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Protocols in Clinical Embryology and ART
Pankaj Talwar, Surveen Ghumman Sindhu
CHAPTER 1:
Setting up of an ART Center
LOCATION OF THE LABORATORY
BASIC INFRASTRUCTURE
Electricity
Air Conditioning
Water Supply
Air Quality
Temperature
Light
Floors, Walls and Ceiling
Doors
Furniture
FLOOR PLAN
Nonsterile Area
Sterile Area
LABORATORY PERSONNEL
EQUIPMENT
Incubators
Laminar Flow Hoods (Clean Air Work Station)
Microscopes
Micromanipulator
Ovum Aspiration Pump
Laboratory Purification System
Heating Appliances
Programmable Biological Freezer with Cryocans
Laser System
Spindle Imaging System
Sperm Counting Chamber
Computer-assisted Semen Analyzer (CASA)
Centrifuge
Ultrasound Machine
Miscellaneous
CONSUMABLES
CULTURE MEDIA
PROTOCOLS IN THE LABORATORY—DO's AND DONT's
QUALITY CONTROL
RISK MANAGEMENT
CHAPTER 2:
Selection and Preparation of an IVF Patient
INFECTION SCREEN
IMMUNIZATION STATUS
MEDICAL AND SURGICAL DISEASE
ASSESSMENT OF BMI AND WEIGHT AND LIFESTYLE CHANGES
Assessment of Nutritional Status and Role of Supplements
ROLE OF PELVIC ULTRASONOGRAPHY IN THE SELECTION AND PREPARATION OF PATIENTS FOR IVF
PRETREATMENT HORMONE ASSESSMENT TO OPTIMIZE IVF OUTCOMES
EVALUATION OF THE OVARIAN RESERVE
PRE-IVF LAPAROSCOPY/HYSTEROSCOPY ASSESSMENT
ASSESSMENT AND TREATMENT OF SPECIFIC PATHOLOGY BEFORE AN IVF CYCLE
Uterine Abnormality: Should it be Treated Before IVF?
Hydrosalpinx
Endometriosis
Ovarian Cyst
Polycystic Ovary Patients—Is there a Role for Metformin in Women Undergoing IVF?
Goals of Treatment
Problems of Controlled Ovarian Stimulation in PCOS
Several Modes of Inducing Ovulation are Used (Flow chart 2.1)
ROLE OF ORAL CONTRACEPTIVE PILLS BEFORE ART CYCLE
ASSESSMENT OF THE MALE
PATIENT COUNSELING AND EDUCATION
CHAPTER 3:
Semen Analysis and Assessment of the Male Partner
SAMPLE COLLECTION AND DELIVERY
PHYSICAL PARAMETERS
Coagulation
Liquefaction
Appearance
Odor
Volume
Cause of Low Volume
Cause of High Volume
Viscosity
Causes of Increased Viscosity
pH
INITIAL MICROSCOPIC EXAMINATION
Agglutination and Aggregation
SPERM CONCENTRATION
Method of Assessment of Sperm Concentration
SPERM MOTILITY
SPERM MORPHOLOGY
Method of Preparation
Staining
Examining the Slide
WHO Criterion for Sperm Morphology
Head
Midpiece
Tail
Abnormal Spermatozoa
CELLULAR ELEMENTS OTHER THAN SPERMATOZOA
Leukocytes
Immature Germ Cells
TESTS FOR SPERM MEMBRANE INTEGRITY
Sperm Viability by Dye Exclusion
Hypo-osmotic Swelling Test
Sperm Longevity Test
SPERM MEMBRANE BINDING, CAPACITATION AND PENETRATION
Hemizona Assay
Hamster Egg Human Sperm Penetration Assay (SPA)
ACROSOME DETECTION
BIOCHEMICAL MEASUREMENT OF SPERM FUNCTION
Reactive Oxygen Species
ANTISPERM ANTIBODIES (ASA)
Mixed Antiglobulin Reaction Test
Immunobead Test (IBT)
Clinical Significance of Positive Test (Flow chart 3.3)
ASSESSMENT OF SPERM DNA DAMAGE
CHAPTER 4:
Gonadotropins
GONADOTROPIN PREPARATIONS
CHOICE OF DOSAGE
PROTOCOLS
Step up High Dose Protocol
Step up Low Dose Protocol
Step Down Protocol
Sequential Step up, Step Down Regimen
Mild Stimulation Protocol
Role of GnRH Antagonist in Mild Stimulation
Clomiphene in Mild Stimulation Protocol
MONITORING
Serum Estradiol Levels
Ultrasonography
SIDE EFFECTS AND RISKS
RECOMBINANT FSH
Advantages of Recombinant FSH
Disadvantages
RECOMBINANT FSH-CTP IN IVF
RECOMBINANT LH
HUMAN CHORIONIC GONADOTROPIN
CHAPTER 5:
Role of GnRH Agonists and Antagonists in ART
GnRH AGONIST (GnRH-A)
Mechanism of Action
Route of Administration
Use of GnRH Agonist in COH Protocols
Protocols for Administration
Long Protocol
Short Protocol
Ultrashort Protocol
Disadvantages of GnRH Agonists
Advantages of GnRH Agonists
Side Effects and Risks of GnRH Agonists
GnRH Agonist for Ovulation Trigger
Advantages
Disadvantages
GnRH ANTAGONISTS
Contraindications
Protocols
Advantages of GnRH Antagonists over GnRH Agonist in ART
Disadvantages of Antagonists
GONADOTROPIN RELEASING HORMONE
CHAPTER 6:
Embryo Transfer and Troubleshooting
INTRODUCTION
FACTORS AFFECTING EMBRYO TRANSFER
RELEVANT ISSUES TO BE ADDRESSED BEFORE EMBRYO TRANSFER
RELEVANT ISSUES TO BE ADDRESSED DURING EMBRYO TRANSFER
RELEVANT ISSUES TO BE ADDRESSED POST EMBRYO TRANSFER
EMBRYO TRANSFER STEP BY STEP (FIGS 6.1 TO 6.10)
CHAPTER 7:
Ovum Pickup and Troubleshooting
INTRODUCTION
RELEVANT ISSUES
Timing
Preparation of Vagina
Anesthesia/Analgesia
Equipment
Ultrasound and Probe
Suction Apparatus
Ovum Pickup Needle (Figs 7.4 to 7.7)
Temperature Control
Follicle Flushing
OPU STEP BY STEP
COMMON PROBLEMS ENCOUNTERED DURING OVUM PICKUP
Failure to Aspirate the Follicular Fluid
No Oocytes Retrieved
COMPLICATIONS
LEARNING CURVE
CHAPTER 8:
Embryo Selection
INTRODUCTION
EMBRYO ASSESSMENT
FERTILIZATION ASSESSMENT
PRONUCLEI SCORING
EVALUATION OF EMBRYO QUALITY
BLASTOCYST SCORING
Blastocyst20
CHAPTER 9:
Culture Media in IVF and Embryo Culture
INTRODUCTION
ESSENTIAL CONCEPTS
pHo and pHi
Buffer System
Sodium Bicarbonate
HEPES [4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid]
Phosphate Buffer
MOPS [3-(N-morpholino)propanesulfonic Acid]
Quality of Water
THE PHYSIOLOGY OF SEQUENTIAL CULTURE MEDIA
Physiology of IVF Fertilization Medium
Physiology of Cleavage Media
Physiology of the Blastocyst Media
USE OF OIL OVERLAY IN ART
QUALITY CONTROL TESTING FOR THE CULTURE MEDIA
pH, Osmolality, Viscosity Measurement
Mouse Embryo Assay (MEA)
Endotoxin Level
Sterility Test
Hybritest™
Sperm Survival Test
Sperm Immobilization Test
Functionality Test
ESSENTIALS OF HANDLING MEDIA AND LAB EQUIPMENT
MEDIA PREPARATION AND PREEQUILIBRATION
STORAGE OF THE MEDIA
SHELF LIFE AND PACKAGING
CONCLUSION
DISPOSABLES IN ART
QUALITY CONTROL TESTS
TYPES OF DISPOSABLES
CHAPTER 10:
Andrology and Beyond
SEMEN PREPARATION FOR IVF
Introduction
Essentials of Sperm Preparation2,3
Nomenclature—Semen Variables (WHO)
Techniques of Semen Preparation
Swim-up and Wash
Density Gradient Centrifugation Method
Double Wash and Swim-Up
Peculiar Conditions
Obstructive Azoospermia: Epididymal and Testicular Sperm
Retrograde Ejaculation
Basis of Centrifugation
Improvement of Motility and Sperm Function
Pentoxifylline
Platelet-Activating Factor
Hypo-osmotic Swelling Test (HOS)
The Ideal Sperm Separation Technique Should
OOCYTE ASSESSMENT
INSEMINATION PROTOCOLS IN ART
Introduction
Time of Insemination
Sperm Concentration
Media for Sperm Preparation and Insemination
Protocols for Insemination and Embryo Culture (Figs 10.3A and B)
Methods of Insemination
Role of Oil Overlay
DENUDATION
Short Denudation Method
ASSESSMENT OF FERTILIZATION (FIGS 10.4 to 10.18)
Washing Denuded Oocytes
Transfer to Culture Dish
Embryo Assessment on Day 2 (Table 10.5)
Embryo Assessment on Day 3
Embryo Assessment on Day 5 (Blastocyst Culture) (Tables 10.6 to 10.8)
CONCLUSION
CHAPTER 11:
Intracytoplasm Sperm Injection and Troubleshooting
INTRODUCTION
INDICATIONS FOR ICSI
ICSI MEDIA
Types of Media Needed for ICSI
Handling
PIPETTES (FIGS 11.1 TO 11.3)
PREPARATION OF ICSI DISHES (FIGS 11.4 TO 11.9)
Precautions
DENUDATION
Steps of Denudation During ICSI (Figs 11.10 to 11.12)
MANIPULATION OF SPERMATOZOA
MANIPULATION OF OOCYTES
MICROINJECTION OF OOCYTES WITH MATURE SPERMATOZOA (FIGS 11.13 TO 11.20)
DIFFICULT ICSI CASES
CONCLUSION
CHAPTER 12:
Gamete Cryopreservation
OOCYTE CRYOPRESERVATION
Introduction
Indications
Essential Principles of Controlled Rate Cooling
Essential Principles of Thawing
Evaluation of Oocyte Quality
Oocyte-denudation—To do or not?
Factors Affecting the Clinical Efficiency of Oocyte Cryopreservation
Cryopreservation of Immature Oocytes at Prophase I (GV Stage)
Recent Advances in Cryopreservation Media
Procedure of Oocyte Slow Freezing (Table 12.1)
Procedure of Oocyte thawing (Table 12.2)
OOCYTE VITRIFICATION AND THAWING
Vitrification Simplified (Figs 12.1 to 12.7)
Steps of Oocyte Warming (Figs 12.17 to 12.27)
SEMEN CRYOPRESERVATION
Introduction
Indications of Semen Cryopreservation
Common Indications for Autologous Semen Banking
Indications for Donor Semen Banking
Should Semen Sample be Prepared Before Freezing?
Outline of Cryoprotectants in Semen Cryopreservation
Packaging of Semen After Addition of Cryopreservative
Cooling and Warming Rates
Step by Step Methodology in Semen Freezing
Freezing
Storage
How Long can Sperms be Stored
Thawing
Assessment of Post-thaw Fertility
Cross-infection in the Semen Banks
Donor Screening Prior to Semen Banking
Security and Maintenance
Cryopreservation of Epididymal Sperms
Cryopreservation of Testicular Sperms
Cryopreservation of Sperm in Empty Human Zona Shell
Vitrification Technique for Ultrarapid Freezing
CONCLUSION
CHAPTER 13:
Embryo Vitrification
INTRODUCTION
PRINCIPLES OF VITRIFICATION
PRINCIPLES OF WARMING
UNIQUE VITRIFICATION DEVICES
SUPERIORITY OF VARIOUS VITRIFICATION METHODS
VITRIFICATION STEP BY STEP
Protocol for Embryo Vitrification using Cryoloop and Vitrolife Vitrification Media (Figs 13.1 to 13.21)
Warming Steps (Figs 13.22 to 13.39)
PREVENTING POTENTIAL CONTAMINATION FROM LIQUID NITROGEN
CONCLUSION
CHAPTER 14:
Endometrial Receptivity and Luteal Support
HISTOLOGICAL CHANGES
BIOCHEMICAL AND MOLECULAR CHANGES
IMMUNOLOGICAL ASPECTS OF ENDOMETRIAL IMPLANTATION
ENDOMETRIAL VASCULAR CHANGES
CURRENT STRATEGIES TO ASSESS ENDOMETRIAL RECEPTIVITY
Endometrial Histology
Endometrial Culture and Histopathology for Infections
Ultrasound and Color Doppler
Hormonal Levels
Hysteroscopy
Markers of the Embryo-endometrial Dialogue
ENDOMETRIAL PREPARATION FOR FROZEN EMBRYO TRANSFER
TREATMENT OF POOR UTERINE RECEPTIVITY
Estrogens
Drugs to Improve the Endometrial Blood Flow
Immunosuppression
Reducing Uterine Contractility
Surgical Treatment
Medical Treatment of Endometritis
Luteal Phase Support
Why Luteal Support is Needed in ART Cycles?
Treatment of Luteal Phase Defect
Progesterone Supplementation
Human Chorionic Gonadotropin (hCG)
Estradiol Supplementation in Luteal Phase
Single-dose GnRH Agonist in the Luteal Phase
CHAPTER 15:
Ovarian Hyperstimulation Syndrome
INCIDENCE
CLASSIFICATION
PATHOPHYSIOLOGY OF OHSS
CLINICAL FEATURES
FATAL COMPLICATIONS
MANAGEMENT OF OHSS
Prevention
Investigation and Monitoring of an OHSS Patient
Treatment
Mild OHSS
Severe OHSS
Critical OHSS
CHAPTER 16:
Multifetal Pregnancy Reduction
METHODS
Transcervical Aspiration
Transabdominal Reduction
Transvaginal Fetal Reduction
COMPLICATIONS OF PREGNANCY REDUCTION
ETHICAL ISSUES
CHAPTER 17:
Ultrasound Assessment of Endometrial Receptivity and Oocyte and Embryo Quality
PHYSIOLOGICAL AND BIOCHEMICAL BASIS OF IMPLANTATION
LESSONS FROM IVF AND OVUM DONATION CYCLES
GRAY SCALE, POWER DOPPLER AND 3D ULTRASOUND
OOCYTE AND EMBRYO QUALITY
CHAPTER 18:
Ethical and Legal Aspects in ART
LEGAL ISSUES OF ART IN MARRIAGE
Adultery in Case of ART
Consummation of Marriage in Case of AIH
RIGHTS OF A CHILD BORN BY DONOR GAMETE
Legitimacy
Right to Information
Divorce
SEMEN BANKS
Who can Set it?
Who can be a Donor?
Age of Donors
Requirements for a Sperm Donor
DNA Fingerprinting
Donor Rights
Donor Anonymity
Is Anonymity of the Donor Justified?
Records
Who can Use the Semen?
Cryopreservation
Charges of Semen Banking
Consanguinity and Limiting Donor
Payment of Donors
Death of Donor
Collection of Sperm from a Dying Person
Posthumous AIH through a Sperm Bank
OOCYTE DONATION
How Long should Gametes be Stored?
SURROGACY
INDEX
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