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

Abstract: 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.