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 prospective study, we employed targeted liquid chromatography–multiple reaction monitoring–mass spectrometry (LC-MRM-MS) with stable isotope-labeled (SIS) standards to characterize the serum proteome of 83 women with uncomplicated singleton pregnancies between 11+2 and 13+6 weeks’ gestation. Robust analysis quantified 115 proteins (83% of targets), 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, and 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 pregnancy-associated plasma protein A (PAPP-A, 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 valuable foundation for future research. The optimized analytical framework and protein signatures identified in this work not only enable the development of next-generation screening approaches but also offer new insights into the molecular adaptations occurring during early pregnancy.