Keywords
soccer - menstrual cycle - premenstrual syndrome - inflammation - cytokine
Palavras-chave
futebol - ciclo menstrual - síndrome pré-menstrual - inflamação - citocina
Introduction
The number of female athletes who participate in high performance sports has increased
in the last years.[1] Weight lifting, wrestling, soccer and other sports are examples of modalities in
which women have expressive performance. Soccer, in particular, is considered a sport
that presents stressful situations and it may generate anxiety responses from the
players because it demands motor, technical, tactical and psychological skills.[2] Thus, it is important to observe the impact of exercise on musculoskeletal and neuroendocrine
systems and the influence of the hormonal fluctuations of the menstrual cycle in female
athletes during their performances.[3]
There are several studies relating physical activity with menstrual changes, but only
a few evaluate the influence of the phases of the menstrual cycle in sports performance,
as well as disorders involved in the menstrual cycle, like premenstrual syndrome (PMS).[4]
Premenstrual syndrome is defined as a group of physical and behavioral changes that
can affect some women. In general, PMS occurs during the luteal phase of the cycle,
after ovulation, until the next menstruation.[5]
[6]
[7]
[8] The prevalence of PMS has a large range and, in Brazil, studies show that 8 to 86%
of women have some kind of symptoms.[9] The symptoms include breast tenderness, increased breast size, weight gain, legs
and whole body swelling, leg pain, increased abdominal size, headache, fatigue, anxiety,
irritability, depression and mood swings.[10]
[11]
[12] The symptoms may manifest with mild to moderate intensity and be repeated for several
cycles.[10]
[11]
[12]
Thus, it is critical to emphasize the importance of accurate detection of PMS, making
use of complete physical examinations and specific questionnaires for the evaluation
of the syndrome. Freeman et al[13] created a questionnaire of daily symptoms, the daily symptom report (DSR), for detecting
the occurrence of PMS, which is composed of 17 symptoms, with scales ranging from
0 to 5, according to the severity of each symptom described.
Another important issue in monitoring female athletes refers to psychological aspects
and its influence on performance. In high performance sports, the psychological assessment
seeks the qualification and quantification of the emotional state, occurrence of psychic
processes and interpersonal relations during training and competitions, prioritizing
teamwork optimization. Indeed, stress caused by strenuous training can lead to a condition
of psychophysical inadaptability, triggering negative effects on athletic performance.[14]
Anxiety is a typical “emotional event” of stress; it is directly related to the athlete
and can be decisive for his/her sports performance.[15] In fact, studies show that women have higher levels of anxiety when compared with
men and, and menstrual cycle fluctuations and PMS are aggravating factors.[16] The Beck Anxiety Inventory (BAI) was created by Beck et al,[17] and it is composed of a self-report scale to measure aspects of anxiety. It consists
of 21 questions that must be answered by the person in reference to him or herself.
The inflammatory mechanism resulting from exercise is well established in the literature;
including tissue remodeling and repair.[18]
[19]
[20]
[21] Inflammation plays an essential role when exercise is conducted in a systematic
and regular manner. Inflammatory pathways include the action of hormones, neurotransmitters
and cytokines, which are responsible for the overall coordination of inflammatory
events and their effects associated with exercise.[18]
[19]
[20]
[21]
Cytokines are produced and released by cells of the immune system, muscle in activity,
adipose tissue and endothelial cells.[22] Interleukin 10 (IL-10) is a cytokine that plays an anti-inflammatory role in response
to exercise, as well as acting in the brain and behavior, improving mood states.[22]
[23]
[24]
[25]
In an attempt to understand these aspects, we proposed to evaluate the levels of anxiety
and their connection with IL-10 in a female soccer player group, presenting or not
PMS, in four moments: before (pre) and after (post) a simulated game and during the
two phases of the menstrual cycle: follicular and luteal.
Methods
This is a longitudinal case-control study. Fifty-two (52) competitive female soccer
players (aged: 19.8 ± 4.7 years) who had played professionally in championships in
the state of São Paulo, Brazil, in 2012 were recruited for the study.
Exclusion criteria included: athletes who were pregnant, exhibited irregular menstrual
cycles, fractures, severe ligament injuries, presented genetic or acquired kidney
disease; used hormonal contraceptive or antidepressants, anxiolytics, diuretics, steroids
or illegal drugs, or did not regularly attend training sessions. All athletes were
aware of the potential risks involved in the study and signed the informed written
consent form. All experiments were performed in accordance with the Declaration of
Helsinki. The Ethics Committee of Universidade Federal de São Paulo approved both
the study protocol and consent form (number 1604/10). All athletes performed the same
tactical/ technical/ resistance training. The soccer specific training was realized
five times a week and, after that, the simulated game took place.
The presence of PMS was determined using the DSR questionnaire validated for the Brazilian
population by Vieira Filho et al.[26] All volunteers kindly answered the DSR questionnaire for 3 consecutive months. Menstruation
phases (follicular and luteal) were monitored to define urine collection dates more
precisely. The follicular phase was estimated to occur from the 1st through the 10th day of the menstrual cycle, and the luteal phase from the 20th day through the beginning of the next period.
Anxiety levels were determined through the BAI anxiety questionnaire validated for
the Brazilian population by Cunha.[27] All athletes answered the questionnaire during both phases of the menstrual cycle
and in two periods: before and after the simulated game, totalizing four questionnaires.
The guidelines about the questionnaire were explained by a sport psychologist.
Urine samples were collected on four occasions: before and after the simulated game,
and during both phases of the menstrual cycle. Sterile containers were used to collect
the urine samples and they were subsequently centrifuged at 1,000 rpm for 10 minutes
to remove sediment. Urine aliquots were stored at - 80°C for cytokine evaluation.
Interleukin-10 was evaluated using the HCYTOMAG-60K kit (EMD Millipore, Billerica,
MA, USA), according to the manufacturer's instructions, in four moments: before, after
the simulated game and during the follicular and luteal phases. A flow cytometer performed
with Luminex xMAP Technology (EMD Millipore, Billerica, MA, USA) was used to read
and analyze the data. The urine osmolarity was determined with the freezing procedure
using a wide range osmometer 3W2. All the results obtained from the urine samples
were normalized by the values obtained from the urine osmolarity analysis (OSM UR).
Fifty-eight athletes participated in this study. However, considering 80% of sample
power and possible loss of adherence in the study, the final sample was defined with
52 athletes. To check the normality of the data, the Kolmogorov-Smirnov test was used.
Data that did not present normal distribution were transformed into square root (√)
values. Thus, t test was used to analyze the data that presented a normal distribution. Analysis
of variance (ANOVA) was used to evaluate data that did not present normal distribution.
For the correlation analysis, we used the Pearson correlation test and the GraphPad
Prism software version 4 (GraphPad Software Inc., La Jolla, USA) for the construction
of the graphs. All data collected on urine samples were previously modified according
to the values obtained from OSM UR by the formula: (gross value / value of OSM UR)
x 1,000. The significance level was set at 5% (p < 0.05).
Data on age, height, weight and BMI were presented as the mean and standard deviation
(SD). The Student t-test was used to analyze differences in age, height, weight and BMI. Analysis of
variance was used for repeated measures and Pearson correlation was used to determine
the correlation between IL-10 cytokine and anxiety.
Results
According to the DRS questionnaire, 59.6% of the volunteers (31 athletes) presented
PMS and 40.4% (21 athletes) did not present PMS. As shown in [Table 1], no differences were observed in the physical characteristics of the volunteers,
presenting or not PMS. Moreover, [Table 1] also depicts the frequency of the main symptoms associated with PMS occurrence observed
in the DRS questionnaire.
Table 1
Physical characteristics (the mean ± standard deviation) of the female soccer players
recruited for this study and the most frequent symptoms in the volunteers with PMS
(%)
|
Characteristics
|
Volunteers (n = 52)
|
|
with PMS (N = 31)
|
without PMS (N = 21)
|
p
|
|
Age (year)
|
18.7 ± 3.99
|
20.68 ± 3.70
|
0.08
|
|
Height (cm)
|
165.1 ± 6.75
|
165.2 ± 5.07
|
0.97
|
|
Weight (kg)
|
60.8 ± 7.83
|
58.51 ± 6.48
|
0.28
|
|
BMI (Kg/m2)
|
22.25 ± 2.14
|
21.41 ± 1.38
|
0.12
|
|
Symptoms (%)
|
|
Irritability
|
80.6
|
−
|
|
|
Mood swings
|
77.4
|
−
|
|
|
Cramps
|
67.7
|
−
|
|
|
Anxiety
|
64.5
|
−
|
|
|
Breast tenderness
|
61.3
|
−
|
|
|
Nervous tension
|
54.8
|
−
|
|
Abbreviations: BMI, body mass index; N, number of subjects; p, p value; PMS, premenstrual
syndrome.
The analysis of the BAI questionnaire revealed that the group with PMS has a higher
level of anxiety when compared with the group without PMS (p = 0.002). Analysis of the urine concentration of IL-10 in the volunteers with and
without PMS, during the two phases of the menstrual cycle, before (pre) and after
(post) a game, revealed a significant decrease of IL-10 levels before the game and
during the luteal phase, when compared with pre-game levels during the follicular
phase in players without PMS ([Fig. 1-A]). There was no significant difference in IL-10 levels in the PMS group in any of
the 4 moments evaluated. The correlation analysis between IL-10 and anxiety showed
a negative correlation post-game in the luteal phase in the group without PMS ([Fig. 1-B]) and a positive correlation post-game in the luteal phase in the PMS group ([Fig. 1-C]).
Fig. 1 Analysis of anxiety and urinary concentration of cytokines in female soccer players.
(A) concentration of IL-10 in the group without PMS. * denotes differences in the values
observed before (pre) the game during the luteal phase and before (pre) the game in
the follicular phase (p < 0.05). (B) negative correlation in the group without PMS, luteal phase, post-game (p = 0.02, r = -0.50). (C) positive correlation in the group with PMS, luteal phase, post-game (p = 0.04, r = 0.36). Abbreviations: osm, osmolarity; PMS, premenstrual syndrome.
Discussion
This study is original in the evaluation of female soccer players with and without
PMS, correlating anxiety and immune response before and after acute exercise performed
in two phases of the menstrual cycle. Factors such as stress and accumulation of daily
functions have contributed to the increase of PMS in recent decades.[10] However, the mechanism of action of multiple factors involved in PMS is still unknown.
Estradiol, progesterone, serotonin and gamma-aminobutyric acid (GABA) participate
in the pathophysiology of women susceptible to PMS.[10] Indeed, hormonal fluctuations in the luteal phase lead to 5-hydroxytryptamine system
(5-HT) alterations in the brain. Five-hydroxytryptamine is a serotonergic neurotransmitter
involved in mood swing modulation, anxiety, appetite, sleep and arousal. Gamma-aminobutyric
acid is a primary regulator of affection, cognition and the expression of its receptor
is regulated by estrogen and progesterone.[16] Thus, probably due to the interaction between female hormones and these neurotransmitters,
it is expected that female soccer players may have PMS, as well as the general female
population.[28]
[29] Indeed, the result of the prevalence of PMS found in our group (59.6%) and the most
frequent symptoms, including anxiety, is similar to that observed in the Brazilian
and international population studies.[3]
[5]
[9]
[10]
[30] A study conducted by Teixeira et al[31] evaluated the correlation between PMS prevalence and physical activity level (PAL)
in a group of healthy women presenting regular menstrual cycle (18 to 35 years old).
Their most frequent symptoms were anxiety, nervous tension, irritability, crying and
insomnia. A negative correlation between the presence of PMS and PAL (lower PAL in
women with PMS compared with women without PMS) was observed in the PAL group.
According to Nillni et al[16] disorders that cause the emergence of anxiety are twice as prevalent in women. Important
hormonal fluctuations that occur in the different phases of the menstrual cycle, particularly
in the premenstrual – or luteal – phase, may constitute a possible neuromodulatory
effect that contributes to the maintenance of anxiety in women.
Our study reveals that the PMS group presents higher levels of anxiety than the group
without PMS. Anxiety is very personal and, in the sport context, it arises in a variety
of situations, having direct influence on the athlete's performance. For instance,
in a situation of “pressure from supporters,” the players who are suffering the pressure
may present doubtful (debilitating) or challenging (facilitative) feelings. Organic
anxiety reactions can cause behavioral and physical changes (athlete's movement and
reaction time, for example), leading to altered results in competitions. In fact,
there are many variables that influence anxiety levels: type of sport, weather, environment,
preparation for competitions, route, etc. In this way, it is very important that the
athlete perceives that he/she is suffering from anxiety, the intensity of this feeling
and the direction (facilitative or debilitating) in order to channel this sensation
positively and to prevent diminished performance.[32]
The balance between pro and anti-inflammatory cytokines in physical exercise-related
inflammation is extremely important, since it promotes tissue repair.[18] Sports performed inadequately can lead to a condition named “sickness behavior.”
Sickness behavior is known by the increase in cortisol and catecholamine secretion
as well as the rise of pro-inflammatory cytokines, such as IL-1β and TNF-α, resulting
in significant behavioral changes, such as depression, mood swings and anxiety.[18] On the other hand, the practice of regular and moderate exercise restores the balance
of the process just described.[33] In this study, a decrease in the levels of IL-10 was observed in the group without
PMS, pre-game, in the luteal phase, when compared with the follicular phase pre-game.
This result indicates the possible action of hormonal fluctuations, which are more
exacerbated in the luteal phase, contributing to the reduction of the levels of IL-10.
This result is consistent with the study of Lee et al,[34] who reported lower IL-10 levels in healthy women during the luteal phase when compared
with the follicular phase. In addition, the increase in IL-10 observed in the follicular
phase supports previous findings in the literature, in which regular exercise promotes
an anti-inflammatory response.[35] It is important to note that the PMS group showed no significant differences in
IL-10 levels in neither phase of the menstrual cycle nor before or after the game.
The correlation between anxiety and IL-10 observed in both groups (with and without
PMS), post-game, in the luteal phase, also highlights the knowledge that menstruation
is a complex inflammatory process involving intracellular and extracellular signaling
events, comprising pro-inflammatory cytokines, prostaglandins, and immune system cells.[36] The positive correlation observed in these female soccer players with PMS demonstrate
that this group, which presents higher levels of anxiety, produces more IL-10 in an
effort, apparently not efficient, to promote the control of this behavioral condition.
In addition, the negative correlation found in the PMS-negative group shows that there
must be a physiological attempt to control anxiety via IL-10 generation.
A recent study developed by our group[25] evaluated the relationship among cytokines, hormones and mood swings in marathon
runners. The results showed that runners presented elevated levels of IL-10, IL-8
(chemokine associated with tissue repair) and growth hormone (GH) before the race,
suggesting that runners probably display mechanisms associated with body preparation
for prolonged and strenuous exercise. It is important to note that IL-10, in addition
to its anti-inflammatory immune function, also has effects on the brain and behavior,
participating in the modulation of mood, anxiety and depression symptoms.[26]
A limitation of our study was the use of urine as a biological sample. We opted for
the collection of urine due to the difficulty of collecting blood, since the athletes
did not agree to perform the venipuncture. Despite this limitation, the results presented
in this study were consistent and relevant.
This study highlights the importance of PMS effect in sports practice and it can be
considered as a warning to physicians, coaches and physiologists. However, further
research is required to elucidate the mechanism of PMS action on the performance and
health of female athletes.
