Cardiometabolic risk profile in non-obese children with obstructive sleep apnea syndrome

Obstructive sleep apnea syndrome (OSAS) in childhood is a complex disease primarily due both to adenotonsillar hypertrophy and pediatric obesity. Notably, inflammation has been recognized as one of the most important shared pathogenic factor between obesity and OSAS resulting in an increased cardiometabolic risk for these patients. To date, evidence is still limited in non-obese population with OSAS. We aimed to evaluate the cardiometabolic risk profile of a pediatric population of non-obese subjects affected by OSAS. A total of 128 school-aged children (mean age 9.70 ± 3.43) diagnosed with OSAS and 213 non-OSAS children (mean age 9.52 ± 3.35) as control group were enrolled. All subjects underwent a complete clinical and biochemical assessment (including white blood cell count (WBC), platelet count (PLT), mean platelet volume (MPV), % of neutrophils (NEU%), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), uric acid, fasting insulin, iron, ferritin, and transferrin levels). A significant association between inflammation markers (including WBC, PLT, MPV, NEU%, ferritin, CPR, and ESR) and OSAS was found (all p < 0.001). Children with OSAS also showed increased transaminase, glucose, uric acid, and insulin levels (all p < 0.001) compared to healthy controls. Conclusion: Taken together, these findings suggested a worse cardiometabolic profile in non-obese children with OSAS. Given the pivotal pathogenic role of inflammation both for hypoxiemia and metabolic derangements, therapeutic strategies for OSAS might also counteract the increased cardiometabolic risk of these patients, by improving their long-term quality of life. What is Known: • Pediatric OSAS has shown a close relationship with obesity and its cardiometabolic comorbidities. • Inflammation represents the hallmark of both obesity and OSAS. What is New: • Non obese children with OSAS presented with a worse cardiometabolic risk profile. • OSAS treatment might serve as an effective approach also for the increased cardiometabolic risk of these children. What is Known: • Pediatric OSAS has shown a close relationship with obesity and its cardiometabolic comorbidities. • Inflammation represents the hallmark of both obesity and OSAS. What is New: • Non obese children with OSAS presented with a worse cardiometabolic risk profile. • OSAS treatment might serve as an effective approach also for the increased cardiometabolic risk of these children.

From a pathophysiological perspective, inflammation has been largely accepted as one of the major pathogenic mechanisms underlying both obesity and OSAS. Over the last decades, several studies have focused on the role of potential biomarkers of pediatric OSAS [10][11][12][13], but no specific determinants in this field have been currently identified, likely due to the overlap of various comorbidities potentially acting as confounding factors [14,15]. Noteworthy, evidence has linked sleep duration to different cardiometabolic markers in a pediatric cohort [16]. Moreover, changes in sleep duration and quality have been related to rapid serum increase of C-reactive protein (CRP) [17][18][19] insulin [20,21] and lipids [22].
Adipose tissue is one of the main sources of inflammatory cytokines (including IL-6). CRP is produced by the liver in response to raising levels of IL-6, derived from adipose tissue and closely related to fat mass [23].
Moreover, some studies have also suggested that OSAS might be associated with body fat deposition in specific areas, directly linked to the severity of clinical features and to visceral fat deposits, in turn closely related to metabolic derangements than subcutaneous fat [24].
Of note, a significant association has been highlighted between increased CRP levels in children with OSAS and both severity disease and treatment administration (e.g., CPAP) [25][26][27]. Indeed, CRP might be considered as a predictor of cardiovascular morbidity [28], with a direct effect on the development of atheromatic plaques [29,30]. Given that, children with high CRP levels may be considered at greater risk of developing long-term cardiovascular complications. Interestingly, vascular injury markers and endothelial activation factors such as adhesion molecules, fat-binding protein, and circulating molecules have been shown to be increased in children with OSAS and associated with endothelial dysfunction [30][31][32]. In addition, the major role of obesity as cardiovascular risk factor has been studied already in childhood [33,34].
In light of this, pediatric OSAS represents a tangled disease with multiple interrelated pathogenic factors. Besides, the shared inflammatory pathway between OSAS and obesity could make this latter as a confounder for sleep breathing problems.
To fill this gap, we aimed to investigate the cardiometabolic risk profile (defined as a cluster of biomarkers such as serum glucose, insulin, acid uric, and transaminase) in a population of non-obese children affected by OSAS.

Ethical approval study design
The present study was conducted according to the Declaration of Helsinki [37], and all parents of the enrolled children (both OSAS and Control group) gave their informed consent for participating to study. The Departmental Ethic Committee of our Institution approved the study.

Study population
We enrolled 128 school-aged children diagnosed with OSAS consecutively attending the Sleep Laboratory for Pediatric Age at Child and Adolescent Neuropsychiatry Clinic of our University between September 1, 2015, and November 30, 2017 ( Fig. 1).
OSAS was diagnosed by overnight nocturnal polysomnographic examination according to the diagnostic ICSD-3 criteria [38]. Non-obese children were diagnosed with OSAS because of the presence of nasal turbinate hypertrophy or non-genetic craniofacial alterations (e.g., retrognathy, prognathism).
As control group, 213 healthy children without OSAS (AHI < 1.0 event/h) were enrolled at the same university clinic. The control group was recruited among inpatient subjects (admitted for assessment of recurrent episodes of headache and abdominal pain) resulted negative for neuropsychiatric evaluation and pediatric screening during hospitalization in our Clinic.

Polysomnography data collected selection
In order to establish the presence of OSAS in the experimental group, all the polysomnography (PSG) data collected from inpatients children between September 1, 2015, and November 30, 2017, were reviewed and analyzed.
An airflow cease for at least two breaths associated to paradoxical ribcage and abdominal movements identified obstructive apnea. The hypopnea index was defined as a nasal flow curve signal reduction > 50% associated to oxygen desaturation or arousal. Central apnea was defined as airflow absence at both nose and mouth with no inspiratory effort within the duration of the event for 20 s or longer, or two missed breaths with an oxygen desaturation ≥ 3%, an arousal, or an awakening. The apnea-hypopnea index (AHI) was determined as the number of apneas and hypopneas per hour of sleep and the lowest oxygen saturation value, and the number of desaturation events ranging from 4 to 90% was computed. Moreover, the oxygen desaturation index (ODI), classified on the basis of events per hour, was calculated. OSAS severity was determined according to the current guidelines specified by the American Academy of Sleep Medicine (AASM) [39]: mild OSAS was defined by an obstructive apnea/hypopnea index (AHI) of 1 to < 5 events per hour; moderate OSAS was defined as ≥ 5 to < 10 events/h, and severe OSAS as ≥ 10 events/h.

Statistical analysis
Data were expressed as mean ± standard deviation. Kolmogorov-Smirnov test was used to examine normal distribution of the population. The independent t-test was used to compare the main parameters between patients with and without OSAS. Not-normally distributed variables were log-transformed before the analysis, but raw means are shown. Age, sex, and insulin were used as covariates, when appropriate.
Considering the relatively limited number of our sample and in order to rule out possible type II errors, the effect sizes using Cohen's d value was calculated. Cohen's d is defined as the difference between two means divided by their pooled standard deviation. According to Cohen, 0.2 is indicative of a small effect, 0.5 of a medium effect size, and 0.8 of a large effect size.
To reduce the chances of false-positive results (type I errors) when multiple pairwise tests were performed on a single set of data, Bonferroni corrections (Bonferroni type adjustment) were applied by dividing the p-value by the number of comparisons being made.
Statistical analyses were performed using the STATIS-TICA software (data analysis software system, version 6, StatSoft, Inc. 2001).
As expected, in children with OSAS, all the parameters evaluated by PSG (AHI, ODI, SpO 2 %, and mean desaturation O 2 ) were significantly lower than controls (p < 0.001) ( Table 1).
Inflammation marker levels (including WBC, PLT, MPV, NEU%, CRP, ESR, and ferritin) were significantly higher in the OSAS group compared to non OSAS group (all p < 0.001) ( Table 2). These associations were confirmed even after adjustments (all p < 0.001).
Moreover, subjects suffering from OSAS showed increased serum glucose, insulin, uric acid, ALT, and AST levels than healthy controls, even after adjustments (all p < 0.001) ( Table 2).
According to the effect size calculation, the Cohen's d appeared to be with large effect for the following param  Table 2).

Discussion
In our study, we provided intriguing evidence for a worse cardiometabolic profile in a cohort of non-obese children with OSAS. In fact, robust statistical data regarding the differences in the main cardiometabolic biomarkers (such as serum insulin, glucose, uric acid, and transaminase) between the two examined groups have been observed. Sleep-related breathing disorders (SRDB) are common in children and adolescents [40][41][42], with a different severity ranging from primary snoring to OSAS [40]. OSAS is a complex disease in which several risk factors (e.g. inflammation, obesity) are interrelated [42,43]. To date, available scientific findings in this field are still conflicting and mainly focused on subjects with obesity. Given the well-recognized role of obesity as major cardiometabolic risk factor [34,35], studies examining OSAS subjects with obesity might suffer from some limitations due to the potential pathophysiological overlap between these diseases.
In this perspective, findings from our non-obese population allow to expand knowledge in this research area. In fact, it could be supposed that gas exchange abnormalities and sleep disturbance characterizing OSAS promote inflammatory responses, as supported by the increased CRP levels observed in our cohort. The association between SRDB and cardiovascular disease in pediatric age has been well documented, particularly in children with endothelial function impairment [44][45][46]. Moreover, these patients experienced recurrent episodes of hypoxiemia leading to an increase in sympathetic activity, oxidative stress, and inflammation (mainly expressed as elevated serum C-reactive protein levels) that enhanced endothelial dysfunction [44][45][46].
Evidence also supported a close relationship between the night-time breathing habits and non-specific biochemical markers of inflammation with particular reference to the findings of neutrophilia in OSAS children [47][48][49][50], by suggesting also potential therapeutic options based on the pathogenic role of specific proinflammatory mediators [51,52]. In this regard, results from our study not only confirmed these findings but provided further evidence in a non-obese pediatric cohort, by underscoring the pivotal pathogenic role of the inflammation also in a such selected population.
Interestingly, our data seem to draw a worse cardiometabolic profile also in non-obese children with OSAS. According to previous findings [56][57][58], pediatric OSAS also showed a close relationship with fatty liver independently of the presence of a metabolic dysfunction as obesity status.
In addition to the pathogenic role of inflammation, dysregulation of other metabolic pathways involving insulin signaling and hepatic homeostasis might be supposed as further harmful players in the tangled puzzle of OSAS pathophysiology [59].
Considering the design of the study (including a selected population such as non-obese children with OSAS and a control group), our results might add to the existing knowledge on the pediatric OSAS development. Based on the current limited data on cardiometabolic outcomes in nonobese children diagnosed with OSAS, our findings might also have significant clinical implications. Indeed, these subjects may benefit from a wider management taking into account also the impact of the hypoxemia correction on the metabolic impairments. Moreover, lifestyle programs might also improve the overall quality of life of non-obese children with OSAS by reducing their cardiometabolic risk.
However, our study has some limitations that deserve mention. Firstly, our population, although well-characterized, is limited. The lack of a more comprehensive metabolic evaluation (e.g., lipid profile, glucose metabolism assessment) did not allow to provide evidence for a wider cardiometabolic risk in these patients. On the other hand, the presence of a control group enhances the strength of our findings.
In conclusion, a worse cardiometabolic profile has been found also in non-obese children with OSAS. In light of this, therapeutic strategies for hypoxemia correction might also pay the way for a better cardiometabolic management, by improving the long-term quality of life of non-obese children with OSAS [53,60]. Further studies are needed to provide a better characterization of these selected patients.