Rapid clearance of bacteria from maternal bloodstream after delivery in pregnancies complicated by pre-labor premature rupture of the membranes.

Objective: We investigated whether birth in patients whose pregnancies are complicated by pre-labor premature rupture of the membranes (PPROM) is associated with translocation of microbes in maternal circulation and a systemic inflammatory process. Research Design and Methods: 66 pregnant patients admitted for PPROM [gestational age (GA) median [IQR]: 32±1 weeks] had maternal blood (MB) retrieved prior to birth and within 1-hour post-partum. Immediately after birth, fetal membranes (FM) and placental tissue were frozen in sterile fashion. Bacterial load was assessed by 16S rDNA gene amplification by qPCR. Multiplexed 16S amplicon libraries were sequenced on the Illumina platform and enrichment analysis performed using LfSe and EdgeR. Pro- and anti-inflammatory cytokines were measured in MB using multiplex immunoassay. Histological chorioamnionitis (HCA) and cord blood haptoglobin and IL-6 indicated fetal exposure to antenatal inflammation (Triple I). Results: Bacterial DNA was identified in MB both before and after birth. A significant decrease in MB bacterial DNA was observed within 1-hour post-delivery (p=0.004). Conversely, MB cytokines IL-6 and Il-10 increased significantly (p<0.05 for all) especially in pregnancies exposed to Triple I. Compared to placenta, FM carried a higher bacterial load (p<0.001) regardless of presence or absence of antenatal inflammation. FM had higher bacterial biodiversity with top representation from Mycoplasma spp although Mycoplasma was not found in MB either pre- or post-delivery. While no taxa were systematically enriched in MB post-delivery, sample-by-sample analysis identified three cases where a MB OTU had a match in FM and four cases with a match in placenta. Yet, the most abundant MB OTUs had no match in either tissues. Conclusion: In patients with PPROM, bacterial DNA is present in MB prior to birth but is rapidly removed after delivery in association with a robust immediate postpartum cytokine surge. Overall design: Using a prospective observational study design, we enrolled 66 consecutive pregnant patients who were admitted on the antepartum service of our academic university hospital for management of pre-labor premature rupture of the membranes (PPROM) at =34 weeks gestational age (GA). Subjects were eligible for enrollment if older than 18 years of age, pregnant between 230/7 to 336/7 weeks gestational age and at high risk of preterm birth secondary to PPROM. Exclusion criteria were anticipated delivery within 2 hours, need for close medical supervision (e.g. cardiac and renal disease, congestive heart failure, history of asthma), maternal viral infection (HIV, hepatitis B or C), known active infections or antibiotic use 7 days prior to admission, need for emergent delivery (e.g. cord prolapse, abruption) and known fetal malformations. Maternal blood (MB) was retrieved via sterile venous puncture prior to birth and within 1-hour post-partum (PP). An alcohol swab was used to prepare the skin area before venipuncture. The blood was collected in serum and plasma citrate tubes (BD Vacutainer, Franklin Lakes, NJ) and transported to the laboratory. An aliquot of whole blood from the plasma tube was first saved for bacterial DNA isolation. The remainder blood volume was spun at 1,500 g for 15 min and plasma and serum aliquoted for cytokine measurements. Cord blood was collected at delivery from all newborns of mothers enrolled in this study. After delivery of the placenta, a loop of the umbilical cord was placed on a sterile field and cord blood was collected by sterile puncture of the umbilical vein. The cord blood was processed as described above for MB. In a subset of deliveries (n=31 newborns: 23 singletons and 4 pairs of twins), a biopsy from FM (full thickness amniochorion) and from the villous tissue in center of the placental disk were retrieved in strict sterile fashion within minutes from delivery. Collection of delivery tissues was contingent on availability of research staff at delivery. Following dissection of the decidua basalis, the villous trophoblast tissue was frozen immediately in liquid nitrogen. The fetal membrane biopsy was rinsed briefly in sterile saline to remove any AF and adherent blood prior to freezing in sterile DNA-free tubes.