Childhood obesity is a major public health challenge that often tracks into adulthood. Infancy and early childhood (birth to 24 months) may be a critical period in the development of overweight and obesity, and early nutrition may play an important role. Breastfeeding has a consistent protecting effect from obesity in childhood and adolescence. However, which of the bioactive components in human milk contribute to the effect on infant metabolic phenotype and growth remains mostly unknown. Human milk oligosaccharides (HMO) are a key bioactive component of human milk. HMO act as human milk prebiotics and help shape a healthy infant gut microbiome. An imbalance in the gut microbiome composition can have functional consequences that can increase susceptibility for weight gain and/or metabolic complications. However, associations between HMO composition and infant growth, body composition and obesity risk have not been studied. This gap in knowledge stems primarily from a lack of suitable longitudinal cohorts to study these associations (i.e. with available breast milk samples, precise clinical diagnoses, and detailed maternal phenotyping) and the absence of technology for high-throughput HMO analysis required for large cohorts. Our proposed project will address these deficiencies by pairing existing datasets and bio-banked milk samples from the Finnish mother-infant STEPS cohort with new state-of-the-art technology for HMO analysis. HMO amount and composition are highly variable between different women, and we hypothesize (1) that HMO composition in mother's milk is associated with growth, body composition and obesity risk in infancy and early childhood, and (2) that maternal factors influence HMO composition. To test these hypotheses, Aim 1 proposes to analyze inter-individual variation in HMO composition in mother's milk and evaluate associations with growth, body composition and overweight/obesity in 811 mother-infant dyads from the STEPS cohort. Aim 2 proposes to identify fixed and modifiable maternal factors that influence HMO composition and apply structural equation modeling to determine the direct and indirect effect of HMO on disease development. Leveraging resources from the landmark STEPS study and using new high-throughput technology for HMO composition analysis provides a unique and powerful opportunity to study the maternal determinants of HMO production and the effects of HMO on child health. Discoveries from the proposed exploratory project will inform new approaches for disease prevention, including (1) HMO supplementation strategies with the aim to add specific `protective' HMO to an infant's diet to reduce the risk of obesity in infancy and early childhood, and (2) recommendations on dietary or lifestyle modifications for breastfeeding mothers to `optimize' HMO composition and enrich specific `protective' HMO.