Collagen changes in the human uterine cervix at parturition

Collagen changes in the human uterine cervix at parturition H. P. KLEISSL, M.D.* MICHEL VAN DER REST, PH.D. FREDERICK NAFTOLIN, M.D., PH.D. FRANCIS H. GLORIEUX, M.D., PH.D. ALBERTO DE LEON, M.D. Montreal, Quebec, Canada Five specimens were taken from the uterine cervix immediately post parium, one specimen was taken before onset of labor, and one was taken at 16 weeks' pregnancy for comparison with five surgical specimens taken from the cervix during the proliferative phase of the menstrual cycle. Cervical collagen, including the reducible cross-links in the acetic acid-soluble and insoluble fractions, was studied. Samples taken from nonpregnant women and from women during delivery could not be differentiated using the ratio of hydroxyproline/total protein as an index of collagen in the soluble fractions. However, after electrophoresis, intense bands corresponding to collagen chains were seen in the proliferative phase and during early pregnancy, whereas only small amounts of intact collagen chains could be detected ante· and intrapartum, indicating the prevalence of collagen breakdown products. In the insoluble fractions the hydroxyproline/total protein ratio drops from 0. 75 in the samples from the nonpregnant group to 0.35 in the intrapartum samples. The typing of collagen in the insoluble fraction showed the predominance of type I collagen. Twenty to 38 per cent of collagen present was type Ill. No striking difference was seen in the analysis of reducible cross-links between the groups. These data show that cervical collagen undergoes significant biochemical changes that might play an important role in cervical dilatation. (AM. J. 0BSTET. GYNECOL. 130: 748, 1978.)

THE DILATATION OF THE CERVICAL CANAL during normal and abnormal labor is still a largely unexplained phenomenon. Unlike the muscular uterine corpus, the cervix consists mainly of connective tissue with only a thin outer layer of smooth muscle. 1 Since it was shown that collagen fibers are separated and widely scattered at full dilatation, 2 a study by Danforth and From the Departments of Obstetrics and Gynecology and Surgery, Royal Victoria Hospital and McGill University, and the Genetics Unit, Shriners Hospital for Crippled Children. This study was done during the tenure rtf a fellowship to Dr. Kleisslfrom the Deutsche Forschungsgemeinschaft. Received for publication August 1, 1977. Accepted September 2, 1977. Reprint requests: Dr. F. Naftolin, Women's Pavilion, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, P. (f. H 3A 1A 1, Canada. *Present address: Universitaetsstr. 21-23, Universitaet1jrauenklinik, 8520 Erlangen, Federal Republic of Germany.

748

associates3 concentrated on changes in the ground substance. This demonstrated a great increase in the synthesis of glycosaminoglycans and the synthesis of large quantities of a "new component" at full dilatation. The same authors noted a decrease in the collagen/ total protein ratio on the basis of the hydroxyproline content. 4 Maillot and Zimmermann, 5 however, found no significant change in the collagen/tissue dry-weight ratio. The solubility of collagen undergoes a huge increase at full dilatation with no significant change in the water content. 5 All these data provided evidence of more than a passive mechanical process occurring during parturition and led to the reconsideration of the role of collagen and its metabolism. The knowledge of the biochemistry of collagen has increased rapidly over the last few years. 6 • 7 The present studies demonstrate the presence of both type I and type III collagen in the cervix and indicate that cervical dilatation may be preceded by a degradation of collagen in this tissue.

0002-9378/78/07130-0748$00.60/0

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V. Mosby Co.

Collag~n

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Materials and methods Twelve subjects were selected for this study. Blocks of anterior cervix were obtained from five women (aged 16 to 33 years) immediately after normal vaginal delivery which followed labor in which contractions and dilatation of the cervix lasted four to seven hours. These are designated as intrapartum specimens. After manual extraction of the placenta, the anterior cervical lip was grasped by use of forceps and a wedge-shaped whole-thickness sample was removed. One specimen was taken at the same site from a "ripe" cervix per vaginam at elective repeat cesarean section (40th week of pregnancy) in a 31-year-old woman. This will be referred to as the antepartum sample. The samples were dissected as described below. In addition, blocks of anterior cervix were removed from abdominal hysterectomy specimens from five women during the reproductive age (29 to 41 years, all but one with proven fertility) who were in the proliferati\e phase of the menstrual cycle as determined by endometrial histology. In one case, the specimen was removed from the cervix of a 44-year-old woman who had had a hysterectomy at 16 weeks' gestation in a pregnancv complicated by a fundal fibroid tumor. Each of these patients underwent surgery for benign uterine disease not involving the cervix. Block specimens of the anterior cervix were taken immediately after surgery. The chosen site of removal was the lower part of the cervix, which contains the least amount of muscle and is supposed to represent the rim at full dilatation. Cervical glands and muscle tissue in the outer layer were discarded and a central strip was analyzed as follows. The homogeneity of connective tissue in the strip was checked by histologic sections (hematoxylin and eosin and Masson trichrome stains). All samples were stored at -:.wo C. for not more than four weeks. They were milled for four minutes at liquid nitrogen temperature in a Spex freezer mill. Portions of I 0 mg. were saved h>r reducible cross·link analysis. A fraction of about 150 mg. was extracted for 24 hours in 5 mi. of 0.175M acetic acid at 4° C. and centrifuged (30 minutes at 37,000 G): the pellet was then extracted again under the same conditions and the supernatant was retained. After another centrifugation the pellets and the pooled supernatants were lyophilized and the dried residues weighed and retained. The small size did not allow complete biochemical analysis in all samples. Amino acid analyses were perfi:>rmed with a Durrum D-500 amino acid analyzer after 20 hours of hydrolysis under nitrogen in constantly boiling II 0° C. 6N HCI. Gel electrophoresis of the soluble collagen was done in urea/sodium dodecvl sulphate/polyacrylamide gel

changes in cervix at parturition 749

Table I. Hydroxyproline concentratiom in acetic acid-soluble fractions

Proliferative phase (n = :J) 16th week of pregnancv (n Antepartum (n = I) Intrapartum (n = 4)

::?H. I ± 6.8t I)

17.2

5H.H ~2.5 :t 5.1 t

*Number of residues/ I ,000 amino acid residues. t Standard deviation. Table II. Percentage of protein staining attributable to they, {3, and a collagen chains in the acetic acid-soluble fractions Percentage of collagen chams in sunned proteins

Cervix stage

Proliferative phase (n 5) 16thweekofpregnancy(n Antepartum (n I) Intrapartum (n = 5)

:1~.~~ ±

=I)

3.4*

:l.'i !0

15.0 ± 8.2*

*Standard deviation; p < 0.005: proliferative partum phase.

vs.

intra-

using the technique of Goldberg and colleagues. 8 Destaining and microdensitometry were performed as described earlier. 9 Cyanogen bromide cleavage of collagen was carried out according to Scott and Veis. 10 The released polypeptides were analyzed bv use of polyacrylamide electrophoresis as described by these authors. The reduced collagen cross-links were separated by chromatography in the standard column of the Durrum amino acid analyzer after reduction with tritiated NaB~ and after 48 hours of hydrolysis under nitrogen in 3N HCl at 105" C.U The radioactivity incorporated in the eluted fractions was determined by scintillation counting.

Results Histologic sections of all specimens showed that our samples consisted of homogeneous·appearing connective tissue. Masson trichrome staining demonstrated the previously described loosening of tht~ collagen network (blue staining) in the intrapartum samples. 2 The specimens showed no striking difference in the relation of the abundant blue-stained area to a small area of red stain (muscle). As already described by Maillot and Zimmermann, 5 we lound a large increase in the solubility of the hydroxyproline containing peptides, which almost solely represent collagen or collagen breakdown products. Of the hydroxyproline peptides 15.8 and 29.9 per cent of two intrapartum samples were soluble as compared to

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Fig. l. Gel electrophoresis patterns of the acetic acid-soluble fractions of cervical tissues. The migrations of y, f3 1t. {3 12 , "'" and a 2 bands of purified human type I collagen are indicated for reference. (a) Cervices at proliferative phase and 16 weeks' pregnancy. (b) Intrapartum and antepartum cervices.

Fig. 2. Gel electrophoresis patterns of cyanogen bromide peptides of the acetic acid-insoluble fractions of the cervical tissue. The migrations of a 1 (I) CB 6 (1) and a 1 (III) CB 8 (2) indicated. (a) Cervix at proliferative phase and 16 weeks' pregnancy. (b) Intrapartum cervix and antepartum cervix.

5.6 ± 1.7 per cent (n = 5) for the nonpregnant group and l.l per cent for the 16 week pregnancy. By comparing the weights of our samples before and after freeze-drying, we estimated water content to be between 73.1 and 79.6 per cent for intrapartum and nonpregnancy samples, respectively, with no significant variations in the groups. The hydroxyproline concentration of the soluble fractions is shown in Table I. Since hydroxyproline constitutes approximately 10 per cent of the amino acid content of collagen,6 it can be estimated that less than 30 per cent of the proteins were derived from collagen in all except the antepartum sample. The total amino acid compositions of the samples conform to this interpretation. We have studied the electrophoretic behavior of

these soluble fractions under dissociating conditions. The samples taken during the proliferative phase of the cycle and those obtained at 16 weeks' pregnancy were indistinguishable. They show intense y, {3, ab and a 2 bands and other components of lower molecular weight (Fig. !, a). This indicates the presence of some type I collagen. Table II gives an evaluation of the intensity of the soluble collagen bands compared to the total protein staining. The intrapartum samples contain significantly fewer y, {3, and a bands than the samples taken from nonpregnant women (p < 0.005) (Fig. 1, b). Th,· soluble fraction of the cervix at 16 weeks of pregnancy is indistinguishable from that in the nonpregnant state, whereas the ante- and intrapartum samples show the same pattern.

Collagen changes in cervix at parturition

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751

75

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Fig. 3. Chromatography indicates reducible collagen cross-links from a sample of proliferative phase cervix. Fractions 34 to 40, dihydroxynorle ucine; 50 to 54, hydroxynorleucine: 120 to 126, dihvdroxylysinonorleucine: I 30 to 135, hydroxylysinonorleucine: I 4R to I 5:!, histidinohvdroxymerodesmosine.

Table III. Hydroxyproline concentrations in acetic acid-insoluble fractions Cervix stage

Hydroxyproline concentration *

Proliferative phase (n = 5) 16th week of pregnancy (n = I) Intrapartum (n = 2)

75.3:!: 4.9t 83.0 33.9, 37.7

*Number of residues/1,000 amino acid residues. t Standard deviation.

The relative hydroxyproline content of each insoluble fraction is given in Table II I. A marked decrease of the ratio of collagen to other proteins is seen in the ante- and intrapartum samples. This confirms the observations of Danforth and Buckingham. 4 Table IV summarizes data from other investigators as well as our findings . The cyanogen bromide-derived peptides of these insoluble fractions vary very little from one sample to the other in each group of samples. They demonstrate the predominance of type I collagen in these tissues with the presence of 20 to 38 per cent of type III collagen as estimated by the area of the bands corresponding to the a 1 (Ill) (:yanogen bromide (CB) 8 fragment (Fig. 2).

Table IV. Relative amounts of components in specimens from nonpregnant and intrapartum human cervices

INonprpgnant llnJrapartum

Sfiecimen: Water HP*/total protein• HP/dry weight" HP soluble/HP total Acetic acid-soluble fra ction: HP!total protein Intact collagen stained protein Acetic acid-insoluble fraction: HP/total protein

73'}( 82'}( 46'}( tj'}(

2H'}(

80% 52% 48% 23%t

32'}(

23% 1!1%

75%

36%t

*Hydroxyproline peptides. t Mean of two values.

The band corresponding to the O\ (I) CB 6 fragment almost completely disappears in the ante- and intrapartum samples (Fig. 2). The a 1 (I) CB 6 fragment is known to be involved in the intermolecular crosslinkage of collagen. 12 Other minor differences in the chromatographic pattern may be related to the same phenomenon. The determination of the reducible cross-links shows that the most abundant cross-links are dihydroxy-

752 Kleiss! et al.

April I, 197/l

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lysinonorleucine and histidinohydroxymerodesmosine in both groups (Fig. 3).

Comment The major differences between ante- and intrapartum and other specimens are found in the acetic acidsoluble fractions. Although there is an increase in the solubility of hydroxyproline in the cervix at delivery (this is generally regarded as an index of increased collagen solubility), 5 this occurs at a time when the cervix contains only a small amount of intact soluble collagen chains. This indicates that parturition is accompanied by a degradation of collagen in the cervix. The breakdown products that migrate faster on gel electrophoresis would account for the higher hydroxyproline content. Indications that the less-cross-linked and mostsoluble collagen is probably digested first are (l) the disappearance of the characteristic collagen bands from the electrophoretic patterns of the soluble fractions as described above and (2) the cyanogen bromide cleavage products of the insoluble materials. The fact that there is a decrease in the intensity ofthea 1 (I) CB 6 peptide band confirms that the most heavily crosslinked collagen remains. When this peptide is involved in intermolecular cross-linking, 12 it migrates more slowly on electrophoresis. Precise timing of the collagen transformation is not possible from these data. However, with these techniques specimens from a cervix at 16 weeks' pregnancy cannot be distinguished from cervix samples at the proliferative phase. The ripe cervix prior to term labor had a collagen profile much the same as that of our intrapartum specimens. It is tempting to propose that the described changes in collagen occur during cervical softening. This is supported by the high content of soluble hydroxyproline peptides and almost complete absence of intact collagen in the ripe cervix before labor. Toward the end of labor there is a considerable drop in hydroxyproline, suggesting that the small peptides present after collagen breakdown have left the cervix. Closer sampling in the last weeks of pregnancy will be required before a definitive conclusion can be drawn. Preliminary attempts to find collagenase activity in primary cultures of cervix failed to demonstrate a clearly increased activity in the intrapartum samples (Delvin and Kleiss)*). This fits with the idea that the collagen breakdown occurs earlier. However, the difficulties of the procedures involved make an interpretation of this "negative" result hazardous and *Unpublished observations.

J. Obstet. Gynecol.

further attempts at collagenase evaluation are in order. Because of the present findings, studies should include the antepartum period. It has been shown by Danforth and co-workers 3 • 4 that in the intrapartum cervix the collagen content decreases relative to the total protein present. This is also apparent from Table II. Obviously, the present findings cannot disclose absolute total differences between tissues since the total weight of the postpartum cervix is not known. Rimmer, 13 studying the effect of pregnancy on the cervical collagen of the mouse, found a fourfold increase in the dry weight of the whole organ with only a 30 per cent rise in total collagen. If this applies to women, the decrease in the collagen/ total protein ratio could reflect a rate of synthesis that is slower for collagen than for other proteins as well as degradation of collagen. Our data nevertheless suggest that there is a degradation of collagen in the cervix. Since all of the hydroxyproline present in the cervix after parturition does not correspond to tropocollagen molecules, it certainly cannot be reutilized per se. as suggested previously.u We conclude that the hydroxyproline or hydroxyproline peptides are either excreted or subsequently metabolized. The studies of Klein and Yen 15 · 1'6 demonstrated a slight elevation in the urinary excretion of these compounds in the days and weeks around delivery. However, because many organs can contribute to this increase and because of the lack of very close sampling before and during labor, this work should be extended before it can be assumed that blood or urine analysis might be helpful. Before the onset of labor there may be a fall of progesterone and rise in estradiol serum levelsY Cervical softening precedes labor and can be induced by exogenous estrogens. 18 During estrus in the mouse, changes in the cervix were observed which are similar to those described at the end of pregnancy . 19 These data strongly suggest that estrogens play an important role in inducing the cervical transformation which, at least in part, can be attributed to the collagen changes we described. In any case, the lack of knowledge of the mechanisms involved in cervical incompetence, premature labor, induced labor, and normal cervical function during pregnancy, labor, and delivery is considerable. Perhaps these and subsequent studies will be helpful in understanding these important clinical entities. We would like to thank the members of the Royal Victoria Hospital, Department of Obstetrics and Gynecology, who helped us to collect the specimens required for this study. This work was supported in part by the Shriners of North America and the Fraser Memorial Fund.

Collagen changes in cervix at parturition

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REFERENCES l. Danforth, D. N.: AM.

J.

OBSTET. GvNECOL. 65: 1261,

1954. 2. Danforth, D. N., Buckingham, J. C., and Roddick, J. W., Jr .. AM. J. Ossn:T. GYNECOL. 89: 939, 1960. 3. Danforth, D. N., Veis, A., Breen, M., Weinstein, H. G., Buckingham, J. C., and Manalo, P.: AM. J. OssTET. GYNECOL. 120: 641. 1974. 4. Danforth. D. N., and Buckingham, J. C.: In Blandau, R. .J.. and Moghissi, K., editors: The Biology of the Cervix, Chicago, !973, University of Chicago Press, p. 351 ff. 5. Maillot v., K., and Zimmermann, B. K.: Arch. Gynaekol. 220: 275, 1976. 6. Miller, E. j.: Mol. Cell. Biochem. 13: 165, 1976. 7. Gross. J.: Harvey Lect. 68: 351, 1974. 8. Goldberg, B., Epstein, E. H., and Sherr, C. J.: Proc. Natl. Acad. Sci. U.S. A. 69: 3655, 1972. 9. Van der Rest, M., Cole, W. G., and Glorieux, F. H.: Biochem. J. 161: 527, 1977.

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10. Scott, P. G .. and Veis. A.: Connect. Ti'>'ll<' Res. 4: 107 1976. 11. Housley, T. J., Tanzer, M. L., and lknmsan. H. H.: Biochim. Biophys. Acta 365: 405. 197 4. 12. Scott, P. G., and Veis, A.: Connect. Ti-;,uc Res. 4: 117. 1976. 13. Rimmer, D. M.: J. Endocrinol. 57: 41~'- 1973. 14. Klein, L.: Proc. Nat!. Acad. Sci. U.S. A 62: 920, 1969. 15. Klein, L: Metabolism 13: 386, 1964 . 16. Klein, L, and Yen, S.C.: Metabolism 19: 1\1, 1970. 17. Turnbull, A. C., Patten, P. T., Flint. A. P. F .. Keirse, M. J N.C .. Jeremy, .J. Y., and Anderson.:\. B. M.: Lancet 1: 101, 1974.

18. Pinto, R. M., Fisch, L., Schwarcz. R. L., and Moutuori. E.: AM.J. 0BSTET. GYNECOL. 90:99, 1964. 19. Rimmer, D. M.: .J. Endocrinol. 55: 21:~. 1\172.

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