Abstract Body: Congenital single-ventricle heart defects (SVHDs) are life-threatening conditions in newborns, where one of the heart's ventricular chambers is severely underdeveloped. While medical advancements have improved survival rates, many SVHD patients still suffer lifelong cardiac complications and comorbidities. Clinical diagnosis of fetal SVHD occurs at around 20 weeks of gestation through echocardiogram. In this study, we aimed to identify novel maternal blood biomarkers that could signal SVHD development in fetuses. The study's premise is based on the limited regenerative capacity of adult human cardiomyocytes, like those in pregnant mothers, compared to the extensive proliferation seen in fetal cardiomyocytes that form the functional heart. We hypothesized small signaling molecules secreted by proliferating fetal cardiomyocytes enter the maternal circulatory system and could indicate abnormal ventricular growth in SVHD-affected fetuses. Both clinical and in vitro approaches were employed to identify potential cell-free miRNAs released by the developing fetal heart into maternal circulation. Blood plasma samples from pregnant women carrying healthy and SVHD-affected fetuses were screened for differentially expressed cell-free miRNAs and were compared with those from the supernatants of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) grown from healthy and SVHD donors. This comparative approach led to the identification of an miRNA panel (miR-487b, miR-433, mi-134, and miR-889) that were elevated in pregnant women carrying SVHD-affected fetuses and in proliferating iPSC-CMs from SVHD patients compared to healthy controls (p.adj<0.05). From this panel, forced-expression of miR-487b and mir-134 modulated the proliferation profiles of human cardiomyocytes in vitro. Results from this study establish these circulating cell-free miRNAs in maternal blood as potential non-invasive biomarkers for predicting abnormal fetal cardiac development leading to SVHDs. These findings could potentially revolutionize SVHD screening as an alternative diagnostic tool to echocardiogram.