BACKGROUND: The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-Associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancyassociated complications. This proof-of-concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting. METHODS: This study was a retrospective case- control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-Time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP. RESULTS: Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P< 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity. CONCLUSIONS: Pregnancy-Associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.