Assisted Conception Unit, Birmingham Women's Hospital, Birmingham, UK
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Abstract |
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Key words: counting chambers/semen analysis/sperm concentration
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Introduction |
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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, 1990; Shiran et al., 1995
) 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., 1997). 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., 1992
; Armant and Ellis, 1995
; Kraemer et al., 1998
).
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, 1994), 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).
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Materials and methods |
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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.
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Results |
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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 IV).
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Discussion |
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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., 1995). 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., 2000
), 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., 1997). 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., 1993; Farrell et al., 1996
). 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.
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Notes |
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References |
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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, 8694.
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, 446453.[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, 882887.[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, 680686.
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, 611619.[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, 14171421.[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, 340345.[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, 662665.[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, 434442.[ISI][Medline]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical 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 SemenCervical Mucus Interaction, 4th edn, Press Syndicate of the University of Cambridge, Cambridge.
Submitted on June 27, 2000; accepted on September 22, 2000.