Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in elite women athletes

PHB-10816; No of Pages 7 Physiology & Behavior xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Physiology & Behavior journal homepage: www.elsevier.com/locate/phb

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Blair T. Crewther a,⁎, Dave Hamilton b, Kathleen Casto c, Liam P. Kilduff d, Christian J. Cook a,d,e

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Keywords: Androgen Adrenal Sport Adaptive Training

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Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in elite women athletes Hamlyn Centre, Imperial College, London, UK Director of Performance Science, USA Field Hockey, Lancaster, USA Department of Psychology, Emory University, USA d A-STEM, College of Engineering, Swansea University, Swansea, UK e Sport and Exercise, Bangor University, Bangor, UK b

a b s t r a c t

Article history: Received 24 February 2015 Received in revised form 25 March 2015 Accepted 8 April 2015 Available online xxxx

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This study examined the salivary testosterone (T) and cortisol (C) responses of elite women hockey players across 4 activities (light and heavy training, club and International competitions). The players formed an oral contraceptive (OC) group (n = 10) and a Non-OC (n = 19) group for analysis. The Non-OC group had higher T levels (by 31–52%) across all activities, whilst the OC group showed signs of reduced T and C reactivity when data were pooled. As a squad, positive T and C changes occurred with heavy training (45%, 46%), club competitions (62%, 80%) and International competitions (40%, 27%), respectively. Our results confirm that OC use lowers T levels in women athletes whilst reducing the T and C responses to training and competition activities within the sporting environment. Differences in the physical and/or psychological demands of the sporting activity could be contributing factors to the observed hormone responses. These factors require consideration when applying theoretical models in sport, with broader implications for women around exercising behaviours and stress physiology. © 2015 Published by Elsevier Inc.

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1. Introduction

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In the past decade, there has been growing interest in the role of testosterone (T) and cortisol (C) in women's athletic competition [14,16, 17,22,25,36]. This research is often based on theoretical models relating to the gaining, maintaining and losing of social status, which has both dominance and stress components [33,44]. According to the biosocial status hypothesis [33], a bidirectional relationship exists between T and dominance such that T levels increase to encourage behaviour aimed at dominating others, and an experience of dominance might itself increase T secretion to reinforce these behaviours. Cortisol provides an indexed marker of stress activation (of the hypothalamic– pituitary–adrenal [HPA] axis) in preparing for, and responding to, sports training and competition [28]. Research on women in competition is consistent with these theoretical models. Women's salivary T and C levels often rise before a competition [2,14,36] with these changes linked to playing abilities [17], bonding, aggressiveness and focus [2]. Sports competition (e.g. rugby, tennis, soccer, volleyball, wrestling) can promote further hormonal increases [2,14,16,17,22]. The percent changes in C are generally much

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⁎ Corresponding author at: The Hamlyn Centre, Imperial College, South Kensington Campus, London SW7 2AZ, UK. E-mail address: [email protected] (B.T. Crewther).

larger than T and possibly due to physical exertion, the psychological stress of competition, or some combination of the two. Winning in sport has also been associated with an elevated T response (vs. losing) [25,36], subsequently leading to positive changes in athlete mood state and anxiety levels [36]. There are instances when T has not responded to sports competition in women [19,28–30]. This may be explained by the shorter duration and individual nature of some competitions (e.g. 2 km rowing trial, power lifting). In addition, simulated events are often examined and these are likely to be less threatening to social status and less stressful than actual competition [12,37]. Still, practice matches in sport can promote hormonal changes comparable to real events [14], perhaps arising from greater physical exertion and psychological aspects around this (e.g. mental effort, motivational drive). To better understand the stressors of elite sport and the resultant hormonal change, it would be informative to compare the T and C responses to training sessions that differ in physical intensity and to competitions of varying social importance. To our knowledge, no research of this nature has been conducted on elite women athletes. Oral contraceptive (OC) use may impact women's baseline T and perhaps, her T response to training and/or competition. Many reports indicate that OC usage can decrease the blood or salivary levels of T and other androgens (e.g. dehydroepiandrosterone, dehydrotestosterone, androstenedione) in healthy women [9,21,42,43,45]. In fact, postmenopausal

http://dx.doi.org/10.1016/j.physbeh.2015.04.017 0031-9384/© 2015 Published by Elsevier Inc.

Please cite this article as: B.T. Crewther, et al., Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in eli..., Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.017

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Twenty-nine elite women hockey players were recruited, with a mean age of 25.3 ± 2.1 years, height of 176.9 ± 9.4 cm, body mass of 71.7 ± 19.8 kg and body mass index of 22.4 ± 1.3 kg/m2. The players were part of an International training squad at the start of the 2011 regular season. A field hockey team is made up of 11 players and 5–7 substitutes covering 4 generic positions: goalkeepers, defenders, midfielders and attackers. All of these positions were represented within the study cohort. As part of the consent procedures, the athletes were asked about current hormone contraceptive use (e.g. oral pill, injection, implantable, or patch-delivered) [14,15]. Only OCs were reported (n = 10), with the remaining players classified as Non-OC users (n = 19) for the purpose of this study. The Non-OC group reported having regular menstrual cycles, between 26 and 32 days without self-noticed problems. This study was performed with ethical approval from the Swansea University Research Ethics Committee, Swansea, UK, and participants provided written informed consent.

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2.2. Experimental procedures

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A 2-group, quasi-experimental design with repeated measures was used to address the study hypotheses. The OC and Non-OC groups were monitored over an intensive 15-day training block in which 13 training sessions were completed (with data taken from 7 training sessions) with 3 club and 4 International competitions played. Table 1 outlines the type and timing of each activity. Saliva samples were collected before and after selected training sessions (i.e. light n = 4, heavy n = 3) and all club and International competitions to monitor T and C levels and changes from baseline. The experimental procedures were performed under normal sporting conditions to improve the ecological validity of the study findings.

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The training sessions were performed at a National centre for elite sport. These sessions lasted approximately 2 h and involved a combination of skill (e.g. hockey team training) and/or physical (e.g. weight training, cardiovascular) conditioning. The weight training and cardiovascular sessions were generically classified as gym training sessions. In total, 50% of athlete training focused on skill/team development and the remaining 50% on physical conditioning. All training activities were pre-planned to optimize the training load and thus, athlete performance and recovery, relative to the competition schedule. The prescription of “light” and “heavy” sessions was part of this process, and confirmed by subjective ratings of perceived exertion (RPE) on a 1–10 scale, with a lower RPE (p b 0.001) after light training (4.7 ± 0.4) than heavy training (7.1 ± 0.2). This is a standard approach in sport to ensure that the training loads for each athlete can be easily quantified and managed [20]. A 10-minute warm-up was performed before any training session. The club and International competitions each lasted 80 min played in 2 periods of 35 min with a 10-minute interval. A more intensive warm-up was performed before each competitive game, lasting around 45 min. The club competitions were played at various locations, as part of a National premier hockey league tournament involving 10 teams, with the recruited athletes distributed amongst these teams. The RPE scores from the club (6.9 ± 0.3) and International competitions (7.3 ± 0.2) were similar to each other (p = 0.344), and heavy training (p N 0.521), but superior to light training (p b 0.001). The International competitions were played against 2 different opponents (2 games each) at the home venue of the current squad, thereby eliminating the possible influence of playing venue (i.e. home vs. away) on the hormonal outcomes, as seen in men [7,34]. The International team in this work had a higher European field hockey ranking (number 3 in European ranking) than their opponents (numbers 5 and 8) at the time of this study. This is reflected in the game outcomes, with the International competitions won by the current team (in order) with scores of 2–1, 2–0, 4–0 and 6–0. The number of athletes completing each activity varied according to prior exercise programming, game selections and the timing of competition, as well as any unforeseen injuries and illnesses. Most participants completed the heavy training sessions (n = 26–28), but the numbers varied during the club (n = 8–24) and International competitions (n = 16–18). Player selections for the International games were also limited by governing rules. The light training sessions were performed on the same day as the International competitions by a sub-group (n = 8–11) of athletes not selected to play. Being an elite squad, a consistent nutritional programme was followed on a daily basis to ensure that the macro- and micro-nutrient intake for each athlete was similar, with water consumed ad libitum across training and competitions.

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2.3. Training and competition schedule

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women with prior OC use had a lower T profile than non-users, suggesting long-term changes in hormone status [8]. On average, the OC-related decreases in free T levels are much greater (61%) than that observed for total T (31%) and likely due to OC-related increases in steroid binding proteins (e.g. sex-hormone-binding globulin) [9,45], thereby reducing the bioactive free steroid, along with the suppression of ovarian and adrenal androgen synthesis [45]. In studies of women athletes, OC users commonly exhibit lower blood or salivary T levels than nonusers (Non-OC) [3,15]. Still, OC women are responsive to sports competition with comparable T increases (percent and delta change) to Non-OC women [14,15]. Others have identified similar T changes to prolonged exercise in untrained (OC, Non-OC) and trained (OC) women [18], although the latter group had lower circulating T before and after exercise. Whilst the female T response appears relatively stable across different concentration ranges, some evidence suggests that OC usage can inhibit the free C responses to physical [4,27] and psychological stress [5,26]. These effects have yet to be validated in an ecologically valid setting when women are exposed to a mixture of physical and/or psychological stress (e.g. sports training and competition). This study examined the salivary T and C responses of elite women hockey players across different sporting activities (i.e. light and heavy training sessions, club and International competitions) in a naturalistic setting and possible interactions with OC use. Based on prior research we formulated the following hypotheses: first, the OC group would exhibit lower T (but similar C) levels than the Non-OC group across all activities; second, the OC group would also show a smaller C (but similar T) response across these activities; third, overall the T and C responses would vary by activity type, being higher in International than club competitions, followed by heavy training and then light training sessions.

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The light training sessions were performed in the morning (9–11 AM) 191 and the heavy sessions in the afternoon (2:30–4:30 PM), as part of a split 192 t1:1 t1:2

Table 1 Training and competition schedule over the 15-day monitoring period. DAY

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Notes: R = rest, GT = gym training, LT = light training, HT = heavy training, CC = club t1:4 competition, IC = International competition. The shaded cells indicate those activities t1:5 with hormone data taken. t1:6

Please cite this article as: B.T. Crewther, et al., Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in eli..., Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.017

B.T. Crewther et al. / Physiology & Behavior xxx (2015) xxx–xxx

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2.6. Statistical analyses

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The hormonal data were assessed using a generalized estimation equation model with a dependent (AR1) correlational structure [31]. This model allows for research designs with non-normal response variables, unbalanced or incomplete data sets, and more efficient parameter estimates over time [1]. Main effects and interactions were determined by significance testing of the Wald chi-square statistic (χ2). To account for differences in hormone levels due to circadian variation, time of day was entered as a covariate when examining T and C levels or reactivity. Where appropriate, post hoc testing was conducted using the Bonferroni sequential procedure. Paired sample T-Tests were also used on the T and C change scores (Post-activity–Pre-activity) to assess the within-session hormone responses. Significance was set at an alpha level of p ≤ 0.05.

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3. Results

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3.1. Group demographics

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The demographics of the study groups are shown in Table 2. Oral contraceptive use in the field hockey squad (34%) was similar to that reported (31–44%) in other studies on women athletes [15,22]. There were no differences in the age (t(27) = 1.26, p = 0.220), height (t(27) = 0.21, p = 0.834), body mass (t(27) = − 0.19, p = 0.849) and body mass index (t(27) = −0.81, p = 0.427) of the OC and NonOC groups.

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3.3. Hormonal changes, OC use and activity type

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Within-session analysis revealed positive T changes with heavy training (45%), club (62%) and International (40%) competitions (p b 0.003, Fig. 5). There was a negative C change across light training (−52%), but positive C changes with heavy training (46%), club (80%) and International (27%) competitions (p b 0.002, Fig. 6). These responses were compared with a GEE model including activity type (light training, heavy training, club competitions, International competitions), group (OC, Non-OC) and activity × group interactions. The effect of activity type on T was significant, χ2 (3) = 65.0, p b 0.001, with heavy training and both competitions promoting a larger T response than light training (p b 0.01). Moreover, the club games produced a greater T change than the International games (p = 0.028). We also found a significant group effect, χ 2 (1) = 5.34, p = 0.021, with a larger T change in Non-OC women. The effect of activity type on C was significant, χ2 (3) = 114.9, p b 0.001. The C responses to heavy training and both competition types were all significantly different from light training (p b 0.01), with club competitions also producing a larger C increase than heavy training and International games (p b 0.03).

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3.4. Hormonal changes, OC use and pooled activities

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A saliva sample (~1 mL) was collected prior to warming up before any training session (10 min) or competition (45 min) to account for a pre-event rise in salivary hormones [2,14,36]. A second sample was taken within 5 min of activity completion. The time between sampling points was similar for training (135 min) and competition (130 min), due to the different activities and warm-up periods. The samples were taken by passive drool into sterile containers and stored at − 30 °C before assay. No food or hot drinks were taken 1 h prior to sampling and fluid intake was restricted 5–10 min beforehand to prevent sample dilution. After thawing and centrifugation (2000 rpm for 10 min), the samples were assayed in duplicate by a commercial lab (HFL Sport Science, UK). The T assay used had a detection range of 6.1 to 600 pg/mL with inter-assay coefficients of variations (CV) of b12%. The C assay detection range was 0.12 to 30 ng/mL with inter-assay CV of b8%. The samples for each player were run on the same assay to eliminate inter-assay variance.

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To compare hormone levels across each activity, the GEE model included sample (Pre, Post), group (OC, Non-OC) and sample × group interaction as categorical factors. With light training sessions we found a significant main group effect for T, χ2 (1) = 23.5, p b 0.001 (Fig. 1A), being 52% higher in Non-OC women. For C, a main sample effect was identified, χ2 (1) = 21.8, p b 0.001 (Fig. 1B), representing a 51% decrease from pre- to post-training. During heavy training we found a main sample effect for both T, χ2 (1) = 57.8, p b 0.001 (Fig. 2A), and C, χ2 (1) = 17.4, p b 0.001 (Fig. 2B). These hormones respectively increased by 37% and 46% from pre- to post-training. A significant group effect was also identified for T, χ2 (1) = 25.0, p b 0.001, being 47% higher in Non-OC women. There were no other significant group effects or interactions during training. Analysis of the club competitions revealed a significant main sample effect for T, χ2 (1) = 37.7, p b 0.001 (Fig. 3A), and for C, χ2 (1) = 32.6, p b 0.001 (Fig. 3B). Both hormones increased by 49% and 75% respectively from pre- to post-competition. Testosterone levels also differed between the study groups, χ2 (1) = 8.77, p = 0.003, being 41% higher in the Non-OC women. Across the International competitions we again found a significant main sample effect for both T, χ2 (1) = 109.8, p b 0.001 (Fig. 4A), and C, χ2 (1) = 7.41, p = 0.006 (Fig. 4B). These hormones increased by 42% and 30% respectively from pre- to post-competition. A significant main group effect was also identified for T, χ2 (1) = 11.5, p = 0.001, with 31% more T in the Non-OC women. No other significant effects or interactions were found during the club or International competitions.

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daily programme. All competitions were played in the afternoon (start times were 3:30 PM for club games and 2:00 PM for International games), except for the 2nd International game which started at 10:30 AM. Cortisol secretion exhibits a clear circadian rhythm, with higher levels in the morning before decreasing across the waking day [26,32,42], whilst T secretion appears to be stable across the waking day [32,41,42]. Consistent with the literature, C levels before light training (3.92 ± 0.39 ng/ml) were higher than heavy training (2.09 ± 0.12 ng/ml), as was C prior to morning competition (3.79 ± 0.32 ng/ml) compared to the afternoon events (2.94 ± 0.18 ng/ml) (p b 0.055). Athlete T levels were more variable, with similar T profiles prior to light (69.0 ± 3.2 pg/ml) and heavy training (62.3 ± 2.2 pg/ml), whereas the morning competition had lower T levels (59.3 ± 4.4 pg/ml) than the afternoon events (70.4 ± 2.6 pg/ml) (p b 0.01). The observed differences in pre-competition and pretraining hormones are addressed in the statistical procedures.

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Within-session analysis revealed that T increases (p b 0.05) in both 297 groups across all training (Non-OC 30%, OC 17%) and competitions 298 (Non-OC 48%, OC 48%). Cortisol was also elevated (p b 0.01) in both 299 t2:1 t2:2

Table 2 Group demographics in the squad of elite female athletes (mean ± SD).

Athletes (number) Age (years) Height (cm) Body mass (kg) Body mass index (kg/m2)

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10 24.6 ± 2.4 168.4 ± 8.5 64.2 ± 6.4 22.6 ± 0.8

t2:4 t2:5 t2:6 t2:7 t2:8

Notes: OC = oral contraceptives.

Please cite this article as: B.T. Crewther, et al., Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in eli..., Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.017

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Fig. 1. Estimated marginal means (± SE) for salivary testosterone (1A) and cortisol (1B) levels pre and post the light training sessions in the OC and non-OC groups. OC = oral contraceptives. 1Significant sample effect p b 0.001, 2Significant group effect p b 0.001.

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This study presented a unique opportunity to examine hormone dynamics in elite women athletes and interactions with OC use during a short, intensive training block. The key findings were as follows: (1) lower absolute T levels in the OC (vs. Non-OC) group across all training sessions and competitions, (2) comparable T and C changes in the OC and Non-OC groups across individual activities, (3) evidence of reduced T and C responses in the OC group when the training and/or competition data were pooled, and (4) different hormone responses (as a squad) depending on the intensity of training and competition status. As hypothesized, the salivary T levels of the OC group were consistently lower than the Non-OC group irrespective of the activity performed, the sample collected and the time of testing. This is consistent

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groups when competing (Non-OC 56%, OC 37%), but not training (NonOC 15%, OC − 16%). A final GEE analysis was conducted with pooled activity (training, competitions), group (OC, Non-OC) and their interaction as categories. The T changes differed by activity type, χ2 (1) = 15.9, p b 0.001, being higher in competition (48%) than training (24%). A group effect was also identified, χ2 (1) = 6.79, p = 0.009, with NonOC women producing a larger T response (39%) than OC women (33%). Activity type had a significant C effect, χ2 (1) = 26.3, p b 0.001, with competition producing a larger C change (46%) than training (− 1%). Overall, C was more responsive in Non-OC women increasing by 36% (p b 0.001), whereas the OC women had a small (10%) nonsignificant change (p = 0.307). The group effect on C approached significance, χ2 (1) = 2.92, p = 0.087.

Fig. 2. Estimated marginal means (±SE) for salivary testosterone (2A) and cortisol (2B) levels pre and post the heavy training sessions in the OC and non-OC groups. OC = Oral contraceptives. 1Significant sample effect p b 0.001, 2Significant group effect p b 0.001.

with prior studies (using saliva or blood T measures) on athletes [3, 15] and healthy populations [9,21,23,32,41–43]. The lower T levels seen in OC users could be explained by corresponding decreases in blood (total and free) T levels with synthetic steroid use, due to the inhibition of ovarian and adrenal androgen synthesis [45], along with increasing sex-hormone binding globulin levels [3,45]. The T differences between the OC and Non-OC groups in this work (31–52%) are consistent with the reported decreases in blood-free T (61%) and total T (31%) [45]. Cortisol levels in the OC and Non-OC groups were similar across this study. Other evidence indicates that OC usage does not influence baseline (or non-stimulated) salivary or blood-free C levels in healthy women [5,26,27,32,42]. Some time-dependant effects have however been reported, with OC use associated with greater overall C secretion in the morning (before midday), a reduced awakening response and/or a delayed early morning peak [4,5,38]. The morning C data taken from the light training sessions (and the 2nd International competition) do not support these findings, perhaps due to the small number of players tested and the sampling schedule, particularly when more frequent sampling (e.g. every 30 min) over several hours is often employed to detect these subtle patterns. The OC and Non-OC women had similar T responses when all activities were examined separately, which is consistent with the testing of collegiate athletes across sports competition [14,15], as well as untrained and trained women across an exercise intervention [18]. We did find a reduced T response in OC women after combining the training and competition data. It is possible that the cited studies lack sufficient sensitivity to identify these differences due to a smaller sample size, the testing of a single event and/or the pooling of data across many athletic groups. Furthermore, we tested an elite population with much higher

Please cite this article as: B.T. Crewther, et al., Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in eli..., Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.017

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salivary T levels (2–3 times more) than prior reports on younger collegiate athletes [14,15], which is consistent with the finding that elite women have higher T levels than non-elites [11]. Several studies indicate that OC use can dampen the free C responses to physical, psychological and/or pharmacological stress [4,5,26,27,38], but there is little supporting data on elite athletes within the sporting environment. We found some evidence of diminished C reactivity in OC women when data were combined across the entire training block. Such a finding could be due to a concomitant rise in cortisol-binding globulin (CBG) levels in blood [9,43], leading to more bound C (but lower free C) when the HPA axis is activated during stress in OC users [38]. Moreover, an increase in CBG levels could potentially decrease free C availability through a negative feedback mechanism involving the glucocorticoid receptors [38]. The hormonal responses generally followed expected patterns of change. The C decline (−52%) with light training could reflect normal circadian variation, especially in the early morning [3,5,26,32,42]. The positive T (45%) and C (46%) responses to heavy training could be explained by greater physical exertion (mean RPE = 7.1) over the same time period, as per light training (mean RPE = 4.7). The hormonal responses to heavy training were comparable to the International competitions, as reported elsewhere [14], and possibly explained by a longer exercising period (120 and 70 min, respectively) at a similar level of exertion during these games (mean RPE = 7.3). Some training sessions in this work were also designed to mimic competitive match play and thus, could engage a high level of competitive spirit and dominance behaviours to activate T availability [14,15]. Our results also confirm a rise in female T and C levels across teamsport competition [2,13–17]. It was surprising that the club games promoted greater T (62%) and C (80%) responses than the International

Fig. 4. Estimated marginal means (±SE) for salivary testosterone (4A) and cortisol (4B) levels pre and post the International competitions in the OC and non-OC groups (with time of day as a covariate). OC = oral contraceptives. 1Significant sample effect p b 0.05, 2 Significant group effect p b 0.01.

games (40% and 27%, respectively). Both events produced similar levels of physical exertion (mean RPE = 6.9 and 7.3 respectively), but the International squad players typically start club games and play for much longer periods (to change the outcomes) than they would during International games. Speculatively, premier club games can be psychologically demanding if players have greater expectations to perform at a higher level, as per their representative status, especially if these matches are contributing to important selections. Other psychological factors relating to sporting performance (e.g. individual contribution to team outcomes, coping styles, personal control) could also regulate hormone reactivity [39], along with actual performance achieved [36].

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Fig. 3. Estimated marginal means (±SE) for salivary testosterone (3A) and cortisol (3B) levels pre and post the club competitions in the OC and non-OC groups. OC = oral contraceptives. 1Significant sample effect p b 0.001, 2Significant group effect p b 0.01.

Fig. 5. Estimated marginal means (±SE) for the changes in salivary testosterone levels across all activities (with time of day as a covariate). *Significantly different from baseline p b 0.05, 1Significantly different from light training p b 0.05, 2Significantly different from International competitions p b 0.05.

Please cite this article as: B.T. Crewther, et al., Effects of oral contraceptive use on the salivary testosterone and cortisol responses to training sessions and competitions in eli..., Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.017

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This work confirms that OC usage can lower T levels in elite women athletes, whilst the pooling of data provided further evidence of reduced T and C responses in the sporting environment. Irrespective of OC use, training and competition promoted different hormone responses likely

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We would like to thank the athletes and coaching staff that contributed to this project. This project was partly supported by a grant from the Engineering and Physical Sciences Research Council UK and the UK Sports Council, as part of the Elite Sport Performance Research in Training (ESPRIT) with Pervasive Sensing Programme [EP/H009744/1].

[1] G.A. Ballinger, Using generalized estimating equations for longitudinal data analysis, Organ. Res. Methods 7 (2004) 127–150, http://dx.doi.org/10.1177/ 1094428104263672. [2] H.S. Bateup, A. Booth, E.A. Shirtcliff, D.A. Granger, Testosterone, cortisol, and women's competition, Evol. Hum. Behav. 23 (2002) 181–192, http://dx.doi.org/10. 1016/S1090-5138(01)00100-3. [3] S. Bermon, P.Y. Garnier, A. Lindén Hirschberg, N. Robinson, S. Giraud, R. Nicoli, N. Baume, M. Saugy, P. Fénichel, S.J. Bruce, H. Henry, G. Dollé, M. Ritzen, Serum androgen levels in elite female athletes, J. Clin. Endocrinol. Metab. 99 (2014) 4328–4335, http://dx.doi.org/10.1210/jc.2014-1391. [4] N. Boisseau, C. Enea, V. Diaz, B. Dugué, J.B. Corcuff, M. Duclos, Oral contraception but not menstrual cycle phase is associated with increased free cortisol levels and low hypothalamo-pituitary-adrenal axis reactivity, J. Endocrinol. Investig. 36 (2013) 955–964, http://dx.doi.org/10.3275/8971. [5] E.M. Bouma, H. Riese, J. Ormel, F.C. Verhulst, A.J. Oldehinkel, Adolescents' cortisol responses to awakening and social stress; effects of gender, menstrual phase and oral contraceptives. The TRAILS study, Psychoneuroendocrinology 34 (2009) 884–893, http://dx.doi.org/10.1016/j.psyneuen.2009.01.003. [6] M. Cardinale, M.H. Stone, Is testosterone influencing explosive performance? J. Strength Cond. Res. 20 (2006) 103–107. [7] J. Carré, No place like home: testosterone responses to victory depend on game location, Am. J. Hum. Biol. 21 (2009) 392–394, http://dx.doi.org/10.1002/ajhb. 20867. [8] M.F. Chan, M. Dowsett, E. Folkerd, N. Wareham, R. Luben, A. Welch, S. Bingham, K.T. Khaw, Past oral contraceptive and hormone therapy use and endogenous hormone concentrations in postmenopausal women, Menopause 15 (2008) 332–339, http:// dx.doi.org/10.1097/gme.0b013e31806458d9. [9] C.M. Coenen, C.M. Thomas, G.F. Borm, J.M. Hollanders, R. Rolland, Changes in androgens during treatment with four low-dose contraceptives, Contraception 53 (1996) 171–176, http://dx.doi.org/10.1016/0010-7824(96)00006-6. [10] C.J. Cook, M.C. Beaven, Salivary testosterone is related to self-selected training load in elite female athletes, Physiol. Behav. 116–117 (2013) 8–12, http://dx.doi.org/10. 1016/j.physbeh.2013.03.013. [11] C.J. Cook, B.T. Crewther, A. Smith, Comparison of baseline free testosterone and cortisol concentrations between elite and non-elite female athletes, Am. J. Hum. Biol. 24 (2012) 856–858, http://dx.doi.org/10.1002/ajhb.22302. [12] B.T. Crewther, T. Heke, J.W.L. Keogh, The effects of training volume and competition on the salivary cortisol concentrations of Olympic weightlifters, J. Strength Cond. Res. 25 (2011) 10–15, http://dx.doi.org/10.1519/JSC.0b013e3181fb47f5. [13] D.A. Edwards, K.V. Casto, Women's intercollegiate athletic competition: cortisol, testosterone, and the dual-hormone hypothesis as it relates to status among teammates, Horm. Behav. 64 (2013) 153–160, http://dx.doi.org/10.1016/j.yhbeh.2013. 03.003. [14] D.A. Edwards, L.S. Kurlander, Women's intercollegiate volleyball and tennis: effects of warm-up, competition, and practice on saliva levels of cortisol and testosterone, Horm. Behav. 58 (2010) 606–613, http://dx.doi.org/10.1016/j.yhbeh.2010.06.015. [15] D.A. Edwards, L.J. O'Neal, Oral contraceptives decrease saliva testosterone but do not affect the rise in testosterone associated with athletic competition, Horm. Behav. 56 (2009) 195–198, http://dx.doi.org/10.1016/j.yhbeh.2009.01.008. [16] D.A. Edwards, J. Waters, A. Weiss, A. Jarvis, Intercollegiate athletics: competition increases saliva testosterone in women soccer, volleyball, and softball players, in: L.I. Ardis (Ed.), Testosterone Research Trends, Nova Science Publishers Inc. 2007, pp. 195–209. [17] D.A. Edwards, K. Wetzel, D.R. Wyner, Intercollegiate soccer: saliva cortisol and testosterone are elevated during competition, and testosterone is related to status and social connectedness with teammates, Physiol. Behav. 87 (2006) 135–143, http://dx.doi.org/10.1016/j.physbeh.2005.09.007. [18] C. Enea, N. Boisseau, M. Ottavy, J. Mulliez, C. Millet, I. Ingrand, A. Diaz, B. Dugue, Effects of menstrual cycle, oral contraception, and training on exercise-induced changes in circulating DHEA-sulphate and testosterone in young women, Eur. J. Appl. Physiol. 106 (2009) 365–373, http://dx.doi.org/10.1007/s00421-009-1017-6. [19] E. Filaire, G. Lac, Dehydroepiandrosterone (DHEA) rather than testosterone shows saliva androgen responses to exercise in elite female handball players, Int. J. Sports Med. 21 (2000) 17–20, http://dx.doi.org/10.1055/s-2000-8851.

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The direct measurement of these behavioural and performance factors would advance studies in this area. Theoretical models relating to the gaining, maintaining and losing of social status (e.g. challenge hypothesis, biosocial status) [33,44] have provided a strong framework to examine and interpret T dynamics in sporting competition (and training), as well as C as a stress biomarker in this environment. Our work highlights the need to additionally consider the challenge demands (e.g. physical, psychological) of the sporting activity and extraneous factors (e.g. OCs) influencing female hormonal physiology when applying these models. Being able to challenge athletes with exercise stressors that differ in their social importance and/or physical intensity also partly supports sport as a possible model to examine dominance and stress physiology, but additional assessments are necessary to quantify these outcomes within a sporting framework. In terms of practical applications, a reduction in T availability with OC usage could possibly influence explosive performance [6], perceived playing abilities [17], and voluntary training loads [10]. Synthetic steroids used in contraception can also modify various metabolic processors (e.g. lipid and carbohydrate metabolism) [40] and thus, access to energy resources under training and competition stress. These findings have broader implications for women when choosing and performing physical activity, along with their ability to cope with and respond to stressful encounters. Although OCs are associated with negative cardiovascular and venous risks [40], they may also provide some health benefits (e.g. lower risk of ovarian cancer, improve pelvic inflammatory disease and acne, reduce premenstrual symptoms) [24]. The risks and benefits are likely to depend upon the molecular structure, type and dose of steroid(s), and their route of administration [40]. We do acknowledge the study limitations. The match demands for different positional groups could elicit different hormonal responses, but this level of analysis was not feasible. Likewise, no psychological data were taken to quantity the emotional state and behaviour of players. Playing strategies and substitutions during competition add to these complexities. We further acknowledge other methodological issues relating to the study design (i.e. missing data, unbalanced groups, no randomisation to treatments, self-report measures), but these are inherent limitations when conducting research on elite athletes. Finally, we did not monitor the menstrual cycle in the Non-OC group, but the T and C profiles of naturally-cycling women appear to be largely unaffected by this cycle [3–5,18,32,35].

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Fig. 6. Estimated marginal means (±SE) for the changes in salivary cortisol levels across all activities (with time of day as a covariate). *Significantly different from baseline p b 0.01, 1Significantly different from light training p b 0.05, 2Significantly different from heavy training and International competitions p b 0.05.

to reflect the physical and/or psychological demands of these activities. These findings highlight the need to consider other factors influencing female hormonal physiology when using theoretical models in sport, with broader implications for women regarding exercising behaviours and related stress physiology.

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