1 The Assisted Conception Unit and 2 Education Resource Centre, Birmingham Womens Hospital, Birmingham B15 2TG and 3 Division of Reproductive and Child Health, The University of Birmingham, Birmingham B15 2TH, UK
4 To whom correspondence should be addressed at: Obstetrics and Gynaecology, The Jessop Wing, Sheffield Teaching Hospital NHS Trust, Tree Root Walk, Sheffield SI0 2SF, UK. e-mail: BolarindeOla{at}aol.com
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Abstract |
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Key words: accuracy/hyaluronic acid/likelihood ratio/methyl cellulose/spermmucus penetration tests
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Introduction |
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The in-vitro spermmucus penetration test (SMPT) is a sperm function test which measures the ability of sperm in the semen to swim up into a column of cervical mucus or substitute. If it can be proven to be as good as semen analysis in assessing progressive sperm motility, then arguably, its additional benefit as a test of functional competence may make it a suitable and cheaper alternative to the present combination of semen analysis and sperm separation procedures (see decision tree in Figure 1). This attraction has led to the re-emergence of research into SMPT for the assessment of semen. Sperm migration into cervical mucus, or any of its substitutes, is based on the same principle as the Kremer (1965) test. Some of the parameters used to reflect the degree of penetration include: the migration or vanguard distance (distance between the foremost sperm in the capillary tube to the end of the tube in the semen reservoir); swim-up sperm count or sperm velocity at 10, 20, 30 or 40 mm at high power field and migration reduction (the decrease in migration density from 10 to 40 mm). Of these, the most commonly used parameter is the vanguard distance of 30 mm measured after incubation at 37°C for 90 min, although other workers have used different vanguard measurements (Ulstein et al., 1972
; Matthews et al., 1980
; Alexander, 1981
). Three groups, David et al. (1979
), Aitken et al. (1992
) and Ivic et al. (2002
), however, used swim-up sperm count per high power field at 10, 20 and 30 mm in a flat capillary tube as the diagnostic criteria for SMPT. Amit et al. (1982
) used average swim-up sperm velocity, which is indirectly related to sperm count per high power field, at 10, 20 and 30 mm after 60 min.
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This systematic review was conducted to answer the clinical question: is in-vitro sperm penetration into cervical mucus, or its substitutes, an accurate diagnostic tool in the evaluation of semen? Our aim was to conduct a systematic quantitative review of all primary diagnostic studies that compared SMPT with semen analysis in a manner that allowed meaningful assessment of test quality and usefulness. This objective was to be achieved by identifying the number, scope and quality of primary studies that measured the diagnostic accuracy of SMPT or sperm penetration through known cervical mucus substitutes, by comparing with a reference standard of progressive or total sperm motility in the seminal plasma, as measured manually or by computerized methods. To our knowledge, this is the first such quantitative systematic review of the diagnostic accuracy of SMPT.
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Materials and methods |
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Identification of studies
We searched the general bibliographic databases: Medline (19662001) and Embase (19882001). We also searched the specialist computer databases of the Cochrane library (2000:4) and SciSearch (19812001). Furthermore, we conducted manual searching of conference papers of the American Society for Reproductive Medicine (19972000), the European Society for Human Reproduction and Embryology (19972000) and bibliographies of known primary and review articles.
The search strategy was based on the clinical question: can in-vitro sperm penetration of cervical mucus and mucus substitutes be used to evaluate the quality of sperm motility in the semen? The search terms (sperm or sperm; penetration or interaction or migration; cervical mucus or cervical mucus substitutes; sperm motility or sperm motion or computer-assisted image processing; fertility or infertility) used in all databases were designed to be initially very sensitive (include as many hits as possible) before they were combined using the Boolean and. For example, the following search query was employed for electronic databases: sperm$.mp or exp spermatozoa/;((cerv$ adj mucus) or mucus$).mp or (exp mucus/ and substit$.mp); penetrat$.mp or interact$.mp or migrat$.mp; exp sperm motility/ or motil$.mp. or motility.mp or sperm motion.mp; exp image processing or computer-assisted/; exp fertility/ or exp infertility, male/.
Study selection
The inclusion of primary studies was conducted in two stages. The first involved scanning of titles and abstracts, which were selected provisionally unless clearly not comparing diagnostic accuracies of sperm penetration into cervical mucus or its substitutes with a stated reference standard. Articles that generated doubts regarding accuracy were retained until the full text documents were retrieved. An account was made of the number of titles identified, retained and excluded in order to ensure reproducibility of this stage. The second stage involved final selection of papers from the list obtained in the first stage. The selection was based on a checklist of inclusion criteria. Studies conducted and reported in non-English language were interpreted and dealt with in the same way as papers written in English.
Inclusion criteria were satisfied if the population comprised fertile and infertile men and if the study tested sperm penetration or migration into cervical mucus or its alternatives. Furthermore, the reference standard required was the World Health Organization reference values for motile, or progressively motile sperm, according to the date of study. Finally, the accuracy measurements based on 2x2 table construction had to be possible either from the text, tables, scattergraph or receiver operator characteristics (ROC) curves. For all included studies, the following were recorded and double-checked: authors, year of publication, study design, setting, population characteristics, details of test and gold standard used. The study by Ivic et al. (2002), which has now been published, was included as unpublished data.
Quality assessment
Methodological quality was defined as the confidence with which the study design, conduct, and analyses minimized biases. Based on existing checklists, we performed quality assessment by scrutinizing study designs and relevant features of the population, test and reference standard (Cochrane working group, 2000; Khan et al., 2001b). We considered a study to be of good quality if it used a prospective design, consecutive enrolment, adequate test description, and blinding of the test result (Lijmer et al., 1999
; Khan et al., 2001b
). Where no explicit information was offered in the paper, this was categorized as unreported.
Diagnostic interventions had to be described in sufficient details to ensure reproducibility. Kremer-type tests using flat or round capillary tubes were considered ideal, but other well-described modifications were also acceptable. Human cervical mucus obtained around the time of natural and drug-induced ovulation, or hormone replacement cycles was ideal, but other well-justified cervical mucus substitutes such as hyaluronic acid or methyl cellulose were considered acceptable.
Despite the problems with interpretation due to its non-dichotomous outcome measures, and inter- and intra-observer errors, semen analysis remains the benchmark test for male infertility (World Health Organization, 1999). The accuracy of the SMPT was assessed in respect of its ability to correctly predict normal or abnormal sperm motility, whether measured by semen analysis or computer-aided sperm analyses (ESHRE, 1996). For the SMPT, any or the entire four outcome measures in common use was considered suitable.
Data abstraction and synthesis
We searched carefully for duplication of data that can result from multiple publications of part or whole research work. For clarity and objectivity, data sheets were used for abstraction, based on study characteristics and quality. To minimize errors, two reviewers (B.O. and A.C.) independently performed the data extraction and measured the degrees of agreement for inclusion and quality criteria. The overall agreement was 93.3%, with a Kappa value () of 0.87 [95% confidence interval (CI) 0.691.04]. Kappa statistics assess agreement beyond chance and allow credit for partial accord (Landis and Koch, 1977
).
Categorical data were summarized into a standard 2x2 table and where the outcome measures were continuous variables, 2x2 tables were derived from scattergram and on one occasion from the text and ROC plot. Statistical analyses and data synthesis were performed on Arcus Quickstat (Biomedical Version 1.0, 1997) and Stata Version 7.0 (Stata Corp., USA). Forest plots and pooled LR+ and LR statistics were derived using the DerSimonian and Laird (1986) random effects models.
Heterogeneity was tested graphically (Forest plots) and statistically. In our review, although no significant statistical heterogeneity was detected, we found that heterogeneity existed clinically, which resulted in our decision to perform sub-group meta-analysis of the primary studies based on SMPT diagnostic criteria. The reference test had the same end point (sperm motility) but varied slightly in thresholds. We measured effect by pooled LR+ and LR instead of summary ROC curves because these thresholds had been classified clearly in each primary study and such minor threshold variations do not significantly modify long-term prognosis (Dickey et al., 1999; Deeks, 2001
).
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Results |
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Discussion |
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The use of LR as measures of test usefulness have been well described (Radack et al., 1986; Sackett et al., 2000
; Khan et al., 2001a
;b). The LR for a positive test quantifies the probability that a truly positive, rather than a falsely positive, test result is to be seen when disease state, as defined by the reference test, is present. Similarly, the likelihood ratio for a negative test quantifies the probability that a false negative rather than a truly negative test result is presented when the reference test is negative. As a general guide to understanding the usefulness of a test, a LR+ >10 and LR <0.1 generally indicate a highly useful test because they generate large and usually conclusive changes in the pre-test probability of disease. LR+ of 510 and LR 0.10.2 indicate a moderately useful test, LR+ of 25 and LR of 0.20.5 slightly useful, and LR+ of 12 and LR 0.51 are considered indices of a non-valuable test (Sackett et al., 2000
; Khan et al., 2001a
). The pooled LR+ and LR of 2.29 (1.822.87) and 0.52 (0.440.63) therefore show that the method of using vanguard distance as diagnostic criterion (i.e. SMPTvd) has a low accuracy.
The studies by David et al. (1979), Amit et al. (1982
), Aitken et al. (1992
) and Ivic et al. (2002
) are different from all others because they use the number of swim-up sperm per high power field at 10, 20 or 30 mm as the test diagnostic criterion (i.e. SMPTsc). There was a substantial difference between the pooled results for these four primary studies; LR+ and LR respectively are 5.24 (3.368.18) and 0.15 (0.060.39), and the other 14 studies in which vanguard distance was used. These findings suggest that swim-up sperm count per high power field is a more accurate diagnostic criterion than vanguard distance for SMPT.
Likelihood ratios from primary studies using slightly different thresholds can be pooled if the end points had been classified clearly and such threshold variations do not significantly modify long-term prognosis (Deeks, 2001). Dickey et al. (1999
) have shown that variations above the diagnostic thresholds of 5x106/ml for total sperm count, and 30% for progressive motility, produced only minimal and insignificant changes in fertility rates. Dickeys group studied 1841 couples undergoing 4056 cycles of intrauterine inseminations and demonstrated that there was very little difference in the effect on fertility rates between normal values of total and progressive motility. They went further to demonstrate that only when initial values of total motile sperm count were <5x106/ml, and progressive motility <30%, was fertility significantly affected. In our review, only 6.9% of the samples in our study had a reference threshold of 25% progressive motility, therefore the only valid ground for sub-grouping was based on the SMPT diagnostic criteria (i.e. whether vanguard distance or swim-up sperm concentration).
Cervical mucus plays an important role in selecting motile, mostly morphologically normal sperm for fertilization. For this reason, the SMPT has always held a potential as an important in-vitro sperm function test. However, human cervical mucus (hCM), apart from being difficult to obtain in large quantities, possesses wide variations in viscosity (Karni, 1971; World Health Organization, 1987; Eggert-Kruse et al., 1989a
;b). As a result, cervical mucus substitutes in many forms are now commonly used in these tests. Bovine cervical mucus (bCM) is particularly common because it is easier to obtain in large quantities, has similar rheological and biochemical properties to hCM and can be stored in the frozen stage with only minimal changes in rheological properties (Meyer, 1977
; Gaddum-Rosse et al., 1980
; Lee et al., 1981
). However, bCM has the disadvantage of between-batch variability in consistency. Hyaluronic acid (HA) has been described as a suitable alternative. It is a muco-polysaccharide with a structure similar to that of cervical mucus (Bothner and Wik, 1987
) and a viscosity that can easily be varied to that described for hCM (Karni 1971
; Ishijima et al., 1986
). More recently, however methylcellulose (MC) has been evaluated as an effective sperm swim-up medium, which is comparable with HA in efficacy (Ivic et al., 2002
). Most included primary studies tested sperm penetration of either bCM or hCM, using sperm motility or progressive motility as the reference standard. It was somewhat surprising that not many eligible primary studies were identified that tested artificial substitutes for cervical mucus such as HA and MC.
There were, however, early problems with accurate quantification of swim-up sperm concentration into capillary tubes and micropipettes with circular cross-sections (Kremer, 1965; Ulstein, 1973
; Kerin et al., 1976
). This problem, well described by Kremer and Kroeks (1975
) is the optical effect due to curvature of the circular walls, which rendered sperm underneath grossly distorted. It was partially solved with the subsequent introduction of generations of flat capillary tubes. The most popular for many years had external measurements of either 0.3x3x50 mm (De Geyter et al., 1988
) or 0.3x3x100 mm (Kotoulas et al., 1984
; Pandya et al., 1986
; Morrow et al., 1992
). However, loss of accuracy in counting within the sides of the flat tubes had continued to be problematic partly due to distortion and also because sperm tended to swim against the surface of the circular walls. This may have accounted partly for the continued adoption of vanguard distance, which is more easily measured, as SMPT threshold. More recently, the accuracy of quantifying sperm migration into cervical mucus substitutes in flat capillary tubes has been improved by newer designs with central counting chambers, away from the circular walls. One such design (Camlab Ltd, UK) has outer dimensions of 1.2x4.8x50 mm, an inner depth of 0.4 mm, and engraved counting frames at intervals of 10, 20, 30 and 40 mm on the tube. Using this design, it was shown that mean count of 39 sperm per high power field at 10 mm, after 30 min incubation at 37°C, is the optimum threshold criterion (Ivic et al., 2002
).
In conclusion, SMPTvd has a low accuracy in the evaluation of semen. However, SMPTsc was found to be moderately accurate in assessing sperm motility in semen. This method of using sperm concentration, instead of vanguard distance, as diagnostic criteria of in-vitro SMPT has potential as a useful laboratory-based sperm function test.
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Acknowledgements |
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Appendix A. List of excluded studies |
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Amit, A., Bergman, A., Yedwab, G., David, M.P., Homonnai, T.Z. and Paz, G. (1982) Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. II. Pattern and velocity of penetration. Int. J. Fertil., 27, 160165.
Bergman, A., Amit, A., David, M.P., Homonnai, T.Z. and Paz, G. (1981) Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. I. Correlation between penetration values. Fertil. Steril., 36, 363367.
Biljan, M.M., Taylor, C.T., Manasse, P.R., Joughin, E.C., Kingsland, C.R. and Lewis-Jones, D.I. (1994) Evaluation of different sperm function tests as screening methods for male fertilization potentialthe value of the sperm migration test. Fertil. Steril., 62, 591598.
Blasco, L., Sokoloski, J.E. and Wolf, D.P. (1979) A practical, objective approach to the evaluation of sperm and cervical mucus in humans. Fertil. Steril., 32, 5560.
Clarke, G.N. (1997) A simplified quantitative cervical mucus penetration test. Hum. Reprod., 12, 11841187.
Clarke, G.N., Garrett, C. and Baker, H.W. (1998) Quantitative sperm mucus penetration: modified formulae for calculating penetration efficiency. Hum. Reprod., 13, 12551259.
De Geyter, C., Bals-Pratsch, M., Doeren, M., Yeung, C.H., Grunert, J.H., Bordt. J., Schneider, H.P. and Nieschlag, E. (1988) Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization. Hum. Reprod., 3, 948954.
Eggert-Kruse, W., Leinhos, G., Gerhard, I., Tilgen, W. and Runnebaum, B. (1989) Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus. Fertil. Steril., 51, 317323.
Eggert-Kruse, W., Schwalbach, B., Rohr, G., Klinga, K., Tilgen, W. and Runnebaum, B. (1993) Evaluation of polyacrylamide gel as substitute for human cervical mucus in the sperm penetration test. Fertil. Steril., 60, 540549.
Eggert-Kruse, W., Reimann-Andersen, J., Rohr, G. Pohl, S., Tilgen, W. and Runnebaum, B. (1995) Clinical relevance of sperm morphology assessment using strict criteria and relationship with spermmucus interaction in vivo and in vitro. Fertil. Steril., 63, 612624.
Engel, S. and Petzoldt, R. (1999) Human sperm penetration in different media. Andrologia, 31, 233239.
Engel, S., Schachschal, G. and Petzoldt, R. (1990) Significance of linear cervix mucus penetration for the evaluation of spermatozoa quality. Andrologia, 22, 347353.
Esfandiari, N., Rouzrokh, A. and Saremi, A.T. (2000) Effects of cryopreservation on penetration ability of human spermatozoa into bovine cervical mucus. Arch. Androl., 45, 233238.
Farhi, J., Valentine, A., Bahadur, G., Shenfield, F., Steele, S.J. and Jacobs, H.S. (1995) In-vitro cervical mucussperm penetration tests and outcome of infertility treatments in couples with repeatedly negative post-coital tests. Hum. Reprod., 10, 8590.
Ikuma, K., Saito,Y., Takeda, M., Koyama, K. and Isojima, S. (1988) Bovine cervical mucus as a substitute of human cervical mucus. Nippon Sanka Fujinka Gakkai Zasshi, 40, 888894.
Ikuma, K., Suno, S., Hasegawa, A., Koyama, K. and Isojima, S. (1989) Role of sperm passage through cervical mucus: fertilizing capacity tested by in vitro fertilization with zona-free hamster eggs. Nippon Sanka Fujinka Gakkai Zasshi, 41, 167172.
Jonsson, B., Eneroth, P., Landgren, B.M. and Wikborn, C. (1986) Evaluation of in vitro sperm penetration testing of 176 infertile couples with the use of ejaculates and cervical mucus from donors. Fertil. Steril., 45, 353356.
Jonsson, B., Landgren, B.M. and Eneroth, P. (1989) Repeated mid-cycle tests of in-vitro sperm penetration (Kremer tests) in healthy women during three menstrual cycles. Hum. Reprod., 4, 670673.
Katzorke, T., Propping, D. and Kolodziej, F.B. (1985) Crossed sperm-penetration-meter test. Prognostic value in male subfertility. Andrologia, 17, 234240.
Kerin, J.F., Matthews, C.D., Svigos, J.M., Makin, A.E., Symons, R.G. and Smeaton, T.C (1976) Linear and quantitative migration of stored sperm through cervical mucus during the periovular period. Fertil. Steril., 27, 10541058.
Kolodziej, F.B., Katzorke, T. and Propping, D. (1986) Prognostic value of the sperm-penetration-meter test according to Kremer. Andrologia, 18, 539544.
Kotoulas, I.G., Burgos-Briceno, L.A., Arana, J., Balmaceda, J.P. and Asch, R.H. (1984) Human sperm penetration in bovine cervical mucus clinical studies. II. Use of split ejaculates. Fertil. Steril., 42, 268273.
Kremer, J. and Kroeks, M.V. (1975) Modifications of the in vitro spermatozoal penetration test by means of the sperm penetration meter. Acta Eur. Fertil., 6, 377380.
Lee, W.I., Gaddum-Rosse, P. and Blandau, R.J. (1981) Sperm penetration into cervical mucus in vitro. III. Effect of freezing on estrous bovine cervical mucus. Fertil. Steril., 36, 209213.
Morrow, A., Drudy, L., Gordon, A. and Harrison, R.F. (1992) Evaluation of bovine cervical mucus penetration as a test of human spermatozoal function for an in vitro fertilization programme. Andrologia, 24, 323326.
Neuwinger, J., Cooper, T.G., Knuth, U.A. and Nieschlag, E. (1991) Hyaluronic acid as a medium for human sperm migration tests. Hum. Reprod., 6, 396400.
Niederberger, C.S., Lamb, D.J., Glinz, M., Lipshultz, L.I. and Scully, N.F. (1993) Tests of sperm function for evaluation of the male: Penetrak and Tru-Trax. Fertil. Steril., 60, 319323.
Pandya, I.J., Mortimer, D. and Sawers, R.S. (1986) A standardized approach for evaluating the penetration of human spermatozoa into cervical mucus in vitro. Fertil. Steril., 45, 357365.
Randall, J.M. and Templeton, A. (1991) Cervical mucus score and in vitro sperm mucus interaction in spontaneous and clomiphene citrate cycles. Fertil. Steril., 56, 465468.
Sharara, F.I., Beatse, S.N., Bailey, S.A., Neal G.S., Coddington, C.C. 3rd and Scott, R.T. Jr (1994) Characterization of Tru-Trax in-vitro penetration testing of cervical mucus. Hum. Reprod., 9, 20272031.
Sharara, F.I., Illions, E.H., Coddington, C.C. 3rd and Scott, R.T. Jr (1995) Evaluation of the Tru-Trax cervical mucus penetration test in predicting fertilization and pregnancy rates in in-vitro fertilization. Hum. Reprod., 10, 14811485.
Shibahara, H., Naito, S., Hasegawa, A., Mitsuo, M., Shigeta, M. and Koyama, K. (1997) Evaluation of sperm fertilizing ability using the Sperm Quality Analyzer. Int. J. Androl., 20, 112117.
Sokol, R.Z., Madding, C.I., Handelsman, D.J. and Swerdloff, R.S. (1986) The split ejaculate: assessment of fertility potential using two in vitro test systems. Andrologia, 18, 380386.
Tang, S., Garrett, C. and Baker, H.W. (1999) Comparison of human cervical mucus and artificial sperm penetration media. Hum. Reprod., 14, 28122817.
Ulstein, M. (1972) Sperm penetration of cervical mucus as a criterion of male fertility. Acta Obstet. Gynecol. Scand., 51, 335340.
Ulstein, M. (1973a) In vitro sperm penetration of cervical mucus and male fertility. Andrologie, 5, 189191.
Ulstein, M. (1973b) Fertility, motility and penetration in cervical mucus of freeze-preserved human spermatozoa. Acta Obstet. Gynecol. Scand., 52, 205210.
Urry, R.L., Carrell, D.T., Hull, D.B., Middleton, R.G. and Wiltbank, M.C. (1983) Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa. Fertil. Steril., 39, 690694.
Urry, R.L., Middleton, R.G. and Mayo, D. (1986) A comparison of the penetration of human sperm into bovine and artificial cervical mucus. Fertil. Steril., 45, 135137.
Zhu, J.J., Barratt, C.L.R. and Cooke, I.D. (1992) Effect of human cervical mucus on human sperm motion and hyperactivation in vitro. Hum. Reprod., 7, 14021406.
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Appendix B. Definitions of some commonly used terms in diagnostic accuracy studies using examples from a 2x2 table |
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Submitted on April 26, 2002; resubmitted on January 9, 2003; accepted on January 15, 2003.