Project Summary/Abstract Expanded cannabis legalization nationwide and increased use among reproductive-aged women have exposed gaps in our scientific knowledge on whether and/or how cannabis use during lactation affects human milk composition. Human milk is considered optimal nutrition for infants for the first 6 months postpartum. Whereas recent evidence has shown that cannabinoids transfer into human milk, there is only one preliminary study that indicates that cannabis use may be associated with changes in milk factors and no studies that assess how cannabis use patterns or cannabinoid concentrations in milk impact holistic milk composition. This lack of knowledge is particularly concerning as 1) human milk is frequently the sole source of infant nutrition in the first months of life; 2) cannabinoids are lipophilic and, given the high lipid content of the mammary gland and human milk, both may be repositories for these compounds, and 3) delta-9-tetrahydrocannabinol (∆9-THC) levels in cannabis products have increased in the past several decades, suggesting that the milk of mothers who use cannabis and their recipient infants may be exposed to higher doses of cannabinoids than ever before. The overall objectives of this project are to 1) characterize and compare holistic human milk composition (metabolome, lipidome, and proteome profiles) among mothers who use cannabis and those who do not use cannabis and 2) investigate the relationships between and among human milk cannabinoid concentrations, maternal characteristics, cannabis use patterns, and milk metabolome, lipidome, and proteome profiles among breastfeeding women who use cannabis. Our central hypotheses are that 1) cannabis use alters human milk metabolome, lipidome, and proteome profiles, 2) cannabinoid concentrations in milk are related to milk metabolome, lipidome, and proteome profiles, and 3) differences in pre- and postnatal cannabis use patterns and selected maternal characteristics [e.g., body mass index (BMI), time postpartum] are related to variation in milk metabolome, lipidome, and proteome profiles, even when controlling for cannabinoid concentrations in milk. To test these hypotheses, we will compare bioarchived milk samples from 20 healthy women (≤ 6 months postpartum) who use cannabis (≥ 1x weekly) and 20 healthy women who do not use cannabis. We will employ a multi-omics approach to characterize and understand holistic milk composition (i.e., metabolome, lipidome, and proteome profiles). Results from this work are expected to provide data on whether cannabis use during lactation impacts human holistic milk composition and, importantly, constitute the foundation for a NIH R01 proposal. It also takes the initial steps toward providing mothers and healthcare providers data to enable more informed advice and evidence-based decisions during lactation.