Epigenetics Modifications in Morbid Obesity and Obstructive Sleep Apnea Patients (EPIMOOSA): Continous Positive Airway Pressure (CPAP) and Bariatric Surgery Impact. EPIMOOSA Study
Conditions: Obstructive Sleep Apnea, Morbid Obesity, Epigenetic Disorder
Sponsor: Aragon Institute of Health Sciences

Official Title: Epigenetics Modifications in Morbid Obesity and Obstructive 
Sleep Apnea Patients (EPIMOOSA): CPAP and Bariatric Surgery Impact. 
EPIMOOSA Study    
 
PRINCIPAL INVESTIGATOR: JAVIER LAZARO  
DATE: May, 23th, 2014 
 
NCT ID: NOT YET ASSIGNED 
 
 
Unique Protocol ID: 23/2014  
 

 
Study design. This is a 2-year prospective, longitudinal, noninterventional cohort 
study.  It will be conducted in the Sleep-Disordered Breathing Units at the Miguel Servet 
University and Royo Villanova hospitals in Zaragoza. 
Participant selection and follow-up. Patients will be selected from the bariatric 
surgery (BS) waiting list based on the inclusion criteria set out in Table 1. At the first visit, 
which will include a polysomnography, the study team will decide whether to begin 
continuous positive airway pressure (CPAP) therapy in accordance with Spanish 
standards for the treatment of obstructive sleep apnea/hypopnea syndrome (OSAHS) 
(1). All participants will be asked to attend a 6-month follow-up visit in which adherence 
to CPAP therapy (if applicable) will be assessed and a date for BS will be set, if the 
procedure is still recommended.  Patients will then attend 3, 6 and 12-month 
postoperative follow-up visits.  
INCLUSION CRITERIA 
EXCLUSION CRITERIA 
Age 18–60 years 
Obesity hypoventilation syndrome or 
treatment with positive pressure devices 

BMI consistently > 40 kg/m2 for 3–
5 years, following more than 1 year of 
unsuccessful controlled medical 
treatment 
Diagnosis of systemic inflammatory 
disease  
BMI 35–40 kg/m2 with comorbidities 
associated with MO susceptible to 
improvement with weight loss (high BP, 
DM, dyslipidemia, OSA, etc.), following 
more than 1 year of unsuccessful 
controlled medical treatment 
Neoplastic diseases in the last 5 years 
A signed informed consent form 
A cardiovascular event in the last 
6 months 
Pregnancy 
Tabla 1. Selection criteria 
The control group will comprise subjects from the EPIOSA study that is currently 
underway (2). EPIOSA is an observational cohort study conducted by the Miguel Servet 
University Hospital in Zaragoza that aims to investigate the natural history of OSA in 
relation to potential epigenetic changes. Patients with and without OSA, adjusted for 
sex and age ( 2 years), will be selected from the EPIOSA study population, which 
excludes individuals with a BMI > 35 kg/m2, for comparison with patients in the MOOSA 
study. 

Sample size.  
The aim is to form three patient groups: a) subjects with MO and without OSA; b) 
patients with MO and OSA who do not need CPAP therapy; and c) patients with MO and 
OSA requiring CPAP therapy. Each subgroup must include at least 12 subjects who 
complete the 2-year follow-up after their inclusion in the baseline visit. The sample size 
has been calculated to reveal significant differences in the epigenetic changes between 
the groups with OSA and the control group (main objective). In a previous study, Kim et 
al. (3) already demonstrated significant differences in DNA methylation of 
proinflammatory genes among two subgroups of eight children with OSAHS with high- 
or low CRP. In our cohort, we observed significant differences in circulating exosome 
levels between groups of 12 patients with OSA versus controls. Therefore, considering a 
potential loss to follow-up of 10%, we proposed the inclusion of 15  subjects per 
subgroup analyzable after a minimum 2-year follow-up, which equates to a final sample 
size of 45 patients. 
These 45 patients will subsequently be compared with another 45 cases from the 
EPIOSA study. Therefore, 90 patients in total will be included: 45 from the EPIMOOSA 
study and 45 from the EPIOSA study.    

 
Figura 1. Protocol Design 
Table 2 shows the schedule for collecting data on each variable during the 
various study visits. 
 
Tabla 2. Study Variables 
Clinical data. The following clinical data and complementary tests will be 
recorded at each visit: a) sociodemographic data, clinical, surgical and family history, 

and regular medications; b) level of daytime sleepiness based on the Epworth scale (4); 
c) weight (kg), height (cm), body mass index (BMI = weight (kg)/height (m)2), and neck, 
waist, and hip circumferences (all in cm); d) blood pressure, measured according to the 
European Society of Hypertension (ESH) and the European Society of Cardiology (5) 
clinical practice guidelines (6); and e) spirometry, measured in line with European 
Respiratory Society (ERS) standards (7). 
Sleep studies. We will use a validated home polysomnography system 
(ApneaLink vs10.20 ResMed®). The process consists of a continuous recording of airflow 
using a nasal cannula, chest movement, oxygen saturation, snoring, and body position. 
We define apnea as a lack of airflow for more than 10 seconds, hypopnea as the 
reduction of airflow (> 50%) for over 10 seconds accompanied by a decrease in oxygen 
levels of greater than 4%, and the apnea–hypopnea index (AHI) is expressed as the sum 
of instances of apnea and hypopnea per hour over the period studied. The principal 
investigator (PI) will interpret the results of the polysomnography. 
  Each patient will be administered CPAP therapy or not based on 
recommendations from the Spanish Society of Pulmonology and Thoracic Surgery 
(SEPAR, its acronym in Spanish). This decision will be made by the PI on a patient-by-
patient basis according to severity of OSAHS, cardiovascular comorbidity, and/or 
daytime symptomatology. Patients treated with CPAP therapy will receive an extra visit 
to carry out a pressure titration at home using AutoCPAP (AutoSet ResMed®) (8).  
Blood pressure and electrocardiogram (ECG) Holter. All patients will complete a 
24-hour blood pressure and electrocardiogram study. This shall be performed using a 
Labtech®EC-3H/ABP that combines a 3-channel ECG Holter and an ambulatory blood 

pressure monitor. These studies will be conducted the day after the home 
polysomnographies during the first and final visits, and over the 24-hour period before 
collecting blood samples for all other visits. They will interpreted according to the 
guidelines published by the Spanish Society of Cardiology (9). 
Blood tests. Blood samples will be collected at each visit. Venous blood samples 
will be obtained with 21G Abbocaths and arterial samples with 23G ProVent® kits for 
blood gas analysis. Glucose, triglycerides, total cholesterol, HDL, LDL, and apolipoprotein 
A and B blood levels will be analyzed by spectrophotometry (IMMAGE® 800 Protein 
Chemistry Analyzer, Beckman Coulter). High-sensitivity CRP will be determined within 
2 hours of collecting blood samples using flow nephelometry. 15 mL of blood will be 
used to obtain serum and plasma, and 5 mL with EDTA to conduct genetic and epigenetic 
studies; these samples will be stored in a freezer at -80 °C until they are analyzed.  
Exosome and microRNA analysis. Circulating exosomes and the encapsulated 
microRNA will be studied at the Translational Unit of the Miguel Servet Hospital in 
Zaragoza. Blood samples will be collected at each visit and frozen at -80 °C on the 
premises of the Royo Villanova Hospital. Once patient enrollment is complete, the blood 
samples will be transferred using containers filled with dry ice to the Translational 
Research Unit at the Miguel Servet Hospital.  MicroRNA will be obtained using the 
PAXgene Blood miRNA Kit (PreAnaltiX) following the manufacturer’s protocol. RNA 
integrity will be ensured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa 
Clara, California, USA). After extracting the RNA samples and ensuring their integrity, 
they will be reverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit 
(Applied Biosystems, Life Technologies, Carlsbad, California, USA). Mature miRNA will 

then be quantified by means of real-time CRP (TaqMan® MicroRNA Assays). Finally, 
miRNA expression shall be analyzed using BioMark 96.96 Dynamic Array™ (Fluidigm 
Corporation, San Francisco, California, USA). The results will be expressed following the 
method 2Ct (10).Table 3 shows the miRNA that will be studied in the MOOSA study. 
 
PANEL OF miRNA TO BE STUDIED IN THE MOOSA PROTOCOL 
UniSP2 
miR-320a 
miR-16-5p 
UniSP5 
miR-145-5p 
miR-126-3p 
cel-miR-39 
miR-146A-5p 
miR-133a-3p 
let 7a-5p 
miR-223-3p 
miR-34a-5p 
miR-21-5p 
miR-155-5p 
 
Tabla 3. Panel of miRNA to be studied in the EPIMOOSA protocol. 
Schedule. The study has already been initiated thanks to support from SEPAR 
and will overlap with the Bariatric Surgery Unit’s care strategy. Patients are being 
enrolled from the waiting list for BS. The recruitment period will last for 2 years, while 
follow-up will run for 6 months after the polysomnography and 1 year after surgery. As 
seen in Figure 2, we plan to conduct six visits over a 2-year period, excluding the surgery 
during which no samples will be collected. 

 
Figure 2. The study schedule includes two clearly distinct stages. The first stage 
features patients who have not yet undergone surgery for MO but who are being treated 
for OSA based on the initial polysomnography. Three months after the initial visit, 
patients with OSA who are receiving CPAP therapy will complete an AutoCPAP pressure 
titration. This will provide information on the impact of OSA treatment in patients with 
MO.  The second stage will start 3 months after bariatric surgery. This phase involves a 
12-month postoperative follow-up period with three visits, the last conducted 12 months 
after the BS because this is when weight loss is thought to stabilize. We shall study the 
impact of BS throughout this second follow-up period.  
 
Intervention allocation: CPAP therapy will be decided according to the PI’s 
clinical judgment in line with SEPAR’s clinical practice guidelines (1). 
Statistical analysis: We have predefined the following statistical procedures: a) 
Description of populations. They will be expressed as mean values ± SD when variables 
are continuous. Frequencies distribution will be obtained for qualitative variables. b) 
Difference between groups: parametric Student’s t-test or ANOVA—if they follow a 
normal distribution—or Mann–Whitney U-test or Kruskal–Wallis H-test—if we do not 
work with said hypothesis. For the qualitative variables, we will use the normal chi-

square test with standardized residuals. c) Relationship between systemic inflammation 
and clinical variables: Pearson’s or Spearman’s correlation depending on the variables’ 
normality.  d) Predictors of systemic inflammation and subclinical atherosclerosis: 
stepwise logistic regression.  e) Effect of treatment: paired samples t-test, if we assume 
normality, or the Wilcoxon nonparametric test for variables that do not follow a normal 
distribution. f) GOstats software will be used to analyze epigenetic markers (Falcon S et 
al., Bioinformatics 2007;23:257). g) The interindividual variability of miRNA will be 
determined through the coefficient of variation. We will use GraphPad Prism 6 
(GraphPad Software) and SPSS version 23.0 (IBM) statistical packages. 
 
 
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