Olivier Salamin

Context Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. Objective To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. Design T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. Setting Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). Intervention(s) Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. Main outcome measure(s) Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. Results An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. Conclusions Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.

Liver-specific microRNA-122 (miR-122) is involved in the replication of hepatitis C virus (HCV) and its potential as a target for antiviral intervention was recently assessed. However, the use of circulating miR-122 in the evaluation of liver function has never been reported. In the present study, changes of serum miRNA levels were first evaluated in acute human hepatotoxicity due to paraquat exposure. Serum samples were collected and analyzed using real-time reverse transcription PCR. The results showed a positive correlation between serum miR-122 and alanine aminotransferase, a clinical biomarker for liver function. Furthermore, serum miR-122 was assessed in patients with hepatitis B and hepatocarcinoma, resulting in distinct miR-122 profiles in these two closely related diseases. In addition to miR-122, another small RNA, U6 small nuclear RNA, was downregulated in hepatocarcinoma patients, suggesting its prognostic significance in this disease. Taken together, these lines of evid...

Introduction Sodium-glucose cotransporter (SGLT) 2 inhibition is a well-known target for type 2 diabetes treatment, chronic heart failure and chronic kidney disease. SGLT2 is encoded by SLC5A2 (Solute Carrier Family 5 Member 2) gene, which is highly expressed in the kidney cortex(x36.9). Notably, SLC5A2is also overexpressed in the testes (x8.1), where glucose uptake could be crucial for androgen production and spermatogenesis. We hypothesized that the SGLT2 inhibitors use could interfere with testosterone production in healthy men. Method This is a post-hoc analysis of a double-blind, randomized, placebo-controlled study, in 27 healthy men who received empagliflozin 10mg once a day during 4 weeks. All weight categories were included: 15 normal weight, 15 overweight, 15 obese. In each group, 5 were randomized to the placebo and 10 to the empagliflozin. Serum samples were analyzed using an ultra-high-performance LC (UHPLC)–MS/MS method for quantification of total testosterone (TT) and...

The steroidal module of the Athlete Biological Passport (ABP) targets the use of exogenous androgenous anabolic steroids (EAAS) in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive OCP cycles (8 weeks) in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and/or epitestosterone (E) were below the limit of quantification of 1 ng/mL in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E (41%)): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5alpha-androstane-3lapha,17beta-diol/5beta-androstane-3,17beta-diol (p < 0.001). In serum, markers variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19% and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by ...

The implementation of the Athlete Biological Passport (ABP) represented a milestone in the fight against doping and unveiled a paradigm shift, namely the indirect detection of prohibited substances or methods through an individual, adaptive and longitudinal monitoring of biomarkers. While this tool has improved detection capability, it still suffers from sensitivity issues. To improve the discriminative performance of ABP, additional and complementary biomarkers are thus required and the blood matrix offers an immense reservoir for biomarkers discovery using emerging and sensitive –omics technologies. In the first part of the thesis, the impact of blood doping on the reticulocytes transcriptome was investigated. Three candidate genes could be highlighted as promising markers of altered erythropoiesis following an autologous blood transfusion using two different quantitative methods, with a magnitude of changes more significant than that of markers currently used in the ABP. The tran...

The haematological module of the Athlete Biological Passport (ABP) monitors longitudinal haematological variations that could be indicative of blood manipulation. This study applied a multi-parametric model previously validated in elite cyclists to compare inferred and actual PV variations while the potential influence of the oral contraceptive pill (OCP) cycle on the ABP blood biomarkers and plasma volume (PV) in 14 physically active women taking OCPs was also investigated. Blood and serum samples were collected each week for 8 weeks, and the ABP haematological variables were determined according to the World Anti-Doping Agency guidelines. Transferrin (sTFN), ferritin (FERR), albumin (ALB), calcium (Ca), creatinine (CRE), total protein (TP), and low-density lipoprotein (LDL) were additionally computed as 'volume-sensitive' variables in a multivariate analysis to determine individual estimations of PV variations. Actual PV variations were indirectly measured using a validated carbon monoxide rebreathing method. We hypothesised ABP markers to be stable during a standard OCP cycle and estimated PV variations similar to measured PV variations. Measured PV variations were in good agreement with the predictions and allowed to explain an atypical passport finding (ATPF). The ABP biomarkers, Hbmass, and PV were stable over 8 weeks. Significant differences occurred only between week 7 and week 1, with lower levels of haemoglobin concentration ([Hb]), haematocrit (HCT), and red blood cell count (RBC)(-4.4%, p < 0.01; -5.1%, p < 0.01; -5.2%, p < 0.01) and higher levels of PV at week 7 (+9%, p = 0.05). We thus concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.

<p><b>(A and B)</b> Two examples of longitudinal monitoring of plasma miR-122 with personalized threshold (indicated by continuous black line) for two volunteers with different <i>UGT2B17</i> genotypes (ins/del and ins/ins) after oral and transdermal intake of T. Thresholds are calculated as the mean of the control phase results + 3 × SD. In these examples, longitudinal monitoring of plasma miR-122 levels after oral T administration is more sensitive than of transdermal T administration. Data were normalized against the corresponding levels of endogenous miR-486-5p.</p

<p>Levels of miR-342 (A) and miR-150 (B) in plasma samples collected from healthy volunteers after administration of oral (gray line) or transdermal (dashed line) T or in the control phase (black line). Collection time points are indicated on the x-axis. Data were normalized against the corresponding levels of endogenous miR-486-5p. Values are expressed as means (±SE) of 19 independent samples. (*) indicates statistically significant difference relative to time = 0 h.</p

In the light of recurring anti-doping news, this article discusses some special cases and extends to the future prospects of developing effective anti-doping strategies. After recalling the current principles of the fight against doping, the use of substances tole-rated at a certain threshold, and the therapeutic use exemptions (TUE) are discussed. Authorized substances with a health risk for athletes are discussed before concluding on the development of the athlete's biological passport as a future common ground for anti-doping and medical follow-up of athletes. In conclusion, this approach emphasizes the imperative dialogue between anti-doping organizations and sports medicine in order to defend good practices preserving the intrinsic value of sport.

Background Tramadol is a synthetic opioid not banned by WADA, but included in the Monitoring Program since 2012. The prevalence of use of tramadol in competition was approximately 5% in cycling. These data clearly suggest that there was a misuse of tramadol in cycling. Opioids have several adverse effects, including nausea, dizziness and reduced vigilance. Such effects might be a potential cause of falls during races. Moreover, the psychoactive effects of tramadol are known to lead to abuse and then to drug dependence. Objective In order to decrease the risk of falls and prevent the occurrence of opioid-related side effects, the UCI has decided to ban the use of tramadol in competition from the 1st March 2019. This regulation was part of a vast program of injury prevention in cycling, and was included in the UCI medical rules. Main outcome measurements The tramadol controls are done in dried blood spots (DBS). The capillary blood is taken from a finger using a specific device that a...

Erythropoietin (EPO) is the main hormone regulating red blood cell (RBC) production. The large-scale production of a recombinant human erythropoietin (rHuEPO) by biotechnological methods has made possible its widespread therapeutic use as well as its misuse in sports. Since the marketing of the first epoetin in 1989, the development has progressed to the third-generation analogs. However, the production of rHuEPO is costly, and the frequent administration of an injectable formula is not optimal for compliance of therapeutic patients. Hence, pharmaceutical industries are currently developing alternative approaches to stimulate erythropoiesis, which might offer new candidates for doping purposes. The hypoxia inducible factors (HIF) pathway is of particular interest. The introduction of new erythropoiesis-stimulating agents (ESAs) for clinical use requires subsequent development of anti-doping methods for detecting the abuse of these substances. The detection of ESAs is based on two di...

Blood manipulations (e.g. blood transfusion, recombinant human erythropoietin (rHuEPO) injections) are currently being used by athletes to enhance their sporting performance despite these methods being prohibited by the World Anti-doping Agency (WADA). Recent advances in “omics” technologies have provided new high-throughput techniques that may facilitate the discovery of new biomarkers for anti-doping purposes [1, 2]. This study aimed to test the application of whole transcriptome analysis to the detection of autologous blood transfusion (ABT). 15 healthy Caucasian male (20-35 yrs old, BMI ≤ 30 kg/m2) donated 500 mL of blood that was stored and then infused 36 days later. Whole blood samples were collected 4 days and 1 day before blood transfusion and at 3, 6 and 12 hours, 1,2,3,6,9 and 15 days after blood transfusion. Total RNA was extracted and 100 ng/ul of total RNA used for gene expression analysis using the GeneChip HTA 2.0 from Affymetrix UK Ltd ( > 285,000 full-length transcripts). One-way repeated measure analysis of variance (ANOVA) was used to identify differential expressed genes following ABT. Changes considered significant at ANOVA p-value < 0.05 and a fold change (FC) of 1.5. As the study is ongoing, we present the results of 8 subjects and only 5 time points (baseline one day before, 3, 6, 9 and 15 days after ABT). Compared to baseline, 202 genes were differentially expressed 3 days after ABT, 170 genes after 6 days, 218 genes after 9 days and 51 genes after 15 days. Approximately 91 genes were differentially expressed among time points, with 23 genes overlapping and upregulated 3, 6 and 9 days after ABT; fold changes varied from 1.5 to 2.9. Of these differentially expressed genes, three overlapped with 45 previously identified rHuEPO genes. Specifically, ALAS2 was downregulated 6 days (FC = −1.77, ANOVA p = 0.003), 9 days (FC = −1.84, ANOVA p = 0.007) and 15 days after ABT (FC = −1.5, ANOVA p = 0.011). CCR7 was upregulated (FC = 1.83, ANOVA p = 0.049) 3 days after ABT and SLC4A1 was downregulated (FC = −1.65, ANOVA p = 0.023) 6 days after ABT. Although preliminary, these results are encouraging and support the detection of autologous blood transfusion using whole genome expression. References Pottgiesser T, et al. Gene expression in the detection of autologous blood transfusion in sports-a pilot study. Vox Sang, 2009. 96(4):333–6 Durussel J, et al. The blood transcriptional signature of recombinant human erythropoietin administration and implications for anti-doping strategies. Physiol Genomics, 2016: physiolgenomics.00108.2015

The concentration of hepcidin, a key regulator of iron metabolism, is suppressed during periods of increased erythropoietic activity. The present study obtained blood samples from 109 elite athletes and examined the correlations between hepcidin and markers of erythropoiesis and iron metabolism (i.e., haemoglobin, erythropoietin (EPO), ferritin, erythroferrone (ERFE), and iron concentration). Furthermore, an administration study was undertaken to examine the effect of recombinant human EPO (rhEPO) delta (Dynepo™) on hepcidin concentrations in healthy male volunteers. The effects on hepcidin were then compared with those on reticulocyte percentage (Ret%) and ferritin concentration. There was a significant positive correlation between hepcidin and ferritin, iron, and haemoglobin levels in athletes, whereas hepcidin showed an inverse correlation with ERFE. Administration of rhEPO delta reduced hepcidin levels, suggesting that monitoring hepcidin may increase the sensitivity of the Athl...

Aim: Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC–MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. Results: The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20 %). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. Conclusion: The UHPLC–MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.

BACKGROUND Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5′-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS When measured over a 7-week period, the intra- and interindividual va...

Autologous blood transfusion is a powerful means to improve performance and remains one of the most challenging method to detect. Recent investigations have identified three candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1) and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the three candidate genes are reliable predictors of bone marrow's response to blood t...

Erythropoietin (EPO) is the main hormone regulating red blood cell (RBC) production. The large-scale production of a recombinant human erythropoietin (rHuEPO) by biotechnological methods has made possible its widespread therapeutic use as well as its misuse in sports. Since the marketing of the first epoetin in 1989, the development has progressed to the third-generation analogs. However, the production of rHuEPO is costly, and the frequent administration of an injectable formula is not optimal for compliance of therapeutic patients. Hence, pharmaceutical industries are currently developing alternative approaches to stimulate erythropoiesis, which might offer new candidates for doping purposes. The hypoxia inducible factors (HIF) pathway is of particular interest. The introduction of new erythropoiesis-stimulating agents (ESAs) for clinical use requires subsequent development of anti-doping methods for detecting the abuse of these substances. The detection of ESAs is based on two different approaches, namely, the direct detection of exogenous substances and the indirect detection, for which the effects of the substances on specific biomarkers are monitored. Omics technologies, such as ironomics or tran-scriptomics, are useful for the development of new promising biomarkers for the detection of ESAs. Finally, the illicit use of ESAs associates with multiple health risks that can be irreversible, and an essential facet of anti-doping work is to educate athletes of these risks. The aim of this review is to provide an overview of the evolution of ESAs, the research and implementation of the available detection methods, and the side effects associated with the misuse of ESAs.

Biomarkers Despite being prohibited by the World Anti-Doping Agency, blood doping through erythropoietin injection or blood transfusion is frequently used by athletes to increase oxygen delivery to muscles and enhance performance. In contrast with allogeneic blood transfusion and erythropoietic stimulants, there is presently no direct method of detection for autologous blood transfusion (ABT) doping. Blood reinfusion is currently monitored with individual follow-up of hematological variables via the athlete biological passport, which requires further improvement. Microdosage is undetectable, and suspicious profiles in athletes are often attributed to exposure to altitude, heat stress, or illness. Additional indirect biomarkers may increase the sensitivity and specificity of the longitudinal approach. The emergence of "-omics " strategies provides new opportunities to discover bio-markers for the indirect detection of ABT. With the development of direct quantitative methods, transcriptomics based on microRNA or messenger RNA expression is a promising approach. Because blood donation and blood reinfusion alter iron metabolism, quantification of proteins involved in metal metabolism, such as hepcidin, may be applied in an " ironomics " strategy to improve the detection of ABT. As red blood cell (RBC) storage triggers changes in membrane proteins, proteomic methods have the potential to identify the presence of stored RBCs in blood. Alternatively, urine matrix can be used for the quantification of the plasticizer di(2-ethyhexyl)phthalate and its metabolites that originate from blood storage bags, suggesting recent blood transfusion , and have an important degree of sensitivity and specificity. This review proposes that various indirect bio-markers should be applied in combination with mathematical approaches for longitudinal monitoring aimed at improving ABT detection.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and thus influence many cellular and physiological processes. miRNAs are also present in cell-free body fluids such as plasma or serum, and these circulating miRNAs are very stable, sensitive , and specific biomarkers of pathophysiological states. In this study, we investigated whether circulating miRNAs could serve as biomarkers of exogenous testosterone administration. Misuse of testosterone as a performance-enhancing drug is thought to be widespread in sports. Detection of testosterone through the urinary steroid profile of the Athlete Biological Passport faces several obstacles, indicating that new biomarkers are required. To this end, we analyzed plasma miRNA levels by high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after transdermal and oral testosterone administration. Screening identified three potential candidate miRNAs that were altered by both routes of testosterone administration. Longitudinal monitoring of these candidates revealed that variation in two of them (miR-150 and miR-342), relative to the corresponding levels in control samples, was testosterone-independent. However, levels of the liver-specific miR-122 increased 3.5-fold 1 day after drug intake. Given that testos-terone is metabolized by the liver, this observation suggests that miR-122 in cell-free fluids may be used as a sensitive biomarker of testosterone misuse via multiple dosing routes and could therefore be integrated into a blood-based multiparametric follow-up.