Exploratory Proteomics of Amniotic Fluid Extracellular Vesicles in Congenital Cytomegalovirus

Introduction Congenital cytomegalovirus (cCMV) is the most common congenital viral infection and a leading non-genetic cause of neurodevelopmental impairment. Current diagnostic methods using fetal biofluids provide limited insight into fetal pathophysiology. Extracellular vesicles (EVs) in amniotic fluid (AF) are a promising source of stable biomolecules that reflect real-time fetal physiology. This proof-of-concept study compared amniotic fluid EV (AF-EV) characteristics in fetuses with severe CMV infection with those of uninfected fetuses and aimed to develop hypotheses about fetal response to cCMV in utero . Methods AF samples were collected from pregnancies with symptomatic CMV infection and gestational-age-matched uninfected controls (4 pairs, n=8 total). EVs were isolated and characterised by Western blotting, cryo-electron microscopy, and nanoparticle tracking analysis. Label-free quantitative proteomics identified CMV-associated changes in the AF-EV proteome. Results CMV-infected AF showed higher vesicle levels (Hedges’ g = 1.55), indicating inflammation and virus-induced changes in EV biogenesis. Proteomic analysis found 8.6% of proteins dysregulated. Upregulated proteins included haemoglobin subunits, immunoglobulin heavy chain mu, and myeloperoxidase (Hedges’ g = 1.51 to 1.88), indicating haemolysis and immune activation. Eleven host proteins related to neurodevelopment, mitochondrial function, lipid metabolism, and Golgi trafficking were absent in infected cases, indicating viral disruption of host pathways. Protein enrichment analysis revealed differences in neurological, haematological, and immune pathways, aligning with severe cCMV pathology. Conclusion This study acts as a proof-of-principle investigation of the AF-EV proteome in cCMV. Although the results highlight key protein signatures associated with severe fetal outcomes, they primarily serve to generate hypotheses and inform larger prospective studies.