Projets de recherche

The proposed project will identify and characterize sportspecific stressors contributing to doping vulnerability among athletes, to include exploring their impact, frequency, severity, timing, and duration. Additionally, it seeks to investigate how psychological flexibility moderates the relationship between these stressors and doping vulnerability, thereby protecting athletes from doping.

A qualitative approach and informed by a co-design process with national anti-doping and sporting organizations. This project seeks to interview women in three countries to 1) investigate the role of support personnel (e.g., coaches, trainers, partners) in facilitating or preventing PIED use, and 2) to understand the broader psycho-socio-cultural factors that contribute to facilitating or preventing PIED use among female athletes. Investigating when, why, and how women choose to use or avoid PIEDs this project will gain important insights on women’s lived experiences with PIEDs.

A cross-sectional survey design and recruit a large sample of elite athletes (1500) from the said countries. The survey will include a number of validated screening tools in Spanish about self-compassion, mindfulness, sport anxiety, perfectionism, and doping susceptibility. Participants will also be invited to take part in a podcast series and to share their insights and experiences on doping in sport. In order to contribute towards more informed decision-making playing a crucial role in the prevention of doping behavior in the PANRADO Region, while focusing on a positive psychology approach emphasizing the role of mindfulness and selfcompassion.

Code: 23GD04KL

The abuse of gene transfer methods in sports is a real and growing concern, the more successes one realizes in gene therapy treatment of hereditary diseases, the more likely the abuse in healthy individuals becomes to improve sports performance. Although the administration manner remains unclear, one can speculate that the most likely method would be a local injection of the transgene(s). Published PCR based methods target cDNA sequences of these transgenes, more specifically the exon-exon junctions. In collaboration with the group of Haisma et al. (University of Groningen, Netherlands), we successfully transferred their published targeted NGS approach to detect exon-exon junctions of 5 potential doping genes to the UZ-Ghent NGS core. However, these proof-of-concept (POC) studies were performed for only a limited number of potential doping genes and with high quality DNA extracted from cell lines. The quality of the input material is similar as the quality from DNA extracted from a fresh blood sample. We would like to optimize this protocol on cfDNA extracted from urine samples so noninvasive samples can be obtained from an athlete. In the POC study, probes were designed to capture the exon-exon boundaries of all transcripts of EPO, GH1, GH2 and IGF. We will now expand the target region with additional potential doping genes, including the coding regions of these genes to evaluate other relevant gene doping marks. A genomic fingerprint will be added so samples can be assigned to correct individual. This method ensures the accurate identification and tracking of biological samples in various fields, including genetics, forensics, and clinical research. Single nucleotide Polymorphisms (SNPs) are genetic variations that involve a single nucleotide change at a specific position in the DNA sequence. They are commonly used as genetic markers due to their abundance and stability. These SNPs should exhibit high variability among individuals or populations to differentiate samples accurately. The unique genetic profiles generated by SNP genotyping allow for accurate sample identification, preventing errors and ensuring the integrity of data and results. In a second work package we will reinvent the data analysis part. Manual counting of the reads (as published by De Boer et al. 2019) is not feasible, especially not if the number of samples to be tested increase. We will further optimize the data analysis to an (semi) automated pipeline for read counting and program an additional module to take non-human traces and gDNA variants into account. Overall, these adjustments to the initial proof of concept study will prepare this analysis to be implemented as a gene doping test.

Code: 23A10XT

Isotope ratio mass spectrometry's (IRMS) efficiency for detecting pseudo-endogenous steroids depends on the difference between the isotopic labeling of the formulations available in the market and the natural isotopic labeling of the endogenously produced steroids by the athletes. The more frequent presence of pharmaceutical formulations with values close to the endogenous ones is becoming challenging and in some populations makes the current detection approach useless. This phenomenon already observed for testosterone preparations lead WADA to establish some countermeasures as the ABP endocrine module (for their indirect detection) or the analysis of the actual testosterone esters (the most frequent way of administration) in blood samples or dried blood spots (DBS). We are here proposing a study directed to verify the confirmation capacity of nandrolone esters in DBS or blood and the effect of the administration over the parameters of the endocrine module. This will be compared with the current approach by GC-MS/MS (for the Initial testing procedure- ITP) and GC-C-IRMS (for the confirmation procedure- CP). Formulations not detectable with the current approach will be included in the study.

Code: 23GD03HS

Gene doping is defined as “the non-therapeutic use of genes, genetic elements and/or modified cells that have the capacity to enhance athletic performance” and is categorized as a threat to the integrity of sport and the health of athletes by the World Anti-Doping Agency (WADA). The only direct approaches for detecting gene doping are PCR assays, so introducing a novel alternative approach for rapid and accurate identification of gene doping is vital. In this project, a novel ultrasensitive detection platform will be designed based on the combination of 2 different technologies i.e. CRISPR and Microfluidics. CRISPR/CAS systems can survey the whole genome and detect transgenic DNA (tDNA) using a single-stranded guide RNA (gRNA). In the presence of a target gene, a ternary complex (Cas12/gRNA/tDNA) will be formed and the quenched fluorescence probe will be degraded by collateral cleavage activity of the ternary complex. Microfluidics fluorescence detection can provide exquisite sensitivity. The combination of CRISPR/CAS as a biological gene doping detection system and a microfluidics system can provide multiplex gene doping detection (up to 96 samples with 96 detection assays per chip). Pre-amplification technologies such as recombinase polymerase amplification will be applied in order to reach single molecule (1 aM) sensitivity. Here, we will investigate more than 20 genes including CSH1, CSH1,CSH2, CSHL1, EPO, GDFB, GH1, GH2, IGF1, IGF2, MYOG, PPARd, VEGF, etc. as a proof of concept. However, the number of assay combinations could be 96 samples with 96 detection assays (96 different Cas/gRNAs). The gRNAs will be designed to target exon-exon junctions, specific sequences of viral vectors, such as CMVp (cytomegalovirus promoter), TkpA (thymidine kinase polyA from herpes simplex virus), hexon (major virus capsid protein from adenovirus), and CMVp (cytomegalovirus promoter) etc. The use of combinations of different gRNAs for the same gene (several gRNAs for different exon-exon junctions) with virus targeted-gRNAs will allow us to detect doping genes and different vectors simultaneously and dramatically increase the successful detection of tDNAs. Moreover, specific CRISPR-reference materials (C-RM) containing modified transgenic sequences will be designed to avoid false positive results due to cross-contamination. Based on the WADA guidelines for gene doping, appropriate no template controls (NTC) and positive template controls (PTC) will be used to determine the specificity, efficiency, sensitivity and limit of detection of the assay. The performance efficiency of the detection platforms will be tested in spiked blood samples. These multiplexed gene doping detection CRISPR-equipped microfluidic platforms could be an ideal and rapidly deployable detection approach, which is accurate, inexpensive and rapid, allowing for anti-gene doping assays in WADA-accredited laboratories.

Code: 23A20MP

GC-C-IRMS is used to confirm the exogenous origin of endogenous anabolic steroids. Samples are analyzed with a fast initial testing procedure to isolate suspicious samples and, afterwards, the suspicious samples are analyzed with an IRMS confirmatory method. High throughput IRMS will allow a substantial expansion of CIR analyses, enabling a considerable increase in doping violation detections and the set-up of an efficient CIR ABP module. High throughput IRMS is also highly desirable in those cases where a large number of samples needs to be tested on IRMS in relative short period of time e.g., preseason/regular season testing and Olympic Games.

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.