Exploratory Proteomics of Amniotic Fluid Extracellular Vesicles in Congenital Cytomegalovirus

Congenital cytomegalovirus (cCMV) is the most prevalent congenital viral infection and a major non-genetic contributor to neurodevelopmental impairments. Current diagnostic methods 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. We compared amniotic fluid EV (AF-EV) characteristics in severe CMV-infected fetuses with those in uninfected fetuses to study fetal responses to cCMV in utero. AF samples were collected from CMV-infected, symptomatic pregnancies and gestational-age-matched uninfected controls (4 pairs, n = 8 total). We isolated EVs and characterised them by Western blotting, cryo-electron microscopy, and nanoparticle tracking analysis. Label-free quantitative proteomics identified CMV-associated changes in the AF-EV proteome. CMV-infected AF showed higher vesicle concentrations, indicating inflammation and virus-induced changes in EV biogenesis. Proteomic analysis identified upregulated proteins associated with haemolysis and immune activation in the fetoplacental unit. Host proteins absent in infected cases indicated viral disruption of host cellular pathways. Protein enrichment changes in neurological, haematological, and immune pathways aligned with severe cCMV pathology. This study provides proof of principle for exploring the AF-EV proteome in cCMV. While the findings identify important protein signatures linked to severe fetal outcomes, their main purpose is to generate hypotheses and guide prospective research.