PROJECT SUMMARY In the US alone, 1.5% of pregnant individuals self-report misuse of prescription opioids, and almost 80 newborns a day are diagnosed with neonatal opioid withdrawal syndrome. In utero opioid exposure causes fetal growth restriction, alters fetal brain development and causes deficits in cognition, motor skills and behavior in affected children. However, the mechanisms by which opioids dysregulate neurodevelopment are currently unknown. The placenta is a critical regulator of fetal development, and emerging evidence suggests that opioids also impair placental development. The overarching aim of this project is to define the developmental processes in the placenta and fetal brain that are dysregulated by opioid exposure, and develop strategies to mitigate the resulting pathology. Oxycodone (oxy) is one of the most commonly abused opioids and crosses the placenta to a greater extent than morphine, heroin fentanyl and methadone. This proposal will test the hypotheses that in rats, in utero oxy exposure leads to alterations in maternal circulating factors, placental structure and function, and fetal neurodevelopment and behavior, and that these changes can be mitigated using melatonin supplementation to correct the abnormal pathophysiology. Placental and fetal brain ultrastructure and gene expression will be comprehensively assessed at mid pregnancy (embryonic (E) day 12.5, when the placenta is fully formed), and just before birth (E19.5), using state of the art technologies including single cell RNA sequencing and X-CLARITY whole organ imaging. Evidence of inflammation, oxidative stress and dysregulated hormone production in maternal plasma, fetal and placental tissues will be also be evaluated. In parallel, changes in the expression of growth factors, inflammatory molecules, immune cells and neurotransmitters in the brains of 4- and 14-day old rat pups exposed to oxy in utero will also be measured, which will be correlated with behavioral studies undertaken at the same time points. These observations will provide the first integrated analysis of the key structural and functional abnormalities, hormonal, and neurodevelopmental changes occurring at the maternofetal interface in response to oxy exposure, and link them to alterations in offspring behavior. The ability of melatonin, a naturally occurring, anti-inflammatory, antioxidant and neuroprotective hormone, to reduce the degree of placental and/or fetal pathophysiology observed, will also be assessed. Successful completion of this proposal, using a translationally-relevant rodent model, will provide a robust framework on which to identify key developmental processes that are susceptible to dysregulation by oxy. These studies, which are not possible in humans, have the potential to inform the first biomarker, fetal monitoring and interventional studies in human pregnancy, providing improved clinical guidelines and treatments for individuals affected by opioid use disorder, thereby aligning with the missions of the NICHD, NIDA and NINDS.

NARRATIVE The dramatic increase in the abuse of prescription opioids has led to a significant increase in the number of babies exposed to opioids while in the womb, which can slow fetal growth, alter fetal brain structures and cause neurological impairments that persist through childhood and beyond. The current proposal uses a rat model to identify pathophysiological mechanisms underlying the effects of oxycodone on placental and fetal brain development, and the resulting behavioral deficits observed in early infancy, and to assess whether melatonin supplementation can reduce this pathology. This work will identify key placental and fetal developmental processes that are affected by oxycodone, and has the potential to inform biomarker, monitoring and interventional studies in humans, to better support and treat pregnancies affected by maternal opioid use disorder.