The misuse of erythropoiesis-stimulating agents (ESAs), such as recombinant human erythropoietin (rhEPO), remains a critical threat to the integrity of equine sports due to their performance-enhancing effects. Monitoring biomarkers associated with these effects offers a promising complementary strategy for doping control. This research aimed to discover blood-based transcriptomic and proteomic biomarkers indicative of rhEPO administration- specifically Mircera® (methoxy polyethylene glycol epoetin beta, a long acting rhEPO)-in Thoroughbred horses.
Increased haematocrit, haemoglobin and red blood cell (RBC) levels were observed as early as four days post-Mircera administration in three treated horses. Transcriptomic analysis of whole blood using RNA-sequencing (RNA-seq) revealed 46 significantly upregulated genes 4-11 days post-administration. Functional enrichment analysis identified 15 erythroid-specific genes (e.g., ALAS2, CA1/CA2, HBA/HBB, SLC4A1), suggesting an increased release of residual RNA-containing reticulocytes (or RBC precursor cells) into circulation. Concurrently, label-free plasma proteomics identified seven significantly altered proteins: haptoglobin (HP) and hemopexin (HPX) were downregulated, while transferrin receptor 1 (TFR1), phospholipid transfer protein (PLTP), tenascin C (TNC), vascular cell adhesion molecule 1 (VCAM1), and galectin 3 binding protein (LGALS3BP) were upregulated. These proteins are involved in erythropoiesis, iron metabolism, and vascular or inflammatory responses. Multivariate analysis of the blood transcriptome and plasma proteome effectively differentiated samples from those control horses.
This work provides the first comprehensive multi-omics biomarker panel associated with rhEPO misuse in horses. The findings provide insights into the biological mechanisms of ESA-induced erythropoiesis and establish a foundation for applying transcriptomic and proteomic biomarkers to control the misuse of ESAs in equine sports. Further validation in larger cohorts is warranted to optimise biomarker panels and analytical models for routine anti-doping implementation.