Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts

The aims of the present study were to compare the levels of mRNA and protein expression of matrix metalloproteinase (MMP)-1, -3, -8 and -9 in human cervical tissue in preterm and term labor as well as not in labor and to determine if corticotropin-releasing hormone (CRH) has an effect on MMP-1, -3 and interleukin (IL)-8 secretion in both preterm and term cervical fibroblasts. Cervical biopsies were taken from 60 women: 18 at preterm labor, 7 at preterm not in labor, 18 at term labor and 17 at term not in labor. ELISA and Immulite were used for protein and real-time RT–PCR for mRNA analysis. Cervical fibroblast cultures were incubated for 18 h with different CRH concentrations (10−13–10−6 M). The mRNA expression of MMP-1, -3 and -9 was higher in laboring groups compared with term not in labor. Protein levels of MMP-8 and -9 were higher in term in labor group compared with non-laboring groups. There were no significant differences in mRNA and protein expression between the preterm and respective term control groups. CRH significantly increased secretion of IL-8 in preterm and term cervical fibroblasts compared with controls. The secretion of IL-8 and MMP-1 was significantly higher and MMP-3 secretion lower in preterm cervical fibroblasts. In conclusion, cervical ripening at preterm seems to be a similar inflammatory process as at term with CRH involved. However, preterm and term cervical fibroblasts might have different phenotypes based on different secretion patterns of IL-8, MMP-1 and MMP-3

The preterm cervix reveals a transcriptomic signature in the presence of premature pre-labor rupture of membranes

BACKGROUND: Premature prelabor rupture of fetal membranes accounts for 30% of all premature births and is associated with detrimental long-term infant outcomes. Premature cervical remodeling, facilitated by matrix metalloproteinases, may trigger rupture at the zone of the fetal membranes overlying the cervix. The similarities and differences underlying cervical remodeling in premature prelabor rupture of fetal membranes and spontaneous preterm labor with intact membranes are unexplored. OBJECTIVES: We aimed to perform the first transcriptomic assessment of the preterm human cervix to identify differences between premature prelabor rupture of fetal membranes and preterm labor with intact membranes and to compare the enzymatic activities of matrix metalloproteinases-2 and -9 between premature prelabor rupture of fetal membranes and preterm labor with intact membranes. STUDY DESIGN: Cervical biopsies were collected following preterm labor with intact membranes (n = 6) and premature prelabor rupture of fetal membranes (n = 5). Biopsies were also collected from reference groups at term labor (n = 12) or term not labor (n = 5). The Illumina HT-12 version 4.0 BeadChips microarray was utilized, and a novel network graph approach determined the specificity of changes between premature prelabor rupture of fetal membranes and preterm labor with intact membranes. Quantitative reverse transcription-polymerase chain reaction and Western blotting confirmed the microarray findings. Immunofluorescence was used for localization studies and gelatin zymography to assess matrix metalloproteinase activity. RESULTS: PML-RARA-regulated adapter molecule 1, FYVE-RhoGEF and PH domain-containing protein 3 and carcinoembryonic antigen-ralated cell adhesion molecule 3 were significantly higher, whereas N-myc downstream regulated gene 2 was lower in the premature prelabor rupture of fetal membranes cervix when compared with the cervix in preterm labor with intact membranes, term labor, and term not labor. PRAM1 and CEACAM3 were localized to immune cells at the cervical stroma and NDRG2 and FGD3 were localized to cervical myofibroblasts. The activity of matrix metalloproteinase-9 was higher (1.22 ± 4.403-fold, P < .05) in the cervix in premature prelabor rupture of fetal membranes compared with preterm labor with intact membranes. CONCLUSION: We identified 4 novel proteins with a potential role in the regulation of cervical remodeling leading to premature prelabor rupture of fetal membranes. Our findings contribute to the studies dissecting the mechanisms underlying premature prelabor rupture of fetal membranes and inspire further investigations toward the development of premature prelabor rupture of fetal membranes therapeutics

Macrophage gene expression associated with remodeling of the prepartum rat cervix:Microarray and pathway analyses

As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor

Preterm and term cervival ripening : Studies on CRH, HMGB1, toll-like receptors, cytokines and matrix metalloproteinases

Objective: Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity. Despite the existing treatment, the frequency of PTB has not changed in the past thirty years. Incomplete understanding of the biological and pathophysiological mechanisms underlying preterm delivery is the major obstacle to preventing PTB. Cervical ripening is necessary for vaginal delivery, for which reason understanding of preterm cervical ripening is required for developing new treatment strategies. The overall aim of the work presented in this thesis was therefore to determine possible differences between preterm and term cervical ripening. Methods: Transvaginal cervical biopsies were obtained from women undergoing spontaneous delivery or elective caesarean section at preterm and term, and from non-pregnant women. Real-time RT-PCR was employed for analysis of mRNA, and immunohistochemistry, ELISA and Immulite for protein analysis. Corticotropin-releasing hormone (CRH), its binding protein (CRH-BP), its receptors (CRH-R1 and CRH-R2), matrix metalloproteinases (MMP) -1, -3, -8, -9, high-mobility group box protein 1 (HMGB1), receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), TLR4, interleukin (IL)-1alpha, IL1-beta, IL-12, IL-18, IL-4, IL-10 and IL-13 were analyzed in cervical tissue. Preterm and term cervical fibroblast cultures were established and the secretion of IL-8, MMP-1 and MMP-3 was measured after stimulation with CRH. Results: CRH, CRH-BP, CRH-R1, CRH-R2 and HMGB1 were identified in human cervical tissue for the first time. TLR2, TLR4, IL-10 and IL-12 were identified in the cervix for the first time in relation to pregnancy and labor. The distinct changes were determined in the cervix in labor irrespective of gestational age. There was downregulation of mRNA for CRH-BP, CRH-R2, RAGE, IL-12, IL-18, but upregulation of mRNA for TLR2, IL-10, IL-1beta, MMP-1, MMP-3 and MMP-9. More extranuclear staining of HMGB1 in stroma and empty nuclei in squamous epithelium were observed in labor. TLR2 and TLR4 tissue expression was lower in labor. IL-4 and IL-12 concentrations were lower, but soluble RAGE, IL-18, MMP-8 and MMP-9 were higher in labor. Differences between preterm and term cervical ripening were found: mRNA expression of TLR2, TLR4 and IL12 was lower in preterm labor, while IL-10 protein expression was higher in the cervical epithelium in preterm labor. Furthermore, preterm and term cervical fibroblasts showed different secretion patterns with higher levels of IL-8 and MMP-1, but lower levels of MMP-3, at preterm. CRH significantly increased the secretion of IL-8 in cervical fibroblasts. Subgroup analysis revealed some differences in association with preterm premature rupture of membranes (PPROM) and positive vaginal and/or urinary cultures. Conclusions: Preterm cervical ripening is an inflammatory process similar to cervical ripening at term. However, some differences still exist: these include downregulation of TLR2, TLR4 and IL-12 and higher levels of IL-10 in cervical epithelium. CRH and HMGB1 are probably involved in cervical ripening. Our results indicate that PPROM and PTL with infection could partly involve different mechanisms

Differences in heparan sulfate production in cervical fibroblast cultures from women undergoing term and preterm delivery.

Objective. An extensive remodeling of the human cervical connective tissue occurs throughout pregnancy, with a decrease in the total concentration of collagen and proteoglycans. We hypothesized that the profound changes in proteoglycan production in the cervix would be seen in corresponding cervical fibroblasts as well. Methods. Cervical biopsies were obtained from five non-pregnant women, five women undergoing elective Cesarean section, six women directly after spontaneous term parturition and four directly after spontaneous preterm parturition. By explant technique, fibroblasts were cultured from the biopsies. Subcultures of the primary fibroblasts were treated with antibodies to heparan sulfate proteoglycans and labeled with radioactive sulfate. The labeled proteoglycans were purified by ion-exchange chromatography and separated by gel electrophoresis. Results. Proteoglycan production was reduced by 50% in fibroblasts obtained from term and preterm women. In comparison to equivalent control cultures from non-pregnant women, this decline was significant. Production of the proteoglycans biglycan and perlecan was similar in term partal and preterm partal cell cultures. Biglycan production was significantly reduced (by 40%) and perlecan production was significantly induced (by 60%) compared to control cultures. Fibroblast cultures established from women with preterm delivery had significantly higher production of heparan sulfate proteoglycans than those obtained from non-pregnant donors. Heparan sulfate proteoglycans were localized to cell membranes and intracellular compartments. Conclusions. The changes in proteoglycan production in the human pregnant cervix can also be seen in corresponding cervical fibroblasts. Term partal and preterm partal cells differed from their non-pregnant counterpart, which suggests a role for proteoglycans in cervical ripening

Does Low Molecular Weight Heparin Shorten Term Labor? Editorial Comment

Dalteparin, a low molecular weight heparin (LMWH), is given to pregnant women with thrombotic disorders. Clinical observations together with the documented changes of heparan sulfate proteoglycans in normal and protracted labor fostered the idea that LMWH shortens delivery time. Labor time was retrospectively determined among nulliparous pregnant women treated with dalteparin because of previous venous thromboembolism (VTE), thrombophilia or acute VTE during current pregnancy. Their labor time was compared to matched untreated controls. The proportion of instrumental deliveries and neonatal outcome was also compared. The dalteparin-treated group showed a significantly (30%) shorter labor time compared to matched controls. Total instrumental deliveries were the same in the two groups but operative intervention due to protracted labor was significantly less common in dalteparin-treated women. There was no difference in neonatal outcome. Dalteparin most likely shortens parturition time and may decrease the number of operative interventions due to protracted labor