Preterm and term cervical ripening : studies on CRH, HMGB1, toll-like receptors, cytokines and matrix metalloproteinases
Author: Dubicke, Aurelija
Date: 2009-06-12
Location: Nanna Svartz Auditorium, A7:00, Karolinska Universitetssjukhuset Solna
Time: 09.00
Department: Institutionen för kvinnors och barns hälsa / Department of Women's and Children's Health
View/ Open:
thesis.pdf (5.945Mb)
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.
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.
List of papers:
I. Klimaviciute A, Calciolari J, Bertucci E, Abelin-Tornblöm S, Stjernholm-Vladic Y, Byström B, Petraglia F, Ekman-Ordeberg G (2006). "Corticotropin-releasing hormone, its binding protein and receptors in human cervical tissue at preterm and term labor in comparison to non-pregnant state." Reprod Biol Endocrinol 4: 29
Pubmed
II. Dubicke A, Akerud A, Sennstrom M, Hamad RR, Bystrom B, Malmstrom A, Ekman-Ordeberg G (2008). "Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts." Mol Hum Reprod 14(11): 641-7. Epub 2008 Oct 15
Pubmed
III. Dubicke A, Andersson P, Fransson E, Andersson E, Sioutas A, Malmström A, Sverremark-Ekström E, Ekman-Ordeberg G (2009). "High-mobility group box protein 1 and Toll-like receptor cervical expression is influenced by labor and differs in human preterm and term cervical ripening." (Submitted)
IV. Dubicke A, Fransson E, Centini G, Andersson E, Byström B, Malmström A, Petraglia F, Sverremark-Ekström E, Ekman-Ordeberg G (2009). "Pro-inflammatory and anti-inflammatory cytokines in human preterm and term cervical ripening." (Submitted)
I. Klimaviciute A, Calciolari J, Bertucci E, Abelin-Tornblöm S, Stjernholm-Vladic Y, Byström B, Petraglia F, Ekman-Ordeberg G (2006). "Corticotropin-releasing hormone, its binding protein and receptors in human cervical tissue at preterm and term labor in comparison to non-pregnant state." Reprod Biol Endocrinol 4: 29
Pubmed
II. Dubicke A, Akerud A, Sennstrom M, Hamad RR, Bystrom B, Malmstrom A, Ekman-Ordeberg G (2008). "Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts." Mol Hum Reprod 14(11): 641-7. Epub 2008 Oct 15
Pubmed
III. Dubicke A, Andersson P, Fransson E, Andersson E, Sioutas A, Malmström A, Sverremark-Ekström E, Ekman-Ordeberg G (2009). "High-mobility group box protein 1 and Toll-like receptor cervical expression is influenced by labor and differs in human preterm and term cervical ripening." (Submitted)
IV. Dubicke A, Fransson E, Centini G, Andersson E, Byström B, Malmström A, Petraglia F, Sverremark-Ekström E, Ekman-Ordeberg G (2009). "Pro-inflammatory and anti-inflammatory cytokines in human preterm and term cervical ripening." (Submitted)
Issue date: 2009-05-22
Rights:
Publication year: 2009
ISBN: 978-91-7409-453-4
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