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Abstract
Objective: The purpose of this randomized controlled trial (RCT) was to evaluate the effect of omega‑3 fatty acid supplementation on the gut–brain axis in children with autism spectrum disorder (ASD) admitted to a tertiary hospital in Pakistan. The study specifically aimed to determine whether supplementation with omega‑3 fatty acids (DHA + EPA) would lead to improvements in gastrointestinal (GI) symptoms, gut microbiota composition, markers of intestinal permeability and systemic inflammation, and core and associated behavioural features of ASD.
Method: We conducted a double‑blind, placebo‑controlled RCT in the paediatric neurodevelopmental unit of a tertiary‑care hospital in Pakistan. Eligible children aged 4–12 years with a confirmed diagnosis of ASD were randomly assigned to receive either omega‑3 fatty acid supplementation (daily doses of DHA 500 mg + EPA 300 mg) or matching placebo for a period of 24 weeks. Primary outcomes included changes in gut microbiota diversity (alpha and beta diversity), abundance of selected taxa (e.g., Bifidobacterium, Clostridia), intestinal permeability (serum zonulin, lipopolysaccharide‑binding protein), and systemic inflammatory markers (IL‑6, TNF‑α). Secondary outcomes included changes in GI symptom scores, social communication and interaction scores (measured via the Autism Diagnostic Observation Schedule – Second Edition (ADOS‑2), Social Responsiveness Scale (SRS)), and repetitive behaviour scores. Data were collected at baseline, 12 weeks and 24 weeks. The analysis employed intention‑to‑treat methods with mixed‑model repeated‑measures analyses.
Results: A total of 80 children were randomised (40 in the omega‑3 group, 40 in placebo). Eight children (3 in omega‑3, 5 in placebo) withdrew prior to study completion. At 24 weeks, the omega‑3 group demonstrated a statistically significant increase in gut microbiota alpha‑diversity compared with placebo (mean change +1.2 vs +0.3, p = 0.02) and a relative increase in Bifidobacterium spp. (mean fold change 1.8 vs 1.1, p = 0.03). Serum zonulin levels decreased significantly in the omega‑3 group (mean change –22 ng/mL vs –5 ng/mL in placebo, p = 0.01) and IL‑6 levels declined (–1.5 pg/mL vs –0.4 pg/mL, p = 0.04). GI symptom scores improved more in the omega‑3 group (mean change –6.4 vs –2.8 on GI‑Symptom Scale, p = 0.01). Behavioural outcomes showed modest but statistically significant improvement in SRS total score in the omega‑3 group (mean change –8.2 vs –3.5, p = 0.03); however, changes in ADOS‑2 and repetitive behaviour scores did not reach statistical significance (p > 0.05). No serious adverse events were reported; minor gastrointestinal upset occurred in three children in the omega‑3 group.
Conclusion: In children with ASD treated at a tertiary hospital in Pakistan, 24‑week supplementation with omega‑3 fatty acids resulted in favourable modulation of gut microbiota composition, reduction of intestinal permeability and systemic inflammation, and improvement in gastrointestinal symptoms and social responsiveness measures. These findings suggest that omega‑3 fatty acids may have adjunctive benefit via the gut–brain axis in ASD. Larger multicentre trials with longer follow‑up are warranted to confirm the behavioural and neurodevelopmental impact and to define optimal dosing.