1 Prince Henry's Institute of Medical Research, Clayton, Victoria 3168 and 2 University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Carlton, Victoria 3053, Australia
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Abstract |
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Key words: cervical mucus/mucus substitute/sperm autoimmunity/sperm motility/spermmucus penetration test
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Introduction |
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An important clinical use of the spermmucus penetration test is in the diagnosis of sperm autoimmunity (Baker et al., 1983; Clarke et al., 1985a
,b
; Wang et al., 1985
; Kremer and Jager 1988, 1992; WHO, 1999). Spermatozoa heavily coated with antisperm antibodies are unable to penetrate cervical mucus. Thus, cervical mucus penetration can be used to determine the clinical importance of positive sperm antibody tests. Men with positive sperm antibody tests but with spermcervical mucus penetration values of >2 cm at 1 h produce pregnancies in their partners at reasonable rates, whereas those with no mucus penetration have a very poor prognosis for producing pregnancies (Baker et al., 1983
; Clarke et al., 1985b
; Wang et al., 1985
; Kremer and Jager, 1988, 1992; Eggert-Kruse et al., 1991
; Barratt et al., 1992). The latter benefit from treatment with glucocorticoids or intracytoplasmic sperm injection (Baker et al., 1983
; Wang et al., 1985
; Hendry et al., 1990
; Clarke et al., 1997
). The WHO manual states the continuing need to use a mucus penetration test to assess the significance of a positive sperm antibody test (WHO, 1999). In addition to sperm antibodies, sperm concentration and motility influence spermcervical mucus penetration results (Insler et al., 1979
; Katz et al., 1980
, 1990
; Overstreet et al., 1980
; Aitken et al., 1985
; Ford et al., 1992
; Clarke, 1997
, 1998). Sperm morphology is also an influential factor (Fredricsson and Bjork, 1977
; Insler et al., 1979
; Jeulin et al., 1985
; Katz et al., 1990
; Eggert-Kruse et al., 1995
, 1996
).
There are several problems associated with the use of human cervical mucus for laboratory tests. In particular, the menstrual cycle-related variability of the visco-elasticity and composition of mucus necessitates collection of samples only at mid-cycle or after treatment with oestrogens. The mixing of vaginal fluid with mucus during collection may make the sample unsuitable because acid (pH <6.5) immobilizes spermatozoa. There is also limited availability of high quality cervical mucus because of the small amounts available for collection and the variability of mucus quality with storage. In addition, there may be technical difficulties with filling capillary tubes with cervical mucus. For these reasons there have been attempts to establish suitable human cervical mucus substitutes, including bovine cervical mucus, hen egg white, hyaluronate and gels of polyacrylamide (Kummerfeld et al., 1981; Lorton et al., 1981
; Goldstein et al., 1982
; Urry et al., 1986
; Marshburn et al., 1989
; Eggert-Kruse et al., 1990
, 1993
; Mortimer et al., 1990
; Aitken et al., 1992
; Morrow et al., 1992
; Niederberger et al., 1993
; Sharara et al., 1994
; Perry et al., 1996
). In general, the ideal mucus substitute should be cheap, easily prepared, stable and, most importantly, possess the same sperm penetrability characteristics as human cervical mucus. Furthermore, to be useful for clinical management of male immunological infertility, the substitute must show a similar sensitivity of the penetration test to antibody-coated spermatozoa as does cervical mucus (Baker et al., 1983
; Wang et al., 1985
; Kremer and Jager 1988; WHO, 1999).
The aim of this study was to compare hyaluronate solution and hen egg white with human cervical mucus with respect to sperm penetration in capillary tube tests. Albumin Tyrode solution was used as a control because it contains no hydrated polymers and should reflect sperm penetration in an isotropic medium.
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Materials and methods |
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Penetration media
Cervical mucus was collected with consent from a number of different spontaneously ovulating women having artificial insemination. All samples were collected during midcycle within a 3 day interval before ovulation. Mucus was aspirated from the cervical canal into a polyethylene tube. After collection the cervical mucus was tested for spinbarkeit, clarity and pH (WHO, 1999). Samples of normal cervical mucus (pH >7.0) were stored in small tubes at 4°C and used within 7 days. Only mucus with normal penetration by fertile donor spermatozoa was used for the tests.
The hyaluronate was from Sigma (St Louis, MO, USA; H-5388, lot 121H3918). It was supplied as the sodium salt of hyaluronic acid having been extracted and purified from rooster combs, with the 2x106 average molecular weight fraction prepared by chemical degradation. The hyaluronate was used at a concentration of 5 mg/ml in Tyrode buffer supplemented with 25 mg/ml bovine serum albumin (BSA Commonwealth Serum Laboratories, Melbourne, Australia) immediately before use. The final pH was 7.4.
Egg white was aspirated with a syringe from the layer surrounding the yolk of a fresh egg for each test.
Tyrode solution (Commonwealth Serum Laboratories Melbourne Australia) with 25 mg/ml BSA was used as a control migration medium.
Capillary tube tests
Unless otherwise specified, all media were pre-equilibrated to 37°C. Circular cross-section capillary tubes, 100 mm in length and inner diameter 0.798 mm (50 µl Drummond microcaps; Edwards Instruments, Melbourne, Australia) were used. Each tube was filled by aspiration using a 1 ml syringe and a plastic tube attached to the upper end of the capillary while the lower end was dipped into a pool of one of the media. A 78 cm column of medium was aspirated into the tube so that the upper meniscus was 23 cm from the top of the tube. Great care was taken to avoid trapping air bubbles within the column. The top of the tube was sealed with plasticine and any trailing medium was cut off the lower end to produce a flat interface.
Approximately 100 µl of liquefied semen was placed in the bottom of a small conical plastic tube and a capillary tube containing one of the migration media placed with its open end in the semen. Each of the four media was tested at the same time with the same semen sample. Where there was insufficient semen, the egg white and Tyrode solution tests were not performed. Two semen reservoirs and capillary tubes were mounted on a microscope slide as shown in the WHO manual (WHO, 1999). The slides were then placed in a Petri dish containing damp sponges to maintain humidity and prevent drying of the semen and media. Capillary tubes were incubated in the horizontal position.
After 60 min incubation, the slides were removed from the Petri dishes. The capillary tube was viewed under bright field illumination with a x20 phase contrast objective lens and x10 oculars. The stage was adjusted to select a focal plane incorporating the central axis of the capillary and, at this magnification, the microscope field width approximated the inner diameter of the capillary tube. The length of the tube was then scanned to establish the distance furthest from the semen reservoir attained by spermatozoa. The maximum distance of migration of spermatozoa after 1 h of incubation was defined as the migration distance and reported to the nearest 1 cm. Sperm penetration concentrations were assessed at half the migration distance using the same magnification and counting spermatozoa whilst focusing from the lower to upper wall of the capillary in a single pass. The volume of medium sampled during counting was estimated to be 0.34 µl using the equation 16r3/3 for the volume of intersection of two cylinders of radius r. The penetration score was calculated by multiplying half the migration distance by the number of spermatozoa counted at half the migration distance. Sperm motility was assessed by examining at least 200 spermatozoa at half the migration distance.
To check that this procedure was reproducible, two observers assessed penetration tests for 22 semen samples independently. An additional 16 samples were tested after incubation at both room temperature (20°C) and 37°C.
Statistical analysis
Results are presented as mean ± SD unless otherwise specified. The significance of differences between results of two observers and two incubation temperatures was examined by Wilcoxon tests. The significance of differences between migration distances, sperm numbers, penetration scores and sperm motility was determined for the four penetration media by two-way analysis of variance. Relationships between variables were evaluated by non-parametric correlation analysis (Spearman tests). The effect of the semen analysis results, age of medium and other factors were tested by multiple regression analysis.
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Results |
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Sixteen duplicate penetration tests for semen samples with a range of results were incubated at both room temperature (20°C) and 37°C. The average difference in migration distance (3.0 and 3.8 cm, 0.79 ± 1.51 cm) appeared higher at 37°C but was not statistically significant. The sperm concentration at half the migration distance was significantly higher at 37°C than at 20°C (134x103/ml and 309x103/ml, mean difference 175 ± 228x103/ml, P = 0.007). The penetration score was also significantly higher at 37°C (195 and 556, mean difference 360 ± 452, P = 0.004). There was no significant difference in progressive sperm motility (41 and 46%, mean difference 5 ± 38%). Subsequent studies were conducted at 37°C.
Comparison of sperm penetration in different media
The mean migration distances and sperm concentrations were not significantly different in cervical mucus and hyaluronate solution (Table I). Although the difference in penetration scores was significant (P < 0.05), the magnitude of the mean difference was only ~10%. Both cervical mucus and the hyaluronate solution had much higher penetration than the control albumin Tyrode solution, while egg white had lower penetrability. Sperm motility was highest in albumin Tyrode and hyaluronate solutions and lower in cervical mucus and egg white. Table II
illustrates the strong correlations between the results for cervical mucus and hyaluronate solution and weaker correlations with those for egg white and albumin Tyrode solution. The close agreement between the penetration scores in hyaluronate and cervical mucus is shown in Figure 1.
There were three samples which for unknown reasons had higher penetration scores in mucus than in the hyaluronate and appear to be outliers.
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Factors affecting sperm penetration
Multiple linear regression analysis was used to determine which groups of factors influenced the various measures of sperm penetration in each of the media (Table III). Sperm morphology was found to be a significant factor (P < 0.002) influencing the migration distance in both cervical mucus and hyaluronate. The percentage of motile spermatozoa bound in the IgG immunobead test was significantly negatively correlated (P = 0.008) with the migration distance in cervical mucus only. Age of cervical mucus was associated with lower migration distances (P = 0.015). Age of hyaluronate affected all three measures of penetration: migration distance, sperm concentration and progressive motility in the medium. Sperm concentration and motility in semen influenced sperm penetration of egg white and albumin Tyrode solution. Positive IgG immunobead tests were associated with greater migration in albumin Tyrode solution. The results for samples from the 10 sperm donors did not differ from those of the patients when other covariates were included in the regression models.
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Discussion |
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Both cervical mucus and hyaluronate solution had higher sperm penetration than did an albumin-containing buffer. When drawn into a capillary tube, the alignment of the polymeric structure of both mucus and hyaluronate solution channels sperm movement parallel to the axis of the tube. This results in greater sperm migration than occurs with an unstructured buffer. Although egg white has been used as a sperm penetration medium (Eggert-Kruse et al., 1990), in this study it had even lower penetrability than the control solution and is therefore not recommended as a useful medium for sperm penetration tests.
An important use of sperm mucus penetration tests is assessment of the clinical significance of positive sperm antibody tests. No substitute for human cervical mucus has previously been found for this purpose. While bovine cervical mucus has been promoted for testing human sperm penetration, it has been shown that spermatozoa heavily coated with antisperm antibodies which fail to penetrate human cervical mucus can still penetrate bovine cervical mucus (Urry et al., 1986). Previously (Mortimer et al., 1990
), no relationship was reported between the presence of sperm antibodies and sperm penetration of hyaluronate, but only three of their 51 samples had a strongly positive immunobead test. Thus, the present results are encouraging. The close relationship between the cervical mucus and hyaluronate penetration results in the presence of sperm antibodies suggests that a more detailed study of this aspect of the use of hyaluronate for assessing sperm autoimmunity would be worthwhile.
A variety of methods of assessing sperm mucus penetration have been devised including the penetration index (Kremer, 1965; WHO, 1999), quantitative mucus penetration (Clarke, 1997
), the mathematically derived percentage of successful collisions (Katz et al., 1980
) and a recent modification of this (Clarke et al., 1998
). The latter approaches require recovery of all the spermatozoa from the cervical mucus and assessment of sperm concentration and straight line velocity in the semen. A simpler method was used for the current comparison of different penetration media, involving assessment of the migration distance to the nearest 1 cm and the total sperm number in a section of the capillary at half the migration distance. The product of distance and sperm concentration produces a penetration score similar to that of several other simple assessment schemes (Kremer, 1965
; WHO, 1999). More quantitative methods (Clarke et al., 1998
) could be used for sperm penetration into these media.
Factors affecting sperm penetration of mucus, particularly sperm morphology and sperm antibodies, were as expected. The effect of age of the cervical mucus and hyaluronate solution was statistically significant but would be relatively unimportant clinically. This effect could be avoided or compensated for by preparing the hyaluronate solution fresh, or using normal donor spermatozoa as a control for all penetration assessments. There were reproducible results between technicians although one systematically recorded slightly higher concentrations of sperm in the capillaries than the other. This would be clinically insignificant and could probably be corrected with training. The relatively large differences in sperm penetration associated with incubating the mucus and hyaluronate solution at 37°C compared with 20°C are consistent with the increase in sperm motility and velocity, and also reduced viscosity of the media with higher temperature (Ford et al., 1992). This sensitivity to temperature indicates that penetration tests should be carried out at a regulated temperature.
Because of the minor difference in the sperm penetration score between hyaluronate and cervical mucus, further studies to determine the influence of the molecular weight range and concentration of the hyaluronate medium on sperm penetration would be worthwhile. Such studies may also lead to understanding of the mechanism by which sperm characteristics and sperm antibodies impair penetration in media containing hydrated polymers.
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Notes |
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4 To whom correspondence should be addressed at: University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Carlton, Victoria 3053, Australia
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References |
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Submitted on July 1, 1999; accepted on August 26, 1999.