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.

Visceral adipose microbial and inflammatory signatures in metabolically healthy and unhealthy nonhuman primates

“…Recent experimental evidence also suggested that within‐host evolution of microbiota may occur, leading to different immune responses [27]; thus, a common GI microbiome can diverge over time, leading to distinct and repeatable shifts in bacterial species and changes in local immune strategies. It is also plausible that Pseudomonas and other gram‐negative microbes are deposited in utero ; Pseudomonas prevalence has been identified in amniotic fluid [28, 29]. Regardless of how the microbes evolve to inhabit the visceral adipose niche, gram‐negative bacterial accumulation in this extraintestinal site drives a robust immune response through nuclear factor‐κB (NF‐κB), inciting proinflammatory M1 macrophage infiltration and Th1 cytokine release [30].…”

“…Recent experimental evidence also suggested that withinhost evolution of microbiota may occur, leading to different immune responses [27]; thus, a common GI microbiome can diverge over time, leading to distinct and repeatable shifts in bacterial species and changes in local immune strategies. It is also plausible that Pseudomonas and other gram-negative microbes are deposited in utero; Pseudomonas prevalence has been identified in amniotic fluid [28,29].…”

The role of upper and lower genital tract microbiota alterations in term chorionamnionitis: A prospective study

“…The debate about whether the intrauterine environment is sterile has gained prominence with many arguing that bacterial DNA contamination from sampling sites, clinical or laboratory environments, as well as reagents and consumables, can greatly influence the results of microbiota studies, especially for low-biomass specimen types ( Salter et al, 2014 ). Consequently, several investigators have examined the effects of strict controls for contamination on subsequent sequencing demonstrating a sterile intrauterine environment ( Leiby et al, 2018 ; de Goffau et al, 2019 ; Kuperman et al, 2020 ; Olomu et al, 2020 ; Winters et al, 2022 ). Furthermore, de Goffau et al (2021) re-analyzed the data from Rackaityte et al (2020) and provided evidence that the identification of Micrococcus in fetal gut samples was contaminated and several findings were caused by an unrecognized batch effect.…”

Alterations and potential roles of microbial population of pregnant mouse saliva and amniotic fluid

“…The amniotic fluid samples were meticulously collected using a sterile procedure following a previously described protocol 18,19 . The abdomen of the pregnant mouse was shaved and sterile by 70% ethanol.…”

“…The amniotic fluid samples were meticulously collected using a sterile procedure following a previously described protocol. 18,19 The abdomen of the pregnant mouse was shaved and sterile by 70% ethanol. Sterile disposable scissors and forceps were utilized to remove the uterine horns and placed in a sterile dish.…”