Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder.

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4ß7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1ß and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4ß7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4ß7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4ß7 mAb in EIA male mice was associated with colonic tissue IL-1ß, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4ß7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1ß and decreased CD68 and TGF-ß mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4ß7 mAb treatment restored the expression of BDNF, TGF-ß and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4ß7+) lymphocytes expressing IL-17A.

Developmental exposure to polycyclic aromatic hydrocarbons (PAHs): Focus on benzo[a]pyrene neurotoxicity.

Polycyclic Aromatic Hydrocarbons (PAHs) are a class of ubiquitous organic compounds produced during the incomplete combustion or pyrolysis of organic material. Dietary source is the main route for PAH human exposure by environmental contamination, food industrial processing or domestic cooking methods. The most studied PAH is benzo[a]pyrene (B[a]P), due to its harmful and multiple effects on human health: in addition to its well-known carcinogenic effects, emerging evidence indicates that B[a]P also induces neurotoxicity earlier and at lower doses than B[a]P-induced carcinogenicity making B[a]P neurotoxicity relevant to human health risk assessment. Developmental neurotoxicity of B[a]P has indeed received increasing attention: both human and experimental studies provide evidence of detrimental effects of prenatal or early postnatal B[a]P exposure, even at low doses. Indeed, in some of the multi-dose animal studies, maximal adverse effects were observed at lower B[a]P doses, according to a non-monotonic dose-response curve typical of endocrine-disrupting compounds. In substantial agreement with epidemiological studies, both rodents and zebrafish developmentally exposed to B[a]P exhibit long-term changes in multiple behavioural domains, in the absence of overt toxicological effects at birth (e.g. body weight and morphologic abnormalities). Notably, most targeted behavioural responses converge on locomotor activity and emotional profile, often, but not always, leading to a disinhibitory/hyperactive profile.

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study.

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.

Differential Expression of Endogenous Retroviruses and Inflammatory Mediators in Female and Male Offspring in a Mouse Model of Maternal Immune Activation.

Maternal infections during pregnancy and the consequent maternal immune activation (MIA) are the major risk factors for autism spectrum disorder (ASD). Epidemiological evidence is corroborated by the preclinical models in which MIA leads to ASD-like behavioral abnormalities and altered neuroinflammatory profiles, with an increase in pro-inflammatory cytokines and microglial markers. In addition to neuroinflammatory response, an abnormal expression of endogenous retroviruses (ERVs) has been identified in neurodevelopmental disorders and have been found to correlate with disease severity. Our aim was to evaluate the transcriptional profile of several ERV families, ERV-related genes, and inflammatory mediators (by RT real-time PCR) in mouse offspring of both sexes, prenatally exposed to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3 that mimics viral maternal infection during pregnancy. We found that prenatal exposure to Poly I:C deregulated the expression of some ERVs and ERV-related genes both in the prefrontal cortex (PFC) and hippocampus, while no changes were detected in the blood. Interestingly, sex-related differences in the expression levels of some ERVs, ERV-related genes, and inflammatory mediators that were higher in females than in males emerged only in PFC. Our findings support the tissue specificity of ERV and ERV-related transcriptional profiles in MIA mice.

Maternal immune activation induces autism-like changes in behavior, neuroinflammatory profile and gut microbiota in mouse offspring of both sexes.

Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.

Short- and Long-Term Effects of Suboptimal Selenium Intake and Developmental Lead Exposure on Behavior and Hippocampal Glutamate Receptors in a Rat Model.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

The principle of the 3Rs between aspiration and reality.

The Principle of the 3Rs is widely recognised as the methodological and ethical backbone of contemporary animal research. Different authors also stress the reciprocal links among the 3Rs, and how these often complement and reinforce each other. We very much agree with this point, but in this contribution we would like to raise some problems related to the application of the "3Rs". There is an obvious link among "Replacement, "Reduction" and "Refinement", but it is worth to notice also that each "R" has its own conceptual characteristics, as well as its own level of applicability. For example, a realistic "methodological inertia" has to be expected more in the case of "Replacement" than in the case of "Refinement". This also leads to a second order of issues, and here we will offer our experience as projects evaluators. The "3Rs" differ also in the possibility to verify how are applied by the proponents of research protocols involving the use of animal models. Sometimes it appears that the application of the Principle still resolves itself in the use of formulaic sentences, from which it is difficult to really understand the reality of the laboratory decisional and procedural processes. However, the demanding characteristics of the "3Rs" can vary greatly, and this is something that has to be considered. We propose that a network, or a virtual platform, of evaluators could help both researchers and evaluators for a more satisfactory understanding and pragmatic application of the Principle of the 3Rs.

Ultrasonic vocalizations in laboratory mice: strain, age, and sex differences.

Mice produce ultrasonic vocalizations (USVs) in different social contexts across lifespan. There is ethological evidence that pup USVs elicit maternal retrieval and adult USVs facilitate social interaction with a conspecific. Analysis of mouse vocal and social repertoire across strains, sex and contexts remains not well explored. To address these issues, in inbred (C57BL/6, FVB) and outbred (CD-1) mouse strains, we recorded and evaluated USVs as neonates and during adult social encounters (male-female and female-female social interaction). We showed significant strain differences in the quantitative (call rate and duration of USVs) and qualitative vocal analysis (spectrographic characterization) from early stage to adulthood, in line with specific patterns of social behaviors. Inbred C57BL/6 mice produced a lower number of calls with less internal changes and shorter duration; inbred FVB mice displayed more social behaviors and produced more syllables with repeated internal changes; outbred CD-1 mice had an intermediate profile. Our results suggest specific vocal signatures in each mouse strain, thus helping to better define socio-communicative profiles of mouse strains and to guide the choice of an appropriate strain according to the experimental settings.

Critical Role of Maternal Selenium Nutrition in Neurodevelopment: Effects on Offspring Behavior and Neuroinflammatory Profile.

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content-i.e., optimal, sub-optimal, and deficient-and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.

Low-level lead exposure during development differentially affects neurobehavioral responses in male and female mouse offspring: A longitudinal study.

Early life low-level lead (Pb) exposure is still an alarming child health issue. To date, animal studies investigating the effects of low doses of Pb since early stages of life to adulthood are scarce. We investigated in a mouse model the behavioral effects of developmental exposure to low-level Pb yielding blood levels similar to those observed in child clinical literature. CD1 outbred mouse dams received Pb (25- or 100-ppm) via drinking water from two weeks pre-mating until the end of lactation. Offspring of both sexes underwent a longitudinal assessment of motor, socio-emotional, and cognitive endpoints from neonatal to adult stage. Pb levels were determined in several matrices (blood, brain and bone) up to six months after the end of exposure. We found that new born pups exposed to Pb have slightly altered motor patterns and reduced preference for the nest odor. Offspring of both sexes exposed to the lowest Pb dose showed diminished interest for social novelty stimuli as adults. Moreover, sex-dependent effects of Pb exposure were observed in the spatial learning and memory task, where males were selectively impaired. Finally, blood, brain and bone Pb levels were elevated in a dose dependent fashion up to six months after termination of exposure. We observed marked accumulation of Pb in bones, with higher Pb levels in 100-ppm exposed females than in males at 7 months of age. In conclusion, developmental Pb exposure caused mild alterations in early- and late-life behavioral domains, particularly involving olfactory and cognitive responses. These findings confirm the importance of animal models to understand how early chronic low-level lead exposure impacts on health in a life-course perspective.

Postweaning social isolation and autism-like phenotype: A biochemical and behavioral comparative analysis.

Adolescence is a critical period for brain development. In most mammalian species, disturbances experienced during adolescence constitute a risk factor for several neuropsychiatric disorders. In this study, we compared the biochemical and behavioral profile induced by postweaning social isolation (PWSI) in inbred C57BL/6 N mice with that of BTBR mice, a rodent model of autism spectrum disorders. Male C57BL/6 N mice were either housed in groups of four or isolated from weaning (postnatal day 21) for four weeks before experimental analyses. After weaning, male BTBR mice were housed four per cage and analyzed at 48 days of age. PWSI reduced hippocampal levels of type 2 metabotropic glutamate (mGlu2) receptors, and glucocorticoid and mineralocorticoid receptors. A similar reduction was seen in group-housed BTBR mice. Plasma corticosterone levels in basal conditions were not influenced by PWSI, but were increased in BTBR mice. Social investigation (total and head sniffing) and the number of ultrasonic vocalizations were reduced in both PWSI mice and age-matched group-housed BTBR mice, indicating a lower social responsiveness in both groups of mice. These results suggest that absence of social stimuli during adolescence induces an endophenotype with social deficit features, which mimics the phenotype of a mouse model of autism spectrum disorders.

Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

Ultrasonic vocalizations as a fundamental tool for early and adult behavioral phenotyping of Autism Spectrum Disorder rodent models.

In rodent models of Autism Spectrum Disorders (ASD), the study of ultrasonic vocalizations has provided the unique opportunity to evaluate social communication and interaction in ethologically-appropriate contexts, behavioral domains relevant to the first core symptom of ASD. In the present review, we selected and evaluated ultrasonic vocalizations' data collected in rodent models of ASD in different experimental settings, either in the neonatal phase or in adulthood. Both quantitative (calling rates) and qualitative (range and shape of the vocal repertoire) abnormalities have been evidenced. The aim of our work was to highlight several promises and a few caveats in the use of ultrasonic vocalizations for behavioral phenotyping of ASD models and give some suggestions to maximize the translational value of these studies.

Cnf1 Variants Endowed with the Ability to Cross the Blood-Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma.

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood-brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

Maternal Immune Activation in Mice Only Partially Recapitulates the Autism Spectrum Disorders Symptomatology.

Prenatal viral/bacterial infections are considered risk factors for autism spectrum disorders (ASD) and rodent models of maternal immune activation (MIA) have been developed and extensively used in preclinical studies. Poly inosinic-cytidylic acid (Poly I:C) was injected in C57BL6/J dams to mimic a viral infection on gestational day 12.5; the experimental design includes 10/12 litters in each treatment group and data were analysed always considering the litter-effect; neonatal (spontaneous motor behaviour and ultrasonic vocalizations) and adult [open field, marble burying, social approach, fear conditioning, prepulse inhibition (PPI)] offspring of both sexes were tested. In vivo magnetic resonance imaging/spectroscopy (MRI-MRS) and high-performance liquid chromatography (HPLC) to quantify both aminoacid and/or neurotransmitter concentration in cortical and striatal regions were also carried out. In both sexes high levels of repetitive motor responses and sensory gating deficits in PPI were the more striking effects of Poly I:C, whereas no alteration of social responses were evidenced. Poly I:C treatment did not affect mean values, but, intriguingly, increased variability in the levels of four aminoacids (aspartate glycine and GABA) selectively in males. As a whole prenatal Poly I:C induced relevant long-term alterations in explorative-stereotyped motor responses and in sensory gating, sparing cognitive and social competences. When systematically assessing differences between male and female siblings within each litter, no significant sex differences were evident except for increased variability of four aminoacid levels in male brains. As a whole, prenatal Poly I:C paradigms appear to be a useful tool to investigate the profound and translationally-relevant effects of developmental immune activation on brain and behavioural development, not necessarily recapitulating the full ASD symptomatology.

Sex-Dependent Effects of Developmental Lead Exposure in Wistar Rats: Evidence from Behavioral and Molecular Correlates.

Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains.

Beneficial Effects of Fingolimod on Social Interaction, CNS and Peripheral Immune Response in the BTBR Mouse Model of Autism.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by social communication deficits and repetitive/stereotyped behaviours. We evaluated the effects of a chronic treatment with the immunomodulator drug Fingolimod (FTY720 - a non-selective Sphingosine 1-Phosphate Receptor ligand) in an ASD model, the BTBR T+tf/J (BTBR) mouse strain. In adult BTBR males, chronic FTY720 treatment (4 weeks) increased social and vocal response during a male-female interaction and hippocampal expression of BDNF and Neuregulin 1, two trophic factors reduced in BTBR when compared to control C57 mice. FTY720 also re-established the expression of IL-1ß and MnSOD in the hippocampus, whereas it did not modify IL-6 mRNA content. In addition to its central effect, FTY720 modulated the activation state of peripheral macrophages in the BTBR model, both in basal conditions and after stimulation with an immune challenge. Furthermore, IL-6 mRNA colonic content of BTBR mice, reduced when compared with C57 mice, was normalized by chronic treatment with FTY720. Our study, while indicating FTY720 as a tool to attenuate relevant alterations of the BTBR neurobehavioural phenotype, emphasizes the importance of gut mucosal immune evaluation as an additional target that deserve to be investigated in preclinical studies of anti-inflammatory therapeutic approaches in ASD.

Differential Expression of Hippocampal Circular RNAs in the BTBR Mouse Model for Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with unknown etiology. Recent experimental evidences suggest the contribution of non-coding RNAs (ncRNAs) in the pathophysiology of ASD. In this work, we aimed to investigate the expression profile of the ncRNA class of circular RNAs (circRNAs) in the hippocampus of the BTBR T + tf/J (BTBR) mouse model and age-matched C57BL/6J (B6) mice. Alongside, we analyzed BTBR hippocampal gene expression profile to evaluate possible correlations between the differential abundance of circular and linear gene products. From RNA sequencing data, we identified circRNAs highly modulated in BTBR mice. Thirteen circRNAs and their corresponding linear isoforms were validated by RT-qPCR analysis. The BTBR-regulated circCdh9 was better characterized in terms of molecular structure and expression, highlighting altered levels not only in the hippocampus, but also in the cerebellum, prefrontal cortex, and amygdala. Finally, gene expression analysis of the BTBR hippocampus pinpointed altered biological and molecular pathways relevant for the ASD phenotype. By comparison of circRNA and gene expression profiles, we identified 6 genes significantly regulated at either circRNA or mRNA gene products, suggesting low overall correlation between circRNA and host gene expression. In conclusion, our results indicate a consistent deregulation of circRNA expression in the hippocampus of BTBR mice. ASD-related circRNAs should be considered in functional studies to identify their contribution to the etiology of the disorder. In addition, as abundant and highly stable molecules, circRNAs represent interesting potential biomarkers for autism.

Pregnancy exposome and child psychomotor development in three European birth cohorts.

Characterization of the exposome, the totality of all environmental factors that one is exposed to from conception onwards, has been recommended to better evaluate the role of environmental influences on developmental programming and life-course vulnerability to major chronic diseases. In the framework of the Health and Environment-wide Associations based on Large population Surveys (HEALS) project we considered the pregnancy exposome exploiting two databases (PHIME and REPRO_PL) that include birth cohorts from three EU countries (Croatia, Slovenia and Poland). The databases contained information on several chemical exposures, socio-demographic, lifestyle and health related factors from conception to child birth, and neuropsychological scores assessed by the Bayley Scales of Infant and Toddler Development in the first two years of life. Our main goal was to assess consistency of environmental influences on neurodevelopment, if any, across European countries differing for geographical, socio-demographic characteristics and levels of chemical exposures to metals such as lead (Pb), mercury (Hg), cadmium (Cd) and trace elements, including micronutrients such as zinc (Zn) and selenium (Se). To this aim, we first selected variables common to the different databases, then applied univariate and multivariate regression analyses to identify factors linked to neurodevelopment, and finally performed meta-analysis to detect potential heterogeneity among cohorts and pooled estimates. Significant differences in exposure levels among the three sub-cohorts were observed as for Hg and Se; exposure levels under study were relatively low and within the range described in existing EU biomonitoring studies. The univariate analyses did not show any common pattern of association as only in the Polish cohort chemical exposure had an impact on neuropsychological outcome. In the meta-analysis, some consistent trends were evident, relative to the adverse influence of Pb on children's language and cognition and the positive influence of Se on language abilities. The effects of the neurotoxic metal Hg positively influenced the motor scores in the Polish cohorts, while it decreased the motor scores in the Slovenia and Croatian sub-cohorts. The only socio-demographic factor consistently associated to the outcome among cohorts was child's sex, with females performing better than males on cognitive and language scores. These findings point to the need of harmonizing existing cohorts or creating prospective study designs that facilitate comparisons in the exposome over time, places and kind of environmental exposures.