Dynamic shift in trophoblast nucleos(t)ide metabolism, transport, and adenosine signaling during gestion and preterm birth

This study investigates the role of nucleos(t)ide metabolism, transport, and adenosine signaling in placental development and spontaneous preterm birth (PTB). We analyzed gene expression in human placentas, primary trophoblasts, BeWo cells and rat placentas across different gestational stages to understand metabolic adaptations and stress responses To understand the role of nucleos(t)ide metabolism (de novo synthesis and salvage patways), transport, and adenosine signaling in placental development and the adaptive response during spontaneous preterm birth (PTB), we analyzed gene expression in first-trimester and term human placentas, PTB placentas, primary human trophoblasts, and BeWo cells. Additionally, we examined rat placentas at different gestation days (GD12, GD15, and GD20) to provide developmental context and validate findings from human samples. Our results highlight the upregulation of nucleos(t)ide metabolism and adenosine signaling during placental growth and trophoblast differentiation (cytotrophoblast to syncytiotrophoblast), with further metabolic shifts in PTB placentas, particularly in pyrimidine de novo synthesis, purine salvage, and adenosine metabolism, suggesting an adaptive stress response. Additionally enhanced and also adenosine signaling and nucleoside transport was observed. This integrated approach provides novel insights into the metabolic regulation of placental function under both normal and pathological conditions This study includes n = 10 for each human pregnancy stage first-trimester, term, and preterm birth (PBT) placentas. In the animal model, rat placentas (n = 5) were analyzed at gestation days (GD) 12, 15, and 20. For in vitro models, BeWo cells (n = 5) and primary trophoblast cells (n = 3) were examined. Each sample was run in technical triplicate to ensure reproducibility. Gene expression data were processed using the ΔΔCt (delta-delta Ct) method. An interplate calibrator reaction was included in each run, analyzed in triplicates, and its mean value was subtracted from the Ct values in all analyses to generate ΔCt values. For normalization, the mean of the ΔCt values of the reference genes B2M, GAPDH, and YWHAZ was used. The normalized ΔCtNORM values were obtained by subtracting the ΔCt value of the target gene from the ΔCt value of the reference gene. These values were then re-scaled to the median of the reference group and presented as log₂ expression, corresponding to the difference between the ΔCtNORM of the sample and the median ΔCtNORM of the reference group.