Comparison of three staining methods for the

morphological evaluation of human spermatozoa

Ralf Henkel, Ph.D.,a Gerhard Schreiber, M.D.,b Anne Sturmhoefel, M.D.,c Uta-Christina Hipler,
Ph.D.,b Dirk Henrik Zermann, M.D.,c and Roelof Menkveld, Ph.D.d
a
Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; b Department of Dermatology, and
c
Department of Urology, Friedrich Schiller University, Jena, Germany; and d Department of Obstetrics and Gynaecology,
Tygerberg Academic Hospital and University of Stellenbosch, Tygerberg, South Africa

Objective: To compare different staining methods to evaluate human sperm morphology.

Design: Prospective study.
Setting: Patients at the Departments of Dermatology and Urology, University of Jena, Germany.
Patient(s): A total of 94 randomly collected patients attending the andrological outpatient clinics of the Depart-
ments of Dermatology and Urology, University of Jena, Germany.
Intervention(s): None.
Main Outcome Measure(s): Statistical comparison of resultant standard morphological parameters (mean percent-
ages) after staining according to Papanicolaou and Shorr methods and with Testsimplets prestained slides.
Result(s): All morphological parameters investigated (percent normal morphology, percent head, midpiece, and
flagellar abnormalities) correlated statistically significantly positively, however with markedly lower correlation
coefficients for the Testsimpletsresults. As compared with the mean Papanicolaou (4.78%  2.54%) and Shorr
staining (4.75%  2.64%) results, a statistically significantly lower percentage of morphologically normal sperma-
tozoa was determined after using the Testsimplets slides (3.89%  2.53%). In general, the mean values of all pa-
rameters differed for all comparisons with the Testsimplets slides and especially for the percentage of flagellar
defects but not between the Papanicolaou and the Shorr staining results.
Conclusion(s): The results show an extensive agreement between the Papanicolaou- and Shorr-stained smears,
whereas Testsimplets staining exhibited statistically significant deviations. Because the correct evaluation of
sperm morphology is of essence within the scope of assisted reproduction and in andrological diagnostics, the
use of rapid staining methods cannot be recommended. (Fertil Steril 2008;89:449–55. 2008 by American So-
ciety for Reproductive Medicine.)
Key Words: Human sperm, morphology, Papanicolaou, Shorr, rapid staining methods

Despite a few negative reports (1–3), repeated and indepen-
dent studies in assisted reproduction programs showed that
sperm morphology is significantly correlated with fertiliza-
tion and pregnancy after different techniques of assisted re-
production (4–9). Thus, in general there is only little doubt
that normal sperm morphology, as evaluated by strict crite-
ria, is a good predictor for assisted reproduction outcome
(10). As a result, sperm morphology represents a corner-
stone in modern andrological diagnostics on which the
therapeutic concept for the treatment of such patients is de-
veloped.
For the evaluation of human sperm morphology, different
staining methods have been described of which the technique
according to Papanicolaou (11) with subsequent evaluation
according to strict criteria (12) has been accepted internation-
ally as standard procedure (13). Apart from the Papanicolaou
stain, the World Health Organization (WHO) also mentions/
Received January 19, 2007; revised March 2, 2007; accepted March 5,
2007.
Reprint requests: Ralf Henkel, Ph.D., Department of Medical Biosciences,
University of the Western Cape, Bellville, South Africa (FAX:
27-21-9593125; E-mail: rhenkel@uwc.ac.za).
allows the staining procedure according to Shorr (14) and
a rapid staining technique such as Diff-Quik stain (15).
The Diff-Quik procedure was introduced by Kruger et al.
in 1987 (15), and it was found to be comparable with the
results of the Papanicolaou staining method. On the basis
of these results, the 1992 WHO manual (16) introduced the
Diff-Quik method together with a number of other staining
techniques as an alternative to the Papanicolaou technique.
Since then the Diff-Quik staining technique has been com-
pared repeatedly with other staining techniques as reference
for the purpose of a more rapid staining procedure or for ob-
taining optimal contrast for the computer-assisted sperm
morphology evaluation in both human and animal semen
samples (17–22). Another rapid sperm staining method, in-
troduced by Schirren et al. (23), is the stain-coated Testsimp-
lets slides, which frequently are used mainly in smaller
centers and practices for assisted reproduction because of
time constraints. In the survey conducted by Ombelet et al.
(24), from 170 respondents worldwide, 33.5% used the Papa-
nicolaou method, 22.9% Diff-Quik, 6.5% Testsimplets,
and 4.7% Shorr stain as their routine staining procedure. Ad-
vantages of the Testsimplets slides are that no fixation step is
necessary and preparation can be done outside a fume cabinet.

0015-0282/08/$34.00 Fertility and Sterility Vol. 89, No. 2, February 2008 449
doi:10.1016/j.fertnstert.2007.03.027 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
 The lack of information in the recent literature about
Testsimplets made necessary a thorough comparison with
standard methods including a comprehensive statistical analy-
sis. Thus, paying tribute to the importance of an accurate
evaluation of sperm morphology in a clinical setup, this study
aimed at comparing the internationally accepted standard
method of strict criteria after staining according to Papanico-
laou with the Düsseldorf criteria (25) after Shorr staining and
the morphology evaluation after the staining procedure with
Testsimplets.
MATERIALS AND METHODS
To compare three commonly used staining techniques for the
evaluation of human sperm morphology, we used freshly
ejaculated semen samples from a total of 94 patients attend-
ing the andrological outpatient clinics at the Departments of
Dermatology and Urology, University of Jena, Germany, ac-
cording to standard ethical guidelines. After performing
standard semen analysis according to WHO guidelines (13)
three smears of each ejaculate were made to be stained ac-
cording to Papanicolaou (12, 13) and Shorr (13, 14) and
with a rapid staining technique, Testsimplets (Waldeck,
Münster, Germany). In brief, droplets of 20 mL ejaculate
were smeared on precleaned glass slides air dried for 24
hours after which Papanicolaou and Shorr staining was per-
formed. In contrast, for the rapid staining technique using the
Testsimplets, the 20 mL droplet of ejaculate was spread over
the Testsimplets slide by putting on a coverslip, which is in-
cluded in the kit, and left standing for 30 to 60 minutes at
room temperature.
The evaluations were performed according to strict criteria
(12) for Papanicolaou- and Testsimplets-stained smears
and according to Düsseldorf criteria (25) for Shorr-stained
smears. Slides were evaluated for the percentages of morpho-
logical normal spermatozoa, head defects, midpiece defects,
and flagellum defects with a bright-field microscope (Zeiss,
Jena, Germany) under 1,000 magnification and included
evaluation of 200 spermatozoa in the four corners and center
of each slide.
Statistical analysis was performed with use of MedCalc
software (version 9.2.0.1; MedCalc, Mariakerke, Belgium).
After testing for normal distribution of the results by means
of the c2 test, the statistical comparison of the three methods
was performed by means of the Wilcoxon test, as well as the
methods according to Bland and Altman (26) and Passing and
Bablok (27) who described a linear regression method with
no special assumptions regarding the distribution of the sam-
ples and the measurement errors. In addition, mountain plots,
which are created by computing a percentile for each ranked
difference between a method and a reference method, were
performed (28).
The three latter statistical methods are methods that com-
pare two or more measurement techniques. The Bland and
Altman plot is a simple graphic method by which the differ-
ences between two techniques are plotted against the average
450 Henkel et al. Comparison of staining methods for sperm morphology
of the two techniques. This procedure identifies methods that
are interchangeable if the differences within mean  1.96
SD are not clinically important. The plot is useful in reveal-
ing relationships between the differences and the averages,
looking for systematic biases and identifying possible out-
liers. In comparison with the Bland and Altman plot, the
mountain plot has the advantages of comparing distributions
more easily and of finding the central 95% of the data, even
in case the data are not normally distributed. On the other
hand, the Passing and Bablok regression does not need these
special assumptions regarding distribution and measurement
errors.
RESULTS
Figure 1 shows the typical staining patterns for the three dif-
ferent staining techniques used. The increased background
staining on the Testsimplets slides is also clearly visible
(Fig. 1C). The mean sperm concentration, total motility
(WHO AþBþC), and progressive motility (WHO AþB)
were 79.1  125.5  106/mL, 43.1%  20.7%, and 33.2%
 19.0%, respectively. A summary of mean, SD, median,
and the coefficient of variation (CV) of the parameters for
the staining methods investigated is depicted in Table 1. Gen-
erally, although the results of the Papanicolaou and Shorr
stain were closer to each other, the Testsimplets results de-
viated. Except for the head defects, the CVs for the Testsimp-
lets stain were markedly higher.
All morphological parameters (percent normal morphol-
ogy, percent head, midpiece, and flagellar abnormalities)
correlated statistically significantly positively among the
different techniques (P<.004) (r ¼ 0.318 to r ¼ 0.797), yet
with lower correlation coefficients for correlations with the
Testsimplets (r ¼ 0.318 to r ¼ 0.704). However, comparing
the mean for percentage normal sperm morphology deter-
mined by means of the Papanicolaou (4.78%  2.54%) and
the Shorr stain (4.75%  2.64%), the Testsimplets proce-
dure resulted in a significantly lower result (3.89% 
2.53%) (P¼.0044 and P¼.0055, respectively). Except for
the comparison of the midpiece defects between Shorr and
Testsimplets methods (P¼.1565), all other comparisons
showed statistically significant (P<.01) deviations between
the Testsimplets method results and the results of the Papa-
nicolaou and Shorr methods. On the other hand, Papanico-
laou- and Shorr-staining results did not differ in any respect
for any of the analyzed parameters (Table 1).
The deviations from the standard techniques by using the
Testsimplets method were even more evident after perform-
ing Bland-Altman plots (Fig. 2A–2C ¼ normal sperm
morphology; Fig. 2D–2F ¼ flagellar defects). For the
comparison of the Papanicolaou with the Shorr stain for nor-
mal sperm morphology (Fig. 2A) the distribution of the data
points is more or less evenly spread along the abscissa. How-
ever, a much more triangular shape of the distribution pattern
of the data is obvious for the comparisons with the Testsimp-
lets (Fig. 2B, 2C). This triangular shape of the distribution
pattern is indicative that the variation depends on the
Vol. 89, No. 2, February 2008
Fertility and Sterility

simplets methods with the Papanicolaou stain as reference

(Fig. 2B, 2C, 2E, 2F).

simplets method showed distinct deviations from the mean
the Papanicolaou with the Shorr stain (Fig. 2A, 2D), the Test-
there is no deviation from the mean for the comparison of
flagellar morphology (Fig. 2E, 2F). In addition, although
magnitude of the measurement. This is even more obvious for

FIGURE 1
Henkel. Comparison of staining methods for sperm morphology. Fertil Steril 2008.

on the Testsimplets slide is clearly visible.

with Testsimplets. Increased background staining
according to (A) Papanicolaou and (B) Shorr and (C)
Human sperm morphology after staining smears
Mountain plots for the comparison of the Shorr and Test-

TABLE 1
Summary statistics of morphology evaluation of 94 samples stained according to Papanicolaou and Shorr and with Testsimplets slides.
Papanicolaou stain Shorr stain Testsimplets slides Differences between
Testsimplets and
Papanicolaou/Shorr
Parameter Mean SD Median CV Mean SD Median CV Mean SD Median CV staining (P values)
Normal morphology 4.78 2.54 4.20 53.3 4.75 2.64 4.45 55.5 3.89 2.53 3.62 65.0 .0044/.0055
Head defects 85.64 10.40 88.00 12.2 86.52 9.00 88.75 10.4 88.75 7.28 90.05 8.2 .0047/.0120
Midpiece defects 38.20 12.38 37.00 32.4 36.50 11.86 35.50 32.5 33.78 13.43 32.00 39.8 .0074/.1565
Flagellum defects 22.67 14.89 19.00 65.7 21.78 13.38 18.60 61.4 10.49 11.80 6.10 112.5 < .0001/< .0001
Note: Although significant differences between the rapid staining technique (Testsimplets) and the standard techniques are found for all parameters, there were no
differences between the Papanicolaou and the Shorr stains.
Henkel. Comparison of staining methods for sperm morphology. Fertil Steril 2008.
451
method revealed good correspondence between Papanico- (P<.01). Thus, only the Papanicolaou and the Shorr staining
laou and Shorr stains (Fig. 3). This is obvious by the narrow procedures are unbiased.
symmetrical distribution of the plot around zero. However,
for the Testsimplets stain a marked deviation from zero, DISCUSSION
particularly for flagellar defects, is obvious (Fig. 3B),
The results of this study show an extensive agreement
indicating that the two methods are not unbiased. Moreover,
between the morphology evaluation on Papanicolaou- and
the long tail to the right-hand side in the Testsimplets
Shorr-stained smears evaluated according to strict criteria
plot is indicative of large differences between the two
and the Düsseldorf classification, respectively, with relatively
methods.
high correlation coefficients. The good correlation obtained
Calculation of a Passing-Bablok regression revealed a between Shorr-stained smears and Papanicolaou-stained
significant deviation from linearity for flagellar defects smears could be expected, even if evaluated according to
when comparing the Papanicolaou stain with Testsimplets the Düsseldorf and strict criteria methods, respectively.

FIGURE 2
Bland-Altman plots for the comparison of the three techniques regarding normal sperm morphology (A–C)
and flagellar defects (D–F). The deviation for the Testsimplets method is obvious, especially for flagellar defects.
In addition, B, C, E, and F for the comparison with the Testsimplets show that the variation strongly depends
on the magnitude of the measurement. N ¼ 94.
8 10

6
A Papanicolaou % - Testsimplets % 8
B
Papanicolaou % - Shorr %

+1.96 SD +1.96 SD
4 6
4.1 5.9
2 4
Mean
0 2 Mean
0.0
-2 0 0.8
-1.96 SD
-4 -2
-4.0
-6 -4 -1.96 SD
-4.2
-8 -6
-10 -8
0 2 4 6 8 10 12 0 2 4 6 8 10 12
AVERAGE of Papanicolaou and Shorr (%) AVERAGE of Papanicolaou and Testsimplets (%)

10 25
C D
8 20
Papanicolaou % - Shorr %
Shorr % - Testsimplets %

+1.96 SD
6 +1.96 15
14.3
5.6 10
4
5
2 Mean Mean
0
0 0.7 -0.1
-5
-2
-10
-1.96 -1.96 SD
-4 -15
-4.1 -14.6
-6 -20
-8 -25
0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70
AVERAGE of Shorr and Testsimplets AVERAGE of Papanicolaou and Shorr (%)

70
Papanicolaou % - Testsimplets %

40
60
E F
+1.96 SD
Shorr % - Testsimplets %

30
50 29.2
20
40
+1.96 SD Mean
30 10 11.2
31.8
20 0
Mean
-1.96 SD
10 12.1 -10 -6.9
0
-1.96 SD
-20
-10 -7.7
-20 -30
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
AVERAGE of Papanicolaou and Testsimplets (%) AVERAGE of Shorr and Testsimplets (%)

Henkel. Comparison of staining methods for sperm morphology. Fertil Steril 2008.

452 Henkel et al. Comparison of staining methods for sperm morphology Vol. 89, No. 2, February 2008

FIGURE 3
Mountain plots for the comparison of the Shorr and
Testsimplets methods with the Papanicolaou stain.
(A) Normal sperm morphology; (B) flagellar defects.
Whereas the Shorr and Papanicolaou methods
correspond quite well with each other, the deviation
from zero for the Testsimplets stain results
compared with the Papanicolaou stain results as
reference shows that the two methods are not
unbiased. Moreover, the long tail to the right-hand
side of the Testsimplets plot in B is indicative of
large differences between the two methods,
underlining the underscoring of flagellar defects with
the Testsimplets method.
50
A postacrosomal forms and spermatozoa with acrosome areas
40
30
Percentile
was too deep to allow sharp definition of the outlines of epithe-
lium cells, especially if the smears were thick (11). This is also
true for the staining of semen smears where a more intense
staining is obtained with the Shorr stain and the outlines of
spermatozoa may appear a little fuzzy if the smears are over-
stained, but otherwise it gives the same differentiations of the
different spermatozoa regions as Papanicolaou staining.
That the Düsseldorf classification was used should also
pose no problem, because for the identification of morpholog-
ically normal spermatozoa this method is basically the same
as that for strict criteria. Hofmann et al. (25) describe the ini-
tial Düsseldorf definition of morphological normal spermato-
zoa as more accurate than the WHO definition and close to the
succeeding strict Tygerberg criteria, but it included sperm
heads with slightly reduced acrosomal areas as still normal.
Clinical observations led to the improved Düsseldorf classifi-
cation where spermatozoa with normal heads but pointed
Shorr <40% are considered as being abnormal, bringing it even
closer to strict criteria. Our results are also in line with those
Testsimplets
found by Meschede et al. (29) who observed that the mean
percentage of morphologically normal spermatozoa was

identical (31.1%  12.9% and 31.1%  12.6%) with the

Papanicolaou and Shorr staining methods, respectively.
20
The Diff-Quik method as a rapid staining technique
repeatedly has been compared with various other methods,
including the Papanicolaou stain, to evaluate sperm morphol-
10
ogy manually and with computerized methods in humans and
animals (19–21, 30). In all these reports, the Diff-Quik stain
0
was found to be a suitable method, especially for the com-
-10 -5 0 5 10 puter-assisted sperm morphology analysis (20), because the
Difference with Papanicolaou (%) computer appears to recognize Diff-Quik–stained sperm
better than using other techniques. However, a direct compar-
50 ison of the Diff-Quik method with the other stains appears to
B result in higher percentages of morphologically normal sper-
matozoa (17, 31). Coetzee et al. (32) found a significant bias
40
Shorr of 1.6% with Diff-Quik versus the Papanicolaou stain in fa-
vor of higher percentages of morphologically normal sperm
Testsimplets
using the Hamilton Thorne morphology analyzer integrated
30
visual optical system (IVOS; Hamilton Thorne, Beverly,
Percentile

MA). Because the two methods produced different normal

morphology profiles, it is essential to establish new normal
20
morphology standard thresholds when using any alternate
method, even computerized analyses. The reason for this
could be that visualization of details using these rapid stain-
10
ing techniques is not perfect and undoubtedly the higher per-
centage of normal forms diagnosed is due to difficulties
studying the fine details (33).
0
-40 -20 0 20 40 60 80
On the other hand, for the results of the Testsimplets rapid
Difference with Papanicolaou (%)
staining technique statistically significant positive correla-
Henkel. Comparison of staining methods for sperm morphology. Fertil Steril 2008. tions with both standard procedures, Papanicolaou and Shorr
staining, were obvious, though with lower correlation coeffi-
cients. These initial results confirm previous reports by Hof-
Basically, the Shorr and Papanicolaou staining methods are mann et al. (25) that the Tygerberg strict criteria as
based on the same principles. Papanicolaou modified the established by Menkveld et al. (12) and the Düsseldorf clas-
Shorr staining method because staining with the Shorr method sification of sperm morphology are quite comparable.

Fertility and Sterility 453

Previous reports by Schirren et al. (23), Calamera and Vilar
(34), and Hellenkemper and Cooper (35) indicating the Test-
simplets method as a good alternative for the evaluation of
normal sperm morphology in an assisted reproduction pro-
gram cannot be confirmed in this report because of its signif-
icant deviations from the two standard methods.
The results of our statistical evaluation revealed the rapid
Testsimplets staining method exhibiting statistically signif-
icant deviations by all the statistical tests applied and thus
the method not being unbiased, particularly regarding the
evaluation of flagellar defects. A possible reason for this
result could be that in this staining procedure no step of fix-
ation is implemented. Because of the wet mount, spermato-
zoa could be lying on their sides rendering confusion with
tapering forms by inexperienced observers (36). Another
reason could be that the staining procedure itself is causing
the sperm to swell, for which there is some indication (17).
Other disadvantages are that these slides cannot be stored
(36, 37), as well as their high costs (33). Nevertheless, be-
cause of the difference in granulation the Testsimplets ap-
pear to be useful for a good differentiation between
leukocytes, spermatids, and spermatocytes (36, 38) and are
also useful in forensic medicine because they appear to be
a practical method to identify sperm in seminal stains
from vaginal swabs (39).
The choice of a staining method should depend on the
purpose of the investigation. For routine purposes the Pa-
panicolaou staining method should be used, for detailed
structural view and photography Spermac (40) is very
useful, and if a quick indication of the patient’s sperm
morphology is needed one of the rapid staining methods
such as Diff-Quik, Hemacolor, or Testsimplets can
be used. However, in view of the increasing importance
placed on sperm morphology evaluation results it should
be kept in mind that the values obtained with each staining
method differ, and a laboratory’s normal values must be
based on the specific staining method used in the labora-
tory (33). This fact should be considered when evaluating
a couple for possible treatment options, especially the pa-
tient with values fluctuating between the poor (P-group)
and good (G-group) prognosis morphology groups (4). It
is therefore important that the staining method used for
the evaluation of the morphology smear should be men-
tioned on the laboratory’s report as also stressed by Ragni
et al. (33).
In conclusion, because the correct evaluation of sperm
morphology is of essence within the scope of assisted repro-
duction and in andrological diagnostics to keep high stan-
dards (41, 42), the use of the Testsimplets method, or any
other rapid staining method, cannot be recommended for
the evaluation of human sperm morphology.
Acknowledgments: The authors express their gratitude to Ms. D. Ledig, De-
partment of Dermatology, and Ms. G. Weigand, Department of Urology,
Friedrich Schiller University of Jena, Jena, Germany, for expert laboratory
assistance and selecting the semen samples.
454 Henkel et al. Comparison of staining methods for sperm morphology
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