WHO LABORATORY MANUAL
for the examination of human semen
and sperm-cervical mucus interaction

FOUKTH EDITION

Published on behalf of theW O R L D IIEALTH ORGANIZATIONbyCAMBRIDGE
UNIVERSITY PRESSP I I R 1 , I S H B D B Y T H E P R E S S S Y N D I C A ' S E 01' T H E l l N T V B R S l T Y O F C A M B R 1 I ) G B

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Q World HealthOrgar~iaation1980, 1987. 1992, 1999

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WHO laboratory ~ n a n n aforl the examination of human semcn and spermc e r v i c a l mucus interaction. - 4th ed.
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Includes bibliographical refcrcuces.
ISBNU 521 h4599 9 (pbk.)
1 . Semen - Examination - 1,aboratory ma~mals. 2. Cervix mucus Examination - Laboratory manuals. 3. Spcrrll-ovum interactionsLaboratory 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 . Spermaloaoachemistry laboratory manuals. 3. Cervix mucus c h e m i s t r ylaboratory manuals. 4. Spernl-Ovum Interactions laboratory manuals.
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Special Programme of Research, Development andPrepared by t h WHO
Research T r a ~ ~ l i nIng Human Reproductloo.
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are distinguished by initial capital letters.

Aclrnowledgcrnc~~ls
Abbreviations
1

Introduction

2

Collection and examination of human semen
STANDARD PROCEDURES
Sample collection and delivery
Safe handling of specimens
Initial macroscopic examination
Liquefucliun
Appearanca
Vol~trne
Viscosily

2A
2.1
2.2

2.4.3
2.4.4
2.4.5
2.5
2.5.1
2.5.2

2.10
2.7 0.1

2.10.22.10.3
2.1 1

Initial microscopic investigation
PreparationJor rauline semen analysis
Preliminary estimation of sperm concentratiorl
Assessment oJ sperm1 motility
Cellular elements olhrr than spermatozoa
Agglutination
Further microscopic examinations
Sperm vitality by dye exclusiun
Assessment of ,sperm r;oncentration
Assessment oJ spernf morphology
Classification of sperm morphalugy
Perfurrninq a sperm morpholngy countTesting for antibody-coating of spermatozoa
Imn~ur~obead
test
Mixeduntiqlobulin reaction test
0I"I'IONAI.TESTS
Calculatioil of indices of multiple sperm dcfccts
Hypo-osmotic swelling (HOS) tcsl
Sc~rlcr~cullureBiochemical assays for ac,cessory sex organ function
Secretory cupacity of the prostate
Secretory cupucity of Ihe seminal vesicles
Secrztor,y capacity of the! epididymis
Computer-aided sperm analysis

pagP viii

2.1 L2C
2.1 3
2.1.3.1

2.13.22.13.3
2. l 4
3

Xona-free hamster oocyte test
RESEARCH TESTSSperm function tests
Keactive ox!jflen species and male infertility
Humnn zonapellucida bindi~rgtests
Assessrner~tof the acrosorrle reactiorr
Computer-aided sperm analysis: morphology
Sperm preparation techniques
Quality control i n the andrology laboratory
Introduction
Nature of errors in measurement
Statistical counting errors in spmen analysis
Other errors i n 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

IA
I&
I1
111
IV

v
v1
v11
VIII

Appendices
Refereuce values of selneii variables
Nomenclature fur some scrrlcrl variables
Safety guidelines for the andrology laboratory
Methods for detecting leukocytes
Sperm vitality techniquesl'apanicolaou staining pruccdurc rriodilicct for spermatozoa
Shorr staining proc,ednre for sperm morphology
Rapid staining procedure for sperm morphology
Immunobead lcsl

34

IX
X
XI
XI1
XI11
XIV

xv
XVI
XVIl
XVlII
XIX
XX
XXI
XXII

Mixed antiglobulin reactiou lcsl (MAR test)
Measurement or zinc in seminal plasma
Mcasurement of fructose in seminal plasma
Measurement of neutral a-glucosidase in seminal plasma
Sample ir~slructionsand record form for serncrl analysis
Computer-aided sperm analysis (CASA)
Protocols for the zurla-free hamster oocyte test
Measurement of reactive oxygen species generated by sperm
suspensions
Induced acrosome reacliuri assay
Sperm preparalion techniques
Quality control (QC) procedures
Sample record form for cervical mucus scoring and postcoital testSper~llcervicalmucus interaction: the capillary tube lest
Basic requirements fbr a n andrology laboratory
Bibliography
Indcx

vii

The IJNDP/UNFPA/WHO/World Bank Special Programme of Research, Developmeril and Research Training in Human Reproduction
wishes to acknowledge the participation, in the preparation andediting of the fourth edition of the WHO Lnborutory Mar~uaIfor the

Examination of Hurnun S ~ r n e nand Sperm-Cervical Mucus Interaction, ofthe following:
Dr R. J. Aitken
MRC: Keproduclivc Biology Unit
Ccrltre for Reproductive Biology
3 7 Chalmers Street
Itdinburgh EH3 9EW
United Kingdom

Dr F. Comhaire
Ilienst Voor Inwendige Zielcten
Endocrinologie-Stofwisselingszicklcrl
DePintelaan 1 8 59000 Gent
Belgium

Dr H. W. G . Baker
University of Melbonrnc
Royal Womcn's Hospital
Department of Obstetrics and(:ynaecology
Carlton 305 3, Victoria
Australia

Dr T. G . Cooper
Institute of Reproductive Medicine of
the Urliversity
Domagkstrasse 11
D-48 129 Miinster
Germany

Dr C. L. R. Barratt
Ilniversity Department of Obstetrics
and Cyllaecology
Birmingham Women's Hospital
Edgbastorl
Birmingham B 1 5 2'1'G
United Kingdom

Dr H. M. Behre
Institute or Rcproductive Medicine of
the University
Domagkstrasse l l
D-48 129 Miinster
GermanyIviii

Dr C. De Jonge
Department of Obslctrics &Gynaecology
University of Nebraska Medical
Center600 South 42 Street,Omaha, NE 68198-3255
llnited States of America
Dr R. Eliasson
Head, A~ldrologyUnit
Sophiahemmet Hospital
SE 1 14 86 Slockholm
SwedenI
I

Ilr T. M. M. Farley
UNDP/lINI:t'A/WHO/World Rank
Special Programme of Research,
1)evelopment and Rcscarch Training
in Human Reproduction
World Health Organization
1 2 1 1 Geneva27
Switzerland

Dr J. W. Overstreet
Division of Reproductive Biology
Deparlment of Obstetrics &Gynaecology
One Shields Avenue
Ur~iversityof California, Davis
Davis, CA95616
llnited States of America

Mr P.D. Griffin
UNDP/UNFI'A/WHO/World Bank
Special Prograrnme of Kesearch,
Development and Research Training
in Human Reproduction
World Health Organization
1211 Geneva27
Switzcrland

Mrs L. Sellaro,
UNDP/IINI:PA/WHO/WorId Banlr
Special Programme of Kesearch,
Development and Rescarch Training
in Human Keproduction
World Health Organization
1 2 1 1 Geneva27
Swilzerland

Dr I. Huhtaniemi
Departmen1 of Physiology
llniversity of Turltu
Kiinamyllynkatn 1 0
SF-20520 Turku
Finland

Dr J. Suominen
Deparirrlcnt of Anatomy
University of Turlcu
Kiinamyllynkatu 1 0
SF-20520 Turku
I:inlandDr T. E Kruger
Reproductive Biology Unit
Tygerberg Hosp~tal
1 ' 0 llox 19058
Tygcrberg 7505
South Africa

Dr G. M. H. Waites
c10 Clinique de Sterilite etd'Endocrinologie (;yn6c,ologique
HBpital Cantonal
Universitairr de Genkve
20 rue Alcidc Jentzer
121 1Geneva l 4
Swilzerland

Dr M. T. Mbizvn
1)epartment of Obstclrics andGynaecology
University of Zimbabwe
1'0 IloxA 1 7 8Avorldale
HarareZimbabwe

Dr C. C'. L. Wang
Clm~calSludy Center (Box 16)
Harbor-UCLA Med~calCenter1000 West Carson Street
Torrancc, CA 90509
United States of AlnericdAbbreviations

BSA
CASA
UMSO
DTT
EDTA
EQA
HIV
HOPT
HOS
HPF
HSA
IBT
IQC
IU
IVF
MAK

MA1
PAS
PBS
PCT

oc
ROS
SCMC
SDI
SPA
rrAl

U
WBC

bovine serutn albumin (Cohn fraction V)
computer-aided sperni analysis
dirnethyl sullbxide
dithiothrdlol
ethylenediaminetelrdacetic acidexternal quality assessment
human irnmunodeficiency virns
hamster oocyte pcrictration test
hypo-osmotic swelling test
high-power field
human serum albumin (Cohn frac,tion V)
innnunobead test
internal quality control
international unit
in vitro fertilization
mixed antiglobulin reaction
multiple ariornalies index
periodic acid SchiCL
phosphate buffered saline
post-coital test
quality control
reactive oxygen species
sperm-cervical mucus contact
sperm deformity index
sperm penetration assay (synonymous with HOP'I')
kratozoospermia index(international) unit
white blood cell (leulocylc)

In responsc to a growing need for the standardization of proceduresfor the examination of human semen, the World Health Organization(WHO) tirst published a Laboratory Manual for the Examination oj'
Hzirizan Semen and Se~nen-Cerviml Mucus Interaction in 1980. The
second and third editions ol the manual followed in 198 7 and 1992.
These publications have been used extensively by clinicians andresearchcrs worldwide. Indeed, the third edition was translated into
the following languages: Arabic, Chinese, French, German,
Indoncsian, Ttalian, Porluguese and Spanish. However, the field of
andrology has corilinued to advance rapidly, and this, togethkr withincreascd awareness of the need for standardized measurements of allsemen variables, has prompted the present revision.
This fourth edition comes at a time of new developments and concerns. The Ianrlmark demonstration of contraceptive efficacy withhornio~iallyinduced suppression of spermatogenesis (World HealthOrgani~alionTask Force on Methods for the Kegulation of Male
Fertility, 1990, 1996) should lead to the development of practical,
efficient and safer hormonal methods of male fertility regulation.
Advances in understanding the genetic basis of male infertility,
together with the successes of assisted reproductive technology,
including intracytoplasmic sperm injection, have regenerated intcrcst
in the field and brought hope for men previously considered infertilc.
These advances in male subfertility treatment have also led to a resurgerice of interest in the assessment of sperm function. Reports ofdeclining sperm counts and increasing incidence of urogenital abnormalities and testicular cancer in some regions of the world havc
aroused public concern. The deterioration of semen quality is not geographically uniform as shown by studies in Denmark, Finland,
France, the United Kingdom and the United States of Amcrica.
Government environmental agencies and national and international
scientific societies, as well as WHO, have conducted discussions on this
topic withoul a clear consensus emerging. Further studies, perhaps
on varied populations in areas with and without known environmental pollutants or toxins, are in progress. The advances in male contraceptivc development and in the treatment of male subfertility,
togcthcr with increasing concerns about the environment andputative cnnscqucnces on male reproductive function, add fresh
urgetlcy to thc scarch for new methods to achieve better standardization and irnprovcmcnt of semen analysis.
For these reasons, the IJNDPIUNFPAIWHOIWorld Bank Special
Programme of Rcscarch, Development and Kesearch Training in
Human Reproduction, after seeking advice from experts throughout
the world via Icttcrs, journals and national andrology societies,
formed a working group (see Acknowledgements) to revise themanual. The prcscnt edition is a companion to the WHO Man~lalfor
the Stnndurdized Irrv~stigntiorznrtd Diagnosis of the infertile Couple published in 1993 by thc Cambridge [Jniversity Press.
Chapler 2 of the present manual, which deals with the examinationof human scmcn, is divided into three major parts. The first partdescribes proccdurcs that are considered to be essential in semen evalualion (Section 2 A , 2.1 to 2. h).The second part comprises proceduresconsidered by most laboratories to be optional but which may be ofclinical diagnostic value (Section 2B, 2.7 to 2.12). The third par1
(Section 2C, 2.1 3 and 2.14) includes methods that assess sperm funclional capacity and developments in computer-aided analyses forsperm morphology. These techniques may not be available in all laboratories but may be of value in the assessment of male subfertility, in
reproduclive toxicology studies or as research tools. In the standard
procerlure section describing sperm morphology, this manual recommends t.he Papanicolaou stain as the preferred method and the use ofa simplified classitication of sperm morphology according to the socalled 'strict critcria'. Computer-aided sperm analysis (CASA) [orsperm motion and the zona-free hamster oocyte test have been
includcd in thc section of optional tests, as these tests may have somediagnostic applications. The section on research tests has been rewritten and updated to reflect the current consensus on the assessment ofsperm functional capacity The statistical basis of counting errors
involved in semcn analysis has been added and the chapter on quality
control has bccn cxpanded to include discussions on practicalmethods of implementing quality control in any andrology laboralory.
In Appcndix I, reference values of semen variables are listed. It,should be noted t,hat it is not the purpose of the manual to establish
lhe rninirnum or lowest semen values compatible with achieving apregnancy, in vivo or in vitro. It proved difficult to reach agreement onsome aspects of the assessment of sperm morphology and on the provision of reference ranges since morphology assessment remains subjective. Reference ranges for human semen presenl some conccptual
difficulties.The relationship of semenquality to fertility is complicatedby many other factors, including female fertility. Thus rncn withabnormal semen may still be fertile while men with bcttcr than

average semen quality produce pregnancics at higher than average
rates. This manual is not designcd only for laboratories that deal with
subfertile couples. It also addrcsscs the needs of laboratories investigating methods for male contraception and studies of reproductive
toxicology. In this contcxt, it is important for this manual to give refpopulation studies of normal men
erence values bascd on nl~~lticentre
and not the mini~nurrlrequirements for fertilization. In addition, thismanual suggests that. it is preferable for each laboratory to determine
its own reftrcnce ranges for each variable (e.g., with samples evaluated for men who have recently achieved a pregnancy).
Finally, it should be emphasized that the major purpose of thismanual is to encourage the use of standard procedures to establish
reference valucs (previously called 'normal' values) for semen analysis. This will permit improved comparability of results between laboratories and thc amalgamation of data from different sources foranalysis. Attcnt,ion to the details of standard procedures should also
sharpen the precision of results and their reproducibility. Above all,the prime objective of the earlier editions has remained: to provide alaboratory manual that will serve the needs of researchers and clinicians in dcvclopiilg countries.

7 Collection and examination of humansemen

Normal semen is a mixture of spcrnmtozoa suspended in secretions
from the testis and epidirlymis which, at the time of ejaculation, are
combined with secretions Srom the prostate, seminal vesicles, and bulbourethral glands. The final composition is a viscous fluid that comprises the ejaculate.
Measurements made on the wholc population of ejaculated spermatozoa cannot define the Sertilixing capacity of the few that reachthe site of fertilization. Neverlheless, scmcn analysis provides essential information on the clinical slatus of thc individual. Clearly, thecollection and analysis of semen must bc undertaken by properly
standardized procedures if the results arc to provide valid information, and this chapter and its associal.ed Appendices (111-XVII) offer
methods for this purpose. These metliods arc divided into standard,
optional, and research procedures.
2A

S T A N D A R D PROCEDURES
The tests described in this part or the manual are accepted proceduresthat constitute the essential steps in semen cvaluation.

Sample collection and delivery
The subject should be given clear, wril.ten or oral instructions concerning the collection and, if rerluirerl, t,ransport of the semen sample(see Appendix XIII).
(a) The sample should be collected aker a minimum of 48 hours but notlonger than seven days of sexual abslinence. To reduce the variabilityof semen analysis results, the number of days of sexual abstinence
should be as constant as possible. The name of the man, the period of
abstinence, the date and time of collecl.ion, completeness of collection, difficulties in producing the sample, thc interval between collection and analysis should be recorded or1 the form accon~panyingeachsemen analysis (see Appendix XIII).
(b) Two samples should be collected for initial evaluation. The interval
between the two colleclions shonld not be less than 7 days or morethan 3 weeks. If the results of thcsc two assessments are markedly
different, additional samples should be examined because the resullsof a man's semen analysis can vary considerably (Fig. 2.1).

2.1

Pig. 2.1. Sperm cunwnlrations inthe semen of one man collcctcd
biweekly over 120 weeks. During
this period Lhe Inan received no
medication and expcricnccd nofebrile illness. The dotted line
irltlicates 20 X 10Vm1 (seeAppendix IA). The data illilstrate
the marked variations in spcrnl
concentration that can occur in thesemen of some men. (Unpublisheddata from C.A. Paulsen.)

180 -I 60 -

.
-

2

140 -

p 120-= 100-2 80 -

.

€
Y1

60

-

40-

20 - .-- - - - - - - -10

-

0 - ,
0

10

20

30

40

50

60
7C
Weeks

80

90

100 110 120(c) Ideally, the sample should be collected in a private room near the laboratory. If this is not possible, it should he delivered to the laboratory
within 1hour of collection. When t.he motility of the spermatozoa isabnormally low (less than 2 5% of spermatozoa showing rapid, progressive motility, see Section 2.4.3), the interval between collection
and analysis of the second sample should be as short as possible
bccause motility declines with time (Eliasson, 1981). If tests of sperm
function are to be performed, it is critical that the spermatozoa be separatcd fi-om the seminal plasma within one hour of ejaculation (seeAppendix XVIII).
(d) The sample should be obtained by masturbatiorl and ejaculated into aclean, wide-mouthed container made of glass or plastic. It should bewarm (20-40°C) to avoid reduction in sperm motility and from abatch that has been checked for toxic effects on sperm motility (seeAppendix XXII). If a microbiological analysis is to bc done, the subject
should pass urine and then wash and rinse his hands and penis before
ejaculating into a sterile container (see Section 2.9).
(e) When circumstances prevent collection by masturbation, special
condoms are available for semen collection (Milex products Inc.,
Chicago, Illinois h063 1,USA; Male Factor PackR,HygieneR,FertiPro
N.V., Belgium). Ordinary latex condoms must not be used for semencollection because they interfere with the viability of the spermatozoa. Coitus irrtt-rruptus is not acceptable as a means of collection
because the tirst portion of the ejaculate, which usually contains
the highest concentration of spermatozoa, may be lost. Moreover,there will bc cellular and bacteriological contamination of thesample and the acid pH of the vaginal fluid adversely affects sperm
molilitv.
(Q It is important to emphasize to the subject that the semen sample
should be complete.

(g) The sample should be protccted from extremes of temperature (lessthan 20 "C and more than 40 "C) during transport to the laboratory.
(h) The container must be adcqnately labelled with the subject's name(andlor identification numbcr) and with the date and time of collection.
2.2

Safe handling of specimens
Laboratory technicians should be aware that semen san~plesmay
contain harmful int'ectious agents (e.g., viruses causing acquired
immunodeficiency ~yndrome,hepatitis, and herpes simplcx) andshould therefore bc handled as a biohazard with extreme care. If
semen culture is to bc performed or the sample is to be processed forbioassays, intrautcrinc insemination, or in vitro fertilization, sterilematerials and techniques must be used in handling the samples.
Safety guidelines as outlined in Appendix I1 should be strictly
observed. Good laboratory practice is fundamental to laboratory
safety (WHO, 1983).

2.3

Initial macroscopic examination

2.3.1

Liquefaction
A normal semcn sample liquefies within 60 minutes at room lemperature, although usually lhis occurs within 1 5 minutes. In some cases,
complete liquefaction does not occur within 60 minutes, and thisshould be recorded. Norrnal semen samples may contain jclly-likegrains (gelatinous bodies) which do not liquefy and do not appear tohave any clinical significance. The presence of mucous strcalts may
interfere with semen analysis.
The sample must bc well mixed in the original container and mustnot be shaken vigorously. During liquefaction, continuous gentle
mixing or rotation of the sample container may reduce errors indetermining sperm concentration (de Ziegler et al., 1987). If sperm
motility is to be asscssed a1 37% the sample should be equilibrated tothis temperature during liquefaction and mixing.
Occasionally, samples may not liquefy, in which case additional
treatment, mechanical ~nixingor enzyme digestion (e.g., bromelain1gll) may be necessary. Some samples can also be induced to liquefy
by the addition of' an equal volume of medium followed by repeatedpipetting. All thcsc n~anipulationsmay affect seminal plasma biochemistry, sperm motility and sperm morphology and their usc must
be recorded.

2.3.2

Appearance
The semen samplc should be examined immediately after liqucfaction
or within one hour of ejaculation, first by simple inspection at room

temperature. A normal sample has a homogenous, grey-opalescentappearance. It may appear less opaque if the sperm concentration isvery low, red-brown when red blood cells are present or ycllow in apatient with jaundice or taking some vitamins.
2.3.3

VolumeThe volume of the ejaculate may be measured i~singa graduatedcylinder with a conical basc or by weighing standard containers withand without semen. Plastic syringes should not be used because they
may affect sperm motility and l~ypodermicneedles are unsafe.

2.3.4

Viscosity
The viscosity (sometimes refcrrcd to as 'consistency') of the liquefied
sample should be recognized as bcing different from coagulation. It
can be estimated by gcntlc aspiration into a wide-bore 5-m1 pipetteand then allowing thc scrncn to drop by gravity and observing thelength of the thread. A normal sample leaves the pipette as small discrete drops. In cascs of abnormal viscosity the drop will form a thread
more than 2 cm long. Alternatively, the viscosity may be evaluated byintroducing a glass rod into the sample and observing the length of
the thread that forms on withdrawal of the rod. Again, the thread
should not exceed 2 cm.
High viscosity can interfere with determinations of sperm motility,
concentration and antibody coating of spermatozoa. The methods toreduce viscosity are the same as those for delayed liquefaction (seeSection 2.3.1 .).

2.3.5

pH
The pH should be measured at a uniform time within one hour of
ejaculation. A drop of semen is spread evenly onto the pH paper(range: pH 6.1 to 10.0 or 6.4 to 8.0). After 30 seconds, the colour of
the irnprcgnated zone should be uniform and is compared with thecalibration strip to read the pII. Whatever type of pH paper is used forthis analysis, its accuracy should be checked against lrnown standards.
If the pH is less than 7.0 in a sample with azoospermia, there may
bc obstruction of the ejaculatory ducts or bilateral congenital absenceof thc vasa.

2.4

Initial microscopic investigation
During the initial n~icroscopicinvestigation of the sample, estimates
are made of the concentration, motility, agglutination of spermatozoa, and presencc of cellular elements other than spermatozoa.
A phase-contrast microscope is recommended for all examinationsof unstaincd preparations of fresh semen or washed spermatozoa. An

ordinary light rnicroscope can be used for unstained preparations,particularly if the condcnscr is lowered to disperse the light.
2.4.1

Preparation for routine semen analysis
The volume of semen and the dimensions of the coverslip must be
standardixed so that the analyses are always carried out in a preparation of fixed depth of about 20 pm. This allows a rough estimate ofsperm concentration to be made in ordcr to determine how to prepare
the semen for lhe accurate dctcrmination of sperm concentration.
Depths less than 20 pm may constrain the rotational movement ofspermatozoa.
A fixed volume of 1 0 ~1 semen is delivcrcd onto a clean glass slidewith a positive displacement pipcttc and covered with a 22 mm X 22mm coverslip. The weight of thc coverslip spreads the sample for
optimum viewing and care should bc taken to avoid forming and trapping bubbles between the covcrslip and the slide.
The freshly made wet preparation is left to stabilize for approximately one minute. Since sperm motility and velocity are highly
dependent on temperalure, the assessrncnt of motility should preferably be performed at 3 7 'C, using a warmed stage. The examination
may be carried out at room temperature, bctween 20 and 24 "C, buttemperature must be standardixed in the laboratory as it will affect theclassification of the grades of motility. Initial evaluation at 100 X
total magnification (i.e., 1 0 X objective and 1 0 X ocular) provides an
overview for determining mucus strand formation, sperm aggregation, and the evenness of spread of spermatozoa on the slide. The
preparation is then examined at a magnification of 400 X total
magnification.

2.4.2

Preliminary estimation of sperm concentration
With different microscopes the 400 X magnification field of view
varies in diameter, usually in the range bctween 250 and 400 pm corresponding to 1 to 2.5 nl with a 20 pm depth of sample. The diameter of the field can be delermined with a micrometer or the grid of acounting chamber. Scanning the slide and estimating the number ofspermatozoa per field or par1 of a field cquivalent to 1 nl gives anapproximate sperm concentration in 106/ml.This estimate is used todecide the dilution for determining the sperm concentration byhaemocytometry: < 15 spermatozoa, dilution 1:5; 15-40 spermatozoa, dilution 1:10; 40-200 spermatozoa, dilution 1:20; >200 spermatozoa, dilution 1:50 (see Table 2.1 ).
If the number of spermatozoa pcr visual field varies considerably, it
indicates that the sample is not homogeneous. In such cases, thesemen sample should be mixcd again thoroughly. Lack of homogeneity may also result from abnormal consistency, abnormal liquefaction,

Table 2.1. Dilutions and conversion frrctola for the iinpmved Neubauer

Conversion factors
Numbcr of large squares counted
Spermatozoa pcr
400 X field

Dilution(semen +diluent)

25

10

5

aggregation of spermatozoa in mucous threads, or from sperm agglutination. These abnormalities should bc mentioned in the semen
analysis report.
If the number of spermatozoa is low (<l-2/field, 4 0 0 X
magnification), it is possible to determine thc sperm concentration
after centrifugation to concentrate the sample. Onc millilitre of semen(or as much of the ejaculate as possible) is centrifugcd at 6008 for 1 5
minutes. A known voluine of the seminal plasma is thcn removed andthe remainder is thoroughly mixed and countcd as described in
Section 2.5.2. The final concentration is corrected to include thevolume of the supernatant removed. Sperm motility and morphology
asscssn~entscan also be performed on the sample after ccntrifugation.
If thc number of spermatozoa is low (<l-2/field), it is suflicient forclinical purposes to report the concentration as 1 2 X lO"m1 with anot,c on whether or not motile spermatozoa were seen.
All samples in which no spermatozoa are detected by microscopy
should bc ccntrifuged to detect the presence of spermat.ozoa in thesediment. Centrifugation at >3000gfor 1 5 minutes is recommended.
Only when no spcrmatozoa are found after a complete and systematic
search of all of the resuspended precipitate should samplcs beclassified as azoospcrmic.
2.4.3

a,

Assessment of sperm motility

A simple grading systcm is recommended which provides an assessment of sperm motility without the need for complex equiprnent.
Other mel.hods of assessment of sperm motility, inc,luding cornputcraided sperrn arlalysis (CASA), are given in Section 2.11.
At. least fivc microscopic fields are assessed in a systematic way toclassify 200 spcrmatozoa (Fig. 2.2). The motility of each spermatozoon is graded 'a', 'h', 'c', or 'd', according to whether it shows:
rapid progrcssivc motility (i.e., 2 2 5pm/s at 3 7 O C and 2 20 prnls at
20°C; notc that 25 kin is approximately equal to five head lengths orhalf a tail Icngth);Fig. 2.2. (a)Tramlines in the field dview of the ~nicroscopeto aid
assessment of sperm motility. (b)Systematic selection of eight fields
for assessment of sperm lnotility atleast 5 mm from the edges of thecover slip.

b.
c.
d.

slow or sluggish progressive motility:
nonprogressive motility (<5 km/s);
immotilily.
Within a defined area of the field indicated by lines (Fig. 2.2(a) and ( b ) )forrned by a graticule in the focal plane of the rnicroscopc, or in thewhole field if the sperm concentration is low, all spermatozoa withgrade a and b motility are counted first. Subsequently spermatozoa
with nonprogressive motility (grade c) and immotile spermatozoa(graded) art: counted in the same area. Thenumber of spermatozoa ineach category can be tallied with the aid of a laboratory counter. The
1count of 200 spermatozoa is repeated on a separate 1 0 ~ speci~ncn
from the same semen sample and the percentages in each motility
grade from Lhe two independent counts are compared. Figurc 2.3
shows the range of differences between the percentages that arc
expected to occur in 95% of samples due to counting error alonc.
Largcr differences would suggest that miscounting had occurred orthat the spermalozoa were not randomly distributed on the slide. In
this case two new slides should be prepared and sperm motility
reassessed.

2.4.4

Cellular elements other than spermatozoa
Thc cjaculale invariably contains cells other than spermatozoa collectively rekrred to as 'round cells'. These include epithclial cells fromthe gerlitourinary tract, prostate cells, spermatogenic cells, and Icukocytcs (see Fig. 2.4). As a general guide, a normal ejaculate should notcontain more than 5 X 10hround cells/ml.

L~ukocytes Leukocytes, predominantly neutrophils, are present inmost human ejaculates (Tomlinson et al., 1 9 93 ) . Excessive numbers
may be associated with infection andof these cells (le~~kocytospermia)

Fig. 2.3. Approximate 95%
confiderlce interval ranges fordifferences bclween two
percentages detcrmir~txlfroln
duplicate counts of 100, 100 or
400 spermatozoa. To assess thepercentages from two counts of200, calculate their average anddifference.If the difference isgreater than indicated by thc
N= LOO curve,the sample must bc
reanalysed. The lower curve shows
the increase in precisior~br two
assessments of 400 sperrnaturuu(N=400). For an average result of
5 ' X , the 95% confidence interval is
3%. The large statisticdl counting
crrors associated with counting
only 100 spermatozoa are apparent
from the top curve (N-100). For a
result of 5%. the two single
asscssments of l00 spermatozoa
could be 3% and 9% by chancealone. (See Appcncli XXII for alarger version of this graph.)

0%

10%

20%
30%
Averdgc percentage

40%

50%

Exnmnl11~1:
Spern~motility in duplicate counts of 200 spernlatozoa are: grade a = 3%
and 20'Y":b = 32% and 40%; c = 5% and 5%; and d = 60% and 35%. The
most conlmon category is grade d motility (60% and 35%), with avcrage
47.5% and differencc 25%. From ITig. 2.3 it can be seen thal, for N=20O
spermatozoa in each sample, and fur a n average of 47.5%, a difference of up
to 10%would be expected l u occur by chance but a differenc,eof 25% would
not. Thus the results are discarded and a fresh preparation estimated. Note
that,, if thc percentage difference of one grade of motility is larger than
expected, then often that of another grade is also larger, as here for grade a.
Only the difference in the perr~ntagesof the most commorl grade need becompared.

Eeamplr L:
Sperm motility in duplicalc counts of' 200 spermatozoa are: grade a = 32%
and 2 5%; b = 4% and 3%:c = 4% and 4% and d = GO'L and 68%. The mostcommon category is grade d, will1 average 64% and difference 8%. Since thc
avcrage percentage 64% is not in Fig. 2.3, lhe expected difference for a n
average percentage 36% (= 100%)-64%) is looked up. For N = 200 in eachsample, differences 11pto 9.5% occur by chance alone and the observed difference is less than this. The mean percentages for each gradc are calculated:
2X.5%, 3.5%,4%,and 64%.Nolcthat theconventionis to round 0.5% to tllc
r~caresteven numher, i.c., 28.5% down to 28% but 3.5% t,o 4%. The percentages reportcd should add up to 100%by adjusting thc most common
group if necessary, i.e., a = 28%. b=4%, c = 4 % andd- 64%).

poor sperm quality (Wolff et al., 1990). The number of leukocytes
should not exceed 1 X 10h/ml.
Several techniques have bee11 devised for quantifying the leukocytc
population in semen. Two cytochcmical techniques based on the presence of intraccllular peroxidase and on leukocyte-specific antigens)
are given in Appendix 111. The pcroxidase technique (Fig. 2 . 4 ~givesPig. 2.4. Leukocytes ill humansemen (400x1. (a)Stained with theprroxidasemcthod. (From C. De
Jonye; sce Appendix 111.1.) (b)Stained with a molloclunal
antibody against the common
leukocyte antigen CD45 (seeAppendix 111.2).

estimates that are lower than those obtained using pan-leukocyte
monoclonal antibodies (Fig. 2.4b).
When the number of leukocytes in semen is high, rriicrobiologicaltests should be performed to investigate if there is an accessory gland
infection. Such tests include the examination of the first voided urine,
the second voided urine, the expressed prostatic fluid, and the postprostatic massage urine (Mearcs & Stamey, 1972). They also include
the biochcmical analysis of seminal plasma, sincc infection of theaccessory glands often causes abriormal secretory function (Section
2.10; Comhaire et al., 1980). However the absence of leul<ocytesdoes

not exclude the possibility of an accessory gland infection (see World
Health Organization, 1993).

Immature germ cells 'l'he round cells other than Icul<ocytesinclude
round spermatids, spermatocytes, spermatogonia, and exfoliatedepithelia1 cells. These are often degenerating and difficult to identify.
The different types of immature germ cells appcaring in semen (Fig.
2.11) are usually indicative of disorders of spermatogenesis; theiridentification can be aided by the use of thc Rryan-Leishman stain(see Appendix V111 of the third edition of this manual). They may be
distinguished from leul<ocytesby thcir cytological fealures and theabsence of intracellular pcroxidase and lack of leukocyte-specificantigens (see Appendix 111). Round spermatids may be identified bystaining the developing acrosome, e.g., with periodic acid Schiff stain.
Excessive shedding of immature germ cells usually results from
impaired seminifcrous tubule function as in hypospermatogenesis,
varicocele, and Scrtoli cell dysfunction and is associated with reduced
et al., 1992).
success of in vitro fertilization (T~~mlinson
Counting cells other than sperrnntozoa The concentration of such cells
can be estimated in wet preparations using a suitable haemocytometer in the same way as spcrmatozoa (see Section 2.5.2). Since only
spermatozoa arc included in the sperm count, the concentration ofother types of germ cclls or leulocytes can be calculated relative tothat of spermatozoa. If N is the number of a given cell type counted in
the same field@)as 1 00 spermatozoa and S is the sperm concentrationin millionsiml, then the concentration C of the given cell type in millions/ml can bc calculated from the formula

c=-NXS100

For example, if the number of immature germ cells or leukocytes
counted is 1 0 per 100 spermalo~oaand the sperm count is 120 X
106/ml,the concentratiori of such cells is
1 0 X 120 X 106
per millilitre = 12 million per ml.
100
If no cells are secn for the whole slide, it is appropriate to report that
there are fewer than 3.7 cellslunit volume examined (the upper 9 5%
confidencelimits of 0 is 3.7 in the Poisson distribution).Thus for a 1p1
sample the count is < 3 700 cellsiml, which is the lower limit of detection. The counting errors are large when only a few leulcocytes are
seen. In research studics of seminal leulocytes or other cells, larger
volumes of semen arc nccded t,ocounl200 cells of interest in order toachieve acceptably low count.ingerrors (see Section 2.5.2).

2.4.5

Agglutination
Agglutinalion of spermatozoa means that motile spermatozoa stick toeach other head to head, tail to tail or in a mixed way, e.g., head to tail.
The adherer~reeither of immotile spermatozoa to each other or ofrnotile spermatozoa to mucous threads, cells other than spermatozoa,
or debris is considcred to be nonspecific aggregation rather than
agglutination and should be recorded as such.
The presence of agglutination is suggestive of, but not sufficient evidence for, an immunological cause of infertility. Agglutination isassessed at the time of dctcrmining sperm motility. The type of agglutination should be recordcd, c.g., head to head, tail to tail or mixed. A
semiquantitalive grading from - (no agglutination) to + + (severe
clumping in which all the ~notilcspcrmatozoa are agglutinated), canbe used.

+

2.5

Further microscopic examinations

2.5.1

Sperm vitality by dye exclusion
Sperm vitality is reflcctcd in the proportion of spermatozoa that are'alive' as determiried by cithcr dye exclusion or hypo-osmotic swelling(see Section 2.8). This should be determined if the percentage ofimmotile spermatozoa excccds 50%. The proportion of live spermatozoa can be determined by using staining techniques that are based on
the principle that dead cclls with a damaged plasma membrane takeup certain stains. Details of the protocols for performing these techniques are given in Appendix IV.
Two hundred spermatozoa are counted with the light or phasecontrast microscope, differentiating the live (unstained) spermalo~oafrom thc dead (stained) cells.
Sperm vitality assessments provide a checlr on the accuracy of the
motility evaluation, since the percentage of dead cells should notexcccd (within counting error) the percentage of immotile sperrnatozoa. The presence of a large proportion of vital but immolile cells may
bc indicative of structural defects in the flagellum.

2.5.2

Assessment of sperm concentration
The concentration of spermatozoa should be determined using thc
haemocytometer method on two separate preparations of thc semen
sample, one for each side of the counting chamber. The dilution is
dctcrmined (1:5, 1:10, 1:20, 1:50) from the preliminary estimationof sperm concentration (Section 2.4.2). For example, a 1:20 dilution
is madc by diluting 5p1 of liquefied semen with 95p1 of diluent. The
dilucnt i~ prepared by adding to distilled water 50 g sodium bicarbonate (NaHCO,), 1 0 m1 35% (vlv) formalin, and either 0.25 g
trypan blue (Colour Index C.1. 23850) or 5 rnl saturated aqueousPig. 2.5. The central grid of the
improvcd Neubauer
haemocytometcr cur~lai~ls
25squares in which the spermaloeoa
arc Lobe coullted (see Section 2.5.1
and Tablc 1.l).

gentian violet, and making up the solution to a tinal volume of 1
litre. The stain need no1 be included if pha~e-~ont,rast
inicroscopy is
uscd.
White-blood-cell pipettcs and automatic pipettcs relying upon air
displacement are not accurate enough for maliing volumetric dilutions of semen. A positive-displacementtype of pipette should be used(see Appendix XXII).
Secure the coverslip on the counting c,hambers of the improved
Neubauer haemocytornetcr by lightly wetting either side of the wells(use a drop of water on the finger).Press the coverslip firmly onto thechambers so that iridescence (Newton's rings) is observed between
the two glass surfaces. Transfer approximately 10 p1 of the thoroughly mixed diluted specimen from each duplicate dilution to eachof the counting chambers of the haemocytometer. This is done bycarefully touching the edge of the cover glass with the pipette tip andallowing each chamber to till by capillary action. The chambers
should not be over or underfilled and the cover glass should not be
moved. The haemocytomcter is allowed to stand for about five minutes
in a humid chamber to prcvent drying out. The cells sediment during
this time and are then counted, preferably with a phase-contrast
microscope. at a magnification of 200 to 4 0 0 X . The count should bemade of complete spermatozoa (heads with tails). Defective spermatozoa (pinheads and tailless heads) should also be counted but
recorded separately.
The procedure for counting the spermatozoa in the haemocytometer chamber is as follows. The central square of the grid in an
improved Neubauer haemocytometer contai~ls2 5 large squares, eachcontaining 1 6 smaller squares (Fig. 2.5). For samples containing
fewer than ten spermatozoa per large square, spermatozoa in thewhole grid of 25 large squares should be assessed; for samples

Fig. 2.6. Approximate 95%

confidence interval for dsfercnces
between two counts. 'lo assess thesecounts, cdlculate their sum anddillcrcnce.If the diffel.enCeis
greater than indicated by lhc curve
there may be a syste~naliccrror (seeChapter 4) and Lhc sample must hereanalyscd. The large statisticalcuunting errors associated withcounting fewer than 200
spermatozoa are apparent. Por
example,b r a mean count of 60
spermatozoa (sum 120),the twocounts could he 50 and 70 bychance alone. (Sec Appendix XXIT
for a larger vcrsion of this graph.)
100

m

100

m

Suin nf Lhe two counts

conlaining ten to 40 spermatozoa per large square, ten large squares
should be assessed; and, for samples containing more than 40 spermatozoa per large square, spermatozoa in five large squarcs should beassessed. If a spermatozoan lies on the line dividing two adjacentsquares, it should be counted only if it is on the upper or the left sideof the square being assessed.
Duplicate counts of 200 spcrrnatozoa must be performcd to achieve
an acceptably low counting crror. To check the two counts, calculate
their sum and difference. Figure 2.6 shows the range of differencesthat are expected to occur in 9 5%)of samples from statistical counting
error alone. Larger differences suggest a n error of dilution, a miscounting, or a nonrandom distribution of spermatozoa in the diluted
semen. When differences in counts are greater than those indicated by
the curve in Fig. 2.6, two fresh duplicate dilutions of semen must be
prepared and recounted.

Example Z :The courils of ten squares in each chanibcr give 2 50 and 198 spermatozoa.
'I'he sum is 448 a ~ i lllc
d difference 52 is greater than Ihc number expected bychance (Pig. 2.6). The results arc thus discarded, the sample thoroughly
remixed, dilution performed in duplicate agairl and each counte,d.
Exarr~pIeL:
Counts of ten squarcs in each chamber yield 247 arid 2 13 spermatozoa. The
sum is 4 h 0 and the difference is 34. Fig. 2.6 shows that, for two counts witha total of 4 6 0 spermatozoa, differences lcss than 42 frequently occur bychance alone. and thc observed difference is below this limit. The average
couril is calculated (46012 = 230) and the concentration estimatedlrom the1
factors in Tahle 2.1.

Fig. 2.7. Making smears forslairli~lgfor sperm morphology. (a)'Feathering' n~elhodfor undiluted
semen. The semen drop (S) spreads
along the back edge of the angled
slidc U I I is
~ pi~lledfr>r~vard
over theflat slide to form the smear. (h)Pipette method for washed samples.
Adrop of the suspension (S) is
sprcad over the surface of the slideby thc hori~ontallyheld pipette (P).

In order to determine the concentratiorl of spermatozoa in the original semen sample in millions/ml, the average number of spermat,ozoa is divided by the appropriate conversion factor shown inTable 2.1.
For example, for an average count of 230 on a 1: 2 0 dilution and ten
squares counted per chamber, the conversion factor is 2 and thesperm concentration 1 1 5 X 10h/ml.
Chambers, other than the haemocytometer, are available for determining sperm c,oncentration, e.g., the Malilcr chamber (Makler,
1980) and the Microcell (Cinsburg & Armant,, 1990). Such c,hambcrs, while convenienl in that they can be user1 without dilution of thespecimen, may lack the accuracy and precision 01' the haemocytometer technique. When used, it is recommended that their validity beestablished by comparison with the haemocytonletcr method.
Assessment of sperm morphology
Although the morphological variability of lhe human spermatozoon
rnalics sperm morphology assessment difficull, observations on spernlatozoa recovered from the female reproductive tract (especially inpostcoital cervical mucus) or from the surfacc of the zona pellucida
have helped to define the appearance of a normal spermatozoon(Frcdricsson & Bjork, 1977; Mortimer et al., 1982; Menkveld et al.,
1990; Liu &Baker. 1992) .

Preparation of smears At least two smears should be made from lhe
fresh semen sample for duplicate assessment and in case of problems
with staining. The slides should first be thoroughly cleaned, washedin 70% ethanol and dried, before a small drop of semen (5 to 20 p1) isapplied to the slide. If the sperm concentration is over 2 0 X 1Ob1ml.
thcn 5 +l of seliien can be used; if the sperm corlcentration is less than
20 X 10h/ml,then 1 0 to 20 ~1of semen should be used.
The 'feathering' technique (whereby the edge of a second slide isused to drag a drop of semen along the surrace of the cleaned slidc(Fig. 2.7(a)) may be used to make smears of spermatozoa, bul carc
rrjust be taken not to makc the smears too thick. Feathering works wellwhen viscosity is low but is often unsuitable for viscous semen.
Alternatively, a drop of semen can be placed in the middle of a slide
and then a second slide, face down, placed on top so that the semenspreads bct,ween them; the two slides are then gently pulled apart tomake two smears simultaneously
Somelimes good smears are difficult to prepare because of thevarying viscosity of scminal plasma resulting in uneven thicliness ofthe smear. Debris and a large amount of particulate material may
for low sperm
cause spermatozoa to lie with their heads on edge. Tl~us,
concentrations or viscous or debris-laden samples, or when cornputer-assisted morphology is to be done, seminal plasma may bediluted and removed after centrifugation (Liu & Baker. 1992;
Menkveld & ICruger, 1996). Thc sperm pellet is resuspended in a nappropriate volume to obtain the highcst sperm concentration possible, but not exceeding 80X 10Gl/ml.An aliquot of 0.2 to 0.5 m1 ofsemen, depending on sperm concentration, is diluted to 1 0 m1 withnormal saline at room temperature. Thc tube is centrifuged at 800g
for 10 minutes and most of the supcrnatant tipped off. The pellet isresuspended in the remaining saline, typically 20-40 ~ 1 by, gently
tapping the tube. Then 5-10 p1 oS this suspcnsion is placed on a glass
microscope slide and the drop is spread across the slide with a pipetteas illustrated in Fig. 2.7(b). The slide is thcn scanned at 4 0 0 X total
magnification to ensure that a smear has an even spread of spermatozoa and that there are at least 40 spermatozoa per 4 0 0 X field
without sperm clumping or overlapping. Tf t,hc smear is too dense, asmaller volume is used or the sample is f~irthcrdiluted and anothersmear made. If the spermatozoa are too sparse on the slide, moresemen is used to obtain a greater number of spcr~natozoa.
These slides
are allowed to dry in air and then fixed. The fixation procedure
depends on the staining method.
Staining rnethods Papanicolaou slain is thc nlethod most widely usedin andrology laboratories and is the mcthod recommended by thismanual. It gives good staining of the spcrrnatozoa and other cells. It
permits staining of the acrosomal and post-acrosomal regions of thehead, the cytoplasmic droplet, the midpiece, and the tail (Appendix V
and Fig. 2.9). The Shorr stain (Appendix V1 and Fig. 2.10) gives
similar results to those of the Papanicolaou stain for normal forms(Meschede et al., 1993).In some laboratories, a rapid staining method
such as the Uiff-Quik is used (Icruger et al., 1987, and Appendix VII).
Some smears stained by rapid procedures may have backgroundstaining and may not always givc the same quality as the
Papanicolaou stain. Also, the sixe of the head of the spermatozoa
stained by Diff-Quik is larger than that stained by Papanicolaou or
Shorr stains (Icruger et al., 1988).
With these stains, the head is staincd pale blue in the acrosomal
region and dark blue in the post-acrosomal region. The midpiece may
show some red staining. The tail is also stained blue or reddish.

Cytoplasmic droplets, usually located behind the head and around themicipiece, are stained green with the Papanicolaou stain (Pig. 2.9).
2.5.4

Classifcation of sperm morphology

The heads of stained human spermatozoa are slightly smaller thanthe heads of living spermatozoa in the original semen, although theirshapes are not appreciably different (Katz et al., 1986). Strict criteria
should be applied when assessing the morphological ilormality of the
spermatozoon (Menlrveld et al., 1990).For a spermatozoon to be considered normal, the sperm head, neck, midpiece, and tail must be
normal. The head should be oval in shape. Allowing for the slightshrinkage that fixation and staining induce, the length of the headshouldbe4.0-5.0 Km and the width 2.5-3.5 ~ml'helength-to-width
ratio should be 1.50 to 1.75. These ranges are the 95% confidence
limits for Papanicolaou-stained sperm heads (Katz et al., 1986).
Estimation of the length and width of the spermatozoon can be madewith an ocular micrometer. There should be a well-defined acrosomal
region comprising 4U-70% of the head area. The midpiece should be
slender, less than 1p m in width, about one and a half times the lengthof the head, and attached axially to the head. Cytoplasmic droplets
should be less than half the size of the normal head. The tail should
be straight, uniform, thinner than the midpiece, uncoiled and approximately 45 p m long. This classification scheme requires that all 'borderline' forms be considered abnormal (ICrugeret al., 1 986; Menlrveld
et al., 199U). Using these criteria of classification, there are data toshow the predictive value of sperm morphology for fertilization invitro (Kruger et al., 1986, 1 988; ICobayashi et al., 1991; Enginsu etal., 1991; Liu &Baler, 1992; Ombelet et al., 1995).
Since the recommended morphological assessment considers thefunctional regions of the spermatozoon, it is considered unnecessary
routinely to distinguish between all the variations in hcad size andshape or between the various midpiece and tail dcfects. Howcvcr, an
additional comment should be madc regarding the prevalent defects.
The following categories of defects should bc noted (see cxamplcs in
Figs. 2.8-2.10).
(a) Head defects,namely large, small, tapcrcd, pyriform, round, and amorphous heads, vacuolated heads (>20% of thc hcad arca occupied byunstained vacuolar areas), heads with small acrosomal arca (<40%of head area) and double heads, or any combination of thcsc.
(b) Neck and midpiece defects, namely 'bent' neck (the neck and tail form an
angle of greaterthan 90%to thelong axis of thehead),asymmetricalinsertion of the midpiece into the head, thick or irregular midpiece, abnormallythin midpiece (i.e.,no mitochondrial sheath),or any combination of these.
(c) Tail defects, namely short, multiple, hairpin, brolren tails, bent tails(>90°), tails of irregular width, coiled tails, or any combination of these.

l17ig.2.8. Scherrlal~cdrawings ulsonle abnormal forms of humanspermatozoa. (Adapted kom
Kruper et al., 1991.)(illHead clel'ccls. (11)Tapered, (b)I'yriform. (c) Round, small andacrosome either absent. or present,(d) Aninvphoos, (P) Vdcnolated. (1)A c r u s u ~ ~area~ a l small. (B) Neck andmidpiece defects. (g) Bcnt nccli. ( h )Asymmetrical insertion of midpiece, (i)'l'hicli midpiece. ( j ) Thin
111ic1-piece.(C) Tziil defects. (l<)S1iol.t
tail, ( l )Bcnt tail. ( m )Coilcd lail. (U)Cytoplasmic droplet defect. ( n )Drnplrt gl-eater than nne third thearea uf the ~lornialspernl head.

A. Head defects(a) Tapered(c)Round(b) PyriformI

No acrosome( d ) Amorphous

-

Small

( e )Vacuolated( f ) Small
acrosomal area

B. Neck and midpiece defects(g) Bent neck(h) Asymmetrical(i) Thickm Thininsertion

D. Cytoplasmic droplet

C.Tail defects(k) Short(I) Bent( m ) Coiled

WHO 98039

Fig. 2.9. Photomicrographs(-1 700 X ) of Papanicolaou-stained
spcrmaloaoa (Prom J. Suominen:.
see Appendix V). (A) ( l )Abnormal,
large head, too wide (>3.5 pm),(2)Abnormal, vacuolated and small
acrosun~alarea, (3) Abnormal.
amorphous hcad. (4)Normal, (5)Abnormal, small acrosomal arca.
(6) Abnormal, amorphous headand cyLoplasmic droplet, (7)Normal, (8)Abnormal, small
acrosomal area and vacuolcs.

C!jtnplasmic droplets greater than one-half of t,hc area of a normal
spcrm hcad. The droplets are usually locat.ed in t,hc midpiece.
Only recognizable spermatozoa with a tail arc considered in adiffcrcntial morphology count; immature cells up to and including theround spermatid stage are not counted as spcrrnatozoa (Fig 2.11).
Loosc or free sperm heads are not c,ounted as spcrmatozoa but arerecorded scparately (see Section 2.5.2). Coiled tails may be associatedwith low spcrm motility or indicate that the spermatozoa have been
exposcd to hypo-osmotic stress. Occasiorially, many of the spermatozoa may have a specific structural defect such as failure of the acrosome to develop, causing the 'small round-head defect' or'globozoospermia' (Pig 2.8). Failure of the basal plate to attach to the
nucleus at the opposite pole to the acrosorue causes the heads and tails
to detach on spermiation. The heads arc absorbed and only tails arefound in the semen giving the 'pirlhead' defect. Pinheads (free tails)
are not counted as head defects since they only rarely possess any
chromatin or head structures anlerior to the basal plate. If many pinheads are seen, this should also be noted scparately.

Pig. 2 . 9 .(cont.)(E) (1)Abnormal, thick mid-piece.
(2) Ahnnrmal, amorphousasyrn~netricalhead, (3) Abnormal,
asymrnclrical hcad. (4)Abnormal,
amorphous round head withasymmetrical postacrosomal
region. (5) Abnormal, cytoplas~nic
druplcl, (6) Abnurmal, round hcad,(7) Nurmal (dcspilc slightasymmetry of the head). (8)Abnormal, small head with small
acrosomal area.

Performing a sperm morphology countWith stained preparations, a 100 X oil-immersion bright-field objective and at least a 10X ocular should be used. Morphological evaluation should be performed in several systematically selected areas ofthe slide (Fig. 2.2(b)).As the slide is examined systematically from one
microscopic field to another, all normal spermatozoa are assessed andscored, and the defects of the abnormal spermatozoa are noted.
Overlapping spermatozoa and those lying with the head on edge
cannot be assessed. The latter can be recognized by focusing up anddown. It is essential to use an ocular micrometer to estimate the sizeof the spermatozoa. Even experienced observers should use the builtin micrometer to check sperm head size with each batch of slides to be
examined.
At least 200 consecutive spermatozoa are counted (assessing 200
once is better than l00 twice). Although it is preferable to count 200
spermatozoa twice to reduce counting error and variability,it may not
be practicable to do this for every sample in every laboratory. However,when the diagnosis and treatment of the patient crucially depends on

Fig. 2.9. (cant.)(C) (1)Normal. (2) Abnormal,
pyriform head, (3)Normal (despiteslightly tapering head), (4)Abnor~ral,amorphous head, (5)Abnormal, snlall acrosomal arca.
thick mid-piece and asymmetricalinsertion, (6) Abnormal,
amoryllous head with thick midpiecc.

the percentage of spermatozoa with normal morphology, 200 spermatozoa should bc assessed twice to incrcase the precision (sec Fig.
2.3).
2.6

Testing for antibody-coating of spermatozoa
Sperm antibodies in semen belong almost exclusively to two
immunoglobulin classes: IgA and IgG. TgA antibodies might havegreater clinical importance than do IgG antibodies (Kremer & Jager,1980). IgM antibodies, because of thcir large molecular size, arerarely found in scnien.
The screening test for antibodies is performed on the fresh semen
sample and makes use of either the immunobead test (IBT) (Rronson
et al., 1982; Clarke et al., 1982) or the mixed antiglobulin reaction(MAR) test (for rcview see Bronson et al., 1984). For these te3ts to bevalid, at least 200 n~otilespermatozoa must be available for countmg.
The results from the immunobead test and the MAR test do notalways agree (Scarsclli et al., 1987; Hellstrom et al., 1989). The
immunobead test correlates well with immobilization tests carried out
on serum. When thcse iests are positivc, additional tests (spcrrnccrvical mucus contact test, sperm-cervical mucus capillary tubetcst) should be done (see Chapter 5).

Fig. 2.9. (cont.)(D) (1)Abnormal, slnall arrosomal-

area. too wide ileneth-to w~dth
ratio < l . 5 ) and round, (1)Abnormal, small round head, noacrosomc, (3) Abnormal, smallhead and thick mid-piece, (4)Abnormal, rnund head and Lhick
mid-piece, (E) (1)Abnormal,
tapered hcad, (2) Abnormal,
pyriform head, (3)Abnormal,
nonoval amorphous head withbroken neck, (4)Normal.
~

Fig. 2.9. (cont.)(F) (1) Abnormal, large head andthick mid-piece, (L) Normal, (7)Normal, (4) Abnormal, vacuolated
amorphuus head withasymmetrical postacrosomal
region, (5) Abnormal, pyrifonn
hcad and vacuolated acrosu~nal
area, (6) Abnormal, small
amorphous hcad, (7) Ahnormal.
amorphous head and ltlrge
cyluplasmir droplet, (8) Normal.

Immunobead test
Antibodies presenl oil the sperm surface can be detected by the direct
immunobead test (AppendixVIII).Immunobeads are polyacrylamide
spheres with covalently bound rabbit antihuman immunoglobulins.
Tbc presence of IgG and IgA antibodics can be assessed simultaneously with this test.
Spcrmatozoa are washed free of seminal fluid by repeated centrifugation and resuspended in buffer. The sperm susperision is mixed
with a suspension ol Immunobeads. Thc preparation is examined at
400 Xmagnification with a phase-contrast microscope. Immunobeads adhere to the motile spermatozoa that have surface-bound antibodies. The percentage of' motile spermatozoa with surfacc antibodies
is determined, the pattern of binding is notcd and the class (IgG,IgA)
of thesc antibodies can be identified by using different sets of
Immunobeads (Appendix VIII).
Sperm penetration into the cervical mucus and in vivo fcrtilization
tend not to be significantly impaired unless 50%or more of the motile
spermatozoa have anlibody bound to them (Ayvaliotis et al., 1985;
Clarlte et al., 1985). On this basis, at least 50%of the motile spermatozoa must be coated with Immunobeads before the test is consideredto bc clinically signiticant. Furthermore, lmmunobcad binding
restricted to the tail tip is not associated with impaired lertility and canbe prescnt in fertile men.

Fig. 2.10.
Photo~~~icrographs(- 2 300 X ) of
Shorr-stained spermatozoa. (FromC. Garrett,& H.W.G. Baker; see
Appendix VI.) (l)Abnormal, thickneck. (2) Normal. (3) Abnonnd.
thick neck. (4)Abnormal,
amorphous, small acrosome area,
thick neck. (S) Abnormal, ronnd,
small head, acrosome ahsent.
(6) Abnormal, pyriform head,cytoplasmic droplet. (7) Abnormal,
amorphous, s~nallhead, small
acrnsonral area, coiled tail.
(8) Abnormal, pyriform h a d , tailabsent. (9) Ahnornlal, amorphous.
(10)Ahnor~nal,pyriform head.

2.6.2

Mixed antiglobulin reaction test
The IgG and IgA MAR tests (Appendix IX) are performed by mixing
fi-esh, untreated semen with latex particles or red blood cells coatedwith human IgG or IgA. To this mixture is added a lnonospecific antihuman-IgG antiserum. The formatiori of mixed agglutinates between
particles and motile spermatozoa indicates the presence of IgG or IgA
antibodies on the spermatozoa. The diagnosis of immunologicalinfertility is possible when 50% or more of the motile spermatozoa haveadherent particles (Barratt et al., 1992), but the diagnosis must be
confirmed by spcrm-mucus interaction tests (see Chapter 5).

Fig. 2.11

Photomicrographs of
Papanicolaou-stained immature
germ cells and of cells ofr~oriLeslicularorigin ruutld insemen. (FromJ. Suominen.) (A)Squamous epithelia1cells (- 540 X )(B) Two neiitrophils (leukocytes)
and one rnonucylc (- 2500 X ) (c)Round spermatids (- 1400 X ) (D)Spermatids (.- 1 4 0 0 X ) (E)Macrophage (- 1100 X)

2B

O P T I O N A L TESTS

Thc tests in this part of the manual are not usually recommended forroutine semen analysis but may be of clinical diagnostic value.
2.7

Calculation of indices of multiple sperm defects
Morphologically abnormal spermatozoa often have multiple defects.
In earlier protocols, when multiple defects were present, only one wasrecorded - with priority given to defects of the sperm head over thoseof the midpiece and to defects of the midpiece over those of the tail. It
is now customary to record the number of defects divided by the
number of defective spermatozoa, a measure called the teratozoospermia index (TZI) or multiple anomalies index (MAl) (jouannet
ct al., 1988; Morlimer et al., 1990),and the number of defects divided
by the total number of spermatozoa, called the sperm deformity index(SDI) (Aziz et al., 1996). These indices are predictors of sperm funct.iorl both in vivo and in vitro.
A laboratory cell counter, commonly used for leulcocyte counts(e.g., Clay Adams Lab Counter, Fisher Scientific, Springfield, NJ, USA,catalogue number 02-670-13) with multiple entry keys labelled'normal', and 'head defect', 'midpiece defect', and 'tail defect', can beused. A spermatozoon with a single defect, e.g., head defect, is scoredby depressing that single lrey and the spermatozoon is counted as one
cell. A spermatozoon with head, midpiece and tail defects is scored bydepressing the three appropriate keys simultaneously; the defects(tolal 3) in each category are counted, but the spermatozoon iscourlled as one cell.
Alternatively, if a cell counter is not available, a scoring sheet canbe subslituted. The following is a n example of the calculation ofindices of multiple sperm defects:
200
Number of spermatozoa counted
Number of normal spermatozoa
78
39%
Pcrccntage of normal spermatozoa (781200 X 100)
Number of spermatozoa with defects ( 2 0 0 78)
l 2 2 (61%)
Number with head defectl 1 0 (55%)
Number with midpiece defectl 8 (9%)Number with tail defect
1 6 (8%)
Total number of defects (ll 0 + l 8 + 1 6 )144
Teratozoosperrnic index (total number of defects1
number or spermatozoa with defec,ts)= l441122
1.18
Spcrm deformity index (lolal number of defects1
number of spermatozoa counted) = 1441200
0.72
The teratoxoospermic index values should read between 1.00 (eachabnormal spcrmatoxoon has only one defect) to 3.00 (each abnormal
spermatozoon has head, mi~lpiece,and tail defects). Previous reports

suggest that a TZI of more llian 1.6 is associatcd with lower prcgnancyrates in untreated infertile couples (Jouannctet al., 1988) andthat an SDI of l .h is the threshold for failure of fertilization in vitro(Aziz et al., 1996).
2.8

Hypo-osmotic swelling (HOS) test
This is a simple tcst based on the scmipermeability of the intact cell
membrane, which causes spermatozoa to swell undcr hypo-osmotic
conditions, when an influx of water results in an expansion of cellvolume (Drevius & Erilrsson, 1966). Introduced as a clinical test byJcyendran et al. (1984),the HOS test may be used as an nptional, additional vitality tesl. It is easy to score and gives additional informationon the integrity arid compliance of thc cell membrane of the spermtail. The method is given in Appendix IV

2.9

Semen culture
Thc culture of seminal plasma to assess the presence of both aerobic
and anaerobic orgariisms may help in t,hcdiagnosis of male accessory
gland infection (Purvis & Christiansen, 199 3). IIowevcr, special precautions are needed to avoid contamination during the collectioll andhandling of the semcn sample for microbiological investigation. The
subject should observe sexual abstinence for 5 to 7 days. Before
obtaining the samplc, the subjec,t should pass urine. Immediately
afterwards, he should wash his hands and penis with soap. He should
rinse away the soap and dry with a fresh, clean towel. The semen conlainer must be sterile.The time between collection and the start of theinvestigation by the rnicrobiological laboratory should not exceed 3
hours.

2.10

Biochemical assays for accessory sex organ function
Tlicre are various biochemical markers of' accessory gland function,e.g., citric acid, zinc, y-glutamyl transpeptidase and ac,idphosphatase
for the prostate gland; fructose and prostaglandins for the seminal
vesicles; free L-carnitine, glycerophosphocholine, and neutral cc-glucosidase for the epididymis. An infection can sometinlcs cause adecrease in the secret,ionof these markers, but despite Ibis, the total
arrlount of markers present may still be within the normal range. An
infection can also cause irreversible damage to the secretory epithelium so that even after treatmen1 the secretion will remain low(Cooper et al., 1990;von der Kammer ct al., 1991).The scmen of men
with ejaculatory duct obstruction or agenesis of the vasa deferentia
and seminal vesicles is characterized by low fructose and, in addition,
by low volume, low pH, no coagulation, and no characteristic semenodour.

2.10.1

Secretory capacity of the prostate
The contents of zinc, citric acid (Gruber & Mollering, 1966) and acidphosphatase (Heite & Wetteraucr, 1979) in semen give reliable measures of prostate gland secretion and there are good correlations
between these markers. A spectrophotometric assay for zinc is
described in Appendix X.

2.10.2

Secretory capacity of the seminal vesicles
Fructose in semen reflects the secretory function of the seminal vesicles, and a method for its estimatiori can be found in Appendix XI.

2.1 0.3

Secretory capacity of the epididymis
L-Carnitine and neutral a-glucosidase are epididyrnal markers usedclinically. There are two isoforrns ol a-glucosidasc in the seminal
plasma: the major, neutral one originates solely from the epididymis
and the minor, acidic one mainly from the prostate. Neutral a-glucosidase has been shown to be more specific and sc~lsitivefor epididymal disorders Lhan x a r n i t i n e and glycerophosphocholiile. Its
measurement is described in Appcndix XII.

2.11

Computer-aided sperm analysis
The use of computer-aided sperm arlalysis (CASA) should provide twoadvantages over nlanual methods: (i) high precision and (ii) provisionof quantitative data on sperm liincmalics. Reliable and repeatable
results are possiblc if appropriate procedures are followed (Davis 8Katz, 1992). Many factors are now known to affect the pcrforrnanceof CASA instruments, e.g., sample prcparation, frameratc, and sperm
concentration (Davis & ICatz, 1 9 92; Mortimer, 1994a, b). Guidelines
for the use of CASA (Mortimer et al., 1 9 9 5; European Society of
Human Reproduction and Embryology, 1996) should bc consulted(sce Appendix XIV).
Provided that adequate care is taken in specimen preparation andinstrument use, CASA can be used for routine diagnostic applications
to monitor the conccntration of progressively motile spermatozoa.
Strict quality control procedures are necessary to establish and maintain a high standard of instrument operation (see Appendix MV).
Even with quality control and adherence to rigorous procedures, it
was previously not Scasible to determine sperm concentration by
CASA because of difficullies in distinguishing spermatozoa fi-om particulate debris (European Society of Human Reproductio~landEmbryology, 1996). However, advances i11 technology, particularly inthe use of fluorescent DNA stains with CASA, may now allow sperm
concentration to be dctcrmined (Zinaman et al., 1996).
Some studies havc suggested that CASA estimates of concentrationand movement charactcrislics of progressively motile spermatozoa

arc significantly related to fertilization ratcs in vitro and to time toconception (Barratt et al., 1993;Tomlinson ct al., 1993; Trvine et al.,
1994; Icrause, 1995).
2.12

Zona-free hamster oocyte test
Sperm-oocyte fusion in the hamster oocyte penetration lest is identical lo that in the human since fusion with the vitelline membrane ofthe oocyte is initiated by the plasrna membrane overlying llie equatorial scgment of acrosome-reacted human spermatozoa. The test
differs fi-on1 the physiological situation in that the zona pellucida isabsent. A standardized protocol for this test is give11in Appendix X%
Thc conventional hamster oocyte test depends upon the occurrence of spontaneous acrosome reactions in populations of spermatozoa incubated for prolonged periods of time in vitro. Since thisprocedure is less efficicllt than the biological process and may involve
different mechanisms, false-negative results (patients whose spcrmatozoa fail in the hamster oocyte test but whic,h succeed in fertilizing
human oocytes in vitro or in vivo) have frequently been recorded(World Health Organization, 1986).
Thc intracellular signals that initiate the acrosome reactio~ifollowing sperm-zona pellucida interaction are an influx of calcium andcytoplasmic allialinization. Both can be generated artificially with thedivalcnt cation ionophore, A231 8 7 (Aitlren et al., 1993; Appendix
XV).

2C

RESEARCH TESTS
A widc range of research tests currently under investigation wereconsidcred for inclusion in this section. Of these, the following wereselecled as approaching diagnostic application in the clinic.

2.13

Sperm function tests
Reactive oxygen species and male infertility

The excessive generation of reactive oxygen species (ROS) and thepresence of high concentrations of cytoplasmic cnzymes such as creatine phosphokiilase may both rctlect abnormal or immaturc spermatozoa with excessively retained cytoplasm in the midpiece (Rao etal., 1989: Gomezet al., 1996).
Reactive oxygen species are metabolites of oxygen and include
supcroxide anion, hydrogen peroxide, hydroxyl radical, hydroperoxyl
radical and nitric oxide. When present in excess, such reactivc oxygen
species call initiate pathological damage by inducing oxidative
damage to cellular lipids, prutcins and DNA (Griveau & I,e Lannou.
1997). Most cells are equipped with either enzymatic antioxidant
systems (superoxide dismutase, glutathione peroxidase and catalase)or norienzymatic antioxidant systems (uric acid, vitamin C, vitaminE). When these defences are ovcrwhelmed, cell function is affected.
h1 the human ejaculate, reactive oxygen species are produced byboth spermatozoa (Aitlcen & Clarkson, 1987: Alvarez et al., 1987;
Iwasaki & Gagnon, 1992) and leultocytes (Aitken & West, 1990).
Seminalplasma possesses antioxidant scavengersand enzymes which
may he deficient in some patients (Jones et al., 1979; Smith ct al.,1996). The removal of seminal plasma during the preparation ofspernlatozoa for assisted conception may rendcr lhese cells vulnerable to oxidative attack. About 40%of men attending infertility clinics
and 9 7% of spinal cord injured men exhibit dctectable levels of ROS
generation in thcir semen (Ivvasaki & Gagnon, 1992). High ROS production may cause peroxidativc damage and loss of sperm function.
Chemiluminescent procedures employing probes such as luminolor lucigenin may be used to measure ROS production (AppendixXVI).
2.13.2

Human zona pellucida binding tests

Thc process of binding of spermai.ozoa to the zona pellucida leads toinitiation of the acrosome reaction, release of lytic acrosomal coruponents and penetration of the spernlatozoon through the zona matrix.
To cvaluate these events, nonviable, nonfertilizable human oocytes
from autopsy or surgically removed ovaries or from in vitro fertilization programmes may be used. These oocytes can be kept in high salt
solutions for several rnonths (Yanagimachi et al., 1979). One zorla
pellucida binding assay, termed the hemizona assay (Burkman ct al.,
1988; Oehninger ct al., 19891, involves microdissection of the zona
pellucida into equal halves and the cxposure of each matching half tothe same concentration of test and control spermatozoa. Anothersperm-zona binding assay involves differentially labelling the test
samplc spermatozoa with one fluorcscent dye (e.g.,fluorescein) and acontrol sperm sample with another dye (e.g., rhodamine; Liu ct al.,
1988, 1989). The number of spermatozoa from thc test and control
samples bound to the sanie intact zona are counted and reported as aratio. Both zona binding tests have becn shown to be correlated withfertilization rates in vitro. These tests can be performed using saltstored oocytes (Kruger et al., 1991)hut are limited by the availabilityof human oocytes.
It may be clinically useful to evaluate the number of bound spermatozoa in cases of low or failed in vitro fertilization (FranBn et al.,
1989; Liu &Baler, 1992).Few or no spermatozoa bound to the zona
pellucida usually indicates a sperm defect.
2.13.3

Assessment of the acrosome reaction

The assessment of acrosomal status after induction of the acrosome
reaction by calcium ionophore (Cummins et al., 199 1; Appendix

XVII) identifies acrosome reaction dysfunction (European Society of
Human Reproduction and Embryology, 1996). Various slaining
methods are available for assessing the acrosomal status of humanspermatozoa using light or fluorescence microscopy (Cross et al.,
1986;De Jonge, 1994; Cross, 1995).
Both microscopy and flow c,ytometry (Fcnichel et al., 1989; Henley
et al., 1994) can be used with fluorescent-labelled lectins such as
Pisurn sativurn (pea agglutinin) and inonoclonal antibodies to
examine the acrosome (Cross, 1995).The presence of the outer acrosomal membrane, the acrosomal contents, and the inner acrosomal
melnbrarlc can be detected by different probes, arid the choice of probe
influences the time course of detection of acrosomc-reacted spermatozoa (Aitlen & Brindle, 1993). Viability of llle spermatozoa can be
dctcrmined at the same time with a fluorescent dye (H 33258 bisbenzaminc, Hoechst) or by the hypo-osmotic swelling test (see Section
2.8).
The physiological acrosome reaction occurs at the zona pellucida
aft,ersperm binding. The development of a valid bioassay for the physiological acrosome reaction of spermatozoa that fertilize oocytes
remains difficult, although calcium ionophores or progesterone(Blacl~morcet al., 1990; Aitken et al., 1996)may be used to assess thecompet,encc of capacitated sper~natozoato ini1iat.e normal acrosome
reactions (sec Scction 2.12). The clinical relevarlce of assessments of
the irlrl~rcedacrosome reaction remains to be esl.ablishcd.
2.14

Computer-aided sperm analysis: morphology
Several studies have suggested that assessment of sperm morphology
using corilputcrizcd methods may provide clinically uscful information (I<at~et al., 1986; Davis & Gravance, 1993; Garrctt & Baker,
1995; Garrett et al., 1997; lrvine et al., 1994; Kruger ct al., 1995,
1996). Sorne CASA companies offer packages to examine sperm morphology bul few arc fully automated and there are problems with the
preparation of samples for analysis and with discrimiriation between
spermatozoa and debris. Further development is thereforc needed
before computer-aidcd sperm analysis can be recommended forroutine assessment of sperm morphology.

3 Sperm preparation techniques

Thc scparation of human spermatozoa from scminal plasma toachicve a final preparation with a high percentagc of morphologicallynormal arid motile spermatozoa, free from dcbris and dead spermatozoa, is important for several therapeutic and diagnostic techniquesin clinical andrology. Many procedures may be used but there are
two main methods of separation (see Appendix XVITI; Mortimer,
1994a, b).
Firstly, spermatozoa may be selected on their ability to swim,
lcnown as the 'swim-up' technique. Centrifugation of spermatozoa(including cell debris and leukocytes) prior to swim-up should be
avoidcd because it can result in damage to the spcrm membranes,
probably by the production of reactive oxygen spccies (see Section
2.1 3.1 ). A direct swim-up from semen is the prefcrrcd method lor the
separation of motile spermatozoa. This technique is pcrforrned by layering culture medium over the liquefied semen. Motile spermatozoa
then swim into the culture medium (see Appendix XVIII.2).
The second method of selecting spermatozoa is by thc use ofdensity gradients. A simple two-step preparation is the most cxtensively used of such methods (AppendixXVIII.3). In general, thc directswim-up technique is used when the semen samples are considered to
be norinal. For semen with suboptimal characteristics, alternative
preparations are generally preferred.
It may be nccessary to select the sperm preparation techniqueaccording to the individual semen samples (see Canale et al., 1994;
Smith et al., 1996). The efficiency of the various techniques is usually
expresscd as the absolute number or the relative yield of morphologically normal motile spermatozoa. In some cases, the functional capacity of the prepared spermatozoa may be determined in, for example,
the zona-frcc hamster oocyte test (AppendixXV) to identify the mostsuitable mcthod of preparation.
In the past, clinical studies have concentrated on the advantages
and disadvantages of using Percoll. Ilowever, Percoll was recently
withdrawn for the separation of spermatozoa for use in human clinical applications and now may be used only for research purposes.
Instead, other methods are becoming available. One is the use of
preparations of colloidal silica coated with silane. Another is the use

of density gradients (Gcllert-Mortimer et al., 1958; Sbracia et al.,
1996; see Appendix XVIII). In addition, glass wool columns arereported to be as effective as Pcrcoll gradients were for the separationof spermatozoa from semen with suboptimal characteristics(Rhemrev et al., 1989;Johnson et al., 1996).

4 Quality control i n the andrology
laboratory

4.1

Introduction
Quality control (QC) of semen analysis is essential for the detection
and correction of systematic errors and high variability. Regular OC
assessment by thc laboratory supervisor is required for laboralory
accreditation and t,omaintain accuracy, precision and competence inthe andrology laboralory. QC activities performed within one laboratory are referrcd to as internal quality control (IQC). External quality
assessment (EQA)is the evaluation of results for thc same samples inseveral laboratories. Ouality assurance is a wider concept including
optimization of the services provided.
The management of OC procedures requircs a n understanding of
the sourcc and magnitude of measurement errors. Semen analysis issubject to relatively large random errors associated with counting
limited numbers of spermatozoa. The large disagreements foundbetween the assessments of sperm concentration and morphology bydifferent laboratories (Neuwinger et al., 1990; Matson, 1 9 9 S) underlines the nccd for improved QC and standardization. Until there areuniversally accepted standard methods and definitions of motility andmorphology, it will not be possible to compare rcsult,s from different
laboratories. Results from each laboratory will nccd to be interpreted
against that laboralory's own reference ranges.
Several publications deal with QC in thc andrology laboratory(Mortimer et al., 1986, 1989; Llunphy et al., 1989; Icnuth et al.,
1989: Coopcr et al., 1992; Mortimer, 1994a, b; Clements et al.,1995). The statistical aspects of OC are found in sonic textbooks andstatistical packages (Barnett, 19 79). Apractical approach lo QCfor an
andrology laboratory is outlined in this chapter.

4.2

Nature of errors in measurement
Any mcasurement has a degree of error, thc magnitude of which is
described by a confidence interval with an upper and a lower limit. A
precise measurement is one in which the limits lie close togcther, andthe result is accurate when there is minimum departure fro111the true
value. Therc are two classes of error: 'random' and 'systematic'.
Random errors, causing lack of precision, arise from chancedifferences in readings or sampling; they can be assessed by repeatcd

measurements by the same observer and equipment. Systematic
errors (sometimes referred to as 'bias') are more insidious since theyarise from factors that alter the result in one direction only and thus
create dcpartures that cannot be detected by repeated rneasuremcnts.
QC procedures are required to detect and assess both random and systematic errors in routine semen analysis.
4.2.2

Statistical counting errors in semen analysis

The rand0111distribution of spermatozoa, even when the sample iswell mixed, accounts for most of the variability or lack of precision insemen analysis results. The measurement of spcrm concentration,
motility and morphology all involve counting a limited number ofspermatozoa presumcd to be representative or the whole sample. The
sampling variation crcated by selecting either a fixed volume for estimating concenlralion or a fixed number of sperrnatozoa for classifying motility or morphology is a random error commonly referred to as
the (statistical) counting crror. The relative magnilude of the counting error is inversely relatcd to the square root of the number of spermatozoa assessed. I<nowlcdge of counting error is crucial forassessing the precision of semen analysis and for implementing QC
procedures to identify othcr sources of error.
Counting errors in measurement of sperm concentration

To measure sperm concentration, the spermatozoa in a fixcd volumeof diluted semen are counted in a haemocytometer. If randomly distributed throughout the chambcr, the exact number of spermatozoa
in a given volume follows Lhe Poisson distribution, whose variance iscqual to the number counted. Thus, when N spermalozoa are, thecounted, the sampling variance is N, the standard error is d ~andpcrcentage error 1 0 0 d N / ~ The.
95% confidence interval for thenumber of spermatozoa in lhe volume of semen is approximately
N? 1.96 dhr.Therefore, if 100 spermatozoa are counted, the standard error of the count is 1 0 (10%)and the Y 5% confidence interval
for thc number of spermatozoa in thc volume of semen counted is80-120. Similarly, if 200 spermatozoa are counted, the standard
error is 14 (7%) with a 95% confidcnce interval of 172-228. To
achicve aprecision represented by a perccntage error of 2.5%it is necessary to count 1600 spermatozoa.
Counting errors in measurement of sperm motility and morphology

When spermatozoa are classified into two or more classes (such as'normal' or 'abnormal' morphology, 'progressive' or 'nonprogrcssive'
motility) the standard error of thc estimated percentage (p)within aclass depends on the true, but unknown percentage as well as on thenumber of spermatozoa countrd ( N ) .The estimated standard error is

.\/P(lOO-p)l~.Thus if 100 sperrr~atozoaare counted and the true
percentage with normal morphology is 20%.the standard error of theestimated percentage of normal spermatozoa is 4.0%. The corresponding 95% confidence interval is 12.2%-27.8%. If 200 spermatozoa are counted, the standard error becomes 2.8% with a 9S0/0
conGdence interval of 14.5%-2 5.5%. If 4U0 spermatozoa arecounted, the standard error is further reduced to 2.0% with a 95%
confidence interval of 16.1?h-23.9%.
Minimizing statistical counting error
While counting error can be reduced by assessing greatcr numbers ofspermatozoa, a balance most be struclr between the gain in statisticalprecision and the possible loss of accuracy in the technician's work
due to fatigue.
4.2.2

Other errors in semen analysis
Repeated analyses by the same technician and procedure will yield
greater variability than the counting error alone, bccanse additionalrandom errors are usually present. These may arise from inadequate
mixing (common with viscous and agglutinated sanlples), iechnlcian
stress (erratic counting or recording error), poor technique (e.g.,careless pipetting or handling during slide or chamber preparation), or
instrument variation (e.g., worn automatic pipettes, which may
reduce reproducibility during sampling and dilution). In contrast,
results with less variability than the expected counting error suggest
the possibility of recording errors or of bias induced by ltnowledge ofprevious measurements.
Measurements repcated by different technicians may differ becauseof systematic differences, or biases from consistent calculation errors,
or differences in the classification of morphology and motility. For
example, the percentage of motile spermatozoa is cornrnonly overestimated because the eye is attracted to movement. This error can be
reduced by systematically counting all motile spermatoxoa Grst andthen counting the innnotile spermatozoa in the same area.

4.2.3

The importance of quality control
The precision or reproducibility of a semen analysis can be assessedfrom the results of repcater1 measurements of the sarne sample, amethod which, togcthcr with regular calibration of equipment, forms
the basis of IQC. Tntratechnician precision is determined from thereplicate estimates or repeated analyses of the same masked sample.
Intertechnician intralahoratory precision is determined by independent analyses of the sarne sample. Interlaboratory variability is evaluated by EQA. IQC should ensure that optimalprecision is maintainedin the andrology laboratory and that there is no gradual shiR in stan-dards. IQC is essential both when a single technician maltcs infrequentassessments or several technicians work together. EOA is essential toensure that different laboratories produce cornparable results and can
indicate when a laboratory's methods should bc adjusted.

4.3

Practical approaches to quality control

The QC procedures described below are the minimum that sllould be
implcnlented in an andrology laboratory. The QCprocedures for otheraspects of good laboratory practice, such as maintaining a constanttemperature in incubators and calibrating balances, are assumed to
be routinely conduct,ed.
At all times - surveillance and correlation of results within samples.
Weeltly analysis of replicate measurements of thc main semen variables by different technicians.
Monthly - analysis of mean results of' tests.
Ouarterly - participation in an EQA scheme, calibration of pipettes.
Yearly - calibration of counting chambers and other equipmenl (seeAppcndix XIX).
Thc QC activity in a laboratory will vary depending on the work
load and experiencx of the technicians. In busy laboratories between
1%and 5% of samples should be for IQC. It is important for the QC
samples to be masked, and they should be analysed as part of routine
laboratory worli. In this way IQC will help to maintain accuracy andprecision in routine semen analysis.
4.3.1

Routine surveillance and correlation of results within samples
Resulls must be c,heckcd for errors of transcription and palient
identilication. Unusual results that do not match othcr aspects or thesemen analysis may suggest such errors. Estimates of sperm concentration, motility, and morphology from the initial microscopic examination of the undiluted semen can be used as a rough checlr on l l ~ cfinal result. The percentage of live a r ~ drnotile spermatozoa should beconsist,cnt.Alarm results are unusual occurrences requiring urgent
attention. Examples of alarm resulls for the laboralory include zero or
unexpected loss of sperm motility, which may be caused by temperature change or contamination with sperm toxins. Alarm resulls forthe c,lirlicianinclude a patient who previously had spermatozoa in thesemen becoming azoospermic or being unable to produce a sample.
For sperm concentration, independent duplicate counts in the twoparts of the counting chamber are compared (see Section 2.5.2, Fig.
2.6). The difference between independent counts on equal volumes
from the same sample is expected to bczero. with standard error equal
to the square root of the sum of the two counts. In about 9 5%of duplicate samples the ciiffcrence between the two counts will be less t,han
1.96 standard errors. If not, a systcnlatic error is suspected and thesample is remixed, diluted again and the procedure repcated. In about
5% of samples, differences greater than 1.96 standard errors will
occur as a result of chance variation alone, and the count will havebeen repeated unnecessarily. IIorvever, this small additional work is anassurance that there have been no systematic errors.
Similar procedures are followed for duplicate estimatcs of the percentages of spermatozoa withdifferent motility grades (Scctiun 2.4.3,
Pig. 2.3) and morphological characteristics (Section 2.5.4).Two independent estimates, p, and p,, are made on N spermatozoa in eachsample and compared. The limit of expected difference d is given
by d = Ipl - p , I < 1 . 9 6 2 / 2 p ( 1 0 0 - ) l ) / ~ where p = (p, +p,)/2 is theaverage pcrcentage. Using these limits, 5% of duplicates will berejected from chance variation alone.
4.3.2

Weekly internal quality control: assessing intra- and inter-technician
variability
A simple method of IQC involves replicate measuren~crltsperformedon separate aliquots of the sample and carried through all stages of
the analysis. Assessing thc slide or chamber twice on thc same occasion is not true replication. It does not assess errors of preparation or
dilution. The replicate assessments should be performed in the same
way as routine samples with technicians unaware that IQC samples
are being assessed. IQC should include assessments of sperm concentration, sperm motility grades, sperm n~orphology,the irnmunobead
test, the mixed agglutination reaction, and any other results reportedby the laboratory. Stored scmen samples (see Appendix XIX) can beused for IQC and have the advantage that the true or target value isknown, or can be estimated. An easier approach is for thc same fresh
sample, or pool of several samples, to be analysed independently bydifferent technicians, which will enable thc variability between theset,echnicians to be assessed; however, the true value is not known andthus systematic error or bias of the group cannot be estimated. The[QC of sperm motility presents special problems since motility may
decline over tirne and thus nceds to be assessed first and at about thesame time by all the technicians.
The statistical proc,eduresfor analysing and reporting the IQC results
are presented below. Some of these procedurcs depend on the true ortarget value being known (or estimated from the first ten or so determirrations). Statis1,icalpackages for laboratory QC are available and can beused for analysis and the presentation of results. The procedures areeasier to interprct.and compute if the same number of technicians participate in each QC determination. If this is not possible, one or more of
the technicians can assess the sasnple a second time so that thc numberof determillations on each QC sample is the same. Some computer programmes can accom~nodatea variable numbcr of observers.

Table 4.1. Fuctors for determirrir~gcontrol lirnits for Xhaland S charts
hased on the uverage stai.ldard deviation (So,,.)Xharchart
coritrol limits

S chart corltrol limits

Lower Lower
Upper
Upper
Number of SD
technicians estimate Warning Actiori action warllirlg warning actioll
(71)

(c,,)

(A,)

2
3
4
5
6
7
8
9
10

1.253
1.128
1.085
1.064
1.051
1.042
1.036
1.032
1.028

1.772
1.303
1.085
0.952
0.858
0.788
0.733
0.688
0.650

(so.qq9)(S".Y~S)('0.025)(~0001)2.659 0.002
1.954 0.036
1.628 0.098
1.427 0.160
1.287 0.215
1.182 0.263
1.099 0.30.3
1.032 0.338
0.975 0.368

0.039
0.180
0.291
0.370
0.428
0.473
0.509
0.539
0.563

2.809
2.167
1.916
1.776
1.684
1.618
1.567
1.527
1.495

4.124
2.966
2.527
2.286
2.129
2.017
1.932
1.864
1.809

IQC with stored samples: sperm concentration
The rcsults obtained by each technician on the QC sample are tabulated and plotted on a graph againsl the sample (or week) number. The
mean and standard dcviation of the rcsults on each sample are computed and also plotted against the sample (or week) number on Xbar
and S control charts, which are used to determine whether the results
obtained on the new QC sample differ from the previous determinat i o n ~or whether the differences betwecn technicians are greater thanwould bc expected as a result of random variation alone.
When at least five, preferably ten, &C samples have been analysed,
the average of the means (Xbar)and standard deviativns (Sbar)are used
to establish control limits on the Xbarchart at 2 and 3 standard errors(warning and action control limits) either side or the target value.
These are given by XbariA2,,, X Sbarand Xbr? AJSnX S where thevalues of the coefficie~lts A,,n and A3,nare read from Table 4.1 for thecorresponding number of technicians ( r l ) . The control limits are used
to morlitor the results obtained on future QC samples. An alternalivc
is to compute the control limits directly as Xbari.2s/-\in and
Xbar53s/.\jn from the pooled between-technician standard devialion( S ) . This pooled standard deviation is greater than the average of thesample standard deviations Sbar and can be computed direc,tly or
obtained by multiplying S,;,rby the factor cm(Table 4.1). This factor isthe amount by which the average shndard deviation underestimat,cs
the pooled standard dcviation in samples of size n from a normal distribution. A worked example is given in Box 4.1.

-Box 4.1:Determining cuntrnl limits for the Xbarchart
The table below shows the sperm concentrations measnred by each of four
technicians on the first ten QC san~plesand the calculation of the mean andthe standard deviation of each sample.
Sperrn concentrntiorl (million/rnl)
1

Sample

2

3

4

5

6

7

8

9

1

0

'TechnicianA38
35 4 034 38
36
4 4 433 9 43TcchnicianB 42
Zh 42 40 4 0 4 0 4 3 43 46 4 0
TechnicianC 38 43 40 51 38
33
39
45
15 39
TechnicianD 34
36
36
37
16 3 9 42 43 46 3 4
Mean
38.0 37.5 $9.5 40.5 38.I) 37.0 42.0 43.5 41.5 39.0
SD
3.27 3.70 2.52 7.42 1.63 3.16 2.16 1.00 5.45 3.74

For thc first ten OC samples the average of the means ( X , & is:(38.0+37.5+ ...+39.0)/10= 39.7,

and the avcrage of the standard deviations(3.27+3.70+

is:...+ 3.74)/10-3.40.

l'he values of the coefficients A,,,, and A,,, in Table 4.1 for samples of sizen = 4 are 1.085 and 1.ALX. respectively. Thus the inner (warning) control
limits (two standard errors from the mean) are given by:
Xb,rZA,,nXSbar=39.7t1.085X3.400r 36.0alld43.3millionlrnl.

Similarly the outer (action) conlrol limits are givcn by:

Xb,,r ? A ,,,,X S b r = 3 9 . 7 t 1 . 6 2 8 X 3 . 4 0 0 r 4 4 1 and45.2millioniml.
These control limits are added to the XhSrchart (Fig. 4.1) to monitor the
results of future OC samples.

Fig. 4.1. XbUlchart
9,

az :
.-

E

.

2

.
a-

;
I

Control limib---------------------p-----.
4

Warning

4

0
v

O

gB .'
I
2

Act~on.....................................................
0......................................................

Warmng------P--------------------.
Action

30,

0

Xaa.

4

4

1

1

3

4

1

6

QC snmplc

7

8

9

1

0

Box 4.1:Determining control limits for theXkarchart (continued)
The estimate ol llle between-technician standard deviation car1be obtainedby mulliplyi~lgSbarby the factor c, (= 1.085 for samples of size n=4) to give
3.69, which is close to the directly computed value 3.84 of the pooled slandard deviation, S = ~ l ( [ s , ~s12++ ... ~,,~],'10)
where g is the standard deviation ol the ith OC sample in the lable above, i = 1,2...10. This result can beused to cornpute the warning and action control limits directly at L and 3
standard errors (sidn)either side of the mean. In this example lhcse warning
limits are 35.8 and 43.5 million/ml, and the actionlimits are 33.9 and45.4
millioniml, respeclivcly,very close to those ohlairled using S,, and the coefficientsand A,,,,.

+

Table 4.2. Basic control rulrsfor (/C chart
Control rule

Error sensitivity

One rcsult outside action limits

Random orsystematic error
Systematic error'I'wo consecutive results both above the upper warning limit
or both below the lower warning limit
Two consecutive results, one above the upper and one below Kandorn error
the lower warning limit
Systematic error
Eight consecutive results ull ubove or all below the mean

There arc basic control rules to monitor performance of future QC
assessments on the QC chart (Table 4.2). If the OC sample is 'rejected',
the sensitivity of the alarm to lhe different types of error (random orsystematic) should direct the investigation into possible causes.
Systematic error is also suspected if there is a succession of sevenpoints that are either all falling or all rising.
The Xbarchart is mainly desigrled to detect changes from the target
value, or an overall increase in variability. It is less sensilive in detecting whetlier technicians are producing highly variable results or systematically under- or overestimaling. For this purpose, t,hc range ofvalues on each QC sample can be monitored on an Rbarchart in asimilar way to the Xhr chart, with warning and action limits set
accordingly. A similar chart with more sensitivity is the S chart, basedon the sample standard deviations. Since the distribution of the standard deviation is not symmetrical, the warning and action limits arechosen in such a way that the probability of a new observation fallingoutside the control limits is the same as for the Xbarchart if there are
no changes in accuracy or precision. Thus the warning and action
limils will be crossed in 5% and 0.2% of future samples as a result ofrandom variation alone. These limits are determined from the x2 dis-tribution, and the factors sN,,,required to multiply the average slandard deviation Sb,, are given in Table 4.1. A worked examplc is given
in Box 4.2. Similar rules to those in Table 4.2 for monitoring performance on future OC samples are applied to the S chart. Results thatfall below thc lower limits on the S chart suggest unexpectedly sinall
variation, which may indicate a genuine improvement in thc level ofagreement between technicians, or possible collusion.
The estimates of the mean and the standard deviation can berecomputed aner every 10 samples and the control limits updated
using thc new values for X,,, and S,,,, provided there havc been no
problems with QC. Before the QC samples run out, a new pool should
be prepared and the first 10 samples of the new batch analysed
togethcr with the remaining samples of the old batch to cstablish the
new control limits.
Assessing systematic differences between technicians

The consistency of results between technicians within the laboratory
is an important aspect of IQC. However, some technicians may systematically over- or under-cstin~tllethe sperm conccntration. Thisshould be assessed after every group of 5 or 10 QC sanlples by twoway analysis of variance with factors for QC samples and technicians(sec below). Since the QC sanlples are all from the same stored pool.
significant differences between samples are not expected, andsignificarll differences betwecn technic,ianswould suggcst,systematic
bias in t,he assessment by one or more technicians.

IQC with stored samples: sperm motility and morphology
The QC procedures for the assessment of sperm motility and morphology follow the same steps as tl~oseoutlined above for sperm concentration, except that percentages are being assessed. Since the total
numbcr of spermatozoa countcd is large, the limits for the control
charts can be set using the normal distribution. The approximation isadequatc for percentages that lie in the range 20% to 8096, but lessgood if the percentages are low (<10%)or high (>90%).
For sperm morphology, many slides can be made fi-nm a single
semen sample. These slides can he prepared from samples with good,
medium, or poor quality spermatozoa and one slide from cach introduced into the QC assessment. The slides must be masked to prevent
recognition as QC samples.Thc slides can be reused and ncw ones prepared once they deteriorate. Video tapes can be used fbr motility QC
assessinenl.
The same basic principles for IQC described above are followed.
Once a series of QC determinations has been made, thc X,,,, and S
charts can be constructed and warning and action litnits set. The
standard deviation of the estimated percentage p is d p ( 1 0 0 - ~ ) l ~ ,Box 4.2: Determining control limits for the S chart
Using the results from Bux 4.1, the average sa~nplestandard deviation Sbaris
3.40 million/ml. The factors S,, for saniples of size n = 4 arc read fromTahle
4.1 to give the lower actiori limit SbarXso4,= 3.40 XO.OY8=0.33
milIion/ml, the lower warning limit S,,,X s j,,= 3.40 X 0 . 2 9 l 1 0.99
million/ml, thc upper warning limil SbarXs, 0 2 5 , 4 = 7.40 X 1 . 9 1 6 = 6.52
millionlml, and the upper action limit S,,,Xs= 3.40 X2.527= 8.60~~~illion/ml.
These limits are plotted on the S chart (Fig. 4.2) to monitor the
performance of future QC samples.

,,,,,,,,,,

Fig. 4.2. S chart
L0

2
-

:
E
*

I

5

-

Acclus80.

......................................................

Warning

#.
0

.
0

2:

b

.

V)

Ui
U

C

Control limits

.

.=.

0

O

sbnr

0

0

........................!. ............... +. ..........
1

2

3

4

5

6

7

8

9

1

Warning
Action0

QC sample

where Nis the number of spermatozoa classified. For values of p in therange 20% to 80%and a tolal of 400 spermatozoa classified, the standard deviation lies in the rarlgc 2.0% to 2.5% with the maximum
value when p is 50%.The slandard deviation of individual readings
should be close to these values. However, the average standard deviation Sbarwill be greater than 2.5% as a result of the additional variation betrnrcen technicians, and reducing S,,,. to t,he theoreticalminimum will be the goal. Only cxperienced technicians who haveachieved a high degree of consistency and standardixat,ionwill be able
to reach this limit. If performance is close to the theoretical limit, then
the warning and action limits on the X,,, and S conlrol charts can be
set using the theoretical standard deviation in prefercnce to theobserved standard deviation Sbar
If the QC samples have perceritages less than 20% or greater than
80%, thcn a modification lo thc above procedure may give more
appropriat,~
control limits.The angular (or arc sin square root) transformat.ion z = sin-]has the property that the standard devi)and depends only on theation of z is given by 1 / ( 2 2 / ~ radians
nunlbcr of spermatozoa counted, N, and not on Lbe true percentage.
This Incans that the probability of crossing the warning and action
lilnils as a result of random variation alone is closer to the theoreticallevel. For sperm rnotility or morphology based on classifying 400 spermatozoa, the standard deviation of z is 0.02 5 radians, which corresponds to the lowest theoretical limit.
As with thc QC of sperm concentralion, the two-way analysis ofvariance is an important step in identifying any consistcnt under- orover-estimation by certain technicians and in identitying ways inwhich quality and consistency can be improved.

IQC with fresh samples: sperm concentrotion, motility and morphology
The QC procedures based on the assessment of fresh scmen samples
are similar to those described above for the stored sample, with the
exception that.the X,,=chart cannot be used as there is no target meanvalue. Howcver, the primary QC procedures are the S char1 for assessing varial~ilitybetween technicians, which is plotted after each QC
sample, and [.hetwo-way analysis of variance for assessing systematicdifferences between technicians alter every five or tcn QC samples.
The two-way analysis of variance is described in many statisticaltextbooks (for example, Altman, 1991) and is available in packages
for computers, together with statistical tests for thc significance ofdifferences between technicians. The worlced example (Box 4.3) illustrates how to compute the standard error of the differences between
technicians directly and assess whether these are grcater than would
be expectcd from chance variatior~alone. When performing computations directly from the observations, a sufficient number uf decimal
places must be kept to avoid any rounding errors. A forrnal statistical
test for diEcrences between tech~iiciansis based on thc F-test from thetwo-way analysis of variance tahle, which can be obtained directlyfrom most statistical c,omputerprograms. The error root mean square(U) is the square root of the residual, or error, mean square from theanalysis of variance table. Mean differences greater than about 2.5
standard errors are unlilcely to result from chance variation alone.
Whether the differences between technicians are significant or not, itis necessary t,o review the technicians' means or mean differences to
identify which are greater than expected. Not all computer packages
provide the starldard error of thc differences between tcchnicians,se(mj), which rnay have to he computed separately. Substantial
differences between technicians should prompt a review ol all procedures to discover W-aysin which thcir consistency can be improved.
4.4

Monitoring monthly means

While the primary IOC procedurcs are based on the assessment ofdifferences between and within technicians, useful additional information can be obtained from moniioring trends in scmen analysis
results from patients. The mean values of each variablc for all thepatients examined over a certain period (e.g., monthly) can be plottedllnx 4.3: Assessing systematic differences between technicians

The table below shows sperm concentrations cstinlated by each of lllrce
technicians 011 five QC samples.
Sperm roraatrtratinr?(millionlrnl)
Sample

1

2

3

4

5

Technician A'I'echnician B
Technician C
Sample mean

108
103

45
47
46
46.0

100
102
XY
97.0

50
50
41
47.0

9L

104
195.0

96
88
92.0

'The diffcrences from the sample meari (djj)are computed by sublracting thesemen sample mean from each observation:

Differences (dii)frorn sample rrlean (milliortlml)
SampleTechnician h'rechnician B
Technician C

l

2

3

4

5

3.0
-2.0
-1.0

1 .O

3.0
5.0
-8.0

3.0
3.0
-6.0

0.0
4.0
-4.0

1.0
0.0

-

The mean, m, = C,d,i/n , and standard devialiun, S, \/wl(n-1),of thcscdiffcrences are computed for each technician, where n is the number ofsemen samples.

Mean and standard deviation of dijJ+rerlces(million/ml)

Tcchuician A
Technician 13'Ibchnician C

Mean
(mi)

SD
(S,)

Mean/standard error(rni/sc(rrtl))

l .h002.200-3.800

1.949
2.775
3.347

1.301
1.788
-3.08L)

Technician C's meandifference is 3.8 millionlml Gelow lhc mean of each QC
sample, or 5.7 (=- 3.8 - 11.6+ 2.2112) millionl~nlless than the average ofthe other two technicians. 'hasscss whether the degree of undcrcstimationis compatible with chance variation, the error root mean square.
ci= ~ , s I L / ( tl),
- where t is the number of technicians, is computedfrurn thestandard deviations of lhc technicians' differences. In Ibis example it is3.369 millionlrrrl. The standard error of each Lcchnician's mean differenceis given by se(rnl)=ci\/ii-1lt)In and is 1.230 millionlml. The absolutevalue of 'l'echnician C's mean difference (.<.X million/ml) is greater than 3
standard errors and is significantly dilfcrent from the expected value of zero
if there are no systematic diffcrences between the technicians.

Box 4.3: Assessing systematic differences between technicians (continued)
A formal statistical test of differences betwccn technicians is based on theF-test from the two-way analysis of variance with factors for technicians and
QC samplcs. The analysis of variance lable, using the above sperlrl concentrations, is given below.

Two-way unu1,ysis of variance

SourceSum ulsquares

Degrees of
freedomMeansquareQC samples
Technicians
Error
Total

9 807.6
109.2
90.8
10007.6

422451.90
54.60
11.35

8
14

F-ratioP-value

216.03
4.81

10.001
0.04%

The errorrool mean square is V11 . 3 5 ~ 3 . 3 6 9million/ml, the same as tlrat
obtained above. As expected, the differences betwecrl QC samples are verylarge (P<0.001) since they are taken from different fresh serrlcn samples.
The F-test for differences between technicians (F = 4.8 1 with 2 and 8 degrecs
of freedom, P = 0.042) is significant at the 0.05 level and suggests that thesediffererlccsare greater than would be expected from random variation alone.

on an Xbar chart with warning and action limits 2 and 3 standard
errors either side ot the mean. The standard error is estimated fromthe standard deviation of the original observatioris divided by thesquare root of the number of semen analyses in each interval, or
direclly from the observcd distribution of the mean (seeBox 4.4). The
conlrol limits should be determined using at least 6 months' observations and revised regularly. There should be at lea-t20 results for eachmcarl, and a small laboratory may have to pool results frommore than
one month. Deviations outside the control limits may indicate uncontrolled changes in laboratory practlce or shifting trends in assessment.
provided that the patient characteristics did not change markedly overthe period studied (secKnuthet al., 1989).Refinements to the method
include monitoring monthly means of patients with normal values
and Lhe use of cumulative sum (CUSUM) charts (Rarnett, 1979) for
the rapid detection of any syslematic departures fi-orn the mean.
4.5

Response to results outside control Limits
The results of QC must be reviewed regularly by the laboratory supervisor, and the report must be signed and dated. When results outsidecontrol limits appear, the probable cause and action taken should also
bc recorded. If the problem is not obvious, the QC samples should bereanalysed to checlc if the first result was unusual. IC the QC result
remains outside control lirnils when repeated, the analysis of routine

Box 4.4: Monitoring monthly means using the X,,, chart
Figure 4.3 shows themean percentage immotile spermatozoa (gradedmotilily) in semen samples ailalysed in the samc laboratory over the period April
1 9 8 7 to April 1989. The mean and standard deviation of the means in llic
first 6 months (April-September 1987) are 47.3% and 2.87'%,respcctively.
The warning limits (47.3?2 X2.87, or 41.6 and 53.0) and action limits(47.3 + 3 X2.87, or 38.7 and 55.9) areused tomonitor s~~bsequentmonthly
means using the control rules in Table 4.2.
The increase in 111e mean proportio~lof immotile spermatozoa fromJanuary 1988 (persistently abovc the action limit) prorrlpted an investigalion
into possible causes. 'l'he change was later attributed to a modificalion to theroutine techniquc for measuring senien volume. Once the laboratory
reverted to the previous technique, the motility levels were similar to thoseobscrved earlier. See Knulh et al. (1989)for further details.
The mean and standard deviation of the 6-monthly mcans from August
1988 were 49.5% and 1.74% respectively. Thc revised warning (46.0%.
53.0%) and action (44.3%. 54.7%) limits are added to the Xbarchart tomonitor subsequent performance.

-

Fig. 4.3. Monthly mcan assessments of graded sperm motility (%)
Grul. d .mcility- M*."..... ...
(Us-

- - - --

Wnrniul limit
Art,"" limit....................~ ~ .....
. . . ~ ~. ~ ~ .........................
. . . ~~...........

105;-*pr M.l

,"l

U
,.

l,$,

AYb

LP n, *,S.

Im

I,- rrc M U *F

May

.
P

1188

l i *S bcp Dr,

NOY

Da lu, F*I

Mar I p r

,989

samples performed that day should be repeated if possible and thereason investigated.
Technician factors,such as pipetting errors and misclassification of
sperm morphology or rnot,ility,changes in procedures or equipment,
particularly pipettes and counting chambers, and deterioration of the
QC sarnple are all possible rcasons for results outside control limits. For
example, in Box 4.4, such results occurred after a change in the procedure for measuring semen volume and, once the original techniquefor preparation was reinstituted, the QC results were similar to previous values. Factors associated with patients may also affect monthly
means. These include changes in collection jars or facilities, seasonaltemperature variations or changes in the pattern of referral of men
with different severities of infertility or a change in the number ofrepeated tests in thc same men. Sometimes, no cause can be discovcred and the resull may be attributed to random variation if subsequent QC results are within range.
4.6

External quality assessment
EQA is an integral part of the QC process. A number of formal EQA
schemes exist (see Appendix XIX). EQA is important for assessing thecffccts of standardizatjon of procedures. EOA also allows a group tocompare its results with t,hoseof others. It also pcrinits evaluation andcomparisons between different methods on a scale not possible in asingle laboratory. Diffcrenccs in the interpretation of sperm motilily
and morphology are probably mainly responsiblc fbr the current poor
results of EQA for these semcn variables.
EQA and 1QC are complementary processes. EQA may reveal problcms with precision that are not apparent from I(/C because thecontrol samples are not adequately masked or selected. EQA lestsamples should be masked and processed in 111e same way as routine
samples.
EQAschemes provide laboratories with inlormation on their resullsin cornparison with those from other participating laboratories.Thesemay include the target values derived from accurate measurement,
for cxample, multiple haemocytometer counls of sperm concentrat,ion or results of computer-aided sperm analysis of sperm motility.
Alternatively, the mean results obtained from a group of reference laboratories may be used. The mcan and range of [.heother participating
ccntres are often given with the extreme top and bottom 10°h of
rcsults trimmed. Sonie graphical presentation, such as a bar chart, isusually also provided. Tf the same EQA sample is used on several occasions,the laboratory's bias and variability for thc results of this samplc
will also be reported.
Laboratories with results persistently higher or lower than theassigned value or mean of the EQA scheme rieed to conduct a reappraisal of their methods. For example, sperm concentration may beincorrectly calculaterl or the haemocytometer chamber may not be oft,hc dimensions expected. EQA results with widc variability are also
usually associated wit,h wide variation in IOC and indicate that there
are inconsisteilcies in the assessment proccdures from sample tosample.Technical procedures should be carcfully reassessed to ensurethat they conform with those recommerldcd in this manual. Exchangeof scientific staff bctween laboratories is often helpful. A consultant
from alaboratory with goodEQAresulls will often be able to see where
methods should be changed to improve rcprodncibility. Conversely,
the training ol t,echnicians in laboralorics with good EOA results
should be beneficial.

15 Sperm-cervical mucus interaction

5.1

Introduction
The epithelium of the human cervix cc~mprisesdifferent types ofsecretory cells, ancl thc nature and abundance of sccretory granules
vary in different parts of the cervix. Secretions rrom these cells contribute to the cervical mucus. Ovarian hormones regulate the secretion of cervical mucus; 1 7P-oestradiol (oestrogen) st,imulates the
production of copious amounts of watery mucus, and progesterone(progestogen) inhibits the sccretory activity of the epithclial cells. The
amount of cervicalmucus secreted shows cyclic variations. Innormal
women of reproductive agc, the daily mucus production varies from500 p,1 during mid-cycle, to less than 100 p,1 during other periods ofthe cycle. A small amount of endometrial, tuba1 ancl possibly follicular fluid may also conlribute to the cervical mucus pool. In addition,
leukocytes and cellular debris fiwm the uterine and cervical epithelia
are present.
Cervical mucus is therefore, a heterogeneous secretion containing
over 90% water. It exhibits a number of rheological properties - consistency, spinnbarkeit and rerning.
Corlsistency is influenced by molecular arrangement and by theprotein and ionic concentrations of the cervical mucus. Mucus varies
in consistency during Lhe cycle from the highly viscous premenstrual
form (which is often cellular) to a watery consistency a1mid-cycle justbefore ovulation. By the time ovulation is completed, lhe viscosity ofthe mucus has already begun to increase again.
Spinnbarkeit is the term used to describe the fibrosity, lhe threadability, or the elasticity charactcristics of cervical mucus.
Ferning refers to the degree and pattern of crystallization obscrved
when cervical mucus is dried on a glass surface (Fig. 5.1).
Cervical mucus is a hydrogcl comprising a high viscosity component and a low viscosity component made up of electrolyles, organic
compounds and soluble proteins. The high viscosity component is amacromolecular network of mucin, which influences the rheologicalproperties of the mucus. Ccrvical mucin is a fibrillar system consisting of subunits made of a peptide core and oligosaccharide side
chains. Cyclic alteration in the constituents of cervical mucus may
influence sperm penetrability and survival. Spermatozoa can begin totl. q i l r l r t ~ ~ r\1rl11
ls
1/11I'rimilry ailr! l,,-;s'..a,nrl;~ry ~ I V I I I score I I ni,,illl!::>I%Ipi.rli~illytcr1I;irv ~ICIIW prc<vlii
I I .\typi.xl !er11 t ~ v ? l , l i I : ~ , . i ~ t ) ~ ~s.")rt. I . 111 Y.) x y s ~ i ~ l l i z a t i u n,.Rwc> L, .'Ill:t l ~ i c l i - ~ i ~ ~ r#,1111,lI.i~.l',trui cur:s arc, iiir hllhl~le,penetrate human cervical mucus at approximately the ninth day of anormal 28-day cycle and the penctrability increases gradually to apeak just before ovulation. Sperm penetration then begins to diminish, even belure large changes in mucus properties arc apparent.
Individual variations in time and degree of sperm penetrability arecommon. Motile spermatozoa may he guided by strands of ccrvicalmucus to thc cervical crypts, wherc they may be retained and released
at a slow ratc into the uterus and Fallopian tubes.

The follorving properties may be ascribed to the cervix and it,ssccretions: (a) receptivity to sperm penelration at, or near, ovulation andinterference with penetration at other times, (b) protection of spcrmatozoa from the hostile environment of the vagina and from being
phagocytosed, (c) supplementation of the energy requirements ofspermatozoa, (d) filtration (i.e., sperm selection on the basis ofdifferential motility and morphology), (c) provision of a short-lerm
sperm reservoir, and (0 initiation of sperm capacitation.
Sperlnalozoa within the mucus are, at all times, suspended in a
fluid medium. The interaction of spermatozoa with the secretions of
the female reproductive tract is of critical importance for the survival
and functional ability of spermatozoa.There is no practical method atpresent of evaluating thc effects of human uterine and tuba1 fluids onspermatozoa, but,cervical mucus is readily available for sampling andstudy. Evaluation of' sperm-cervical mucus intcraction therefore, isan important measurc to be included in any complete investigation ofinfertility. A finding of abnormal sperm-cervical ~llucusinteraction
may be an indication for artificial insemination or other forms ofassisted reproduction.
5.2
5.2.1

Collection and preservation of cervical mucus
Collection procedure

The cervix is exposed with a speculum and the mlernal os is gently
wiped with a cotton swab to remove the external pool of vaginal contaminants. The exocervical mucus is removed with the swab or withforceps. Cervical mucus is collected from the eridoccrvical canal by
any one of the following methods. The mucus can bc aspirated with atuberculin syringe (without a needle), a mucus syringe, a pipette, or apolyethylene tube. Whcnever possible, the quality of the nlucus
should be evaluated irnmcdiately on collection. If this is not possible,
the mucus should be preservcd (Section 5.2.2) until it can be tested.
When mucus is collected by aspiration, it is important to standardize the manner in which suctioil pressure is applied to thc collection
device (syringe, catheter, etc.). Suction is initiated after t,hc tip of thedevice has been advanced approximately 1cm into the cervical canal.
Suction is then mainlaincd as the device is withdrawn. Just prior towithdrawal of lhe device from the external cervical os, suction pressure is released. It is then advisable to clamp the catheter to protect
against accumulation of air bubbles or vaginal material in the collected mucus when the device is removed from thc cervical canal.
When cervical mucus is to be collected at a time other than at midcycle, its production can be increased by the administration of 20 to
80 ~ g l d a yethinyloestradiol, beginning, e.g., on the fifth day of agiven cycle for a period of up to 10 days. The mucus may be collectedwithin the period 7-10 days after the start of administration of
ethinyloestradiol. This procedure will producc a more hydrated, andtherefore less viscous, mucus secretion (Eggert-Kruse et al., 1989).
While this approach may be useful in assessing sperm-mucus interaction in vitro, it will not necessarily reflect the in vivo situation for the
couple when hormones are not administered.
Storage and presenlation
Mucus can be preserved either in the original tuberculin syringe orpolyetlylene tube or in small test tubes and sealed with a stopper orwith paraffin papcr to avoid dehydration. Care should be taken to minimize the air spacc in the storage container. Thc samples should bepreserved in a refrigerahr at 4'C for a period not exceeding 5 days. If
possible, mucus specimens should be utilized within 2 days of collecLion, and the interval between collection and use should always be
noted. Rheological and sperm penetration tests should not be performed on mucus specimens that have been frozen and thawed.

Evaluation of cervical mucus
Evaluation of thc properties of cervical mucus includes assessment of
spinnbarkeit, ferning (crystallization),consistency and pH. Appendix
XX gives a sample form for recording the postcoital test in which these
cervical mucus properties are scored according to the syslem devised
by Moghissi (1976), based on the original proposal of Insler et al.
(19 72). The maximum scorc is 15. A score greater than l 0 is usually
inclicative of a good cervical mucus favouring sperm penetration, anda score less than 10 may rcpresent unfavourable cervical mucus. The
score is derived from the volume of cervical mucus collectcd (Section
5.3.1) and four variables (Sections 5.3.2 to 5.3.5) describing its characleristicsand appearancc. The pH of the mucus is not included in the
total cervical mucus scorc, but should be measured as an imporlant
determinant of sperm-mucus iriteraction (Eggert-Kruseet al., 1993).
VolumeVolume is scored as follows:0 = 0 ml,l = 0 . l ml,
2 = 0.2 ml,
3 = 0.3 m1 or more.
Consistency

The consistency of cervical mucus is the most important f'actor
influencing sperm penetration. There is little resistance to spcrm
migration through the cervical mucus in mid-cycle, but viscous
mucus such as that observed during the lutealphase forms a morc for-

midable barrier. Cellular debris arid leulocytes in cervical mucus
impede sperm migration. Gross endocervicitis has been alleged to be
associated n7itt1 reduced fertility.
Consistency is scorcd as follows:
0 =thick, highly viscous, premenstrual mucus,
1=mucus of intermediate viscosity,
2 =mildly viscous mucus,
3 =watery, minimally viscous, mid-cycle (preovulatory) mucus.
5.3.3

Ferning
Ferning (Pig. 5.1) is scored by examination of cervical mucus which
is air-dried on glass microscope slides. Such preparations reveal
various patterns of crystallization which may have a fern-lilte appearance. Depending on the composition of the mucus Lhe 'ferns' mayhavc only a primary stem or the st,crnmay branch once, twice or threelirnes to produce secondary, tertiary and quaternary stems, respectively Scveral fields around the preparation are observed and the scoreis expresscd as the highest degree of ferning that is typical of the specimen according to the following definitions:
0 = n o crystallization,
1=atypical fern formation,
2 =primary and secondary stem ferning,
3 = terliary and quarternary stem ferning.

5.3.4

Spinnbarkeit
The cervical mucus on a microsc,ope slide is touched with a c,overslip,
or a secorid slide held crosswise, which is lifted gently. The length ofthe c,ervical mucus thread stretched in bet,ween is estimated in centimetres and is scored as follows:
O = < l cm,
l = 1-4 cm,
2 = 5-8 c,m,
3 = 9 cm or more.

5.3.5

Cellularity
It is recommended that all cell counts be expressed in cells/mm3(i.e.,
per pl). An estimate of the number of leulcocytes arid other cells in thecervical mucus is traditionally based on the number counted per highpower microscope Geld or HPE One combination of microscope optics
to produce the I-IPF is a 10X wide-field ocular lens (aperture size 20mm) and a 4 0 X objective lens. Because the diameter of the microscope field is equal to the diameter of the the aperture of the ocularlens divided by the magnification of the objective lens, these optics
result in a microscope field with a diameter of approximately 500 ym.
The depth of the preparation can also be standardized by supportingthe coverslip 011 silicone grease containing 1 0 0 pm glass beads(Drobnis ct al., 1988). If the field is 1 0 0 fir11 cleep, its volume must be
0.02 mm3. Thus, a count of 1 0 cells1HPP is equivalent to approximately 500 cells/mm3 under these condit,ions.
The ranlt scores for cells are:0- >20 cells/HPI: or > l 0 0 0 cells/mm3
1= 11-20 cells1IIPF or 501-1000 cells/rnmJ
2 = 1-10 cclls/HPF or 1-500 cells/mm3
3 = 0 cclls
5.3.6

pH
The pH of cervical mucus from t,he endocervical canal should beobtained with pH paper, range 6.4-8.0, in situ or immediately following collection. If the pH is mcasured in situ, care should be taken t,oincasure it correctly, since thc pH of exocervical mucus is always
lower than that of mucus in the endocervical canal. Care should also
be taken to avoid contamination with secretions of the vagina, whichhave an ac,idicpH.
Spermatozoa are susceptible to changes in pH of thc cervicalrriucus. Acid mucus iminobilizes spermatozoa, wl~crcasallialine
mucus may enhance motility. Excessive alkalinity of the cervicalmucus (pH greater than X. 5) may, however, adversely affect. the viability of spermatozoa. Thc opt.irnumpH value for sperm migration andsi~rvivalin the cervical nilicus is between 7.0 and 8.5, which represents the pH range of normal, mid-cycle cervical mucus. However, apH value between 6.0 and 7.0 rnay still be compatible with spcrrn penetration.
Tn some cases cervical mucus may be substantially morc acidic.
This can be due either to al>normalsecretion or to the prescnce of abacterial infection.

5.4

Interaction between sperm and cewical mucus
Cervical mucus is receptive to sperm migration for a limited lime
during the cycle. Oestrogen-rnfluenced mucus favours pcnctrat~on.
The length of time during which spermatozoa can penctrate cerv~cal
mucus varies considerably from one woman to another, and may varyin the same individual from one cycle to another. Abnormality ofsperm-cervical mucus inleraction should not be i~lfcrredwithout
repeated tests in separatc cycles.

5.4.1 I n vivo (postcoital) test(i) Tirning Postcoital tcs1.sshould be performed prior to ov~ilationandas c,loselyas possible to the l.imeof ovulation as determined by clinicalcriteria, i.e., usual cyclc length, basal body tenlperaturc, cervicalmucus changes, vaginal cytology, and, when available, scrum or
urinary oestrogen assays and an ovarian ultrasound examination. It
is important for all laboratories to evaluate the mucus at a standard
time after coitus. This time should be from 9 to 24 hours.

(a)
(b)

(c)

(d)

(ii) Suggested instructions topaticr~tsinpreparutionfor thepostcoital test
You and your partner should abstain from irlt,ercourse for at least 2days bcfnre the test.
The most,suitable day for your tcst is (day)(month) (year).Intercourseshoulrl takc place the night before this day acc,ording to your normal
practice.
Do not usc any vaginal lubricants during intercourse and do notdouche aft,crintercourse. You Inay take a shower alter intercourse but
do not takc a h11 bath.
Reporl to the clinic for the test at (time), on (day) (month) (year).
of postcoital test A nonlubricated speuulum is inserted(iii) Te~:l~r~iqueinto the vagina and a sample ol tlic tluid pool in the posterior vaginal
fornix is aspirated with a tuberculin syringe (without needle), a
pipette, or a polyethylene tube. Using a different syringe or catheter, asample of mucus is then aspirated ti-om the endocervical canal
placed on a glass microscope slide, covered with a(Section 5.7.1).
cover slip, and examined at a standard depth under a phase-contrast
microscope (Scction 5.3.5).

(iv) Vaginulpool sanlple Spermatozoa are usually killed in the vagina
within two hours. The vaginal pool sample is examined to ensure thatsemen has been deposited in the vagina.
(v) Cervical mucus snrvlyle The number of spermatozoa in thc lower
part of the cervical canal varies with tirnc elapsed after inlercourse.
Some 2-3 hours aftcr coitus, there is a large accumulation of spermatozoa in the lower part of the cervical canal.
It is recommended that the concentration of spermatozoa within
the mucus be expressed in standard units (number of spermatozoa/mmi), which is analogous to the rncasurement of mucus cellularity (Section 5.3.5).
Sperm motility in cervical mucus is graded as follows: a. =rapid
progressive motilit,~;b, =slow or sluggish progressive motility; c, =non-progressive motility; and d, =irnmotiIe spermatozoa (Scction
2.4.3). The most important indicator of normal cervical h~nctionis
the presence ol any spermatozoa with rapid progressive motility.
(vi) Lnterpretution The purpose of a postcoital test is not only to determine the number of active spermatozoa in the cervical mucus but alsoto evaluate sperm survival and behaviour many hours alter coitus(reservoir role). Therefore, a test. performed 9-24 hours postcoitallyprovides information on the longevity and survival of spcrmalozoa.
The prcsence of any spermatoxoa with rapid progressive motility at
this stagc in the endocervix argues against significant ccrvical factors
as possible causes of infertility (Oei et al., 1995 ).
Thc postcoital test should be repeated if the initial rcsult is negative
or abnormal. When no spermatozoa are found in the cervical canal orvagina, the couple should bc aslied to confirm that ejaculation anddeposit,ion of spermatozoa into the vagina have occurred. A negative
test may also be due to incorrect timing. A test performed too early or
too late in the menstrual cycle may be negative in a n otherwise fertile
wornan. In some women the lest may be positive for only 1 or 2 days
during tlie entire menstrual cycle. When ovulation cannot be timed
with a reasonable degree of accuracy, it may be necessary to repeat
thc poslcoital test several times during a cycle or to perform repeatedtests in vitro. Repeated abnormal postcoital tcst,s in cycles with
optimal timing are required to establish cervical faclors as a possible
cause of infertility.
5.4.2

I n vitro tests

A detailed assessment of sperm-cervical mucus interaction may beundertaken using in vitro penetration tests. These tests are usuallyperlormed after there is an abnormal postcoital test, and are mostinformative when carried out with crossover testing using donor
semcrl and donor cervical mucus.
When the purpose of thc sperm-cervical lllucus interaction test is
to compare the quality of various cervical mucus specimens, a single
sample of semen with optimum count, motility, and morphology
should be used. On the othcr hand, when the interest is to evaluate thequality of several semen specimens, the same sample of cervicalmucus should be used to assess the ability of spermatozoa to penetrate
it. When an abnormal rcsult is obtained using t,he husband's semen
and the wife's mucus, crossover testing using dorlor semen and donor
cervical mucus can be performed to identify whether the semen
andlor cervical mucus is responsible for the abnormal result. Donor
cervical muc,us can be obtained at mid-cycle from women who arescheduled for artificial inscmirlation. The cervical mucus should becollected prior to insernirialion in natural cycles or cycles in which
ovulalion is induced by treatment with gonadotrophins. Women who
arc receiving clomiphene citrate for induction of ovulation should not
be used as cervical mucus donors because of the possible effects of thisantioestrogen on the cervix.
In vitro tests should be done within 1hour of sernen collection andmid-cycle human cervical tnucus should be used. Surrogate gels suchI

as bovine cervical mucus or synthetic gels cannot be regardcd asequivalent to human cervical mucus for in vitro testing of sperm-ccrvical mucus interaction.
(i) Sir77plij5ed slide test A drop of cervical rnucus is placed on a slideand flattened by a cover slip (22 m m x 22 mm). The depth of thispreparat,ioncan be standardized by supporting the cover slip with silicone greasc containing 100 pm glass beads (Section 5.3.5). A dropof semen is deposited at each side and in corllact with the edge of thecover slip so that the semen moves under the cover slip by capillary
force. In this way a clear inlerface is obtained between the cervicalmucus and the semen.
The slide preparation is incubated at 3 7 'C in a moist chamber for
30 minutes.
At the interfacc, finger-like projections (phalanges) of seminal fluid
develop within a few minutes and penetrate into the mucus. Most
spermatozoa penetrate the phalarlgeal canal before entering the
mucus. In many instances, a singlc spermatozoorl appears to lead acolumn of sperm into the mucus. Once in the cervical mucus, thespermatozoa fan out and move at random. Some return to the seminal
plasma, while most migrate deep into the cervical mucus until theyruect resistance froni ccllular debris or Icul<ocytes.
(ii) l~lterpretation 1nt.erpretationof the simplified slide lest is subjective, because it is impossible to standardize the size and shape of thesemen-mucus interface in a plain slide preparation. Consequently,it
is recommended that lhc test be used only as a qualitative assessmentof spcrm-mucus interaction.
Useful observations from the test are as follows:
Spermatozoa
penetrate into the mucus phasc and more than 90% are(a)motile with definite progression (normal result).
(b) Spermat,ozoa penetrate into the muc,us phase, but most do not
progress further than 500 pm (i.e., about 10 sperm lengths) from thesemen-mucus interface (poor result).
,c) Spermalozoa penetrate into the mucus phase but rapidly become
either imrnotile or show the 'shaking' pattern of movement (abnormal result.,suggesting the presence of antisperrnatozoal antibodics).
(d) No penetration of spermatozoa through the semen-mucus interface
takes placc. Phalanges may or may not be formed, but the spermatozoa congrcgate along the semen side of Lbc interface (abnormal
result).
(iii) Thr cupillarg tube test (sccAppendix X X I )Appendix I A

Reference values of semen variables

Each laboratory should determine its own reference range for eachvariable. For referencc semen variables, specimens should be evaluated from men who have recently achieved a pregnancy, preferably
within 1 2 months of the couple ceasing contraception, or prospective
studies of fertility should be undertaken. The need for largc numbers(around 1000) and the complex relationship between semen analysis
results and fertilization, together with the time taken to achieve pregnancy, make thesc studies difficult to perform. 'Thus, true reference
ranges have not been established as they have for othcr laboratory
tests. To date, no significant differcnces in semen variables have beenfound between racl-S.
It should be crnphasized again that this manualis not only intended
for laboratories dealing with infertility but also addresses the needs oflaboratories investigating potential methods for male fertility regulation or studying rnale reproductivc toxicology. In this conlext the following referencc ranges are given, based on the clinical experience ofmany investigators who have studied populations of healthy fertile
men. Because these values are not the minimum semcn values needed
for conception, e.g., obtained by evaluation of in vitro or in vivo fertility in a subfertile population. their categorization has been changed
from 'normal' values to 'referencc' values. Thus men with semen variables lower than those indicated in this manual may be fertile.
The following reference values give the description of a semen
sample analysecl according to the methods described in this manual.
Reference values
VolumepH
Sperm concentration
Total sperm number
MotilityMorphology
Vitality

2.0 ml or more
7.2 or more
2 0 X l 0%permatozoa/ml or more
40 X 10' spermatozoa pcr ejaculate or more
50% or more motile (grades a + b) or 25% ormorc wilh progressive niotility (grade a )within 6 0 minutes of ejaculation%

50% or more live, i.e., excluding dye,

1Fewer than l X lOVml
Fewer than 50% motile spcrlnatozoa withbeads bound
MAR test
Fewer than 50% motile spermatozoa withadherent particles* Multicentre population-based studies utilizing the methods of morphology assessment in this manual are now in progress.
Data from assisted reproductive technology programmes s ~ ~ g g c s tthat, as sperm morphology falls below 15% normal forms using themethods and defiriitions described in this manual, the fertilization rate
in vitro decreases.
Wllitc blood cells
Immunobead test

Appendix I B

Nomenclature for some semen variables

Since it is often useful to describe deviations frvm reference semenvariables with words instead of numbers, a nomenclature was introduced (Eliasson et al., 1970). It is importarit to recognize that thisnornenclaturc describes only some semen variables and does not
imply any causal relationship. With this stipulation, the nomenclature should he used as follows:
Normozoospcrrnia
Oligozoospermia
Aslllenozoospermia
Teratozoospcrmia
Oligoasthenoteratozoospermia
Azoospermia
Aspermia

Normal ejaculatc as defined by the reference valucs
Sperm concentration less than the reference valueLess than the reference value for motility
T,PSS than the refcrcr~cevalue for morphology
Signifies disturbance of all thrcc variables(combinations of only two prefixes may also be used)
No spermatozoa in tllc ejaculate
No cjaculate

Reference

Eliasson, R., Hcllinga, F., Lubcke, F,, Meyhofer, W., Niermann, H.,
Steeno, 0.& Schirren, C. (1970) Empfehlungenzur Nomenldatur inder Andrologie. Andrologia, 2: 125 7.

Appendix 11

Safety guidelines for the andrology
laboratorya

Conduct of laboratory personnel
Ilurnan fluids such as semen and blood must be regarded as potentially infectious and should therefore be handled arid disposed of withspecial care.
Semen samples could be contaminated with infectious microorganisms or pathogens, and for the andrology laboratory, the most important are the Human Inlrnunodeficiency Virus (HIV) and the Hepalitis
B virus (HBV).
All personnel working in the andrology laboratory should be vaccinated against hepatitis B.
Strict precautions musl be taken to avoid accidental wounds fromsharp instruments conlaminated with semen and the contact ofsemen with open skin, cuts, abrasions, or Icsions.
All sharp objects (needles, blades, etc.) should be placed in a marked
container, which is sealed bcfore being too full and disposed of in thesame way as other dangerous laboratory items. Other disposableitems(gloves,semen containers) should be collected for disposal.
Infection in the andrology laboratory could also occur following
spillage or splashing of infected semen or blood samples. Steps should
be taken to prevent and contain spillages.
The last drops of the specimens should not be forcibly expelled
because this may create droplets or aerosols. Surgical maslts rnust beworn when procedures are conducted that have a potential for creating aerosols or droplets. These include vortexing and centrifuging of
open containers.
Disposable rubber or plastic gloves must be worn when handling freshand frozen semen or seminal plasma and any containers that havecome into contact with semen or seminal plasma. Gloves must be
removed and discarded when leaving the laboratory or when handling the telephonc and door handles. Gloves must not be reused.
A laboratory coat or disposable gown must be worn in the andrology
laboratory. This coat or gown must be removed on leaving the audrology laboratory. Such clothing should not be worn outside the laboratory, especially in social rooms or cafeterias.
" Adapted from: Schrader, S.M. (1989) Safety guidelines for the andrology laboratnry.
FertilityandSterilitg, 51: 387-9.

Disinfectarll soap or antiseptic skin clcarlser should bc readily available in the laboratory, and personnel should wash their hands regularly, cspecially before leaving thc laboratory, after handlingspecimens and after rcrrloving gowns and gloves.
If thc o ~ ~ t s i dofe a scrrlen collection jar is contaminated, it must bc
washed with a disinfcclant solution (e.g., 5.25 gil sodium hypochlorite, or household bleach diluted 1:10).
Laboratory work surhces, which st~ouldbe impcrrneable, must be
decorltaminated with a disinfectant (e.g., 5.25gll sotli~lmhypochlorite, or household bleach diluted 1 :10) immediately after any spills
occur and also 011 completing thc analyses each day.
Disposable laboratory supplies should be used whe~ieverpossible andcarefully disposcd of.
Mechanical pipctting devices must be used for the manipulation ofliquids in the laboratory. Pipetting by mouth is never permitted.
An aildrology lahoratory should carry equipment for rinsing thc eyes.
Reagents, chcrnicals, or dyes of a toxic nature s l l o ~ ~bel dB p t in a fume
cupboard.
Eating, drinking, smoking, applying cosmetics, storing food, etc., mustnot be permitted in the andrology laboratory.
For further rlelails, the reader is referred to two WHO publicalions:
Laboratory Riasaj'ety Manrral, Geneva. Worl~lHealth Organization,
1983;and Guidelines on Sterilization and High-lrvrl Disinfcctiorl Mdhods
Eflective Against IIuinan Irr~rn~tnodeficicncy
Virus, 2nd ed., Geneva,
World Health Organization, 1989 (AIDS Series, No. L.) p. 10.

Appendix 111

Methods for detecting leukocytes

The traditional method for counting leulwcytcs in human semen is touse a histochemical procedure to identify the peroxidase enzyme thatcllaracterizes polymorpllonuclear granulocytes (Fig. 2.4((r)). This
lechnique has the advantage of being relatively casy to perform, but
il does not detect activaterl polymorphs that have rcleased their granules; nor does it detect other species of leulcoocyte, such as lymphocytes, that do not contain peroxidase. Such cells can be detected byimmunocytochemical means.
111.1
111.1.2
1.

2.
3.
4.

111.1.2
(i)
(ii)

(iii)
(iv)
(v)
(vi)

Peroxidase stain using ortho-toluidinea

Reagents
Saturated NH,Cl solution (250 g l l )Na,EDTA, 50 g11 in phosphate buffer (pH 6.0)
Ortho-toluitlinc, (0.25 mg/ml)hH ,O,, 30°h in distilled water'The working solution consists of: 1r n l of reagent 1; 1m1 of reagent
2; 9 m1 of reagent 3; and one drop of reagent 4.
'This solutiorl can be used for 24 hours after preparation.

ProcedureMix 0.1 m1 semen with 0.9 m1 working solution.
Shalre for 2 minules.
Leave for 20-30 mir~utcsat room temperature.
Shake again.
Pcroxidase-positive cells are stained brown, while peroxidase-negalive
cells are unstained.
Count in duplicate 200 lcukocytes in a haemocytometer chamber andestimate the percenlagc of peroxidase positive and negative cells.

From Nahoum, C.R.D. & Cardoso. D. (1980) Staining for volu~lletri' colint of leuliocylcs
in semen and plmstate-vesicular fluid. Fertility arid Sterility. 3 4 : 6 8 9 .
h The Intcrnulionnl Agenry for Research on Carlcer (IARC) has stated that ortlro-toluidine'should be regarded, for practical purposes, as iT it presented a carcinogcriic risk tohumans' (IARC Monogrupi~sun the Evaluntion of the Corcirrogenir Risk of Chemicuh to
Hurrmns (1982). vol. 27, suppl. 4, pp. 169-70).

"

Immunocytochemistry
All classes of human leulocytes express a spccific antigen ( 0 4 5 )that
can be detected using an appropriate monoclonal antibody (see Fig.
2.4(b)).By changing the nature of the first antibody, this general procedure can be adapted to allow detcction of the different typcs olleuliocyte such as rnacrophages, neutrophils, B- or T- cells.

Reagents
Dulbecco's phosphate buffered salinc (PBS).
Constituents of PES solution
CaC1,.2H20
0.1 32 g
KC1
0.2 g
KH,PO,
0.2 g
MgC1,.6H20
01g
NaCl
8.0 g
Na,HP04
1.15 gMake up to 1litrc with water.

Tris buffered saline (TBS): a 1OX stock solution is prepared anddilutcd 1:l0 immediately before use.
Constituents of 10 X TBS solution
Triznla base60.55 gNaCl
85.2 g
Add water, adjust to pH 8 . 6 wilh l (molll) HCI
Malre up to 1litre with water.

Alkaline phosphatase substrate is prepared as described below andfiltered.
Corlstituents of alkalinc phosphatase substrate
Naphthol AS-MX phosphate
DiinethylCormamide
0.1 MTris buffer, pH 8.1"
1M Lcvamisolr
Fast Red TR salt, added just before use

2 mg
0.2 m1
9.7 1111
0 . 1 m1
10 mg

" 1.21 g uf Trizmabasedissnlvedin water. pHadjosted to 8.2 with 1MHC1. madc up to 100
1111with water.

Primary antibody. A mouse monoclonal antibody against thecommon leultocyte antigen, encoded CD45, and widely available
commercially.
Secondary anlibody. Antimouse irnrriunoglobulins raised in a rabbit;
the dilution used will depend on antibody titre and source (e.g., 1 2 5
dilution of the 225 9 antibody niarlufactured by DAKO Corp., 6392
Via Real, Carpinteria. CA 9 3013, lJSA).

Alltaline phosphatase:antialkaline phosphalase complex (APAAP).
Again the dilution will depend 011 antibody titre and source (e.g., 1:50
dilution of the D651 complex produced by DAICO Corp., h392 Via
Real, Carpinteria, CA 9301 3, USA).

(ii)

(iii)

(vii)
(viii)

Cell preparation
Procedure
An aliquot of liquctied semen (approximately 0.5 ml) is mixed withfive volumes of phosphale-buffered saline (PBS) and centrifuged at500.9 for 5 minutes at room temperature.
This procedure is repeated two times and the cell pellet resuspended
in PBS to the original volume of the semen sample. The suspension isthen diluted with two to five times its volume of PBS depending on theconcentration of spermatozoa.
Two 5 p1 aliquuts of this cell suspension are then air-dried onto aclean glass slide, Gxed and stained immediately or wrapped in aluminium foil and stored at - 70 "C: until subsequent. analysis.
The air-dried cells are fixed in absolute acetone for 1 0 miilutes or in a
mixture of acetone, methanol, and 3 7% formaldehyde (in volumetricproportions 95:95:10) for 90 seconds, washed twice with Tris
buffered saline (TES: see III.2.1), and allowed to drain.
Each aliquot of fixed cells is covered with 10 p1 of primary monoclonal antibody and incubated in a humidified chamber for 3 0
millut,cs at.room temperature. The slidcs are then washed a further 2
X withTBS and allowed to drain.
The cells are covered with 10 ~1 of secondary antibody, incubated for
30 minules in a humidified chamber at room temperature, washed 2
X with TES and drained.
To each specimen is added 10 ~1 alltaline phosphatase:anti-alltaline~~hosphatasecomplex (APAAP),and the specimen is incubated for 1
hour in a humidified chamber at room temperature before hcirlg
washed 2 X inTBS and drained.
In order to intensify the reaction product, staining with the secondary
antibody and AR4AP can be repeated, with a 15-minute incubation
period for each reagent.
The cells are washed 2 X wilhTES, drained, and iilcubated with 10 p1of alkaline phosphatase substrate for 18 minutes.
After the alkaline phosphatase colour reaction has developed, theslides are washed with TBS and finally counterstaincd for a few
scconds with haematoxylin bcfore being washed in tap water andmounted in an aqueous mounting medium.

Appendix I V

Sperm vitality techniques'I'hese tests provirle a good internal control of the estimate of motility.
The sum of dead ancl rnotile spermatozoa should not excced 100%.
IV.l
TV. 1.2

Eosin alone

Reagents
EosinY; make a 5 g11 solution of EosinY (Colour Index, C.I. 45380) in
a 9 glL aqueous sodium chloride solution. Alternatively, the standard
stain can be obtained fro111a number of companies in different countries.

IK 1.2 Procedure(i) Either: Mix one drop of lresli scmen with one drop of the eosin solu-

(ii)

IV.2

I%2.1
(i)
(ii)

IV.2.2
(i)
(ii)
(iii)

tion on a microsc,ope slide, cover with a cover slip ant1 examine after
30 seconds at 4 0 0 X wilt1 a light microscope.
These slides have to be asscssed immediately: live spermatozoa are
unstained (white): dead cells are stained red.
Or: Mix the semen and eosin on a microscope slide and after 1minutemake a smear and air dry. This can be examined later undcr oil immersion (1000 X ) with a negative phase contrast microscope.
Live spermatozoa appear blaclr and the dead spermat,ozoa arestained yellow.
Count unstained (live)and stained (dead) spermatozoa as described in
the text (Section 2.5.1).
Eosin-nigrosin (a modification of BLom's technique)

Reagents
EosinY (C.1. 45380), 1 0 gll, in distilled water, i.e., 1%
Nigrosin (C.I. 5042O), 1 0 0 gll, in distilled water, i.e., 10%

ProcedureMix one drop of semen with two drops of 1%EosinY.
Aftcr 30 seconds, add three drops of 10% nigrosin solution and mix.
Place a drop of the semen-eosin-nigrosin mixture on a microscopeslide and make a smear within 30 seconds of adding the nigrosin. The

Fig. IV1. Scl~e~natic
represenlationof typical morphological changes US
human spermatozoa subjected tohypo-osonlotic stress: (a) = rlochange: (b-g) = varioi~stypes oI Lailchanges. Tail region showing
swelling is indicated by tbr llatrhedarea.

smears sho~lldnot be too thick. Allow to air-dry and examine under
oil immersion (1U00x) with a light microscope.
The live spermatozoa are white and the dead arc stained red. The~ ~ i g r o sprovides
ina dark background which nlalces the slides easier toassess.
IV.3

Hypo-osmotic swelling (HOS) test

IK3.1

Swelling solution
Dissolve 0.735 p sodium citrate dihydrate Na,C,H,O,.LEI,O and1.3 5 1 g fructose in 100 m1 distilled walrr. Store aliquots of this solution fi-ozen at -20°C. 'Thaw and mix well before use.

IK3.2

MethodWarrn 1m1 s ~ e l l i n gsolution in a closed Eppendorf tubc at 3 7'C foraboul 5 minutes. Add 0.1. m1 liquefied semen and mix gently with thepipetle. Keep at 3 7 'C for at least 30 minutes (but not longer than 120
minut.cs) and exanline the sperrn cells with a phase-contrast mic,roscope. Swelling of sperm is iderilified as c,hanges in the shape of thetail, as shown in Fig. IV.1. Co~lritin duplicate the number of swollerl
cells ill a total of 200 spermatozoa counted and calculat,~the meanpercer~t,age.
3.

Interpretation of results

The 110s test is considered nornlal for a semen sample il rnore than
60%ol the spermatozoa undergo tail swelling. Tf less than .SO0!, of the
spermatozoa show tail swelling, the semen specimen is corisidered to
be abnormal.
Since some semen samples will have spermat,ozoa with curled tails
before exposure to thc HOS solulion, it is essential that an ejaculate beobserved before exposure to the HOS solution. The percentage of spermatozoa with curled tails in the untreated sample should be sub-tracted from the percentage obtained after treatment to obtain thetrue percentage of spermatozoa that reacted in the HOS test.
IK3.4

Quality control
New batches of HOS solution should be checked against thc old batchand scores should not be significantly different ( p 0 . 0 5 by paired
t-test) before being accepted for clinical use. If the differenceis significant, discard the new batch and prepare another soli~tion.
References
Eliasson, R. CG- Treschl, L. (19 71) Supravital staining of human spermatozoa. Fertilily and Sterilitg,22:134-7.
Eliasson, R. (1981) Analysis of semen. In The Testis, ed. H. Burger &D. de Kretser, pp. 38 1-99. New York: Raven Press.
Jeyendran, R.S., Van der Vcn, II.H., Perez-Pelaez, M., Crabo, B.C. &Zaneveld, L.J.D. (1984) Development of an assay to assess the functional integrity of the hurnan sperm mcrnbrane and its relationshipto other semen characterislics. ]ournu1 oj Reproductintz and Fertility,
70:219-28.

Appendix V

Papanicolaou staining procedure modified
for spermatozoa

The Papanicolaou stain distinguishes clearly between basophilic andacidophilic cell compo~icntsand allows a detailed examination of thenuclear chromatin pattern. Although this method has been used forroutine diagnostic cytology, the standard Papanicolaou method forvaginal cytology gives poor results when applied to spermatozoa. The
present modified staining technique has proved useful in the analysisof sperm ~nnrphologyarid in the examination of immature germ cells(see Figs. 2.9 and 2 . 11).
V.l

Preparation of specimen
The smear (see Section 2.5.3) should be air-dried and then fixed inequal parts of 95% (950 mlll) ethanol and ether for 5-1 5 minutes.

V.2

Staining procedure
Fixed smears should be stairled by the following procedure:
ethanol RC)'%"ethanol 70%
etharlol50%
distilled wat,er (dcionized,demineralized)
Harris's or Mayer's haematoxylin
running wateracid ethanol
running water
Scult's solution'
distilled water
ethanol 50%
ctllallol 70'Yn
ethanol 80%
ethano1 90'%
Orange Ghn
ethanol95%
etha11019 5%
EA-50"
ethanu1 95%
cthanol9 5%ethanol9 5%1
etha110199.5%
Xyleneeor Kotisolf (three stainingjars)

1 0 dipsb
1 0 dipsl I)dips
10 dips
3 minutes exactly
3-5 minutes
2 dips
3-5 minutcs
4 minutes
1dip
10 dips
10 dips
10 dips
10 dips
2 minutes
1 0 dips
1 0 dips
5 minutes
5 dips5 dips5 dips2 minutes
1rrlinute in each" Chrrk the acidity of the water before preparing Lhc diflerent grades of ethanol. The pHshould bc 7.0.
Onc dip corrcspo~ldsto an immersion ol about l second.
Scott's snlutiur~(see Section V.3.4) is used when the ordinary tap water is 'hard'.
" Stains arid solutions: tlle prepared Papanicolai~ustain (EA-50 and Orange G h ) may beobtair~edcomtncrclally 'The same rnn~par~ics
usually manufacture the haen~atoxylin
prcparation.
" Change thexylcne if it turnsmillcy Mount at once wi1hI)PX (BDH 36029) or any monnting medlu~n(e.g., Bukitt, Riedcl de IIaen, 11-30926. Germany ). Xylrnc is not permittedins u n ~ countrieschecause of the health risk on inhalation.
J Car1 Roth GmbH & Co. Schoeniperstrassc l - j , 1 ) - 7 h l S 5 , Karlsruhe, Grrmaiiy.
"v. 3

Preparation of stains
The commercially available stains are ~ ~ s u a l satisfactory,ly
but thc
stains may be preparcd in the laboratory at a substarilia1 saving asfollows:

K3.1

Constituents of €A-36 equiva!ent to EA-50
Eosirl Y ((:olourIndex, C.1. 45380)
I3ismarck Brown Y (C.I. 21000)
Light-Green SF, Yellowish (C.I. 42095)
Distilled water
1:thanol 95'1,
Pliosphotungstic acid
Saturated lithium carboriate solution(in distilled water)

K3.1.1

"Jg

10 g
10 g
'300 m1
2000 ml

4g

0.5 1x11

Procedure
Stock solutions

Preparc separate 10%solutions of each of the stains as follows:
10 g of' Eosin Y in 100 m1 of distilled water.
10 g of' Bismarck BrowriY in 100 ml of distilled water.
10 g of' Light-Green SF in 100 m1 of dislilled water.
(ii) To prcpare 2 litres of stain, mix thc above stock solutions as follows:
50 m1 of Eosin Y
10 ml of Bismarck Brown Y
1 2 . 5 1111of Light-Grccn SF(iii) Malce up to 2 litres with 9 5% ethanol; add 4 g of phosphotungstic arid
and 0.5 m1 of saturated lithium carbonate solution.
(iv) Mix well and store solutio~lat room temperature in dark-browntightly capped bottles. The solution is stable for 2 to 3 months. Filler
before using.
(i)

K3.2

Constituents of Orange G6Orange i; crystals (C.I.1b270)
Ilistilled water
9 S'X, ethanol
Phospllotungstic acid

10 Y

100 1111
IOU0 m1
0.15 gProcedure
Stock solution No. 1 (Orange G6, 10%solution)
Prepare 10%aqueous solution by dissolving 10gof OrangeG crystals
in 100 m1 of distilled water. Shalre well and allow to stand io a darlrbrown bottle at room temperalurc for 1week before using.
Stock solut~onNo. 2 (Omnge G6, 0.5% solut~on)
Prepare as follows:(i) Stock solution No. l , 50 ml.
(~i) Make up with 9 5% ethano1 to 1000ml.
(iii) Add 0.15 g of phosphotungstic acid.
(iv) Mix well and store in dark-brown stoppered bottles at room temperature. Filter before use. The solution is stable for 2 to 3 months.

K3.2.1

K3.3

Constituents of Harris's haematoxylin without acetic acid
Haeniatoxyl~n(dark crystals; C.I. 75290)
95%ethannl
AINIi, (S0,),.12H20
D~stilledwater
HgO

8g
80 m1
160 g
1500 mlhg

Procedure for preparation of the staining mixture
Dissolve 160 g aluminium ammonium sulfate in l600 m1 distilledwater by heating.
(ii) Dissolve 8 g hae~llatoxyliucrystals in 80 m1 9S1%ethanol.
(iii) Add haematoxylirl solution to the aluminium ammonium sulfate
solution.
Heat the mixture to 9 5 "C.
Remove mixture frorn heat and slowly add the mercuric oxide whilestirring. Solution will bc dark purple in colour.
Ilnniediately plunge thc container ini.0 a cold water bath and filter
when the solution is cold.
(vii) Store in dark-brown bottles at room tenlperature and allow to standfor 48 hours.
(viii) Dilule the required amount with an equal part of distilled water andfiller again.
K3.5

Constituents of Scott's solution
N aHCO,
MgS0,. 7H,ODistilled water

3.5 g
20.0 g1000 1111

Scott's solution is to be used only when the ordinary tap water is 'hard'and should be changed frequently, e.g.. after rinsing 20 to 25 slides.

K3.6

Constituents of acid ethanol solution
Ethanol 99.5%
Cwcentratcd HCIDistilled water

300 ml2.0 ml100 m1

Appendix V1

Shorr staining procedure for spermmorphology

See Pig. 2.10
VI.1

Preparation of smear

The sniear (see Section 2.5.3) sllould be air-dried and thcn fixed in
75% etllanol for about 1minute.
VI.2

Staining procedure

Smears should be stained by the following procedure:
running walcr
haemaloxylin
running waterammonium alcohol
running water
50% ethanol
Shorr stair1
SO'%, cthanol
7j0/o ethanol
9 5% etharlol
absolute ethanol
xylene"One

VI.3

12-1 5 dips"
1-2 minutcs
12-1 5 dips
5 passages of 5 seconds each
12-1 5 dips
5 minutes
3-5 minutes
5 minutes
5 minutes
5 minutes
2 passaycs of 5 minutes each
2 passages of 5 minutes eachdip corresporlds to an immersion of ahout 1scconcl.

Reagents

1. Haematoxylin Papanicolaou No. 1 (Mercli. Cat. No. 92 5 3).
2. Ammonium alcohol
9 5 ml 7 5% ethanol+ 5 m1 L 5% ammonium hydroxide
3. Either (i) Shorr solution (Mercli, Cat. No. 9275)(ii) 4 g BDII Shorr powder (RDH 34147-26) dissolved in 220
orm1 of warm 50%ethanol and allowed to cool, with additionof 2.0 m1 glacial acetic acid (in fume cupboard) and subsequent filtration.

Appendix V11

Rapid staining procedure for spermmorphology

A differential stairling set, called Diff-Quik is available for this method(Cat.No. B41 32-1, Allegiance Healthcare Corp., McGaw Park, Illinois
50085-5787, USA).

VII.1
(i)
(ii)
(iii)

VII.2

Reagents

Fixative: 1.8 mgll triarylinelhane in mettlyl alcohol
Solution 1: l g l l xanthene in bodium azide-preserved hulfer
Solution 2: 1.2 5 g11 thiaxirle dye mixture (0,525 g11 Azure A and
0.62 5 g11 mct1)rlene blue) in buffer
Procedure(i) Fix the slidc for 1 5 seconds in fixative.
(ii) Drain the cxcess solution by placing slides vertically on absorbent
paper.
(iii) Stain the slides in solution 1 for 10 seconds and thcn in solution 2 for
5 seconds. Drain the exccss solution bctween steps.
Dip the slides in running water 10 to 15 times to remove excess stain.
Place the slides vertically to drain watcr and to allow them to air-dry
completely.
The
slides can be mountcd.
(vi)(vii) If there arc background problems, a n aliquot of the semen sample
should be washed and new slides prcpared and stained (see Section
2.5.3).
References

Icruger, T.F., Aclierman, S.B., Simmons. K.F,, Swanson, K.J., Rrugo,
S.S. & Acosta, A.A. ( l 987) A quick, reliable staining technique forhuman sperin morphology. Archiv~sof Andrology, 18: 2 75-7.
Enginsu, M.E., Dumoulin, J.C.M., Picters. M.H.E.C., Bras, M., Evers,
J.L.H. & Geraedts, J.P.M. (1991) Evaluation of human sperm morphology using strict criteria after Diff-Ouilcstaining: correlation ofmorphology with fertilization in vitro. Hurnun Reproduction, 16:854-8.

Appendix VIII

Immunobead test

Reagents
1. Immunobeads: anti-lgG, -1gA and -1gM beads arc obtainable from
Irvirle Scientific, Santa Ana, California 92705, USA, and Laboserv
GinbH, Am Bodrn 2 5-2 6, 3 5460 Staufenberg, Germany. For screening purposes, anli-Tg combined beads, which will identify all Llic isotypes, can be used. Keconstilutc the i~nmunobeadsaccording to the
manufacturer's inst,ructions. These can be kept for sevcral months at+ 4" C with preservative.
2. Stock buifer: Tyrodc's solution or Dulbecco's phosphate-bufferedsaline (PRS) can be used (both available co~nmerciallyfrom several
suppliers, e.g., Irvine Scientific).

VIII.l

Tvrodc's solution
CaCI,
ICC1
NaH,PO,
MgCl,.hli,O
NaCl
NaHCO,
Clucnse

(811)
0.2
0.2
0.05
0.2
8.0
1.0
l .O

I~ulbecco'sPBS
(gfl)

CaCl,
KC1

I<H,PO,
Mg('l,.6H,O
NdCl
Na,HP0,.7H2O

0.1
0.2
0.2
01
8.0
2.16

Buffer l: conlains 0.3%bovine serum albumin (BSA: Cohn fraction V):
wcigh out 0.3 g BSA and make up to 100 m1 with stoclr buffcr.
4. Bufier 11: conlains 5 %BSA: rweigh out 5.0 g BSA and make up to 100
ml with stoclc buffer.
5. All solutions should be passed through a 0.22 p m or 0.45 p m filter
and warmed (2 5-35 "C) before use.
3.

Procedure(i) For each Immunobead typc, add 0.2 m1 of stock bead suspension to
10 m1 of Buffer 1in separat,~
conical-base centrifilgc tubes.
Transfertherequiredamountof semen to a conical-base centrifuge(ii)tube and make up to 10 1111 with Buffer 1. The anlount ol semen
required is delermined from the sperm concentration and rnotility
according to thc following t,able.

VIII.2

Concentration(lOh/ml)

Motility(grades a + b)%tL

> 50
20-50
20-50
120
c20
<l0
a

>40
140

>40
i40

Semen required"
(m11
0.2
0.4
0.8
1.0
2.0
22.0

Scc Section 2.4.3.
Aliquots of > l . O ml requirc 3 washes.

(iii) Centrifuge all the tubes at 5009 for 5-10 minutes.
(iv) Sperm tube(s):decant and discard the supernatant. Gently resuspend
the sperm pellet in 1 0 m1 of freshBuffer I and centrifuge again at 500g
for 5-10 minutes. Decant and discard the supernatant. Gently resuspend the sperm pellet in 0.2 m1 of Buffer 11.
Immilnobead
tube(s): decant and discard the supernatant. Gently(v,
resuspend the beads in 0.2 m1 of Buffcr 11.
(vi) Place 5 p1 drops of each type of immunobead on one or Inore glass
slides. Add 5 p1of the washed sperm suspension to each of the bead
drops and mix m7ell using either a pipette tip or the edge of a cover slip.
Place a cover slip (20 to 24 mm square) on each of the mixtures, andafter leaving the slide for 10 minutes in a moist chamber, observe at
4OOX to 500X magnification with a phase-contrast microscope.
(vii) Score separately the percentage of inotile spermatozoa that have two
or more attached immurrobeads (ignore tail-tip binding). Count atleast 200 motile spermato~oain duplicate per prcparation. Rccord theclass (IgG or IgA) and thc site of binding of the immunobeads to thespermatozoa (head, mid-piece, tail).
Interpretation. The test is regarded as clinically significant if 50% ormore of motile (progressive and nonprogressive) spermatozoa arecoated with beads. Binding to the tail-tip is not considered to be clinically relevant.

VIII.3

Indirect immunobead test

The indirect immunobcad test is used to detect antisperm anlibodies
in heat inactivated serum, seminal plasma, or bromelain-solubilized
cervical mucus.
0) Wash normal donor spermatozoa twice with Buffer I as describedabove, steps (ii), (iii), (iv).
(ii) Arljust the washed sperm suspension to a concentration of
50 X 10"inl in Buffer 11.
(iii) Dilute 1 0 p1 of the body fluid to be tested with 40 p1 of Ruffer 11 andthen mix with 50 p1 of the waslied sperm suspension. Incubate at
3 7 "C for l hour.

(iv)VIII.4

Wash the spermatozoa twice again as described above, steps (iii) and(iv), and perform the tests as described in steps (vi) and (vii).

Controls
A posilive control and a negative control should be included in eachtest run. A positive control can be prepared using serurn from a donor(e.g., a vasectomized man) with high titres of serum sperrn antibodies
as detected by the indirect immunobead test. This serum is prepared
as in VI11.3 and assayed in parallel with each test run.

References
Bronson, R.A., Coopcr, G.W. & Rosenfeld, D. (1982) Detection of
sperm specific antibodies on the spermatozoa surface byimmunobead binding. Archives of Andrologg, 9: 61.
Bronson, R.A., Cooper, G.W. &Kosenfeld, D. ( l984) Sperm antibodies:
their role in infertility. Fertility and Sterility, 42: 171-83.
Clarlre, G.N., StojanoB, A . & Cauchi, M.N. (1982) Immunoglobulinclass of sperm-bound antibodies in semen. In Immunology of
Reproduction, ed. K. Bratanov, pp. 482-5. Sofia,Bulgaria: Bulgarian
Academy of Sciences Press.
Clarkc, G.N. (1990) Detection of antisperm antibodies using
Immunobeads. In Hundbook of the Labarutory Diagnosis andTwntrnent of Infert~lity,erl. R.A. Keel & B.W. Webstcr, pp. 177-92.
Boca Raton, Florida: CRC.

Appendix I X

Mixed antiglobulin reaction test (MAR test)

Since IgA antibodies almost never occur without IgG antibodies,
testing for the latter is sufficient as a routine screening method.
Reagents for the MAR test are available from Fertility 'I'echnologies,
Natick, Massa'liusetts 017601, lJSA and from Fertipro N.V.,
Beernem, Belgium.
IX.1
(i)

(ii)

IX.2

Procedure
1 0 ~1 of unwashed fresh semen, 1 0 ~1 of IgG- and IgA-coated latex
particles (kom Fertility 'I'echnologies Inc.) and 10 ~l of antiserum tohuman TgG (from Hoechst-Bchring ORCM-04/05, or Dakopatts
A089, Denmark) or human IgA are placed on a microscope slide.
The drops of semen and IgG (or TgA)-coated particles arc mixccl first,
and thcn the drop of antiserum is added using a larger cover slip (e.g.,
40 mlnX 24 mm), which is thcn laid on the mixture. The wet preparation is observed in the microscope at 400X or 6 0 0 X rnagriification
either using bright field or phase contrast optics after 2-3 minutes andagain after 1 0 minutes.
Interpretation
In the absence of coating antibodies, the sperinatozoa will be seen
swimming freely between tllc pprlicles, which themselves adhere toeach other in groups, proving the effectiveness of the preparation.
If sperm antibodies are prcserll on the spermatozoa, the motile
spermatozoa will have latex particles adhering to them. The motile
sperrnatozoa are initially sccrl moving around with a few or even abunch of particles attached. Eventually the agglutirrales become so
massive that the moverne~its of the spermatozoa are severely
restricted.
At least 200 motile spcrrnatozoa should be counted. The percentage of the motile spermatozoa that have particles attached is calculated.

Appendix X

Measurement of zinc in seminal plasma

X.lBackgroundA calorimetric assay for tlie determination of zinc in body fluids wasshown to be useful for the dcterminatioil of zinc in seminal plasma.
The nlethod given is modified from thal of Johnson K. Eliasson (1987).
A kit for the estimation of zinc in semen is available (from Wako
Chemicals GmbH, Nissanslrasse 2, 41468 Neuss 1,Germany). The
assay described is for users of a plate reader with 96 wells. The
volumes of semen and reagents can be proportionally adjusted forspect.rnphotometers using 3-ml or l-m1 cuvettes. The appropriate corrections must be nlade for the calculatiori of results.

X.2

Principle
5-Br-PAPS +ZnL+-------t 5-Br-PAPS-Zn complex, which absorbs at
wavelength 5 60 nm.
5-Br-PAPS is an abbreviation for 2-(5-brorno-2-pyridylazo)-5-(Npropyl-h'-su1phopropylamino)-pllcnol.

Reagents
1. Zinc kit (from Walw Chemicals), which is stable at room tcmperature
for a year. IJse only colour reagent A ( L X 60 m1 bottles) and colour
reagent E (1 X 3U m1 bottle).
2. Zinc standard: dissolve 0.144 g ZnS04.7H,0 in 5 0 m1 water anddilute
this l 0 O X by adding l m1 to 99 m1 water to achieve a concentrationof 0.1 mM (0.1mmolll); store at room temperature or - 20 'C.

X.3

X.4 Methodl. Prepare a standard curve: dilute 1 0 0 p M zinc standard with water toyield additional standards of 80, 6 0 , 4 0 , 2 U and l 0 PM.
2. Prepare colour reagenl: mix 4 parts colour reagent A with 1 partcolour reagent B (about 2 5 m1 is needed for one 96-well plate). This
chromogen solution is stable for 2 days at room lemperature or 1week
at4"C
3. Sample preparation:(i) Centrifuge llle semen sample for 10 minutes at 1000g, decant thescnlinal plasma and store until anaIysis at - 20 'C.
(ii) Thaw the sperm-free seminal plasma and mix well on a vortex mixer.

(iii)

(iv)

(v)
(vi)

(Internal quality control samples containing high, medium, and low
concentrations of zinc are also assayed.)
Dilute each sample of seminal plasma in duplicate: to 300 p1 water in
a 1.5 m1 tube add 5 p1 semirlal plasma (with a positive displacement
pipette) and mix.
To the 96-well plate add duplicate 4 0 ~ diluted
1semen samples.
Include duplicate blanlcs (40 kl water) and standards.
Add 200 p1 colour reagent and mix for 5 minutes on 96-well-plateshaker.
Read plate at 560 nm wavelength.

X.5 Calculation
1. Rcad the concentration or zinc in the sample from the standard curve(mM).
2 . Multiply by the dilution factor (61) to obtain the concentration of zinc(mM) in undiluted seminal plasma.
3 . Multiply by ejaculate volume to obtain pmol/ejaculate.
4. Reject samples that lie above the top standard and reassay thesesamples at greater dilution.
X.6

Reference value
2.4 pm01 or more per ejaculate.
ReferenceJohnson, 0.& Ellasson, R. (1Y 8 7) Evaluation of a commercially available kit for the calorimetric determination of zinc in humanseminal plasma. lnter~zationalJournal of Andrology, 10:43 5-40.

Appendix XI

Measurement of fructose i n seminal plasma

Background
The mcthod given is modified from that of Karvonen & Malm ( l 9 5 5). A
kit for the estimation of fructose in semen is available (from FertiPro
N.V, Lotenhulle, Belgium).The assay described is for users of a 9 6-wellplate reader.The volumes of semen and reagents can be proportionallyadjusted for spcctrophotometers using 3-m1 or l -m1 cuvettes. The
appropriate corrections must be made for the calculation of results.
Principle

Fructose + indole
length 470 nm.

heat, acidAcomplex, which absorbs at wave-

Reagents
Deproteinizing agents: 63 pM (pmolll) XnS04.7H,0 (dissolve 1.8 g in
1 0 0 m1 water) and 0.1 h4 (mol/l)-NaOH (dissolie 0.4 g in l 0 0 m1
water).
Colour reagent (indole 2 pM in benzoate preservative 1 6 PM): dissolve 200 mg benzoic acid in 90 m1 water by shaking in a watcr bathat 60°C; dissolve 2 5 mg indole in this and make up to 100 ml; filterand store at 4 "C.
Fructose standard (2.24 mM): dissolve 40.32 mg fructose in l 0 0 m1
water; store at 4 'C or freeze in aliquots.
MethodPrepare a standard curve: dilute thc 2.24 mM standard with water toyield four additional standards of 1 .l 2,0.56, 0.28 and 0.14 mM.
Sample preparation:(i) Centrifuge the semen sample for l 0 minutes at 1000g, decant
the seminal plasma and store at -20 "C until analysis.
(ii) Thaw the sperm-free seminal plasma and mix well on a vortex
mixer. (Internal quality control samples containing high,medium and low concentrations of fructose are also assayed.)(iii) llilute each semen sample in duplicate: to 50 p,1 water in a 1 . 5
m1 tube add 5 p1 seminal plasma (with a positive displacement
pipette) and mix.
83

(iv) Deproteinize each semen sample: to the 5 5 p1 diluted sample
acid 1 2 . 5 p1 63 p M ZnS04 and 12.5 p1 O.1M-NaOH, mix, allow
to stand for 1 5 minutes at room temperature and then centrifuge samples at 80009 for 5 1ninut.e~.
(v) Remove 50 pl supernatant t,o test tubes. Include duplicate
blanks ( 5 0 p1 water) and duplicate 50 p1 standards.
(vi) Add 5 0 p1 indole reagent to each h b e , mix: add 0.5 m1 concentrated (3236)HCl to each samplc, cover with Parafilm, mixcarefully in fume cupboardlhood; heat for 20 minutes at 50°C inwater bath, mix: cool in ice-water (15 minutes)".
(vii) Transfer 2 5 0 p1 with a positive displacement pipette to 9 6-wellplates carefully in fume cupboard/hood.
(viii) Read the plate (with thc lid on to protect the spectrophotometer) in a 9 6-rvell plate rcader at 4 7 0 nm wavelength.
XI.5 Calculation
1. Read the concentration of fructose in the sample from the standard
curve (mM).
2. Multiply by the dilution factor (16) to obtain the concentration offructose (mM) in undiluted seminal plasma.
3 . Multiply by the ejaculate volume to obtain pn-ollejaculate.
4. Reject samples that lie above the top stanlard and reassay thesesamples at greater dilution.
XI.6

Reference value

1 3 &m01or more per ejaculate
Reference

Icarvonen, M.J. & Malm, M. (1gC5)Colorirnetric determination offructose with indol. Scundinr ~ i a nJournal of Clinical Laboratorg
Investigation, 7: 305-7.
" 11'the use of concentrated WC1 is cor~sidcrcdunpleasant, an alternative but more expensive enzylnatic assily using ultraviolet spectrophotometrg (e.g., Boehrirrger Munnhcim Itit
No. lJYlU6) may be performed.

Appendix XI1

Measurement of neutral a-glucosidase i nseminal plasma

Background
Seminal plasma contains both a neutrala-glucosldase isoenzyme thatoriginates from the epididymis and an acid isoenzyme contributed by
the prostate. The latter can be belcctively inhibited (Paquin et al,1984) to permit measurement of the neutral a-glucosldase reflecting
cpididymal function. Kits for the esllrr~ationof glucosidasc in semenarc available (from Bochringer Mannheim, Cat. No. 1 742 02 7, andfrom Fertik'ro N.V, Becrnem, Belgium). The method given is fromCooper et al. (1990).The assay described is for users of a 9 6-well-plateof semen and reagents can be proportionallyreader. l'he vol~~niesadjusted for spectrophotometers usirig 3-1111 or l-m1 cuvettes. The
appropriate corrections must be made for the calculation of results.
Principle

a-glucosidase
Na,CO,
p-nitrophenol-a-glucopyranoside ---+
p-nitrophenol +absorbs at 405 nm
Reagents
0.2M (molll) phosphate buffer, pH 5.8: prepare from 0.2MI<,IIP0,.3H20 (dissolve 4.56 g in 1 0 0 ml) and 0.2M- KH,PO, (dissolve 2.72 g in l 0 0 ml) and [nix approximately equal volumes of each
until the pH is 6.8.
EuKer containing l'%sodiumdodecyl sulphate (SDS):dissolve 1g SDS
in 1 0 0 m1 of the above buffer. This precipitates on storage at 4 'C, but
redissolvcs upon gentle warming.
Colow rcagent l (for stopping the reaction): 0 . l M Na,CO,.H,O.
Dissolve 6.20 g Na ,CO,.H,O in 500 m1 water.
Colour rcagent 2 (for diluting the product): colour reagent 1conlaining 0.1 0/ (wlv) SDS.
Subslratc pnitrophenol glucopyranoside (PNPG), 5 g/]: make fresllly
for each assay by dissolvi~lg0.1 g PNPG (Sigma N1377) in 2 0 rril
buffer containing 1% SDS and warm the solution on a hot plate atabout 50°C with stirring for about 10 minutes. A few crystals rrlay
remain undissolved. The solution s h o ~ ~bel dkept at 3 7 "C during use.
Glucosidasc inhibitor for sernen blanks (castanospcrmine): prepare al 0 mM sloclr by dissolving 0.01 89 g castanospermine (Sigma C3 784)
in l 0 m1 water and dilute further in water to give a 1 mM working
solution. Freeze in aliquots at -20 "C.
7. Standard, 5 mM p-nitrophenol (PNY): dissolve 0.0695 g PNP (Sigma
104-8) in 100 m1 water. Warm the solution if necessary and store at
4 "C in a brown bottle. Make up a fresh standard solution every threemonths.
XII.4 Method
1. Prepare a standard curve (within the last hour of incubation):(i) place 4 0 0 ~1 5 mM stock PNP in a l 0-m1 volumetric flaslc arid
make up to 10 m1 with colour reagent 2 (200 FM).
(ii) dilute the 200 pM standard with c o l o ~reagent~r
2 to yield four
additional standards of 16U, 120, 8 0 and 4 0 pM PNP.
2. Prepare samples:(i) Centrifuge the semen sample for 1 0 minutes at 1000g, decant
the seminal plasma and store until analysis at -20°C.
(ii) Thaw the sperm-free seminal plasma and mix well on a vortex
mixer. (Internal quality control samples containing high,medium and low activities of ncutral a-glucosidase are also
assayed.)(iii) Place duplicate 1 5 p1 semen samples with a positive displacement pipettc in 1.5 m1 tubes. Include duplicate blanks (15 p1
water), duplicate internal quality coritrol semen pools containing medium and low activities of glucosidase and quadruplicate
internal quality control semen pools containing high-aclivity
glucosidasc.
(iv) To two of thc high-activity quality-control semen pools add 8 p1
of 1mM cast,anospermineto provide the semen blank value.
(v) Place 1 0 0 pI PNPG substrate solution (at about 3 7°C) in each
Eppendorf tube.
(vi) Vortex each tube and incubate at 3 7 'C for 2 hours (exact temperature and time control are crucial).
(vii) Stop incubation after L hours by the addition of 1 m1 colour
reagent 1and mix.
(viii) Transfer 250-p1 samples and standards to the 9 6-well-plate.
(ix) Read thc plate in a 96-well-plate reader at 405 nm wavelength
within 60 minutes, using the water blank to set zero.

XII.5

Calculation
1unit (U) of glucosidase activity =the production of 1 pmol product(PNP)per minute at 3 7 'C. In this assay the activity is derived from 1 5
p1 semen in a tolal volume of 1.11 5 ml over 1 2 0 minutcs, so a correction factor of 1115/151120 (=0.6194) is required.

1. Read the concentration of PNP produced by the sample from the standard curve (FM).
2. Multiply by the correction factor (0.6194) to obtain the activity ofneutral glucosidase in undiluted seminal plasma (1JlI).
3 . Subtract the activity (Ull) of the castanospermine semen blanlc fromeach sample to obtain the corrected (glucosidase-related) activity.
4. Multiply the corrected activity by the ejaculate volume to obtain glucosidase activity (mu) per ejaculate.
5. Reject samples that lie above the top standard and reassay thesesamples after dilution.
X11.6

Reference value
2 0 mU per ejaculate (minimum).
References
Cooper, T.G.. Yeung, C.H., Nashan, I)., Joclcenhovel,E & Nieschlag, E.
(1990)Improvement in the assessment of human epididymal function by the use of inhibitors in the assay of a-glucosidase in seminal
plasma. Ir~ternatiorlalJourrlal of Andrologg, 13: 29 7-305.
Paquin, R., Chapdelaine, P., Dube, J.Y.&Tremblay,K.R. (1984)Similar
biochen~icalproperties of human seminal plasma and epididymal
a-1,4-glucosidasc. Journal of Andrology, 5: 277-82.

Appendix XI11

Sample instructions and record form forsemen analysis

Sample instructions for the collection and delivery of a semen
sampleRefrain from sexual intcrcourse and masturbation for between 2 days
and 7 days.
Produce the sample by n~asturbationwithout artificial lubrication.
When collection by masturbation is not possible, special condoms (notordinary latex condoms; scc Scctiorl2.1 (e))may be used.
Collect the samplc in a clcan, wide-moutliconlainer of glass or plastic.
It is important that t,hc whole ejaculate is collected. If not, the sample
should bc labcllcd 'incomplete'.
Within 1hour of collection, bring the container and sample to the laboratory, lcccping it warn1 in a p o c k t near to your body.
Samplcs may also be produced in a room near the laboratory.
Labcl thc specirneri with name (andfor identifimtion number), date,
and t i ~ n cof collect,ion.
Sample record form for semen analysis
This samplc record form is offered as a model. It contains a layout forrccording the observations made during the semen analysis, using themethods dcscribed in this manual. When used for c l i n i d purposes, it
may be uscful to add certain derived variables, which are combinations of rcsults frorn the data. An example of such a variable is thetotal motilc sperm count (obtained by multiplying the sperm c,oncentration, the sernen volume, and the percentage of motile spermatozoa). Such derived variables have not been included on the sample
form since their use depends on the clinical circumstances and theclinician's views of their relevance. When used for research purposes,
data from lhe sample record form can be entered directly into a computer database, and any derived variables can be computed electronically.
Thc sample record form has been printed with multiple columns forrccording t,he resulls of semen analyses collected on different dates.
This is a convenient way of presenting serial semen sample results.
Similarly, additional space may be required in certain plac,es to allow
the recording of comments and observations that cannot be coded on
the form.

SAMPLE RECORD FORM FOR SEMEN ANALYSIS
Day
Date of sample
Duration of abstinence (days)
Interval between ejaculation andstart of analysis (min)Appearance(l -normal, 2 - abnormal)
Liquefaction( l - nonnal, 2 - abnormal)
Consistency(l normal, 2 - abnormal)-

Volume iml)

Motility (U/, spermatozoa)(a) rapid progression(b) slow progression(c) non-progressive motility(d) immotile
Agglutination ( X )Vitality (%live)
Concentration (106/ml)
Morphology (%)- normal-head defects-neck or midpiece defects
t a i l defects- cytoplasmic droplets
White blood cells (106/ml)
Immature germ cells (106/ml)
Irnmunohead/MAR test
( X with adherent Ig beads)
MAR test(% with adherent particles)
Biochemistry
zinc (mmol/l)fructose (mmol/l)
a-glucosidase (neutral) (U/l)

L

I

Month

I

I

Year

I

I

Day

I

U

I

I

Month

I

I

Year

I

I

L)ay

I

U

I

I

Month

I

I

Year

I

I

I

U

Appendix XIV

Computer-aided sperm analysis (CASA)

XIV. 1 Systems available
Several commercial manuPxturers provide CASA systems, e.g.,
Hamilton Thorne (Hamilton Thorne Research, Beverly MA, IJSA),
Hobson Sperm Traclrer (Hobson Sperm Tracking, Sheffield, ITnited
Kingdom) and several versions of each systcni are available.
XIV.2

Parameter settings
The correct setup for each CASA instrument is vital for optimum performance and will depend on its anticipatcd use. The manufacturers
providc suitable settings but the user is responsible for checking thateach instrument is performing to the required degree of repcatability
and reliability. Use of appropriate quality control material, c.g.,videotapes, is essential (sce Chapter 4). Thc following authors have discussed CASA settings in a general context (Davis C(r ICatz, 1992;
Mortimer, 1994b).

XIV.3

Preparation of samples
The criteria for semen collection and preparation for CASA applications are identical to those in Chapter 2. The CASA systcrns must
maintain the specimen temperature at 3 7 "C because the variables ofsperm motion are temperature-sensitive.Motility characteristics andsperm cvncentration can be assessed in undiluted semen. Often,
however, as in samples with high sperm concentrations (i.e., greaterthan 50X 1U6/ml),collisions may occur with increasing [requency
and are likely to induce errors. In such cases, dilution of the sample isrecommended. If dilution in homologous seminal plasma is not practical, dilution to a final standard concentration (25-50X10h/ml)
with media is acceptable. Dulbecco's PBS-glucose-BSA medium canbe used (for discussion of media supporting sperm motility, see Farrell
et al., 1996). In orcler to standardize results, the same dilution
medium should be used throughout.

Composition of Uulbecco's PBS-glucose-BSA
(all)

NaCl
KC1
CaC1, (anhydrous)"
Na,HPO, (anhydrous)
KH,PO,
MgC1, ( a n h y d r ~ u s ) ~D-Glucose
Phenol rcd
S o d i ~ ~pyruvate
rl~
BSAb

8.000
0.200
0.1 001.150
0.200
0.04 7
1.000
0.005
0.06
3.000

(mmol/l)
137
3
1

8
1
<J
6
-

<l

-

Also added: 0.4'?h1antibiotic mixture(GIBCO, Grand Island, New York, I ISA)

" Added separatcly to prevent precipitation.
h

BSA Bovine serum ulbumen (Cat. Nu. AhOO3. Sigma Chemical Co.. St. Louis, Missouri),essentially fatty acid frce, is added lasl.

Specialized chambers for use in CASA machines are available. A 20
ym-deep Microcell counting chamber (ConceptionTechnologies, San
Diego, CA, USA)gives reliable results when analysing spermatozoa insemen. Both chambers should be filled and assessed. Several representative fields sho~ildbe examined: 6 fields per chamber (12 fields intotal) give reliable results. At least 200 spermatozoa in each chambershould be assessed.The same principles of quality control apply as forstandard estimations of concentrat,ion (see Section 2.5.2). If hyperactivated spermatozoa separated from seminal plasma are to be examined, the depth of' chamber should be a minimum of 20 km.
XIV.4

Use of the CASA instrument
Samples can either be analysed directly or by means of a video recording. Analysis from a videot,ape improves standardization and allows
implementation of quality assurance procedures (Chapter 4). The
manufacturer will advise on the choice of videotape recorder and on
the optimum sett,ing of the illumination to achieve maximum cuntrast between sperm heads and the baclrground.
The time necessary to follow the spermatozoa to achieve accurate
resu1t.sis controversial.The Hamilton Thorne and the Hobson traclier
will follow spermatozoa for a minimum of 1second, which should be
sufficient to give reliable detcrminations of the basic CASA measurements in semen (see Mortinier, 1994b).

XIV. 5CASA terminology
There is a standard terminology for variables measured by CASA
systems, some of which are illustrated in Fig. XIV.1.

Pig.XIV.1. Standard t e r ~ ~ ~ i n o l o g y
ibr variabics measarecl by CASA
systems.

Curvilinear

V"

-7,

VCL = curvilinear velocity (km/s). Time-average velocily of a sperm
head along its actual curvilinear path, as perceived in two dimensions
in the microscope.
VSL=straight-line velocity (p,m/s). Time-average velocity of a sperm
head along the straight line between its Brst detected position arld its
last.
VAP = averlzge path ve10~:ity(knl/s). Time-average vclocity of a sperm
head along its average path. This path is computed by smoothing theactual path according to algorithms in the CASA inslrument; t.he,sealgorithms vary between instruments.
AIjII=ampBtude of luternl head displacement (km). Magnitude oflateral displacement of a sperm head about its average path. It can be
expressed a s a maxirrlum or an averagc of such displacemcnts.
Different CASA instruments compute ALH using differerit algorithms,
so [hat values are not strictly coniparable.
LIN = 1inenrit.y.The linearity of a curvilinear path, VST,/VCL.
WOB= wobble. Measure of oscillation of the actual path about lhe
average path, VAP/VCL.
STR =straightness. Linearity of the average path, VSIJVAP.
RCF = beat-crossfi.equency (beatdsecond). The average rate at which
the sperm's curvilinear path crosses its average path.
MAD=rr~ean nnguIar displacemmt jdegrces). The time average ofabsolute values of the instantaneous turning anglc ol the sperrn headalong its curvilinear trajectory.
Different CASA instruments use different mathematical algorithms
to compute many of these movcmnent variables. The degree of cormparability of ~rieasurementsacross all instruments is not yet linown.

XIV. 6

Statistical analysis
It is recomrnended that computer-aided sperm analysis be used toobtain movement parameters for at least 200 motile sperrn tracks per
specimen. Note that this will require detec,tion of many more spermatozoa. If il is desired to sort spermatozoa into subcategories of motion
or to make other analyses of variability within a specimen, the traclisof at least 200 and, if possible, 400 motile spermatozoa will be~iccded.Experimental designs should standardize the number of spermatozoa analysed per specimen. It is desirable to interface (he CASA
instruments with computer software that permits data organizationand statisticxi analysis. The distributions of nlarly of the movement
parameters within a spec,inlen are not normal. Thercfore the rncdian,rather than the mean value, should be used to surri~narizethe central
tendency of each movement variable. 11may be necessary to performm a t h e m a t i d t,ransformat.ionsof the measurements for singlc spermatozoa before certain statistical analyses are donc.
References(see Ribliography for the references which also appear in Sections
2.1 1and 2.14)
Davis, R.O. & Icatz, D.E (1992) Standardization and comparability of
CASA instruments. ]olrrn~Jof' Andrology, 13: 81-6.
Farrell, P.B., Foote, R.H., McArdle, M.M., Trouern-Trend. V.L. &Tardif,
A.L. (1996) Media and dilution procedures tested lo minimize handling effects on human, rabbit and bull spcrm for computer-assisted
17: 293-300.
sperm analysis (CASA).Jmrrnal of A~~drolog!~,Mortimer D. (1994b) Praclical Lnboratorg Androloyy. Oxford: Oxford
University Press.

Appendix XVProtocols for the zona-free hamster oocyte
test

XV.1

Standard protocol

Procedure
Allow 3 0 to 60 minutes for full liquefaction of the semen sample tooccur.
(ii) The culture medium for the test is medium BWW (Biggers et al.,1971). This medium is prepared as a stock solution (see below) thatcan be storcd at 4 "C for several weclis without delerioration. On the
day of the tcst, 100m1 of the stock solution is supplemented with 2 10
mg sodium bicarbonatc, 1 0 0 mg glucose, 0.3 7 m1 of a 600 g/l sodium
lactate syrup, 3 mg sodium pyruvate, 350 mg Fraction V bovine
serum albumin, 10 000 units penicillin, 10 mg streptomycin sulfate
and 20 mM IIEPES salts. The mcdium should he warmed to 37°C
before use, preferably in a n atmosphere of 5% CO,,- 95% air.

XI/. 1.1(i)

Chmponents o ~ j " the medium BWW stock solution
Compound

N aCl
KC1
CaCl2.2H,O
KH,PO,
MgSO ,.7H,O
Phenol red, 1.0m111

Ouantity
g/l
5.540
0.356
0.250
0.1 62
0.294(iii) Semen samples are prepared using the spcrrn preparation techniques
described in Appendix XVIII. I f the 'swim-up' procedure is used, anumbcr of tubes (three to ten depending on the volumc of the sample
and the concentration of the spermatozoa in semen) can be prepared.
Thc tubes are incubated at 3' 7 'C for 1hour m an atmosphere of 5%CO,, 9 5% air. If such a n incubator is not available, thc tubes may bc
tightly rapped and maintained at 3 7 ' C in air. During the incubation
period the most motile spermatozoa migrate from the seminal plasma
into the overlying medium.
(iv) The sperm suspension is centrifuged at 5009 for 5 minutes and thenresuspcrided at approximately 1 0 X 106spermatozoa/ml in a volume

of not less than 0.5 m1 and irlcubated for 18-24 hours at 37 "C in a n
atmosphere of 5% CO,, 9 5% air. If a CO2incubator is not available, thetubes can be capped tightly and incubatcd at 3 7°C in air. During their~cubationperiod, the tubes should be inclined at a n angle of 20" tothe horizontal in order to prevent settling of the spermatozoa into apellet and to increase the surface area for gaseous cxchange.
(v) Oocytes can bc obtained from immature hamsters injected at random
or from rnaturc hamsters injected on day one of the oestrous cycle.
Pregnant mare's scrum (PMS) and human chorionic gonadotrophin(hCG) are injected intraperitoneally at a dose of 30 to 4 0 IIJ, 48 to 72
hours apart. The oocytcs should then be recovered within 18 hours
after the injection of hCG and prepared at room temperature using
0.1% hyaluronidase and 0.1% trypsin to remove the cumulus cells
and zonae pellucidae, respcctively. Each enzyme treatment should be
followed by two washes in mcdium BWW. The isolated oocytes can bewarmed to 37 "C and introduced immediately into the sperm suspcnsions or stored at 4 ° C for up to 2 4 hours.
(vi) At the end of the capacitation phase, the incubation tubes arc
returned to a vertical positiorl for 2 0 minutes to allow settling of any
immotile cells, after which the motilc spermatozoa are aspiratedin the
supernatant and adjusted to a concentration of 3.5 X 10' motile spermatozoa/ml. The spermatozoa are thcn placed under liquid paraffinin 50 to 100 ~1droplets, and 30 zona-kce hamster oocytes are introduccd, incorporating at least 1 5 oocytes pcr droplet. The gametes aret,hen incubated at 37°C in an atmosphere of 5% CO,, 9 5% air for 3
hours.
(vii) The number of spermatozoa that have entered the oocytes is thenassessed by carefully removing the oocyles and washing them free ofloosely adherent spermatozoa; after which thcy are compressed to adepth of about 30 p m beneath a 22 mmX22 mm cover slip andexanlined by phase-contrast microscopy. (Fixation and storage of theoocytes in, for cxample, 1%glutaraldehyde, hllowcd by staining withlacmoid or acctn-orcein, is a possibility.)(viii) The oocytes should then be examined to determine the percentagethat have spermatozoa within their cytoplasm and the mean numberof incorporated spermatozoa per oocyte (Pig XB1). Thc presence of
spermatozoa remaining bound to the surface of the oocyte after the
initial washing procedure should also be rec,orded, sincc this may givesome indicat,ion of the proportion of the sperm population that has
undergone t,hc acrosome reaction.
XV.2

Protocol incorporating Ca ionophore (A23187)
A highly motile sperm population is prepared by Percoll gradient centrifugation employing a two-step discontinuous gradicnt as describedin Appendix XVTTT. The pellet at the bottom of the 80% fraction is thenFig. .
]'
X
Azona-free hamster
oocyte containing humanspel-matozoa. as seer1 by phasecontrosl ~nicrvsvpy.The arrows
ir~dicatcthe presence of
decondensing sperm heads w~itbir~
the ooplas~n(scale: X = 500).
From Ailken cl al. (1983).

taltcrl LIP into 8 m1 of medium BWW, cenlriruged at 5008 for 5
mir~utesand finally resuspended at a concetltralion of 5 X 10"notile
sprrmatozoa/mL.
A2 3 1 8 7 is prepared as follows: a 1 mM suspension of the free acidol A2 318 7 is prepared from a 10 rnM stoclc in dimethylsulfoxide(DMSO) by a l in 10 dilution with medium EWW. This suspensioil isliepl at 4 "C for at least 3 days before use.
On the day of the test, the A 2 31 8 7 is added to the spermatozoa toachieve two final concentrations of 1.25 and 2.5 FM. The

dose-response curve for ionophore lrcatment varies from individual
to individual, and as a resu It it is preferable to test each patient at both1.25 and 2.5 ~LM.
The spermatozoa are incubated with the ionophore
lur 3 hours, aftcr which the cells are pelletcd by centrift~gationat 5008
and resusperlded in the sarnc volume of fresh lnedi~~rn
BWW.
At this point t,he percentage of motile spermatozoa is assessed, andIhc concentration of spermatozoa is readjusted to 3.5 X 10hmolilc
spermatozoalml before being dispersed as 50-100 p1 droplets underparaffin oil. IIowcver, valid results can still bc obtained using concentrations as low ay l X 10hmotile spermalozoa/ml (Ailltcn L(. Elton,
19861.
The zona-free hamster oocytes, prepared as ciescribed by
Yarlagi~nachiet al. (1 9 7 h ) ,are dispensed into the droplets with about
5 oocytes/drop and 20 oocyteslsample.
Aker a further 3 l~oursthe oocytcs are recovered from the droplets,
washed ti-ee of loosely adherent spermatozoa, cornpressed lo a depthof about 3 0 pm under a 22 X 22 rnm cover slip on a glass slide andassesscd for the presence of decondensing sperm heads with anattacllcd or closely associated tail, using phase-contrast microscopy.
The number of spermatozoa penetrating eac,hegg is assessetl and theresulls expressed as the mean number of spermatozoa penctratingeach oocyte.
XV.3

Quality control
The assays lllust be performed with an adequate degree of quality
control. The within-assay coefficient of variation should be established hy replicating the analysis of a single sample at least 10 times
in one assay. Under sucll circumstances the within-assay coefkicnt
of variation should not exceed 15%. Tlic between-assay coefficient ofvariation should not exceed 2 5'%;it can be ascertained using a cryostored pool of spermatozoa with a ltnown level of penetration. Any
assay in which the value obtained for the standard preparation ismore than two standard deviatior~saway from the mean value
should be rejected as unreliable. Any analysis in which a penetration
score of zero is obtained should be repeated on a separate serncn
sample.
References
Aitlien, R.J. & Elton, R.A. (1986) Application of a Poisson-garnma
model lo study the ~~lfluenceof gamete concentration on
sperm-oocvte fusion In the zona-frec hamster egg penetratlorl tcst.
lourninl of Reproduct~orrand Fcrtlhty. 7 8 . 733-9.
Aitlien, R.J., Templeton, A., Schats. R., Best, F.. Rlchardsor~,D.,Ujahanbalthch, 0 & Let-S,M. (198 3) Methods of assessing thelunctional capacity of humarl spermatozoa: their role in the selection ofpatients for in vitro fertilization. In Fertilization of the Hurnan Egg invitro, ed. H. Beier R H. Lindner, pp. 147-65. Berlin: Springer.
Biggers, J.D., Whitten, W.K. 8r Whiltingham, D.G. (1971)The cultureof' rnouse embryos in vitro. In Methods in Mummalian Enlbryology,
ed. J.C.Daniel, pp. 86-116. San Francisco: Frceman.
Yanagimachi, K., Yaoagimachi, H. & Rogers, R.J. (1976) Thc use of
zona-free animal ova as a test system for the assessment of fertilizing capacity of human spermatozoa. Biology of Reproduction, 15:471-6.

Appendix XVI

Measurement of reactive oxygen species
generated by sperm suspensions

XVI.1

(i)

(ii)

(iii)
(iv)

(v)

ProtocolThis procedure requires the use of a sensitive luminometer (e.g., theBerthold LR 9505) to measure low amounts of light generated byhuman spermatozoa in the presence of a cl~emiluminescentprobe
such as luminol or lucigenin. The mcthodology described employs a
mixture of luminol and horseradish peroxidase to achieve sensitive
rlleasurements of hydrogen peroxidc generation. However, otherprobes have also been used to monitor the production of reactive
oxygen species (ROS)by the washed human ejaculate including lucigenin which is believed to measure the extracellular release of' superoxide anion (Aitken et al., 1 992: McKinney et al., 1996).
Pipette 400 p1 of washed human spermatozoa (10X10"permatozoalml) suspended in a simple ICrebs Ringer medium lacking phenol
red into a disposable lunlinometer container being careful to avoid thecreation of air bubbles.
Add 4 ~1 of 5-amino-2,3 dehydro-1,4 phthaladnedione (luminol;
Sigma Chemical Company, St. Louis, MO) prepared as a L 5 mM stockin DMSO (dimethyl sulfoxide), to the container t.ogether with 8 p1
horseradish pcroxidase (12.4 U of horseradish peroxidase Type Vl,
3 1 0 U/mg: Sigma Chemical Company).
Monitor the chcmiluminescent signal at 37OC for about 5 minutes
until it has stabilized.
FMLP -provocation tesl for leuliocytes
Add 2 p1 of the leukocyte-specific probe formyl-methionyl-leucylphenylalaninc (FMLP), prepared as a 1 0 rnM stock in DMSO, to thereaction mixture in order to stimulate a chemiluminescent signal
from any polymorphs that are present in the sperm suspension(Krausz et al., 1992).Sirlce FMLF receptors are not present on thesurface of human spermdt,ozoa,this signal is specific for the le~~ltocyte
population and can be calibrated with suspensions containing known
numbers of polymorphonuclear leul~ocytes.
PMA provocation of ROS generation by leulcocytes and spermatozoa
After the FMLP signal has subsided, treat the sperm suspension
with 4 p1 phorbol 12-myristate 13-acctate (PMA), preparecl as a 1
rrlM stock solution in DMSO and diluted 1 in 100 to give a l 0 p M
working stock solution and a final concentration of 100 nM. This

Fig. XVI.1. Patterr~sof
chemilumi~lesccnceobserved withhuman sperm suspenslolls using
the lurninol-peroxidase system. (a)In Lhc presence of leukocyte
contaminatio~la burst of ROS
generation is observed on additionof the leukocpte-sperificprobe
FMLP (formyl-methionyl-leucglpllcnylalanine). Thc subsequent
addition of PMA (12-myristra~c
13-acetate phorbol ester) generates
a sustained, inteiise
chemilun~inescentsignal from boththe spermatozoa and lenkocylc
populations. (b) In the abscnce of
Irokucyte contamination, the FMLP
resonse is lost while PMA elicits apronounced ch~miluminesced
signal fro111the spermatozoa(Krausect al.. 1992).

Time (min)
(b)
L

.

.

.

I

I

I

.

.

,

I

.

1.00-

PMA

1

.

.
5

.

.

.
10

.

.

.

.

.

15

.
20

.

.
25

Time (min)reagent gives an indication of the contributions rnade by each of theleulcocyte and sperm subpopulations to the reactive oxygen generating capacity of the suspension (Fig. XVI.1).
Note: The capacity of a leul~ocytclo generate reactive oxygen species
is at least 100 times greater than that of a sperniatozoon. A low levelof leukocyte contamination can therefore havc a major influence on
the chen~iluminescentsignals gencrated by a sperm suspension.
References
Aitlen, R.J., Buckingham, 1l.W.&West, 1C.M. (1992) Reactive oxygen
species and human spermatozoa: analysis of the cellular nlecha-

nisms involved in luminol- and lucigenin-dependent chemiluminescence. Journal of Cellmlar Phgsio10q.y~ l 5 l :466-77.
Krausz, C., West, I<,Bucliingham, D. llr Aitlien, R.J. (1992) Analysis of
thc interaction between N-formylmethionyl-IeucyI phenylalanineand human sperm suspensions: devcloplnent of a technique formonitoring the contamination of human semen samples withleukocytcs. Fertility and Stenlity, 5 7: 1 31 7-25.
McI<inney, K.A., Lewis, S.E.M. & Thompson, W. (1996) Reactive
oxygen specics generation in human sperm: lurninol and lucigenin
chernilurnincsccnce probe. Archives of Andrologg, 36: 119-2 5.

Appendix XVII
-

p
-

P

P

Induced acrosome reaction assay

The acrosome reaction (AR) IS an exocytotic process that occurs after
the spermatozoa bind to the zona pellucida and must take place before
the spermatozoon can penetrate the oocyte vestments and fertilize lhe
oocyte. Calcium influx is believed to be an initiating event in thenormal AR. Inducing calcium influx by using a calcium transporting
agent, e.g., ionophore A 2 3 1 87, or by progesteronc treatment, areways of teqting the competence of capacitatcd sperrnatozoa toundergo thc AR. However, testing acrosome status needs further evaluation bcfore it can be considered a routine clinical assay.
Acrosome reaction inducer
Prepare a stock solution of 2-5 mM calcium ionophore A23 187 (freeacid) in dirnethylsulfoxide (DMSO) and store in small aliquots at-20°C. Nolr: A23187 is light sensitive, so precautions should betaken to prevent exposure to light.
Prepare a working solution by dilution of the stock solution with
culturc medium such that a small volume (e.g., 2.5-10 1 ~ 1 )may be
added to tubes to yield a final concentration of A 2 3 1S 7 in the assayof 1 0 p M arid a DMSO concentratioil of <1%.
XVII.2

General procedure
Allow
30-60 minutes for complete liquefaction of semen.
(i)A
techniquefor sperm preparation to obtain a highly motile popula(ii)tion frcc from contaminants such as leukocytes, germ cells, and deadsperrnatozoa is recommended (see Appendix XVIII) but is not mandatory.
A
bicarbonate-buffered medium, such as BWW (see Appendix XV),
(iii)
Harn's P-10, or synthetic human tuba1 fluid (HTF),containing 10-35
gll human serum albumin should be uscd in the test for culturing thespermatozoa to induce capacitation. Warm the medium before use to
37 'C, preferably in a 5% CO,, 95% air incubator. It is recommended
that lhe capacitation-inducing mediurn be prepared freshly for eachassay.
(iv) Prepare a control and a n experimental tube each containing 1X 106
motile spermatozoa/ml and incubate for 3 hours to induce capacitation.

(v) To the cxperimental tube add a suficient volume of A2 3 18 7 worlcing
solution to yield a tinal concentration of 10 FM. To the control tube,
add the same volurne of DMSO.
(vi) Illcubate both tubes at 37 "C for 1 5 minutes. Remove a small aliquot
from i.he control and experimental tubes for motility determination
before stopping the reaction, e.g., sing 3% glutaraldehydeor elhanol.
(vii) Transler the fixed spermatozoa lo precleaned microscope slides andair dry.
(viii) Stain the spcrrnatozoa using fluoresccrll labels evaluated bytluorescence microscopy.
(ix) Assess the percentage of acrosome-reacted spermatozoa in the experimental samplcs (test % AR) and the control samples (control O/o AR).
XVII.3Results
The acrosome reaction after ionophore challenge (ARK) is the test %AR minus the control % AR. The normal value is 15%.Values <10%
are considered abnormal. Values between 10%and 1 5% suggest thatsperm function may be abnormal. If >20% of spermatozoa in thc
control tube show a spol~taneousAR after 3 hours' incubation thenpremature AR has occurretl.

XVII.4

Quality control
A positive coritrol samplc rnust be run cach time the test is performcd.
To ensure that each new preparation of stain has bcerl made properly,a crossover test should be performed with the old stain using positive
control spermatozoa with a linown rcsponse.
Reference
European Society of Human Reproduction and Ernbryology ( 1 996)
Conscrlsus workshop on advanrrd diagnostic andrology. Hlirnan
Keyrod~~ction,
11: 1463-79.

Appendix XVIII

Sperm preparation techniques

XVIII.l

Procedures
Several simple preparation techniques are described. For all of themthe culture medium suggested is supplemented Earle's balanced salt
solution, although other media that will support sperm motility andviability for at least 18 hours at 37 'C may be used, e.g., Ham's F10,
Humall tuba1 fluid (HTF).

XVIII.2

Swim-up
Supplemented Earle's medium (1.2 ml) is gently layered over semen (1ml) in a sterile 1 5 m1 conical-based centrifuge tube. The tube is
inclined at an angle of 45" and incubated for 1hour at 3 7 'C. lt is thengently returned to the upright position and the uppermost 1 m1
removed. This aliquot of motile cells is then diluted with eight volumesof supplemented Earle's, centrifuged at 500g for 5 minutes, andfinally resuspendell in 0.5m1 of Earle's medium for the assessment ofor for other procedures.
sperm concentration or sperm ru~~ction,

Constituents of supplemented Enrle's rrledium
46 m1 Earle's balanced salt solution
4 m1 heat-inactivated ( 5 G ° C for 20 minutes) palicnt's serum
1.5 mg sodium pyruvate
0.18 mlsodium lactate (60%syrup)
100 m g sodiurr~bicarbonate

50 1x11 Earle's balanced salt solutivrl
3 0 0 mg h u m a n serum albumina
1 . 5 mg sodium pyruvate
0.18 ml sodium lactate (6O'Kb syrup)l 0 0 m g sodium bicarbonate" Fur assisted reproduction procedures such as in vitro fertilization (IVF), artificial insemination, or gamete intrafallopian transfer, it is imperative that thc human serum albumin
be highly purified and kee from viral, bacterial. and prior1 contaminatiol~.Some preparutions of albumin have been designed for such procedures (e.g.. from lrvine Scientific,
Santa Ana. CA, ClSA: Armour Pharmaceuticals, Fastbournc, United Kingdom).

Discontinuous density gradients
Pipette 3 ml of 801X,PercoIl or alternative product into a 15 1111sterile
conical-botl.orned tube.
(ii) Gently pipettc 3 m1 of 40% Percoll or alternalivc product on top of the
80% layer, taking care not lo disturb the inlerface between the twolayers.
(iii) Gently layer 1-2 ml semen over the gradient arid centrifuge al 50Og
for 20 minules.
(iv) Resuspended thc pellet at the bot,tom of the 80°h fraction in 5-10 m1
of Earle's medium and centrifuge at 5008 for 5 mitiutes before resuspending it in 1ml of Earle's medium.
Estimate
the sperrn concentratiorl and motility or spcrln function.
(v)
It should be notcd that Percoll has been withdrawn from use in
human clinical applications and car1 now be used only for research
purposes. IIowevcr, other products are available for human clinicalapplications, e.g., PureSperm (NidaCon Labs, Gotlienburg, Sweden)or Isolate (Irvirie Scientific, NidaCon, Santa Ana, CA, IJSA). To dale,
only the manulacturer's instructions for these techniques are available. There are few publications comparing their eficicilcy to that ofother density gradient centrifugation techniques although someclinics have found the techniques to bc equal to those useing Percoll(Perezet al.. 199 7). RothPureSperm andIsolate areisotonic; thus volunzetric dilutions (S + 2 and 4 + 6) arc required. PureSperm is in a
gradients should thus be rnade using a
Hepes-buffered merli~~m;
Hepes-buffered medium.

XVIII.3(i)Corrstituents of isuLonic Percol1 or olterrrati\?eprodl4~t
10 m1 of 10x concentrated Earle's medium (c.g., from Flow Laboratories)
90 m1 density gradient material
300 mp human serum albumin
3 mg sodium pyruvalc
0.37 ml sodiurn lactate (60%syrup)
LOO mg sodiu~nbicarhonalc

Constituents of 80% dunsitq gradient rnuterial
40 m1 isotonic material
10 ml supplemented Barle's medium

Cr1nstituents of 40%der~sitggradient material
2 0 m1 isotonic material
30 1111 supplemented Earle's medium

XVIII.4

Preparation of poor quality samples

In cases of severc oligozoospermia andlor asthenozoospermia,
dcnsil y gradients arc preferable to the 'swim-up' method because of
the improved recoverics obtained. Moreover the scale and composition of the gradient can be altered to mcct the specific needs of individual samples. One possibility, for example, is to apply the sample toseveral minidensity gradients (Mortimcr, 1 994b) containing or~ly0.3
ml volumes of the 40% and 80%density gradient material.
References

Mortimer, L). (1994b) Pr~cticalLaborutory Andrology. p.393. Oxford:
Oxford University Press.
Perez, S.M., Chan, P.J., Patton, W.C. &King, A. (1997). Silane-coatedsilica particle colloid processing of hurnan sperm. lournal of Assisted
Reproduction arzdGenetics, 14: 368-93.

Appendix XIX

Quality control (QC) procedures

XIX.l

QC test samples
QC spccimens should be representative of the range of bemen samples
processed In thc laboratory. If only a small number of OC samples arc
to be analysed, those with results in clinically significant ranges (concentrat~on5 X 106-30 X 106/ml,motility 10-4016, and normal morphology bclow 20%) should be chosen. Centres supplying QC samples[or assessment by others need to ensure that they are negalive forhepalilis arid HTV.

I

XIX.2

Materials for quality control
Aliquols of poolcd scmen samples can be stored frozen or liquid witha preservalive (1 0% formalin, 100 p1 per m1 of semen) at 4OC andanalysed at intervals fbr sperm concentration. Cryopreserved pooledsemen may also be used for QC of motility. Videotapes can be used forsperm motility and photographs, video tapes and stained or unstained
fixed smears on slides can be used for sperm morphology. IIowever,some problerns wilh thesc materials should be recognized. Mixing ofpooled semen may be incomplete, especially before or after thawing.
Video tapes are parlicularly useful for training in motility and morphology assessment. However, t,hcir use for QC tends to give a n optimistic impression and they should not he used to the exclusion of
replimte assessmenls of semen spccimens. Stained slides for spermmorphology tend to fade, and prctixed smears tend to deteriorate.
Spermatozoa may not survive cryoprcscrvation sufficiently well to bea useful source of internal and external QC materials for motility andsperm antibody tests. Sperm antibody positive serum may be used for
QC of indirect immunobead tests but is suboptimal for direct
immunobead tests. Thus OC practice where the same VC pools areincluded in every assay, is not always feasible.

XIX.3

Calibration of equipment
Pipettes can be calibrated by wcighing distilled water from single ormultiple transfers wit11 replication to allow both accuracy and precision to be determined. It is ncccssary to correct for the temperatureeffect on the density of water, i.c., at 2 0 ° C 1 m1 of distilled water
weighs 0.9982 g. Alternatively, rnicropipcttes may be calibrated bydiluting a coloured substance that can bc measured accurately with aspectrophotometer. Counting chambers can be calibrated with beadsuspensions of lcnown particle dcnsity. Counling chamber depths canbe determined with a Vernier scale on thc tine focus of a rriicroscopeby focusing on the grid and then on an ink mark on the underside of
the cover slip. Measurements should be repcatcd a nurnber ol times,
e.g., ten times. Kesults should agree with thc stated volume or depth
within the error of the measuremcnts (+ 2SD). Balances sho~lldbe
checked regularly with internal calibrators and by external calibration at the time of regular laboratory maintenance service.
Temperatures of incubators and warn1 stages should be checlred withthermometers which arc in turn calibraled regularly. Gas mixtures
should be checlred daily with the incubator readout, by Pyrite or othersystems weekly to monthly, and by gas sarrlplirlg al the time of servicing. Other laboratory equipment and reagenls such as p11 meters
should also be checked with standards at 3- lo 6-month intervals.
XIX.4

Some external quality assessment schemes
AUSTRALASIA
1:ertility Society of Australia
Exlcrr~alQuality Assurance Schemesfor Reproductive Medicine
PO&ox l l 0 1
West Leedcrvillc
Western Australia 6901, Australia

EIIKOt'l!
UKNEQAS S c l ~ c r ~for~ c AndrologysDepartment of Reproductive
Medicine
St. Mary's Hospital
Whitworth Park
Manchester M1 3 OJH, Urlilcd
Kingdom

SCANDINAVIA
NAFA (Nordic Associatiorl br
Andrology)
Androluyy IJnit.
Reproductive Medicine Ccnti-c
Karolinslza Hospital
PO Box 140
SE-1 71 76 Stocltholm. Sweden

USA
American Association of
Bioanalysts
Proficiency Testing Service,
205 West Levee
Brownsville.
Texas 78520.5596, USA

Notes:
National external quality control programmes have been created in Bclgium,
the Netherlands and France, and are being developed in other c,ountries.

References
Altman, U.G. (1991) Prartir,al Stutisticsj~wMrrliltal Ruearcll. London:
Chaplnan and Hall.
Barnett, R.N. (1979) Clin~raILaburc~tur-yStatist~cs,2nd ed. Boston:
Little, Brown.

I

1

Appendix XX

Sample Record Form for Cervical Mucus
Scoring and Postcoital Test

Day

Month

Year

U

Date of last menstrual period
Daily cervical mucus score

Day
Date

Month

~

L

Day

Month

-

,

l

l

Month

L

U

l.-L

VolumeU

U

U

U

Consistency

U

U

U

U

Ferning

U

U

U

U

Spinnbarkeit

L.A

U

U

U

Cellularity

U

U

U

U

Total score

U

U

l-L.J

U

P1-I

U

U

U

U

Day
Date
Time after coitus (h)

Month

U
U
Vaginalpool

Cndocervicalpool

L L L L l u
Motility (% spermatozoa)(a) Rapid progression(d) Immotile

Day

U

Postcoital test(C) Nonprogressive

Month

U

Day of cycle(b) Slow progression

Day

AAppendix XXI

Sperm-cervical mucus interaction: thecapillary tube test

The capillary tube test was originally designed by Icremer (1965). It
measures the ability of spermatozoa to penetrate a column of cervical mucus in a capillary tube. Various modifications of capillary tube
tests have bccn proposed. The proccdurc recommended here is basedon the original test derrcloped by Krcmcr, which most clinicians haveadopted.
XXI.1

(i)
(ii)

XXI.2

Equipment
Various types of capillary tubes have been used but flat capillarytubes, 5 cm long and 3 mm wide by 0.3 mm deep in cross section arerecommended. Such tubes can be obtairiecl lrom Carnlab T,td.,
Cambridge, United Kingdom.
The Icremer sperm penetration meter (Fig. XXI. 1)may be constructedin the laboratory as follows:
Glue onto a glass slide three reservoirs (R) of semicircular crosssection (radius about 3.5 mm).
A second glass slide is then glued onto the first. The second slide is 1 . 5
cm shorter and is positioned at a distance of 5 mm lromthe reservoirs.
This construction prevent,^ creeping of the seminal fluid between thecapillary tube and the glass slide. A centimetre scalc is prcscnt on theslides.
MethodFresh semen, not older than 1hour after ejaculation, should be used.
Cervical mucus is aspirated into a capillary tube, making sure that no
air bubbles are introduced. One end of the tube is sealed with plasticine, modelling clay, or similar agents. Enough sealer should beapplied that the mucus column protrudes slightly out of the open end
ol the tube. The open end or the capillary tube is then placed on a slide
so that it projects about 0.5 cm into the reservoir containing thesemen sample. During the process of sperm penetration, the slide
should be placed in a covered Petri dish with damp sponge on the sides,
to maintain humidity and prevent drying of the semen and mucus.
The measurements are carried out preferably in a chamber with anambient temperature of 3 7 "C.

Fig. XXl.l. The Kremer sperm
penetration meter (see text).

XXI.3

Assessment of the test
After 2 hours, the migration distance, penetration density, migration
reduction, and presence of spermatozoa wilh forward motility areread.
The capillary tubes may be inspected again after 24 hours for thepresence of forward moving spermatozoa only.
Variables to be assessed after two hours are defined as follows.

1

Migration distanceThe distance from the end of the capillary tube in the semen reservoir
to the foremost spermatozoan in the tube.

MI.3.

Penetration density
This is determined at distances of 1 and 4.5 cm from the end of thecapillary tube in the semen reservoir. At each distance the mean
number of spermatozoa per low power field (LPF: 10X 10) is determined. The mean number is obtained from estimations of five adjacent low power fields. The mean of the estimations is expressed in oneof the penetration density classes (see Table XXI.1). For theclassification of the test, the highest penetration density class isrecorded.

3

Table XXI. 1.Order of' classes I$penetration density
Penetration density class

XX1.3.3

Rank order

Migration reduction
This indicates the decrease in penetration density at 4.5 cm comparedto the penetration density a t 1 cm. It is expressed as the difference inrank order numbcr.

Examples
1. Penetration density at 1 cm is 51-100 and at 4 . 5 cm is 6-10. The
migration rcduction value is 3 (rank order 6 to rank order 3 ; Table
XX1.1).
2. Penetration derlsity a1 1cm is 21-50 and at 4.5 cm is 51-100.Thc
migration reduction value is zero because there is no decrease in pcnetration dcnsity.

XY1.3.4

Duration of progressive movements
The prcscnce in the cervical mucus of spermatozoa with forward
motility rnay be determined after 2 and 24 hours.
The results arc classified according to Table XX1.2.

Table XXT.2. C1mssific:cntionof the test results
Migration
distance(cm)

Pcnetration
Migration reduction Duration ofClassification
densityfrom l l o 4.5 cm
progressiverr~ovcments(spermatozoa (decrease in ra~llc
pcr loworder numher)
in cervicalmucus (hours)
power field)

10-<10or:3
orAll combinatio~lsof' test results thatcari~iotbe classified as: negative,
poor or good
>SO and
<3
and

<3 or

4.5 andL

Negative
Poor
Fair

>24

Good

Reference

Kremer, J. (1965) A simple penelralion lest. International Jaurnul
Fertility, 10: 209-1 5.

Appendix XXII

Basic requirements for an andrology
laboratory

The following is a list of the supplies and equipment that would enablea laboratory to function as an andrology laboratory and to performthe basic tests suggested in this manual. This assurnes that a refrigerator, a centrifuge (swing-out rotor), and an incubator are alreadyavailable.
Safety i n the andrology laboratory
Rubber glovcs
Face masks
Laboratory coats
Sodium hypochlorite, 5.25 g11 (e.g., household bleach diluted 1:10
with water)
Protective safety glasses
First-ard liit
Eyc-washing solution
Showcr (optional)
Container for sharp objects
B. Semen analysis
1. W H O Laboratory ik'fnnual fi7r the Exurrlirrution ~ l fH~irnrnn Sernrn nnd
Sperrn- Cervical Mucus 1nterai:tion (fourth edition)
2 . Record forms for selncn analysis
3. Wide-mouth disposable eontairlers with lids. These should be demonstrated not to have toxic effects on sperrnalozoa as follows:(i) select semen samples with high sperm concentration and good
spcrm mot,ility;(ii) divide each sarnple and put hall in a glass container linown to be
non-toxic (control) and the other half in the test container;(iii) assess sperm motility and vitality at hourly intervals for 4 hours;(iv) repeat (i) - (iii) for several samples. If there are no differences
between control and test assessments, the test containers are
considered to be nontoxic.
4. Pasteur pipettes with lalex droppers or plastic disposable transfer
pipettes
5 . Automatic pipettes (e.g., Eppendorf, Oxford, Finnpipet, Pipetman,available from many suppliers)Positive displacement pipette to measure 50 kl, such as the Wiretrol I1
W-100 (Drurnmond Scientific Co., 500 Parliwaq: Broomhall, PA
19008, USA), thc Gilson pipette, (Cilson Medical Electronics SA, F95400, Villiers Ic Bel, France); and the SMI Digital Adjust
MicroIPettor Model P5068-20D for volumes of 2 0 to 100 p1 (SMI
liquid Handling Products, American Hospital Supply Corporation,
Miami, FL 33152, USA)
Laboratory counter, such as the Clay Adams Lab Counter (Fisher
Scientific, Springfield, NJ, USA, catalogue No. 02-670-1 3) and the'Assistant' (Karl Willcrs, Laborbedarc D4815 1Miinster, Gcrmany).
Hacmocytometers (improved Neubauer)
Warming plate (bench top)
Vort,cx mixer
Phasc-contrast microscope, to include X 1 0 , X20, X40 phase objectives, X 100 oil-immersion objective, and X 10 (wide-field) eycpiece,
The microscope should be checlced regularly to ensure that the phaserings arc adjusted for optimal use.
50-wat,t light source
Microscope slides
Appropriate quality control
C. Sperm-cervical mucus interaction
1. Record furtns for scoring
2. Mucus sampling syringe, e.g., Aspiglaire (BICEE I:Aigle, France)
3 . Glass rap~llarytubes for the capillary tube test, 3 mm wide, 0.3 mrrideep. 5 cm long (Camlab Ltd. Cambridge, Un~tedKingdom).
D.

Reagents for Immunobead or MAR test
See Apperidiccs VIII and IX

E.

Reagents for sperm morphology
See Apperidiccs 111to VII.

F.

GraphsFigs. 2.3 and 2. h of this manual for display in the laboratory

Fig. 2.3. Approximate 95%
confidence interval ranges fordifferences between two
percentages determined from
dnplicate counts of 100, 2013 or
400 spermatozoa. To assess tllc
percentages frnm two counts of200. calrulate their average anddifference. II thc dinerence isgreater than indicated by the
N= L00 curve, the sample must bereanalysed. The lower curve shows
the increase ill precision ibr twn
assessmenls of 400 spermatozoa(A1= 400). For an average result ofS'%, the 9.5%co~lfiderlccinterval is
3%. The large stalistical counting
errors associated with col~nting
orlly 100 spermatozoa are apparent
from the top curve (N= 100).For a
result of 5% Lhc two single
asseshrrlcntsof' 100 sperlnatoaoa
could be 3% and 9% by chancealone.

-

,

,

1

:

*

' I - - - ',
8

$

1

'

- - -

/

.

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,

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:

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,

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,
,,

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:
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,,

i

m

,

,

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-

,

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'

/

-,-

,

8

,

- -

,-

a

~-

,
8

,

,

,

,

1

- - m

m

i-

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,

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8

S

h--.'-,,,- - b - - ~ ; - - - - .'--. - '

,

-->

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sazequaa~aduaaM]aq aauaaaa!a0Fig. 2.6. Approximate '35%
confiderlce interval for differrnc~s
bet~'ee11twu C U U K I ~ STO. assess Lliesecounts, calculate their surn anddifference. If the difference isgreater than iudicated by the curve
thcrc may bc a systcrnatic crror (scc
Chapter 4) and the sample must be
I-ranalysed.The large statisticalcounting errors associated withcounting fcwcr than LOO
spermatozoa are apparentPor
example, for a mean count of 60
spernlati~zoa( s u ~ n120).the Lwu
counts could be 50 and 70 bychance alone.

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Yanagimachi, R., Lopala, A.. Odom. (:.I%., Bronson, R.A.. Mahi. C.A. & Nicolson,
A.I.. (1 9 79) Retention of biologic cl~aracteristicsof sona pellucida in highly
corlcenlrated salt solution: the use of salt stored eggs Tor assessing the fertilizing capacity of spcrrnalozoa. I+rtiIity and Sterility, 31: 562-74.
Yeung, (:.I I., Cooper, T.G. & Nieschlag, E. (199 7) A technique for standardisationand qualily control of sirbjectivespermmotility assessnlerllsin semen analysis. Fcrtilit~and Sterility. 67: 1156-8.
dc Ziegler, l)., Cedars, M.I., Hamilton. E, Morcno, T. & Meldrum, D.R. (1987)
Factors influencing maintenance of sperm motility during in vilro processing. Fertility and Sterility, 48: 816-10.
Zinaman, M,]., Ilhler, M.L., Vertuno, E., Fisher, S.G. & Clegg, 11.1). (1996)
Evaluation of cornpuler-assisted semen analysis (CASA)with IDENT slain lo
determine sperm concentration. Journal of Andrologq, 17: 288-92.

A2 3 1X 7, svu calcium ionophore
ubsli~~er~ce,
sexual 4
accessory glands, mulebiochemical assays of function 29-3O,
81-7
inleclior~s10-1 3, 29
accuracy 36
acid ethanol solution 74
acid phosphatase 30
acroso~~ial
region, sperm 19. 20
acrosomc rcuc1iu11
alter ionophore challenge (AKIC)
102-1
assessment 32-3, 102-3
agglutinulior~,s p e r r ~14~
aggregation. sperm 1 4
alarm 1.es111ts1 9
allialine p11osphatase:antiaIkaline
phosphi~lasccumplex (APAAP) 67
alkaline phosphatase substratc 6 b
analysis of variance, two-way 4 4 . 4 6 , 4 8
ilnliuxidant systems 3 1-2
appearance. serrlrrl 6-7
aspermia h2
asthenozoospermia 62
azoospermia h2balances, calibration llJS
hias 37
Blom's techniqyr, modified(cusirl-~~igrosin)
68-9
blood, safe handling 63-4
hromelain 6
Bryan-Leisllman stain 1 1
calcinm ionophorc (A2 3 18 7)in acrosolne reaction assay 32-3,
102-3
in zona-free 11alnstr1-oncyte test 3 1 ,Y 5-7
capillary tube test 110-1 3, 115
I.-carnitine 30
CASA. see computer-aided sperm analysis
castanosperr~~ine
85-6CD45 monoclonal antibody 1 2 , h6
cells
in cervical mucus 5 5-6
in semen inon-sperm) 10-1 1,2 7
counting 13
see ~ l s leukocytesocentrifilgatinn, semen 9. 1X
cervical rnucus 51-9

cellularity 5 5-6collection 53-4
consistency 51. 54-5
donor, for in v i t r o tests 58
cvaluuliur~54-6
krning (crystallizuliur~)51. 52, 5 5
pH 54.56
sample record ibrm 109
sperm interaction, P P sperm-cervicalmucus inleraclion
sperm penetrabilitylsurvival 51-3
Spinnbarkeit 5 1 , 55
storage and preservation 54
surrogalc gcla 58 9
volume 54
chemil~rminescenceassay, reactive oxygcrr
species 99-101
citric acid 30
clothing, protective 6 3
coitus iritvrrrrptus 5
corr~puler-aidedsperm analysis (CASA)
30-1, 90-3
morphology 33
parameter scttinys 40
san~plepreparation 90-1
slalislical analysis 9 3
terminology 9 1-1
use of instrument 9 1
corldo~ns5
confidcncc ir~lervals36, 3 7
containers, semen collccliorr 5, 114
cnntraception, male 1
co~itrollimits
action 41, 42. 43-6
monthly means 48.49
response to results outside 48-5O
warning 41,42,41-6
weekly cl~iirts41-6
countcr, lilborulury cell 2 8 , 115
counting
cells other than sperm 1 3
errors, statistical 16, ? 7-8
sperm 15-1 6
sperm morphology 12-3
conntingchambers 1 5 , 1 7 , Y lcalibration 108
see iilsu 1l;~emocytometermethod
counls, diffcrcnccs beiween two 16, 11 7
creati~iephospllokirlase 5 1
crvopresrrvation, sperm 1 0 7
cumulalive su111(CIJSITM) charts 48
cytoplasmic droplcts 1 9 , 20. L 1

density gradients, sperrn separation 34-5.
105. 106
Uiff-Vuik stain 18, 76
disinfection 64
Dulbecco's phosphate buffered salirle (PM)66
ilye exclusion tests 14, 68-9
CA-50 72
Earle's medium, supplcmcnted 1 0 4
ejaculatory duct obstruction 7.29
enzyme digestion, semen 6
eosin exclusion test 68
eosin-nigrosiu (rnodificd Blom) exclusion
Lest 68-9
cpididynlis, secretory function 29. 30.
85-7epithelia1 cells, exluliatcd 13
eyuipmcnt
basic requirements 114-1 5calibration 107-8
error root Ineau square 46.47-8
errorsmcasurement 36-9
random 36
in semen analysis 37-8
systernulic 36-7
cthinyloestradiol 53-4
external quality asseusment (EQA) 36,
38-9,50
schemes 108
F-test 46, 48"feather~ng"techn~que1 7 1 8fernlng 51, 52, 55
flow cytornelry 3.3
FMLP-provocat~ontest 99. 100
Iructose, in seminal plauma 30, X 3 4gentian violet 1 5
gerr!~cells, immature 13, 2 1,27
glass wool colun~ns1 5"glohnnoospennia" 2 1
gloves 63
a-glucuaidasc, neutral 30, 85-7
graphs. for laboratory display 11 6-17
haemoCytolneter meltrod 14-1 7, 37
Harnillun l'horne CASA system 90, 91
hand washing 64
Harris's haematnxylin 73
hemiaona assay 32
hepatitis B virus (HBV) b3
Hobson tracker CASA system 90,9 1
horseradish peroxidase 99
human immunodeficiency virus (HIV) 63
hypo-oslnotic swellirlg (HOS) test 29,
69-70
hypospermatogenesis 1 3
TgA, sperm antlbodles 2 3 . 2 5 . 2 6

IgG, sperrrl dntibodics 23, 25, 26
IgM, sperm antibodies 23
immunobead test 2 1 , 2 5,77-9
controls 79

indirect 78-9
procedure 77-8
qualily control 1 0 7
reagents 77
reference values 6 1
immunncytochemistrv, leukocytes insemen 11,12. 66-7
ir~fcclions,accessory gland 12-1 3 , 2 9
infectious agents. safety precautions 6, 63
infertility treatments, male 1
instructions to yulienls
povlcoital test 5 7senlen collection 8 8internal qnality control (IQC) 36, 38 9
monthly 4 6 8 , 4 9prucedurcs 107-8
response to results outside control limits
48-50
with stored sarnplcs
sperrrl collccntration 41-4
sperm motility and morphology 44-6
weekly 40-6
Isolate 105
Krcmcr sperm penetration meter 110,111
laboratory
basic requirements 114-1 7graphs for display 1 16-1 7
quality control 36-50
safety guidelines 63-4
variability between, see external quality
assurance
lectins. fluorescent-labelled 33
leukncytrs
in cervical ~rlucus55-6
PMLP-provocation test 99, 1 0 0
insemen 10-13.27,61
counting 1 1
detection methods 11-12. 65-7
preparation 6 7
leukocytospermia 10-1 1liquefaction, semen hlucigenin 99
lurnir~ul99
Makler chamber 1 7
malecoritraccption 1
subfcrtility treatments 1
MAR, see mixed antiglobulin reaction
masks, surgical 63
means, average of, ace Xba,
medium BWW 94
microbiologicaltests
in leukocytospcrmia 12-1 3
semen 29
Microccll counting chamber 1 7 , 9 1
microscope
light 8phase-rontrast 7-8, 115
microscouv. semen 7-23
mixed an&lobulin reaction (MAR) 23,
26, 61. 80

mucin, cervical 51
multiple anomalies index (MAI) L8
Neubauer haemocylor~leter15, 1 15
nentral a-glucosrdase 30, 85-7
p-nitrophenol glucopyranoside (PNPG) 85
normozoosper~ri~a62

practical approacl~es39-46
procedures 107-8
response to results outside limits 48-50
routine surveillance 3 9 4 0
test samples 1 0 7
weekly. intra- and inter-technician
varrabilily 40-6

oestrogen 51
oliguasthenokratozoospermia 62
oligvaouspermia 62
oocytessalt-stored human 32
sona-free hamster 94, 95, 9 7Orangc G6 72
ortho-toluidiue h5
oxygen specles,reactive (ROS) 31-2.
99-101

Rba, c11al.t. 4 1
reaclive oxygen species (ROS) 3 1-2,
99-101
record f o r mcervical mucus scoringlposlcuital test
109
semcn arralysis 88-9
replication, measurement 40
reprnducibility 38"round cells" 10-1 ?

Papanicolaou stain for sper~natozoa
l 8-1 9.71-4
percentages, differences between two 10,
11,116
Percoll gradients 34-5. 105
peroxidase technique 11-12. 65
pHcervical mucus 54, 56
semen 7 , 6 0
phorbol 12-myristate 13-acetate (PMA)
99 1 0 0
phosphatc buffered saline (PBS) 6 h"pinhead defect 2 1
pipettes 8 , 15, 114-15
cillibration 107-8
safety 64
Poisson distribution 3 7
postcoital test 54, 56-8
cervical mucus sample 5 7
inslructio~lsto patients 57
interpretation 5 7-8
repeated 58
sample record form 109
tcchniyue 57
timing 56-7
vaginal pool sample 57
precision 36
asscssme~ll3 8progesterone 33. 51, 102
prostate gland, secretory function 29. 30
prostatic fluid, ~llicrohiologicaltests 12
PurcSpcrm 105

S controlcharts 41.43-6
safetyequipment 114
guidelines B, 63-4
sample collection
cervical mucus 5 3 4se111e114-6, 88
Scott's solution 73
semen
appearance h-7
cellular elements (non-sperm) 10-13
centrifugation 9, 1 8
collection and delivery 4-6, 8 8
culture 29
dorior, for in vitro tests 58
homogeneity 8-9
initial macroscopic cxamir~alion6-7
liquefaction 6
microbiologicaltests 12-13
micruscopy
further 14-2 3
initial 7-14
pH 7 , 6 0
quality decline 1
safe handling 6, 63-4
smears 17-18
sperm antibody tests 23-6
sperm preparation techniques 34-5,
104-6
variables, nomenclature 62
viscosity (consistency) 7
volume 7. 60
semen analysis
hasic laboratory equipment 114-1 S
errors in 17-8
opliorlal tests 28-31
preparation Cur routine 8
reference values 60-1
research tests 3 1-3
sa~nplerecord form 88-9
standard procedures 4-2 6
seminal plasmabiochemical tests 12. 81-7
phalanges 59
sperrn separation techniques 34-5,
104-6

quality assessment, external. see exlernal
quality assessment
quality assurance 36
quality co~llrul(QC) 30, 36-50
calibration of equipmonl 107-8
charts 41-8,49
correlation of resnlts within samples
39-40
importance 38-9
internal, see internal quality control
limits, s88 control limitsmaterials for 107
monitoring munlhly mealls 46-8, 49

sernirral vesicles. secretory function 29. 30
Scrtoli cell dysfunction 1 3
sharps disposal 63
Shol.rstai11 1 8 , 26, 75
silane-coated colloidal silica 34-5
simplified slide test 59
smears, senien 17-18
sperm
acrosornal region 19. 2 0
agglutination 1 4
cryopreservation 1 0 7
cytoplasmic droplsls 1 9 . 1 0 . 2 1
functiou lesls 3 1-3length and width 1 9neck arid rnidpiece defects 19. 1 0 , 2 1
tail defects 19, 20, 21
total nu~nbcr6O
sperm antibodies 23-6
immunobead test, sec immunobead Lest
mixed antiglobulir~reaction (MAR), see
mixed antiglobin (MAR) (analogous
with immunobead test)in simplified slide test 59
sperm-ervical mucus interaction 5 1-3,
56 9
basic laboratory equipment 115
in vitro tests 58-9, 110-1 3
in vivo, sccpostcoital Lost
sperm co~~ccntration60
assessment 14-1 7
counting errors 37
preliminary estirlialion 8-9
quality conlrol 39-40. 41-4, 46, 107
sperrrl deformity index (SDI) 28-9
sperm head
defects 19, 20, 21
normal 1 9
sperni rrlurphology 17-2 1. 60
classification 19-21
computer-aided analysis 33
count 22-3
counting errors 3 7-8
irldiccs of multiple defects 28-9
quality control 40, 44-6, 107smear preparatiurl 17-18
stniuir~gmcthods 18-19. 71-6
sperm motility 60
assessment 9-1 0. 11
computer-aided analysis (CASA) 30-1.
90-3
coumling errors 3 7-8
in postcoital test 5 7
quality control 40.44-6, 1 0 7
sperni preparation techniques 34-5,
104-6

discur~linuousdensity gradients 34-5.
105
poor quality samples 106
swim-up technique 34, 1 0 4
sperru vilality tests 14, 33, 60. 68-70
dye exclusion method 14, 68-9
hypo-osmotic swelli~lg(HOS) 19.69-70
spennatids, ruur~d13.L1.27
sprrmatocytes 1 3
spermatogonia 1 3
Spinnbarkeit S 1 , 5 5standard deviat~on,average (Sbn)41.42,
414-5
standard error 3 7-8
between technicians 4 6 , 4 7
swim-up technique 34, 104
technicians
assessing variahility between 38, 40 6.
4 7-8
ossessir~gvariability within 38, 40-6
tcmpcrature, semen examination 8
teratozoospermia h2
teratnaoospermia index (TZI) 28-9
Tris buffered saline (TBS) 66
Lrypan blue 14
urine, microbiological tests 12
varicocoele 1 3
rrasa deferentia, bilateral couge~lital
absence 7, 29
viscosity, serrlerl 7
volurnc
cervical mucus 54
semen 7, 60
white bluod coils, see leukocytes
Xbdr4 1 . 4 2
Xbarcontrol chartsmonthly 48.49
wcckly 41-6

zinc, in seminal plasrua 30. 81-2
zona-free 21smstcr oocyte test 3 1,34,
Y 4-8
protocol using Ca ionophore (A23187)
95-7
quality control 9 7
standard protocol 94-5
zona pellucida binding tests, human 32