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External Quality Control of Semen Analysis
Reveals Low Compliance with WHO Guidelines
E. Nieschlag, T. Pock, B. Hellenkemper,
QuaDeGA (Quality Control Programme of the German Society of Andrology)
Introduction
In 2011 the German Federal Medical
Board (BÄK) integrated semen analy-
sis into its compulsory external quality
control program for medical laboratories
[1]. Since then all laboratories perform-
ing semen analysis for human medi cal
purposes are obliged to partici pate twice
yearly in ring trials (“Ringversuche”) to
obtain a certificate as a pre-requisite for
charging patients or insurances for se-
men analysis. The BÄK guidelines re-
quire participating laboratories to use
methodology as described in the current
World Health Organization (WHO). Lab-
oratory Manual for the Examination and
Processing of Human Semen. At the pre-
sent time this is the 5th edition of WHO
Manual [2] for which a German transla-
tion has been published [3]. The Quali-
ty Control Programe of the German So-
ciety of Andrology ( QuaDeGA GmbH)
was licensed by the BÄK as official Ref-
erence Institution to conduct the compul-
sory ring trials.
QuaDeGA was established in 2002 and
had performed ring trials on a voluntary
basis until 2011. Before participation be-
came mandatory in 2011 250 laborato-
ries had taken part in this external quality
control program. Since then the number
has increased to over 700 (Fig. 1).
While the number of participants in the
programme has amost tripled since it
became compulsory, over the years the
Youden plots continued to result in broad
windows and the rate of obtaining the cer-
tificate has remained around 80 % with
only a small tendency for improvement
(Fig 2). In an attempt to find out why the
failure rate remains high, we conducted a
survey among the participants asking for
details of their laboratory techniques, es-
pecially in regard to guidelines provided
by the WHO Manu al [2, 3].
Received: October 12, 2017; accepted after revision: October 26, 2017 (responsible Editor: Prof. H. Behre, Halle)
From the Center of Reproductive Medicine and Andrology, University Hospital Münster, Germany
Correspondence: Univ.-Prof. em. Dr. med. Dr. h.c. Eberhard Nieschlag, FRCP, Center of Reproductive Medicine and Andrology, Domagkstraße 11, D-48149 Münster,
e-mail: Eberhard.Nieschlag@ukmuenster.de
Semen analysis is the oldest and most commonly used surrogate parameter for male fertility. Like all other clinical laboratory tests, semen analysis should
be under strict internal and external quality control. WHO has pioneered standardisation and quality control of semen analysis. The external Quality Control
Program of the German Society of Andrology (QuaDeGA) is based on WHO guidelines and since 2011 participation in ring trials is compulsory for all labora-
tories performing semen analysis in Germany. However, many laboratories fail to follow these guidelines so that it remains unclear whether the wide scatter
of results from ring trials is caused by protocol failures or is inherent to semen analysis. In order to resolve this question we performed a survey among 624
participants and received valid answers from 256 (42.5%). The answers revealed that many laboratories lack basic equipment such as mixers, vortex, posi-
tive displacement pipettes and appropriate microscopes, do not use the recommended improved Neubauer chamber for counting, use inappropriate staining
methods and do not evaluate sufficient sperm. Most surprisingly, 23% of the laboratories apply no internal quality control and these have a higher failure
rate than those with internal control (28 vs 8%). Laboratories performing more than 20 semen analyses per month score better than those with lower sam-
ple frequency. Strict observation of WHO guidelines and more intensive teaching and practical training of technicians appear to be the most important meas-
ures to improve results. Until better agreement of results within and between laboratories is achieved, arguing over normal reference and threshold values
remains a secondary problem. J Reproduktionsmed Endokrinol_Online 2017; 14 (6): 306–10.
Key words: Quality Control Program of the German Society of Andrology, QuaDeGA, semen analysis
Figure 2. Development of total number of participants and the percentage of those
obtaining a certificate from ring trial 20/2011 to 31/2017.
Figure 1. Participants in the QuaDeGA program 2002–2016 (ring trial 1–30).
J Reproduktionsmed Endokrinol_Online 2017; 14 (6)
External Quality Control of Semen Analysis Reveals Low Compliance with WHO Guideline
307
Methods
Ring trials
The QuaDeGA program has been de-
scribed in previous publications [4, 5]
and the reader is referred for details to
these publications. In short, QuaDeGA
carries out external quality control tri-
als twice a year, consisting of two fixed
sperm preparations (sample A and B).
These samples allow for the measure-
ment of sperm concentration as well as
for preparation and staining of a smear
for the assessment of morphologically
normal forms. Film sequences of two dif-
ferent native semen samples are provid-
ed on the QuaDeGA platform for analy-
sis of sperm motility. Each participant in-
serts the results on the online platform.
For the three parameters (1) sperm con-
centration, (2) normal morphology and
(3) progressive motility medians of the
values obtained by those laboratories
indicating that they adhere strictly to
WHO guidelines the 2.5 and 97.5 per-
centiles are used to construct Youden
plots (Fig. 3). Those participants whose
results for all parameters lie within the
Youden plot windows receive certificates
of passing the external quality control. In
addition, QuaDeGA provides a ranking
for each result indicating whether results
lie within the Youden plot (rank 1), or
whether a systematic (rank 2) or a non-
systematic (rank 3) or a random error
(rank 4) has been noted.
Method used for the Survey
A questionnaire comprising 35 items
concerning technical and methodologi-
cal details of semen analysis as per-
formed in the individual laboratories was
drafted in German and sent electronical-
ly to participants using the online sur-
vey platform SurveyMonkey (Registered
trade mark). The 624 participants in the
ring trial 30/2016 in Germany, Austria
and Switzerland were addressed, and a
deadline of 3 weeks was set for return-
ing answers. The questions are not de-
scribed here in detail as their content
becomes evident from the results in the
next section.
Results
Responders
273 answers were received of which 256
(93.8%) could be evaluated. Data were
saved and summarized by the online
platform for further analysis. The 256
laboratories represent 42.5 % of those
who had received the questionnaire and
reflected the spectrum of participating
laboratories (andrologists, urologists,
ART centers, general clinical laborato-
ries, hospitals, university clinics and pri-
vate surgeries). Data were saved for fur-
ther analysis with Microsoft Excel.
In order to find out whether the respon-
ders were biased concerning their per-
formance in the ring trials tests, their re-
sults in run 30/2016 were compared with
those from the non-responders. While
83% of the 624 addressed participants
had received a certificate, 86% of the
256 responders and 82% of the 359 non-
responders had received certificates, in-
dicating that there was no significant
difference in performance between re-
sponders and non-responders. As not all
responders answered all questions, the
number of replies varies from question
to question. On average 7% of the ques-
tions were not answered.
Availability of the WHO Manu-
al, Lab equipment and Tech-
niques
When asked whether the WHO Manu-
al was available in the individual labora-
Figure 3. QuaDeGA ring trial 30/2016: Youden plots for sperm parameters in samples A and B of 663 participants constructed from medians of the WHO compliant participants (2.5
to 97.5 percentiles).
J Reproduktionsmed Endokrinol_Online 2017; 14 (6)
External Quality Control of Semen Analysis Reveals Low Compliance with WHO Guideline
308
tory, 93% answered “Yes”. When asked
whether they followed WHO guidelines
for the individual parameters, 93% an-
swered “Yes” for sperm concentration,
94% for motility and 90% for morpho-
logy.
However, when asked for the available
basic equipment in their laboratory, 28%
had no Vortex (or comparable mixing de-
vice), 45% no simple counting aid and
34% no laboratory counter. Of 254 an-
swering laboratories 36% used a phase
contrast microscope and 22% a high-
quality light microscope; the rest used
medium (33%) or standard (9%) mi-
croscopes not considered sufficient by
WHO guidelines.
Only 38.2% used the WHO recommend-
ed diluent, 23.1% no diluent and the re-
maining 38.7% used either NaCl, water
or other media.
Only 59% of 249 laboratories used the
WHO recommended improved Neu-
bauer chamber for counting sperm. 27%
used the Makler chamber, and the re-
maining 14% various but not recom-
mended chambers.
Only 55 of 256 laboratories (22%) used
the WHO recommended positive dis-
placement pipettes for concentration. All
49 laboratories using the correct cham-
ber and correct pipettes achieved rank 1,
while only 93% of those using the cor-
rect chamber, but incorrect pipettes, and
95% of those using Makler chambers
achieved rank 1 (Fig. 4).
Only 55% of 256 labs used the WHO
recommended Papanicolaou, Shorr or
DiffQuik staining method (Fig. 5). The
61 of 244 (25%) laboratories evaluating
more than 200 sperm cells achieved the
best results.
Internal Quality Control
The WHO manual considers it manda-
tory that all laboratories perform inter-
nal quality control and the BÄK provid-
ed Excel tables for this purpose which
can be obtained from QuaDeGA upon
request. However, only 35% perform
their internal quality control by using
these tables, while 42% use other pro-
grams for internal quality control. Sur-
prisingly 23% of the 244 laboratories
answering this question used no inter-
nal quality control. While 92% of those
practicing internal quality control ob-
tained a certifi cate, only 74% of those
without internal quality control reached
that goal.
Frequency of Semen Analysis
Investigating the impact of the number of
semen analyses on the results of the indi-
vidual laboratories, it became clear that
those laboratories performing more than
20 semen analyses per month obtain cer-
tificates to a higher proportion than those
laboratories with fewer than 20 semen
samples (Fig. 6).
Discussion
Despite a battery of sophisticated sperm
function tests, semen analysis remains
the basic standard investigation to as-
sess male fertility and infertility. Semen
analy sis plays a central role in the work-
up of the infertile couple, but it is also
important in toxicological, ecological
and epidemiological studies. Recent-
ly, semen parameters were even found
to reflect general health and – as a bio-
marker – to predict life expectancy [6].
5% 3%
2% 2%
93%
100%
95%
100%
98%
96%
94%
92%
90%
88%
WHO compliant Chamber WHO compliant /
but different pipee
Makler chamber
n = 49 n = 132 n = 66
Rank 1 Conc.
Rank 2 Conc.
Rank 3 Conc.
Figure 4. Impact of counting chambers and pipettes on determination of sperm concentrations (from 247 responses) as
reflected by results from ranking. Left: using improved Neubauer chamber and positive displacement pipettes; middle:
using improved Neubauer chamber, but no positive displacement pipettes; right: using Makler chamber.
Papanicolaou
Shorr
DiffQuik
Others
0% 10% 20% 30% 40% 50% 60% 70%
45%
37%
4%
14%
Figure 5. Different stainings used by 257 laboratories for sperm morphology. Green: WHO compliant; red: Not WHO
recommended.
J Reproduktionsmed Endokrinol_Online 2017; 14 (6)
External Quality Control of Semen Analysis Reveals Low Compliance with WHO Guideline
309
In the light of this eminent role of semen
parameters, it is surprising that semen
analysis has long escaped quality assur-
ance programs which are an obligatory
exercise for all other measurements in
the medical laboratory. Even the WHO
manual (1st edition 1980) was not over-
ly concerned with external quality con-
trol, and it was only in the 4th edition in
1990 that a small paragraph was dedicat-
ed to this topic. The 5th edition of 2010
[2, 3] expanded external quality control
into a chapter. In parallel, several nation-
al and international external quality con-
trol programs have been developed in
order to improve the validity and inter-
labora tory comparability of results. In
a few countries participation in external
quality control programs became legal-
ly obligatory, and in Germany health in-
surances will not refund costs for semen
analysis without a valid certificate from
the quality control reference institution
issued to the investigating laboratory.
Nevertheless, some sceptics continue to
doubt the value of quality control and the
adherence to generally accepted guide-
lines in order to guarantee reliable and
reproducible results for sperm counts,
motility and morphology [7, 8]. Others
question the predictive value of sperm
parameters in terms of chances for con-
ception and pregnancy [9–13]. However,
this remains an elusive discussion as
long as the suggested and largely accept-
ed guidelines are not strictly followed.
With its manual, WHO provides such
guidelines which have been accepted as
the international standard, although they
remain not undisputed.
As long as participation in an external
quality control program for semen analy-
sis was voluntary , we recognized that
only 8% of laboratories participating in
the QuaDeGA program adhere strictly to
the WHO manual [5], and other external
quality control schemes reported a simi-
lar low adherence to WHO guidelines
[14–16]. Since external quali ty con-
trol and use of WHO guidelines became
compulsory in Germany over 90% of
labs claim to adhere to WHO guidelines,
but as our current survey has shown, in
reality the proportion of WHO followers
is much lower. This failure to adhere to
the guidelines ranges from inappropriate
equipment and techniques to the lack of
internal quality control. Until this situa-
tion changes and uniform methodology
is used, it will be impossible to judge the
value of quality control programs and the
predictive value of semen parameters as
such. The high failure rate in obtaining
the certificate by those not performing
internal quality control, and high suc-
cess rates of those using proper counting
chambers and pipettes, provide visible
examples of how adherence to guidelines
can impact results positively. Also the
impact of different staining techniques
on the evaluation of sperm morphology
has been well documented, as not only
the chemicals used for coloring, but also
the osmolarity of the solutions strong-
ly influence sperm appearance and pre-
dispose to divergent results. In order to
overcome this problem, use of only one
staining technique to be used in all labo-
ratories has been suggested [17, 18], but
the editors of the WHO manual could not
agree to such a strict requirement.
Only if all participants adhere to the
same technical template, can the mag-
nitude of an adherence problem of
semen analysis be properly assessed. It
is a fact that since the first discovery of
sperm under the microscope of Anthony
Leeuwen hoek in 1678, semen analysis
has remained a subjective method de-
pending on the training and the skills of
64
44
61
23
64
16
8
3
32
90
80
70
60
50
40
30
20
10
0
1-10 Semen
analysis
11-20 Semen
analysis
21-50 Semen
analysis
51-100 Semen
analysis
>100 Semen
analysis
Passed Failed
Figure 7. Performance in QuaDeGA ring trial 30/2016 with 663 laboratories divided into three groups of 221 each
according to time of starting participation between 2002 and 2016.
Figure 6. Impact of number of semen analyses per lab and month on awarding the certificate based on answers from
218 participants.
11% 18% 26%
250
200
150
100
50
0
1–221 222–443 444–663
2001–2011 2011–2013 2014–2016
Cerficate
No cerficate
J Reproduktionsmed Endokrinol_Online 2017; 14 (6)
External Quality Control of Semen Analysis Reveals Low Compliance with WHO Guideline
310
the investigator. As most semen analy-
ses are performed by medical laboratory
technicians it is deplorable that at most
of their schools semen analysis is not in-
cluded in their curriculum and most un-
dergo training on the job when confront-
ed with semen analysis. Therefore post-
graduate semen analysis courses are of
utmost importance for improvement of
this situation [13]. In addition, participa-
tion in external quality control programs
has an educational effect on its own [19],
as demonstrated by an increasing rate of
obtaining certificates with the duration
of participating in the QuaDeGA ring tri-
als (Fig. 7). In support of stricter use of
guidelines, journals should request proof
of proficiency by the laboratories sub-
mitting data from semen analysis [20].
Furthermore it is astonishing that despite
30 years of computer-assisted semen
analysis (CASA) research, semen anal-
ysis remains a subjective method. Al-
though technology has advanced to the
extent that individual human faces can be
identified among thousands of subjects, it
remains a puzzle why sperm at low con-
centrations cannot be differentiat ed ex-
actly from debris and sperm morphology
cannot be recognized accurately by elec-
tronic means. Hopefully, once the neces-
sary technology has been develop ed,
all quality control problems will be re-
solved – or not, if it should then become
evident that reproducibility and interlabo-
ratory comparability of results from se-
men analysis depend on other factors in-
trinsic to the object under investigation.
Acknowledgements
The authors gratefully acknowledge co-
operation with Dr. T. J. Panholzer for
maintaining the QuaDeGA platform at
the Institute for Medical Biometry, Epi-
demiology and Information Technolo-
gy of the University Medicine Mainz,
as well as language editing of the manu-
script by Susan Nieschlag MA.
Conflict of Interest
The authors are employed part-time
by the Quality Control Programme
of the German Society of Andrology
( QuaDeGA GmbH).
References:
1. BÄK. Richtlinie der Bundesärztekammer zur Qualitäts-
sicherung laboratoriumsmedizinischer Untersuchungen:
Ejakulatuntersuchungen. Dt Ärztebl 2011; 108: 2298–304.
2. World Health Organization (WHO). Laboratory manual for the
examination and processing of human semen. 5th Edition,
Geneva, 2010.
3. WHO Laborhandbuch zur Untersuchung und Aufarbeitung des
menschlichen Ejakulates. Übersetzt von Nieschlag E, Schlatt S,
Behre HM, Kliesch S (Hrsg.) unter Mitarbeit von Bongers R,
Gottardo F, Greither T, Hellenkemper B, Nieschlag S, Nordhoff V,
Schalkowski M, Zitzmann M. 5. Auflage, Springer Heidelberg,
2012.
4. Cooper T, Hellenkemper B, Nieschlag E. External Quality
Control for Semen Analysis in Germany - Qualitätskontrolle der
Deutschen Gesellschaft für Andrologie (QuaDeGA). The First 5
Years. J Reprod Med Endocrinol 2007; 4: 331–5.
5. Mallidis C, Cooper TG, Hellenkemper B, Lablans M, Ückert F,
Nieschlag E. Ten years’ experience with an external quality con-
trol program for semen analysis. Fertil Steril 2012; 98: 611–6.
6. Latif T, Kold Jensen T, Mehlsen J, Holmboe SA, Brinth L, Pors
K, et al. Semen quality as a predictor of subsequent morbidity:
A Danish cohort study of 4,712 men with long-term follow-up.
Am J Epidemiol. 2017; 279:1–8.
7. Tomlinson MJ. Uncertainty of measurement and clinical value
of semen analysis: has standardisation through professional
guidelines helped or hindered progress? Andrology 2016;
4:763–70.
8. Jequier AM. Is quality assurance in semen analysis still really
necessary? A clinician’s viewpoint. Hum Reprod 2005; 20: 2039–
42.
9. Van Der Steeg JW, Steures P, Eijkemans MJC, Habbema JDF,
Hompes PGA, Kremer JAM, et al. Role of semen analysis in
subfertile couples. Fertil Steril 2011; 95:1013–9.
10. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker
HWG, Behre HM, et al. World Health Organization reference
values for human semen characteristics. Hum Reprod Update
2009; 16: 231–45.
11. Van Geloven N, Van Der Veen F, Bossuyt PMM, Hompes PG,
Zwinderman AH, Mol BW. Can we distinguish between infertili-
ty and subfertility when predicting natural conception in couples
with an unfulfilled child wish? Hum Reprod 2013; 28: 658–65.
12. Hamilton JAM, Cissen M, Brandes M, Smeenk JMJ, De
Bruin JP, Kremer JAM, et al. Total motile sperm count: A better
indicator for the severity of male factor infertility than the
WHO sperm classification system. Hum Reprod 2015; 30:
1110–21.
13. Barratt CLR, Björndahl 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: 3207–12.
14. Penn HA, Windsperger A, Smith Z, Parekattil SJ, Kuang
WW, Kolettis PN, et al. National semen analysis reference
range reporting: Adherence to the 1999 World Health Organiza-
tion guidelines 10 years later. Fertil Steril 2011; 95: 2320–3.
15. Björndahl L. Compliance with recommendations for reliable
semen analysis results – a matter of importance for patients
and scientific development. Andrology 2016; 4: 771–2.
16. Punjabi U, Wyns C, Mahmoud A, Vernelen K, China B,
Verheyen G. Fifteen years of Belgian experience with external
quality assessment of semen analysis. Andrology 2016; 4:
1084–93.
17. Gatimel N, Moreau J, Parinaud J, Léandri RD. Sperm mor-
phology: Assessment, pathophysiology, clinical relevance, and
state of the art in 2017. Andrology 2017; 1–18.
18. Meschede D, Keck C, Zander M, Cooper TG, Yeung CH,
Nieschlag E. Influence of three different preparation techniques
on the results of human sperm morphology analysis. Int J
Androl 1993; 16: 362–9.
19. Brüggemann, M. Qualitätssicherung laboratoriumsmedizini-
scher Untersuchungen. In: Jonitz G, Mansky T, Scriba PC (Hrsg).
Ergebnisverbesserung durch Qualitätsmanagement. Deutscher
Ärzteverlag, Köln, 2014; 109–114.
20. Carrell DT, De Jonge CJ. The troubling state of the semen
analysis. Andrology 2016; 4: 761–2.
J Reproduktionsmed Endokrinol_Online 2017; 14 (6)