Potential contribution of age-related and methodological factors to limited reproducibility in autism spectrum disorder blood miRNA biomarker studies: an exploratory meta-analysis

Cross-study inconsistencies in autism spectrum disorder (ASD) blood microRNA biomarker studies suggest that methodological heterogeneity may substantially limit reproducibility. We conducted an exploratory meta-analysis of publicly available ASD blood miRNA datasets from the Gene Expression Omnibus, applying rigorous inclusion criteria and standardized analytical protocols. Three datasets were included (GSE89596, GSE67979, GSE222046) comprising 614 miRNAs across 90 participants (45 ASD, 45 controls). Random-effects meta-analysis was performed using Hedges' g effect sizes, with comprehensive heterogeneity assessment and leave-one-dataset-out cross-validation. No miRNAs survived multiple testing correction (Benjamini-Hochberg FDR < 0.05), though seven candidate signals showed consistent evidence with unadjusted p < 0.01 and large effect sizes. These candidates demonstrated near-zero between-study heterogeneity and consistent directionality across validation analyses. Potential age-related and platform-related differences were observed, with near-zero correlation between adult and pediatric effect sizes (Kendall's τ = -0.022); however, these two sources of variability were fully confounded in the available data and could not be separated. Some miRNAs exhibited extreme between-study variability (I² > 80%), indicating substantial methodological differences. Cross-validation revealed that excluding the single adult dataset reduced sign consistency from 89.9% to 68.9%. Our findings suggest that age-related and methodological factors, including technical platform differences, may contribute to limited reproducibility in ASD blood miRNA research, and that blood-derived signals should be interpreted as potentially reflecting peripheral physiological states rather than central disease mechanisms. A supplementary cross-tissue analysis using post-mortem prefrontal cortex data (GSE59286; n = 45) provided direct empirical support for this interpretation: the majority of blood candidate miRNAs showed no corresponding expression in brain tissue, with only hsa-miR-29c-5p demonstrating directional concordance across both tissues. These findings suggest that age stratification, platform harmonization, and cross-tissue validation should be considered essential prerequisites for reliable ASD miRNA biomarker discovery, rather than optional refinements.