ATLAS OF ASSISTED REPRODUCTIVE TECHNOLOGIES AND CLINICAL EMBRYOLOGY

Section 1
Setting up of an Assisted Reproductive Technology
Center and Quality Assurance in the Laboratory
Chapter 1 Setting up of an Assisted Reproductive Technology Center 3
Surveen Ghumman
Location of the Laboratory 3
Basic Infrastructure 4
Ancillary Laboratory Facilities 8
Laboratory Personnel 10
Culture Media 16
Protocols in the Laboratory 17
Chapter 2 Plastic Ware in Assisted Reproductive Technology 20
Pankaj Talwar
Quality Control Tests 21
Quality Control Acceptance Criterion 22
Material Used 22
Types of Disposables 24
Chapter 3 Culture Media and Assisted Reproductive Technology 31
Pankaj Talwar
History 31
Types of Media for Embryo Culture 32
Composition of Embryo Culture Media 33
Physiology and Sequential Culture Media 39
Embryo Handling Guidelines 43
Media Preparation and Pre-equilibration 45
Quality Control Testing for the Culture Media 45
Measuring ph of the Culture Media 47
Precautions to be Taken while Dispensing Media and
Opening the Media Bottles 48
Storage of the Media 50
Shelf Life and Packaging 50
Chapter 4 Air in the Laboratory 52
Pankaj Talwar
Modalities of Air Filtrations 53
Procedure of Air Purification 53
Air Composition 54
Volatile Compounds in the Air 54
Air Filtration Systems 55
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Jaypee’s Video Atlas of Assisted Reproductive Technologies and Clinical Embryology
xQuality Check of Air in the Assisted Reproductive
Technology Laboratory 57
Quality of Air in the Clean Rooms 57
Quality of Air in the Incubators 58
Quality of Air in the Biological Safety Cabinets 59
Biological Safety Cabinet Classes 59
Quality Control of Air in the Assisted Reproductive
Technology Laboratory 62
Air Handling Unit of an Assisted Reproductive
Technology Center 62
Chapter 5 Classical CO2
Incubator: Heart and Soul of an Ivf Laboratory 66
Dilip Patil
Temperature Control 66
Humidity Control 68
Carbon Dioxide Control 68
Role of Oxygen Tension in an Incubator 70
Contamination Control in the Incubator 71
Factors to be Considered in Choosing a CO2
Incubator 71
Practical Tips to Ensure Proper Functioning of CO2
Incubators 73
Incubator of the Future 74
Chapter 6 Modular IVF Facility 75
Sarabpreet Singh
Oocyte Aspiration and Embryo Transfer Room 75
In Vitro Fertilization Laboratory—
“Sanctum Sanctorum” of the Project 76
Section 2
Ovum Pickup and Ovulation Induction Complication
Chapter 7 Ovum Pickup 83
Pankaj Talwar
Brief History 83
Relevant Issues Regarding Ovum Pickup 83
Oocyte Pickup Step by Step 89
Common Problems Encountered During Ovum Pickup 91
Complications 91
The Learning Curve 93
Chapter 8 Embryo Culture: Laboratory Protocols 95
Pankaj Talwar
Preparing the Culture System 97
Principles of Embryo Culture 98
Tips for Embryo Culture 98
Step-by-Step Fertilization Assessment on Day 1 99
Step-by-Step Scoring of Human Pronuclear Embryos 101
Embryo Assessment on Day 2 102
Step-by-Step Embryo Assessment on Day 3 102
Step-by-Step Embryo Assessment on Day 5
(Blastocyst Culture) 103
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Contents
Chapter 9 Insemination and Denudation 105
Pankaj Talwar
Sperm Concentration 105
Media for Sperm Preparation and Insemination 105
Duration of Sperm-Egg Contact 105
Method of Insemination 106
Tips for Denudation 108
Chapter 10 Analysis of Fertilization 110
Pankaj Talwar
The Oocyte 110
Assessment of Fertilization 114
Pronuclear Scoring Systems 115
The Centrosome in Fertilization 116
Embryo Assessment 116
Chapter 11 Multifetal Pregnancy Reduction 122
Pankaj Talwar
Incidence of Multifetal Pregnancy 122
Risks Associated with Multifetal Pregnancies 122
Prevention of Multifetal Pregnancies 123
Counseling 124
Options 124
Multifetal Pregnancy (Embryo) Reduction 124
Selective Termination versus Multifetal Pregnancy Reduction 124
Rationale 124
Time of Procedure 124
Various Approaches for Multifetal Pregnancy Reduction 125
Embryo Reduction 125
Selection of Gestational Sac/Fetuses for Reduction 125
Transvaginal Embryo Reduction Procedure 126
Complications of Multifetal Pregnancy Reduction 128
Benefits of Multifetal Pregnancy Reduction 128
Chapter 12 Ovarian Hyperstimulation Syndrome 130
Anjali Tempe
Definition, Incidence and Classification 130
Etiopathology 131
Prevention 131
Investigations and Monitoring in a Case of Ohss 132
Treatment of Severe OHSS 132
Complications of Ohss 133
Section 3
Embryo Transfer
Chapter 13 Embryo Transfer 137
Pankaj Talwar
Factors Affecting Embryo Transfer 137
Embryo Transfer Step by Step 141
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Section 4
Intracytoplasmic Sperm Injection: Step-by-Step
Chapter 14 Intracytoplasmic Sperm Injection: Step-by-Step 151
Pankaj Talwar
Intracytoplasmic Sperm Injection Media and Pipettes 151
Preparation of Icsi Dishes 152
Denudation 153
Manipulation of Spermatozoa 157
Manipulation of Oocytes 157
Microinjection of Oocytes with Mature Spermatozoa 158
Difficult Intracytoplasmic Sperm Inject Cases 160
Chapter 15 Intracytoplasmic Sperm Injection: Basics 163
Sarabpreet Singh
Setting up Olympus: Narishige Micromanipulator 163
New Installation 163
While the Microinjector is Already Functional 164
How to Align the Pipettes 164
Intracytoplasmic Sperm Injection 164
Intracytoplasmic Sperm Injection Procedure 165
Chapter 16 How to Set up Olympus: Narishige Micromanipulator 166
Sarabpreet Singh
Components 166
Objective 167
Eye Piece 167
Centering the Condenser 167
Narishige Micromanipulator 168
Micromanipulator 168
Motorized Movement Joystick 168
Hydraulic Fine Movement Controller 168
Manipulator Components Mounted on the Microscope 168
IM-9B/Microinjector 169
Setting up Olympus: Narishige Micromanipulator 169
New Installation 170
While the Microinjector is Already Functional 170
How to Align the Pipettes 170
Chapter 17 Surgical Sperm Retrieval Techniques 172
Pankaj Talwar
Methodologies 172
Sperm Preparation on the Day of Icsi 174
Preparation of Sperm Samples for Cryopreservation 175
Testicular Biopsy Banking 175
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Contents
Section 5
Intrauterine Insemination
Chapter 18 Semen Preparation for Intrauterine Insemination 179
Rk Sehgal
Semen Collection 179
Methods of Semen Preparation 179
Selection of Semen Processing Technique 180
Semen Processing Kits 181
The Setup for Iui Laboratory 181
Equipments Required for Iui Laboratory 182
Standard Operating Procedures for Various Semen
Processing Techniques 184
Section 6
Semen Preparation in IVF
Chapter 19 Semen Preparation in IVF 189
Pankaj Talwar
Essentials of Sperm Preparation 189
Nomenclature—Semen Variables (Who) 190
Techniques of Semen Preparation 190
Basis of Centrifugation 199
Improvement of Motility and Sperm Function 199
The Ideal Sperm Separation Technique 200
Section 7
Controlled Ovarian Stimulation and Luteal Support
Chapter 20 Ovarian Stimulation Regimens 205
Leena Wadhwa, Pankaj Talwar
Various Types of Stimulation Cycles 205
Challenges in Controlled Ovarian Stimulation 208
Section 8
Cryobiology in Assisted Reproductive Technologies
Chapter 21 Basics of Cryobiology 213
Pankaj Talwar
Basic Definitions 214
Thermodynamics 214
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Physics 214
Concept of Latent Heat 214
Biology of Cryofreezing 215
Cryoprotectants 215
Principles of Cryobiology 216
Steps of Cryopreservation 216
Events during Freezing 218
Freeze Injuries 219
Events during Thawing 219
Thaw Injuries 219
Chapter 22 Embryo Vitrification and Warming 221
Pankaj Talwar
Overview 221
Principles of Vitrification 221
Common Carrier Devices and Methods 222
Superiority of Various Vitrification Methods 223
Protocol for Embryo Vitrification Using Cryoloop and
Vitrolife Vitrification Media 224
Embryo Warming 229
Preventing Potential Contamination from Liquid Nitrogen 234
Chapter 23 Oocyte Slow Freezing and Thawing 236
Pankaj Talwar
Background 236
Evaluation of Oocyte Quality 237
Essential Principles of Controlled Rate Cooling 237
Survival of Frozen-Thawed Oocytes and Sucrose Concentrations 238
Oocyte Denudation–To Do or Not? 238
Factors Affecting the Clinical Efficiency of Oocyte Cryopreservation 238
Procedure of Oocyte Freezing 239
Chapter 24 Embryo Slow Freezing and Thawing Protocol 247
Pankaj Talwar
Historical Background 247
Indications 247
Principles of Cryofreezing of Human Embryos 248
How to Grade and Select Embryos for Cryofreezing? 248
Embryo Freezing Simplified 249
Risk of Cryofreezing 254
Duration of Freezing 257
Embryo Thawing Protocols 258
Mode of Injuries 258
Types of Cryoprotectants 259
Principles of Embryo Thawing 260
Timing of Embryo Thawing and Endometrial Preparation 261
Post-thaw Grading of the Embryos 261
Cryoprotectants Used for Embryo Thawing 261
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Contents
Embryo Thawing Simplified 261
Potential Danger of Disease Transmission 266
Chapter 25 Oocyte Vitrification and Warming Protocol 267
Pankaj Talwar
Oocyte Cryopreservation Methods 267
Peculiarities of Oocyte Vitrification 267
Principles of Vitrification 267
Vitrification Simplified 270
Timing of Cryopreservation of Oocytes 272
Unique Vitrification Devices 273
Cryodamage 274
Type of Cryoinjuries 274
Oocyte Warming Protocol 277
Background of Oocyte Freezing 277
Warming Made Easy Using Medicult Devitrification Medium Using Cryoleaf 278
Injuries to Oocyte: Zona Damage and Cell Lysis 281
Oocyte Vitrification (Using Irvine Vitrification Media) 282
Equipment 282
Reagents 282
Procedure: Vitrification 283
Chapter 26 Semen Freezing: Practical Aspects 284
Pankaj Talwar
Background of Sperm Banking 284
Principles of Cryobiology 285
Indications of Semen Cryopreservation 285
Sample Collection 286
Physiology of Semen Cryopreservation 287
Overview of Semen Banking: Step-by-Step 288
Recovery of Spermatozoa Post-thaw 296
Chapter 27 Ovarian Cortex Freezing 299
Pankaj Talwar
Indications for Ovarian Cryopreservation 301
Assessment of Ovarian Reserve 302
Current Techniques of Fertility Preservation 302
Ovarian Cortex Cryopreservation 303
Limitations for Ovarian Tissue Cryopreservation 304
Collection of Ovarian Tissue 304
Cryoprotectant Preparation 304
Tissue Preparation 304
Histological Analysis 307
Steps of Ovarian Cortex Freezing 308
Peculiarities of Ovarian Tissue Freezing 310
Methods of Thawing 311
Transplant 311
Ethical Concerns in Ovarian Tissue Cryopreservation 311
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Jaypee’s Video Atlas of Assisted Reproductive Technologies and Clinical Embryology
Section 9
Newer Equipment in Assisted Reproductive Technologies
Chapter 28 Laser in Assisted Reproductive Technology 317
Sarabpreet singh
Applications of Laser in Assisted Reproductive Technology 318
Chapter 29 Intracytoplasmic Morphologically Selected Sperm Injection 320
Sarabpreet singh
Msome (Motile Sperm Organelle Morphology
Examination) Criteria 320
Setting up Olympus—Narishige Micromanipulator 321
New Installation 321
While the Microinjector is Already Functional 321
How to Align the Pipettes 321
How to Prepare an Imsi Dish 322
Intracytoplasmic Morphologically Selected Sperm Injection 323
Section 10
Ultrasound in Assisted Reproductive Technologies
Chapter 30 Normal Female Pelvis 327
Kuldeep Singh, Narendra Malhotra
Uterus 327
Fallopian Tube 329
Ovaries 329
Pouch of Douglas 336
Doppler Evaluation of Pelvic Viscera 336
Three-Dimensional Evaluation of Pelvic Viscera 336
Chapter 31 Vaginal and Cervical Factor Infertility Evaluation 337
Kuldeep Singh, Narendra Malhotra
Chapter 32 Uterine Factor Assessment and Evaluation 339
Kuldeep Singh, Narendra Malhotra
Leiomyomas 339
Congenital Anomalies 341
Sonohysterography 341
Adenomyosis 342
Evaluation of Endometrial Growth 343
Chapter 33 Tubal Evaluation 346
Kuldeep Singh, Narendra Malhotra
Sonosalpingography 346
Chapter 34 Ovarian Evaluation by Ultrasound 348
Kuldeep Singh, Narendra Malhotra
Ovarian Study 348
Follicular Development 348
Prediction of Ovulation 350
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Contents
Confirming Ovulation 351
Ovary in Anovulatory Cycles 351
Corpus Luteum 351
Polycystic Ovaries 352
Predicting Ovarian Reserve 352
Chapter 35 Color Doppler in Female Infertility 353
Kuldeep Singh, Narendra Malhotra
Physiological Changes of Menstrual Cycle by Color Doppler 353
Endometrial Changes 353
Ovarian Vascularity 354
Luteal Phase Defect 356
Section 11
Endoscopy in Assisted Reproductive Technologies
Chapter 36 Principles of Diagnostic Laparoscopy and Setting up of
an Endoscopy Unit 359
Malvika Sabharwal, Kuldeep Jain
Chapter 37 Infertility (Laparoscopic: Fertility Enhancing Surgeries) 363
Malvika Sabharwal
Chapter 38 Laparoscopy for Endometriosis 364
Malvika Sabharwal
Chapter 39 Operative Hysteroscopy 366
Malvika Sabharwal
Chapter 40 Ectopic Pregnancy: Laparoscopic Management 369
Malvika Sabharwal
Index 371

Section 1 Setting up of an Assisted Reproductive Technology Center and
Quality Assurance in the Laboratory
Chapter
No.
Chapter Title Author Video Link/Folder Video
Contributor
1. Setting up of an Assisted
Reproductive Technology
Centre
Surveen
Ghumman
V.1. Ideal Art Center Set Up
V.2.1(a) Modular Ivf Set up
V.2.1(b) Modular Ivf Set up
V.2.1(c) Modular Ivf Set up
V.2.2. Modular Ivf Facility
V.3.1. Tour of a Cryobiology Laboratory
V.3.2. Cryobiology Accessories
V.4.1. Basic Lab Cleaning
V.4.2. Digital Co2
Measurement
V.4.3. Co2
Measurement: Fyrite Kit
V.4.4. Digital Temperature Recording
V.4.5. Air Sampling
V.5. Plastic Ware in Art
V.6. Media in Art
V.7.1. Work Station and Accessories
V.7.2. Incubators
Pankaj Talwar
ashish modi,
Kersi Avari
Sarabpreet
Singh
Pankaj talwar
Pankaj talwar
Pankaj talwar
Pankaj talwar
Pankaj talwar
Video Link V.1 V.3.1 V.3.2 V.4.1-V.4.5 V.7.1
DVD No. 1 1 1 2 3
2. Plastic Ware in Assisted
Reproductive Technology
Pankaj Talwar
Video Link V.5
DVD No. 2
3. Culture Media and Assisted
Reproductive Technology
Pankaj Talwar
Video Link V.6
DVD No. 3
4. Air in the Laboratory Pankaj Talwar
Video Link V.4.5
DVD No. 2
5. Classical CO2
Incubator:
Heart and Soul of an IVF
Laboratory
Dilip Patil
Video Link V.7.2
DVD No. 3
6. Modular IVF Facility Sarabpreet
Singh
Video Link V.2.1(a) V.2.1(b) V.2.1(c) V.2.2
DVD No. 1 1 1 1
Video References
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Jaypee’s Video Atlas of Assisted Reproductive Technologies and Clinical Embryology
Section 2 Ovum Pickup and Ovulation Induction Complication
Chapter
No.
Chapter Title Author Video Link/Folder Video
Contributor
7. Ovum Pickup Pankaj Talwar V.8.1. Lab Preparation Prior to Opu
V.8.2. Opu Needles
V.8.3. Opu Procedure and Embryology
Post Opu
V.8.4. Insemination Procedure
V.9.1. Denudation Plate Preparation
V.9.2. Denudation Procedure and
Second Look
V.10.1. Embryo Reduction Procedure
V.10.2. Embryo Reduction and PostReduction Follow Up.
Pankaj talwar
Pankaj talwar
Pankaj talwar
Video Link V.8.2 V.8.3
DVD No. 4 4
8. Embryo Culture: Laboratory
Protocols
Pankaj Talwar
Video Link V.8.1 V.8.3
DVD No. 4 4
9. Insemination and
Denudation
Pankaj Talwar
Video Link V.8.4 V.9.1 V.9.2
DVD No. 4 5 5
10. Analysis of Fertilization Pankaj Talwar
No Video Link
No DVD
11. Multifetal Pregnancy
Reduction
Pankaj Talwar V.11.1. Ultrasound in Ohss
V.11.2. Paracentesis Procedure
Pankaj talwar
Video Link V.10.1 V.10.2
DVD No. 5 5
12. Ovarian Hyperstimulation
Syndrome
Anjali Tempe
Video Link V.11.1 V.11.2
DVD No. 6 6
Section 3 Embryo Transfer
13. Embryo Transfer Pankaj Talwar V.12.1. Embryo Transfer Catheters
V.12.2. Embryo Transfer Procedure
V.12.3. Embryo Loading and Transfer
Pankaj talwar
Video Link V.12.1 V.12.2 V.12.3
DVD No. 7 7 7
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Video References
Section 4 Intracytoplasmic Sperm Injection: Step-by-Step
Chapter
No.
Chapter Title Author Video Link/Folder Video
Contributor
14. Intracytoplasmic Sperm
Injection: Step-by-Step
Pankaj Talwar V.13.1. ICSI Plate Preparation
V.13.2. Sperm Aspiration and Icsi
Procedure
V.13.3. (a) Set Up of Narishighe
Micromanipulator
V.13.3. (b) Set Up of Narishighe
Micromanipulator
V.13.4. Set Up of an Ri Inverted
Microscope
V.13.5. Set Up of an Eppendorf Inverted
Microscope
V.14.1. PESA (Percutaneous Epididymal
Sperm Aspiration)
V.14.2. Tese (Testicular Sperm
Extraction)
Pankaj Talwar
sarabpreet
Singh
Dilip Patil
Rajvi Mehta,
ashish modi,
Kersi Avari
Pankaj talwar
Video Link V.13.1 V.13.2 V.13.4 V.13.5
DVD No. 8 8 8 8
15. Intracytoplasmic Sperm
Injection: Basics
Sarabpreet
Singh
No Video Link
No DVD
16. How to Set up Olympus:
Narishige Micromanipulator
Sarabpreet
Singh
Video Link V.13.3 (a) V.13.3 (b)
DVD No. 8 8
17. Surgical Sperm Retrieval
Techniques
Pankaj Talwar
Video Link V.14.1 V.14.2
DVD No. 9 9
Section 5 Intrauterine Insemination
18. Semen Preparation for
Intrauterine Insemination
RK Sehgal V.15.1. Overview
V.15.2. Semen Analysis
V.15.3. About Us
V.15.4. Neat Sample Swim Up
V.15.5. Centrifugation and Swim Up
V.15.6. Single Density Technique
V.15.7. Double Density Technique
V.15.8. Ideal Equipment
RK sehgal
Video Link V.15.1 V.15.2 V.15.3 V.15.4 V.15.5 V.15.6 V.15.7 V.15.8
DVD No. 10 10 10 10 10 10 10 10
Section 6 Semen Preparation in IVF
19. Semen Preparation in IVF Pankaj Talwar V.16.1. Swim Up and Wash Method
V.16.2. Density Gradient Method
V.16.3. Double Wash and Swim Up
Method
Kuldeep Jain
Kuldeep Jain
Pankaj Talwar
Video Link V.16.1 V.16.2 V.16.