Does the Amniotic Fluid of Mice Contain a Viable Microbiota?

Andrew D Winters,Roberto Romero,Jose Galaz,Kevin R Theis,Zachary D Shaffer,Nardhy Gomez-Lopez,Jonathan M Greenberg,Valeria Garcia-Flores,David J Kracht

doi:10.3389/fimmu.2022.820366

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.

Highlights

IntroductionThe mammalian amniotic cavity is filled with a protective liquid (i.e., amniotic fluid) that surrounds the fetus throughout gestation
The presence of microorganisms in the amniotic fluid is associated with adverse maternal and neonatal outcomes [61– 68], and the traditional view in obstetrics has been the “sterile womb hypothesis”, which posits that the fetal environment is sterile and that the neonate first acquires a microbiota during the birthing process [69]
The 16S rRNA gene signal did not differ between paired proximal and distal samples (V = 89, p = 0.571) (Figure 2A). These results indicate that the murine amniotic fluid contains 16S rRNA gene copies, and that their concentrations do not depend on proximity to the cervix

Summary

Introduction

The mammalian amniotic cavity is filled with a protective liquid (i.e., amniotic fluid) that surrounds the fetus throughout gestation. Recent investigations have posited that the placenta [70–82], the amniotic fluid [75, 83– 85], and the developing fetus [86–88] harbor resident microbiotas, and that the amniotic fluid microbiota functions as a primary source of microorganisms for initial colonization of the offspring in utero [75, 83, 85, 89, 90]. These juxtaposed views have sparked much debate [69, 84, 91–95]

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