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Female Reproductive Tract |
Department of Farm Animal Health,2 Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands
Department of Biochemistry,3 Cell-Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
Gaubius Laboratory,4 TNO Prevention and Health, Department of Vascular and Connective Tissue Research, 2301 CE Leiden, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (µg/mg dry wt) as well as the insoluble collagen concentration (µg/mg wet wt) could be derived. The water content of both layers of the cervix significantly increased between midpregnancy and parturition (P < 0.01). The insoluble collagen content and the insoluble collagen concentration were significantly increased at term (P < 0.01 and P < 0.05, respectively) but were significantly decreased at calving (P < 0.05 and P < 0.01, respectively). Both parameters showed no significant differences between the superficial and deep stromal layer, and they were significantly correlated with each other. A significant increase in the percentage denaturation of the deep stromal layer occurred between the second trimester and term pregnancy (P < 0.01), whereas at calving, the percentage denaturation had not significantly increased compared to term. The percentage of collagen denaturation of the superficial stromal layer did not significantly change with stage of gestation or at parturition. Our findings indicate that cervical ripening is a combination of increased collagen synthesis and increased percentage of collagen denaturation, whereas at calving, an increased digestion of the denatured collagen leads to increased collagen loss from the cervical connective tissue. The finding that cervical ripening mainly takes place in the deep stromal layer of the cervix emphasizes the importance of a detailed description of the tissue sampling sites for a proper interpretation of the results obtained from biochemical studies of the cervix.
cervix, female reproductive tract, parturition, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changing of the collagen structure starts at the beginning of the third trimester of gestation, as has been shown in several species, such as the rat [5, 7, 8], sheep [4], and cow [6]. However, cervical softening or ripening, which results from the collagen fibers becoming dispersed and less aggregated compared to those of the nonpregnant cervix, does not necessarily coincide with the loss of tensile strength [9]. Microscopic and electron-microscopic studies have shown that in the collagen network of the ripened pregnant cervix, the dense fiber alignment has changed into a partly degraded, loosely organized network with shorter fibers [3, 10, 11] that can be extracted more easily from the tissue [12, 13]. Because intact triple-helical collagen molecules are highly resistant to proteolytic enzymes, whereas denatured (unwound) collagen is easily digested and, therefore, easily extracted from the tissue [14], we hypothesized that the percentage of denaturation of the collagen network may serve as a quantitative measure of cervical ripeness. This was investigated in the present study. In a previous experiment involving nonpregnant cycling cows, we demonstrated that regional differences in collagen biochemistry exist along the circular axis of the cervix [15]. Therefore, we performed our measurements in both the superficial stromal layer and the deep stromal layer of serial biopsy specimens obtained from the caudal cervix of cows halfway through the second trimester, at Day 275 of gestation (term), and shortly after spontaneous calving. In addition to the percentage of collagen denaturation, we also assessed the insoluble collagen content as well as the insoluble collagen concentration and the water content in the two stromal layers of these biopsy specimens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Guanidium chloride (GuHCl), EDTA, iodoacetamide, and -chymotrypsine (CT) were obtained from Sigma (St. Louis, MO). Hydrochloric acid (37% or 12 M), sodium acetate trihydrate, sodium hydroxide (analytical grade, p.a.), acidic acid, 2-propanol, chloramine-T, dimethylaminobenzoaldehyde (DMBA), perchloric acid (60%), and hydroxyproline were obtained from Merck (Darmstadt, Germany), and citric acid was obtained from Fluka (Buchs, Switzerland).
Incubation buffer consisted of 1 mM iodoacetamide and 1 mM EDTA in PBS (pH 7.5). A solvent of 4 M GuHCl in incubation buffer was used for extraction of proteoglycans and soluble collagen. Digestion buffer was made by dissolving 1 mg/ml of CT in incubation buffer. Stock buffer (pH 6.1) contained 50.44 g/L of citric acid, 117.76 g/L of sodium acetate trihydrate, and 34 g/L of sodium hydroxide p.a. Assay buffer was made by mixing 100 ml of stock buffer with 30 ml of 2-propanol and 20 ml of deionized water. Chloramine-T reagents contained 0.141 g of chloramine-T dissolved in 1 ml of 2-propanol, 1 ml of deionized water, and 8 ml of stock buffer. The DMBA reagents contained 4 g of DMBA in 2.5 ml of 2-propanol and 5.5 ml of 60% perchloric acid. The 200 µM hydroxyproline standard contained 26.23 µg/ml of hydroxyproline.
Animals and Collection of Samples
Cervical biopsy specimens were collected between January and November of 1999 from nine Holstein Friesian or Holstein Friesian/Dutch Friesian cross-bred, pluriparous cows at the two different stages of gestation and at calving. The cows were housed at the experimental farm of the Faculty of Veterinary Medicine of the Utrecht University and belonged to the commercially kept, high-yielding dairy herd. A dry-off period of 8 wk before the expected day of parturition was a standard procedure at the farm. The experimental procedure was approved by the Committee for the Use of Animals in Research of the Utrecht University. The cows were fed according to their individual nutritional needs as defined by their level of milk production and stage of gestation.
Cervical biopsy specimens were obtained transvaginally using a skin biopsy punch of 6 mm in diameter (Kai Industries Co. Ltd., Oyana, Japan). The biopsies were collected at the inside of the cervical canal, approximately 2 cm cranial to the vaginal cervical opening. Care was taken that the deep stromal layer was always included in the sample. The first biopsy specimen was collected between Days 149 and 157 of gestation (second-trimester biopsy), the second specimen at 275 days of gestation (term biopsy), and the third within 2 h after spontaneous calving (calving biopsy). The day of artificial insemination was Day 1 of gestation. Before collection of a biopsy specimen, 2.5 ml of lidocaine HCl (2%; Apharmo, Arnhem, The Netherlands) was applied in the first intercoccygeal area as epidural anesthesia to avoid abdominal straining during the vaginal manipulations.
Immediately after the biopsy, specimens were cleaned from mucus and blood. The outer muscle layer was removed, and the cervical stromal tissue was divided into a superficial part, containing the epithelium and soft fibrous tissue directly underneath, and a deep part, containing the more peripheral fibrous tissue. The samples were then snap-frozen in liquid nitrogen and stored at -80°C until further analysis.
In addition, venous blood samples were collected from the tail vein, on the days when the cervical biopsy specimens were collected, to measure plasma progesterone (P4) concentrations by means of a validated, direct, solid-phase, 125I RIA [16] with a sensitivity of 47 pg/ml, an interassay coefficient of variation of 11% (n = 16), and an intraassay coefficient of variation of 7.5% (n = 20).
Water Content
After thawing in isotonic saline for an hour, the samples were lyophilized for 24 h. The water content of the samples was derived from the weight of the samples before and after lyophilization. The wet weight of the samples varied between 105 and 1579 mg, and the dry weight varied between 11 and 246 mg.
Analysis of Collagen Content, Collagen Concentration, and Denaturation
After lyophilization, the samples were rehydrated in isotonic saline. Proteoglycans and soluble, noncross-linked collagen were extracted from the samples by washing them six times with 1 ml of 4 M GuHCl in incubation buffer at 4°C for 48 h. The tissue samples were then washed three times for 6 h at 4°C with 1 ml of incubation buffer to remove the GuHCl. After this, they were digested for 24 h at 37°C with 1 ml of digestion buffer. The supernatant, containing the CT-digested collagen fragment, was removed, and 500 µl were diluted 1:1 with 12 M HCl. The remaining tissue was immersed in 800 µl of 6 M HCl, after which supernatant and remaining tissue were hydrolyzed at 110°C for 24 h and dried. The dried hydrolysates were dissolved in 1.5 ml of deionized water. The hydroxyproline concentration of the hydrolysates from the supernatant and the remaining tissue was measured by a colorimetric method according to the principles of Stegeman and Stadler [17] and as described by Creemers et al. [18]. In short, the dissolved hydrolysates of the supernatant and of the remaining tissue were diluted 6-fold. From these diluted samples, 60 µl were pipetted into a well of a polystyrene microtiter plate (Greiner 655101, Frickenhausen, Germany), after which 20 µl of assay buffer and 40 µl of chloramine-T reagents were added. After a 20-min incubation at room temperature, 80 µl of DMBA reagents were added to the samples and carefully mixed. Subsequently, the plate was closed with a lid and placed in a small water bath, which was then placed in an incubator at 60°C for 25 min. The plate was cooled by placing it in a water bath containing cold water for 5 min, during which time the water was refreshed once. Subsequently, the extinction was measured at 570 nm on a Titertek multiscan MCC/340 (Labsystems, Finland). A hydroxyproline standard series (200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 µM) and blanks (water) were included in the measurements. The extinction measured for the standards and the samples was corrected for the blanks. Samples that had an extinction higher than that of the maximum dilution of the standard series were diluted further and measured again.
The hydroxyproline concentration of the hydrolyzed supernatant and remaining tissue samples was interpolated from the standard curve. It was assumed that collagen contains 300 hydroxyproline residues per triple helix and that the molecular weight of collagen is 300 kDa. Therefore, the amount of collagen (µg) of the cervical biopsy samples that was denatured but still cross-linked (the insoluble collagen) could be calculated from the hydroxyproline concentration of the supernatant and the tissue hydrolysates. The collagen content was calculated by expressing collagen relative to the dry weight of the cervical tissue, whereas the collagen concentration was expressed relative to the wet weight of the tissue.
The percentage collagen denaturation of the samples was calculated as
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Statistical Analysis
Results are expressed as the mean ± SEM. A general linear model (GLM) procedure of the SAS software (SAS Institute, Inc., Cary, NC) was used to test all interactions, after which significant effects were reanalyzed in more detail with the following model:
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Differences between depths of the tissue layers and variation resulting from gestational age were tested against Animal x Depth and Animal x G interaction, respectively. In addition, the Pearson correlation test was used to analyze if P4 levels were correlated to gestational age and if the different biochemical parameters were related with each other. Significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All nine cows calved spontaneously and delivered healthy calves. The gestational age at calving varied from 277 to 292 days, which means that the interval between the term biopsies and the calving biopsies varied from 2 to 17 days. Plasma P4 concentrations in the individual cows at first sampling (second trimester) were 6.3 ± 0.3 ng/ml (range, 5.0–7.6 ng/ml). On Day 275 of gestation (second biopsy), these concentrations were 4.7 ± 0.7 ng/ml (range, 1.0–7.7 ng/ml), whereas at calving (third biopsy), they were 0.2 ± 0.1 ng/ml (range, 0.04–0.10 ng/ml). The plasma P4 concentrations were significantly correlated with gestational age (P < 0.0001).
Water Content
A significant effect of gestational age (P < 0.0001) and of depth of the tissue layer (P < 0.01) was found on the water content of the cervix (Fig. 1). Within the same gestational age, no significant differences were found in water content between the superficial and the deep stromal layers. Within one layer (superficial or deep), the water content increased significantly with gestational age. The water content of the superficial stromal layer increased significantly between the second trimester and term and between term and calving (P < 0.01). Similarly, the water content of the deep stromal layer increased significantly from the second trimester to term and calving (P < 0.01).
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Insoluble Collagen Content and Insoluble Collagen Concentration
The insoluble collagen content and the insoluble collagen concentration of the cervix (P < 0.001) were significantly influenced by gestational age, without any effect of depth of the tissue layer. Therefore, the values of the superficial stromal layer and the deep stromal layer were pooled for further statistical analysis of the effect of gestational age on both parameters. Between the second trimester and term, the mean insoluble collagen content of the cervix increased significantly (P < 0.01) by 36%, but at calving, it had returned to its second-trimester value (Fig. 2A). The insoluble collagen concentration of the cervix also increased significantly (P < 0.05) between the second trimester and term by 23% of its second-trimester value and decreased between term and calving to values not different from that in the second trimester (Fig. 2B). The insoluble collagen content and insoluble collagen concentration were positively correlated (R = 0.8, P < 0.0001) with each other.
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Percentage Denaturation of Collagen
The percentage of collagen denaturation in the cervix was significantly affected by gestational age (P < 0.0001) and depth of the tissue layer (P < 0.001) (Fig. 3). A significant increase (P < 0.05) was found in percentage denaturation in the deep stromal layer from the second trimester to term, but not between samples taken at term and at calving. During the second trimester, the percentage of collagen denaturation in the superficial stromal layer was significantly higher (P < 0.05) than in the deep stromal layer, whereas during the other two stages, the differences between the two layers were nonsignificant (Fig. 3).
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Overall, a positive but weak correlation was observed between the percentage denaturation and the water content (R = 0.46, P < 0.001), but this was only significant in the deep stromal layer (R = 0.53, P < 0.01) (Fig. 4). In the deep stromal layer, the increase in water content between the second trimester and calving was correlated with the increase in percentage denaturation (R = 0.70, P < 0.05, data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results also demonstrate that collagen degradation in the deep stromal layer of the bovine cervix at term is already significantly increased compared to the second trimester, and from then on, it does not increase until calving. During the second trimester, the percentages of collagen denaturation in the superficial and deep stromal layers were much higher than the values we found during the nonpregnant state [15]. However, similar to the cervix of nonpregnant cows [15], significant differences in the percentage of denaturation were found between both stromal layers of the cervix. The fact that during the second trimester the percentage of denaturation in the superficial layer is already much higher than that in nonpregnant cows [15] indicates that changes in the superficial and deep stromal layers may have different functions during the pregnant state. The observation that such differences are no longer apparent during term pregnancy and calving, and that the percentage denaturation in the superficial layer does not increase significantly after the second trimester, indicates that cervical ripening mainly takes place in the deep stromal layer. This is in accordance with the data in the rat reported by Yu et al. [9], who described three distinct regions along the cross-sectional axis and found that during cervical softening, the microscopic loss of structure in the middle layer of the cervix was more significant than in the other two layers. Similarly, for the sheep, it has been reported that changes during late pregnancy are predominantly located in the deeper connective tissue layer [4].
In the present study, both the insoluble collagen content and the collagen concentration of the cervix significantly increased between the second trimester and term, but both dropped again significantly at parturition. These results are not in agreement with those of others, who reported that collagen decreased from early to term pregnancy and that no further change occurred between term and labor in women [12, 13, 19, 20], sheep [4, 21], and cows [6]. The discrepancy between our data, reporting an increase of (insoluble) collagen concentrations between the second trimester and term pregnancy, and the decrease reported by others may partly be explained by methodological differences. Our values with respect to insoluble collagen content and concentration reflect the sum of degraded collagen, still incorporated within the network, plus intact collagen, whereas others have measured both soluble and insoluble collagen. In pregnant women, the soluble fraction of collagen significantly increases at 10 wk of pregnancy compared to nonpregnant values, but these percentages are very low and remain at approximately 1.5% during the rest of pregnancy [12]. If one takes into account the considerable decrease of insoluble collagen that occurs between term and calving, as was found in the present study, then it is to be expected that we would have found a larger increase in the soluble fraction than in the above-described study had we measured the amount of collagen present in the GuHCl fraction. On the other hand, species differences may also explain some of the differences in results.
Our results also show that throughout gestation, a significant correlation exists between the insoluble collagen content and the insoluble collagen concentration. In most of the studies performed by other groups, collagen was expressed either relative to the dry weight or total protein content of the cervix or relative to the wet weight. However, for the case in which both parameters were used, it was reported that the total collagen content of the cervix increased during gestation, whereas the collagen concentration of the cervix gradually decreased, as shown in the cow [6], sheep [21], and rat [7]. These authors measured the collagen content of the entire cervix, and as such, their results reflect the growth of the entire cervix rather than an increase in the collagen content per unit dry weight of connective tissue. Therefore, these results cannot be compared to the present results. However, it cannot be excluded that the increase in both insoluble collagen content and concentration likely is caused by increased collagen synthesis. Thus, the increased synthesis may lead to an increased collagen density of the tissue, but it may also contribute to the increase in size, as reported by others.
We conclude from our results that between the second trimester and term pregnancy, cervical ripening involves an increased synthesis of collagen and, at the same time, an increased rate of collagen degradation. This is in accordance with the claim of Kleisl et al. [10] that partly degraded collagen is retained within the tissue, but it does not support the suggestion made by Leppert [3] that the loss of structure of collagen during ripening is not the result of an increased degradation but, rather, of an increase in loosely bound, newly synthesized collagen.
The decrease in both insoluble collagen content and insoluble collagen concentration between term pregnancy and calving, as found in the present study, could be explained by further digestion of the degraded, though still cross-linked, collagen into soluble, noncross-linked collagen fragments. These fragments are easily washed out during the GuHCl extraction. Likewise, newly synthesized collagen molecules that might have been present during all three sampling periods have also been washed out with the GuHCl. If this had not been done, these fragments and molecules would have been included in the CT supernatant, and this would have led to overestimation of the percentage of collagen denaturation, which in turn could have led to overestimation of the degradative processes in the tissue.
In the present experiment, the water content was significantly increased at term pregnancy compared to the second trimester, but in contrast to the percentage of collagen denaturation, it had increased even further at calving. This was true for both the superficial and the deep stromal layers. In contrast to our earlier findings in the nonpregnant cervix [15], no significant differences in water content were found between the two layers. A possible explanation for this could be that the water content of the deep stromal layer before the second-trimester sampling had already started to increase relative to its nonpregnant value. Although other investigators have not differentiated between the superficial and deep layers of the cervix, the increase in water content, as measured in the present study, is of the same magnitude as that in women [12] and rats [22]. However, in cows [6], sheep [21], and women [13], others have not found significant differences in water content with increasing gestational age. Meanwhile, the actual increase in water content is relatively small and, therefore, can hardly explain the histological findings of the widely dispersed collagen fibers at parturition as compared to the more densely packed fibers of the nonpregnant and pregnant (term) cervix [19]. Neither can it explain the weight gain of the cervix, as was suggested for the rat by Cabrol et al. [22]. Although part of the decrease in the insoluble collagen concentration, as found in the present study between term pregnancy and calving, may have been caused by the increase in water content of the cervix, the amount of insoluble collagen related to the wet weight shows a strong, positive correlation with the amount of insoluble collagen related to the dry weight. This observation favors the explanation already given above, that collagen loss because of increased digestion was responsible for the decrease in both insoluble collagen content and concentration as measured in the present study. Additional mechanisms, such as an increased content of other proteins, namely glycosaminoglycans, may also be responsible for the decrease in insoluble collagen content at calving.
Indirect evidence also suggests that the regulation of collagenolytic events during cervical ripening differs from that during cervical dilatation. A massive influx of leukocytes occurs during active labor in sheep [23], women [24–26], and rats [27], but leukocytes are almost completely absent during early and term pregnancy. This indicates that different proteinases may be active at the two stages. In fact, the increasing diameter of the cervix is positively correlated with the concentrations of matrix metalloproteinases (MMPs) as well as the leukocyte count rather than with the duration of labor [26]. Our recent findings, that cervical dilatation in the cow starts 15 h after uterine contractions have started to increase, indicates that the connective tissue of the cervix still has to undergo considerable changes before it can give in to the strong uterine contractions [28]. A differential expression of several MMPs during ripening and parturition may also play a role. Some authors report that collagenolytic activity is already increased during term pregnancy [12, 29] or only during active labor [26, 30]. Others have not found any evidence for increased activity at all [31, 32]. However, the assays that were used in these studies were not specific for the different proteases, and this may have influenced the interpretation of their results. Different MMPs are known to act on different substrates or on different regions of the same substrate [33–35]. For example, collagenase-1 (MMP-1), which is of fibroblastic origin, is said to preferentially cleave type III collagen, whereas collagenase-2 (MMP-8), which is of neutrophilic origin, is more active against type I collagen [36]. Collagenases cleave interstitial collagen triple helices, whereas gelatinases (MMP-2 and MMP-9), another subfamily of MMPs, digest unwound collagen molecules and gelatin [33–35]. It is possible that during cervical ripening of late pregnancy, fibroblast-collagenase plays a more active role by cleaving the triple-helical structure of the collagen molecule, leading to an increase in collagen denaturation, as found in the present study. Subsequently, the neutrophil-collagenase, the gelatinases, and possibly other proteolytic enzymes may further cleave and digest the collagen into small, extractable fragments during cervical dilatation. Further study is needed to differentiate the actions of the different collagenolytic enzymes involved in cervical softening and dilatation. The biopsy technique used in the present study may be a useful tool for such research.
In none of the cows of this experiment study did we observe any clinical illness or premature labor associated with the moment that the biopsy specimens were collected. In the present study, the gestational age of the pregnancies determined the days of the first two biopsies. It is important to realize that animals with the same gestational age are not necessarily at the same stage relative to parturition. This is illustrated by the rather large variation in the interval between the term biopsies, which were performed at a fixed gestational age of 275 days, and the biopsies that were performed directly after calving. This might explain at least part of the variation in collagen data between cows. The functional changes in the cervix that lead to the loss of tensile strength, as needed to achieve cervical dilatation, most probably start very shortly before or at the moment when parturition is initiated. This calls for a more detailed study of the period between term pregnancy and calving, in which the timing of the collection of biopsy specimens is related to the stage of parturition. In the case of spontaneously calving cows, this would hardly be possible. Therefore, efforts should be made to find a suitable protocol for induction of parturition that closely mimics the physiological events during calving. Induction of parturition by way of synthetic prostaglandin F2, as described previously [28], may prove to be a suitable protocol.
In summary, the results of the present study suggest that cervical ripening is a process that probably takes place during gestation and that involves both increased collagen synthesis and increased collagen degradation. Cervical dilatation during parturition (labor), on the other hand, is associated with increased collagen loss, most likely as a result of increased proteolytic activity. The findings that characteristics of cervical ripening are more pronounced in the deep stromal layer than in the superficial stromal layer of the cervix emphasize that a detailed description of the origin of the tissue samples is necessary when interpreting the results of studies regarding cervical ripening and softening.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 24 October 2002.
First decision: 15 November 2002.
Accepted: 24 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced parturition in cows. Biol Reprod 2003 68:536-542This article has been cited by other articles:
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  B. C. Timmons and M. S. Mahendroo Timing of Neutrophil Activation and Expression of Proinflammatory Markers Do Not Support a Role for Neutrophils in Cervical Ripening in the Mouse Biol Reprod, February 1, 2006; 74(2): 236 - 245. [Abstract] [Full Text] [PDF]
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