Title: Inflammatory response of human gestational membranes to Ureaplasma parvum using a novel dual-chamber tissue explant system 1 Running title: Response of gestational membranes to U. parvum Summary sentence: Ex vivo human gestational membranes produce a dose- dependent inflammatory response upon exposure to Ureaplasma parvum. Keywords: cytokines, gestational membranes, tissue explant, Ureaplasma parvum, preterm premature rupture of membranes

Preterm premature rupture of membranes (PPROM) is often associated with intraamniotic inflammation and infection. Current understanding of the pathogenesis of PPROM includes activation of pro-inflammatory cytokines and proteolytic enzymes leading to compromise of membrane integrity. The impact of exposure to bacterial pathogens, including Ureaplasma parvum, on gestational membranes is poorly understood. Our objective was to develop a dual-chamber system to characterize the inflammatory response of gestational membranes to U. parvum in a directional nature. Full-thickness human gestational membrane explants, with either choriodecidua or amnion oriented superiorly, were suspended between two washers in a cylindrical device, creating two distinct compartments. Brilliant Green dye was introduced into the top chamber to assess integrity of the system. Tissue viability was evaluated after 72 h using a colorimetric cell proliferation assay. Choriodecidua or amnion was exposed to three doses of U. parvum and incubated for 24 h. Following treatment, media from each compartment were used for quantification of U. parvum (qPCR), interleukin (IL)-8 (ELISA), and matrix metalloproteinase (MMP)-2 and MMP-9 activity (zymography). We observed that system integrity and explant viability were maintained over 72 h. Dosedependent increases in recovered U. parvum, IL-8 concentration, and MMP-2 activity were detected in both compartments. Significant differences in IL-8 concentration and MMP-9 activity were found between the choriodecidua and amnion. This tissue explant system can be used to investigate the inflammatory consequences of directional