Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes

Abstract

Preterm birth is a major cause of neonatal mortality and morbidity worldwide. Bacterial infection and the subsequent inflammatory response are recognised as an important cause of preterm birth. It is hypothesised that these organisms ascend the cervical canal, colonise placental tissues, cause chorioamnionitis and in severe cases infect amniotic fluid and the foetus. However, the presence of bacteria within the intrauterine cavity does not always precede chorioamnionitis or preterm birth. Whereas previous studies observing the types of bacteria present have been limited in size and the specificity of a few predetermined organisms, in this study we characterised bacteria found in placental tissues from a cohort of 1391 women in rural Malawi using 16S ribosomal RNA gene sequencing. We found that specific bacteria found concurrently on placental tissues associate with chorioamnionitis and delivery of a smaller newborn. Severe chorioamnionitis was associated with a distinct difference in community members, a higher bacterial load and lower species richness. Furthermore, Sneathia sanguinengens and Peptostreptococcus anaerobius found in both matched participant vaginal and placental samples were associated with a lower newborn length-for-age Z-score. This is the largest study to date to examine the placental microbiome and its impact of birth outcomes. Our results provide data on the role of the vaginal microbiome as a source of placental infection as well as the possibility of therapeutic interventions against targeted organisms during pregnancy.

Fig 1. Study participant flow diagram.

Fig 2. The effect of sample collection on detection of bacterial DNA.

(A) Histogram of the number of hours between delivery and the placenta being sampled. (B) Box-and-whisker plot showing the association between the number of hours after delivery sampling took place and whether the placental tissue had detectable levels of bacterial DNA. Correlation between the number of hours after delivery the placenta was sampled (C), and the observed number of OTUs and median inter-individual unweighted UniFrac distance (D).

Fig 3. Identification of a core, distinct placental microbiome.

(A) Rank abundance curve showing the 25 most common organisms recovered from participants’ placental tissue (n = 476) and fetal membranes (n = 738) that were positive for bacterial DNA. (B) Principal coordinate analysis of unweighted UniFrac distances computed for matched participant placenta (n = 445), fetal membrane (n = 719), oral (n = 725) and vaginal (n = 747) samples from 1107 participants. PC1 and PC2 refer to principal coordinate two and principal coordinate three respectively. Each individual point refers to a single participant’s microbiome for that body site, with samples in similar positions on each axis assumed to have similar microbiomes and samples further apart in the plot are assumed to have more divergent microbiomes. (C) Principal coordinate analysis of unweighted UniFrac distances using the same participants as (B) but plotting axes PC2 and PC3.

Fig 4. High bacterial load in placental tissues associated with a restricted number of phylogenetically diverse organisms.

Multivariate plot of participant’s (A) placental and (B) fetal membrane bacterial load, observed number of OTUs and median intra-individual unweighted UniFrac distances.

Fig 5. Specific combinations of bacteria found in fetal membranes that associate with each other, severe chorioamnionitis and adverse birth outcomes.

Heat map of Spearman’s correlations between the 6 most abundant bacterial phyla with each other (A) and 20 most abundant bacterial families with each other (B) recovered from fetal membranes. Hierarchical clustering was computed by complete linkage of Euclidean distances. Heat map is annotated with mean difference in bacterial load between participants with and without severe chorioamnionitis, preterm birth (<37 weeks), low birth weight (<2500g) and neonatal stunting (LAZ < -2) and small head circumference (HCZ < -2) for each bacterial phyla or family. Asterisks indicate p<0·05 association between higher load of that bacterial phyla or family and prevalence of severe chorioamnionitis, preterm birth, low birth weight, neonatal stunting or small head circumference. The mean different and confidence intervals of these associations can be found in the main results text. Adjusted model P values were calculated using linear regression adjusting for the nutritional intervention, maternal BMI at enrolment, maternal age, proxy for socioeconomic status, number of previous pregnancies, anaemia, site of enrolment, mode of delivery and time between delivery and placenta sampling.

Fig 6. Severe chorioamnionitis is associated with lower species richness and higher phylogenetic diversity.

Comparison between observed number of OTUs and median intra-individual unweighted UniFrac distances in placental tissue (A) and fetal membranes (B) and presence of severe chorioamnionitis. Adjusted model P values were calculated using linear regression adjusting for the intervention, maternal BMI at enrolment, maternal age, proxy for socioeconomic status, number of previous pregnancies, anaemia, site of enrolment, mode of delivery and time between delivery and placenta sampling (*p<0.05, **p<0.01).

Fig 7. OTUs identified in placental tissues are also identified in participant’s mouth and vagina.

(A) The mean ± SEM estimated proportion of OTUs in placenta and fetal membranes sourced from vaginal, oral cavity or an unknown source. (B) Comparison between the proportion of OTUs found in placenta and fetal membranes sourced from the vagina stratified by participants who delivered vaginally or by caesarean section.

Fig 8. Presence of OTUs in both vagina and placental tissue associated with a lower length-for-age z-score.

(A) Heat map showing presence of vaginal organisms in all individuals’ vaginal and placental samples. Hierarchical clustering was computed using average linkage of Euclidean distances. (B) Mean ± 95%CI LAZ score for participants with individual bacterium not present in their vagina, present in their vagina only and present in both vagina and placental tissue (*q<0.05).