Effects of Long Term Supplementation of Anabolic Androgen Steroids on Human Skeletal MuscleConceived and designed the experiments: Contributed to the writing of the manuscript: The effects of long-term over several years anabolic androgen steroids AAS administration on human skeletal muscle are still unclear. In this study, seventeen strength training athletes were recruited and individually interviewed regarding self-administration of banned substances. Ten subjects admitted having taken AAS or AAS derivatives for the past 5 to 15 years Doped and the dosage and type of banned substances were recorded.
Effects of Long Term Supplementation of Anabolic Androgen Steroids on Human Skeletal Muscle
Conceived and designed the experiments: Contributed to the writing of the manuscript: The effects of long-term over several years anabolic androgen steroids AAS administration on human skeletal muscle are still unclear. In this study, seventeen strength training athletes were recruited and individually interviewed regarding self-administration of banned substances.
Ten subjects admitted having taken AAS or AAS derivatives for the past 5 to 15 years Doped and the dosage and type of banned substances were recorded. The remaining seven subjects testified to having never used any banned substances Clean.
For all subjects, maximal muscle strength and body composition were tested, and biopsies from the vastus lateralis muscle were obtained. Using histochemistry and immunohistochemistry IHC , muscle biopsies were evaluated for morphology including fiber type composition, fiber size, capillary variables and myonuclei. Compared with the Clean athletes, the Doped athletes had significantly higher lean leg mass, capillary per fibre and myonuclei per fiber. In contrast, the Doped athletes had significantly lower absolute value in maximal squat force and relative values in maximal squat force relative to lean body mass, to lean leg mass and to muscle fiber area.
In Doped athletes, AAS dose-dependent increases were observed in lean body mass, muscle fiber area, capillary density and myonuclei density. In conclusion, long term AAS supplementation led to increases in lean leg mass, muscle fiber size and a parallel improvement in muscle strength, and all were dose-dependent. Administration of AAS may induce sustained morphological changes in human skeletal muscle, leading to physical performance enhancement. Testosterone and other anabolic androgen steroids AAS are used by increasing population of professional and recreational athletes with the intention to increase muscle size and improve muscle strength  — .
Even though athletes using AAS claim significant gain in performance, a large number of academic studies investigating the performance-enhancing effects of AAS have described discordant and often contradictory outcomes  , . Such conflicting results have been attributed to poor study design including non-blinded condition, no placebo control, small sample size and AAS dose variation. Above all, in most studies, out of ethic consideration, AAS administration was usually no longer than 6 months.
Such a short period of AAS administration obviously could not reflect the reality of AAS abuse in athletes and sport enthusiasts. In reality, AAS usage was estimated to sustain for several years or the whole competition period in athletes . Thus, the difference in AAS administration period between AAS abusers and subjects in most academic studies might be one of the major reasons leading to the different conclusions. Short term AAS administration has been shown to induce muscle strength enhancement.
The increased muscle strength has been attributed to increased muscle mass which was associated with muscle fiber hypertrophy of both type I and type II fibers  , . Effects of long term AAS administration on muscle morphology in relation with muscle strength as well as with body composition are, however, still unclear.
In addition, in the steroid users, significant increase in frequency of fibers expressing developmental myosin heavy chain MyHC isoforms was also observed compared to the non-steroid users  , . On basis of the results, we concluded that intake of anabolic steroids in combination with strength training induced both fiber hypertrophy and fiber hyperplasia formation of new muscle fibres , in which the activation of satellite cells is a key process.
However, the studies did not reveal whether the changes in muscle morphology were accompanied by improvement in muscle strength as well as body composition. In anti-doping campaign, blood and urine samples are the major materials to be tested . However, due to the fast metabolic character of most AAS, remnants of AAS or its metabolites are traceable only for a short time in blood or urine after AAS intake, while the effects of AAS on skeletal muscles will remain for a long period, perhaps lifetime .
So far, no study has compared muscle morphology and strength between long-term AAS abusing, and clean athletes. It has been proposed that the effects of AAS on muscle are dose-dependent  ,  ,  , . Twenty weeks of testosterone administration increases skeletal muscle mass, leg strength and power in a dose-dependent fashion, but did not improve muscle fatigability or physical function .
However, the effects of AAS dosage on skeletal muscles have never been studied over a period of several years. The present study will investigate the effects of long term supplementation of AAS on muscle strength and morphology, and explore the relationships between AAS dosage, muscle strength and morphology in elite athletes.
We proposed that strength training athletes using AAS will have a higher enhancement in muscle strength through morphological adaptations compared with strength training athletes without using AAS. In addition, the effects of long term AAS supplementation on skeletal muscles will be dose-dependent. Thus, the muscular responses to long term AAS supplementation can be detected and used to separate Doped from Clean athletes.
All participants were informed about the design of the study and written informed consent was obtained from all participants. This was not an intervention study and no actions were taken to influence the participants' exercise training regime, diet, AAS administration or other activities.
Manuscript data is confidential and protected by the Swedish personal integrity law Personuppgiftslagen To investigate the long term effects of AAS supplementation on athletes, we recruited 17 strength training elite athletes through personal contact. All subjects were individually interviewed regarding doping substances, physical activity, smoking habits, known illnesses and medication intake. Clean subjects had signed a contract with their local clubs and the Swedish Power Lifting Federation, committing them to never use any drugs, under sever monetary punishment.
The subjects have been continuously doping-tested with negative results. The ten Doped subjects were asked to report all banned-substances including doses and intervals taken for the past years. Detailed information of the banned substances and dosage is shown in Table 1.
Intake usually follows a pyramid schedule with increased intake over time to avoid equation of AAS levels. All the subjects reported that they had trained regularly between 4—6 times per week for at least five years.
The physical training was defined as self-reported mean hours of exercise training each week during the past five years, and consisted mainly of high intensity resistance training. The Doped group consists of a mixture of bodybuilders, strongmen competitors and weightlifters whereas the Clean group consists of weightlifters only. We have to emphasize that this is the only ethically feasible approach to study long term effects of AAS abuse on athletes.
Maximal force one single recording , Mean of the highest 50 recordings and Mean of 0. Personal records PR from competition without tight suits or equivalent not all participants had competed in all disciplines for Bench press, Squat lift and Deadlift were also used for comparisons. Blood sample of 10 ml was collected from all subjects the same time in the morning after overnight fasting by venipuncture from the cubital vein.
Skeletal muscle biopsies were obtained from the vastus lateralis muscle using standard needle or forceps biopsy technique  , . Due to technical problems, one biopsy from the Clean group was discarded, leaving 6 biopsies in the Clean group for analysis.
For fiber phenotype type classification, serial sections were stained with monoclonal antibodies mAbs against different MyHC isoforms: The IHC staining process is the same as described earlier . All antibodies were diluted in 0. Control sections were treated similarly except that the primary antibodies were exchanged with non-immune serum.
For each muscle sample, more than 50 fibres mean were individually analysed in order to obtain a robust morphometric analysis . Detailed description of fiber type classification has been described in our earlier study .
Estimation of fiber area and number of capillary has been described in detail in a publication from our laboratory . An average of capillaries range — per muscle sample cross section was counted.
The number of capillaries around each fiber relative to fiber cross sectional area CAFA was calculated according to the formula: Nuclei in each fiber NIF were calculated as all nuclei within each muscle fiber. The number of nuclei in each fiber relative to fiber area NIFA was calculated as: This variable measures the nuclear domain in each fiber. Normal distribution of data was tested using the Shapiro-Wilk's test and visually inspected through normal quantile plot.
Accordingly, mean and standard deviation SD or median and range were used for descriptive statistics. Correlation analysis between AAS dosage and other variables was performed using Pearson correlation and linear regression, and skew data was log-transformed. One predictive component was calculated Y , where R2Y display the cumulative percent of the modelled variation in Y, using the X model. Q2Y values display the cumulative percent of the variation in Y that can be predicted by the model according to cross validation leave one out methods and seven groups , using the X model.
The variation modelled of X, using all predictive components and orthogonal components in X, R2X cum is a measure of fit, i. Group values of maximal muscle strength and anthropometry were presented in Table 2. Therefore, results were presented separately with or without the data of the subject. Group values of measurements were presented in Table 2. The vastus lateralis muscle was predominated by fibers expressing slow MyHCI and fast MyHCIIa fibers in both groups, and there was no difference in fiber type proportion in the muscle between the two groups.
No significant difference in mean fiber area of either type I or type IIa was observed between the Doped and the lean athletes.
Internal nuclei are marked with arrows in A. When capillary measurements were expressed as CD, no difference between the Doped and the Clean groups was observed; however, when expressed as CAF, the Doped group had significantly higher values in both type I and type IIa fibers.
Because most blood hormone concentration were not normally distributed, data was analysed by non-parametric statistics Wilcoxon signed rank, Chi 2 approximation , and presented as median and minimum - maximum Table 3. Most notably were LH, where all, and FSH where all but one, Doped subjects had below the clinical range indicating disturbed pituitary gland function.
Seven Doped subjects had testosterone levels above clinical range, but as a group not significantly different from Clean. Table 4 was the results of correlation analysis between AAS dosage and all the other measurements. Some linear regression models were presented in Figure 2.
Correlations between AAS weekly intake and muscle performance: Of the eight variables, four morphological measurements were higher and the other four of relative maximal squat force were lower in the Doped than in the Clean athletes.
All nine Doped subjects and six of seven Clean are correctly classified, leaving one Clean un-classified. Variables of importance are displayed in Figure 4. Bars indicate scaled ratios between the Doped and the Clean groups, with higher ratios of the Doped group to the left, and lower ratios to the right. The main findings of the study were that the doped athletes had higher lean mass, capillary density and myonuclei density, but lower maximal squat force relative to muscle mass and to fiber area, compared to the clean athletes.
The Doped group also had a tendency towards larger fibers, although not significant, most likely due to large variations in fibre area. Low levels of LH and FSH, and high levels of prolactin in some individuals indicate a disturbed pituitary gland function with possible negative effects on reproductive function. However, no correlations between AAS intake and hormone levels was observed. Thus, testosterone levels at time of sampling cannot explain alternations in these variables, rather concentrations outside clinical limits must stem from long-term supplementation of AAS.
Multivariate statistics showed that a combination of eight morphological parameters could clearly separate the doped from the clean athletes. Correlation analysis revealed significant positive correlations between AAS dosage and relative muscle force. The results support previous findings that AAS administration could induce enhancement in both muscle mass and muscle strength, and that the improvements are AAS dose-dependent  ,  ,  ,  , .
This is the first study to confirm previous laboratory findings in active doping athletes.