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					               JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, Suppl 6, 719-726



            Institute of Pathological Physiology, Jessenius Faculty of Medicine,
                          Commenius University, Martin, Slovakia

           Studies in healthy adult volunteers and patients with chronic cough have shown that
           women have increased cough sensitivity to inhaled tussigenic stimuli, including
           capsaicin. The explanation for this phenomenon remains unknown, although the
           influence of pubertal changes (dimensional, hormonal) may play a role. In the
           present study we set out to examine the effect of the pubertal status on cough reflex
           sensitivity in a population of male and female children and adolescents. The study
           consisted of cough reflex sensitivity (CRS) testing, spirometry, and a children-
           completed pubertal status questionnaire. The inclusion criteria were no current
           symptoms of respiratory disease, no respiratory infection in the preceding 2 weeks,
           no allergic disease, and no other disease that could modulate CRS. All children were
           non-smokers. For assessing the CRS we used a compressed air-driven nebuliser
           controlled by a dosimeter with an inspiratory flow regulator valve. Each subject
           inhaled up to 12 capsaicin aerosol concentrations (0.61-1250 µmol/l) during 400 ms
           at 1 min intervals. CRS was defined as the lowest capsaicin concentration that
           evoked minimally 2 coughs. 225 children (median age 13 yr, 103 girls/122 boys)
           were divided according to the pubertal status (prepubertal, early pubertal, and late
           pubertal) and gender. We found that CRS [geometric mean (95%CI)] was similar in
           prepubertal and early pubertal girls and boys. However, CRS was significantly
           higher in late pubertal girls (n=56) than in boys (n=26) [53.57 (35.62-80.64) µmol/l
           vs. 119.7 (70.74-208.55) µmol/l; respectively; P=0.017]. We conclude that puberty
           and gender have a significant influence on the cough threshold to capsaicin. It seems,
           however, hard to determine what factors are responsible for the observed gender
           differences in cough reactivity adolescents.

 K e y w o r d s : age, capsaicin, cough reactivity, gender differences, puberty


    Information about the cough threshold to tussigenic agents in children is
sparse. Studies in adults revealed that the cough threshold (CT) is gender-
dependent. In general, females cough twice as much as males to any given cough
challenge and CT is consistently lower in female subjects (1). These observations
were made in healthy subjects (2, 3), patients attending cough clinic due to
chronic persistent non-productive cough (4), and also patients with ACE-
inhibitors induced cough (5).
    Most cough reactivity studies are realized in adults. Information about the
influence of developmental changes during childhood and adolescence on the
cough reflex pathway is missing. According to Chang and Widdicombe (6),
maturation of the cough pathways from a premature infant to mature newborn is
probable, but whether age-related differences in cough exist among neonates,
children, and adults has by far been unexplored. Children change in many aspects
during puberty. The most prominent are dimensional and hormonal changes that
could play a role in the above-mentioned gender-related differences in cough
reactivity in adults (1).
    The aim of the present study was to investigate cough reactivity in school-age
children and to find out whether gender differences exist also in children and
adolescents, and whether cough reactivity to capsaicin is influenced by pubertal

                                  MATERIAL AND METHODS

   The study was approved by the Ethics Committee of Jessenius Medical School in Martin
Slovakia and the children's parents gave informed consent.

    Two hundred twenty five healthy, non-atopic, non-wheezing children and adolescent volunteers
(age range 7–18 yr, median age 13 yr (interquartile range: 12-16; 103 females/122 males) from
elementary and secondary schools in the Martin Region, Slovakia participated in the study. The
inclusion criteria were: non-smoker, no respiratory morbidity, and no symptoms of respiratory
diseases in the preceding 2 weeks before testing, no history of allergic diseases, and no history of
other diseases that could modulate CT (diabetes mellitus, gastroesophageal reflux disease).

Cough sensitivity testing
    Capsaicin challenge was performed in agreement with the ERS guidelines on assessment of
cough (1). We used a compressed air-driven nebuliser (model 646; DeVilbiss Health Care, Inc.,
Somerset, PA, USA) controlled by a dosimeter (KoKo DigiDoser-Spirometer; nSpire health Inc,
Louisville, CO, USA) with an inspiratory flow regulator valve added (RIFR; nSpire health Inc,
Louisville, CO, USA) to assign identical inspiratory flow rate during capsaicin inhalations in all
subjects. Each subject inhaled saline randomly interspersed with 12 incremental capsaicin aerosol

concentrations (0.61-1250 µmol/l). Each administration of saline and capsaicin aerosol was
performed at 1 min intervals with the inhalation time set at 400 ms.
    Spirometry was performed before and after capsaicin challenge (KoKo DigiDoser-Spirometer;
nSpire health Inc, Louisville, CO, USA). Subjects performed three forced expiratory manoeuvres
from total lung capacity to residual volume and the best record was used for an analysis.

Pubertal development rating
     Classification of a pubertal category was made according to Carskadon and Acebo (7) by filling
in a self-administered rating scale for the pubertal development. To test the effect of a pubertal stage
on CRS, the subdivision was made based on the gender for each pubertal stage according to the
Puberty Category Score obtained:
     1.    20 girls aged 8.0 (8.0-9.5 yr) (median, interquartile range) and 14 boys aged 9.5 (8.0-11.0
           yr) were classified as subjects at ‘prepubertal stage’ (1st pubertal stage - PS).
     2.    14 girls aged 11 (10-13) yr and 82 boys aged 13 (12-14) yr were classified as ‘early
           pubertal stage or midpubertal stage’ (2nd pubertal stage).
     3.    56 girls (aged 15 (13-16) yr and 26 boys aged 16 (15-17) yr were classified as subjects at
           ‘late pubertal stage’ (3rd pubertal stage).
     4.    13 girls aged 16 (14-17) yr, but no boys, were classified as subjects at ‘postpubertal
           stage’, and were not included in the statistical analysis.

Statistical analysis
    Analysis was performed using Systat 11 software. C2 values were normally distributed after
natural logarithm (ln) transformation and are expressed as geometric means (95% confidence
interval). Two-way ANOVA was used to examine the effects of two independent variables
concurrently: pubertal status and gender and to determine whether the two independent variables
interact with respect to their effect on the dependent variable. One-way ANOVA was used to
compare average C2 values at three pubertal stages in female and male subjects separately, followed
by a post hoc Tukey test. Comparisons between the gender subgroups were performed with a two-
sample t-test. Multiple linear regression was performed to assess contributions of several factors
(age, height, weight, FEV1, FVC, and pubertal stage) to cough sensitivity to capsaicin. P<0.05 was
regarded as statistically significant.


   Spirometric results, median age, height, and weight of studied subjects divided
according to the pubertal development rating are shown in Table 1. FEV1 and
FVC were significantly lower in girls compared with boys at each pubertal stage.
At the late pubertal stage, girls had significantly lower FVC and FEV1 expressed
in %predicted. Girls at each pubertal level were significantly younger and shorter
compared with boys.
   Two-way ANOVA using the lnC2 as the dependent variable, and pubertal
stage and gender as between-group factors revealed that gender showed no
significant effect on the cough threshold (P=0.33), pubertal status showed a
significant effect (P=0.001), and there was no interaction of the two on the cough
threshold (P=0.2).

    One-way ANOVA table with the pubertal stage as a factor and lnC2 as a
dependent variable showed a significant difference among the three lnC2 means
when all subject were taken into account (P=0.002); and also in female (P=0.035)
and male subjects (P=0.009) separately (Fig. 1). The results of the post hoc Tukey
test are described in Table 2.
    A two-sample t-test, performed between boys and girls at consecutive pubertal
stages revealed that CS was similar in both groups at the ‘prepubertal stage [19.53
(7.94-47.99) µmol/l vs. 26.28 (8.78-78.65) µmol/l; P=0.66]. Girls at the ‘early
pubertal or midpubertal’ stage had a lower cough sensitivity compared with boys,

                                                            Fig. 1. Cough threshold to inhaled
                                                            capsaicin expressed in natural
                                                            logarithm values in boys and girls at
                                                            separate pubertal stages. Results of
                                                            boys are represented by crosses linked
                                                            with full lines and girls are represented
                                                            by dots linked with dotted line.
                                                            *Significant difference in lnC2
                                                            between boys and girls at the late
                                                            pubertal stage.

Table 1. Proband characteristics.
                                                   Early pubertal or
                     Prepubertal stage                                       Late pubertal stage
                                                  midpubertal stage
                    F             M               F               M            F               M
      n             20            14              14              82          57              26
   Age (yr)     8 (8-9.5)*    9.5 (8-11)    11 (10-13)*      13 (12-14) 15 (13-16)**      16 (15-17)
 Height (cm) 136 (129-144)* 141 (137-150)* 154 (145-158) 160 (152-162) 168 (161-170)** 180 (174-185)
 Weight (kg) 31.5 (27-39)     36 (30-40)    42.5 (40-46)     49 (41-60) 53 (50-60)** 69.5 (63-76)
  FEV1 (L)     1.8 ±0.3**      2.4 ±0.6      2.5 ±0.7*        3.1 ±0.8   3.1 ±0.4**        4.3 ±0.6
  FEV1, %
               90.1 ±12.7     98.0 ±10.9     92.9 ±12.3      94.6 ±14.8  88.9 ±9.9**     100.1 ±15.0
  FVC (L)       1.9 ±0.3*      2.6 ±0.7       2.8 ±0.6*       3.5 ±0.9    3.4 ±0.4**       4.7 ±0.9
 FVC, %pred    93.1 ±9.8*     99.9 ±10.6     97.4 ±12.7     100.0 ±12.2   94.3 ±0.8*     101.0 ±14.3
Data are expressed as median (IQR) (for categories: age, height, weight) or mean ±SD (for
categories: FEV1,FVC). *P<0.05 and **P<0.001 compared with the corresponding values for boys.

Table 2. Post hoc Tukey test - matrix of pairwise comparison probabilities in lnC2 among different
pubertal stages.
                     All children                          Girls                                 Boys
            1st PS     2nd PS       3rd PS    1st PS      2nd PS       3rd PS        1st PS      2nd PS      3rd PS
   1st PS P=1.000                             P=1.000                            P=1.000
   2nd PS P=0.005 P=1.000                     P=0.061     P=1.000                P=0.180       P=1.000
   3rd PS P=0.001 P=0.790 P=1.000             P=0.056     P=0.795     P=1.000    P=0.008       P=0.079    P=1.000
PS – pubertal stage

Table 3. C2 (geometric mean (95%CI)) values in separate subgroups of subjects.
                                         Geometric mean of C2 (95% CI) µmol/l
            All children                                            Pubertal stage
                                        1st                   2nd                    3rd           postpubertal
      M   61.2 (46.3-80.8)      26.3 (8.8-78.7)        55.7 (40.1-77.3)     119.7 (70.7-208.5)
      F   45.2 (31.6-64.7)      19.5 (7.9-48.0)        74.4 (27.4-201.9)    53.9 (35.6-81.7)       19.5 (6.6-57.6)
PS – pubertal stage

but the difference was insignificant [74.37 (27.38-201.88) µmol/l vs. 55.70
(40.12-77.32) µmol/l; P=0.56]. In contrast the ‘late pubertal’ girls had a
significantly higher cough sensitivity compared with the boys in this group [53.89
(35.55-81.69) µmol/l vs. 119.7 (70.74-202.55) µmol/l; P=0.02] (Fig. 1, Table 3).
    As none of boys were at the post-pubertal stage at the time of testing, we were
not able to follow whether the sex-related differences determined in our study had
a tendency to accentuate after puberty had ended. The information obtained from a
small sample of post-pubertal girls (n=13) suggests a further significant reduction
of the cough threshold compared with the late pubertal girls (P=0.04) (Table 3).
    A stepwise multiple regression analysis was performed with lnC2 as a
dependent variable and age, height, weight, FVC, FEV1, and pubertal stage as
independent variables in girls and boys separately. In the boys, the pubertal stage
was the most important predicting factor with its significant contribution to
capsaicin cough sensitivity (partial regression coefficient was 0.754; P=0.002). In
the girls, both age and pubertal stage significantly contributed to capsaicin cough
sensitivity (partial regression coefficient for age was 0.296 (P=0.0001) and for the
pubertal stage -0.571 (P=0.009). Other independent variables did not significantly
contribute to cough threshold.


   The aim of this study was to clarify whether there are gender differences in
cough reactivity during childhood and adolescence and to test the impact of the
pubertal development on capsaicin cough threshold.

    According to the ERS recommendations we tested the cough threshold to
capsaicin using standardized methodology in terms of equipment, method of
administration, nebulizer output, and inspiratory flow rate (1). Two exceptions
were made, each arising from the differences between children and adults: 1) we
used different capsaicin concentrations, first applied by Chang et al (8) for cough
sensitivity testing in children; 2) we applied a different dose of aerosol per breath,
necessitated by inhalation time reduction to 400 ms. These changes were made
according to our previous experience with cough reactivity testing in children and
were proved to be adequate for CT testing in comparative clinical studies in
children (9-11).
    Our results show that changes represented by puberty had a strong impact on
capsaicin cough sensitivity occurring throughout childhood. A highly significant
increase in cough threshold to capsaicin was observed in children at the 2nd PS
(P=0.005) and 3rd PS (P=0.001) compared with children at the 1st PS. Therefore,
it is likely that the cough reflex pathway undergoes a maturation process toward
a lower cough reactivity in adolescence.
    Gender differences in cough reactivity, however, were not present when
comparing the whole group of tested children. These results are similar to those
previously published (12). After dividing subjects according to the pubertal stage,
our analysis revealed that cough threshold was similar in boys and girls before the
start of puberty. In the course of puberty, a 2-fold increase of the cough threshold
occurred in boys and almost a 4-fold increase was noted in girls at the 2nd PS
compared with the corresponding gender at the prepubertal stage.
    Analysis of the late pubertal stage determined a further increase in the cough
threshold in boys, whereas there was a tendency to reduce it in girls. This
divergence caused a statistically significant difference in cough reactivity
observed between boys and girls at the late pubertal stage.
    There is no definite answer why there are gender differences in airway
behavior, although according to review of Becklake and Kauffmann (13), the
determinants of biological nature (dimensional, immunological, and hormonal),
and also environmental and sociocultural factors are likely to play a role.
    In agreement with other published studies (3, 4, 14), we observed that forced
vital capacity (FVC) and forced expiratory volume in one second (FEV1) did not
significantly contribute to the cough threshold. Even though there are significant
differences in spirometric parameters between boys and girls, such differences in
the airway size are not likely to affect CT.
    The most tempting explanation for the sex-related differences in cough
reactivity to capsaicin would be an influence of different sexual hormones. As the
late pubertal stage in females is characterized by starting of menarche, female
sexual hormones could play a role in reduction of the cough threshold that was
observed in our study. On the other hand, no significant difference in CT between
girls at pre-menarche pubertal stages (1st and 2nd PS) compared with girls with
menarche (late pubertal and postpubertal stage) (P=0.45; results not shown)

argues against this hypothesis. Similarly, an analysis of the cough threshold in
postmenopausal women showed significantly reduced CT compared with pre-
menopausal women (3). According to these observations we could conclude that
female sexual hormones are not likely to cause a decrease in cough reactivity in
female gender.
    The question about gender differences in cough reactivity to tussigenic agents
still remains unexplained. According to our results we can only conclude that the
pubertal development plays a significant role in changing the cough threshold
during childhood and adolescence. This change has, however, an opposite trend
in females (towards enhancement of cough reactivity) and in males (towards
reduction of cough reactivity) resulting in sex-related differences in cough reflex
sensitivity in adulthood.

   Acknowledgements: This work was supported by Science and Technology Assistance Agency
under contract No. APVT-20-005304 and by Project of European Social Fond No. SOP LZ
20005/NP1-027, 11230100433.

   Conflicts of interest: The authors had no conflicts of interest to declare in relation to this article.


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   R e c e i v e d : May 30, 2008
   A c c e p t e d : August 22, 2008

   Author’s address: S. Varechova, Institute of Pathological Physiology, Jessenius Faculty of
Medicine, Sklabinska 26 St., 037 53 Martin, Slovakia; phone: +42 143 4238213, fax: +42 143 4134807;

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