Study design
This study was a prospective, randomized, assessor- and statistician-masked, controlled trial comparing the effect of 0.01%A+AAS and 0.01% A alone on myopia progression and choroidal thickness in Chinese children. The protocol and informed consent were approved by the Institutional Ethical Committee Review Board of Fudan University Eye & ENT Hospital and also was registered with Chinese Clinical Trial Registry (identifier: ChiCTR1900021316). All subjects were treated in accordance with the tenets of the Declaration of Helsinki.
Eligibility Criteria
The study recruitment was announced via an official account on social media. The guardians of the subjects were instructed to read the informed consent and those who responded positively were scheduled for the screening visit. Inclusion criteria were: aged 7 to 12 years with myopic refraction between -6.00 D and +0.50 D, astigmatism of less than 1.50 D, anisometropia of less than 1.50 D, intraocular pressure between 10 to 21 mmHg. Excluded were those who had other ocular diseases (e.g., cataract, uveitis, amblyopia), auricular diseases or systemic diseases, allergy to atropine, previous use of atropine or any other myopia control treatment within 1 month. Informed consent was obtained from both subjects and their guardians.
Randomization and masking
In a permuted block design stratified by age (7 to 9, 10 to 12), each subject was randomly assigned with equal probability to receive 0.01%A treatment or 0.01%A+AAS treatment. The randomization sequence was generated by a third party using SPSS 24.0. The assessors and the statisticians were masked to the treatment allocation, while open to the acupuncturists. In the 0.01%A group, subjects and their guardians were told to remove the magnetic plasters before each assessment and not to discuss any issues related to auricular acupoint with the assessors.
Interventions
Subjects in 0.01%A group were treated with a single drop of 0.01% atropine eye drops (0.4ml:0.04mg, Shenyang Xingqi Pharmaceutical Co., Ltd., Shenyang, China) every night for 6 months. Subjects in 0.01%A+AAS group received 0.01% atropine treatment plus auricular acupoint stimulation for 6 months. Seven auricular acupoints on the ear, including Eye (LO5), Anterior Intertragic Notch (TG2l), Posterior Intertragicus (AT1l), Heart (CO15), Liver (CO12), Kidney (CO10), Shenmen (TF4) were selected as shown in Figure 1 according to traditional Chinese medicine theory based on review of the literature [14, 20, 21]. A magnetic bead plastered with 7mm tape (Hwato, Suzhou Medical Appliance Factory Co., Ltd., Suzhou, China) was used for acupoint stimulation. Pressing stimulation was administered 3 times per day (6:30-7:30, 15:30-16:30, 20:00-21:00) and 30 times for each acupoint. In order to avoid the decrease of acupoint sensitivity and skin allergy, only unilateral auricles were pressed every week, and bilateral auricles were carried out alternately. The acupoint plaster was changed weekly by licensed acupuncturists with more than 2 years’ experience. At the same time, the teaching videos and maps describing AAS therapy were distributed to the guardians for learning, so that the magnetic beads fell from the acupoints by chance (e.g., bathing) could be pasted in time. Quality control was performed by the acupuncturists through checking the instant photos sent by the guardians.
Outcome measurements
After the screening visit (baseline), subjects were reassessed at 1, 3, and 6 months. At each visit, spherical equivalent (SE), axial length (AL) and choroidal thickness were measured.
Cycloplegic autorefraction was measured using an open-field autorefractor (WAM-5500, Grand Seiko Ltd, Japan) [26] 30 minutes after one drop of 0.5% Alcaine and two drops of 1% cyclopentolate HCL in 5-minute interval. This equipment allowed a sensitivity of 0.01 D, which could detect subtle diopter differences. The subjects were instructed to fixate on a Maltese cross at 5 m during measurements. If the subject’s uncorrected visual acuity was < 6/12, a green spotlight was utilised to minimise eye movements. SE was calculated for autorefraction readings. Five SE outcomes were obtained and averaged.
AL was measured by a swept source optical coherence tomography based IOL-Master 700 (Carl Zeiss Meditec, Inc., Germany) [27]. Three readings of repeated measurements and difference no greater than 0.02 mm were taken and averaged.
Images of the choroid in the macular region were obtained using a spectral domain OCT (RS-3000, NIDEK, Japan). This OCT device uses a super luminescent diode of central wavelength 880 nm for OCT imaging, with an axial resolution of 7 μm and transverse resolution of 20 μm. A three-dimensional scanning procedure was performed with a 6 × 6 mm raster scan centered on the fovea, which was composed of 128 B-scans. Each image is the average of 10 scans and images with motion artifacts, blinking, or segmentation failure were not included in the data analysis.
Data analyses
The analysis for OCT imaging in terms of choroidal volume was processed in three steps: attenuation compensation, choroid segmentation, and en-face mapping. The attenuation compensation algorithm was proposed [28] to enhance the visibility of the sclera-choroid interface and to minimise the projection shadows of retinal vessels in previous publications [29, 30]. We then employed the U-shape convolutional network (U-Net) [31, 32] to automatically segment the choroid in OCT. The trained U-Net for the choroid segmentation here achieved an AUSDE of 2.65 pixels. We deployed it to segment the data used in this paper and manually checked the results afterwards. The segmentation results were used to calculate the subfoveal choroidal thickness (SChT) and average choroidal thickness (AChT) by manually localizing the fovea and averaging over the entire 6×6 mm2 field of view, respectively. More methodological details are shown in Additional file 1.
Statistical analyses
Based on a previous study [20], the enhanced effect of AAS was assumed to be at least 0.17 D, with a common standard deviation of 0.28 D in each treatment group. The sample size was estimated to be 88 (44 per group) to achieve 80% power at a 0.05 significance level (two-sided). Considering a 10% non-adherence to treatment and a 5% loss to follow-up, a sample size of 104 subjects (52 per group) would be needed.
Full analysis set was performed based on the intention-to-treat principle. Since no statistically significant differences were found in baseline data between the two eyes, only data from right eyes were used for statistical analyses. Baseline characteristics between the two treatment groups was evaluated by unpaired t-tests for continuous data meeting normality assumptions and the chi-square test or the Fisher exact test for categorical data.
Generalised estimating equations (GEE), with one within-subject factor (time), one between-subject factor (treatment: 0.01%A or 0.01%A+AAS) and their interactions, was used to compare changes in all outcomes. Age, sex, baseline SE, number of myopic parents and outdoor time were included in the GEE model to determine the changes of SE and AL in the two study groups. Bonferroni adjustments were used for pairwise comparisons. Pearson correlation tests were used to evaluate the association between changes in SE and AL. We undertook ancillary analyses to confirm the treatment effects across potentially prognostic subgroups. These subgroups were sex, age, and baseline SE. The models also included the subgroup as a factor and the interaction between treatment and subgroup to test the significance of any difference in treatment effects across the subgroups. The significance level was set at P < 0.05.