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The effect of progestins on TNFα induced MMP9 activity and mRNA expression in primary chorion and amnion cells in term fetal membranes
Abstract Number: BP-05
Abstract Type: Original Research
Mechanisms to prevent PPROM are limited but progestins may play a role. We have previously demonstrated that progestins attenuate TNFα induced matrix metalloproteinase-9 (MMP9) activity in a cytotrophoblast cell line. TNFα induced MMP9 activity and expression in fetal membranes leads to membrane weakening and rupture and is a key pathophysiologic process in PPROM. However whether progestins have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. The objective of this study was to evaluate the effect of progestins on basal and TNFα-induced MMP9 activity and mRNA expression in primary chorion and amnion cells harvested from the fetal membranes of term non-laboring patients.
Methods: Fetal membranes were harvested from term uncomplicated non-laboring patients following elective cesarean delivery (n=6). Primary amnion and chorion cells were isolated using our established protocol. Harvested cells were incubated and grown to confluence in serum containing cell culture media. Cell cultures were pretreated with control (vehicle), progesterone (P4), 17 alpha-hydroxyprogesterone caproate (17P) or medroxyprogesterone acetate (MPA) at 10-6 M concentration for 6 h followed by stimulation with TNFα at 10 ng/ml for an additional 24 h. Cell culture media were harvested for MMP9 activity quantification using gelatin zymography. Total RNA was extracted from cell lysates and reverse transcribed into cDNA. MMP 9 and GAPDH mRNA expression were quantified by RT-qPCR using prevalidated Taqman probes. MMP9 mRNA expression was normalized to GAPDH. Both MMP9 activity and mRNA data were normalized to control or TNFα only stimulation, summarized as mean ± SEM and analyzed by 2way ANOVA.
Results: Compared to the control level, TNFα increased mRNA expression and MMP9 activity (fig A&B). MPA, but not P4 and 17P, reduced basal MMP9 activity in primary chorion and amnion cells (fig C), but only reduced basal mRNA expression in primary amnion cells when compared with controls (fig D). MPA also reduced TNFα-induced MMP9 activity in both primary chorion and amnion cells (fig E), but only reduced TNFα-induced MMP9 mRNA expression in primary amnion cells (fig F) when compared with TNFα only stimulation.
Conclusion: Our findings demonstrate a possible mechanism by which progestins may prevent PPROM but also suggest that different molecular mechanisms are involved in the attenuation of basal and TNFα induced MMP9 activity by MPA in the chorion and the amnion.