2.1 Subjects and screening
50 children (5-9 years old) with hyperopia anisometropic amblyopia were recruited from the optometry and pediatric ophthalmology clinic of West China Hospital. The inclusion criteria are as follows: (1) the interocular difference of hyperopic spherical diopter ≥ 1.50D, hyperopic spherical diopter ≥ 2.00D for high-diopter eyes, and hyperopic spherical diopter ≥ 0.50D for low-diopter eyes; (2) the interocular difference of cylinder diopter ≤ 1.00D, and cylinder diopter ≤ 0.50D. (3) Each subject shall be reexamined once every three months, four consecutive reexaminations for 12 months, of which only 25 subjects with normal BCVA after wearing glasses for six months were included in the improved group. The rest 25 subjects were treated by glasses and six months’ patching. Their interocular difference of visual acuity was still more than or equal to LogMAR 0.2, and the eyes with poor visual acuity did not reach the normal level in the twelfth month. These subjects were included in the non-improved group. In the non-improved group, we divide amblyopia patients into two groups according to the difference of the BCVA between the eyes: one group was moderate-to-severe amblyopia group, the BCVA difference between the two eyes of the subjects in the group is higher than LogMAR 0.2; the other group was mild amblyopia group, the BCVA difference between the two eyes of the subjects is equal to LogMAR 0.2.
The exclusion criteria were: (1) The best-corrected visual acuity had fluctuation during the follow-up period. (2) During the follow-up period, there were other eye diseases, or other diseases affecting BCVA. (3) Patients with poor compliance with patching or wearing glasses. (4) The results of eye movement test, light reflex test, cover test and , the prism and alternate cover test had no sign of strabismus, and the strabismus degree from distance or near prism was less than 2△; (5) 4 △ was used to ruled out the subjects, who with micro strabismus or macular inhibition blind spot.
The trial analysis data were collected at the fourth follow-up visit, but all subjects received routine pediatric ophthalmic examination at each follow-up visit. Routine pediatric ophthalmic examination including slit lamp test, ophthalmoscope test and examination like fundus photography was performed by the same ophthalmologist to exclude any other ocular disease.
The compliance was guaranteed by parents who monitored children’s daily habits on a record list. Only children who were willing to wear glasses and eye patches according to the treatment plans were recruited because of their excellent compliance. Three subjects dropped out because they stopped wearing glasses for over three days in the ninth month. The study was approved by the West China Medical Ethics Committee, Sichuan University, and complied with the Declaration of Helsinki's guidelines. Informed consent was obtained from each patient and their guardian.
2.2 Main data
The optical and biometric data, including axial length and higher-order aberrations (3 to 6 orders), were measured for both eyes in 50 subjects.
2.2.1 Refraction
After total cycloplegia (1% Tropicamide eyedrop), the refraction dioptre of all participants was done randomly by anyone of the two optometrists at each visit. The subjective refraction was measured by phoropter. The objective refraction was measured by retinoscopy and autorefraction, which could help measure subjective refraction. The spherical equivalent refractive error (SER)is the sum of spherical diopter and half-cylindrical diopter. The EDTRS chart was used to measure the participants’ best-corrected visual acuity with their corrected glasses18. The visual acuity (VA) was recorded as a log MAR. The subjective refraction of the twelfth month was used as the results.
2.2.2 Axial length (AL)
The data were measured by non-contact instrument IOL Master (Carl Zeiss Meditec, Germany) for all the subjects19. The Signal to Noise Ratio of each subject was more than 3 (3.2±0.3).
2.2.3 Higher-order aberrations
As the measurement diameter affects the change of higher-order aberrations of the eyes, each subject should recheck the pupil diameter before measurement to ensure that the pupil diameter is not less than the required diameter during measurement.
All higher-order aberrations were collected in the data collection stage of this experiment, because no statistical difference was found in the fifth or above order aberrations, and their contribution to visual effect was weak 26, so they were not listed in the results. The data was measured by the i-Trace (Carl Zeiss Meditec, Germany). I-Trace uses laser ray tracing technology and describes aberrations by the Zernike coefficient. Zernike polynomials of the irregularities aberrations were computed over a circular pupil of 5mm diameter, associating to the America National Standards Institute20. Compared with other instruments. i-Trace showed good repeatability performance21. All the subjects were tested between 10:00 AM and 2:00 PM to reduce the daily variation17. In view of enantiomorphism (mirror symmetry between fellow eyes of an individual), the appropriate coefficient of left eyes was changed so that all eyes could be considered as right eyes22.
The RMS is an acronym for the root mean square of each order aberrations. It is a mathematical expression of the effective value of higher-order aberration that affects the light wave in each order.
2.3 Data analysis
Four eye groups’ data was included in the analysis, including the high-diopter (amblyopia) eyes and the low-diopter eyes in the amblyopia group, the high-diopter eyes, and the low-diopter eyes in the control group. Each dataset of higher-order aberrations, visual acuity, and axial length showed normal distributions in these four eye groups (by Kolmogorov-Smirnov test). Firstly, the comparison between the four groups’ eyes was analyzed by one-way ANOVA. These normal distribution datasets all showed homogeneity of variance except the visual acuity. Then the multiple comparisons after ANOVA among normal datasets were tested by Bonferroni Test (the LSD-t Test with Bonferroni correction). Dunnett’s T3 Test was used to test the visual acuity. Secondly, the Paired Sample T-test was used to find the interocular differences between the high-diopter and low-diopter eyes. The Paired Sample T-test is much more effective than the LSD-t Test because the dataset of each subject for interocular comparison is paired.
All data of two amblyopia subgroups , and the refractive data (sphere, cylinder and SER) were non-normally distributed. The analyses were performed by Kruskal-Wallis H Test firstly. Furthermore, Nemenyi Test (with correction) was used to analyze the results of the pairwise comparison between groups. All the statistical analyses were performed by IBM SPSS version 19.0 software, SPSS (Chicago, IL, USA).