SIST EN ISO 23162:2021
(Main)Basic semen examination - Specification and test methods (ISO 23162:2021)
Basic semen examination - Specification and test methods (ISO 23162:2021)
This document will describe pre-examination, examination and post-examination processes for medical laboratory examination of human semen. It is intended to be used for diagnostics in small and large medical laboratories performing semen analysis. Preparation of semen for therapeutic use is not included.
Grundlegende Samenanalyse - Spezifikation und Testmethoden (ISO 23162:2021)
Dieses Dokument legt die Mindestanforderungen an die Laborausstattung und entscheidende Aspekte der Prüfmethoden für bewährte Vorgehensweisen in Laboratorien fest, die eine grundlegende Analyse von durch Ejakulation gesammelten menschlichem Samen durchführen.
Dieses Dokument ist anwendbar auf den gesamten Prozess der manuellen grundlegenden Samenanalyse und auch auf die Probenvorbereitung für die computerassistierte Spermienanalyse (CASA).
Dieses Dokument gilt nicht für Bewertungen nach einer Vasektomie.
ANMERKUNG Angesichts der medizinisch-rechtlichen Folgen, die mit der Evaluierung von Ejakulaten nach einer Vasektomie einhergehen, reicht die Methodik in diesem Dokument aller Wahrscheinlichkeit nach nicht aus, um ein Ejakulat als vollständig „frei“ (d. h. ohne Spermien im Ejakulat) zu bezeichnen.
Analyse de base du sperme - Spécifications et méthodologie analytique (ISO 23162:2021)
Le présent document spécifie les exigences minimales applicables aux équipements et aux points critiques des méthodes d’essai pour de bonnes pratiques dans les laboratoires réalisant une analyse de base du sperme humain obtenu par éjaculation.
Le présent document est applicable à l’ensemble du processus d’analyse de base du sperme, mais aussi à la préparation de l’échantillon en vue d’une analyse assistée par ordinateur du sperme (CASA).
Le présent document ne s’applique pas aux évaluations post-vasectomie.
NOTE Du fait des implications médico-légales entourant l’évaluation des éjaculats post-vasectomie, la méthodologie du présent document est selon toute vraisemblance inadéquate pour établir qu’un éjaculat est totalement «exempt de spermatozoïde» (c’est-à-dire aucun spermatozoïde dans l’éjaculat).
Osnovne preiskave semena - Specifikacija in preskusne metode (ISO 23162:2021)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 23162:2021
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
SIST EN ISO 23162:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 23162:2021
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SIST EN ISO 23162:2021
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.
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SIST EN ISO 23162:2021
EN ISO 23162:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 23162:2021
EN ISO 23162:2021 (E)
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.
3
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SIST EN ISO 23162:2021
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
ISO 23162:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
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 2021 – All rights reserved v
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
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).
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SIST EN ISO 23162:2021
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.
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
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
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SIST EN ISO 23162:2021
ISO
...
SLOVENSKI STANDARD
oSIST prEN ISO 23162:2020
01-september-2020
Osnovne preiskave semena - Specifikacija in preskusne metode (ISO/DIS
23162:2020)
Basic semen examination - Specification and test methods (ISO/DIS 23162:2020)
Grundlegende Samenanalyse - Spezifikation und Testmethoden (ISO/DIS 23162:2020)
Analyse de base du sperme - Spécifications et méthodologie analytique (ISO/DIS
23162:2020)
Ta slovenski standard je istoveten z: prEN ISO 23162
ICS:
11.100.01 Laboratorijska medicina na Laboratory medicine in
splošno general
oSIST prEN ISO 23162:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 23162:2020
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oSIST prEN ISO 23162:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23162
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
2020-07-13 2020-10-05
Basic semen examination — Specification and test methods
ICS: 11.100.01
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 23162:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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oSIST prEN ISO 23162:2020
ISO/DIS 23162:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 23162:2020
ISO/DIS 23162:2020(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
4.3 Maintenance of Competence . 4
5 Semen Characteristics, Sampling and Pre-Examination Handling .4
5.1 General Characteristics . 4
5.2 Physical and Chemical Characteristics . 5
5.3 Sample Collection and Initial Handling . 5
5.4 Subject Information and Data Collection . 5
5.4.1 Information to be Provided to Subjects . 5
5.4.2 Data Collection from the Subject. 6
5.5 Initial Sample Handling . 6
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 . 8
6.3.3 Direct Microscopy of the Wet Preparation . 8
6.3.4 Sperm Motility Assessment . 9
6.3.5 Sperm Concentration Assessment . 9
6.3.6 Assessment of Absence of Spermatozoa .10
6.3.7 Sperm Vitality Assessment .10
6.3.8 Sperm Morphology Evaluation .10
7 Post-Examination Handling and Test Report .10
7.1 General .10
7.2 Results Calculations and Presentation .10
7.2.1 Total Amount in the Ejaculate .10
7.2.2 Other Calculations .11
7.3 Presentation of Results .11
7.3.1 General.11
7.3.2 Contents of the Semen Examination Report .12
7.4 Practical Aspects of Quality Assurance .12
7.4.1 Internal Quality Control .12
7.4.2 Intra-laboratory comparisons .13
7.4.3 Inter-laboratory Comparisons .13
Annex A (informative) The statistical basis for determination of absence of spermatozoa .14
Annex B (informative) High power field .15
Annex C (informative) Motility assessment training .16
Annex D (informative) Diluent for sperm concentration assessment .19
Annex E (informative) Estimation of suitable dilution for the assessment of sperm
concentration .20
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oSIST prEN ISO 23162:2020
ISO/DIS 23162:2020(E)
Annex F (informative) Comparison of concordance between two replicate assessments that
report percentages .21
Annex G (informative) Comparison of concordance between two replicate counts of sperm
concentration .23
Annex H (informative) Sperm vitality assessment .26
Annex I (informative) Sperm morphology assessment .28
Bibliography .30
iv © ISO 2020 – All rights reserved
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oSIST prEN ISO 23162:2020
ISO/DIS 23162:2020(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.
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The committee responsible for this document is Technical Committee ISO/TC 212, Clinical laboratory
testing and in vitro diagnostic test systems.
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Introduction
This document was developed in response to worldwide demand for standards for reliable examination
of human semen. The WHO has published a manual on human semen examination in five editions
between 1980 and 2010 providing general recommendations for laboratory procedures. These
[13]
recommendations have become increasingly detailed and succinct .
However, a formal standard, based on best available evidence and global consensus, would facilitate
any laboratory seeking accreditation for semen examination using laboratory procedures most likely
to give reliable results. Subjects would benefit from fewer random factors influencing the choice
of treatment modality. Costs for using suboptimal treatments could be expected to decrease. Also,
the development of clinical science would prosper by the use of proper procedures allowing the
development of both diagnostic and therapeutic strategies to benefit from well-defined laboratory
techniques. This includes both the symptomatic treatment of infertility of the couple (Assisted
Reproductive Technology – ART; In Vitro Fertilization - IVF) and the follow-up of often causative
treatments of disorders in the male (e.g. hypogonadism, varicocele). Furthermore, for validation and
evaluation of new methods to improve diagnosis and treatment of infertility the here defined standard
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 the Computer-Aided Sperm Analysis (CASA). The standardized handling and
preparation of semen samples is essential to the quality of the data obtained.
In this document, the following verbal forms are used:
• ‘shall’ indicates a requirement;
• ‘should’ indicates a recommendation;
• ‘may’ indicates a permission;
• ‘can’ indicates a possibility or capability.
Further details can be found in the ISO/IEC Directives, Part 2:2018, 7
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DRAFT INTERNATIONAL STANDARD ISO/DIS 23162:2020(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).
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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 or semen sample obtained by masturbation, intercourse, vibratory stimulation or electro-
ejaculation
Note 1 to entry: The ejaculate is a mixture of spermatozoa and secretions, mainly from the seminal vesicles, the
prostate and the epididymides.
3.5
ejaculate collection container
primary sample container
Note 1 to entry: Ejaculate collection container should 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.
3.6
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.
3.7
interlaboratory comparison
external quality control
proficiency testing
organization, performance and evaluation of measurements or tests on the same or similar items by
two or more laboratories in accordance with predetermined conditions
3.8
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).
3.9
immotile
total lack of active tail movements
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.
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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 > 5 µm/s
Note 1 to entry: See also slow progressive sperm motility (3.17) and rapid progressive sperm motility (3.14).
3.14
rapid progressive sperm motility
forward motility of a spermatozoon of at least 25 µm/s
3.15
seminal plasma
mixture of secretions, mainly from the seminal vesicles, the prostate and the epididymides, without
spermatozoa
3.16
sexual abstinence
time between ejaculate (3.4) collection and the most recent previous ejaculation
3.17
slow progressive sperm motility
forward motility of a spermatozoon of 5 µm/s to24 µm/s
3.18
sperm concentration
number of spermatozoa per unit volume
Note 1 to entry: Sperm concentration is expressed as millions or thousands/millilitre).
Note 2 to entry: It shall not to be confused with sperm density (mass/volume).
3.19
sperm vitality
percentage of vital spermatozoa, independent of their ability to move
3.20
total sperm number
calculated total number of spermatozoa in the ejaculate (3.4)
Note 1 to entry: Total sperm number = sperm concentration × ejaculate (3.4) volume.
Note 2 to entry: Total sperm number is not the same as sperm concentration.
3.21
Tygerberg strict criteria
sperm morphology criteria based on the morphology of spermatozoa able to penetrate into and migrate
within cervical mucus
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3.22
typical spermatozoon
spermatozoon with the morphology typical of spermatozoa able to penetrate into and migrate within
cervical mucus and reach the site of fertilization
[7] [8]
[SOURCE: Menkveld, et al., 1991 , Menkveld and Kruger, 1995 ]
3.23
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 15189:2012. How these
requirements are applied to human semen analysis are covered here.
4.2 Training
Semen examination involves many analytical steps that require operator training to minimize operator
[6]
subjectivity in order to provide accurate reliable results (MacLeod and Gold, 1951 , Mortimer,
[10] [1]
1994 ,Barratt, et al., 2011 ).
All personnel 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.
NOTE Effective goal-oriented reiterative training procedures for these assessments have been published
[10] [2]
(Mortimer, 1994 , Björndahl, et al., 2010 ); 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).
4.3 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 As per subclause 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.
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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 may help with sample
collection. For subjects with ethical or religious objections to masturbation a non-spermotoxic (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 may be necessary by some subjects; such products shall be validated as
[11]
non-toxic to spermatozoa (Mortimer, et al., 2013 ).
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 minutes after ejaculation. Incomplete liquefaction at 60 minutes 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
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.
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
• T
...
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