Cytotoxicity and induction of proinflammatory cytokines in placental explant cells following Azathioprine exposure

Immunosuppressive drugs offer hope for the survival and motherhood of women with various disorders. Although the systemic side effects of these drugs on mothers have been studied, their impact on the placenta remains poorly understood, which can negatively affect fetal and postnatal development. This study investigated the effects of Azathioprine, an immunosuppressive drug, on the chorionic villi of human placentas from healthy pregnancies, focusing on cellular vitality and pro-inflammatory cytokines expression. Chorionic villi from term placentas were cultured and treated with Azathioprine at concentrations ranging from 0 to 100 ng/mL for 24 h. Azathioprine reduced mitochondrial metabolic activity (MTT assay) at all concentrations tested (p < 0.05) and increased cellular injury rates (LDH assay) at 50 and 100 ng/mL (p < 0.05). It also elevated protein (at 12.5 and 25 ng/mL, p < 0.05) and gene expression levels of interleukin (IL)-1β (12.5 ng/mL, p < 0.05). Protein levels of IL-18 increased significantly following Azathioprine exposure (p < 0.05), along with cleaved Caspase-1 (p = 0.003) and phosphorylated NF-κB levels (p < 0.05), compared to controls. IL-18 gene expression was elevated only at 12.5 ng/mL (p < 0.0005). These findings suggest that Azathioprine creates a cytotoxic microenvironment that disrupts the metabolism of chorionic villi, leading to injury and activation of pro-inflammatory cytokine expression. Such changes may contribute to an imbalance in placental homeostasis, potentially associated with adverse maternal and neonatal outcomes that warrant further investigation.