SPECIFC AIMS
In the United States, the rate of opioid use during pregnancy has dramatically increased, paralleling the crisis
observed in the general population. Maternal opioid use results in higher incidence of adverse outcomes for both
mother and offspring, notably preterm birth, delivery complications, and neonatal opioid withdrawal syndrome
(NOWS), which can lead to major, life-threatening sequelae if left untreated. The incidence of NOWS has more
than quadrupled since 2000 and yearly medical costs are now close to $1 billion. Therefore, there is a clear and
urgent need to improve medical strategies for these vulnerable unique patients. Better approaches for early
detection of infants who will most likely withdraw and identification of targets of safe treatments for an effective
recovery are sorely needed. In this application, we will test the central hypothesis that biomarkers that can
predict onset and severity of NOWS, length of hospitalization and neurodevelopmental outcomes can
be detected in fetal circulation prior to symptom onset. We are uniquely positioned to address this
hypothesis by leveraging the infrastructure of the Perinatal Assistance and Treatment Home (PATHways)
program, and the Neonatal Abstinence Care Unit (NACU), unique resources at the University of Kentucky that
serve pregnant women with substance use disorder and their neonates. We propose to collect 60 paired umbilical
cord blood (UCB) at delivery and neonatal blood samples at 24 hours of life and during NACU hospitalization of
neonates born to women with a history of chronic opioid use disorder as well as perform neurodevelopmental
tests to complete the following specific aims:
Aim 1: Identify biomarkers of NOWS onset and severity. Approximately 50% of neonates exposed in utero
to opioids develop NOWS, however symptoms do not predict the length of hospitalization. Current treatment
paradigm for NOWS involves waiting to see signs of withdrawal before initiation of medical treatments, which
themselves may have detrimental effects on the newborn’s development. Thus, identification of early biomarkers
of NOWS that would predict disease onset and severity are urgently needed. We will determine levels of
inflammatory mediators, markers of brain injury and neurotrophic factors in UCB and 24 hours post birth plasma
by Luminex and ELISA and use statistical tools such Random Forest to identify predictive biomarkers.
Furthermore, NOWS severity is variable and determining indicators of NACU stay would help the clinical care
team adjust interventions. Thus, we will also carry out a similar analysis in blood samples obtained from neonates
admitted to the NACU to identify biomarkers of severity and NACU stay length.
Aim 2: Measure the role of the neonatal immune system in NOWS development. Recent data strongly
suggest a complex bi-directional interaction between immune system and opioids. In non-gravid adult individuals,
opioids have been shown to dysregulate immune responses while, a significant increase in fragile-like regulatory
T cells (Tregs) that cross the blood-brain barrier, alter neuronal signaling, and contribute to withdrawal
symptoms. Therefore, this aim will test the hypothesis that alteration in neonatal immune cell frequency and
function can be predictive of NOWS onset and severity. Therefore, we will determine the frequency, phenotype,
and functional potential of UCB and neonatal blood leukocytes by flow cytometry and use artificial intelligence
tools to extract predictive biomarkers.
Aim 3: Determine the implications of altered inflammatory changes on neurobehavior. In utero opioid
exposure is associated with neurocognitive deficits in attention, executive function, visual-motor function, and
neurobehavior development in the offspring. Microglia, the macrophages of the brain, are key regulators of
central nervous system (CNS) development through their ability to regulate neuronal survival, differentiation, and
axonal outgrowth. Thus, in this aim, we will test the hypothesis that functional alteration in microglia align with
neurobehavioral outcomes in neonates exposed to opioids in utero. To that end, UCB cells will be differentiated
into microglia-like cells as surrogates to assess opioid-associated changes in CNS-resident microglia. These
results will be integrated with the NeoNatal Neurobehavioral Scale (NNNS-II) performed on day of life (DOL) 5
and at the time of NACU discharge, as well as data from Aims 1 and 2.
The success of this application is bolstered by access to the NACU and PATHways clinic at the University of
Kentucky (UK), the innovative experimental approaches, and the use of paired clinical samples. Moreover, this
proposal brings to bear the combined expertise of Dr. Messaoudi, an immunologist with an established track
record investigating immunity at the maternal-fetal interface, Dr. Giannone, a neonatologist with extensive
expertise in necrotizing enterocolitis and brain injury in preterm infants and Dr. O’Brien, a maternal-fetal medicine
specialist with comprehensive expertise in high-risk pregnancies.