Circulating Transcriptomic signatures uncover persistent thyroid hormone signaling dysfunction in Levothyroxine-treated thyroidectomized patients

Abstract Background: Levothyroxine (LT4) monotherapy is the standard replacement therapy following a total thyroidectomy. This is based on the assumption that the conversion of thyroxine (T4) to triiodothyronine (T3) by peripheral type II deiodinase (DIO2) ensures adequate intracellular thyroid hormone (TH) signalling. However, a significant proportion of patients treated with LT4 following thyroidectomy report persistent hypothyroid-like symptoms despite biochemical euthyroidism. Previous proteomic studies from our group have demonstrated inflammatory and thrombo-inflammatory alterations, particularly in patients with reduced postoperative FT3 levels, suggesting incomplete restoration of tissue-level TH action. It is not known whether these downstream proteomic signatures reflect coordinated upstream transcriptional changes. Methods: This prospective study involved performing paired pre- and postoperative circulating mRNA transcriptome profiling in 12 patients with biochemically euthyroidism undergoing total thyroidectomy for benign nodular disease. Plasma mRNA expression was quantified using the NanoString nCounter® Human Metabolic Pathways Panel. The patients were also genotyped for the DIO2 Thr92Ala polymorphism and completed longitudinal SF-36 questionnaires. We used differential gene expression (DEG), gene set enrichment analysis (GSEA) and unsupervised clustering of quality-of-life (QoL) scores to integrate clinical, genetic and molecular data. Results: Paired analysis revealed postoperative upregulation of genes involved in extracellular matrix remodelling (THBS1 and ITGB1), inflammatory and chemokine signalling (CCL5 and CTSA), lipid metabolism (PTGS1) and modulation of the PI3K/AKT pathway. DIO2 Thr92Ala carriers displayed a distinct transcriptional phenotype characterised by the downregulation of mitochondrial detoxification (ALDH2), stress-response kinases (MAPK8) and ECM components (LAMB1), as well as the upregulation of metabolic regulators (RPS6KB1 and NDUFB4). Unsupervised clustering of SF-36 scores identified two quality of life (QoL) subgroups: patients with poorer postoperative QoL exhibited transcriptional signatures enriched for oxidative stress, neuro-immune activation (CTSS and GLUL) and astrocytemetabolic pathways. Conclusions: This study provides the first evidence from paired human circulating transcriptomes that levothyroxine monotherapy does not consistently restore tissue-level euthyroidism after total thyroidectomy, even when serum TSH levels are normalised. The postoperative mRNA landscape revealed sustained activation of inflammatory, metabolic and extracellular matrix (ECM)-integrin remodelling pathways, which were further modulated by DIO2 genotype and patient-reported symptom burden. These findings highlight the limitations of monitoring only biochemical markers and provide strong evidence in favour of integrating transcripts-based biomarkers to identify patients at risk of persistent dysfunction. This would enable more accurate stratification of postoperative vulnerability and guide truly precision tailored thyroid hormone replacement therapy.