You are on page 1of 10

ANDROLOGY

ISSN: 2047-2919

ORIGINAL ARTICLE

Correspondence:
Usha Punjabi, Centre for Reproductive Medicine,
Antwerp University Hospital, Wilrijkstraat 10, 2650
Edegem, Belgium.
E-mail: usha.punjabi@uza.be

Keywords:
quality control, sperm concentration, sperm
morphology, sperm motility, sperm quality
parameters
Received: 28-Mar-2016
Revised: 2-May-2016
Accepted: 6-May-2016

Fifteen years of Belgian experience


with external quality assessment of
semen analysis
1

U. Punjabi, 2C. Wyns, 3A. Mahmoud, 4K. Vernelen, 4B. China


and 5G. Verheyen

Centre for Reproductive Medicine, Antwerp University Hospital, Edegem, 2Cliniques Universitaires
Saint Luc, Brussels, 3University Hospital of Ghent, Ghent, 4Scientific Institute of Public Health,
Brussels, and 5Centre for Reproductive Medicine, UZ Brussel, Brussels, Belgium

doi: 10.1111/andr.12230

SUMMARY
Semen analysis is difficult to standardize, quality control and quality assurance are necessary to ensure that results are accurate
and precise. This Belgian EQA survey over a 15-year period, involving 121 laboratories, attempted to reduce interlaboratory variability
and at the same time, encouraged participating laboratories to implement correct techniques as advised by the WHO. Over the total
period, the median coefficient of variation (CV) for sperm count, irrespective of the method used was 19.2%, while using improved
Neubauer chamber resulted in a significantly (p < 0.001) lower median CV (14.4%). The overall median CV for rapid progressive
motility was high (37.1%), but progressive motility (15.1%) and total motility (13.8%) were acceptable. Sperm morphology revealed a
large variability in 79.4% irrespective of the staining procedures or evaluation criteria used. Participation in the Belgian EQA is on voluntary basis. Both, participation and implementation of the correct techniques should be made mandatory for accreditation and
benefit of patient treatment. The existing Belgian EQA program should now be harmonized with other existing EQA schemes in
Europe.

INTRODUCTION
Accurate semen analysis is the cornerstone of male infertility
evaluation. However, the large variations in the analysis data
obtained within and between laboratories (Jequier & Ukombe,
1983; Ayodeji & Baker, 1986; Neuwinger et al., 1990; Walker,
1992; Matson, 1995; Keel et al., 2000) have made semen analysis
inaccurate and unreliable (Chong et al., 1983; Mortimer et al.,
1986; Ombelet et al., 1997).
In response to the growing need for standardization of procedures, the World Health Organization (WHO) has published a
series of laboratory manuals to encourage this (WHO, 1980,
1987, 1992, 1999, 2010). Later on, the Nordic Association for
Andrology (NAFA) and the Special Interest Group Andrology
(SIGA) of the European Society of Human Reproduction and
Embryology (ESHRE) have provided more detailed instructions
rnfor the techniques recommended by the WHO (Kvist & Bjo
dahl, 2002).
Although these manuals are widely recognized as the golden
standard for semen testing worldwide, not all laboratories have
implemented the recommended techniques and the repeatedly
updated normal or reference values. Cut-off values (normal vs.
2016 American Society of Andrology and European Academy of Andrology

abnormal) are still highly variable between laboratories, and differences in assessment procedures between laboratories jeopardize the comparability of test results and conclusions regarding
the male fertility status.
As semen analysis is difficult to standardize, quality control is
essential to detect and correct systematic errors and an internal
quality assurance program should ensure that results are both
accurate and precise. Mortimer et al. (1986) introduced the concept of quality assurance in semen analysis, which was included
only from the fourth edition of the WHO manual (1999) and further emphasized in the last edition (WHO, 2010). Quality control
and quality assurance should be an integral part both within [internal quality control (IQC)] and between [external quality control (EQC)] laboratories. While IQC monitors precision, EQC
monitors the accuracy and stability of the methods (Plaut &
Westgard, 2002), thus both are complementary processes.
At present, EQC for the determination of sperm concentration,
motility, and morphology is being widely appreciated in Europe
(Neuwinger et al., 1990; Cooper, 1996; Jrgensen et al., 1997;
Ochsendorf & Beschmann, 1998; Auger et al., 2000; Alvarez
et al., 2005; Mallidis et al., 2012; Jedrzejczak et al., 2012;
Andrology, 110

U. Punjabi et al.

Filimberti et al., 2012) and other parts of the world (Keel et al.,
2000; Brazil et al., 2004; Zuvela et al., 2011).
In 1997, the Scientific Institute of Public Health (IPH) of
the Belgian Ministry became aware of the need for an EQC in
semen analysis because of the large costs ( 116509.92 for
41260 concentration/motility tests and 69410.17 for 26631
morphology tests on yearly basis) covering these analyses and
the lack of standardization in the semen parameters analyzed.
This led to the establishment of an EQC program to monitor
interlaboratory proficiency, aiming for a growing awareness of
the need to implement the correct procedures by participating laboratories. In 1999, the IPH was officially mandated for
the organization of EQC in clinical biology laboratories in Belgium (Demotte, 1999). The IPH was accredited under the
International Organization for Standardization (ISO) guide 43
(Proficiency testing by interlaboratory comparisons, 1997.
Geneva, Switzerland) and The International Accreditation
cooperation (ILAC) G13 (Guidelines for the requirements for
the competence of provider of proficiency testing schemes,
2000. Silverwater, Australia) and since 2011 under ISO17043
(Conformity assessment General requirements for proficiency testing, 2010. Geneva, Switzerland) standard for the
organization of EQC in human semen quality. The present
paper describes 15 years of Belgian experience with external
quality assessment of semen analysis.

MATERIALS AND METHODS


Coordinating committee
The first step in the establishment of an EQC program was the
foundation of an ad hoc coordinating committee (CC), composed of eight representatives in the field of assisted reproduction from different university or non-university hospitals. The
CC was responsible for the content of the global reports sent out
to the participating laboratories after each survey.
Participants
Information over participant laboratories was obtained
through different questionnaires. In 1997, an initial questionnaire was sent to all clinical laboratories (private and hospitalbased) all over the country (approximately 400) in order to
obtain relevant information regarding the used procedures. A
second questionnaire was sent in 2006 to gather additional information concerning the type of laboratory, the education/training undertaken, and the number of technicians involved in
semen analysis. Participation to the EQC program has always
been on a voluntary basis, from the start up to date.
Scheme organization
Since 1998, three schemes per year have been organized
including two semen aliquots for sperm concentration and two
smears for sperm morphology. One scheme each year additionally included a video/DVD recording of four samples for sperm
motility. Each participating laboratory received different aliquots/slides from the same batch of samples and returned their
results to the IPH. The homogeneity and the stability of the samples for both sperm count and morphology were independently
assessed according to ISO13528 (Statistical methods for use in
proficiency testing by interlaboratory comparisons, 2005, Geneva, Switzerland) norms.
2

Andrology, 110

ANDROLOGY
The results of the participants were analyzed by the IPH and
discussed with the CC. Feedback to the participating laboratories based on WHO recommendations was provided as well as
advice for improvement, with possibility of specific training
when available (Basic semen analysis course in collaboration
with the ESHRE SIGA). Upon request, laboratory technicians
from participating centers could benefit from onsite training in
the laboratories of the members of the CC.
Sample preparation and distribution
One single Andrology laboratory prepared all the samples for
concentration and morphology for each scheme. For sperm
count, human semen for two different pools was collected, fixed
in formalin, and aliquots of 500 lL were prepared. The participating laboratories were instructed to mix the samples gently by
a few aspirations with a glass pipette or using a vortex at low
speed. Spermatozoa were counted according to the laboratorys
routine procedure. Results were recorded as 9106 spermatozoa/
mL. The laboratory was requested to indicate the type of counting chamber used. Since 2007, the use of positive displacement
pipette was also reported.
For morphology, semen smears were prepared from two different pools of semen and air-dried. Two air-dried smears were
distributed per scheme. The participants were instructed to fix
and stain the smears and perform morphological analysis adopting their routine methods. Results were reported as percentage
of normal forms and the morphology smears were as such also
scored as normal or abnormal. The participants were requested
to indicate the type of stain and the criteria used to evaluate
sperm morphology.
For sperm motility, a series of video/DVDs containing four different samples were prepared. Participating laboratories
reported the four motility grades (rapid progressive, slow progressive, non-progressive, and immotile, WHO, 1999) or three
motility grades (progressive, non-progressive, and immotile,
WHO, 2010). Only one video/DVD was distributed per year.
After preparation, the samples were distributed to the participating laboratories by the IPH. The laboratories were asked to
perform the analyses and return the results within 2 weeks of
reception.
Data processing for each survey
Results were received by the IPH and each laboratory was
coded before data processing by the IPH. After data processing,
the results of the survey were discussed with the CC and recommendations were formulated. Per scheme, the results for each
parameter were reported as the overall mean, median, standard
deviation, p10p90, p25p75, minimum and the maximum
value, and the coefficient of variation (CV) of all the participating
laboratories and of those performing the analysis with the same
method. In addition, box and whisker plots were included for
concentration, motility, and morphology. For morphology, in
addition to the percentile ranges, an overall score as normal/abnormal was given to each smear based on the classification used
by each laboratory.
Statistical analyses
Over the total period of 15 years, the evolution of the coefficients of variation for each parameter were presented in figures.
For each parameter, for the whole period (19982012), the
2016 American Society of Andrology and European Academy of Andrology

ANDROLOGY

BELGIAN EQA OF SEMEN ANALYSIS

overall median CV was calculated as well as the lower and upper


quartiles (Q1Q3). Statistical analysis was performed using the
program MEDCALC Software version 13.0.6.0. For the comparison
of median CV values of the count using all chambers and the
count using the improved Neubauer chamber, the Wilcoxon
signed-rank test was used. A p-value of <0.05 was considered as
statistically significant.

(laboratories analyzing samples only after vasectomy did not


participate).
The number of the diagnostic analyses per laboratory varied
from 1 sample/month to 75 samples/week. Only six laboratories
analyzed >10 samples/week. The number of technicians
involved varied between 1 and 10.
One hundred and twenty-one answers were received on 143
(84.6%) for the second questionnaire sent in 2006. Information
regarding the type of laboratory, the number of samples analyzed, the number of technicians involved, and the training
undertaken are shown in Table 1.
Up to 2008, there has been a steady increase in the number of
participating laboratories for all three semen parameters concentration, motility, and morphology (Fig. 1).

RESULTS
Participating laboratories
Out of the 200 replies (50%) to the initial 1997 questionnaire,
181 laboratories (90.5%) were performing diagnostic semen
analysis and were in favor of participating in an EQC scheme

Scheme organization and distribution


Between 1998 and 2012, forty-five distributions were organized by the IPH. One complete distribution (including two samples for sperm count and two smears for sperm morphology)
was lost by the national postal department. Of the 44 successful
distributions, one sample for sperm count because of extremely
low numbers, one non-homogenous air-dried smear, and one
motility sample because of poor quality of the recorded images
were rejected.
In total, 87 suspensions for sperm count, 87 smears for sperm
morphology, and 59 recordings for sperm motility were included
in this report.

Table I Answers to the questionnaire in 2006 regarding the type of laboratories, number of samples analyzed, number of technicians involved and
training undertaken by the laboratory staff
Type of laboratories involved
General Clinical laboratories in a hospital set-up
Laboratories linked to an IVF Centre in a hospital set-up
General Clinical laboratory in a private set-up
Specialized andrology laboratories
Mean number of samples analyzed/month (range)
Sperm concentration
Sperm motility
Sperm morphology
Number of technicians involved in semen analysis
Laboratories were analysis was performed by dedicated
technicians
Laboratories were analysis was performed by technicians
involved in other clinical tests
Information unavailable
Training undertaken by the laboratory staff
Laboratory technicians only performing semen analyses after
training
Laboratory technicians combining semen analysis and other
clinical tests after training
Head of the laboratories (Clinicians/Clinical biologists/
embryologists) with training

72/121 (59.5%)
14/121 (11.6%)
29/121 (24.0%)
6/121 (5.0%)
15 (1250)
10 (1250)
10 (1250)
20/121 (16.5%)

Sperm count
Regarding the 87 samples for sperm count, the median CV
over the whole period (19982012) was 19.2% (Q1Q3: 16.2
22.6), irrespective of the method used (Fig. 2). The use of the
improved Neubauer chamber resulted in a significantly
(p < 0.001) lower median CV of 14.4% (Q1Q3: 12.218.3; Fig. 3)
compared with the global results (Fig. 4).
There was a clear evolution in the use of the different counting
chambers (Fig. 5). The use of the improved Neubauer increased

83/121 (68.6%)
18/121 (14.9%)
26/65 (40.0%)
83/401 (20.7%)
60/88 (68.2%)

Figure 1 Evolution in the number of participants for sperm concentration, motility, and morphology over the whole period (19982012).
160

Number of participants

140

120

100

80

60

12
20

11
20

10
20

09
20

07

08
20

06

20

05

20

03

02

01

00

99

04

20

20

20

20

20

20

98

19

19

19

97

40

Year
Concentration

2016 American Society of Andrology and European Academy of Andrology

Morphology

Motility

Andrology, 110

ANDROLOGY

U. Punjabi et al.

Figure 2 Evolution in the mean coefficient of variation (CV) of sperm concentration over the whole period (19982012) using all types of counting
chambers.
90
80
70

CV (%)

60
50
40
30
20
10

20
13

20
12

20
11

20
10

20
09

20
08

20
07

20
06

20
05

20
04

20
03

20
02

20
01

20
00

19
99

19
98

19
97

Year

Figure 3 Evolution in the mean coefficient of variation (CV) of sperm concentration over the whole period (19982012) using the improved Neubauer
counting chamber.
90
80
70

CV (%)

60
50
40
30
20
10

13
20

12
20

11
20

10
20

09
20

08
20

07
20

06
20

05
20

04
20

03
20

02
20

01
20

00
20

99
19

98
19

19

97

Year

from 32 to 68%, whereas the Burker chamber and Makler chamber decreased from 29 to 10% and 12 to 5%, respectively. The
number of participants using the Thoma chamber (8%) and the
Fuchs-Rosenthal chamber (6%) remained stable. The combined
use of the improved Neubauer chamber with a positive displacement pipette (recorded since 2007) is still increasing.
Sperm motility
For sperm motility analysis of the 59 recordings, the overall
median CV was 37.1% for rapid progressive motility grade a (Q1
Q3: 28.259.8), 15.1% for progressive motility grade a + b (Q1Q3:
12.120.4), 13.8% for total motility, grade a + b + c (Q1Q3:
4

Andrology, 110

10.816.9), and 23.3% for immotility grade d (Q1Q3: 19.128.1).


Although CVs for rapid progressive motility have been highly variable, the CVs for progressive and total motility were much lower
and have remained relatively constant over the years (Fig. 6).
Sperm morphology
When fixing and staining the slides and returning the results
within 2 weeks of reception, we observed that this time interval
did not affect the reported results. There was no influence of the
time of analysis on the target value. When the medians of
encoded values were plotted in function of the day of analysis
after sending, the slope of the curve was not significantly
2016 American Society of Andrology and European Academy of Andrology

ANDROLOGY

BELGIAN EQA OF SEMEN ANALYSIS


Figure 4 Box plots showing differences in the coefficient of variation (CV)
of sperm count using all types of counting chambers and improved Neubauer counting chamber.

Figure 6 Box plots showing coefficient of variation (CV) (%) variability in


rapid progressive (a), progressive (ab), total motility (abc), and immotility
(d) during the whole period (19982012).
n = 87

n = 87
p < 0.001

different of 0, indicating that there was no significant effect of


the time of the analysis when performed within the requested
2 weeks after reception. For sperm morphology, the median CV
over the whole period (Fig. 7) was 79.4% (Q1Q3: 53.994.4),
irrespective of the staining methods or evaluation criteria used.
While the CV was very high in the early years, there is a clear
improvement over the years. The use of the WHO recommended
(modified) Papanicolaou staining method has increased over
time from 44 to 65%, while the use of the recommended Shorr

staining has remained constant (around 5%). On the other hand,


use of rapid staining methods, namely Diff quick, has increased
from 6 to 13% and Spermac increased from 9 to 18%. Conversely,
there was a decrease in the use of Giemsa from 30 to 2% (Fig. 8).
At the end of 2012, 72% of the laboratories were using the recommended Papanicolaou, Shorr, or Diff-Quick staining, compared
with 50% in 1998.
Over the years, most laboratories adapted their criteria for sperm
morphology according to the WHO recommended approach

Figure 5 Evolution in the use of different counting chambers for sperm concentration during the whole period (19982012).

80

70

Number of participants

60

50

40

30

20

10

0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

2009 2010 2011 2012

Year
Improved Neubauer + positive displacement pipette
Burker
Makler
2016 American Society of Andrology and European Academy of Andrology

Improved Neubauer
Thoma
Fuchs Rosenthal

Andrology, 110

ANDROLOGY

U. Punjabi et al.
Figure 7 Evolution in the coefficient of variation (CV) of sperm morphology during the whole period (19982012).
250

CV (%)

200

150

100

50

19
97
19
98
19
99
20
00
20
01
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
20
11
20
12
20
13

Year

Figure 8 Evolution in the use of different staining procedures for sperm morphology during the whole period (19982012).
70

Number of participants

60

50

40

30

20

10

0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Year
Papanicolaou

GIEMSA

(Fig. 9). In 2012, 89% of the laboratories had adopted the WHO
2010 or the equivalent Strict criteria (Menkveld et al., 1990).
Irrespective of the staining procedures or the criteria used,
there was consensus between the participants in morphology
scoring as normal/abnormal for 69/87 smears (79.3%). In 14/87
(16.1%) smears, there was no agreement and in 4/87 (4.6%)
smears, the borderline score was adopted as consensus.

DISCUSSION
In spite of the clear laboratory guidelines addressing the techniques and quality control procedures for semen analysis, as
published by the WHO and NAFA-ESHRE, the results of the Belgian EQC program reveal a large variability, as also observed by
the other groups (Neuwinger et al., 1990; Matson, 1995; Cooper
6

Andrology, 110

SHORR

Spermac

Diff quick

et al., 1999; Auger et al., 2000; Gandini et al., 2000; Keel, 2004;
Alvarez et al., 2005; Mallidis et al., 2012; Filimberti et al., 2012).
However, the variability observed among the Belgian participants was much more acceptable than what has reported in literature as far as concentration and motility are concerned.
For the first time, we present here the results of an EQC program over an extended period (15 years), including a large number of participating laboratories, and involving a substantial
number of distributions. Over this period, participants were continuously advised by the CC to adopt the latest WHO recommended procedures. Moreover, an additional step toward
standardization was teaching the right procedures during basic
semen analysis courses organized by members of the CC (Pun rndahl et al.,
jabi & Spiessens, 1998; Vreeburg & Weber, 1998; Bjo
2016 American Society of Andrology and European Academy of Andrology

ANDROLOGY

BELGIAN EQA OF SEMEN ANALYSIS


Figure 9 Evolution in the criteria used to evaluate sperm morphology during the whole period (19982012).
100
90
80

CV (%)

70
60
50
40
30
20
10
0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Year
WHO 1987

WHO 1992

2002) and, upon request, onsite training in laboratories under


the supervision of CC members. These approaches were effective in emphasizing the many details that contribute to reliable
semen analysis and at the same time increasing the skills and
experience of the participants.
Although participation in the Belgian EQC scheme was always
on voluntary basis, the awareness on the part of the laboratories
for the need to improve standardization was demonstrated by
the number of participants enrolled in the EQC program. Of the
181 laboratories which initially performed semen analysis, only
some of them showed interest in the Belgian EQC scheme and
actually registered in the program in 1997. Indeed, on an average, 69% participated for concentration, 62% for morphology,
and 57% for motility. This could be related to the general
decrease in the total number of medical laboratories in Belgium
(from 400 in 1997 to 235 in 2012, as registered by the IPH) mainly
because of the fusion of small laboratories into bigger entities or
because of activity cancelation. Moreover, not all the three basic
parameters were carried out in the same laboratory with some
only performing sperm counts after vasectomy. The frequency
of performing semen analysis per month was also highly variable
as in most general clinical laboratories, semen analyses represents only a small proportion of their activities, and little or no
resources were spent for training. In general, most laboratories
had very low numbers of semen analyses with several technicians involved. Only in a small number of laboratories (16.5%)
were the technicians privileged to do exclusively semen analyses,
which increases precision. However, only 40.0% of their staff had
followed theoretical and practical training via organized semen
courses.
For sperm count, a low mean CV value (14.4%) was obtained
with the improved Neubauer hemocytometer (the recommended counting chamber in all five editions of the WHO manual), which was significantly lower than the mean CV using all
counting methods (20.9%). When compared with EQC schemes
in other European countries, the mean CV observed in our study
group was much lower. Neuwinger et al. (1990) evaluated eight
samples between 10 German laboratories and obtained a mean
CV of 37.5%. Data of the EQC under the auspices of the British
2016 American Society of Andrology and European Academy of Andrology

WHO 1999

TYGERBERG + WHO 2010

Andrology Society reported a CV for sperm concentration to be


64.7% for 20 participating laboratories (Matson, 1995). Alvarez
et al. (2005) reported coefficients of variation for concentration
ranging from 20.8 to 33.8% (including nine surveys in 1840 laboratories between 1998 and 2002). Gandini et al. (2000) noted
CVs between 29.67 and 52.08% in two trials of seven samples.
Later, another Italian group (Filimberti et al., 2012) reported CVs
of 46.65, 59.03% up to 75.14% in a survey including three trials.
The high CVs observed could be because of the number of laboratories involved, the number of samples included in the surveys, or the length of the survey period studied.
To corroborate our observations, Mallidis et al. (2012) using
the Youden plot rankings in their survey of 19 runs found that
those participants who used the WHO recommended procedure
for sperm counting (i.e. Neubauer chamber and recommended
diluents) recorded more consistent results within the target
range than those who did not follow the WHO recommendations
(Neubauer chamber with alternative diluents, Makler chamber
or Thoma, or no use of diluents).
The earlier editions of the WHO manual (1980, 1987, 1992,
1999) recommended the use of Neubauer hemocytometers,
other cytometers, and the Makler chamber. At the start of the
EQC program in 1997, more than 80% of our participants used
hemocytometers that were applied for other clinical tests in
the general clinical biology laboratories. In 1997, 32% used the
Neubauer chamber ,while only a small percentage (12%)
applied the Makler chamber. This is in contrast to the observation by Alvarez et al. (2005) where 100% of their participants
used the Makler in the initial survey in 1998. But, by the end
of their survey period in 2002, only 32.5% used the Neubauer
hemocytometer but 67.5% still used the Makler. So did the
26% of the participants to the American Association of Bioanalysts National Proficiency Program (Keel et al., 2000). Use of
the Makler chamber was in compliance with the techniques
recommended in the WHO 1999 manual. The fifth edition of
the WHO manual (2010), however, discouraged the use of the
Makler chamber because of its poor precision and underestimation (Bailey et al., 2007). Nevertheless, 50% of the contributes to the Italian program (Filimberti et al., 2012) and 35%
Andrology, 110

U. Punjabi et al.

of the German Program (Mallidis et al., 2012) persisted in its


use.
As many additional sources of error beyond the counting
chamber may result in unreliable sperm count data, participants
were asked to specify not only the type of counting chamber but
also the dilution factor and since 2007 the use of a positive displacement pipette. Correct maintenance and quality control of
the counting chamber, correct use of diluents, correct mixing of
the sample, correct sampling, use of replicate counts, correct
statistical procedures, and internal quality control all contribute
to accurate assessment of sperm numbers. EQC allows the laboratory to have an idea of the type of errors present (systematic or
random) as well as the level of certainty of their result (Pacey,
2006).
It is generally accepted that the distinction between rapid and
slow progressive motility is subjective as visual assessment of
the velocity (a > 25 lm/sec or b < 25 lm/sec) is difficult. A large
variability was indeed found in the classification of rapid progressive motility in our EQC program. Moreover, velocity is temperature dependent, and not all participants did the assessments
at 37 C. This problem was largely solved when combining
sperm grade a and b (progressive motility), which resulted in
much lower CV values. The total progressive and total motility
have remained relatively constant in our program over the years.
The fifth edition of the WHO manual has positively contributed
to solve this problem by signifying the importance of progressive
motility over the subjective assessment of rapid progressive
motility. In addition, it was no longer advised to perform the
assessment at 37 C.
Filimberti et al. (2012) observed during the three trials organized, no differences between mean CV values of progressive,
non-progressive, and immotile spermatozoa. However, the highest CV values (>60%) were observed for the non-progressive
motility, while those for progressive motility and immotile spermatozoa were the lowest (<30%). The authors concluded that
the results may be influenced by the fact that the samples (sent
as a DVD) did not need any procedural step, thereby limiting
errors caused by sample handling.
Auger et al. (2000) assessed % motile spermatozoa and
observed an interindividual CV of 21.8%, similar to that found in
an earlier study by Neuwinger et al. (1990). Our results of the
total motile spermatozoa were better than the two former groups
(Q1Q3, 10.816.9%). Gandini et al. (2000) obtained a range of
CVs for forward motility between 39 and 71% in two trials of four
samples each, while the Belgian participants revealed a better
Q1Q3 range of 12.120.4%.
Sperm motility CV reported by Neuwinger et al. (1990) and
Alvarez et al. (2005) showed a high between laboratories variability when cryopreserved semen was used. The authors report
that experience of motility assessment on a videotape is obviously different from microscopic examination, and that the
rndahl (2002)
obtained results are very imprecise. Kvist & Bjo
suggested using monitors for routine sperm motility determination, to avoid the bias involved in the use of monitors in external
quality control and microscopy in the daily routine. Few laboratories seem to have adopted this approach.
For sperm motility, no specifications about the routine practice have been asked to the participants in our program.
Sperm morphology is considered to be the most subjective
parameter in sperm analysis, resulting in large discrepancies.
8

Andrology, 110

ANDROLOGY
The changes in assessment criteria as well as changes in cut-off
normal values in the consecutive editions of the WHO manual,
have undoubtedly contributed to this problem. In the initial
years of our EQC program, large CVs were observed. In the later
years, there was a tendency toward improvement, although the
CV was still unacceptably high. Different authors ascribed these
variations to a lack of standardization (Chong et al., 1983; Dunphy et al., 1989), to a difference in techniques of smear preparation and staining procedures (Davis & Gravance, 1993) and to
the level of technical expertize (Dunphy et al., 1989; Neuwinger
et al., 1990).
Keel et al. (2000) reported a wide variation in the CV of percent
normal morphology ranging from 15 to 93%. These authors
claimed that premade semen smears eliminate the potential variation associated with individual slide preparation, thereby excluding its potential effect on individual laboratory performance.
Results of Filimberti et al. (2012) suggest that the variability
concerning morphology is independent of the staining procedure as mean CV values for the various methods employed by
their participating laboratories were not different. However, the
mean CV noted in three trials was very high and ranged between
88.6 and 105.6%. Moreover, the participating laboratories
adopted staining procedures not recommended by the WHO
(Haematoxilin-Eosin, May-Grunwald and use of Test Simplets).
According to Filimberti et al. (2012), sperm morphology teaching courses may offer the solution to decrease the variability in
rndahl et al.
the results, confirming the observations of Bjo
(2002). The same authors, however, finally observed that teaching was insufficient to limit variability in the morphology results
at the end of their study period.
In our scheme, we observed a trend toward a reduced variability over the period of 15 years, which might mainly be explained
by the adoption of the WHO 2010 or Strict criteria by most of the
laboratories.
Keel et al. (2000) pointed out that one must be cautious when
computing and comparing CVs among morphology criteria with
different cutoffs for normality. During one of our surveys in
2003, participating laboratories were asked to provide the cut-off
values used to define normality for the specific morphology criteria adopted by them. The laboratories using WHO 1992 criteria
handled a cutoff for normality ranging between 25 and 60%.
Those using WHO-1999 criteria had a cutoff between 10 and
70%, and for the strict criteria cutoffs ranged between 5 and
25%. The change in the normal morphology cutoff of 50% in the
WHO 1987 edition to 30% in the WHO 1992 edition, and the
omission of a normal value in the WHO 1999 edition has certainly added to the ambiguity regarding morphology assessment
and to the increased confusion regarding its clinical relevance
(Riddell et al., 2005). These authors also note that only 5% of the
laboratories who participated in their survey were compliant
with all current WHO guidelines regarding sperm morphology.
Alvarez et al. (2005) also believe that differences in the application of morphologic criteria are the main cause of variations
between laboratories (CV 33.575.0%). The authors, however,
believe that the use of micrometers, as recommended in the
1999 WHO manual, could help to reduce this difference.
In our program, participants were asked to specify the staining
procedure and assessment criteria for sperm morphology at
each survey. However, other sources of error were beyond our
control.
2016 American Society of Andrology and European Academy of Andrology

BELGIAN EQA OF SEMEN ANALYSIS

It is critical to keep in mind that semen analysis is a diagnostic tool, and as such, the results are used to guide
patients treatment. Inappropriate material and procedures
may undoubtedly lead to the wrong diagnosis and inappropriate therapy. In addition, in Belgium, semen analysis costs are
reimbursed by the Health Care System, possible errors in the
procedures arising from incorrect semen analysis may result
in a great waste of public money. Quality survey should meet
the needs of all disciplines that are involved in semen analysis. Participation in an EQC survey should be made mandatory in order to fulfill the requirements for accreditation and
for the benefit of patient treatment. This Belgian EQC survey
over a 15-year period has attempted to reduce interlaboratory
variability. At the same time, participating laboratories have
been encouraged to implement correct techniques as advised
by the WHO. While an overall trend to improve standardization over the years was observed, continuous efforts and new
initiatives should aim to further harmonize the procedures
among the laboratories. This unique long-term EQA program
emphasizes that quality assessment must be a continuous
process paving the way to semen analysis standardization.
The next step toward standardization is to harmonize the Belgian EQC program with an existing EQC scheme of other
European countries or societies.

ACKNOWLEDGEMENTS
The authors acknowledge Jean Claude Libeer (past member of
the IPH), Frank Comhaire and Bernard Van Abelle (past members of the CC).

DECLARATION OF INTEREST
The authors declare no conflict of interest.

FUNDING
IPH departmental funds were used to support the authors
throughout the study period in sample preparation and
distribution.

AUTHOR CONTRIBUTIONS
U.P. substantial contribution to conception, acquisition of
data, analysis and interpretation of data, and drafting the article.
G.V., C.W. revising it critically for important intellectual content.
A.M. involved in sample collection and preparation, contributed
toward the critical discussion of the manuscript. K.V., B.C.
involved in coordinating the scheme distributions, making global reports, critical discussion of the manuscript. All authors
approved the final version of the manuscript.

REFERENCES
Alvarez C, Castilla JA, Ramirez JP, Vergara F, Yoldi A, Fernandez A &
Gaforio JJ. (2005) External quality control program for semen analysis:
Spanish experience. J Assist Reprod Genet 22, 379387.
Auger J, Eustace F, Ducot B, Blandin T, Daudin M & Diaz I. (2000) Intra
and inter individual variability in human sperm concentration,
motility and vitality assessment during a workshop involving ten
laboratories. Hum Reprod 15, 23602368.
Ayodeji O & Baker HW. (1986) Is there a specific abnormality of sperm
morphology in men with varicoceles? Fertil Steril 45, 839842.
Bailey E, Fenning N, Chamberlain S, Devlin L, Hopkisson J & Tomlinson
M. (2007) Validation of sperm counting methods using limits of
agreement. J Androl 28, 364373.
2016 American Society of Andrology and European Academy of Andrology

ANDROLOGY
rndahl L, Barratt CL, Fraser LR, Kvist U & Mortimer D. (2002)
Bjo
ESHRE basic semen analysis courses 19951999: immediate
beneficial effects of standardized training. Hum Reprod 17, 1299
1305.
Brazil C, Swan SH, Tollner CR, Treece R, Drobnis EZ, Wang C, Redmon JB
& Overstreet JW, the study for future families research group. (2004)
Quality control of laboratory methods for semen evaluation in a
multicenter research group. J Androl 25, 645656.
Chong AP, Walters CA & Weinrieh SA. (1983) The neglected laboratory
test. The semen analysis. J Androl 4, 280282.
Cooper TG. (1996) News from the European Academy of Andrology
(EAA). Implementation of quality control in the andrology laboratory.
Int J Androl 19, 6768.
Cooper TG, Atkinson AD & Nieschlag E. (1999) Experience with external
quality control in spermatology. Hum Reprod 14, 765769.
Davis RO & Gravance CG. (1993) Standardization of specimen
preparation, staining, and sampling methods improves automated
spermhead morphometry analysis. Fertil Steril 59, 412417.
Demotte R. (1999) Minist
eres des affaires sociales et de la sant
e publique.
^t 
cembre 1999 relatif a
 lagre
ment des laboratoires
Arre
e royal du 3 de
de biologie clinique par le Ministre qui a la sant
e publique dans ses
attributions. Moniteur Belge 5021750231.
Dunphy BC, Kay R, Barratt CLR & Cooke ID. (1989) Quality control
during the conventional analysis of semen, an essential exercise.
J Androl 10, 378385.
Filimberti E, DeglInnocenti S, Borsotti M, Quercioli M, Piomboni P,
Natali I, Fino MG, Caglieresi C, Criscuoli L, Gandin L, Biggeri A,
Maggi M & Baldi E. (2012) High variability in results of semen
analysis in andrology laboratories in Tuscany (Italy): the
experience of an external quality control (EQC) programme.
Andrology 1, 401407.
Gandini L, Menditto A, Chiodo F & Lenzi A. (2000) From the European
Academy of Andrology. Italian pilot study for an external quality
control scheme in semen analysis and antisperm antibiotics detection.
Int J Androl 23, 13.
Jedrzejczak P, Talarczyk J, Taszarek-Hauke G, Berger A, Hauke J &
Pawelczyk L. (2012) External quality assessment of semen analysis in
Poland. Ginkol Pol 83, 835840.
Jequier AM & Ukombe EB. (1983) Errors inherent in the performance of a
routine semen analysis. Br J Urol 55, 434436.
Jrgensen N, Auger J, Giwercman A, Irvine DS, Jensen TK, Jouannet P,
Keiding N, Le Bon C, MacDonald E, Pekuri AM, Scheike T, Simonsen
M, Suominen J & Skakkebaek NE. (1997) Semen analysis performed by
different laboratory teams: an intervariation study. Int J Androl 20,
201208.
Keel BA. (2004) How reliable are results from the semen analysis. Fertil
Steril 82, 4144.
Keel BA, Quinn P, Schmidt CF, Jr, Serafy NT, Jr, Serafy NT & Schalue
TK. (2000) Results of the American Association of Bioanalysts
national proficiency testing programme in andrology. Hum Reprod
15, 680686.
rndahl L. (2002) ESHRE Monographs: Manual on Basis
Kvist U & Bjo
Semen Analysis. Oxford University Press, Oxford, UK.
Mallidis C, Cooper TG, Hellenkemper B, Lablans M, Uckert F & Nieschlag
E. (2012) Ten years experience with an external quality control
program for semen analysis. Fertil Steril 98, 611616.
Matson PL. (1995) External quality assessment for semen analysis and
sperm antibody detection: results of a pilot scheme. Hum Reprod 10,
620625.
Menkveld R, Stander FS, Kotze TJ, Kruger TF & Zyl JA. (1990) The
evaluation of morphological characteristics of human spermatozoa
according to stricter criteria. Hum Reprod 5, 586592.
Mortimer D, Shu MA & Tan R. (1986) Standardization and quality control
of sperm concentration and sperm motility counts in semen analysis.
Hum Reprod 1, 299303.

Andrology, 110

U. Punjabi et al.
Neuwinger J, Behre HM & Nieschlag E. (1990) External quality control in
the andrology laboratory: an experimental multicenter trial. Fertil
Steril 54, 308314.
tssicherung im
Ochsendorf FR & Beschmann HA. (1998) Qualita
andrologischen Labor. Reproduktionsmedizin 14, 97105.
Ombelet W, Pollet H, Bosmans E & Vereecken A. (1997) Results of a
questionnaire on sperm morphology assessment. Hum Reprod 12,
10151020.
Pacey AA. (2006) Is quality assurance in semen analysis still really
necessary? A view from the andrology laboratory Hum Reprod 21,
11051109.
Plaut DA & Westgard JOW. (2002) QC external quality assessment. In:
Basic QC Practices: Training in Statistical Quality Control for
Healthcare Laboratories (ed. JO Westgard), pp. 125163. QC
Publishing, Madison, WI.
Punjabi U & Spiessens C. (1998) Basic semen analysis courses:
experience in Belgium. In: Modern Art in the 2000s (eds W
Ombelet, E Bosmans, H Vandeput, A Vereecken, M Renier & E
Hoomans), pp. 107113. Parthenon Publishing Group, New York/
London.
Riddell D, Pacey AA & Whittington K. (2005) Lack of compliance by UK
andrology laboratories with World Health organization
recommendations for sperm morphology assessment. Hum Reprod 20,
34413445.

10

Andrology, 110

ANDROLOGY
Vreeburg JTM & Weber RFA. (1998) Basic semen analysis courses:
experience in Belgium. In: Modern Art in the 2000s (eds W Ombelet, E
Bosmans, H Vandeput, A Vereecken, M Renier & E Hoomans), pp.
103106. Parthenon Publishing Group, New York/London.
Walker RH. (1992) Pilot surveys for proficiency testing of semen analysis.
Arch Pathol Lab Med 116, 422424.
World Health Organization. (1980) Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Mucus Interaction,
1st edn. Cambridge University Press, Cambridge, UK.
World Health Organization. (1987) Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Mucus Interaction,
2nd edn. Cambridge University Press, Cambridge, UK.
World Health Organization. (1992) Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Mucus Interaction,
3rd edn. Cambridge University Press, Cambridge, UK.
World Health Organization. (1999) Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Mucus Interaction,
4th edn. Cambridge University Press, Cambridge, UK.
World Health Organization. (2010) Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Mucus Interaction,
5th edn. Cambridge University Press, Cambridge, UK.
Zuvela E, Junk S, Moska N & Matson Ph. (2011) The use of latex beads in
external quality assurance and internal quality control for routine
semen analysis. Reprod Biol 11, 264275.

2016 American Society of Andrology and European Academy of Andrology