WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction

231 Pages·39.141 MB·English

WHO Laboratory Manual for the examination of human semen
and sperm-cervical mucus interaction

WHO LABORATORY MANUAL
for the examination of human semen
and sperm-cervicaL mucus interaction
FOURTH EDITION
Published on behalf of the
WORLD HEALTH ORGANIZATION
by
~CAMBRIDGE
<: UNIVERSITY PRESS
PUlnISI-IED BY THE PRESS SYNDICr\T1·: OF THE Ui\:IVERSITY 01' C,\,\iIHRIIHa:
The Pitt Building. Trumpington Street. Cilmbridge. United Kingdom
CAMBRIDGE UNIVERSITY PHI·:SS
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©World Health Organization 1980. 1987. 1992. 1999
This book isincopyright. Subject tostCitutory exception and tothe provisions
or relevilnt collective licensing agreements. no reproduction of any part may
take place without the wrilten permission or Cilmbridge University Press.
First published byPress Concern. Sing<lpore 1980
Second edition published byCCimbridge University Press 1987
Reprinted 1988. ]990
Third edition published byCambridge University Press 1992
Reprinted] 993. 1995. 1998
Fourth edition published byCambridge University Press 1999
Reprinted 2000
Typeset in 10.51] 3pt Monotype Photina inQuarkXPress™ IsEI
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WHO laboratory manual rorthe examinution or human semen and sperm
- cervicul mucus interaction. - 4th cd.
p. em.
Includes bibliographical rererences.
ISBN0521 645999 (pbk.)
1.Semen - Examination - Laboratory manuals. 2. Cervix mucus-
Examination - Laboratory manuals. 3.Sperm-ovum interactions-
Laboratory manuals. I.World I-IcillthOrganization. II.Title:
Laboratory manual forthe examination of human semen and sperm
- cervical mucus intemclion.
IDNLM: 1.Semen -chemistry laborawry manuals. 2.Spermatozoa-
chemistry laboratory manuals. 3.Cervix mucus - chemistry
laboratory manuals. 4. Spcrm~Ovum Inteructions laboratory manuals.
QY] 90 W628 1999J
RB54.W4H 1999
612.6']-<lc21 98-468911 Cll'
DNLMIDLC
forLibrary or Congress
ISBN0521645999 paperback
Prepared bythe WHO Special Programme of Research, LJevelopment and
Research Training inHuman Ikproduction.
The mention or specific companies ororcertain manufacturers' products does
not imply that they are endorsed or recommcnded bythe World Health
Organizution inpreference toothers of asimilar nature that ilrc not
mentioned. Errors and omissions cxcepted. the names of proprietCiry products
are distinguished byinitial capitHlletters.
Contents
Acknowledgements page viii
Abbreviations x
1 Introduction 1
2 Collection and examination of human semen 4
2A STANDARD PROCEDURES 4
2.1 Sample collection and delivery 4
2.2 Safe handling of specimens 6
2.3 [nitial macroscopic examination 6
2.3.1 LiqueJilction 6
2.3.2 Appearance 6
2.3.3 Volume 7
2.3.4 Viscosity 7
2.3.5 pH 7
2.4 Initial microscopic investigation 7
2.4. I Preparation Jor routille semen analysis 8
2.4.2 Preliminary estimation of sperm concentration 8
2.4.3 Assessment oj sperm motility 9
2.4.4 Cel/ular elelne/'lts other than spermatozoa 10
2.4.5 Agglntillation 14
2.5 Further microscopic examinations 14
2.5. I Spenn vitality by dye exclusion 14
2.5.2 Assessment of spenn COllCelltration 14
2.5.3 Assessment oj sperm morphology 17
2.5.4 Classificatioll oj sperm morphology 19
2.5.5 PerJormillg asperm morphology co!mt 22
2.6 Testing forantibody-coating of spermatozoa 23
2.6.1 Imlllllllobead test 25
2.6.2 Mixed antiglobulill reactioll test 26
28 OPTIONAL TESTS 28
2.7 Calculation of indices of multiple sperm defects 28
2.8 Hypo-osmotic swelling (HOS) test 29
2.9 Semen culture 29
2.10 BiochemicClIassays foraccessory sex organ function 29
2.10.1 Secretory capacity of the prostate 30
2.10.2 Secretory capacity of the seminal vesicles 30
2.10.3 Secretory capacity of the epididymis 30
2.11 Computer-aided sperm analysis 30
v
2.12 Zona-free hamster oocyte test 31
2C RESEARCHTESTS 31
2.13 Sperm function tests 31
2.13.1 Reactive oxygen species and male inJertility 31
2.73.2 Human zona pellucida bindillg tests 32
2.13.3 Assessment oj tileacrosome reactioll 32
2.14 Computer-aided sperm analysis: morphology 33
3 Sperm preparation techniques 34
4 Quality control in the andrology laboratory 36
4.1 Introduction 36
4.2 Nature of errors inmeasurement 36
4.2.1 Statistical counting errors illsemen analysis 37
4.2.2 Other errors insemen analysis 38
4.2.3 Tileimportance oj quality control 38
4.3 Practical approaches to quality control 39
4.3.1 Routille surveillallce and correlation oj results witll samples 39
4.3.2 Weekly internal quality control: assessing intra- and inter-tecllnician variability 40
4.4 Monitoring monthly means 46
4.5 Response to results outside control limits 48
4.6 External quality assessment 50
5 Sperm-cervicaL mucus interaction 5]
5.1 Introduction 5]
5.2 Collection and preservation of cervical mucus 53
5.2.1 Collectioll procedure 53
5.2.2 Storage alld preservation 54
5.3 Evaluation of cervical mucus 54
5.3.7 Volume 54
5.3.2 Consistency 54
5.3.3 Ferning 55
5.3.4 Spinl1barkeit 55
5.3.5 Cellularity 55
5.3.6 pH 56
5.4 [nteraction between sperm and cervical mucus 56
5.4.7 In vivo (postcoital) test 56
5.4.2 In vitro tests 58
Appendices 60
fA Reference values of semen variables 60
IB Nomenclature forsome semen variables 62
[[ Safety guidelines forthe andrology laboratory 63
III Methods fordetecting leukocytes 65
IV Sperm vitality techniques 68
V Papanicolaou staining procedure modified forspermatozoa 7]
VI Shorr staining procedure forsperm morphology 75
VII Rapid staining procedure forsperm morphology 76
VIII Immunobead test 77
vi
IX Mixed antiglobulin reaction test (MAR test) 80
X Measurement of zinc in seminal plasma 81
XI Measurement of fructose inseminal plasma 83
XII Measurement of neutral a-glucosidase inseminal plasma 85
XIII Sample instructions and record form forsemen analysis 88
XIV Computer-aided sperm analysis (CASA) 90
XV Protocols forthe zona-free hamsler oocyte lest 94
XVI Measurement of reactive oxygen species generated bysperm
suspensions 99
XVII Induced acrosome reaction assay 102
XVfll Spcrm preparation techniques 104
XIX Quality control (QC)procedures 107
XX Sample record form forcervical mucus scoring and postcoital test l09
XXI Spcrm-{;ervical mucus interaction: the capillary tube test 110
XXII Basic requirements foran andrology laboratory 114
Bibliography 118
Index 125
vii
AcknowLedgements
The UNDP/UNFPA/WHO/World Bank Special Programme of Res-
earch. Development and Research Training in Human Reproduction
wishes to acknowledge the participation. in the preparation and
editing of the fourth edition of the WHO Laboratory Manual Jor the
Examination oj Human Semen and Sperm-Cervical Mucus Interaction, of
the following:
Dr R.J.Aitken Dr F.Comhaire
MRCReproductive Biology Unit Dienst Voor Inwendige Ziekten
Centre for Reproductive Biology Endocri nologie-Stofwissel ingsziekten
37 Chalmers Street DePintelaan 185
Edinburgh EH3 9EW 9000 Gcnt
United Kingdom Belgium
Dr H. W.G.Baker Dr T.G.Cooper
University of Melbourne Institutc of Reproductive Medicine of
Royal Women's Hospital the University
Department of Obstetrics and Domagkstrasse 11
Gynaecology D-48] 29 Munster
Carlton 3053, Victoria Germany
Australia
Dr C.De Jonge
Dr C.L.R. Barratt Department of Obstetrics &
University Department of Obstetrics Gynaecology
and Gynaecology University of Nebraska Medical
Birmingham Women's Hospital Center
Edgbaston 600 South 42 Street.
Birmingham B15 2TG Omaha, NE68198-3255
United Kingdom United States of America
Dr H. M. Behre Dr R.Eliasson
Institute of Reproductive Medicine of Head, Andrology Unit
the University Sophiahemmet Hospital
Domagkstrasse 11 SE114 86 Stockholm
D-48129 Munster Sweden
Germany
viii