Chronic in utero oxycodone exposure alters placental small EV proteome and fetal cardiomyopathy-linked pathways

Aim: The rising prevalence of opioid use during pregnancy poses serious public health concerns. The placenta is a critical organ during gestation, and opioid exposure can disrupt its function and fetal development. However, the molecular mechanisms through which opioids such as oxycodone affect feto-placental development remain poorly understood. This study aimed to investigate the effects of chronic in-utero oxycodone exposure on the composition and signaling functions of placenta-derived small extracellular vesicles (PSEVs) using a rat model. Methods: Extracellular vesicles (EVs) were isolated from placental tissue and characterized through nanoparticle tracking analysis, transmission electron microscopy, western blotting, and label-free quantitative proteomics. Bioinformatic enrichment analyses were conducted to evaluate changes in EVs biophysical properties and protein cargo. Results: Chronic oxycodone exposure significantly altered PSEV characteristics, including particle size distribution and proteomic composition. Among the 456 identified EV proteins, 107 proteins were significantly dysregulated. We found key downregulatory proteins including Atp2a2, Lmna, Tgfb3, Agt, and Sgce, which are crucial for myocardial calcium cycling, nuclear integrity, extracellular matrix remodeling, and blood pressure regulation. These findings indicate disruptions in fetal cardiac programming, particularly hypertrophic and dilated cardiomyopathy pathways. Additionally, enrichment analyses revealed notable perturbations in metabolic processes (e.g., citrate cycle, fatty acid degradation, N-glycan biosynthesis), along with upregulation of vesicle transport and neurodevelopment-related proteins, indicating broader systemic effects on fetal development. While these proteomic findings are robust, further independent validation (e.g., via targeted assays or Western blotting) will be necessary to confirm individual protein-level changes. Conclusion: These results highlight PSEVs as sensitive molecular indicators linking maternal oxycodone use to disrupted fetal cardiovascular, metabolic, and neurodevelopmental pathways. This study provides a novel systems-level framework for understanding opioid-induced placental signaling alterations and lays the groundwork for developing EV-based diagnostic biomarkers and targeted interventions.