Project Summary/Abstract This is a revised competing renewal to continue work in our birth cohort of mother-infant Hispanic dyads. Through repeated sampling and analysis of breastmilk, along with detailed and repeated infant assessments from birth to 2y of age, we have made significant contributions to the field of maternal-infant nutrition. This work extended our knowledge on the role of human milk oligosaccharides (HMOs), and their dynamic changes during the course of breastfeeding, on infant growth, obesity, eating behaviors, and cognitive development. Based on novel findings, we propose a series of new rigorous assessments at age 5y, with a focus on examining the role of HMOs on adiposity (abdominal MRI), appetite regulation (eating in the absence of hunger), brain structure and function (multimodal MRI), and cognitive outcomes (NIH toolbox). Aim 1 will examine the impact of infant exposure to HMOs during breastfeeding on obesity and appetite regulation, with a focus on 5 HMO’s that have demonstrated significant associations in prior/ongoing studies. We hypothesize that these HMOs will be associated with reduced adiposity (subcutaneous, visceral, and liver fat), and this will be mediated by reduced eating in the absence of hunger. Aim 2 will examine the impact of infant exposure to HMOs that provide a source of fucose and sialic acid that are important for brain development. We will use multimodal MRI for assessment of brain structure (anatomical MRI), function (resting state fMRI), blood flow (arterial spin labeling), and myelination and tissue microstructure (diffusion tensor imaging) as well as cognitive outcomes. Based on preliminary data, we hypothesize that 2’FL during the first month of breastfeeding, and continued exposure to 3FL and 3’SL (the only HMOs which we have found to increase during breastfeeding), will be associated with improved brain outcomes. This will include: a) thicker cortical mantle and reduced fractional anisotropy within cortical gray matter (representing greater dendritic arborization); b) reduced resting perfusion of cortical gray matter (representing greater neuronal metabolic efficiency); c) reduced diffusivity in white matter tracts (representing more myelination); d) stronger measures of resting functional connectivity (representing more efficient information transfer), and e) enhanced cognitive outcomes. Aim 3 will examine the impact of breast feeding on eating in the absence of hunger and structural and functional differences in key areas of the brain involved with appetite regulation. We hypothesize that longer-term duration breastfeeding, and therefore greater exposure to 3FL and 3’SL, will be associated with reduced eating in the absence of hunger, and this will be mediated by a thicker cortical mantle and reduced fractional anistrophy in brain regions associated with appetite regulation. This study will move the field forward by identifying how early-life dietary exposures (breastfeeding, HMOs and HMO c...