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A Workbook on Human Spermatozoa and Assisted Conception
Sonia Malik, Ashok Agarwal
1:
Equipment and Culture Media
INTRODUCTION
EQUIPMENT
Incubators
Benchtop Incubators
Laminar Flow
Clean Benches
Culture Lab Air Cleaner (Coda Air Filtration System)
In Vitro Fertilization (IVF) Workstation
Microscopes
Micromanipulation
Ovum Aspiration Pump
Controlled Rate Freezing System/Cryosystems
General Equipment
Centrifuge
Refrigerator/Freezer
Dry Heat Oven for Sterilizing
Makler's Chamber/Phase Contrast Microscope
Laser-Assisted System for PGD and Hatching
Labware and Disposables
Ovum Pickup Needles
Embryo Transfer Catheter
Oocyte Preparation Pipettes
Denudation Pipettes
Holding Pipettes
Injection Pipette
CULTURE MEDIA FOR IVF
Composition
pH and Osmolality
Temperature
Culture Conditions
Water
Protein Source
Sources for Protein Supplements (serves today for research only)
Commercial IVF Media
Salt Solution in MTF
Carbohydrates
Amino Acids
Vitamins
Antibiotics
Chelators
HTF Hepes
Enhance HTF
Enhance Day 1
Enhance Day 3
Enhance Day 5
Human Serum Albumin (HSA)
Mineral Oil
Polyvinyl Pyrrolidone (PVP)
Hyaluronidase
2:
Computer-assisted Semen Analysis (CASA)
INTRODUCTION
3:
Semen Analysis
INTRODUCTION
Terminology
Background Data
Container
Abstinence
Method for Production
Physical Parameters
Microscopic Examination
Method
4:
Sperm Morphology
THE WHO CRITERIA (1987)
THE WHO CRITERIA (1992)
THE STRICT CRITERIA
Clinical Relevance of Strict Criteria
Papanicolaou Stained Smear (Figs 4.5 to 4.9)
5:
Semen Preparation: Ejaculated Sperm for ICSI
INTRODUCTION
MATERIALS, EQUIPMENT AND REAGENTS
Material and Equipment
Reagents
PROCEDURES
Double-density Gradient Centrifugation
Prepare Reagents
Analyze and Wash Specimen
Swim-up (Figs 5.1A to F)
Swim-up from a Sperm Pellet
Direct Swim-up (Figs 5.2A to E)
6:
Semen Preparation: Sperm Extraction for ICSI
INTRODUCTION
MATERIALS, EQUIPMENT, REAGENTS AND IN VITRO FERTILIZATION (IVF) LABORATORY SET-UP
Material and Equipment
Reagents
Laboratory Set-up
PESA/MESA SPERM PROCESSING (FIG. 6.1)
PESA-MESA-TESA-TESE SPERM PROCESSING
TESA SPERM PROCESSING (FIG. 6.2)
TESE SPERM PROCESSING (FIG. 6.3)
CRYO-THAWED EPIDIDYMAL/TESTICULAR SPERM PROCESSING
METHODS FOR SELECTING VIABLE IMMOTILE SPERM FOR ICSI
Hypo-osmotic Swelling Test (HOST)
Sperm Tail Flexibility Test (STFT)
Motility Stimulant Sperm Challenge (MSC)
Preparation of Microdroplets
APPENDIX
GLOSSARY
7:
Semen Preparation: Infected Sample (HIV)
INTRODUCTION
RISK OF TRANSMISSION
LABORATORY ASPECTS
SCREENING FOR SEROPOSITIVE MALE PARTNERS
SPERM SEPARATION TECHNIQUE
SEMEN PREPARATION FROM INFECTED SAMPLE
Double Density Gradient Centrifugation with Swim-up
EQUIPMENT AND DISPOSABLES
MEDIUM FOR SPERM PREPARATION
PROCEDURE
8:
Oxidative Stress Tests
INTRODUCTION
What are Free Radicals?
Types of Free Radicals
Generation of Free Radicals in the Seminal Ejaculate
What is Oxidative Stress?
Measurement of Free Radicals
Luminometers
Chemiluminescence Measurement
Equipment and Material
Reagent Preparation
Specimen Preparation and ROS Measurement
Types of Samples for ROS Measurement
ROS Measurement by Luminometer
Enter the Information in the Luminometer
Calculation of ROS Results
Reference Values for Abnormal ROS
Probes for Extracellular and Intracellular ROS Measurement
SUMMARY
9:
TUNEL Test
INTRODUCTION
ASSAY PRINCIPLE
SPECIMEN COLLECTION
STAINING PROTOCOL
STAINING SOLUTION (SINGLE ASSAY)
Negative Control
Positive Control
FLOW CYTOMETRY
FACTORS AFFECTING THE ASSAY RESULTS
REFERENCE RANGE OF SPERM DNA DAMAGE
10:
Reactive Oxygen Species Measurement
INTRODUCTION
METHODOLOGY
Sample Collection
Sample Analysis
Instrument Setup
Preparation of Tubes
ROS Estimation
Blank
Negative Control
Positive Control
Measurement of ROS in Neat Semen
Measurement of ROS in Washed Semen
Data Analysis
Calculation of ROS
Materials Required
Chemicals Required
PREPARATION OF REAGENTS AND SOLUTIONS
11:
Assessment of DNA Fragmentation (Halo Test)
INTRODUCTION
LAB PROTOCOL
Principle of the Method3*
Material and Equipment
SPERM SAMPLE
Procedure
SPERM CLASSIFICATION
Sperm without Fragmented DNA
Sperm with Fragmented DNA
Sperm Chromatin Dispersion Patterns Observed After Staining (Fig. 11.1)
INTERPRETING THE RESULTS
SAFETY AND THE ENVIRONMENT
PRECAUTIONS
12:
Sperm Function Tests
HYPO-OSMOTIC SWELLING (HOS) TEST (PATENT FILED)1
Protocol
NUCLEAR CHROMATIN DECONDENSATION TEST(PATENT FILED)
Protocol
ACROSOME STATUS AND FUNCTION (PATENT FILED)2
Protocol
13:
Micromanipulator Set-up and ICSI
MICROINJECTION
INTRACYTOPLASMIC SPERM INJECTION (ICSI)—PROCEDURE AND EQUIPMENT
Materials and Methods
Devices
Consumables and Media
Microinjection Dish Preparation
Preparation of the Microinjection Capillaries
Procedure of Microinjection
Assessment of Fertilization
INTRACYTOPLASMIC MORPHOLOGICALLY SELECTED SPERM INJECTION (IMSI)
PETRI DISH ICSI (PICSI)
14:
IMSI and PICSI Sperm Selection
IMSI PROTOCOL
PICSI PROTOCOL
15:
Oocyte Spindle Imaging System
INTRODUCTION
16:
Semen Cryopreservation and Banking
INTRODUCTION
INDICATIONS OF SEMEN CRYOPRESERVATION
OUTLINE OF CRYOPROTECTANTS
ESSENTIALS OF FREEZE-THAW CYCLE
LEGISLATION PERTAINING TO THE SEMEN BANKING
DONOR SCREENING PRIOR TO SEMEN BANKING
CROSSINFECTION IN THE SEMEN BANKS
SECURITY OF THE SEMEN BANK
17:
Semen Vitrification
INDEX
TOC
Index
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