The Bacterial DNA Profiling of Chorionic Villi and Amniotic Fluids Reveals Overlaps with Maternal Oral, Vaginal, and Gut Microbiomes

Abstract

The in utero microbiome hypothesis has been long debated. This hypothesis will change our comprehension of the pioneer human microbiome if proved correct. In 60 uncomplicated pregnancies, we profiled the microbiome of chorionic villi (CV) and amniotic fluids (AF) in relation to maternal saliva, rectum, and vagina and the soluble cytokines cascade in the vagina, CV and AF. In our series, 12/37 (32%) AF and 10/23 (44%) CV tested positive for bacterial DNA. CV and AF harbored bacterial DNA of Streptococcus and Lactobacillus, overlapping that of the matched oral and vaginal niches, which showed a dysbiotic microbiome. In these pregnant women, the immune profiling revealed an immune hyporesponsiveness in the vagina and a high intraamniotic concentration of inflammatory cytokines. To understand the eventual role of bacterial colonization of the CV and AF and the associated immune response in the pregnancy outcome, further appropriate studies are needed. In this context, further studies should highlight if the hematogenous route could justify the spread of bacterial DNA from the oral microbiome to the placenta and if vaginal dysbiosis could favor the likelihood of identifying CV and AF positive for bacterial DNA.

Keywords: amniotic fluid; chorionic villi; in utero microbiome; maternal-fetal microbiota axis; pregnancy; sterile womb hypothesis.

Figure 1

(A,B) Alpha diversity in AF and CVS samples. The alpha diversity metrics measured by means of the evenness and observed ASVs metrics. CVS = chorionic villus samples; Vag.CVS = vaginal swabs matched to CVS; Rect.CVS = rectal swabs matched to CVS samples; Sal.CVS = saliva samples matched to CVS; AF = amniotic fluid samples; Vag.AF = vaginal swabs matched to AF; Rect.AF = rectal swabs matched to AF samples; Sal.AF = saliva samples matched to AF. The comparisons among groups were performed by the Kruskal–Wallis test. (C,D) Alpha diversity in different body sites. The alpha diversity metrics measured by means of the evenness and observed ASVs metrics. The comparisons among groups were performed by the Kruskal–Wallis test.

Figure 2

The beta diversity. Principal coordinate analysis (PcoA) based on Bray–Curtis dissimilarity matrix of bacterial communities in the analyzed groups. (A) CVS positive for bacterial DNA (light green) and matched vaginal (pink), rectal (dark green), and saliva (yellow) samples. (B) AF samples (yellow) and matched vaginal (light blue), rectal (orange), and saliva (purple) samples. Not all the analyzed samples are visible; hiding samples in an ordination can be misleading.

Figure 3

Bacteria in positive AF samples (A) and CVS (B) and their matched samples. The identified bacteria in the amniotic fluid (AF) samples and chorionic villi samples (CVS) were positive for the presence of bacterial DNA and in the matched vaginal (VAG), rectal (RECT), and saliva (SAL) samples. Data are presented as relative abundances.

Figure 4

(A,B) Significantly different bacterial genera among samples matched to positive and negative CVS and AF samples. (C,D) Significantly different bacterial species among samples matched to positive and negative CVS and AF samples. Biomarkers identified by the LEfSe test. Data are shown as median relative abundances.

Figure 5

(A) The significantly modulated immune soluble factors between AF samples positive for bacterial DNA and AF samples negative for bacterial DNA. (B) The significantly modulated immune soluble factors between vaginal swabs matched to negative and positive AF samples for bacterial DNA. Differences were calculated by means of a non-parametric T test for pairwise comparisons (GraphPad Prism v. 5). * p < 0.05, ** p < 0.01, *** p < 0.001.