Quality control workshops in standardization of sperm concentration and motility assessment in multicentre studies

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1 international journal of andrology, 28: (5) doi:.1111/j x Quality control workshops in standardization of sperm concentration and motility assessment in multicentre studies GUNNAR TOFT,* ANNA RIGNELL-HYDBOM, EWA TYRKIEL,à MARYNA SHVETS and ALEKSANDER GIWERCMAN *Department of Occupational Medicine, Aarhus University Hospital, Aarhus, Denmark, Department of Occupational and Environmental Medicine, Lund University, Lund, Sweden, àdepartment of Environmental Toxicology, National Institute of Hygiene, Warsaw, Poland, Kharkov Regional Clinical Centre of Urology and Nephrology, Ukraine, and Fertility Centre, Scanian Andrology Centre, Malmö University Hospital, Malmö, Sweden Summary Semen quality has been reported to vary markedly between different regions. To properly assess the differences among countries a minimization of the variation among centres in the assessments of sperm quality is essential. We here report on the training and two subsequent follow-up workshops on assessments of sperm concentration and motility. A total of 26 fresh ejaculates were analysed by four persons who were collecting these data in a multicentre study on sperm quality. In addition, two trained technicians from one of the laboratories analysed the samples. At the first training workshop, the median coefficient of variation among centres in sperm concentration was 27.7%, but decreased markedly to 17.% at the second workshop and 8.1% (p ¼.48) at the third workshop. The CV for evaluation of the proportion of progressive motile sperm decreased less and not statistically significant from 16.5 to 14.1% and 11.% (p ¼.94). At the third workshop there were no statistical significant differences among centres in assessments of sperm concentration or motility (p >.57). Furthermore the coefficient of variation in assessments of sperm concentration and motility among centres were at the same level as between two trained technicians (p >.72). This study indicates that training and subsequent follow-up workshops can assure minimum variability among centres in assessments of sperm concentration and motility. Keywords: multicentre studies, quality control, sperm concentration, sperm motility, variance Introduction Recent multicentre studies have indicated that semen quality varies among regions in Europe and the USA (Jørgensen et al., 2; Swan et al., 3). The cause of the Correspondence: Gunnar Toft, PhD, Department of Occupational Medicine, Aarhus University Hospital, Noerrebrogade 44, Build 2C, DK-8 Aarhus C, Denmark. gutof@as.aaa.dk differences among regions is not known. To exclude that the differences are simply the result of differences among technicians in the assessments of sperm quality and to maximize the power in comparison among centres, a standardized method of sperm cell analysis with a minimum variation is needed. The variation among laboratories has been reported to be substantial if the laboratory staff has not been trained in the same programme (Matson, 1995). In a Ó 5 Blackwell Publishing Ltd.

2 Quality control in semen studies 145 multicentre set-up, morphology assessment can be centralized. However, both concentration and motility measurements need to be performed by the local laboratories and these methods call most urgently for interlaboratory standardization. To asses the variability among centres, previous studies have mainly used external quality control programmes including sending samples to the participating centres during the data collection period, preceded by a training prior to the onset of the study (Jørgensen et al., 1; Swan et al., 3; Punab et al., 2). However, in the daily routine in the laboratories, sperm concentration and motility are always assessed on fresh semen samples and especially for motility assessment the samples must be analysed shortly after collection. External quality control programmes have been developed based on use of video recording of sperm cells or shipments of cryopreserved ejaculates as fresh semen samples will not be motile for more than a few hours and can therefore not be used (Cooper et al., 2; Neuwinger et al., 199). These modifications of the daily routine are known to introduce artefacts in the semen samples assessed and may, thereby, not give a true picture of the interlaboratory variation. Furthermore, the external quality control programme based on sending the material to the participating centres, do not give any possibility to detect the source of variation and thereby minimizing it. Therefore, to reduce the level of interlaboratory variation in assessment of sperm concentration and motility, a new strategy was introduced. This was based on repeated training/quality control workshops and use of a standardized protocol joined to all laboratories. The results of this standardization programme being a part of a European project on human fertility (INUENDO) are presented in the present paper. Methods Logistics Three workshops (W1, W2 and W3), each 3 days long, were organized at the Fertility Centre, Malmö University Hospital (Malmö, Sweden) with approximately 6-month interval. In order to minimize the variation, only one person from the four participating centres, was analysing semen samples in the INUENDO project. These four persons attended the workshops. Prior to W1, the four investigators were asked to become familiar with the manual on performing semen analysis jointly developed by the Nordic Association for Andrology and European Society of Human Reproduction and Embryology (NAFA-ESHRE). The NAFA-ESHRE manual (Kvist & Björndahl, 2) is based on the WHO (1999) guidelines, but is more strict regarding specification of the equipment and procedures to be applied. Furthermore, the participants were asked to purchase the specified equipment, including positive displacement pipettes and improved Neubauer chambers (Paul Marienfeld, Bad Mergentheim, Germany), and bring their own pipettes and dilution buffers to the venue of the workshops. At W1, training in the fundamental principle of sperm concentration and motility assessment was given. Thereafter, three semen samples were analysed by each participant for estimation of the variation among analysers in assessment of sperm concentration and four samples were analysed for sperm motility. The participants were asked to practice semen analysis in their home laboratories, according to the skills they learned during W1. In order to further reduce the variance among centres, W2 included continuous training on six samples for sperm concentration and five of these were analysed for motility. This session was followed by evaluation and individual correction and then five additional samples were analysed for sperm concentration and motility on a separate day. W2 was completed prior to the onset of the major part of the data collection for the main study. W3 was held in the middle of the data collection period of the INUENDO study. During W3, 11 semen samples were analysed for sperm concentration and motility without any additional training. Two experienced laboratory technicians from the Fertility Centre, Malmö University Hospital analysed some of the same semen samples for sperm concentration and motility at the workshops, in order to compare the variation between expert and more novice investigators. One technician analysed in total 22 samples and the other analysed 13 samples. Semen samples In total 26 fresh semen samples were obtained from semen donors at the Fertility Centre, University Hospital in Malmö. The samples were collected in the privacy of a room close to the laboratory and delivered to the laboratory within 5 min after collection. Semen analysis The semen samples were analysed for motility and sperm concentration according to the NAFA-ESHRE manual for basic semen analysis (Kvist & Björndahl, 2). In order to shorten the time of analysis to make it feasible under field conditions the motility assessment of 2 cells only was performed. Therefore, the acceptance levels for the difference between the two counts were based on the WHO (1999) recommendations given for a total number of spermatozoa scored equal to. If the difference between the two counts of any of the four motility categories exceeded the tabulated acceptance limits the sample was recounted. All semen samples were analysed after liquefaction for a minimum of min at 37 C. The motility was assessed as recommended by WHO (1999) by placing one drop of undiluted semen on a clean glass slide covered with a cover slip. With the aid of an ocular grid, the proportion of sperm cells in each of four motility groups was assessed: a, rapidly Ó 5 Blackwell Publishing Ltd, International Journal of Andrology, 28,

3 146 G. Toft et al. progressive sperm ( 25 lm/s); b, slowly progressive sperm (5 24 lm/s); c, non-progressively motile sperm (<5 lm/s) and d, immotile sperm. All motility analyses were performed at 37 C. Sperm concentration was assessed on diluted semen samples (1 : or 1 : ) using an improved Neubauer haemocytometer. Typically sperm cells were counted in each of two chambers and, if the difference between the two chambers exceeded the acceptance limits specified in the WHO (1999) manual a new sample was counted. Statistical analysis As a result of relative small sample sizes and possible skewed distributions, nonparametric tests were used to compare the data. The coefficient of variation (CV ¼ standard deviation/mean ) was calculated on the individual semen samples as a measure of the variation among analysers. Kruskal Wallis test was applied to test for differences in the CV of concentration and motility assessments between the three workshops, followed by Mann Whitney tests for pairwise comparisons between workshops. Wilcoxons signed rank test was used to test differences in CV of sperm concentration and motility assessments between the INUENDO analysers and trained technicians. Friedman s test was used to test if the results of assessment of sperm concentration and motility differed among the individual analysers. The above tests were conducted using SAS (version 8.2, SAS Institute Inc., Cary, NC, USA). Results As seen in Fig. 1, the median coefficient of variation in assessment of sperm concentration dropped markedly from 27.7% at W1 to 17.% at W2 (17.2% at day 1 and 16.7% at day 2) and 8.1% at W3. As the CV at W2 was quite similar on the 2 days, the data from these 2 days were combined in the statistical analysis of differences between workshops. Pairwise comparisons showed most significant difference between W2 and W3 (p ¼.48). Probably because of the low sample size at W1, the median CV was not statistically significant from that for W2 (p ¼.29) and it only tended to be statistically significantly different from the median CV at W3 (p ¼.9). The CV among the trained technicians, when compared with the INUENDO investigators, was lower at W1 (p ¼.11; Fig. 1). At the following two workshops the CV among trained technicians and among the INUENDO staff was quite similar (p >.72). The median variation in the percentage of progressively motile sperm (grade a + b) decreased from 16.5% at W1 to 14.1% at W2 (15.8% at day 1 and 13.9% at day 2) and 11.1% at W3 (Fig. 2). Again the data from W2 was combined in statistical analysis. The differences in CV of sperm motility assessment were not statistically significant between workshops (p ¼.94). The variation among the CV (concentration) W1 W2 W3 Figure 1. Coefficient of variation of sperm concentration assessment on individual semen samples among the four INUENDO analysers d and among two trained technicians at three workshops [W1 (three samples) W2 (11 samples) and W3 (11 samples)]. Median coefficient of variation is presented by. CV (motility) W1 W2 W3 Figure 2. Coefficient of variation of sperm cell progressive motility assessment on individual semen samples among the four INUENDO analysers d and among two trained technicians at three workshops [W1(four samples) W2 ( samples) and W3 (11 samples)]. Median coefficient of variation is presented by. trained technicians seemed to be in the same range as the variation among the INUENDO semen analysers (p ¼.48). The CV for assessment of the total percentage of motile sperm (grade a + b + c) was 8.% at W1,.4% at W2 and 8.6% at W3 not showing any trend over time. The assessments of the different categories of sperm motility varied more with a median CV of.1% for motility group a, 35.8% for motility group b, 49.1% for motility group c and 29.% for motility group d across all workshops, with no apparent trend across workshops (data not shown). At W1 the results of assessment of sperm concentration (three samples) and motility (four samples) performed by the INUENDO investigators did not differ (p ¼.615 and.55, Ó 5 Blackwell Publishing Ltd, International Journal of Andrology, 28,

4 Quality control in semen studies 147 Workshop 1 Workshop 2 day 2 Workshop 2 day 1 Workshop 3 Figure 3. Differences from mean sperm concentration assessment by the four analysers at the three workshops. Each line represent one of the 25 semen samples analysed for sperm cell concentration and individual analysers are denoted by letters a d. respectively). At the first day of W2 there seemed to be systematic differences among analysers in the measurements of sperm concentration (p ¼.2) and motility (p ¼.8) whereas there appeared to be no difference at day 2 (p ¼.18 and.39, respectively). At W3 there was no systematic difference among analysers in the quantification of sperm concentration (p ¼.57) or motility (p ¼.86). The variation of the individual observers from the mean of the analysed samples is presented in Fig. 3 for sperm concentration and Fig. 4 for progressive motility. As an example of the actual numbers obtained, data from the third workshop representing the middle of the data collection period is presented in Table 1. Discussion In the present study, we have demonstrated that by applying a joint protocol for assessment of semen quality and training based on repeated workshops, it is possible to achieve an interlaboratory variation of approximately % for evaluation of sperm concentration and proportion of progressively motile sperm. Furthermore, at the end of the training, there was no systematic difference between the centres as considers the concentration and motility scores. The value of training courses introduced by ESHRE in order to minimize interlaboratory variation in semen quality assessment has been reported by others (Björndahl et al., 2). However, our study demonstrated that by follow-up workshops combining training and quality control, the interlaboratory variation was reduced from 28 to 8%, for sperm concentration and less distinct for the percentage of progressively motile sperm, the reduction from 17 to 11% not being statistically significant. However, in evaluation of the results of the statistical analysis, its relatively low power should be kept in mind. Thus, as two of the workshops (W1 and W2) combined training and quality control, the number of samples used for the analysis was relatively low, in particular during W1. Previous studies have demonstrated a CV among trained technicians within one laboratory to be.6% for sperm concentration assessments (Cooper et al., 2) and the intra-individual CV for analysis of sperm concentration at a workshop including 13 participants was 28% for untrained analysers and 9.8% for persons who have been performing semen analysis for more than 3 years (Auger et al., ). Our results of an interindividual CV of 8.1% for sperm concentration assessment at W3 indicates that after sufficient training the variation among analysers within the INUEN- DO project was not larger than what would be expected within or among trained technicians from one laboratory. Furthermore, when compared with the trained technicians, the CV of sperm concentration assessment only tended to be higher among the INUENDO analysers at the first workshop whereas at W2 and W3 there seemed to be no difference between INUENDO analysers and trained technicians. The fact that the variation among INUENDO analysers was within the same range as the trained technicians both in this study and in other studies indicates that additional training cannot reduce the variation among INUENDO analysers any further. In the studies performed to date, a larger variation has always been reported on manual motility assessments Ó 5 Blackwell Publishing Ltd, International Journal of Andrology, 28,

5 148 G. Toft et al Workshop Workshop 2 day 2 Workshop 2 day 1 Workshop Figure 4. Differences from mean progressive sperm motility assessment by the four analysers at the three workshops. Each line represent one of the 25 semen samples analysed for sperm cell motility and individual analysers are denoted by letters a d. Table 1. Mean and median sperm concentration and motility on 11 samples assessed at the third workshop representing the middle of the data collecting period Analyser Concentration (million/ml) % Progressive motile sperm Mean (SE) Median Mean (SE) Median A 67.7 (11.4) (5.6) 62. B 66.2 (11.4) (6.9) 69. C 65.6 (12.6) (7.3) 68. D 63.8 (.4) (5.3) 62. compared with the concentration assessments when fresh samples are analysed (Auger et al., ; Jørgensen et al., 1997), possibly because of the inherent higher degree of subjectivity in motility assessments. In the study by (Auger et al., ) an interobserver CV of 21.8% was found in assessment of motility grade a + b + c. In our study the median interindividual coefficient of variation of motility grade a + b + c was 8.6% at the third workshop. The median interindividual coefficient of variation on the assessment of motility grade a + b was 11.1% indicating that the proportion of motility grade a + b can be assessed with approximately the same accuracy. Furthermore, the CV among INUENDO analysers and trained technicians seemed to be in the same range, indicating that even by additional training any significant reduction of the interobserver variation can hardly be expected. In the present study, the assessments of sperm cell motility on 2 cells were performed and not 2 as recommended by the newest WHO (1999) manual. Counting on more sperm cells would probably reduce the variation further, but it would not change the main conclusion of this study, of an achievement of an acceptable low interlaboratory CV through use of a joint, strictly defined protocol and repeated training. As a result of the considerable variability in assessment of sperm cell motility, computer-assisted semen analysis (CASA) methods have been developed to eliminate the variation because of subjective assessment (Verstegen et al., 2). However, CASA assessment of motility may vary between different systems and even within one system because of differences in the set-up of the system. The basic principles of sperm concentration assessment, implies a 95% probability of assessing the sperm number within 15% of the true value, because of measurement error alone, if the guidelines of the NAFA-ESHRE manual based on the WHO (1999) manual for semen analysis are strictly followed. Additional variation will however, most likely appear, because individuals vary from day to day in their performance. In addition to the random variation among analysers, systematic differences contribute to the interindividual variation. It is of outmost importance to detect such systematic differences among analysers. In this study we focused on, during W1 and W2, detection and correction of such systematic differences in performing semen analysis between the observers. At W3 there were no significant systematic differences among the semen analysers and also the interobserver CV for assessment of sperm concentration decreased significantly between W2 and W3 indicating that this strategy proved efficient. Ó 5 Blackwell Publishing Ltd, International Journal of Andrology, 28,

6 Quality control in semen studies 149 As the variation among technician in assessment of sperm cell morphology is known to vary considerably (Jørgensen et al., 1997), no attempt was made to standardize these measurements in this multicentre study, but instead the assessment of morphology of all semen samples collected in the four countries has been centralized. Our results do also indicate a somewhat limited value of quality control programmes based on mailed samples. In a study by Neuwinger et al. (199) the interlaboratory variation in assessment of sperm concentration was estimated to be 23 73% depending on the sperm concentration. For the motility, the corresponding figure was 21%. In another study, also based on mailed samples, the CV for concentration assessment was between 23 and 29% (Giwercman et al., 1999). These figures seem to be rather high when compared with those obtained by us. It might indicate that the procedures introduced for preservation of quality control sample addition of sodium azide or formaldehyde for concentration and cryopreservation for motility does interfere with their quality, thereby introducing falsely high estimates of interlaboratory variation. In conclusion, our study demonstrated that multicentre studies can be performed with sufficient low variation in assessment of sperm concentration and percentage of motile sperm among centres when specific training and quality control programmes are set up. Workshops with participation of all involved analysers have the advantage that differences among analysers can be corrected not just reported. Furthermore, use of fresh sperm samples might give a more realistic picture of the variation to be assessed. Acknowledgements We wish to thank the technicians Katarina Jepson and Cecilia Tingsmark for organizing this study at the Fertility Centre in Malmö. Financial support for the INUENDO project ( has been given by the European Commission, quality of life and Management of Living Resources, Key action four on environment and health (contract no. QLK4-CT-1-2) and in part by INTAS Poll call 1 (contract no. 1-25). Auger, J., Eustache, F., Ducot, B., Blandin, T., Daudin, M., Diaz, I. et al. () Intra- and inter-individual variability in human sperm concentration, motility and vitality assessment during a workshop involving ten laboratories. Human Reproduction 15, Björndahl, L., Barratt, C. L., Fraser, L. R., Kvist, U. & Mortimer, D. (2) ESHRE basic semen analysis courses : immediate beneficial effects of standardized training. Human Reproduction 17, Cooper, T. G., Björndahl, L., Vreeburg, J. & Nieschlag, E. (2) Semen analysis and external quality control schemes for semen analysis need global standardization. International Journal of Andrology 25, Giwercman, A., Spano, M., Lahdetie, J. & Bonde, J. P. (1999) Quality assurance of semen analysis in multicenter studies. Asclepios. Scandinavian Journal of Work, Environment and Health 25(Suppl. 1), Jørgensen, N., Andersen, A. G., Eustache, F., Irvine, D. S., Suominen, J., Petersen, J. H. et al. (1) Regional differences in semen quality in Europe. Human Reproduction 16, Jørgensen, N., Auger, J., Giwercman, A., Irvine, D. S., Jensen, T. K., Jouannet, P. et al. (1997) Semen analysis performed by different laboratory teams: an intervariation study. International Journal of Andrology, 1 8. Jørgensen, N., Carlsen, E., Nermoen, I., Punab, M., Suominen, J., Andersen, A. G. et al. (2) East-West gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Human Reproduction 17, References Kvist, U. & Björndahl L. (2) ESHRE Monographs. Manual on Basic Semen Analysis. Oxford University Press, Oxford. Matson, P. L. (1995) External quality assessment for semen analysis and sperm antibody detection: results of a pilot scheme. Human Reproduction, Neuwinger, J., Behre, H. M. & Nieschlag, E. (199) External quality control in the andrology laboratory: an experimental multicenter trial. Fertility and Sterility 54, Punab, M., Zilaitiene, B., Jorgensen, N., Horte, A., Matulevicius, V., Peetsalu, A. & Skakkebaek, N. E. (2) Regional differences in semen qualities in the Baltic region. International Journal of Andrology 25, Swan, S. H., Brazil, C., Drobnis, E. Z., Liu, F., Kruse, R. L., Hatch, M., Redmon, J. B., Wang, C. & Overstreet, J. W. (3) Geographic differences in semen quality of fertile U.S. males. Environmental Health Perspectives 111, Verstegen, J., Iguer-Ouada, M. & Onclin, K. (2) Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 57, World Health Organisation (WHO) (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK/ New York. Received May 4; revised 1 July 4; accepted 2 July 4 Ó 5 Blackwell Publishing Ltd, International Journal of Andrology, 28,