One-step disposable chambers for sperm concentration and motility assessment: how do they compare with the World Health Organization's recommended methods?

M. Tomlinson1, J. Turner, G. Powell and D. Sakkas

Assisted Conception Unit, Birmingham Women's Hospital, Birmingham, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The World Health Organization suggests that `one-step' type disposable chambers lack the accuracy and precision of the haemocytometer method for assessing sperm concentration. The purpose of this particular study was to compare sperm concentration measurements and motility using the Neubauer® haemocytometer with those obtained using three `one-step' methods: the Microcell® slide, Leja® slide and a plain glass slide with a 22x22 mm coverslip. A total of 200 sperm concentration measurements and 100 motility assessments were performed on all chambers. Paired comparisons showed enormous discrepancies between the counts, particularly between the Neubauer® and other chambers (P < 0.0001). This discrepancy was less pronounced in oligozoospermic samples, and samples with low (<30% progression) motility but more pronounced in normozoospermic samples and those with good motility (>50% progression). In addition, concentration assessments from a fresh undiluted and unfixed semen sample on the Microcell® slide were found to be significantly lower than both fixed counts on the same slide (P = 0.011) and the initial laboratory reading on the Neubauer® chamber (P = 0.009). No differences were observed in progressive motility between the different chambers and a plain glass slide. There appears to be little comparison between the haemocytometer and either re-useable or disposable one-step chambers. The unfortunate consequence of this is that diagnostic semen analysis and guidelines for allocation of patients to appropriate treatment groups will vary from centre to centre, depending on the method used and may, on occasion, be erroneous.

Key words: counting chambers/semen analysis/sperm concentration


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For many years, counting on a haemocytometer such as the Improved Neubauer® chamber has been widely accepted as the most accurate method for the estimation of sperm concentration [World Health Organization (WHO), 1992, 1999; Mortimer, 1994]. The flexible approach of fixation/immobilization; dilution of highly concentrated samples; and counting of spermatozoa in a single 10 µm plane, are all thought to contribute to its accuracy and ease of use.

Alternative one-step methods using reusable glass chambers such as the Makler® (Sefi Medical Instruments, Haifa, Israel) and Horwell® (Horwell Ltd, London, UK), which recommend counting undiluted, moving spermatozoa have been available for some time. These have proven particularly popular in IVF laboratories, where the keeping of fixatives is clearly undesirable. However, both chambers have been shown to produce inconsistent results and an overestimate of sperm numbers when compared with other methods (Ginsburg and Armant, 1990Go; Shiran et al., 1995Go) and in contrast to IVF laboratories, have generally failed to gain acceptance by the andrology community.

Disposable one-step chambers such as the Cell Vu® (Fertility Technologies, Natick, MA, USA), Microcell® (Conception Technologies, San Diego, CA, USA) and the Leja slide® (Gynotec Malden, Nieuw-Vennep, the Netherlands) are now becoming increasingly popular and are now the preferred choice for many IVF laboratories. These are fixed-depth chambers which, when used with an eyepiece graticule and calibrated using a stage micrometer, can be used to assess both concentration and motility, without the need for fixing or diluting the specimen. Consistent and accurate counts with latex beads and static homogenized sperm suspensions have already been reported (Mahmoud et al., 1997Go). There also appear to be clear advantages to using a chamber with consistent depth of 20 µm for the assessment of sperm motility, rather than a plain glass slide with a 22x22 mm coverslip, as is currently recommended, particularly as sperm motion characteristics are highly dependent on chamber material and depth (Le Lannou et al., 1992Go; Armant and Ellis, 1995Go; Kraemer et al., 1998Go).

The WHO manual states that `such chambers lack the accuracy and precision of the haemocytometer method for assessing sperm concentration and require validation alongside this method before full acceptance for use in routine andrology' (WHO, 1999). With this in mind, further clarification was sought. Therefore, the present study aims firstly to assess two of the one-step type disposable chambers, the Microcell® and Leja® slide, in the determination of sperm concentration using fresh undiluted and unfixed semen samples (rather than latex bead suspensions) and compare these counts with the recommended haemocytometer method. As the depth under a 22x22 mm coverslip on a plain glass slide (and a sample volume of 10 µl) approximates to 20 µm (Mortimer, 1994Go), another comparison was also made in order to determine whether a plain glass slide would give sperm concentration measurements comparable to those of the precision-made disposable chambers. Secondly, sperm motility, assessed on both chambers, was compared with that on a plain glass slide with a 22x22 mm coverslip, again the method currently recommended by the WHO (WHO, 1992WHO, 1999).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semen samples and semen analysis
In total, 50 semen samples were obtained from patients attending the diagnostic andrology service of the Assisted Conception Unit (ACU), Birmingham Women's Hospital, Edgbaston, Birmingham. Samples were produced into a sterile 60 ml container (Sterilin, Stone, Staffs, UK) and allowed to liquefy for 1 h prior to analysis at 37°C. All standard semen analyses were carried out according to WHO (1999) guidelines, using phase-contrast optics at x200 and x400 final magnification. The laboratory performs regular internal quality control and is a participant in the UK National External Quality Assurance Scheme for Andrology (UKNEQAS).

All sperm counting chambers were used strictly according to the recommended protocol of either the WHO manual (WHO, 1999) or according to manufacturer's recommendations and no attempt was made to optimize their use beforehand.

Sperm concentration measurements using the haemocytometer
Haemocytometer chambers were prepared for counting according to the WHO (WHO, 1999). Positive displacement pipettes (Gilson Microman, Anachem, UK) were used throughout for the transfer of semen. Both sides of two improved Neubauer® chambers were read for each of the 50 samples leading to a total of 200 observations.

Sperm concentration measurements using the Microcell®, Leja® and plain glass slide
Counts were made for Microcell® and Leja® slides using a 10x10 eyepiece graticule after calibration according to manufacturer's instructions. Again, positive displacement pipettes were used to fill all three chambers. A minimum of 100 spermatozoa was recorded from five random fields with both sides of two chambers read for each sample. In total, 200 readings were made from Microcell® and Leja® slides and 140 from plain glass slides.

An additional observation was made on further samples to examine whether sperm concentration, assessed on fresh undiluted and unfixed semen samples, differed from that of immobilized spermatozoa. A total of 10 semen samples was assessed using the Microcell®. Sperm concentration was carried out either neat or diluted 1:1 in 4% formal saline. Diluted counts were then re-adjusted accordingly.

Sperm motility
Sperm motility was assessed using the WHO classification system (WHO, 1999), with only the first two grades (a and b) reported, i.e. rapid forward progression and medium/slow forward progression. Each sample was assessed twice, a total of 100 observations per chamber. For consistency, all readings were carried out at 37°C, using a heated microscope stage (Linkham Scientific, Tadworth, UK).

Statistics
All statistical analyses were carried out using SPSS for windows (version 9.0). Repeatability of counts from individual chambers was assessed using analysis of variance (ANOVA). As most of the data distributions were non-parametric, data were reported as median and range with paired comparisons of counts and motilities performed using the Wilcoxon signed rank test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sperm concentration
In all, 740 sperm concentration and 300 motility measurements were made on all chambers. Coefficients of variation (CV) for the four repeat readings were 7.4, 10.15 and 10.6% for the haemocytometer, Microcell® and Leja® slides respectively. It was apparent that data distributions for sperm concentration measurements varied enormously from chamber to chamber. Using paired comparisons for non-parametric data, haemocytometer counts were found to be significantly higher than those produced by all other methods (P < 0.0001). Microcell® counts were closest to those of the haemocytometer and indeed were also significantly higher than either the Leja® (P < 0.0001) or plain glass slide readings (P < 0.0001). Plain glass slide readings showed the poorest agreement with haemocytometer counts. To determine whether this was consistent throughout the data distribution, groups were formed of normozoospermic samples (>20x106/ml) and oligozoospermic samples (<20x106/ml), using the haemocytometer count as a reference. The pattern repeated itself, although with oligozoospermic samples, the difference between haemocytometer and Microcell® counts was not statistically significant (Table IGo). The discrepancy increased in counts over 20x106/ml, indicating a relationship between sperm concentration and agreement with the haemocytometer counts (Table IIGo).


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Table I. Test statistics for oligozoospermic samples as measured on the haemocytometer on glass slides and the Microcell® and Leja® slides
 

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Table II. Test statistics for normozoospermic samples as measured on the haemocytometer on glass slides and the Microcell® and Leja® slides
 
Sperm motility
A total of 98 motility readings, WHO grades a and b, was available for analysis. All three methods gave similar results, with no significant differences found between any chamber (Table IIIGo).


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Table III. Rapid and medium progressive sperm motility (WHO, grades a and b) as measured on glass slides and the Microcell® and Leja® slides
 
Sperm concentration measurements in relation to motility
The huge discrepancy in sperm concentration between the haemocytometer and other chambers was also analysed to determine whether it altered at high and low sample motility. The 35th and 65th progressive motility percentiles, relating to 30 and 50% forward progression respectively, were selected for examination. The discrepancy in concentration between haemocytometer versus Microcell® and haemocytometer versus Leja® was then compared at motilities of <30 versus >50%. At >50% forward progression, haemocytometer counts were 73.3 and 79% higher (P < 0.0001) than Microcell® and Leja® counts respectively. In contrast, there was no statistical difference in sperm concentration by the three methods when progressive motility was <30%. Interestingly, CV for the four repeat counts from both Microcell® and Leja® slides in samples with high concentration (>70x106/ml) and high motility (>50% progression) were 13.1 and 11.5% respectively, which is higher than they were for all samples. CV from the haemocytometer for this group of samples remained low at 5.7%.

An additional experiment using fixed versus unfixed spermatozoa performed on the Microcell® appeared to corroborate the above findings. Motile sperm concentration measurements were significantly lower than both fixed counts on the same slide (P = 0.011) and the initial laboratory reading on the haemocytometer chamber (P = 0.009). Counts of fixed spermatozoa on the Microcell® chamber were still somewhat lower than those of the haemocytometer but this difference was not statistically significant (Table IVGo).


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Table IV. Sperm concentration assessed using motile and fixed samples by the Microcell® method, compared with the haemocytometer
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The haemocytometer method has long been the accepted standard for the assessment of sperm concentration. More recently described one-step methods for use on undiluted and fresh samples have been put forward as convenient and possibly safer alternatives. The study described above, based on over 700 observations, suggests that there is little agreement between the two methods. When compared case by case, sperm concentration measurements made using the haemocytometer were significantly higher than those using the Microcell®, Leja® and plain glass slides utilizing the same eyepiece graticule counting method. This difference was amplified with increasing sperm concentration and sperm motility. Conversely, better agreement was observed with low concentration, low motility samples.

Amongst the one-step methods, Microcell® appeared to agree best with the haemocytometer, with a plain glass slide being the poorest. The differences between one-step methods are somewhat difficult to explain, although they may well relate to chamber depth. We know that both Microcell® and Leja® slides are produced with a fixed, consistent chamber depth. Despite an initial 20 µm depth, glass slide depth undoubtedly varies, as sample may leach from under the coverslip and is therefore highly dependent on the time lag between slide preparation and reading. However, motility readings appear to be perfectly adequate using a plain glass slide. The differences between Microcell® and Leja® counts are perhaps more difficult to understand, particularly as they are put forward as identical methods.

This is not the first study which has highlighted enormous inconsistencies between sperm counting methods. Horwell® chambers have been reported to read consistently higher, and Makler® consistently lower, when compared specifically to the haemocytometer (Shiran et al., 1995Go). In addition, recent results from the American Association of Bioanalysts proficiency testing for andrology demonstrated similar results, showing haemocytometer counts to be 40% higher than those of Microcell® and Cell Vu® chambers (Keel et al., 2000Go), a finding which appears to agree with the present study.

The questions that now arise are: why are there such discrepancies and which method is most representative of the `true' sperm concentration? They surely are not all right.

In answer to the first question, this study appears to provide a partial explanation. It is clear that both sperm concentration and motility significantly affect the count from the Microcell® and Leja® slide, in relation to that obtained from the haemocytometer chamber. Indeed, not only were significantly higher counts produced on the Microcell® by simply immobilizing the spermatozoa in formal saline but better agreement with the haemocytometer was obtained in samples with poorer concentration and motility. This is further illustrated by the coefficients of variation from Microcell® and Leja® slide readings, which are increased in samples with increased concentration and progressive motility. The relatively low volumes used on both Microcell® and Leja® slides compared with those on a haemocytometer (5 versus 10 µl) may contribute to a lack of precision, particularly if the spermatozoa are not distributed homogeneously throughout the sample. Improved agreement with the haemocytometer may be achieved by dilution of samples read on the Microcell®, Leja® slide etc. Indeed manufacturer's instructions suggest that highly concentrated samples should be viewed at higher magnification or diluted. It may be useful in the future if advice were given as to the concentration at which this should be done, and perhaps the most suitable diluent. Previous studies have shown these chambers to give extremely reproducible results with latex beads or immobilized sperm preparations (Mahmoud et al., 1997Go). In a similar study, Seaman et al. (1996) compared chambers using known concentrations of latex beads and found that both haemocytometers and the Makler® chamber overestimated concentration. Again, counts using the Microcell® and Cell Vu® chambers were both accurate and consistent. Unfortunately, the vital piece of information missing from both studies appears to be exactly how comparable are the counts on motile spermatozoa? The data we present here are from fresh motile spermatozoa rather than quality control media, and as a consequence produced highly contrasting results.

The more comparable results from samples with low concentration and motility and from fixed/diluted samples illustrate this further and strongly suggest that the counting of motile spermatozoa is inherently prone to a degree of error. This begs the question: should sperm concentration always be assessed on immobilized/diluted spermatozoa?

The second problem remains unanswered, i.e. which is the most appropriate and accurate method for sperm counting? Is the WHO justified in continuing to recommend the haemocytometer as the exceptional method? Automated methods such as computer-assisted semen analysis (CASA) and flow cytometry have been used successfully but are simply too expensive for use in most laboratories (Evenson et al., 1993Go; Farrell et al., 1996Go). Significant discrepancies between virtually all available manual methods exist, yet we continue to use them. The unfortunate consequence of this is that diagnostic semen analysis and subsequent allocation of patients to appropriate treatment groups, e.g. intrauterine insemination (IUI), IVF or intracytoplasmic sperm injection (ICSI), may be erroneous.

In conclusion, the lack of agreement between the three chambers studied here is certainly cause for some concern for andrology and assisted reproduction laboratories. If the WHO continues to recommend the haemocytometer as the gold standard method, then on current information, we should be using no other method. Perhaps in the future we can work with manufacturers to establish guidelines to optimize sperm counting on fixed depth disposable chambers.


    Notes
 
1 To whom correspondence should be addressed at: Assisted Conception Unit, Birmingham Women's Hospital, Metchley Park Lane, Edgbaston, Birmingham B15 2TG, UK. E-mail: matthew.tomlinson{at}bham-womens.thenhs.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Armant, D.R. and Ellis, M.A. (1995) Improved accuracy of sperm motility assessment using a modified Micro-Cell sperm counting chamber. Fertil. Steril., 63, 1128–1130.[ISI][Medline]

Evenson, D.P., Parks, J.E., Kaproth, M.T. and Jost, L.K. (1993) Rapid determination of sperm cell concentration in bovine semen by flow cytometry. J. Dairy Sci., 76, 86–94.[Abstract/Free Full Text]

Farrell, P.B., Foote, R.H. and Zinamen, M.J. (1996) Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342. Fertil. Steril., 66, 446–453.[ISI][Medline]

Ginsburg, K. and Armant, D.R. (1990) The influence of chamber characteristics on the reliability of sperm concentration and movement measurements obtained by manual and videomicrographic analysis. Fertil. Steril., 53, 882–887.[ISI][Medline]

Keel, B.A., Quinn, P., Schmidt, C.F. et al. (2000) Results of the American Association of Bioanalysts national proficiency testing programme in andrology. Hum. Reprod., 15, 680–686.[Abstract/Free Full Text]

Kraemer, M., Fillion, C., Martin-Pont, B. and Auger, J. (1998) Factors influencing human sperm kinematic measurements by the Celltrak computer-assisted sperm analysis system. Hum. Reprod., 13, 611–619.[Abstract]

Le Lannou, D., Griveau, J.F., Le Pichon, J.P. and Quero, J.C. (1992) Effects of chamber depth on the motion pattern of human spermatozoa in semen or in capacitating medium. Hum. Reprod., 7, 1417–1421.[Abstract]

Mahmoud, A.M., Depoorter, B., Piens, N. and Comhaire, F.H. (1997) The performance of 10 different methods for the estimation of sperm concentration. Fertil. Steril., 68, 340–345.[ISI][Medline]

Mortimer, D. (1994) Practical Laboratory Andrology. Oxford University Press, Oxford.

Seaman, E.K., Goluboff, E., BarChama, N. and Fisch, H. (1996) Accuracy of semen counting chambers as determined by the use of latex beads. Fertil. Steril., 66, 662–665.[ISI][Medline]

Shiran, E., Stoller, J., Blumenfeld, Z. et al. (1995) Evaluating the accuracy of different sperm counting chambers by performing strict counts of photographed beads. J. Assist. Reprod. Genet., 12, 434–442.[ISI][Medline]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction, 3rd edn, Press Syndicate of the University of Cambridge, Cambridge.

World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction, 4th edn, Press Syndicate of the University of Cambridge, Cambridge.

Submitted on June 27, 2000; accepted on September 22, 2000.