Novel biomarkers to assess in utero effects of maternal opioid use: First steps toward understanding short- and long-term neurodevelopmental sequelae

Abstract

Maternal opioid use disorder is common, resulting in significant neonatal morbidity and cost. Currently, it is not possible to predict which opioid-exposed newborns will require pharmacotherapy for neonatal abstinence syndrome. Further, little is known regarding the effects of maternal opioid use disorder on the developing human brain. We hypothesized that novel methodologies utilizing fetal central nervous system-derived extracellular vesicles isolated from maternal blood can address these gaps in knowledge. Plasma from opioid users and controls between 9 and 21 weeks was precipitated and extracellular vesicles were isolated. Mu opioid and cannabinoid receptor levels were quantified. Label-free proteomics studies and unbiased small RNA next generation sequencing was performed in paired fetal brain tissue. Maternal opioid use disorder increased mu opioid receptor protein levels in extracellular vesicles independent of opioid equivalent dose. Moreover, cannabinoid receptor levels in extracellular vesicles were upregulated with opioid exposure indicating cross talk with endocannabinoids. Maternal opioid use disorder was associated with significant changes in extracellular vesicle protein cargo and fetal brain micro RNA expression, especially in male fetuses. Many of the altered cargo molecules and micro RNAs identified are associated with adverse clinical neurodevelopmental outcomes. Our data suggest that assays relying on extracellular vesicles isolated from maternal blood extracellular vesicles may provide information regarding fetal response to opioids in the setting of maternal opioid use disorder. Prospective clinical studies are needed to evaluate the association between extracellular vesicle biomarkers, risk of neonatal abstinence syndrome and neurodevelopmental outcomes.

Keywords: cannabinoid; extracellular vesicles; fetal; maternal opioid use disorder; micro RNA.

Figure 1.

Fetal exposure to opiates is associated with up-regulation of the μ opioid receptor (μOR) in FCEs (Panel A, p=0.002) and down-regulation in matched fetal brain synaptosomes (Panel B, p=0.01). Changes are most extreme with pure opioid agonists with an intermediate response with mixed agonist/antagonists.

Figure 2.

Fetal exposure to opiates is associated with up regulation of the cannabinoid CB1 receptor in fetal CNS-derived FCEs (p=0.02).

Figure 3.

In utero buprenorphine exposure elicits distinct brain miRNA profiles in male and female fetuses. The first two principal components (PC1 and PC2), which account for the majority of the variance are depicted on the x- and y-axes, respectively. Here we show that miRNA expression data cluster based on sex and treatment. In males and females, PC1 seem to be attributable to buprenorphine exposure.

Figure 4.

Validation of change in miRNA expression in fetal brain following in utero opioid exposure using qPCR. A. Expression of miR-196b-5p was reduced in female cases exposed to buprenorphine in utero, which matches our sequencing results. B. Expression of the miRs examined trended in the same direction as we observed using the sequencing approach. (p values for male data; miR-196a-5p = 0.1937; mir-196b-5p = 0.2084; miR-183-5p = 0.0789; miR-10a-5p = 0.1429; miR-10b-5p = 0.1269l miR-128-3p = 0.0574). * p< 0.05