Abstract Background Fistulising Crohn’s disease (CD) is a severe phenotype lacking reliable biomarkers for prediction and stratification. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally, while isomiRs, sequence variants of miRNAs, may provide enhanced specificity and functional relevance. This study aimed to characterise plasma miRNAs and isomiRs associated with fistula formation in CD. Methods Plasma small RNA-seq was performed on 57 samples from 20 patients with CD. Samples included both baseline specimens obtained prior to surgical intervention and follow-up samples collected throughout the postoperative course, and seven non-IBD controls. Patients were classified depending on the presence of fistulas in the affected zone. miRNAs and isomiRs were sequenced and quantified using the XICRA pipeline, and differential expression was analysed with DESeq2. Predicted targets of significant miRNAs/isomiRs were identified using miRDB. Functional enrichment (GO and KEGG) was conducted with ClusterProfiler, and protein–protein interactions were explored using STRING. Results We identified 41 miRNAs and 211 isomiRs showing expression differences across the three pairwise comparisons (fistulising CD or non-fistulising CD vs non-IBD controls, and fistulising vs non-fistulising CD). After applying predefined selection criteria, isomiR profiling provided higher resolution than canonical miRNAs. Fistulising CD showed distinct isomiR expression patterns potentially linked to inflammation, TGF-β signaling, epithelial–mesenchymal transition, and extracellular matrix remodeling. Network analysis highlighted functionally relevant clusters: miRNA biogenesis (AGO1, DICER1), fibrosis/ECM remodeling (COL3A1, LOX), and epithelial integrity/signaling (TGFBR1, NOTCH2). Based on expression specificity and network relevance, one miRNA (hsa-miR-181a-5p) and four isomiRs (derived from hsa-miR-191-5p, hsa-miR-103a-3p, hsa-miR-423-5p) were prioritized as candidate biomarkers. Conclusion Plasma isomiR profiling provides higher resolution than canonical miRNAs for molecular stratification of fistulising CD. The identified isomiRs highlight fibrotic and extracellular matrix-remodeling pathways, supporting their potential as circulating biomarkers for fistulising phenotypes. Conflict of interest: Bernal, Carla: No conflict of interest Mr. Suau, Roger: No conflict of interest Naves, Juan E.: No conflict of interest Clua, Laura: No conflict of interest Pluvinet, Raquel: No conflict of interest Benaiges-Fernandez, Robert: No conflict of interest López Balastegui, Marta: No conflict of interest Sánchez Herrero, José Francisco: No conflict of interest Ginés Mir, Iris: No conflicts Mañosa Ciria, Miriam: Personal Fees: Abbvie, FAES Pharma, Ferring, Jannsen, MSD, Pfizer, Tillots and Lilly Sumoy, Lauro: No conflict of interest Serena, Carolina: None conflict of interest Domènech Moral, Eugeni: Personal Fees: I have served as a speaker, or has received research or education funding or advisory fees from AbbVie, Adacyte Therapeutics, Biogen, Celltrion, Gilead, Janssen, Kern Pharma, MSD, Pfizer, Roche, Samsung, Takeda, Tillots. Other: I have served as a speaker, or has received research or education funding or advisory fees from AbbVie, Adacyte Therapeutics, Alfasigma/Galapagos Biogen, Celltrion, Ferring, Gilead, GoodGut, Imidomics, Janssen, Kern Pharma, Lilly, MSD, Pfizer, Roche, Takeda, Tillots. Manyé Almero, Josep: no conflict of interest
Bernal et al. (Thu,) studied this question.