Study design
The SUN Project is a dynamic prospective cohort study formed of university graduates. It started in Spain in December 1999 and the recruitment of new participants is permanently open. Participants’ information is collected biennially through mailed or electronically mailed self-reported questionnaires. Upon completion of the first questionnaire (Q-0), including a total of 554 items used as baseline information, participants receive follow-up questionnaires biennially. These follow-up questionnaires contain questions to evaluate changes in lifestyle and health-related behaviours, anthropometric measures, incident diseases, and medical conditions. The study methods have been previously published 14.
Study participants
We preselected a total of 22 475 subjects who had responded the baseline questionnaire Q-0 before September 2 015. We used the information collected after the first two years of follow-up (Q2), and after every two subsequent years (Q4, Q6, Q8, Q10, Q12, Q14). Participants were followed up until the diagnose of glaucoma or until the last follow-up questionnaire available at the time of these analyses, (Q16) that collected information after 16 years of follow-up. Data analysis excluded 1 983 individuals without follow-up questionnaires (90.8% retention rate). Participants who reported extremely low or high total energy intake 15 (1 925), and participants with a previous diagnosis of glaucoma (59) were also excluded. Finally, 18 420 participants were included in the analysis (Fig. 1). The study was approved by the Human Research Ethics Committee at the University of Navarra (091/2008), and followed the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. Voluntary completion of the first questionnaire was considered by this Commitee to imply informed consent; this handling of consent was approved by the ethics committee.
Exposure assessment: Healthy Lifestyle Score variables
Dietary exposure was gathered using a semi-quantitative food frequency questionnaire (FFQ) with consume information on 136 items, that has been repeatedly validated in Spain 16. To Trichopoulou score (score 0–8, excluding alcohol), was used to assess the adherence to the Mediterranean diet 17. To collect information on alcohol consumption, data were obtained through the FFQ and also though other additional alcohol-intake related items present in the baseline questionnaire.
Mediterranean lifestyle was assessed with the SHLS. For the SHLS calculation, one point was given to each participant for each of these 10 habits: never smoking, moderate-to-high physical activity (> 20 MET-h/wk), Mediterranean diet (≥ 4 adherence points), body mass index (BMI) ≤ 22, moderate alcohol consumption (women, 0.1-5.0 g/d; men, 0.1–10.0 g/d; abstainers excluded), low television exposure (< 2 h/d), no binge drinking (≤ 5 alcoholic drinks at any time), taking a short afternoon nap (< 30 min/d), regularly meeting up with friends (> 1 h/d), and working at least 40 h/wk. The SHLS scale could range between 0 points (worst lifestyle) and 10 points (best lifestyle).
Outcome assessment
Glaucoma was assessed through a specific question included in the follow-up questionnaires. Participants responded to the question: “Have you ever been diagnosed of glaucoma by a health professional?”, along with the date of diagnosis. Validity of self-reported glaucoma diagnosis was assessed in a subgroup of our cohort by an experienced ophthalmologist, blinded to the questionnaires. This validation study showed a Kappa value of 0.85 (95% coefficient interval [CI], 0.834–0.872). The sensitivity found was 0.83 and the specificity 0.99. Prevalent cases of glaucoma at baseline were excluded from this analysis.
Ascertainment of covariates
Baseline questionnaire also gathered information of multiple potential confounding factors such as socio-demographic characteristics (i.e. sex, age, educational level), lifestyle and health-related characteristics (i.e. smoking, physical activity, adherence to the Mediterranean diet, total energy intake, consumption of a special diet, caffeine intake, Omega 3/6 ratio), anthropometric measures (i.e. BMI), and prevalent diseases (i.e. hypertension, cardiovascular disease, cancer, diabetes).
Statistical analyses
According to the baseline score obtained with the SHLS, participants were classified into four groups, to ensure an appropriate sample distribution and a sufficient number of incident cases within each category. These four categories were SHLS 0–2, 3–4, 5–6 and 7–10 points. We estimated hazard ratios (HR) and 95% confidence interval (CI) for glaucoma, for every category of SHLS, using the Cox regression model, defining the first category (0–2) as the reference category, and adjusting for multiple potential confounding factors, such as sex, age, calorie intake, caffeine intake, alcohol intake, omega-3/omega-6 ratio, prevalence of cancer, prevalence of hypertension, prevalence of diabetes mellitus type 2, educational level, and special diets. Linear trends tests were calculated by assigning the median score of each category to all participants in that category and treating this variable as continuous.
To analyse the individual contribution of each specific factor of the SHLS score to the risk of glaucoma, Cox regression models were fitted for each of the ten items of healthy life habits, adjusting for the effect of the rest of the items that constituted the index. The reference category was the absence of the healthy habit (score 0 on the specific item).
Sensitivity analyses were also performed to ensure the robustness of the results in different scenarios. We repeated the analyses stratifying by age (≥ 50), sex and smoking.
All p values presented are two-tailed; p < 0.05 was considered statistically significant. Analyses were performed using STATA/SE version 12.0 (STATA Corp LP, College Station, TX, USA).