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Archive > Year 2011, Number 2

The effects of successive official competitions on salivary cortisol and immunogolobulin responses in women handballers


INTRODUCTION
Daily engagement of individual athletes may compromise their ability to recover from the earlier competition. Based on the temporary immunosuppressive changes that have been observed for some hours after exercise sessions or competitions, the “open window” theory has been proposed to explain the transient changes [14]. Where official competitions are completed on the successive days, incomplete recovery from the primary competition could affect the responses to stress in the subsequent competitions. Therefore, the higher incidence of the upper respiratory tract infection (URTI) reported in elite athletes [15] may be due, at least in part, to the repetition of exercise sessions and competitions without allowing sufficient time for recovery. It has been suggested that increased frequency of URTI episodes [14], recurrent URTI, or reduced protection against certain infections [1] are associated with lowered concentrations of salivary immunoglobulin A (IgA) or chronic salivary IgA deficiencies. Salivary IgA, a key component of the mucosal immune system, which is locally synthesized and has a short half-life, can bind selectively to antigens, such as microbes, thereby preventing such antigens from attaching to or penetrating the mucosal surface [2].
Salivary IgA provides a major defence against potential pathogens by preventing colonization and replication on the mucosal surfaces of the upper respiratory tract. Competition has been also considered an acute stress. However, it has been suggested that the psychological stress of competition may modulate secretory immunity and result in increased susceptibility to URTI in athletes [14]. During the competition process, the sympathetic nervous system is activated [18, 32], which is known to modulate the immune system. Various studies have examined how salivary IgA concentration or secretion rate is modulated by competition stress. There are reports demonstrating that salivary IgA concentrations are suppressed [12] or elevated [3, 22, 27, 30] in response to competition. In the field of sport and exercise, moreover, there is some evidence that chronic exercise (i.e. overtraining) is also associated with reductions in salivary IgA [e.g. 15, 31]. Additionally, reductions in mucosal immune function have been observed during a competition period among elite female athletes [12].
However, in a study on boy players, Tharp [30] provided support for positive effects of basketball training. He examined salivary IgA levels before and after three games and three practice sessions during a basketball season among two age groups. The results indicated that basketball exercise can increase salivary IgA levels and that chronic exercise over the basketball season may increase the resting levels of salivary IgA. In conclusion, current evidence on immunity changes in response to chronic stress would support a view that intense stress increases, whereas moderate stress reduces the susceptibility to illness, predominately URTI. Nevertheless, to our knowledge, the cumulative effects of successive official competitions have not been examined. Cortisol is also thought to play a role in inhibiting mobilization of salivary IgA and influencing its synthesis [5]. Administration of dexamethasone (a potent synthetic glucocorticoid) has been shown to cause a decline in the salivary IgA concentration but with a delay of 24 hours, which is more likely attributable to decreased IgA synthesis and/or induction of lymphocyte apoptosis. Several studies have reported elevated concentrations of cortisol in saliva after competitions in team sports [9, 17] and individual sports [7, 11].
In light of this current understanding, concentrations of IgA and cortisol in saliva may be used to reflect changes in the immune function [29]. The cited studies have been conducted using boys on team sports such as basketball. However, it remains unclear if this effect on salivary cortisol and IgA responses exists: 1) in handball and 2) in girls.
Therefore, the main aim of the current study was to investigate the cumulative effect of successive official competitions on salivary cortisol and IgA in women handball players.

MATERIALS AND METHODS
SUBJECTS

The subjects were carefully informed about the experimental procedures and the possible risk and benefits of the project approved by the local committee of ethics and gave written consent to the participation in this study. Twelve girl players (Table 1) on a regional team were asked to participate in this study. Subjects were preparing to compete in the 3rd Iranian Sports Olympiad. After familiarization with the procedure, they provided written informed consent and completed the preliminary measurements. The subjects were all in good health and took part in handball exercise regularly (four times a week). They were all non-smokers, were not in the menstrual cycle phase, reported no significant hormonal disorders or oral, dental or other symptoms of infection and had not been taking any medication in the month leading up to the experiment. The team under investigation won five successive games during the competitions. The physical characteristics of the subjects are presented in Table 1 (Mean ± SD).



PRELIMINARY MEASUREMENTS
Each subject’s height was determined using a stadiometer (SECA, Germany) with a precision of 2 mm and a range of 130-210 cm and nude body mass (kg) was measured using calibrated precision-weighing scales (SECA, Germany). The player’s Frankfort Horizontal plane and mass (kg) were recorded by a weighing machine model (SECA, Germany), with a precision of 0.2 kg and a range of 2 kg to 130 kg. The body mass index (BMI) was calculated and percent body fat was estimated from four skinfold thickness measurements using the method by Durnin and Womersley [8]. The aerobic power of the participants was assessed using an incremental test to determine maximal oxygen consumption (VO2max) on a motor-driven treadmill. The guidelines of the British Association for Sport and Exercise Sciences were followed to confirm that VO2max was achieved.

Saliva Collection and Analysis
Saliva samples were collected at the same time of day (5:00 pm) during a resting day and the competitions. The “passive expectoration method” was used according to the directions given by Navazesh and Christensen [25]. The subjects were required initially to rinse out their mouths with distilled water to prevent potential sample contamination that might affect IgA and/or cortisol levels. Then, 8 ml of unstimulated saliva was collected by expectoration into sterile laboratory tubes, with the eyes open, the head tilted slightly forward and making minimal orofacial movement. The samples were then aliquoted and stored at –20 ºC for subsequent analysis. The quantitative measurement of IgA in saliva samples was done by means of the indirect competitive enzyme immunoassay (EIA) kit (Salimetrics, State College, PA, USA). Briefly, a constant amount of goat anti-human salivary IgA conjugated to horseradish peroxidase was added to the tubes containing specific dilutions of standards or the saliva samples vortexed and centrifuged at 3000 rev•min–1 for 15 min. The antibody conjugate bound the salivary IgA in the standard or saliva samples. The amount of salivary IgA present was inversely proportional to the quantity of free antibody remaining. Following incubation and mixing, an equal solution from each tube was added in duplicate to a microtitre plate coated with human salivary IgA. Therefore, the free or unbound antibody conjugate bound to the salivary IgA on the plate. Then the unbound components were washed away after incubation and the bound conjugate was measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). The amount of peroxidase was inversely proportional to the quantity of salivary IgA in the samples [4]. Optical density was read on a standard automated plate reader at 450 nm (Triturus, IBL, Barcelona, Spain). The concentration of salivary IgA was calculated upon the linear curve fit of the related four parameters. Also, the DRG salivary cortisol ELISA kit (DRG Diagnostics, Marburg, Germany) was used for the quantification of cortisol in the saliva samples. The coefficients of variation for the analytical methods were 2.2% for cortisol and 1.6% for salivary IgA. The test-retest correlations of the salivary IgA and cortisol procedures in this laboratory were 0.99 and 0.98 respectively.

Assessment of the Game Difficulty
In order to control the difficulty of the games, all subjects were asked to rate their perceived difficulty on a 10-point Likert scale, ranging from 1 (very easy) to 10 (very difficult) 15 minutes before the games and immediately after the first and second half times. Total scores of perceived difficulty for each game were calculated by averaging three sets of related scores. The alpha coefficient for the five games was achieved between 0.89 to 0.94. To assess the game difficulty, the scores were classified into three categories, where scores 1 to 4 represented a light competition, scores 4.1 to 7 represented a moderate competition and scores 7.1 to 10 represented a heavy competition. According to this classification and scores rated by the subjects, Games 1, 2 and 4 were considered moderate competitions while Games 3 and 5 were considered heavy competitions.

STATISTICAL ANALYSIS
The data are presented as Mean ± SD. They were analyzed using repeated measures analysis of variance (ANOVA). Normality of distribution for all variables was checked with the Shapiro–Wilk test. The assumptions of homogeneity and sphericity in the data were checked and, where appropriate, adjustments to the degrees of freedom were made for the ANOVA. Post hoc analysis was carried out by paired t-tests with an appropriate Bonferroni correction factor. The effect size was classified according to the system proposed by Cohen [6], where an effect size of 0.2 represents a small effect, one of 0.5 represents a moderate effect, and one of 0.8 or above represents a large effect. Statistical significance was set at an alpha of p < 0.05. The data were analyzed using the statistical package SPSS, PC program, version 15.0 (SPSS Inc., USA).

RESULTS
SALIVARY CORTISOL

The main effect of time on salivary cortisol concentrations was significant (F(3.8, 38.3)=260.62, p<0.001, η2 = 0.963). Salivary cortisol concentrations increased significantly from 3.2 ± 0.35 ng•mL-1 at the baseline to 4.26 ± 0.7 ng•mL-1 before the first competition. Salivary cortisol concentrations for the five competitions increased significantly (105.4%, 116.8%, 129%, 114% and 143.1%, respectively) compared with pre-competition, and decreased significantly (51%, 39.9%, 66.4% and 44.6%, respectively) after the first four competitions compared with the pre-competition values of the next event (Table 2 and Figure 1).
The main effect of the succession of competitions on pre-competition concentrations of salivary cortisol was significant (F(4, 40)=6.69, p<0.001, η2 = 0.719). Post hoc comparisons showed that the pre-competition cortisol concentrations of Games 3 and 5 were significantly higher than others (p<0.05). There was a significant main effect of successive competitions on the post-competition concentrations of salivary cortisol (F(4, 40)=56.14, p<0.001, η2 = 0.849). Further analyses indicated that post-competition cortisol concentrations for Games 3 and 5 were significantly higher than the others (p<0.05) (Table 2 and Figure 1).






SALIVARY IMMUNOGLOBULIN A
The main effect of time on salivary IgA concentrations was significant (F(3.1, 31.8)=91.01, p<0.001, η2 = 0.901). The salivary IgA concentrations did not change significantly from 227.1 ± 27.3 mg•L-1 at the baseline to 218.9 ± 24 mg•L-1 before Game 1. The salivary IgA concentrations increased in response to Games 1, 2, and 4 by an average of 51.3%, 38.2%, and 19.7%, respectively, and fell to below the baseline in response to Game 3 (20.6%) and Game 5 (8.5%). Additionally, the salivary IgA concentrations decreased significantly (28.5%, 31.9% and 16%, respectively) after Games 1, 2, and 4 compared with the pre-competition levels of the next game, and remained unchanged after the third competition (Table 2 and Figure 2).
The main effect of successive competitions on pre-competition concentrations of salivary IgA was significant (F(4, 40)=25.53, p<0.001, η2 = 0.718). Post hoc comparisons showed that pre-competition salivary IgA concentrations for Games 4 and 5 were significantly lower than the others (p<0.05). Also, there was a significant main effect of successive competitions on post-competition concentrations of salivary IgA (F(4, 40)=131.4, p<0.001, η2 = 0.929). The Paired t test revealed that post-competition salivary IgA concentrations for Games 3 and 5 were significantly lower than the others (p<0.05) (Table 2 and Figure 2).



DISCUSSION
This is first study that focused on salivary cortisol and IgA responses to successive handball competitive games. The official women handball competition actually increased salivary cortisol concentrations and heavy competitions induced higher salivary cortisol responses than the moderate ones. Salivary cortisol concentration was not influenced by the succession of the competitions nor did its response depend on the difficulty of the game.
In our study, the post-competition salivary cortisol concentrations increased significantly compared with the pre-competition levels by an average of 116.7% (range 101-128%), and the pre-competition levels were averagely 50.7% (range 33-77%) higher than the resting baseline. The overall effect size of 0.963 would suggest a large difference between the baseline, pre- and post-competition levels. Previous studies have also consistently indicated an increase in salivary cortisol concentration during handball games and other team sports competitions [9, 17]. The rise in cortisol before the competitions may be a positive, appropriate, and adaptive response to prepare the competitors’ physiology for extreme physical exertion. Alternatively, it may be associated with anxiety and distress building in anticipation of the impending challenge. In addition, the degree of the rise in cortisol has been linked to the increased intensity of the physical challenge via association with lactate production which is known among female athletes. It is possible also that the change in cortisol exhibits a circadian variation, but the 1.5-h time difference between measurements might not be enough to influence the post-competition values [17].
Although the main effects of successive games on pre- and post-competition levels of salivary cortisol concentration were significant and the relative effect sizes of 0.719 and 0.849 would indicate medium and large differences, it nevertheless seems that the existing differences may not be induced by the cumulative effects of the successive games. In other words, non-significant differences between the pre-competition levels of Games 4 and 1 as well as between the post-competition levels of these games could not reflect the cumulative effect of successive competitions. The same observations could be found between the levels of Games 3 and 5 indicating no succession effect. These findings suggest that the resting durations between the competitive games may not have been short enough to influence the pre- and post-competition levels and they allowed salivary cortisol concentrations to recover effectively. On the other hand, the heavy competitions compared with the moderate competitions elicited higher levels of salivary cortisol concentrations for pre-competition (76.2% vs. 33.7%, respectively) and post-competition (123.8% vs. 110.8%, respectively). Most likely the psychological stress of a heavy match provides an extra stimulus for hypothalamic pituitary adrenal axis activation and the subsequent cortisol secretion [20].
In the current investigation, the salivary IgA concentration was elevated in response to Games 1, 2 and 4 by an average of 36.4% (range 19.7-51.3%). These observations are compatible with the results from a number of other investigations demonstrating positive effects of informal competition [30], or formal competition without presence of physical exertion [22] on the immune function. In addition, previous studies carried out in the laboratory settings have also shown transient increases in salivary IgA using standard psychological stress task, mental arithmetic [27], and acute psychological challenge [3]. The reason for this increase may be partly attributable to a higher sympathetic activity which plays a role in immune enhancement in the short term. Furthermore, the increase in salivary IgA concentrations after each game seemed to result, at least in part, from the reduction of the saliva flow rate. In contrast, with a different pattern, the salivary IgA concentration was suppressed in response to Games 3 and 5 by an average of 14.5% (range 8.5-20.6%), indicating immunosuppressive effects of heavy competitions. Critically, the current results supported the hypothesis that competition would lead to changes in secretory immunity [12].
It is known that cortisol plays a role in inhibiting mobilization of salivary IgA [19] and in influencing its synthesis [5], which is also affected by the autonomic nervous system and the hypothalamic-pituitary adrenal axis [5]. In the current study, the heavy competitions elicited higher levels of salivary cortisol concentrations than moderate competitions. Therefore, the observed decrease in salivary IgA concentrations immediately after heavy competitions suggests that cortisol affects salivary IgA levels. On the other hand, the competition process activates the sympathetic nervous system [18, 32]. The salivary glands in the mouth, which secrete both fluid and protein, are innervated by both sympathetic and parasympathetic nerves [26]. Therefore, it is possible that the studied competitive games were associated with different levels of neural stimulation of these glands.
In exercise and training contexts, the immune and endocrinological responses to exercise are affected by the modality, intensity, and duration of exercise as well as the performance level of the population under investigation. Moreover, a possible reason for the different pattern of salivary IgA responses found in the literature could be the effects of competitive task difficulty. In our study, Games 3 and 5 were judged to be more challenging and difficult than the others, and it is therefore possible that these two competitive games were sufficiently demanding to produce a decrease in salivary IgA.
The salivary IgA concentration responded significantly to the succession of the five competitive games. Its pre-competition levels decreased 21.1% after Game 3 and did not change significantly during the final competition. The post-competition level of salivary IgA decreased significantly 76.4% after Game 2, and subsequently increased 19.7% and then decreased 23.1% during the next games compared with the previous post-competition level. The magnitudes of the total decrease in salivary IgA concentration for pre- and post-competition were 13.7% and 50.7% compared with the initial baseline, respectively. Both pre- and post-competition levels of salivary IgA fell below the baseline during Game 3 and did not return significantly to the primary level. These results suggest that after a heavy official competition during a daily succession of games, salivary IgA concentrations fail to return fully to the initial baseline level. The decrease in the salivary IgA concentration seemed to result, at least in part, from the increased psychological stress of heavy competitions and incomplete recovery. Similar findings have been reported in elite female athletes during a competition period [12, 13]. These results may extend to successive competitive games when vulnerable players may experience clinically relevant reductions, given that they were regional players; this could be one limitation of the study. Moreover, a major limitation of this study was the subjectively determined intensity of the game.

CONCLUSION
In conclusion, official handball competition actually increased salivary cortisol concentrations and heavy competitions induced higher salivary cortisol responses than moderate ones. The salivary cortisol concentration was not influenced by the succession of competitions and its response depended on the difficulty of the games. Moderate handball competitions elevated salivary IgA concentration whereas heavy competitions suppressed salivary IgA concentration; one day rest after a heavy competition seemed to be insufficient for the secretory immune function to recover fully for subsequent games and led women handball players to increased susceptibility to URTI. Future research should examine whether this relationship occurs during preseason and during the preparatory and tapering periods.

PRACTICAL APPLICATION
Our findings suggest that for the improvement of the women handball players’ performance, one day rest between successive competitive games seemed to be insufficient for the secretary immune function to recover for subsequent games. The purpose of this study was to present scientific information about physiological characteristics of the handball game, which should be more interesting to practicing coaches, strength and conditioning coaches, physiotherapists, or sport physicians in order to gain insight of the physiological responses during the games.

ACKNOWLEDGEMENTS
The Khorasgan Azad University in the Islamic Republic of Iran supported this investigation.