Comparative Label-Free Proteomics of a High-Glucose Syncytialised Trophoblast Model Identifies Mitochondrial Fission Factor (MFF) as a Candidate Serum Biomarker Associated with Hyperglycaemic Pregnancy

Placental protein markers of maternal hyperglycaemia in gestational diabetes mellitus (GDM) remain scarce. We aimed to find trophoblast proteins that change under high glucose and to test whether a lead candidate is also raised in maternal serum. In our established insulin-resistant model, we used label-free liquid chromatography–tandem mass spectrometry (LC-MS/MS) to compare BeWo cells syncytialised with forskolin/8-Br-cAMP in high-glucose (25 mM) medium, with or without vascular endothelial growth factor (VEGF), with undifferentiated control cells in normoglycaemic (5 mM) medium (n = 3 per group); glucose and differentiation therefore varied together. Of 3528 protein groups, 388 were differentially abundant (24 at Benjamini–Hochberg q < 0.05). Mitochondrial fission factor (MFF), the outer-membrane receptor for dynamin-related protein 1 (DRP1), increased 4.5-fold (q = 0.040), whereas DRP1 was unchanged; mitochondrial calcium-uniporter components, pyruvate dehydrogenase kinase 3 and glycogen synthase 1 also increased. VEGF did not alter this signature (MFF: 0.91-fold, p = 0.71). Enzyme-linked immunosorbent assay (ELISA) confirmed higher MFF in high-glucose cell lysates (+48%) and, independently of in vitro differentiation, in gestational-day-7 serum of streptozotocin-treated hyperglycaemic pregnant rats (+41%; both p < 0.001; n = 3 per group), consistent with reported increases of MFF in GDM placentae. As serum was sampled after hyperglycaemia developed, these data show association, not prediction. MFF is a candidate serum biomarker of hyperglycaemic pregnancy; studies separating glucose from differentiation and validation in women are needed.