An observational human study investigating the effect of anabolic androgenic steroid use on the transcriptome of skeletal muscle and whole blood using RNA-Seq.

BACKGROUND: The effects of Anabolic Androgenic Steroids (AAS) are largely illustrated through Androgen Receptor induced gene transcription, yet RNA-Seq has yet to be conducted on human whole blood and skeletal muscle. Investigating the transcriptional signature of AAS in blood may aid AAS detection and in muscle further understanding of AAS induced hypertrophy. METHODS: Males aged 20-42 were recruited and sampled once: sedentary controls (C), resistance trained lifters (RT) and resistance trained current AAS users (RT-AS) who ceased exposure ≤ 2 or ≥ 10 weeks prior to sampling. RT-AS were sampled twice as Returning Participants (RP) if AAS usage ceased for ≥ 18 weeks. RNA was extracted from whole blood and trapezius muscle samples. RNA libraries were sequenced twice, for validation purposes, on the DNBSEQ-G400RS with either standard or CoolMPS PE100 reagents following MGI protocols. Genes were considered differentially expressed with FDR < 0.05 and a 1.2- fold change. RESULTS: Cross-comparison of both standard reagent whole blood (N = 55: C = 7, RT = 20, RT-AS ≤ 2 = 14, RT-AS ≥ 10 = 10, RP = 4; N = 46: C = 6, RT = 17, RT-AS ≤ 2 = 12, RT-AS ≥ 10 = 8, RP = 3) sequencing datasets, showed that no genes or gene sets/pathways were differentially expressed between time points for RP or between group comparisons of RT-AS ≤ 2 vs. C, RT, or RT-AS ≥ 10. Cross-comparison of both muscle (N = 51, C = 5, RT = 17, RT-AS ≤ 2 = 15, RT-AS ≥ 10 = 11, RP = 3) sequencing (one standard & one CoolMPS reagent) datasets, showed one gene, CHRDL1, which has atrophying potential, was upregulated in RP visit two. In both muscle sequencing datasets, nine differentially expressed genes, overlapped with RT-AS ≤ 2 vs. RT and RT-AS ≤ 2 vs. C, but were not differentially expressed with RT vs. C, possibly suggesting they are from acute doping alone. No genes seemed to be differentially expressed in muscle after the long-term cessation of AAS, whereas a previous study found long term proteomic changes. CONCLUSION: A whole blood transcriptional signature of AAS doping was not identified. However, RNA-Seq of muscle has identified numerous differentially expressed genes with known impacts on hypertrophic processes that may further our understanding on AAS induced hypertrophy. Differences in training regimens in participant groupings may have influenced results. Future studies should focus on longitudinal sampling pre, during and post-AAS exposure to better control for confounding variables.

The MMAAS Project: An Observational Human Study Investigating the Effect of Anabolic Androgenic Steroid Use on Gene Expression and the Molecular Mechanism of Muscle Memory.

OBJECTIVE: It remains unknown whether myonuclei remain elevated post anabolic-androgenic steroid (AAS) usage in humans. Limited data exist on AAS-induced changes in gene expression. DESIGN: Cross-sectional/longitudinal. SETTING: University. PARTICIPANTS: Fifty-six men aged 20 to 42 years. INDEPENDENT VARIABLES: Non-resistance-trained (C) or resistance-trained (RT), RT currently using AAS (RT-AS), of which if AAS usage ceased for ≥18 weeks resampled as Returning Participants (RP) or RT previously using AAS (PREV). MAIN OUTCOME MEASURES: Myonuclei per fiber and cross-sectional area (CSA) of trapezius muscle fibers. RESULTS: There were no significant differences between C (n = 5), RT (n = 15), RT-AS (n = 17), and PREV (n = 6) for myonuclei per fiber. Three of 5 returning participants (RP1-3) were biopsied twice. Before visit 1, RP1 ceased AAS usage 34 weeks before, RP2 and RP3 ceased AAS usage ≤2 weeks before, and all had 28 weeks between visits. Fiber CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm 2 ; 7854 vs 5677 µm 2 ) while myonuclei per fiber remained similar (3.5 vs 3.4; 2.5 vs 2.6). Respectively, these values increased for RP3 between visits (7167 vs 7889 µm 2 ; 2.6 vs 3.3). CONCLUSIONS: This cohort of past AAS users did not have elevated myonuclei per fiber values, unlike previous research, but reported AAS usage was much lower. Training and AAS usage history also varied widely among participants. Comparable myonuclei per fiber numbers despite decrements in fiber CSA postexposure adheres with the muscle memory mechanism, but there is variation in usage relative to sampling date and low numbers of returning participants.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Risk factors and future directions for preventing and diagnosing exertional rhabdomyolysis.

Exertional rhabdomyolysis may occur when an individual is subjected to strenuous physical exercise. It is occasionally associated with myoglobinuria (i.e. "cola-colored" urine) alongside muscle pain and weakness. The pathophysiology of exertional rhabdomyolysis involves striated muscle damage and the release of cellular components into extracellular fluid and bloodstream. This can cause acute renal failure, electrolyte abnormalities, arrhythmias and potentially death. Exertional rhabdomyolysis is observed in high-performance athletes who are subjected to intense, repetitive and/or prolonged exercise but is also observed in untrained individuals and highly trained or elite groups of military personnel. Several risk factors have been reported to increase the likelihood of the condition in athletes, including: viral infection, drug and alcohol abuse, exercise in intensely hot and humid environments, genetic polymorphisms (e.g. sickle cell trait and McArdle disease) and epigenetic modifications. This article reviews several of these risk factors and proposes screening protocols to identify individual susceptibility to exertional rhabdomyolysis as well as the relevance of proteomics for the evaluation of potential biomarkers of muscle damage.

Anti-doping and other sport integrity challenges during the COVID-19 pandemic.

The coronavirus disease (COVID-19) pandemic has had an unprecedent impact on the world of sport and society at large. Many of the challenges with respect to integrity previously facing competitive sport have been accentuated further during the pandemic. Threats to the integrity of sporting competition include traditional doping, issues of technological fairness, and integration of transgender and intersex athletes in elite sport. The enforced lull in competitive sport provides an unprecedented opportunity for stakeholders in sport to focus on unresolved integrity issues and develop and implement long-lasting solutions. There needs to be a concerted effort to focus on the many technological innovations accelerated by and perfected during COVID-19 that have enabled us to work from home, such as teaching students on-line, applications for medical advice, prescriptions and referrals, and treating patients in hospitals/care homes via video links and use these developments and innovations to enhance sport integrity and anti-doping procedures. Positive sports integrity actions will require a considered application of all such technology, as well as the inclusion of "omics" technology, big data, bioinformatics and machine learning/artificial intelligence approaches to modernize sport. Applications include protecting the health of athletes, considered non-discriminative integration of athletes into elite sport, intelligent remote testing to improve the frequency of anti-doping tests, detection windows, and the potential combination with omics technology to improve the tests' sensitivity and specificity in order to protect clean athletes and deter doping practices.

Analysis of Anti-Doping Rule Violations That Have Impacted Medal Results at the Summer Olympic Games 1968-2012.

INTRODUCTION: Since 2004, the International Olympic Committee (IOC) store all samples collected at summer Olympic Games (OG) for retrospective re-analysis with more advanced analytical techniques to catch doping athletes. METHODS: All announced Anti-Doping Rule Violations (ADRVs) from IOC re-tests of the 2004, 2008 and 2012 OG (via IOC, International Federations and Athletics Integrity Unit public data) and other ADRVs confirmed to impact OG results from 1968 to 2012 (via the list of Doping Irregularities on olympedia.org) were collated to investigate how many medals have been impacted by ADRVs, when the ADRV was identified relative to the OG in question and its cause. RESULTS: One hundred and thirty-four medals were impacted by ADRVs but only 26% of these ADRVs were identified at the time of the OG. Most ADRVs impacting medal results (74%) were identified retrospectively, either from events prior to the OG (17%) or via IOC re-tests of samples from 2004, 2008 and 2012 (57%). ADRVs impacting medal results from these re-tests took a mean of 6.8 ± 2.0 years to be announced relative to the end of the OG in which the medal was originally won. Exogenous Anabolic Androgenic Steroid metabolites were present in 90% of all athlete (n = 142) samples from IOC re-tests with dehydrochloromethyltestosterone and stanozolol accounting for 79% of detected substances. Athletics (n = 64) and weightlifting (n = 62) were the most affected sports. CONCLUSION: This analysis shows the frequency of targeted pre-OG Out-of-Competition testing should increase. We advocate for long-term sample storage to continue and additionally incorporate novel and potentially complementary technologies/sample matrices.

Integrating Transwomen and Female Athletes with Differences of Sex Development (DSD) into Elite Competition: The FIMS 2021 Consensus Statement.

Sport is historically designated by the binary categorization of male and female that conflicts with modern society. Sport's governing bodies should consider reviewing rules determining the eligibility of athletes in the female category as there may be lasting advantages of previously high testosterone concentrations for transwomen athletes and currently high testosterone concentrations in differences in sex development (DSD) athletes. The use of serum testosterone concentrations to regulate the inclusion of such athletes into the elite female category is currently the objective biomarker that is supported by most available scientific literature, but it has limitations due to the lack of sports performance data before, during or after testosterone suppression. Innovative research studies are needed to identify other biomarkers of testosterone sensitivity/responsiveness, including molecular tools to determine the functional status of androgen receptors. The scientific community also needs to conduct longitudinal studies with specific control groups to generate the biological and sports performance data for individual sports to inform the fair inclusion or exclusion of these athletes. Eligibility of each athlete to a sport-specific policy needs to be based on peer-reviewed scientific evidence made available to policymakers from all scientific communities. However, even the most evidence-based regulations are unlikely to eliminate all differences in performance between cisgender women with and without DSD and transwomen athletes. Any remaining advantage held by transwomen or DSD women could be considered as part of the athlete's unique makeup.

Doping practices in international weightlifting: analysis of sanctioned athletes/support personnel from 2008 to 2019 and retesting of samples from the 2008 and 2012 Olympic Games.

BACKGROUND: The pervasiveness of doping and findings of anti-doping corruption threaten weightlifting's position at the 2024 Olympic Games. Analysing the practices of doping in weightlifters could identify patterns in doping that assist in future detection. METHODS: We analysed publicly available data on sanctioned athletes/support personnel from the International Weightlifting Federation between 2008 and 2019 and announced retrospective Anti-Doping Rule Violations (ADRVs) from the 2008 and 2012 Olympic Games. RESULTS: There were 565 sanctions between 2008 and 2019 of which 82% related to the detection of exogenous Anabolic Androgenic Steroid (AAS) metabolites and markers indicating endogenous AAS usage. The detection of exogenous AAS metabolites, markers of endogenous AAS usage and other substance metabolites varied by IWF Continental Federation (p ≤ 0.05) with Europe (74%, 11%, 15%) and Asia (70%, 15%, 15%) showing a higher detection of exogenous AAS compared to Pan America (37%, 30%, 33%) and Africa (50%, 17%, 33%). When looking at the 10 most detected substances, the nations with the highest number of sanctions (range 17-35) all had at least one overrepresented substance that accounted for 38-60% of all detected substances. The targeted re-analysis of samples from the 2008 and 2012 Olympic Games due to the discovery of long-term metabolites for exogenous AAS resulted in 61 weightlifters producing retrospective ADRVs. This includes 34 original medallists (9 gold, 10 silver and 15 bronze), the highest of any sport identified by Olympic Games sample re-testing. The exogenous AAS dehydrochloromethyltestosterone and stanozolol accounted for 83% of detected substances and were present in 95% of these samples. CONCLUSION: Based on these findings of regional differences in doping practices, weightlifting would benefit from the targeted testing of certain regions and continuing investment in long-term sample storage as the sensitivity and specificity of detection continues to improve.

The Fluidity of Gender and Implications for the Biology of Inclusion for Transgender and Intersex Athletes.

One of the most contentious issues in modern day sport arises when sports are divided into male and female categories. The International Association of Athletics Federations' (IAAF) previous policy regulating intersex athletes was suspended by the Court of Arbitration for Sport (CAS), resulting in a new policy. The challenge faced by the governing body of athletics is to formulate a policy that upholds both international law and the Olympic charter that stipulates athletes compete without discrimination of any kind. Implementation of the policy has been delayed until after a verdict, expected no later than March 26, 2019, in the Semenya versus IAAF trial in the Court of Arbitration for Sport. If the policy is enacted, it will restrict athletes from competing in the female athletics category with specific differences of sex development (DSD) in races from 400 m up to the mile in international level competitions unless they lower their natural testosterone (T) levels below 5 nmol·L. To thoroughly assess this new IAAF policy, one needs to appreciate its legal, sociological, and scientific underpinnings but also the history of previous policies attempting to define precisely how athletes should be divided into male and female categories. We previously proposed a system to deal with gender variant athletes that relied on a determination of an "athlete/athletic gender." The concept of "athlete gender" was presented to multiple audiences, and the resulting survey is included. A large majority of participants (71% of 153) who answered the survey agreed with the idea of an athlete gender. This position also was accompanied by the request for more studies (20% of those who agreed) and concern over the process of hormone monitoring (32% of those who agreed) to avoid doping misuse. The primary argument of those participating in the survey that disagreed with the position (23% of 153) was that biological differences between males and females remained even after the transition (47% of opposing comments). Mixed gender/sex competitions provide unique opportunities for athletes to compete against one another outside of the traditional male/female divide and pave the way for a more flexible approach for dealing with gender variant athletes.