The aim of this research was to investigate the role of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the etiopathogenesis of VSD using urine samples as an innovative, non-invasive approach, and to determine if their concentrations in urine can be used as biomarkers for predicting the spontaneous course of this congenital heart defect. This evidence concerns the gene TIMP2 and ventricular septal defect.