The present study investigated the potential of methylated cell-free DNA (mcf-DNA) and methylated extracellular vesicle-derived DNA (mev-DNA) as an early, non-invasive epigenetic approach, specifically focusing on fetal-specific methylated regions (FSMRs) of <i>RASSF1A, ERG,</i> and <i>UMODL1 (U1 and U2)</i> to assess Trisomy 21 risk in maternal plasma.<h4>Methods</h4>Blood samples were collected from pregnant women (<i>n</i> = 120) between 10th and 24th weeks of gestation who were at higher risk for Trisomy 21. The gene discussed is RASSF1; the disease is trisomy 21.