An integrated analysis of amniotic fluid reveals a dynamic prenatal microbial landscape and regulatory axis

Challenging the sterile womb dogma, our study investigated amniotic fluid (AF) microbial colonization and its potential role in foetal immune development. We analysed microbial presence in 154 AF samples without clinical and analytical suspicion of AF infection using optimised bacterial culture methods and full-length 16S rRNA gene sequencing. Shotgun metagenomic sequencing was also performed on a subset of culture-positive samples, and antimicrobial peptide levels were also measured. Remarkably, 29.5% of amniocentesis-derived and 55.0% of caesarean-derived samples yielded positive cultures, predominantly Bacillus , Cutibacterium , Micrococcus , and Staphylococcus genera, with Cutibacterium acnes and Staphylococcus epidermidis being most common species. Both sequencing methods revealed a low-biomass, low- diversity microbial community with high inter-individual variability. Higher HBD-1 levels correlated with an absence of culturable bacteria or detectable microbial DNA. While sequencing showed no significant microbial differences based on gestational age or pregnancy outcome, bacterial species identified through culture varied with reproductive treatments. Of most interest, diamniotic twins showed intra-individual microbial discordance between sacs, directly challenging intrauterine homogeneity. Our findings indicate that viable bacteria and/or their DNA can transiently access the prenatal environment. This highlights the importance of a possible microbial balance in the prenatal setting, suggesting a novel perspective of a potential microbial regulatory axis.