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.

The authors have declared no competing interest.

This work was supported by grants from the Federacion de Asociaciones de Celiacos de Espana (FACE) (SUBN/2019/005) and US-15332/I+D+I FEDER Andalucia 2014-2020. Maria Gonzalez-Rovira received a fellowship from the University of Seville, Spain (PIF del VI Plan Propio de Investigacion y Transferencia II.2A).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was reviewed and approved by the Sevilla Sur Provincial Research Ethics Committee (ID: 0452-N-22) for samples from Virgen de Valme University Hospital (Sevilla, Spain), Virgen del Rocio University Hospital (Sevilla, Spain) and QuironSalud Sagrado Corazon Hospital (Sevilla, Spain). Written informed consent was obtained from all the participants. The participants consented for the use of their data and/or samples for health-related research purposes.

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Datasets generated during the current study can be found in the Sequence Read Archives, under accession PRJNA1288569.