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Regular Article |
a Division for Reproductive Endocrinology
b Division for Obstetrics and Gynecology, Department of Woman and Child Health, Karolinska Institutet, 17176 Stockholm, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cervix, female reproductive tract, parturition, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The activity of MMPs is controlled at transcriptional and posttranscriptional levels by various factors [1]. Most of the MMP genes are inducible, and their expression can be regulated by growth factors, hormones, and cytokines via induction and suppression of promoter regions. Proteolytic activity of synthesized MMPs is modulated during activation from their precursors (pro-forms) and by specific tissue inhibitors of metalloproteinases and by plasma inhibitors [1, 3].
The human uterine cervix is a fibrous organ high in collagen, which is arranged in fibers. Decorin, biglycan, and versican are proteoglycans identified in the human cervix [4]. Ripening of the cervix is an important biological and clinical event required for a normal parturition. The process is gradual during pregnancy, with a fast final remodeling of connective tissue prior to parturition [5]. The final ripening precedes the biomechanical dilatation of the cervix during parturition. Increased collagen degradation and synthesis and high activity of collagenases have been observed in the human cervix during final ripening [6], which was described more than 20 yr ago as an inflammatory-like reaction [7]. The suggested role of inflammatory cells in cervical ripening has been supported by studies showing that cervical collagenases at parturition originate from resident fibroblasts of the cervical stroma and from leukocytes invading the cervix [8]. The number of activated leukocytes in the human cervix greatly increases in late pregnancy [9].
The ECM reorganization in the cervix before parturition is a complicated multistep process of which the precise mechanism is not known. To increase our knowledge of cervical ripening, we studied the cervical production and localization of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). These proteases can cleave collagen IV of basement membranes and other denatured collagens and are presumed to be important components of the cervical ripening process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three groups of patients were included in the study. The first group consisted of nonpregnant women (n = 6) with a regular menstrual cycle (25–35 days), a mean age of 42 yr (range: 32–49 yr), and a mean parity of 1.8 (range: 0–3). All underwent hysterectomy because of benign disorders not involving the cervix. All surgeries were performed when the women were in the proliferative phase of the menstrual cycle. The women had received no hormonal treatment prior to the surgery.
Term pregnant women with a mean age of 34 yr (range: 28–38 yr), a mean gestational age of 38 wk (range: 37–40 wk), and a mean parity of 1.3 (range: 1–2) that were undergoing elective cesarean section were enrolled in the second group (n = 8). These women were not in labor and had unripe cervices with a Bishop score of <5 points.
The third group included postpartum women from whom biopsies were obtained within 15 min after spontaneous delivery (n = 6). The mean age in this group was 30 yr (range: 25–34 yr), and the mean parity was 1.0 (range: 1–1). The women had uncomplicated pregnancies, and deliveries took place at a mean gestational age of 40 wk (range: 39–42 wk). The study was approved by the local ethical committee of the Karolinska Hospital (reference no. 99-099). Informed consent was obtained from all women before they were included in the study.
Tissue Collection
Cervical biopsies were obtained transvaginally from the anterior cervical lip at the 12 o'clock position at a depth of 10–20 mm. The biopsies were similar in their morphology. The stroma of the vaginal portion of the cervix was covered with stratified squamous epithelium and consisted predominately of ECM, fibroblasts, and a small amount of smooth muscle. Each biopsy was divided in two pieces. One piece was fixed in 4% formaldehyde at 4°C overnight, stored in 70% ethanol, and embedded in paraffin. Paraffin-embedded sections were cut at 5 µm, mounted on positively charged slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany), and dried at 50°C overnight before use. The other piece was frozen in liquid nitrogen and stored at -70°C until total RNA extraction and a reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed.
Antibodies
Monoclonal antibodies for detection of MMP-2 and MMP-9 were purchased from NeoMarkers (Fremont, CA). These antibodies recognize proteins of both pro- and active forms of human MMP-2 and MMP-9, respectively, and do not cross-react with pro- and active forms of other MMPs. Monoclonal antibodies to 
-actin and desmin were obtained from DAKO (Carpinteria, CA). Specifications of the antibodies are listed in Table 1.
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Immunohistochemistry
All tissue sections were dewaxed with Bio-Clear (Bio-Optica, Milan, Italy), rehydrated in graded ethanol, and washed consecutively in double-distilled water and 0.01 M PBS (pH 7.4). After washing, the sections were transferred to plastic jars containing 0.01 M citrate buffer (pH 6.0) and were boiled in a microwave oven twice for 5 min each at 700 W. Samples were allowed to cool for 20 min before washing in PBS. Nonspecific blocking was performed for 30 min with 1.5% normal horse serum. Excess liquid was removed, and slides were incubated with the respective primary antibody (anti-MMP-2, diluted 1:200 in PBS; anti-MMP-9 diluted 1:200 in 1.5% BSA in PBS; anti-actin, diluted 1:50 in PBS; and anti-desmin, diluted 1:100 in PBS) for 60 min at room temperature. The primary antibody was replaced with a normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) to obtain negative controls. After washing in PBS, sections were incubated for 60 min at room temperature with horse anti-mouse biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:200 in PBS. After an additional wash in PBS, the sections were incubated for 30 min at room temperature with avidin and biotinylated horseradish peroxidase macromolecular complex (Vectastain Elite ABC Reagent; Vector Laboratories) prepared according to a protocol recommended by the manufacturer.
After further washing in PBS, the sections were developed with 3,3-diaminobenzidine (DAKO) for 15–20 sec and then washed with tap water for 5 min and counterstained with hematoxylin. The sections were then rewashed in water, dehydrated in graded alcohol solutions, cleared with Bio-Clear, and permanently mounted using Pertex mounting medium (Histolab Products, Gothenburg, Sweden).
RNA Isolation and RT-PCR Analysis
Total RNA from frozen cervical tissue samples was purified with the SV Total RNA isolation system (Promega, Madison, WI) according to a procedure recommended by the manufacturer. One microgram of total RNA from each sample was reverse transcribed at 42°C for 45 min in a final volume of 40 µl with a reaction mixture containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 7.5 mmol/L dithiothreitol, 0.5 mmol/L dNTPs, 1 µg random hexamers, and 400 U of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Paisley, U.K.).
Oligonucleotide primers were based on the sequences of the human MMP-2 and MMP-9 genes (GenBank nos. BC002576 and BC006093, respectively). The primer pairs (Table 2) were designed with Primer3 software [10] and evaluated using the Amplify 1.2 PCR simulation program [11]. The predicted size of the PCR products was 198 base pairs (bp) for both MMP-2 and MMP-9.
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For PCR, the cDNAs corresponding to 50 ng RNA were added to the reaction mixture of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 1.25 U Taq DNA polymerase (Gibco-BRL), 0.2 mM dNTPs, and 50 pmol of each oligonucleotide primer pair in a final volume of 25 µl. The samples were overlaid with mineral oil and subjected to PCR consisted of 24 cycles (MMP-2) or 34 cycles (MMP-9) at 94°C for 30 sec, 63°C for 30 sec, and 72°C for 60 sec, with a final incubation at 72°C for 3 min in the DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, CT). The amount of PCR product increased linearly up to 26 cycles for MMP-2 and up to 36 cycles for MMP-9 (data not shown).
To standardize the quantification method, an endogenous 18S rRNA was used as an internal standard. Multiplex PCR was performed with the internal standard amplified in the same tube with the mRNA of interest in each PCR reaction. The 18S rRNA primers and Competimers (modified at their 3' ends to block extension by DNA polymerase) were obtained from Ambion (QuantumRNA 18S Internal Standards Kit; Ambion, Austin, TX). The Competimer technology, represented by a mixture of 18S primers and 18S Competimers (1:9), was used to modulate amplification efficiency of 18S rRNA to the same linear range as MMP-2 and MMP-9 RNA when amplified under the same conditions. The predicted size of the PCR product for 18S rRNA was 315 bp. The levels of MMP-2 and MMP-9 PCR products were normalized against the product from 18S rRNA.
PCR products were electrophoresed on a 1.5% agarose gel and poststained with ethidium bromide. Bands were captured and analyzed using Gel Doc 2000 Gel Documentation System (Bio-Rad Laboratories, Hercules, CA).
Statistical Analyses
Data from the relative quantification of RT-PCR products were analyzed with ANOVA and a Kruskal-Wallis test. Significance levels were calculated using a Dunn test. Differences at P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MMP-2 protein was detected in samples from all groups as a brown stain in the cytoplasm and extracellularly (Fig. 1, A–C). Positive immunostaining was observed in the stroma but not in the squamous epithelium. Many stromal cells with a phenotype of smooth muscle cell were positive for MMP-2 (Fig. 1D). A combination of anti-smooth muscle actin and anti-desmin antibodies was used in consecutive tissue sections to confirm a smooth muscle cell phenotype. The first antibody reacts with the -smooth muscle isoform of actin and does not react with actin from fibroblasts. The anti-desmin antibody was applied to the next tissue section to distinguish smooth muscle cells from activated fibroblasts, which may be positive for -actin but lack significant amounts of desmin [12]. MMP-2-positive cells with an appearance of smooth muscle cells were positive for -actin (Fig. 1E) and desmin (Fig. 1F). Some stromal cells were positive for MMP-2 but negative for -actin and desmin, suggesting that those cells were not myofibroblasts but may have been stromal fibroblasts (Fig. 1, D–F; arrow). A small proportion of intravascular leukocytes showed positive immunostaining for MMP-2 (data not shown).
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The intensity of MMP-2 immunostaining and the number of positive cells increased gradually at term pregnancy and postpartum, particularly in the subepithelial cervical stroma and in the smooth muscle cells (Fig. 1, B and C), as compared with the nonpregnant state.
MMP-9 immunostaining in the human cervix was only observed in leukocytes in both intravascular and extravascular locations. Positively stained cells were seldom detected in the samples from the nonpregnant group but were frequent at term pregnancy and abundant postpartum (Fig. 1, G–I). The number of MMP-9-positive cells appears to increase in the term pregnant and the postpartum groups (Fig. 1, H and I) than in the nonpregnant group (Fig. 1G), with a pattern previously described for invading leukocytes in human cervix [9, 13].
Negative controls using normal mouse IgG instead of primary antibodies to MMP-2 and MMP-9 showed no immunostaining (Fig. 1J).
Reverse Transcription-Polymerase Chain Reaction
MMP-2 and MMP-9 mRNAs were examined in 16 samples by RT-PCR (nonpregnant, n = 4; term pregnant, n = 8; postpartum, n = 4). MMP-2 mRNA (Fig. 2A, lower lane) and MMP-9 mRNA (Fig. 3A, lower lane) were detected in all samples. The level of 18S rRNA did not differ between the samples (Figs. 2A and 3A, upper lanes). Ratios of relative intensity scores for each band pair (MMP-2:18S rRNA and MMP-9:18S rRNA) were compared in the three study groups. The MMP-2 mRNA level in the term pregnant group was increased to approximately 2.5-fold that of the nonpregnant group. A similar increase of the MMP-2 mRNA level was observed in the postpartum group (approximately 3-fold the level in the nonpregnant group) (Fig. 2B).
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The level of MMP-9 was approximately 3-fold higher in the term pregnant group than in the nonpregnant group (Fig. 3B), and the same tendency was seen in the postpartum group (in the postpartum group statistical significance was not reached; one sample was lost during the experiment).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cervical stromal fibroblasts were the main source of MMP-2, but some leukocytes and smooth muscle cells were also positive for MMP-2 at the time of parturition. Difficulties in identifying smooth muscle cells by phenotype and in distinction between smooth muscle cells and activated fibroblasts have been reported [15]. The cervix, unlike the uterus body, contains only 4%–12% smooth muscle tissue, with the lowest level in the distal part of the cervix [16–18]. To distinguish activated fibroblasts from smooth muscle cells, we used a combination of anti-smooth muscle actin and anti-desmin antibodies in consecutive tissue sections.
In contrast to MMP-2, the MMP-9 protein was not found in fibroblasts and was detected only in blood leukocytes. This finding is in agreement with that of another study in which MMP-2, but not MMP-9, was produced by isolated cervical fibroblasts from human cervix, which suggested that fibroblasts are not the source of MMP-9 in the nonpregnant human cervix [14]. However, during early pregnancy positive immunostaining for MMP-9 was observed in cervical stroma, surface epithelium, and blood vessels, but MMP-9 protein and its activity was not detected in cervical explants by Western blotting and gelatinase zymography [14]. In addition, human fibroblasts secrete MMP-9 in vitro after stimulation with cytokines [19, 20]. Our in vivo data indicate that even during parturition, when inflammatory mediators and cytokines are produced and released in the cervical tissue [21], leukocytes remain the main and possibly the only source of MMP-9.
The present study showed a low level of MMP-9 mRNA in the cervix from nonpregnant women. The MMP-9 protein was present in sparsely observed leukocytes within the cervical tissue. During cervical ripening, the concentrations of collagens and proteoglycans are decreased by 30%–50%, which indicates activity of different MMPs, such as MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10 [3]. The maximal increase in the level of MMP-9 mRNA and the increased number of MMP-9-positive leukocytes at term pregnancy indicate that MMP-9 is involved in cervical ripening and probably contributes to the remodeling process with a further degradation of the collagens already denatured by collagenases (e.g., MMP-1 and MMP-8). However, because the antibodies used in this study recognize both pro- and active forms of gelatinases, the localization and the level of MMP-2 and MMP-9 can be inferred but the posttranslational regulation of activity cannot.
MMP-9 may facilitate invasion of leukocytes into the cervical tissue by digestion of collagen IV, a major ECM protein in basement membranes. However, we observed no MMP-9 staining in the ECM. Considering this observation, the breakdown of basement membranes would be a more plausible role for MMP-9 in cervical ripening. Invasion of blood leukocytes into the cervical tissue in term pregnancy is a well-known phenomenon in humans [9]; however, not much is known about the mechanism and regulation of leukocyte invasion. One probable reason for the lack of knowledge is that this reaction is not observed in common laboratory animal species, and therefore there is no good animal model for in vivo studies. Eosinophilic infiltration associated with collagenolysis has been described in the rat uterine cervix at term pregnancy [22]. In the human cervix at term pregnancy, neutrophils and macrophages are the most frequently observed leukocytes [9, 13].
Gonadal steroids modulate eosinophilic invasion and collagenolysis in the rat cervix [23]. Estradiol stimulated and progesterone inhibited eosinophilic infiltration. In other studies, both MMP-2 and MMP-9 genes were regulated by estrogen [24, 25]. In humans, there is no major change in the serum level of gonadal steroids before parturition [26–28], as observed in other species such as the rat [29]. However, in clinical studies of progesterone antagonists reports of cervical softening during pregnancy, when an increase in plasma 17ß-estradiol was observed, indicate that leukocyte invasion and the ripening process in humans may be regulated by gonadal steroids [30, 31]. One possible mechanism for a modified effect of a hormone could be regulation of the hormone receptor. In previous studies, the levels of estrogen receptor (ER) subtypes and ß in the cervix are different at term pregnancy compared with the nonpregnant and the postpartum state [32]. Before parturition, ERß expression is increased and ER is downregulated. Since the ERß subtype was discovered [33], new estrogen target tissues and estrogen effects are being identified and studied. Estrogens may have an indirect action on leukocytes by modifying production of leukocyte chemotactic cytokines in the cervix and may have a direct effect, as suggested by the presence of ERß in polymorphonuclear leukocytes and macrophages in the human cervix during parturition [13]. Estrogens may, via an upregulated ERß at term pregnancy, affect activation of leukocytes and thereby production of cytokines and proteases required for tissue remodeling.
Levels of MMP-2 and MMP-9 in the human cervix increase at term pregnancy, suggesting involvement of these proteases in the extracellular matrix reorganization during cervical ripening. In the human cervix, MMP-2 and MMP-9 are produced by different cell types: MMP-2 was immunolocalized mainly to stromal fibroblasts and smooth muscle cells, whereas MMP-9 was observed exclusively in leukocytes. Further studies are required to investigate the contribution of each cell type to the cervical ripening process and to test the hypothesis of the ERß-mediated regulation of leukocyte functions in the cervix.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the Magn. Bergvalls Foundation and the Swedish Medical Research Council (grants 3972 and 9508). D.S. had a scholarship from the Swedish Institute. 
2 Correspondence: Lena Sahlin, Division for Reproductive Endocrinology, Karolinska Hospital, L5:01, S-171 76 Stockholm, Sweden. FAX: 46 8 5177 3485; lena.sahlin{at}kbh.ki.se
Accepted: April 15, 2002.
Received: March 1, 2002.
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REFERENCES |
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and ß in the human cervix at term pregnancy. Mol Hum Reprod 2001 7:293-300This article has been cited by other articles:
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  B. C. Timmons, A.-M. Fairhurst, and M. S. Mahendroo Temporal Changes in Myeloid Cells in the Cervix during Pregnancy and Parturition J. Immunol., March 1, 2009; 182(5): 2700 - 2707. [Abstract] [Full Text] [PDF]
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 D. Stygar, B. Masironi, H. Eriksson, and L. Sahlin Studies on estrogen receptor (ER) {alpha} and {beta} responses on gene regulation in peripheral blood leukocytes in vivo using selective ER agonists J. Endocrinol., July 1, 2007; 194(1): 101 - 119. [Abstract] [Full Text] [PDF]
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E. Malmstrom, M. Sennstrom, A. Holmberg, H. Frielingsdorf, E. Eklund, L. Malmstrom, E. Tufvesson, M. F. Gomez, G. Westergren-Thorsson, G. Ekman-Ordeberg, et al. The importance of fibroblasts in remodelling of the human uterine cervix during pregnancy and parturition Mol. Hum. Reprod., May 1, 2007; 13(5): 333 - 341. [Abstract] [Full Text] [PDF]
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 S. A. Tornblom, F. A. Patel, B. Bystrom, D. Giannoulias, A. Malmstrom, M. Sennstrom, S. J. Lye, J. R. G. Challis, and G. Ekman 15-Hydroxyprostaglandin Dehydrogenase and Cyclooxygenase 2 Messenger Ribonucleic Acid Expression and Immunohistochemical Localization in Human Cervical Tissue during Term and Preterm Labor J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2909 - 2915. [Abstract] [Full Text] [PDF]
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