Comprehensive quantification of human milk oligosaccharides (HMOs) & sparse-but-true microbes in 2nd-trimester amniotic fluid (AF)

There has been recent controversy as to whether mammalian fetuses normally develop in sterile intrauterine environments, or rather amidst sparse & low-abundance/low-diversity beneficial microbial communities. Despite supportive evidence from cultivation, metagenomics, molecular tagging & functional immune experiments, this controversy has persisted because of (1) presumptive contaminants, and (2) a lack of identifiable intrauterine substrates which could support beneficial microbial growth. Milk oligosaccharides are evolutionarily preserved complex glycans which are indigestible by mammals which produce them, but provide a growth advantage to beneficial microbes & promote a healthy infant gut. Based on initial detection in an unbiased AF metabolomics screen, we hypothesized that HMOs may be ubiquitous & serve as intrauterine beneficial microbial substrates . Our aims were to use unequivocal methodology to detect & correlate HMOs and metagenomes from 2nd trimester AF. n=610 AF samples from 731 consenting subjects with ongoing and uncomplicated 2nd trimester gestations met QC for whole genome shotgun (WGS) metagenomics (microbiome) and HPLC-Chip/TOF-MS (HMOs). Robust and validated sequencing and computational approaches quantified and resolved high resolution microbial metagenomes and individual glycan structures, matched by accurate mass & retention time. All known 34 HMOs and multiple non-pathogenic microbial metagenomes were detected in AF, with a strong correlation between HMO abundance and prokaryotic gene count (Fig.1). Metagenomes were distinct from contaminant controls, and microbial genomes from beneficial strains known to populate infant gut could be reconstructed (Fig.2) We have demonstrated a strong correlation between HMOs and microbial metagenomes in 2nd trimester AF samples from a cohort of gravidae, of whom >85% go onto have largely uncomplicated term deliveries. We speculate that HMOs may serve as conserved bacterial substrates for sparse beneficial microbial communities in the intrauterine and postnatal environment of lactating mammals.View Large Image Figure ViewerDownload Hi-res image Download (PPT)