Projets de recherche

Code: ISF16D11XD

The detection of the exogenous administration of synthetic androgens (the called 'pseudo-endogenous" steroids) having the same chemical structure of the compounds produced endogenously (i.e.  testosterone, 5α-dihydrotestosterone or androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles. 
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories will be collected starting 1st January 2014 in a global database integrated in the endocrinological module of the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges of every athlete. Once the ABP detects an atypical profile, an isotope ratio mass spectrometric (IRMS) confirmation must be applied.
The ABP will be effective once a sufficient number of data of a given individual will be collected. In normal conditions, almost two years are needed to collect such information. This will delay in any case the investigations and the time to take the appropriate decisions. Although new specific steroid metabolites have been described, the gap between the real confirmation capacity by IRMS and a suspicious finding is still too large.
IRMS data have demonstrate to be much more stable than the parameters of the ABP and a Bayesian approach similar to the one already in force for the ABP could be applied on the IRMS values. By this combined approach, both the effects on the ABP and the direct detection of pseudoendogenous steroids not produced by the athlete are possible.
The main goal of this project is to define and include in the ABP the more relevant and specific IRMS data of pseudoendogenous steroids metabolites. This should reduce the gap between the suspicion and confirmation capacities of laboratories.

Code: ISF17E11GJ 

Erythropoietin (EPO) is a natural glycoprotein hormone in the body that controls red blood cell production, and there is conclusive evidence that administering exogenous EPO results in significant performance enhancement in endurance sports. Current methods of EPO detection are unable to reliably detect micro-dosing where the drug is given at lower doses but more frequently. Exosomes are small cell derived particles in the blood and urine which contain protein. It is only recently that the importance of exosomes containing miRNAs and other proteins in cell-to-cell communication has become apparent in physiological processes, and the functions are complex and still largely unknown. Exosomes appear to have an intricate and important role in a range of blood processes related to hematopoiesis (creation of new blood cells) which is highly relevant to oxygen carrying capacity via red blood cells and endurance performance. Recent developments in sample preparation will allow us to isolate exosomes from blood and urine, and perform proteomics (simultaneous determination of protein levels across a large number of proteins using LC-MS/MS and bioinformatics) on athletes before and after treatment with EPO at low doses. Due to the intricate involvement of exosomes in red blood cell production, it is anticipated that this project will lead to identification of a number of candidate exosome proteins that will be indicative of administration of micro-dose exogenous EPO.

Code: 16B02JB 

Under this project, Operational Technologies Corporation will develop high affinity and highly specific DNA aptamers to bind the GH-releasing peptides GHRP-6, Ipamorelin and the GHRP-2 main metabolite AA-3 (D-Ala-D-(beta-naphthyl)-Ala-Ala-OH). The targets will be covalently attached to magnetic microbeads (MBs) to select for the highest affinity DNA aptamer candidates in commercially available certified disease-free human serum and urine to ensure highly specific aptamers for their cognate targets in real samples.  Following 8-10 rounds of aptamer selection and PCR amplification, aptamer candidates will be cloned and sequenced.  DNA sequences will be analyzed for partial and full-length consensus sequences.  All candidate aptamers will also be screened by ELISA-like (ELASA) colorimetric microplate assays to rank their relative affinities and specificity for their intended targets as well as related and unrelated targets.  The top candidate aptamers from ELASA screening will also be characterized by Surface Plasmon Resonance (SPR) analyses to determine Ka/Kd values versus their intended targets.

The top aptamer candidates will then be attached to MBs and used to probe spiked human serum and urine samples for the three different GHRP or metabolite targets.  Validation of aptamer-MB pulled down methods will be accomplished by mass spectral analysis at the core proteomics laboratory of the University of Texas Health Sciences Center in San Antonio, TX.  The aptamer-MB pull down protocol will be optimized to include aptamer-MB concentration, capture and elution times and chemical conditions such as pH, ionic strength and the addition of various detergents or other potential additives.  Successful development of aptamer-MB pull-down methods may enhance WADA’s ability to concentrate GHRPs or their metabolites from body fluids and enhance mass spectral GHRP detection capabilities.

Main Findings: 

Although not entirely successful for each of the 3 GHRPs, the basic requirements of this grant have been fulfilled in that several aptamers were developed in a 1:10 diluted human serum or undiluted human urine environment. Most of the 6 lead aptamers were subsequently shown to at least somewhat pull down their cognate target GHRPs in whole human serum or urine with successful detection by ESI-TOF mass spectrometry (MS). In some cases, detection by MS following aptamer-MB pull down and acid-elution failed, but could be corrected by adding 5-fold more aptamer-MBs. In another case, the pull down failure may be due to binding of the G6U-18R to a higher MW (45-60 kD) protein which interfered or competed with binding of the spiked G6 peptide. This same G6U-18R aptamer did, however, detect G6 in urine by MS as it was developed to do. The negative controls lacking aptamers on SAv-MBs demonstrated that the pull down assays were dependent on the specific aptamers tethered to the MBs and that non-specific binding of the GHRPs to the SAv-MBs was essentially non-existent.
It is noteworthy, that with the exception of the 45-60 kD protein pulled down in serum by the two lead anti-G6 aptamer candidates, the Coomassie Blue-stained electrophoresis gels of the pull down acid-eluates are clear. This observation suggests very specific and high affinity binding by the aptamers to their cognate GHRPs because very little else was pulled down on the surface of the aptamer-MBs in serum or urine. Of course, these aptamers were intentionally selected in 10% pooled serum and 100% pooled urine to minimize non-specific binding of the final selected lead aptamers.  It is rather surprising that these 6 lead aptamers bound their cognate GHRP targets with low ng detection limits in buffer by ELASA, but failed to bind in serum and urine matrices by ELASA.  Fortunately, these same aptamers on MBs were mostly able to detect their peptide targets in serum and urine by MS. This suggests that: 1) MS is more sensitive than ELASA and/or 2) active probing of a liquid sample using mobile aptamer-MBs binds more target than a static well surface. Taken together, albeit not perfect, the use of aptamers on MBs for pull down, purification or concentration of target peptides in serum or urine is a promising technique especially when coupled to MS detection which could significantly aid WADA in its search for doping athletes. 

Code: ISF16D07NN

In this 3-year international collaborative project, we will consolidate and expand the existing Athletes Biological Passport (ABP) approach as well as evaluate new strategies for detection of blood manipulation. 
The project consists of two clinical trials where samples are collected from four week doping regimes expected to be currently used by cheating athletes: A) autologous blood transfusion of volumes <150 ml packed red blood cells;
B) frequent intravenous injections of <10 IU per kg bw of recombinant human erythropoietin.          Samples collected from these main trials will be subjected to different analytical approaches, comprising ABP analyses including the abnormal blood profile score (ABPS); evaluation of reticulocyte percentage as a standalone marker; evaluation of iron-homeostatic markers potential for revealing blood manipulation including analyses of the newly discovered hormone erythroferrone; metabolomics and proteomic analyses. 
Thus the project covers a broad range of both well-known and new analytical strategies. Another unique and highly required part of the proposed project, is addressing if gender should be taken into account when interpreting fluctuations of existing and novel markers of blood volume manipulation.

Main Findings: 

With the present study we were able to demonstrate that the inclusion of the iron regulatory hormones, hepcidin and erythroferrone, can aid in the Athlete Biological Passport in the indirect detection of a small volume autologous blood transfusion. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise but intra-individual variability is of concern and additional studies are required. Furthermore, we demonstrate that small, frequent doses of recombinant human erythropoietin (rHuEPO) treatment administered intravenously provides sufficient erythropoietic response to increase aerobic-dominated performance. We investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rHuEPO treatment. With this study we demonstrate that a low dose rHuEPO treatment alters IRF and IR/RBC compared with placebo, and that such changes can aid in the indirect detection of rHuEPO misuse. When combining the current markers in the ABP with IRF and IR/RBC ~79% of the rHuEPO treated subjects were flagged at least once. 

Code: R16R03GJ 

Beta2-agonists are among the most commonly used drugs by athletes, which is related to the high prevalence of asthma and exercise induced bronchoconstriction (EIB) in this population.
This project will determine analytical urine thresholds for two beta2-agonist asthma drugs, salmeterol and terbutaline. 
Salmeterol is used to treat asthma and is permitted in sport when taken by inhalation in accordance with the manufacturers’ recommended regimen. There is currently no urine threshold for an adverse analytical finding (AAF) and athletes can freely administer supratherapeutic doses for doping purposes with impunity. The project will provide data on urine levels of salmeterol and other compounds related to its use found after inhaled dosing at both permitted (therapeutic) and prohibited (supratherapeutic) doses over a seven day treatment period.
Urinary thresholds and decision limits are a way to avoid excessive use of beta2-agonist asthma drugs by athletes and to lessen the burden associated with therapeutic use exemption (TUE) applications. One of the most common beta2-agonists, terbutaline, is widely used in Europe but is currently prohibited unless a TUE has been granted. Terbutaline is responsible for over three quarters of AAFs associated with beta2-agonists in doping control. The project will develop a urine threshold for terbutaline to discriminate between normal therapeutic use via inhalation, high dose (supratherapeutic) inhalation and oral ingestion of terbutaline.
Both salmeterol and terbutaline are chiral compounds administered as 50:50 mixtures of two enantiomers (stereoisomers) which are molecules with non-superimposable mirror images (analogous to right and left hands). Differences in the way the body excretes enantiomers of the same drug can be used to improve discriminatory capability of urine doping detection methods – this requires the use of advanced stereoselective UPLC-MS/MS assays to distinguish between enantiomers which will be used in this project.

Main Findings: 

Background. Terbuutaline, a short acting β2-agonist, is widely used in Europe and accounts for 76% of adverse analytical findings (AAFs) of β2-agonists in doping control. Terbutaline is a chiral drug consisting of a 1:1 ratio of two enantiomers ®-terbutaline and (S)-terbutaline, and given differences in metabolism between both enantiomers, the ratio of enantiomers may afford the ability to distinguish between oral and inhaled routes of administration.

Objective. To discriminate between therapeutic inhaled dosing and both supratherapeutic inhaled administration and oral dosing of terbutaline using a chiral assya for terbutaline applied to urine.

Methods. Part Ia: The study was crossover design, with 14 well-trained men and women undertaking three intervention arms; Inhalation regimen (low dose therapeutic) consisting of 1 mg twice daily for 7 days (2 mg/day); inhalation regimen (high dose supratherapeutic) consisting of 2 mg twice daily for 7 days (4 mg/day); prohibited oral regimen consisting of 10 mg daily for 7 days; prohibited oral regimen consisting of 20 mg daily for 3 days. Urine was collected 2 h post dose on each treatment day and (R)- and (S)-terbutaline determined using a chiral UPLC-MS/MS method. Part Ib: A further investigation of 25 mg/d oral terbutaline (15 mg morning + 10 mg afternoon) was undertaken in nine trained men for 4 weeks for comparison with urine collected 1-3 hours after administration on the first day, half-way through the intervention, and the final day. Part II: The study was an acute (24 h) PK study examining urine levels and R:S terbutaline ratio following dosing with 10 mg oral or 4 mg inhaled terbutaline and 1½ h exercise in highly-trained men with asthma.  

Results. Part Ia: Racemic terbutaline urine levels were highest in the supratherapeutic inhaled group. The sulfate metabolite was the main metabolite with some evidence of enantioselective metabolism. The urine log(R:S) terbutaline enantiomer ratio demonstrated excellent diagnostic performance at 2 h post dose with the ability to distinguish between oral and inhaled dosing, with sensitivity and specificity both greater than 98% based on an arbitrary cut-off value of 0.1.  Further discrimination between therapeutic inhaled (1 mg twice daily) and supratherapeutic inhaled (2 mg twice daily) dosing, was demonstrated using a urine threshold approach set at an arbitrary 1000 ng/ml with sufficient specificity (>98%), and while sensitivity was modest at 19%, adoption of this approach could potentially disincentivize supratherapeutic inhaled doping. Part Ib: resulted in similar findings to Part 1a with respect to log(R:S) ratio, noting that maximum levels were not achieved until week 4. Part II: In the acute PK study, urine levels were higher following inhaled administration than oral but the predictive value of R:S ratio was not repeated from Part Ia. 

Conclusion. The urine enantiomer ratio method shows some promise to discriminate between permitted inhalation and oral dosing of terbutaline in sport, and if combined with a rac-terbutaline threshold for supretherapeutic inhaled dosing, could pave the way for terbutaline in sport without the need for TUEs. Further work is required to investigate why this approach did not work in an acute single dosing scenario.

Code: ISF16D20FB 

The Athlete Biological Passport (ABP) represents an integral part of the anti-doping analyses. In the fight against doping, laboratories rely on monitoring blood and steroid profile data to set up long-term, individualized profiles of athletes. To uncover a prohibited administration of pseudoendogenous steroids (class S1.1 of the WADA “List of Prohibited Substances and Methods”) laboratories monitor concentrations and ratios of various endogenously produced steroidal hormones, their precursors, and metabolites for almost 25 years. Several studies reported only very small naturally occurring intra-individual variations of urinary endogenous steroid ratios like testosterone/epitestosterone (T/EpiT), androsterone/etiocholanolone (And/Etio), And/T, and 5α-/5β-androstane-3α,17β-diol (Adiol/Bdiol), that have been shown to be stable over months and even years in adult humans. In cases of a misuse of pseudoendogenous steroids as doping substances, these ratios are altered. Thus, the ABP is used to uncover a prohibited administration of class S1.1 steroids. However, it was shown recently that some other (non-prohibited) drugs may also influence the urinary steroid profile. In this project we focus our attention on the class of non steroidal anti inflammatory drugs (NSAIDs) that are frequently used as pain killers and antiphlogistics. Specifically, NSAIDs have been shown to inhibit the steroid metabolizing aldo-keto-reductases (AKR) 1C, namely the 3α-hydroxysteroid dehydrogenase (AKR1C2) and the 17β-hydroxysteroid dehydrogenase (AKR1C3). As no scientific data on the influence of these inhibitory NSAIDs on urinary steroids are available from literature, the project aims to close this gap.

Main findings

The Athlete Biological Passport (ABP) represents an integral part of anti-doping analyses. In the fight against doping, WADA accredited laboratories rely on monitoring blood and steroid profile data to set up long-term, individualized profiles of atheletes using the so called "hematological module" (in whole blood samples) and "steroidal module" (in urine samples). The alteration of one or more parameters of the passport can indeed be the effect of the recourse to doping substances and/or methods. More specifically, the monitoring of the hematological module can reveal the recourse to different forms of "blood doping" (transfusion, synthetic hemoglobins, illicit intake of erythropoiesis stimulating agents) while the steroidal module is used to unearth the administration of testosterone, its precursors and other "pseudo-endogenous" steroids (that are endogenous steroids when administered exogenously). The correct evaluation and interpretation of data obtained in the analysis of the ABP modules require the identification of any "confounding factors", that is non-prohibited substances that can alter the reliability of the ABP measurement.

In this project, we specifically considered the potential effects on markers of the steroidal module of the ABP of the use of "pain killers", that is non-prohibited drugs belonging to the class of non-steroidal anti-inflammatory drugs (NSAIDS). These substances are very commonly used by the athletes of virtually all sports/sport disciplines so it seemed to us of the utmost importance to verify whether their intake could cause an alteration in one or more parameters of the steroidal module of the ABP.

The project was structured along two parallel research lines:

• the in vitro evaluation of the inhibitory effect of NSAIDs (assessed on the prototype compound ibuprofen) on the activity of hydroxysteroid reductases, that is enzymatic systems involved in the biometabolic pathways of testosterone and other endogenous androgenic anabolic steroids;

• the analysis of urine samples obtained from controlled administration studies, performed collecting the urine samples before, during, and after a two-week period of intake of a daily, therapeutic dose of either ibuprofen or indomethacin, to verify the effects over time on the urinary steroid profile, and, at the same time, to identify and quantitate suitable markers of NSAID intake, i.e. the parent drug itself and/or its main metabolite(s).

Our findings confirm that both ibuprofen and indomethacin can act as "confounding factors" of the steroidal module of the ABP, altering the measured concentrations and concentration ratios of key markers of the steroid profile. This finding was more evident in the female subject than in the males; such an inter-gender difference was more evident for ibuprofen than for indomethacin. Should the results obtained in the present project be confirmed on a more statistically significant number of subjects, of both sexes and ideally of different ethnicities, NSAIDs could be considered for inclusion among the "confounding factors" of the steroidal module of the ABP.

Code: 16C02CR 

The aim of the proposed project is the determination of reference distributions for cobalt in urine of elite athletes. Two cohorts will be compared: samples from athletes performing in endurance sports (i.e. sports prone to misuse of erythropoiesis stimulating agents), and samples from non-endurance sports. The reference distributions will help defining thresholds of cobalt in urine for doping control purposes, above which erythropoiesis was illegally stimulated by cobalt according to chapter S2.2 “Hypoxia-inducible factor (HIF) stabilizers” of the WADA Prohibited List 2016.[3]  
In order to stimulate erythropoiesis, athletes have to apply Co2+ ions (e.g. CoCl2) at relatively high doses, which results in urinary concentrations, which are clearly distinguishable from cobalt in negative controls and after e.g. cobalamin administration. In total, ca. 600 urine samples (females/males equally distributed) will be analysed by ICP-MS as well as regarding their statistical properties (distribution type, outliers). 

Main Findings: 

The first part of the project (“Establishing doping-related reference distributions for cobalt in human urine”) studied cobalt concentrations in urine samples of athletes. In total, 894 samples were collected worldwide and analysed by ICP-MS. About half of the athletes were from endurance sports (frequency of ESA-testing 30% or above according to WADA TD2014SSA), and about half from non-endurance sports (ESA-frequency < 30%). Furthermore, approx. 50% of the samples were from female, approx. 50% from male athletes. Non-parametric statistical analyses revealed for endurance-sports a median Co-concentration of 0.5 (0.8) μg/L (without and with specific gravity correction) for females (n=220) and 0.4 (0.6) μg/L for males (n=215). For non-endurance sports, the medians for females and males were 0.4 (0.6) μg/L (n=232) and 0.4 (0.4) μg/L (n=227), respectively. Based on the 5th and 95th percentiles the range of Co-values was 0.1 (0.2)-2.1 (2.4) μg/L for non-endurance and 0.1 (0.3)-4.9 (8.9) μg/L for endurance sports. A significant difference between the medians of Co measured in endurance and non-endurance samples (without or with SG correction) as well as the medians of Co in male and female samples (without or with SG correction) was found (p < 0.01). Seventeen samples (w/o specific gravity correction; all IC, 15 from endurance sports) showed Co-concentrations above 10 μg/L with a highest observed value of 948.0 (653.8) μg/L. Two of the 17 athletes declared cobalamin intake. Their values were 55.1 (78.7) and 10.5 (9.5) μg/L, respectively. In order to define possible decision limits (DL) at the 99.99% level, data were log-transformed and “outliers” removed to achieve normal distribution. Different DLs were obtained for males and females, as well as endurance and non-endurance sports and for data without and with SG correction.

The second part (“Reference values for cobalt in serum and urine after cobalamin /vitamin B12) administration”) investigated Co-concentrations in urine and serum samples after application of cobalamin (50/1000 μg cyanocobalamin oral, 1000 μg cyanocobalamin intramuscular, 1000 μg hydroxylcobalamin intramuscular). Only hydroxylcobalamin led to an increase in urinary and serum Co-concentrations. Maximum values were 11.7 (9.8) and 3.9 μg/L, respectively. No influence of cobalamin on ABP-blood parameters (HGB, Ret%) was found. However, the blood drawing system needs to be carefully selected and validated in order to not contaminate serum samples with cobalt as observed with the “butterfly needle system”.

In case a threshold for Co in athletes' samples is defined in the future, it will be necessary to remove cobalamin in confirmation samples as recently shown by Knoop et al. (Rapid Commun Mass Spectrom. 2020;34(7):e8649).

Code: ISF16D12JS

The steroidal module of the Athlete Biological Passport has now been in use for 24 months. The biomarkers used are testosterone, four of its metabolites and epitestosterone. Using these, five steroid ratios are calculated and monitored in the adaptive model. An atypical passport finding will be generated if any of these ratios are outside the individual reference limits, and it is the task of the Athlete Passport Management Unit to evaluate whether further confirmation (i.e. IRMS or follow up testing) is needed. With the exception of the extensively studied T/E ratio, the impact of doping or other confounding factors on the other biomarkers needs further investigation. The largest confounders of the steroid biomarkers are genetic factors, bacterial contamination, alcohol and certain non-prohibited drugs. These confounders are well known and the adaptive model compensates for these, or, in the case of alcohol and bacterial contamination, this is reported by the laboratory. However, it is evident after two years of monitoring steroid ratios in the biological passport that there is still large unexplained variation in the ratios. The origin and extent of this variation in actual doping test results and longitudinal profiles in athletes have not been investigated to date. 
This will be a retrospective study analyzing the variation in steroid profiles (single samples) for > 6000 urine samples, as well as longitudinal profiles (passports) in athletes with >10 samples in their steroidal passport (n ≈ 200), from the beginning of 2014 to date. In order to improve the future interpretation of steroid profiles, we will study the behavior of the biomarkers in e.g. different types of sport, in/out of competition samples, seasonal variations, in athletes using non-prohibited drugs as well as in reported AAFs for prohibited drugs (anabolic agents and/or hormones and metabolic modulators).

Main Findings: 

The steroid module of the Athlete Biological Passport has now been in use for about four years. Its usefulness has been proven when detecting doping with endogenous steroids, e.g. testosterone, especially in individuals with naturally low T/E ratios due to the UGT2B17 deletion polymorphism, with an increased number of positive IRMS analyses in samples with T/E < 4.0. However, the steroid profiles are complicated to interpret and there is still much variation in the five ratios that is difficult to explain. A large systematic study of natural variation and confounders of the steroid ratios and concentrations is needed in order to provide the scientists evaluating the passport with sharper tools, not only to select the profiles suspicious of doping, but also to be able to reject and not spend unnecessary time and resources on profiles showing atypical results due to natural causes. The ultimate goal is to be able to proceed with a passport case, where the steroidal passport is the only evidence of doping.

In this study, we used over 11 000 steroid profiles from Swedish and Norwegian athletes to determine both the inter- and intra-individual variations of all steroids and ratios in the steroidal passport. Furthermore, we investigated if these variations could be associated with factors such as gender, age, type of sport, collection time of day and time of year as well as if the sample was taken in competition or out of competition.

We show that there are factors reported in today’s doping tests that significantly affect the steroid profiles. There were large interindividual variation in the steroid profiles and only part of this variation, up to 16 %, could be explained by the factors studied. The factors with the largest influence on the steroid profile was what type of sport classification the athlete belonged to and if the urine was collected In or Out of Competition. For women all steroids showed higher levels when collected IC than OOC except for 5βAdiol. T/E, A/Etio and 5αAdiol/5βAdiol show higher levels in competition whereas A/T and 5αAdiol/E are lower. For men all ratios but A/T were affected but to a lesser extent. There were also significant differences based on what time of day and time of year the urine sample was collected.

If these significant changes are relevant when longitudinally monitoring athletes in the steroidal module of the ABP, must be further evaluated.

Code: 16E12HH 

Gene doping represents a threat to the integrity of sport and the suitable for publication on WADA's website health of athletes. The anti-doping community has been focusing efforts on developing a test for its detection. The current methodology to detect doping genes in an athletes’ blood uses the polymerase chain reaction (PCR) that targets unique sequences in a doping gene, which correspond to exon-exon junctions in the intronless transgene. These so-called real-time PCR assays detect unique sequences in the complementary DNA (cDNA) for human erythropoietin (EPO) and other doping genes such as insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. 
As the sequences of cDNA of Epo and other doping genes are known, it is relatively easy to aggravate these tests, which will then result in a false-negative result. Recently, we developed a new gene doping detection assay that will overcome this problem. The test is based on targeted sequencing of doping genes with potential to detect any doping gene in any context with a very high sensitivity. Using an in-house designed next generation sequencing assay, we developed a gene doping detection assay for cDNA of EPO which targets all potential exon-exon junctions of all possible EPO-transcripts. 
We propose to evaluate and further develop a multiplex ‘gene doping detection panel’ which targets genes for, among others, insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. The panel allows simultaneous detection of several ‘sport-specific’ genes in one sample, reducing the test’s cost and turn-around-time. This research is crucial in the development of a reliable routine method for detection of gene doping that may be potentially used in all sports. 

Main Findings: 

The main aim of the project was to evaluate and further develop a next generation sequencing-based multiplex ‘gene doping detection panel’. First we developed probes for the detection by sequencing of Erythropoietin, Insulin-like growth factor and Growth Hormone. Second, we evaluated the developed probes in the multiplex sequencing of these genes. Finally, we optimized and fine-tuned the developed method and determined the sensitivity of the developed method.

Our results show that, using the developed probes for next generation sequencing, we were able to simultaneously detect plasmid-derived cDNA copies of Erythropoietin, Insulin-like growth factor and Growth Hormone in a background of genomic DNA with 100% specificity. We were able to detect EPO GH1, GH2, IGF1 and IGF2 cDNA in concentrations below 0.01 percent gDNA at all exon-exon boundaries. For quantification of the amount of cDNA we spiked a GFP plasmid into the samples and found stable numbers of GFP across samples, enabling quantification of gene-doping cDNA levels.

Code: ISF16D21LE 

The administration of recombinant human growth hormone (rhGH) and/or small peptides, i.e. GH releasing factors (GHRF) that stimulate the endogenous production of GH have increased recently as a result of the availability and lack of sensitive tests. Two independent immunoassay methods are currently being employed to detect rhGH doping as well as mass-spectrometric approaches to find GHRFs.  
Our research proposal encompasses projects designed to investigate how the use of rhGH and GHRFs affect the traditional markers as well as putative markers such as miRNA. We will conduct a study in healthy male volunteers that will be given rhGH (Somatropin – two different doses) daily for one week. Some participants will be given two doses of sermorelin (Geref), a GHR peptide, for one week. Urine and serum samples will be collected several times prior to the administration in order to study the different markers longitudinally. Moreover, the steroid profile will be monitored in relation to hrGH/GHRP administration in order to see how the biomarkers of the different ABP modules interact. The use of small GH-releasing peptides is difficult to study in controlled settings since small peptides are not available as traditional drugs. Here we will use samples from patients, both men and women, self-reporting doping with peptides (as well as other doping agents) in order to see which peptides can be identified with the different approaches. 

Main Findings: 

It was found that the GH isoform ratio may detect rhGH intake when 1 and 4IU/day were administered for two weeks to healthy men. Using the biomarker test, none of the participants (n=9) displayed a GH2000 score above the population-based score of 9.98. However, when longitudinally monitored, the GH2000 score and its components IGF-I and P-III-NP, most of the participants showed values outside their individual calculated thresholds (mean four baseline values ±3 SD). Also, the longitudinally testing approach ws studied in four individuals in relation to 5 days GHRH(1-44) administration. It was found that monitoring of IGF-I may be useful for identifying also the intake of GHRH. Additionally, it was investigated if rhGH/GHRH administration exert an impact on ABP biomarkers. A minor increase i RET% and OFF-score after rhGH treatment was found, not resulting in any atypical passport findings. The urinary steroid profile, as well as serum concenctrations of androgens were not affected by rhGH. Moreover, the validity of putative biomarkers was assessed. It was concluded that miRNAs previously associated with GH supplementary treatment were not affected by the rhGH doses given here. Previous findings that fibronectin 1 may be a promising additive protein for detection of rhGH was confirmed.

Longitudinally monitoring of IGF-I and P-III-NP in an endocrine module may be a promising method in the future to increase the chances to detect rhGH/GHRH doping in men. The inclusion of additionaly biomarkers such as fibronectin 1 may increase the effect and/or detection window in some individuals.