91: Human milk oligosaccharides are present in midgestation amniotic fluid & associated with a sparse microbiome

HMOs comprise a diverse group of indigestible complex glycans in sialy- (SL) or fuco-(FL) sylated forms thought to only be found in human milk. HMOs play a beneficial role in infant gut health by favoring proliferation of beneficial bacteria, acting as “decoys” for pathogenic microbes, & establishing the gut epithelial barrier. During a full spectral metabolomics screen on n=731 midgestation AF samples, we identified a peak consistent with a glycan oligosaccharide. Hypothesizing that this spectral peak was actually a HMO, we sought to definitively identify & quantify. All 731 subjects AF samples were analyzed on the 1290 Infinity (6545 Q-TOF in positive/negative ion modes). Lowess normalization enabled deconvolution, and correction for multiple comparisons employed an FDR=0.1 with significance set a priori at q< 0.001. Validation & quantificaiton of HMOs in a subgroup (n=279) occurred via oligosaccharide extraction over C18/carbograph microcolumns, followed by fluorescent labeling with 2-aminobenzamide, & analysis via amide-80 HPLC with separation & annotation by ion trap mass spec. For metagenomics, DNA was extracted from all 731 amniotic fluid samples with parallel “contaminant” controls & subjected to 16S V4 & shotgun metagenomic sequencing (Illumina & BGI). We confirmed isolated 3’SL HMO in amniotic fluid at 589.34 ± 18.65 ng/ml (normalized distribution; Fig.1); no other HMO species was detected. Metagenomic sequencing revealed an association with microbiome taxonomy & phylogenetic diversity (Fig.2). We report for the first time that 3’SL HMOs are found in midgestation AF at physiologic levels, albeit log-lower than breast milk. Parallel metagenomics analysis demonstrated microbiome composition & functional variation in a sparse community which was distinct from contamination controls. Collectively, these findings suggest that both a known beneficial bacterial substrate (3’SL HMO) and a sparse but consistently present microbiome can be detected in “sterile” amniotic fluid in midgestation pregnancies which will go on to deliver months later.View Large Image Figure ViewerDownload Hi-res image Download (PPT)