Early gestational prediction of spontaneous preterm birth using a validated three-protein serum biomarker panel

Abstract Background Spontaneous preterm birth (sPTB) remains a major contributor to neonatal morbidity and mortality, with limited reliable early prediction tools. Existing biomarkers, such as the insulin-like growth factor-binding protein 4 (IBP4) to sex hormone-binding globulin (SHBG) ratio, offer modest predictive performance and are restricted to mid-gestation use (18–20 weeks), limiting their utility for timely intervention. We aimed to develop and validate a novel serological test based on early-gestational sampling to predict the risk of sPTB. Methods We conducted a meta-analysis of 18 placental transcriptomic datasets to identify candidate genes associated with sPTB, resulting in 21 protein candidates tested by targeted proteomics. We developed a three-protein panel (glutathione peroxidase 3, GPX3; nidogen-1, NID1; and pappalysin-2, PAPPA2) and validated it in four independent cohorts (456 subjects and 1048 serum specimens) from the USA and Asia. Longitudinal serum samples were collected from 5 weeks and were analyzed using mass spectrometry and ELISA platforms. Predictor performance was compared to the IBP4/SHBG ratio. Results The three-protein predictor (GPX3, NID1, and PAPPA2) demonstrated reproducible and superior performance across cohorts: AUC 0.74 (95% CI 0.59–0.88) in Alabama, 0.93 (95% CI 0.88–0.99) in California, 0.80 (95% CI 0.75–0.85) in Asia 1, and 0.83 (95% CI 0.70–0.95) in Asia 2. This outperformed the IBP4/SHBG ratio, which achieved AUCs of 0.68 (95% CI 0.50–0.89), 0.77 (95% CI 0.67–0.88), 0.59 (95% CI 0.52–0.65), and 0.61 (95% CI 0.50–0.75), respectively. Across obstetric trimesters, the three-protein panel maintained high predictive accuracy in the first and second trimesters (AUROC 0.82–0.97), the window when preventive interventions such as progesterone, cerclage, and low-dose aspirin are most effective. Kaplan–Meier analyses confirmed significantly earlier delivery among high-risk pregnancies identified by the three-protein panel. Conclusions This maternal serum test provides a reliable approach for early risk assessment of sPTB. The three-protein panel demonstrated reproducible performance across cohorts and across PPROM-positive and PPROM-negative phenotypes, with the strongest discrimination in the first and second trimesters, when preventive therapies such as progesterone or cerclage are most effective. These findings support its potential as an early, clinically actionable screening tool for improving pregnancy outcomes.