WHO Laboratory Manual For The Examination And Processing .
WHO laboratory manual for theExamination and processingof human semenFIFTH EDITION
WHO laboratory manual for theExamination and processingof human semenFIFTH EDITION
WHO Library Cataloguing-in-Publication DataWHO laboratory manual for the examination and processing of human semen - 5th ed.Previous editions had different title : WHO laboratory manual for the examination of humansemen and sperm-cervical mucus interaction.1.Semen - chemistry. 2.Semen - laboratory manuals. 3.Spermatozoa - laboratory manuals.4.Sperm count. 5.Sperm-ovum interactions - laboratory manuals. 6.Laboratory techniquesand procedures - standards. 7.Quality control. I.World Health Organization.ISBN 978 92 4 154778 9(NLM classification: QY 190) World Health Organization 2010All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, WorldHealth Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: 41 22 791 3264; fax: 41 22 7914857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications—whether for sale or for noncommercial distribution—should be addressed to WHO Press, at the above address(fax: 41 22 791 4806; e-mail: permissions@who.int).The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of anycountry, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.The mention of specific companies or of certain manufacturers’ products does not imply that they areendorsed or recommended by the World Health Organization in preference to others of a similar nature thatare not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished byinitial capital letters.All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind,either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader.In no event shall the World Health Organization be liable for damages arising from its use.Printed in Switzerland.Cover photo: courtesy of C. Brazil
iiiCONTENTSAcknowledgementsAcronyms and abbreviations used in this manualChapter 11.11.21.3PART I.Chapter nThe fifth editionScope of the manual113SEMEN ANALYSISStandard proceduresIntroductionSample collectionPreparationCollection of semen for diagnostic or research purposesSterile collection of semen for assisted reproductionSterile collection of semen for microbiological analysisCollection of semen at homeCollection of semen by condomSafe handling of specimensInitial macroscopic examinationLiquefactionSemen viscosityAppearance of the ejaculateSemen volumeSemen pHInitial microscopic investigationThorough mixing and representative sampling of semenMaking a wet preparationAggregation of spermatozoaAgglutination of spermatozoaCellular elements other than spermatozoaSperm motilityCategories of sperm movementPreparing and assessing a sample for motilityWorked examplesLower reference limitSperm vitalityVitality test using eosin–nigrosinVitality test using eosin aloneVitality test using hypo-osmotic swellingSperm numbersTypes of counting chamberThe improved Neubauer haemocytometerUsing the haemocytometer gridCare of the counting chamberFixative for diluting semenImportance of counting sufficient 92121222225262627293032343435353636
utine counting procedureDetermining the required dilutionPreparing the dilutions and loading the haemocytometer chambersAssessing sperm numbers in the counting chambersCalculation of the concentration of spermatozoa in semenWorked examplesLower reference limit for sperm concentrationCalculation of the total number of spermatozoa in the ejaculateLower reference limit for total sperm numberLow sperm numbers: cryptozoospermia and suspected azoospermiaWhen an accurate assessment of low sperm numbers is not requiredTaking no further actionExamination of centrifuged samples to detect spermatozoaExamination of non-centrifuged samples to detect motile spermatozoaWhen an accurate assessment of low sperm numbers is requiredAssessing low sperm numbers in the entire improved Neubauer chamber(phase-contrast microscopy)Assessing low sperm numbers in large-volume disposable chambers(fluorescence microscopy)Counting of cells other than spermatozoaCalculation of the concentration of round cells in semenSensitivity of the methodWorked examplesSperm morphologyThe concept of normal spermatozoaPreparation of semen smearsStaining methodsTraditional fixation and sequential stainingPapanicolaou staining procedure for sperm morphologyShorr staining procedure for sperm morphologyRapid staining procedure for sperm morphologyExamining the stained preparationClassification of normal sperm morphologyClassification of abnormal sperm morphologyMorphology platesAnalysing a sperm morphology smearAssessment of normal sperm morphologyWorked examplesLower reference limitAssessment of abnormal sperm morphologyWorked exampleAssessment of specific sperm defectsAssessment of leukocytes in semenStaining cellular peroxidase using ortho-toluidineAssessment of immature germ cells in semenTesting for antibody coating of spermatozoaThe mixed antiglobulin reaction testThe direct immunobead testThe indirect immunobead 8109111113
vChapter ter 44.14.1.14.1.24.24.34.44.4.14.4.24.54.5.14.6PART II.Chapter tional procedures115Indices of multiple sperm defectsCalculation of indices of multiple morphological defectsWorked examplePanleukocyte (CD45) immunocytochemical stainingPrincipleReagentsProcedureInteraction between spermatozoa and cervical mucusIn-vivo (postcoital) testIn-vitro testsIn-vitro simplified slide testCapillary tube testBiochemical assays for accessory sex organ functionMeasurement of zinc in seminal plasmaMeasurement of fructose in seminal plasmaMeasurement of neutral D-glucosidase in seminal plasmaComputer-aided sperm analysisIntroductionUse of CASA to assess sperm motilityUse of CASA to estimate sperm concentrationComputer-aided sperm morphometric 30132134136136137140140Research procedures142Reactive oxygen speciesIntroductionMeasurement of reactive oxygen species generated by sperm suspensionsHuman sperm–oocyte interaction testsHuman zona pellucida binding testsAssessment of the acrosome reactionProcedure for the fluorescence assessment of acrosomal statusInduced acrosome reaction assayZona-free hamster oocyte penetration testProtocolAssessment of sperm chromatin142142143146146147147150152152157SPERM PREPARATIONSperm preparation techniques161IntroductionWhen spermatozoa may need to be separated from seminal plasmaChoice of methodEfficiency of sperm separation from seminal plasma and infectious organismsGeneral principlesSimple washingReagentsProcedureDirect 4164164
vi5.55.5.15.5.25.65.75.7.15.7.25.7.35.85.9Chapter 66.16.26.2.16.2.26.2.3Discontinuous density gradientsReagentsProcedurePreparing HIV-infected semen samplesPreparing testicular and epididymal spermatozoaEnzymatic methodMechanical methodProcessing sperm suspensions for intracytoplasmic sperm injectionPreparing retrograde ejaculation samplesPreparing assisted ejaculation ion of spermatozoa169IntroductionSemen cryopreservation protocolsStandard procedureModified freezing protocols for oligozoospermia and surgically retrievedspermatozoaLabelling of straws and records169172172175176PART III.QUALITY ASSURANCEChapter 7Quality assurance and quality control179Controlling for quality in the andrology laboratoryThe nature of errors in semen analysisMinimizing statistical sampling errorThe quality assurance programmeLaboratory procedures manualInternal quality controlPurchased QC samplesLaboratory-made QC samplesStored samples (purchased or laboratory-made)Fresh QC samples (laboratory-made)Statistical procedures for analysing and reporting within- andamong-technician systematic errorsThe Xbar chartThe S chartQC for percentagesAssessing Xbar and S chartsHow to recognize out-of-control valuesCauses of out-of-control valuesResponses to out-of-control valuesStatistical procedures for analysing and reporting among-technicianvariabilityComparing results from two or more techniciansMonitoring monthly meansExternal quality control and quality assuranceAssessment of EQC resultsResponses to out-of-control resultsFrequency and priority of quality 5188189189189190191191191194194196197197
vii7.137.13.17.13.27.13.37.13.4TrainingPractical hints when experiencing difficulty assessing sperm concentrationPractical hints when experiencing difficulty assessing sperm morphologyPractical hints when experiencing difficulty assessing sperm motilityPractical hints when experiencing difficulty assessing sperm ndix 1 Reference values and semen nomenclatureA1.1A1.2Reference valuesNomenclatureAppendix 2 Equipment and safetyA2.1A2.2A2.3A2.4A2.5Basic supplies needed in an andrology laboratoryPotential biohazards in an andrology laboratorySafety procedures for laboratory personnelSafety procedures for laboratory equipmentSafety precautions when handling liquid nitrogenAppendix 3 MicroscopyA3.1A3.2A3.3A3.4A3.5A3.6A3.7A3.8Loading the sampleAdjusting the ocularsFocusing the imageFocusing the ocularsFocusing the light condenserCentring the condenserAdjusting the phase ringsFluorescence microscopyAppendix 4 Stock Biggers, Whitten and WhittinghamDulbecco’s phosphate-buffered salineEarle’s mediumHam’s F-10 mediumHanks’ balanced salt solutionHuman tubal fluidKrebs–Ringer mediumTris-buffered salineTyrode’s solutionPapanicolaou stainAppendix 5 Cervical mucusA5.1A5.2A5.3Appendix 6A6.1A6.2IntroductionCollection and preservation of cervical mucusEvaluation of cervical mucusRecord forms for semen and cervical mucus analysesTemplate for a semen analysis recording formTemplate for a cervical mucus recording 247251251253
viiiAppendix 7 Sampling errors and quality controlA7.1A7.2A7.3A7.4A7.5A7.6A7.7A7.8Errors in measurement of sperm concentrationThe importance of understanding sampling errorsErrors in measurement of percentagesProduction of semen samples for quality controlPreparation of a video-recording for internal quality control of analysis ofsperm motilityPreparation of diluted semen for internal quality control of determinationof sperm concentrationPreparation of slides for internal quality control of assessment of spermmorphologyCalibration of equipmentAppendix 8 National external quality control programmes for semen analysis254254256257260261265268269271FIGURESFig. 2.1Fig. 2.2Fig. 2.3Fig. 2.4Fig. 2.5Fig. 2.6Fig. 2.7Fig. 2.8Fig. 2.9Fig. 2.10Fig. 2.11Fig. 2.12Fig. 2.13Fig. 2.14Fig. 3.1Fig. 3.2Fig. 3.3Fig. 4.1Fig. 4.2Fig. 4.3Fig. 4.4Fig. 7.1Fig. 7.2Fig. 7.3Fig. 7.4Fig. A2.1Fig. A5.1Fig. A7.1Variation in total number of spermatozoa and sperm concentrationover a one-and-a-half-year periodNon-specific aggregation of spermatozoa in semenSchematic diagram of different extents of sperm agglutinationAids to assessing sperm motilityEosin–nigrosin smear observed in brightfield opticsSchematic representation of typical morphological changes of humanspermatozoa subjected to hypo-osmotic stressThe improved Neubauer haemocytometerWhich spermatozoa to count in the grid squaresScanning the entire coverslip for the presence of motile spermatozoaMorphologically “normal” spermatozoaSemen smearing methods for sperm morphologyPreparing a normal semen smearSchematic drawings of some abnormal forms of human spermatozoaPeroxidase-positive and -negative cells in human semenLeukocytes in semenThe Kremer sperm penetration meterStandard terminology for variables measured by CASA systemsChemiluminescence generated in response to opsonized zymosan treatmentRelative contributions made by leukocyte and sperm subpopulations tothe reactive-oxygen-generating capacity of the cell suspensionFluorescent Pisum sativum agglutinin (PSA) staining of human spermatozoaPhase-contrast micrograph of a zona-free hamster oocyte containinghuman spermatozoaAn Xbar chart for sperm concentrationAn S chart for sperm concentrationA Bland–Altman plot of manual and CASA estimates of percentageprogressive sperm motilityA Youden plot of estimates of the concentration of spermatozoaNomogram for reading relative centrifugal force (RCF) from rotor radiusand rotation speedExamples of fern formation in cervical mucus air-dried on a glass slideThe acceptable differences between two replicate counts as a functionof the total number of spermatozoa 6149157187189192193231246255
ixFig. A7.2Fig. A7.3Fig. A7.4Fig. A7.5The acceptable differences between two replicate assessments of percentage asa function of the true percentage and the total number of spermatozoa assessedAid to assessing sperm motilityView through an ocular with reticle (red grid)View of the videotaped image of the stage micrometer on the monitorand the drawn overlay258263264264BOXESBox 2.1Box 2.2Box 2.3Box 2.4Box 2.5Box 2.6Box 2.7Box 2.8Box 2.9Box 2.10Box 2.11Box 2.12Box 2.13Box 2.14Box 3.1Box 3.2Box 4.1Box 4.2Box 6.1Box 6.2Box 7.1Box 7.2Box 7.3Box 7.4Box 7.5Box 7.6Box 7.7Box 7.8Box 7.9Box A2.1Box A3.1Box A5.1Box A5.2Confirming the compatibility of semen collection vesselsPreparation of bromelainThorough mixing of semenDepth of wet preparationsErrors in estimating percentagesComparison of replicate percentagesErrors in estimating numbersAchieving 200 spermatozoa per replicate in the central three grids ofthe improved Neubauer chamberVolume observed per high-power field of a 20-Pm-deep wet preparationComparison of replicate countsAchieving 200 spermatozoa per replicate in all nine grids of the improvedNeubauer chamberAchieving 200 spermatozoa per replicate in a 100-Pm-deep, large-volumedisposable chamberVolume observed per high-power field in a 100-Pm-deep, large-volumedisposable chamberMounting mediaPreparation of a wax–petroleum jelly mixtureVolume observed per high-power field in a 100-Pm-deep mucus preparationInduction of ovulation in hamstersPreparation of glass pipettesReasons for cryopreservation of spermatozoaRisk assessment of cryopreservation and storage of human semenTerminology in quality assurance and quality controlDetermining the values for the warning and action control limits of an Xbar chartAlternative method for calculating the Xbar control limits from the pooledstandard deviationDetermining the values for the warning and action control limits of an S chartBasic control rules for QC chartsAssessing systematic differences among techniciansMain features of IQC proceduresTime schedule for quality controlSummary of QC testsCalculating centrifugal forcesThe objective lensDetermining the volume of mucus collectedVolume observed per high-power field in a 100-Pm-deep mucus 5170171180186187188190195196198198230235247249
xTABLESTable 2.1Table 2.2Table 2.3Table 2.4Table 2.5Table 2.6Table 2.7Table 3.1Table 3.2Table 3.3Table 3.4Table 7.1Table 7.2Table 7.3Table 7.4Table 7.5Table A1.1Table A1.2Table A1.3Table A7.1Table A7.2Table A7.3Table A7.4Acceptable differences between two percentages for a given average,determined from replicate counts of 200 spermatozoa (total 400 counted)25Rounded sampling errors (%) according to total number of spermatozoacounted37Semen dilutions required, how to make them, which chambers to use and potentialareas to assess39Acceptable differences between two replicate counts for a given sum42Acceptable differences between two counts for a given sum: low concentrations50Explanations used in commentaries to the morphological plates (1–14)71How much semen to use for an immunobead test111Calculation of indices of multiple sperm defects116Sperm defect indices for men from fertile and infertile couples117Rank order of sperm penetration density129Classification of the capillary tube test results129Factors for determining control limits for Xbar charts and S charts based onthe average standard deviation (Sbar)186Sources of variation (error) in assessing sperm concentration and proposedsolutions199Sources of variation (error) in assessing sperm morphology and proposedsolutions200Sources of variation (error) in assessing sperm motility and proposed solutions201Sources of variation (error) in assessing sperm vitality and proposed solutions202Lower reference limits (5th centiles and their 95% confidence intervals)for semen characteristics224Distribution of values for semen parameters from men whose partners becamepregnant within 12 months of discontinuing contraceptive use225Nomenclature related to semen quality226Acceptable differences between two replicate counts for a given sum255Acceptable differences between two percentages for a given average,determined from replicate counts of 100 spermatozoa (total 200 counted)259Acceptable differences between two percentages for a given average,determined from replicate counts of 200 spermatozoa (total 400 counted)259Acceptable differences between two percentages for a given average,determined from replicate counts of 400 spermatozoa (total 800 counted)260
xiAcknowledgementsThis publication was produced by the UNDP/UNFPA/WHO/World Bank Special Programme ofResearch, Development and Research Training in Human Reproduction (HRP), WHO Department of Reproductive Health and Research (RHR). The participation of the following individualsin the preparation and editing of this manual is gratefully acknowledged:Editor-in-chiefDr Trevor G CooperCentre of Reproductive Medicine andAndrology of the University,Münster, Germany(WHO Collaborating Centrefor Research in Male Reproduction)Editorial teamDr John AitkenBiological SciencesSchool of Life and Environmental SciencesUniversity DriveCallaghan, New South Wales, AustraliaDr Jacques AugerService de Biologie de la RéproductionPavillon Cassini Hôpital CochinParis, FranceDr HW Gordon BakerUniversity of MelbourneDepartment of Obstetrics andGynaecologyRoyal Women’s HospitalCarlton, Victoria, AustraliaDr Chris LR BarrattDivision of Maternal andChild Health SciencesThe Medical SchoolNinewells HospitalDundee, ScotlandDr Hermann M BehreCentre for ReproductiveMedicine and AndrologyMartin-Luther-UniversityHalle, GermanyDr Lars BjörndahlAndrology Centre,Karolinska University Hospitaland Institute,Stockholm, SwedenMs Charlene BrazilCenter for Health andthe EnvironmentUniversity of CaliforniaDavis, CA, USADr Christopher De JongeUniversity of Minneso
1.2 The fi fth edition 1 1.3 Scope of the manual 3 PART I. SEMEN ANALYSIS Chapter 2 Standard procedures 7 2.1 Introduction 7 2.2 Sample collection 10 2.2.1 Preparation 10 2.2.2 Collection of semen for diagnostic or research purposes 11 2.2.3 Sterile collection of semen for assisted reproduction 11
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