Early maternal plasma protein signatures identify preeclampsia risk in twin pregnancies

Twin pregnancies carry a two- to three-fold higher risk of preeclampsia, but early prediction remains poor because current biomarkers and models are largely derived from singleton pregnancies. This study aimed to characterize maternal plasma protein changes preceding preeclampsia in twin pregnancies and to test whether early circulating proteins could improve risk stratification. It was a nested case-control study within a prospective twin pregnancy cohort at Peking University Third Hospital, including 84 women: 40 with preeclampsia and 44 normotensive controls, with longitudinal maternal plasma samples from the first, second, and third trimesters.

In the discovery cohort, maternal plasma proteins were profiled by mass spectrometry to identify trimester-specific protein changes associated with preeclampsia. Candidate proteins were then validated in an independent validation set. The researchers also assessed functional relevance in trophoblast and endothelial cell models and constructed prediction models integrating biomarkers with maternal clinical characteristics.

Twin pregnancies complicated by preeclampsia showed distinct trimester-dependent protein alterations. First- and second-trimester changes mainly involved immune regulation and glycolipid metabolism, while later changes were enriched in cardiovascular-related pathways. Early discriminatory patterns included increased annexin A3, chitotriosidase-1, and ferritin light chain, together with decreased annexin A2 in the first trimester. In contrast, the conventional singleton markers sFlt-1 and PAPP-A did not differ. ELISA validation of the candidate biomarkers yielded an AUC of 0.93 for twin preeclampsia prediction, and integration with standard maternal clinical factors improved discrimination to an AUC of 0.97. A multivariable model combining annexin A2, annexin A3, chitotriosidase-1, FTL, and maternal clinical factors achieved strong internal performance for early discrimination of preeclampsia in twin pregnancies (AUC, 0.97; 95% CI, 0.93-1.00).

Functionally, annexin A2 knockdown impaired trophoblast migration and invasion and reduced inflammatory cytokine secretion in endothelial cell models. These findings suggest that twin-specific plasma protein signatures may enable earlier risk stratification for preeclampsia and point to immune, metabolic, and vascular mechanisms that differ from singleton pregnancies.