Projets de recherche

Ce document n'est disponible qu'en français

Ce document n'est disponible qu'en anglais.

WADA has repeatedly encouraged social science research projects to take a regional perspective. The Caribbean is one region where systematic research on doping has been absent, despite calls for action (Acevedo et al., 2011). The paucity of doping research on Caribbean countries is surprising given the region’s strong sporting tradition and history. Sport is part of the Caribbean’s regional identity (CARICOM Commission on Youth Development, 2010), as witnessed by their success internationally. For instance, at the 2016 Olympic Games, 5% of Gold medals were won by Caribbean countries (Jordens, 2016).

Description coming soon.

Code: T20M03RV

Recent research has demonstrated that the criterion of discrimination between allowed and prohibited administrations of GCs needs to be compound specific. For some GCs, largely detected in doping controls, no sufficient data is available, and the objective of this project is to perform excretion studies with these GCs to generate the data needed to define the reporting levels and the washout periods.

The project will be focused on dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF).

The research will be focused on the following specific objectives:

• To perform excretion studies of DEX, MP and DEF with administration of one single oral dose. For DEX, multiple oral doses will be also studied.
• To measure concentrations in urine of the parent compounds and the main metabolite in case of DEF, to evaluate the reporting lebels and washout periods.
• To measure concentrations of the compounds and cortisol in plasma, to evalulate the concordance of the urinary reporting levels and washout periods proposed with plasma concentrations of the active drug and the systemic effect

Main findings

Glucocorticoids (GCs) are prohibited in-competition by all injectable routes (including intraarticular and periarticular routes) in addition to oral and rectal administrations. In out-ofcompetition periods, there is no restriction of use. Since most GCs are marketed in different administration forms, the distinction between administration routes is needed to ensure safe treatments by allowed administration routes and to detect the use of prohibited administration routes during competitions. The regulations of GCs in sports were revisited in 2022, and new criteria were established for some of the compounds. In the present study, the discrimination criteria was evaluated for three GCs: dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF).

Five clinical studies which involved oral administration of GCs to healthy volunteers were performed: single oral dose of DEX (4 mg, n=8), multiple oral dose of DEX (2mg/12h/5 days, n=8), single oral dose of MP (12 mg, n=8), multiple oral dose of MP (12 mg/3 days, n=8) and single oral dose of DEF (30 mg, n=8). Urine and plasma samples were collected before, during and after the treatments, and both the urinary and plasmatic excretion profiles of each GCs and their metabolites were evaluated using liquid chromatography-tandem mass spectrometry. Overall, the results of the project demonstrate that the minimum reporting levels (MRL) of 60 ng/mL for DEX, 30 ng/mL of MP and 30 ng/mL of desacetyldeflazacort (DEF metabolite) are suitable to detect oral administration of the compounds and the washout-period of 3 days for oral administration is also adequate for these GCs. Results obtained in this project provide additional data that supports that criteria based on substance-specific MRL improve the discrimination between prohibited and permitted use of GCS in sports.

Code: T20M02DM

The differential immunoassays to detect doping with recombinant human growth hormone (hGH) have been in use at WADA accredited laboratories for more than 10 years now. From the beginning, the assays were produced in the coated tube (CT) format. In the past, this format, where the specific antibodies are coated to plastic tubes, and the final assay signal is determined by a tube luminometer, was very popular for immunoassays used in laboratory medicine and research applications. However, it meanwhile has been largely abandoned, and the respective assays were switched to the so-called microtiter plate (MTP) format. Here, the antibodies are coated to 96 wells of a plastic plate, and the final assay signal is read by a plate luminometer. The microplate format has advantages in terms of production, but also in terms of workflow in the laboratory. As a consequence of the grossly reduced use of CT assays in laboratories worldwide, production capacities for such CT assays become rare, and might become unavailable in the near future. To ensure continued production and availability of the differential immunoassays it is suggested to convert these assays to the MTP format now. While assay ingredients (and particularly the monoclonal antibodies involved) remain unaffected by the conversion to the MTP format, and it has been demonstrated for many assays used in clinical routine that key parameters such as assay specificity, linearity, reproducibility etc. remain unaffected by the change, the MTP format nevertheless has to be optimized and validated for each individual assay. The current project aims to establish the MTP format for the differential immunoassays.

Code: R20M01MS

Primarily, the objective of this project is to conduct an exploratory study to monitor the blood steroid profile of women subjects over two menstrual cycles to compare the intra- and inter-individual variability of the blood steroid profile of women subjects during a menstrual cycle. Secondly, transdermal testosterone gel (Androgel 1%, AbbVie, North Chicago, IL) will be administered to all volunteers once. The study will enable the pharmacokinetic study of transdermal testosterone gel in women in addition to its detection and will allow to determine putative differences of steroid metabolism between men and women. Simultaneously, the urinary steroid profile will be monitored to allow comparison of the steroid profile between both matrices. This study should help to develop further the blood steroidal of the ABP and to refine the identification of doping behaviours in females with adequate investigations allowing to define reference values for specific female athletic populations. This study will also allow to lay the foundations for a further untargeted metabolomics project for the discovery of new steroidomic biomarkers for steroid detection in female athletes. The design of the study was modified and extended to three menstrual cycles and the treatment regimen was modified. Instead of a single application, testosterone gel (Tostran 20mg/g, 0.5g applied corresponding to 10mg of testosterone) is applied daily for a menstrual cycle (corresponding to 28 days).

Main Findings

In women, hormonal fluctuations related to menstrual cycle may impose a great source of variability for some urinary biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. Additional biomarkers and alternative matrices need therefore to be investigated to improve the detection capability for doping practices with T, especially in female athletes. The aim of this study was therefore to investigate the impact of menstrual cycle combined with T gel administration on the biomarkers of the ABP (steroidal and haematological module) and on serum steroid biomarkers in females. It allowed to directly compare the sensitivity of T gel detection between urinary and blood steroid profiling either for targeting samples for IRMS or for longitudinal evaluation. To achieve this, a clinical trial involving fourteen healthy women subjects was conducted over three consecutive menstrual cycles with the second cycle combined with a daily administration of T gel for 28 days. The sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers was investigated for the monitoring of the T gel treatment. Additionally, endogenous fluctuation of these parameters were monitored within the menstrual cycle.

Code: 20D09OP

In previous WADA funded projects (11A9RV, 12A13OP, 14A29OP) our research group discovered two endogenous steroid (EAAS) metabolites, namely, 6β-hydroxyandrosterone-3-glucuronide (6bOHA-3G), and 6β-hydroxyetiocholanolone-3-glucuronide (6bOHEtio-3G) particularly selective markers of the administration of testosterone. Small quantities of the compounds were synthesized and fully characterized. They showed to be resistant to enzymatic hydrolysis, a reason for its late discovery.

An analytical method has already been developed, fully validated and published for the direct analysis of those substances together with other regular steroid profile glucuronide markers. One way to improve the the application of the method would be to have access to labelled internal standards which will decrease the variability of the basal values.

These two new markers have proven to be sensitive to testosterone administration (oral, intramuscular and even transdermal, improving its detection window, as compared to the current T/E ratio. We are  currently testing their specificity to differentiate testosterone administration from ethanol consumption or the combination (WADA project ISF18D13OP). Preliminary results of this on-going project show that while TG/EG increases both with T and EtOH administration, the new markers increase specifically after T administration while they do not increase after EtOH administration. This differential behavior adds specificity and improves the steroid profile. 

The current project (inter-ALMA) aims at finally incorporating these new markers into a routine screening by performing an interlaboratory validation incorporating 5 different laboratories. For that purpose, analytical methodology, standards (already synthesize in the frame of the 2019 PCC project entitled “Essential reference materials to advance the long term detection of testosterone abuse”) and their deuterated analogues (synthesized in the frame of iner-ALMA) will be provided to the laboratories. The outcome after using common or comparable methodologies to analyze selected samples will be compared.

Code: 20D03JH

The use of information technology within sport has significantly increased over recent years. The availability of data for longitudinal tracking of athletes over the course of their careers is critical to identify an individual’s performance progress. As such, a key component of this longitudinal monitoring is to be able to differentiate between “normal” increases in performance caused by maturation and training, from an “unnatural” improvement caused by doping. Our initial work demonstrates that it is possible to identify specific performance profiles characteristics for athletes with previous ADRVs, which appear to be consistent across the track and field disciplines we have investigated (100-800m, jumps & throws). However, there is a need to extend the current model and translate it into a useful tool for anti-doping authorities. Therefore, the purpose of this study is to refine and further develop our existing Bayesian modelling approach. Specifically, this project will enable us Page 2/8 to explore confounding factors for modeling performance data, and at the same time explore the effects of these confounders on the probability level needed to flag a suspicious individual, balancing false positives (clean athletes identified as doping) and false negatives (doping athletes which are not flagged). We will therefore develop a “risk” score for suspicious performance profiles. A key aspect of the method will be its impact on decisions made via the traditional Athlete Biological Passport (ABP). Working in conjunction with the AIU, we will retrospectively explore whether performance data adds value to decisions made by expert witnesses in historical passport cases. Using this approach, we will evaluate whether adding performance data to existing ABP data is more effective than current methods for identifying athletes who demonstrate may present adverse, or uncertain passport profiles findings.

Main findings

As the aim of any doping regime is to improve sporting performance, it has been suggested that analysis of athlete competitive results might be informative in identifying those at greater risk of doping. The aim of this research project was to investigate the utility of a statistical performance model to discriminate between athletes who have a previous anti-doping rule violation (ADRV) and those who do not.

We analysed performances of male and female 100 – 10,000m runners obtained from the World Athletics results database using a Bayesian spline model. Measures of unusual improvement in performance were quantified by comparing the yearly change athlete's performance (delta excess performance) to quantiles of performance (50%, 75% and 90%) in their age matched peers from the database population. Sudden or unexpected changes in an athlete's level of excess performance the exceed these quantiles might therefore be indicative of doping. The discriminative ability of these risk measures was investigated using the area under the ROC curve (AUC) with the highest values being observed using the 75% quantile (AUC = 0.78-0.80). To assess the specificity of the model using the 75% quantile at different age points we assessed the False Positive rate across different probability levels for delta excess performance. The true positive rate ranges between 0.20 and 0.67 across the ages due to the changes in the number of observed true positives (i.e. ADRVs) recorded at each age, and athletes within the database.

Further, we investigated the ability of delta excess performance to discriminate between athletes with and without adverse analytical findings (AAFs), adverse passport findings (APFs) and Anti-Doping Rule Violations (ADRVs). The 75% quantile for delta excess performance demonstrated AUC values of ~0.60 at age points with the highest numbers of APFs. However, by comparison, the model showed a better ability to discriminate the ADRV status of athletes by AUC values of ~0.65 to 0.75 at the same corresponding age points.

The findings of this project demonstrate the utility of performance monitoring to discriminate between athletes on the basis of their doping status. However, it is important to recognise that high levels of delta excess performance are not sufficient to prove an athlete is doping, and that information obtained from this type of analysis should be integrated with other data as part of a wider intelligence gathering approach to anti-doping.

Publication:

Hopker JG, Griffin, JE. Hinoveanu, LC. Saugy, J. Faiss, R. (2023) Competitive performance as a discriminator of doping status in elite athletes. Drug Testing and Analysis. 16:473-81. doi: 10.1002/dta.3563.

Code: 20D02FP

Volumetric Absorptive Microsampling (VAMS) is a recent microsampling technique used to obtain dried specimens of biological fluids that could represent in the near future a valid alternative to urine, whole blood and serum sampling for anti-doping purposes. Although similar to Dried Blood Spots (DBS), VAMS promise to bring a number of significant advantages over them: i) improving quantitative performance thanks to higher sample volume acccuracy; ii) obtaining the extraction yield and reproducibility without and impact of hematocrit (HCT) value; iii) enhancing the overall analytical performance, resulting in better correlatio with plasma values; iv) simplifying collection procedures by homogeneous samples with a lower reject rate; v) availability of 96-well formats, opening the access to automation. In this study, the suitability of VAMS, in 30µL format, as an innovative matrix for the measurement of novel highlighted blood markers of Endogenous Anabolic Androgenic Steroids (EAAS) doping will be evaluated. A UHPLC-MS/MS method for the quantification of a significant panel of circulating steroid hormones and their phase II metabolites will be developed. Mass spectrometric parameters and transitions will be fine-tuned to achieve the maximal sensitivity needed to detect ad quantify as many markers as possible. Chromatographic conditions will also be thoroughly optimized in terms of separation of isomers with the aim of finding the best compromise between resolution and analysis time. Validation procedure will be carried out following the WADA requirements. Various parameters, such as limits of detection and quantification, precision and accuracy, matrix effects, selectivity, repeatability and robustness will be investigated for each measured analyte. The developed analytical method will be transferred to a WADA-accredited laboratory and an inter-laboratory comparison will be performed. Finally, results will be discussed putting particular emphasis on the systematic comparison with more conventional hematological matrices, such as serum and plasma.

Main findings

A novel LC-MS/MS method for the measurement of 18 steroids panel, including main endogenous steroid hormones and androgen phase II metabolites, was developed and validated in compliance with WADA requirements for quantitative methods. A simple sample preparation procedure was optimized and resulted in extraction recoveries ranging from 69.8% to 92.5%, while showing ion suppression-related matrix effect around 30% for most of target analytes. The validation protocol allowed demonstrating the satisfactory performance of developed method in terms of selectivity (separation of isomers and exclusion of interferences), trueness, repeatability, precision, combined uncertainty, linearity range, LLOQ, carry-over and overall robustness. Finally, the method was successfully transferred to the Lausanne WADA-accredited laboratory. A stability study involving 20 healthy subjects (10 males and 10 females) demonstrated that steroid concentrations measured in VAMS samples stored at room temperature, 4°C, -20°C and -80°C, do not deviate from the values measured in baseline samples, with calculated mean percentage differences being always lower than Reference Change Value threshold which estimated method’s analytical variability. Moreover, the influence of up to three consecutive freeze and thaw cycles was evaluated as not significant. VAMS proved to be a valid collection strategy for measuring steroid hormones in blood that could be employed in doping control analysis with the goal of increasing sampling frequency dedicated to the newly implemented Blood Steroid Profile (BSP). The stability of steroid hormones in blood microsampling collected on VAMS support opens the way of interesting advantages for blood samples’ transportation and storage for anti-doping purposes. Nevertheless, prior to the introduction of VAMS sampling for BSP analysis, further studies aiming at evaluating the correlation between steroid concentration levels measured in VAMS (capillary whole blood) and in currently employed serum samples should be performed in the future to gather an exhaustive overview of VAMS potential in anti-doping context.

Publications/Presentations related to the project

Publications:

- F. Ponzetto, M. Parasiliti Caprino, L. Leoni, L. Marinelli, A. Nonnato, R. Nicoli, T. Kuuranne, E. Ghigo, G. Mengozzi, F. Settanni, LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes, Clin. Chim. Acta (2024), under review.