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

Abstract

Preterm pre-labor rupture of membranes (PPROM) increases maternal and neonatal sepsis risk, yet its association with maternal microbial translocation and systemic inflammation remains unclear. We investigated whether PPROM is linked to microbial DNA in maternal blood (MB) and inflammatory responses around delivery. In 66 PPROM patients (median GA: 32±1 weeks), MB was collected pre-delivery and within 1 hour postpartum. Fetal membranes (FM) and placental tissues were sampled immediately after delivery. Bacterial load and diversity were analyzed via 16S rDNA qPCR and sequencing. Maternal cytokines were quantified by multiplex immunoassay, and fetal inflammatory exposure (Triple I) assessed using histological chorioamnionitis (HCA), cord blood haptoglobin, and IL-6. Bacterial DNA was detected in maternal blood (MB) pre- and post-delivery, with a significant postpartum decline (p=0.004). FM carried higher bacterial load and biodiversity than placenta (p<0.001), dominated by Mycoplasma spp. Maternal IL-6 and IL-10 levels rose postpartum (p<0.05), particularly in cases with fetal inflammatory exposure. Limited overlap was found between MB and tissue microbial taxa. In conclusion, bacterial DNA is detectable in maternal circulation in patients with PPROM before birth but rapidly clears postpartum alongside a robust cytokine surge, suggesting efficient clearance and dynamic inflammatory changes.

Figure 1

Flowchart of patients, newborns and biological samples analyzed part of this study

Figure 2

Bacterial load in maternal blood (MB) before (n=66) and after birth (n=66) measured by 16S rRNA PCR and normalized to the volume of blood (A), total DNA (B) or the human housekeeping gene glucose-6-phosphate isomerase (GPI) amplified in the same sample (C). Level of change in MB bacterial load normalized to GPI for patients where a decrease (D, n=45) or an increase in bacterial load was observed (E, n=21) after delivery. F: change in MB bacterial load peri-partum expressed as a ratio after:before delivery for mothers with fetuses non-exposed (NEXP, n=35) or exposed (EXP, n=31) to Triple I. Of the five twin pregnancies included in the study, two pregnancies were classified as EXP based on one of the fetuses presenting the cord blood biomarkers of antenatal exposure to infection/inflammation (Triple I). A-C & F: Scatterplots with group median (red thick horizontal line) and interquartile range. Groups are compared with Wilcoxon signed rank test with Yates correction. D&E: Symbols represent levels before and after birth for each patient connected by a line.

Figure 3

Scatterplots of maternal serum concentrations of interleukin-6 (IL-6, A), interleukin-8 (IL-8, B), interleukin-10 (IL-10, C) and tumor necrosis alpha (TNFα, D) before and 1-hour after delivery. Scatterplots comparing the maternal serum concentrations of IL-6 (E), IL-8, (F), IL-10, (G) and TNFα, (G) between non-exposed (NEXP, n=35) and exposed (EXP, n=31) mothers. Exposure status of the mother was determined by laboratory analysis of cord blood for haptoglobin and IL-6 as described in Methods. Mothers with twin gestations were considered EXP if at least one fetus was determined as EXP. A-D: The two-time points are compared by Wilcoxon signed rank test with Yates correction (n=66 mothers). E-G: The two groups are compared by Mann Whitney U-test. The group median (red thick horizontal line) and interquartile range are shown overlaid on each scatterplot.

Figure 4

Relationship between the peripartum changes in maternal circulating bacterial DNA versus concentration of interleukin-6 (IL-6) as determined by ELISA (x-axis, log transformed) among exposed (EXP, A, n=31) and non-exposed (NEXP, B, n=35) mothers. Exposure status of the mother was determined by laboratory analysis of cord blood for haptoglobin and IL-6 as described in Methods. Mothers with twin gestations were considered EXP if at least one of their newborns was determined as EXP. The peripartum change was calculated from the ratio of the analyte after:before delivery. The blue dotted line represents a ratio of 1 (no change). The regression line is shown by the red thick line along with its confidence interval (dotted red lines).

Figure 5

A: Quantitative relationships among bacterial load in fetal membranes, placental villous tissue and maternal blood (MB) before and after delivery in a subset of deliveries with matched samples (n=27 mothers and their 31 newborns). Bacterial load among different types of samples is appreciated by dCt for bacterial 16S RNA normalized to the human housekeeping gene glucose-6-phosphate isomerase (GPI) amplified in the same sample. B: Qualitative relationships in α-diversity appreciated by the Chao-1 index of operational taxonomic units (OTU, genus level) index among fetal membranes, placental villous tissue and MB before and after delivery. Exposure status of the newborns was determined by laboratory analysis of cord blood for haptoglobin and IL-6 as described in Methods. Of the 31 newborns 17 were identified as exposed (EXP) and 14 as non-exposed (NEXP). Data is shown as mean + SEM. Groups were compared using 2-way ANOVA after log transformation; Correlation between twins was adjusted using a Generalized Estimating Equation (GEE) model. *** p<0.001; * p<0.05; Qualitative relationships among samples appreciated by the relative representation of bacterial families (C) and phyla (D) among sequencing reads. E: Dendogram clustering of genus level operational taxonomic units (OTU) among samples from each delivery. The color intensity represents the number of reads in each sample.