Analysis of axial length, retinal nerve fiber layer, macular area and estimated retinal ganglion cell count in myopic preperimetric glaucomatous eyes: an observational study

BACKGROUND: The aim of the study is to evaluate the diagnostic ability of OCT parameters and retinal ganglion cells (RGCs) count in identify glaucomatous disease in myopic preperimetric eyes. METHODS: This was a cross-sectional observational study. The study group consisted of 154 eyes: 36 healthy, 64 preperimetric (PPG), and 54 primary openangle glaucoma (POAG) eyes. Each group was divided into three subgroups based on axial length: emmetropic, myopic with axial length (AL) <25 mm, and myopic with AL>25 mm, to analyze the effect of myopia. The RGCs count was obtained using a model described later. As regard the influence of myopia on OCT parameters and RGC count, we performed Pearson’s correlation. The Area Under Receiver Operator Characteristics Curves (AUROC curves) evaluated which parameter had the best sensitivity and specificity in identifying glaucoma in myopic eyes. RESULTS: In Pearson’s test, all Ganglion Cell Complex (GCC) thicknesses showed the weakest and less significant correlation with AL in all groups. All the AUROCs were statistically significant, and above 0.5. Inferior GCC and Global Loss Volume (GLV) showed the highest AUCs in all myopic group and the best diagnostic ability in distinguishing healthy from glaucomatous eyes. RGCcount showed good AUROC in all groups, with sensitivities of about 83% in myopic eyes, and specificity over 91% in all groups. CONCLUSIONS: GCC is the parameter less influenced by the AL, and the inferior GCC and the GLV have the best diagnostic performance. The RGCcount has good sensitivity and specificity, so it can be used as a complementary test in the diagnosis of glaucoma in myopic preperimetric eyes. and Standard Automated Perimery performed with Swedish Interactive Threshold Algorithm (SITA) Field Analyzer Fixation losses less than or equal to 20%, false positives and false negatives less than or equal to 33% were established as the reliability criteria. The study included three groups of subjects: preperimetric glaucomatous (PPG), glaucomatous, and a control group recruited from the healthy population. The latter were required to have no family history of glaucoma, highest daily IOP less than 21 mm Hg, a normal visual field (VF) test and a normal optic nerve head (ONH) appearance. Patients affected by PPG had the highest daily IOP greater than 21 mm Hg, normal VF test, and ONH changes (cup–disc ratio alteration, disc hemorrhages, rim notching, diffused or localized RNFL defects). Primary open-angle glaucoma (POAG) was diagnosed based on the presence of highest daily IOP greater than 21 mm Hg, abnormal VF according to Hodapp-Parrish-Anderson criteria for diagnosing glaucomatous damage (14), and glaucomatous optic disc changes. Optic nerve head appearance was evaluated by slit-lamp biomicroscopy of the posterior segment using a 78-D lens.

CONCLUSIONS: GCC is the parameter less influenced by the AL, and the inferior GCC and the GLV have the best diagnostic performance. The RGCcount has good sensitivity and specificity, so it can be used as a complementary test in the diagnosis of glaucoma in myopic preperimetric eyes.

Background
Myopia and glaucoma are two of the commonest causes of impaired vision in the world population.
Myopia is projected to affect 5 billion people in the world by 2050 (1), and glaucoma is a leading cause of blindness and irreversible visual impairment, affecting more than 100 million people worldwide (2,3) . Myopic eyes have an increased risk of glaucoma (4). The link between myopia and glaucoma seems to be the greater deformability of the lamina cribrosa in myopic eyes. Myopic changes include longer axial lengths and vitreous chamber depths together with alterations in connective tissue which might contribute to a higher susceptibility to glaucomatous optic disc changes (5).
With the ophthalmoscopic evaluation alone, it may be hard to tell apart myopia from glaucoma for a combination of reasons (6). The use of structural imaging or visual field testing in glaucoma is fraught with multiple potential pitfalls in myopia, such as the presence of posterior staphylomas or macular atrophy. Thus, myopic eyes may be falsely overdiagnosed with glaucoma if care is not taken to distinguish between glaucomatous and myopic pathology (7).
The aim of this cross-sectional observational study is to test whether Retinal Nerve Fiber Layer (RNFL) and Ganglionar Cell Complex (GCC) thicknesses are thinner in myopic PPG than emmetropic PPG and myopic healthy eyes, and whether this thickness reduction corresponds to a decrease in the estimated retinal ganglion cells (RGCs) number, so the RGCs count could be useful to identify early glaucomatous disease in myopic eyes. Another purpose of the study was to evaluate and compare the diagnostic ability of OCT parameters and RGC count in emmetropes and myopes.

Methods
This cross-sectional observational study was conducted at the Glaucoma Center of the Eye Clinic, Department of Surgical Sciences, University of Torino, Italy. The methods were applied in accordance to the tenets of the Declaration of Helsinki; informed consent was obtained from all subjects and the Ethics Committee (University & General Hospital San Giovanni Battista of Torino) gave approval.
To be included in the study, participants had to meet the following criteria: age between 18 and 80 years, best-corrected visual acuity (BCVA) greater than or equal to 20/30, spherical equivalent between +1.00D and -1.00 D for emmetropic subjects, and between -3.00D and -7.00D for myopic subjects, and open angle on gonioscopy. Subjects with ocular surgery, retinal or macular pathology, or systemic or neurologic conditions that could produce visual field defects, were excluded.
All subjects underwent a comprehensive ophthalmic examination consisting of: BCVA, ultrasound pachymetry, slit-lamp biomicroscopy of anterior and posterior segment, Goldmann applanation tonometry (GAT), diurnal tonometric curve, gonioscopy, peripapillary, and macular imaging using Fourier Domain-OCT (FD-OCT RTVue-100 software version A4, 5, 0, 59; Optovue, Fremont, CA, USA), and measurement of axial lenght with low-coherence interferometry system (Aladdin biometer, Topcon). Standard Automated Perimery was performed with Swedish Interactive Threshold Algorithm (SITA) Standard strategy, program 24-2 of the Humphrey Field Analyzer (HFA; Carl Zeiss Meditec, Jena, Germany). Fixation losses less than or equal to 20%, false positives and false negatives less than or equal to 33% were established as the reliability criteria. The study included three groups of subjects: preperimetric glaucomatous (PPG), glaucomatous, and a control group recruited from the healthy population. The latter were required to have no family history of glaucoma, highest daily IOP less than 21 mm Hg, a normal visual field (VF) test and a normal optic nerve head (ONH) appearance.
Patients affected by PPG had the highest daily IOP greater than 21 mm Hg, normal VF test, and ONH changes (cup-disc ratio alteration, disc hemorrhages, rim notching, diffused or localized RNFL defects). Primary open-angle glaucoma (POAG) was diagnosed based on the presence of highest daily IOP greater than 21 mm Hg, abnormal VF according to Hodapp-Parrish-Anderson criteria for diagnosing glaucomatous damage (14), and glaucomatous optic disc changes. Optic nerve head appearance was evaluated by slit-lamp biomicroscopy of the posterior segment using a 78-D lens.
Each group was further divided into three subgroups based on axial length: emmetropic, myopic with AL<25 mm and myopic with AL>25 mm, in order to analyze the effect of mild and moderate myopia.

FD-OCT RTVue-100
The Glaucoma Protocol of FD-OCT RTVue-100 was used to acquire RNFL thickness measurements. The same operator repeated all scans three times. The RNFL thicknesses used for the analysis derived from the scan with the highest signal strength index (SSI). This scan was used to calculate RNFL thicknesses. Scans with motion artifacts and with signal strength index less than 45 were excluded.

Estimate of Retinal and Macular Ganglion Cell Count
The estimated of RGC count was obtained by applying the model developed and described in detail by Medeiros et al (9)(10)(11)15) based on the empirical formulas processed by Harwerth et al (8). We described this formula in detail in our previous article (Rolle et al.). (16)

Statistical analysis
The collection, processing and statistical analysis of the results were carried out through the Microsoft Excel 2016 worksheets and the SPSS statistical program for Windows, version 19.0, SPSS Inc, Chicago, IL. We used the analysis of variance (ANOVA) and χ2 test to assess the comparability of the groups for continuous and dichotomic variables respectively.
For each of the three groups (healthy, PPG and POAG) the Pearson's linear correlation coefficient was calculated to evaluate how the measured parameters are influenced by the AL increasing. Then, each group was further subdivided into 3 subgroups (emmetropes, myopic eyes with AL<25 mm and myopic eyes with AL>25 mm) to analyze the effects of mild and moderate axial myopia by the Mann-Whitney U test. (Figure 1) To investigate the ability of OCT parameters and RGCcount to diagnose glaucoma, we calculated areas under the receiver operating characteristic (AUROC). We compare these AUROCs between subgroups with different axial length (emmetropes, all myopic eyes, and myopes with AL<25mm and >25mm) using the method described by DeLong et al (17). The same method is used to compare the best OCT parameters and RGCs count with all the other parameters. For the calculation of AUROC and for all the comparisons between AUROCs we used a statistical software package (MedCalc v. For all statistical analysis, a p value <0.05 was considered statistically significant.

Results
The study group consisted of 154 eyes: 36 healthy, 64 PPG, and 54 POAG eyes. Demographic characteristics of the study population are illustrated in Table 1.
The ANOVA and χ 2 test showed that the groups are comparable for age, sex, and refraction.
Glaucomatous eyes have significantly worse VF MD and PSD than healthy and PPG eyes (P < 0.0001).
RNFLavg and RGC number reduce with the progression of glaucomatous damage, as demonstrated in previous studies (16,18).
As regard the influence of myopia on OCT parameters and RGC count, we performed the Pearson's correlation of axial length with RGC (Table 3). Almost all parameters have a moderate significant negative correlation with axial length, except GCC thicknesses in healthy group. RGC count in healthy group shows the strongest negative correlation with AL, and all GCC thicknesses the weakest and less significant in all groups.
To better evaluate the influence of myopia, we subdivided each group in three subgroups, basing on refractive error emmetropic eyes with defect between +1.00 sf and -1.00 sf, and myopic eyes with defect between -3.00 sf and -7.00 sf, and in these latter eyes axial length superior or inferior to 25 mm. This cut-off was chosen because it is two standard deviations from the reference average of the main normative databases (19).
In Table 4.a-b-c we resumed the results of Mann-Whitney U Test between PPG and healthy subgroups.
In comparisons between healthy subgroups with different axial length (Table 4.c) we can merely evaluate the influence of myopia on OCT parameters. In all comparisons the MD is not statistically significant, a sign that there is no influence of any myopic damage on the functional aspect. In all the subgroups we can observe a difference between emmetropes and myopes for both macular and papillary OCT parameters, a sign of an influence of myopia on the structural aspect.
In Table 5 we compared emmetropic PPG eyes and all myopic PPG eyes with no distinguishing about axial length, and the difference is statistically significant for all parameters.
The AUCs of MD, OCT parameters and RGCcount of emmetropes, myopes with AL<25mm, myopes with AL>25mm and all myopes subjects are summarized in Tables 6.a-d and Fig. 1-4; Tables 6.a-d also shows SE, IC95%, and sensitivity and specificity of every parameters.
All the AUROCs are statistically significant, and above 0.5. Inferior GCC and Global Loss Volume (GLV) show the highest AUCs in all myopic groups and the best diagnostic ability in distinguishing healthy from glaucomatous eyes. Both GCCinf and GLV have sensibility >95% in all groups, except GLV sensitivity in all myopes' group (92.59%). We compare AUROC of GCCinf and GLV with AUROCs of all others parameters (Table 7) using the method of DeLong (17) to test for statistical significance.
GCCinf and GLV show significant differences with MD in all comparison, with all RNFL parameters in high myopic group and in myopic group without considering AL, and only with RNFLsup if considering mild myopes group. This is probably due to the fact that the other OCT parameters also have very good AUCs and elevated sensibility and specificity. Table 8 shows the results of the comparisons of AUROCs of MD, OCT parameters and RGCcount between groups with different axial length. All comparisons between emmetropes and myopes eyes shows statistically significant difference for all GCC parameters and GLV.
RGCcount shows very good AUC curves in all groups, with sensitivities of about 73% in emmetropic eyes, and 83% in myopic eyes, and very high specificity (over 91% in all groups, 100% in emmetropes eyes). RGCcount has the ability to identify healthy with certainty, and with good safety those with glaucoma, more in emmetropes than in myopes (but the difference of RGCcount AUROCs between groups is not statistically significant).
RNFLavg and MD are used in the formula of RGCcount of Medeiros et al (9) to calculate the RGC number. We compared the diagnostic performance of both parameters with that of RGCcount (see Table 9) to assess whether the retinal ganglion cell count adds more diagnostic information than the respective parameters from which it is derived. RGCcount performs significantly better than MD in all groups, and almost the same than RNFLavg with no statistical significant differences.

Discussion
Patients with high myopia has a sixfold increased odds to develop glaucomatous disease (20), and in this case the early diagnosis is mandatory and needs tests with high sensitivity and specificity (21).
The evaluation of peripapillary RNFL is used in common clinical practice to detect the presence of glaucomatous damage (22), but in high myopia its interpretation is made difficult by the frequent presence of optic nerve tilt. Shin et al (23) showed that optic disc tilt reduce RNFL diagnostic ability in detecting glaucoma, while it doesn't influence ganglion cell-inner plexiform layer (GCIPL) thickness, which is more reliable in the evaluation of glaucoma in high myopia.
In our study all the OCT parameters and RGCs count are correlated to the increase in axial length with a moderate significant negative correlation, except GCC thicknesses that seem to be the less correlated to axial length increase because they have the weakest and less significant correlation in all groups (Table 3). This is this is in agreement with the results of many studies: Shoji et al (24) have shown that GCC parameters are not significantly affected by high myopia, while RNFL measurements have a decreased ability to detect glaucoma in myopic subjects; Considering the results of the comparisons among subgroups (Tables 4.a As regards diagnostic ability of OCT parameters, in our study all parameters has an AUROC > 0.5 (Table 6), and all curves are statistical significant, with high values of sensibility and specificity. While RNFLinf and FLV showed the best AUROCs in emmetropes, GCCinf and GLV showed the best AUROCs in all myopic group. This is in agreement with many studies that demonstrated that GCC thickness have a glaucoma detection ability as effective as that of RNFL parameters (24-30).
In the comparison between GCCinf and GLV and all other parameters, there was a statistical difference with almost all RNFL parameters, especially in high myopes ( Table 7). The study of Seol et al (31) showed that inferotemporal GCIPL has a significantly better diagnostic ability than RNFL parameters, and this is in agreement with our results, and also than average GCIPL parameters, while in our study GCCinf and GLV have a better AUROC than other GCC parameters, but non statistically significant.
In Table 8 the results of the comparison of AUROC of every parameters between different subgroups are illustrated: in all the comparisons with emmetropes there is a statistical significant difference between emmetropes and myopes for GCC parameters, which show better diagnostic abilities in myopes. This means that GCC parameters are more useful to differentiate glaucomatous from healthy eyes in myopic than emmetropic eyes.
However we must take into account some limitations of GCC in the evaluation of glaucoma in myopic eyes. In the eyes in which the head of the optic nerve is deformed and therefore difficult to evaluate, we may assume that the macular region is less distorted, but this is not always true. The studies by Kim et al (32, 33) have suggested that the outline of the entire posterior pole determines the possible configuration of the optic nerve head. So, the presence of irregularities in macular region could invalidate the evaluation of GCC in myopia. Another bias is due to the high axial length which causes a false positive GCC thinning (34). This is because a greater axial length determines a streching of the globe with an increase in the distance between the optic nerve and the macula and consequent false thinning of macular region(35, 36). Furthermore, the presence of macular degeneration can cause GCC thinning (for retinal atrophy) or thickening (for intraretinal fluid due to myopic CNV or to macular retinoschisis) that are independent from glaucoma (37).
To our knowledge, no previous studies have reported on use of RGCs count in identify glaucoma in myopic eyes and its diagnostic ability. There are many studies about RGCcount in non-myopic eyes demonstrating that a combined measure of structural and functional parameters performs better than the single OCT and perimetry parameters (13,15). We wanted to evaluate if the diagnostic ability of the RGC was superior to those of the single parameters used in its calculation formula (MD and RNFLavg) also in myopic groups: both in mild and high myopes, AUROCs of RGCcount are significantly better than those of MD, and approximately similar to those of RNFLavg without statistically significant differences (Table 9). In all groups, both myopes and emmetropes, RGCcount shows very good AUROCs (between 0.873 in emmetropes and 0.929 in mild myopes), with sensitivity > 70% and specificity > 90%.
Based on the results of our study, RGCs count seems to be complementary to OCT parameters in the detection of glaucomatous damage in the myopia, also if GCC parameters show better diagnostic ability. Since the glaucomatous damage in the myopia is more early to be detected at macular level, it could be useful to evaluate the number of macular ganglion cells, as already done by Rolle et al (16), also in myopic subjects. This could be analysed in a further study.
The limitation of this study is that, despite of a good number of the total sample, the subdivision in different subgroups makes the number of each subgroup reduced. However, this is also found in other studies (31, 38, 39). To validate the results obtained it would be indicated to use an even larger cohort of investigation.
Another limitation is related to the fact that the sample of myopic eyes does not perfectly correspond to what we find in clinical practice, because it does not include all the eyes with perimetric alterations (enlarged blind spot, general reduction of sensitivity and superotemporal peripheral defects), which also are very frequent in myopic eyes.

Conclusions
In conclusion, in the OCT analysis of myopic eyes RNFL is the parameter most influenced by the axial length, while the GCC, in particular the inferior, and the GLV are the two OCT parameters with better diagnostic performance. The RGCcount appears to have good sensitivity and specificity, but not higher than the OCT parameters, so it can be used as a complementary test in the diagnosis of glaucoma in myopic eyes.
Identifying the presence of glaucoma in a myopic eye is one of the current diagnostic challenges in ophthalmology, and we must interpret all the instrumental data considering the influence of myopia.
Current OCTs analyze the thicknesses of retinal nerve fibers and ganglion cells using a normative database that includes emmetropic subjects. Our study agrees with other works affirming that the OCT parameters are affected, although to varying degrees, by the axial length. Therefore to increase the reliability of the OCT in the diagnosis it would be appropriate to insert myopic eyes in the normative databases of the instruments and develop algorithms that take into account the axial length to analyze the thicknesses detected with OCT.

COMPETING INTERESTING
The authors declare that they have no competing interests FUNDING This study was not supported by any research grants.

AVAILABILITY OF DATA AND MATERIALS
The data have not been placed in any online data storage. The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

AUTHORS' CONTRIBUTION
Study concept and design (TR, BB, AM); collection, management, analysis, and interpretation of data (BB, AM); preparation, review, or approval of the manuscript (TR, BB, AM, LD). All authors read and approved the final manuscript.