Project Summary/Abstract Electronic nicotinic delivery systems (ENDS) or e-cigarettes are widely used in the nation today, despite a paucity of information regarding their safety. In particular, ENDS usage by pregnant women is prevalent, as cigarette smoking during pregnancy is associated with miscarriage and fetal death. However, little is known regarding the safety of ENDS use during pregnancy. ENDS is made up of a liquid containing a mixture of propylene glycol and vegetable glycerine with additions of flavorings, nicotine, and thickening agents that is heated, aerosolized, and inhaled. Supplementation of ENDS with flavors and thickening agents can dramatically enhance the toxicity level and thus, impair fine-tuned biological processes such as pregnancy. At the start of pregnancy, the uterine stroma and epithelium communicate in a complex orchestration of molecular and hormone signaling events to coordinate the implantation of an embryo. After implantation, the fetal-derived trophoblast cells invade into the maternal tissue to remodel the spiral arteries, initiating the formation of the placenta. My recently published data was the first to describe that ENDS exposure elongated the timing of pregnancy and caused a delay in embryo implantation. I also determined ENDS exposure elicited a dramatic transcriptomic shift at the time of implantation. However, no studies have further examined the mechanism causing implantation delay. ENDS exposure was identified to impair trophoblast cell function in vitro, yet no in vivo studies have examined placental development. Although most ENDS users enjoy the addition of flavors, no studies have examined ENDS with flavor usage or thickening agents on pregnancy. Thus, there is an urgent need to further examine how ENDS can influence pregnancy. This proposal aims to 1.) test how ENDS with additives affects fertility and placental development, 2.) investigate the ENDS-driven mechanisms resulting in delayed implantation using an in vitro human epithelial organoid model, and 3.) determine the timing and cause of in vivo ENDS-induced delayed embryo implantation. This proposal provides a detailed plan to examine how ENDS regulates fertility and early pregnancy success and proposes to validate ENDS-mediated molecular mechanisms delaying implantation. This study can advise the public on the measure of caution observed for ENDS usage in pregnancy and can expand our understanding of mechanisms that regulate implantation success, benefitting women experiencing infertility or recurrent implantation failure.