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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 MinnesotaReproductive Medicine CenterMinneapolis, MN, USADr Gustavo F DoncelCONRADDepartment of Obstetrics andGynecology Eastern VirginiaMedical SchoolNorfolk, VA, USADr Daniel FrankenDepartment of Obstetrics andGynaecologyTygerberg HospitalTygerberg, South AfricaDr Trine B HaugenFaculty of Health SciencesOslo University CollegeOslo, NorwayDr Aucky HintingAndrology Unit,Department of BiomedicineSchool of MedicineAirlangga University,Surabaya, Indonesia

xiiMr Godwin E ImadeDepartment of Obstetrics and GynaecologyFaculty of Medical Sciences University of JosJos, NigeriaDr Thinus F KrugerReproductive Biology UnitStellenbosch UniversityTygerberg, South AfricaDr Hesbon O OdongoDepartment of ZoologyUniversity of NairobiNairobi, KenyaMs Elizabeth NoonanFred Hutchinson Cancer Research CenterStatistical Center for HIV/AIDS Research andPreventionSeattle, WA, USADr Steven M SchraderNational Institute for OccupationalSafety and HealthCenters for Disease Control and PreventionCincinnati, OH, USADr Christina CL WangHarbor-UCLA Medical CenterTorrance, CA, USADr William Shu-Biu YeungDepartment of Obstetrics and GynaecologyUniversity of Hong KongHong Kong SAR, ChinaWHO Secretariat,Department of Reproductive Healthand ResearchDr Kirsten M VogelsongScientistResearch Area ManagerDr Sigrid von EckardsteinFormer Acting Research Area ManagerDr Michael T MbizvoDirector ad interimMs Maud KeizerSecretaryAdditional thanks go to: Ms Cathy Treece, Ms Charlene Tollner and Professor Jim Overstreet(University of California, Davis, CA, USA) for producing the morphology micrographs andchecking the media; Dr Rune Eliasson (Sophiahemmet Hospital, Stockholm, Sweden) for helpin defining non-sperm cells; Dr Timothy Farley (World Health Organization, Geneva, Switzerland) for review of the sections on quality control; and Dr Gary N Clarke (The Royal Women’sHospital, Carlton, Australia), Dr Roelof Menkveld (Tygerberg Academic Hospital and Universityof Stellenbosch, Tygerberg, South Africa), and Professor Pieter Wranz (University of Stellenbosch, Tygerberg, South Africa) for providing additional information used in the compilation ofthe manual.The financial support of the International Society of Andrology is gratefully acknowledged.This edition of the manual is dedicated to the memory of the late Geoffrey Waites (1928–2005),former manager of the WHO Task Force on Methods for the Regulation of Male Fertility and coeditor of the second, third and fourth editions of this laboratory manual. The editorial committee’s devotion to its task was driven by its appreciation of Geoff’s honesty, fairness and concernfor the underprivileged.

xiiiAcronyms and abbreviations used in this AEQAEQCERCFITCFMLPGIFTGPCH 2 O2HBSSHBVhCGHCVHIVHOPHOSHPFHRPantibodyartificial inseminationartificial insemination with donor semenartificial insemination with husband’s semenamplitude of lateral head displacementanalysis of variancealkaline phosphatase–anti-alkaline phosphatase complexacrosome-reactedassisted reproductive technologyanti-sperm antibodyN-benzoyl-L-arginine ethyl esterbeat-cross frequency (Hz)bovine serum albuminBiggers, Whitten and Whittinghamcomputer-aided sperm analysiscomputer-aided sperm morphometric assessmentcongenital bilateral absence of the vas deferenscompact diskcytoplasmic dropletcluster of determination 45 (pan-leukocyte marker)cluster of determination 46 (acrosomal antigen)confidence intervalconfidence limitscarbon dioxidedimethyl sulfoxidedeoxyribonucleic acidDulbecco’s phosphate-buffered salinedigital versatile discethylenediamine tetra-acetic acidexternal quality assuranceexternal quality controlexcess residual cytoplasmfluorescein egamete intrafallopian transferglycerophosphocholinehydrogen peroxideHanks’ balanced salt solutionhepatitis B virushuman chorionic gonadotrophinhepatitis C virushuman immunodeficiency virushamster oocyte penetrationhypo-osmotic swellinghigh-power fieldhorseradish peroxidase

m.SDSDISDSSESOPSTRTBSTGGTZIVAPVCLVSLWHOWOBhuman serum albuminhuman tubal fluidimmunobeadimmunobead testintracytoplasmic sperm injectionimmunoglobulinimmotilityinternal quality controlinternational unitintrauterine inseminationin-vitro fertilizationKrebs–Ringer Mediumlinearitylower limit of quantificationlow-power fieldmean angular displacementmultiple anomalies indexmixed antiglobulin reactionnumerical aperturenon-progressive (motility)phosphate-buffered salineplan, do, check, actphorbol 12-myristate 13-acetatepregnant mare serum gonadotrophinp-nitrophenol glucopyranosideprogressive (motility)Pisum sativum agglutininquality assurancequality controlrelative centrifugal forcerefractive indexribonucleic acidreactive oxygen speciesrevolutions per minutestandard deviationsperm deformity indexsodium dodecyl sulfatestandard errorstandard operating procedurestraightness (VSL/VAP)Tris-buffered salineTyrode’s glucose glycerolteratozoospermia indexaverage path velocitycurvilinear velocitystraight-line (rectilinear) velocityWorld Health Organizationwobble (VAP/VCL)

1CHAPTER 1 Background1.1 IntroductionThe WHO laboratory manual for the examination of human semen and sperm–cervical mucus interaction was first published in 1980, in response to a growingneed for the standardization of procedures for the examination of human semen.It has since been updated three times, and translated into a number of languages.Over the past 30 years, the manual has been recognized as providing globalstandards and has been used extensively by research and clinical laboratoriesthroughout the world.Despite this success, it has become apparent that some recommendations fromprevious editions of the manual needed to be revised in light of new evidence, andthat some concepts needed more explanation and supporting evidence. Promptedby these considerations, WHO established an editorial committee to review all themethods described in the manual, with a view to endorsing, changing or updating them. In many instances, this proved difficult, as insufficient data had beenobtained using the methods described in the manual. In some cases, single wellaccredited laboratories were obtaining consistent results, but these had not beenconfirmed by others. For these situations, the editorial committee developed aconsensus position after evaluating the pertinent literature.Additional recommendations were received from technicians and scientists, notably regarding the need for more detail for many of the methods described. Lack ofdetail in previous editions has meant that some laboratories have preferred to usemethods described elsewhere, or have developed their own versions of methods,while still claiming to perform semen analysis according to the WHO manual. Inorder to make global comparisons easier, this edition of the manual thereforeincludes much greater detail, and the rationale is explained when alternative methods of analysis are presented. It is recommended that, when reporting results inpublished articles, laboratories should indicate which specific method was usedwhen they refer to this manual.1.2 The fifth editionThe fifth edition comprises three parts: semen analysis (Chapters 2–4), spermpreparation (Chapters 5 and 6) and quality assurance (Chapter 7). Part I, dealingwith semen analysis, resembles that in previous editions, but is divided into threechapters: standard methods, which are robust routine procedures for determiningsemen quality; optional tests, which may be used in certain situations or by choiceof the laboratory; and research tests, which are not currently regarded as routine.As semen culture is not normally performed in an andrology laboratory, this ismentioned only in the section on sterile collection of semen. The section on spermpreparation extends beyond the ejaculate to include spermatozoa obtained fromthe testis and epididymis. Interspersed with bulleted methodological instructionsare Notes (explanations of methodology), Comments (interpretation of results) andBoxes (containing additional explanatory material).

2CHAPTER 1 BackgroundThe main features of this fifth edition are outlined below.y The chapters on semen analysis include details of all working solutions, procedures, calculations and interpretation, so that any given methodology is essentially complete, with minimal cross-referencing to other parts of the manual.y The section on sperm preparation has been expanded, and a chapter oncryopreservation of spermatozoa has been added. Procedures related to theanalysis of cervical mucus have been divided between the chapter on optionalprocedures and an appendix on characteristics of mucus.y There are fewer appendices than in earlier editions, and they are restricted tospecialized or only rarely needed information.y Assessment of sperm numbers. The semen dilutions and the areas of thecounting chamber used to assess the number of spermatozoa in a semen sample have been changed to allow 200 spermatozoa per replicate to be counted.The importance of sampling errors, and the certainty of the numerical resultsobtained, is emphasized. The editorial committee considered that total spermnumber per ejaculate provides a more accurate assessment of testicular function than does sperm concentration, but for this semen volume has to bemeasured accurately.y Assessment of azoospermia. Although superficially simple, the diagnosis ofazoospermia is confounded by many factors, including large errors associated with counting few spermatozoa, the large number of microscopic fieldsto be analysed and difficulties in examining debris-laden sperm pellets.Recommended changes include examining fixed, uncentrifuged samples andindicating the sensitivity of the counting methods employed. However, centrifugation methods necessary for accumulating suff

Chapetr 1 Backgor und 1 1.1 Introduction 1 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