The current study adopted a double-blind, randomized, parallel-group, placebo-controlled design to investigate MOFO mfERG as a predictor of response to atropine 0.01% for myopia control, as well as the efficacy of topical atropine 0.01% on overall myopia progression in school-aged children (ClinicalTrials.gov Identifier: NCT03374306, first recorded 12/15/ 2017). Trial registration information is available at https://clinicaltrials.gov/ct2/show/NCT03374306. The primary endpoint of the study was annualized change in equivalent spherical refraction (SER), and the secondary endpoint was annualized change in axial length (AL). The relationships between baseline retinal electrophysiological response and SER and AL were also analyzed such that baseline MOFO mfERG interaction with atropine treatment was assessed. All procedures followed the principles of the Declaration of Helsinki and were approved by the Human Subjects Subcommittee of Hong Kong Polytechnic University. This study followed the guidelines of the Consolidated Test Reporting Standards (CONSORT).
The study was announced and recruitment invitations were sent to parent groups outlining the details of the clinical trial. Prior to any study-specific procedures, written informed consent and verbal consent were obtained from parents and participants, respectively. Participants were 7 to 10 years old (inclusive), had an SER between -0.50 D and -5.00 D inclusive, and an astigmatism error of less than 1.00 DC. Participants were required to have normal visual acuity with best correction (LogMAR 0.00 or better) and normal color vision. Any strabismus, amblyopia, ocular or systemic disease and history of epilepsy were excluded.
Randomization, masking and intervention
After confirmation of eligibility, all participants were randomly assigned in a 1:1 ratio to 0.01% atropine or placebo treatment. The randomization process was conducted using a computer-generated random sequence by one investigator (HHLC) while all other investigators and participants were masked. Atropine eye drops, manufactured by AIM (Aseptic Innovative Medicine Co. Ltd, Taiwan), contained 0.01% atropine, while placebo eye drops contained 0.9% sodium chloride. All packaging was the same for atropine and placebo eye drops to keep participants and other investigators masked. Participants were prescribed a dose of single-dose eye drops over 3 months quarterly and instructed to instill the assigned eye drops once daily in both eyes.
Sample size and power
In a previous study with low concentration atropine, the annual rate of myopia progression (mean ± SD) under 0.01% atropine and placebo control was −0.49 ± 0.63 D and −1.20 ± 0.69 D, respectively, which equates to an effect size of 1.0720. Therefore, 24 participants per treatment group were required to provide 95% power (5% type I error, two-tailed test). Assuming a dropout rate of approximately 20% throughout the study period (based on prior experience), the minimum enrollment goal was 60 participants.
Baseline and follow-up eye examinations were performed at the Hong Kong Polytechnic University Optometry Research Clinic, including refraction, axial length, and mfERG (KYC, WYY, and SZCL) assessments. Additional visits to the eye clinic at Grantham Hospital in Hong Kong were arranged for eye consultations (ALN, BLC or JCHC) and collection of pre-assigned eye drops (atropine 0.01% or placebo). Participants and their parents were instructed to instill 1 drop of the assigned eye drops in both eyes once every 24 h. Participants were asked to return to the hospital eye clinic every 3 months and to the university optometry clinic every 6 months throughout the 18-month study period. Recruitment began in February 2018 and the last follow-up visit was completed in May 2020. Due to COVID-19 related lockdowns, a notable proportion of follow-up assessments were completed outside of the pre-visit window. -specified, which was explained by calculating the annualized variation of SER and AL.
Refraction was measured at least 5 times using an open-field auto-refractor (NVision K5001, Shin-Nippon, Japan) 30 min after instillation of the cycloplegic agent (2 drops of cyclopentolate to 1 %, at 5 min intervals). AL was measured at least 5 times using an IOLMaster (Carl Zeiss, Dublin, CA). Due to the strong correlation between right and left eyes (SER r = 0.82, p
Retinal activity was measured by MOFO mfERG (VERIS Science 6.0.6d19, Electro-Diagnostic Imaging, Milpitas, CA) with a minimum pupil diameter of 7 mm. A Dawson-Trick-Litzkow electrode was used as the active electrode (positioned between the cornea and the palpebral conjunctiva), and surface gold cup electrodes were used as the reference and ground electrodes (positioned at the canthus external of the tested eye and on the forehead, respectively). Participants were exposed to the stimulus pattern displayed on a 22-inch monitor with a frame rate of 75 Hz (VG2239M-LED, ViewSonic, Brea, CA) at a working distance of 40 cm, consisting of 61 hexagons, and subtended 37° horizontally and 33° vertically (Fig. 1A), with a sphero-cylindrical correction adjusted to 40 cm depending on the cycloplegic refraction. The stimulation cycle started with a multifocal frame (M), followed by a dark frame (O), an overall flash frame (F) and finally a second black frame, i.e. MOFO , repeated for 212–1 sequences (Fig. 1B). Luminance for light and dark hexagons was 140 and 48 cd/m2respectively, to achieve 49% contrast with an average background luminance of 94 cd/m2. The recording bandwidth was set from 10 to 200 Hz with a signal gain of 100 k times. The responses were grouped into 5 concentric regions: rings 1 to 5 (Fig. 1A), in which the direct and induced components (DC and IC) were determined, and the peak amplitudes and times were measured (Fig. . 1 C).
Due to the university campus closure related to COVID-19, eye exams were not always performed within the time window defined by the study and were partly delayed. To minimize the effect of delayed visits, changes from baseline in SER and AL were normalized over time and expressed as an annualized change for each participant. All participants included in the analysis had completed the baseline eye exam and at least 2 follow-up eye exam visits during the study period.
The data distribution was approximately normal (Shapiro-Wilk test – SER: p = 0.16; AL: p = 0.27). A univariate general linear model (GLM) was used to compare the annualized change in SER and AL between treatment groups (atropine versus placebo), controlling for baseline SER and age as covariates. Since a weakened central inner retinal response has been reported as a risk factor for the development of myopia18, the relationship between ring 1 IC and annualized change in SER and AL were assessed using GLM and compared between atropine and placebo groups using moderator analysis. The correlation coefficients (Rp to countera) were also compared across treatment types using Fisher’s R-to-Z test in the Ring 1 IC, as well as other mfERG MOFO regions and parameters. All statistical procedures were performed with SPSS22.0 (IBM, Armonk, NY). Hochberg adjustment was applied for multiple comparisons21, with a significance level set at p ≤ 0.05. A receiver operating characteristics analysis was also performed to assess the predictive value of baseline Ring 1 CI on rapid progression (annualized progression ≥ 1.0 D) in each of the atropine and control groups.