PIWI-Interacting RNA Sequencing Profiles in Maternal Plasma-Derived Exosomes Reveal Novel Non-Invasive Prenatal Biomarkers for the Diagnosis of Nonsyndromic Cleft Lip and Palate

Background: Nonsyndromic cleft lip and palate (nsCLP) is a common birth defect with high neonatal morbidity/mortality and complex etiology. Current clinical strategies to diagnose this disorder mainly rely on ultrasound with some limitations. Therefore, we firstly investigated PIWI-interacting RNAs (piRNAs) as potential prenatal biomarkers in plasma-derived exosomes from pregnant women carrying nsCLP fetuses. Methods: In total, 252 pregnant women, including 50 pregnant women carrying nsCLP fetuses, 40 pregnant women carrying fetuses with congenital heart defects (CHDs), 21 pregnant women carrying fetuses with neural tube defects (NTDs), and 141 healthy participants were selected from an ongoing Shengjing Birth Cohort Study. Small RNA sequencing in plasma-derived exosomes was used to analyze five pregnant women carrying nsCLP fetuses and five pregnant women carrying normal fetuses. Findings: We identified a biomarker panel consisting of these three pregnancy-associated exosomal piRNAs (hsa-piR-009228, hsa-piR-016659, and hsa-piR-020496) for distinguishing nsCLP from controls (area under the receiver operating characteristic curve, 0.950; P < 0.0001). They were also significantly downregulated in plasma exosomes from pregnant women carrying NTDs fetuses (P < 0.0001). In addition, hsa-piR-009228 was downregulated, whereas hsa-piR-016659 was upregulated in plasma exosomes from pregnant women carrying CHDs fetuses. Interpretation: Our work highlights the potential clinical application value of piRNAs in predicting the nonsyndromic cleft lip and palate, neural tube defects and congenital heart defects. Funding: This work was supported by the National Key Research and Development Program (2016YFC1000505), the National Natural Science Foundation of China (Grant numbers: 81871219, 81671469), and the LiaoNing Revitalization Talents Program (XLYC1902099). Declaration of Interests: The authors declare that they have no interests that might be perceived as posing conflict or bias. Ethics Approval Statement: Written informed consent was obtained from all pregnant women, and the study was performed in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Shengjing Hospital Affiliated to China Medical University (approval no. 2017PS264K).