Changes in blood parameters after intramuscular testosterone ester injections – Implications for anti‐doping

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the ‘haematological module’ of the athlete biological passport (ABP) used by the World Anti‐Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo‐controlleddouble‐blind design. Urine samples and blood samples were collected twice pre‐treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post‐injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®‐treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope‐ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF‐Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3–10 days after injection. From an anti‐doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow‐up testing and substantiating likely misuse of testosterone. Testosterone treatment stimulates erythropoiesis and alters iron homeostasis. This is the first study to use the adaptive model in the ‘Athlete Biological Passport – Haematological Module’ to infer testosterone ester misuse. The placebo‐controlled design allows evaluation of both sensitivity and specificity. We report that markers in the haematological module are sensitive to testosterone doping and valuable in selecting suspicious samples for follow‐up testing.