
PREIMPLANTATION GENETIC DIAGNOSIS— HISTORICAL PERSPECTIVE
STEPS INVOLVED IN PREIMPLANTATION GENETIC DIAGNOSIS
Getting Embryos: In Vitro Fertilization/Intracytoplasmic Sperm Injection
Embryo Biopsy
Polar Body Biopsy
Cleavage-Stage Biopsy (Blastomere Biopsy)
Blastocyst Biopsy
Genetic Analysis
Fluorescence In Situ Hybridization
Polymerase Chain Reaction (PCR)
EVOLUTION OF PGD FOR ANEUPLOIDIES, STRUCTURAL CHROMOSOMAL REARRANGEMENTS AND PREIMPLANTATION GENETIC SCREENING
EVOLUTION OF PREIMPLANTATION GENETIC DIAGNOSIS FOR MONOGENIC DISORDERS
PREIMPLANTATION GENETIC DIAGNOSIS FOR HUMAN LEUKOCYTE ANTIGEN MATCHING: SAviOUR SIBLING
PREIMPLANTATION GENETIC DIAGNOSIS FOR CANCER PREDISPOSITION
PREIMPLANTATION GENETIC DIAGNOSIS— CURRENT GLOBAL STATUS
PREIMPLANTATION GENETIC DIAGNOSIS— AN INDIAN PERSPECTIVE
CHALLENGES FACED WITH PREIMPLANTATION GENETIC DIAGNOSIS
ETHICAL LEGAL AND SOCIAL ISSUES
CONCLUSION
11:
Assisted Hatching
BACKGROUND
GENERAL PRINCIPLES FOR AH
Indications for AH
Advanced Maternal Age/Elevated D3 FSH/Thick Zonae
Previous IVF Implantation Failures
EXCESSIVE FRAGMENTATION
POOR EMBRYO GROWTH RATE/MORPHOLOGY
Post-thaw Embryos
Fresh and Thawed Blastocysts
ASSISTED HATCHING USING ACIDIC TYRODE'S SOLUTION
Embryo Assessment
Dish Preparation
Micromanipulator Set-up
Procedure
Assisted Hatching Using the Laser
DISCUSSION
12:
Overview of Cryobiology in Assisted Reproductive Technology
INTRODUCTION
BASIC DEFINITIONS
THERMODYNAMICS
PHYSICS
CONCEPT OF LATENT HEAT
Latent (Hidden)
BIOLOGY OF CRYOFREEZING
CRYOPROTECTANTS
Permeating Agents
Mechanism of Action
Nonpermeating Agents
Mechanism of Action
PRINCIPLES OF CRYOBIOLOGY
STEPS OF CRYOPRESERVATION
Step 1
Step 2
Step 3
Step 4
Storage of the Specimen at −196°C
Step 5
Step 6
EVENTS DURING FREEZING
FREEZE INJURIES
EVENTS DURING THAWING
THAW INJURIES
Intracellular Ice Crystals
Osmotic Stress
Gas Expansion
Risk of Storing Biological Materials at Low Temperatures
CONCLUSION
13:
Simplifying IVF Program: Satellite Module
14:
Fertility Preservation in Female Cancer Patients
INTRODUCTION
INDICATIONS OF OFFERING FERTILITY PRESERVATION
VULNERABILITY OF THE REPRODUCTIVE SYSTEM TO CANCER TREATMENT
MODE OF ACTION OF GONADOTOXIC AGENTS
Radiotherapy Induced Gonadal Damage
Chemotherapy Induced Gonadal Damage
ASSESSMENT OF OVARIAN RESERVE
FERTILITY PRESERVATION OPTIONS
Embryo Preservation
Oocyte Cryopreservation
Mature Oocyte Cryopreservation
Immature Oocyte Cryopreservation
Cryopreservation of Ovarian Tissue
Ovarian Tissue Cryopreservation in Humans Background
Limitations for Ovarian Tissue Cryopreservation
Methodology of Ovarian Tissue Collection and Processing of the Tissue
Cortical Tissue Permeation with Cryoprotectants
Freezing and Thawing Protocols
Cryopreservation of the Whole Ovary
Role of Vitrification in Human Ovarian Tissue Cryopreservation
ETHICAL CONCERNS IN OVARIAN TISSUE CRYOPRESERVATION
NOVEL OPTIONS OF FERTILITY PRESERVATION
Ovarian Transposition
Medical Options
CONCLUSION
15:
ICSI—An Introduction
INSTRUMENTATION FOR ICSI
Micromanipulator Set-up
Inverted Microscope
Description of Micromanipulator
Manufacturers
Narishige
Eppendorf
Research Instruments
Air or Oil in Microsyringes
MICROTOOLS
PREPARATION OF CULTURE DISHES
Reminder
SET-UP OF DISHES
SPERM PREPARATION
Procedure
Swim-up Method
Washing with HEPES Buffered HTF
Tips
DENUDING OF OOCYTES FOR ICSI
Recombinant Hyaluronidase
Denuding
SETTING-UP MICROSCOPE
NARISHIGE
RESEARCH INSTRUMENTS
EPPENDORF
SPERM INJECTION
Tips
SPECIAL CIRCUMSTANCES OF APPLYING ICSI
Patients in First Attempt
Patients after Failed IUI
Surgically Retrieved Sperm Samples
Frozen Donor/Husband Semen Samples
RESCUE ICSI
In Vitro Maturation (IVM)
FERTILIZATION IN ICSI
PERSONAL CARE OF ICSI PRACTITIONER
16:
Guidelines and Legislation on Assisted Reproduction Technology in India
SECTION 2: ESSENTIAL PROTOCOLS IN ART
17:
SPERM FREEZING
SEMEN FREEZING
Sample Collection
Liquid Nitrogen Vapor Cooling
Cryovials
Cryostraws
Thawing Procedure
Technique Using Controlled Rate-freezing Method
Straws
Cryovials
Thawing
18:
EMBRYO TRANSFER
DISPOSABLES AND EQUIPMENT REQUIRED
REAGENTS
EMBRYO TRANSFER— PROCEDURE
19:
INTRACYTOPLASMIC SPERM INJECTION PROTOCOLS
INTRACYTOPLASMIC SPERM INJECTION MEDIA AND PIPETTES
PREPARATION OF INTRAOYTOPLASMIC SPERM INJECTION DISHES
DENUDATION
Method of Denudation during Intracytoplasmic Sperm Injection
MANIPULATION OF SPERMATOZOA
MANIPULATION OF OOCYTES
MICROINJECTION OF OOCYTES WITH MATURE SPERMATOZOA
20:
OOCYTE CRYOPRESERVATION
OOCYTE CRYOPRESERVATION— S LOW COOLING
Equipment
Reagents
Freezing
Thawing
Specimen
Procedure
Oocyte Freezing Protocol
Oocyte Thawing Protocol
OOCYTE CRYOPRESERVATION— VITRIFICATION
Equipment
Reagents
Vitrification Media
Thawing
Specimen
Procedure—Vitrification
OOCYTE WARMING PROTOCOL
Making the Warming Dish
Identification of the Straws
21:
TESTICULAR BIOPSY BANKING
TECHNIQUE FOR CRYOPRESERVATION OF TESTICULAR SPERM
Disposables and Equipment Required
Reagents
Preparation of Erythrocyte (RBC) Lysis Buffer
Specimen Collection
Procedure
Discard the Supernatant
Thawing of the Specimen
22:
OVUM PICK-UP
EQUIPMENT REQUIRED
REAGENTS REQUIRED
Culture Media
PROCEDURE: OPU STEP-BY-STEP
23:
FOLLICULAR TREKKING AND USE OF COLOR DOPPLER IN ART CYCLES
EQUIPMENT REQUIRED
Baseline USG
What to Look for at the Basic Scan?
Treatment cycle
INDEX
TOC
Index
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