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MicroRNAs (miRNAs) are dynamically regulated during neurodevelopment, yet few reports have examined their role in spina biifda. In this study, we used an established fetal rat model of spina biifda induced by intragastrically administering olive oil-containing all-trans retinoic acid to dams on day 10 of pregnancy. Dams that received intragastric administration of all-trans retinoic acid-free olive oil served as controls. The miRNA expression proifle in the amniotic lfuid of rats at 20 days of pregnancy was analyzed using an miRNA microarray assay. Com-pared with that in control fetuses, the expression of miRNA-9, miRNA-124a, and miRNA-138 was signiifcantly decreased (> 2-fold), whereas the expression of miRNA-134 was signiifcantly increased (> 4-fold) in the amniotic lfuid of rats with fetuses modeling spina biifda. These results were validated using real-time quantitative reverse-transcription polymerase chain reaction. Hierarchical clustering analysis of the microarray data showed that these differentially expressed miRNAs could distinguish fetuses modeling spina biifda from control fetuses. Our bioinformatics analysis suggested that these differentially expressed miRNAs were associated with many cytological pathways, in-cluding a nervous system development signaling pathway. These ifndings indicate that further studies are warranted examining the role of miRNAs through their regulation of a variety of cell functional pathways in the pathogenesis of spina biifda. Such studies may provide novel targets for the early diagnosis and treatment of spina biifda.