Human Reproduction, Vol.31, No.2 pp. 227– 232, 2016
Advanced Access publication on December 18, 2015 doi:10.1093/humrep/dev305
OPINION
‘How to count sperm properly’:
checklist for acceptability of studies
based on human semen analysis
1
Centre for Andrology and Sexual Medicine, Karolinska University Hospital and Karolinska Institutet, Huddinge C2:94, S-141 86 Stockholm,
Sweden 2Reproductive Medicine, University of Dundee, Dundee, UK 3Oozoa Biomedical Inc., Vancouver, BC, Canada 4Université Paris
Descartes, Paris, France
*Correspondence address. E-mail: Lars.Bjorndahl@ki.se
Submitted on October 19, 2015; resubmitted on October 19, 2015; accepted on November 17, 2015
study question: Can a tool be developed for authors, reviewers and editors of the ESHRE Journals to improve the quality of published
studies which rely on semen analysis data?
summary answer: A basic checklist for authors, reviewers and editors has been developed and is presented.
what is known already: Laboratory work which includes semen analysis is burdened by a lack of standardization. This has significant
negative effects on the quality of scientific and epidemiological studies, potential misclassification of patients and the potential to impair clinical
treatments/diagnoses that rely on accurate semen quality information. Robust methods are available to reduce laboratory error in semen analysis,
inducing adherence to World Health Organization techniques, participation in an external quality control scheme and appropriate training of
laboratory personnel. However, journals have not had appropriate systems to assess if these methods have been used.
study design, size, duration: After discussion at a series of Associate Editor Meetings of the ESHRE Journals the authors of the
present text were asked to propose a tool for authors, reviewers and editors of the ESHRE Journals to ensure a high quality assessment of
submitted manuscripts which rely on semen analysis data, including a detailed verification of the relevance and the quality of the methods used.
participants/materials, setting, methods: N/A.
main results and the role of chance: A basic checklist for authors, reviewers and editors is presented. The checklist contains
key points which should be considered by authors when designing studies and which provides essential information for when the submitted manuscript is evaluated. For published articles the answers in the checklist are suitable to be available as supplementary data, which will also reduce the
space necessary for technical details in the printed article.
limitations, reasons for caution: Guidelines such as these should not be used uncritically. It is therefore important that submitting authors, in situations where their study does not comply with the basic requirements for semen analysis, not only explain all methodological
deviations but also declare the level of uncertainty in their analyses and how it complies with, or might confound, the aims of the study.
wider implications of the findings: The fundamental importance of appropriate and robust methodology to facilitate advances
in scientific understanding and patient management and treatment, is now accepted as being paramount. Use of the semen analysis checklist
should be part of this process, and when completed and signed by the corresponding author at the time of submitting a manuscript should
result in greater transparency, and ultimately uniformity. It is hoped that this initiative will pave the way for wider adoption of the methodology/reporting by other biomedical, epidemiological and scientific journals, and ultimately become the standard of practice for papers reporting
semen analysis results obtained in laboratory and clinical andrology. Systems to assist referees, authors and editors to present high quality findings
should have a significant impact on the field of reproductive medicine.
study funding/competing interest(s): No funding was obtained for this work. The authors have no competing interests in
relation to the present publication and checklist.
trial registration number: N/A.
Key words: semen analysis / standardization / laboratory quality / evidence-based medicine / check-list / study design / manuscript review
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
Lars Björndahl1,*, Christopher L.R. Barratt 2, David Mortimer 3, and
Pierre Jouannet 4
228
Introduction
General considerations of the quality
of science
Laboratory investigations performed with a high level of uncertainty—i.e.
subject to a large influence of random errors—can conceal true biological relationships. At the simplest level, a lack of significant correlation with
an investigated factor might be due to a high level of uncontrolled errors
that led to inaccurate, and hence highly variable, observations. The fundamental importance of proper and robust methodology for sound scientific development in medicine and biomedical science is gaining
substantial interest and being widely discussed (Plant et al., 2014).
Much attention has already been focused on the appropriate selection
and size of experimental and control groups (in terms of achieving necessary statistical power) but all efforts to obtain these essential requirements are foredoomed if the laboratory investigations lack sufficient
robustness, reliability and repeatability.
Implementation of new methods in other
centres
Laboratory methods must be described properly to permit other centres
to implement the techniques and/or repeat a study to verify its conclusions. This also includes the proper participation—by both the publishing
and implementing centres—in EQA programmes that test the critical
parts of semen analysis involved.
Consistency in reporting embryology and
andrology laboratory results
Consistent requirements for the stringent reporting of embryology and
andrology laboratory data will hopefully emphasize the necessity of
implementing adequate laboratory techniques in all centres.
Furthermore, the awareness of, and compliance with, modern laboratory andrology standards and techniques is often lacking. For instance, it
appears that many authors are unaware that the WHO main recommendation for the assessment of human sperm morphology is actually based
on the Tygerberg Strict Criteria (World Health Organization, 2010),
quite often resulting in incorrect comments in manuscripts on what are
supposed to be the same assessment criteria but described and presented as separate methods. Furthermore, the present WHO recommendations do not include motility and concentration assessments
carried out on raw semen by computer-assisted sperm analysis
(CASA) (ESHRE Special Interest Group in Andrology, 1998; World
Health Organization, 2010), although statements like ‘semen analysis
was performed according to WHO using CASA’ occur frequently in submitted manuscripts – despite their being clear non sequiturs.
The purpose of this paper is to provide guidelines for all scientists
designing, performing and writing reports on investigations that rely on
semen analysis data that are to be submitted to any of the ESHRE Journals. The concept is that the checklist in the Appendix should be compulsory, and must be completed and signed by the corresponding author at
the time of submitting the manuscript. Any deviation from this guideline
should be declared and explained in the Materials and Methods section of
the manuscript (see Appendix). The checklist would also help reviewers
and Editors in evaluating the submitted manuscripts.
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
Laboratory andrology, which includes semen analysis, is a branch of
reproductive science burdened by severe lack of standardization
(Harvey and Jackson, 1945; Tomlinson, 2010; Walczak-Jedrzejowska
et al., 2013) that has caused it to lag behind in the development of
sophisticated testing methods and has also led to suboptimal care
for childless couples. It is therefore immensely important for scientific
journals publishing studies in the field of human reproduction to
promote methodological standardization that will lead to more accurate and robust information, leading directly to improved quality in laboratory andrology, and hence in scientific studies and reproductive
services. This is even more urgent when considered from the perspective of evidence-based medicine: with poor laboratory methodologies, not even the best-designed clinical study can provide reliable
and valuable results. The simple explanation for this is that RCTs
and other clinical studies designed to produce evidence are rarely
focused on laboratory services; their aim being primarily to evaluate
the most efficacious treatment options, not the quality or reliability
of the laboratories providing basic diagnostic testing. However, if laboratories produce results that often suffer from substantial, more
or less random, errors this will lead to the more or less random diagnostic classification of patients, and clearly even well designed basic or
clinical studies will fail to produce useful information.
The Special Interest Group in Andrology (SIGA) was formed in
1992 during the European Society of Human Reproduction and Embryology (ESHRE) Annual Meeting in the Hague under the leadership
of Professor Lynn Fraser. In addition to the usual activities for an
ESHRE special interest group (organizing Campus Courses and PreCongress Courses), the SIGA presented in 1994 a course on Basic
Semen Analysis (‘BSA Course’) that has since been standardized
and repeated in a many countries (Sweden, The Netherlands,
Belgium, Denmark, Norway, Finland, Ukraine, Spain, Greece, Portugal, Poland, Italy, South Africa, Canada, and the UK) in a range of languages (Björndahl et al., 2002). The BSA course has been revised to
comply with relevant recommendations from the World Health Organization (WHO) (World Health Organization, 2010) and SIGA
policy decisions (Björndahl et al., 2010; Barratt et al., 2011). In addition to the BSA Course, the SIGA has operated an external quality assurance (EQA) scheme since 1999, and a comprehensive guideline for
practically all aspects of designing, accomplishing and publishing scientific studies involving assessments of semen quality ‘SEMQUA’ was
published recently (Sanchez-Pozo et al., 2013). The comprehensive
SEMQUA document clarifies the necessary steps and considerations
for investigations aiming at robust and reliable determination of semen
parameters. There are, however, studies using semen analysis results
for other purposes, for example the evaluation of treatment modalities or prognostic usefulness of new techniques and/or different
methods.
During the 2012 Associate Editor Meetings of the ESHRE Journals in
Istanbul the authors of the present text were asked to propose a tool
for authors and reviewers of the ESHRE Journals to ensure a high
quality assessment of submitted manuscripts which rely on basic
semen analysis data, including a detailed verification of the relevance
and the quality of the methods used. There are several important
reasons for the ESHRE Journals to strengthen their requirements for
data on semen analysis, as described below.
Björndahl et al.
229
Obtaining accurate data from human semen analyses
International standards for
medical laboratory testing
In all areas of medical laboratory testing the principles of ISO 15189
(International Standards Organization, 2012) define the basic requirements for quality as well as competence. Consequently, any semen analysis that is performed for diagnostic reasons, or during a fertility
treatment (e.g. IVF in an embryology lab), must be expected to
conform to these international standards.
Of particular importance with regard to EQA activities, which can include
the analysis of materials such as verified reference materials, exchange of
samples with other laboratories, and control materials from interlaboratory comparison programmes, are the requirements of ISO/IEC
17043:2010 (International Standards Organization, 2010). A typical
feature of EQA programmes is to provide education to participants
and promote quality improvement: hence advisory and educational comments should comprise part of the report returned to participants to
achieve this aim. For semen analysis this means that the distributed
EQA specimens must have assigned values that allow assessment of accuracy (trueness and precision) as well as the measurement uncertainty
required or expected for each measurand.
When a consensus value is used as the assigned value (see Annex B of
ISO/IEC 17043:2010), the proficiency testing provider (EQA scheme
operator) must document the reason for that selection and also estimate
the uncertainty of the assigned value in order to establish a reasonable
target range (Palacios et al., 2012). The ESHRE SIGA EQA scheme
assigns such reference values based on the average of results and variation reported by its reference laboratories—those that have been
recognized as operating to high standards in accordance with the recommended methodology (Björndahl et al., 2010) and staff training and proficiency. Consequently, if a participating laboratory discovers that its
values deviate significantly from the reference values, then corrective
action can be implemented using the EQA scheme reference values as
the target. However, a scheme that only reports participating laboratories’ performance in comparison to each other, with no reference to any
reference values (e.g. using a statistic such as the ‘all laboratories trimmed
mean’), embodies no capacity for real world quality improvement in
terms of trueness. The system of operation of the EQA scheme is
therefore essential for quality of the results obtained in the individual
participating laboratories. Thus, participation in schemes running with
suboptimal methodology will require more detailed considerations of uncertainty (correctness and variability) if the centre wishes to publish data.
Adaptation to investigations
of different types
The checklist provided in the Appendix to this paper is based on the
WHO and ESHRE-SIGA recommendations for basic semen analysis,
and conforms to the standards taught in the ESHRE-SIGA BSA Course
(Barratt et al., 2011). While the level of uncertainty (error) that is acceptable can certainly vary, depending on the purpose of the investigation, it
should not—as a general rule—exceed +10%.
But guidelines such as these should not be used uncritically. For
example, in large epidemiological population studies a high number of
patients or samples can compensate for the influence of random
errors in each individual assessment when calculating average values
for populations, in contrast to the case where a single result from one individual is obtained with the intent of determining further investigations
or selecting the best treatment modality.
However, in an experimental study where different aliquots of each
semen sample are treated in different ways and compared, poor laboratory methods can conceal true differences: paired analyses will be subject
to comparable uncertainty of measurement errors. Here each sample
acts as its own control and pair-wise or similar statistics with higher
power should be used, sometimes allowing the observation or (discovery) of even quite small effects in spite of suboptimal laboratory investigations. On the other hand, in the clinical situation where laboratory data
from the investigation of one—or a few—semen analyses will be used to
decide a treatment modality, there may be a need for even higher reliability to avoid the wrong treatment being given to the couple. An everyday
example to illustrate this would be where a clinical laboratory makes
decisions on whether to perform routine IVF or ICSI based on a sperm
concentration obtained after sperm preparation by counting in a
shallow chamber (e.g. a Makler chamber) using a ‘standard’ procedure
leading to too few observations in order to obtain a reliable result.
Such simplified procedures are still common in clinical practice although
they are not in compliance with published recommendations or even
those of the manufacturer of the device (Makler, 1978, 1980). For
instance, if ICSI might be deemed necessary when the sperm concentration after preparation is ,0.8 million/ml (M/ml), ordinary IVF treatment when the concentration is .1 M/ml, and in cases where the
concentration is believed to be between 0.8 and 1 M/ml, half of the
oocytes are injected (ICSI) and the other half given ordinary IVF treatment (an ‘IVF/ICSI split’). This may sound reassuring, but how reliable
is the basis for this decision? In such counts, the uncertainty of the
result depends on how many spermatozoa were observed. Consider a
case where the assessment gives the result 0.9 (M/ml) (Fig. 1). This
would be a clear case for an IVF/ICSI split. However, taking into consideration the huge influence of random errors when only a few
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
Section 5.1 of the ISO 15189 elaborates on Personnel expectations that
adequate training shall have been provided, and that competence shall
also have been assessed and established according to established criteria, and also will be re-assessed on a regular basis.
Section 5.4 covers Pre-examination Processes, including sample collection and handling (along with the pertinent pre-collection and collection instructions to patients), specimen transportation, specimen
reception and handling, preparation and storage.
Section 5.5 covers Examination Procedures, including their validation
and the need to establish the measurement uncertainty of results.
Section 5.6 considers how to ensure the quality of results, including
requirements concerning internal quality control (IQC) and EQA.
IQC activities among laboratory staff need to be anchored by a reference staff member, whose training and competency have been verified
against a properly validated EQA scheme that incorporates effective
quality improvement capabilities, as described above.
Until all laboratories performing semen analysis conform to these
standard international expectations for medical testing laboratories,
semen analysis data reported from studies on human semen ‘quality’
will remain of unknown quality and hence continue to be suspect.
230
Björndahl et al.
spermatozoa are counted, only one in four patients will be offered the
treatment that is believed to be appropriate based on the assessment
and the set limits. In over 40% of the couples with the measurement
0.9 M/ml, the true value is .1 M/ml and these couples should
have been given IVF only. In over 30% of these couples the true sperm
concentration would be ,0.8 M/ml for which the intended treatment
was ICSI only. A clinic using WHO and ESHRE standards for semen analysis would give 97% of all patients the intended treatment and the influence of random error would be kept under 5% (Fig. 1), calculations that
are not always obvious to those lacking specialist insights into quantitative
laboratory science. Clearly this is an area for improvements concerning
patient security and efficiency in the treatment of infertility; comprehensive
education in laboratory andrology should therefore be an essential component in the development of reproductive medicine. Furthermore,
data obtained using suboptimal techniques is not warranted in scientific
studies.
It is therefore important that submitting authors, in situations where
their study does not comply with the basic requirements for semen analysis, not only explain all methodological deviations but also declare the
level of uncertainty in their analyses and how it complies with—or
might confound—the aims of the study. When recommendations on
clinical decision limits are given in relation to an investigation, information
should be provided as to not only the positive and negative predictive
values but also the uncertainty of recommended analytical methods
around the suggested clinical decision limit. It is not sufficient to
present a simple cut-off obtained from a receiver operating characteristic
curve (ROC curve) analysis.
Conclusions
To support scientific development in Reproductive Medicine, as well as
improve patient diagnosis, management and treatment, it is essential that
standards be raised in regard to the publishing of results from studies involving semen analyses. We therefore propose that the basic checklist
shown in the Appendix of this paper be used by submitting authors as
well as peer reviewers and Editors of all pertinent journals.
Based on discussions with Editors and Associate Editors of the three
ESHRE journals there are several strategies for actually implementing
this within the submission and review process. Clearly the Questionnaire
would need to be completed and signed by at least the submitting author at
the time of initial manuscript (MS) submission. It should probably also be
made clear that making a false declaration would constitute scientific fraud.
The process for review of a submitted MS could then be carried out in
different ways, depending on the individual circumstances of the MS. In
some cases, a technical review of the MS could be done by an Expert
Panel appointed by the journal to verify conformity (akin to statistical
review such as is undertaken for meta-analyses) before sending the MS
to reviewers, while in other cases an Expert Panel view may be pertinent
before the Associate Editor’s decision. Simple expediency would suggest
doing this step post-triage, to reduce the load on the Expert Panel. To do
a technical review before sending an MS to peer reviewers (i) would allow
authors to resolve perceived non-conformities prior to review, and (ii)
facilitate the reviewers’ work.
For the future, we hope that this initiative will be adopted by other biomedical, epidemiological and scientific journals, and ultimately become
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
Figure 1 A schematic representation of the uncertainties associated with different numbers of spermatozoa counted in shallow or deep counting chambers, based on examples of different methods actually used in some IVF centres to choose between different treatment strategies: IVF only, ICSI only or—
with results of sperm counting in the borderline zone (grey box)—50:50 IVF/ICSI. The proportion of each area-under-the-curve that falls within the ‘grey
box’ represents the proportion of results that will lead to the intended treatment modality. The curves show the probable distribution (Poisson) of the true
sperm concentration in a sperm preparation for assisted reproductive techniques, based on the number of counted spermatozoa. For the shallow (e.g.
Makler) chamber (red curve), the 9 spermatozoa were counted in 10 squares, a practice quite common among many IVF centres, although not according
to published recommendations (Makler, 1978, 1980). When 100 spermatozoa are counted in a haemocytometer (yellow curve) the uncertainty decreases
as illustrated by the narrower peak. Assessment of 400 spermatozoa (green curve) would mean that 95% of counts giving the result 0.9 millions/ml (M/ml)
would have the true value within the interval 0.8– 1.0 M/ml. In the case with 9 counted spermatozoa only, a minute fraction of the true values will be within
the expected interval (grey box), resulting in a more or less random selection of treatment. A majority of couples would, in this example, actually receive a
treatment that was not intended.
231
Obtaining accurate data from human semen analyses
the standard of practice for papers reporting results obtained in laboratory and clinical andrology.
Authors’ roles
All authors have contributed equally to the conception and writing of the
manuscript.
Funding
No funding was obtained for this work.
None declared.
References
Barratt CL, Bjorndahl L, Menkveld R, Mortimer D. ESHRE special interest group
for andrology basic semen analysis course: a continued focus on accuracy,
quality, efficiency and clinical relevance. Hum Reprod 2011;26:3207–3212.
Björndahl L, Barratt CL, Fraser LR, Kvist U, Mortimer D. ESHRE basic semen
analysis courses 1995– 1999: immediate beneficial effects of standardized
training. Hum Reprod 2002;17:1299 – 1305.
Björndahl L, Mortimer D, Barratt CLR, Castilla JA, Menkveld R, Kvist U,
Alvarez JG, Haugen TB. A Practical Guide to Basic Laboratory Andrology,
1st edn. Cambridge: Cambridge University Press, 2010.
ESHRE Special Interest Group in Andrology. Guidelines on the application of
CASA technology in the analysis of spermatozoa. In: Mortimer S,
Mortimer D, Fraser L (eds). Hum Reprod, 1998,142– 145.
Harvey C, Jackson MH. Assessment of male fertility by semen analysis—an
attempt to standardise methods. Lancet 1945;2:99 – 104.
International Standards Organization. ISO/IEC 17043:2010 Conformity
Assessment—General Requirements for Proficiency Testing, Geneva, 2010.
International Standards Organization. ISO 15189:2012 Medical Laboratories—
Requirements for Quality and Competence, Geneva, 2012.
Makler A. A new chamber for rapid sperm count and motility estimation.
Fertil Steril 1978;30:313 –318.
Makler A. The improved ten-micrometer chamber for rapid sperm count and
motility evaluation. Fertil Steril 1980;33:337 – 338.
Mortimer D, Barratt CLR, Björndahl L, de Jager C, Jequier AM, Muller CH.
What should it take to describe a substance or product as ‘sperm-safe’.
Hum Reprod Update 2013;19:i1 – i45.
Palacios ER, Clavero A, Gonzalvo MC, Rosales A, Mozas J, Martinez L,
Ramirez JP, Björndahl L, Morancho-Zaragoza J, Fernandez-Pardo E et al.
Acceptable variability in external quality assessment programmes for
basic semen analysis. Hum Reprod 2012;27:314 – 322.
Plant AL, Locascio LE, May WE, Gallagher PD. Improved reproducibility by
assuring confidence in measurements in biomedical research. Nat
Methods 2014;11:895– 898.
Sanchez-Pozo MC, Mendiola J, Serrano M, Mozas J, Bjorndahl L, Menkveld R,
Lewis SE, Mortimer D, Jorgensen N, Barratt CL et al. Proposal of guidelines
for the appraisal of SEMen QUAlity studies (SEMQUA). Hum Reprod 2013;
28:10– 21.
Tomlinson M. Is your andrology service up to scratch? Hum Fertil (Camb)
2010;13:194 – 200.
Walczak-Jedrzejowska R, Marchlewska K, Oszukowska E, Filipiak E,
Bergier L, Slowikowska-Hilczer J. Semen analysis standardization: is
there any problem in Polish laboratories? Asian J Androl 2013;15:616– 621.
World Health Organization. WHO laboratory manual for the examination of
human semen and sperm-cervical mucus interactions, 4th edn. Cambridge,
UK: Cambridge University Press, 1999.
Appendix
A check list for authors, reviewers
and editors
Any deviation from this guideline (i.e. a box without a tick) must be
declared and explained in the Materials and Methods section of the manuscript, including explaining its effect on the measurement uncertainty of the
data, in order to allow the reader to evaluate the qualityof the analyses performed. Investigations that would be subject to this requirement can
roughly be classified as clinical (evaluating patient treatment, diagnostic
classification or predictive powers of certain assessments), experimental
(e.g. exposure of sperm to different compounds or in vitro treatments
(Mortimer et al., 2013)), or epidemiological (evaluating variations in
semen characteristics or effects of exposure populations to certain compounds or other circumstances). Any scientific rationale for not complying
with the guidelines, which is not included in the Materials and Methods
section of the manuscript, must be substantiated to Editor and reviewers.
Patients
A
A
For clinical studies: The patient population (e.g. patients, volunteers, students) has been declared in the manuscript, together
with the recruitment method and inclusion and exclusion criteria.
If the study concerns couples being investigated for infertility then
the following must be specified in the manuscript: fertility status of
female partner; and primary, secondary or other level of investigation of the man.
If used in the manuscript, the term ‘male factor’ must be completely defined.
General aspects
A
A
A
A
A
A
A
A
Patients were instructed to maintain 2–7 days of sexual abstinence before collecting a sample for investigation.
Patients were informed about the importance of reporting any
missed early ejaculate fractions, and men’s answers were noted
on the laboratory form.
For specimens not collected at the laboratory, patients were
instructed to avoid cooling or heating of the semen sample
during transport to the laboratory.
Samples were kept at 378C before initiation of and during the analysis in case of sperm motility assessment.
For samples collected adjacent to the laboratory, analysis was
initiated after completion of liquefaction and within 30 min after
ejaculation. If this was not done—and more importantly when
some of the samples are collected in the laboratory and others
are collected at home—it should be checked that this did not influence the data (and, if yes, that this effect must be included as a
confounding factor in the statistical analysis).
Liquefaction was first checked within 30 min after ejaculation.
Volume was determined either by weighing or using a wide-bore
volumetric pipette.
Viscosity was measured using either a wide-bore pipette or a glass
rod.
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
Conflict of interest
World Health Organization. WHO laboratory manual for the examination and
processing of human semen, 5th edn. Geneva: World Health
Organization, 2010.
232
A
A
Björndahl et al.
All staff members who performed the analyses have been trained
in basic semen analysis (ESHRE Basic Semen Analysis Course—or
equivalent—and further in-house training) and participate regularly in internal quality control.
If more than one method can be recommended for a particular
characteristic (e.g. to measure volume), only one should be used
in a given study.
Sperm concentration assessment
A
A
A
A
A
Sperm motility assessment
A
A
A
A
A
A
A
Motility assessments were performed at 378C + 0.58C.
Motility assessments were done using phase contrast microscope
optics (200–400×).
Sperm motility was classified using a four-category scheme: rapid
progressive, slow progressive, non-progressive, and immotile
(World Health Organization, 1999; Björndahl et al., 2010;
Barratt et al., 2011).
Motility assessments were done in duplicate and compared;
counts were re-done on new preparations when the difference
between duplicates exceeded the acceptance limits.
The wet preparation was made with a drop of _____ ml and a
____ x ____ mm coverslip to give a depth of _____ mm (must
be at least 10 mm, but not too deep so as to allow spermatozoa
to move freely in and out of focus; typically ca. 20 mm).
At least 200 spermatozoa were assessed in each duplicate motility count.
At least 5 microscope fields of view were examined in each duplicate count.
Sperm vitality assessment
A
A
A
A validated supravital staining, appropriate to the type of microscope optics utilized, was used to assess sperm vitality.
At least 200 spermatozoa were evaluated in each sample.
Assessments were done under high magnification (×1000–
1250) using a 100× high resolution oil immersion objective and
bright field microscope optics (Köhler illumination).
A Tygerberg Strict Criteria were used for the evaluation of human
sperm morphology.
Note: Another classification could be used for scientific studies
with specific aims if the classification is described or referenced.
Depending on the aim of the study, the evaluation of particular abnormal forms might be useful.
A Abnormalities are recorded for all four regions of the spermatozoon (head, neck/midpiece, tail and cytoplasmic residue) and the
Teratozoospermia Index or ‘TZI’ was calculated (Björndahl et al.,
2010; Barratt et al., 2011).
A If the laboratory claims to use Tygerberg Strict Criteria for the
evaluation of human sperm morphology, then the laboratory
must participate in an external quality assurance scheme to
verify that its assessments comply with these criteria.
A The Papanicolaou staining method adapted for the assessment of
human sperm morphology was used. For specific aims other staining
methods could be used, but must then be declared and explained.
A At least 200 spermatozoa were assessed in each ejaculate.
A Assessments were done under high magnification (×1000–
1250) using a 100× high resolution oil immersion objective and
bright field microscope optics (Köhler illumination).
Other findings
A The presence of abnormal clumping (aggregates and agglutinates)
was recorded.
A Abnormal viscosity was recorded.
A The presence of inflammatory cells was recorded and reported if
more than 1 million/ml.
A For the purpose of classifying infertility status (World Health Organization, 2010), antisperm antibodies were examined with a
validated screening test (state which method was used:
________________).
Analysing data
A The actual duration of sexual abstinence (in ‘hours’ or ‘days’) was
recorded for each sample and included in the data reported in
the manuscript.
A As a minimum in clinical studies, semen volume, sperm concentration, total number of spermatozoa/ejaculate, and abstinence
time are given to reflect sperm production and output; only
samples identified as having been collected completely can be
included in the study.
A Confounding factors have been considered for statistical analysis:
e.g. abstinence time and age, to evidence secular or geographical
variations in sperm concentration or sperm count.
A If appropriate, optional biochemical markers for prostatic,
seminal vesicular and epididymal secretions were analysed and
reported both as concentration and total amount.
A Signs of active infection/inflammation were noted and considered in the analysis of data in the study (e.g. inflammatory cells,
impaired sperm motility, possibly also antisperm antibodies and
reduction of secretory contributions).
Downloaded from https://academic.oup.com/humrep/article/31/2/227/2380030 by guest on 01 September 2021
A
A
Semen aliquot to be diluted for sperm concentration assessment
was taken with a positive displacement pipette (i.e. a ‘PCR
pipette’) using a recommended diluent (state which diluent:
_____________).
Only standard dilutions were used (1:50, 1:20, or 1:10).
Sperm concentration was assessed using haemocytometers with
improved Neubauer ruling.
Haemocytometers were allowed to rest for 10–15 min in a
humid chamber to allow sedimentation of the suspended spermatozoa onto the counting grid before counting.
Sperm counting was done using phase contrast microscope optics
(200–400×).
Comparisons were made between duplicate counts, and counts
re-done when the difference exceeded the acceptance limits.
Typically at least 200 spermatozoa were counted in each of the
duplicate assessments.
Sperm morphology assessment