Influence of Female Sex Hormones on Ultra-Running Performance and Post-Race Recovery: Role of Testosterone

years, increasing numbers of women have participated in extremely long races. In adult males, there is a clear association between physiological levels of endogenous sex hormones and physical performance. However, the in uence of plasmatic sex hormones and the effects of different types of hormonal contraception (HC) on the modulation of physical performance in adult females remain to be fully clari ed. Eighteen female ultra-endurance athletes were recruited to participate in the study. Different variables were studied, including hematological parameters, body mass index, and

sex hormones and physical performance. However, the in uence of plasmatic sex hormones and the effects of different types of hormonal contraception (HC) on the modulation of physical performance in adult females remain to be fully clari ed. Eighteen female ultra-endurance athletes were recruited to participate in the study. Different variables were studied, including hematological parameters, body mass index, and body composition. Strength measurements were obtained using the squat-jump and hand-grip test. A repeated-measures analysis demonstrated signi cant differences in hematological values of CK and LDH pre-race as compared to immediately post-race and after 24/48 h. Furthermore, statistical differences were found in squat-jump and hand-grip test results after the ultramarathon. Testosterone, estradiol, and the testosterone/estrogen ratio were signi cantly correlated with muscle fatigue and were found to be indirect markers of muscle damage. A multivariate analysis demonstrated the protective role of testosterone against muscle damage and severe fatigue. Fluctuations in endogenous testosterone levels were correlated with greater fatigability and muscle damage after the competition. Adjusting the menstrual cycle with HC would not provide any further bene t to the athlete's competitive capacity. Keywords: ultra-endurance; hormones; female athletes; physical performance; muscle damage; muscle fatigue; hormonal contraception 1. Introduction Ultramarathon races are de ned as sporting events that involve running and/or walking for distances greater than the of cial marathon distance of 42.195 km. In recent years, these competitive events have increased in popularity [1]. These extremely long races defy our physiological systems, inducing muscle injuries, respiratory fatigue, and cardiac and renal damage. They, thus, provide an outstanding model to evaluate the effects of ultra- endurance activities on the human body. The percentage of women participating in ultra- endurance sports has greatly increased in recent years [2]. In elite athletic competitions, males and females are usually separated, thus avoiding disadvantages for females in terms of strength, power, and speed as compared to their male counterparts. It has been demonstrated that sex differences in sports performance are associated with higher circulating testosterone concentrations in males during puberty. In fact, results from Int. J. Environ. Res. Public Health2021,18, 10403.

and females are usually separated, thus avoiding disadvantages for females in terms of strength, power, and speed as compared to their male counterparts. It has been demonstrated that sex differences in sports performance are associated with higher circulating testosterone concentrations in males during puberty. In fact, results from Int. J. Environ. Res. Public Health2021,18, 10403.

Int. J. Environ. Res. Public Health2021,18, 10403 2 of 12 sports competitions show no sex differences prior to puberty. Postpubertal testosterone- induced changes occur with regard to muscle mass, strength, bone composition, and hemoglobin levels, conferring a physical advantage in sporting events [3]. Male sex hormones such as testosterone, dihydrotestosterone, and dehydroepiandros- terone modulate the different physiological mechanisms that are responsible for athletic performance improvement [4,5]. Testosterone binding to the androgen receptor at the surface of the muscle ber increases calcium release from the intracellular stores, activating different mechanisms that increase the number of muscle bers and satellite cells, as well as the size of motor neurons [6]. Thus, skeletal muscular activity is enhanced, eliciting improved athlete performance and recovery [7]. Although there is a clear association between physiological levels of endogenous testosterone and physical performance in men [3,8], the in uence of plasma concentrations of female androgens on sports performance needs to be clari ed. The paucity of research on female athletes can be attributed to the cultural marginalization of women in sport and a lack of female participation in both the research and sporting contexts [9]. It is also the result of the cofounding variables of female sex hormone uctuations, for example in terms of the endogenous hormone pro le and differences in this pro le under the use of hormonal contraception [10]. Further, the greater incidence of anovulation and luteal phase de ciency in female athletes complicates research in this regard [11]. Athletic amenorrhea is common in elite female athletes. It has been postulated to be an adaptative mechanism to a negative energy balance (hypometabolic state), and is associated with low testosterone levels [12]. Conversely, Hagmar [13] reported that most cases of menstrual disturbances observed among female elite athletes were due to polycystic ovary syndrome (PCOS). Interestingly, this syndrome is characterized by an elevated ovarian production of testosterone and is associated with greater muscle mass [14,15]. This increase in testosterone levels may positively modulate physical performance in athletes, especially in ultra-endurance events. Estrogen is thought to be another hormonal factor modulating muscle strength, metabolism, and stiffness [16].

elite athletes were due to polycystic ovary syndrome (PCOS). Interestingly, this syndrome is characterized by an elevated ovarian production of testosterone and is associated with greater muscle mass [14,15]. This increase in testosterone levels may positively modulate physical performance in athletes, especially in ultra-endurance events. Estrogen is thought to be another hormonal factor modulating muscle strength, metabolism, and stiffness [16]. Moreover, the testosterone/estrogen ratio (T/E ratio) has been recently considered as a predictor of over-training syndrome in male athletes [17]. Hence, basal estrogen levels and the testosterone/estrogen ratio (T/E) are critical to a better understanding of ultra-trail performance and post-race recovery in female runners. It has been reported that many elite female athletes use hormonal contraception, with gures varying from 20% to 70% depending on the country and sport [18–21]. However, little is known about the impact and prevalence of HC use and the effects of intermittent treatment. Furthermore, the physiological aspects of HC use are not always considered when monitoring the health of athletes. As such, we hypothesized that the menstrual cycle phase and, in particular, its in u- ence on testosterone levels could modulate muscular physiological response and recovery after intense sporting activity in ultra-trail events. Thus, we aimed to elucidate the in uence of basal testosterone and the T/E ratio on the post-race loss of skeletal muscle strength and muscle damage in female ultra-trail runners. The objectives of this study were extended to consider whether HC could in uence these variables. 2. Materials and Methods 2.1. Study Design This was a cross-sectional observational study that formed part of the Penyagolosa Trail Healthy Women project and involved a sample of 18 amateur runners who participated in the Penyagolosa Trail CSP ultra-trail race on 12 May 2018. The trail track was of 107.4 km, with an incline of 5604 m and a decline of 4356 m. All subjects were fully informed of the procedure and gave their written consent to participate. For more detailed information on the methods, see the study registration information in the ClinicalTrails.gov database (registration number NCT03990259).

track was of 107.4 km, with an incline of 5604 m and a decline of 4356 m. All subjects were fully informed of the procedure and gave their written consent to participate. For more detailed information on the methods, see the study registration information in the ClinicalTrails.gov database (registration number NCT03990259).

Int. J. Environ. Res. Public Health2021,18, 10403 3 of 12 2.2. Study Population and Ethical Approval The sample was composed entirely of amateur runners. In total, 35 women nished the race. Our sample size included 18 female athletes, representing 51.42% of the total sample nishers. This sample can be considered representative, with a 95% con dence interval, a precision of 6%, and a proportion of 3%. The mean age was 41+/–6 years, with an average height of 1.61 0.05 centimeters and weight of 56.92 4.36 kilograms. In relation to training habits, the runners underwent 4.87 0.9 days of weekly training, with an average training time of 9.07 2.54 h and distance of 73.07 43.32 kilometers. The average length of menstrual bleeding was 3.56 1.04 days, and a total of 6 participants in the study used some type of hormonal contraception (2 used oral contraceptives, 1 used a vaginal ring, and 3 used hormonal IUDs). The investigation was conducted according to the Declaration of Helsinki, and the project was approved by the Research Ethics Committee of the university ( le number CD/007/2019). Informed consent was obtained from all subjects participating in the study. 2.3. Hematological Variables Blood samples were collected from an antecubital vein by venipuncture on the same day of the race prior to starting, after crossing the nishing line, and 24 and 48 h later. The serum hormones related to the menstrual cycle used in the present investigation were estradiol and testosterone. Lactate dehydrogenase (LDH) and creatine kinase (CK) were used as indicators of muscle membrane disruption resulting from tissue injury. The hematological variables included ferritin, hemoglobin, hematocrit, and red blood cell count. The biochemical results obtained immediately post-race were adjusted by employing the Dill and Costill method [22], using hematocrit and hemoglobin to determine the magnitude of plasma volume changes after the race in each participant. 2.4. Body Mass Index (BMI) and Body Composition Assessment Prior to starting on the same day as the race we measured the height and weight of all volunteers. They were also subjected to a body composition evaluation test (Tanita BC-780MA, Tanita Corp.,

using hematocrit and hemoglobin to determine the magnitude of plasma volume changes after the race in each participant. 2.4. Body Mass Index (BMI) and Body Composition Assessment Prior to starting on the same day as the race we measured the height and weight of all volunteers. They were also subjected to a body composition evaluation test (Tanita BC-780MA, Tanita Corp., Tokyo, Japan). 2.5. Loss of Strength Measurement To assess the force, speed, and power of the extensor muscles in the lower extremities we used the squat-jump (SJ) test. The SJ test has been validated and is based on 3 simple parameters (body mass, jump height, and push distance) [23]. Grip strength (HG) is a direct measure of hand skeletal muscle strength. It is an index of endurance and general muscle capacity, and can re ect the association between peripheral strength and exercise capacity [24]. Previous studies have also suggested that the strength decline index (SDI), calculated as the decline in strength as a proportion of baseline values (measured through tests such as the HG and SJ), is a useful assessment of muscle fatigue [25]. Brie y, volunteers were rst familiarized 3 to 5 times with the technical aspects of the testing procedures. The HG and SJ tests were performed before the race and 15 min after the race. For the HG assessment, volunteers remained in standing position, holding the grip dynamometer (T.K.K. 5401 GRIP-D, Takei Scienti c Instruments Co., Tokyo, Japan) in their dominant hand. They were asked to squeeze the dynamometer for 5 s and the test was performed twice, with 30 s of rest in between attempts. The peak value for each individual was retained for statistical analysis. For the SJ, the participants were asked to jump as high as possible. In the starting position, hips and knees were exed 80 and hands were immobilized on hips to avoid arm swing. Jump height was estimated by the ight time as measured with a contact platform (Chronojump, Barcelona, Spain). The test was performed twice, with 90 s of rest between attempts. Each individual's best performance value was retained for

high as possible. In the starting position, hips and knees were exed 80 and hands were immobilized on hips to avoid arm swing. Jump height was estimated by the ight time as measured with a contact platform (Chronojump, Barcelona, Spain). The test was performed twice, with 90 s of rest between attempts. Each individual's best performance value was retained for statistical analysis [26].

Int. J. Environ. Res. Public Health2021,18, 10403 4 of 12 2.6. Statistical Methods The level of signi cance was established atp< 0.05. The data are presented as the mean standard error of the mean (SEM). We opted for non-parametric analysis [27] due to the sample size (n< 30). Spearman's correlation analysis was used to assess whether the baseline values of sex hormones were interrelated or related to the loss of upper (HG) and lower limb (SJ) strength, hematological variables representing muscle membrane disruption (CK, LDH), and the percentage of muscle mass (MM). For each subject, values for the variables HG, SJ, MM, CK, and LDH post-race and 24 and 48 h later were related to the individual baseline levels to de ne the delta scores (D):D(fold increase) = (post-race value—baseline value)/baseline value [28]. Test outcome meaningfulness was estimated through the size of the estimated effect of the correlation: strong, moderate, and small (>0.5, 0.3–0.5, and <0.3, respectively). Regarding the use of HC method, the strength and muscular damage variables (ex- pressed as a delta score of the baseline values for each subject) were compared using the Mann–Whitney U test. The test outcome meaningfulness was estimated through Cohen's d effect size pairwise comparisons. A Cohen's d value < 0.5 was considered small, while a value between 0.5 and 0.8 was considered moderate, and a value greater than 0.8 was considered as large. Finally, multiple regression analysis was performed using the forward stepwise method. Only normally distributed variables were used as dependent variables. Among the models obtained, the parsimony principle was applied [29]. Given the limited simple size and the non-normal distribution of independent variables, residual errors from the resulting models were inspected to ensure their normal distribution and, thus, the reliability of our regression models [30]. To identify the predictive value of the model, the Cohen criterion [31] was applied to one-way ANOVA models. This criterion indicates that R 2 values less than 0.10 do not present a relevant explanatory value, while R 2 values between 0.10 and 0.25 indicate a dependence of the explanation of the variance of the analyzed

of our regression models [30]. To identify the predictive value of the model, the Cohen criterion [31] was applied to one-way ANOVA models. This criterion indicates that R 2 values less than 0.10 do not present a relevant explanatory value, while R 2 values between 0.10 and 0.25 indicate a dependence of the explanation of the variance of the analyzed variable on the identi ed factors, and with R 2 values above 0.25, we can af rm that the explanatory model is very clinically relevant. 3. Results The average race nish time was 22 h 20 min 2 h 24 min. Data regarding sex hormones and variables of muscle fatigue, systemic in ammation, and damage to muscle tissues are shown in Table. Repeated-measures analysis demonstrated signi cant differ- ences in CK and LDH values pre-race as compared to immediately post-race and 24/48 h later. Similarly, signi cant differences were found between the loss of muscle strength measured with the HG, SJ, and loss of muscle mass after the ultramarathon. Baseline values of sex hormones, fatigue, and muscle strength were within the normal clinical range. Table 1. Descriptive data on sex hormones and variables of muscle fatigue, loss of muscle mass, and injury to muscle tissues (mean SD). Baseline Data Finish Line 24 h Post-Race 48 h Post-Race Estradiol (pg/mL) 89.06 87.99 Testosterone (ng/dL) 19.34 9.34 T/E ratio 1.25 1.99 CK (ui/L) 137.59 54.73 5075.76 3871.18 *2036.61 1389.83 * # 905.30 534.95 * # LDH (ui/L) 185.88 25.29 380.59 112.80 * 320.93 93.97 * # 303.38 86.67 * # MM (%) 39.20 3.45 38.00 4.86 * HG (kg) 31.33 3.69 28.67 4.10 * SJ (cm) 20.76 2.72 18.47 2.38 * Abbreviations: T/E ratio: testosterone/estradiol ratio; CK: creatine kinase; LDH: lactate dehydrogenase; MM: muscle mass; HG: hand grip; SJ: squat jump. * Signi cantly different from the preceding time point (p< 0.05); # signi cantly different from the pre-race value (p< 0.05).

MM: muscle mass; HG: hand grip; SJ: squat jump. * Signi cantly different from the preceding time point (p< 0.05); # signi cantly different from the pre-race value (p< 0.05).

The study investigates the role of testosterone in muscle damage and fatigue in female ultra-endurance athletes.