Infant gut microbiota colonization is important for supporting normal development and long-term health of children. Human milk oligosaccharides (HMOs) influence the composition of the gut microbiota, but their specific effects, particularly after breastfeeding, remain poorly understood.

We aimed to deepen the understanding of how HMOs associate with the gut microbiota composition at 3 months and at 13 months of age. Additionally, we assessed the role of HMOs as microbiome-rebalancing agents in cesarean delivered infants.

We analyzed fecal samples from infants at 3 months (n=517) and 13 months (n=522), along with human milk samples at 3 months, from a population-based cohort. Gut microbiota was profiled by 16S rRNA sequencing, and 19 HMOs quantified by high-performance liquid chromatography with fluorescent detection. Dirichlet Multinomial Mixtures clustering was used to identify bacterial fecal community types (FCTs) and multinomial logistic regression models to study the association between HMOs and FCTs. PERMANOVA and linear regression models were used to associate HMOs with gut microbiota diversity measures and Spearman correlation to bacterial genera.

HMOs associated with gut microbiota FCTs, diversity measures, and bacterial genera at 3 and 13 months of age. At 3 months, disialyllacto-N-tetraose (DSLNT) and the structurally related lacto-N-sialyllactose b (LSTb) showed notable associations with the gut microbiota while at 13 months, fucodisialyllacto-N-hexaose (FDSLNH) associated with multiple gut microbiota metrics. Maternal secretor status was associated with the gut microbiota beta diversity (R2=0.003, p<0.05) and decreased Shannon diversity (b=-0.24, p<0.05) at 3 months, with diminishing associations at 13 months (Observed richness, b=-11, p<0.05). While no individual HMOs showed microbiome-rebalancing effects in cesarean-born infants, infants fed by non-secretor mothers exhibited stronger cesarean-related microbiota patterns compared to those fed by secretors.

HMOs exhibit age-dependent and structure-specific associations with infant gut microbiota, extending beyond breastfeeding.