kSIST FprEN ISO 23162:2021
(Main)Basic semen examination - Specification and test methods (ISO/DIS 23162:2020)
Basic semen examination - Specification and test methods (ISO/DIS 23162:2020)
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/DIS 23162:2020)
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/DIS 23162:2020)
Osnovne preiskave semena - Specifikacija in preskusne metode (ISO/DIS 23162:2020)
General Information
Standards Content (sample)
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
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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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
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ii © ISO 2020 – All rights reserved
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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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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
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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 on 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 the following URL: www .iso .org/ iso/ foreword .html.
The committee responsible for this document is Technical Committee ISO/TC 212, Clinical laboratory
testing and in vitro diagnostic test systems.© ISO 2020 – All rights reserved v
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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 methodsThe 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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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 competenceISO/TS 20914, Medical laboratories — Practical guidance for the estimation of measurement uncertainty
3 Terms and DefinitionsFor 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 2020 – All rights reserved 1
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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-
ejaculationNote 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 conditions3.8
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.2 © ISO 2020 – All rights reserved
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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 chamberNote 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.
3.13progressive 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).
3.14rapid 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
spermatozoa3.16
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© ISO 2020 – All rights reserved 3
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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 spermatozoaNote 1 to entry: This index is, by definition, never outside the interval of [1.00;4.00].
4 Staff Training and Competence4.1 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 CompetenceOngoing 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.4 © ISO 2020 – All rights reserved
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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 Collection5.4.1 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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