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Practical Laboratory Andrology

Practical Laboratory Andrology

Practical Laboratory Andrology

David Mortimer, Ph.D.

Oxford University Press, New York, 1994
ISBN 0-19-506595-6

This practical manual on sperm analysis presents the diagnostic and therapeutic procedures that are used in andrology laboratories to analyze and assess male infertility. Diagnostic areas include: semen analysis and the biochemical, immunological and microbiological examination of human semen and spermatozoa; computer-aided sperm motility analysis; sperm ultrastructure; and assessment of sperm transport through the female reproductive tract and sperm fertilizing ability. The clinical relevance of the various diagnostic procedures is also discussed. Therapeutic topics include sperm washing techniques, semen cryopreservation, and insemination procedures. The volume also covers safety in the andrology laboratory, technician training and quality control.

The text is extensively illustrated and will be an invaluable resource to all scientists and technicians working in the diagnosis of male infertility. It will also be of interest to researchers working in human gamete biology and reproductive physiology. The detailed methods described in the book are relevant to all hospital, commercial, and university laboratories involved in infertility diagnosis and treatment. Each Chapter has a list of references and recommended reading.

Table of Contents

Introduction

Conventions used in this book
Concepts of fertility, infertility and sterility
Defining normal ranges
Terminology used by andrologists

Sperm physiology

  Sperm production:  testis; epididymis; accessory glands
  Ejaculation and insemination
  Spermatozoon:  sperm morphology and morphometry; sperm motility
  Sperm transport in the female tract:  spermatozoa in the vagina; spermatozoa in the cervix; “rapid” sperm transport, spermatozoa in the uterus; isthmic sperm reservoir
  Gamete interaction and fertilization:  capacitation; acrosome reaction; physiological regulation of sperm fertilizing ability; sperm-egg interaction; sperm-oocyte fusion and syngamy

Semen analysis

General principles:  patient instructions; sample collection; split ejaculates; liquefaction; mixing
Physical characteristics:  appearance; liquefaction and viscosity; odor; ejaculate volume; semen pH
Assessment of the wet preparation:  sperm motility; sperm aggregation and agglutination; other cellular components and debris
Identification of leukocytes in semen:  peroxidase staining using benzidine-cyanosine; peroxidase staining using o-toluidine blue; immunocytochemical identification
Sperm concentration:  hemocytometry; specialized counting chambers
Sperm vitality, integrity and longevity:  sperm vitality assessment using eosin-nigrosin; sperm vitality assessment using the H33258 fluorochrome; sperm osmotic fragility: the hypo-osmotic swelling test; sperm longevity assessments
Sperm morphology:  Papanicolaou staining; Bryan-Leishman staining; Shorr staining; sperm morphology classification systems

Biochemistry of spermatozoa and seminal plasma

  Biochemistry of spermatozoa:  acrosin; ATP content; chromatin condensation assessments; creatine kinase content; glutamic-oxaloacetic transaminase; hyaluronidase; lactate dehydrogenase isozymes; nuclear chromatin decondensation test; reactive oxygen species production; malonaldehyde production
  Biochemistry of seminal plasma:  acid phosphatase; carnitine; citric acid; fructose: quantitative analysis; fructose: spot test; -glucosidase; glycerophosphocholine; magnesium; prostaglandins; zinc

Antisperm antibodies

  Mechanisms of action
  Diagnostic tests for antisperm antibodies:  overview; spermagglutinating antibody titration: the Friberg test; spermotoxic antibody detection/titration; direct Immunobead test; indirect Immunobead test
  Clinical application

Semen microbiology and virology

  Pyospermia
  Microbiological examination of semen:  collection of semen specimens; examination of ejaculate
  Organisms found in human semen:  Ureaplasma and Mycoplasma; Chlamydia; Neisseria gonorrhoeae; other organisms; viruses
  Infection control and semen donors:  initial screening; repeat screening; each ejaculate; actions for positive tests

Computer-aided sperm analysis and sperm kinematics

  Limitations of CASA technology:  fundamental principles of digital image analysis; sources of error in CASA systems
  Application of CASA systems to semen analysis:  validation of CASA semen analysis results; possible solutions to problems of CASA
  Sperm kinematics:  sperm movement characteristics; methods of measuring sperm movement; basic principles of sperm kinematics

Ultrastructure of spermatozoa

Electron microscopy of human spermatozoa:  scanning electron microsocpy; transmission electron microscopy; surface replica preparations
Preparation of human spermatozoa for electron microscopy:  TEM: thin sections; TEM: surface replicas; SEM

Assessment of sperm transport

Cervical mucus sampling and assessment:  timing the procedure; sampling cervical mucus; assessment of cervical mucus quality: the Insler score; measurement of cervical mucus pH; storage of cervical mucus
In vitro sperm-mucus interaction tests:  background; Kremer test; Kurzrok-Miller test; sperm-cervical mucus-contact test; crossed-hostility testing; mucus substitutes
In vivo sperm-mucus interaction testing:  background; post-coital test
Sperm transport in vivo:  background; laparoscopic sperm recovery

Sperm fertilizing ability testing

Sperm migration testing:  hyaluronate migration test
Assessment of capacitation:  sperm surface lectin labelling; chlortetracycline fluorescence; hyperactivated motility
Assessment of the acrosome reaction:  triple-stain technique; fluorescent lectin labelling; acrosome reaction induction protocols
Assessment of sperm-zona pellucida binding:  hemizona assay; competitive zona binding test; fluorescent labeling of living human spermatozoa; competitive sperm-zona binding test procedure; expression of zona binding test results; recovering zonae pellucidae from human ovarian tissue; salt storage of human zonae pellucidae
Zona-free hamster egg penetration test:  overview; recommended sperm preparation protocol; preparation of fresh zona-free hamster eggs; preparation of cryopreserved zona-free hamster eggs; gamete coincubation conditions; reading the HEPT; cryopreservation of zona-intact hamster eggs
Comprehensive sperm function testing protocol

Clinical relevance of diagnostic procedures

Semen analysis:  extrinsic effects; aspects of sperm quality; relationships with fertility
Antisperm antibodies:  effects of ASABs in men; effects of ASABs in women; prevalence and significance of ASABs
Sperm-cervical mucus interaction testing:  post-coital test; in vitro tests
Sperm migration tests
Zona-free hamster egg penetration test:  HEPT and in vivo conception; HEPT and IVF; influence of HEPT methodology; future use of HEPT
Sperm-zona pellucida binding tests
Sperm acrosome reaction assessments
Sperm movement analysis and CASA:  semen analysis; sperm kinematics and SMIT results; sperm kinematics and HEPT; sperm hyperactivation; sperm kinematics and IVF
Laparoscopic sperm recovery
Evaluation of a sperm function test

Sperm washing

Yield calculations:  relative yield; absolute yield; yield quality
Culture media for human spermatozoa:  preparation of HTF culture medium
Dilution and washing techniques
Migration techniques:  swim-up from semen; swim-up from a washed pellet; swim-up from a washed sperm preparation; swim-down migration; SpermSelect; combined migration-sedimentation; trans-membrane migration; migration across meshes
Selective washing techniques:  Percoll; Nycodenz
Adherence methods:  glass wool column filtration; glass beads; Sephadex columns
Clinical applications:  sperm washing and microbiology; choice of sperm washing method; viscous semen samples; stimulation of sperm motility

Therapeutic insemination procedures

Sperm preparation for therapeutic insemination procedures:  sterile technique; protein supplementation of culture media; sperm preparation techniques
Retrograde ejaculation: retrograde ejaculation evaluation
Pericervical insemination
Intrauterine insemination
Gamete intrafallopian transfer
Intraperitoneal insemination
Insemination of human oocytes in vitro
Mechanically assisted fertilization
Treatment of antibody-coated spermatozoa:  attempts to reduce ASAB binding to spermatozoa; attempts to separate antibody-free spermatozoa; attempts to remove ASABs from the sperm surface; clinical treatment of ASABs

Semen cryopreservation

Historical perspectives
Clinical applications:  semen autoconservation; donor semen cryopreservation
Technical background:  cryoprotectants; interindividual variability; packaging; cryopreservation; inventory control systems
Cryopreservation of human semen:  glycerol-egg yolk-citrate method; other cryoprotectant media recipes
Organization of a sperm bank
Transportation of cryopreserved semen

Safety in the andrology laboratory

Basic laboratory safety: accident prevention; protective clothing and equipment; fire safety; hazardous chemicals; radioactive materials; compressed gases
Cryogenics
Infection control:  specific recommendations
Implementation of laboratory safety
Conclusions

Technician training and quality control aspects

Technician training:  goal-oriented training scheme for semen analysis
Quality control and quality assurance:  basic concepts; quality control of semen analysis
Conclusions

Appendices

Semen analysis procedural schedule and sample laboratory form
Microscope field calibration
Epifluorescence
Sample antisperm antibody laboratory report form
Sample donor screening forms
Sample sperm-mucus interaction testing laboratory and report forms
Semen cryopreservation forms
Equipment required for a basic andrology laboratory

Index

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