Identification of gestational-age-dependent cell-free fetal mRNA transcriptional changes in human amniotic fluid

Cell-free fetal mRNA (cff-mRNA) in amniotic fluid has emerged as a valuable source of molecular information reflecting fetal development. We aimed to identify cff-mRNA transcripts showing gestational-age-dependent expression patterns in human amniotic fluid and to characterize their expression changes across gestation. Cell-free mRNAs from amniotic fluid samples at 16 and 38 weeks' gestation (n = 1 each) were analyzed by RNA sequencing. Candidate mRNAs showing gestational-age-dependent transcriptional changes were selected based on an expression level of ≥300 transcripts per million (TPM) at either 16 or 38 weeks' gestation and a 38-week/16-week expression ratio of ≥20. As confirmation analysis, RT-qPCR was performed using independent amniotic fluid samples collected at 16 weeks' gestation (n = 20) and 38 weeks' gestation (n = 20). Overall, 1587 mRNAs were identified by RNA sequencing analysis. Among mRNAs showing gestational-age-dependent increases in expression, six candidate genes (SFTPC, NAA10, CALML5, SMIM29, IFITM3P6, and ARF-1) were selected for RT-qPCR validation based on expression changes and tissue representation. RT-qPCR confirmed that all six genes were significantly more highly expressed at 38 weeks than at 16 weeks (all p < 0.0001; Mann-Whitney U test). This study identified candidate cff-mRNAs in human amniotic fluid that exhibit gestational-age-dependent expression patterns. These findings provide preliminary insights into developmental transcriptional changes during fetal development and contribute to our understanding of the amniotic fluid transcriptome. Further validation of the findings obtained here in larger, independent cohorts is required.