Label-Free Imaging of Red Blood Cells in Gestational Diabetes: Autofluorescence Captures Oxidative Stress beyond Membrane Fluidity

Gestational diabetes mellitus (GDM) is a transient pregnancy disorder marked by insulin resistance and oxidative stress. In type 1 and 2 diabetes, reduced red blood cell (RBC) membrane fluidity, measured by Laurdan-GP, associates with vascular risk, but its role in GDM is unclear. This study compared GP with intrinsic RBC autofluorescence (green-to-red ratio, AFG,R) to identify a better marker of metabolic stress in GDM. Forty-eight pregnant women were enrolled (31 GDM, 17 controls). High-resolution multispectral confocal imaging quantified RBC GP and AFG,R. A machine-learning pipeline analyzed core, membrane, and whole-cell metrics, alongside clinical and biochemical data. Logistic regression assessed associations with GDM. GP did not differ between groups (0.60 ± 0.04 versus 0.61 ± 0.04; p = 0.168), unlike the reductions reported in chronic diabetes. Conversely, AFG,R values were higher in GDM across compartments (core: -0.073 ± 0.202 versus -0.154 ± 0.060; membrane: 0.013 ± 0.198 versus -0.066 ± 0.075; p = 0.049). In multivariable analysis, AFG,R Core independently predicted GDM status (p = 0.018), outperforming clinical and hematological markers. In GDM, membrane fluidity remains unchanged, but RBC autofluorescence reflects acute oxidative stress, supporting its use as a non-invasive, repeatable complement to OGTT for early risk assessment and monitoring.