OBJECTIVE: Polycystic ovary syndrome (PCOS), a disease of unknown etiology, is characterized by a variable elevation of luteinizing hormone. We previously showed presence and activity of autoantibodies (AAbs) to the second extracellular loop (ECL2) of other G-protein receptors (similar to GnRH). Our objective was to compare the presence and activity of AAbs directed to the ECL2 of the GnRH receptor (GnRHR) in PCOS patients compared to ovulatory controls. DESIGN: Case-cohort study MATERIALS AND METHODS: Infertile PCOS subjects based on Rotterdam criteria and infertile ovulatory controls seen at an academic fertility clinic 2012-2016 were included in the study if they had stored serum prior to beginning treatment. Serum was screened by ELISA for AAbs to GnRHR using a synthetic 28-mer peptide (GenScript, Piscataway, NJ) from the ECL2 of human GnRHR as coating antigen. Optical density (OD) values were read at 405 nm at 60 minutes. Activity of GnRHR AAb in IgG purified from sera of 4 subjects with PCOS and 4 controls was analyzed with a GnRHR-transfected Chem-1 cell-based calcium flux assay (Eurofins, St Charles, MO). AAb specific effect was tested by GnRHR blockade. Group data are presented as mean SD or percent. Groups were compared using Student t or Pearson chi-squared tests. OD values were converted to z-scores for analysis, and are reported as such. An ROC curve was used to assess OD as a diagnostic test for PCOS. RESULTS: 79 PCOS patients and 73 controls were included. There were no significant (p>0.05) differences between the groups in age (overall 29 3), race (74% white) or BMI (29 8). Standardized OD in PCOS patients (0.53 1.00) was significantly higher (p<0.001) than in ovulatory controls (-0.55 0.62). Using OD to determine a ROC curve for PCOS the Area Under the Curve was 0.83 ( 0.03;p<0.001). As a predictor of PCOS, OD alone had 74% sensitivity and 84% specificity for PCOS. There was a significant calcium flux response to IgG isolated from PCOS patients compared to controls (67.1 6.4 vs 30.9 1.5, % of maximum response, p<0.01). This PCOS (but not control) IgG-induced GnRHR activation was effectively suppressed by specific GnRHR blockade with cetrorelix (p<0.01). CONCLUSIONS: We developed a sensitive and specific biomarker for PCOS, activating AAb to the ECL2 of the GnRHR. At the hypothalamic/pituitary level, AAbs will likely be contributive and possibly causative of the menstrual dysfunction and metabolic disturbances demonstrated in PCOS subjects, and may represent the long-desired identifying diagnostic test for PCOS. We plan to evaluate the association between these AAbs and the PCOS-associated metabolic abnormalities in humans, in animal models and in pregnancy outcomes with ovulation induction.