Project Summary/Abstract, Tsepamo Plus (Project 1) Antiretroviral treatment (ART) guidelines depend upon safety and efficacy data in pregnancy, especially in resource-limited settings where programs rely on a single ART regimen as first-line treatment for all pregnant and non-pregnant adults. Current considerations for using modern ART regimens in pregnancy – especially those containing dolutegravir (DTG) and tenofovir alafenamide (TAF) – include understanding the risks for neural tube defects (NTDs) and for the effect of weight gain on pregnancy outcomes. The Tsepamo Study (R01HD080471, R01HD095766) is the largest pregnancy surveillance system in Africa, and has evaluated over 180,000 deliveries in Botswana since 2014. The study has provided critical safety data and treatment guidance related to specific ART regimens that have been adopted for early use within the Botswana ART program. In 2018, the Tsepamo Study demonstrated a potential association with DTG and NTDs, and while this concern has decreased over time following expanded surveillance, it has not disappeared. Ongoing NTD surveillance is required both for DTG and for other new antiretroviral agents. New considerations regarding weight gain with modern ART regimens have also been analyzed successfully in Tsepamo, but further data stratified by baseline maternal weight groups are needed. The existing Tsepamo award was funded to continue birth surveillance through December 2022, and the P01 mechanism can extend surveillance for 4 additional years from January 2023 through December 2026. This will allow the completion of aims that include: 1) ongoing surveillance to understand and characterize the neural tube defect risk associated with DTG over a longer period, and with additional evaluation of dietary folic acid intake; 2) a new aim to conduct an emulated clinical trial comparing the most relevant ART regimens when used at different weight strata; and 3) new aims that utilize a unique identifier to capture data across multiple pregnancies for the first time in our surveillance. These novel aims take advantage of the unique ART landscape in Botswana, and build on the scientific findings from our highly productive surveillance system. Data generated from this study will be critical for updating international guidelines for the use of ART in pregnancy.