Multiplexed Quantification of First-Trimester Serum Biomarkers in Healthy Pregnancy

The maternal circulating proteome reflects critical physiological adaptations during pregnancy, yet standardized reference profiles for early gestation are lacking. In this pro-spective study, we employed targeted liquid chromatography-multiple reaction monitor-ing-mass spectrometry (LC-MRM-MS) with stable isotope-labeled (SIS) standards to char-acterize the serum proteome of 83 women with uncomplicated singleton pregnancies be-tween 11+2 to 13+6 weeks' gestation. Robust analysis quantified 115 proteins (83% of tar-gets), with 101 meeting ICH M10 standards. These included 38 FDA-approved, 19 CVD-related, and 25 CLIA-approved biomarkers. We identified 43 proteins significantly associated (p< 0.05) with gestational age, maternal factors (BMI, age, parity, myomas), and fetal sex. Key findings included identification of 12 proteins significantly associated with trisomy risk (|R|=0.21–0.45, p< 0.05), and extreme physiological variability in Pregnancy zone protein (PZP, 123.9-fold), followed by Apolipoprotein(a) (LPA; 9.9-fold), and Preg-nancy-associated plasma protein A (PAPPA, 9.3-fold). In contrast, Hemopexin (HPX) demonstrated remarkable stability (CV=8.5%), suggesting its utility as a reference marker. The study successfully implemented multiples of the median (MoM) transformation for clinical standardization of protein profiles, with RobNorm proving particularly effective for batch-effect correction in our dataset. These methodological advances, combined with the establishment of comprehensive pregnancy-specific reference ranges, provide a valua-ble foundation for future research. The optimized analytical framework and protein sig-natures identified in this work not only enable development of next-generation screening approaches but also offer new insights into the molecular adaptations occurring during early pregnancy.