Disease-related and age-related changes of anterior chamber angle structures in patients with primary congenital glaucoma: An in vivo high-frequency ultrasound biomicroscopy-based study.

OBJECTIVES: To provide in vivo measurements of anterior chamber angle structures and their relationship with age as evaluated by high-frequency ultrasound biomicroscopy (UBM) in patients with primary congenital glaucoma (PCG).
METHODS: High-frequency UBM was done for 51 PCG eyes from 40 patients (aged from 3 to 96 months) and 11 unaffected contralateral eyes. Parameters, including the proportion of observable abnormal tissue membrane and Schlemm's canal, the largest cross-sectional area (CSA) of Schlemm's canal (SC), SC meridional diameter, trabecular-iris angle (TIA), trabecular meshwork (TM) thickness, iris thickness, ciliary process length, and corneal limbus thickness were compared between the two groups and their relationship with age was explored in PCG eyes.
RESULTS: Abnormal tissue membrane was detected in 27.5% of PCG eyes and none in unaffected eyes. SC was observed in 73.1% of PGC eyes compared to 100% in unaffected eyes (P<0.001). The largest CSA of SC, SC meridional diameter, iris thickness, and corneal limbus thickness were all significantly smaller in PCG eyes compared to unaffected eyes (all P<0.05). TIA and ciliary process length in unaffected eyes were smaller than PCG eyes (both P<0.05). The largest CSA of SC, TM thickness, iris thickness, and ciliary process length were all significantly correlated to age in PCG eyes (P<0.05).
CONCLUSIONS: The anatomical information evaluated by high-frequency UBM may provide glaucoma specialists a useful tool to aid in understanding the dysgenesis and changes with age of anterior chamber angle in PCG.

Introduction

an class="Disease">Primary congenital glaucoma (PCG) is the most common types of the an class="Disease">childhood glaucoma and is thought to be secondary to developmental defects in Schlemm’s canal (SC) and the trabecular meshwork (TM), the main aqueous humor outflow structures [1]. In vivo physiological features of these structures would provide insight into the ocular structures involved in PCG; however, previous studies of SC and TM in PCG have been limited to histologic specimens in vitro [2-4].

The invention of ultrasound biomicroscopy (UBM) provides a noninvasive, real-time, dynamic, continuous, and in vivo assessment of the morphology of the anterior segment structure in PCG an class="Species">patients, and more importantly, it is suitable for examination in an class="Species">infants and children under general anesthesia in the supine position [5-8] compared with standard anterior segment optical coherence tomography (OCT), which can only be performed in a seated, awake child. Standard 50 MHz UBM is limited by its low resolution and cannot provide quantitative measurements of SC and TM [5,6,9]. A newer 80 MHz high-frequency UBM, the iUltrasound imaging system (iScience Interventional Inc., Menlo Park, CA), affords a more detailed view of the anterior segment than previous 50 MHz UBM with an improvement in axial resolution. Quantitative assessment of SC and TM in adults were recently reported, [10-12] and its application in PCG was limited to one published report about the measurement of SC diameter in a small case series [13]. Here, we measured in vivo anterior chamber angle structure dimensions and evaluate their relations to age in patients with PCG using high-frequency UBM, which are essential in understanding the pathogenesis of congenital glaucoma [6-8,13-15].

Materials and methods

This prospective study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Tongren Eye Center. Each an class="Species">patient’s legal guardian or representative signed an informed consent. The clinicne">al trial was registered under the Chinese Clinicne">al Trials Registry (ChiCTR-an class="Chemical">OCC-15005789).

Subjects

an class="Gene">All an class="Species">patients with newly diagnosed PCG between February 2015 and March 2018 at Beijing Tongren Eye Center were enrolled. Patients underwent complete ophthalmologic examination under general anesthesia. The diagnosis of PCG was based on the presence of at least 2 of the following clinical features: (1) increased corneal diameter (>12 mm) together with elevated intraocular pressure (IOP) (>21 mm Hg), (2) Haab’s striae, (3) corneal edema and (4) increased cup-to-disc ratio. Exclusion criteria included other ocular or systemic anomalies or any previous intraocular surgery. Those with severe trabeculodysgenesis under high-frequency UBM, corresponding to those with insertion of both the iris and ciliary processes before the scleral spur as we previously reported, were excluded due to the difficulty in identifying the scleral spur and measuring TM [15]. And those with no identified SC in all four quadrants under high-frequency UBM were also excluded.

IOP was measured using the Icare tonometer (Icare Tan class="Gene">A01i, Icne">are Finland Oy, Espoo, Finland). For an class="Species">patients with bilateral disease, both eyes were included in the analysis (Group 1). For those with unilateral disease, the unaffected contralateral eyes served as the control group (Group 2). Parameters including gender, age, IOP, and corneal diameter were recorded.

Imaging of the anterior chamber angle

The anterior chamber angle was examined in the supine position under general anesthesia using the iUltrasound imaging system by the same investigator (YS). an class="Gene">A low-viscosity gel was placed to assist in transduction. The self-contained probe was placed directly on the eye for imaging in the 3, 6, 9, and 1an class="Gene">2 o’clock meridians. Images were obtained using the following settings: transducer frequency, 80 MHz; axial resolution, 25 μm; lateral resolution, 50 μm; electronic resolution, 10 μm; tissue penetration depth, 2 mm; scan rate, 7 frames/second; and imaging window size, 4.5 × 4.5 mm. In each subject, at least 20 ultrasound images for each position were obtained.

Image processing

Two investigators (YS, CX) independently identified Schlemm’s canal (SC), and the scleral spur (SS). When the investigators disagreed on the delineation of SC, a mutual conclusion was reached after discussion. These parameters were then measured by a masked, experienced investigator (YS) using ImageJ software (version 1.47, National Institutes of Health, an class="Chemical">Bethesda, Maryland, USan class="Gene">A) transformed from pixel to anatomic values as previously described [16]. SS was defined as the end point of the curved interface between the ciliary body and the sclera/TM based on previous studies [17]. SC was defined as observable when a thin, black, lucent space adjacent to the SS and considered detected when observed on two consecutive images [12]. Presence or absence of an abnormal tissue membrane in each quadrant was recorded for analysis (Fig 1).

Fig 1

Angle of a primary congenital glaucoma eye with presence of abnormal tissue membrane (dotted arrow) and absent Schlemm’s canal.

Details of the measurements evaluated are depicted in Fig 2. SC area was calculated using the automated area function in ImageJ. Measurements of the cross-sectional area (an class="Gene">CSA) of SC were taken at four different positions (the 3, 6, 9, and 1an class="Gene">2 o’clock meridians), the largest of which was used for analysis to account for any variability related to visualization of SC. The percentage of eyes with observable SC was calculated for each quadrant (superior, inferior, nasal, temporal). SC meridional diameter was defined as the distance between the most posterior and anterior part of SC directly adjacent to the TM [12].

Fig 2

High-frequency ultrasound biomiscroscopy image showing Schlemm’s canal (SC) and scleral spur (SS).

Representative schematic lines and angles are shown including standard measurements of the ciliary process length (CPL), iris thickness (IT), trabecular meshwork thickness (TMT), corneal limbus thickness (CLT), and trabecular-iris angle (TIA, white angle) as previously described in other studies. Upper: image of an affected eye of a 40 months old child with primary congenital glaucoma (PCG); Lower: image of an unaffected contralateral eye of a 26 months old child with PCG.

Representative schematic lines and angles are shown including standard measurements of the ciliary process length (CPL), an class="Disease">iris thickness (IT), trabecular meshwork thickness (an class="Chemical">TMT), corneal limbus thickness (CLT), and trabecular-iris angle (TIA, white angle) as previously described in other studies. Upper: image of an affected eye of a 40 months old child with primary congenital glaucoma (PCG); Lower: image of an unaffected contralateral eye of a 26 months old child with PCG.

Considering the retrodisplacement of SC in PCG, the thickness of TM was measured at the anterior end point of SC, as more posterior measurements of TM might not truly represent the thickness of the TM itself but rather the ciliary muscle behind the scleral spur [3,12,18]. an class="Disease">Iris thickness was measured across a verticne">al line which was perpendicular to the posterior iris plane locne">ated 500 μm centrally from the iris root. The trabecular-iris angle (TIan class="Gene">A) was measured as the angle between the arms passing through a point on the inner surface of trabecular meshwork 500 μm from the scleral spur and the point perpendicularly opposite on the iris [9]. Ciliary process length (CLP) was measured along the line starting from the point of most-anterior tip of the ciliary body to the beginning of the zonules [6]. Corneal limbus thickness (CLT) was measured from scleral spur to the outer surface of the corneal limbus, perpendicular to the limbus tangent. Apart from the CSA of SC, measurements of other parameters were all taken at four quadrants on the image with largest SC and the average values of other anterior chamber angle parameters were used for analysis.

Statistical analysis

an class="Gene">All statisticne">al analyses were performed using SPSS (V.16.0; SPSS, Chicne">ago, Illinois, USan class="Gene">A) or GraphPad Prism (V.7; GraphPad Software, Inc. La Jolla, CA, USA) with p<0.05 considered significant. Frequency histograms and the one-sample Kolmogorov–Smirnov test were used to assess the distribution of numerical data for parametric characteristics. Qualitative and categorical data were counted by frequency, while the median (range) was used for those quantitative data which did not obey normal distribution. The Pearson χ2 test was used to compare right and left eyes between groups. A linear mixed model was used to adjust for the correlation between eyes in patients with bilateral disease and compare various parameters between Groups 1 and 2 including age, gender, IOP, corneal diameter, the distribution of quadrants with the largest SC, the proportion of observable Schlemm’s canal, and other anterior chamber angle parameters. Generalized Estimating Equations was used to analyze the precise relation of age to anterior chamber angle parameters and the relation of IOP to the largest area of SC. Fifty percent of eyes (31 eyes) were randomly selected to assess observer variability, intraobserver and interobserver variability using a coefficient of repeatability and 95% limits of agreement and intraclass correlation coefficients (ICC). Interobserver agreement was calculated by comparing initial values of Observer 1 (YS) to those of Observer 2 (CX).

Results

Subject characteristics

Fifty-one an class="Species">patients with 75 eyes underwent the UBM measurement, while 13 eyes were excluded for either hne">aving severe trabeculodysgenesis (4 eyes) or hne">aving no identified SC (4 eyes), or both (5 eyes). Therefore, a total of 40 an class="Species">patients with 62 eyes were included: 51 eyes with PCG and 11 unaffected contralateral eyes. Thirty-one patients (78%) were male. The median age was 36 months (range: 3–96). The ratio of unilateral/bilateral disease was approximate 1:2.6 with 11 unilateral and 29 patients with bilateral PCG. There were 22 patients (38 PCG eyes and 6 unaffected contralateral eyes) had both eyes and 18 patients (13 PCG eyes and 5 unaffected contralateral eyes) had one eye recruited in this study. Comparison of subject characteristics between Group 1 and Group 2 are presented in Table 1. The number of eyes in Group 2 was significantly lower due to the lower incidence of unilateral PCG, while age, gender, and laterality was matched in the two groups (all P>0.05). The IOP and corneal diameter were significantly greater in Group 1 (both p<0.001).

Table 1

Comparison of subject characteristics and observable Schlemm’s canal proportion between groups.

Group	Group 1	Group 2	P
No. of eyes (N, %)	51	11	<0.001*
OD/OS	27/24	6/5	0.595
Gender (male/female)	39/12	8/3	0.853
Age(months; median, range)	36 (3–96)	26 (5–79)	0.858
IOP (mmHg; median, range)	33 (22–48)	15 (12–20)	<0.001*
Corneal diameter (mm; median, range)	13.0 (12.0–16.0)	11.1 (10.5–11.5)	<0.001*
Proportion of eyes with the largest CSA of SC in each quadrants (superior/nasal/inferior/temporal, %)	9.8/35.3/33.3/21.6	0/54.5/36.4/9.1	0.686
Observable SC proportion	-	-	-
Total (n,%)	152/204 (74.5%)	44/44 (100%)	<0.001*
Superior region (n,%)	30/51 (58.8%)	11/11 (100%)	0.008*
Nasal region (n,%)	40/51 (78.4%)	11/11 (100%)	0.142
Inferior region (n,%)	40/51 (78.4%)	11/11 (100%)	0.095
Temporal region (n,%)	42/51 (82.4%)	11/11 (100%)	0.137

* Statistical significance; Group 1, primary congenital glaucoma eyes; Group 2, unaffected contralateral eyes. OD, right eye; OS, left eye; IOP, intraocular pressure; CSA, cross-sectional area; SC, Schlemm’s canal.

* Statistical significance; Group 1, an class="Disease">primary congenital glaucoma eyes; Group 2, unaffected contralateral eyes. OD, right eye; OS, left eye; IOP, intraocular pressure; an class="Gene">CSA, cross-sectional area; SC, Schlemm’s canal.

Qualitative anterior chamber angle parameters

The largest an class="Gene">CSA of SC were identified more frequently in the nasal and inferior quadrants in Group 1 (P = 0.036) but equally distributed in Group an class="Gene">2 (P = 0.178) with no significant difference between groups (P = 0.686). SC was observed in 73.1% of eyes with PCG compared to 100% in unaffected contralateral eyes (P<0.001). The major difference was noted in the superior quadrant (57.7% in PCG versus 100% in normal eyes, P = 0.008) while all other quadrants had similar visibility of SC between groups (all P>0.05) (Table 1). In affected eyes, the number of eyes with abnormal tissue membrane in superior, nasal, inferior, and temporal quadrants were 11, 7, 6, 9, respectively (P = 0.545). The total percentage of the presence of abnormal tissue membrane in all quadrants was 16.2%, and the percentage of PCG eyes with abnormal tissue membrane in any quadrant was 27.5% (14 in 51 eyes). No abnormal tissue membrane was detected in unaffected eyes.

Intraobserver and interobserver reproducibility of quantitative anterior chamber angle measurements

an class="Gene">Analysis of the reproducibility of quantitative measurements from a randomly selected subset of 31 eyes using the intraclass correlation coefficient (ICC) is shown in Table 2. an class="Gene">All quantitative measurements of the anterior chamber angle had good reproducibility.

Table 2

Reproducibility of quantitative measurements of the anterior chamber angle in a randomly selected subset of 31 eyes.

Anterior chamber angle parameters	Intraobserver repeatability			Interobserver reproducibility
	Mean	Difference	ICC (lower 95% CI)	Mean	Difference	ICC (lower 95% CI)
Largest area of SC (μm2)	3625.71	23.49	0.875 (0.756)	3567.09	140.71	0.851 (0.714)
SC meridional diameter (μm)	258.87	13.80	0.895 (0.794)	252.05	27.43	0.814 (0.649)
TIA (μm)	59.51	3.67	0.832 (0.680)	59.63	3.43	0.804 (0.632)
TM thickness (μm)	105.05	4.06	0.912 (0.825)	110.56	6.9	0.946 (0.891)
Iris thickness (μm)	201.59	1.23	0.842 (0.698)	199.53	2.89	0.872 (0.752)
Ciliary process length (μm)	1684.50	103.93	0.842 (0.698)	1679.04	114.86	0.821 (0.661)
Corneal limbus thickness (μm)	712.20	17.40	0.846 (0.705)	709.10	12.93	0.841 (0.696)

SC: Schlemm’s canal; TIA: Trabecular-iris angle; TM, trabecular meshwork; ICC: Intraclass correlation coefficient; CI: confidence interval.

SC: Schlemm’s canal; TIan class="Gene">A: Trabecular-iris angle; TM, trabecular meshwork; ICC: Intraclass correlation coefficient; CI: confidence interval.

Quantitative anterior chamber angle parameters

Quantitative parameters of the anterior chamber angle for groups 1 and an class="Gene">2 are shown in Table 3. Notably, an class="Gene">CSA of SC, SC meridional diameter, iris thickness, and corneal limbus thickness were all significantly smaller in eyes with PCG (Group 1) compared to unaffected eyes (Group 2, all P<0.05). While TIA and ciliary process length in Group 2 was smaller than those in Group 1 (both P<0.05). There were no statistical differences between groups in TM thickness (P = 0.072). IOP was not related to the largest area of SC in either group by Generalized Estimating Equations (Group 1: P = 0.510; Group 2: P = 0.455) (Fig 3).

Table 3

Comparison of quantitative parameters of the anterior chamber angle between groups.

Group	Group 1	Group 2	P
Largest CSA of Schlemm’s canal (μm2, mean±SD)	3363.91±1082.98	5130.66±1231.90	<0.001*
SC meridional diameter (μm, mean±SD)	257.70±66.70	335.09±104.76	0.009*
TIA (μm, mean±SD)	64.52±15.28	48.02±15.62	0.002*
TM thickness (μm, mean±SD)	111.52±40.79	95.37±25.72	0.072
Iris thickness (μm, mean±SD)	188.10±40.62	235.25±65.34	0.002*
Ciliary process length (μm, mean±SD)	1498.88±300.02	1278.37±130.28	0.018
Corneal limbus thickness (μm, mean±SD)	702.73±100.37	771.62±78.59	0.030*

* Statistical significance; Group 1, primary congenital glaucoma eyes; Group 2, unaffected contralateral eyes. CSA, cross-sectional area; SC, Schlemm’s canal. TIA, trabecular-iris angle; TM, trabecular meshwork.

Fig 3

Generalized Estimating Equations analysis of intra-ocular pressure (IOP) and the largest cross-sectional area (CSA) of Schlemm’s canal (SC) in 51 primary congenital glaucoma (PCG) eyes (A) and 11 contralateral unaffected eyes (B), and IOP was not related to the largest CSA of SC in either group.

Generalized Estimating Equations analysis of intra-ocular pressure (IOP) and the largest cross-sectional area (an class="Gene">CSA) of Schlemm’s cne">anal (SC) in 51 an class="Disease">primary congenital glaucoma (PCG) eyes (A) and 11 contralateral unaffected eyes (B), and IOP was not related to the largest CSA of SC in either group.

* Statistical significance; Group 1, an class="Disease">primary congenital glaucoma eyes; Group 2, unaffected contralateral eyes. an class="Gene">CSA, cross-sectional area; SC, Schlemm’s canal. TIA, trabecular-iris angle; TM, trabecular meshwork.

Anterior chamber angle parameters and age in PCG eyes

The largest an class="Gene">CSA of SC, TM thickness, an class="Disease">iris thickness and ciliary process length were significantly correlated to age in 51 PCG eyes. The slopes and intercepts for the regression analysis, with correlation coefficients with p values, R2 values and regression quotation are shown in Fig 4. IOP was not correlated with age in PCG eyes (P = 0.234).

Fig 4

Generalized Estimating Equations analysis of age and anterior chamber angle parameters in 51 PCG eyes.

(A) The largest cross-sectional area (CSA) of Schlemm’s canal (SC) in relation to age (months) with mean and 95% prediction limits. (B) Iris thickness in relation to age (months) with mean and 95% prediction limits. (C) TM thickness in relation to age (months) with mean and 95% prediction limits. (D) Ciliary process length in relation to age (months) with mean and 95% prediction limits. The slopes and intercepts for the regression analysis, with correlation coefficients with p values, R2 values and regression quotation are shown.

(an class="Gene">A) The largest cross-sectional area (an class="Gene">CSA) of Schlemm’s canal (SC) in relation to age (months) with mean and 95% prediction limits. (B) Iris thickness in relation to age (months) with mean and 95% prediction limits. (C) TM thickness in relation to age (months) with mean and 95% prediction limits. (D) Ciliary process length in relation to age (months) with mean and 95% prediction limits. The slopes and intercepts for the regression analysis, with correlation coefficients with p values, R2 values and regression quotation are shown.

Discussion

Precise in vivo measurements of the developing anterior segment are essential in understanding the pathogenesis of an class="Disease">congenital glaucoma [6-8,14]. It also provides useful information in planning an class="Disease">glaucoma surgery [13,15]. Measurements of the TM and SC in vivo using spectral-domain optical coherence tomography (SD-OCT) or swept-source optical coherence tomography (SS-OCT) have been reported in older children [19-21]; however, this modality is not possible in infants and younger children due to the amount of cooperation needed to complete the test, which can only be performed in a seated, awake child. The iUltrasound imaging system is suitable for examination in infants and children under general anesthesia in the supine position and both TM and SC can be clearly identified [10-12]. To our knowledge, only one prior study has measured SC diameter in PCG eyes using this modality [13]. Our study is the first to provide an in vivo quantitative assessment of the TM and SC, and explore their relationship to age in patients with PCG.

Previous study of Schlemm’s canal in younger an class="Species">patients is notably limited. Using the 80-MHz UBM, Tandon and colleagues found Schlemm’s cne">anal could be identified in 62.5% of an class="Species">patients with PCG (age range: 3 days to 3 years) and in all 19 normal eyes (age range: 7 weeks-17 years) [13]. Since we excluded 9 eyes with no identified SC in any quadrants, we were able to observe Schlemm’s canal in 73.1% of all quadrants with PCG and in 100% of normal eyes. Lack of proper development of SC has been postulated to play a role in the pathogenesis of primary congenital glaucoma [22-25], and its absence has been associated with a severe form of goniodysgenesis [26]. Inability to detect SC might be a result of segmental outflow as previously described in healthy adult eyes [27-29] or may affected by variation in IOP in patients with primary open-angle glaucoma [30]. For these reasons, we cannot fully extrapolate our finding of lower visibility of SC to the pathogenesis of PCG yet. However, the lack of SC was reported to be associated with poor prognosis of angle surgeries in PCG [4,26] identification of SC under 80-MHz UBM might be useful to plan angle surgery in these eyes.

Most current quantitative measurements of SC are confined to histologic study in vitro and have reported SC diameter from 180 to 250 μm in an class="Disease">non-glaucomatous pediatric eyes and 9an class="Gene">2 to 250 μm in pediatric eyes with glaucoma [2]. The SC meridional diameter in our study was slightly larger in both groups compared to histological studies though smaller in eyes with PCG than with normal eyes (257.70±66.70 μm in PCG; 335.09±104.76 in normal eyes, P = 0.009). This could represent a difference in in vivo versus in vitro measurement as fixation and preparation methods may significantly alter the size and shape of SC from natural settings. Our investigation of Schlemm’s canal in vivo may better reflect the canal’s physiologic and functional dimensions. Moreover, SC diameter may dynamically vary depending on the amount of aqueous fluid. In this study, we chose the largest CSA of SC among 4 quadrants, which may lead to measure the largest possible diameter of SC. Using the 80 MHz anterior segment ultrasound, only Tandon et al. reported the single meridional canal diameter in pediatric nonglaucomatous eyes (142 ± 33.2 μm) was larger than average meridional canal diameter of 4 quadrants in pediatric glaucomatous eyes (64.9 ± 10.90 μm; P = 0.007), but they only recruited 10 subjects in each group and 50% glaucoma eyes failed to identify the SC [13].

Similarly, the largest an class="Gene">CSA of SC in PCG eyes was significne">antly smaller thne">an thne">at of unaffected eyes (3363.91±1082.98 μman class="Gene">2 compared to 5130.66±1231.90μm2, P<0.001). No previous studies have reported the CSA of SC in PCG eyes, but in healthy adults using SD-OCT and SS-OCT, the SC CSA varies rapidly within short distances along its arc, ranging from 4064±1308 μm2 to 13991±1357 μm2 and may decrease with age [19,31-33]. Eyes with primary open angle glaucoma have a reduced CSA compared with normal healthy controls [33], and reduced SC size may be associated with elevated IOP because the size of SC is related to outflow facility [33,34].

The smaller dimensions of Schlemm’s canal in an class="Species">children, as seen in our study, are well known [35,36]. We detected a significne">ant increase with age in PCG eyes, and significne">antly smaller area in eyes with PCG compared to unaffected eyes. However, we didn’t find a correlation between SC an class="Gene">CSA and IOP in either group. We speculate that the smaller largest-measured SC area we observed in PCG eyes compared with unaffected eyes may not be a result of elevated IOP but related to disease pathogenesis. Also, the elevation of IOP may related to the degree of the maldeveloped SC, but we didn’t have enough data in this study to prove this. We only performed UBM at 4 o’clock, not 360-degree. The future development of circumference SC image would help solve this question.

The trabecular beams in PCG have been described using microscopy as thickened and compacted, increasing outflow resistance [25,37]. In our study, trabecular meshwork thickness in eyes with PCG was thicker than unaffected eyes, but this difference was not statistically significant. This is consistent with prior studies showing trabecular meshwork thickness of 107±51 μm from trabeculotomy specimens of an class="Species">patients with an class="Disease">infantile glaucoma (average age 8 years) [3]. Similar to ours results, that study shows a decrease in trabecular meshwork thickness with age in patients with glaucoma in contrast to the increase in trabecular meshwork thickness with age reported in healthy individuals [19]. However, the presence of an anterior chamber membrane may have affected our ability to accurately measure trabecular meshwork thickness. Twenty seven percent of glaucoma patients had identifiable abnormal tissue in our cohort. The rate of identifying the anterior chamber membrane varies in the literature, ranging from 100% to 12%.5 One study identifying abnormal tissue in 100% of eyes using SD-OCT suffered from poor inter-grader agreement (κ = 0.61, P < 0.005) [21]. The lower rate of membrane identification in this study may be due to our strict criteria requiring agreement between two glaucoma specialists where equivocal cases were excluded. Further studies comparing histological and UBM results may better determine if high-resolution ultrasound is a precise method to measure trabecular meshwork thickness in vivo.

The trabecular-iris angle was larger in eyes with an class="Disease">primary congenital glaucoma compared to unaffected eyes, and an class="Disease">iris thickness was thinner and ciliary process length was longer in PCG eyes compared with unaffected eyes, consistent with prior studies [5,6,8,38]. Although ridges or crypts of the iris surface may affect the measurement of iris thickness, loss of normal iris configuration in PCG similar to our findings has been reported [5]. Iris thickness increases with aging, while ciliary process length decreases with aging. Although it might denote that the thinning of the iris and ill-defined ciliary process could be the result of progressive stretching of the globe in these eyes, it could also indicate dysplasia of the anterior chamber angle in PCG with concomitant abnormal iris and ciliary process, which could still develop with age in PCG eyes.

We detected that the corneal limbus thickness was thinner in PCG eyes compared to unaffected eyes. Both thinner [39-41] and thicker [42,43] central cornea thickness (CCT) have both been reported in PCG an class="Species">patients, explained by corneal stretching and/or scne">arring for thinner CCT or an inherent component of the pathophysiology related to racial and genetic bne">ackground or an class="Disease">edema for thicker CCT. Since scarring and edema rarely affect the limbus in PCG eyes, we believe that the thinner corneal limbus thickness may better illustrate the corneal stretching due to enlargement of eye ball under high IOP.

The current study reports a bilateral PCG incidence of about 72%, consistent with rates in the literature: 60%-99.3% [44]. This higher incidence of bilaterality in other reports may be related to disease severity or underlying an class="Disease">genetic abnormality in different ethnic groups. Although we did find the anterior segment structures were significantly different between affected PCG eyes and unaffected eyes, we still cannot determine if severe goniodysgenesis is the cause or the result of the disease. Differences between affected and unaffected eyes in patients with unilateral PCG patients shed insight into this issue; however, there were only 6 unilateral PCG patients with both affected and unaffected eyes recruited in this study, so further study is warranted to explore this relationship.

The present study has certain limitations. Firstly, observer and measurement an class="Disease">errors might exist in thne">at all measurements were taken by a single masked observer, though we attempted to minimize this effect by obtaining a consensus of two observers on the identificne">ation of key angle structures. an class="Gene">Analysis of the intraobserver and interobserver reproducibility was high. Secondly, our study has a small sample size, but is acceptable given the incidence of primary congenital glaucoma. Thirdly, with regards to measurement of the largest cross-sectional area of Schlemm’s canal, it is possible that this varies from quadrant to quadrant with natural variation at different anatomic locations with the largest cross-sectional area not comprehensively reflecting the anatomy of the whole eye, and the inability to identify SC in the superior quadrant alone could be due to imaging technique for those enlarged PCG eyes and not a consistent anatomical finding. Fourthly, PCG is a genetic disorder and it is likely that the “unaffected” eye is not truly normal [44]. Further studies should address the differences of anterior chamber angle structures between unaffected eyes of PCG patients and normal age-matched eyes. Finally, the cross-sectional nature of our study did not allow us to comprehensively elucidate age-related changes in the development of anterior chamber angle in PCG eyes, and longitudinal studies are warranted to further explore this relationship.

Conclusions

Notwithstanding the limitations, we believe that this study is the first of its kind to provide quantitative information about anterior segment morphology and its relationship with age in vivo using high resolution UBM in eyes with PCG. The anatomical information gleaned in the study demonstrates the usefulness of high-frequency UBM in understanding the angle dysgenesis, and it might also be useful in planning anterior segment surgery in these eyes based on the development of the anterior chamber angle.

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16 Sep 2019

an class="Chemical">PONE-D-19-17802

Disease-related and age-related changes of anterior chamber angle structures in an class="Species">patients with an class="Disease">primary congenital glaucoma: An in vivo high-frequency ultrasound biomicroscopy-based study

PLOS ONE

an class="Gene">Dear M.D., Ph.D. Wne">ang,

Thank you for submitting your manuscript to PLOS ONE. an class="Gene">After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised by the reviewers and the comments below..

The study investigates features of an class="Disease">primary congenital glaucoma (PGC) using ultrasound biomicroscopy.

1. The small sample size for unaffected eyes (controls) may impact significance of the findings. Please justify.

2. The controls were all contralateral eyes of unilateral cases of PGC. One study recently suggested that the normal eyes in unilateral PGC may not be anatomically normal (Bayoumi 2017) - can the authors please comment, and also indicate if they have  compared their findings to data for an class="Species">patients with both eyes unaffected.

3. The issue of unilateral versus bilateral PGC is interesting and should be commented upon further. What is the prevalence of each condition in China and across other countries?  Is there any data on the underlying causes of PGC for the study an class="Species">participants? Have genetic mutations for example, been investigated?

4. The methods used to assess the anterior chamber angle characteristics are unclear. Please revise.

5. The age-related effects for PGC eyes are presented in Figure 3, but there is limited discussion as to what these graphs mean.. How does this compare to normal unaffected eyes (noting very small sample size) (both contralateral eyes, and also both eyes unaffected).

==============================

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data an class="Gene">Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly shne">aring data—e.g. an class="Species">participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Please see comments to be addressed below. In addition to the below comments the only other concern with regard to statistical analysis is the relative small sample size of control "normal" eyes compared to affected eyes. If possible, someone more specialised in statistical analysis might be useful to check the validity of the analysis.

Line 45: Last sentence in conclusion should also state “and changes with age”

Line 48: The sentence “an class="Disease">Primary congenital glaucoma is the leading cause of an class="Disease">blindness worldwide” is incorrect.

PCG only accounts for 3-5% of an class="Disease">blindness in an class="Species">children worldwide. A greater number are affected in developing countries. It is the leading cause of blindness in Saudi Arabia and the Rom population of Slovakia. It is however a very rare condition that is difficult to treat and detect and can often be missed which is why this study is interesting.

Line 84: Sclera spur should be changed to scleral spur

Line 91: For imaging of the anterior chamber angle were the scans acquired by a technician and then interpreted by the two investigators? an class="Gene">And if so, was it the same an class="Species">person that did all the scans?

Line 161: 51 an class="Species">patients with 75 eyes were underwent the UBM measurement (remove were)

For statistics:

1. Comparing 51 affected eyes to 11 “normal/control” eyes not sure how reliable it is to do hypothesis testing here as there is a small comparative sample size

For results:

1. Table 1: Group 1 column, under “observable SC proportion”

a. Total (n, %). 152/208 (*where does the value of 208 come from? If the SC is observed in 4 quadrants per eye should it not be 204?)

b. For each of the remaining regions (superior, nasal, inferior and temporal) should the fraction be /51 instead of 52?

Line 319: should read “which was consistent with the results in other studies”

Line 334: In addition of the study limitations mentioned I wonder if the following two points should also be included:

1. Small comparison sample size. Only 11 contralateral unaffected eyes compared to 51 affected eyes, this does make comparison between groups difficult

2. Is the supposedly unaffected eye in unilateral disease actually normal? PCG is a an class="Disease">genetic disorder and it is likely that the “unaffected” eye is not truly normal.

It would be interesting to know if the eyes that were excluded with complete absence of SC had angle surgery or went straight to either a trabeculectomy or an class="Disease">glaucoma drainage device? Presumably absence of SC would mean thne">at angle surgery including trabeculotomy would be contraindicne">ated. In your conclusion an additional point to be mentioned would be thne">at UBM could be used to guide surgicne">al options.

Reviewer #2: This study on the application of UBM to an class="Disease">primary congenital glaucoma is an interesting one. This work has several key messages that would contribute significantly to the field. My main comment is regarding the clarity of the methods and the unfortunate dilution of some of the key messages of the paper. I have some other comments for the authors to address about their work.

Introduction:

Line 52-53: The introduction clearly outlines a need to understand the mechanism of PCG. To be clear though, the use of UBM as an instrument would provide insight into the ocular structures involved in an class="Disease">primary congenital glaucoma, though the mechne">anism by which an class="Disease">primary congenital glaucoma occurs is something that would probably remain elusive, e.g. a genetic cause. It may be useful for the authors to clarify this.

Line 61-62: It would be useful to be more critical in the description of UBM use. In adult an class="Disease">glaucoma, it is more commonly used to verify the presence of an class="Disease">plateau iris syndrome for example rather than routine use, and importantly it contrasts with the greater utility of anterior segment OCT in general practice. Part of the issue here is the statement regarding "precise measurements", which is simply not comparable to high resolution instruments such as AS OCT (e.g. Liebmann & Ritch 1996). Although it is stated later one, it is worthwhile clarifying that there is an improvement in axial resolution compared to 50 MHz UBM.

Lines 66-67: there is a disconnect between this sentence and the above paragraphs as the authors make the leap of logic from understanding the mechanism of PCG to "pathogenesis and management".

Methods:

- Line 82: "corresponding"

- Lines 98-99: it is not clear if the ultrasound recordings were in video form or in image frame form when it is expressed in "20 ultrasound recordings". It is worthwhile clarifying here.

- Lines 108-111: Could the authors comment on the issue identified by Tandon et al 2017 J an class="Gene">AAPOS and Yan et al 2016 PLOS who found that 50% of the time Schlemm's canal could not be identified in PCG?

- The methods in lines 114 onwards are a little bit confusing an lack sufficient detail. For example, the cross-sectional area "taken at four different positions" is not clear. Is it four per quadrant or four in total? I'm not sure how the "largest of which was used for analysis to account for any variability" would reduce variability and not in fact introduce a systematic bias? The trabecular-iris angle is an interesting choice of parameter. What happens when there are irregularities in the anterior iris surface, for example, the presence of ridges or crypts? It would be more informative to state at which distance, similar to the way that an class="Gene">AOD is measured for example, the TIan class="Gene">A was taken. Corneal limbus thickness is poorly defined: is the the shortest distance or the perpendicular to the limbus tangent? Line 131 "average values" -- of what?

- Statistical analysis: intraobserver variance was only measured in 18 eyes... at this point of the manuscript, it is not clear how significant this number is relative to the proportion of the sample size. It is more informative to state, e.g. 20% of the eyes were randomly selected for re-evaluation. I note that this was only for a single observer - was this just for the measurements and not the delineation? What if there were issues with landmarks? The fidelity of the measurements is highly dependent upon accurate delineation of landmarks and so that is also important to assess for inter- and intraobserver variability.

Results:

- Table 1: how come total is out 208 for Group 1 when there are 51 eyes? Should it not be 204? an class="Gene">Also, would it not be more informative to have proportion in terms of "number of eyes with the largest CSA of SC in each quadrants" as well? Otherwise, it is currently confusing and without context.

- Line 186: this is a very key and interesting finding but it is lost amidst this paragraph and the use of aban class="Chemical">breviations. My suggestion is to rename "group 1" and "group 2" as they are currently meaningless and consider instead "an class="Disease">Bilateral disease" and "Unilateral disease" instead.

- Given the large number of parameters being examined in this study, I would suggest a Table in the methods to list out the relevant parameters as well.

- It might be worthwhile combining Figure 3 and Table 4 which state the same thing, with the regression equations being put in as insets.

- The relationship between IOP and other parameters was sparingly mentioned in the Results. It may be worthwhile showing these figures so that the reader can contrast these results with the work of Yan et al 2016 PLOS

Discussion/conclusions:

- Lines 238-24an class="Gene">2 should really be put in the introduction to highlight the importance of UBM.

- Lines 243-246: I'm not sure if this claim is fully supported. There are other papers in the literature that report on quantitative assessment of the anterior chamber structures in PCG (e.g. Gupta et al 2007 J an class="Gene">Aan class="Gene">APOS, Hussein et al 2014 Clin Ophthalmol)

- Lines 250 onwards: what is an interesting question here is whether other meaningful parameters can be assessed in an class="Species">patients in whom SC cne">annot be visualised. This is worth discussing and even reporting if the data is available. Given thne">at such a large proportion of an class="Species">patients fit into this criteria of SC non-visibility, it would be highly informative and contributory to the literature.

- The discussion is generally very long and perhaps unnecessarily so given the length of the results. I refer specifically to the paragraphs between lines 287-324.

- What I feel is a very interesting result in the unilateral versus bilateral comparison group was not really discussed.

- Conclusions (line 352) the idea of age needs to be mentioned throughout the manuscript if this claim is to be made. Right now, it is relatively sparse.

Miscellaneous comments:

- The overall writing is generally clear. There are minor grammatical an class="Disease">errors that should be carefully reviewed.

- Data availability: I don't see where the data is/will be made available at this stage of the review process.

**********

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight an class="Gene">Analysis and Conversion Engine (an class="Chemical">PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 an class="Chemical">Oct 2019

The study investigates features of an class="Disease">primary congenital glaucoma (PGC) using ultrasound biomicroscopy.

1. The small sample size for unaffected eyes (controls) may impact significance of the findings. Please justify.

Thank you for the comment. We realize our sample size is small due to the low incidence of an class="Disease">primary congenital glaucoma. We have included a detailed statistical power calculation as it relates to our sample size in our response to Reviewer 1 below.

2. The controls were all contralateral eyes of unilateral cases of PGC. One study recently suggested that the normal eyes in unilateral PGC may not be anatomically normal (Bayoumi 2017) - can the authors please comment, and also indicate if they have compared their findings to data for an class="Species">patients with both eyes unaffected.

We agree completely that unaffected eyes of an class="Species">children with unilateral PCG are not totally normal. In future studies, we plan to address the differences of anterior chne">amber angle structures between unaffected eyes of PCG an class="Species">patients and normal age-matched eyes. Given the small number of unaffected eyes in patients with unilateral PCG in this study, we have further discussed this in the limitations portion of our manuscript. (Lines 359 to 362)

3. The issue of unilateral versus bilateral PGC is interesting and should be commented upon further. What is the prevalence of each condition in China and across other countries?  Is there any data on the underlying causes of PGC for the study an class="Species">participants? Have genetic mutations for example, been investigated?

The current study reports an incidence of bilateral PCG of 72%, in line with prior published reports: 60%-99.3%. We included this information in our revised manuscript. (Lines 338 to 347) While outside the scope of this paper, we are currently researching the genetic background of unilateral and bilateral PCG.

4. The methods used to assess the anterior chamber angle characteristics are unclear. Please revise.

We have revised them according to the reviewers’ comments.

5. The age-related effects for PGC eyes are presented in Figure 3, but there is limited discussion as to what these graphs mean. How does this compare to normal unaffected eyes (noting very small sample size) (both contralateral eyes, and also both eyes unaffected).

We had discussed the age-related effects for PCG eyes (Line 290, Line 295, Lines 305 to 308, Lines 324 to 329, Lines 36an class="Gene">2 to 365). Unfortunately, there were so limited previously published data about this issue. Moreover, the cross-sectional nature of our study did not allow us to comprehensively elucidate age-related chne">anges in the development of anterior chne">amber angle in PCG eyes, and longitudinal studies are warranted to further explore this relationship. We plan to evaluate this in future studies, and even to compare our findings to data for an class="Species">patients with both normal eyes, as the unaffected eyes in unilateral PCG may not be anatomically normal as discussed above (Bayoumi 2017).

Reviewers' comments:

Reviewer #1: Please see comments to be addressed below. In addition to the below comments the only other concern with regard to statistical analysis is the relative small sample size of control "normal" eyes compared to affected eyes. If possible, someone more specialised in statistical analysis might be useful to check the validity of the analysis.

Thank you for your advice. We had a statistician check the validity of the analysis and have included additional details below.

Line 45: Last sentence in conclusion should also state “and changes with age”

This has been corrected. (Line 44)

Line 48: The sentence “an class="Disease">Primary congenital glaucoma is the leading cne">ause of an class="Disease">blindness worldwide” is incorrect. PCG only accounts for 3-5% of blindness in children worldwide. A greater number are affected in developing countries. It is the leading cause of blindness in Saudi Arabia and the Rom population of Slovakia. It is however a very rare condition that is difficult to treat and detect and can often be missed which is why this study is interesting.

This has been corrected. (Line 47)

Line 84: Sclera spur should be changed to scleral spur

This has been corrected. (Line 85)

Line 91: For imaging of the anterior chamber angle were the scans acquired by a technician and then interpreted by the two investigators? an class="Gene">And if so, was it the same an class="Species">person that did all the scans?

an class="Gene">All the scans were acquired by the same investigator (YS) and then interpreted by two investigators (YS and CX). We have added this information to the manuscript. (Line 94)

Line 161: 51 an class="Species">patients with 75 eyes were underwent the UBM measurement (remove were)

We have corrected this sentence. (Line 167)

For statistics:

1. Comparing 51 affected eyes to 11 “normal/control” eyes not sure how reliable it is to do hypothesis testing here as there is a small comparative sample size

Literature on imbalanced designs indicates that a 1:1 ratio may not be optimal in terms of statistical precision or costs.1,an class="Gene">2 This is especially true for situations where the exposure is rare and for stronger relationships between the exposure and the outcome under study.3 Since the PCG is commonly bilateral, the unequal sample size of affected eyes and unaffected control eyes is inevitable. Generally, the formula for cne">alculating sample size of an unmatched cne">ase–control study is

n_1=(〖〖(Z〗_(1-α/2)+Z_(1-β))〗^2×(σ_1^2+σ_2^2)×(1+1/K))/δ^2

n_1: sample size for group 1. : sample size for group 2.

K: the ratio of cases in two groups

σ_1: the standard deviation of group 1

σ_2: the standard deviation of group 1

δ: the mean difference between groups

The main outcome of this study is the largest an class="Gene">CSA of Schlemm’s cne">anal, which were 3363.91±1082.98 μman class="Gene">2 in group 1 and 5130.66±1231.90 μm2 in group 2. The ratio of affected/unaffected eyes was 4.6. Using a two-sided test with significance of 0.05 and a power of 80%, the minimum required sample size of affected eye was 16.5 and 3.6 unaffected eyes. Since we included both eyes of patients with bilateral disease, we tripled the minimum sample size in each group, comparing 51 affected eyes to 11 unaffected eyes.

1. Nam JM. Optian class="Gene">mum sample sizes for the comparison of the control and treatment. Biometrics. 1973;29(1):101-8.

2. Liu X. Statistical power and optian class="Gene">mum sample allocation ratio for treatment and control having unequal costs per unit of randomizne">ation. J Edu Behav Stats. 2003;28(3):231-48.

3. Groenwold R an class="Chemical">H H , Smeden M V . Efficient Sampling in Unmatched Case-Control Studies When the Total Number of Cases and Controls Is Fixed.[J]. Epidemiology, 2017, 28(6):834.

For results:

1. Table 1: Group 1 column, under “observable SC proportion”

a. Total (n, %). 152/208 (*where does the value of 208 come from? If the SC is observed in 4 quadrants per eye should it not be 204?)

b. For each of the remaining regions (superior, nasal, inferior and temporal) should the fraction be /51 instead of 52?

Thank you for finding these an class="Disease">errors – we have corrected them.

Line 319: should read “which was consistent with the results in other studies”

This has been corrected. (Line 321)

Line 334: In addition of the study limitations mentioned I wonder if the following two points should also be included:

1. Small comparison sample size. Only 11 contralateral unaffected eyes compared to 51 affected eyes, this does make comparison between groups difficult

The issue of sample size was addressed above. The small number of an class="Species">patients with unilateral PCG an class="Species">patients created the disparity between the number of affected and unaffected eyes. We added this to our discussion as well. (Lines 344 to 347)

2. Is the supposedly unaffected eye in unilateral disease actually normal? PCG is a an class="Disease">genetic disorder and it is likely that the “unaffected” eye is not truly normal.

an class="Gene">As above, we have added this to the study limitations. (Lines 359 to 362)

It would be interesting to know if the eyes that were excluded with complete absence of SC had angle surgery or went straight to either a trabeculectomy or an class="Disease">glaucoma drainage device? Presumably absence of SC would mean thne">at angle surgery including trabeculotomy would be contraindicne">ated. In your conclusion an additional point to be mentioned would be thne">at UBM could be used to guide surgicne">al options.

We do currently use UBM to guide surgical options, especially for consideration of microcatheter-assisted trabeculotomy (Man class="Gene">AT). We have previously reported on the correlation of goniodysgenesis as evaluated by UBM and outcomes of Man class="Gene">AT in eyes with PCG.1

an class="Gene">All PCG an class="Species">patients in this study underwent the MAT. However, in 9 patients with complete absence of SC on UBM, we can still catheterize Schlemm’s canal 360-degree (2 eyes) or partially (3 eyes). Schlemm’s canal could not be identified in 4 eyes during the surgery, and then they underwent the traditional trabeculotomy using Harm’s trabeculotome. Predicting the degree of successful catheterization based on UBM SC visualization can be challenging. We only tested four positions of the SC using UBM, while a lack of SC visibility in these four positions may result from segmental outflow of SC, as detected in healthy eyes2-4 or may be affected by the IOP, as found in primary open-angle glaucoma.5 So diminished visualization of SC under UBM may suggest a decreased possibility of full 360° catheterization, but further studies were warranted. We mention the role of UBM in surgery in our discussion. (Lines 265 to 268)

1. Shi Y, Wang HZ, Han Y, Cao K, Vu V, Hu M, et al. Correlation between trabeculodysgenesis assessed by ultrasound biomicroscopy and surgical outcomes in an class="Disease">primary congenital glaucoma. an class="Gene">Am J Ophthalmol. 2018;196:57-64.

2. Keller KE, an class="Chemical">Bradley JM, Vranka Jan class="Gene">A, Acott TS. Segmental versican expression in the trabecular meshwork and involvement in outflow facility. Invest Ophthalmol Vis Sci. 2011 Jul 7;52(8):5049-57.

3. Yang CY, Liu Y, an class="Chemical">Lu Z, Ren R, Gong H. Effects of Y27632 on aqueous humor outflow facility with changes in hydrodynamic pattern and morphology in human eyes. Invest Ophthalmol Vis Sci. 2013 Aug 28;54(8):5859-70.

4. Hann CR, an class="Disease">Fautsch MP. Preferential fluid flow in the human trabecular meshwork near collector channels. Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1692-7.

5. Tandon an class="Gene">A, Watson C, an class="Gene">Ayyala R. Ultrasound biomicroscopy measurement of Schlemm's canal in pediatric patients with and without glaucoma. J AAPOS. 2017;21(3):234-237.

Reviewer #2: This study on the application of UBM to an class="Disease">primary congenital glaucoma is an interesting one. This work has several key messages that would contribute significantly to the field. My main comment is regarding the clarity of the methods and the unfortunate dilution of some of the key messages of the paper. I have some other comments for the authors to address about their work.

Introduction:

Line 52-53: The introduction clearly outlines a need to understand the mechanism of PCG. To be clear though, the use of UBM as an instrument would provide insight into the ocular structures involved in an class="Disease">primary congenital glaucoma, though the mechne">anism by which an class="Disease">primary congenital glaucoma occurs is something that would probably remain elusive, e.g. a genetic cause. It may be useful for the authors to clarify this.

Thank you for this insightful comment. We have updated our language to reflect this important point. (Lines 50 to 52)

Line 61-62: It would be useful to be more critical in the description of UBM use. In adult an class="Disease">glaucoma, it is more commonly used to verify the presence of an class="Disease">plateau iris syndrome for example rather than routine use, and importantly it contrasts with the greater utility of anterior segment OCT in general practice. Part of the issue here is the statement regarding "precise measurements", which is simply not comparable to high resolution instruments such as AS OCT (e.g. Liebmann & Ritch 1996). Although it is stated later one, it is worthwhile clarifying that there is an improvement in axial resolution compared to 50 MHz UBM.

Thank you for your reminder. We have updated our manuscript to include these points. (Lines 57 to 58, Lines 6an class="Gene">2 to 63, 65)

Lines 66-67: there is a disconnect between this sentence and the above paragraphs as the authors make the leap of logic from understanding the mechanism of PCG to "pathogenesis and management".

We have revised this. (Line 68)

Methods:

- Line 82: "corresponding"

We have corrected it. (Line 83)

- Lines 98-99: it is not clear if the ultrasound recordings were in video form or in image frame form when it is expressed in "20 ultrasound recordings". It is worthwhile clarifying here.

These were images – we have clarified this. (Line 100)

- Lines 108-111: Could the authors comment on the issue identified by Tandon et al 2017 J an class="Gene">AAPOS and Yan et al 2016 PLOS who found that 50% of the time Schlemm's canal could not be identified in PCG?

We address identification of Schlemm’s canal in the discussion section. (Lines 254 to 268)

- The methods in lines 114 onwards are a little bit confusing an lack sufficient detail. For example, the cross-sectional area "taken at four different positions" is not clear. Is it four per quadrant or four in total?

We have updated the methods to provide additional clarity. (Lines 118 to 119)

I'm not sure how the "largest of which was used for analysis to account for any variability" would reduce variability and not in fact introduce a systematic bias?

We have included a discussion of variation of the cross-sectional area of Schlemm’s canal in the discussion section. We agree with you that the largest cross-sectional area may not be representative of the anatomy of the whole eye, but it did serve as a standardized measurement for the purposes of our study. (Lines 353 to 359)

The trabecular-iris angle is an interesting choice of parameter. What happens when there are irregularities in the anterior iris surface, for example, the presence of ridges or crypts?

Loss of normal iris configuration has been reported in PCG eyes1, though we did not observe any ridges or crypts of the iris surface in our cohort of PCG eyes. We have added a comment to address this in the discussion section. (Lines 32an class="Gene">2 to 324)

1. Hussain T, Shalaby S, Elbakary Man class="Gene">A, Elseht R, Gad R. Ultrasound biomicroscopy as a diagnostic tool in infants with primary congenital glaucoma. Clin Ophthalmol. 2014:1725-1730.

It would be more informative to state at which distance, similar to the way that an class="Gene">AOD is measured for example, the TIan class="Gene">A was taken.

This was measured 500 μm from the scleral spur. We have updated the methods with a detailed description. (Lines 128 to 130)

Corneal limbus thickness is poorly defined: is the shortest distance or the perpendicular to the limbus tangent?

This was measured perpendicular to the limbus tangent. Methods have been updated to reflect this. (Lines 133 to 134)

Line 131 "average values" -- of what?

This has been updated. (Line 136)

- Statistical analysis: intraobserver variance was only measured in 18 eyes... at this point of the manuscript, it is not clear how significant this number is relative to the proportion of the sample size. It is more informative to state, e.g. 20% of the eyes were randomly selected for re-evaluation. I note that this was only for a single observer - was this just for the measurements and not the delineation? What if there were issues with landmarks? The fidelity of the measurements is highly dependent upon accurate delineation of landmarks and so that is also important to assess for inter- and intraobserver variability.

We have re-evaluated the intraobserver and interobserver variances using a randomly selected 50% of the eyes. an class="Gene">As the accurate delineation of landmarks was quite important for our measurement, the landmarks (SC and SS) were delineated by two observers together to ensure a similar understanding of the anatomicne">al landmarks. When the observers disagreed on the delineation of landmarks, a mutual conclusion was reached after discussion. For this reason, we didn’t analyze the interobserver variability, since the landmarks was agreed up by the two observers. Our result showed a good interobserver and intraobserver reproducibility. (Table 2, Lines 160 to 164, Lines 351)

Results:

- Table 1: how come total is out 208 for Group 1 when there are 51 eyes? Should it not be 204? an class="Gene">Also, would it not be more informative to have proportion in terms of "number of eyes with the largest CSA of SC in each quadrants" as well? Otherwise, it is currently confusing and without context.

We have updated this table to incorporate these suggestions. (Table 1)

- Line 186: this is a very key and interesting finding but it is lost amidst this paragraph and the use of aban class="Chemical">breviations. My suggestion is to rename "group 1" and "group 2" as they are currently meaningless and consider instead "an class="Disease">Bilateral disease" and "Unilateral disease" instead.

Thank you for this excellent suggestion – we have made the change to emphasize the importance of this finding. We have also updated our abstract to reflect this distinction. (Line 28, Lines 34 to 50, Line 192, Line 197)

- Given the large number of parameters being examined in this study, I would suggest a Table in the methods to list out the relevant parameters as well.

Thank you for your suggestion. We agree that the large number of parameters is difficult to convey. Our hope is that our changes to Table an class="Gene">2 and 3 clarify this.

- It might be worthwhile combining Figure 3 and Table 4 which state the same thing, with the regression equations being put in as insets.

Thank you for your suggestion. We have deleted Table 4 and put the regression equations in Figure 4.

- The relationship between IOP and other parameters was sparingly mentioned in the Results. It may be worthwhile showing these figures so that the reader can contrast these results with the work of Yan et al 2016 PLOS

We have added this to Figure 3.

Discussion/conclusions:

- Lines 238-24an class="Gene">2 should really be put in the introduction to highlight the importance of UBM.

We have highlighted this point in the introduction. (Lines 57 to 58)

- Lines 243-246: I'm not sure if this claim is fully supported. There are other papers in the literature that report on quantitative assessment of the anterior chamber structures in PCG (e.g. Gupta et al 2007 J an class="Gene">Aan class="Gene">APOS, Hussein et al 2014 Clin Ophthalmol)

Our study is the first to provide an in vivo quantitative assessment of the TM and SC, and explore their relationship to age in an class="Species">patients with PCG. We have revised this sentence to reflect that. (Lines 251 to 253)

- Lines 250 onwards: what is an interesting question here is whether other meaningful parameters can be assessed in an class="Species">patients in whom SC cne">annot be visualised. This is worth discussing and even reporting if the data is available. Given thne">at such a large proportion of an class="Species">patients fit into this criteria of SC non-visibility, it would be highly informative and contributory to the literature.

Thank you for your suggestion. Our main outcomes were to measure SC and TM. When SC cannot be visualized, then SC and TM cannot be measured, which was why we excluded these cases.

- The discussion is generally very long and perhaps unnecessarily so given the length of the results. I refer specifically to the paragraphs between lines 287-324.

We have condensed this section to make it more concise.

- What I feel is a very interesting result in the unilateral versus bilateral comparison group was not really discussed.

We have updated our discussion to address this. (Lines 338 to 347)

- Conclusions (line 352) the idea of age needs to be mentioned throughout the manuscript if this claim is to be made. Right now, it is relatively sparse.

Thank you for this feedback. The results section “an class="Gene">Anterior Chne">amber an class="Gene">Angle Parameters and Age in PCG Eyes” addresses this. (Line 226) Additionally, we have updated our discussion to include additional mention of this. (Line 290, Line 295, Lines 305 to 308, Lines 324 to 329, Lines 362 to 365)

Miscellaneous comments:

- The overall writing is generally clear. There are minor grammatical an class="Disease">errors that should be carefully reviewed.

Thank you for this feedback. We had our native English-speaking author (Julius Oatts) revised the manuscript.

- Data availability: I don't see where the data is/will be made available at this stage of the review process.

We updated the data and submitted it as the supporting information according to the PLOS Data policy during the process of submitting our revised manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Dec 2019

an class="Chemical">PONE-D-19-17802R1

Disease-related and age-related changes of anterior chamber angle structures in an class="Species">patients with an class="Disease">primary congenital glaucoma: An in vivo high-frequency ultrasound biomicroscopy-based study

PLOS ONE

an class="Gene">Dear M.D., Ph.D. Wne">ang,

Thank you for submitting your manuscript to PLOS ONE. an class="Gene">After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Thank you for the detailed revision of the submitted manuscript.

Please note the following minor comments (and the Reviewer an class="Gene">2 comments below) and please address all points:

1. Please confirm that the an class="Disease">iris thickness measurements were taken perpendicular to the posterior iris plane  and include this information in the Methods of the manuscript.

2. Please indicate if trabecular iris san class="Chemical">pace area (TISan class="Gene">A) measurements (mm2) were included during the study - these are usually reported for UBM studies (either at 500um or 700um from the scleral spur). If the TISA measurements were taken, please include in the manuscript and discuss the outcomes.

3. Line 54: please amend to 'physiological.

4. Line 95: 'For those with unilateral disease, the unaffected contralateral eyes served as the control group (Group 2).'

5. Line 175: 'Interobserver agreement was calculated by comparing initial values of Observer 1 (YS)

to those of Observer 2 (CX).'

6. Line 382: 'an class="Gene">Analysis of the intraobserver and interobserver reproducibility was high.'

7. Line 390: 'Fourthly,...'

=============================

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an class="Gene">Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the an class="Gene">Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "an class="Gene">Accept" recommendation.

Reviewer #2: (No Response)

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. an class="Gene">Any typographicne">al or grammaticne">al errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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Please use the san class="Chemical">pace provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I thank the authors for their comprehensive response. I have a few minor comments below.

Line 127: I presume that the an class="Disease">iris thickness was measured perpendicularly to the retroiridal iris plane?

My point about trabecular-iris angle was related more to why trabecular-iris san class="Chemical">pace area was not used to account for iris surface anatomy. TISA is commonly used and I wonder why it has been omitted here.

Line 359: spelling an class="Disease">error

With regard to intraocular pressure, how do the authors explain the big difference in pressure between Groups 1 and an class="Gene">2 even though there was no relationship found cross sectional area of Schlemm's canal (Figure 3)? Could it be related to the degree to which the canals were closed/narrow?

**********

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Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight an class="Gene">Analysis and Conversion Engine (an class="Chemical">PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Dec 2019

1. Please confirm that the an class="Disease">iris thickness measurements were taken perpendicular to the posterior iris plane and include this information in the Methods of the manuscript.

The an class="Disease">iris thickness measurements were taken perpendicular to the posterior iris plane, we have included this information in the Methods of the manuscript. (Lines 127-128)

2. Please indicate if trabecular iris san class="Chemical">pace area (TISan class="Gene">A) measurements (mm2) were included during the study - these are usually reported for UBM studies (either at 500um or 700um from the scleral spur). If the TISA measurements were taken, please include in the manuscript and discuss the outcomes.

In an class="Species">patients with PCG, the dysgenesis of trabecular meshwork mainly affects the angle recess, which makes angle measurement involving the labeling of scleral spur difficult. So TISan class="Gene">A measurement may not be accurate. On the other hand, TIA better illustrates the dysgenesis of trabecular meshwork and has been broadly used for patients with PCG. (Kobayashi H, Ono H, Kiryu J, Kobayashi K, Kondo T. Ultrasound biomicroscopic measurement of development of anterior chamber angle. Br J Ophthalmol. 1999;83(5):559-562)

3. Line 54: please amend to 'physiological.

This has been corrected. (Line 50)

4. Line 95: 'For those with unilateral disease, the unaffected contralateral eyes served as the control group (Group 2).'

We have corrected this sentence. (Line 89-90)

5. Line 175: 'Interobserver agreement was calculated by comparing initial values of Observer 1 (YS)

to those of Observer 2 (CX).'

We have corrected this sentence. (Lines 163-164)

5. Line 382: 'an class="Gene">Analysis of the intraobserver and interobserver reproducibility was high.'

We have corrected this sentence. (Line 355)

7. Line 390: 'Fourthly,...'

This has been corrected. (Line 363)

Reviewers' comments:

Reviewer #2: I thank the authors for their comprehensive response. I have a few minor comments below.

Line 127: I presume that the an class="Disease">iris thickness was measured perpendicularly to the retroiridal iris plane?

The an class="Disease">iris thickness measurements were taken perpendicular to the posterior iris plane, we have included this information in the Methods of the manuscript. (Lines 127-128)

My point about trabecular-iris angle was related more to why trabecular-iris san class="Chemical">pace area was not used to account for iris surface anatomy. TISA is commonly used and I wonder why it has been omitted here.

See above.

Line 359: spelling an class="Disease">error

This has been corrected. (Line 363)

With regard to intraocular pressure, how do the authors explain the big difference in pressure between Groups 1 and an class="Gene">2 even though there was no relationship found cross sectional area of Schlemm's canal (Figure 3)? Could it be related to the degree to which the canals were closed/narrow?

We agree with the reviewer that the IOP may affect the cross-sectional area of Schlemm’s canal (SC) as previous study revealed in POan class="Gene">AG (Mu L, Yin Z, Yan X, Hong Z. The Relationship between the 24-hour Fluctuations in Schlemm’s Canal and Intraocular Pressure: an class="Gene">An Observational Study using High-Frequency Ultrasound Biomicroscopy. Curr Eye Res. 2017;42(10):1389-1395.) But in this study, IOP was not related with the size of the largest-measured SC area in pediatric patients with PCG. One explanation is that patients with PCG have maldeveloped SC, which could be segmental. The degree of the maldeveloped SC may lead to elevated IOP, which we don’t have enough data in this study to prove this. We only performed UBM at 4 o’clock, not 360 degree. The future development of circumference SC image would help solve this question. We have discussed it in the Discussion section. (Lines 296 to 303)

Submitted filename: Response to reviewers.docx

Click here for additional data file.

26 Dec 2019

Disease-related and age-related changes of anterior chamber angle structures in an class="Species">patients with an class="Disease">primary congenital glaucoma: An in vivo high-frequency ultrasound biomicroscopy-based study

an class="Chemical">PONE-D-19-17802R2

an class="Gene">Dear Dr. Wne">ang,

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Michele Madigan

an class="Gene">Academic Editor

PLOS ONE

an class="Gene">Additional Editor Comments (optional):

Reviewers' comments:

9 Jan 2020

an class="Chemical">PONE-D-19-17802R2

Disease-related and age-related changes of anterior chamber angle structures in an class="Species">patients with an class="Disease">primary congenital glaucoma: An in vivo high-frequency ultrasound biomicroscopy-based study

an class="Gene">Dear Dr. Wne">ang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until an class="Gene">2 pm Eastern Time on the date of publicne">ation. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

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on behalf of

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PLOS ONE