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Who Laboratory Manual WHO LABORATORY MANUAL for the examination of human semen and sperm-cervical mucus interaction FOUKTH EDITION Published on behalf of the WORLD IIEALTH ORGANIZATION by CAMBRIDGE UNIVERSITY PRESS PIIR1,ISHBD BY THE PRESS SYNDICA'SE 01' THE llNTVBRSlTY OF CAMBR1I)GB The Pitt Ruilding. Trumpington Street. Cambridge, [Jnited Kingdom CAMRKIUGB UNIVERSITY PKESS The Edinburgh Building. Cambridge CB1 2RU. UK http:iiwww.cup.cam.ac.uk 40 West 20th Street, Ncw York. NY 10011-421 1. USA hltp://www.cup.org 10 Stunford Road, Oakleigh. Melbourne 3166. Australia Ruiz dc Alarcan 13. 28014 Madrid. Spain Q World HealthOrgar~iaation1980, 1987. 1992, 1999 This book is in copyright. Subjccl to statutory exception and lo the provisions of relevant collective licensing agreemenls, no reproduction of any part may take place without the written permission of Cambridge IIniversity Press. First published by Press Concern, Singapore 1980 Second edition published by Cambridge tJniversity Press 1987 Reprinted 1988. 1Y90 Third edition published by Cambridge [Jniversity Press 1992 Reprinltvl 199 3. 1995,1998 Fourth edition published by Cambridge University Press 1999 Reprinted 2000 Typcsel in 10.5i1 ?pt Monotypc Photina in QuarkXl'rcssrM [SE] A catalogue record Jur illis hook is availablefrum thr British Librarg Lihriir!j of Corlgrpss Cataloguing in Pirhlicatiol~data WHO laboratory ~nannalfor the examination of human semcn and sperm cervical mucus interaction. - 4th ed. p. cm. Includes bibliographical refcrcuces. ISBNU 521 h4599 9 (pbk.) 1.Semen - Examination - 1,aboratory ma~mals. 2. Cervix mucus - Examination - Laboratory manuals. 3. Spcrrll-ovum interactions- Laboratory manuals. 1. Wurld Heall h Organizatior~. 11. Title: Laboratory manual for the examirriltion of human semcn and sperm -cervical mucus interaction. [l>NT,M: 1.Semcr~ chemistry laboratory ~nanuals. 2. Spermaloaoa- chemistry laboratory manuals. 3. Cervix mucus chemistry laboratory manuals. 4. Spernl-Ovum Interactions laboratory manuals. QY 190 W628 19991 RR54.W48 1999 612.6'1-dc21 98-46890CIP DNLMIDLC for Library oT Congress lSBN 0 521 64599 9 paperback Prepared by th~WHO Special Programme of Research, Development and Research Tra~~lingIn Human Reproductloo. l'he nlention of specific cun~paniesor of certain manufacturers' products does not in~plythat they are endorsed or recommended by the World Health Organizaliun in preference to olhers of a similar nature that are nnt mentioned. Errurs and omissions excepted, the names of proprietary products are distinguished by initial capital letters. Aclrnowledgcrnc~~ls pagP viii Abbreviations 1 Introduction 2 Collection and examination of human semen 2A STANDARD PROCEDURES 2.1 Sample collection and delivery 2.2 Safe handling of specimens Initial macroscopic examination Liquefucliun Appearanca Vol~trne Viscosily Initial microscopic investigation PreparationJor rauline semen analysis Preliminary estimation of sperm concentratiorl 2.4.3 Assessment oJ sperm1 motility 2.4.4 Cellular elements olhrr than spermatozoa 2.4.5 Agglutination 2.5 Further microscopic examinations 2.5.1 Sperm vitality by dye exclusiun 2.5.2 Assessment of ,sperm r;oncentration Assessment oJ spernf morphology Classification of sperm morphalugy Perfurrninq a sperm morpholngy count Testing for antibody-coating of spermatozoa Imn~ur~obeadtest Mixeduntiqlobulin reaction test 0I"I'IONAI.TESTS Calculatioil of indices of multiple sperm dcfccts Hypo-osmotic swelling (HOS) tcsl Sc~rlcr~cullure 2.10 Biochemical assays for ac,cessory sex organ function 2.7 0.1 Secretory cupacity of the prostate 2.10.2 Secretory cupucity of Ihe seminal vesicles 2.10.3 Secrztor,y capacity of the! epididymis 2.1 1 Computer-aided sperm analysis 2.1 L Xona-free hamster oocyte test 2C RESEARCH TESTS 2.1 3 Sperm function tests 2.1.3.1 Keactive ox!jflen species and male infertility 2.13.2 Humnn zonapellucida bindi~rgtests 2.13.3 Assessrner~tof the acrosorrle reactiorr 2.l 4 Computer-aided sperm analysis: morphology 3 Sperm preparation techniques 34 Quality control in the andrology laboratory Introduction Nature of errors in measurement Statistical counting errors in spmen analysis Other errors in sernerl analysis The importuncle uf quulily control Practical approaches to quality control Routirle surveillance arld correlatiol~of results with sarnples Weekly internal y~lulilycontrol: assessing intra- and inter-technician variability Monitoring monthly means Response to results outside control limits Exlcrrlal quality assessment Sperm-cervical mucus interaction Introductio~l (:ollection and preservation of cervical mucus Collection procedure Storage arrd preservatior~ Evalualivn ul cervical rrlucus Volrnnre Consistency Fernlng Spinnhark~it Celhdarity YH Interaction between sperm and cervical mucus In vivo (postcoitul) test In oilro lesls Appendices I A Refereuce values of selneii variables I& Nomenclature fur some scrrlcrl variables I1 Safety guidelines for the andrology laboratory 111 Methods for detecting leukocytes IV Sperm vitality techniques v l'apanicolaou staining pruccdurc rriodilicct for spermatozoa v1 Shorr staining proc,ednre for sperm morphology v11 Rapid staining procedure for sperm morphology VIII Immunobead lcsl IX Mixed antiglobulin reactiou lcsl (MAR test) X Measurement or zinc in seminal plasma XI Mcasurement of fructose in seminal plasma XI1 Measurement of neutral a-glucosidase in seminal plasma XI11 Sample ir~slructionsand record form for serncrl analysis XIV Computer-aided sperm analysis (CASA) xv Protocols for the zurla-free hamster oocyte test XVI Measurement of reactive oxygen species generated by sperm suspensions XVIl Induced acrosome reacliuri assay XVlII Sperm preparalion techniques XIX Quality control (QC) procedures XX Sample record form for cervical mucus scoring and postcoital test XXI Sper~llcervicalmucus interaction: the capillary tube lest XXII Basic requirements fbr an andrology laboratory Bibliography Indcx vii The IJNDP/UNFPA/WHO/World Bank Special Programme of Res- I earch, Developmeril and Research Training in Human Reproduction I wishes to acknowledge the participation, in the preparation and editing of the fourth edition of the WHO Lnborutory Mar~uaIfor the Examination of Hurnun S~rnenand Sperm-Cervical Mucus Interaction, of the following: Dr R. J. Aitken Dr F. Comhaire MRC: Keproduclivc Biology Unit Ilienst Voor Inwendige Zielcten Ccrltre for Reproductive Biology Endocrinologie-Stofwisselingszicklcrl 3 7 Chalmers Street DePintelaan 185 Itdinburgh EH3 9EW 9000 Gent United Kingdom Belgium Dr H. W. G.Baker Dr T. G. Cooper University of Melbonrnc Institute of Reproductive Medicine of Royal Womcn's Hospital the Urliversity Department of Obstetrics and Domagkstrasse 11 (:ynaecology D-48 129 Miinster Carlton 305 3, Victoria Germany Australia Dr C. De Jonge Dr C. L. R. Barratt Department of Obslctrics & Ilniversity Department of Obstetrics Gynaecology and Cyllaecology University of Nebraska Medical Birmingham Women's Hospital Center Edgbastorl 600 South 42 Street, Birmingham B15 2'1'G Omaha, NE 68198-3255 United Kingdom llnited States of America Dr H. M. Behre Dr R. Eliasson Institute or Rcproductive Medicine of Head, A~ldrologyUnit the University Sophiahemmet Hospital Domagkstrasse l l SE 1 14 86 Slockholm D-48 129 Miinster Sweden Germany I viii Ilr T. M. M. Farley Dr J. W. Overstreet UNDP/lINI:t'A/WHO/World Rank Division of Reproductive Biology Special Programme of Research, Deparlment of Obstetrics & 1)evelopment and Rcscarch Training Gynaecology in Human Reproduction One Shields Avenue World Health Organization Ur~iversityof California, Davis 1211Geneva27 Davis, CA95616 Switzerland llnited States of America Mr P. D. Griffin Mrs L. Sellaro, UNDP/UNFI'A/WHO/World Bank UNDP/IINI:PA/WHO/WorId Banlr Special Prograrnme of Kesearch, Special Programme of Kesearch, Development and Research Training Development and Rescarch Training in Human Reproduction in Human Keproduction World Health Organization World Health Organization 1211 Geneva27 1211 Geneva27 Switzcrland Swilzerland Dr I. Huhtaniemi Dr J. Suominen Departmen1 of Physiology Deparirrlcnt of Anatomy llniversity of Turltu University of Turlcu Kiinamyllynkatn 10 Kiinamyllynkatu 10 SF-20520 Turku SF-20520 Turku Finland I:inland Dr T. E Kruger Dr G. M. H. Waites Reproductive Biology Unit c10 Clinique de Sterilite et Tygerberg Hosp~tal d'Endocrinologie (;yn6c,ologique 1'0 llox 19058 HBpital Cantonal Tygcrberg 7505 Universitairr de Genkve South Africa 20 rue Alcidc Jentzer 121 1 Geneva l4 Dr M. T. Mbizvn Swilzerland 1)epartment of Obstclrics and Gynaecology Dr C. C'. L. Wang University of Zimbabwe Clm~calSludy Center (Box 16) 1'0 IloxA 178 Harbor-UCLA Med~calCenter Avorldale 1000 West Carson Street Harare Torrancc, CA 90509 Zimbabwe United States of Alnericd Abbreviations BS A bovine serutn albumin (Cohn fraction V) CASA computer-aided sperni analysis UMSO dirnethyl sullbxide DTT dithiothrdlol EDTA ethylenediaminetelrdacetic acid EQA external quality assessment HIV human irnmunodeficiency virns HOPT hamster oocyte pcrictration test HOS hypo-osmotic swelling test HPF high-power field HSA human serum albumin (Cohn frac,tion V) IBT innnunobead test IQC internal quality control IU international unit IVF in vitro fertilization MAK mixed antiglobulin reaction MA1 multiple ariornalies index PAS periodic acid SchiCL PBS phosphate buffered saline PCT post-coital test oc quality control ROS reactive oxygen species SCMC sperm-cervical mucus contact SDI sperm deformity index SPA sperm penetration assay (synonymous with HOP'I') rrAl kratozoospermia index
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