ObjectivesTo evaluate the scientific validity of the International Olympic Committee's (IOC) 2021 framework principle of “No Presumption of Performance Advantage,” which suggests that circulating testosterone levels alone does not confer a competitive advantage.DesignA critical review of existing scientific literature concerning the physiological effects of testosterone and male puberty on athletic performance, complemented by a forward-looking proposal for integrating multi-layered biological and performance data.MethodRelevant peer-reviewed studies were examined, focusing on the role of endogenous testosterone, the long-term effects of male puberty, and performance outcomes among transgender women and athletes with differences in sexual development (DSD). Emphasis was placed on strength, power, and endurance metrics. In addition, we outline the emerging potential of combining wearable sensor monitoring with omics profiling to generate a comprehensive, dynamic evidence base.ResultsEvidence consistently shows that male puberty is associated with lasting increases in muscle mass, strength, speed, and endurance due to elevated testosterone. Current evidence also suggests such physiological adaptations may endure even after testosterone suppression, potentially mediated by muscle memory mechanisms such as myonuclei permeance and/or epigenetic changes. If verified, transgender women who experienced male puberty and later reduced testosterone levels retain physical advantages, particularly in strength-based sports. Similarly, athletes with DSD exhibiting male-range testosterone levels also show performance advantages in select events.ConclusionsThe IOC's principle of “No Presumption of Performance Advantage” contradicts a substantial body of scientific evidence. While inclusion and fairness are important goals, the framework's rejection of testosterone as a key determinant of athletic performance risks undermining competitive fairness and athlete safety in sex-segregated sports, particularly where strength and power are critical. Acknowledging testosterone as a key determinant of performance, our study proposes a novel multi-layered evaluation framework that integrates real-time sensor data with omics analyses. This innovative approach supports the development of adaptive, context-specific, and ethically grounded policies that promote fair, inclusive, and safe competition while offering a forward-looking roadmap for future policy and research.

Androgens act through androgen receptor (AR) to maintain muscle mass. Evidence suggests that this pathway is influenced by "the gene for speed," ACTN3 (α-actinin-3). Given that one in five people lack α-actinin-3, it is possible that they may respond to androgens differently. Here, we show that α-actinin-3 deficiency decreases AR in muscles of mice and humans (in males and females) and that AR positively correlates with α-actinin-3 expression in a dosage-dependent manner. α-Actinin-3 deficiency exacerbates gastrocnemius mass loss with androgen deprivation in male mice and stunts the muscle growth response to dihydrotestosterone in female mice at the onset of puberty. This is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, autophagy, mitochondrial activity, MAPK, and calcineurin signaling, likely driven by seven key genes that are both androgen sensitive and α-actinin-3-dependent in expression. Our results highlight a role for ACTN3 as a regulator of muscle mass and a genetic modifier of androgen action in skeletal muscle.

Recent identification of new human muscle glycogen phosphorylation sites has renewed interest in understanding human variations in the regulation of glycogen metabolism and glucose homeostasis. This paper presents a detailed method for the measurement of glycogen phosphorylase (GPh) activity in skeletal muscle. Our approach incorporates modifications to existing radiolabelling assays, optimizing specificity and sensitivity while enabling the assessment of both active and total enzyme activity levels. The utilization of radioisotope tracers and scintillation counting ensures accurate quantification of GPh activity, which we use to validate a previously published reduction in GPh activity in an Actn3 deficient mouse model. Moreover, we introduce a step-by-step guide for data acquisition, highlight the use of appropriate homogenization, discuss the need for allosteric activators/inhibitors and the importance of assay optimization to record a GPh activity assay for skeletal muscle. In conclusion, our refined method not only contributes to a deeper understanding of glycogen metabolism in muscle tissue but also provides a framework for future investigations, underscoring its role in advancing research on glycogen utilization and glucose homeostasis.NEW & NOTEWORTHY The study optimizes the glycogen phosphorylase radiolabelled activity assay, unveiling nuances in muscle homogenization, sample dilution, and caffeine inclusion. The research introduces standardized conditions, enhancing assay reliability and reproducibility across mouse strains to reveal sex specific variations in GPh activity and underscore novel distinctions in an Actn3 deficient mouse model. These findings advance our understanding of muscle glycogen metabolism, offering a crucial tool for researchers and facilitating meaningful inter-laboratory comparisons.### Competing Interest StatementThe authors have declared no competing interest.

A nonsense polymorphism in the ACTN3 gene (R577X, rs1815739) results in the loss of the fast skeletal muscle fiber protein α-actinin-3 in an estimated 1.5 billion humans worldwide. Homozygosity for this common polymorphism (ACTN3 577XX) does not cause disease but is detrimental to sprint performance in elite athletes. Recently, we reported that ACTN3 577XX humans and Actn3 knockout mice show improved cold tolerance. This was not due to an increase in brown adipose tissue activity or a greater muscle shivering response, but was associated with an increased abundance of slow myosin heavy chain muscle fibers in XX individuals, which in turn increased muscle tone to improve cold tolerance. Using the Actn3 knockout mouse we have now analyzed the molecular and physiological impacts of acute cold exposure (4oC, for 5 hours) on skeletal muscle. In addition, we used statistical inference of publicly available archaic (Neanderthal and Denisovan) and modern human DNA data to estimate the age of the ACTN3 X-allele and to identify a novel haplotype associated with this variant that provides support for genetic selection in some populations of modern humans. Our results indicate that skeletal muscle of Actn3 knockout mice exhibit higher mitochondrial oxidative phosphorylation abundance and activity than wild-type controls. Following acute cold exposure, we identified reductions in gene ontology pathways associated with metabolism and protein breakdown, which impact muscle mass loss and improved muscle fatigue resistance. Together this provides further evidence for improved cold tolerance in Actn3 knockout mice and ACTN3 577XX individuals. Furthermore, we confirm that the X-allele first appeared in modern humans ~135,000 years ago, with this variant increasing in frequency in European and Asian populations from as early as 42,000 years ago, which aligns with modern human migration out of Africa. Taken together these data provide additional molecular and functional evidence for a protective advantage of α-actinin-3 deficiency following cold exposure, highlighting the importance of skeletal muscle in mammalian thermogenesis and its potential role in the evolution of modern-day humans. ### Competing Interest Statement The authors have declared no competing interest.

Androgens are vital for the maintenance of muscle mass and their anabolic effects are primarily exerted through the androgen receptor (AR). Accumulating evidence in humans and mice suggests that circulating androgens, AR and androgen response are influenced by ACTN3 ( α- actinin-3), also known as “the gene for speed”. One in 5 people worldwide are α-actinin-3 deficient due to homozygous inheritance of a common null polymorphism (577X) in ACTN3 . In this study, we show that α-actinin-3 deficiency decreases baseline AR in skeletal muscles of mice and humans, in both males and females, and that AR expression directly correlates with ACTN3 in a dosage dependent manner. We further demonstrate in Actn3 knockout mice that α- actinin-3 deficiency increases muscle wasting induced by androgen deprivation and reduces the muscle hypertrophic response to dihydrotestosterone and this is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, MAPK signalling, autophagy, mitochondrial activity and calcineurin signalling. Gene set enrichment and protein analyses indicate that the absence of α-actinin-3 results in a failure to coactivate many of these pathways in response to changes in androgens, and relies on leveraging mitochondrial remodelling and calcineurin signalling to restore muscle homeostasis. We further identified 7 genes that are androgen sensitive and α-actinin-3-dependent in expression, and whose functions correspond to these processes. Our results highlight the pivotal role of α- actinin-3 in various processes associated with the regulation of protein turnover and muscle mass, and suggest that ACTN3 genotype is a genetic modifier of androgen action in skeletal muscle.### Competing Interest StatementF.J.R. receives institutional support as a coinvestigator and subcontracted by the Peter MacCallum Cancer Centre for an investigator-initiated trial which receives funding support from Sanofi/Regeneron Pharmaceuticals.

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 μm2; 7854 vs 5677 μm2) 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 μm2; 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.

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

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.

Homozygosity for the common ACTN3 null polymorphism (ACTN3 577X) results in α-actinin-3 deficiency in ~20% of humans worldwide and is linked to reduced sprint and power performance in both elite athletes and the general population. α-Actinin-3 deficiency is also associated with reduced muscle mass, increased risk of sarcopenia, and altered muscle wasting response induced by denervation and immobilization. Here, we show that α-actinin-3 plays a key role in the regulation of protein synthesis and breakdown signaling in skeletal muscle and influences muscle mass from early postnatal development. We also show that α-actinin-3 deficiency reduces the atrophic and anti-inflammatory response to the glucocorticoid dexamethasone in muscle and protects against dexamethasone-induced muscle wasting in female but not male mice. The effects of α-actinin-3 deficiency on muscle mass regulation and response to muscle wasting provide an additional mechanistic explanation for the positive selection of the ACTN3 577X allele in recent human history.

Rapid advances in technologies in the field of genomics such as high throughput DNA sequencing, big data processing by machine learning algorithms and gene-editing techniques are expected to make precision medicine and gene-therapy a greater reality. However, this development will raise many important new issues, including ethical, moral, social and privacy issues. The field of exercise genomics has also advanced by incorporating these innovative technologies. There is therefore an urgent need for guiding references for sport and exercise genomics to allow the necessary advancements in this field of sport and exercise medicine, while protecting athletes from any invasion of privacy and misuse of their genomic information. Here, we update a previous consensus and develop a guiding reference for sport and exercise genomics based on a SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis. This SWOT analysis and the developed guiding reference highlight the need for scientists/clinicians to be well-versed in ethics and data protection policy to advance sport and exercise genomics without compromising the privacy of athletes and the efforts of international sports federations. Conducting research based on the present guiding reference will mitigate to a great extent the risks brought about by inappropriate use of genomic information and allow further development of sport and exercise genomics in accordance with best ethical standards and international data protection principles and policies. This guiding reference should regularly be updated on the basis of new information emerging from the area of sport and exercise medicine as well as from the developments and challenges in genomics of health and disease in general in order to best protect the athletes, patients and all other relevant stakeholders.

α-Actinin-3 (ACTN3 R577X, rs.1815739) polymorphism is a genetic variation that shows the most consistent influence on metabolic pathway and muscle phenotype. XX genotype is associated with higher metabolic efficiency of skeletal muscle; however, the role of ACTN3 polymorphism in oxygen transport and utilization system has not yet been investigated. Therefore, the aim of this study was to determine the influence of ACTN3 polymorphisms on hematological and iron metabolism response induced by marathon race. Eighty-one Brazilian amateur male endurance runners participated in the study. Blood samples and urine were collected before; immediately after; and 1, 3, and 15 days after the marathon race. Urine, hematological parameters, iron metabolism, and ACTN3 genotyping analyses were performed. The marathon race induced a decrease in erythrocytes, Hb, and Ht, and an increase in hematuria, creatinine, myoglobin, red cell distribution width, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, direct and indirect bilirubin and erythropoietin. Moreover, an elevation immediately or 1 day after the marathon race follows a reduction 3 or 15 days after the marathon race were observed on transferrin saturation and iron and transferrin levels. Hematological parameters and iron metabolism changes induced by marathon race were not observed in XX genotypes. Hematuria and decreased erythrocytes, Hb, Ht, and iron and transferrin levels were observed only in RR and/or RX genotypes but not in XX genotypes. The percentage of runners with hematuria, leukocyturia, iron deficiency, creatinine, myoglobin, and bilirubin imbalance was higher in RR compared to XX genotypes. ACTN3 polymorphism is associated with iron metabolism and hematological responses after endurance exercise. Despite these results being based on a small sample, they highlight a protective role of the XX genotype on hematological and renal changes induced by long-distance exercise. Therefore, these findings should be further replicated.

The α-actinin-3 gene (ACTN3) R577X polymorphism is one of the most highly studied genetic variations associated with human skeletal muscle function and performance. This chapter summarizes the current research in ACTN3 R577X and discusses the molecular mechanisms that underlie the consequences of a-actinin-3 deficiency, with new insights into implications for the general population beyond athletic performance — in healthy ageing, muscle disuse, and disease. The α-actinins are a family of four actin-binding proteins that have evolved from repeated gene duplication events to perform similar roles in different cell types. The role of sarcomeric α-actinins in the maintenance of ordered myofibrillar arrays in skeletal muscle make them prime candidate genes for human muscle disease. The varied allele frequencies observed between the different ethnic groups and specialized protein expression in fast glycolytic muscle fibers suggest that selective forces may be acting on the ACTN3 577X allele during modern human evolution.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

A common null polymorphism in the ACTN3 gene (rs1815739:C>T) results in replacement of an arginine (R) with a premature stop codon (X) at amino acid 577 in the fast muscle protein α-actinin-3. The ACTN3 p.Arg577Ter allele (aka p.R577* or R577X) has undergone positive selection, with an increase in the X allele frequency as modern humans migrated out of Africa into the colder, less species-rich Eurasian climates suggesting that the absence of α-actinin-3 may be beneficial in these conditions. Approximately 1.5 billion people worldwide are completely deficient in α-actinin-3. While the absence of α-actinin-3 influences skeletal muscle function and metabolism this does not result in overt muscle disease. α-Actinin-3 deficiency (ACTN3 XX genotype) is constantly underrepresented in sprint/power performance athletes. However, recent findings from our group and others suggest that the ACTN3 R577X genotype plays a role beyond athletic performance with effects observed in ageing, bone health, and inherited muscle disorders such as McArdle disease and Duchenne muscle dystrophy. In this review, we provide an update on the current knowledge regarding the influence of ACTN3 R577X on skeletal muscle function and its potential biological and clinical implications. We also outline future research directions to explore the role of α-actinin-3 in healthy and diseased populations.

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

Obesity is a worldwide health crisis, and the identification of genetic modifiers of weight gain is crucial in understanding this complex disorder. A common null polymorphism in the fast fiber-specific gene ACTN3 (R577X) is known to influence skeletal muscle function and metabolism. α-Actinin-3 deficiency occurs in an estimated 1.5 billion people worldwide, and results in reduced muscle strength and a shift towards a more efficient oxidative metabolism. The X-allele has undergone strong positive selection during recent human evolution, and in this study, we sought to determine whether ACTN3 genotype influences weight gain and obesity in mice and humans. An Actn3 KO mouse has been generated on two genetic backgrounds (129X1/SvJ and C57BL/6J) and fed a high-fat diet (HFD, 45% calories from fat). Anthropomorphic features (including body weight) were examined and show that Actn3 KO 129X1/SvJ mice gained less weight compared to WT. In addition, six independent human cohorts were genotyped for ACTN3 R577X (Rs1815739) and body mass index (BMI), waist-to-hip ratio-adjusted BMI (WHRadjBMI) and obesity-related traits were assessed. In humans, ACTN3 genotype alone does not contribute to alterations in BMI or obesity.

Although it is well established that exogenous androgens have anabolic effects on skeletal muscle mass in humans and mice, data from muscle-specific androgen receptor (AR) knockout (ARKO) mice indicate that myocytic expression of the AR is dispensable for hind-limb muscle mass accrual in males. To identify possible indirect actions of androgens via the AR in neurons to regulate muscle, we generated neuron-ARKO mice in which the dominant DNA binding-dependent actions of the AR are deleted in neurons of the cortex, forebrain, hypothalamus, and olfactory bulb. Serum testosterone and luteinizing hormone levels were elevated twofold in neuron-ARKO males compared with wild-type littermates due to disruption of negative feedback to the hypothalamic-pituitary-gonadal axis. Despite this increase in serum testosterone levels, which was expected to increase muscle mass, the mass of the mixed-fiber gastrocnemius (Gast) and the fast-twitch fiber extensor digitorum longus hind-limb muscles was decreased by 10% in neuron-ARKOs at 12 weeks of age, whereas muscle strength and fatigue of the Gast were unaffected. The mass of the soleus muscle, however, which consists of a high proportion of slow-twitch fibers, was unaffected in neuron-ARKOs, demonstrating a stimulatory action of androgens via the AR in neurons to increase the mass of fast-twitch hind-limb muscles. Furthermore, neuron-ARKOs displayed reductions in voluntary and involuntary physical activity by up to 60%. These data provide evidence for a role of androgens via the AR in neurons to positively regulate fast-twitch hind-limb muscle mass and physical activity in male mice.