Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM)

Abstract

Background Preterm premature rupture of membranes (PPROM) is a leading contributor to maternal and neonatal morbidity and mortality. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently PPROM. Although blood is suspected as the likely source of thrombin in fetal membranes and amniotic fluid of patients with PPROM, this has not been proven. Ureaplasma Parvum (U. parvum) is emerging as a pathogen involved in prematurity, including PPROM, but until now, prothrombin production directly induced by bacteria in fetal membranes has not been described. Objectives This study was designed to investigate whether U. parvum exposure can induce prothrombin production in fetal membranes cells. Study Design Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live U. parvum (1 × 105, 1 × 106, or 1 × 107 colony forming units (cfu)/ml) or lipopolysaccharide (Escherichia coli J5, L-5014, Sigma, 100 ng/ml or 1000 ng/ml) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and mRNA were harvested from the cells and tissue explants for Western blot or qRT-PCR to quantify thrombin/prothrombin protein or mRNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using one-way ANOVA with post hoc Dunnett’s test. Results Prothrombin production and localization was confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin mRNA and protein expression in fetal membranes was significantly increased by U. parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, U. parvum at a dose of 1×107 cfu/ml significantly increased both prothrombin mRNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; FM: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared to untreated controls. U. parvum at a dose of 1×106 cfu/ml significantly upregulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin mRNA expression in decidua cells (fold change: 4.4±1.9). Conclusions Our results demonstrate that prothrombin can be directly produced by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by U. parvum exposure in fetal membranes. These findings represent a potentia...

Detection of Vaginal Metabolite Changes in Premature Rupture of Membrane Patients in Third Trimester Pregnancy: a Prospective Cohort Study

“…All of the metabolite changes discussed above may work together to impair the healthy vaginal microecology and lead to the development of vaginal dysbiosis and asymptomatic vaginitis. Interestingly, the ascending vaginitis can destroy the cervical fetal membrane through upregulating activation of matrix metalloproteinase-9 (MMP9), an enzyme involved in the remodeling of the fetal membranes and the cervix before the onset of labor, result in a weaken fetal membranes [51] and thus increase the risk of PROM during pregnancy.…”

Good clinical practice advice: Prediction of preterm labor and preterm premature rupture of membranes

“…Furthermore, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of U. parvum ‐induced P‐PROM and the potential use of prothrombin as a predictor of this unfavorable event …”

“…These findings represent a potential novel underlying mechanism of U. parvum-induced P-PROM and the potential use of prothrombin as a predictor of this unfavorable event. 56 Timing of delivery is often unknown; approximately half of women who are admitted to hospital with threatened preterm birth remain undelivered after 7 days. 57,58 Several other studies have reported increased odds ratios and/or predictive efficiency for preterm birth when two or more biomarkers (sometimes derived from different tissues) are combined compared to single biomarkers alone.…”

Purinergic, P2X3 positive cells in amniotic membranes

“…We congratulate Dr Feng et al on their innovative studies demonstrating infection-induced thrombin production in preterm premature rupture of the membranes (PPROM). 1 Are similar results demonstrable in vivo using noncultured tissues, and which comes first: the infection or thrombin production? Or does prior denervation of the lower genital tract promote opportunist infection and a diverse cascade of pathologic changes in preterm labor/ PPROM?…”

“…We appreciate the thoughtful comments of Dr Quinn and colleagues regarding our recent article. 1 In response to their first question, we demonstrated only the results in vitro cell or tissue explants. The cause-effect is an important question; however, our model is not designed to answer a cause-effect relationship but primarily to show how infection can exaggerate a response with thrombin, a known agent that weakens membranes.…”