Projets de recherche

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.

Code: 15A21MP 

As per list of the World Anti-Doping Agency (WADA) 2015 Oral-Turinabol (4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one), which was extensively misused by GDR athletes is prohibited in sports. In recent years it has been rediscovered by producers of "dietary supplements" and gained new importance in doping control (continuously increasing numbers of adverse analytical findings since 2003). Following its administration long-term metabolites with 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrost-13-ene structure have been identified. However these metabolites are not available as reference substance up to now. Even though the use of post-administration urines instead of purified reference material has been accepted in confirmatory analyses, the athletes in question may challenge the results of the anti-doping laboratories resulting in prolonged trials. The goal of the present project is to produce Oral-Turinabol long-term metabolites with 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrost-13-ene structure that cannot be synthesised via classical chemical synthesis. Therefore a joint chemical synthesis and biotechnological approach will be applied. The substrate (17,17-dimethyl-18-nor-13-ene intermediate) will be converted by single-step hydroxylation to the desired product in a whole-cell biotransformation assay using recombinant strains of fission yeasts that express the human cytochrome P450 enzymes CYP 3A4 or CYP 21 as already successfully performed for the analogue metandienone long-term metabolite by our group, that is now available in worldwide anti-doping laboratories. As already published by our group both enzymes catalyze the respective reaction in Oral-Turinabol as well. Prior to the biotechnological hydroxylation, the substrate will be chemically derived from Oral-Turinabol by Wagner-Weerwein rearrangement. Purification and subsequent NMR structure confirmation will be performed and the product distributed to the anti-doping community. 

Main Findings: 

Anabolic androgenic steroids (AAS) are misused very frequently in sport competitions as performance enhancing agents. One of the doping compounds that has been detected with increased frequency in the last few years is dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one; brand name Oral Turinabol). Since the parent drug DHCMT (10) is eliminated relatively fast from the human body, efforts to detect its illicit use have concentrated on its metabolites for many years. In recent years, some metabolites with 17β-hydroxymethyl-17α-methyl-18-norandrost-13-ene structure were detected as metabolites following the administration of different 17α-methyl steroids. 
The long-term DHCMT metabolites 20βOH-NorDHCMT (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one) and its A-ring reduced analogue 20βOH-NorTHCMT (4ξ-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5ξ-androst-13-ene-3ξ-ol) were reported earlier, however without providing reference material and assigning the stereochemistry of the latter. Very recently the stereochemistry of the latter was reported as 3α,4α,5α.

In this study we investigated the applicability of a combined chemical and biotechnological approach for the synthesis of reference material. A combination of Wagner-Meerwein rearrangement of DHCMT to NorDHCMT (4-chloro-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) and subsequent whole-cell biotransformation with a recombinant fission yeast strain expressing human cytochrome P450 enzymes subtype 21A2 (CYP21A2) was successfully used for the synthesis of mg amounts of 20βOH-NorDHCMT.

Unexpectedly, this approach failed in the synthesis of 20βOH-NorTHCMT. It was observed that substrates with 3-hydroxy groups are neither hydroxylated on C20 by CYP21A2 nor by CYP3A4. Further confirmation of this finding was obtained by testing other 17,17-dimethyl-18-norandrost-13-ene steroids. Thus, insights into the structure-activity relationship of these enzymes were provided.

Furthermore, post administration urines of DHCMT were analyzed for the presence of 20βOH-Nor-metabolites. Both metabolites, 20βOH-NorDHCMT and 20βOH-NorTHCMT, were detected in very low abundances in the urines.

Code: 15C01DS 

Tramadol is an analgesic medication for the treatment of moderate to severe pain. Recently, there have been concerns about the abuse of tramadol causing falls in the pro-cycling peloton. Only few studies have examined the effects of Tramadol on performance and none with athletes or healthy subjects. The impact of Tramadol on cycling performance might be mediated by its effect on sustained attention, which is defined as a capacity to detect of relevant stimuli that are rare and unpredictable. 
The aims of this project are: 1) to investigate the impact of Tramadol on physical performance in cycling with a Functional threshold power test (1st year); 2) to investigate the effect of Tramadol on sustained attention at rest (1st year) and during exercise (2nd year) with the Continuous Performance Task; 3) to study whether sustained attention might mediate the effect of Tramadol on cycling performance with an ecologically-valid task (3rd year). All experiments to reach the objectives will be performed using a placebo-controlled, double blind, randomized counterbalance methodology. Tramadol (a single dose of 100 mg of orally administered will be used) or Placebo will be administered to participants 120 min before starting each test. We will recruit 30 subjects for each experiment (15 males and 15 females). Together with objective measures of physical and cognitive performance, we will obtain measures of subjective performance such as perceived effort and mental fatigue. 
This research project will be the first to test the effects of Tramadol on sustained attention during cycling. This is particularly relevant since Tramadol has been placed on WADA’s Monitoring Program from 2012 to 2015 (Narcotics: in competition only) in order to detect potential patterns of abuse. Our findings would certainly help the WADA to decide whether to include Tramadol into the Prohibited List or not. 

Main Findings: 

The use of tramadol, an analgesic medication, in professional cycling is generating particular interest and concern. Tramadol might enhance cycling performance at the cost of reducing the ability to stay focused. The aim of the present study was to test the hypothesis that acute oral administration of tramadol improves exercise performance during a 20-min cycling Time-Trial (TT) (Experiment 1) in a group of cyclists and whether sustained attention would be impaired during exercise (Experiment 2).

This study is a clinical trial (EudraCT number: 2015-005056-96) using a placebo-controlled, double blind, within-participants, counterbalanced methodology.  We administered a single oral dose of Tramadol (100 mg), or placebo 120min before starting the TT. Electroencephalography measures (EEG) were recorded throughout the cycling exercise and at rest. In Experiment 2, the methods were the same as in Experiment 1 except that participants performed an Oddball (cognitive) sustained attention task during exercise.

We recruited 56 cyclists in total. In Experiment 1, overall power output was higher in the tramadol condition than in the placebo condition. This result was partially replicated in Experiment 2, as the power output during the second half of the TT was higher under tramadol, while no differences were observed in the first part of the TT. No effects of condition were shown either in the PVT or the oddball task.  Tonic EEG analysis at baseline revealed opposite results when comparing Experiments 1 and 2. However, EEG data showed a significant effect at the neural-related activity related to stimulus processing during exercise.

Futures studies should include a pre-fatigue protocol at a fixed load before the TT to address this issue, and in addition a more ecological task.

Code: ISF15D03MG 

Autologous blood doping is applied in order to increase red blood cell (RBC) mass and thus oxygen transport capacity of the blood and to improve exercise performance.The International Olympic Committee (IOC) banned blood boosting after the 1984 Olympics and indirect markers for blood doping, including total haemoglobin mass measurements, or to test for the excretion of metabolites of bag plasticizers in the urine have been developed. 
The Athlete Biological Passport was introduced a couple of years ago and consists of a longitudinal monitoring of biologic measures to identify patterns suspicious of doping and evaluation of such abnormal patterns by a panel of experts. Parameters included are the hemoglobin concentration and the reticulocyte percentage but the latest incidents (e.g. doping charge Claudia Pechstein) proved that the interpretation of hematological parameters are difficult because of wide inter-individual differences. Thus, at present no reliable method exists to detect autologous blood doping. We have previously investigated how blood storage affects RBC deformability, which is an indispensable characteristic of RBC to ensure the oxygen supply to the tissues, and found that during hypothermic storage of liquid blood units RBC deformability decreases with increasing storage duration. This was associated with cell senescence and reduced nitric oxide bioavailability. Nowadays blood bags are cryopreserved for later re-infusion and thus the aim of the study is to investigate the impacts of cryopreservation on RBC deformability and associated nitric oxide signaling pathways. We hypothesize an impact of cryopreservation on these parameters. Thus, the investigation of RBC function and related cellular pathways seems to be a promising attempt in order to detect autologous blood doping.

Main Findings: 

Structure and function of red blood cells (RBC) may be affected during cryopreservation (freezing, storage, thawing of RBC) and identification of respective cell markers might aid to hint towards reinfusion of stored blood. In vitro and in vivo tests with cryopreserved RBC were conducted to investigate the effects of cryopreservation on parameters of RBC aging, integrity, (nitric oxide) metabolism, deformation and viscosity. Results revealed that cryopreservation does not affect basal blood parameters or cell metabolism but reduces cell aging which was reported to be a major problem in liquid preserved blood samples stored at 4°C. Overall ability of RBC to deform was reduced after cryopreservation but sensitivity to shear stress was
increased which might be related to remaining freezing medium in the cells that might affect the membranous structure of the RBC. Cryopreservation also alters osmotic stability of RBC further indicating that the membrane and/or the membrane-cytoskeleton interaction is affected by cryopreservation. Finally, viscosity of RBC was reduced which again, hints towards an effect of freezing medium on the viscoelastic properties of RBC. Nitric oxide metabolism, which accounts – at least in part –for RBC deformation process, was not affected by cryopreservation.

Reported effects were less measurable in vivo compared to in vitro suggesting that blood volume that was reinfused to respective donors in vivo was too little to detect any measurable changes in the tested parameters and which might not precisely reflect doping praxis. In conclusion, the data of the present study were the first to investigate changes of cryopreservation on RBC structure and function in more detail. Cryopreservation affects RBC structure leading to functional changes which needs further investigation to assess its effects in vivo.

Code: ISF15C15PV

Saliva is a well-established matrix in toxicology and clinic testing. However, for doping control purposes, it was shown that saliva analysis has very interesting pharmacokinetic properties after application of small doses of testosterone gel. Salivary testosterone concentrations exceeded normal values with a 100 fold and can provide good discriminating properties after low-dose application of testosterone. Currently, literature is short on salivary analysis for doping control purposes and data are absent on salivary concentrations and behavior of other androgen metabolites after intake of endogenous anabolic steroids. Consequently, this project aims to explore the possibility of steroid profiling in saliva and to relate its outcome with urinary analysis for future implementation in routine anti-doping screening. Consequently, a sensitive salivary steroid profiling method will be developed for the quantitative monitoring of testosterone and other androgen metabolites in the pg/ml-range. In a second stage, population thresholds for the monitored salivary compounds will be established in a cohort of 400 men and 400 women. Intra-individual variance shall be evaluated by analyzing longitudinal data of 10 male and 10 female volunteers. Within-subject variance on long term shall be evaluated in a period of six month with 10 oral fluid samples per volunteer. Intra-day variance shall be evaluated by collecting three aliquots per day during one week. Finally, it is planned to organize a series of administration studies with single doses of oral, gel and intra-muscular testosterone formulations and the intake of the prohormone dehydroepiandrosterone. The salivary steroid profile data will be compared with urinary steroid profiling data.

Main Findings

A quantitative method has been developed for the detection of 5 endogenous steroids (testosterone, androst-4-ene)-3,17-dione, dehydroepiandrosterone, cortisone and cortisol) in oral fluid. The method was validated and was capable of detecting accurately steroid concentrations in a reference population (387 males and 439 female volunteers). Based upon this reference population thresholds were established. In the future, these can be used to identify outliers. Intra-individual variation was investigated to evaluate the potential of saliva for a passport-like approach. Testosterone gel was administered to one volunteer and the changes in the salivary concentrations were evaluated. The concentration of T itself showed a dramatic increase, followed by a strong fluctuating pattern. For T-gel administration T itself and T/DHEA is provided the highest sensitivity/longest detection window. Hence, the method is suitable to detect misuse with synthetic analogues of natural steroids and shows a strong response to T use even in a formulation that is difficult to detect when using urine.