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Background: The exact pathogenesis of autism is still unknown. Both thyroid hormones and 25(OH)D are important for brain development, in addition to CD5; all have immunomodulatory actions by which their dysregulation may have a potential role in autism pathogenesis. Objectives: The objectives of this study were to assess the thyroid profile, serum 25(OH)D levels and CD5 expression levels among autistic patients and to find out the correlations between the measured biomarkers with each other on one side and with the disease severity on the other side. Patients and methods: This cross-sectional case-control study has been conducted on 60 children with autism and 40 controls, recruited from Qena Governorate, Upper Egypt. Childhood Autism Rating Scale (CARS) score was used to assess the included patients. Biochemical assays of thyroid function in the form of free triiodothyronine (FT3), free tetraiodothyronine (FT4), thyroid-stimulating hormone (TSH) and 25(OH)D were done using commercially available enzyme-linked immunosorbent assay (ELISA) kits, while CD5 expression levels were measured using flow cytometry (FCM) analysis for all the included patients and controls. Results: The overall measurement results show significant higher mean serum TSH levels, mean CD5 expression levels with significant lower mean serum 25(OH)D levels among autistic children when compared with the control group (p,0.05 for all). Significant negative correlations between CD5 with FT3, FT4 and 25(OH)D were observed. CARS score showed significant negative correlations with both FT3 and 25(OH)D, while it was positively correlated with CD5 in a significant manner (p,0.05 for all). Conclusion: Elevated CD5 expression and decreased 25(OH)D stores could play a potential role in the pathogenesis of autism via their immune-modulator actions. High TSH serum levels among autistic children, although within the physiological range, reflect the presence of thyroid dysfunction among such children, which needs further assessment. [ABSTRACT FROM AUTHOR]