Does the Amniotic Fluid of Mice Contain a Viable Microbiota?

Abstract

The existence of an amniotic fluid microbiota (i.e., a viable microbial community) in mammals is controversial. Its existence would require a fundamental reconsideration of fetal in utero exposure to and colonization by microorganisms and the role of intra-amniotic microorganisms in fetal immune development as well as in pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. The profiles of the amniotic fluids of pups located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 dams). These profiles were compared to those of technical controls for bacterial and DNA contamination. The load of 16S rRNA genes in the amniotic fluid exceeded that in controls. Additionally, the 16S rRNA gene profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured from amniotic fluid. Of the 42 attempted bacterial cultures of amniotic fluids, only one yielded bacterial growth - Lactobacillus murinus. The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in the 16S rRNA gene load, 16S rRNA gene profile, or bacterial culture were observed between the amniotic fluids located Proximally and distally to the cervix. Collectively, these data indicate that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. While this means that amniotic fluid is not a source of microorganisms for in utero colonization in mice, it may nevertheless contribute to fetal exposure to microbial components. The developmental consequences of this observation warrant further investigation.

Keywords: amniotic fluid; in utero colonization; low microbial biomass study; microbiome; microbiota; mouse model; sterile womb hypothesis.

Figure 1

Study design to test for the presence of bacteria in murine amniotic fluid. Created with BioRender.com.

Figure 2

16S rRNA gene qPCR and sequencing results for amniotic fluid and blank control samples. (A) Cycle of Quantification (Cq) values from qPCR of proximal and distal amniotic fluid and blank control (BLK) samples. (B) Principal coordinate analysis (PCoA) illustrating variation in 16S rRNA gene profiles among proximal and distal amniotic fluid and blank control samples. Similarities in the 16S rRNA gene profiles were characterized using the Bray-Curtis similarity index. (C) Taxonomic classifications of the 20 amplicon sequence variants (ASVs) with highest relative abundance across all proximal and distal amniotic fluid and blank control samples. Bars of identical color within the same sample indicate multiple ASVs with the same bacterial taxonomic classification. The DNA extract of the proximal amniotic fluid sample from Dam #2 did not yield a 16S rRNA gene sequence library.

Figure 3

Differentially abundant amplicon sequence variants (ASVs) in proximal and distal amniotic fluid and blank control samples. (A) proximal and (B) distal amniotic fluid samples compared to blank DNA extraction controls as determined by Linear discriminant analysis effect size analyses. (C) Dendrogram of the three differentially abundant Corynebacterium ASVs in amniotic fluid samples and partial 16S rRNA gene sequences of closely related bacterial type strains. Numbers at the nodes are maximum-likelihood bootstrap values. Scale bar indicates the number of nucleotide substitutions per site.

Figure 4

Amniotic fluid sequencing results after the removal of likely contaminating sequences. (A) Bar graph showing the taxonomy of the 45 amplicon sequence variants (ASVs) with highest relative abundance across all proximal and distal amniotic samples. Bars of identical color within the same sample indicate multiple ASVs with the same bacterial taxonomic classification. The DNA extract of the proximal amniotic fluid sample from Dam #2 did not yield a 16S rRNA gene sequence library. (B) Principal coordinate analysis (PCoA) illustrating variation in 16S rRNA gene profiles among proximal and distal amniotic fluid samples. The 16S rRNA gene profiles were characterized using the Bray-Curtis similarity index.

Figure 5

Amniotic fluid culture and blank control 16S rRNA gene qPCR and sequencing results. (A) Bacterial cultivation results for proximal and distal amniotic fluid samples. (B) Cycle of quantification values from qPCR on amniotic fluid culture samples and BHI culture medium controls. (C) Principal coordinate analysis (PCoA) of bacterial relative abundance data from amniotic fluid samples and BHI culture medium controls. (D) Relative abundance of bacteria in the 16S rRNA gene profiles of amniotic fluid samples and BHI culture medium controls. Bars of identical color within the same sample indicate multiple amplicon sequence variants with the same bacterial taxonomic classification.