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Histochemical changes in cervical mucus-secreting epithelium during the normal menstrual cycle
FERTILITY AND STERILITY
Vol. 61, No.2, February 1989 Printed in U.S.A.
Copyright <> 1989 The American Fertility Society
Histochemical changes in cervical mucus-secreting epithelium during the normal menstrual cycle
Cyril B. Gilks, M.D. Philip E. Reid, Ph.D. Philip B. Clement, M.D. David A. Owen, M.B., B.Ch.* Department of Pathology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Recently developed specific histochemical techniques were applied to sections of human endocervix to investigate possible cyclic changes in stainable intracellular mucin glycoproteins. During the secretory phase, there is a decrease in the sialic acid/0-sulfate ester ratio, a decrease in the neutral sugar/acidic sugar ratio and increased amounts of 0sulfate ester, compared with during the proliferative phase. Endocervical epithelia from prepubertal, pregnant, and postmenopausal females also show decreased sialic acid/0sulfate ester and neutral sugar/acidic sugar ratios, compared with during the proliferative phase. The authors conclude that proliferative phase mucin is chemically different from secretory phase mucin, and that this difference may be significant in allowing sperm penetration in the periovulatory period. Fertil Steril51:286, 1989
Mucin glycoproteins secreted by the human endocervical mucosa form the predominant solid component of cervical mucus. This mucus protects the sterility of the uterine cavity and is permissive for sperm penetration for a period of approximately 5 days in the periovulatory period. During this period, the mucus, in response to estrogenic stimulation, becomes copious in amount and shows striking changes in physical characteristics, becoming much less viscous and less acidic. 1- 3 The visco-elasticity of cervical mucus (CM) has been shown to be a function of its mucin glycoprotein component.4 Mucin glycoproteins are characterized by a high proportion of carbohydrate prosthetic groups linked to the serine or threonine residues of a peptide backbone by 0-glycosidic linkages. Attempts to correlate the physical Received May 12, 1988; revised and accepted October 12, 1988. * Reprint requests: David A. Owen, M.B., B.Ch., Associate Professor, Department of Pathology, Vancouver General Hospital, 855 West 12th Avenue, Vancouver, British Columbia V5Z 1M9, Canada.
286
Gilks et al.
Cyclic changes in cervical mucins
changes in CM with chemical changes in the mucin have shown that the protein backbone does not change during the menstrual cycle.6 Changes in the carbohydrate composition of cervical mucin corresponding to the changes in visco-elasticity have not been consistently demonstrated. Some studies have shown cyclic changes in sialic acid content,6•7 whereas others have been unable to demonstrate strch changes.6 •8 A promising approach to the study of possible cyclic changes in the carbohydrate component of mucin is to use specific histochemical methods to examine the relative amounts of stainable sugars within endocervical epithelial cells. The stainable sugars within endocervical epithelium are part of the carbohydrate side chains of the mucin glycoprotein molecules; thus, changes in their histochemical staining reflect changes in the composition of the intracellular mucin. Recently we have shown, using a new histochemical method, that the neutral sugar/acidic sugar ratio decreases during the secretory phase. 9 In a continuation of these studies, we now present the re-
Fertility and Sterility
Table 1
Structural Elements Stained by the Histochemical Technique Used in the Investigation of Cervical Mucins• Sialic acids with side chain 0acyl substituent& b
KOH/PA*/ABLO/S KOH/PA*/Bh/ AB2.5/PAS PA*/S PA/Bh/KOH/PAS ABl.O
0-sulfate esters
Neutral sugar vicinal diols •
None
c7
CsorCod
References
A A
0 R
R 0
R 0
R 0
15 9
0 0
0 0 0
R 0 0
R R 0
0 R 0
11,12 13,14 13
A
• A, aqua; R, red; P, purple; 0, no staining. (In procedures in which two dyes are used, mixtures of the structural elements stain in shades of purple). b Data presented in Table assumes that C9 0-acyl sialic acids
are not oxidized under the conditions employed.11•12 • Hexose, 6-deoxyhexose and N -acetyl hexosamine. d For histochemical purposes, this class of sialic acids also includes those with 2 or 3 side chain 0-acyl substituent&.
sults of an expanded study of cervical epithelial glycoproteins during the normal menstrual cycle and in prepubertal and postmenopausal patients.
odic acid/borohydride/potassium hydroxide/periodic acid Schiff's/alcian blue pH 1.0 (PA/Bh/ KOH/PAS/AB 1.0). These abbreviations indicate that the following reactions have been carried out: KOH, saponification with 0.5% (wt/vol) potassium hydroxide at room temperature for 15 minutes, which will remove 0-acyl esters; AB 2.5, staining with alcian blue 8Gx in 3% acetic acid pH 2.5 at room temperature for 20 minutes; AB 1.0, staining with alcian blue 8Gx in 0.1 M hydrochloric acid pH 1.0 at room temperature for 30 minutes; PA, oxidation with 1% (wt/vol) periodic acid; P A*, oxidation at 4 ·c for 1 hour with a solution of 0.4 mM periodic acid in 1 M hydrochloric acid to produce total oxidation of sialic acids, but without significant oxidation of neutral sugars; S, staining with Schiff's reagent for 1 hour at room temperature; Bh, reduction with sodium borohydride. The experimental details and specificities of these methods have been described elsewhere.9•11- 17 Histochemical observations were made by two observers using a co-observation bridge and the consensus result recorded. The histochemical stains were assessed without prior knowledge of the patient's age or menstrual status. In the pediatric cases, however, the small size of the cervices was obvious, and the results of histochemical staining could not be assessed "blindly." The colors observed in the combined alcian blue/periodic acidSchiff (PAS) procedures were converted to numerical values as follows: aqua (the color of alcian blue alone) = 1; blue = 2; purple = 3; red purple = 4; and magenta (the color of the PAS alone) = 5. The intensity of staining with the alcian blue pH 1.0 and P A* /S techniques was recorded on a scale of 0 (no staining) to 4 (maximum staining). A previous
MATERIALS AND METHODS
Specimens of formalin -fixed, paraffin -embedded cervix from 86 cases were obtained from the surgical pathology files of the Vancouver General Hospital and the autopsy files of the British Columbia Children's Hospital. These included 11 prepubertal cervixes, 20 proliferative phase cervixes, 33 secretory phase cervixes, 3 pregnancy cervixes, and 19 postmenopausal cervixes. Endometrium from women in the reproductive years was independently phased according to accepted criteria10 by two of the authors (C.B.G. and P.B.C.). The menstrual cycle was divided into six parts: ( 1) early proliferative; (2) mid to late proliferative; (3) early secretory; (4) mid-secretory; (5) late secretory; and (6) menstrual. The postmenopausal patients were divided into two groups; those with atrophic endometria and those with endometrial hyperplasia. Sections of cervix cut at a thickness of 5 JLm were stained with hematoxylin and eosin for morphologic assessment and with specific histochemical techniques to assess the chemical composition of the cervical mucins (Table 1). The techniques employed included potassium hydroxide/periodic acid and hydrochloric acid/alcian blue pH 1.0/Schi:ff's (KOH/PA*/AB 1.0/S; PA* indicates oxidation with periodic acid and hydrochloric acid), potassium hydroxide/periodic acid and hydrochloric acid/borohydride/alcian blue pH 2.5/periodic acid Schiff's (KOH/PA*/Bh/AB 2.5/PAS), periodic acid and hydrochloric acid/Schiff's (PA* /S), peri-
Vol. 51, No.2, February 1989
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Cyclic changes in cervical mucins
287
5
3.5
5
..........
3.5
.......
...... .... 2
""
......
2
Proliferative
Figure 1 Results of semiquantitative assessment of sections of cervix stained with the KOH/PA• I AB 1.0/Schiff method, to demonstrate the ratio of sialic acid/0-sulfate ester. The higher numerical grades correspond to higher sialic acid/0-sulfate ester ratios. The sialic acid/0-sulfate ester ratio is significantly higher during the proliferative phase than in prepubertal, secretory phase, or postmenopausal patients (P < 0.001 for all comparisons).
study18 has demonstrated a good correlation between results using such histochemical scales and results obtained by chemical analysis. Statistical analyses were performed using the Mann-Whitney test with appropriate correction for multiple comparisons.19
....
_
Figure 2 Results of semiquantitative assessment of sections of cervix stained with the KOH/PA */Bh/AB 2.5/PAS method, to demonstrate the ratio of neutral to acidic sugars. The higher numerical grades correspond to higher neutral/acidic sugar ratios. The neutral/acidic sugar ratio is significantly higher during the proliferative phase than in prepubertal or postmenopausal patients (P < 0.001 for both comparisons).
hyperplasia or atrophic endometria and therefore, the results from these groups were combined (Figs .. 2 and 3). The prepubertal group included five preterm stillborn infants as well as six individuals ranging in age from 21 months to 6.5 years. Only the latter were included in the data shown in Fig5
RESULTS The results of the semiquantitative histochemical studies of the cervical mucins are illustrated in Figures 1, 2, and 3. These studies were performed on specimens that contained endocervical glands at the squamocolumnar junction, showed no morphologic abnormality of the cervix except minor degrees of inflammation, and had endometrium suitable for phasing. Histochemical assessments were confined to glands superior to the anatomic squamocolumnar junction, as glands in the transformation zone do not respond normally to hormonal stimuli. 20 Because preliminary data analysis demonstrated no histochemical differences in the mucins within the subdivisions of the proliferative and secretory phases, the results from all early, mid, and late secretory and menstrual specimens were combined as secretory in Figures 1 to 3, while specimens from earJy and mid to late proliferative phases were combined as proliferative. No significant differences were observed in the cervical mucins of postmenopausal women with endometrial
288
Gilks et al. Cyclic changes in cervical mucins
4
Pro~ftralive
StcreiOiy
PA*/S
Proliferative
Secrttory
Ab 1.0
Figure 3 Results of semiquantitative assessment of sections of cervix stained with the P A*/S method to demonstrate amounts of sialic acid, and with the AB 1.0 method to demonstrate amounts of 0-sulfate ester are shown. The higher assessed numerical grades correspond to the presence of more stainable mucin. There is no significant difference in the amount of sialic acid during the proliferative and secretory phase. There is more 0-sulfate ester present during the secretory phase compared with during the proliferative phase (P < 0.02).
Fertility and Sterility
ures 1 and 2. There was insufficient tissue to perform all of the histochemical stains on all tissue specimens; thus, fewer cases were stained with the PA*/S and AB 1.0 techniques (Fig. 3). Sections of cervix from the prepubertal and postmenopausal cases were only stained with the KOH/PA*I AB 1.0/S, KOH/PA*/Bh/AB 2.5/PAS and PA*/Bh/ KOH/PAS techniques. As shown in Figure 1, the ratio of sialic acid to 0-sulfate ester, as determined by the KOH/PA *I AB 1.0/S procedure, was significantly higher (P < 0.001) in the proliferative phase mucins than in mucins from the secretory phase or in mucins from prepubertal (P < 0.001) or postmenopausal (P < 0.001) individuals. This difference between the two phases of the menstrual cycle can be attributed to increased sulfation in the secretary phase because the intensity of the staining of the secretory phase mucins with AB 1.0 was significantly greater (P < 0.02) than in the proliferative phase (Fig. 3). The ratio of neutral to acidic sugar, as demonstrated with the KOH/PA*/Bh/AB 2.5/PAS method, was lower in mucins from prepubertal (P < 0.001) and postmenopausal patients (P < 0.001) than in mucins from the proliferative phase. We have previously demonstrated that this ratio is also lower in the secretory phase compared with the proliferative phase. 9 Presumably, this difference reflects the increased sulfation seen in secretory phase, prepubertal, and postmenopausal mucins; however, the possibility of concomitant reductions in the quantities of neutral sugars has not been eliminated. No staining of the cervical mucins was observed with the PA */Bh/KOH/PAS technique, indicating that side chain 0-acylated sialic acids are absent in normal cervical mucins at all times of life. However, this staining procedure gave positive results with the glycogen in the exocervical squamous epithelium. This staining did not complicate interpretation, as the columnar epithelium of the endocervix does not contain glycogen stainable with the PAS method. Staining of the exocervical squamous epithelium with the PA*/Bh/KOH/ PAS procedure could be eliminated by prior diastase digestion or by prolonging the initial period of periodic acid oxidation used in the staining technique. In addition to the results of semiquantitative analysis, interesting qualitative changes were seen. In five of seven cases from early secretory phase (postovulatory days 2 to 4), a zonal distribution of
Vol. 51, No.2, February 1989
staining was seen within the glands with KOH/ PA*I AB 1.0/S staining. In these cases, there was a lower ratio of sialic acid to 0-sulfate ester at the base of the glands; that is, the mucin at the base of the glands was chemically like the secretory phase mucin, whereas the superficial parts of the glands contained mucin similar to that seen during the proliferative phase. In cervixes from preterm stillborn infants, mucin staining first appears at 26 weeks gestational age {2 cases) and was localized to less than ten cells at the squamocolumnar junction in these cases. Intracellular mucin was not seen in less mature infants (21, 22, and 24 weeks gestational age). In contrast, mucin appears in the fetal gastrointestinal tract by 10 weeks gestational age. 21 Cervixes from three pregnant patients were examined. Although the number of cases was too small for statistical analysis, the mucin staining pattern was similar to that seen during secretory phase, with a lower sialic acid/0-sulfate ester ratio and lower neutral/acidic sugar ratio than during the proliferative phase. DISCUSSION
Early histochemical studies of cervical mucins failed to establish cyclic changes in cervical mucin composition (reviewed by Wakefield and Wells 22 ). However, they did demonstrate the presence of abundant intracellular mucin in both menstrual and postmenopausal cervixes and showed that sialic acid and sulfated mucins both may be found in the cervix. Wakefield and W ells 22 were the first to employ more specific histochemical techniques. Using the high iron diamine/alcian blue technique, they found an increased ratio of sialic acid/0-sulfate ester in intracellular endocervical mucins during the proliferative phase compared with during the secretory phase. We have confirmed and expanded Wakefield and Wells' results, demonstrating definite differences between proliferative phase and secretory phase, prepubertal, and postmenopausal cervical mucins using specific histochemical techniques. The intracellular mucins of the proliferative phase are characterized by an increased ratio of sialic acid/0-sulfate esters, due to an absolute reduction in 0-sulfate esters, and an increased ratio of neutral/acidic sugars. It is not possible to correlate precisely the structure of intracellular mucins with the structure of
Gilks et al.
Cyclic changes in cervical mucins
289
mucin glycoprotein in extracellular cervical mucus at any given time. There is a variable delay between synthesis and secretion; this delay is shortest in the periovulatory period, when the secretory rate is greatest. At other times during the menstrual cycle, there may be a significant delay between the synthesis of the mucin and its secretion. We believe the intracellular mucins seen during the proliferative phase (days 5 to 15 of a 28-day cycle) correspond to the less viscous, copious secretory product of the periovulatory period, while the intracellular mucins during secretory and menstrual phase (days 16 to 28 and 1 to 4) and in prepubertal and postmenopausal cervices correspond to the thick tenacious secretory product seen at other times during the menstrual cycle and during the nonreproductive years. A transition between these two types of mucin was seen in most cases during the early secretory phase. Our results can all be explained by an increase in the sulfation of mucin during the secretory phase and during postmenopausal and prepubertal periods or, conversely, by a decrease in the sulfation of cervical mucins during the proliferative phase. Sulfation of glycoproteins is a terminal event in the synthetic sequence, and involves the transfer of a sulfate group from 3'-phosphoadenosine 5'phosphosulfate under the control of one or more sulfate transferase enzymes.23 It is not known how the enzyme or enzymes that catalyze this reaction are regulated. Our results suggest that they may be under hormonal control in endocervical cells, which contain hormone receptor proteins. 24•25 Alternately, it could be argued that sulfation occurs at a constant rate, and the degree of sulfation is related to the rate of secretion, with proliferative phase mucin being less sulfated because it is secreted more rapidly. Further experimental work is necessary to determine if sulfation is under hormonal control. Chemical analysis of the sulfate content of CM, aspirated from the endocervical canal, using a colorimetric method, showed only a small cyclic change in sulfate concentrations (0.35 gm/100 gm preovulatory, .42 gm/100 gm postovulatory). 7 Because of the small molecular weight of sulfate relative to sialic acid, histochemical stains will, theoretically, be more sensitive to small changes in the ratio of sialic acid to sulfate than chemical analysis. Further, the histochemical method used assesses only sulfate bound as an ester, which is the chemical form of sulfate in mucin glycoproteins.
290
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Cyclic changes in cervical mucins
Sialic acid concentrations during the menstrual cycle also have been studied by chemically analyzing aspirated cervical mucus. Some studies showed an increase in the concentration of sialic acid during the periovulatory fertile period,6 •7 while others showed no such difference. 5 •8 Our result, an increase in stainable sialic acid residues during the proliferative phase that does not reach statistical significance, is consistent with these results, and suggests that a cyclic change in sialic acid concentration may occur. The fact that this change cannot be consistently demonstrated suggests that it is unlikely to be responsible for the striking alterations in the physical properties of cervical mucus observed during different phases of the menstrual cycle. In conclusion, we have found significant differences in the histochemistry of cervical mucins during the proliferative phase compared with during the secretory phase or during nonreproductive years. These changes are, to date, the only consistently demonstrable cyclic changes in the structure of cervical mucin glycoprotein, which forms the . predominant solid component of cervical mucus. These chemical changes may contribute to the changes in the physical properties of the CM, which allow sperm penetration during the periovulatory period. REFERENCES 1. Fordney-Settlage D: A review of cervical mucus and sperm interactions in humans. lnt J Fertil26:161, 1981 2. Chantler E: Structure and function of cervical mucus. In Mucus in Health and Disease II, Edited by E Chantler, J Elder, M Elstein. New York and London, Plenum Press, 1982,p 251 3. Daunter B: Biochemical and functional structural aspects of human cervical mucin. Scanning Electron Microscopy 1: 345,1984 4. Gibbons RA: Molecular shape and the chemical properties of mucin. Nature 184:610, 1959 5. WolfDP, Sokolaski JE, Litt M: Composition and function of human cervical mucus. Biochim Biophys Acta 630:545, 1980 6. Moghissi KS, Syner FN: Cyclic changes in the amount of sialic acid in cervical mucus. Int J Fertil Steril 21:246, 1976 7. Iacobelli S, Garcea N, Angeloni A: Biochemistry of cervical mucus, a comparative analysis of the secretion from preovulatory, postovulatory and pregnancy periods. Fertil Steril22:727, 1971 8. Van Kooij RJ, Roelofs HJM, Kathmann GAM, Kramer MF: Human cervical mucus and its mucous glycoprotein during the menstrual cycle. Fertil Steril 34:226, 1980 9. Gilks CB, Reid PE, Clement PB, Owen DA: A simple histochemical procedure for the assessment ofthe relative quan-
Fertility and Sterility
10. 11.
12.
13.
14.
15.
tities of neutral and acidic sugars in mucin glycoproteins and its use in assessing cyclical changes in human cervical mucins. J Clin Pathol. 41:1021, 1988 Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, 1950 Volz D, Reid PE, Park CM, Owen DA, Dunn WL: A new method for the selective periodate oxidation of total tissue sialic acids. Histochem J 19:249, 1987 Volz D, Reid PE, Park CM, Owen DA, Dunn WL: Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain 0acyl variants or 0-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids. Histochem J 19:257, 1987 Culling CF A, Reid PE, Dunn WL, Clay MG: The histochemical demonstration of 0-acylated sialic acids in gastrointestinal mucins: their association with the potassium hydroxide-periodic acid-Schiff effect. J Histochem Cytochem 22:826, 1974 Reid PE, Culling CFA, Dunn WL: Saponification induced increase in the periodic acid-Schiffreaction in the gastrointestinal tract: mechanism and distribution of the reactive substance. J Histochem Cytochem 21:473, 1973 Reid PE, Volz D, Park CM, Owen DA, Dunn WL: Methods for the identification of sidechain 0-acyl substituted sialic acids and for the simultaneous visualization of sialic acid, its 0-acyl variants and 0-sulphate ester. Histochem J 19: 396,1987
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16. Barger JD, DeLamater ED: The use of thionyl chloride in the preparation of Schiff reagent. Science 108:121, 1948 17. Lillie RD, Pizzolato P: Histochemical use of borohydrides as aldehyde blocking reagents. Stain Technol43:13, 1972 18. Reid PE, Ramey CW, Owen DA, Dunn WL, Lazosky DA, Clay MG: Chemical and histochemical studies of normal and diseased gastrointestinal tract. IV. A comparison of histochemical and chemical methods for the estimation of side chain 0-acylated sialic acids. Histochem J 17:183, 1983 19. Zar JH: Biostatistical Analysis, 2nd edition. Englewood Cliffs, New Jersey, Prentice Hall, 1983, p 138 20. Singer A: Dynamic anatomy of the cervical epithelium. In Mucus in Health and Disease I, Edited by M Elstein, DV Parke. New York, Plenum Press, 1976, p 77 21. Reid PE, Owen DA: Unpublished observations 22. Wakefield EA, Wells M: Histochemical study of endocervical glycoproteins throughout the normal menstrual cycle and adjacent to cervical intraepithelial neoplasia. Int J Gynecol Pathol 4:230, 1985 23. Orten JM, Neuhaus 0: in Human Biochemistry, lOth edition. St. Louis, CV Mosby Company, 1983, p 408 24. Sanborn BM, Held B, Kuo S: Specific estrogen binding proteins in human endocervix. J Steroid Biochem 6:1107, 1975 25. Sanborn BM, Held B, Kuo S: Hormone action in human cervix. II. Specific progestogen binding proteins in human cervix. J Steroid Biochem 7:665, 1976
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Vol. 61, No.2, February 1989 Printed in U.S.A.
Copyright <> 1989 The American Fertility Society
Histochemical changes in cervical mucus-secreting epithelium during the normal menstrual cycle
Cyril B. Gilks, M.D. Philip E. Reid, Ph.D. Philip B. Clement, M.D. David A. Owen, M.B., B.Ch.* Department of Pathology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Recently developed specific histochemical techniques were applied to sections of human endocervix to investigate possible cyclic changes in stainable intracellular mucin glycoproteins. During the secretory phase, there is a decrease in the sialic acid/0-sulfate ester ratio, a decrease in the neutral sugar/acidic sugar ratio and increased amounts of 0sulfate ester, compared with during the proliferative phase. Endocervical epithelia from prepubertal, pregnant, and postmenopausal females also show decreased sialic acid/0sulfate ester and neutral sugar/acidic sugar ratios, compared with during the proliferative phase. The authors conclude that proliferative phase mucin is chemically different from secretory phase mucin, and that this difference may be significant in allowing sperm penetration in the periovulatory period. Fertil Steril51:286, 1989
Mucin glycoproteins secreted by the human endocervical mucosa form the predominant solid component of cervical mucus. This mucus protects the sterility of the uterine cavity and is permissive for sperm penetration for a period of approximately 5 days in the periovulatory period. During this period, the mucus, in response to estrogenic stimulation, becomes copious in amount and shows striking changes in physical characteristics, becoming much less viscous and less acidic. 1- 3 The visco-elasticity of cervical mucus (CM) has been shown to be a function of its mucin glycoprotein component.4 Mucin glycoproteins are characterized by a high proportion of carbohydrate prosthetic groups linked to the serine or threonine residues of a peptide backbone by 0-glycosidic linkages. Attempts to correlate the physical Received May 12, 1988; revised and accepted October 12, 1988. * Reprint requests: David A. Owen, M.B., B.Ch., Associate Professor, Department of Pathology, Vancouver General Hospital, 855 West 12th Avenue, Vancouver, British Columbia V5Z 1M9, Canada.
286
Gilks et al.
Cyclic changes in cervical mucins
changes in CM with chemical changes in the mucin have shown that the protein backbone does not change during the menstrual cycle.6 Changes in the carbohydrate composition of cervical mucin corresponding to the changes in visco-elasticity have not been consistently demonstrated. Some studies have shown cyclic changes in sialic acid content,6•7 whereas others have been unable to demonstrate strch changes.6 •8 A promising approach to the study of possible cyclic changes in the carbohydrate component of mucin is to use specific histochemical methods to examine the relative amounts of stainable sugars within endocervical epithelial cells. The stainable sugars within endocervical epithelium are part of the carbohydrate side chains of the mucin glycoprotein molecules; thus, changes in their histochemical staining reflect changes in the composition of the intracellular mucin. Recently we have shown, using a new histochemical method, that the neutral sugar/acidic sugar ratio decreases during the secretory phase. 9 In a continuation of these studies, we now present the re-
Fertility and Sterility
Table 1
Structural Elements Stained by the Histochemical Technique Used in the Investigation of Cervical Mucins• Sialic acids with side chain 0acyl substituent& b
KOH/PA*/ABLO/S KOH/PA*/Bh/ AB2.5/PAS PA*/S PA/Bh/KOH/PAS ABl.O
0-sulfate esters
Neutral sugar vicinal diols •
None
c7
CsorCod
References
A A
0 R
R 0
R 0
R 0
15 9
0 0
0 0 0
R 0 0
R R 0
0 R 0
11,12 13,14 13
A
• A, aqua; R, red; P, purple; 0, no staining. (In procedures in which two dyes are used, mixtures of the structural elements stain in shades of purple). b Data presented in Table assumes that C9 0-acyl sialic acids
are not oxidized under the conditions employed.11•12 • Hexose, 6-deoxyhexose and N -acetyl hexosamine. d For histochemical purposes, this class of sialic acids also includes those with 2 or 3 side chain 0-acyl substituent&.
sults of an expanded study of cervical epithelial glycoproteins during the normal menstrual cycle and in prepubertal and postmenopausal patients.
odic acid/borohydride/potassium hydroxide/periodic acid Schiff's/alcian blue pH 1.0 (PA/Bh/ KOH/PAS/AB 1.0). These abbreviations indicate that the following reactions have been carried out: KOH, saponification with 0.5% (wt/vol) potassium hydroxide at room temperature for 15 minutes, which will remove 0-acyl esters; AB 2.5, staining with alcian blue 8Gx in 3% acetic acid pH 2.5 at room temperature for 20 minutes; AB 1.0, staining with alcian blue 8Gx in 0.1 M hydrochloric acid pH 1.0 at room temperature for 30 minutes; PA, oxidation with 1% (wt/vol) periodic acid; P A*, oxidation at 4 ·c for 1 hour with a solution of 0.4 mM periodic acid in 1 M hydrochloric acid to produce total oxidation of sialic acids, but without significant oxidation of neutral sugars; S, staining with Schiff's reagent for 1 hour at room temperature; Bh, reduction with sodium borohydride. The experimental details and specificities of these methods have been described elsewhere.9•11- 17 Histochemical observations were made by two observers using a co-observation bridge and the consensus result recorded. The histochemical stains were assessed without prior knowledge of the patient's age or menstrual status. In the pediatric cases, however, the small size of the cervices was obvious, and the results of histochemical staining could not be assessed "blindly." The colors observed in the combined alcian blue/periodic acidSchiff (PAS) procedures were converted to numerical values as follows: aqua (the color of alcian blue alone) = 1; blue = 2; purple = 3; red purple = 4; and magenta (the color of the PAS alone) = 5. The intensity of staining with the alcian blue pH 1.0 and P A* /S techniques was recorded on a scale of 0 (no staining) to 4 (maximum staining). A previous
MATERIALS AND METHODS
Specimens of formalin -fixed, paraffin -embedded cervix from 86 cases were obtained from the surgical pathology files of the Vancouver General Hospital and the autopsy files of the British Columbia Children's Hospital. These included 11 prepubertal cervixes, 20 proliferative phase cervixes, 33 secretory phase cervixes, 3 pregnancy cervixes, and 19 postmenopausal cervixes. Endometrium from women in the reproductive years was independently phased according to accepted criteria10 by two of the authors (C.B.G. and P.B.C.). The menstrual cycle was divided into six parts: ( 1) early proliferative; (2) mid to late proliferative; (3) early secretory; (4) mid-secretory; (5) late secretory; and (6) menstrual. The postmenopausal patients were divided into two groups; those with atrophic endometria and those with endometrial hyperplasia. Sections of cervix cut at a thickness of 5 JLm were stained with hematoxylin and eosin for morphologic assessment and with specific histochemical techniques to assess the chemical composition of the cervical mucins (Table 1). The techniques employed included potassium hydroxide/periodic acid and hydrochloric acid/alcian blue pH 1.0/Schi:ff's (KOH/PA*/AB 1.0/S; PA* indicates oxidation with periodic acid and hydrochloric acid), potassium hydroxide/periodic acid and hydrochloric acid/borohydride/alcian blue pH 2.5/periodic acid Schiff's (KOH/PA*/Bh/AB 2.5/PAS), periodic acid and hydrochloric acid/Schiff's (PA* /S), peri-
Vol. 51, No.2, February 1989
Gilks et al.
Cyclic changes in cervical mucins
287
5
3.5
5
..........
3.5
.......
...... .... 2
""
......
2
Proliferative
Figure 1 Results of semiquantitative assessment of sections of cervix stained with the KOH/PA• I AB 1.0/Schiff method, to demonstrate the ratio of sialic acid/0-sulfate ester. The higher numerical grades correspond to higher sialic acid/0-sulfate ester ratios. The sialic acid/0-sulfate ester ratio is significantly higher during the proliferative phase than in prepubertal, secretory phase, or postmenopausal patients (P < 0.001 for all comparisons).
study18 has demonstrated a good correlation between results using such histochemical scales and results obtained by chemical analysis. Statistical analyses were performed using the Mann-Whitney test with appropriate correction for multiple comparisons.19
....
_
Figure 2 Results of semiquantitative assessment of sections of cervix stained with the KOH/PA */Bh/AB 2.5/PAS method, to demonstrate the ratio of neutral to acidic sugars. The higher numerical grades correspond to higher neutral/acidic sugar ratios. The neutral/acidic sugar ratio is significantly higher during the proliferative phase than in prepubertal or postmenopausal patients (P < 0.001 for both comparisons).
hyperplasia or atrophic endometria and therefore, the results from these groups were combined (Figs .. 2 and 3). The prepubertal group included five preterm stillborn infants as well as six individuals ranging in age from 21 months to 6.5 years. Only the latter were included in the data shown in Fig5
RESULTS The results of the semiquantitative histochemical studies of the cervical mucins are illustrated in Figures 1, 2, and 3. These studies were performed on specimens that contained endocervical glands at the squamocolumnar junction, showed no morphologic abnormality of the cervix except minor degrees of inflammation, and had endometrium suitable for phasing. Histochemical assessments were confined to glands superior to the anatomic squamocolumnar junction, as glands in the transformation zone do not respond normally to hormonal stimuli. 20 Because preliminary data analysis demonstrated no histochemical differences in the mucins within the subdivisions of the proliferative and secretory phases, the results from all early, mid, and late secretory and menstrual specimens were combined as secretory in Figures 1 to 3, while specimens from earJy and mid to late proliferative phases were combined as proliferative. No significant differences were observed in the cervical mucins of postmenopausal women with endometrial
288
Gilks et al. Cyclic changes in cervical mucins
4
Pro~ftralive
StcreiOiy
PA*/S
Proliferative
Secrttory
Ab 1.0
Figure 3 Results of semiquantitative assessment of sections of cervix stained with the P A*/S method to demonstrate amounts of sialic acid, and with the AB 1.0 method to demonstrate amounts of 0-sulfate ester are shown. The higher assessed numerical grades correspond to the presence of more stainable mucin. There is no significant difference in the amount of sialic acid during the proliferative and secretory phase. There is more 0-sulfate ester present during the secretory phase compared with during the proliferative phase (P < 0.02).
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ures 1 and 2. There was insufficient tissue to perform all of the histochemical stains on all tissue specimens; thus, fewer cases were stained with the PA*/S and AB 1.0 techniques (Fig. 3). Sections of cervix from the prepubertal and postmenopausal cases were only stained with the KOH/PA*I AB 1.0/S, KOH/PA*/Bh/AB 2.5/PAS and PA*/Bh/ KOH/PAS techniques. As shown in Figure 1, the ratio of sialic acid to 0-sulfate ester, as determined by the KOH/PA *I AB 1.0/S procedure, was significantly higher (P < 0.001) in the proliferative phase mucins than in mucins from the secretory phase or in mucins from prepubertal (P < 0.001) or postmenopausal (P < 0.001) individuals. This difference between the two phases of the menstrual cycle can be attributed to increased sulfation in the secretary phase because the intensity of the staining of the secretory phase mucins with AB 1.0 was significantly greater (P < 0.02) than in the proliferative phase (Fig. 3). The ratio of neutral to acidic sugar, as demonstrated with the KOH/PA*/Bh/AB 2.5/PAS method, was lower in mucins from prepubertal (P < 0.001) and postmenopausal patients (P < 0.001) than in mucins from the proliferative phase. We have previously demonstrated that this ratio is also lower in the secretory phase compared with the proliferative phase. 9 Presumably, this difference reflects the increased sulfation seen in secretory phase, prepubertal, and postmenopausal mucins; however, the possibility of concomitant reductions in the quantities of neutral sugars has not been eliminated. No staining of the cervical mucins was observed with the PA */Bh/KOH/PAS technique, indicating that side chain 0-acylated sialic acids are absent in normal cervical mucins at all times of life. However, this staining procedure gave positive results with the glycogen in the exocervical squamous epithelium. This staining did not complicate interpretation, as the columnar epithelium of the endocervix does not contain glycogen stainable with the PAS method. Staining of the exocervical squamous epithelium with the PA*/Bh/KOH/ PAS procedure could be eliminated by prior diastase digestion or by prolonging the initial period of periodic acid oxidation used in the staining technique. In addition to the results of semiquantitative analysis, interesting qualitative changes were seen. In five of seven cases from early secretory phase (postovulatory days 2 to 4), a zonal distribution of
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staining was seen within the glands with KOH/ PA*I AB 1.0/S staining. In these cases, there was a lower ratio of sialic acid to 0-sulfate ester at the base of the glands; that is, the mucin at the base of the glands was chemically like the secretory phase mucin, whereas the superficial parts of the glands contained mucin similar to that seen during the proliferative phase. In cervixes from preterm stillborn infants, mucin staining first appears at 26 weeks gestational age {2 cases) and was localized to less than ten cells at the squamocolumnar junction in these cases. Intracellular mucin was not seen in less mature infants (21, 22, and 24 weeks gestational age). In contrast, mucin appears in the fetal gastrointestinal tract by 10 weeks gestational age. 21 Cervixes from three pregnant patients were examined. Although the number of cases was too small for statistical analysis, the mucin staining pattern was similar to that seen during secretory phase, with a lower sialic acid/0-sulfate ester ratio and lower neutral/acidic sugar ratio than during the proliferative phase. DISCUSSION
Early histochemical studies of cervical mucins failed to establish cyclic changes in cervical mucin composition (reviewed by Wakefield and Wells 22 ). However, they did demonstrate the presence of abundant intracellular mucin in both menstrual and postmenopausal cervixes and showed that sialic acid and sulfated mucins both may be found in the cervix. Wakefield and W ells 22 were the first to employ more specific histochemical techniques. Using the high iron diamine/alcian blue technique, they found an increased ratio of sialic acid/0-sulfate ester in intracellular endocervical mucins during the proliferative phase compared with during the secretory phase. We have confirmed and expanded Wakefield and Wells' results, demonstrating definite differences between proliferative phase and secretory phase, prepubertal, and postmenopausal cervical mucins using specific histochemical techniques. The intracellular mucins of the proliferative phase are characterized by an increased ratio of sialic acid/0-sulfate esters, due to an absolute reduction in 0-sulfate esters, and an increased ratio of neutral/acidic sugars. It is not possible to correlate precisely the structure of intracellular mucins with the structure of
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mucin glycoprotein in extracellular cervical mucus at any given time. There is a variable delay between synthesis and secretion; this delay is shortest in the periovulatory period, when the secretory rate is greatest. At other times during the menstrual cycle, there may be a significant delay between the synthesis of the mucin and its secretion. We believe the intracellular mucins seen during the proliferative phase (days 5 to 15 of a 28-day cycle) correspond to the less viscous, copious secretory product of the periovulatory period, while the intracellular mucins during secretory and menstrual phase (days 16 to 28 and 1 to 4) and in prepubertal and postmenopausal cervices correspond to the thick tenacious secretory product seen at other times during the menstrual cycle and during the nonreproductive years. A transition between these two types of mucin was seen in most cases during the early secretory phase. Our results can all be explained by an increase in the sulfation of mucin during the secretory phase and during postmenopausal and prepubertal periods or, conversely, by a decrease in the sulfation of cervical mucins during the proliferative phase. Sulfation of glycoproteins is a terminal event in the synthetic sequence, and involves the transfer of a sulfate group from 3'-phosphoadenosine 5'phosphosulfate under the control of one or more sulfate transferase enzymes.23 It is not known how the enzyme or enzymes that catalyze this reaction are regulated. Our results suggest that they may be under hormonal control in endocervical cells, which contain hormone receptor proteins. 24•25 Alternately, it could be argued that sulfation occurs at a constant rate, and the degree of sulfation is related to the rate of secretion, with proliferative phase mucin being less sulfated because it is secreted more rapidly. Further experimental work is necessary to determine if sulfation is under hormonal control. Chemical analysis of the sulfate content of CM, aspirated from the endocervical canal, using a colorimetric method, showed only a small cyclic change in sulfate concentrations (0.35 gm/100 gm preovulatory, .42 gm/100 gm postovulatory). 7 Because of the small molecular weight of sulfate relative to sialic acid, histochemical stains will, theoretically, be more sensitive to small changes in the ratio of sialic acid to sulfate than chemical analysis. Further, the histochemical method used assesses only sulfate bound as an ester, which is the chemical form of sulfate in mucin glycoproteins.
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Sialic acid concentrations during the menstrual cycle also have been studied by chemically analyzing aspirated cervical mucus. Some studies showed an increase in the concentration of sialic acid during the periovulatory fertile period,6 •7 while others showed no such difference. 5 •8 Our result, an increase in stainable sialic acid residues during the proliferative phase that does not reach statistical significance, is consistent with these results, and suggests that a cyclic change in sialic acid concentration may occur. The fact that this change cannot be consistently demonstrated suggests that it is unlikely to be responsible for the striking alterations in the physical properties of cervical mucus observed during different phases of the menstrual cycle. In conclusion, we have found significant differences in the histochemistry of cervical mucins during the proliferative phase compared with during the secretory phase or during nonreproductive years. These changes are, to date, the only consistently demonstrable cyclic changes in the structure of cervical mucin glycoprotein, which forms the . predominant solid component of cervical mucus. These chemical changes may contribute to the changes in the physical properties of the CM, which allow sperm penetration during the periovulatory period. REFERENCES 1. Fordney-Settlage D: A review of cervical mucus and sperm interactions in humans. lnt J Fertil26:161, 1981 2. Chantler E: Structure and function of cervical mucus. In Mucus in Health and Disease II, Edited by E Chantler, J Elder, M Elstein. New York and London, Plenum Press, 1982,p 251 3. Daunter B: Biochemical and functional structural aspects of human cervical mucin. Scanning Electron Microscopy 1: 345,1984 4. Gibbons RA: Molecular shape and the chemical properties of mucin. Nature 184:610, 1959 5. WolfDP, Sokolaski JE, Litt M: Composition and function of human cervical mucus. Biochim Biophys Acta 630:545, 1980 6. Moghissi KS, Syner FN: Cyclic changes in the amount of sialic acid in cervical mucus. Int J Fertil Steril 21:246, 1976 7. Iacobelli S, Garcea N, Angeloni A: Biochemistry of cervical mucus, a comparative analysis of the secretion from preovulatory, postovulatory and pregnancy periods. Fertil Steril22:727, 1971 8. Van Kooij RJ, Roelofs HJM, Kathmann GAM, Kramer MF: Human cervical mucus and its mucous glycoprotein during the menstrual cycle. Fertil Steril 34:226, 1980 9. Gilks CB, Reid PE, Clement PB, Owen DA: A simple histochemical procedure for the assessment ofthe relative quan-
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tities of neutral and acidic sugars in mucin glycoproteins and its use in assessing cyclical changes in human cervical mucins. J Clin Pathol. 41:1021, 1988 Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, 1950 Volz D, Reid PE, Park CM, Owen DA, Dunn WL: A new method for the selective periodate oxidation of total tissue sialic acids. Histochem J 19:249, 1987 Volz D, Reid PE, Park CM, Owen DA, Dunn WL: Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain 0acyl variants or 0-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids. Histochem J 19:257, 1987 Culling CF A, Reid PE, Dunn WL, Clay MG: The histochemical demonstration of 0-acylated sialic acids in gastrointestinal mucins: their association with the potassium hydroxide-periodic acid-Schiff effect. J Histochem Cytochem 22:826, 1974 Reid PE, Culling CFA, Dunn WL: Saponification induced increase in the periodic acid-Schiffreaction in the gastrointestinal tract: mechanism and distribution of the reactive substance. J Histochem Cytochem 21:473, 1973 Reid PE, Volz D, Park CM, Owen DA, Dunn WL: Methods for the identification of sidechain 0-acyl substituted sialic acids and for the simultaneous visualization of sialic acid, its 0-acyl variants and 0-sulphate ester. Histochem J 19: 396,1987
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16. Barger JD, DeLamater ED: The use of thionyl chloride in the preparation of Schiff reagent. Science 108:121, 1948 17. Lillie RD, Pizzolato P: Histochemical use of borohydrides as aldehyde blocking reagents. Stain Technol43:13, 1972 18. Reid PE, Ramey CW, Owen DA, Dunn WL, Lazosky DA, Clay MG: Chemical and histochemical studies of normal and diseased gastrointestinal tract. IV. A comparison of histochemical and chemical methods for the estimation of side chain 0-acylated sialic acids. Histochem J 17:183, 1983 19. Zar JH: Biostatistical Analysis, 2nd edition. Englewood Cliffs, New Jersey, Prentice Hall, 1983, p 138 20. Singer A: Dynamic anatomy of the cervical epithelium. In Mucus in Health and Disease I, Edited by M Elstein, DV Parke. New York, Plenum Press, 1976, p 77 21. Reid PE, Owen DA: Unpublished observations 22. Wakefield EA, Wells M: Histochemical study of endocervical glycoproteins throughout the normal menstrual cycle and adjacent to cervical intraepithelial neoplasia. Int J Gynecol Pathol 4:230, 1985 23. Orten JM, Neuhaus 0: in Human Biochemistry, lOth edition. St. Louis, CV Mosby Company, 1983, p 408 24. Sanborn BM, Held B, Kuo S: Specific estrogen binding proteins in human endocervix. J Steroid Biochem 6:1107, 1975 25. Sanborn BM, Held B, Kuo S: Hormone action in human cervix. II. Specific progestogen binding proteins in human cervix. J Steroid Biochem 7:665, 1976
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