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

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

High-mobility group box protein 1 and its signalling receptors in human preterm and term cervix

The objective of this study was to identify possible changes in mRNA and protein expression of high-mobility group box protein 1 (HMGB1) and its suggested receptors - receptor for advanced glycation end-products (RAGE) and Toll-like receptor 2 (TLR2) and TLR4 - in human cervix during pregnancy, term and preterm labor. Cervical biopsies were taken from 58 women: 20 at preterm labor, 24 at term labor, 10 at term not in labor and 4 from non-pregnant women. Real-time RT-PCR was used to quantify mRNA expression, and immunohistochemistry and ELISA for protein analysis. HMGB1, RAGE, TLR2 and TLR4 proteins were localized and their mRNA expression was detected in the cervix. There was more extranuclear HMGB1 in the cervical epithelium and stroma in preterm and term labor compared to the term not in labor. TLR2 mRNA expression was upregulated 5-fold in term labor and 3-fold in preterm labor compared to term not in labor and non-pregnant controls. There was lower expression of TLR2 and TLR4 mRNAs in preterm labor compared to term. Lower mRNA expression of HMGB1 was found in the subgroup with preterm premature rupture of membranes than in the rest of the preterm group, where levels were significantly higher than in term labor. In conclusion, extranuclear expression of HMGB1 during labor suggests a possible role of HMGB1 during the process of cervical ripening. Changes in expression of mRNAs encoding HMGB1, TLR2 and TLR4 in preterm labor suggest differences in the mechanism of cervical ripening at preterm and term delivery. (C) 2009 Elsevier Ireland Ltd. All rights reserved

Pro-inflammatory and anti-inflammatory cytokines in human preterm and term cervical ripening

Cervical ripening is necessary for successful delivery. Since cytokines are believed to be involved in this process, the aim of this study was to investigate possible changes in the mRNA and protein expression of pro-inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-12, IL-18) and anti-inflammatory cytokines (IL-4, IL-10, IL-13)in the human cervix during pregnancy, term and preterm labor. Cervical biopsies were taken from 59 women: 21 at preterm labor, 24 at term labor, 10 at term not in labor and 4 from non-pregnant women. mRNA was analyzed with real-time RT-PCR and protein expression and/or secretion with immunohistochemistry and ELISA. There was an upregulation of mRNA for IL-10, IL-13, IL-1 alpha and IL-1 beta in the laboring groups, while mRNA for IL-12 and IL-18 was downregulated. IL-4 mRNA was detected more frequently, while IL-12 mRNA expression was lower, in the preterm labor group than in the term labor group. The protein levels of IL-4 and IL-12 were lower and IL-18 tended to be higher in the labor groups, while IL-10 protein levels were unaffected by labor. IL-4 protein levels were significantly higher in the preterm subgroup with bacterial infection than in the non-infected group. IL-10 had higher expression in squamous epithelium at preterm labor than at term. In conclusion, the major changes in pro-inflammatory and anti-inflammatory cytokine mRNA and protein expression in cervix occur during the labor process irrespective of the length of gestation. Our results indicate that dysregulation of anti-inflammatory cytokines in the human cervix could be involved in the pathogenesis of preterm labor

Pro-inflammatory and anti-inflammatory cytokines in human preterm and term cervical ripening

Cervical ripening is necessary for successful delivery. Since cytokines are believed to be involved in this process, the aim of this study was to investigate possible changes in the mRNA and protein expression of pro-inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-12, IL-18) and anti-inflammatory cytokines (IL-4, IL-10, IL-13)in the human cervix during pregnancy, term and preterm labor. Cervical biopsies were taken from 59 women: 21 at preterm labor, 24 at term labor, 10 at term not in labor and 4 from non-pregnant women. mRNA was analyzed with real-time RT-PCR and protein expression and/or secretion with immunohistochemistry and ELISA. There was an upregulation of mRNA for IL-10, IL-13, IL-1 alpha and IL-1 beta in the laboring groups, while mRNA for IL-12 and IL-18 was downregulated. IL-4 mRNA was detected more frequently, while IL-12 mRNA expression was lower, in the preterm labor group than in the term labor group. The protein levels of IL-4 and IL-12 were lower and IL-18 tended to be higher in the labor groups, while IL-10 protein levels were unaffected by labor. IL-4 protein levels were significantly higher in the preterm subgroup with bacterial infection than in the non-infected group. IL-10 had higher expression in squamous epithelium at preterm labor than at term. In conclusion, the major changes in pro-inflammatory and anti-inflammatory cytokine mRNA and protein expression in cervix occur during the labor process irrespective of the length of gestation. Our results indicate that dysregulation of anti-inflammatory cytokines in the human cervix could be involved in the pathogenesis of preterm labor

Low molecular weight heparin stimulates myometrial contractility and cervical remodeling in vitro

Objectives. The low molecular weight heparin, Dalteparin, shortens human labor time. The aim of this study was to investigate if the mechanism behind this effect involves myometrial contractility and cervical ripening and if the anticoagulative activity is necessary for its effect. Design. Experimental in vitro study. Setting. Lund University and Karolinska Institute, Sweden. Methods. The effect of low molecular weight heparins with or without anticoagulative properties on myometrial contractility was measured in vitro on smooth muscle strips from biopsies obtained at elective cesarean sections. The effects on cervical ripening were assessed in cervical fibroblasts cultured from explants of cervical biopsies obtained at delivery. Main outcome measures. Mean force and number of contractions in uterine smooth muscle strips and interleukin-8 (IL-8) secretion in cervical fibroblasts. Results. Myometrial smooth muscle strips pretreated with low molecular weight heparins showed increased contractile activity compared to untreated smooth muscle strips. Secretion of IL-8 from cultured cervical fibroblasts was significantly increased after treatment with low molecular weight heparin. Both these effects were independent of anticoagulative activity of the low molecular weight heparin. Conclusions. A possible underlying mechanism for the shortened labor time after low molecular weight heparin treatment is enhanced myometrial contractility and an increased IL-8 secretion in cervical fibroblast, mimicking the final cervical ripening in vivo. Our data support the notion that anticoagulant activity is not required to promote labor