Association between the neutrophil-to-lymphocyte ratio and obstructive sleep apnea: a meta-analysis

This meta-analysis is aimed to investigate the association between the neutrophil-to-lymphocyte ratio (NLR) and obstructive sleep apnea (OSA). The PubMed, Web of Science, Google Scholar, and Cochrane Library databases were searched to collect all relevant articles. The pooled standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated using the random effects model. In addition, subgroup analysis and meta-regression analysis were performed. Eleven eligible articles containing 2,259 patients with OSA were included in this study. Pooled outcomes revealed that the NLR was significantly higher in patients with OSA than in controls (SMD 0.62, 95% CI 0.29–0.94, P = 0.002). In subgroup analyses, differences in the NLR between patients and controls increased with worsening OSA grades. Furthermore, meta-regression analysis showed that differences in mean BMI exerted a significant effect on differences in the NLR (P = 0.0003). In summary, our meta-analysis demonstrated that the NLR in OSA patients was significantly higher than that in controls, and the difference was larger in patients with severe OSA. These results indicate that the NLR may be a reliable marker for detecting systemic inflammation and predicting disease severity in patients with OSA.

Statistical analysis. The meta-analysis of enrolled studies was performed using R 3.4.3 version statistical software (R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https ://www.R-proje ct.org/.). The difference in the NLR between the OSA and control groups was assessed using the standardized mean difference (SMD). Heterogeneity across the enrolled studies was calculated using the I 2 test, where I 2 > 50% indicated significant heterogeneity between studies and prevented reliance on combined study results. In these cases, the random effects model was used to generate pooled effects. By contrast, outcomes without a significant level of heterogeneity (I 2 < 50) were analyzed using the fixed effects model. Subgroup analysis was performed to evaluate the effects of disease severity, BMI, country, and sex. We also conducted meta-regression analysis to identify possible sources of heterogeneity. Differences in mean age (years), proportion of male patients (%), and mean BMI were the sources of heterogeneity assessed. A funnel plot and Egger test were used to detect publication bias 25 . Additionally, the Duval and Tweedie trim-and-fill method was used to adjust for missing studies and correct the overall effect size according to publication bias 26 . Sensitivity analyses were performed to estimate the influence of each study on the overall meta-analysis results 27 .

Results
Literature search and study characteristics. The database search using the aforementioned keywords and search strategies returned 2,405 articles, 36 of which were excluded because of duplication. After the title and abstract screening, we excluded 2,353 articles and read the full text of only 16 articles. Finally, 11 articles involving 2,259 OSA patients were included in our meta-analysis [13][14][15][16][17][18][19][20][21][22]28 . The process of study selection is presented in Fig. 1. Among the 11 studies, nine were conducted in Turkey and two were conducted in East Asia (Japan and China). The NOS scores of enrolled studies ranged from 6 to 7, and the publication year ranged from 2015 to 2019. All enrolled studies were case series and hospital-based studies. Eight articles [13][14][15][17][18][19]21,22 provided data based on OSA severity (mild: 5 ≤ AHI < 15; moderate: 15 ≤ AHI < 30; and severe: 30 ≤ AHI) and one article 20 provided data based on BMI (normal weight: BMI < 25; overweight: 25 ≤ BMI ≤ 30; and obesity: 30 < BMI). Two studies 18,28 provided NLR value before and after CPAP treatment. The characteristics of the enrolled studies are summarized in Table 1.
Associations between the NLR and the presence of OSA. Since 10 studies [13][14][15][16][17][18][19][20][21][22] fully provided NLR values of control subjects and patients with OSA, we included those studies for the meta-analysis to investigate association between NLR and the presence of OSA. We used the random effects model due to significant heterogeneity between the enrolled studies (I 2 = 95%). A significant difference in the NLR was observed between the OSA and control groups (SMD = 0.62, 95% confidence interval (CI) = 0.29-0.94, P = 0.002) (Fig. 2).
Meta-regression analysis. We conducted a meta-regression analysis to further assess the effect of confounding factors on differences in the NLR between OSA patients and controls. The outcome variable was the SMD of the NLR, and the covariates included differences in age (years), BMI, and the proportion of male patients (%). Only the difference in BMI (P = 0.0003) was found to exert a significant effect on the difference in the NLR, whereas differences in age (P = 0.3849) and the proportion of male patients (%) (P = 0.1275) showed no significant effect ( Supplementary Fig. 1a-c).
publication bias. Visual inspection of funnel plots revealed the existence of asymmetry ( Supplementary   Fig. 2). Egger's regression test also suggested evidence of publication bias (t = 2.2406, P = 0.0372). However, the trim-and-fill method showed that no study required to ensure symmetry and the adjusted SMD was not changed (adjusted SMD = 0.62, 95% CI 0.29-0.94, P = 0.002).
Sensitivity analysis. Stability of the results was evaluated through sensitivity analysis. The corresponding pooled SMD values were not substantially altered when single studies were sequentially removed, with an effect size ranging between 0.46 and 0.66, suggesting that the results of the meta-analysis were stable ( Supplementary  Fig. 3).
The effect of CPAP treatment on NLR. In our primary search results, two studies 18,28 compared NLR before and after CPAP treatment. We performed a meta-analysis of those studies to investigate the effect of CPAP treatment on NLR. There was no significant difference in NLR before and after treatment with CPAP (P = 0.20) (Supplementary Fig. 4).

Discussion
Although the association between the NLR and OSA has drawn considerable attention and has been widely investigated, previous results have been controversial. In the present study, we comprehensively analyzed the results of 10 studies through a meta-analysis and found that the NLR value was higher in OSA patients than in control subjects. These results indicate that neutrophilic inflammation may play a key role in the pathogenesis of OSA.
In recent years, both local and systemic inflammation as well as changes in immune response have been reported in patients with OSA. Previous studies have indicated that intermittent hypoxia selectively activates nuclear factor-κB (NF-κB) pathways 29,30 . In addition, the levels of several inflammatory markers, including www.nature.com/scientificreports/ C-reactive protein, interleukin-6, and tumor necrosis factor, have been widely recognized as being higher in OSA patients [31][32][33] , suggesting that inflammatory activation is critical in the pathogenesis of OSA. CPAP treatment significantly reduces elevated inflammatory markers in OSA patients 34 . Several studies have also demonstrated that systemic inflammation may contribute to a higher risk of cardiovascular diseases in patients with OSA 7,35 and elevated levels of inflammatory markers are associated with a higher cardiovascular risk 36 . Because neutrophils and lymphocytes play a major role in inflammatory responses through the release of various inflammatory mediators, their absolute counts or relative values may reflect the status of systemic inflammation. NLR is superior to other individual leukocyte parameters (e.g. neutrophil, lymphocyte, and total leukocyte counts) in terms of stability, because it is a ratio of two different immune pathways 37,38 . Hence, the NLR has been proposed as a novel inflammatory marker associated with many chronic diseases, including coronary heart disease, hypertension, pulmonary thromboembolism, psoriasis, and various malignancies 12,[39][40][41] . A previous study also reported an association between an elevated NLR and mortality in a group of non-ST-elevation myocardial infarction patients 42 . Similarly, our meta-analysis demonstrated that the NLR was associated with OSA. However, NLR may be influenced by certain factors, including bacterial infection 43 and medications 44,45 . Therefore, the NLR is a particularly reliable marker in patients without conditions that may affect inflammatory response. The mechanism underlying the elevation of the NLR in OSA patients may be related to hypoxia-induced chronic inflammation. Neutrophils from patients with moderate and severe OSA demonstrate prolonged survival, which is associated with higher NF-κB levels and worse balance of pro-apoptotic and anti-apoptotic proteins 30,46,47 . In the case of lymphocytes, some researchers have proposed that the decrease in lymphocytes reflects a higher degree of physiological stress 48,49 .
In subgroup analyses, we found that the difference in the NLR between patients and controls increased gradually with the severity of OSA. These findings strongly suggest that the NLR may reflect disease severity, and the activation of the inflammatory process is linked to the disease activity of OSA. In addition, considering that the difference in the NLR between patients and controls was larger in obese patients and the difference in BMI was correlated with the NLR in meta-regression analysis, obesity may play a key role in systemic inflammation in OSA patients via unknown mechanisms. Furthermore, several investigators reported that the NLR value was higher in obese individuals than in control subjects with normal weight 50,51 . Therefore, comparison with BMI-matched controls is needed to avoid the confounding effect of obesity on NLR. Notably, no statistically significant differences in the NLR were observed between patients and controls in studies with only male patients and studies from East Asia. Previous studies using a large US national data set reported that females and non-Hispanic black were associated with lower NLR 52,53 , indicating that gender and ethnicity may affect NLR value. In addition, given that racial difference in inflammatory responses has been proposed 54 , increase of inflammatory biomarker levels in patients with OSA may be different between races. Similarly, differential inflammatory responses between male and female subjects might explain the discrepancy in the results among each subgroup according to sex  Calculation for studies from East Asia and Turkey was based on the fixed and random effects models, respectively. (b) Studies were divided into two subgroups (male and mixed sex) according to the sex composition of the study patients. Calculation for studies with only male patient groups and studies with mixedsex patient groups was based on the fixed and random effects models, respectively. The results were expressed as the standardized mean difference and 95% confidence intervals. OSA, obstructive sleep apnea; SD, standard deviation; SMD, standardized mean difference; CI, confidence interval.
Scientific RepoRtS | (2020) 10:10862 | https://doi.org/10.1038/s41598-020-67708-w www.nature.com/scientificreports/ composition. It has been described that females show a more pronounced response to inflammatory stimuli 55 . Furthermore, pro-inflammatory cytokine responses and NF-κB activation of mononuclear cells following sleep disturbance were more exaggerated in females than in males 56,57 . However, concluding a relationship between these parameters and the NLR in OSA patients may be premature because the results were derived from few studies. Future studies with a large number of patients may be required to obtain more definitive results. This study had several limitations. First, there might be a referral bias because all the studies enrolled in this meta-analysis were hospital-based studies. Population-based studies may provide more accurate information on the general population, compared to hospital-based studies. Second, we excluded articles in foreign languages, which may have biased the results. Third, the medication status and co-morbidities of OSA patients were unknown, which may represent uncontrollable factors in our analysis. Fourth, all enrolled studies were case series, which generally have a lower level of evidence than prospective cohort studies. Fifth, a potential bias may be derived from the geographical background of included studies. The majority of included studies investigated Turkish population. Further studies conducted in other countries are required to validate our results. Despite these limitations, this meta-analysis has several considerable strengths. First, we included the largest number of studies available. Second, our study was the first to conduct analyses to investigate the effects of potential modifiers.
In summary, our meta-analysis demonstrated that the NLR of patients with OSA was significantly higher than that of controls. Furthermore, the difference in the NLR between patients and controls gradually increased with the severity of OSA. These findings indicate that the NLR may be a reliable marker for detecting systemic inflammation and predicting disease activity in patients with OSA.