There is a lack of clinical information available on early human placentation when many pregnancy pathologies originate. To address this gap and to begin the development of robust diagnostic tools to manage pregnancy complications we propose a pilot study to analyze disease specific proteins in placental trophoblast cells. Using a safe pap smear between 5 to 20 wks of gestation we can non-invasively capture hundreds of homogeneous, HLA-G-and hCG expressing trophoblast cells. We propose that these placental cells are useful for assessing pregnancy status and assessing risk of perinatal disease in vivo. Our premise is based on our published data demonstrating that isolated placental cells obtained express extra villous trophoblast lineage markers (e.g., hCG, HLA-G), and have molecular profiles associated with pathology. Immunocytochemical (ICC) protein ex- pression analysis of the cells demonstrates altered levels of several key proteins in pregnancies that later develop miscarriage, fetal growth restriction (FGR) or preeclampsia. Since the applied method are difficult to translate into a clinical tool (standardization and multiplex capabilities), we will use now a robust and highly sen- sitive single cell mass cytometry assay that does not require prior cell isolation from the clinical sample. We hypothesize that, based on robust ICC data obtained in our published studies, that AFP and PGF levels are significantly altered in EVT cells from pregnancies linked to FGR, a key indicator of placenta-based perinatal disorders. Therefore, we will rigorously determine if expression of these proteins identified in our preliminary studies in fetal cells from cervical specimens correlate with fetal growth rates, considering fetal sex and other relevant factors. The specific aim of this application is to establish the fundamental relationship of cervical extra-villous trophoblast biomarker levels to pregnancy outcomes. We will quantify AFP and PGF and cell type specific proteins in placental cells in cervical specimens in the first trimester, comparing cohorts of subjects with normal term pregnancies to those with adverse outcomes associated with low birth weight for GA at birth. This goal will be accomplished in three milestones. Milestone 1, we will validate a superior high- sensitivity single cell mass spectrometry assay (MSA) to quantify AFP and PGF in trophoblast cells and opti- mize the assay with clinical specimens. Milestone 2, we will collect up to 100 specimens of trophoblast cells in the first trimester, along with de-identified medical records to determine pregnancy outcomes. Milestone 3, the remaining patient trophoblast samples will be assayed for AFP and PGF and other protein levels and compared to patient outcomes, particularly reduced birth weight for gestational age. These experiments will validate the testing platform and provide preliminary data for a larger clinical study to establish a perinatal risk scores and a test for placental disord...