Unveiling balanced prenatal microbial colonization in amniotic fluid through an integrated culture and sequencing approach.

BACKGROUND: The evidence of a low-biomass microbial community in the amniotic fluid (AF) is challenging the traditional concept of a sterile womb. To clarify microbial presence and host responses, a comprehensive, multi-methodological approach is required. METHODS: We designed an optimized culturing strategy that maximized microorganism recovery by implementing differential centrifugation and concentration of AF samples, followed by plating onto four distinct selective media types and incubation under both stringent aerobic (up to two weeks) and prolonged anaerobic (up to four weeks) conditions, including an initial pre-enrichment step in Brain Heart Infusion (BHI) broth for low-abundance organisms. These results were combined with PacBio 16S rRNA gene sequencing, Illumina shotgun metagenomics, and antimicrobial peptides (AMP) detection. Using this approach, we characterized microbial presence in 154 AF samples across gestational stages. Data normality was assessed with the Shapiro-Wilk test, guiding the selection of both parametric and non-parametric tests, and a-value of < 0.05 was considered statistically significant. RESULTS: We detected culturable microorganisms in 33.1% of samples, with a higher proportion in elective caesarean Sect. (55.0%) compared to amniocentesis (29.5%), suggesting increased microbial load toward term. We applied stringent contamination controls, and repeatedly recovered viable microorganisms,,, and, withandcommon. Both sequencing methods revealed a low-biomass, low-diversity microbial community with high inter-individual variability. Notably, striking microbial discordance in diamniotic twin pregnancies, challenged intrauterine homogeneity. Higher Human Beta Defensin (HBD) -1 levels correlated with absence of culturable bacteria or microbial DNA, while levels of HBD-1, HBD-3, and LL-37 were reduced in-positive samples, suggesting a dynamic interplay between specific bacteria and host defences. CONCLUSIONS: Our findings indicate that viable bacteria and/or DNA can transiently access the prenatal environment microbial balance. We propose a novel perspective of a potential regulatory axis between microorganisms and AMP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-025-07601-0.