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From the Centre of Reproductive Medicine and Andrology of the University of Münster, Münster, Germany.
| Correspondence to: Dr T. G. Cooper, Centre of Reproductive Medicine and Andrology of the University, Domagkstrasse 11, D-48129 Münster, Germany (e-mail: TrevorG.Cooper{at}ukmuenster.de). |
| Received for publication May 14, 2008; accepted for publication June 30, 2008. |
Comparison of methods that estimate viability of human spermatozoa by
monitoring head membrane permeability revealed that wet preparations (whether
using positive or negative phase-contrast microscopy) generated significantly
higher percentages of nonviable cells than did air-dried eosin-nigrosin
smears. Only with the latter method did the sum of motile (presumed live) and
stained (presumed dead) preparations never exceed 100%, making this the method
of choice for sperm viability estimates.
All versions of the World Health Organization (1980, 1987, 1992, 1999) Handbook for the Examination of Human Semen and Semen-Cervical Mucus Interaction have presented 2 tests of sperm viability, involving examination of either wet preparations or air dried smears of semen to which eosin has been added. For the wet preparations the World Health Organization (1980) suggested the use of a negative-phase objective with which dead spermatozoa appeared yellow and live spermatozoa bluish, contrasting with bright-field (nonphase) optics in which dead cells were red and live cells white. The World Health Organization (1987, 1992) suggested the use of either bright-field or phase-contrast optics for the wet preparation, whereas the World Health Organization (1999) recommended bright-field optics for wet preparations and negative-phase for smears of eosin-only wet preparations. A recent critical analysis of the alternative method, eosin-nigrosin smears, concluded that a modified 1-step method using isotonic media should be incorporated into the laboratory routine (Björndahl et al, 2004).
Because both bright-field and phase-contrast lenses are recommended for semen analysis (the former for sperm morphology and the latter for motility assessments), the confusion over which lenses should be used for vitality assessment remains to be clarified. Negative-phase lenses are both difficult to acquire and expensive. Furthermore, it is possible that the "haloes" that surround sperm heads in wet preparations examined under positive phase-contrast conditions interfere with the assessment of weak pink color, reducing the percentage of stained cells assessed. The current study was done to compare the use of different optics (positive and negative phase contrast) and different methods (wet preparations and dried smears) on the outcome of sperm viability tests that rely on eosin dye exclusion.
Materials and Methods
Semen Samples—
Semen samples from 112 patients attending the Institute of Reproductive
Medicine were analyzed for vitality by 2 methods using 3 different optics.
After liquefaction and routine semen analysis, begun 30 minutes after
production, the samples were brought to the laboratory and assessed for
vitality as follows. In method 1, wet preparations
(World Health Organization,
1999) in which 5 µL eosin solution (aqueous 5% [wt/vol] to
which NaCl, 0.9% [wt/vol] had been added; Sigma Chemie, Taufkirchen, Germany)
was added to 5 µL semen on a slide and covered by a 21 x 26-mm
coverslip. Estimates of the percentage of stained (presumed dead) and
unstained (presumed live) sperm heads were made with both positive and
negative phase x40 objectives. The order of assessment with the
negative- and positive-phase lenses was alternated with each sample. In method
2, the 1-step eosin-nigrosin method
(Björndahl et al, 2003)
was employed, but because only aliquots of semen were available, smaller than
recommended volumes were used. Twenty microliters of semen were mixed with 20
µL 0.67% (wt/vol) eosin and 10% (wt/vol) nigrosin in 0.9% NaCl in an
Eppendorf tube and a smear was made after 60 seconds. Assessments were made
with a bright-field x100 objective. Because the eosin-nigrosin smears
were analyzed with a x100 lens, whereas the wet preparations were
analyzed with x40 objectives, the possible effect of magnification on
the percentage of stained cells was assessed by reanalyzing the eosin-nigrosin
smears with a x40 objective lens.
From each sample, 200 spermatozoa were scored in duplicate, and the differences between replicates were accepted if they fell within the acceptable 95% confidence limits (World Health Organization, 1999). Additional information on the percentage of immotile spermatozoa (World Health Organization, 1999, grade d) measured on separate aliquots of the same samples at 37°C was obtained before the vitality measurements. To avoid intertechnician variability, all estimates were made by 1 technician.
Microscopy—
An Olympus BX 40 microscope (Olympus, Hamburg, Germany) was used with a
positive phase-contrast objective (UPlanFl 40x/0.75 Ph2 
/0.17), a
negative phase-contrast objective (UPlanFL N40x/0.75 Ph2 /0.17/FN
26.5), and a Plan 100x/1.25 oil Ph3 /– objective, the
latter used without the phase ring.
Statistics— The SigmaStat program (version 3.5; SYSTAT, Erkath, Germany), was used for the methods explained in the text. Statistical significance was accepted with P < .05.
Results
Comparison of Different Optics on Vitality Assessment in Wet Preparations—
One hundred and twelve semen samples were assessed in wet preparations for
the percentage of nonvital spermatozoa by positive and negative phase-contrast
microscopy. With both positive- and negative-phase lenses and with
bright-field assessment of eosin-nigrosin–stained smears sperm heads
appeared either red, white, or pink (not shown). There was a strong positive
correlation between the percentage of dead spermatozoa estimated with both
optical systems (Figure 1,
upper panel), and the line of linear regression was close to, and parallel
with, the line of identity. A Bland-Altman plot of the difference between the
2 methods against the average (Figure
1, lower panel) revealed that the differences were independent of
the magnitude of the assessment and that positive phase contrast overestimated
stained cells compared with the negative-phase method.
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Comparison of Wet Preparations With Semen Smears— Seventy-four of the above samples were also analyzed by the eosin-nigrosin method on air-dried smears. There were positive correlations between the percentage of stained spermatozoa estimated by positive- and negative-phase microscopy and the eosin-nigrosin method (Figure 2, upper panel). The Bland-Altman plot revealed that the differences between methods increased as the percentage of stained cells increased and that larger percentages of stained cells were generated by the wet preparations, regardless of optics, compared with those provided by the smears (Figure 2, lower panel).
There were statistically significant differences (Friedman repeated measures analysis of variance [ANOVA] on ranks) among estimates of the percentage of stained cells from the wet preparations (both positive and negative phase-contrast microscopy: medians 29.4% and 28.9%, respectively) and that from eosin-nigrosin smears (23.8%): absolute differences of 5.6% and 5.1%, or 18%–19% of the maximum assessed value.
Comparison of Viability With Estimated Sperm Motility— Results of 58 of the above samples were compared with the percentage motile (ie, live) spermatozoa in the same samples. Because the sum of the percentage of motile and immotile cells should equal 100%, and because some immotile spermatozoa may be alive, the sum of motile and stained cells should be, within counting error, equal to or less than 100%, depending on the percentage of immotile but live cells. The percentage of immotile spermatozoa in each sample (median 41%) was significantly greater (Friedman repeated measures ANOVA on ranks) than the percentage of stained cells estimated by each of the 3 methods: medians 33% positive phase, 32% negative phase, and 27% eosin-nigrosin.
The Bland-Altman plot in Figure 3, upper panel, shows that as the percentage of stained spermatozoa increased, irrespective of method, the percentage of motile cells decreased and that only with the viability estimates from the wet preparations did the difference from the immotile cells become positive (ie, there were more stained than immotile cells): with positive phase 12 values and with negative phase 7 values were greater than the percentage of immotile cells. This is demonstrated clearly in the scatter plot in Figure 3, lower panel, in which the sum of motile and stained cells clearly exceeds 100% for the viability estimates derived from positive and negative phase-contrast optics on wet preparations.
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There was no statistically significant difference (signed rank test) between the percentage of stained cells assessed by the x40 lens (median 22) and the x100 lens (median 21.6).
Discussion
The present study has shown that the use of eosin alone in wet seminal
preparations produces higher estimates of the percentage of stained cells than
does the method employing eosin-nigrosin smears. Although there was always a
close agreement between methods in the estimates of stained cells, with
essentially parallel lines of identity and regression with each method, the
differences highlighted in the Bland-Altman plots showed a consistent bias
towards higher values from the wet preparations. Contrary to expectation, a
positive-phase lens that created a "halo" around the sperm head,
anticipated to mask lightly pink-stained cells, generated more stained cells
than did the negative-phase lens. Furthermore, the statement by the World
Health Organization (1980) that
yellow and blue sperm heads may be seen with a negative-phase lens was not
corroborated; these colors may have an origin in chromatic aberration of
inferior quality lenses.
Because the sum of the percentage of motile and stained cells should be equal to or less than 100%, the values from the wet preparations exceeding 100% indicate that these methods are registering more stained than immotile cells, that is, that they are categorizing some vital cells as stained. Because this sum estimated from the eosin-nigrosin smear data did not exceed 100%, the latter procedure should be the method of choice for gauging sperm viability from the extent of eosin exclusion, confirming the conclusion of Björndahl et al (2003).
Footnotes
Supported by the German Andrology Society's external quality control scheme for semen analysis, QuaDeGA.
* Andrology Lab Corner welcomes the submission of unsolicited
manuscripts, requested reviews, and articles in a debate format. Manuscripts
will be reviewed and edited by the Section Editor. All submissions should be
sent to the Journal of Andrology Editorial Office. Letters to
the editor in response to articles as well as suggested topics for future
issues are encouraged. 
References
Björndahl L, Söderlund I, Johansson S, Mohammadieh M,
Pourian MR, Kvist U. Why the WHO recommendations for eosin-nigrosin staining
techniques for human sperm vitality assessment must change. J
Androl. 2004;25: 671
–678.
Björndahl L, Soderlund I, Kvist U. Evaluation of the one-step
eosin-nigrosin staining technique for human sperm vitality assessment.
Hum Reprod. 2003; 18: 813
–816.
Cooper TG, Yeung CH. A flow cytometric technique using peanut
agglutinin for evaluating acrosomal loss from human spermatozoa. J
Androl. 1998;19: 542
–550.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 1st ed. Singapore: Press Concern; 1980.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 2nd ed. Cambridge, United Kingdom: Cambridge University Press; 1987 .
World Health Organization. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 3rd ed. Cambridge, United Kingdom: Cambridge University Press; 1992 .
World Health Organization. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed. Cambridge, United Kingdom: Cambridge University Press; 1999 .
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