Table 5.

Animal clinical studies on the role of folic acid in ASDrisk.

No	Study	Type of Animal	Treatment Used	Study Outcome Parameters	Sample Size	Outcomes
1	Degroote S et al., (2018) [64]	Female Wistar rat	0.2 mg/kg FA	Blood homocysteine levels. Behavioral alterations relevant to autism-like in offspring	n = 12 female, n = 8 male	Maternal periconceptional deficit in folate provokes alterations in the behavior of offspring relevant to the autistic-like phenotype [64].
2	Barua S et al., (2016) [65]	Female C57BL/6J Mice	2 mg/kg control diet FA (CD) 20 mg/kg high maternal FA (HMFA)	Expression of genes in the cerebral hemispheres (CHs) of 1-day-old pups, FA concentration in CH Tissue	n = 12 CD female, n = 12 HMFA female	The results showed that HMFA supplementation alters offsprings’ CH gene expression in a sex-specific manner. These changes may influence infants’ brain development. In addition, it was also found that HMFA had no impact on global DNA methylation levels of the offspring epigenome [65].
3	Barua S et al., (2015) [67]	C57BL/6 J mice	2 mg/kg FA,20 mg/kg FA	Expression of genes in the cerebellum of offspring from day old pups	n = 6,n = 6	Results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups [67].
4	Kezurer N et al., (2013) [66]	Mice on a Balb/cAnNCrlBR backgroundMTHFR heterozygote	Mild neonatal stress (25 μL PBS at postnatal days 4–10)	Offspring genotyped of DNA isolated from toe clips. Reflex analysis and newborn development. Reflex development in newborn mice. Anxiety-related behavior.Adult behavior. Corticosterone levels	n = 97 mice (7–14 in each group) were tested	Overall, the results support an interaction between mild neonatal stress, the MTHFR genotype, and sex [66].
5	Orenbuch A et al., (2019) [27]	Mice with a Balb/cAnNCrlBR backgroundheterozygous for the Mthfr-knockout	9 mg/mL FA in the enriched food group	Tissue concentrations of C1 metabolites in the liver, cerebral cortex, and basal forebrainMthfr/ Genotypingbehaviors	5 groups of mice from each sex	The study suggests that MTHFR deficiency can increase the risk of ASD-like behavior in mice and that prenatal dietary intervention focused on MTHFR genotypes can reduce the risk of ASDs-like behavior. Findings emphasize the critical role of in utero C1 metabolism in developmental trajectories that lead to the presentation of autistic behavior. Aberrations in both the GABAergic and glutamatergic pathways suggest that Mthfr deficiency is linked to deleterious alterations in the basal cortical circuit activities in the affected mice [27].
6	Sadigurschi N et al., (2019) [68]	Mice on a Balb/cAnNCrlBR backgroundheterozygote Mthfr-KO mice	No treatment	Genotyping of DNA isolated from toe clips. Immuno-fluorescence analysis of brain tissue. Morphogenic and behavioral assessments	N.S (not specified)	The study provides evidence for the profound impact of a genetic deficiency in the MTHFR gene on the induction of autistic features. In the mouse model of ASDs, this deficiency directly regulates metabolite availability and indirectly controls the environment of the developing embryonic brain [68].
7	Sequeira JM et al., (2016) [69]	Long Evans hooded Rat	Normal diet containing 2 mg/kg FAFRα- Ab at a dose of 4μg/embryo mixed intra-peritoneal (IP) injection FRα-Ab (35μg per pup per day) on post-natal days (PND) 10, 11 and 12 (GST-PRW group). Single dose of antibody at 4.0 μg/embryo on GD8. Antibody (50μg in 0.2ml rat serum IP) on PND 26, 27, and 28 (POW group)	Behavioral tests investigated the effect of exposure to FRα antibodies (Ab) during gestation (GST), the pre-weaning (PRW), and the post weaning (POW) periods on learning and behavior in adulthood	N.S	Deficits in rats exposed to Ab during gestation and pre-weaning (GST+PRW) included indications of increased levels of anxiety. None of these rats learned the active place avoidance task, indicating severe learning deficits and cognitive impairment. Similar but less severe deficits were observed in rats exposed to Ab during GST alone or only during the PRW period, suggesting the extreme sensitivity of the fetal as well as the neonatal rat brain to the deleterious effects of exposure to Ab during this period [69].
8	Chu D et al., (2019) [70]	ICR mice	NIH-31 open formula diet containing 2 mg /kg FA.Deionized water for 1week containing: no FA (control) 3.75 mg/L FA (MFA) 22.5 mg/L FA (HFA)	Behavioral testsRNA sequencing for postnatal day 21 mice brain. Expression of genes in adult mice brain 5 months postpartum of each group.Body weight of the offspring for 5 months	n = 6 male, n = 12 female	Results demonstrated a change in gene expression profile in weaning mice during early life FA supplementation. Long-term behavioral effects were more evident in adult male mouse offspring in a dose-sensitive mode [70].
9	Desai A et al., (2017) [71]	Rat	Normal diet containing 2 mg/kg FAFRα Ab at a dose of 4 or 12 μg per embryo in 1 mL normal rat serum was administered by (IP) injection1 mg of folinic acid (GD7–GD12) IP and/or 0.5 mg	Folate uptake and FRα Ab localization studies. Behavioral tests	50	Findings suggest severe behavioral and cognitive changes mirroring ASD symptoms following gestational Ab exposure in a rat model and protection afforded by folinic acid and dexamethasone treatment [71].