Introduction: Autism is a neurological disability that often appears after the age of three in children, also known as an Autism Spectrum Disorder (ASD). Several studies have examined the influence of some environmental factors, and many parameters related to the behavior of autistic patients have been measured in order to find ways to reduce ASD. This study investigates the relationship between ASD and serum levels of vitamin D3, B12, folic acid, and the intestinal microbiome. Material and methods: The serum levels of vitamin D3, B12, and folic acid in ASD patients were measured by the ELISA method and compared to healthy groups. DNA was extracted from stool samples of ASD patients and the control group, and then the gut microbiome was investigated via a metagenomics approach. Statistical analysis was done by Graphpad Prism software. Metagenomics sequencing was performed to analyze the 16S rRNA gene sequencing for species and strain-level microbiome. Result and Conclusion: The result showed no significant change in the VitD3and folate levels of ASD patients compared to the control group (p=0.157 and p=0.0505, respectively). There was a significant difference in the VitB12 level between control healthy individuals and ASD patients, in which the serum VitB12 concentration was significantly lower than the control group (p=0.0001). Gut metagenomics showed that the abundance of the bacterial familes Erysipelotrichaceae and Ruminococcaceae were higher, and Prevotellaceae, Actinomycetaceae, Streptococcaceae, Bifidobacteriaceae were lower in ASD. Conclusion: Patients with ASD have lower serum levels of vitamin B12 and different gut microbiomes compared to healthy controls. Low vitamin B12 levels and the altered gut microbiome seem to have clinical relevance as a predictor of severe outcomes in ASD.
