Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as NAFLD, is a leading cause of chronic liver disease worldwide. Current diagnostic methods, including liver biopsies, are invasive and have significant limitations, emphasizing the need for non-invasive alternatives. This study aimed to evaluate extracellular vesicles (EV) as biomarkers for diagnosing and staging steatosis in MASLD patients, utilizing machine learning (ML) and explainable artificial intelligence (XAI). Methods: This prospective, single-center cohort study was conducted at the GI-Liver Unit, Hippocration General Hospital, Athens. It included 76 MASLD patients with ultrasound-confirmed steatosis and at least one cardiometabolic risk factor. Patients underwent transient elastography for steatosis and fibrosis staging and blood sampling for EV analysis using nanoparticle tracking. Twenty machine learning models were developed. Six to distinguish non-steatosis (S0) from steatosis (S1-S3), and fourteen to identify severe steatosis (S3). Models incorporated EV measurements (size and concentration), anthropomorphic and clinical features, with performance evaluated using AUROC and SHAP-based interpretability methods. Results: The CB-C1a model achieved, on average on 10 random splits of 5-fold cross validation (5CV) of the train set, an AUROC of 0.71/0.86 (train/test) for distinguishing S0 from S1-S3 steatosis stages, relying on EV metrics alone. The CB-C2h-21 model identified severe steatosis (S3), on average on 10 random splits of 3-fold cross validation (3CV) of the train set, with an AUROC of 0.81/1.00 (train/test), demonstrating superior performance when combining EV with anthropomorphic and clinical features such as diabetes and advanced fibrosis. Key EV features, including mean size and concentration, were identified as important predictors. SHAP analysis highlighted complex non- linear relationships between features and steatosis staging. Conclusions: EV metrics are promising non-invasive biomarkers for diagnosing and staging MASLD. The integration of ML-enhanced EV analysis with clinical features offers a scalable, patient-friendly alternative to invasive liver biopsies, advancing precision in MASLD management. Further research should refine these methods for broader clinical application. Keywords: Metabolic dysfunction-associated steatotic liver disease (MASLD), extracellular vesicles (EVs), non-invasive biomarkers, machine learning (ML), explainable artificial intelligence (XAI), steatosis staging, transient elastography, chronic liver disease, cardiometabolic risk factors, SHAP analysis, diagnostic precision, advanced fibrosis, severe steatosis (S3), liver biopsy alternatives, hepatology diagnostics.
