key: cord-0745660-jjdc2jbg
authors: Smith, Brittany L.
title: Improving translational relevance: The need for combined exposure models for studying prenatal adversity
date: 2021-07-06
journal: Brain Behav Immun Health
DOI: 10.1016/j.bbih.2021.100294
sha: 2c40ab2f4bd5aee58e226b0a5a48d5e6ebd94a38
doc_id: 745660
cord_uid: jjdc2jbg

Prenatal environmental adversity is a risk factor for neurodevelopmental disorders (NDDs), with the neuroimmune environment proposed to play a role in this risk. Adverse maternal exposures are associated with cognitive consequences in the offspring that are characteristics of NDDs and simultaneous neuroimmune changes that may underlie NDD risk. In both animal models and human studies the association between prenatal environmental exposure and NDD risk has been shown to be complex. Maternal overnutrition/obesity and opioid use are two different examples of complex exposure epidemics, each with their own unique comorbidities. This review will examine maternal obesity and maternal opioid use separately, illustrating the pervasive comorbidities with each exposure to argue a need for animal models of compound prenatal exposures. Many of these comorbidities can impact neuroimmune function, warranting systematic investigation of combined exposures to begin to understand this complexity. While traditional approaches in animal models have focused on modeling a single prenatal exposure or second exposure later in life, a translational approach would begin to incorporate the most prevalent co-occurring prenatal exposures. Long term follow-up in humans is extremely challenging, so animal models can provide timely insight into neurodevelopmental consequences of complex prenatal exposures. Animal models that represent this translational context of comorbid exposures behind maternal obesity or comorbid exposures behind maternal opioid use may reveal potential synergistic neuroimmune interactions that contribute to cognitive consequences and NDD risk. Finally, translational co-exposure models can identify concerning exposure combinations to guide treatment in complex cases, and identify high risk children starting in the prenatal period where early interventions improve prognosis.

The prenatal period is a critical neurodevelopmental window, with maternal environmental experiences shaping offspring brain development and subsequent behaviors (Bale et al., 2010) . Maternal experiences affect the developing fetus and can program a risk for neurodevelopmental disorders (NDDs), with perturbations in the maternal-fetal environment playing a well-recognized role in this risk (Allswede et al., 2020) . While the prenatal period represents a window of vulnerability, it is also a window of opportunity. Concerted efforts in research and healthcare are continually optimizing prenatal care to promote maternal and infant health. The frequency of routine prenatal healthcare visits gives providers unique opportunities to identify and treat concerns in the United States. Over 75% of women in the U.S. receive appropriate prenatal care (Osterman and Martin, 2018) . Prenatal care varies widely based on factors like maternal age, race, education, or geographic location but approximately 50% of U.S. women in groups with low rates of reported care still receive it. (Osterman and Martin, 2018) .

While many barriers and challenges exist in prenatal care, pregnant women are often a motivated patient population (Economidoy et al., 2012) and preventative measures taken during pregnancy can directly reduce the number of risk factors a child will face. Once the child is born, follow-up for high risk infants is imperative to identify neurodevelopmental concerns and intervene prior to the manifestation of behavioral health disorders. In contrast to pregnancy, longitudinal childhood assessments are challenging because they require substantial resources and lack standardization (Doyle et al., 2014) . This emphasizes the need to identify predictive factors in the prenatal and early postnatal period that categorize a child as high-risk, which can include efforts to reach mothers at risk for delivering without prenatal care and in countries where access to prenatal care is limited.

Based on the literature, maternal environmental risk factors that warrant childhood neurobehavioral follow-up include maternal malnutrition, obesity, substance abuse, infection, and stress (Bale et al., 2010; B€ olte et al., 2019; Debost et al., 2017; Smith and Reyes, 2017a) . Since early interventions are key to improving child developmental outcomes (Tough et al., 2010) , understanding maternal risk factors can guide follow-up strategies to ensure high risk children receive appropriate frequency and depth of care. This ideally should include prenatal history. Understanding prenatal environment is vital because maternal risks are associated with an increased likelihood of neonatal complications that significantly exacerbate NDD risk such as preterm birth, low birth weight, and being small for gestational age (Lee and Lin, 2010; Tedesco et al., 2020; Copper et al., 1996; Baer et al., 2019; Forray, 2016; Hofheimer et al., 2020; Indredavik et al., 2010; Arpino et al., 2010) . Then as the number of risk factors increase, there is a compounding risk for adverse consequences for the child (Doyle et al., 2014) . This makes it imperative to identify, track, and intervene starting in the early prenatal period to understand combined environmental exposures that likely produce a direct increase in the risk for NDDs. While many single prenatal exposures have been identified as increasing risk, combinations of prenatal exposures further escalate risk of NDDs (Kong et al., 2020) and may identify high risk children.

The immune system is the integrated response unit for addressing the full complexity of environmental challenges (Bennett et al., 2018 ). The immune system responds to many stimuli that represent potential danger, which can range from infection, trauma, physical stress, deviations in tissue homeostasis, and even psychological stress. In the brain, microglia are the resident immune cells that respond to changes in the environment via multiple pathways, one being via intrinsic activation of toll-like receptors (TLRs) (Bennett et al., 2018; Bachtell et al., 2015) . Microglial activation by the environment provides a route for these stimuli to alter brain function and synaptic development, due to the role of microglia in synaptic pruning (Zhan et al., 2014; Squarzoni et al., 2014; Schafer et al., 2012; Frost and Schafer, 2016; VanRyzin et al., 2019; McCarthy et al., 2015) . Mounting an immune response is required to effectively adapt, overcome, and protect the organism from environmental challenges. However, compounding environmental risk factors can cause immune activation that exceeds the adaptive range in magnitude and/or time, leading to the development of chronic disease (Bennett et al., 2018) .

Environmental adversity in early life is thought to have an embedding effect on immune cells, increasing their responsivity to subsequent stimuli and reducing their ability to resolve inflammation, shifting towards an overall pro-inflammatory state (Miller et al., 2011) . Common outcomes of early life adversity, such as elevated stress reactivity and poor lifestyle habits (Miller et al., 2011) , can increase the risk of encountering further adversity over the lifespan. This can result in a state of non-resolving inflammation, especially with an increased load of environmental adversity encountered during critical neurodevelopmental periods (Nathan and Ding, 2010) . While immune responses are adaptive, this non-resolving inflammation associated with multiple adversities is likely a key contributor to NDDs (Han et al., 2021) , however relatively little is understood about combinations of exposures exclusively in the prenatal period.

Prenatally, fetal exposure to a proinflammatory environment via maternal adversity provides a proposed means for altered neuroimmune and behavioral function to increase NDD risk (Smith and Reyes, 2017a) . Elevations in markers of maternal inflammation are consistently associated with offspring inflammation, behavioral impairments, and NDDs (Allswede et al., 2020; Rudolph et al., 2018; Lee et al., 2015) . This association is evident in the epidemiology of NDDs and has a strong mechanistic premise via the known role of microglia in brain development (Zhan et al., 2014; Squarzoni et al., 2014; Schafer et al., 2012; Frost and Schafer, 2016; VanRyzin et al., 2019; McCarthy et al., 2015) .

Maternal inflammation by adverse environmental stimuli has manifold ways to reach the developing fetus. Peripheral maternal cytokine elevations propagate to the placenta and mammary glands, and can result in increases in offspring peripheral and central cytokines (Crew et al., 2016; Reynolds et al., 2015; Frias et al., 2011; Hernandez et al., 2012; Bilbo and Tsang, 2010; Grissom et al., 2016; Sanders et al., 2014; Kang et al., 2014) . With numerous routes for inflammation to travel from mother to fetus, a greater burden of prenatal exposures may result in a graded immune response and subsequently amplified impact on offspring cognitive development. A single prenatal exposure may produce a mild immune response that resolves quickly while combined prenatal exposures may produce higher grade immune responses that lead to chronic cytokine elevations (Miller et al., 2011) . Single prenatal exposures that may be subthreshold for eliciting an immune response may have the capacity to produce a more severe response when combined with other subthreshold exposures. For example, combined prenatal diesel exhaust particle exposure and prenatal stress elicits an increase in male offspring TLR4 expression in the brain, while either stimulus alone is insufficient to cause such a change (Bolton et al., 2013) . Additionally, maternal high fat diet produces minimal changes in basal offspring cytokine or chemokine gene expression (Smith et al., 2020) , unless offspring are challenged in adulthood with LPS or a stressor . Finally, individuals that may have appropriately graded and timed immune responses to a single exposure could have excessive or chronic responses to combined exposures.

Animal models are essential for advancing research on prenatal programming with a single risk factor, and even more so with compounding exposures. To control for and begin to understand these complex exposures, animal models offer control over prenatal variables to an extent not possible with human studies. The short life span of rodents accelerates the pace for studying long term neurobehavioral consequences, which is necessary to gain information fast enough to develop meaningful interventions. Then animal models provide access to fetal and neonatal samples, and realistically the only access to brain samples from this population.

This accentuates the need for continuous evaluation of existing prenatal exposure models, development of new models, and a deep understanding of the translational context or background behind each prenatal exposure. Simplifying a model to a single exposure is required to build a foundational understanding, but to successfully translate findings and improve child health, models need to be relevant to the human population that they are intended to study. This means that at some point, models must directly confront the complexity encountered in humans in a systematic fashion. Furthermore, single exposures may not be sufficient to cause NDDs but combined exposures can act synergistically to confer vulnerability (Debost et al., 2017) .

Animal models have demonstrated compounding effects of multiple environmental exposures in altering neuroimmune and behavioral outcomes in the offspring. However, most of these are sequential in nature, and focus on the effects of a single prenatal exposure followed by a second exposure in adolescence or adulthood. These two-hit models demonstrate that a single prenatal exposure alone may not reveal behavioral deficits or could even be advantageous, while a second exposure later in life can result in impairment (Giovanoli et al., 2013 (Giovanoli et al., , 2016 Bilbo et al., 2005; Makinson et al., 2019; Zhao et al., 2021) . With the sequential two-hit approach in mice, maternal immune activation acts synergistically with pubertal stress to reveal behavioral impairments and an increase in hippocampal microglia (Giovanoli et al., 2013 (Giovanoli et al., , 2016 . Additionally, recent evidence shows that maternal immune activation improved performance on a visual discrimination task, although exposed offspring had impaired discrimination and reversal learning if they were challenged with a stressor in adulthood (Zhao et al., 2021) . Models less frequently address combined exposures during the prenatal period but the few that do suggest significant interactions. For example, prenatal stress and diesel exhaust particle exposure in mice acted synergistically to increase neuroinflammatory tone in the offspring brain and impair cognitive function, particularly in male offspring (Bolton et al., 2013) . A review on animal models of combined prenatal exposures suggests mild synergistic effects on impairing fetal growth, but they are sparse and subject to bias based on blinding, concealment, and incomplete outcome reporting (Vesterinen et al., 2017) . Furthermore, this was only related to fetal growth while cognitive outcomes remain understudied.

Clinical literature is focused on understanding synergistic exposures because humans almost always experience multiple exposures (Debost et al., 2017; Siddika et al., 2019; Clarke et al., 2009; Elliott et al., 2020) . Animal models can provide systematic investigation of each exposure alone and in combination, starting with the most prevalent comorbidity and using 2 Â 2 experimental designs to identify synergistic effects. Then as long-term behavioral follow-up to identify neurodevelopmental issues is challenging in humans, animal models can give an insight on where to focus resources and identify exposure combinations that are the most concerning. This would enable clinicians to leverage the existing prenatal healthcare structure to track exposures prior to birth or shortly after. Understanding common co-occurring exposures may reveal novel interactions, which could relate to a graded neuroimmune response as the number of exposures increase. Then from a clinical perspective, it can help identify and track children who are high risk. Using the translational context behind prevalent prenatal environmental exposures to guide animal models may help identify causative factors or combinations of factors that contribute to NDD risk. As an example, maternal obesity/overnutrition and maternal opioid use are current epidemics that each have a unique constellation of co-occurring exposures. This review highlights these comorbidities, to guide future work on understanding how they may interact.

Approximately 20-25% of women are obese at the onset of pregnancy, with 50% being classified as at least overweight (Chen et al., 2018; Branum et al., 2014) . Maternal obesity is consistently problematic in the U.S. but has been sharply increasing in other countries, making it a global issue (Chen et al., 2018) . Increased maternal body weight is associated with a greater risk of the child developing schizophrenia, autism spectrum disorders (ASD), and attention deficit/hyperactivity disorder (ADHD) (Schaefer et al., 2000; Buss et al., 2012) . Maternal overnutrition plays a causal role in maternal overweight status and obesity. The risk for offspring NDDs is associated with maternal nutritional status, even after adjusting for prevalent risk factors like maternal age or nicotine use (Schaefer et al., 2000; Rodriguez et al., 2008) . Maternal overnutrition and obesity are associated with impairments in offspring cognitive function, which may explain the risk for NDDs (Smith et al., 2020; Smith and Reyes, 2017b; Grissom et al., 2015; Veena et al., 2016; Iessa and B erard, 2015) .

In animal models, maternal obesogenic HF diets increase cytokine levels at the maternal-fetal interface, with elevated cytokines peripherally in the dam, placenta, and mammary glands (Crew et al., 2016; Reynolds et al., 2015; Frias et al., 2011; Hernandez et al., 2012; Edlow et al., 2019) . Consequently, HF offspring display peripheral and central cytokine elevations and reactivity (Bilbo and Tsang, 2010; Grissom et al., 2016; Sanders et al., 2014; Kang et al., 2014) , along with evidence of increased microglial activation in the brain (Bilbo and Tsang, 2010; Kang et al., 2014) . Increases in inflammatory cytokines and chemokines in HF offspring coincide with cognitive deficits (Graf et al., 2017; McKee et al., 2017) and ablation of microglia during adolescent neurodevelopment can reduce behavioral impulsivity in HF male progeny (Smith et al., 2020 ) (seeFig. 1).

Since neuroimmune interactions are hypothesized mechanisms for offspring cognitive deficits after maternal overnutrition/obesity, comorbid exposures may lead to non-resolving inflammation and an exacerbated risk for NDDs. Maternal obesity prior to and during gestation is associated with a constellation of health problems and pregnancy complications, many of which are characterized by chronic immune activation. Maternal obesity increases the risk of heightened gestational weight gain, type II diabetes, gestational diabetes, heart disease, hypertension, preeclampsia (Leddy et al., 2008) , and even increases the severity of infections (Karlsson et al., 2012; McCartney et al., 2020) (Fig. 2) . Children with NDDs are more likely to have a mother that had a Fig. 1 . Brittany L. Smith: My research goal is to understand how adverse prenatal environments influence neuroimmune and executive function, and how this may be associated with neurodevelopmental disorders. I have a specific interest in prenatal substance exposure and am actively examining the effects of prenatal opioid exposure. I am working to build a translational research program to understand the molecular basis of prenatal programming after opioid exposure, search for biomarkers to identify children who are high risk, and develop interventions. I completed my dissertation under the mentorship of Dr. James Herman at the University of Cincinnati. While studying chronic stress exposure, I found that microglia and the neuroimmune environment within the prefrontal cortex respond to chronic stress in a manner that depends on stressor modality. Then for my dissertation work, I developed a novel and ethologically relevant stress paradigm based on loss of an enriched environment. As a postdoctoral fellow, I am working under the mentorship of Dr. Teresa Reyes at the University of Cincinnati. I am studying how prenatal experiences affect neuroimmune and executive function. I began my postdoctoral fellowship by studying how maternal high fat diet in mice programs offspring behavioral function, with a focus on the role of microglia in behavioral impulsivity. I went on to obtain K99/R00 funding from NIDA to examine how prenatal opioid exposure affects offspring brain development. I am using rodent models to study offspring behavioral function in domains frequently impaired in opioid exposed children, along with identifying molecular changes in the brain that may contribute to deficits. In humans, I am working to understand the epidemiological context and epigenetic consequences of prenatal opioid exposure. metabolic condition during pregnancy (Krakowiak et al., 2012) , so it is important to understand NDD risk in the context of obesity-related comorbidities. Combinations of these maternal comorbidities increase the risk of offspring NDDs beyond the magnitude seen with a single exposure, such as maternal obesity and diabetes together exacerbating autism risk and more subtly ADHD risk (Kong et al., 2020) .

NDD risk after maternal obesity may be exacerbated by comorbid maternal inflammatory conditions. For example, preeclampsia is described as an immune imbalance that leads to persistently elevated immune activation (Cornelius, 2018) . Prenatal preeclampsia exposure impairs offspring cognitive function and increases the risk and severity of NDDs such as ASD, ADHD, and schizophrenia (Gumusoglu et al., 2020) . Similarly, maternal diabetes can increase inflammation and lead to an increased risk of NDDs in the offspring (Krakowiak et al., 2012; Van Lieshout and Voruganti, 2008; Zhao et al., 2019; Xu et al., 2014) . With maternal obesity, comorbid conditions such as hypertension and diabetes exacerbate the risk of developmental delays or ASD in the child (Kong et al., 2020; Krakowiak et al., 2012) .

Furthermore, maternal obesity increases the severity of viral infections like influenza and COVID-19 in pregnant women (Karlsson et al., 2012; McCartney et al., 2020) , and increases the incidence of maternal infection-related asthma complications (Murphy et al., 2014) . Maternal infection is associated with offspring NDDs, particularly with additional second hit exposures (Debost et al., 2017; Giovanoli et al., 2013) . Obesity in pregnancy also increases the risk of offspring infections both in early life and long-term (Gutvirtz et al., 2019; Gutierrez et al., 2021; Griffiths et al., 2016) , which could predispose these offspring to a second-hit immune challenge.

In rodents, HF diet exposure to induce maternal obesity can impair glucose tolerance and increase blood pressure (Ge et al., 2013; Yokomizo et al., 2014) , although these endpoints are not routinely studied in the context of offspring behavior. Maternal overnutrition, obesity, diabetes, and hypertension each are associated with NDDs in humans, but the impact of combined exposures is lacking (Han et al., 2021) . In an attempt to parse out effects in animal models, maternal diabetes can be studied in the absence of maternal obesity. Maternal diabetes impaired offspring neurodevelopment in the early postnatal period and reduced object recognition in females (Sousa et al., 2020) . Interestingly, maternal diabetes did not affect offspring microglial morphology but maternal insulin treatment for diabetes did increase microglial number and processes (Sousa et al., 2020) . In contrast, maternal dietary intervention reduces offspring microglial activation in response to maternal HF diet (Kang et al., 2014) . Treatment strategies for maternal obesity and related comorbidities may impact offspring neuroimmune outcomes.

Collectively, maternal adversity such as obesity increases inflammation and is associated with an increased risk of offspring NDDs, but that association is variable (Meyer, 2019) . Obesity-related comorbidities may confer NDD vulnerability by increasing the severity of inflammation at the maternal-fetal interface. While many factors during the prenatal and postnatal periods can increase or decrease susceptibility (Meyer, 2019) , the challenge of postnatal long term follow-up emphasizes the need to understand factors during the prenatal period. Studying the impact of maternal obesity together with obesity-related conditions that increase disease severity will enable clinicians who treat pregnant women with complex experiences to take into account the long term cognitive health of the child, especially if maternal treatments can influence offspring outcomes. Fig. 2 . Modeling prenatal comorbidities to understand NDD risk: Maternal obesity and maternal opioid use have distinct comorbidities that have the capacity to increase neuroinflammatory signaling in the offspring and result in behavioral impairments to increase neurodevelopmental disorder (NDD) risk. Multiple prenatal comorbidities are present in the human population, but exposure combinations are seldom assessed in animal models. Due to the ability of the immune system to have a graded response to increasing stimuli severity and the multiple routes for inflammatory cytokines to reach the developing fetus, combined prenatal exposures may act synergistically. Understanding these possible synergistic interactions in rodent models will guide how to treat mothers with complex comorbidities, identify offspring consequences to facilitate interventions, and improve long term follow-up by identifying high risk children. (Created with BioRender.com).

Opioid use during pregnancy has increased by over 130% in the past decade, contributing to an average 80% increase in neonatal opioid withdrawal syndrome (NOWS) across the U.S. (Tolia et al., 2015; Desai et al., 2014; Pan and Yi, 2013; Hirai et al., 2021) . Opioid drugs readily cross the placenta and the blood brain barrier to reach the fetal brain (Gabrielsson and Paalzow, 1983; Farid et al., 2008) , where they have been shown to affect neurodevelopment. Children born to opioid-using mothers have cognitive problems related to NDDs, including hyperactivity, inattention, impulsivity, poor planning, lowered adaptability, and impaired working memory (Hickey et al., 1995; Nygaard et al., 2016; Ornoy et al., 1996; Slinning, 2004; Levine and Woodward, 2018; Wahlsten and Sarman, 2013) . These problems persist into adolescence and early adulthood and are present even if the mother was on methadone or buprenorphine treatments for opioid use disorder (Levine and Woodward, 2018; Wahlsten and Sarman, 2013; Nygaard et al., 2017) . Recently, maternal opioid use has also been associated with an elevated risk of ADHD and ASD symptoms and diagnoses in the child (Azuine et al., 2019; Rubenstein et al., 2019; Haugland et al., 2018) .

In rodent models, offspring exposed prenatally to opioids displayed learning and memory deficits in basic cognitive tasks ( Slamberov a et al., 2001; Wang and Han, 2009; Lu et al., 2012; Zagon et al., 1979) . Recently, Jantzie et al, 2020 found that prenatal methadone exposure impaired executive function and cognitive flexibility in the visual discrimination reversal task . In conjunction with behavioral deficits, methadone increased peripheral inflammation maternally and in the offspring Newville et al., 2020) . Notably, methadone exposed offspring had increased morphological activation of microglia, increased cortical expression of TLR4 and TLR4 adapter protein Myd88, along with increased protein levels of cytokine IL-1b and chemokine Cxcl1 . Furthermore, peripheral blood mononuclear cells of offspring exposed prenatally to methadone had increased proinflammatory activity, both at baseline and after stimulation (Newville et al., 2020) .

The proinflammatory effect of prenatal opioid exposure on the offspring brain may seem paradoxical given that opioids are considered immunosuppressive (Eisenstein, 2019; Roy et al., 2011) . However, this evidence has focused largely on peripheral immune cells while in microglia, morphine has been shown to induce proinflammatory signaling (Merighi et al., 2013; Wang et al., 2012) . Morphine (the immediate metabolite of heroin), along with a wide range of opioids including methadone, buprenorphine, oxycodone, and fentanyl can activate TLR4 in the CNS environment (Wang et al., 2012; Hutchinson et al., 2010) . Given the role of microglia in brain development, microglial activation may provide a direct route for maternal opioid use to interfere with offspring neurodevelopment and subsequent behavioral function.

Based on rodent models, maternal opioid use may induce a neuroinflammatory state in the offspring that is associated with impaired cognitive function. In addition to the neuroinflammatory potential of opioids, it is critical to note that in humans, the maternal opioid using population has high rates of other exposures that could potentially act synergistically to increase neuroinflammation, impair cognitive function, and increase NDD risk (Fig. 2) .

Pregnant women who use opioids are more likely to use other substances and combinations of multiple substances (Smith et al.) . Polysubstance use of nicotine, cocaine, methamphetamine, and benzodiazepines are disproportionately higher in pregnant women who test positive for opioids (Smith et al.) . Cocaine and other drugs can activate TLR4 (Northcutt et al., 2015) , so it is possible that polysubstance use could heighten TLR4 activation of microglia and further increase NDD risk. Polysubstance use in the context of opioid use is particularly concerning due to the synergistic effects on addictive properties, overdose risk, and increased severity of NOWS (Compton et al., 2021; Choo et al., 2004; Desai et al., 2015; Jansson et al., 2012 Jansson et al., , 2017 Sanlorenzo et al., 2019; Kaltenbach et al., 2012) . NOWS treatment is notoriously challenging for infants exposed prenatally to multiple substances, who frequently require longer hospital lengths of stay (Choo et al., 2004; Jansson et al., 2012; Sanlorenzo et al., 2019; Kaltenbach et al., 2012) . However, long-term neuroimmune status and cognitive function in polydrug exposed infants remains unknown.

Opioid use during pregnancy is highly comorbid with nicotine use to the extent that co-treatment for dual use of these two substances has been recommended as standard of care (Morris and Garver-Apgar, 2020) . Up to 95% of opioid-using mothers also use nicotine, especially women taking methadone or buprenorphine, medications that are used to treat opioid use disorder Chisolm et al., 2013) . In humans, prenatal nicotine exposure was associated with offspring cognitive and behavioral problems (Daseking et al., 2015; Tiesler and Heinrich, 2014) , along with an increased risk for ADHD (Wang, 2018; Ross et al., 2015) and schizophrenia (Niemel€ a et al., 2016) . In rodents, offspring exposed prenatally to nicotine had executive function deficits (Bryden et al., 2016; Schneider et al., 2011) , increased peripheral cytokines as newborns (Mohsenzadeh et al., 2014) , and increased brain cytokine levels as adults (Chan et al., 2016) . Furthermore, prenatal nicotine exposure has been shown to be an important mediating variable for the cognitive deficits reported in children exposed prenatally to opioids (Nelson et al., 2020) . This evidence supports the idea that inclusion of prenatal nicotine exposure in preclinical rodent models of maternal opioid use would greatly enhance the translational validity of these studies.

Beyond polysubstance use, pregnant women who use opioids are at a greater risk for a range of infections. Intravenous drug use is a primary causal route because it increases the chance of exposure, particularly to HIV or hepatitis C. Additionally, opioid use is associated with immunosuppression that impairs host defense, leading to increased incidence and severity of opportunistic infections such as pneumonia and influenza, in addition to HIV and hepatitis C (Roy et al., 2011) . Hepatitis C exposure is particularly high in the context of opioid use, present in over 40% of pregnant women who use opioids (Fajemirokun-Odudeyi et al., 2006) . The neurodevelopmental consequences of prenatal or perinatal hepatitis C exposure are unknown, let alone in combination with prenatal opioid exposure. While prenatal exposure to opioids, hepatitis C, or nicotine have not been shown to cause overt structural damage to the developing brain, white matter lesions have been recently reported in human infants exposed prenatally to all three of these stimuli (Merhar et al., 2019) . In rodents, Iba-1 expression was increased after exposure to a combination of nicotine, morphine, and HIV but not with any of these stimuli alone (Cornwell et al., 2020) . This emphasizes the importance of leveraging animal models to understand how these exposures interact in the prenatal period.

HIV has been surging in the context of opioid use disorder where treatment strategies are working to combine medication distribution for opioid use disorder with antiretroviral therapy (Bach et al., 2015; Tofighi et al., 2019) . The consequences of prenatal exposure to opioids and HIV are also unknown, but children exposed perinatally to HIV experienced developmental delays and had impaired neurobehavioral development as toddlers (Wu et al., 2018; Wedderburn et al., 2019) . At the neuroimmune level, morphine acts synergistically in the context of HIV and pneumonia co-infection to enhance cytokine synthesis and TLR activation in microglia (Dutta et al., 2012) . However, this interaction is unknown after prenatal co-exposure. There is a need to understand the neurodevelopmental consequences of prenatal opioid exposure combined with comorbid maternal infections, because the little that is known suggests these exposures may act synergistically.

Although obesity has been associated with increased prescription opioid use in the general population (Stokes et al., 2020) , this association is not evident in pregnant women (Whiteman et al., 2014) . Conversely, pregnant women with opioid use disorders are more likely to be underweight (Nagarajan and Goodman, 2020) . Poor nutrition is pervasive in drug using populations and some adverse effects of prenatal drug exposure are possibly attributed to malnutrition (Tonkiss et al., 1996; Wright and Walker, 2007) . Undernutrition and nutritional deficiencies are just beginning to be recognized and addressed in pregnant women with opioid use disorders (Nagarajan and Goodman, 2020) . Nutritional deficiencies are common across undernutrition and overnutrition, as both result in a lack of key nutrients either via low food access or excess consumption of nutritionally poor quality foods. Consequently, maternal undernutrition is also associated with offspring NDDs with a likely contribution of the neuroimmune environment (Smith and Reyes, 2017b) .

While prenatal care is largely accessible across the United States, pregnant substance using women are more likely to deliver without prenatal care (Maupin et al., 2004) . Substance using women frequently have significant social, psychological, economic, and legal barriers that prevent them from getting adequate care during pregnancy (Stone, 2015) . Understanding common co-exposures in this population may lead to improved evidence-based treatment strategies that help dissolve barriers and stigma, along with efforts to increase global access to prenatal care. Finally, the prevalence of psychiatric illness is extremely high in the maternal opioid using population, an important comorbidity that warrants its own review (Noose Glovak et al., 2020; Arnaudo et al., 2017) .

Clinically, comorbid maternal exposures make it difficult to definitively test associations between a single exposure and the risk for offspring NDDs. These comorbidities are especially prevalent in the context of maternal obesity and maternal opioid use, and a number of them are associated with neuroimmune and behavioral consequences. Animal models can eliminate confounds to help disentangle whether a single exposure alone contributes to NDD risk.

Comorbid exposures may act synergistically to exacerbate the risk for complications, possibly via a graded neuroimmune response to an increased number of stimuli. However, these synergistic outcomes have yet to be systematically assessed in relation to cognitive function and NDD risk. Complex co-occurring exposures are associated with an increased risk of neonatal complications, making mothers and babies difficult to treat from early on. Clinical practice lacks a preclinical understanding of co-exposure contributions to long term neurodevelopment, which is particularly problematic due to the challenges of long-term follow up.

Animal models have the capacity to address these complex problems and ideally should capture some of the complexity seen in the human populations that they are intended to study. Studying the most concerning exposures and exposure combinations can establish a means for predicting risk versus resilience starting in the prenatal period. In complex cases, this would help allocate long term resources to the most high risk children. Simultaneously, it would use the existing U.S. prenatal healthcare system or encourage increased access to prenatal care worldwide to adjust prenatal and early postnatal interventions to encourage resiliency.

Interestingly, there are some children exposed to extremely complex prenatal adversities that thrive against all odds. Neuroimmune and behavioral responses to adverse prenatal environments alternatively could serve a protective role as necessary adaptations to these early life stimuli. It is important to assess the context and duration of these responses to help determine their valence, in conjunction with continued work on the role of microglia in shaping the development of higher level cognitive processes. As the field progresses, the translational context of prenatal exposures will promote the clinical relevance of preclinical models.

None.

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Suboptimal nutrition in early life affects the inflammatory gene expression profile and behavioral responses to stressors

Suboptimal nutrition in early life affects the inflammatory gene expression profile and behavioral responses to stressors

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Psychosocial and medical adversity associated with neonatal neurobehavior in infants born before 30 Weeks' gestation

Evidence that opioids may have toll-like receptor 4 and MD-2 effects

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Exposure to in utero inflammation increases locomotor activity, alters cognitive performance and drives vulnerability to cognitive performance deficits after acute immune activation

Characteristics of women who deliver with no prenatal care

Surprising origins of sex differences in the brain

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Methyl donor supplementation alters cognitive performance and motivation in female offspring from high-fat diet-fed dams

White matter injury and structural anomalies in infants with prenatal opioid exposure

Morphine mediates a proinflammatory phenotype via m-opioid receptor-PKCε-Akt-ERK1/2 signaling pathway in activated microglial cells

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Prenatal exposure to nicotine in pregnant rat increased inflammatory marker in Newborn Rat

Nicotine and opioids: a call for Co-treatment as the standard of care

The role of maternal obesity in susceptibility to respiratory viral infection and exacerbation rate in pregnant women with asthma

Not just substance use: the critical gap in nutritional interventions for pregnant women with opioid use disorders

Nonresolving Inflammation

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Perinatal opioid exposure primes the peripheral immune system toward hyperreactivity

Prenatal nicotine exposure and risk of schizophrenia among offspring in a national birth cohort

Pregnant women misusing opioids: depression, anxiety, stress, and neonatal opioid withdrawal syndrome

DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling

Behavior and attention problems in eight-year-old children with prenatal opiate and poly-substance exposure: a longitudinal study

Cognitive function of youths born to mothers with opioid and poly-substance abuse problems during pregnancy

The developmental outcome of children born to heroin-dependent mothers, raised at home or adopted

Timing and adequacy of prenatal care in the United States

Prevalence of hospitalized live births affected by alcohol and drugs and parturient women diagnosed with substance abuse at liveborn delivery: United States

Maternal high fat and/or salt consumption induces sex-specific inflammatory and nutrient transport in the rat placenta

Maternal adiposity prior to pregnancy is associated with ADHD symptoms in offspring: evidence from three prospective pregnancy cohorts

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Opioid drug abuse and modulation of immune function: consequences in the susceptibility to opportunistic infections

Brief report: maternal opioid prescription from preconception through pregnancy and the odds of autism spectrum disorder and autism features in children

Maternal IL-6 during pregnancy can be estimated from newborn brain connectivity and predicts future working memory in offspring

Maternal obesity and IL-6 lead to aberrant developmental gene expression and deregulated neurite growth in the fetal arcuate nucleus

Increased severity of neonatal abstinence syndrome associated with concomitant antenatal opioid and benzodiazepine exposure

Maternal prepregnant body mass and risk of schizophrenia in adult offspring

Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner

Prenatal exposure to nicotine impairs performance of the 5-choice serial reaction time task in adult rats

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Prenatal morphine exposure differentially alters learning and memory in male and female rats

Foster placed children prenatally exposed to poly-substances: attention-related problems at ages 2 and 4 1/2

Offspring neuroimmune consequences of maternal malnutrition: potential mechanism for behavioral impairments that underlie metabolic and neurodevelopmental disorders

Offspring neuroimmune consequences of maternal malnutrition: potential mechanism for behavioral impairments that underlie metabolic and neurodevelopmental disorders

Rates of Substance and Polysubstance Use through Universal Maternal Testing at the Time of Delivery

Adolescent microglia play a role in executive function in male mice exposed to perinatal high fat diet

Sex differences in offspring neurodevelopment, cognitive performance and microglia morphology associated with maternal diabetes: putative targets for insulin therapy

Microglia modulate wiring of the embryonic forebrain

Obesity and incident prescription opioid use in the

Pregnant women and substance use: fear, stigma, and barriers to care

The role of maternal infection in preterm birth: evidence from the brazilian multicentre study on preterm birth (EMIP)

Prenatal nicotine exposure and child behavioural problems

Perspectives on the HIV continuum of care among adult opioid users in New York City: a qualitative study

Increasing incidence of the neonatal abstinence syndrome in U.S. neonatal ICUs

Differential effects of prenatal protein malnutrition and prenatal cocaine on a test of homing behavior in rat pups

Maternal well-being and its association to risk of developmental problems in children at school entry

Diabetes mellitus during pregnancy and increased risk of schizophrenia in offspring: a review of the evidence and putative mechanisms

Microglial phagocytosis of newborn cells is induced by endocannabinoids and sculpts sex differences in juvenile rat social play

Association between maternal nutritional status in pregnancy and offspring cognitive function during childhood and adolescence ; a systematic review

Cumulative effects of prenatal-exposure to exogenous chemicals and psychosocial stress on fetal growth: systematic-review of the human and animal evidence

Neurobehavioural development of preschool-age children born to addicted mothers given opiate maintenance treatment with buprenorphine during pregnancy

Maternal smoking and attention-deficit/hyperactivity disorder in Offspring : a meta-analysis

Prenatal exposure to heroin in mice elicits memory deficits that can be attributed to neuronal apoptosis

Morphine activates neuroinflammation in a manner parallel to endotoxin

Neurodevelopment of HIV-exposed uninfected children in South Africa: outcomes from an observational birth cohort study

Maternal opioid drug use during pregnancy and its impact on perinatal morbidity, mortality, and the costs of medical care in the United States

Management of women who use drugs during pregnancy

Neurodevelopmental outcomes in young children born to HIVpositive mothers in rural Yunnan

Maternal diabetes and the risk of autism spectrum disorders in the offspring: a systematic review and meta-analysis

Maternal high-fat diet induces insulin resistance and deterioration of pancreatic β-cell function in adult offspring with sex differences in mice

Learning ability in adult female rats perinatally exposed to methadone

Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior

p>The association of maternal diabetes with attention deficit and hyperactivity disorder in offspring: a meta-analysis</p>

Hidden Talents: poly (I:C)-induced maternal immune activation improves mouse visual discrimination performance and reversal learning in a sex-dependent manner

I would like to thank Teresa Reyes, PhD for helping to review this article. I would also like to thank funding to Brittany L. Smith, K99DA049908 (NIH/NIDA). Fig. 2 was created using BioRender.com.