Human Reproduction, Vol. 17, No. 1, 143-149,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Critical evaluation of methylcellulose as an alternative medium in sperm migration tests
1 The Assisted Conception Unit, Birmingham Women's Hospital, Birmingham B15 2TG, 2 Reproductive Biology and Genetics Group, The University of Birmingham B15 2TH and 3 School of Mathematics and Statistics, The University of Birmingham, Birmingham B15 2TT, UK
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Abstract |
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BACKGROUND: The aim of this study was to evaluate the ability of human spermatozoa to penetrate methylcellulose (MC) and to compare this with penetration in hyaluronic acid. METHODS: Spermatozoa from normal (
20x106 sperm/ml, 50% progressive motility, 5% normal forms) and oligozoospermic (<20x106 sperm/ml) semen samples were allowed to swim into glass capillary tubes containing methylcellulose with a viscosity of 15 centipoise (cp) (MC15) and 4000 cp (MC4000), hyaluronic acid (rooster comb) or Sperm Select. Penetration of the spermatozoa at 1, 2, 3 and 4 cm were correlated with basic semen parameters (concentration, motility and morphology). The effects of temperature on penetration into MC4000 were explored at 17–37°C. RESULTS: Higher numbers of spermatozoa penetrated MC4000 (10 mg/ml) compared with MC15 and the hyaluronic acid preparations. There was a highly significant correlation between the number of spermatozoa at all migration distances in MC4000 (10 mg/ml) and semen parameters. Increases in temperature from 17–37°C were accompanied by significantly higher numbers of spermatozoa at each penetration distance. MC4000 at 10 mg/ml was at least as favourable to sperm penetration as human cervical mucus. Effective discrimination between normal and abnormal samples was achieved using MC4000 (10 mg/ml). CONCLUSION: Our results suggest the potential use of methylcellulose (MC4000, 10 mg/ml) as a reproducible and effective alternative to hyaluronic acid in sperm migration tests.
Key words: cervical mucus/hyaluronic acid/methylcellulose/sperm penetration test
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Traditional semen parameters (concentration, motility and morphology) only provide a limited degree of prognostic and diagnostic information for the infertile couple (Tomlinson et al., 1999
). Consequently, studies have focused on developing simple and effective tests of sperm function to allow more accurate data to be obtained. However, despite the plethora of assays available, progress has been disappointing (ESHRE, 1996; Mortimer, 1999). Penetration of spermatozoa into human cervical mucus (HCM) in vitro is known to provide important predictive information about sperm function (Barratt et al., 1989; Eggert-Kruse et al.,1989, 1996; Abu-Heija et al., 1996). Yet, because of the known problems associated with HCM (described below), the use of these tests has been very limited and alternative media have been sought. The aim of our experiments was to evaluate critically methylcellulose (MC) as a potential medium for sperm penetration. Comparison was made to hyaluronic acid which is the most widely used HCM alternative.
A major problem with the use of CM in in-vitro tests is the difficulty in standardizing its quality. The constituents of HCM are glycosylated sialoglycoprotein molecules, the hydration of which changes during the menstrual cycle (Katz et al., 1997). The variability of HCM, problems with availability and difficulties in collecting sufficient quantities, as well as instability and storage problems, have led to the development of substitutes for use in in-vitro (Kremer-type) tests.
For an HCM substitute to be clinically useful it should behave in a similar manner to HCM. In particular, penetration of spermatozoa into the media should be significantly correlated with semen quality. In addition, HCM substitutes should be readily available, easily prepared, cheap and stable. Substitutes have included bovine CM (Keel and Webster, 1988), which has a variable consistency, is not easy to obtain or store and has poor predictive value (Barratt et al., 1992). Polyacrylamide gel has been used in a number of studies (Lorton et al., 1981; Urry et al., 1986) but is less efficient at allowing sperm penetration and has the added disadvantage of being toxic. Hyaluronic acid has been extensively used as an HCM substitute and penetration of spermatozoa into it is highly correlated with semen characteristics. It is regarded as an effective alternative to HCM (Mortimer et al., 1990; Neuwinger et al., 1991; Aitken et al., 1992).
Our studies have focused on MC as an alternative medium to HCM for the sperm penetration test. MC is a long-chain substituted cellulose with ~30% of its hydroxyl groups in the form of methyl ether. Various grades of MC have viscosities varying between 10–10 000 centipoise (cp) for a 2% solution, which cover the range of that found in HCM, where viscosities of 2800–10 000 cp have been described (Karni, 1971). MC has a uniform quality and consistency, is stable over long periods of time, easy to obtain and significantly cheaper than hyaluronic acid. This inert, non-toxic substance is widely used in the pharmaceutical and food industries, and has been used as a sperm diluent in ICSI procedures (Ray et al., 1995).
The aim of the present study was to critically evaluate MC as a potential medium for sperm penetration, comparing it with hyaluronic acid. The study parameters were based on the standard sperm characteristics of concentration, motility and morphology. The effect of temperature on sperm migration was also examined. To determine that penetration into MC was comparable with HCM, a preliminary comparison was undertaken. Finally, the discriminatory properties of MC in identifying abnormal semen samples were explored.
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Materials and methods |
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Study Population
Semen samples used in this study were obtained from patients attending the andrology laboratory at the Birmingham Women's Hospital, UK [Human Fertilisation and Embryology Authority (HFEA) centre no. 0119] as part of fertility investigations and from fertile research donors. Semen samples were taken in accordance with HFEA Code of Practice.
Evaluation and preparation of samples
Semen samples were collected at the laboratory after 2–4 days sexual abstinence, by masturbation into sterile plastic containers, and left to liquefy at 37°C for 30 min. Semen analyses were performed according to World Health Organization (WHO) criteria (World Health Organization, 1999). Sterile disposable 5 ml pipettes were used to determine the volume of the semen sample. Sperm motility was assessed on a freshly prepared wet preparation, which was examined at a total magnification of x400 using an Olympus BH2 microscope with phase contrast optics. Motility was classified according to WHO guidelines into four categories—rapid progressive (grade a), slow progressive (grade b), non-progressive (grade c) and immotile (grade d). An improved Neubauer haemocytometer was used to determine the concentration of spermatozoa. The definition of a normal spermatozoon was based on the WHO guidelines. Slides were prepared using a thin and well spread smear, which was air-dried, fixed and stained using Papanicolaou staining. Examination of the slides was performed using bright field illumination at a total magnification of x1000 under oil-immersion.
From the available samples, two groups were identified based on WHO criteria: (i) normal samples: these contained 20x106sperm/ml, 50% motility (grades a+b, or 25% progressively motile grade a) and 5% normal forms. Morphology of 5% normal forms was used as a threshold value, as it is generally accepted that below this limit IVF rates are significantly affected (Kruger et al., 1999). (ii) Oligozoospermic samples: these were samples which contained <20x106 sperm/ml.
Penetration media
MC with a viscosity of 15 cp (MC15) (2% solution at 22°C) was obtained from Sigma (Poole, Dorset, UK) and with a viscosity of 4000 cp (MC4000) from Aldrich (Gillingham, Dorset, UK). MC15 was used at 10 and 4 mg/ml and MC4000 at 10 and 1 mg/ml. Several other concentrations and viscosities of MC were examined in preliminary studies (MC25, MC400 and MC1500 were tested at 10, 5, 2 and 1 mg/ml—data not presented).
Two different preparations of hyaluronic acid were used. Commercially available hyaluronic acid was obtained as Sperm Select (Select Medical Systems, Williston, VT, USA), containing 2 mg/ml hyaluronic acid. Sperm Select was used at a final concentration of 1 mg/ml. Hyaluronic acid from rooster comb (MW range = 1–4x106 Da) was purchased from Sigma and used at a final concentration of 1 mg/ml.
All penetration media were prepared in Earle's balanced salt solution (Gibco-BRL, Paisley, Scotland) containing 0.45% human serum albumin (BioProducts Laboratory, Elstrea, UK) and 10 mmol/l HEPES (ICN Biomedicals Inc., Aurora, USA). MC and rooster comb hyaluronic acid were dissolved at ambient temperature with gentle shaking for 1 h and then left overnight at 4°C to ensure solubilization.
Sperm penetration tests
The penetration media were introduced by capillary action into 5 cm long flattened glass capillary tubes (Camlab Limited, Cambridge, UK) (dimensions of 1.2x4.8 mm and an inner depth of 0.4 mm). Care was taken to prevent air bubbles from being trapped in the column. One end of the capillary tube was sealed with PlasticineTM and the open end of the tube placed in a 1.5 ml microfuge tube (Appleton Woods, Birmingham, UK) containing 100 µl of the liquefied semen sample. Motile spermatozoa were allowed to migrate into the penetration medium for 30 min, and the capillary tube was then removed from the semen sample, wiped to remove residual spermatozoa from the surface of the glass, and then viewed using phase contrast optics on an Olympus BH-2 microscope at a total magnification of x200. The number of spermatozoa per high-power field (HPF, area = 0.785 mm2) were recorded at 1, 2, 3 and 4 cm distances from the base of the capillary tube. All the observations were analysed by the same two observers (A.I. and H.O.) throughout the study. Reliability and repeatability studies showed good within and between observer variability.
Experimental design and statistical analysis
Comparison between MC15 and MC4000
The two concentrations of MC15 and MC4000 were compared in the sperm penetration test, which was carried out at ambient temperature (24 ± 3°C).
Temperature study
The influence of temperature on the penetration of spermatozoa into MC4000 at 10 mg/ml was examined. Penetration tests were carried out at 17, 22, 30 and 37°C.
Comparison between MC4000, hyaluronic acid and Sperm Select
Sperm penetration tests were performed at 37°C for 30 min, as described above. Each semen sample was split into three 100 µl aliquots and the number of spermatozoa penetrating MC4000 at 10 mg/ml, Sperm Select and rooster comb hyaluronic acid were recorded. Samples were not split into normal and oligozoospermic groups for the purposes of this comparison.
Comparison between MC4000 and HCM
To determine whether penetration into MC was comparable with HCM, a preliminary comparison was undertaken. Sperm penetration tests were performed at 37°C for 30 min, as described above. Each semen sample was split into two 100 µl aliquots and the number of spermatozoa penetrating MC4000 at 10 mg/ml and HCM were recorded. Samples were not split into normal and oligozoospermic groups for the purposes of this comparison. Cervical mucus was collected from patients attending the infertility clinic for assisted conception (Barratt et al., 1989).
Classification of abnormal samples using receiver operating characteristic (ROC) analysis
This experiment was performed at 37°C using MC4000 at 10 mg/ml. Normal samples were defined as being above the stated normal (World Health Organization, 1999) reference ranges for concentration, motility and morphology while abnormal samples were defined as any semen sample below these limits.
Statistics
Comparisons between penetration media were conducted using the Sign test and the Wilcoxon Signed Rank test, and standard correlation methods were applied to assess the influence of sperm characteristics on penetration numbers. Two-way analysis of variance was used to isolate the temperature effect. The comparison of receiver operating characteristic (ROC) curves in Figure 6 followed the method proposed by Delong et al. (Delong et al., 1988). The calculations were carried out in the MINITAB (Release 12.1) and Statistics Package for Social Sciences (Release 7.5.1) statistical programs.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Comparison of MC15 and MC4000
Summary statistics for the semen samples used in the sperm penetration tests are shown in Table I
and the penetration of spermatozoa into various concentrations of MC15 and MC4000 are shown in Table II.
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For both normal and oligozoospermic samples, the mean number of spermatozoa was inversely related to penetration distance, and appeared to be systematically higher in some media than in others (Figure 1
—only normals shown). As expected, the standard deviations were greatest for those combinations of height and penetration media which generated the largest numbers of spermatozoa (Table II
). Nevertheless, an increase in mean value was generally associated with a less than proportional increase in standard deviation, so that the coefficient of variation of sperm numbers tended to decline as the mean number increased (Figure 2). The percentage error in a mean value was therefore smallest when that mean was large. This phenomenon is consistent with a Poisson model for sperm penetration.
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Formal statistical comparison of the different penetration media was carried out through paired analyses, based on those sperm samples which were tested in more than one media, in each case under identical laboratory conditions. This was done by applying two non-parametric procedures—the Sign test and the Wilcoxon Signed Rank test—to the differences between the observed numbers of penetrating spermatozoa for the same sample in two different media. Results for the Sign test are presented in Table III
. Similar results were obtained at 3 cm and at 1 cm, where the difference between the two concentrations of MC15 was also identified as statistically significant (P = 0.012). The Wilcoxon results were qualitatively similar in every case.
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Strong associations were observed between the number of penetrating spermatozoa and the conventional semen parameters of concentration, motility, concentration of motile cells and morphology. At 1 cm the Rho values were 0.84 (concentration x106/ml – P < 0.001), 0.70 [motility (%) – P < 0.001], 0.89 [concentration of motile cells (x106/ml) – P < 0.001] and 0.70 [morphology (%) – P < 0.001]. Similar results (not shown) were obtained at sperm penetration depths of 2, 3 and 4 cm where the correlation coefficients were slightly reduced but still highly significant.
Temperature study
Criteria of semen quality of patients used in this study are shown in Table IV.
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Semen from 67 samples was employed to assess the effect of temperature on penetration, using MC4000 at 10 mg/ml as the penetration medium. Table V
shows the mean numbers penetrating at each distance for four different temperatures (17, 22, 30 and 37°C).
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For technical reasons, it proved difficult to obtain reliable penetration counts in certain cases when the numbers of spermatozoa present in the high power microscope field (HPF) were >200. These cases are missing from Table V
, with a consequent distortion in the perceived temperature effect, since higher penetration counts arise predominantly at higher temperatures. The stated values of n are the actual numbers of samples contributing to the calculated means and standard deviations. This problem did not affect the interpretation of the temperature effect arising from the analysis of variance described below, because the method incorporates an adjustment for missing data values.
Two-way analyses of variance (temperature x samples), allowing for differences between samples, were carried out on the logarithms of the data, having first added 1 to each value to cope with zero counts. The log transformation was found empirically to improve the stability of the standard deviation of sperm numbers. In theory, the square root transformation may be superior (Box et al., 1978), but was not found to be so for this set of data and the results from the analysis of square roots were similar in all respects to those reported here (data not shown).
The evidence for a temperature effect is overwhelming. For both normal and oligozoospermic samples, it is highly significant (P < 0.001) at penetration distances of 1, 2 and 3 cm, and also in the multivariate analyses of variance in which all the penetration distances are considered simultaneously. At 4 cm the P-values are 0.001 for normal and 0.012 for oligozoospermic samples. Adjusted temperature effects based on these analyses are presented in Figure 3. For normal samples the data are consistent with a tenfold increase in penetration numbers at each distance over the temperature range 17–37°C. For oligozoospermic samples the proportional increase is less pronounced, estimated as a factor of ~4 at a distance of 1 cm.
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Comparison of MC4000, hyaluronic acid and Sperm Select
Twenty-six semen samples were used to generate pairwise comparisons between MC4000, hyaluronic acid and Sperm Select media. For these samples, the median sperm concentration was 79.5x106/ml (range 3–159x106) and the median motility 63% (range 37–85). A summary of the penetration results is displayed in Figure 4
. At 1 cm, only small differences in penetration numbers were observed, though at greater distances (2, 3 and 4 cm) the numbers achieved in MC4000 were significantly greater (P < 0.001, for each of the six Sign tests and each of the six Wilcoxon tests) than either of the other two media. At these distances, no significant differences between Sperm Select and hyaluronic acid were identified (P > 0.05 for every test). It appears that MC4000 at 10 mg/ml is at least as favourable to sperm penetration as the other two media.
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Comparison of MC4000 and HCM
Twenty-four semen samples were used to generate pairwise comparisons between the MC4000 and human cervical mucus. For these samples, the median sperm concentration was 47x106/ml (range 10–125x106) and the median motility 56% (range 20–82). A summary of the penetration results is displayed in Figure 5
. The penetration numbers were significantly greater for MC4000 than for HCM at 1, 2 and 4cm (Sign test P-values < 0.0001, 0.001 and 0.004 respectively) with no significant difference observed at 3 cm. Overall, MC4000 at 10 mg/ml is at least as favourable to sperm penetration as HCM.
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Classification of abnormal samples
The effectiveness of penetration numbers at 1 and 2 cm for discriminating between normal and abnormal samples is illustrated by the ROC curves in Figure 6
. These are based on the behaviour of 24 normal and 33 abnormal samples at 37°C. The curves represent the proportion of abnormal samples correctly classified (sensitivity), plotted against the proportion of normal samples incorrectly classified (false abnormality rate), as the threshold point is allowed to vary. For example, it shows that a sensitivity of 85% with a false abnormality rate of 17% may be achieved by using a 1 cm penetration count of 
59 as the criterion for abnormality. It appears that the number of spermatozoa at 1 cm provide better discrimination than those at 2 cm. A statistical test based on the area of the region between the two curves just failed to show a significant difference (P = 0.052) in discriminatory performance. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The aim of the present study was to critically evaluate MC as a potential medium for sperm penetration, comparing it with hyaluronic acid. Penetration of spermatozoa into MC is highly dependent on sperm characteristics such as concentration, motility and morphology. Preliminary data show that penetration into MC4000 (10 mg/ml) is comparable with HCM. In addition, ROC analysis illustrates that MC4000 (10 mg/ml) allowed good discrimination between normal and abnormal semen samples (Figure 6
). These results suggest that MC can be considered as a potential alternative to hyaluronic acid as an HCM substitute.
In our preliminary experiments a series of different MC preparations were examined. As the mean number of spermatozoa penetrating MC increased, the coefficient of variation reduced (Figure 2). This suggests that a sensitive test, for example, one that can discriminate between normal and abnormal samples, should employ a medium which allows the greatest number of spermatozoa to penetrate. In this case the difference between both poor quality and high quality samples is magnified, and the percentage error of the difference reduced. Our experiments show that MC4000 at 10 mg/ml was the medium that met these criteria and allowed good discrimination between normal and oligozoospermic samples (Table II). MC4000 (10 mg/ml) was therefore used in all further experiments.
Penetration of spermatozoa into MC4000 (10 mg/ml) was highly dependent on the concentration, progressive motility and morphology of spermatozoa. Many studies have documented the importance of similar semen characteristics in penetrating hyaluronic acid and HCM (Mortimer et al., 1990; Aitken et al., 1992; Tang et al., 1999), suggesting that MC can be considered as a potential alternative to hyaluronic acid as an HCM substitute. Interestingly, the mean number of spermatozoa that penetrated MC4000 was significantly higher than hyaluronic acid. Hyaluronic acid concentration and molecular weight have been shown to affect sperm migration (Neuwinger et al., 1991) and a series of further experiments are required to determine if the number of spermatozoa penetrating MC4000 is higher when other combinations of hyaluronic acid (molecular weights, concentrations, source of preparation) are examined.
The temperature at which the sperm penetration test was performed is a critical factor. Not surprisingly, higher temperatures were accompanied by higher numbers of spermatozoa penetrating MC4000 (Table V). Interestingly, the temperature effect appeared greatest between 17 and 30°C, and less marked between 30 and 37°C. A number of studies have shown that sperm velocity increases as temperature is increased (Milligan et al., 1978; Ford et al., 1992; Kraemer et al., 1998). Tang and colleagues showed that higher temperatures (22–37°C) resulted in higher numbers of spermatozoa penetrating hyaluronic acid (Tang et al., 1999). It is therefore critical to maintain a constant temperature when performing sperm penetration tests.
In conclusion, sperm penetration into MC depends on similar semen characteristics to those reported for HCM (Figure 5) and hyaluronic acid (Figure 4) and reasonable separation between abnormal and normal samples can be achieved (Figure 6). Initial experiments showed similar penetration between MC and HCM. MC is readily available, easily standardized, cheap and stable over a long period of time. Thus, MC can be regarded as a potential alternative to hyaluronic acid for in-vitro sperm penetration tests.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The authors would like to thank patients and donors who took part in this study, staff in the Clinical Andrology laboratory for assistance with semen samples and Julie Edwards for helping with the preparation of the manuscript. A.I. and H.O. contributed equally to this manuscript. This work was funded by grants from Genosis Inc., USA and an NHS programme grant (#0205) to C.L.R.B.
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Notes |
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4 To whom correspondence should be addressed at: The Assisted Conception Unit, Birmingham Women's Hospital, BirminghamB15 2TG, UK. E-mail: c.l.barratt{at}bham.ac.uk
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References |
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Submitted on May 11, 2001; accepted on August 31, 2001.
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