LACK OF EVIDENCE FOR A VIABLE MICROBIOTA IN MURINE AMNIOTIC FLUID

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 the role of intra-amniotic microbes in fetal development and pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. Bacterial profiles of amniotic fluids located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 mice). These profiles were compared to those of technical controls for background DNA contamination. The load of 16S rDNA in the amniotic fluid exceeded that in controls. Additionally, the 16S rDNA 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. Of the 42 total 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 16S rDNA load, 16S rDNA profile, or bacterial culture were observed between amniotic fluids located proximal and distal to the cervix. Collectively, these data show 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. These findings refute the proposed role of amniotic fluid as a source of microorganisms for in utero colonization. IMPORTANCEThe prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during labor and/or the vaginal delivery process. However, it has been suggested that fetuses are consistently colonized in utero. One proposed source of colonizers is the amniotic fluid surrounding the fetus. This concept has been derived primarily from investigations that relied on DNA sequencing. Due to the low microbial biomass of amniotic fluid, such studies are susceptible to influences of background DNA contamination. Additionally, even if there is a microbial DNA signature in amniotic fluid, this is not necessarily reflective of a resident microbiota that could colonize the mammalian fetus. In the current study, using multiple microbiologic approaches and incorporating technical controls for DNA contamination, we show that, although there is a low abundance bacterial DNA signal in amniotic fluid, this does not translate to the presence of viable bacteria.