Objectives
Preterm premature rupture of membranes is associated with intra-amniotic infection and inflammation, and the impact of exposure to bacterial pathogens on gestational membranes is poorly understood. Our objective was to develop an ex vivo system to characterize the inflammatory response of gestational membranes to bacteria. Specifically, we intended to determine whether Ureaplasma parvum traverses gestational membranes and provokes a protease response. Characteristics sought for this system include preservation of tissue viability, limitation of membrane tension that could compromise interpretation of results, and lack of leakage from one compartment to the other.
Methods
Gestational membranes were collected from uncomplicated, term cesarean deliveries of patients receiving care at an academic medical center (n=5). Two-centimeter, full-thickness membrane discs were prepared. Each disc (n=6 per subject) was positioned without stretch between two washers and with maternal decidua oriented superiorly creating two distinct compartments (see Diagram 1). All devices were incubated in culture media at 37°C with media changed every 24 hours. Sustained metabolic activity was evaluated using a colorimetric cell proliferation assay. To assess integrity of the system, Brilliant Green dye suspension was introduced to the superior compartment with assessment of leakage to the inferior compartment. After 48 hours incubation, 105, 106, or 107 colony-forming units of a live genital isolate of U. parvum were inoculated into the superior compartment. Media was collected from both compartments 24 h later. Quantitative real-time PCR was employed to evaluate bacterial migration across membranes, and zymography was performed to assess proteolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9.
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