Capsulotomy opening diameter outcomes in aphakic eyes after primary congenital cataract removal and its association

Aim To observe the change of capsulotomy opening diameter (COD) in aphakic eyes after primary congenital cataract removal and investigate its influencing factors. Methods Ocular parameters, including corneal diameter (CD), axial length (AL), anterior and posterior COD (ACOD, PCOD), and age at surgery were recorded at primary congenital cataract removal and secondary intraocular lens implantation. The concentrations of 15 kinds of cytokines in aqueous humor samples collected at the primary surgery were detected. The change (Δ) of COD between two surgeries were described, and its association was analyzed. Results Fifty eyes from 33 patients with congenital cataract who underwent primary and secondary surgery were enrolled. The changes in ACOD and PCOD were not statistically significant on the whole. ΔACOD was positively correlated with ΔCD and the concentrations of PDGF-AA, VEGF and TGF-β1. The concentration of FGF-2 and the interval between two surgeries showed negative correlations with ΔACOD and ΔPCOD. Conclusion COD in aphakic eyes kept changing after primary surgery. The positive correlation between ΔACOD and ΔCD manifested the enlargement of ACOD was influenced by lateral eye growth. Meanwhile, ΔACOD was also associated with cytokines, indicating postoperative inflammation promoted the ACOD constriction.


Introduction
Congenital cataract, with an overall prevalence of 0.63-9.7 per 10,000 people, is the main cause of childhood blindness worldwide (1,2). As a precondition for visual rehabilitation, surgery is now a safe and effective intervention for infant patients (3). Due to their undeveloped anatomical structure, majority of children <2-year-old routinely undergo secondary intraocular lens (IOL) implantation at an older age (4).
Sulcus and capsular bag are the most common positions to house an IOL during secondary surgery. The procedure of sulcus IOL implantation is relatively easier, but with higher rates of postoperative complications, such as pigment dispersion, elevated intraocular pressure and IOL decentration (5,6). In-the-bag IOL implantation, with the advantage of conforming to physiological structure, better IOL centration and lower rates of postoperative complications, may be a better long-term choice for patients (6,7).
Capsulotomy opening diameter (COD) is a key factor in IOL stability. Published studies had recommended an anterior COD (ACOD) of 4.0-5.0 mm and a posterior COD (PCOD) of 3.0-4.0 mm for optimal capsular outcome for secondary IOL implantation (8,9). Along with the eye ball growth and inflammatory response, COD seemed to go through uncertain changes postoperatively such as capsulotomy opening area opacity and capsular constriction (10). Cases whose ACOD and PCOD changed similarly or inversely were occasionally seen, some of which even lost the chance to undergo in-the-bad IOL implantation due to severe COD change. Thus, it was necessary to figure out the change of COD. However, previously studies rarely focused on COD itself.
This study aimed to observe the change of COD in aphakic eyes after primary congenital cataract removal, and to investigate its influencing factors, in order to discover the change rule of COD.

Patients and enrolment criteria
Patients who were diagnosed with congenital cataract by experienced ophthalmologists and underwent primary cataract removal and secondary IOL implantation at the Eye Hospital of Wenzhou Medical University (Hangzhou branch) were preliminarily enrolled. Complete follow-up data and clear operation videos that showed COD were requisite. Exclusion criteria included ophthalmic diseases (microcornea, microphthalmia, posterior capsule defect and persistent fetal vasculature), and systemic disorders (Marfan syndrome). Patients with severe capsular fibrosis, who needed scissors or a vitrector to remove part of capsule, and whose pupils could not be dilated during surgery, were also excluded.
This study was conducted following the tenets of the Declaration of Helsinki, adhered to the ARVO statement on human subjects. It was approved by the Ethics Review Committee of Wenzhou Medical University. Informed consent forms were obtained from the legal guardians of the patients.

Surgical technique
All surgeries were performed by the same experienced pediatric cataract surgeon (Yune Zhao) under general anesthesia using a 23-gauge microincision vitrectomy system (Accurus, Alcon Laboratories, Fort Worth, TX, United States).
Primary surgery included lensectomy, posterior capsulotomy and limited anterior vitrectomy. Two 1.0-mm clear corneal paracenteses were created at 3 and 9 o'clock using a diamond knife. A 23-gauge irrigating cannula was inserted via one port to maintain the anterior chamber, while the other 23-gauge vitrector was inserted via the other port to create a stab opening in the anterior capsule. The vitrector hole was then extended until an anterior capsulotomy opening of approximately 4.0-5.0 mm diameter was created. The cortex was aspirated in irrigation/aspiration mode. A posterior capsulotomy opening of approximately 3.0-4.0 mm diameter was performed with the vitrector. About one third of the anterior vitreous volume was removed using the same vitrectomy setting.
Compared to primary surgery, an additional scleral tunnel at 12 o'clock was made for IOL insertion at secondary surgery. After reopening the residual capsule with an iris spatula or capsulorhexis forceps and aspirating the cortex in the peripheral bag with a vitrector, the viscoelastic material was filled into the rebuilt capsular bag and a suitable IOL was placed in the bag.

Ocular parameters
The age at surgery was recorded. Axial length (AL) was measured via an A-scan (Axis nano, Quantel Medical, Cournon, France) under sedation before surgery. Corneal diameter (CD) was measured after general anethesia at the beginning of surgery using calipers from the nasal to the temporal inner side of the limbus transition zone.
The measurements of ACOD and posterior OCD (PCOD) were performed using Adobe Photoshop CC 2018 (Adobe Systems Inc., CA, United States) ( Figure 1). First, we captured a legible image that clearly showed ACOD and PCOD from the operation video. In the video of the primary surgery, an image was captured under a stable anterior chamber after suturing two corneal incisions. As for the secondary surgery, images were captured after separating the fused anterior and posterior capsule and when the anterior chamber was well maintained by a 23-gauge infusion cannula with the pressure of 30 cmH 2 O. Second, we measured the length of CD, ACOD and PCOD from the nasal edge to the temporal edge horizontally via Adobe Photoshop CC 2018 in units of pixels. Finally, since the value of CD in units of millimeter had already been measured before, the values of ACOD and PCOD in units of millimeter were calculated by equal-scale conversion.
As all parameters used for the equal-scale conversion were measured manually, we used a fixed optical surface diameter of IOL (6 mm) to test the accuracy of this method ( Figure 2). First, we captured a legible image that clearly showed ACOD and PCOD from the operation video of primary IOL implantation. Second, we measured the length of CD, ACOD, PCOD and IOL in units of pixels from the nasal edge to the temporal edge horizontally using Adobe Photoshop CC 2018. Third, as the value of CD (mm) had already been measured before and the optical surface diameter of IOL was fixed, the values of ACOD and PCOD (mm) could be calculated by equal-scale conversion through CD and IOL. Finally, the values of ACOD and PCOD calculated using CD and IOL were compared.
The change (Δ) of the parameters between two surgeries was defined as the parameter of secondary surgery minus that of primary surgery.

Collection and detection of aqueous humor samples
All samples were collected before any intraocular operation to avoid the influence of surgical stimulation. Considering that the anterior chambers in infant patients were narrow, only 100 μl of aqueous humor was collected. After being transferred to aseptic EP tubes marked with the information of the patients, the samples were immediately stored in −80°C refrigerator until analysis.

Statistical analysis
Data were analyzed using SPSS 22.0 (SPSS, Armonk, NY, United States). Normally distributed data were expressed as mean ± standard deviation. Two-tailed paired t test was used to compare the means between the primary and secondary parameters. The correlations between normally distributed parameters were analyzed using Pearson's correlation analysis. For data that did not fit the normal distribution, values were recorded as median and 25th to 75th interquartile range. Wilcoxon signed-rank test was used to compare the primary and secondary parameters. Spearman correlation analysis was used to analyze the correlations. A two-tailed P value less than 0.05 was considered statistically significant for all tests.

Baseline data
Sixty eyes from 41 congenital cataract patients were preliminarily enrolled. One eye from 1 patient, whose pupil failed to be dilated, was excluded. Six eyes from 5 patients were excluded due to concurrent ophthalmic diseases. Three eyes from 2 patients were excluded for incomplete data. Finally, 50 eyes from 33 patients who underwent primary and secondary surgery between February 2016 and April 2020 were enrolled ( Figure 3). Demographic data for the enrolled patients are summarized in Table 1.

Change of COD between primary and secondary surgery
The values of the ocular parameters were presented as mean ± standard deviation and were summarized in Table 3. No significant difference in ACOD and PCOD between two surgeries was found.

Correlation of ΔCOD with the concentrations of cytokines
A total of 50 samples from 50 eyes of 33 patients were collected. The concentrations of cytokines in the aqueous humor samples are presented as the median and 25th to 75th interquartile range ( Table 4).

Discussion
In consideration of the postoperative complications and the complexity of IOL power calculation, for infants younger than 2 years old, especially under 6 months, lens removal with posterior capsulotomy and limited anterior vitrectomy was the more common surgery choice (4,11). At secondary IOL implantation, only those whose residual peripheral capsule was intact with suitable COD could accept in-the-bag IOL implantation.
In a previous study, COD was measured with a self-made measuring needle with a laser marker (1 mm scale) (12). This Detailed change in capsulotomy opening diameter (COD) between the two surgeries. Frontiers in Pediatrics measurement method ignored the indication error caused by the angel between the capsulotomy opening plane and the measuring needle. In this study, COD was actually calculated by equal-scale conversion. Besides, we verified the accuracy of this method with a fixed optical surface diameter of IOL (6 mm).
In the enrolled aphakic eyes of the current study, both ACOD and PCOD changed, along with age, axial length and corneal diameter. Although on the whole, the changes of AOCD and PCOD were minor and not statistically significant, the range of changes was very wide. Serious changes in COD increase the difficulty of secondary in-the-bag IOL implantation, and related factors need to be furtherly studied. In our study, ΔCD represents lateral eye growth while ΔAL represents axial growth.
The positive correlation between ΔACOD and ΔCD indicated that the change of ACOD was influenced by lateral eye growth rather than axial growth, since no correlation was shown between ΔACOD and ΔAL. While ΔPCOD did not show similar positive correlation with ΔCD, it changed strongly in agreement with ΔACOD. Meanwhile, ΔACOD and ΔPCOD both showed negative correlations with the interval between two surgeries, indicating a constriction trend of COD after primary surgery.
The strength to contract the capsule was more likely due to the proliferation of lens epithelial cells (LECs). Anterior and posterior capsule opacification were the classical fibrotic disease exhibiting LECs hyperproliferation and outgrow after cataract surgery in response to surgical trauma, myofibroblast formation, matrix deposition and constriction, which were considered to be associated with wrinkling/constriction of the residual capsule (13,14). This surgery-induced complication was commonly seen in infants after congenital cataract surgery. In this fibrotic progression, the inflammation response had long been considered to be the inducement (15).
In our study, the correlations between ΔACOD and the concentrations of FGF-2 (r 2 = 0.216, y = −0.04*x + 0.52, P = 0.037) and TGF-β1 (r 2 = 0.225, y = 0.001*×−0.18, P = 0.013) prompted the roles of FGF-2 and TGF-β playing in LECs proliferation and differentiation. It had been shown that FGF-2 plays an important role in the proliferation and differentiation of LECs (16, 17). Low concentrations of FGF-2 could maintain LEC proliferation while high concentrations could promote differentiation and lentoid formation in vitro (17). Meanwhile, in the human lens, LECs transdifferentiated and proliferated into myofibroblasts through an epithelial-mesenchymal transition induced by TGF-β1, and as a result, these myofibroblasts promoted lens capsule wrinkling (18)(19)(20). Studies mentioned   above proved that FGF-2 and TGF-β played roles in the progression of LECs proliferation and differentiation. PDGF-AA (r 2 = 0.108, y = 1.56 × 10 −3 *x − 0.58, P = 0.016) and VEGF (r 2 = 0.080, y = 1.33 × 10 −3 *x − 0.29, P = 0.033) were the other two cytokines that showed correlations with ΔACOD, which indicated the participation of them in the change of residual capsule. Previously, Wu et al. found that the preoperative concentration of PDGF-AA exhibited positive correlations with the PCO scores at 1 month and 3 months after pediatric cataract surgery (r = 0.497, P = 0.030; r = 0.647, P = 0.009) (21). As for VEGF, which was considered strongly implicated in neovascularization, although there was no published study describing its association with the capsule, its existence in some nonvascular tissues, such as the cornea, was confirmed by Gogat et al. (22). To date, the concrete mechanism of the two cytokines in the change of residual capsule has been unknown, but our findings might provide new scope for further basic research.
The main strengths of our study are as follows. First, this study investigated the postoperative capsular outcome from the perspective of COD, and furtherly came up with a hypothesis that the change of ACOD after primary surgery was influenced by the lateral eye growth and postoperative inflammation. After primary surgery, the strength of the lateral eye growth gradually decreased as the patients passing the developmental phase after birth, but the strength to contract the capsule existed persistently. As a result, ACOD showed contraction trend after primary surgery. Second, more patients with congenital cataract were included in this study compared to those in previous studies.
The limitations of our study must be mentioned. First, only 100 μl of aqueous humor was collected from a congenital cataract eye per surgery for security, which limited the number of cytokines that could be detected. Second, the results only presented the change in COD in interval between primary and secondary surgery, longer follow-up was needed.
In conclusion, the results showed that the COD remained changing after primary congenital cataract removal. The correlations between ΔACOD and ΔCD, the interval between two surgeries, as well as the correlations between ΔACOD and the concentrations of FGF-2, PDGF-AA, and TGF-β1, prompted us to first put forward the hypothesis that the change in ACOD was influenced by lateral eye growth and LECs proliferation regulated by cytokines. However, it is hard to summarize the changing rule of COD simply according to the result of this study, and thus further research is needed.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by Ethics Review Committee of Wenzhou Medical Correlation between ΔCOD and the concentrations of FGF-2, PDGF-AA, VEGF and TGF-β1. University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

Author contributions
QHZ designed and conducted the research, and wrote this article. PJC designed the research. YYZ analyzed the data. DDW helped to polish this article. YEZ designed the research and was the corresponding author. All authors contributed to the article and approved the submitted version.

Funding
National Natural Science Foundation of China (grant no. 81870680).