During the 1960s there was a rubella epidemic in the United States that resulted in various pregnancy and birth complications as well as physical and cognitive birth defects in the affected infants (Lindquist et al., 1965; Chess et al., 1979; Berger et al., 2011). While many cases of rubella infection have since been prevented by vaccination, rubella is estimated to still affect around 5% of pregnant persons worldwide (Berger et al., 2011; Hutton, 2016). Early links between rubella and ASD were identified from a New York cohort study of children in the United States that were part of the Rubella Birth Defect Evaluation Project (RBDEP). This study identified a significant correlation between congenital rubella syndrome (CRS) and autism diagnosis during childhood (Chess et al., 1979). Of particular note, CRS and ASD seem to overlap in their manifestation and symptomatology (Desmond et al., 1969; Swisher and Swisher, 1975; Hutton, 2016; Mawson and Croft, 2019). One mechanism thought to underlie the link between maternal rubella infection, particularly during the first trimester, and ASD risk in offspring is via liver dysfunction resulting in fetal exposure to high levels of vitamin A, which can be toxic to brain and other tissues of the developing fetus (Mawson and Croft, 2019).

Another cohort study of the RBDEP found that prenatal rubella exposure was associated with risk for nonaffective psychosis in young adulthood, regardless of hearing loss (Brown et al., 2000a). This association held true during a follow-up study with an updated assessment that allowed for a diagnosis of schizophrenia (Brown et al., 2001), which provided evidence that prenatal rubella is linked with an increased risk for schizophrenia in young adulthood. CRS and schizophrenia also overlap in brain dysmorphology, with both groups having reduced cortical gray matter volume and enlarged lateral ventricle volume, when adjusted for age and head size (Lim et al., 1995). In all, additional research still needs to be conducted to determine the underlying characteristics of prenatal rubella infection that may contribute to symptoms of ASD and schizophrenia, teased apart from other symptoms more specific to CRS.

In a Swedish cohort study, bacterial infection, not associated with a particular trimester of pregnancy, was linked with ASD without comorbid intellectual disability, ID (Lee et al., 2015). A significant association between ASD diagnosis and general bacterial infection during the third trimester was also reported in a Taiwanese case–control study (Fang et al., 2015). A meta-analysis similarly found that bacterial infection, particularly during the second or third trimester, was associated with ASD in offspring (Jiang et al., 2016). Moreover, bacterial infections requiring hospitalization during the second trimester (most commonly including urinary tract infection and genital infection) were linked to ASD diagnosis in a Danish cohort study (Atladóttir et al., 2010). Similarly, in a California case–control study in the United States, bacterial infections (such as urinary tract infection, amniotic infection at delivery, and major puerperal infection) diagnosed during a hospital stay, particularly during the third trimester of pregnancy, were significantly associated with risk for ASD in offspring (Zerbo et al., 2015). Additionally, in a Danish cohort study, genitourinary infections during weeks 33–36 of the third trimester were significantly linked with increased risk for ADHD in offspring (Werenberg Dreier et al., 2016).

Furthermore, a meta-analysis found increased risk of psychosis in offspring linked to general bacterial infections during pregnancy (Zhou et al., 2021). In a Danish cohort study, exposure to bacterial infection (including sinusitis, tonsillitis, pneumonia, cystitis, pyelonephritis, and bacterial venereal infection) during the first trimester of pregnancy was associated with an elevated risk for schizophrenia in offspring (Sørensen et al., 2009). Exposure to maternal genital/reproductive (G/R) infections during the periconceptual period (such as endometritis, cervicitis, pelvic inflammatory disease, vaginitis, syphilis, condylomata, “venereal disease,” and gonorrhea) was also linked with increased risk for schizophrenia in offspring (Babulas et al., 2006). Further, pyelonephritis infection (kidney infection) that required hospitalization was associated with schizophrenia in offspring, but notably, only when there was a family history of psychosis (Clarke et al., 2009). Similarly, in a Swedish population-based cohort study, maternal infection during pregnancy was associated with later psychosis in offspring, only when there was also parental history of a psychiatric disorder (Blomström et al., 2016). While much of the above evidence supports that maternal bacterial infection increases the risk of NDDs, these last two studies, in particular, suggest that there may alternatively be an underlying susceptibility to perinatal infection in families with a history of NDDs, a concept that we will discuss in greater detail below.

Influenza during pregnancy has been linked to an increased risk of schizophrenia in affected offspring (Khandaker et al., 2013). Two cohort studies reported an increased risk of schizophrenia associated with serologically confirmed maternal influenza infection, although this increased risk was ultimately not statistically significant (Brown et al., 2004; Ellman et al., 2009). In a prediction model, the number of influenza deaths in the general population of England was significantly associated with risk of schizophrenia in offspring that were in their 6th or 7th month of gestation at the time (Sham et al., 1992). Similarly, the number of influenza infections in the general population of Denmark were linked with schizophrenia risk in offspring that were in their 6th month of gestation at that time (Barr et al., 1990; Takei et al., 1996). Moreover, using data from influenza epidemics in France between 1949 and 1981, Limosin et al. (2003) also reported that adults with schizophrenia were more likely to have been exposed to influenza during the 5th month of gestation as compared to controls.

Often, a major limitation of these studies is the lack of direct link between prenatal influenza exposure and schizophrenia outcomes within the same subjects, although a few studies have established this link. In a study examining outcomes of the 1957 influenza epidemic in Finland, admissions into psychiatric hospitals for schizophrenia in offspring was associated with a second-trimester gestational age at the time of the epidemic (Mednick et al., 1988). Prenatal infections were later confirmed via medical records, supporting that influenza exposure during the second trimester was associated with an increased risk for schizophrenia as compared to infection during the first or third trimesters (Mednick et al., 1994). Further, a Californian cohort study in the United States found that respiratory infections during the second trimester of pregnancy were significantly associated with schizophrenia spectrum disorder diagnosis in offspring (Brown et al., 2000b).

There is perhaps more limited evidence supporting an association between influenza infection during gestation, not specifically linked to a specific trimester, and increased risk for ASD (Atladóttir et al., 2012). In a United States study using a large patient dataset within Kaiser Permanente healthcare network in Northern California (Zerbo et al., 2017), and in a Norwegian cohort study (Mahic et al., 2017), there were no significant associations found between influenza infection during pregnancy and ASD diagnosis. On the other hand, influenza infection during the second trimester was significantly associated with risk for ASD in a Boston cohort study in the United States (Holingue et al., 2020). Interestingly, this association was only true when antibiotics were not taken at any point during pregnancy, and not necessarily that they were only avoided at the specific time of infection (Holingue et al., 2020). In all, there is limited and conflicting evidence for the link between prenatal influenza and NDDs, particularly schizophrenia and ASD, which suggests that the association may be more complex than initially reported.

Even though ASD and developmental delays were not associated with general influenza exposure in the Northern California cohort, the incidence of ASD and NDDs in offspring were significantly associated with fever during pregnancy in an earlier study (Zerbo et al., 2013). As expected, this fever-associated risk for ASD was attenuated in people that reported taking antipyretics to reduce their fever during pregnancy (Zerbo et al., 2013). Supporting these data, a meta-analysis examining the relationship between prenatal immune activation and ASD diagnosis found a significant association between maternal fever and ASD diagnosis in offspring; this association was not significant for prenatal infections without fever (Tioleco et al., 2021). A United States case–control study also found that there was a significant association between ASD risk and having a fever during the second trimester, even though there was no association between general prenatal infection and ASD risk (Croen et al., 2019). Thus, it is possible that links between maternal influenza during pregnancy and ASD risk may be obscured by unmeasured febrile response and unreported medication use (either antibiotics or antipyretics) in retrospective epidemiological studies, or through the lack of inclusion of such criteria in the original study design or analysis.

Furthermore, there is some evidence that maternal febrile response to influenza infection during pregnancy may be an important factor associated with increased risk for schizophrenia in offspring (Edwards, 2007). A Finnish cohort study found an increased odds ratio for schizophrenia in offspring that was associated with maternal fever during pregnancy, however the data were not statistically significant (Jones et al., 1998). Moreover, a Danish cohort study found that exposure to infections or maternal fever during gestation was associated with offspring having one psychosis-like experience by 11-years of age (Dreier et al., 2018). On another note, a Danish cohort study identified an association between maternal fever during weeks 9–12 of the first trimester and ADHD risk in offspring (Werenberg Dreier et al., 2016). Overall, it seems that the febrile response to infection during pregnancy or during parturition is linked with risk for NDDs in offspring and may serve as a potential mechanism to disrupt neurobiological development in the fetus and/or child.

An additional nuance to the data presented thus far is the severity and duration of an infection or febrile response during pregnancy. A Danish cohort study reported that febrile episodes lasting more than 1 week, and that occurred prior to 32 weeks of gestation (roughly mid-third trimester), were associated with a threefold increase in risk for ASD in offspring (Atladóttir et al., 2012). Supporting this evidence, a study using data from the Norwegian Mother and Child Cohort Study (Magnus, et al., 2006) and Autism Birth Cohort Study (Stoltenberg, et al., 2010) found that maternal fever during the second trimester was associated with increased risk for ASD, and this risk was augmented with increased number of febrile episodes (Hornig et al., 2018).

Furthermore, a Swedish cohort study found an increased risk of ASD diagnosis associated with exposure to severe types of perinatal infections (such as sepsis, pneumonia, pyelonephritis, meningitis or encephalitis, influenza, and chorioamnionitis) versus a non-severe urinary tract infection (Al-Haddad et al., 2019). Having two or more infections during pregnancy, particularly during the third trimester, was also associated with increased risk for ASD (Zerbo et al., 2015). Along these lines, a Danish cohort study found that hospital admissions for viral infection during the first trimester (including influenza, gastroenteritis, rubella, etc.) and for bacterial infection (including urinary tract and genital infections) during the second trimester were significantly associated with an increased risk for ASD diagnosis (Atladóttir et al., 2010). Similarly, a meta-analysis showed that infection during pregnancy that required hospitalization was associated with increased risk for ASD, particularly during the first or second trimester of pregnancy (Jiang et al., 2016).

Psychosis risk in offspring has also been linked to hospital treatment for maternal infection during pregnancy (Zhou et al., 2021). Interestingly, schizophrenia in offspring has also been associated with both maternal and paternal infections that specifically required a visit to the hospital, regardless of whether the visit occurred before, during, or after pregnancy (Nielsen et al., 2013). This suggests that familial history of prior illness or general infection may also be an important factor to consider in the risk for NDDs, which we will discuss more later in this review. Overall, these data indicate that, during gestation, the magnitude of the febrile response and the severity of infection may be specific factors important for consideration in the risk of ASD and schizophrenia diagnoses.

As originally proposed by Dr. Paul Patterson and confirmed by many colleagues since, one mechanism by which influenza (or other infections) increases the risk of NDDs in offspring may be via the maternal immune response and its associated circulating cytokines, rather than via a direct infection of the fetus itself (Shi et al., 2005; Mahic et al., 2017). In the past few decades, human epidemiological studies have provided further evidence of this idea. A Californian case–control study in the United States found that elevated levels of interferon (IFN)-γ, interleukin (IL)-4, and IL-5 in maternal serum mid-pregnancy were associated with increased ASD risk in offspring relative to the general population (Goines et al., 2011). Importantly, cytokine levels were adjusted for covariates during analysis, including gestational age at the time of specimen collection and maternal weight, age, ethnicity and country of birth. Similarly, a Danish case–control study found increased levels of tumor necrosis factor (TNF)-α and TNF-β in amniotic fluid (collected during screening or diagnostic amniocentesis procedures) of individuals later diagnosed with ASD (Abdallah et al., 2013). The researchers also found an association between ASD and elevated levels of IL-4, IL-10, and monocyte chemoattractant protein (MCP)-1 in the amniotic fluid of individuals born after 1993, which is when an updated diagnostic code for autism was introduced (Abdallah et al., 2012, 2013).

Moreover, a Philadelphia case–control study in the United States analyzed maternal serum samples that were collected during various prenatal visits or at birth and found that elevated levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were associated with the diagnosis of a psychiatric disorder (i.e., schizophrenia, schizoaffective disorder, and major depression or bipolar disorder with psychosis) decades later in adult offspring (Allswede et al., 2020). This association was particularly true for samples collected during the first half of pregnancy but was not significant for those collected during the second half of pregnancy. Similarly, a Rhode Island study in the United States found increased levels of TNF-α and IL-8 in serum collected at parturition that were linked with reports of maternal infection during the third trimester (Buka et al., 2001). The elevation in TNF-α was specifically associated with increased risk for psychosis in offspring.

Overall, these studies support that activation of the immune system (via infection) induces a concert of cytokines and chemokines that circulate throughout the body to fight the invading pathogen. In doing so, these pleiotropic immune molecules have powerful effects on the body; they can pass through the placental barrier and enter the fetal compartment, or they can trigger cytokine production in the placenta itself, which can produce similarly powerful effects on the developing fetus (Hsiao and Patterson, 2012). This inflammatory response can thus drive alterations in the fetal brain by disrupting processes important for typical neural development, which may underlie later vulnerability to NDDs and their symptoms.

As mentioned previously, the immune system is highly involved in regulating numerous processes important for neural development. Microglia, the innate immune cells of the brain, are involved in the pruning and maturation of appropriate synapses during development via phagocytosis of axonal terminals and dendritic spines (Schafer et al., 2012). Exposure to early-life immune activation (e.g., MIA) compromises these functions, and can subsequently affect the number and function of microglia, disrupt synaptic maturation and pruning, and result in neural circuit remodeling and deficits in neural function and behavior (Paolicelli et al., 2011; Zhan et al., 2014; Tay et al., 2017). The consequences of this dysregulation may also extend beyond fetal development and affect neurodevelopmental processes during the postnatal period (Matcovitch-Natan et al., 2016; de Cossío et al., 2017), such as disrupting synapse/neural circuit formation important for the ontogeny of specific behavioral phenotypes (e.g., language acquisition, social behaviors, learning, etc.). Thus, rodent models of MIA and NDDs are especially important to help elucidate the neural and molecular processes that can become disrupted by immune activation during sensitive periods of development. In particular, many rodent models have focused on measuring or manipulating the specific cytokines produced during a maternal infection or immune challenge, in order to examine their effects on offspring brain and behavior throughout the lifespan. (For a more detailed review of neurobiological processes associated with NDDs that can become disrupted by MIA, see: Knuesel et al., 2014; Estes and McAllister, 2016; Bergdolt and Dunaevsky, 2019).

Poly I:C is a synthetic double-stranded RNA that is used to stimulate an innate immune response through the Toll-like receptor 3 (TLR3) pathway (Reisinger et al., 2015). As described by Bao et al. (2022), Poly I:C is recognized and internalized by TLR3, which causes downstream activation of nuclear factor-κB (NF-κB), activator protein 1 (AP-1), and interferon regulatory factor 3 (IRF3). This, in turn, induces the expression of inflammatory cytokines and type 1 interferon (IFN) genes which are produced and released by the immune system in order to, respectively, stimulate an innate immune response and inhibit replication of the viral Poly I:C RNA.

Mouse and rat models of MIA report using a range of doses of Poly I:C, from 250 μg/kg to over 20 mg/kg, with doses of 4 mg/kg and 20 mg/kg being the most commonly used (Boksa, 2010; Solek et al., 2018). The gestational timing of Poly I:C administration also varies widely from embryonic days (E)9 to E19, with administration most commonly reported on E12.5 and E15. Some labs also report using multiple exposures Poly I:C, such as across two consecutive days of gestation or on two different days such as E9 and E17 (Boksa, 2010; Solek et al., 2018).

LPS is a cell-wall component of gram-negative bacteria (like Escherichia coli) that can also be used to stimulate an innate immune response via the TLR4 pathway (Reisinger et al., 2015). With the help of cluster of differentiation 14 (CD14), LPS binds to proteins on TLR4 which, similarly to Poly I:C, causes downstream gene expression and production of inflammatory cytokines and type 1 interferons (IFN) (Bae et al., 2010; Bao et al., 2022). Although interferons (particularly IFN-ɣ) are vital for the immune response to viruses, the activity of IFN-α and INF-β can also help the immune system fight off other pathogens such as bacteria (Bae et al., 2010).

Researchers report using dosages of LPS ranging from 25 μg/kg to over 1 mg/kg, with 100 μg/kg being more commonly used (Boksa, 2010; Solek et al., 2018). The timing of LPS administration varies across gestation, from E9 to E19, with administration most commonly reported around E15 and E18. Some models also employ multiple hits of LPS, such as subsequent injections on E15 and E16 or on E18 and E19. The utility of multiple hits of LPS is lessened by evidence that the peripheral cytokine response is attenuated after one previous LPS exposure (Wendeln et al., 2018). However, there is evidence to suggest the neural cytokine response is augmented to a second, but not a third or fourth, “hit” of LPS (Wendeln et al., 2018). Though, in the context of MIA, the maternal peripheral immune system is what influences the cytokine response in the fetal compartment; there is no evidence to support that the maternal neural immune response is implicated in the association between MIA and NDDs. Therefore, it is unclear how multiple hits of LPS during gestation would affect the developing fetal brain any differently than a single hit of LPS.

Several review articles have compiled data from rat and mouse MIA models that examine behavioral and neural outcomes following exposure to prenatal Poly I:C and LPS (see Meyer et al., 2009; Boksa, 2010; Solek et al., 2018; Kentner et al., 2019; Bauman and Van de Water, 2020). Some of the behavioral outcomes of rodent MIA studies that are summarized in these reviews include: deficits of latent inhibition and prepulse inhibition, reduced open-field exploration and novel object recognition, decreased social interactions, altered spatial memory in novel object location and Morris water maze tasks, and increased repetitive behaviors such as grooming or stereotyped behaviors in an open field. These rodent behavioral phenotypes are relevant to humans in that they align with the common overlapping symptoms of NDDs, such as deficits in sensorimotor gating, learning disabilities, and altered social behaviors, as we previously described. Such findings support the RDoC framework. Further, these data highlight how maternal inflammation during pregnancy, even in the absence of overt infection, can produce NDD-associated behavioral changes in offspring, likely through the disruption of neural processes necessary for brain development.

Similar to the human epidemiological data summarized above, the severity of the immune activation—related to immunogen dose in rodent models—may be implicated in neurodevelopmental outcomes related to NDDs. MIA with Poly I:C, and with lower doses of LPS (100–500 μg/kg), can cause subtle neural changes such as long-term alterations in cytokine expression, changes in neurotransmission, reduced proliferation of new neurons, and changes in microglia activation measured in adolescent and/or adult offspring (see Boksa, 2010; Solek et al., 2018; Kentner et al., 2019; Hameete et al., 2021 for more detailed information). On the other hand, larger doses of LPS (more than 1 mg/kg) administered during gestation can produce severe damage to white matter, axons, and dendrites (Fan et al., 2005). These MIA-driven neural changes may occur during fetal development but are often prolonged and measurable throughout offspring postnatal development and into adulthood. Again, many of these neural changes are thought to be driven by the maternal response to MIA, which can interact with the fetal compartment and disrupt immune molecules and immune cells like microglia from performing their essential functions during neural development (i.e., synaptic pruning, neural circuit formation, etc.). Moreover, sex differences have been explored in rodent models of MIA and are detailed in other review articles (Ardalan et al., 2019; Bauman and Van de Water, 2020; Breach and Lenz, 2022), with the takeaway being that MIA differently affects the behavioral and neural phenotypes of male and female offspring. Below, we will further discuss the importance of offspring sex when studying the association between MIA and NDDs.

Taken together, rodent models of Poly I:C and LPS, which mimic viral and bacterial infections, respectively, seem to be effective in modeling the behavioral phenotypes of NDDs, particularly ASD, schizophrenia, and generalized learning deficits. Rather than employing a direct infection, Poly I:C and LPS are typically administered either intraperitoneally or subcutaneously, thereby stimulating the innate immune system in a similar manner to a peripheral infection in humans, but without the full infection (Ashdown et al., 2006). This is particularly relevant given the epidemiological data summarized above, which suggest that the maternal immune response (including fever, cytokine production, altered fetal microglia function, etc.) may precipitate NDDs in offspring rather than the direct infection itself. Interestingly, neonatal administration of LPS actually produces a broader and more robust neuroimmune response in rat hippocampus than E. coli (Schwarz and Bilbo, 2011a). Similarly, Poly I:C is more likely to result in activation of the rodent immune system, given that many common human viruses are not pathogenic in rodents. Though, many human viruses can be adapted for use in a rodent model if part of the immune system (e.g., IFN-ɣ) is knocked out of the rodent genome (Brehm et al., 2013; Sarkar and Heise, 2019). Thus, LPS and Poly I:C in rodent models may allow us to characterize the effects of MIA on neurobiological processes underlying symptoms of NDDs, independent of the infection itself.

One major limitation in our ability to interpret various rodent models of MIA is the variability in experimental design across labs, including: gestational timing and frequency of the immune challenge, serotype/strain/dose of the immune challenge, age at which behavioral measures are tested in offspring, the types of behaviors and neural outcomes measured within a lab, and mouse versus rat models (whose immune systems are quite distinct). More specifically, researchers should determine whether their model of MIA may be better studied in mice versus rats, depending on the intended experimental manipulation and measured outcomes. For instance, mouse models are currently better suited than rat models for manipulations whereby researchers can examine the role of particular genes in NDD risk. On the other hand, rats are often more adept at performing complex learning and behavioral tasks, deficits in which may be associated with NDDs (Parker et al., 2014). Furthermore, researchers should consider that different strains of mice and rats often display distinct biological and behavioral profiles, and that this can even be influenced by the vendor from which the animals are sourced (Kentner et al., 2019). Nonetheless, both mouse and rat models of MIA seem to yield similar patterns of behavioral and neural findings related to NDD risk and symptomatology, even with varying rodent strains, offspring age at the time of measured outcomes, and dosages/gestational timing of the immune challenge. This provides further support that rodent models can provide insight into the neurobiological mechanisms underlying MIA-driven disruptions in offspring neural and behavioral development.

There are also inconsistencies in the reports of maternal death and fetal resorption or pup death that are often an inevitable consequence of many of these models. For example, even administration of an “ultra-low” dose of LPS (0.5 μg/kg) early in gestation (E5) results in significantly smaller litters and resorbed fetuses (Xue et al., 2015). Thus, it is likely that experiments using higher doses of LPS or PolyI:C produce a much more severe immune activation in the dam and fetuses. The question then remains whether this associated fetal mortality is actually modeling simple maternal infection (MIA related to NDDs), or rather modeling more severe infections like chorioamnionitis or endometritis that are associated with serious birth complications like premature birth or stalled labor. On the other hand, one potential benefit of these experimental inconsistencies is that they mirror the in outcomes variability in outcomes that can occur within the human population, even in the severity of the maternal immune response during pregnancy. In all, there are similar neurobiological and behavioral consequences reported across multiple different epidemiological and rodent studies, even with variation in the factors contributing to the models; this provides strong evidence for an association between MIA and outcomes associated with NDDs. Beyond this, researchers have also begun to utilize “two-hit” and “multi-hit” models that incorporate MIA as just one of the multiple “hits” to examine the ontogeny or symptoms of NDDs.

Twin studies have identified a high concordance among monozygotic (MZ) twins that is much lower in dizygotic (DZ) twins, demonstrating that many NDDs—namely ASD or schizophrenia—have a strong genetic link (Tick et al., 2016). That said, while thousands of genes, copy number variants, and de novo mutations have been associated with NDDs, to date there have been no risk loci identified that are common within each type of NDD or across all types of NDDs (Vorstman et al., 2017; López-Rivera et al., 2020). Rather, epidemiological data suggest that genetic risk provides a foundation upon which other factors may precipitate or enhance the risk for many NDDs (Zawadzka et al., 2021). It is possible that this genetic risk could be hereditary in nature, as both a familial history of psychiatric disorders and a parental history of severe infections seem to be involved in the association between MIA and offspring NDD risk, as discussed above.

Accordingly, genes that are implicated in schizophrenia may also impact how the body processes and fights off different pathogens, including influenza, rubella, and T. gondii (Carter, 2009), which suggests that individuals with these genes may be more prone to infections and, in turn, more at risk of NDDs as a consequence of the maternal infection. Similarly, people with ASD have an upregulation in genes that regulate neural cell development, but also in genes that regulate the immune response, the inflammatory response, antigen production and presentation, as well as immune cell signaling (Voineagu et al., 2011; Voineagu and Eapen, 2013). These genetic markers can also increase one’s susceptibility to other inflammation-inducing factors, such as diet, physical stress, psychological stress, etc. There is also evidence that MIA produces transcriptional changes in expression of inflammatory markers, GABAergic signaling proteins, and myelin, and may drive epigenetic changes in the transcription of genes associated with NDDs (Woods et al., 2021). For example, MIA with Poly I:C in mice produced an integrated stress response (ISR) in male offspring, associated with increased phosphorylation of eIF2α which is important for cellular translation (Kalish et al., 2021). Therefore, genetic influences may impact both the maternal immune response as well as the neurodevelopmental and behavioral processes in offspring that are ultimately affected by the immune response.

Maternal and paternal age have also been implicated in the risk for certain NDDs. For instance, older maternal and paternal ages have been linked with increased risk for ASD (Abdallah et al., 2012; Sandin et al., 2012; Carlsson et al., 2021). Advanced paternal and maternal ages have also been associated with increased risk for schizophrenia and psychosis (El-Saadi et al., 2004; Lopez-Castroman et al., 2010; Fountoulakis et al., 2018). On the other hand, younger maternal age has also been implicated in risk for psychosis, when controlling for paternal age (El-Saadi et al., 2004). Younger maternal and paternal ages have been associated with increased risk for ADHD diagnosis overall (Chang et al., 2014; Hvolgaard Mikkelsen et al., 2017). Interestingly, older maternal age and younger paternal age have been associated with hyperactivity/impulsivity symptoms of ADHD, whereas younger maternal and paternal ages have been linked to inattentive symptoms of ADHD (Ghanizadeh, 2014; Sciberras et al., 2017).

Not much is known about how parental age influences the risk of NDD in children. Maternal age may influence the immune response to MIA, given that immune function changes with age (though usually much older ages; see Haynes, 2020). Increased maternal age is also associated with an increased risk of pregnancy and obstetric or birth complications that are often associated with inflammation, such as preeclampsia, gestational diabetes, or general hypertension (Londero et al., 2019). The role of paternal age as a risk for NDDs implies that genetics may also have a role in this relationship. As the body ages, there is a greater risk for genetic mutations in the eggs or the sperm that would contribute to an increased risk of NDDs. Moreover, environmental exposure to toxins or infections throughout the lifespan may also result in de novo genetic mutations that can be passed to offspring and increase their risk for NDDs, an effect that appears to happen more frequently in sperm than in eggs (Kong et al., 2012; Jónsson et al., 2017).

Notably, the age of the dam or sire are not consistently reported or controlled for in animal models, nor how many litters that any one dam has had previously. Furthermore, there are few to no animal studies that have examined maternal or paternal age as a risk factor that may interact with MIA or developmental outcomes in the offspring. As with all controlled rodent studies, reporting the age of the mating pair is important, whether or not it has an effect on MIA or on the behavioral outcomes in the phenotype being examined. Human epidemiological studies often control for age as a covariate when examining the link between MIA and NDDs, however few studies actively include it as a variable in their overall analysis. As future studies characterize the role of aging in the risk of NDDs, they should consider use of a multivariate model that considers parental age, along with infection during or before pregnancy in either the mothers or the fathers, to get a better understanding of how these risk factors interact.

The gut microbiota can be impacted by diet and by metabolic conditions. Maternal metabolic conditions such as obesity, diabetes, and hypertension have been associated with increased risk for ASD (Van Lieshout and Voruganti, 2008; Krakowiak et al., 2012). In mice, maternal high-fat diet has been shown to produce microglia-associated changes in myelination and increase the number of perivascular microglia in the offspring brain (Bordeleau et al., 2021, 2022), as well as cause offspring to have less diverse gut communities, decreased oxytocin production in the paraventricular nucleus of the hypothalamus, and diminished synaptic plasticity in the ventral tegmental area (Buffington et al., 2016). Maternal high-fat diet itself has been used to model MIA in rodents, as it can trigger a chronic inflammatory profile in the dam and can produce behavioral phenotypes in offspring that are related to NDDs, including increased repetitive behaviors and disruptions in social and cognitive behaviors (Sullivan et al., 2015; Buffington et al., 2016; Penna et al., 2020; Bordeleau et al., 2021, 2022). Gestational diabetes may also interact with MIA to impact neurodevelopmental processes in offspring (Van Lieshout and Voruganti, 2008). Prenatal exposure to both gestational diabetes mellitus and Poly I:C in mice resulted in offspring with an altered transcriptional profile of genes that are associated with differentiation of dopamine neurons and the innate immune response (Money et al., 2018). Finally, antibiotic use can also alter the composition of the gut microbiota (Patrono et al., 2021). In humans, second trimester influenza infection associated with ASD risk was only apparent when antibiotics were not taken at any point during the pregnancy (Holingue et al., 2020), which suggests that the antibiotics may have altered the microbiome in a way that prevented the negative consequences of influenza from affecting the developing fetal brain.

The gut microbiota are essential in regulating the immune system, including the proliferation and differentiation of T- and B-cells that drive the maternal cytokine production implicated in MIA (Minakova and Warner, 2018). Certain forms of commensal gut bacteria, like segmented filamentous bacteria, are more likely to induce differentiation of T-cells that produce IL-17a, which is a cytokine that has consistently been associated with behavioral changes (particularly decreased social behaviors) and cortical abnormalities in various models of MIA (Kim et al., 2017). Colonization of Pregnant female mice that were colonized with segmented filamentous bacteria, then challenged with Poly I:C on E12.5, were more likely to produce TH-17 cells and have offspring with distinct behavioral phenotypes characteristics of NDDs (Kim et al., 2017), likely triggered by exposure to the enhanced IL-7 production from the maternal gut’s adaptive immune cells (Kim et al., 2017). Similarly, increased levels of pro-inflammatory cytokines in the gut have also been associated with the positive symptoms of schizophrenia in humans (Patrono et al., 2021). Taken together, the gut microbiome may prove useful in providing additional biomarkers for immune dysregulation associated with NDDs or as targets for therapies against NDDs, particularly if the microbiome changes in concert with, or before the onset of, symptoms for many NDDs.

Maternal diet deficiencies of iron, omega-3 fatty acids, and folic acid may also impact neurodevelopmental outcomes in rodents and humans in the context of MIA. Long ago, researchers determined that folic acid was necessary as part of the maternal diet to ensure proper development of the fetal neural tube (Greenberg et al., 2011). In mice exposed to LPS on E17, omega-3 deficiency in the maternal diet caused increased IL-6 expression in maternal plasma, placenta, and fetal brain (Richardson and Ross, 2000; Labrousse et al., 2018). Adult offspring exposed to both the MIA and omega-3 deficiency during development had spatial memory deficits in a Y-maze task. Furthermore, in humans, anemia, with or without exposure to prenatal infection, is associated with an increased risk for schizophrenia (Nielsen et al., 2016). In rats, dams fed an iron-deficient diet had increased serum levels of IL-6 and TNF-α following prenatal LPS on E15, as compared to typical chow-fed dams (Harvey and Boksa, 2014). Moreover, exposure to iron-deficiency and to MIA independently caused deficits in the offspring’s development of various sensorimotor behaviors. In all, there is limited evidence of multiplicative effects between dietary iron deficiencies and MIA exposure, however both seem to be independently implicated in the risk for NDDs.

Perhaps with growing evidence such as that described here, future research should examine whether the gestational/developmental timing of dietary deficiencies or alterations in the gut microbiota may interact with MIA to increase the risk of NDDs. In turn, studies should examine whether diet-derived supplementations might mitigate the effects of MIA. For example, maternal dietary supplementation with choline, around the time of birth in rats, attenuated the splenic cytokine immune response of 3-week-old offspring to an ex vivo immune challenge (Richard et al., 2017). In addition, several dietary factors—including high maternal iron, zinc, and vitamin D—have been associated with resilience to effects of MIA via anti-inflammatory cytokine production and enhancement of antioxidant systems (Vuillermot et al., 2017; Meyer, 2019).

Prenatal stress has long been associated with an increased risk of various NDDs, most notably schizophrenia, ADHD and autism (Ronald et al., 2011; Diz-Chaves et al., 2012, 2013; Chan et al., 2018; Minakova and Warner, 2018; Makris et al., 2022). More recently, this association has been further characterized by changes in inflammatory biomarkers in the maternal circulation that may increase the risk of various NDDs. For example, even socioeconomic disadvantage is a stressor that is associated with transcriptional indications of greater immune activation and slower tissue maturation in the placenta (Miller et al., 2017). This stress can lead to overproduction of pro-inflammatory cytokines by immune cells in response to additional immunostimulation (Miller et al., 2017). Stress-induced susceptibility to MIA may be linked to changes in baseline maternal cortisol levels (Van den Bergh et al., 2005), resulting in continuously elevated or stimulated pro-inflammatory cytokine levels that may impact fetal neurodevelopment associated with NDD risk.

Animal models of prenatal stress have demonstrated a pro-inflammatory cytokine response, particularly IL-6, with microglial activation similar to that elicited by MIA models. Specifically, prenatal stress in rodents enhanced cytokine levels in the hippocampus and increased the total number of immunoreactive microglial cells in the offspring compared to non-stressed animals, which exacerbated the inflammatory response to LPS (Diz-Chaves, et al., 2012). Behavioral phenotypes of anxiety, learning deficits, and depressive-like symptoms in prenatally stressed rat and non-human primate offspring are further associated with maternal and fetal HPA-axis alterations (Weinstock, 2005; Weinstock, 2008). Gestational stress and excess corticosterone in maternal and fetal plasma can impair feedback regulation of the HPA axis in both infancy and adulthood and can increase corticotropin-releasing hormone (CRH) activity in the amygdala (Van den Bergh et al., 2005; Weinstock, 2005; Weinstock, 2008). Excess amounts of CRH and cortisol that reach the fetal brain during periods of chronic maternal stress could thereby influence how the fetal brain responds in the presence of MIA, or how the brain is programmed to respond to subsequent stressors or immune challenges later in life.

Animal models often fail to report or account for unintended stressors in their models that may interact with MIA to exacerbate the neural and behavioral consequences in dams and offspring. For instance, stress associated with ambient noise levels, bedding levels, handling, injection procedures, behavioral tests, caging conditions, and nearby construction are all factors that may commonly occur throughout the course of an experiment. Researchers should take care to reduce exogenous stressors wherever possible, and when unable to control for such factors, should document and report them in the literature.

The placenta is an important organ that connects mother and fetus, providing oxygen and nutrition to the baby while protecting the delicate fetus from certain factors, most notably infections, that could harm it. That said, while it is well-known that many pathogens and larger immunogenic molecules do not cross the placental barrier, the placenta might also be implicated in the active transfer of immune molecules through the circulation to the fetus (Robbins and Bakardjiev, 2012). Unfortunately, research examining the site of the placental transfer of cytokines associated with MIA is sparse. Nevertheless, it is important to understand the role of the placenta as a site of cytokine transfer during MIA.

Decades ago, research indicated that monozygotic twins concordant for schizophrenia were more likely to have been monochorionic and to have shared a single placenta, whereas discordant monozygotic twins appear more likely to have been dichorionic with separate placentas (Davis et al., 1995). In human twin pregnancies with a conjoined placenta, the dividing membrane between the two placentae can be composed of four layers—the amnion and chorion of each twin—which allows some degree of shared circulation between the two fetuses (Benirschke, 1990). In this case, each twin may be exposed to similar circulating molecules, such as cytokines, from the mother. Maternal immune and endothelial cells come into contact with extravillous fetal cells at the uterine implantation site, allowing for maternal blood to surround the epithelial covering of placental cells, called syncytiotrophoblasts.

The syncytiotrophoblasts have been shown to be resistant to infections and thereby may contribute to the protective function of the placenta. At the same time, they are a type of immune cell that can initiate their own cytokine response in the presence of innate immune receptor activation. Due to their hemochorial nature, the placental buffer in rats and humans function in similar ways. Like in humans, the trophoblast epithelium of the rat placenta is directly bathed in maternal blood (Furukawa et al., 2019). In humans, this occurs at the decidua, the site of uterine implantation, which only has one dividing layer (the syncytiotrophoblasts). However, the rat has three layers at this site, which might imply differences in the fetal-maternal exchange processes between the two species. In both species, uterine natural killer (NK) cells are present in parts of the placenta, and help the uterus to adapt and accommodate for the fetus. In rats, MIA with Poly I:C can increase maternally-derived IL-6 protein directly in the placenta, which activates the JAK/STAT3 pathway and causes expression of acute phase immune genes in the placenta that can enter into the fetal circulation (Hsiao and Patterson, 2011). In humans, IL-6 is transferred bidirectionally between maternal and fetal circulation (Zaretsky et al., 2004). While studies suggest that many immunogenic molecules, like LPS, do not cross the placental barrier (Ashdown et al., 2006; Ning et al., 2008), there remains some debate of whether fetal immune activation by way of MIA occurs via the reception of cytokines from the maternal circulation or via an immune response precipitated in the placenta itself. Thus, additional research should be performed to examine the rat placental barrier and its potential ability to transfer immune molecules from maternal circulation.

Moreover, early work has identified dichorionic monozygotic twins as having a lower rate of concordance for various NDDs (Davis et al., 1995), which should also be considered within the context of rodent pregnancies where each pup has its own placenta and might respond differently to MIA. Hormones can travel through the multiple placentae among fetuses due to the blood flow of the mother. More specifically, in pregnant rats, blood flows from the caudal to distal direction, or from cervix to ovaries. Thus, a rat fetus located at the cervical end of the uterus will receive maternal blood flow prior to fetuses in other uterine positions. In litter-bearing mammals that have multiple pregnancies, effects of intrauterine position on fetal development have been observed (Ryan and Vandenbergh, 2002). For instance, female fetuses that develop downstream from male fetuses have been shown to exhibit slightly masculinized anatomical, physiological, and behavioral characteristics as adults, including altered hormone levels and disrupted endocrine systems (Ryan and Vandenbergh, 2002). This is due to diffusion of testosterone from male fetuses to their uterine neighbors via amniotic fluid and the maternal circulation. Given this mechanism of hormonal transfer, it may be possible for the same type of transfer to occur with immune factors such as cytokines; this may result in differential exposure to MIA-associated molecules between fetuses based on their uterine position. It is also possible that some fetuses may be more exposed to the circulating maternal immune molecules from the pregnant dam in MIA models, particularly those located more caudally as they are the first to receive maternal blood flow.

In all, more research is needed to consider the role of the placenta in the fetal response and susceptibility to the inflammatory effects of MIA. Researchers should consider how differences in rodent and human pregnancies—particularly the number of fetuses, characteristics of the placental barrier, and maternal transference or fetal production of cytokines and other immunogens—may impact the generalizability of their findings to human NDDs and the translatability of rodent models of MIA to humans.