Key Amniotic Fluid miRNAs as Promising Target Molecules for the Antenatal Prevention of Pulmonary Hypoplasia Associated with Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) remains associated with high morbidity and mortality, primarily due to pulmonary hypoplasia and hypertension. Current antenatal diagnostic methods, such as ultrasound and magnetic resonance imaging (MRI), are unable to assess the severity of defects in lung and pulmonary vascular structures, which are critical determinants of the diverse phenotypes of CDH. Aberrant epigenetic regulation of lung development during gestation is believed to play a significant role in the pathogenesis of CDH. In this study, we aimed to identify miRNA patterns in amniotic fluid capable of categorizing CDH-fetuses for the personalized selection of effective treatment strategies at the antenatal and/or postnatal stages. Using deep sequencing and quantitative real-time polymerase chain reaction (PCR), we identified a set of miRNAs-miR-485-3p, miR-320b, miR-320a-3p, miR-221-3p, miR-200b-3p, miR-100-5p, miR-92a-3p, miR-30c-5p, miR-26a-5p, and let-7c-5p-whose reduced expression in amniotic fluid at 19-24 weeks of gestation allowed us to categorize fetuses with CDH into two distinct groups: one significantly different from the control group (non-CDH) and the other closely resembling it. Notably, no significant correlations were found between the content of these miRNAs in amniotic fluid and severity of lung hypoplasia assessed by ultrasound or MRI. However, there was significant positive correlation between the level of each of the miRNAs with that of miR-200b-3p, whose role in ensuring proper bronchopulmonary tissue structure during prenatal development-as well as its therapeutic potential for CDH-associated hypoplastic lungs-has been previously demonstrated. These findings lay the groundwork for the future development of genetically engineered drug formulations designed for antenatal endotracheal administration to correct abnormal miRNA levels in lung tissue and mitigate the progression of pulmonary hypoplasia and hypertension in CDH-fetuses.