The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection

Abstract

Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10−7 M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and chorion cells.

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

“…This study used human fetal membrane tissues collected following planned, uncomplicated cesarean delivery at term without rupture of membranes or labor and a genital isolate U. parvum strain as described previously 45 .…”

“…The reflected amnion layer was removed manually. Amnion epithelial cells were harvested using a modification of a technique which was previously described 45, 59 . Briefly, the amnion tissue was minced and digested in DMEM/F12 containing 0.2% trypsin (Sigma) at 37°C for 30 min with periodic agitation.…”

“…However, to date, the pathogenicity of U. parvum in preterm birth is poorly understood 44 . Using our in vitro fetal membrane infection model, we have demonstrated that U. parvum adheres to fetal membrane cells and induces inflammation 45, 46 .…”

“…There is now evidence that the dose and variation of the MBA of U. parvum might affect the severity of chorioamnionitis in pregnant sheep 95 . Therefore, we optimized the doses of U. parvum and treatment duration to avoid cell death as observed in our previous studies 45, 96 . The optimized dose range was chosen because: 1) the consistent and effective induction of interleukin 8 (IL-8), cyclooxygenase 2 (COX2), prostaglandin E2 (PGE2) and MMP9 in these cultures was observed; 2) cell death was not observed for the treatment duration; 3) the multiplicity of infection (MOI) of 5 (for example, 10 7 cfu U. parvum to 2 × 10 6 fetal membrane cells) was within the range of positive adherence to fetal membrane cells; and 4) the MOI of U. parvum in this study is consistent with other studies in human amniotic epithelial cells 97 .…”

“…In this study, 10 8 cfu U. parvum infected 10 7 amniotic epithelial cells with a MOI of 10 97 . Based on methods previously published, we have established standardized experimental procedures to ensure that the experiments can be directly compared 45 . Overgrowth of U. parvum in cell culture is not a concern because of their unique growth conditions: high concentrations of urea, a variety of amino acid supplementation, and acidic conditions of the culture media.…”