Early life stress (ELS) can disrupt the development of nervous, cardiovascular, metabolic, and immune systems, leading to poor physical and mental health throughout the lifespan. The rising prevalence of ELS in preschool children is a major public health concern, epidemiologically linked with higher rates of youth suicides, dating violence, drinking, and drug abuse. Individuals respond to acute stress by releasing cortisol; thus, hair cortisol concentrations (HCC) can quantify the exposures to recurrent or chronic stress in children. Our pilot data on HCC revealed various factors that are associated with chronic stress in preschool children. We propose hair oxytocin concentrations (HOC) as a summative measure of supportive and/or socially affiliative experiences. Specific aims of the proposed study are: (1) to validate novel assays for measuring HCC and HOC in 1200 preschool children and their parents, in order to quantify their cumulative exposures to stressful vs. supportive early experiences; and (2) to measure hair composition (protein, lipid, water content) to interpret and/or adjust the HCC and HOC values obtained from the preschool children and their parents. We are collecting hair samples from children ages 1-5 years (n=1200) and at least one accompanying parent (n=1200). From parents, we obtain their child’s demographic, developmental, health and environmental data. Hair samples are processed using our novel extraction methods and analyzed using specific enzyme-linked immunosorbent assays (ELISA) for HCC and HOC; these assays are validated using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS-MS). We measure hair composition using direct spectrophotometric detection. On completion, these data will establish the reference norms for HCC and HOC in preschool children and examine the natural variations in HCC, HOC, and hair composition in relation to age, sex, race, ethnicity, parental, socioeconomic, or other factors. HCC and HOC values obtained from the same hair sample will also allow us to examine oxytocin-mediated regulation of the cortisol responses to ELS. Enrollment was halted due to pandemic-associated lockdowns and restrictions on preschool or childcare sites since March 2020. To enable subject enrollment, the study protocol was modified to a “contact-free” approach launched in February 2021, which will also allow us to examine effects of the COVID-19 pandemic on the chronic stress and social affiliation being experienced by preschool children. We have a multidisciplinary team, the equipment, the experience, and the pilot data to successfully complete the proposed project. Completion of this research will provide age-specific reference norms for HCC and HOC in healthy preschool children. This project will address fundamental gaps in our current knowledge and establish a scientific framework to investigate long-term physical and mental health consequences of early childhood stress, following ph...