ArticlePublished: 12 August 2026Chiaki Eishi ORCID: orcid.org/0000-0002-6830-06381,Shoko Miura1,Koh Nagata1,Ai Nagata1,Yuriko Kitajima1,Yuri Hasegawa ORCID: orcid.org/0000-0002-9584-00231,Koh-ichiro Yoshiura ORCID: orcid.org/0000-0003-3209-57802,3 &…Kiyonori Miura ORCID: orcid.org/0000-0001-5396-88811 Journal of Human Genetics (2026) Cite this articleSave articleView saved researchAbstractCell-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