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Drug testing and analysis2025; doi: 10.1002/dta.3899

Transcriptomic Biomarkers in Blood Indicative of the Administration of Recombinant Human Erythropoietin to Thoroughbred Horses.

Abstract: Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied RNA-sequencing (RNA-seq) to discover blood RNA biomarkers in Thoroughbred horses after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera®. A single subcutaneous administration of Mircera® at ~ 4.2 μg/kg was effective in elevating haematocrit, haemoglobin and erythrocyte levels to varying extents in as early as 4 days post-administration in all three horses, which persisted for 40 days post-administration (the last sample collected). RNA-seq was applied to analyse blood transcriptomic changes. Differential gene expression analysis has allowed the identification of 46 genes that showed dramatic and temporary upregulation at 4-11 days after Mircera® administration. STRING analysis has identified the functional enrichment of 15 genes important for erythropoiesis and erythrocyte function, supporting the idea of an increased release into the peripheral circulation of residual RNA-containing reticulocytes after rhEPO exposure, which would otherwise mature normally inside the bone marrow in horses. Machine learning of blood transcriptomes has enabled the discrimination of samples with or without Mircera administration. Therefore, our study has provided new insights into the biological mechanism of erythropoiesis caused by rhEPO administration in horses and has provided evidence supporting the control of misuse of ESAs by monitoring the equine blood transcriptome.
© 2025 John Wiley & Sons Ltd.
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Publication Date: 2025-04-21 PubMed ID: 40256823DOI: 10.1002/dta.3899Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates how to detect erythropoiesis-stimulating agent (ESA) use in racehorses by studying transcriptomic biomarkers. The study discovered that residual RNA-containing reticulocytes entered the bloodstream after the administration of an ESA, leading to potentially detectable markers of ESA misuse.

Introduction to the Problem

  • ESAs, which artificially improve athletic performance, are a significant concern in both human and equine sports. Direct testing may sometimes fail to detect ESA use due to the small window of detection, hence necessitating complementary indirect detection methods.
  • The study focused on identifying blood RNA biomarkers in thoroughbred horses after administering recombinant human erythropoietin (rhEPO), a long-acting form of ESA. This was done to gain insight into the biological processes induced by rhEPO and establish an indirect method for detecting its use.

Methodology

  • The researchers applied RNA-Sequencing (RNA-seq) to analyze the changes in the blood transcriptome (the full set of RNA sequences) of thoroughbred horses upon injection of methoxy polyethylene glycol epoetin beta, also known as Mircera®, a type of rhEPO.
  • A single administration of Mircera® at about 4.2 µg/kg significantly increased the hematocrit, hemoglobin, and erythrocyte levels in all three horses as early as 4 days post injection till 40 days after (the duration of sample collection).

Findings

  • Differential gene expression analysis identified 46 genes that showed sudden and temporary upregulation 4-11 days after Mircera® administration.
  • By using the STRING analysis, the study found a functional enrichment of 15 genes crucial for erythropoiesis and erythrocyte function, supporting the hypothesis that the rhEPO administration induced residuals of RNA-containing reticulocytes to be released into the peripheral blood circulation – these cells would otherwise mature normally inside bone marrow in horses.
  • Machine learning techniques allowed researchers to distinguish between blood sample with and without Mircera administration based on the observed transcriptomic changes.

Conclusion

  • The study provides new insights into the biological mechanism of erythropoiesis due to rhEPO administration in horses and presents preliminary evidence for controlling the misuse of ESAs by monitoring the equine blood transcriptome.
  • This transcriptomic monitoring potentially allows detection of Mircera use beyond the typically available window for direct testing and could help ensure the integrity of equine sports.

Cite This Article

APA
Cheung HW, Wong KS, Cheng PCF, Tsang CYN, Farrington AF, Wan TSM, Ho ENM. (2025). Transcriptomic Biomarkers in Blood Indicative of the Administration of Recombinant Human Erythropoietin to Thoroughbred Horses. Drug Test Anal. https://doi.org/10.1002/dta.3899

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Cheng, Paul C F
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Tsang, Candice Y N
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Farrington, Adrian F
  • Veterinary Clinical Services, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, the Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.

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