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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cervical mucus/hyaluronic acid/methylcellulose/sperm penetration test
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1010 000 centipoise (cp) for a 2% solution, which cover the range of that found in HCM, where viscosities of 280010 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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evaluation and preparation of samples
Semen samples were collected at the laboratory after 24 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 categoriesrapid 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/mldata 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 = 14x106 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.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Temperature study
Criteria of semen quality of patients used in this study are shown in Table IV.
|
|
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 1737°C. For oligozoospermic samples the proportional increase is less pronounced, estimated as a factor of ~4 at a distance of 1 cm.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (2237°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.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aitken, R.J., Bowie, H., Buckingham, D. et al. (1992) Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence. J. Androl., 13, 4454.
Barratt, C.L.R., Osborn, J.C., Harrison, P.E. et al. (1989) The hypo-osmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte. Hum. Reprod., 4, 430434.[Abstract]
Barratt, C.L.R., McLeod, I.D., Dunphy, B.C. et al. (1992) Prognostic value of two putative sperm function tests: hypo-osmotic swelling and bovine sperm mucus penetration test (Penetrak). Hum. Reprod., 7, 12401244.[Abstract]
Box, G.E.P., Hunter, W.G. and Hunter, J.S. (1978) Statistics for Experimenters. Wiley, New York, p. 144.
Delong, E.R, Delong, D.M. and Clarke-Pearson, D.L. (1988) Comparing the areas under two or more competing receiver operating characteristic curves: a nonparametric approach. Biometrics, 44, 837845.[ISI][Medline]
Eggert-Kruse, W., Gerhard, I., Tilgen, W. et al. (1989) Clinical significance of crossed in vitro sperm cervical mucus penetration test in infertility investigation. Fertil. Steril., 52, 1032 1040.[ISI][Medline]
Eggert-Kruse, W., Schwarz, H., Rohr, G. et al. ( 1996). Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception. Hum. Reprod., 11, 139 146.[Abstract]
ESHRE (1996) Consensus workshop on advanced diagnostic andrology techniques. ESHRE (Euopean Society for Human Reproduction and Embryology) Andrology Special Interest Group. Hum. Reprod., 11, 1463 1479.
Ford, W.C.L., Ponting, F.A., McLaughlin, E.A. et al. (1992) Controlling the swimming speed of human sperm by varying the incubation temperature and its effect on cervical mucus penetration. Int. J. Androl., 15, 127 134.[ISI][Medline]
Karni, Z. (1971) Newtonian viscosity of the human cervical mucus during the menstrual cycle. Int. J. Fertil., 16, 185188.[ISI][Medline]
Katz, D.F., Slade, D.A. and Nakajima, S.T. (1997) Analysis of pre-ovulatory changes in cervical mucus hydration and sperm penetrability. Adv. Contracept., 13, 143151.[ISI][Medline]
Keel, B.A. and Webster, B.W. (1988) Correlation of human sperm motility characteristics with an in vitro cervical mucus penetration test. Fertil. Steril., 49, 138143.[ISI][Medline]
Kraemer, M., Fillion, C., Martin-Pont, B. et al. (1998) Factors influencing human sperm kinematic measurements by the Celltrak computer-assisted sperm analysis system. Hum. Reprod., 13, 611619.[Abstract]
Kruger, T.F. and Coetzee, K. (1999) The role of sperm morphology in assisted reproduction. Hum. Reprod. Update, 5, 172178.
Lorton, S.P., Kummerfeld, H.L. and Foote, R.H. (1981) Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil. Steril., 35, 222225.[ISI][Medline]
Milligan, M.P., Harris, S.J. and Dennis, K.J. (1978) The effect of temperature on the velocity of human spermatozoa as measured by time-lapse photography. Fertil. Steril., 30, 592594.[ISI][Medline]
Mortimer, D. (1999) Structured management as a basis for cost-effective infertility care. In Gagnon, C. (ed.) The Male Gamete: From Basic Science to Clinical Applications, Cache River Press, Vienna, IL, USA, pp 363370.
Mortimer, D., Mortimer, S.T., Shu, M.A. et al. (1990). A simplified approach to spermcervical mucus interaction testing using a hyaluronate migration test. Hum. Reprod., 5, 835841.[Abstract]
Neuwinger, J., Cooper, T.G., Knuth, U.A. et al. (1991) Hyaluronic acid as a medium for human sperm migration tests. Fertil. Steril., 58, 396400.[ISI]
Ray, B.D., Howell, R.T., McDermott, A. et al. (1995) Testing the mutagenic potential of polyvinylpyrrolidone and methylcellulose by sister chromatid exchange analysis prior to use in intracytoplasmic sperm injection procedures. Hum. Reprod., 10, 436438.[Abstract]
Tang, S., Garrett, C. and Baker, H.W.G. (1999) Comparison of human cervical mucus and artificial sperm penetration media. Hum. Reprod., 14, 28122817.
Tomlinson, M.J., Kessopoulou, E. and Barratt, C.L.R. (1999) The diagnostic and prognostic value of traditional semen parameters. J. Androl., 5, 588593.
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.[ISI][Medline]
World Health Organization (1999) WHO laboratory manual for the examination of human semen and spermcervical mucus interaction. World Health Organization, 4th edn, Cambridge University Press, Cambridge.
Submitted on May 11, 2001; accepted on August 31, 2001.