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Sialic acid levels and scanning electronmicroscopy of cervical mucus
CONTRACEPTION
SIALIC ACID LEVELS AND SCANNING ELECTRONMICROSCOPY OF CERVICAL MDCUS
B. Daunter, Research Fellow Department of Obstetrics and Gynaecology,* University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Queensland 4029, Australia
ABSTRACT
Sialic acid levels were determined in cervical mucin from women not using contraceptiveprotection (controls), WomeR using Copper Seven IUD containing 200 mm2 of copper wire, and women using an inert Dalkon Shield IUD. The sialic acid levels of the control cervical mucin were found to undergo cyclic variation during the menstrual cycle with higher levels in the preovulatory phase than in the luteal phase. There was no significant difference in sialic acid levels of cervical mucin between the preovulatory and luteal phase of the contraceptive regimes, or when compared to the controls. Renvval of sialic acid by neuraminidase from late preovulatory cervical mucin resulted in a structure similar to that found in the early preovulatory and luteal phase when examined by scanning electronmicroscopy.
* Address for reprint requests.
Accepted
JANUARY
for publication
1978 VOL. 17 NO. 1
November
14, 1977
27
CONTRACEPTION
INTRODUCTION
Cervical mucins have been separated into three fractions on the basis of their sialic acid(s), fucose and sulphate contents (1). Other workers (2) have found that the electrophoneticdistributionof cervical mucin suggests it belongs to a single population of glycoproteinmolecules, their variation based mainly on differences in sialic acid(s) content. The sialic acid(s) are present in a greater proportion than fucose (3) and data suggests that sialic acid(s) undergo cyclic variation during the menstrual cycle (4, 5). When sialic acid(s) are removed from cervical mucin by neuraminidase,or the ionization of the sialic acid(s) are increased, fluidificationresults (6, 7). It would appear therefore, that sialic acid(s) play an important role in the structure and rheologicalproperties of cervical mucin. It has been shown that IUDs, the Cu-7 especially, cause a decrease in cervical mucus trace metals (8). Metal ions are antagonistic to each other in their action, which is a function of their concentration (9) and this can result in changes in metallo-enzymeactivity (10). Steroid binding proteins are altered in the presence of a Cu-7 IUD (11) and hypo- and hyper-estrogenismhas been associated with ItJDs (12, 13). Scanning electronmicroscopystudies suggests that there may be some structural change in cervical mucin from women using Cu-7 IUDs (14). Thus any change in the hormonal environment or enzyme activity in the cervical mucosa, may be reflected in the sialic acid levels of the cervical mucin. MATERIALS AND METHODS Cervical mucus specimens were collected and stored as previously described (8) from women in their active reproductive years using (i) Cu-7 IUD, (ii) D-S. IUD, (iii) no contraceptive protection. The preovulatory and luteal phase of the menstrual cycle was based on basal body temperature. The preovulatory and luteal phase cervical mucus samples investigated for sialic acid were from day -7 to day 0, and day 0 to day +lO,respectively. The control cervical mucus samples examined by scanning electronmicroscopy were from the early and late preovulatory phase, day -9 and -2,respectively, and from the luteal phase day +2. The number of women and cervical mucus samples investigated are presented in Table I.
28
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION Table I Cervical Mucus Investigated
Contraceptive regime
None
Number of womsn
Duration of use
2
Abbreviations
Total number of cycles studied
Number of cervical mucus samples (P) (L)
Controls
2
9
4
IUD
2
2 months
Cu 72
2
5
5
Cu-7 IUD
2
6 months
Cu 7 6
2
6
5
Cu-7 IUD
2
13 months
Cu 713
2
6
6
D.S. IUD
2
2 months
D.Ss2
2
8
3
h-7
(P) = Preovulatory
(L) = Luteal
Sialic Acid Determinations Sialic acid determinations were carried out on gluteraldehyde-fixed, freeze dried cervical mucin using the thiobarbituric acid method (15). Preparation of Cervical Mucus Cervical mucus was washed in physiological saline three times, centrifuging between each washing for 10 minutes at a relative centrifugal force (RCF) of 2,500 g. The cervical mucin was then fixed in 0.6% gluteraldehyde in Sdrensen buffer, pH 7.2, followed by washing and freeze drying (14). Samples of the cervical mucin from the control cycles, day -9 and day +2 were plated with gold and platinum and examined by scanning electronmicroscopy (14). Neuraminidase Treatment Cervical mucus from the control cycles, day -2 was divided into two portions. One half was incubated with 500 units of neuraminidase mucopolysaccharide N-acetylneuraminylhydrolase (Batch No. HE792) (EC 3.2.1.18) ex. Vibrio comma (Cholerae) (Hoecht Pharmaceuticals Ltd., Middlesex, G.B.) for 1.5 hours at 370C in a total volume of 1.5 ml of sodium acetate buffer, pH 5.5 (in which the neuraminidase was supplied). The other half of the cervical mucus was similarly treated, but without neuraminidase.
JANUARY
1978 VOL. 17 NO. 1
29
CONTRACEPTION
After incubation the cervical mucus was prepared as previously described. Samples of the cervical mucin were examined by scanning electrvnmicroswpy (14). The anvunt of sialic acid renvved from the cervical mucin by neuraminidase was determined in the supernatant and by the difference of sialic acid content between the neuraminidase treated and untreated fixed freeze dried cervical mucin. Statistical Analysis The variance ratio (F) test was first applied to see if the parametric 't' test could be used. If the variance ratio was significantly different (p = cO.05) then the non-parametric ranking method was used (16). RESULTS A significant increase in sialic acid(s) levels were found in the preovulatory phase cervical mucin of the wntrols relative to the luteal phase (Table II). This represents a percentage difference between the mean sialic acid levels of approxismtely 53%. No significant difference in sialic acid(s) levels were found between the preovulatory and luteal phase of the contraceptive regimes, or when compared with the wntrols (Table II).
The removal of sialic acid(s) by neuraminidase from wntrol preovulatory cervical mucin (Figure 1), resulted in a structure (Figure 2) similar to that found in the early preovulatory (Figure 3) and luteal phase (Figure 4). The amount of sialic acid(s) removed by neuraminidase treatment was approximately 50 - 60%. Table II Sialic Acid Levels of Cervical Mucin in the Preovulatory and Luteal Phase Mean Values Significant Vg Sialic Acid/mg Dry % difference MUCin Pnxwulatory : Luteal
Significant % difference relative to wntrols Preovulatory : Luteal
Controls
29
13
cu 72
42
26
0.0
0.0
0.0
cu 76
21
17
0.0
0.0
0.0
26
36
0.0
0.0
0.0
31
26
0.0
0.0
0.0
cu 7 13 D.S.
30
53
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
Figure 1.
Preovulatory Cervical Mucin, clay-2 based on basal body temperature. Magnification 900x. Before treatment with neuraminidase.
Figure 2.
Preovulatory Cervical Mucin, day -2 based on basal body temperature. Xagnification 900x. After treatment with neuraminidase.
JANUARY
1978 VOL. 17 NO. 1
31
CONTRACEPTION
Figure 3.
Preovulatory Cervical Mucin, day -9 based on basal body temperature. Magnification 900x.
Figure 4.
32
Luteal Cervical Nucin, day +2 based on basal body temperature. Magnification 900x.
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
DISCUSSION The data of other investigators (3, 4, 5) suggests that sialic acid(s) may be increased in luteal phase cervical mucin, relative to the preovulatory phase, and be responsible for the change in its rheological properties (18). This report suggests the opposite, that is, increased. levels of sialic acid(s) have been found in preowlatory cervical mucin. In addition, the scanning electronmicroscopy results demonstrate that the removal of sialic acid(s) from preovulatory cervical mucin results in a structure similar to that found in the early preovulatory and luteal phase. The amount of sialic acid(s) removed to produce this type of structure was in the order of 50 - 60%. This is close to the percentage difference in sialic acid(s) levels found between the control preovulatory and luteal phase cervical mucin. There was no significant difference in sialic acid(s) levels between cervical mucin from the controls and the contraceptiveregimes. In addition, no significant difference was found in sialic acid(s) levels of cervical mucin between the preowlatory and luteal phase of the contraceptive regimes. It would appear that the apparent increase and decrease of sialic acid(s) levels found in the contraceptive regimes are not significant relative to the controls, but result in similar statistical levels of sialic acid(s) in both phases of the menstrual cycle. This may be due to the heterogeneityof the cervical mucin (1, 2, 5, 17) or the inflammatory response induced by the presence of the IUDs (11) resulting in an influx of phagocytic cells into the cervical mucus. These cells may become trapped in the cervical mucin and may not be removed by the washing procedure described. Alternatively, it might be a reflection of changes in glycosyltransferaseactivities of the cervical mucosa, due to changes in the trace metal balance of the cervical mucus (8). In conclusion, these studies demonstrate the cyclic behaviour of cervical mucin during the menstrual cycle, and the importance of sialic acid to the structural integrity of cervical mucin. The effect of the IUDs on the sialic acid(s) levels of cervical mucin has not been completely elucidated.
JANUARY
1978 VOL. 17 NO. 1
33
CONTRACEPTION REFERENCES
34
1.
Gell, P., Crepin, G., Roussel, P., Degand, P. and naves, R.: Gynec. Obstet. (Paris). 68: 279, 1969.
2.
Masson, P. L., Heremans, J. F. and Ferin, J.: Int. J. Fert., 14: 1: 1969.
3.
Moghissi, K. S. and Blandau, R. J.: J. Reprod. Med. 8: (1): 2, 1971.
4.
Gibbons, R. A.: Biochem.
5.
Iacobelli, S., Garea, N. and Angeloni, C.: Fertil. Steril. 22: 727, 1971.
6.
Gottschalk, A.: Nature. 186: 949, 1960.
7.
Bettelheim, F. A. and Dey, S. K.: Archs. Biochem. 109: 259, 1965.
8.
Daunter, B., Chantler, E. N. and Elstein, Contraception 15: (5): 543, 1977.
9.
Underwood, E. J.: Trace Elements in Human and Animal Nutrition. 2nd Edition. Publ. Academic Press, N.Y. U.S.A. 48, 1962.
J.
72: (3): 217, 1959.
M:
10.
Dixon, M. and Webb, E. C.: Enzymes. 2nd Edition. Publ. Longmans, 1971.
11.
Salgo. M. P. and Webb, F. T.: Res. Review. p 3, 1976.
12.
S-ur, M. B., Iskanders, S. G, and Rifai, S. F.: Am. J. Obstet. Gynec. 98: 946, 1967.
13.
Pakrashi, A. and Ray, G. R.: J. Reprod. Fertil. 357, 1969.
14.
Daurter, B., Chantler, E. N. and Elstein, M.: Brit. J. Obstet. Gynaec. 83: (9): 738, 1976.
15.
Warren,
16.
Wilcoxon, F.: (Biomed. Bul. 1: 80, 1945): Cited by Colquhon, D.: In: Lectures on Biostatistics. Clarendon Press, Oxford, 1976.
17.
Gibbons, R. A. and Roberts, G. P.: Acad. Sci. (NY). 106: 218, 1963.
18.
Masson,
L. :
J. Biol. Chem.
19:
234: 1971, 1959.
P. L.: In: Cervical Mucus in Human Reproduction. W.H.O. Colloquim, September, 1972. (Eds. Elstein, M. and Moghissi, K. S.). Publ. University of Chicago Press, U. S. A., 1973.
JANUARY
1978 VOL. 17 NO. 1
SIALIC ACID LEVELS AND SCANNING ELECTRONMICROSCOPY OF CERVICAL MDCUS
B. Daunter, Research Fellow Department of Obstetrics and Gynaecology,* University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Queensland 4029, Australia
ABSTRACT
Sialic acid levels were determined in cervical mucin from women not using contraceptiveprotection (controls), WomeR using Copper Seven IUD containing 200 mm2 of copper wire, and women using an inert Dalkon Shield IUD. The sialic acid levels of the control cervical mucin were found to undergo cyclic variation during the menstrual cycle with higher levels in the preovulatory phase than in the luteal phase. There was no significant difference in sialic acid levels of cervical mucin between the preovulatory and luteal phase of the contraceptive regimes, or when compared to the controls. Renvval of sialic acid by neuraminidase from late preovulatory cervical mucin resulted in a structure similar to that found in the early preovulatory and luteal phase when examined by scanning electronmicroscopy.
* Address for reprint requests.
Accepted
JANUARY
for publication
1978 VOL. 17 NO. 1
November
14, 1977
27
CONTRACEPTION
INTRODUCTION
Cervical mucins have been separated into three fractions on the basis of their sialic acid(s), fucose and sulphate contents (1). Other workers (2) have found that the electrophoneticdistributionof cervical mucin suggests it belongs to a single population of glycoproteinmolecules, their variation based mainly on differences in sialic acid(s) content. The sialic acid(s) are present in a greater proportion than fucose (3) and data suggests that sialic acid(s) undergo cyclic variation during the menstrual cycle (4, 5). When sialic acid(s) are removed from cervical mucin by neuraminidase,or the ionization of the sialic acid(s) are increased, fluidificationresults (6, 7). It would appear therefore, that sialic acid(s) play an important role in the structure and rheologicalproperties of cervical mucin. It has been shown that IUDs, the Cu-7 especially, cause a decrease in cervical mucus trace metals (8). Metal ions are antagonistic to each other in their action, which is a function of their concentration (9) and this can result in changes in metallo-enzymeactivity (10). Steroid binding proteins are altered in the presence of a Cu-7 IUD (11) and hypo- and hyper-estrogenismhas been associated with ItJDs (12, 13). Scanning electronmicroscopystudies suggests that there may be some structural change in cervical mucin from women using Cu-7 IUDs (14). Thus any change in the hormonal environment or enzyme activity in the cervical mucosa, may be reflected in the sialic acid levels of the cervical mucin. MATERIALS AND METHODS Cervical mucus specimens were collected and stored as previously described (8) from women in their active reproductive years using (i) Cu-7 IUD, (ii) D-S. IUD, (iii) no contraceptive protection. The preovulatory and luteal phase of the menstrual cycle was based on basal body temperature. The preovulatory and luteal phase cervical mucus samples investigated for sialic acid were from day -7 to day 0, and day 0 to day +lO,respectively. The control cervical mucus samples examined by scanning electronmicroscopy were from the early and late preovulatory phase, day -9 and -2,respectively, and from the luteal phase day +2. The number of women and cervical mucus samples investigated are presented in Table I.
28
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION Table I Cervical Mucus Investigated
Contraceptive regime
None
Number of womsn
Duration of use
2
Abbreviations
Total number of cycles studied
Number of cervical mucus samples (P) (L)
Controls
2
9
4
IUD
2
2 months
Cu 72
2
5
5
Cu-7 IUD
2
6 months
Cu 7 6
2
6
5
Cu-7 IUD
2
13 months
Cu 713
2
6
6
D.S. IUD
2
2 months
D.Ss2
2
8
3
h-7
(P) = Preovulatory
(L) = Luteal
Sialic Acid Determinations Sialic acid determinations were carried out on gluteraldehyde-fixed, freeze dried cervical mucin using the thiobarbituric acid method (15). Preparation of Cervical Mucus Cervical mucus was washed in physiological saline three times, centrifuging between each washing for 10 minutes at a relative centrifugal force (RCF) of 2,500 g. The cervical mucin was then fixed in 0.6% gluteraldehyde in Sdrensen buffer, pH 7.2, followed by washing and freeze drying (14). Samples of the cervical mucin from the control cycles, day -9 and day +2 were plated with gold and platinum and examined by scanning electronmicroscopy (14). Neuraminidase Treatment Cervical mucus from the control cycles, day -2 was divided into two portions. One half was incubated with 500 units of neuraminidase mucopolysaccharide N-acetylneuraminylhydrolase (Batch No. HE792) (EC 3.2.1.18) ex. Vibrio comma (Cholerae) (Hoecht Pharmaceuticals Ltd., Middlesex, G.B.) for 1.5 hours at 370C in a total volume of 1.5 ml of sodium acetate buffer, pH 5.5 (in which the neuraminidase was supplied). The other half of the cervical mucus was similarly treated, but without neuraminidase.
JANUARY
1978 VOL. 17 NO. 1
29
CONTRACEPTION
After incubation the cervical mucus was prepared as previously described. Samples of the cervical mucin were examined by scanning electrvnmicroswpy (14). The anvunt of sialic acid renvved from the cervical mucin by neuraminidase was determined in the supernatant and by the difference of sialic acid content between the neuraminidase treated and untreated fixed freeze dried cervical mucin. Statistical Analysis The variance ratio (F) test was first applied to see if the parametric 't' test could be used. If the variance ratio was significantly different (p = cO.05) then the non-parametric ranking method was used (16). RESULTS A significant increase in sialic acid(s) levels were found in the preovulatory phase cervical mucin of the wntrols relative to the luteal phase (Table II). This represents a percentage difference between the mean sialic acid levels of approxismtely 53%. No significant difference in sialic acid(s) levels were found between the preovulatory and luteal phase of the contraceptive regimes, or when compared with the wntrols (Table II).
The removal of sialic acid(s) by neuraminidase from wntrol preovulatory cervical mucin (Figure 1), resulted in a structure (Figure 2) similar to that found in the early preovulatory (Figure 3) and luteal phase (Figure 4). The amount of sialic acid(s) removed by neuraminidase treatment was approximately 50 - 60%. Table II Sialic Acid Levels of Cervical Mucin in the Preovulatory and Luteal Phase Mean Values Significant Vg Sialic Acid/mg Dry % difference MUCin Pnxwulatory : Luteal
Significant % difference relative to wntrols Preovulatory : Luteal
Controls
29
13
cu 72
42
26
0.0
0.0
0.0
cu 76
21
17
0.0
0.0
0.0
26
36
0.0
0.0
0.0
31
26
0.0
0.0
0.0
cu 7 13 D.S.
30
53
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
Figure 1.
Preovulatory Cervical Mucin, clay-2 based on basal body temperature. Magnification 900x. Before treatment with neuraminidase.
Figure 2.
Preovulatory Cervical Mucin, day -2 based on basal body temperature. Xagnification 900x. After treatment with neuraminidase.
JANUARY
1978 VOL. 17 NO. 1
31
CONTRACEPTION
Figure 3.
Preovulatory Cervical Mucin, day -9 based on basal body temperature. Magnification 900x.
Figure 4.
32
Luteal Cervical Nucin, day +2 based on basal body temperature. Magnification 900x.
JANUARY
1978 VOL. 17 NO. 1
CONTRACEPTION
DISCUSSION The data of other investigators (3, 4, 5) suggests that sialic acid(s) may be increased in luteal phase cervical mucin, relative to the preovulatory phase, and be responsible for the change in its rheological properties (18). This report suggests the opposite, that is, increased. levels of sialic acid(s) have been found in preowlatory cervical mucin. In addition, the scanning electronmicroscopy results demonstrate that the removal of sialic acid(s) from preovulatory cervical mucin results in a structure similar to that found in the early preovulatory and luteal phase. The amount of sialic acid(s) removed to produce this type of structure was in the order of 50 - 60%. This is close to the percentage difference in sialic acid(s) levels found between the control preovulatory and luteal phase cervical mucin. There was no significant difference in sialic acid(s) levels between cervical mucin from the controls and the contraceptiveregimes. In addition, no significant difference was found in sialic acid(s) levels of cervical mucin between the preowlatory and luteal phase of the contraceptive regimes. It would appear that the apparent increase and decrease of sialic acid(s) levels found in the contraceptive regimes are not significant relative to the controls, but result in similar statistical levels of sialic acid(s) in both phases of the menstrual cycle. This may be due to the heterogeneityof the cervical mucin (1, 2, 5, 17) or the inflammatory response induced by the presence of the IUDs (11) resulting in an influx of phagocytic cells into the cervical mucus. These cells may become trapped in the cervical mucin and may not be removed by the washing procedure described. Alternatively, it might be a reflection of changes in glycosyltransferaseactivities of the cervical mucosa, due to changes in the trace metal balance of the cervical mucus (8). In conclusion, these studies demonstrate the cyclic behaviour of cervical mucin during the menstrual cycle, and the importance of sialic acid to the structural integrity of cervical mucin. The effect of the IUDs on the sialic acid(s) levels of cervical mucin has not been completely elucidated.
JANUARY
1978 VOL. 17 NO. 1
33
CONTRACEPTION REFERENCES
34
1.
Gell, P., Crepin, G., Roussel, P., Degand, P. and naves, R.: Gynec. Obstet. (Paris). 68: 279, 1969.
2.
Masson, P. L., Heremans, J. F. and Ferin, J.: Int. J. Fert., 14: 1: 1969.
3.
Moghissi, K. S. and Blandau, R. J.: J. Reprod. Med. 8: (1): 2, 1971.
4.
Gibbons, R. A.: Biochem.
5.
Iacobelli, S., Garea, N. and Angeloni, C.: Fertil. Steril. 22: 727, 1971.
6.
Gottschalk, A.: Nature. 186: 949, 1960.
7.
Bettelheim, F. A. and Dey, S. K.: Archs. Biochem. 109: 259, 1965.
8.
Daunter, B., Chantler, E. N. and Elstein, Contraception 15: (5): 543, 1977.
9.
Underwood, E. J.: Trace Elements in Human and Animal Nutrition. 2nd Edition. Publ. Academic Press, N.Y. U.S.A. 48, 1962.
J.
72: (3): 217, 1959.
M:
10.
Dixon, M. and Webb, E. C.: Enzymes. 2nd Edition. Publ. Longmans, 1971.
11.
Salgo. M. P. and Webb, F. T.: Res. Review. p 3, 1976.
12.
S-ur, M. B., Iskanders, S. G, and Rifai, S. F.: Am. J. Obstet. Gynec. 98: 946, 1967.
13.
Pakrashi, A. and Ray, G. R.: J. Reprod. Fertil. 357, 1969.
14.
Daurter, B., Chantler, E. N. and Elstein, M.: Brit. J. Obstet. Gynaec. 83: (9): 738, 1976.
15.
Warren,
16.
Wilcoxon, F.: (Biomed. Bul. 1: 80, 1945): Cited by Colquhon, D.: In: Lectures on Biostatistics. Clarendon Press, Oxford, 1976.
17.
Gibbons, R. A. and Roberts, G. P.: Acad. Sci. (NY). 106: 218, 1963.
18.
Masson,
L. :
J. Biol. Chem.
19:
234: 1971, 1959.
P. L.: In: Cervical Mucus in Human Reproduction. W.H.O. Colloquim, September, 1972. (Eds. Elstein, M. and Moghissi, K. S.). Publ. University of Chicago Press, U. S. A., 1973.
JANUARY
1978 VOL. 17 NO. 1