Projets de recherche

Code: 23B02CR

Chapter S2 of WADA’s Prohibited List 2023 (“Peptide hormones, growth factors, related substances, and mimetics”) lists Erythropoietin Receptor Agonists (ERAs) and Transforming growth factor beta (TGF-ß) signalling inhibitors (luspatercept, sotatercept) under chapter 1 (“Erythropoietins (EPO) and agents affecting erythropoiesis”) as prohibited substances. Currently, SAR- and SDS-PAGE are the most frequently applied techniques for the initial testing and confirmation procedures (ITP, CP) for ERAs in WADA accredited laboratories worldwide (TD2022EPO). While electrophoretic detection methods for TGF-ß signalling inhibitors were also developed, they are not yet part of the technical document. In 2019 and 2020, the first luspatercept-based pharmaceutical (Reblozyl®, “luspatercept-aamt”) was approved by FDA (USA) and EMA (Europe).

Hence, routine testing for TGF-ß signalling inhibitors will become necessary in the near future. For the detection of erythropoietins and TGF-ß signalling inhibitors in blood and urine immunoaffinity purification is required before electrophoretic separation. Due to significant structural differences between these compounds, three different capture antibodies have to be employed, i.e. anti-EPO, anti-activin receptor type IIA (ACVR2A), and type IIB (ACVR2B) antibodies for epoetins, sotatercept and luspatercept, respectively. A protocol for the combined immunopurification of ERAs, sotatercept and luspatercept followed by isoelectric focusing (IEF-PAGE) was published in 2019, another one for ERAs and luspatercept in combination with SDS- and SAR-PAGE in 2021. Both protocols were based on covalent immobilization of relatively large amounts of the capture antibodies on magnetic beads.

Consequently, the beads had to be re-used several times for cost-reduction. Additionally, sotatercept was not included in the protocol for SDS- and SAR-PAGE. We already developed individual protocols for capturing sotatercept and luspatercept in serum samples, which use non-covalent immobilization of very small antibody amounts on anti-antibody coated magnetic beads. Additionally, we presented a similar cost-minimized protocol for the purification of EPOs from blood and urine at the Cologne workshop in March 2023. The plan is to combine these protocols in order to simultaneously purify all three compounds from the three sample matrices (serum/plasma/urine). Another disadvantage of the 2021 protocol was, that EPOs and luspatercept could not be detected in a truly multiplexed way after electrophoretic separation and Western blotting.

The reason were interferences caused by non-specific binding of the two detection antibodies with co-eluted proteins (the protocol used the same polyclonal antibody for capture and detection of luspatercept). Hence, the membrane had to be first incubated with an anti-EPO antibody followed by re-incubation with the anti-ACVR2B antibody. Contrary to that, the proposal of this project is that (1) the target proteins will be simultaneously detected by incubating the blot membrane with a mixture of all three detection antibodies, (2) it will also include sotatercept, and (3) the protocol will also be applicable to urine (it was shown in 2022 that luspatercept is also partly excreted in urine).

Code: 23E04FP

It is well known that the use performance-enhancing substances by athletes provide an unfair advantage and doping is contrary to the ‘the spirit of sport’. However, a randomised response surveys conducted between 2011-2018 indicate that >15% of athletes have doped with banned substances. Yet in 2018, WADA reported only 2% positive samples, with hormone doping accounting for >50% of adverse analytical findings. These statistics suggests that micro-dosing, off-competition doping and cyclical (on/off)/pyramiding patterns of administering anabolic steroids (AAS) have allowed athletes to evade current detection methods, while beneficial for building muscle mass, strength and performance. In addition, drug administration studies in humans have demonstrated that testosterone dose-dependently increases satellite cell and myonuclei number, muscle fibre cross sectional area (CSA) and induces hypertrophy of muscle fibres. These benefits may remain long after cessation of AAS use and/or detraining leading a muscle atrophy, when a new stimulus (i.e., returning to training) is given, the performance is enhanced, a phenomenon known as “muscle memory”. The “muscle memory” associated with AAS could explain why androgen cycling has been so effective for doping athletes. AAS treatment in mice for 14 days has been shown to increase myonuclei number by 66%, and these are retained despite drug withdrawal and contribute to faster muscle growth in response to muscle overload (Egner et al, 2013). These findings indicate that even brief exposure to AAS may provide long-term benefits for muscle performance. Increasing understanding of the mechanisms associated with AAS muscle memory is thus vital for enhancing anti-doping detection. Other mechanisms, besides myonuclei accretion/retention, have also been implicated with the muscle memory phenomenon, and the understanding of this process is crucial for the identification of long-term biomarkers. Therefore, the aim of this research is to identify relevant biomarkers associated with past-AAS use. Building on previous work, we will generate and characterise a mouse model of AAS muscle memory and explore the associated mechanisms using transcriptomic, phosphoproteomic and metabolomic approaches. In our previous research funded by a WADA research grant (16E11FP), we have established a tissue biobank (muscle biopsies, saliva, urine, serum) from powerlifters that either do not use AAS, actively use AAS or have previously used AAS. Guided by findings in mice, we will examine the utility of these markers for identifying current and past-AAS use in our biobank of tissues. Our findings are envisaged to enhance anti-doping detection, provide guidance for appropriate punitive measures in response to positive tests, and contribute to ongoing debates regarding transgender athlete inclusion in elite competition.

Code: 23D10YD

Our main purpose of this study is to evaluate non-analytical factors that may influence the markers in the serum steroid profiles and endocrine profiles, which are indirect methods to detect doping with anabolic androgenic steroids and growth hormone, respectively, and which will be integrated into the WADA Athlete Biological Passport (ABP) in 2023. The new knowledge will support the interpretation and evaluation of serum steroid and endocrine profiles, and may increase the sensitivity of the ABP. In project 1, 27 Swedish athletes with symptoms of long-term low energy availability (LEA) and 27 matched controls are invited to participate in a cross-sectional study evaluating the influence of long-term LEA on the serum endocrine and steroid profiles in athletes. It is hypothesized that male and female athletes with long-term LEA expressed as reproductive dysfunction (amenorrhea/low libido) will have suppressed levels of present and new biomarkers in the serum steroid and endocrine profiles compared with male and female athletes with normal reproductive function without symptoms of eating disorders. In project 2, 22 female national level powerlifters and weightlifters in Norway are recruited to evaluate changes in endocrine and steroid profiles in serum after a familiar resistance training session at two different time points (follicular phase and luteal phase) in the menstrual cycle. Based on existing literature, it is hypothesized that present and new serum steroid biomarkers are unaltered and that endocrine biomarkers are increased and more variable immediately after a resistance training session in female weightlifters and powerlifters during either menstrual cycle phases.

Code: 23C07KD

Tesamorelin is a synthetic growth hormone-releasing hormone (GHRH) analogue, used for the reduction of excess abdominal fat in HIV-infected adults. As a GHRH analogue, it triggers the secretion of growth hormone (GH), which in turn increases serum insulin-like growth factor 1 (IGF-1) levels. The latter exerts an anabolic (growth) effect on tissues throughout the human body. Since this anabolic effect is evidently beneficial for many athletes, the GHRH – GH –IGF-1 axis is a common target in sports doping. All three compounds, as well as their analogues, are banned by the World Anti-Doping Agency (WADA) at all times. Besides curating the list of prohibited compounds, WADA also provides doping control laboratories with a set of minimum required performance levels (MRPL) for these compounds. These MRPLs indicate the sensitivity that a detection method should achieve for a certain compound and are commonly based on administration studies. Since doping often corresponds to the illicit use of prescription medicine, administration studies for many prohibited compounds have only been performed in the context of a clinical trial involving a specific patient population. This research project aims to perform a Tesamorelin administration study on six healthy volunteers. This will allow for a re-evaluation of the current MRPL using a study population more representative of healthy athletes, as well as generate insight into the urinary excretion pattern of Tesamorelin. Both are important factors for the anti-doping community in evaluating the performance of detection methods. For the second objective of this project, the influence of Tesamorelin administration on serum IGF-1 levels will be monitored to investigate the possibility of using IGF-1 as a long-term marker for GHRH doping. Also P-III-NP levels will be assessed to evaluate if the IGF-1/P-III-NP ratio (as will be applied in the endocrine module of the athlete biological passport) is a stronger marker than IGF-1 alone to detect Tesamorelin abuse and GH secretagogues in general in sport.

Code: T23A05GG

The project deal with the synthesis and analytical characterization of an epimeric long-term metabolite of the anabolic steroid Dehydrochloromethyltestosterone (4a-chloro-18-nor-17b-hydroxymethyl-17a-methyl-5a-androst-13-en-3a-ol)

Code: T23A03GG

The project deal with the synthesis and analytical characterization of a long term metabolite of the anabolic steroid oxymesterone (17β-methyl-4,17α-dihydroxy-androst-4-en-3-one)

Code: T23A02GG

The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the stimulant Adrafinil (Diphenylmethyl)sulfinyl]-N-hydroxyacetamide)

Code: T23A01GG

The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the anabolic agent Andarine (2S)-3-(4-acetamidophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide)

Code: 23E05FD

Several polymorphisms/variants of the human EPO gene could be the cause of confounding effects in the application of the analytical methods that are routinely applied for the detection of the abuse of recombinant erythropoietin (recEPO). The most known of them is the c.577del (rs369859204) variant, a single nucleotide deletion in the position 577 of the human EPO gene exon 5. The deletion causes a shifting of the sequence within the terminal exon 5 with consequent loss of the stop codon. At translational level, this results in the formation of a longer mature protein with a higher molecular weight of 3.3 kDa. In doping control, this results in the visualization of a “double band” during the application of the routinary SAR-Page method for the detection of the abuse of recEPO. The double band may be then erroneously interpreted as a false positive sample. As stated in the WADA technical document TDEPO2022, it is suggested to screen for the presence of the c.577del variant whenever a suspected double band is detected by the SAR-Page analysis, in the final aim to avoid the possibility of a false positive finding. Moreover, at least 12 other polymorphisms/variants have been described in the literature that are potentially capable of generating confounding effects in the interpretations of the antidoping analyzes. By following the input given by WADA TDEPO2022, this project involves the development and validation of a genetic test, based on the genomic DNA Sanger sequencing method, that is capable to identify the c.577del variant and all the other polymorphisms/variants present on the human EPO gene (namely: rs1554393458, rs1554393463, rs1419397684, rs773895305, rs763035217, rs1562901775, rs71517124, rs1562902091, rs893404064, rs752755372, rs951322017, rs369859204), and the study of their incidence in determining confounding effects in the interpretation of the routinary tests performed for the purpose of detecting the abuse of recombinant EPO by athletes.

Code: 23D05SV

The aim of this study is to use artificial intelligence (AI) to evaluate blood smears as a potential matrix to detect doping with recombinant EPO (rEPO). Previous publications reported changes in the Red blood Cell Morphology after rEPO administration (e.g. Macrocytes, Stomatocytes). During these times automated haematology analysers and manual microscopy were used for the estimation of these parameters. Recently, digital Morphology is a developing field in Haematology which enables the automated analysis of blood smears by artificial intelligence. With this technology it is possible to evaluate morphological changes with an increased precision, based on a higher number of cells and to discover even minor changes in cellular shapes. AI and deep learning is capable of revealing new insights which conventional approaches were lacking so far, like predicting molecular changes on cytomorphology. Therefore, the goal of this study is to identify relevant changes in cell morphology during rEPO administration which are not addressed using current state of the art techniques. In the long term, a blood smear-based athlete blood passport providing an individual erythrocyte signature might be a prospective application of monitoring athletes by using artificial intelligence based on this postulated deep learning model.

Main findings

A total of thirty healthy athletes participated in a controlled, interventional trial in which they received either rEPO or a placebo over a three-week period. Each participant provided 13 blood samples across an 11-week timeframe, encompassing pre-administration, administration, and post-administration phases. PBS were prepared and digitized at each time point for subsequent analysis.Hematological parameters, including hemoglobin concentration, hematocrit, and reticulocyte indices, showed distinct temporal patterns between the rEPO-treated and placebo groups. Digitized PBS were analysed using a customized deep learning pipeline based on the Haemorasis pipeline, specifically adapted for this study. For the binary classification task comparing PBS samples obtained after iron supplementation but without/before rEPO to those obtained at the peak of reticulocyte response following rEPO administration, the model achieved an area under the receiver operating characteristic curve (AUC) of 0.79 for the entire cohort. Subgroup analyses yielded an AUC of 0.72 for male participants and 0.66 for female participants. These preliminary findings suggest that AI-driven morphological analysis of PBS may serve as a novel and complementary approach for the detection of rEPO doping. However, further validation in larger and more diverse cohorts is necessary to confirm these results and refine the model for potential application in anti-doping efforts.