Date Published: March 6, 2014
Publisher: Public Library of Science
Author(s): Jeroen P. Vanderhoeven, Craig J. Bierle, Raj P. Kapur, Ryan M. McAdams, Richard P. Beyer, Theo K. Bammler, Federico M. Farin, Aasthaa Bansal, Min Spencer, Mei Deng, Michael G. Gravett, Craig E. Rubens, Lakshmi Rajagopal, Kristina M. Adams Waldorf, Paul M. Sullam.
Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion) associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM), we examined the effects of a Group B Streptococcus (GBS) choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received choriodecidual inoculation of either GBS (n = 5) or saline (n = 5). Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site) was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion), Luminex (amniotic fluid, AF), immunohistochemistry, and transmission electron microscopy (TEM). Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6) were detected in GBS versus controls (p<0.05). Choriodecidual infection resolved by the time of cesarean section in 3 of 5 cases and GBS was undetectable by culture and PCR in the AF. A total of 331 genes were differentially expressed (>2-fold change, p<0.05). Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK) and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006). In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002). These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS choriodecidual infection, which may contribute to PPROM.
Preterm premature rupture of membranes (PPROM) occurs in 1–2% of all pregnancies, but complicates 30% of all preterm deliveries . The majority of women (70%) with PPROM deliver within 24 hours after membrane rupture. The fetal membranes (chorioamnion) are composed of two membranes called the amnion and chorion that enclose the amniotic cavity. Inflammation of the chorioamnion and within the amniotic fluid is thought to play a major role in the pathogenesis of premature rupture resulting in preterm delivery. Infection-associated preterm labor is commonly characterized by elevated amniotic fluid cytokine levels in women and animal models of preterm birth , , , , , In addition to mediating inflammation, cytokines have also been associated with increased collagen remodeling and, ultimately, biophysical weakening of the fetal membranes or chorioamnion in vitro. Unfortunately, current therapies to prevent preterm delivery following membrane rupture are of limited efficacy. In the setting of PPROM attributed to infection, there are currently no therapeutic alternatives to immediate delivery . Beyond the prevention of urogenital infection, clinically useful biomarkers for preterm membrane rupture have not yet been identified to aid in meaningful prevention efforts .
We previously reported uterine contraction, microbial culture, and cytokine (amniotic fluid and fetal plasma) results in our model of early choriodecidual infection in association with in utero fetal lung injury (Table S1, Fig. S1) .
Our results demonstrated a large-scale downregulation of amniocyte CK synthesis, retraction of the intermediate filament network from the basement membrane and formation of dense intermediate filament aggregates after GBS exposure or PPROM. CK are components of intermediate filaments vital for maintaining cellular structure, elasticity, and resisting shear stress. Weakening of the intermediate filament network, which occurs in several human disorders with mutated CK genes (e.g. epidermolysis bullosa simplex), increases the risk of membrane dysfunction . Loss of CK also makes cells vulnerable to apoptosis, which is a typical finding in placentas following PPROM or after exposure to infection , , . Prior studies in women with PPROM have focused on the contribution of matrix metalloproteinases to weakening of collagen in the extracellular matrix . Our study suggests that chorioamnion weakening also occurs through a novel mechanism of downregulation of CK gene expression within the amniotic epithelium leaving the amnion vulnerable to apoptosis, shear stress, and rupture (Fig. 5). The observation that the amniotic epithelium provides the greatest tensile strength to the chorioamnion imparts even greater significance to our study .
Many of the methods related to our animals and study groups, uterine activity, GBS and bacterial cultures, quantitation of inflammatory mediators (cytokines, prostaglandins, matrix metalloproteinases), and performance of the microarray, qRT-PCR, microarray and IPA analysis have been previously published , .