Fentanyl and fentanyl-xylazine co-exposure influence mitochondrial transcriptional programs in human trophoblasts

Introduction: Illicit fentanyl exposure during pregnancy is associated with adverse outcomes. Yet its direct effects and their underlying molecular mechanisms in human trophoblasts remain largely unknown. Xylazine and medetomidine, two ɑ2-adrenergic agonists, were recently detected as adulterants in illicit fentanyl-containing products, and have been linked to worsened clinical outcomes. Methods: We assessed the transcriptional responses of human trophoblast stem (TS) cells in their cytotrophoblast state (TS-CTBs) and derived syncytiotrophoblasts (STBs) after 4-day and 9-day exposure to fentanyl alone or combined with medetomidine and xylazine (FMX). Results: Fentanyl alone produced modest gene expression changes in TS-CTBs, but consistently induced suppression of mitochondrial and ribosomal transcriptional programs in STBs regardless of exposure duration, an effect absent in parental TS-CTBs. 9-day FMX co-exposure influenced mitochondrial respiratory chain gene expression in derived STBs, resulting in discordant regulation of nuclear versus mitochondrial genome-encoded respiratory chain genes. Temporal pathway analysis reveals progressive trophoblast remodeling and dysregulation of mitochondrial transcriptional programs following fentanyl and FMX co-exposure. Discussion: Together, these findings illuminate trophoblast pathways affected by fentanyl alone and by FMX co-exposure, and establish a basis for future studies examining the functional and clinical relevance of these transcriptional responses.