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The amniotic fluid (AF) cell-free RNA was shown to reflect physiological and pathological processes in pregnancy, but its value in the prediction of spontaneous preterm delivery is unknown. Herein we profiled cell-free RNA in AF samples collected from women who underwent transabdominal amniocentesis after an episode of spontaneous preterm labor and subsequently delivered within 24 h (n = 10) or later (n = 28) in gestation. Expression of known placental single-cell RNA-Seq signatures was quantified in AF cell-free RNA and compared between the groups. Random forest models were applied to predict time-to-delivery after amniocentesis. There were 2385 genes differentially expressed in AF samples of women who delivered within 24 h of amniocentesis compared to gestational age-matched samples from women who delivered after 24 h of amniocentesis. Genes with cell-free RNA changes were associated with immune and inflammatory processes related to the onset of labor, and the expression of placental single-cell RNA-Seq signatures of immune cells was increased with imminent delivery. AF transcriptomic prediction models captured these effects and predicted delivery within 24 h of amniocentesis (AUROC = 0.81). These results may inform the development of biomarkers for spontaneous preterm birth. [ABSTRACT FROM AUTHOR]