Hepatic steatosis in postmenopausal women is characterized by distinct serum extracellular vesicle proteomic signatures.

backgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is common among midlife women. Circulating extracellular vesicles (EVs) carry bioactive cargo that may mediate or reflect disease processes, but their role in hepatic steatosis in postmenopausal women remains unexplored.

methodsWe conducted liquid chromatography data-independent acquisition-mass spectrometry on serum-derived EVs from 275 postmenopausal women enrolled in the Michigan site of the Study of Women's Health Across the Nation (MI-SWAN). Participants were grouped by hepatic steatosis status (n = 75), assessed via standardized ultrasound at the 2010 follow-up visit. Fasting serum samples were processed using size exclusion chromatography to isolate EVs. Differential EV protein abundance was evaluated by ANCOVA, adjusting for ethnicity and diabetes status, and applying Benjamini-Hochberg correction. Gene Set Enrichment Analysis (GSEA) was performed to identify enriched biological pathways.

resultsAmong 469 detected EV proteins, 60 differed by hepatic steatosis status (p < 0.05), with two proteins remaining significant after multiple testing correction: complement C4A (C4A) and afamin (AFM). GSEA indicated enrichment in lipid metabolism and innate immune activation pathways. Subgroup analyses revealed racial and disease severity-specific differences in EV protein profiles. In Black women (n = 172), AFM, C4A, and APOA1 were significantly elevated, while in White participants (n = 103), no proteins reached significance, although AFM displayed a nonsignificant trend toward higher abundance. In participants with severe hepatic steatosis (n = 43), subgroup analysis showed increased COL18A1, AFM, PRG4, and INHBE and decreased C4A and APOA1. INHBE was the only protein consistently elevated across all three subgroups, whereas others showed subgroup-specific enrichment, such as immunoglobulins in Black women and complement or coagulation proteins in White participants and those with severe steatosis. Analysis of hepatic transcriptomic datasets demonstrated consistently higher INHBE expression across the MASLD spectrum, including metabolic dysfunction-associated steatohepatitis (MASH), while AFM expression was significantly higher in the MASH vs. steatosis comparison.

conclusionsThis study demonstrates that circulating EV proteomes differ by hepatic steatosis status in postmenopausal women. While exploratory, candidate EV proteins such as INHBE and AFM merit validation as biomarkers and potential contributors to MASLD in this high-risk population.