Computed Tomography and Cephalometric Evaluation of Obstructive Sleep Apnea Syndrome

Abstract

To examine the changes of upper airway cross sectional area in each phase of respiration in different degrees of severity of OSAS with computed tomography and cephalometry to decide on further treatment. A Prospective study was done in the Department of Radiology and Imaging, Kasturba Medical College, Mangalore, spanning over a period from March 2017 to December 2019. 50 patients were included in the study including control group. Patients who had at least 2–3 major symptoms of sleep apnea such as snoring, daytime somnolence, and apnea were included in this study. All patients were examined and then subjected to polysomnography(PSG) and upper airway CT. Patients with apnea–hypopnea index (AHI) of < 5 on Polysomnography were included in the control group and those with AHI of > 5 were categorized in to the study group Cross-sectional area of the airway at the level of the nasopharynx, oropharynx and the hypopharynx were obtained. Standard cephalometric measurements were made on a lateral radiograph of skull/ CT scanogram. Of the 36 patients in the study group, 31 patients were males and 5 were females. In the control group of 12 patients, 8 were males and 4 females. The cross sectional area at the lower border of the nasopharynx which is also the level of the nasopharyngeal sphincter was the most affected level in OSAS (p value of < 0.0001). Mean uvular diameter in the control group was 9.6 mm and in the OSAS group it was 11.2 mm. The mean length of the soft palate was 36.4 mm in the controls, 39.5 mm in the mild/moderate OSAS and 41.2 mm in the severe OSAS group. Obstructive sleep apnea is a complex disorder characterized by apneic episodes during sleep. In this study the most common site of obstruction is nasopharyngeal sphincter and the oropharynx. Although PSG is the diagnostic test of choice, imaging plays an important role in planning surgical and conventional treatment.

Introduction

Obstructive sleep apnea/hypopnea (OSAS) syndrome is a sleep disorder characterized by recurrent upper airway collapse and obstruction during sleep associated with recurrent oxygen desaturation and arousals from sleep. Obstructive sleep apnea/hypopnea syndrome is associated with excessive daytime sleepiness and leads to symptoms such as snoring, excessive daytime sleepiness, increased risk of cardiovascular disease, hypertension, insulin resistance, cerebrovascular disease and road traffic accidents [1]. Imaging of the anatomical factors associated with recurrent upper airway collapse and the pathogenesis of OSA provide an opportunity to detect features that are strongly associated with unsuspected OSA and to raise the possibility of this diagnosis. One of the gold standard of treatment is continuous positive airway pressure (CPAP). Its efficacy is frequently limited by poor tolerance and some clinicians and patients are increasingly opting for one of a range of surgical procedures. Imaging can be performed for evaluation of the upper airway to aid surgical planning. Imaging modalities performed in the supine position are recommended to avoid reduction in airway dimensions when changing from erect to supine. Multidetector Computed Tomograhy and Magnetic Resonance Imaging provide optimal information regarding the nature of the soft-tissues constricting the upper airway [2]. Approximately 33–40% of the adult population is affected by OSA and adverse health consequences with significantly increased risk of serious morbidity and mortality are known to occur proportionally to the worldwide rise in obesity [3].

Although loud snoring is seen in all patients with OSAS, not all snorers have OSAS. Understanding the differences between patients with OSAS and simple snorers is important to explain the mechanisms responsible for upper airway obstruction rather than those between OSAS and normal non-snorers [4]. Hence habitual snorers have been used as control subjects in our study. Polysomnography is considered to be the gold standard for diagnosis of OSAS, estimation of severity and measurement of treatment response. It is imperative to locate the site of collapse of airway before deciding upon patient management particularly when surgical intervention is being considered following failure or noncompliance of conservative management.

Before the advent of cross sectional imaging, Radiological assessment of the airway for OSAS was restricted to Fluoroscopy and cephalometry. CT and MRI have made it possible to obtain cross sectional images, the advantages of which include:

• Non invasive technique.

• Patient may be assessed in supine position when or during sleep.

• Dynamic as well as static imaging can be performed.

• The images precisely define the osseous and soft tissue anatomy.

• Cross sectional area and volume of the airway can be accurately shown.

• Multi-planar imaging and image reconstruction are possible.

CT is readily available and is a quick imaging modality. It is also less expensive as compared to MRI. This study was done with the purpose of identifying the role of CT in the diagnosis and management of patients with sleep disordered breathing.

Objectives

1. Quantitative assessment of the naso, oro and hypopharyngeal airway by computed tomography in OSAS.

2. Compare CT of the pharynx in OSAS with that of healthy snorers.

3. Determination of the variable site of pharyngeal narrowing and regional variations of pharyngeal collapsibility among snoring patients during wakefulness by axial CT measurements of the cross sectional area of the pharynx.

4. Pharyngeal size and shape during wakefulness in patients with OSAS.

5. Correlation of cephalometric and select demographic variables in patients with OSAS.

6. Evaluation of the upper airway cross sectional area changes in different degrees of severity of OSAS.

Materials and Methods

This study was organized at the Department of Radiodiagnosis spanning over a period from March 2017 to December 2019. It was prospective study. During this period 50 patients who presented with History of snoring and day time sleepiness were evaluated. 36 patients (31 men, 5 women were included in the study group. 13 habitual snorers (8 men and 5 women were included in the control group. Patients with central or mixed sleep apnea were excluded from the study. Two male children who presented with sleep apnea were not included in the statistical analysis as the patho-physiology and treatment of sleep apnea in children differ significantly from that of adults. The investigation protocol was given clearance by the ethical committee of our institution. An informed consent was obtained from each of the participants in both groups.

The presenting complaints of patients were documented, which mainly comprised of snoring, apneic episodes during sleep and excessive day time sleepiness. Severity of snoring was assessed based on whether snoring was audible in the same room/ adjacent room/anywhere in the house/ neighbor. Excessive day time sleepiness was assessed based on Epworth sleepiness scale (Questionnaire 1).

*Epworth Sleepiness Scale questionnaire:
How likely are you to doze or fall asleep in the following situations in contrast to just feeling tired. Use the following scale to choose the most appropriate number for each situation:
0 = Would never doze
1 = Slight chance of dozing
2 = Moderate chance of dozing
3 = High chance of dozing
Situation	Chance of dozing
Sitting and reading
Watching television
Sitting inactive in public place (theatre or meeting)
As a passenger in a car for an hour without a break
Lying down for a rest in the afternoon
Sitting and talking to someone
Sitting quietly after lunch without alcohol
In a car while stopped for five minutes in traffic
• An ESS score greater than 10 is generally considered sleepy
• The ESS is useful for evaluating responses to treatment; the ESS score should decrease with effective treatment

A thorough local examination was performed on all patients for analysis of nasal, oral, pharyngeal, tonsillar and mandibular abnormalities. Weight and heights of all study subjects were recorded and body mass index was calculated by dividing weight (in kilograms) by square of height (In meters). Patients with a BMI of 25–30 were categorized as overweight and those with a BMI more than 30 were considered as obese. Neck circumference was measured in centimeters and presence of short neck documented. Blood pressure was measured.

Overnight polysomnography was performed in all patients by a computerized system, which included variables such as EEG, ECG, EOG, airflow monitoring, thoracic and abdominal respiratory movements recorded by inductive plethysmography, microphone for monitoring snoring, arterial oxyhemoglobin saturation, monitoring patient position. The overnight study was plotted on a polysomnograph. An oxygen desaturation event was described as a fall in the in oxygen saturation by atleast 4%. An apneic event was described as a signal drop below 20% of the reference amplitude lasting 10 s or more. A hypopneic event was described as signal drop below 70% of the reference amplitude lasting 10 s. The reference amplitude was calculated as mean of peak amplitudes found in a period of 10 min preceding the event. Patients with apnea–hypopnea index (AHI) of < 5 on Polysomnography were included in the control group and those with AHI of > 5 were categorized in to the study group. Further AHI of 5–15 was classified as mild OSA, 15–30 as moderate OSA and an AHI of > 30 as severe OSA.

CT scan of the upper airway in the head and neck was performed on GE high speed scanners. Scans were performed on awake patients, in supine position with head in neutral position. In each patient high resolution spiral axial CT scans were obtained once during deep inspiration and again during forced expiration.

To avoid unnecessary reexposure radiation and loss of data we did not repeat or exclude any study with images which were adequate for measurements as required for the study.

• Patients were given verbal commands to coincide the scan with the two phases of respiration. They were also instructed not to swallow during the scan.

• Gantry was tilted parallel to the intervertebral spaces of C1-C2 and C2-C3.

• Axial CT was obtained with a slice thickness of 5 mm using 120 kV and 150 mA. Field of view of 25.8 × 25.8 was chosen with a 512 × 512 pixel display matrix. The computer window width of 400 and level of 40 were set.

• After a lateral scout view, axial CT sections were taken from the nasopharynx up to the hypopharynx during and at the end of deep inspiration and expiration (Fig. 1). Cross sectional areas of the airway were measured manually by electronic calipers. Measurements of the air way were taken at the lower border of the nasopharynx (at the level of the tip of the uvula), lower border of the oropharynx (just above the tip of the epiglottis) and hypopharynx (at the level of the hyoid bone during inspiration and expiration (Figs. 2, 3 and 4). Retropharyngeal soft tissue thickness was measured 2 cm below the hard palate in all patients. The uvular diameter was measured at the level of maximal thickness (2 cm below hard palate). Cephalometric measurements were made on the lateral scout view using electronic calipers at CT workstation (Fig. 5).

Fig. 1.

Reference lines at the levels of measurement of cross sectional areas of nasopharynx, oropharynx, and hypopharynx

Fig. 2.

Cross sectional area measured at the lower border of the lower of the nasopharynx during inspiration and expiration

Fig. 3.

Cross sectional area of the oropharynx during inspiration and expiration

Fig. 4.

Cross sectional area of the hypopharynx during inspiration and expiration

Fig. 5.

Cephalometric measurements on laternal scout image, PNS-posterior nasal spine, P-tip of the palate, PNS-P-length of the soft palate, SP max-maximum thickness of the soft palate, MP-Mandibular plane, H-Superior margin of the hyoid bone, MPH-Mandibular plane to hyoid bone distance

The cephalometric measurements were.

• MP-H distance- from the mandibular plane to the hyoid bone

• PNS- P- distance from the posterior nasal spine to the tip of the uvula which provides the length of the soft palate.

• SP max- maximum thickness of the soft palate measured perpendicular to PNS–P.

• Statistical analysis was performed using statistical program SPSS version 13. ANNNOVA and Kruskal–Wallis test value were used for comparison between the control group, mild/moderate OSAS group and the severe OSAS group. Paired samples t test and × 2 test were used to analyse the data between groups. Pearson coefficient-(r) and p value were calculated to determine the correlation between variables. A p value of < 0.05 was assumed to be statistically significant and a p value of < 0.01 was assumed to be of high statistical significance.

Results

50 patients with history of snoring presented to our hospital from March 2017 to December 2019. Patients with additional history of excessive day time sleepiness, apneic episodes and apnea hypopnea index of > 5 on polysomnography were included in the study group. Others with AHI < 5 were included in the control group as habitual snorers.

Of the 36 patients in the study group, 31 patients were males and 5 were females. In the control group of 12 patients, 8 were males and 4 females. 17 of the 36 patients in the cases group belonged to the age group of 30–50 years. The youngest patient was 6 years old and the oldest patient was 69 years old. The cases group was further divided in to those with mild/moderate OSAS (Apnea hypopnea index: 5–30) and severe OSAS (Apnea hypopnea index > 30).

42% of the cases were overweight and 25% had grade I obesity. Amongst controls 58% had a normal BMI and 25% were overweight. Mean age and BMI of cases were similar to that of controls.

Mean neck circumference was 38.8 cm in controls and 40 and 42 respectively in the mild/moderate and severe OSAS groups. However despite the fact the mean neck circumference was higher in more severe OSAS patients, this difference was not found to be statistically significant.

Polysomnography

AHI: Apnea Hypopnea Index ranged from a minimum of 6 to a maximum of 79 among the cases. The mean AHI was 12.87 in the mild/moderate group and 55.9 in the severe OSAS group.

OD Events-(Fig. 6). Oxygen desaturation events: Oxygen desaturation event was detected when oxygen saturation fell by at least 4.0%. Mean OD events was 3.0 in the control group, 10.1 in the mild/moderate group and 39.9in the severe OSAS group. P value after Kruskal Wallis test was < 0.001 which is highly significant.

Fig. 6.

Mean OD events was 3.0 in the control group, 10.1 in the mild/moderate group and 39.9in the severe OSAS group. P value after Kruskal Wallis test was < 0.001 which is highly significant

The mean cross sectional areas at the lower border of the nasopharynx were 173.4 mm², 59.8 mm² and 98.4 mm² during inspiration as against 139.5 mm², 90.9 mm² and 54.8 mm² during expiration in the three groups respectively. This indicates that the cross sectional area at the lower border of the nasopharynx which is also the level of the nasopharyngeal sphincter is the most affected level in OSAS. The p value of < 0.0001 indicates this to be a highly significant correlation.

The mean cross sectional areas at the lower border of oropharynx were 286.67 mm², 160.32 mm² and 178.29 mm² during inspiration and 186.99 mm², 139.47 mm² and 163.57 mm² during expiration in the control, mild/moderate and severe groups respectively. The p value of 0.010 is significant correlation.

The mean cross sectional areas of the hypopharynx were 317.89 mm², 263.62 mm² and 243.43 mm² during inspiration and 284.16 mm², 252.89 mm² and 294.00 mm² during expiration in the control, mild/moderate and severe groups respectively.

Cross sectional areas of airway- (Fig. 7).

Fig. 7.

Cross sectional areas of airway

The narrowest cross sectional area of the airway amongst controls had a mean value of 95.9 mm², the corresponding values in mild/moderate cases group was 27.8 mm² and in severe cases group was 24.3 mm². The p value was 0.001 which was highly significant.

The narrowest cross sectional area of the airway was at the level of the oropharynx in 58.3% of controls, 37.9% of mild/moderate group and 57.1% of the severe OSAS group. In 20.7% of mild/moderate group, narrowest cross sectional area was seen at multiple levels of nasopharynx and oropharynx. Another important observation was that 42.9% of patients with severe OSAS had narrowest cross sectional area at the level of nasopharyngeal sphincter which is at the junction of nasopharynx and oropharynx. Narrowest cross sectional area of the airway-(Fig. 8).

Fig. 8.

Narrowest cross sectional area of the airway in mm²

Mean uvular diameter in the control group was 9.6 mm and in the OSAS group it was 11.2 mm. The mean length of the soft palate was 36.4 mm in the controls, 39.5 mm in the mild/moderate OSAS and 41.2 mm in the severe OSAS group. Mean diameter of the soft palate was 10.7 mm in the controls, 11.0 mm in mild/moderate OSAS and 10.7 mm in severe OSAS. There was no significant difference in the mean retropharyngeal soft tissue thickness amongst the three groups. Uvular, soft palate and retropharyngeal measurements (Fig. 9) Fig. 10 Increased retropharyngeal soft tissue thickness. Figure 11 -Increased uvular diameter.

Fig. 9.

Uvular, soft palate and retropharyngeal measurements in mm. UD-Uvular diameter, SP LNTH- Soft palate length, SP Diameter- Soft palate diameter, RPST- Retropharyngeal soft tissue thickening

Fig. 10.

Increased retropharyngeal soft tissue thickness

Fig. 11.

Increased uvular diameter

Figure 12 Complete multilevel obstruction of the oropharyngeal airway.

Fig. 12.

Complete multilevel obstruction of the oropharyngeal airway

The hyoid bone was displaced downwards in 51.7% of those with mild/moderate OSAS and in 85.7% of those with severe OSAS.

Comparison of multiple factorial parameters in imaging evaluation of sleep apnoea (Table 1).

Table 1.

Comparison multiple parameters between male and female group

	SEX	N	Min	Max	Mean	Std. deviation	Median	Mann–whitney Z value	P value
Neck C	Male	31	32	47	41.10	3.600	42.00	− 1.932	.053
	Female	5	35	43	37.80	3.114	37.00		NS
	Total	36	32	47	40.64	3.681	41.00
NASO INS	Male	31	0	218	68.12	63.303	60.00	− 206	.837
	Female	5	19	131	62.60	41.192	55.00		NS
	Total	36	0	218	67.35	60.270	55.00
NASO EXP	Male	30	0	264	83.99	64.130	83.50	− 283	.777
	Female	5	29	160	89.20	46.655	88.00		NS
	Total	35	0	264	84.73	61.378	88.00
ORO INS	Male	31	0	587	160.75	155.627	101.00	− 435	.664
	Female	5	45	314	182.80	130.867	217.00		NS
	Total	36	0	587	163.82	150.920	124.50
ORO EXP	Male	31	0	436	129.83	124.751	111.00	− 1.168	.243
	Female	5	57	490	233.00	187.988	214.00		NS
	Total	36	0	490	144.16	136.703	111.50
HP INS	Male	31	3	620	264.29	159.132	286.00	− 480	.631
	Female	5	80	380	231.20	114.870	196.00		NS
	Total	36	3	620	259.70	152.801	281.00
HP EXP	Male	31	2	575	259.60	176.918	284.00	− 229	819
	Female	5	111	493	268.80	138.922	237.00		NS
	Total	36	2	575	260.88	170.425	266.00
UD	Male	31	6.1	15.0	11.548	2.0583	11.900	− 2.757	.082
	Female	5	8.0	10.4	8.820	1.1498	8.000		NS
	Total	36	6.1	15.0	11.169	2.1675	11.300
SP LNTH	Male	31	31.3	49.0	40.484	5.8370	39.000	− 1.742	.006
	Female	5	33.0	42.0	36.000	4.2426	33.000		HS
	Total	36	31.3	49.0	39.861	5.8081	39.000
SP DIA	Male	31	8.1	18.5	11.265	2.0737	11.000	− 2.076	.038
	Female	5	8.0	11.0	9.340	1.3885	9.200		Sig
	Total	36	8.0	18.5	10.997	2.0885	10.950
RPST	Male	31	4	23	9.35	3.916	8.10	− 2.479	.013
	Female	5	3	7	5.46	3.661	5.30		Sig
	Total	36	3	23	8.81	3.913	8.00
OD events per hr	Male	31	.0	80.2	17.155	19.6313	11.900	− 1.304	.192
	Female	5	5.5	12.8	8.320	2.9064	7.600		NS
	Total	36	.0	80.2	15.928	18.4635	9.750
Narrowest	Male	30	0	95	24.22	27.721	10.00	− 1.597	.110
Diameter	Female	5	19	71	44.60	18.407	45.00		NS
	Total	35	0	95	27.13	27.343	19.00

Neck C-Neck Circumference, Naso Insp-Nasopharynx inspiration Naso Exp-Nasopharynx Expiration, Oro Insp- Oropharynx Inspiration, Oro Exp-Oropharynx Expiration,HP Insp-Hypopharynx inspiration,HP Exp-Hypopharynx Expiration, UD-Uvula Diameter, SP LNTH-Soft palate length, SP DIA- Soft palate diameter,RPST-Retropharyngeal soft tissue, OD-Oxygen desaturation,NS –not significant,HS- Highly significant, Sig- Significant

Discussion

Obstructive sleep apnea syndrome is known to occur in adults as well as in the pediatric population. However the etiologies differ considerably in the two groups. Here we discuss the age and sex distribution, correlation with body mass index, CT features in various degrees of severity of OSAS and cephalometric features of OSAS.

Obstructive sleep apnea is known to be more common in men than in women. Men have been reported to have significantly more predisposing factors than women, which included greater neck circumference, increased uvular diameter, increased length and thickness of soft palate and downward displacement of the hyoid bone. 6 in our study male to female ratio is 6:1. The uvular diameter was significantly greater in males as compared to females. The diameter of the soft palate was also greater in males than in females.

Indians tend to have a larger neck circumference thereby predisposing them to develop sleep apnea even with marginally increased BMI. OSAS in Asian men has been reported to be found more frequently in nonobese patients, despite the presence of severe illness, when compared with white male patients with OSAS [5].

The entire upper airway is smaller than normal in OSAS and the total volume of OSAS pharynx is reduced [6]. Most of the studies done on sleep apnea so far have reported Oropharynx to be the most severely affected and the narrowest level in patients with OSAS.

Upper airway collapse almost always occurred at oropharyngeal level in sleep apnea studies reported thus far [7].

Narrowing of the oropharyngeal area at the end of expiration is seen in severe OSAS [8]. In our study, most significant was the narrowing at the level of uvula or the nasopharyngeal sphincter during inspiration and to lesser extent during expiration. The expiratory hypo pharyngeal airway cross sectional area was greater in severe OSAS group than in the control group. A larger hypopharynx exposes the oropharynx to inspiratory pressure giving rise to its collapse [9]. Our series has shown a marginal increase in the hypopharyngeal cross sectional area in patients with severe OSAS as compared with the control group.

Avrahami et al. [10] reported that a narrowest cross sectional area of about 50 mm² for the oropharynx during quiet breathing should be consistent with severe OSAS. Our study has shown a narrowest cross sectional area of 0–50 mm² (24 ± 28 mm²) in severe OSAS.

Our OSAS cases showed a statistically significant increase in uvular diameter as compared to the controls.

In our study, the retropharyngeal soft tissue thickness was greater in males as compared to females in both cases and controls, probably because of the thickness of prevertebral muscles in men.

Our study has shown a consistent downward displacement of the hyoid bone in patients with severe OSAS. The position of the hyoid bone is important because it serves as a central anchorage for the tongue muscles and thereby partly determines the position of the tongue. The inferior and anterior displacement of the hyoid bone in obese patients may therefore be the result of the greater tongue mass and the deposition of adipose tissue [11]. Length of the soft palate and maximal thickness of the soft palate were increased proportionally in mild/moderate and severe OSAS as compared to controls in our study. These patients were recommended Uvulopalatopharyngoplasty.

Two of the patients were of the pediatric age group and were hence not included in the statistical analysis. These two patients had mild to moderate OSAS. They had grossly enlarged tonsils and adenoids. Adenoid hypertrophy is the most common cause of OSAS in children, other causes being macroglossia, obesity, nasopharyngeal masses [12]. They present with history of loud snoring, obstructive pauses, retractions and mouth breathing [13].

The lateral cephalometric radiograph can be used as excellent screening tool, treatment planning and postoperative assessment of patients who have OSA to delineate two-dimensional anatomy, show excellent hard tissue structures with minimal cost and radiation exposure to the patient [14].

Approach to treating OSA involves recognition of this individual variability. Each of these traits are seen in select individuals and especially in those patients intolerant of continuous positive airway pressure (CPAP). Uvulopalatopharyngoplasty may be a useful strategy for patients who have OSA as a result of anatomic compromise at the level of the velopharynx. Implantable hypoglossal nerve stimulators are effective in a subset of patients with OSA, although there are substantial barriers to this modality including cost, the need for multiple invasive procedures, and the inability to accurately predict responders. Surgical interventions may be ineffective in patients who primarily have an issue with unstable ventilatory control. The use of oxygen or acetazolamide, which are 2 strategies to lower loop gain and have been shown to be effective in a subset of patients with OSA. Nonmyorelaxant hypnotic agents (eg, trazodone or eszopiclone) have been shown to increase the arousal threshold and may be useful in reducing sleep-disordered breathing events in select patients with a low arousal threshold predisposing to OSA. Hypnotics may be ineffective or even deleterious in patients who have other underlying OSA mechanisms. Combination interventions have also been tried with some success to treat OSA in patients with multiple endotypes [15].

CBCT is noninvasive imaging modality that can identify obstructions and constrictions through the nasopharynx, oropharynx, and laryngopharynx. Detailed dimensions and anatomic information can identify risk levels and risk predictors for patients diagnosed with obstructive sleep apnea [16].

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dr. Ajit Mahale, Dr. Pallavi Rao, Dr. Sonali Ullal, Dr. Merwyn Fernandes and Dr. Sonali Prabhu. The first draft of the manuscript was written by Dr. Ajit Mahale and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The authors did not receive support from any organization for the submitted work.

Funding

No funding was received to assist with the preparation of this manuscript. No funding was received for conducting this study. No funds, grants, or other support was received. The authors have no relevant financial or non-financial interests to disclose. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.

Declarations

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethical approval

Ethics committee number: IEC KMC MLR O1-2007.

Human and animal rights

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Approval was granted by the Ethics Committee of University.

Informed consent

Informed consent was obtained from all individual participants included in the study.