SIMPLIFIED MATHEMATICAL MODEL TO EVALUATE SPERM CONCENTRATION IN KREMER S CAPILLARY TUBE TEST: A PRELIMINARY STUDY REPORT

Transcription

1 S. Tang, et al ORIGINAL ARTICLE SIMPLIFIED MATHEMATICAL MODEL TO EVALUATE SPERM CONCENTRATION IN KREMER S CAPILLARY TUBE TEST: A PRELIMINARY STUDY REPORT Stephen Tang, Hung-Sheng Chen, Jau-Nan Lee, Eing-Mei Tsai* Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, and Department of Obstetrics and Gynecology, Guoren Hospital, Kaohsiung, Taiwan. SUMMARY Objective: The in vitro cervical mucus penetration assay, which is rarely used now, plays a role in basic research and is used to determine the clinical importance of cervical hostility and immunologic causes of infertility. Sperm concentration observed in a capillary tube test, where mucus-filled cylindrical tubes are placed with an end immersed in a semen reservoir mounted on a glass slide, indicates general sperm function and has a prognostic value for subsequent in vivo pregnancy. Because cylindrical tubes are optically inferior and may hinder the counting of sperm, the assessment of the efficiency of sperm penetration into cervical mucus is a time-consuming procedure and requires complex calculations. The present study investigated the feasibility of applying a simplified calculation model to estimate sperm concentration using the results of a capillary tube test. The postulated equation (sperm concentration N 1 3 /.339 sperm/ml) was derived from a mathematical model of definite integrals used to calculate the volume of intersection between two cylinders. Materials and Methods: Semen samples from 46 male subjects were allowed to penetrate mid-cycle human cervical mucus contained in cylindrical tubes and incubated at 37 C for 6 minutes. Assessments of sperm concentration in the mucus column were performed using the postulated equation and a control method whereby the total number of sperm was assessed after extracting all contents from the capillary. Results: Estimates of sperm concentration obtained using the postulated equation ( /ml) were similar and correlated with those obtained using the control method ( /ml; r =.79; p <.5). The mean difference in sperm concentration was not significant ( /ml). Conclusion: The strong correlation of sperm concentrations between these two methods suggests that the postulated equation may provide a simplified calculation model to indicate penetration efficiency in the capillary tube test. [Taiwanese J Obstet Gynecol 25;44(1):36 41] Key Words: capillary tube test, cervical mucus penetration, fertility investigation Introduction Sperm function is multifaceted and sperm exhibit a range of different properties to penetrate the cervical mucus and migrate to the site of fertilization. Thus, sperm quality cannot be measured by any single variable. *Correspondence to: Dr. Eing-Mei Tsai, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, 1 Tzyou 1 st Road, Kaohsiung 87, Taiwan. chs@kmu.edu.tw Received: June 16, 24 Revised: August 3, 24 Accepted: October 28, 24 In general, the most important sperm characteristic in routine clinical semen analysis is the number of sperm with normal motility and morphology that have normal functional capacities [1]. Because penetrating the cervical mucus is the first obstacle that sperm must overcome to achieve conception in vivo, evaluation of the interaction between sperm and cervical mucus in vitro has substantial physiologic and clinical significance. The fertilizing capacity and prognosis for pregnancy of sperm are correlated with the degree of cervical mucus penetration [2 4]. Since the sperm mucus interaction is susceptible to the influence of a multiplicity of properties of semen and cervical mucus including hormonal, immunologic, microbial, and even psycho- 36

2 Simplified Mathematical Model to Evaluate Sperm Concentration sexual factors, the evaluation of cervical mucus penetration may be recommended as an integral part of infertility investigation. Various attempts have been made to develop reliable tests of the ability of sperm to penetrate the cervical mucus [5]. Two techniques have been used for the investigation of this type of test: the slide method and the capillary tube system [6 9]. The capillary tube test system measures the ability of sperm to penetrate a column of cervical mucus within a capillary tube [8,9]. Sperm penetration of the media in a capillary tube has been studied for more than 5 years. An early study reported that mid-cycle cervical mucus was optimal for sperm penetration, with a speed of sperm progression ranging from 1 to 3 mm/min [1]. In 1956, Botella-Llusia, using a capillary tube filled with Ringer solution, noticed that the average speed of progression of sperm was 5 mm in 2 hours [11]. In the early 196s, several researchers indicated that the penetration of sperm into different media was correlated with their density, motility, and morphology [12,13]. A number of researchers reported that capillary tests of mucus penetration were useful for determining male infertility [12,13]. In 1965, Kremer developed a method for examining sperm penetration by filling a capillary tube with mid-cycle cervical mucus without air bubbles and closing one end with modeling clay [8]. Sperm penetration of the mucus is assessed at different time intervals, using distances of penetration, concentration of sperm, and character of motility at various levels. Donor sperm and donor cervical mucus or artificial media have also been used for cross-over tests to determine whether the cervical mucus is at fault in cases of apparent cervical hostility. Likewise, reduced sperm penetration of donor cervical mucus and artificial media has been used in assessing the clinical significance of antisperm antibodies [14 18]. Problems associated with the capillary tube system include the menstrual cycle-related variability of the visco-elasticity and the composition of human cervical mucus, limited availability of optimal-quality cervical mucus, and technical difficulties in assessing the characteristics of sperm penetration in an optically inferior round tube. In view of these, attempts have been made to develop a simplified yet reliable calculation model to assess the efficiency of sperm penetration into cervical mucus using the capillary tube test. The postulated equation used in the present study was derived from a mathematical model to calculate the volume of the intersection between two cylinders. The theoretical basis is that revolving a plane area about one line (i.e. axis of revolution) on the plane will generate a solid of revolution, whereas a rectangle rotated about one side will generate a cylinder, which in the present circumstance is the volume inside a round capillary tube. Materials and Methods Human cervical mucus Cervical mucus was collected with consent from women visiting the infertility clinic for artificial insemination. All samples were collected during mid-cycle within a 3-day interval before ovulation, by aspiration from the cervical canal into a sterile tuberculin syringe (Terumo Medical Corp, Elkton, MD, USA) and tested for spinnbarkeit and clarity as described previously [17,19]. Aliquots of normal cervical mucus were stored in small tubes at 4 C and used within 7 days [2]. Semen Semen was collected by masturbation after abstinence from ejaculation for 3 5 days from 46 male patients visiting our infertility clinic. Semen analysis was performed according to the World Health Organization (WHO) criteria. In this study, semen samples were selected on the basis of normal sperm concentration (mean, sperm/ml), progressive motility (mean, %; median, 63%; range, 38 88%), and lack of sperm-bound antibodies (< 5% motile sperm with adherent particles) using the mixed agglutination reaction (MAR) test [19]. Capillary tube test The capillary tube test used circular cross-section tubes 1 mm in length with an inner diameter of.798 mm (5 L Drummond microcaps, Edwards Instruments, Melbourne, Australia). They were filled by aspiration using a 1 ml syringe with a plastic tube attached to the end of the capillary tube, while the other end was dipped into the pool of the mucus. A 7 8 cm column of mucus was aspirated into the capillary tube so that the upper meniscus was 2 3 cm from its upper end. Air bubbles were prevented from being trapped inside the mucus column and the upper end of the capillary tube was sealed with plasticine. Excess mucus trailing at the lower end was trimmed off to produce a flat interface such that it was level with the capillary end, ensuring that the surface area of the sperm mucus interaction was approximately equal to the cross-sectional area of the capillary tube. Approximately 1 L of liquefied semen was placed in the bottom of a semen reservoir of semicircular cross-section (radius, about 3.5 mm). The open end of the capillary tube containing cervical mucus was placed into the semen reservoir, which was glued onto a glass slide, a construction called a sperm penetration meter, as described in the WHO manual [19]. The 37

3 S. Tang, et al sperm penetration meter was then placed in a covered Petri dish containing damp sponges on the sides (to maintain humidity and prevent drying of the semen and mucus) and was incubated in the horizontal position to avoid the effect of gravity on sperm migration. After 6 minutes of incubation at 37 C, the sperm penetration meter was removed from the Petri dish and the capillary tube was viewed under a bright field with a 2 phase contrast objective lens and 1 oculars. The stage was adjusted to select a focal plane incorporating the central axis of the capillary tube and, at the chosen magnification, the microscopic field width approximated the inner diameter of the capillary tube. The distance from the end of the capillary tube in the semen reservoir to the vanguard sperm observed in the tube was defined as the migration distance. Penetration concentration was determined at half the migration distance and within 1 cm of it at the same magnification, while focusing from the lower to upper wall of the capillary in a single pass (Figure 1). At each distance, the mean number of sperm per power field (2 1) was determined as the mean of three adjacent low-power fields. The volume of mucus sampled during the counting was estimated as.34 L, using the formula 16 R 3 /3 for the volume of intersection of two cylinders of radius R, and sperm concentration was estimated using the postulated equation (sperm concentration N 1 3 /.339 sperm/ml, where N = mean number of sperm) derived from the mathematical model of definite integrals for the calculation of the volume of a cylinder. Using a circular cross-section capillary tube with a volume of 5 L, a length of 1 mm, and an inner diameter of approximately mm, the volume of a cross-sectional field was estimated as described below: For a 2 objective (2 magnification), the field diameter was.88 mm. The volume of the intersection of the two cylinders (with radius R and length Z) focusing a right angle cross was calculated. The intersection slice in the place of the cross formed by intersecting cylinders was a square (Figures 2 and 3). R 2 = X 2 + Z 2 X 2 = (R 2 Z 2 ) Area of square section = (2X) 2 Volume = 2 R (2x) 2 dz = 8 R (R 2 Z 2 )dz = 8 R [R 2 Z Z 3 /3] = 8 R 3 [1 1/3] = 16 R 3 /3 Figure 2. Intersection slice in the plane formed by the intersecting cylinders is a square. 1/2 intersecting volume R Z Figure 1. For a 2 objective (2 magnification), the field diameter is.88 mm. The volume of the intersection of the two cylinders (radius R) focusing at right angles across a cylinder. Figure 3. Right-angle cross focused by the volume of intersection of two cylinders with radius R. 38

4 Simplified Mathematical Model to Evaluate Sperm Concentration Since the inner diameter of the capillary tube was mm, the volume of the capillary tube was.339 L. Therefore, the sperm concentration is approximately N 1,/.339 sperm/ml. (For example, if the mean number of sperm per power field is 2, the calculated sperm concentration is /ml). In this study, the sperm extraction method used in the control model was based on a previously described method for the recovery of sperm from cervical mucus to assess sperm concentration in capillary tube tests [17]. After the capillary tube test, the mounted capillary tube was removed from the semen reservoir and placed with its mucus end into a round-bottom test tube containing Bromelain solution (2 mg/ml, diluted with Tyrode s solution) for 1 2 minutes to remove the adherent sperm. The mucus was then expelled into 1 L of Bromelain (1:4 dilution) and the mixture was incubated at 37 C for 3 minutes. After dissolving the mucus, the total number of sperm was assessed using a hemocytometer [17,21]. Statistical analysis Results are presented as mean standard deviation unless otherwise specified. Sperm concentration was analyzed using Student s t test. Correlations were sought by non-parametric correlation analysis (Spearman test). A two-sided p value of less than.5 was considered significant. Differences were not significant unless indicated. Mean concentration of sperm ( 1,/mL) Control method Postulated calculation Number of semen sample Figure 4. Mean concentration of sperm obtained using two calculation models (mean values, n = 46). Sperm concentration ( 1,/mL) obtained by the control method y = Sperm concentration ( 1,/mL) obtained by the postulated method Figure 5. Relationship between sperm concentration obtained using two calculation models (n = 46, r =.79, p <.5). Results A total of 46 capillary tube tests were conducted with 46 semen samples. Figure 4 shows the results obtained using the two calculation models. All data represent the mean values obtained from each calculation model. The mean sperm number assessed using the postulated equation was with a median of 48.5 (range, 26 71). There was no significant difference in sperm concentrations determined using the postulated equation ( /ml) and the control method ( /ml). The mean difference ( /ml) was not significant. Figure 5 illustrates the strong correlation between the results obtained using the two methods (r =.79; p <.5). Discussion The rationale for attempting to develop a simplified calculation model for assessing penetration efficiency of sperm originates from previous work suggesting an association between cervical mucus penetration and the fertilizing ability of semen [22 25]. Some researchers also indicated that sperm motility and the concentration of motile sperm were important indicators of fertility [26,27]. Nevertheless, the assessment of characteristics of motility is difficult, as it depends on sophisticated equipment and expertise to attain accuracy or to avoid subjectivity. The capillary tube test is a principal procedure used to study cervical mucus penetration. It is unique among methods of investigation of sperm cervical mucus interactions in vitro in that it is relatively easy to perform, to quantify, and to determine the longevity of sperm and the distance sperm travel in cervical mucus [13,28 31]. Early in 198, Kremer and Jager found significant correlation between the shaking phenomenon, failure of cervical mucus penetration, and sperm-agglutinin titers in seminal plasma and cervical mucus, signifying that it could also be used for screening for immunologic causes of infertility [7]. Some researchers suggest that assessing the sperm s ability to penetrate cervical mucus 39

5 S. Tang, et al provides useful information on the in vitro fertilization (IVF) performance of the semen [25,32,33]. One researcher reported that some of the same sperm functions involved in cervical mucus penetration were essential in the penetration of the cumulus oophorus and zona pellucida [34]. At present, capillary tube testing is recognized as clinically important in the diagnosis and continued assessment of sperm autoimmunity [16,19,35 39]. In addition to antisperm antibodies, the results of this test are mainly influenced by the concentration of mobile sperm in the semen [17, 21,4 45]. There are a number of parameters of cervical mucus penetration, including the penetration index [8, 19], quantitative mucus penetration, and percentages of successful collisions [21,41]. From the clinical perspective, sperm concentration may be useful as an index of the ability of sperm to penetrate the cervical mucus and may thereby provide a useful marker of potential fertility. There are potential advantages in clinical and research arenas for the development of a simple and reliable method of assessing the concentration of sperm. In this study, we showed that the postulated equation provides a numerical value that reflected a variety of aspects of sperm penetration of the cervical mucus. The mean sperm concentration correlated well with the control calculation model. Although the results of these two approaches were comparable, there was an overall trend toward an increase in sperm concentration with the control method. The results were further complicated by a few potential outliers in the data obtained using the postulated method with inconsistent and higher sperm concentration readings, compared with the control method. This indicated a potential disadvantage in the counting procedure. However, the other samples appeared to fit the predicted model. In this study, the penetration concentration was assessed at half the migration distance traveled by the vanguard sperm 1 cm, focusing on the lower to upper wall of the tube in a single pass. The disadvantage was that a group of sperm may have been missed or counted repeatedly, especially when the postulated approach was based on the assumption that a selected subpopulation of sperm exhibits the overall penetration efficiency of the semen sample. Such errors were readily detectable when the number of sperm assessed by the postulated method was higher than that from the control model. Other obstacles arising from the routine application of the capillary tube test included difficulty in the collection and storage of mid-cycle or estrogen-stimulated cervical mucus and ensuring that sperm motility was not zero as sperm movement is responsible for sperm penetration. Although the capillary tube test cannot replace a properly performed semen analysis, it provides a semiquantitative measure of sperm function, and it may add predictive value to the sperm analysis. Because IVF depends on multiple properties of sperm, many of which are assessed in cervical mucus penetration, the capillary tube test can assist in predicting sperm performance in IVF and in detecting a subgroup of malefactor cases that are not identified using routine semen analysis. The simplicity and objectivity of this test may be useful in situations where immediate information on penetration efficiency is required in the absence of sophisticated facilities and expertise, as in infertility investigations and semen testing during sperm donor selection or artificial insemination. Likewise, it can be used with donor sperm and donor mucus in the crosshostility format and in the diagnosis of immunologic infertility in the male through the screening of antisperm antibody if the immunobead test or MAR test is not available. In conclusion, the existence of a strong correlation in terms of sperm concentration between the two approaches adopted in this study suggests that the postulated equation can serve as a simplified calculation model to estimate the efficiency of cervical mucus penetration in capillary tube tests performed in round tubes. However, it remains to be determined whether the capillary tube test can be used in a flat capillary tube system with a rectangular lumen that may scatter light differently. Further evaluation of a refined model providing greater accuracy may provide insight into the relationships between cervical mucus penetration and fertilizing ability. References 1. Mortimer D. Practical Laboratory Andrology. New York: Oxford University Press, 1994: Alexander NJ. Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril 1972;36: Eggert-Kruse W, Leinhos G, Gerhard I, Tilgen W, Runnebaum B. Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus. Fertil Steril 1989;51: Ulstein M. Sperm penetration of cervical mucus as a criterion of male infertility. Acta Obstet Gynecol Scand 1972;51: Matthews CD, Makin AE, Cox LW. Experience with in vitro sperm penetration testing in infertile and fertile couples. Fertil Steril 198;3: Miller EG, Kurzrok R. Biochemical studies of human semen, factors affecting migration of sperm through the cervix. Am J Obstet Gynecol 1932;24: Kremer J, Jager S. The sperm cervical mucus contact test: a preliminary report. Fertil Steril 1976;27:

6 Simplified Mathematical Model to Evaluate Sperm Concentration 8. Kremer J. Preliminary report: a simple sperm penetration test. Int J Fertil 1965;1: Kremer J. Fertility Investigation Thesis 24. Van Denderen: Groningen, Lamar JK, Shettles LB, Delfs E. Cyclic penetrability of human cervical mucus to sperm in vitro. Am J Physiol 194;129: Botella-Llusia J. Measurement of linear progression of the human spermatozoa as an index of male fertility. Int J Fertil 1956;1: Bergman P, Gennser G. Investigation of sperm migration in artificial medium. Acta Obstet Gynecol Scand 1958;2: Botella-Llusia J, Ruiz-Velasco V. Postcoital test in vitro. Int J Fertil 196;5: Ansbacher R, Keung-Yeung K, Behrman J. Clinical significance of sperm antibodies in infertile couples. Fertil Steril 1973;24: Jager S, Kremer J, Kuiken J. Induction of the shaking phenomenon by pre-treatment of spermatozoa with sera containing anti-spermatozoal antibodies. Fertil Steril 1981; 36: Baker HWG, Clarke GN, Hudson B, McBain JC, McGowan MP, Pepperell RJ. Treatment of sperm auto-immunity in men. Clin Reprod Fertil 1983;2: Clarke GN, Garrett C, Baker HWG. Quantitative sperm mucus penetration: modified formulae for calculating penetration efficiency. Hum Reprod 1998;13: Tang S, Garrett C, Baker HWG. Comparison of human cervical mucus and artificial sperm penetration media. Hum Reprod 1999;14: World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge: Cambridge University Press, Kroeks MVAM, Kremer J. The role of cervical factors in infertility. In: Pepperell RJ, Hudson B, Wood C, eds. The Infertile Couple. Edinburgh: Churchill Livingstone, 198: Clarke GN. A simplified quantitative cervical mucus penetration test. Hum Reprod 1997;13: Mills RN, Katz DF. A flat capillary tube system for assessment of sperm movement in cervical mucus. Fertil Steril 1978;29: De Geyter C, Bals-Pratsch M, Doeren M, et al. Human and bovine cervical mucus penetration test of sperm function for in-vitro fertilization. Hum Reprod 1988;3: Barratt CLR, Osborn JC, Harrison PE, et al. The hypoosmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte. Hum Reprod 1989;4: Berberoglugil P, Englert Y, Van den Bergh M, Rodesch C, Bertrand E, Biramane J. Abnormal sperm-mucus penetration test predicts low in vitro fertilization ability of apparently normal semen. Fertil Steril 1993;59: Francavilla F, Romano R, Santucci R, Poccia G. Effect of sperm morphology and motile sperm count on outcome of intrauterine insemination in oligozoospermia and/or asthenozoospermia. Fertil Steril 199;53: Johnston RC, Kovacs GT, Lording DH, Baker HWG. Correlations of semen variables and pregnancy rates for donor insemination: in 15-year retrospective. Fertil Steril 1994;61: Kurzrok R, Miller EG. Biochemical studies of human semen. III. Factors affecting migration of sperm through the cervix. Am J Obstet Gynecol 1932;24: Jones WR. Immunological aspects of infertility. In: Scott JS, Jones WR, eds. Immunology of Reproduction. New York: Grunne Stratton, 1977: Beer AE, Neaves WB. Antigenic status of semen from the view points of the female and male. Fertil Steril 1978;29: Ingerslev HJ. Sperm-agglutinating antibodies and sperm penetration of cervical mucus from infertile women with sperm agglutinating antibodies in serum. Fertil Steril 198; 34: Englert Y, Vekemans M, Lejenune B, et al. Higher pregnancy rates after in vitro fertilization and embryo transfer in cases with sperm defect. Fertil Steril 1987;48: Keel BA, Webster BW. Correlation of human sperm motility characteristics with an in vitro cervical mucus penetration test. Fertil Steril 1988;49: Katz DF, Morales P, Samuels SJ, et al. Mechanisms of filtration of morphologically abnormal sperm by cervical mucus. Fertil Steril 199;54: Clarke GN, Elliott PJ, Samaila C. Detection of sperm antibodies in semen using the immunobead tests: a survey of 813 consecutive patients Am J Reprod Immunol Microbiol 1985;7: Clarke GN, Lopata A, McBain JC, et al. Effect of sperm antibodies in males on human in vitro fertilization (IVF). Am J Reprod Immunol Microbiol 1985;8: Wang C, Baker HWG, Jennings MG, et al. Interaction between human cervical mucus and sperm surface antibodies. Fertil Steril 1985;44: Kremer J, Jager S. Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies. Hum Reprod 1988;3: Kremer J, Jager S. The significance of antisperm antibodies for sperm-cervical mucus interaction. Hum Reprod 1992;7: Insler V, Bernstein D, Glezerman M, et al. Correlation of seminal fluid analysis with mucus-penetrating ability of spermatozoa. Fertil Steril 1979;32: Katz DF, Drobnis EZ, Overstreet JW. Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res 1989;22: Katz DF, Overstreet JW, Hanson FW. A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 198; 33: Overstreet JW, Coates C, Katz DF, et al. The importance of seminal plasma for sperm penetration of human cervical mucus. Fertil Steril 198;34: Aitken RJ, Sutton M, Warner P, et al. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73: Ford WC, Ponting FA, McLaughlin EA, et al. Controlling the swimming speed of human sperm by varying the incubation temperature and its effect on cervical mucus penetration. Int J Androl 1992;15: