SIST EN ISO 23162:2021

SLOVENSKI STANDARD
01-september-2021
Osnovne preiskave semena - Specifikacija in preskusne metode (ISO 23162:2021)
Basic semen examination - Specification and test methods (ISO 23162:2021)
Grundlegende Samenanalyse - Spezifikation und Testmethoden (ISO 23162:2021)
Analyse de base du sperme - Spécifications et méthodologie analytique (ISO
23162:2021)
Ta slovenski standard je istoveten z: EN ISO 23162:2021
ICS:
11.100.01 Laboratorijska medicina na Laboratory medicine in
splošno general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 23162
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2021
EUROPÄISCHE NORM
ICS 11.100.01
English Version
Basic semen examination - Specification and test methods
(ISO 23162:2021)
Analyse de base du sperme - Spécifications et Grundlegende Samenanalyse - Spezifikation und
méthodologie analytique (ISO 23162:2021) Testmethoden (ISO 23162:2021)
This European Standard was approved by CEN on 11 June 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23162:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23162:2021) has been prepared by Technical Committee ISO/TC 212 "Clinical
laboratory testing and in vitro diagnostic test systems" in collaboration with Technical Committee
CEN/TC 140 “In vitro diagnostic medical devices” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2022, and conflicting national standards shall
be withdrawn at the latest by July 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN websites.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 23162:2021 has been approved by CEN as EN ISO 23162:2021 without any modification.

INTERNATIONAL ISO
STANDARD 23162
First edition
2021-07
Basic semen examination —
Specification and test methods
Analyse de base du sperme — Spécifications et méthodologie
analytique
Reference number
ISO 23162:2021(E)
©
ISO 2021
ISO 23162:2021(E)
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
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Phone: +41 22 749 01 11
Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2021 – All rights reserved

ISO 23162:2021(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative References . 1
3 Terms and Definitions . 1
4 Staff Training and Competence . 4
4.1 General Aspects . 4
4.2 Training . 4
4.2.1 General. 4
4.2.2 Training for quantitative assessments . 4
4.2.3 Training for qualitative assessments. 4
4.2.4 Training for pH assessment . 4
4.3 Maintenance of Competence . 5
5 Semen Characteristics, Sampling and Pre-Examination Handling .5
5.1 General Characteristics . 5
5.2 Physical and Chemical Characteristics . 5
5.3 Sample Collection and Initial Handling . 5
5.4 Subject Information and Data Collection . 6
5.4.1 Information to be Provided to Subjects . 6
5.4.2 Data Collection from the Subject. 6
5.5 Initial Sample Handling . 7
5.6 Sperm Toxicity Testing . 7
6 Examinations . 7
6.1 Required Equipment . 7
6.2 In-house Prepared Reagents . 8
6.3 Assessments . 8
6.3.1 Initiation of Assessments . 8
6.3.2 Macroscopic Assessment . 9
6.3.3 Direct Microscopy of the Wet Preparation . 9
6.3.4 Sperm Motility Assessment . 9
6.3.5 Sperm Concentration Assessment .10
6.3.6 Assessment of Absence of Spermatozoa .10
6.3.7 Sperm Vitality Assessment .11
6.3.8 Sperm Morphology Evaluation .11
7 Post-Examination Handling and Test Report .11
7.1 General .11
7.2 Results Calculations and Presentation .11
7.2.1 Total Amount in the Ejaculate .11
7.2.2 Other Calculations .11
7.3 Presentation of Results .12
7.3.1 General.12
7.3.2 Contents of the Semen Examination Report .12
7.4 Practical Aspects of Quality Assurance .13
7.4.1 Internal Quality Control .13
7.4.2 Intralaboratory Comparisons .14
7.4.3 Interlaboratory Comparisons .14
Annex A (informative) The statistical basis for determination of absence of spermatozoa .15
Annex B (informative) High power field .16
Annex C (informative) Motility assessment training .17
Annex D (informative) Diluent for sperm concentration assessment .20
ISO 23162:2021(E)
Annex E (informative) Estimation of suitable dilution for the assessment of sperm
concentration .21
Annex F (informative) Comparison of concordance between two replicate assessments that
report percentages .22
Annex G (informative) Comparison of concordance between two replicate counts of sperm
concentration .24
Annex H (informative) Sperm vitality assessment .27
Annex I (informative) Sperm morphology assessment .28
Bibliography .31
iv © ISO 2021 – All rights reserved

ISO 23162:2021(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
ISO 23162:2021(E)
Introduction
This document was developed in response to global demand for standards for reliable examination
of human semen. The five editions of a laboratory manual for human semen analysis published by
the WHO between 1980 and 2010 have provided general recommendations for suitable laboratory
[16]
procedures, but even the latest edition (World Health Organization 2010 ) does not constitute a
Technical Standard adequate for use under ISO 15189.
A Technical Standard based on best available evidence and global consensus regarding laboratory
procedures most likely to give reliable results will facilitate any laboratory seeking accreditation for
human semen examination. Subjects, and biomedical science in general, would benefit from fewer
random factors affecting the accuracy of results. Clinically this would support improved diagnoses
as well as provide more objective grounds for choosing between possible management strategies
or alternative treatment modalities. Furthermore, to support the evaluation and validation of new
methods to improve the diagnosis and treatment of infertility, these standardized techniques can serve
as reference methods.
The pre-examination preparation of human semen is important not only in manual basic semen
examination, but also for Computer-Aided Sperm Analysis (CASA). Standardized handling and
preparation of semen samples is essential to the quality of the data obtained.
vi © ISO 2021 – All rights reserved

INTERNATIONAL STANDARD ISO 23162:2021(E)
Basic semen examination — Specification and test methods
1 Scope
This document specifies the minimum requirements for equipment and critical aspects of the test
methods for best practice in laboratories performing basic examination of human semen collected by
ejaculation.
This document is applicable to the entire process of basic manual semen examination and also to sample
preparation for Computer-Aided Sperm Analysis (CASA).
This document does not apply to the post-vasectomy assessments.
NOTE Given the medico-legal ramifications surrounding the evaluation of post-vasectomy ejaculates, the
methodology in this document is in all likelihood inadequate to establish an ejaculate as being completely “clear”
(i.e. no spermatozoa in the ejaculate).
2 Normative References
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 15189, Medical laboratories — Requirements for quality and competence
ISO/TS 20914, Medical laboratories — Practical guidance for the estimation of measurement uncertainty
3 Terms and Definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
air displacement pipette
common laboratory pipette with disposable tips where the volume aspirated is controlled by the
displacement of an equivalent volume of air inside an enclosed chamber inside the pipette handle
Note 1 to entry: An air displacement pipette can only give accurate volumes for liquids with viscosity close to
that of water.
3.2
azoospermia
complete absence of spermatozoa in the ejaculate (3.4)
Note 1 to entry: The term azoospermia is not a clinical diagnosis but a description of a laboratory finding.
Complete lack of spermatozoa is difficult to determine in absolute terms. Since only parts of an ejaculate (3.4)
can be examined, the modern definition is based on probability calculations derived from data obtained from
investigations of random aliquots from an ejaculate (3.4) (See Annex A).
ISO 23162:2021(E)
3.3
CASA
computer-aided sperm analysis
automated examination of ejaculates (3.4) with equipment using imaging technology
Note 1 to entry: Examination based on image analysis of video sequences to obtain information on sperm
concentration (3.18) and motility, more seldom sperm morphology.
Note 2 to entry: There are CASA systems commercially available, but no common standard for validation,
evaluation, reliability in analyses or contents of reports. The scope of this document is not to provide a standard
for CASA, although the pre-examination aspects can be useful also to developers, manufacturers, and users of
CASA equipment.
3.4
ejaculate
semen sample, which is a mixture of spermatozoa and secretions, mainly from the seminal vesicles, the
prostate and the epididymides
Note 1 to entry: The ejaculate can be obtained by various methods including masturbation, intercourse, vibratory
stimulation or electro-ejaculation.
3.5
ejaculate viscosity
property of an ejaculate (3.4) describing its resistance to flow like water after liquefaction (3.10)
Note 1 to entry: Incompletely liquefied semen is not a homogenous liquid due to the contents of gelatinous
structures in the ejaculate fluid.
3.6
high power field
area of a slide which is visible in the microscope under high power magnification (×400)
Note 1 to entry: This is not a standard field area as the size varies according to the type of oculars used (e.g.
standard or wide field) (see Annex B).
3.7
immotile
total lack of active tail movements
3.8
interlaboratory comparison
organization, performance and evaluation of measurements or tests on the same or similar items by
two or more laboratories in accordance with predetermined conditions
[SOURCE: ISO/IEC 17043:2010, 3.4]
3.9
ideal spermatozoon
spermatozoon with the morphology typical of spermatozoa able to penetrate into and migrate within
cervical mucus and reach the site of fertilization
[9] [10]
[SOURCE: Menkveld, et al., 1991, Menkveld and Kruger, 1995 ]
3.10
liquefaction
process of change in the consistency of the ejaculate (3.4) from gel-like or coagulum-like into a liquid
phase
Note 1 to entry: Liquefaction occurs due to degradation of the gel-like or coagulum-like property, by enzymatic
action on macromolecules.
2 © ISO 2021 – All rights reserved

ISO 23162:2021(E)
3.11
non-progressive sperm motility
active tail movements leading to a sperm propagation of less than approximately 5 µm/s
Note 1 to entry: A normal head length is approximately 5 μm.
3.12
positive displacement pipette
common laboratory pipette working by piston-driven displacement within a capillary, not the
displacement of air within an enclosed chamber
Note 1 to entry: The piston in the pipette tip is in direct contact with the liquid specimen.
Note 2 to entry: Use to avoid major volume errors with viscous liquids like semen.
3.13
progressive sperm motility
forward motility of a spermatozoon of at least 5 µm/s
Note 1 to entry: See also slow progressive sperm motility (3.16) and rapid progressive sperm motility (3.14).
Note 2 to entry: Spermatozoa moving in circular paths are considered progressive based on space gain.
3.14
rapid progressive sperm motility
forward motility of a spermatozoon of at least 25 µm/s
3.15
sexual abstinence
time between the collection of ejaculate (3.4) for analysis and the most recent previous ejaculation
Note 1 to entry: Expressed in days or hours as appropriate for the intended use.
3.16
slow progressive sperm motility
forward motility of a spermatozoon of at least 5 µm/s but less than 25 µm/s
3.17
specimen collection container
receptacle used to collect primary samples
Note 1 to entry: Specimen collection container shall be not toxic to spermatozoa.
Note 2 to entry: If an ejaculate (3.4) can only be collected at sexual intercourse, a non-toxic, Silastic™ condom can
be used. The ejaculate (3.4) shall be transferred to an ejaculate sample container upon receipt by the laboratory;
this shall be noted in the report form.
3.18
sperm concentration
number of spermatozoa per unit volume
Note 1 to entry: Sperm concentration is expressed in millions or thousands/millilitre.
Note 2 to entry: It shall not be confused with sperm density (mass/volume).
3.19
sperm vitality
percentage of vital spermatozoa, independent of their ability to move
ISO 23162:2021(E)
3.20
total sperm number
calculated total number of spermatozoa in the ejaculate (3.4)
Note 1 to entry: Total sperm number is the sperm concentration (3.18) multiplied by the ejaculate (3.4) volume.
Note 2 to entry: Total sperm number is not the same as sperm concentration (3.18).
3.21
Tygerberg strict criteria
sperm morphology criteria based on the morphology of spermatozoa able to penetrate into and migrate
within cervical mucus
3.22
Teratozoospermia Index
TZI
average number of defective regions (head, neck/midpiece, tail, and/or cytoplasmic droplet) in
abnormal spermatozoa
Note 1 to entry: This index is, by definition, never outside the interval of [1.00;4.00].
4 Staff Training and Competence
4.1 General Aspects
General requirements for staff training and competence are covered in ISO 15189. How these
requirements are applied to human semen analysis is covered here.
4.2 Training
4.2.1 General
Semen examination involves many analytical steps that require operator training to minimize
[7][12][1]
subjectivity in order to provide accurate reliable results .
4.2.2 Training for quantitative assessments
All assessors performing assessments of sperm motility, sperm concentration, sperm vitality and/
or sperm morphology shall receive training using either commercial, in-house or EQA-derived
validated reference materials to ensure that their results conform to the laboratory’s pre-determined
measurement error limits. Without such training staff cannot be expected to be able to provide accurate
or reliable results for these assessments, and participation in EQA schemes is pointless.
[12]
NOTE Effective goal-oriented reiterative training procedures for these assessments have been published
[14]
; a ± 10 % range of measurement error is expected between novices upon completion of their training and the
laboratory’s experienced staff (see also Annex C).
4.2.3 Training for qualitative assessments
Competency training for qualitative assessments, such as viscosity and round cells, shall achieve
agreement between trainee and expert in at least 90 % of cases.
4.2.4 Training for pH assessment
The ability of assessors to read test strips against the comparator scale shall be verified.
4 © ISO 2021 – All rights reserved

ISO 23162:2021(E)
4.3 Maintenance of Competence
Ongoing verification of competence shall be demonstrated by all personnel performing these
assessments at regular intervals as defined in the laboratory’s quality framework.
NOTE According to 4.2, the same ± 10 % range of measurement error is expected for ongoing verification of
competence by all trained staff performing these assessments.
5 Semen Characteristics, Sampling and Pre-Examination Handling
5.1 General Characteristics
Examination of the ejaculate is in some important aspects different from investigations of other
human bodily fluids. The subject is expected to accomplish the collection of the ejaculate. Results are
dependent on ejaculation frequency before collection, as well as on the time and temperature before
initiation of investigations. In case of infertility diagnosis, clear reference limits are missing due to the
fact that the desired outcome is dependent on the particular clinical situation of each couple trying to
achieve a pregnancy.
5.2 Physical and Chemical Characteristics
There is no internal homeostatic control in an ejaculate collected in a device for laboratory
investigations. Initially the entire ejaculate is incorporated into a gel-like coagulum that is gradually
degraded (liquefaction) into a still viscous but more water-like liquid. During this process carbon
dioxide evaporates causing a change in pH. Enzymatic degradation of gel components causes a
significant increase in osmotic properties of the liquid surrounding the spermatozoa, which in turn
affects sperm performance.
5.3 Sample Collection and Initial Handling
Sample collection shall, except for some men with, for example, disabled limbs, spinal cord injury or
paraplegia, always be done by the subject. If necessary, the subject’s partner can help with sample
collection. For subjects with ethical or religious objections to masturbation a non-spermotoxic
1)
(Silastic™ ) condom can be used to collect an ejaculate during intercourse. However, this collection
method will result in some loss of the overall sample as it is recovered from the condom. Collection of
ejaculates by coitus interruptus (“withdrawal”) is not recommended as the first, sperm-rich, fraction of
the ejaculate is often lost. Use of lubricants can be necessary by some subjects; such products shall be
[13]
validated as non-toxic to spermatozoa .
After ejaculation, the sample shall be kept as close as possible to 37 °C and never higher; cooling or
warming can cause artefacts and sperm dysfunction. Due to all the changes occurring after ejaculation,
investigations shall start as soon as possible after liquefaction, that typically is completed within
30 min after ejaculation. Incomplete liquefaction at 60 min after ejaculation indicates an abnormality.
Initiation of assessments after completion of liquefaction is best achieved if the ejaculate is collected
near the laboratory. Since the duration and level of sexual arousal experienced by the subject will affect
the ejaculation, sample collection could be best performed in a place chosen by the subject in case of
major difficulty. When an ejaculate is collected outside the lab environment it shall be delivered to the
laboratory, preferably within 30 min, but at least within 60 min (circumstance for ejaculate collection
and transport shall be noted in the report). Nonetheless, considerations of temperature and time to
investigation remain important for the quality and robustness of the examination.
1) Silastic™ is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this product.
ISO 23162:2021(E)
5.4 Subject Information and Data Collection
5.4.1 Information to be Provided to Subjects
The following information shall be provided to the subject in writing in a language understandable by
the subject and shall include the following:
a) General information:
— Contact information for the laboratory;
— The reason for the investigation if made available from the requester;
— An outline of what will be investigated;
— How results of the laboratory investigations will be communicated to the subject.
b) Ejaculate collection, handling and transportation:
— How to collect the ejaculate;
— Effect of delay between sample collection and initiation of assessments;
— Importance of avoiding cooling down or warming up of the ejaculate;
— Importance of reporting correct sexual abstinence time;
— Importance of reporting any incompleteness of sample collection.
5.4.2 Data Collection from the Subject
a) Required information
Each subject shall be asked to provide the following information to be recorded by the laboratory:
— Reliable personal identification (at least two unique identifiers attributable to the patient and
specified by the organization);
— Duration of sexual abstinence;
— Time of sample collection;
— Transport of ejaculates should be avoided but if not collected at the premises of the laboratory:
confirmation that during transport to the laboratory the specimen was protected from extremes of
temperature;
— Completeness of sample collection; in case of incomplete collection, with information of which parts
in the sequence of ejaculation that have been missed in collection.
b) Additional information
Information that is of importance to the clinical interpretation and that can be practical to obtain when
the subject visits the laboratory. The collection of this information is, however, not part of the
laboratory work:
— Medical history, which can include:
— Any episode of severe inflammatory process the last three months;
— Any previous surgery (inguinal hernia, varicocele, cryptorchidism or other problems related
to the urogenital sphere) or treatment with chemotherapy, cytostatics or radiation of the
urogenital organs;
6 © ISO 2021 – All rights reserved

ISO 23162:2021(E)
— Any use of pharmaceutical drugs except short term use of non-prescription drugs (e.g. pain
killers, and anti-allergy drugs).
— Any use of recreational drugs, anabolic steroids or other performance enhancing dietary additions
(like protein powders).
5.5 Initial Sample Handling
— Every ejaculate should be considered potentially infectious and handled accordingly (see
ISO 15190:2020, Annex B).
— Information provided by the subject shall be recorded.
— Specimen collection container shall be clean, non-toxic and for single use.
— Specimen collection container should preferably be weighed before sample collection and its weight
recorded in grams with two decimal places.
— Ejaculate volume should preferably be determined by weight. In this case the specimen collection
container is weighed (recorded in grams with two decimal places) before and after specimen
collection and the weight difference used as the volume, assuming 1,0 g of ejaculate equals 1,0 ml
[3]
of ejaculate . If a calibrated serological pipette is used, some semen will always be lost in the
specimen collection container and inside the pipette after making the measurement. The lab should
be aware of the differences of the two methods. The ejaculate volume shall be reported in ml to one
decimal place.
— All documents and the specimen collection container shall be labelled with at least two unique
identifiers.
— As soon as possible after collection the specimen collection container shall be kept at a temperature
between 35 °C and 37 °C to facilitate liquefaction and prepare for motility assessment at standardized
temperature, preferably on a moving tray to enhance mixing during liquefaction (frequent manual
agitation is required when moving tray is not available).
Spermatozoa are affected by the earth gravity and sediment to the bottom of any container (“geotaxis”)
even if they are motile. Consequently, when sampling an ejaculate, it shall be well-mixed to evenly
distribute the spermatozoa and other elements of the ejaculate. Even sitting for a short period of time
will result in an uneven distribution of the cellular elements of the ejaculate. It is therefore important
to gently mix the ejaculate thoroughly before any aliquot is taken for examination, noting that a vortex
mixer shall not be used.
5.6 Sperm Toxicity Testing
To ensure that materials in contact with ejaculates (specimen collection container, pipette tips) are
not toxic to spermatozoa, a basic toxicity test shall be performed on every new batch of material. The
principle is based on comparison of motility of spermatozoa exposed to present material and the new
[6]
material . The time of exposure shall be at least twice the expected time of exposure of sperm to the
material – seconds for pipette tips and 30 min to 60 min for sample collection containers.
6 Examinations
6.1 Required Equipment
Sperm motility is largely influenced by the ambient temperature, especially regarding velocity. The use
of temperature-controlled equipment reduces the influence of variable room temperature.
The following equipment is required:
— Laboratory balance, range 0,00 g to 50,00 g (reading to two decimal places);
ISO 23162:2021(E)
— An incubator or warm plate that can maintain the ejaculate at human body temperature, preferably
including a moving tray (orbital mixer);
— Upright light microscope with phase contrast (10×, 20× and 40× objectives recommended) and
bright field (100× oil immersion, high resolution objective) optics, and 10× oculars and an ocular
micrometer (or an ocular reticle or grid calibrated with a stage micrometer scale), and means of
keeping wet preparations at body temperature (e.g. heated stage);
— Positive displacement pipette for the assessment of sperm concentration, 0 µl to 50 μl or 0 µl to
100 μl capacity;
— Air displacement pipettes (1 µl to 20 μl, 20 µl to 200 μl and 200 µl to 1 000 μl sizes) for concentration
diluent, wet preparations and other preparations;
— Equipment to stain and mount morphology and vitality slides (slide holders, staining jars, disposable
pipettes for mounting medium);
— Centrifuge (e.g. for 15 ml conical centrifuge tubes) – a swing-out bucket is preferred to produce
more discrete pellets – and either sealed buckets or a sealed rotor to protect operators from possible
aerosol contamination should a tube break during centrifugation;
— Haemocytometers with Improved Neubauer ruling (100 µm depth).
NOTE 1 The majority of international experts recommend using haemocytometers with Improved Neubauer
[12][14][16]
ruling . Other patterns of haemocytometers can be used so long as the correct calculation factors are
employed
NOTE 2 Non-disposable haemocytometers need to be checked regularly that wear and tear does not change
the depth of chamber. Disposable haemocytometers can be used provided they are properly validated and
[5]
evaluated .
[17]
NOTE 3 Makler chambers have lower accuracy than haemocytometers .
NOTE 4 Some disposable counting chambers intended for urine analysis have insufficient accuracy for this
[17]
purpose .
NOTE 5 Fixed depth chambers using capillary action are subject to the Segré-Silberberg effect and hence will
[4]
have a variable error .
— Humid chamber for sedimentation of spermatozoa in haemocytometers
— Vortex mixer (for agitating fixed sperm suspensions for assessment of concentration)
6.2 In-house Prepared Reagents
A diluent for sperm concentration assessment is essential and can be prepared in-house (See Annex D).
The purpose is to immobilize (kill) spermatozoa to make counting more reliable. For the ease and
reliability of assessment it is also an advantage if growth of micro-organisms can be prevented.
6.3 Assessments
6.3.1 Initiation of Assessments
For a reliable assessment of ejaculate characteristics liquefaction shall be completed. For most
ejaculates this is achieved within 30 min if kept at 37 °C after collection. If liquefaction is not completed,
the sample can be left in the incubator for a further period of time (maximum 30 min, so that motility
can be assessed within 60 min after collection), after which assessments shall be initiated. Incomplete
liquefaction at the start of examination shall be noted in the report as well as the time between sample
collection and initiation of wet preparation assessment.
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ISO 23162:2021(E)
6.3.2 Macroscopic Assessment
In the absence of metrological standards for physical characteristics of human semen it is not possible
to achieve proper standardization of methods or establish an uncertainty of measurement. However,
some observations regarding colour or perception of its odour can have clinical relevance concerning
the provenance of the specimen or the subject’s medical status. Consequently, any such comments
included in the report are considered as having been reported by exception, i.e. as attempts to describe
observations of characteristics that are outside the expected.
a) Visual appearance:
1) Colour (Normal: a wide range of opalescent, greyish-white, sometimes slightly or even bright
yellowish. Abnormal: brownish or red, transparent; strong yellow);
2) Liquefaction (should be complete within 30 min after ejaculation at 37 °C; abnormal: remaining
gel clumps).
b) Other physical observations:
1) Viscosity – measured by allowing semen to slowly drop by gravity from a wide bore pipette
(e.g. a 5 ml serological pipette or a non-toxic glass or plastic Pasteur pipette). Normal: discrete
droplets with < 2 cm “threads”;
2) Odour (there is no “normal” smell to semen, and its odour is highly subjective. However, a
strong, putrescent smell is often indicative of an active infection; a mild putrescent smell might
indicate prolonged abstinence time or a strong urine smell could indicate contamination of the
semen with urine);
3) Semen pH – measured by placing an aliquot of liquified semen on a validated pH test strip
within 30 min after ejaculate collection, at least in case of absence of spermatozoa and low
semen volume.
6.3.3 Direct Microscopy of the Wet Preparation
A wet preparation shall be made by placing a 10 μl aliquot of well-mixed semen on a labelled pre-
warmed microscope slide and covering it with a pre-warmed 22 mm × 22 mm cover slip (thickness #1½
or #2, to allow full spreading of the droplet) to obtain a preparation depth of about 20 μm (for 18 mm ×
18 mm cover slips only 6,5 μl is required for the same depth).
— Observe the presence of spermatozoa, other cells, debris, crystals.
— Observe the presence of sperm agglutinates and aggregates.
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