Group B Streptococcal Infection of the Choriodecidua Induces Dysfunction of the Cytokeratin Network in Amniotic Epithelium: A Pathway to Membrane Weakening

Abstract

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.

Author Summary

Group B Streptococcus (GBS) is one cause of preterm birth, stillbirth, and fetal brain injury. GBS is present in the vagina and is thought to ascend into the uterus of some women where it can cause placental inflammation and preterm birth. Understanding the earliest events in the placenta that lead to preterm birth is elusive in humans, because the placenta cannot be studied until after birth. Here, we use a nonhuman primate model to show that an early GBS infection can damage the structural support of the fetal membranes, specifically the cytokeratin network in the epithelium of the amnion (one part of the membranes). Next, we obtained human placentas to show that this cytokeratin network was also damaged in human patients that had preterm premature rupture of the membranes, a major cause of preterm birth. Our work is important in understanding why fetal membranes may rupture prematurely, which may lead to early interventions to prevent membrane damage after placental infection and preterm birth.