ABSTRACT Congenital syphilis (CS) rates are rising exponentially in the United State. Syphilis in pregnancy is associated with miscarriage, stillbirth, early infant death, low birth weight, and preterm birth. CS is also associated with other neonatal finding, including skeletal abnormalities, skin rashes, vision and hearing loss, jaundice, severe anemia, and hepatosplenomegaly. There is an urgent need for improved diagnostic tests in pregnancy, when timely and adequate treatment is of crucial importance to reduce the risk of CS. Currently, syphilis diagnosis relies on specific and non-specific serologic assays (treponemal and non-treponemal) that may be inadequate to accurately distinguish between past infection, current infection, or reinfection. In the highest risk patients, serology can be difficult to interpret due to lifelong persistence of treponemal antibodies in those with prior syphilis, incomplete treatment histories, inadequate serologic response after treatment, and reinfection. Furthermore, the current treatment regimen for late latent syphilis or syphilis of unknown duration in pregnancy is three weekly, intramuscular doses of penicillin, which is often challenging to administer in a timely manner in the highest risk patients. Adequate response to treatment is determined by a 4-fold drop in non-treponemal antibody titer in 3-6 months, which in clinical practice, may be after delivery of the infant depending on gestational age of infection. A more time-sensitive biomarker of successful treatment during pregnancy would have a great impact on resource utilization during pregnancy, and aid in the postnatal risk assessment of the infant. Unbiased metabolomics and proteomics approaches have been increasingly applied to discover novel biomarkers of disease status and prognosis, as well as treatment. Molecular-based testing for Treponema pallidum antigens may also be a useful adjunct to traditional serologic testing, especially for more complex clinical scenarios. These approaches have not been widely applied to syphilis in pregnancy. Here, we propose to enroll a prospective cohort of pregnant patients with syphilis in Fresno, CA, where the rates of CS in Fresno are 2.5-times higher than national averages. In the following Aims, we propose to analyze longitudinally collected samples from pregnant patients diagnosed with syphilis to identify novel biomarkers of syphilis stage, characterize the prevalence of T. pallidum in oral and vaginal samples at the time of diagnosis through the application of a new nucleic acid amplification test, and identify proteomic biomarkers in maternal and infant samples with confirmed CS. The overarching goal of these studies is to discover new candidate molecules for novel diagnostic markers for syphilis in pregnancy and CS.