Generally, athletes with high training loads experience impaired physical condition and athletic performance. Upper respiratory tract infection (URTI) engenders sore throat, headache, runny nose, coughing, fever, fatigue, or emesis, leading to impairment of the athletes' physical condition (15,18). Indeed, URTI is a particularly common illness among athletes because of the chronic immunosuppression that occurs during intensive exercise training and major competition.
Salivary secretory immunoglobulin A (SIgA), which neutralizes and prevents pathogens from entering the body via mucosal surfaces by inhibiting their attachment and replication, plays an important role in mucosal immune function. Salivary SIgA is mainly responsible for URTI. Decreased SIgA levels are linked to increased incidence of URTI (7,14). Resting salivary SIgA secretion is reduced by intensive exercise training (1,7,18). Reportedly, a decline in salivary SIgA was found to precede the appearance of URTI during a training period, suggesting that salivary SIgA is useful for evaluation of URTI risk in athletes (14).
In recent years, the female athlete triad, which is a combination of amenorrhea, eating disorders, and osteoporosis, has received considerable attention. Actually, higher incidence of amenorrhea is found in athletes than in the general population (13). Strenuous exercise in female athletes induces endocrine and metabolic changes, engendering severe hormonal disturbances. Therefore, athletes with amenorrhea have low levels of the estrogen (5,11). Estrogen plays a critical regulatory role in the development and maintenance of immune function; it is related to susceptibility to infections (19). Reportedly, the pattern of serum estradiol, which is the major estrogen, resembles that of salivary SIgA during the menstrual cycle (9). Salivary estradiol is significantly correlated with salivary SIgA in healthy women (22). Moreover, female athletes are implicated as susceptible to infection during particular stages of the menstrual cycle (19). Therefore, estrogen deficiency might accelerate downregulation of mucosal immune function and enhancement of susceptibility to infection in female athletes. However, no report has described a study of the effects of amenorrhea in athletes on salivary SIgA secretion and incidence of URTI. Such a finding is expected to contribute to the planning of conditioning programs for female athletes.
This study was designed to examine the effects of amenorrhea on mucosal immune function and susceptibility to URTI in elite female distance runners. We hypothesized that amenorrheic runners (ARs) have lower levels of serum estradiol and salivary SIgA and higher incidence of URTI symptoms than eumenorrheic runners (ERs) have.
Experimental Approach to the Problem
Female athletes with high training loads are likely to experience amenorrhea. Amenorrheic athletes are prone to have low levels of estrogen, which might be related to alteration of salivary SIgA and susceptibility to URTI. Nevertheless, the effects of amenorrhea in athletes on SIgA and URTI have not been elucidated. The present study evaluates salivary SIgA and the appearance of URTI symptoms in elite female athletes by comparing amenorrheic and eumenorrheic runners.
This study examined 21 elite, collegiate, female distance runners. All were of Japanese race and ethnicity. They were classified as ER (n = 8; 19.9 ± 0.8 years) or AR (n = 13; 20.0 ± 0.3 years) based on their menstrual cycles during the prior year. A regular menstrual cycle was defined as 10–14 per year. Amenorrhea was defined as fewer than 4 menstrual periods per year. All subjects had experienced menarche. Based on results of a self-reported questionnaire, no subject had been treated with any drug known to affect immune function, nor was any subject smoking tobacco regularly. This study, which conforms to the principles outlined in the Declaration of Helsinki, was approved by the Ethics Committees of the University of Tsukuba. All subjects signed an informed consent form prior to participating in the study.
The body mass, percentage of fat, fat mass, fat-free mass, and body water were recorded using a digital scale (Bioelectrical Impedance Analysis, MLT-10; Sekisui Chemical Co. Ltd., Tokyo, Japan). Each subject wore light clothing and no footwear.
Saliva samples were collected between 8:30 and 9:30 AM. Participants came to the laboratory without breakfast (except for water). We had previously asked participants to get sufficient sleep the night before. We also asked them to drink water to avoid thirst before measurements. They sat and rinsed their mouth completely with distilled water (30 seconds × 3 times). Then, they rested for at least 5 minutes. Saliva production was stimulated by chewing a sterile cotton ball (Sarstedt, Nümbrecht, Germany) at a frequency of 120/120 seconds. The obtained saliva was separated from the cotton by centrifuging at 3,000 revolutions per minute. After measurement of the sample volume, saliva samples were frozen at −40°C. Salivary SIgA concentrations were measured using enzyme-linked immunosorbent assay according to the procedures described for previous studies (1,18). To analyze SIgA levels, data were expressed as the SIgA secretion rate (μg·2 min−1). The SIgA secretion rate was calculated by multiplying the absolute SIgA concentration (μg·mL−1) by the saliva flow rate (ml·2 min−1).
Blood samples were collected after saliva sampling. Subjects sat and rested for at least 5 minutes. Blood samples were collected in vacutainers containing sodium EDTA. Serum 17β-estradiol concentrations and progesterone concentrations were measured using radioimmunoassay (RIA; Mitsubishi Chemical Medience, Corp., Tokyo, Japan).
Appearance of URTI Symptoms
We asked subjects, “Have you experienced URTI symptoms (sore throat, headache, runny nose, coughing, or fever) during the last month?” using a questionnaire, as described in a previous report (18).
All data are represented as means ± SE. For all analyses, p ≤ 0.05 was inferred as statistically significant. All variables were analyzed using Mann-Whitney U-test. An intraclass correlation (ICC Rs) was calculated for each measurement to assess the reliability of each test. Reliability ICC Rs for the dependent variables was 0.93–0.99. The effect size was calculated as the difference between means. An effect size of r = 0.10–0.23 is considered small, r = 0.24–0.36 is medium, and r = 0.37 or larger is large (3).
Table 1 presents subject characteristics for both groups. Body mass and body mass index were significantly lower in ARs than in ERs (p < 0.05; r = 0.42). The menstrual frequency per year was significantly lower in ARs than in ERs (p < 0.05; r = 0.74). The serum estradiol concentration was also significantly lower for ARs than for ERs (p < 0.05; r = 0.59). The serum progesterone concentration showed no significant difference between groups. The running distance per week was significantly longer in ARs than in ERs (p < 0.05; r = 0.48).
The saliva flow rate averaged 2.07 ± 0.23 ml·2 min−1 in ERs and 2.04 ± 0.29 ml·2 min−1 in ARs (Figure 1A). The saliva flow rate showed no significant difference between groups (r = 0.11). The SIgA concentration averaged 24.1 ± 5.5 μg·mL−1 in ERs and 14.1 ± 1.6 μg·mL−1 in ARs (Figure 1B). The SIgA concentration tended to be lower in ARs than in ERs (p = 0.09; r = 0.35). The SIgA secretion rate averaged 47.3 ± 8.6 μg·2 min−1 in ERs and 28.2 ± 5.0 μg·2 min−1 in ARs (Figure 1C); that rate was significantly lower in ARs than in ERs (p < 0.05; r = 0.44).
Table 2 presents the numbers of URTI symptoms in both groups. In ERs, 2 reports of sore throat (25.0%), 1 of runny nose (12.5%), and 1 of coughing (12.5%) were given for the prior month. In ARs, 3 reports of sore throats (23.1%), 2 of headaches (15.4%), 4 of runny nose (30.8%), 2 of coughing (15.4%), and 3 of fever (23.1%) were given for the prior month.
The primary finding of our investigation was that amenorrheic distance runners had lower levels of serum estradiol and salivary SIgA and higher appearances of URTI symptoms than eumenorrheic distance runners had. These results suggest that athletic amenorrhea accelerates downregulation of mucosal immune function and enhances susceptibility to URTI in female athletes.
Female athletes have a high prevalence of menstrual disorders (17). Especially, exercise-induced amenorrhea is a severe menstrual disorder. Athletes with amenorrhea show disturbances in the release of follicle-stimulating hormone and luteinizing hormone, including the presence of hypothalamic dysfunction, leading to estrogen deficiency (5,13). Results of the present study also show that ARs had lower levels of serum estradiol than ERs. This result concurs with earlier findings (5,13).
Estrogen has been associated with variation of salivary SIgA secretion (9,22) and susceptibility to infection (19). Salivary SIgA, the human body's first line of defense against pathogenic microbial invasion, is linked to the incidence of URTI (7,14). In this study, ARs had lower estradiol and salivary SIgA and more frequent incidence of URTI symptoms than ERs had. These results suggest that athletes with athletic amenorrhea might be related to impairment of mucosal immune function and might be susceptible to infection. Consecutive heavy training induces decreasing resting levels of estradiol (4), engendering athletic amenorrhea. Intensive exercise training also reduces salivary SIgA secretion and increases susceptibility to URTI in athletes (7,14,18). It is therefore possible that exercise-induced deficiency of estradiol might further downregulate salivary SIgA secretion and increase the incidence of URTI in highly trained athletes.
To date, the mechanisms underlying decreased salivary SIgA secretion through athletic amenorrhea in athletes have remained unclear. The immunoglobulin A (IgA) is secreted by plasma cells, which differentiate from B cells residing beneath the acinar epithelium in the salivary glands. Transport of IgA across the epithelial cell barrier requires a polymeric immunoglobulin receptor (pIgR) (20). Reportedly, the secretory component (SC), which consists of a portion of pIgR and the J chain, a constituent of SIgA, changes during a menstrual cycle (2). Estrogen putatively acts via estrogen receptors in both the oral epithelium and salivary glands and modulates saliva secretion and composition (21). Furthermore, results of an in vivo study showed that long-term exposure to high doses of estradiol enhanced B-cell proliferation in mice (16). It is therefore possible that estradiol regulates expressions of SC and IgA secretion by plasma cells, thereby engendering variation of salivary SIgA secretion. Moreover, exhaustive exercise reduces submandibular pIgR expression and salivary SIgA concentration in mice (10). High-intensity exercise might induce downregulation of hormonal secretion including estradiol, leading to degraded SC expression and IgA produced by plasma cells and reduced salivary SIgA secretion in female athletes. Future studies should examine the influences of athletic amenorrhea on SIgA secretion closely by measuring female hormones and other immune parameters including IgA producibility in plasma cells and expression of SC.
Athletic amenorrhea presents risks of becoming refractory (second-grade amenorrhea) and decreased bone density. Moreover, it adversely affects fertility. In this study, ARs had higher appearances of URTI symptoms than ERs. Therefore, female athletes, and their coaches and support staff, must be aware of the potential effect that athletic amenorrhea might have on acceleration of downregulated mucosal immune function and susceptibility to infections and take appropriate precautions against them. Monitoring salivary SIgA levels can be useful for identifying excessive training loads and determining the risk of URTI in athletes (14). Therefore, we recommended monitoring of menstrual status and salivary SIgA levels to modify the training volume and predict the risk of URTI in athletes. From the viewpoint of nutrition, previous studies have shown that vitamin A deficiency decreased SIgA secretion (6) and that regular ingestion of Lactobacillus inhibited decreasing salivary SIgA secretion and reduced the frequency of URTI in athletes during training and competition periods (8). Moreover, acupuncture treatment has an inhibitory effect on exercise-induced decrease of salivary SIgA in athletes during a period of competition (1). Therefore, monitoring nutritional status, ingesting Lactobacillus, and receiving acupuncture treatment might be related to better maintenance of mucosal immune function in athletes.
This study has study limitations. First, the small sample size of subjects limited our ability to conduct meaningful analyses of each parameter. It is related to the stringent inclusion criteria and the difficulty of finding amenorrheic and eumenorrheic athletes who belong to the same team, have similar competitive level, and are sampled at the same period. Further studies must use proper quantities of amenorrheic and eumenorrheic athletes in several teams with similar competitive levels. Second, we did not conduct sampling of ER subjects in the same phase of the menstrual cycle. Five of 8 subjects in ER were sampled in the follicular phase. The other 3 subjects were sampled in the ovulation phase. In this study, serum progesterone in ARs had a similar value in ERs. This result agreed with previous reports that serum progesterone in amenorrheic athletes shows a similar value in eumenorrheic athletes in follicular phase (5). In addition, progesterone induced the increase of plasma cells in mucosal tissues, suggesting that progesterone might regulate SIgA secretion (12). Additional research is necessary to conduct comprehensive sampling in the same phase of menstrual cycle, to compare data among menstrual phases, and to elucidate the contribution of potential mechanisms underlying estradiol- and progesterone-regulated SIgA secretion in athletes.
In conclusion, we demonstrated that amenorrheic distance runners had lower levels of serum estradiol concentration and salivary SIgA secretion and higher appearances of URTI symptoms than eumenorrheic distance runners. These results suggest that athletic amenorrhea with low levels of estradiol accelerate downregulation of mucosal immune function and high susceptibility to infection such as URTI in female athletes.
Female athletes are subject to menstrual disorders such as amenorrhea. In this study, amenorrheic distance runners had lower levels of serum estradiol and salivary SIgA and higher appearance of URTI symptoms than eumenorrheic distance runners. Female athletes and their coaches and support staff must be informed of the potential effects that menstrual disorders might have on the acceleration of downregulated mucosal immune function and susceptibility to infections and take appropriate precautions against them. We recommend modulating the training volume and nutritional status adequately by monitoring menstrual status, salivary SIgA level, and nutritional intake and by applying Lactobacillus ingestion (8) and acupuncture treatment (1) for better maintenance of physical condition including mucosal immune function in female athletes.
We thank all participants in this study, Dr. Takayuki Akimoto (Laboratory of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine Graduate School of Medicine, The University of Tokyo) and Dr. Fuminori Kimura (Graduate School of Comprehensive Human Sciences, University of Tsukuba), for critical comments. This study was supported by a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21700705 to K.S.).
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