Project Summary/Abstract The human placenta plays a major role in maintaining the proper environment for fetal growth, but remains as the most poorly understood organ. In particular, placental function is most difficult to ascertain during an on- going pregnancy. The current maternal serum analytes are poor indicators of placental function; sampling of the placenta itself is invasive and, aside from genotype, offers little toward diagnostic or prognostic information. Many pregnancy complications, including miscarriage, preeclampsia, and fetal growth restriction, are thought to be secondary to abnormal implantation, which is carried out by specialized placental cells, called extravillous trophoblast (EVT). Pap smears have been deemed safe during pregnancy, and endocervical samples from pregnant patients have been shown to contain fetal-derived, EVT-like cells; however, the significance of these cells, and thus their utility for prenatal diagnosis, have been questioned. This project aims to first determine the origin of these cells, by comparing them to placental trophoblast subpopulations. Subsequently, our goal is to collect and evaluate endocervical cells from a cohort of patients across gestation, followed by collection and evaluation of their placenta at delivery. Specifically, we will determine whether these cells originate from the EVT in the placenta, as claimed based on limited marker expression on fixed cells. In Aim 1, we will evaluate freshly isolated cells, from both endocervical samples and placental tissues, obtained at the time of elective pregnancy termination, and perform RNAseq to obtain a detailed transcriptome profile of these cells for a comprehensive analysis. In Aim 2, we will collect endocervical samples from a cohort of 100 uncomplicated pregnant patients once during each trimester, followed by collection of placenta at delivery. We will isolate trophoblast from these samples and subject them to proteomic analysis, using multiplex Tandem Mass Tag technology on the state-of-the-art Orbitrap Fusion Tribrid Mass Spectrometer. This will result in a detailed proteomic profile of these cells across gestation in a normal pregnancy, which can then be used as standards against which endocervical trophoblast profiles of complicated pregnancies can be measured, and specific biomarkers developed. In addition, since ~1/3 of our pregnant population is obese, we will be able to evaluate the effect of BMI on the endocervical trophoblast proteome. The successful completion of this project has the potential, not only to provide a window into placental development and function in real time, but most importantly to establish diagnostic and/or prognostic biomarkers for pregnancy complications.