Immune function in Japanese schoolchildren with sleep-disordered breathing: a preliminary study with analyses of salivary markers

doi:10.4236/ojst.2011.14022

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Open Journal of Stomatology, 2011, 1, 150-157

doi:10.4236/ojst.2011.14022 Published Online December 2011 (http://www.SciRP.org/journal/ojst/

OJST

Published Online December 2011 in SciRes. http://www.scirp.org/journal/OJST

Immune function in Japanese schoolchildren with

sleep-disordered breathing: a preliminary study with analyses

of salivary markers

Hiroshi Ueda, Atsushi Horihata, Eka Matsumoto, Genki Watanabe, Myongsun Koh,

Kotaro Tanimoto, Kazuo Tanne

Department of Orthodontics and Craniofacial Developmental Biology, Graduate School of Biomedical Sciences, Hiroshima Univer-

sity, Hiroshima, Japan.

Email: [email protected]

Received 25 August 2011; revised 6 October 2011; accepted 21 October 2011.

ABSTRACT

The aim of this study was to assess the prevalence of

sleep-disordered breathing (SDB) symptoms among

primary schoolchildren, and to objectively determine

the influence of SDB on the intra-oral environment

through the analysis of saliva. A questionnaire survey

was conducted among approximately 400 children

from a primary school in Hiroshima. Parents were

asked to complete the questionnaire and provide their

contact information if they allowed the collection of

saliva samples from their children. Thirty-eight chil-

dren agreed to participate in the saliva study. Habit-

ual snoring and cessation of breathing during sleep

were found in approximately 8% and 1% of children,

respectively. The present results showed significant

correlations between snoring and mouth breathing. A

significant association between excessive daytime sle-

epiness (EDS) and learning problems was found. Fur-

thermore, among children between the ages of 7 and

12 years, those with EDS and learning problems

tended to be older. SDB symptom scores were statisti-

cally significant only in relation to EDS. The present

study also demonstrated significantly higher levels of

salivary IgA and cortisol in children with sleep-re-

lated disorders. The present study determined the pre-

valence and characteristics of SDB among Japanese

primary schoolchildren and their effects on the oral

environment. Approximately 8% of primary school-

children with habitual snoring might need to be care-

fully monitored for SDB symptoms and immune sta-

tus to ensure proper psychological and physical deve-

lopment.

Keywords: Schoolchildren; Sleep-Disordered Breathing

Symptoms; Immune Function; Salivary Markers

1. INTRODUCTION

Sleep-disordered breathing (SDB), including sleep apnea

syndrome (SAS), is an important cause of morbidity for

both adults and children [1-5]. In children in particular,

SDB is of great concern because it can cause neurobe-

havioral and growth-related problems, and affect aca-

demic performance [6-9]. The estimated prevalence of

SAS in children ranges from 1% to 3% and the incidence

of habitual snoring has been reported to be 7% - 12% in

previous studies [10-13]. Therefore, the prevalence of

SDB and its influence on daily life are significant and

cannot be neglected in growing children.

In the fields of dentistry and orthodontics, mouth

breathing patients are known to have narrow upper den-

tition and long facial height resulting from the backward

and downward displacement of the mandible. This asso-

ciation has been explained by the fact that mouth brea-

thing causes reduced activity of the facial and mastica-

tory muscles, leading to the appearance of skeletal defor-

mities represented by an open bite with a small and dis-

tally located mandible. In addition, the descriptive term

“adenoid face”, consisting of narrow dentition, protrude-

ing teeth, and lip incompetence at rest has been docu-

mented in the literature for at least a century [14-16].

Over the last decades, the effect of SDB on the immu-

ne system has been reported frequently [17]. The meas-

urement of secretory immunoglobulin A (IgA) levels in

the saliva is a convenient and frequently used indicator

of immune status. SDB during childhood differs signify-

cantly from that in adults in terms of various parameters

such as symptoms and pathogenesis. Therefore, an appro-

priate differential study to discriminate SDB symptoms

in children is necessary.

The aim of this study was to assess the prevalence of

SDB symptoms in primary schoolchildren and to object-

tively determine its influence on the intra-oral environ-

ment by means of saliva analysis.

H. Ueda et al. / Open Journal of Stomatology 1 (2011) 150-157 151

2. MATERIALS AND METHODS

2.1. Participants

A questionnaire survey was conducted among 412 chil-

dren (197 boys and 215 girls) from a primary school in

Hiroshima. All children were within normal range in bo-

th physical and psychological development and had no

significant systemic diseases. Each subject gave written

informed consent and the study protocol was approved

by the Ethical Review Board of Hiroshima University

Hospital.

2.2. Questionnaire

A questionnaire on self-reported symptoms for the eva-

luation of SDB was used according to the Tucson Chil-

dren’s Assessment of Sleep Apnea (TuCASA) study re-

ported previously by Goodwin et al. [18]. This screening

questionnaire was designed to assess the severity of sle-

ep-related symptoms irrespective of age and gender. The

questionnaire was translated into Japanese. Parents were

asked to answer 13 questions pertaining to their child’s

sleep habits (APPENDIX). These questions were scored

by the parent on a scale of “never”, “rarely”, “occasion-

ally”, “frequently”, “almost always”, or “don’t know”.

Each questionnaire informed the parent that we would

complete sleep studies on approximately 400 children.

The children who had all 3 symptoms (excessive day-

time sleepiness (EDS), witnessed apnea (WITAP), and

snoring) or more were selected and classified into the

SDB group and the children who had no symptoms at all

served as the control group according to the TuCASA

study.

The complete list of survey questions is shown in the

“APPENDIX”.

2.3. Saliva Sample Collection

Parents were asked to complete the questions and to pro-

vide their contact information if they agreed to allow

study personnel to call and schedule a saliva test for their

children. Of 412 children, 38 children (19 boys and 19

girls, ranging in age from 6.7 to 11.7 years) agreed to

participate in the saliva study. Before the start of the sa-

liva collection and analysis, informed consent in paper

form was obtained from the parents again. The same nu-

mber of children (19 boys and 19 girls) was selected for

the control group with similar distribution of sex and

age.

Saliva was collected from each participant and analy-

zed for IgA and cortisol levels according to the guide-

lines of Hanrahan et al. [19]. All subjects were requested

to collect the saliva sample within the first 20 min after

waking up. The samples were then taken to school on the

same day, collected by the study personnel and immedi-

ately frozen at –20˚C until their subsequent analysis.

2.4. SIgA and Cortisol Assay

Samples were recovered after thawing by centrifugation

at 1500 g for 15 min. Salivary IgA (μg/mL) and cortisol

(μg/dL) were quantified by means of an enzyme-linked

immunosorbent assay (ELISA) using the Salivary Sec-

retory IgA Indirect Enzyme Immunoassay Kit and High

Sensitivity Salivary Cortisol Enzyme Immunoassay Kit,

respectively (Salimetrics, LLC, State College, PA, USA).

A ready-to-use 96-well microtiter plate coated with

highly purified human SIgA was used to assess IgA lev-

els and for generating standard immunoglobulin level

curves. As a detecting antibody, goat anti-human SIgA

conjugated to horseradish peroxide was used. The first 2

columns of each plate contained blank controls and 6

dilutions of purified human SIgA (600, 200, 66.7, 22.2,

7.4, and 2.5 μg/mL) in duplicates. The rest of the plate

was filled with a high and low control, and diluted (1:

120) saliva samples in duplicates. After incubation for

90 min at room temperature with continuous mixing at

400 rpm, the wells were washed 6 times with wash buf-

fer (phosphate-buffered solution) and filled with TMB

solution (tetramethylbenzidine). After 5 min of mixing at

500 rpm and additional 40-min incubation in the dark at

room temperature, the wells were filled with an acidic

formulation stop solution. The plate was mixed at 500

rpm for 3 min and the developed color was measured at

an optical density of 450 nm on a microplate reader

(Model 550, Bio-Rad Laboratories, Hercules, CA, USA).

For the assessment of the salivary cortisol concentra-

tion, all samples were assayed in duplicate. Absorbance

was measured at an optical density of 450 nm on a mi-

croplate reader (Model 550, Bio-Rad Laboratories) and

the quantity was derived from the standard curve.

2.5. Data Analysis

Thirteen questionnaires for the evaluation of SDB sym-

ptoms and school activities were initially scored accord-

ing to frequency of occurrence as never (0), rarely (1),

sometimes (2), frequent (3), or always (4). Blanks were

regarded as missing data and “don’t know” was ne-

glected. Five principal subjective parameters regarding

respiration and sleep were analyzed and the findings were

compared with previous results obtained from the same

school in 2003.

To examine saliva data, the subjects were judged as

experiencing EDS if the parent reported that their child

was sleepy in the daytime “frequently” or more. WITAP

was present if the parent reported that their child stopped

or struggled to breath during sleep “frequently” or more.

Snoring was present if parents reported loud snoring

from their child “frequently” or more.

Data are presented as percentages. A similar study

with the same questionnaire was conducted in 2003, and

H. Ueda et al. / Open Journal of Stomatology 1 (2011) 150-157

152

the statistical significance of the symptom scores in the

previous and current surveys was examined with the

Mann-Whitney U test. A Spearman rank correlation ana-

lysis was used for non-parametric variables. To compare

the control and SDB symptom groups, Student’s t-test

was conducted. A p value < 0.05 was considered signifi-

cant.

3. RESULTS

3.1. Demographics of the Participants

Tables 1 and 2 show the demographic data of the pre-

vious survey in 2003 and of the present study. The num-

ber of participants in the present study was 69 (34 boys,

35 girls) in the first grade, 63 (29 boys, 34 girls) in the

second grade, 65 (30 boys, 35 girls) in the third grade,

72 (34 boys, 38 girls) in the fourth grade, 76 (37 boys,

39 girls) in the fifth grade, and 67 (33 boys, 34 girls) in

the sixth grade. The children ranged in age from 6 (first

grade) to 12 years (sixth grade), and the mean age was

9.6 (0.4) years. In addition, no significant differences in

the distribution of boys and girls were found between the

previous and current demographic data shown in Tables

1 and 2.

Table 1. The number of participants in the previous survey in

2003.

Boys Girls

Grade 1 46 21 25

Grade 2 80 41 39

Male 206

Grade 3 74 32 42

Grade 4 78 40 38

Grade 5 82 43 39

Total

418

Female 212

Grade 6 58 29 29

Table 2. The number of participants in the current survey.

Boys Girls

Grade 1 69 34 35

Grade 2 63 29 34

Male 197

Grade 3 65 30 35

Grade 4 72 34 38

Grade 5 76 37 39

Total

412

Female 215

Grade 6 67 33 34

3.2. SDB-Related Symptoms

The percentage of children reported to snore loudly “al-

most always”, “frequently”, and “occasionally” was 6.8%;

those reported to stop breathing during sleep “almost

always”, “frequently”, and “occasionally” were 1.3%, and

those reported to be daytime mouth breathers “almost

always” and “frequently” were 14.3%. Furthermore, in

association with school activities, the percentage of chil-

dren who answered that they were sleepy during the day-

time “almost always” and “frequently” were 3.9%, and

the percentage in those who had learning problems “almost

always”, “frequently”, and “occasionally” were 2.8%. In

the 2003 data, the percentage of children who answered

these questions similarly was almost the same (Table 3).

Table 4 shows the comparison of the distribution of

SDB problems after the children were divided into 2

groups based on their school grades. In the older group

comprising grade 4 - 6 children, the percentage of chil-

dren who reported daytime sleepiness and learning pro-

blems was higher than in the younger group, whereas the

percentage of children who reported snoring loudly was

lower in the older group than in the younger group.

As shown in Tabl e 5 , statistically significant correla-

tions were found among the 5 main questions. All signi-

ficant correlations were positive (r = 0.11 - 0.27). The

snoring scores were significantly correlated with most of

the other variables, excluding learning problems. On the

other hand, the learning problems score was significantly

correlated with that of EDS in both the previous and the

present surveys (p < 0.01).

With respect to the SDB symptom scores, the only

Table 3. Distribution of common SDB problems in the sleep

survey.

Question Responses

survey in

2003

(N = 418)

Current

survey

(N = 393)

Q1. How often

does your child

snore loudly?

Almost

always/Frequently/

Occasionally

8.8 6.8

Q2. How does

your child stop

breathing during

sleep?

Almost always/Frequently/

Occasionally 1.4 1.3

Q3. Is your child

a daytime mouth

breather?

Almost always/Frequently 14.3 14.3

Q4. Is your child

sleepy during the

daytime?

Almost always/Frequently 4 3.9

Q5. Does your

child ever have

learning prob-

lems?

Almost

always/Frequently/

Occasionally

2.4 2.8

H. Ueda et al. / Open Journal of Stomatology 1 (2011) 150-157

153

Table 4. Distribution of common SDB problems in sleep survey.

Grade 1, 2, 3 Grade 4, 5, 6

Question Responses

Previous survey

(N = 200) Current survey

(N = 197)

Previous survey

(N = 218) Current survey (N =

215)

Q1. How often does your child snore loudly?

Almost

always/Frequently/

Occasionally

9.0 5.1 8.7 7.0

Q2. How does your child stop breathing

during sleep?

Almost

always/Frequently/

Occasionally

2.0 1.5 0.9 0.9

Q3. Is your child a daytime mouth breather? Almost always/

Frequently 14.0 12.7 20.6 14.4

Q4. Is your child sleepy during the daytime? Almost always/

Frequently 1.5 2.5 6.0 14.0

Q5. Does your child ever have learning

problems?

Almost

always/Frequently/

Occasionally

1.0 1.5 3.7 3.7

Table 5. Correlation coefficients between questions. Table 6. Descriptive statistics and comparisons between SDB

symptom scores in the 2 groups.

Q1 Q2 Q3 Q4 Q5

0.23**

0.10

0.27**

0.25**

0.18**

0.13**

0.09

0.07

0.21**

0.08

0.11*

−0.01

0.11*

0.03

0.08

0.25**

0.15**

0.07

0.13**

0.15**

2003

Grade 1, 2, 3 Grade 4, 5, 6

Med. 25% 75% Med. 25% 75% p

Q11 0 2 1 0 2 NS

Q20 0 0 0 0 0 NS

Q32 1 2 2 1 2 NS

Q40 0 1 1 0 1 *

Q51 1 2 1 1 2 NS

Med., median; 25%, 25th percentile; and 75%, 75th percentile. *Statistical

significance was defined as p < 0.05.

EDS question that was used in the previous and current

questionnaires showed a significantly large difference in

the score in the older group (Tables 6 and 7).

Table 7. Descriptive statistics and comparisons between SDB

symptom scores in the 2 groups.

2006

Grade 1, 2, 3 Grade 4, 5, 6

Med. 25% 75% Med. 25% 75% p

Q10 0 1 0 0 1 NS

Q20 0 0 0 0 0 NS

Q30 0 1 0 0 2 NS

Q40 0 1 1 0 2 *

Q50 0 0 0 0 0 NS

3.3. Saliva Analysis

Because no significant differences in IgA and cortisol

levels were found between boys and girls in both the

control and symptomatic groups, the means obtained

from 38 subjects in total were used for the following

statistical comparison.

Measurement of IgA levels showed that the symptom-

matic group had significantly higher levels than the con-

trols did (p < 0.05) (Figure 1). With respect to the corti-

sol levels, the symptomatic group also showed a consid-

erably higher level than the control group, although a sta-

tistically significant difference was not found (Figur e 2). Med., median; 25%, 25th percentile; and 75%, 75th percentile. *Statistical

significance was defined as p < 0.05.

OJST

H. Ueda et al. / Open Journal of Stomatology 1 (2011) 150-157

154

200

400

600

800

Control groupSymptom group

＊

IgA

(μg/mL)

＊

：significantly different (p < 0.05)

200

400

600

800

Control groupSymptom group

＊

IgA

(μg/mL)

＊

：significantly different (p < 0.05)

Figure 1. Comparison of the IgA levels between control and

SDB symptom groups.

5.0

10.0

15.0

20.0

25.0

Control groupSymptom group

Cortisol

(μg/dL)

5.0

10.0

15.0

20.0

25.0

Control groupSymptom group

Cortisol

(μg/dL)

Figure 2. Comparison of the cortisol levels between control

nd SDB symptom groups.

4. DISCUSSION

In the present study, a consciousness survey was con-

ducted for the assessment of SDB symptoms and prob-

lem behaviors among primary schoolchildren. The influ-

ence of these factors on salivary secretory immuno-

globulin A and cortisol levels was also examined. Sleep

apnea syndrome (SAS) has been of increasing concern

among the middle-aged population in Japan in recent

years, and the physical and psychological development

of children is considered to be greatly influenced by sle-

ep quality [2-4]. Mental retardation from chronic hypo-

xia, low stature from hyposecretion of growth hormones,

and slow learning due to daytime sleepiness are listed as

examples of the association between SDB and physical

and psychological development; hence, children in pri-

mary school were defined as the target of the present

study.

4.1. Prevalence of SDB Symptoms

In this study, habitual snoring and cessation of breathing

during sleep were found in approximately 8% and 1% of

children, respectively. Several studies from other coun-

tries have reported the prevalence of sleep problems in a

population-based sample of children. Gozal and Pope re-

ported that the mean prevalence of snoring among 1588

children was 9% in the United States [7]. A similar pre-

valence of snoring among children was reported in Fr-

ance (10%) [20] and in Italy (7.3%) [12]. The overall

snoring prevalence in the previous and current surveys

from our group was at the upper limit of the range re-

ported by others.

With respect to cessation of breathing, the present re-

sults were similar to those of previous studies reporting

that SAS affects approximately 1% - 2% children [2,

10,13]. Cessation of breathing during sleep is not the

same as diagnostic SAS. However, apneic symptoms

such as cessation of breathing, difficulty in periodic

breathing, and nocturnal choking might be the indicators

of the presence of SAS.

The present results showed significant correlations

between snoring and mouth breathing. Mouth breathing

is one of the characteristic SAS symptoms [13]. Fur-

thermore, nasal influences on mouth breathing, snoring,

and sleep apnea have been reported [21]. Therefore, it is

reasonable to assume that mouth breathing caused by

nasal obstruction leads to loud snoring, apneic breathing,

and disturbed sleep.

In this study, a strong association between EDS and

learning problems was detected. This finding suggests

that poor sleep quality such as sleep fragmentation and

nocturnal hypoxemia might induce EDS, which ulti-

mately influences academic performance and ability. In

agreement with our results, EDS, as well as various be-

havioral problems such as inattention, hyperactivity, and

aggression in school were reported in association with

sleep-related disorders in previous studies [7-9]. Inter-

estingly, Gozal demonstrated that improvements in scho-

ol performance could be induced by remediation of SAS

in first-grade children with substantial academic impair-

ment in school-related activities [6].

Furthermore, among children between 7 and 12 years

of age, those with EDS and learning problems were more

likely to be older than those without. Our data regarding

SDB symptom scores were statistically significant only

with respect to EDS. This may be due to the difference

in mean sleep times between older and younger children,

which were 9 h 18 min (32 min) (first grade) and 7 h 38

min (56 min) (sixth grade), respectively (data not sho-

wn). The current lifestyle of Japanese children, which

includes longer study periods after school and the resul-

ting delay in bedtime, might be a significant factor affec-

ting children’s sleep.

The Tucson Children’s Assessment of Sleep Apnea

(TuCASA) study is a prospective cohort study that was

designed to determine the prevalence of objectively do-

cumented SDB in preadolescent children and to investi-

gate its relationship to symptoms, performance on neuro-

behavioral measures, and physiologic and anatomic risk

factors [18]. Although screening questionnaires for chil-

dren can easily show bias, most TuCASA components

H. Ueda et al. / Open Journal of Stomatology 1 (2011) 150-157 155

are regarded as standard questions that are found in

sleep-habit questionnaires pertaining to SDB in children.

4.2. Association of SDB Symptoms with Salivary

Immune Factors

The results of the present study showed significantly hi-

gher levels of salivary IgA and cortisol in children with

sleep-related disorders. Secretory IgA, which is meas-

ured in the saliva, is the predominant antibody detected

in bodily secretions. IgA levels are believed to indicate

the functional status of the entire mucosal immune sys-

tem [22]. As a source of biological fluid for physical

examination, saliva has several advantages. Collection of

saliva is less intrusive and easier than that of urine or

blood, especially for children.

The most likely explanation for the significantly high

concentration of salivary IgA in the symptom group is

that sleep-related disorders negatively affect immune

functions and increase IgA levels for protection against

certain infections. The release of salivary IgA could be

an indicator of intra-oral and upper airway inflammation.

Therefore, salivary IgA provides a major defense against

potential pathogens by preventing colonization and rep-

lication on the mucosal surfaces of the oral cavity and

the upper respiratory tract. Previous studies demonstra-

ted that recurrent upper respiratory infection was associ-

ated with increased prevalence of SDB symptoms in

children [12,23,24].

The use of self-reported stress as a measurement me-

thod can be associated with bias and variation in study

results [25]. The psychological status of patients can be

reflected in their stress level. Cortisol is a stress hormone

that indicates hypothalamic-pituitary-adrenal (HPA) axis

activity and is a reliable biomarker of stress that is easily

measured in saliva [26]. The cortisol awakening re-

sponse, which is measured during the first 30 - 45 min

after waking up in the morning, is an especially reliable

marker of HPA axis activity [27]. Yehuda et al. demon-

strated that a single salivary cortisol sample taken im-

mediately after waking up can provide an accurate esti-

mate of urinary cortisol and mean salivary cortisol thr-

oughout the day [28]. The present results indicated that

SDB symptoms can constitute a stressor capable of ele-

vating cortisol levels, and suggested that children in pri-

mary school with greater adrenocortical reactivity might

have a considerable level of psychological stress.

In the field of dentistry, especially orthodontics, many

studies have shown the influence of breathing disorders

caused by respiratory obstruction with the large soft pal-

ate, tonsils, tongue, and nasal inflammation on craniofa-

cial morphology. Animal studies demonstrated that spe-

cific skeletal and dental changes, including an increased

lower facial height, anterior downward tipping of the

occlusal plane, and dual bites, crossbite, and open bites

occur within 24 months after nasal obstruction in mon-

keys [29,30]. Bresolin et al. investigated the relation-

ship between mouth breathing in allergic children and

dento-facial development [29], and demonstrated that

mouth breathers had longer faces with narrower maxilla

and retrognathic mandibles [31]. These studies show that

breathing disorders are strongly correlated with cranio-

facial morphology. Moreover, a small oral cavity with

narrow dentition associated with retrognathism due to

undergrowth of the mandible could induce SDB. There-

fore, anatomical disorders carry a certain risk in terms of

predisposition to the development of respiratory disor-

ders such as SAS.

In conclusion, the present study determined the pre-

valence and characteristics of SDB among Japanese

primary schoolchildren and its influence on the oral en-

vironment. Approximately 8% of primary schoolchildren

with habitual snoring might need to be carefully moni-

tored for SDB symptoms and immune status to ensure

appropriate psychological and physical development.

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APPENDIX

Screeni n g Questionnaire

1) Does your child stop breathing during sleep?

2) Does your child struggle to breathe during sleep?

3) Do you ever shake your child during sleep to make

him/her breathe again?

4) Do your child’s lips ever turn blue or purple while

he/she is sleeping?

5) Are you ever concerned about your child’s breath-

ing during sleep?

6) How often does your child snore loud?

7) How often does your child have a sore throat?

8) Does your child complain of morning headaches?

9) Is your child a daytime mouth breather?

10) Is your child sleepy during the daytime?

11) Does your child fall asleep at school?

12) Does your child fall asleep while watching televi-

sion?

13) Does your child have learning problems?

Possible Responses

1) Almost always

2) Frequently

3) Occasionally

4) Rarely

5) Never

6) Don’t know