Projets de recherche

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Code: 15C18MP 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported. 
Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study. However, scientific studies in humans are very rarely accessible.  
Thus, our project aims at investigating the effects of ecdysterone containing products on human athletic performance. A 12-week intervention study in young man will be conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements will be administered during the study to evaluate the performance enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement will be conducted. 
To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents regular screening for prohibited compounds is included in the project. Furthermore, the administered supplements will be tested for the absence of anabolic steroid contaminations. 

Main Findings: 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported.

Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible.

Thus, our project aimed at investigating the effects of ecdysterone containing products on human athletic performance. A ten-week intervention study in young man has been conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements have been administered during the study to evaluate the performance enhancing effect. Analyses of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement have been conducted.

To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents screening for prohibited compounds was also performed. Furthermore, the administered supplements have been tested for the absence of anabolic steroid contaminations prior to administration.

The ecdysterone administration led to increased serum IGF1 concentrations in comparison to the control group while thyroxin (T4) concentrations decreased.

Significantly higher increases in muscle mass were observed in those volunteers that were dosed with the ecdysterone supplements. Even more relevant with respect to sports performance, also significantly more pronounced increases in one-repetition bench press performance were observed.
These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. We therefore strongly recommend to include ecdysterone in the list of prohibited substances and methods in sports to improve clean competition in the future. As the exact mechanism of action is not yet fully understood, we suggest to include it in class S1.2 “other anabolic agents”.

Code: 15A29OP 

Screening for endogenous androgenic anabolic steroid (EAAS) misuse remains one of the main challenges in doping control.  Currently, this challenge is addressed by quantification of the seven markers forming the steroid profile included in the steroid module of the Athlete Biological Passport (ABP). The common approach for this quantification involves enzymatic hydrolysis, TMS-derivatization and GC-MS(/MS) analysis. However, several markers might be either lost or underestimated by this approach. 
Preliminary experiments performed by the project team show the occurrence of EAAS excreted as bis-sulfates and highlight their potential usefulness for the detection of testosterone misuse.  
The BIs-Conjugates in the Endogenous Profile of Steroids (BICEPS) project aims to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse. For this purpose, the project will be divided in two parts. 
In Part I, the usefulness of bis-sulfates for the detection of EAAS misuse will be evaluated. Bis-sulfate reference materials will be quantitatively synthesized and characterized, and an analytical approach for the quantification of urinary EAAS bis-sulfates will be developed and validated. The validated method will be applied to several samples from available drug administration excretion studies. 
In Part II, the occurrence and usefulness of other EAAS bis-conjugates will be evaluated. Libraries of steroid bis-glucuronides and glucuronide-sulfates will be synthesized. From these available reference materials an open screening strategy will be developed based on MS behavior of the synthesized compounds enabling the detection of EAAS bis-conjugates. The detection of these bis-conjugates in selected postadministration samples will be evaluated. 
Taken together, the BICEPS project will reveal which bis-conjugate metabolites are useful for screening of EAAS misuse. Further, the project will deliver a range of characterised reference materials derived by chemical synthesis for their further study and quantification. 

Main Findings: 

Several important EAAS might remain undetectable with the current approach used for the determination of the steroid profile i.e. gas chromatography-mass spectrometry (GC-MS) analysis after an enzymatic hydrolysis with E. coli β-glucuronidase, and the silylation of the steroids. Among them steroid bis-conjugates remain unexplored. The main goal of BICEPS is to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse.

Firstly, we have characterized the MS behavior of steroid bis-sulfates and we have developed an open screening method for their detection in urine. We have quantitatively synthesized 12 steroid bis-sulfates and we have developed and validated a quantitative method for their determination in urine. The method has been applied to samples collected after oral administration of testosterone undecanoate. We have evaluated several ratios between the validated analytes and we found that they have limited applicability for doping control purposes. However, we found two additional steroid bis-sulfates which allowed for the screening of the misuse with promising results. We hypothesize that these markers are two isomeric forms of the compound 3,16-dihydroxy-5-androstane-17-one bis sulfate. Synthesis of reference material is required to confirm the identity. Using these two markers, we obtained results comparable with those obtained with the best retrospective markers for oral misuse (resistant glucuronides and cysteinyl conjugates).

In a second part of the project we have synthesized some steroid bis-glucuronides and steroid glucuronide-sulfates. We have demonstrated the occurrence of some of them in human urine samples. Preliminary results showed that one of them (5α-androstane-3β,17β-diol 3-sulfate 17-glucuronide) clearly increased after oral testosterone administration supporting its potential usefulness for doping control.

Taken together, the results of BICEPS provide the first evidence about the potential usefulness of bis-conjugates in the doping control field.

The main results of BICEPS have been published at :
1.- McLeod MD, Waller CC, Esquivel A, Balcells G, Ventura R, Segura J, Pozo OJ*. A constant ion loss method for the untargeted detection of bissulfate metabolites. Anal Chem 2017; 89(3): 1602-1609.
2.- Pranata A, Fitzgerald CC, Khymenets O, Westley E, Anderson NJ, Ma P, Pozo O J, McLeod MD*. Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide Reference Materials: Unearthing Neglected Treasures of Steroid Metabolism. Steroids 2019: doi 10.1016/j.steroids.2018.11.017.
Some results have been presented at: 1.- “Steroid bis-sulfates: a forgotten minority” Oral presentation at SUPA2017, Targeting Steroid Sulfation Pathways, Birmingham April 2017

Code: 15E05AB 

We will develop a new approach to gene doping detection based on the leading edge technology, targeted massively parallel sequencing (MPS). Similarly to PCR-based methodology, doping genes will be detected by identifying sequences that do not feature introns present in natural genes. However, the MPS approach will be more reliable since multiple splice sites will be analyzed simultaneously. 
Its sensitivity is likely to be superior due to target enrichment during library preparation and to greater flexibility in choosing targeted regions. The MPS multiplexing capability will allow simultaneous analysis of many samples and genes, reducing test’s cost and turnaround time. We will develop a reference material for the MPS detection of five genes, most likely candidates for doping, and validate the test using blood samples. 

Main Findings:

In this project we developed and tested the protocol for a targeted MPS-based method for simultaneous detection of five transgenes in solutions of doping genes and genomic DNA that we used to mimic DNA extracts from athletes’ blood samples. Enrichment is achieved by PCR that amplifies a large portion of each transgene covering several exon-exon junctions, which should increase the likelihood of transgene detection. This also allows greater flexibility in selecting regions targeted by primers for enrichment PCR and provides a potential advantage of MPS over real-time PCR methods, where assay design is confined to a small area around the exon-exon border. Enrichment PCR for each transgene is performed separately in simplex, before the materials from five PCR, each for one transgene, are mixed prior to being subjected to library preparation and sequencing. With this experimental workflow, it will be relatively easy to add more genes to the detection panel; this will require designing primers and optimising enrichment PCR for new target genes.

We optimised all wet lab steps and designed, produced and tested a prototype reference material that is suitable for use in positive controls in the test.

We developed a tailored bioinformatics pipeline that reliably distinguishes doping genes from the corresponding endogenous genes and from the reference material. The pipeline enabled step-wise analysis of global distribution of alignments for sample and the RM, checking whether the sample is positive for the RM and doping genes, followed by analysis of distribution of alignments across the individual doping genes and their corresponding fragments in the RM and, lastly, making a final positive/negative call for individual doping genes. The process allows optimum elimination of false positives due to accidental contamination of sample with the RM.

Using ‘mock’ samples, we showed that MPS-based approach can detect down to five copies of doping genes in a background of genomic DNA similar in quantity to that in one mL of blood. We demonstrated that the amount of genomic DNA in a sample affects target enrichment for some transgenes more than others and this can affect the sensitivity of their detection.

As part of the evaluation of the long-term potential of the MPS for transgene detection, we performed preliminary assessment of the method's cost, sensitivity, reliability, turn-around time and required infrastructure and expertise, and compared it with the real-time PCR-base transgene detection.

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Elite athletes can be persuaded not to take banned substances – either by appealing to their sense of morality or educating them about the risks of using performance-enhancing drugs, according to previous research.

The aim of this research project was to develop, implement, and evaluate an evidence-based moral intervention, and determine whether it is more effective than a standard educational (i.e., knowledge-based) intervention, in reducing doping likelihood in young athletes, in the UK and Greece.

Researchers developed two separate intervention programmes – one targeting moral factors associated with doping likelihood, the other introducing doping and providing information about the health consequences of banned substances and the risks of sport supplements.

Methodology

Phase 1 - Intervention Development and Screening Survey:

In this phase, the researchers developed a moral intervention consisting of six one-hour sessions aimed at changing predictors of doping intentions: moral identity, moral disengagement, and moral atmosphere. A screening survey was conducted involving over 1000 athletes across Greece and the UK to identify eligible participants with some likelihood of doping. The interventions were pilot-tested with athlete feedback guiding revisions.

Phase 2 - Intervention Delivery and Evaluation:

A total of 280 athletes from 24 clubs in Greece and the UK were selected for the study. Clubs were randomly assigned to receive either the moral or educational intervention. Participants were aged 16-22, participating in individual and team sports. The interventions were delivered over six-to-eight weeks, with measures collected at four time points. Data analysis employed a repeated measures multivariate approach, assessing outcomes for intervention type, gender, and time.

Phase 3 - Focus Groups:

After the interventions, focus groups were conducted with participants (32 in Greece, 35 in the UK) to gather qualitative insights.

Main finding

Both programs were tested on young elite athletes from the UK and Greece, finding that both approaches were effective at deterring the athletes from taking banned substances over a six-month period.

Impact for Clean Sport

1. Program Evaluation: The project highlights the importance of evaluating the effectiveness and outcomes of antidoping education programs. Understanding the impact of interventions is crucial for refining strategies and ensuring that efforts are yielding the desired results.
2. Promoting Values-Based Education: The findings advocate for the promotion of values-based education programs for doping prevention. By emphasizing values such as honesty, fair play, and ethical behavior, these programs can effectively reduce athletes' likelihood of engaging in doping.
3. Fostering Clean Sport Ideals: Values-based education can contribute to fostering clean sport ideals. By emphasizing the significance of ethical behavior and integrity, athletes are more likely to align with the principles of fair competition and uphold the spirit of clean sport.
4. Long-Term Effectiveness: The research demonstrated that the moral intervention had lasting effects on athletes' attitudes towards doping even six months after completion. This highlights the potential for creating interventions that have a sustained impact over time.
5. Policy and Advocacy: The study underscores the importance of advocating for values-based anti-doping education programs at policy levels. Such programs can contribute to a cultural shift within the sports community towards integrity, ethics, and honesty.

Overall, this research project contributes to the advancement of antidoping efforts by providing evidence-based insights into effective education strategies. By targeting athletes' moral identity and values, the project aligns with the overarching goal of maintaining a level playing field, upholding fair competition, and promoting clean sport principles.

Related Publications:

• A Moral Intervention Reduces Doping Likelihood in British and Greek Athletes: Evidence From a Cluster Randomized Control Trial
• Teaching athletes about morality in sport can help reduce doping

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Code: 15D10XT 

The detection of the exogenous administration of synthetic androgens having the same chemical structure of the compounds produced endogenously (i.e the so called “pseudoendogenous” steroids, like testosterone, 5α-dihydrotestosterone and 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 are being collected since 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 for every athlete. Once the ABP detects an atypical profile, an isotope ratio mass spectrometric 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.  New and long term specific endogenous steroids markers have been detected by our research group and others. The data collected up to now suggest that by monitoring such metabolites, the detection window of the abuse of pseudoendogenous steroids can be enlarged.  The use of such markers will permit to suspect of a steroid misuse and to carry out a confirmation process by IRMS, even in such in cases where the use of the current criteria will not be effective.

The main goal of this project is to define and include in the ABP the more relevant and specific pseudoendogenous steroids metabolites permitting to suspect from a steroid abuse and to proceed with an IRMS confirmation. This should reduce the gap between the suspicion and confirmation capacities of laboratories and antidoping authorities.

Main Findings: 

The detection of pseudoendogenous steroids (testosterone or its precursors) is based on the monitoring of selected markers of the steroid profile after a longitudinal evaluation using the Bayesian adaptive model set up in ADAMS. If an individual profile is found atypical, then the application of isotope ratio mass spectrometry (GC-C-IRMS) is mandatory according to WADA Technical Documents in order to disclose the endogenous or synthetic origin of the target compounds found in urine.

GC-C-IRMS requires a comlex sample preparation in order to guarantee the purity of the extracts and subsequent reliability of the measurements performed. The intrinsic charactertistic of the technique makes that is use is limited to those cases that really deserve its application.

In order to better select the samples for IRMS analysis, we have investigated the potential incorporation of additional markers of the steroid profile into the steroid module of the ABP. We have investigated the modifications of the steroid profile including the current parameters of the ABP and the specific hydroxylated metabolites (positions 2, 4, 6, 7 and 16), after controlled administrations of testosterone, androstenedione and dehydroepiandrosterone (DHEA). The results have been compared to the modifications of 13C (%0) delta values obtained by IRMS.

There is no doubt that the specificity of the hydroxylation of pseudo-endogenous steroids is an additional value to get a better insight of the alterations of the steroid profile provoked after the exogenous administrations of testosterone or its precursors (androstenedione or DHEA). Compared to the current markers of the steroid profile included in the steroid module of the ABP, the hydroxylated compounds allow in some cases a better retrospectivity after the administrations (i.e. 2 hydroxylation for AED and T, 4 and 7 hydroxylation for DHEA). These depends on the compound selected, the settings of the detection method used and to some extent on the inter-individual variability.

Hydroxylated steroids being endogenous compounds are present in all urine samples although this can depend on the detection capacity of the method used. In our Laboratory, when working MS/MS and trying to differentiate by MS some isobars (i.e. 2 and 4 OH-androstenedione by selecting minor but specific ion transitions) some compounds can only be detected above some concentration. This indirectly helps to detect at a glance the exogenous administration by monitoring of such compounds. In other cases (i.e. 6OH metabolites, usually present in larger concentrations), it would be necessary to establish reference ranges and to harmonize their quantification among WADA accredited laboratories (to increase their robustness as biomarkers) before thinking in their incorporation to the ABP. The monitoring of hydroxylated metabolites of pseudo-endogenous steroids is less trivial that what one may a priori think. Most of the current GC methods used to screen synthetic and pseudo-endogenous steroids have been designed for the correct separation and quantification of the compounds currently in the steroid module of the ABP. Hydroxylated compounds are late eluding, in an area of the chromatogram of "less interest", where frequent co-elutions may happen, also with some endogenous corticosteroids and pregnanes. To concentrate in a single GC run the capacity of monitoring all the compounds simultaneously is challenging. In our case we have evaluated as much as possible the data obtained under routine conditions and applied an alternative GC method with a different selectivity in those cases where the routine conditions were not adequate.

Although some hydroxylated compounds, specifically in C2, increased for some administrations the detection capacity compared to the current markers of the ABP, their analysis is challenging and in absolute terms, the analysis by IRMS showed the better retrospectivity. The detection of DHEA doping is the most challenging situation. The more significant metabolites of this compound follow sulfo-conjugation before the excretion into urine. This makes the detection of such compoun challenging by the usual and unique monitoring the metabolites excreted in the free + glucuronated fraction. A better knowledge of the sulfate fraction is needed.

A method based on IRMS analysis at the ITP level, considering also the sulfate conjugates may be an option to be investigated in the future to cope with this situation.

Code: ISF15E09MP 

The detection of illegal applied autologous blood transfusion is an everlasting and unsolved problem in the human doping scene. Since no external drugs are applied, no substance residues can be detected in the body. But there might be traceable physiological changes in the transfused blood cells, introduced by sampling or blood/erythrocyte storage. In such a case the detection of the physiological response of the illegal manipulation will be a promising approach.

In animal husbandry the problem of applied growth promoting substances is also present. We could show that illegal given drugs change the transcriptome level of RNA biomarkers in various tissues.  In the case of autologous blood doping, blood cells are directly affected by sampling or storage and thus blood would be a promising matrix for the detection of such transcriptional changes.  In a previous study we could show, that storage of erythrocyte concentrates in stabilization buffer has a tremendous and stable effect on the miRNA profile.

This study was designed to investigate whether those changes are also visible in subjects after autologous blood transfusion and if the storage conditions, e.g. the storage buffer, induces the same changes in the recipient’s blood miRNA profile. Another point of investigation will be to monitor physiological changes in the blood cells that occur after receiving erythrocyte concentrates on small RNA level.  A holistic approach is planned to detect physiological changes on small RNA level. We want to analyze all kinds of small RNAs (miRNA, piRNA, etc) in different blood fractions (whole blood or plasma) via small RNA sequencing. This technology allows a holistic detection of all small RNAs in the biological sample. The clinical trial with healthy subjects that receive autologous erythrocyte concentrates is already approved. Finally a transcriptional biomarker signature for field analysis via RT-qPCR will be developed.
 

Main Findings: 

The World Anti-Doping Code was established to ensure equality and the athletes' health by listing methods and substances whose usages are considered as rule violations. Whereas the abuse of many performance enhancing applications mentioned therein is already detectable, the definite unveiling of autologous blood doping (ABD), for example, is still unfeasible. Based on forward-looking results in biomarker research in the field of microRNAs (miRNAs). the present study aimed at revealing ABD-dependent changes of miRNA signatures in blood samples after ABD.

Therefore, an ethically approved human study was conducted based on a total of 30 healthy and sportive males, who were equally distributed to three groups with different extent of ABD and several sampling time points. Hematological markers were determined by standard clinical laboratory measures, showing indeed, highly significant physiological changes in response to blood donation as well as erythrocyte concentrate re-transfusion. Especially clinical paramters related to erythocyte and iron metabolism were clearly affected, while coagulation and urine markers remained largely ABD-unaffected. In addition, transcriptomic analyses were performed by small RNA sequencing allowing for high-throughput microRNA (miRNA) screening without the need for a prior knowledge. In the following, ABD-dependent changes in the miRNA profile were identified by bioinformatic-driven data analyses. This generated a set of a minimum of 29 miRNAs that could be used as biomarker signature to discriminate doped and non-doped conditions at a high prediction level. Further, sequencing results were selectively validated by real-time reverse transcription quantitative polymerase chain reaction demonstrating exceedingly high and significant correlation.

Next steps will be focusing on the confirmation of the miRNA biomarker signature by applying it to the complete study dataset on the individual level, since inter- and intra-individual variations are still premanent challenges in unequivocal ABD detection. Nevertheless, the present study already contributed enormously to the overall understaning of ABD-dependent changes on the blood level. Due to its high abundance of data as well as comprehensive and well-constructed study design, this study could also act as impeccable starting position for future studies on ABD detection.