This study adheres to the CONSORT checklist for reporting randomized clinical trials (16). The present controlled randomized parallel clinical trial was conducted in the tertiary Retina and Vitreous Clinic of Labbafinezhad Hospital to compare the outcome of treatment with IVB in combination with vitamin D supplementation to IVB alone in eyes with diabetic macular edema. The study was conducted from May 2018 to February 2020 (~21 months). Patients with insufficient vitamin D levels were randomly assigned to two groups, one receiving vitamin D supplementation and standard IVB treatment and the other receiving only the IVB treatment; the allocation ratio was 1:1. The Ethics Committee of the Ophthalmic Research Center of Shahid Beheshti University of Medical Sciences approved the study protocol, and all participants signed an informed consent letter. The registry number of the present clinical trial is IRCT20200407046978N1.
2.2 Population and definitions
All adult (>18 years) patients with diabetic macular edema, admitted to the Retina and Vitreous Clinic, who required IVB administration, were assessed in terms of eligibility for inclusion in the trial. The inclusion criteria were: I) a central macular thickness (CMT, i.e., within the central 1mm of the macula) of 300 microns or above, as determined by optical coherence tomography (OCT) imaging (Spectralis OCT; Heidelberg Engineering, Heidelberg, Germany), and II) a corrected distance visual acuity (CDVA) of 8/10 (0.1 logMAR) or worse, in the affected eye. Patients were excluded in case of I) any previous ocular surgery (except a complication-free cataract surgery before the last six months), II) any previous intravitreal injections (any anti-VEGF agents, steroids, etc.), III) epiretinal membrane or vitreomacular traction, IV) history of retinal vascular accidents, V) history of uveitis, VI) pregnancy or breastfeeding, VII) consumption of vitamin supplements at the time of recruitment, VIII) failure to adhere to the vitamin D supplementation regimen, IX) severe vitamin D deficiency, X) any macular pathology other than diabetic macular edema, XI) hypercalcemia and hyperphosphatemia, and XII) serum creatinine levels above 3mg/dl. From each eligible individual, one eye was enrolled in this study.
Sufficiency, insufficiency, deficiency, and severe deficiency of serum 25(OH)D levels were defined as levels above 30 ng/ml, 20-30 ng/ml, 10-20 ng/ml, and below 10 ng/ml, respectively. DME was defined as a CMT of 300 microns or above.
2.3 Patient assignment and interventions
Before any therapeutic intervention, all participants were interviewed; demographic information and the complete history of diabetes mellitus, medications, and other comorbid disorders (e.g., hypertension, hyperlipidemia, ischemic heart disease, etc.) were obtained. All recruited eyes underwent complete ophthalmic examinations before the treatment initiation and upon each follow-up session, including CDVA assessment as well as anterior segment evaluation and tonometry using slit lamps. Further, a detailed fundus examination was performed using non-contact 78 D or 90 D lenses; CMT was also measured using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). An ophthalmologist graded the DR severity using three field fundus photographs (optic disc centered, fovea centered, and centered on the temporal edge of the macula) following the International DR Severity Scale (17). In addition, to perform urinalysis (U/A) and laboratory assessments on baseline serum vitamin D levels, fasting blood sugar (FBS), hemoglobin A1C (Hb1Ac), erythrocyte sedimentation rate, C-reactive protein, lipid profile (including triglyceride, total cholesterol, HDL, and LDL), and serum creatinine levels, urine and serum samples were collected from participants before treatment.
Participants with insufficient (10-30 ng/ml) and sufficient (>30 ng/ml) baseline serum 25-hydroxyvitamin D (25(OH)D) levels were divided into two groups. Patients with vitamin D insufficiency were randomly assigned (using a random number table) to two groups receiving the standard treatment with IVB or IVB combined with vitamin D supplementation. Thus, with an allocation ratio of 1:1:1, three groups were formed: 1) vitamin D sufficient group, 2) vitamin D insufficient group not receiving oral vitamin D supplements, and 3) vitamin D insufficient group receiving oral vitamin D supplements.
All eyes underwent three monthly intravitreal injections of bevacizumab (Avastin®, Genentech/Roche, CA, USA) by ophthalmologists blinded to the patients’ groups at the Vitreous and Retina Clinic of Labbafinezhad Hospital. Fundus examination and OCT imaging were repeated before each session. Patients in group 3 started taking oral vitamin D supplements along their IVB injection schedule. The supplementation regimen for patients with serum 25(OH)D levels of 10-20 ng/ml was eight weekly doses of 50000IU vitamin D3 pearls (D-Vigel 50000 IU, Daana Pharmaceutical Company, Iran) for eight consecutive weeks followed by a maintaining daily dose of 800IU. Those with serum 25(OH)D levels between 20 ng/ml and 30 ng/ml received daily doses of 800IU. After achieving a sufficient vitamin D level (30 ng/ml), the daily 800IU dose was continued for both subgroups.
Patients were followed up for six months after the third IVB administration. The primary outcomes were functional improvements in vision, as evaluated by E-charts and expressed through changes in CDVA logMAR and anatomical reductions in CMT, as measured by Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). Comprehensive ophthalmic examinations were performed 1, 3, and 6 months after the third IVB injection. The secondary outcome was serum 25(OH)D level; it was assessed on months 1 and 6 after the third IVB injection. Upon follow-up sessions, additional IVB injections were given if needed. After the study was completed, patients in group 2 received vitamin D supplementation. Considering that patients with severe vitamin D deficiency were not enrolled, a nine-month delay in correcting vitamin D levels above 10 ng/ml was not considered harmful; patients in group 2 were thoroughly informed of and consented to this allocation.
The ophthalmologist who injected the IVB were blinded to the randomized groups. The optometrists who performed the ophthalmic examinations were blinded to the patient’s group and findings from the previous follow-up sessions.
2.6 Sample size and statistical analysis
With no similar previous study available when designing this study, we were to run an initial pilot study with 10 participants in each group and use the obtained data to determine the minimum sample sizes required. After the interim analysis of the initial pilot phase results, we estimated that a minimum of 20 patients was required for each group (one-tailed α = 0.05; 1-β = 0.80; the clinically acceptable margin for CMT changes = 50 microns; the standard deviation of CMT = 56 microns).
Descriptive statistics were expressed through mean, standard deviation (SD), median, range, frequency, and percentage. Analysis of inter-group differences was performed using Chi-square or ANOVA, where appropriate. Paired T-test was utilized to assess changes in DCVA, CMT, and serum 25(OH)D levels between different time points. All statistical analyses were performed using the SPSS software (V.25.0, IBM). P-values below 0.05 were considered statistically significant.