Relationship between diabetic macular edema and choroidal layer thickness.

PURPOSE: To investigate the relationship between diabetic macular edema (DME) and the choroidal layer thickness in diabetic patients.
METHODS: This is a retrospective observation study. Three hundred eighteen eyes of 159 diabetes mellitus (DM) patients and age-matched 100 eyes of 79 healthy controls were enrolled. DME was defined as over 300 μm in the central retinal subfield of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid sector. The central choroidal thickness (CCT), as well as inner and outer layers were determined based on enhanced depth imaging (EDI)-OCT. Diabetic patients with/without systemic diabetic treatments (DT) at the start of this study was defined as DT+ and DT-, respectively. The number of eyes examined was 62 and 256 eyes in DME+and DME-groups, respectively. DM patients were further subdivided into 4 groups with/without DME and DT; DME+DT+(35 eyes), DME-DT+(159 eyes), DME+DT-(27 eyes), and DME-DT-group (97 eyes). Multiple comparisons on CCT layers including control and each DM group were statistically examined.
RESULTS: The total CCT layer was 254±83, 283±88, and 251±70 μm in the control, DME+, and DME-group, respectively. A total CCT layer in DME+was significantly thicker than the DME-group (P < 0.05). The outer CCT layer was 195±75, 222±83, and 193±63 μm in the control, DME+, and DME-group, respectively. The outer CCT layer in DME+ was significantly thicker than the DME-group (P < 0.05). In the subdivided groups, the total CCT layers in the control, DME+DT+, DME-DT+, DME+DT-and DME-DT-groups were 254±83, 274±88, 247±66, 290±84 and 258±75 μm, respectively. The outer CCT layers in each group were 195±75, 214±83, 189±58, 228±77, and 201±70 μm, respectively. Total CCT and the outer layer in DME+DT-was significantly thicker than the DME-DT+group (each P < 0.05). In contrast, there was no significant difference in inner layer between the groups.
CONCLUSIONS: The total and outer CCT layers of diabetic eyes were significantly thickened in the DME+DT-as compared with the DME-DT+group, suggesting that CCT may be related to the pathology of DME.

Introduction

Diabetic macular edema (DME) is a major cause of visual impairment in the working age population [1]. The pathogenesis of DME is mainly caused by retinal vascular hyperpermeability. The choroid is an important vascular tissue responsible for blood supply to the retinal pigment epithelium and photoreceptor cells, which plays pivotal roles in the metabolic exchange to the avascular fovea [2]. It is indisputable that the choroid takes part in not only the physiological homeostasis in the outer retina, but also the pathology of many retinal diseases including diabetic retinopathy (DR). Experimental and clinical analyses have proved that choroidal vascular disorders in diabetes might involve the pathogenesis of DR [3,4]. Histopathological changes in choroidal vessels in diabetic patients are similar to retinal vascular changes seen in DR such as increased vascular tortuosity, extravascular hemorrhage, microaneurysms, nonperfusion areas, vasodilatation and stenosis as well as the neovascularization [3]. It has also been reported that protein liquid leakage to the stromal dropout and choroid interstitium possibly reflects ischemia and vascular hyperpermeability, which is observed in the retinal vasculature of DR as well [5].

With recent advances in imaging technologies, the choroids have been mainly evaluated by indocyanine green angiography [4] and laser doppler flowmetry [6,7]. In more recent years, the visualization of the choroid was improved by the enhanced depth imaging-optical coherence tomography (EDI-OCT), which enables ophthalmologists to obtain macular cross-sectional images with high quality and resolution [8]. Clinical evaluation in the choroids of DM eyes has been conducted; however, the results examined regarding choroid thickness were various, such as thickened, thinned, or no changes [9]. Similarly, the previous results of the choroidal thickness measurement in DME eyes demonstrated thinned [10-13], thickened [14,15], and no change [16-18]. We have investigated patients with DR by dividing them into systemic DM treatment and DR severity, and concluded that the central choroid thickness (CCT) was significantly reduced in early DR without treatment [19]. More recently, we showed that the changes in CCT of DR patients might result from outer choroidal layer thickness [20]. Thus, the most important pathological change in the choroid in DR may extend not only to the inner layer including choriocapillaris but also to the outer layer of the choroid [9]. However, little is known about the relationship between the choroidal layer thickness of DME eyes and DM treatment.

The aim of this study is to investigate the relationship between DME with/without diabetes treatment and the thickness of choroidal layer in diabetic patients.

Methods

Subjects and clinical study protocol

The participants provided their verbal informed consent to participate in this study. In fact, the institutional review committee of Teine Keijinkai hospital (IRB) approved this consent procedure and also gave an opportunity to refuse the participation anytime to the participants by disclosing the study protocol on thewebsite: http://www.keijinkai.com/teine/about/efforts/feature/". This retrospective observation study was approved by the IRB, and informed consent was obtained from all subjects (patients diagnosed with type 2 diabetes and healthy controls). This study followed the principles of the Declaration of Helsinki. Three hundred and eighteen eyes with 159 DM patients comprising 107 males (67.3%) and 52 females (32.7%) (mean age 61.3±11.8 years), and 100 eyes in 79 normal subjects without DM (31 male (39.2%) and 48 females (60.8%); mean age 59.8±13.9 years) were enrolled in this study. All subjects underwent tests including a best-corrected visual acuity (BCVA, decimal scale), Goldmann applanation tonometry, biomicroscopy of the anterior segment using a slit lamp examination, and ophthalmoscopy of the posterior segment, and axial length measured using optical biometry (IOL Master Zeiss; Jena, Germany) and EDI-OCT (Cirrus HD–OCT, Carl Zeiss Meditec) during December 2013 to April 2018. Exclusion criteria were eyes with a history of other ocular disease, previous topical treatments including retinal laser photocoagulation, local vascular endothelial growth factor (VEGF) and/or triamcinolone acetonide injection, or any ocular surgeries including vitrectomy, cataract or glaucoma surgery. Moreover, eyes with spherical power greater than –5 diopters, cylindrical astigmatism with cylinder power greater than 3 diopters, and ocular axial length being greater than 26 mm were excluded. Age-matched healthy subjects were recruited from consecutive populations scheduled for routine ophthalmic examinations to check refractive errors. The selection criteria for control subjects were the same as those for DM subjects, except for the absence of diabetes.

First, all the DM patients were divided into diabetics with/without DME (DME+/–) and compared those with the control group. DM patients further were divided into two groups based on information regarding systemic DM treatment (DT) situation: patients (DM-treated group: DT+) who received systemic pharmacotherapy with oral hypoglycemic or subcutaneous insulin therapy for DM managed by the attending physician, or who did not receive systemic treatments for DM (DM-untreated group: DT–) until the start of this study. Finally, DM patients were classified into 4 groups according to with/without DME and DT+/–; DME+DT+, DME–DT+, DME+DT–, and DME–DT–group. Those 4 groups were eventually compared with the control group. In addition, DME + group was classified into three groups based on the disease stage of DR, that is, international severity classification; mild / moderate non-proliferative DR (mNPDR), severe NPDR (sNPDR), and PDR. Furthermore, DME was classified into 3 groups according to the configuration of DME based on the previous report by Otani et al. [21]; sponge-like retinal swelling (SLRS), cystoid macular edema (CME), and serous retinal detachment (SRD). Each classified DR group was compared with control group, respectively.

Retinal and choroidal thickness measurements using EDI-OCT imaging

OCT facilitates objective quantification of retinal thickness. Central retinal thickness (CRT) was obtained by automatic retina thickness map in the Early Treatment Diabetic Retinopathy Study (ETDRS) grid sector. DME was defined as over 300 μm in the central retinal subfield of the ETDRS grid sector. As described in the previous report, this study analyzed choroid tomograms obtained by the EDI-OCT method spectral domain OCT (Cirrus HD OCT; Carl Zeiss Meditec, Inc., Dublin, CA, USA) [19]. The EDI-OCT scan included 9 mm horizontal lines centered on the fovea, obtained by experienced technicians. They have confirmed that signal strength of all the measured values was more than 6 out of 10. Therefore, images with a signal strength of less than 6 were excluded from this study. Anatomically, the choroidal vascular layers are made up of three layers: choriocapillaris, medium vascular layer (Sattler’s layer) and large vessel layer (Haller’s layer); however, the boundary of the three-layer structure is not clear compared to the retina. For this reason, CCT data from the total choroid layer, inner layer (choriocapillaris + medium vascular layer), and outer layer was manually identified based on findings of EDI-OCT horizontal scan through the fovea, as reported previously (Fig 1) [22]. To identify the measurement position of the choroid thickness, the position where the foveal bulge was confirmed, or the position where the vertical height of the DME was the highest, was defined as the central retinal thickness, and then the choroid thickness was measured beneath the positions. Two evaluators (H.E. and C.S.) independently assessed OCT images in a masked fashion regarding the subject's clinical information.

Fig 1

Identification of choroid layer thicknesses by EDI-OCT in a healthy control (A) and diabetic macular edema (B). The choroid outer layer thickness (red line) was measured from the inner boundary of the choroid scleral junction to the innermost point (green line) of the choroidal large blood vessel observed closest to the fovea centralis. The choroid inner layer thickness (yellow line) was obtained by subtracting the choroidal outer layer thickness (red line) from the total choroid layer thickness (white line).

Statistical analyses

Age, best-corrected visual acuity, refractive error, intraocular pressure, ocular axial length, hemoglobin A1c (HbA1c), systolic blood pressure (SBP), diastolic blood pressure (DBP), estimated glomerular filtration rate (eGFR), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride (TG), between DM patient and control groups were analyzed using Steel test. Retinal thickness, choroid layer thickness between DM patients and control groups were calculated using Kruskal-Wallis test and analyzed using Steel-Dwass test. Data of choroid layer thickness obtained by examiners were analyzed for relative reliability and absolute reliability. Relative and absolute reliability were examined using Spearman's rank correlation coefficient and Bland-Altman plot, respectively. In order to ensure measurement accuracy, the absolute agreement proved by the Bland-Altman plot between measurements is applied for fixed and proportional biases. Correlation between visual acuity in the DME eye and retinal and choroid layer thicknesses was examined using Spearman's rank correlation coefficient. Data were presented as mean±standard deviation. In all studies, a P value of less than 0.05 was considered statistically significant, which was determined using the statistical software commercially available (SPSS version 21.0).

Results

The clinical characteristics of the study group are summarized in Table 1. Three hundred and eighteen eyes with 159 DM patients comprising 107 males (67.3%) and 52 females (32.7%) (mean age 61.3±11.8 years), and 100 eyes in 79 normal subjects without DM (31 male (39.2%) and 48 females (60.8%); mean age 59.8±13.9 years) were enrolled in this study. The number of DME + or DME− eyes was 62 eyes and 256 eyes, respectively. There were no significant differences in age, equivalent sphere, intraocular pressure, axial length, diabetes duration, DBP, eGFR, and LDL-C between DM group and control group. HbA1c, SBP and TG were significantly higher in DM group than in control group, respectively (each, P<0.01). On the other hand, TC and HDL-C were significantly lower in DM group than in control group (P<0.05).

Table 1

Patient’s characteristics of control and diabetes mellitus groups.

	Control group	DM group
		DME+	DME–
Total number of eyes	100	62	256
Age (years)	59.8±13.9	59.3±12.0	61.7±11.7
BCVA–logMAR (snellen)	–0.02±0.16 (20/19)	0.29±0.34 (20/39) **	0.06±0.22 (20/23) **
Spherical error (diopter)	–0.13±1.56	–0.40±1.59	–0.39±1.66
IOP (mmHg)	14.6±3.1	15.3±3.9	15.2±3.1
Axial length (mm)	23.80±0.83	23.57±0.88	23.65±0.93
DM duration (years)	–	9.7±7.3	10.9±9.0
HbA1c (%)	5.6±0.4	8.33±2.17**	8.61±2.19**
SBP (mmHg)	122±16	139±22**	137±21**
DBP (mmHg)	75±13	77±11	78±12
eGFR (mℓ/min/1.73 m2)	76±16	69±32	75±29
TC (mg/dl)	208±34	195±44	193±49*
HDL-C (mg/dl)	63±14	53±16	49±14**
LDL-C (mg/dl)	109±25	119±36	112±37
TG (mg/dl)	97±46	140±58*	172±124**

* :P<0.05

**:P<0.01 vs. control group

DM, diabetes mellitus; DME, diabetic macular edema; BCVA, best corrected visual acuity; IOP, intraocular pressure; HbA1c, hemoglobin A1c; SBP, systolic blood pressure; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; diastolic blood pressure; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TG, triglyceride

The number of DT–patients with systemic DM treatment discontinuation or without history of DM treatments was 27 and 35 cases, respectively. The number of eyes in the DME + DT +, DME-DT +, DME + DT-, and DME-DT- groups classified into 4 groups according to with/without DME and DT were 35, 159, 27 and 97, respectively (Table 2). There was no significant difference in age, equivalent spherical power, intraocular pressure, ocular axial length, duration of diabetes, SBP, DBP, eGFR, TC, HDL-C, LDL-C, and TG in each DM group. On the other hand, the HbA1c value was significantly higher in the DT–group (10.0±2.4%) than in the DT+group (7.5±1.3%) (P < 0.001 in Table 1). There were no significant differences in age, equivalent sphere, intraocular pressure, axial length, diabetes duration, DBP, eGFR, and LDL-C between DM group and control group. SBP and TG were significantly higher in DM group than in control group, respectively (each, P<0.01). On the other hand, TC and HDL-C were significantly lower in DM group than in control group (P<0.05).

Table 2

Patient’s characteristics of control and DM-treated/untreated groups.

	Control group	DM-treated group		DM-untreated group
		DME+	DME–	DME+	DME–
Total number of eyes	100	35	159	27	97
Age (years)	59.8±13.9	61.8±10.8	63.8±10.9	56.1±12.6	58.4±12.1
BCVA–logMAR (snellen)	–0.02±0.16 (20/19)	0.31±0.34 (20/41)**	0.05±0.21 (20/22)**	0.27±0.34 (20/37)**	0.10±0.25 (20/25)**
Spherical error (diopter)	–0.13±1.56	–0.73±1.21	–0.68±1.56	–1.01±1.72	–0.45±1.32
IOP (mmHg)	14.6±3.1	14.6±3.8	15.3±3.1	14.3±3.7	15.0±3.1
Axial length (mm)	23.80±0.83	23.50±1.01	23.61±0.94	23.67±1.01	23.70±0.97
DM duration (years)	–	9.9±8.0	11.6±9.3	9.1±6.0	9.2±7.7
HbA1c (%)	5.6±0.4	7.2±1.0**	7.6±1.3**	9.8±2.4**	10.2±2.4**
SBP (mmHg)	122±16	140±20*	134±18*	138±24	140±23**
DBP (mmHg)	75±13	81±10	76±12	73±10	80±12
eGFR (mℓ/min/1.73 m2)	76±16	73±23	70±24	67±37	80±32
TC (mg/dl)	208±34	191±35	188±47**	198±50	200±52
HDL-C (mg/dl)	63±14	56±12	48±12**	49±20	50±16**
LDL-C (mg/dl)	109±25	112±35	104±32	130±34	122±40
TG (mg/dl)	97±46	141±57	168±122*	139±59	176±128**

* :P<0.05

**:P<0.01 vs. control group

DM, diabetes mellitus; DME, diabetic macular edema; BCVA, best corrected visual acuity; IOP, intraocular pressure; HbA1c, hemoglobin A1c; SBP, systolic blood pressure; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; diastolic blood pressure; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TG, triglyceride

The reproducibility of the CCT measurements for total and outer layers were examined in normal and diabetic eyes (S1 Table). In all the layers tested, the intraclass correlation coefficient (ICC) exceeded 0.9, which reflected high reproducibility (S1 Table). Next, with the limit of agreement determined by Bland- Altman plot for choroidal thickness in normal and diabetic eyes, the systematic bias in the measured values was visualized with dots (S1 Fig). In order to judge the presence of fixed bias, a 95% confidence interval of the average of the difference between two measurements was obtained. The 95% confidence interval was –0.56 to 2.20, –1.22 to 1.74, –0.44 to 1.27, and –0.86 to 1.09, respectively, proving no significant fixed bias in choroidal layers in normal and DM eyes (S1 Table). Next, in order to judge the presence of proportional bias, the significant correlation in the difference and the average measured between the corresponding two groups was tested. The correlation was R = 0.007 (P = 0.94), R = 0.099 (P = 0.33), R = –0.109 (P = 0.053), and R = –0.101 (P = 0.07), respectively, indicating no significant proportional bias in choroidal layers in normal and DM eyes (S1 Table). These data overall suggest that there was no particular tendency to cause the difference between the evaluators.

In this study, the number of eyes examined was 62 and 256 eyes in DME+and DME–groups, respectively. The total CCT layer was 254±83, 283±88, and 251±70 μm in the control, DME+, and DME–group, respectively (Fig 2B). A total CCT layer in DME+was significantly thicker than the DME–group (P < 0.05). Next, the inner CCT layer was 59±13, 60±12, and 57±13 μm in the control group, DME+, and DME–groups, respectively (Fig 2C). There was no significant difference among all groups in the inner CCT layer. The outer CCT layer was 195±75, 222±83, and 193±63 μm in the control, DME+, and DME–group, respectively (Fig 2D). The outer CCT layer in DME+ was significantly thicker than the DME–group (P < 0.05).

Fig 2

Changes in retinal thickness (A) and total choroidal layer thickness (B) with or without diabetic macular edema (DME). DME + group shows significantly thicker retinal thickness than control and DME–groups (A). DME + group reveals a significantly thicker choroidal thickness than DME- group (B). Changes in choroidal inner layer thickness (C) and outer layer thickness (D) with or without DME. There was no significant difference in all groups about inner layer (C). In contrast, DME+group reveals a significantly thicker choroidal thickness than DME- group (D). Mean retinal and choroidal thickness are shown in the three groups. Error bars represent standard deviation. The asterisk (*) indicates a significant difference (P < 0.05).

In this study, the number of eyes examined was 161 and 124 eyes in the DT + and DT- groups, respectively. The retinal thickness was significantly thicker in the diabetic group than in the control group (Fig 3A). Next, there was no significant difference in total CCT, inner CCT, and outer CCT between the control and the diabetic group (Fig 3B–3D). The characteristics of the control group and DM 4 groups are shown in Table 2. The number of eyes in DT+group was 35 and 159 in DME+and DME–, respectively, and the number of eyes in DT–was 27 and 97 in DME+and DME–, respectively. Total CCT layers were 254±83, 274±88, 247±66, 290±84, and 258±75 μm for control, DME+and DME–in DT+, and DME+ and DME–in DT–, respectively (Fig 4B). The total CCT layer in DME+DT–was significantly thicker than the DME–DT+group (P < 0.05). Next, inner CCT layer was 59±13, 60±12, 58±14 and 61±12 and 56±12 μm for control, DME+and DME–in DT+, and DME+ and DME–in DT–groups, respectively (Fig 4C). There was no significant difference between all groups in the inner CCT layer. Finally, outer CCT layer was 195±75, 214±83, 189±58 and 228±77, and 201±70 μm for control, DME+and DME–in DT+, and DME+ and DME–in DT–, respectively (Fig 4D). The outer CCT layer in DME+DT–was significantly thicker than the DME–DT+ group (P < 0.05), whereas there was no significant difference between DT + and DT–groups in outer CCT layer of DME+eyes.

Fig 3

Changes in retinal thickness (A) and total choroidal layer thickness (B) with or without systemic diabetes treatment (DT). DT+and DT–group reveals a significantly thicker retinal thickness than control group (A). There was no significant difference in all groups about choroidal layer (B). Changes in choroidal inner layer thickness (C) and outer layer thickness (D) with or without DT. There was no significant difference in all groups about inner (C) and outer layer (D). Mean retinal and choroidal thickness are shown in the three groups. Error bars represent standard deviation. The asterisk (*) indicates a significant difference (P < 0.05). The double asterisk (**) indicates a significant difference (P < 0.01).

Fig 4

Changes in retinal thickness (A) and choroidal total layer thickness (B) in diabetic eyes based on the history of systemic treatments. Retinal thickness was significantly thicker in DME+groups regardless of DM treatments, and DME- groups than the control group (A). In contrast, DM-untreated DME + group shows a significantly thicker choroidal thickness than DM-treated DME- group (B). Changes in choroidal inner layer thickness (C) and outer layer thickness (D) in diabetic eyes. There was no significant difference in all groups in inner layer (C). DM-untreated DME + group reveals a significantly thicker outer choroidal thickness than DM-treated DME- group (D). Mean retinal and choroidal thicknesses are shown in the five groups. Error bars represent standard deviation. The asterisk (*) indicates a significant difference (P < 0.05). The double asterisk (**) indicates a significant difference (P < 0.01).

Choroidal layer thickness by each stage of DR in DME + group and by each type of DME is shown in Tables 3 and 4, respectively. In stage of DR, inner CCT layer was significantly thicker in sNPDR compared to the control group (Table 3, P < 0.05). In each type of DME, choroidal layer thickness was not significantly different as compared with the control group. CRT was significantly thicker in DME+groups regardless of DM treatments, and DME- groups than the control group (Fig 4A). Correlation between BCVA and parameters on the OCT image in the DME+ group had a significant positive correlation with CRT (R = 0.50、P < 0.01), whereas there was no correlation with each choroid layer thickness (Table 5).

Table 3

Choroid layer thickness of Control and DR stage groups.

	Control group	DR stage groups
		mNPDR	sNPDR	PDR
Total number of eyes	100	19	17	26
Retinal thickness in the central subfield	244±23	355±133**	381±136**	389±207**
Choroidal total layer thickness	254±83	266±122	307±59	279±81
Choroidal inner layer thickness	59±13	56±10	67±10*	59±11
Choroidal outer layer thickness	195±75	210±106	239±56	220±76

*:P<0.05

**:P<0.01 vs. control group

DR, diabetic retinopathy; DME, diabetic macular edema; mNPDR, mild/moderate non-proliferative DR; sNPDR, severe NPDR

Table 4

Choroid layer thickness of Control and DME subtype groups.

	Control group	DME subtype groups
		SLRS	CME	SRD
Total number of eyes	100	24	27	11
Retinal thickness in the central subfield	244±23	327±26**	422±88**	528±261**
Choroidal total layer thickness	254±83	294±109	268±70	294±70
Choroidal inner layer thickness	59±13	60±12	61±12	60±9
Choroidal outer layer thickness	195±75	234±102	207±65	234±66

*:P<0.05

**:P<0.01 vs. control group

DR, diabetic retinopathy; DME, diabetic macular edema; SLRS, sponge-like retinal swelling; CME, cystoid macular edema; SRD, serous retinal detachment

Table 5

The relationship between best corrected visual acuity, retinal thickness, and choroidal thickness in the diabetic macular edema eyes.

Variables (n = 62)	1. BCVA–logMAR	2. Retinal thickness in the central subfield	3. Choroidal total layer thickness	4. Choroidal inner layer thickness	5. Choroidal outer layer thickness
1. BCVA–logMAR	–	0.50**	-0.24	-0.12	-0.25
2. Retinal thickness in the central subfield	0.50**	–	-0.05	0.002	-0.04
3. Choroidal total layer thickness	-0.24	-0.05	–	0.58**	0.99**
4. Choroidal inner layer thickness	-0.12	0.002	0.58**	–	0.51**
5. Choroidal outer layer thickness	-0.25	-0.04	0.99**	0.51**	–

**:P<0.01

BCVA, best corrected visual acuity

Discussion

In this study, the total CCT layer was significantly thicker in the DME+ than in the DME–group in all DME cases examined (P < 0.05). Previous studies demonstrated that the choroidal thickness of untreated DME eyes was thinned [10-13], thickened [14,15], or no change [16-18], and the consequence was different among the researchers. One of reasons in such different results on CCT might be different inclusion criteria even in untreated DME eyes examined. For example, studies showing thinning of CCT in DME eyes were lack of eyes with PDR in the study subjects, and the number of eyes examined was small [10-13]. On the other hand, studies showing no change in CCT included eyes with all the DR stages including PDR. In addition, CCT is influenced by the patient background of the study subject, systemic or topical treatment history, differences in treatment methods, and differences between races. Indeed, CCT has been suggested to be involved with local factors of the eye in DR. Several authors have shown that CCT could be affected by panretinal photocoagulation [23], intravitreal anti-VEGF therapy [24], and intravitreal triamcinolone acetonide injection [25]. As a matter of fact, these have been shown to be effective treatments for patients with DME. In this study, we collected cases without any ocular treatment history, and consequently showed significant thickening of the total CCT layer in the DME eye compared to the eye without DME. Our results are consistent with previous publications including the report by Kim et al., looking at DME eyes without local treatments [14,15].

Mechanisms underlying the thickened choroid in DME eyes has yet to be elucidated. VEGF is an important cytokine that mediates vascular hyper-permeability, and an increase in the level of VEGF protein concentration has also been observed in the DME eyes [26]. In addition, the choroidal thickness was significantly reduced after anti-VEGF therapy in DME eyes [27,28]. Okamoto et al. examined the choroidal blood flow before and after intravitreal ranibizumab injection into DME eyes using laser speckle flowgraphy, and showed that the choroidal blood flow significantly decreased one month after the treatment [15]. An animal study has demonstrated that the choroidal vascular development and homeostasis are highly dependent on VEGF [29]. Taken together, these results suggested that alteration of choroidal thickness and the blood flow in DME depends on VEGF. Therefore, the association between DME and CCT seen in this study may have correlated with increased VEGF expression, leading to increased choroidal blood flow and subsequent CCT thickening. Although it is still unknown whether the onset of DME affects the cause or the result of CCT thickening, further studies are needed to clarify the mechanisms.

Recently, it has been reported that many systemic and physiological conditions involving hemodynamics influence DME and choroidal thickness [30]. Large-scale epidemiological studies indicated that elevated HbA1c increased the risk of the onset of DME [31,32]. Hwang et al. evaluated macular thickness before and after hemodialysis in diabetic patients with end-stage renal disease, and found a significant decrease of choroidal thickness in DME eyes after hemodialysis [33]. In the FIELD study that examined the effect of fenofibrate for the treatment of dyslipidemia in type 2 diabetic patients, the risk of DME progression decreased by 31% in the fenofibrate group compared to the placebo group [34]. These results suggest a complex mechanistic association between DME and systemic factors, providing evidence to manage DME patients by systemic controls. We previously examined subdivided DR patients with/without systemic DM treatment or DR severity, and demonstrated that CCT was significantly reduced in mild/moderate nonproliferative DR without DM treatment [19]. In addition, we showed that changes in CCT of DM eyes possibly depended on choroidal outer layer thickness [20]. These indicated that chronic hyperglycemia may be a further deteriorating factor for choroidal microcirculation disorders.

A previous study analyzing the choroid layer thickness reported that the thickness of the inner layer combining the choriocapillaris and medium vascular layers significantly decreased in the PDR and DME eyes [11]. On the other hand, the most significant changes observed in the choroid not only limited the choriocapillaris, but also extended to larger vessels [9]. In this study, the outer choroidal layer thickness in all the DM patients examined was significantly greater in the DME+ than in the DME–group. In the previous report, VEGF protein concentrations increased in DME eyes [35], which suggest the reason why the outer CCT layer increased in the DME+group. Previously we demonstrated that there was no significant correlation between CCT and DR stages including outer layer thickness in the DM treatment group, and that HbA1c value was significantly lower in treatment group than in DM untreated group [19,20]. These results suggest that systemic DM therapy might play an important role in the stabilization of the outer layer thickness in the choroid. HbA1c levels in this study was also significantly lower in DT+group than in DT–group (P < 0.01, Table 1). In addition, the outer choroidal thickness of DM patients in this study changed depending on with/without DT and DME, and was significantly thinner in the DME–DT+group than in the DME+DT–group. However, there was no significant difference between DT+group and DT–group in total and outer CCT layers of DME+patients, indicating systemic DM treatment alone is not an independent factor involving the choroidal thickness.

Therefore, we speculated the mechanisms underlying significantly thickened outer layer in DME+DT–group as follows: 1) chronic hyperglycemic condition caused by untreated DM deteriorated choroidal microcirculatory system in DME eyes, 2) more increase in VEGF protein associated with DME, led to vasodilatation of outer choroid layer, increased choroidal blood flow and/or vascular hyperpermeability, 3) increased accumulation of several diabetes-related proteins including advanced glycation end products in choroidal tissues, as shown in DM donor eyes with immunohistochemistry [36]. However, the mechanisms underlying thickened inner choroidal layers in sNPDR-DME+ group compared to the controls are unknown. In fact, the major histopathological findings in the inner layer of DM eyes were occlusion of choriocapillaris [3]. Therefore, in order to validate the clinicopathological correlation, it is necessary to investigate morphological changes in choroidal vessels and/or stromal components using recent imaging techniques. Future studies including the lumen/stroma morphology with binarization methods may clarify the pathology of the inner and outer choroidal layers in DME.

It is well known that DME can cause refractory vision disorder in DM patients. In our study, there was a significant negative correlation between BCVA in the DME and CRT determined by OCT findings, but BCVA was not correlated with choroid layer thickness (Table 5). Yiu et al. evaluated the relevance to changes in visual and anatomical outcomes during 6 months following anti-VEGF treatment for DME eyes, and reported that there was no association between BCVA and CCT [27]. In contrast, Eliwa et al. showed a significantly negative correlation between BCVA and CCT in the DME eyes [13]. The differences between the previous and the current studies may be related to different DR stages examined. They limited the NPDR in the DME group [13], whereas we included the DME patients having from NPDR to PDR. In addition, CCT in DME patients in their study showed a significant thinning compared to healthy controls, whereas our results showed rather thickening. These results suggest that CCT in DME might correlate with patients’ vision in the early stage of DR.

The current study has several limitations. First, we measured the layer thickness including the outer thickness manually. It is indisputable to ensure data reproducibility between evaluators. Since Wong et al. have reported that the presence of thickened choroid and subretinal fluid may affect the reproducibility [37], these factors may involve measurements of the thickness of the choroidal layer in this study. Second, the thickness of the choroid layer was evaluated only in the central choroid in this study. It has been shown that parafoveal choroidal thickness may be different in each case [38]. Recently, a binarization method can be used to assess choroidal vascular changes in diabetic eyes [39]; therefore, further study is needed to clarify a wide range of layer thicknesses. Third, classification of systemic diabetes treatment was performed with or without medication treatment. However, detailed information on systemic treatments for diabetes mellitus could not be obtained. Therefore, differences in drug medications given in each subject might affect choroidal thickness. Fourth, changes in choroidal thickness may also be associated with choroidal blood flow. However, since data about indocyanine green angiography and optical coherence tomography angiography are not available in this study, further studies are needed to clarify the association between choroidal thickness and the circulation. Fifth, this study was able to adjust the local confounding factor that could affect the choroid structure. However, systolic blood pressure and serum lipids levels were significantly higher in the DM group than normal controls. These systemic confounding factors might affect the acquisition of data regarding choroidal structure. Finally, the layer thickness was examined based on EDI-OCT imaging in this study. Further studies will also prove differences of data obtained by EDI-OCT or swept source OCT in diabetic eyes.

In conclusion, the total and outer CCT layer of the DM eye was significantly thickened in the DME+DT–group compared to the DME–DT+group. These results indicated that CCT may be related to the pathology of DME.

Band-Altman blot analysis at total choroidal thickness and outer thickness in normal and diabetic eyes.

Total choroidal thickness (A) and outer choroidal thickness (B) in normal eyes. Total choroidal thickness (C) and outer choroidal thickness (D) in diabetic eyes. Solid line indicates the average mean difference, while dotted line shows 95% confidence limit of agreement. There is no specific trend to result in the difference between raters.

(TIFF)

Click here for additional data file.

The support file is available at the file “20191106 Data in supporting information.xlsx.

(XLSX)

Click here for additional data file.

Inter-examiner reliability of choroidal segmentation for control and diabetes mellitus group.

(DOCX)

Click here for additional data file.

28 Oct 2019

PONE-D-19-26407

Relationship between diabetic macular edema and choroidal layer thickness

PLOS ONE

Dear Dr Kase,

Thank you for submitting your manuscript to PLOS ONE. 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.

Both reviewers found this work interesting but in need of improvement. Especially about how concussions are drawn form associations that cannot determine causality.  I believe the points raised can be addressed and we would look forward to a substantially revised version

We would appreciate receiving your revised manuscript by Dec 12 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Demetrios G. Vavvas

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please amend your current ethics statement to address the following concerns:

a) Did participants provide their written or verbal informed consent to participate in this study?

b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure.

3. Thank you for stating the following financial disclosure: No

Please provide an amended Funding Statement that declares *all* the funding or sources of support received during this specific study (whether external or internal to your organization) as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.

Please state what role the funders took in the study.  If any authors received a salary from any of your funders, please state which authors and which funder. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4.  Thank you for stating the following in your Competing Interests section:

No

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

This information should be included in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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 #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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 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 sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space 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 #1: The authors investigated the relationship between diabetic macular edema (DME) and choroidal layer thickness. They also revealed the significance of the systemic condition in diabetes mellitus (DM) patients in CT evaluation. It could be an important contribution. The data is well collected and presented clearly. However, overall, this paper seems somewhat disorganized, especially in Methods and Results section. I have the following concerns.

First and foremost, please recheck and follow our submission guidelines such as double-spaced layout, line number included, etc. (See https://journals.plos.org/plosone/s/submission-guidelines). And I recommend

Major concerns,

(1) Page 4: Subjects and Clinical study protocol: The number of subjects, sex, and eyes should be moved to Result section.

(2) Page 4: Exclusion criteria: Did this study include the subjects with another ocular disease and surgery (cataract, glaucoma, etc.)?

(3) Page 4: Please describe normal subjects clearly. How did the authors collect and choose normal subjects? Age-matched? Were they all healthy subjects? No ocular history?

(4) In Results section, these sections should be revised to improve clarity. I suggest the authors describe the data and analyses of three main groups (control, DME+, DME-) with a new Table (separated from Table 1) and Figure 3 in the 1st paragraph. After that, the subgroup data and analyses should be shown. This flow is the same order the authors stated in Methods section.

(5) Page 6, 1st Paragraph: Why did the authors do the statistical analyses for groups with AND without control (Why twice)? Is it a proper statistical approach?

(6) Table 1: The footnote showed that asterisk means statistically significance "versus control." If so, the authors should state using Steel test (not Steel-Dwass test) for this analysis in Methods section.

(7) Page 7, 2nd Paragraph, and Table 2: This reproducibility analysis is not needed in this paper. The authors did not discuss it at all (they mentioned the reproducibility as the study limitation, though). And above all, it is not essential data for this study aim. I recommend that it should be deleted or moved to Supplementary data.

(8) Page 8, 4th Paragraph: This paragraph is confusing. Please rewrite the paragraph for our readers to easily understand. And how were these subgroups' backgrounds? Was there no significant difference among the subgroups (including control) in their backgrounds?

(9) Table 3: The information should be presented in two separate tables, one focusing on the stage of diabetic retinopathy and the other on type of DME).

Minor concerns,

(10) Was the flare measured in this study? The flare data may support the possible significance of VEGF in DME and choroidal layer thickness. (The relationship between the flare value and the concentration of VEGF in aqueous was reported before)

Reviewer #2: The authors aim to investigate the relationship between diabetic macular edema (DME) and the choroidal layer thickness in diabetic patients. The findings are interesting; however, some points need to be clarified.

1) Although the authors found -in each DM group- that there was no significant difference in systolic blood pressure, this latter was significantly higher in DM group than in control group (P<0.01). Do they think that this could have influenced their results? Please make a comment on it.

2) Authors indicated in the methods that data from the total choroid layer, inner layer (choriocapillaris + medium vascular layer), and outer layer was manually identified based on findings of EDI-OCT horizontal scan through the fovea, as reported previously [22]. Since patients with DME usually do not have foveal depression, which scan did the authors include to measure choroidal layers?

3) I know they considered excluded images with signal strength less than 6. Nevertheless, I believe that in this kind of study (manual measurements) it is important to statistically correct all the OCT images for signal strength index. Also, what is about the presence of significant motion artifacts?

4) I would suggest the authors to include just one eye per patient, as they correlated several parameters referred to a subject (for example: duration of diabetes, age, blood pressure and so on) to retinal parameters that are measured separately in a single eye. Although the numbers will be more limited, it would be more correct to use a single eye in the statistical analyses.

5) The found that in stage of DR, inner CCT layer was significantly thicker in severe non-proliferative diabetic retinopathy compared to the control group (P < 0.05). How many patients with DME in this group?

6) Did they include patients with Type 2 DM? Type 2 and type 1 DM? These latter usually are younger and have fewer confounding factors or systemic comorbidities.

7) Based on their finding, the authors stated that systemic DM treatment alone is not an independent factor involving the choroidal thickness. Also, they speculated that one of the mechanisms underlying significantly thickened outer layer in DME＋DT-group might be correlated to chronic hyperglycemic condition caused by untreated DM.

One could argue why patients with DME- DT- (97 patients and second largest group) do not present any choroidal statistically significant difference as an early sign of the deteriorated choroidal microcirculatory system.

Please make a comment on it.

Minor:

Page 5, please delete the double references #19

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (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.

7 Nov 2019

For your convenience, the reviewers’ comments will appear by a single underline, and then our reply will follow the comments.

Reviewers' comments #1 to the Author:

Reviewer #1:

Major concerns

(1) Page 4: Subjects and Clinical study protocol: The number of subjects, sex, and eyes should be moved to Result section.

Thank you so much for your comments.

We moved the number of subjects, gender, and eyes listed in the Methods section to the appropriate location in the Results section.

(2) Page 4: Exclusion criteria: Did this study include the subjects with another ocular disease and surgery (cataract, glaucoma, etc.)?

Thank you so much for your comments.

The exclusion criteria are listed in the Method section as follows.

Page 6, 1st Paragraph: Exclusion criteria were eyes with a history of other ocular disease, previous topical treatments including retinal laser photocoagulation, local vascular endothelial growth factor (VEGF) and/or triamcinolone acetonide injection, or any ocular surgeries including vitrectomy, cataract or glaucoma surgery. Moreover, eyes with spherical power greater than －5 diopters, cylindrical astigmatism with cylinder power greater than 3 diopters, ocular axial length being greater than 26 mm, and other fundus diseases were excluded.

Therefore, subjects with other eye diseases and surgical history are not included.

(3) Page 4: Please describe normal subjects clearly. How did the authors collect and choose normal subjects? Age-matched? Were they all healthy subjects? No ocular history?

Thank you so much for your comments.

In order to clarify the selection criteria for healthy controls, the following sentence was added in the revised version.

Page 6, 1st Paragraph: Age-matched healthy subjects were recruited from consecutive patients scheduled for routine ophthalmic examinations to check refractive errors. The selection criteria for control subjects were the same as those for DM subjects, except for the absence of diabetes. The statements were added in the Methods section in the revised version.

(4) In Results section, these sections should be revised to improve clarity. I suggest the authors describe the data and analyses of three main groups (control, DME+, DME-) with a new Table (separated from Table 1) and Figure 3 in the 1st paragraph. After that, the subgroup data and analyses should be shown. This flow is the same order the authors stated in Methods section.

Thank you so much for your comments.

In order to improve the clarity of the results, the data and analysis of the three main groups (control, DME +, DME-) were prepared as a new table 1. Therefore, the data and analysis corresponding to the results section were added.

Page 8, 1st paragraph: The clinical characteristics of the study group are summarized in Table 1. Three hundred and eighteen eyes with 159 DM patients comprising 107 males (67.3%) and 52 females (32.7%) (mean age 61.3±11.8 years), and 100 eyes in 79 normal subjects without DM (31 male (39.2%) and 48 females (60.8%); mean age 59.8±13.9 years) were enrolled in this study. The number of DME + or DME− eyes was 62 eyes and 256 eyes, respectively. There were no significant differences in age, equivalent sphere, intraocular pressure, axial length, diabetes duration, DBP, eGFR, and LDL-C between DM group and control group. HbA1c, SBP and TG were significantly higher in DM group than in control group, respectively (each, P<0.01). On the other hand, TC and HDL-C were significantly lower in DM group than in control group (P<0.05).

(5) Page 6, 1st Paragraph: Why did the authors do the statistical analyses for groups with AND without control (Why twice)? Is it a proper statistical approach?

Thank you so much for your comments.

First, the study group was simplified and limited to variables that could affect the results. Therefore, the analysis was performed in three groups: control, DME +, and DME-. CCT is known to be affected by systemic and/or local factors. In this study, we collected and examined cases that had no history of ophthalmic treatments. In addition, we examined diabetic eyes including DME which merged with all the stages of DR. Indeed, previous publications reporting the thinning of CCT included DME eyes without PDR, and the sample size was small. On the other hand, the other studies showed thickening and no change of CCT, which included all stages including PDR. Furthermore, we investigated the relationship between DME and choroidal thickness in diabetic patients in a new classification with and without diabetes mellitus treatments to investigate the impact of systemic diabetes treatment. In this way, we think that it is a proper statistical approach.

(6) Table 1: The footnote showed that asterisk means statistically significance "versus control." If so, the authors should state using Steel test (not Steel-Dwass test) for this analysis in Methods section.

Thank you so much for your comments. As you suggested, the following factors were tested by Steel test or Steel-Dwass test, appropriately.

The revised version also added the following statements in the method section.

Page 8: Age, best-corrected visual acuity, refractive error, intraocular pressure, ocular axial length, hemoglobin A1c (HbA1c), systolic blood pressure (SBP), diastolic blood pressure (DBP), estimated glomerular filtration rate (eGFR), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride (TG), between DM patient and control groups were analyzed using Steel test. Retinal thickness, choroid layer thickness between DM patient and control groups were calculated using Kruskal-Wallis test and analyzed using Steel-Dwass test.

(7) Page 7, 2nd Paragraph, and Table 2: This reproducibility analysis is not needed in this paper. The authors did not discuss it at all (they mentioned the reproducibility as the study limitation, though). And above all, it is not essential data for this study aim. I recommend that it should be deleted or moved to Supplementary data.

Thank you so much for your comments.

As indicated, we did not discuss reproducibility analysis. However, we believe that reproducibility in measuring choroid thickness is important to clarify the reliability of data.

Therefore, we moved the initial Table 3 and Figure 2 to Supplementary table 1 and Supplementary figure 1, respectively.

(8) Page 8, 4th Paragraph: This paragraph is confusing. Please rewrite the paragraph for our readers to easily understand. And how were these subgroups' backgrounds? Was there no significant difference among the subgroups (including control) in their backgrounds?

Thank you so much for your comments.

It is well known that there is a systemic confounding factors in measuring choroidal thicknesses. Therefore, high systolic blood pressure and serum lipids in the DM group may have influenced the results.

Therefore, the revised version added the following in the discussion section.

Page 19, 2th Paragraph: Fifth, this study was able to adjust the local confounding factor that could affect the choroid structure. However, systolic blood pressure and serum lipids were significantly higher in the DM group than normal controls. These systemic confounding factors might affect the acquisition of data regarding choroidal structure.

(9) Table 3: The information should be presented in two separate tables, one focusing on the stage of diabetic retinopathy and the other on type of DME).

Thank you so much for your comments.

A separated table was created, focusing on the stage of the DR and the type of DME (Tables 3 ang 4).

Minor concerns,

(10) Was the flare measured in this study? The flare data may support the possible significance of VEGF in DME and choroidal layer thickness. (The relationship between the flare value and the concentration of VEGF in aqueous was reported before)

Thank you so much for your comments.

It is known that the breakdown of the blood-retina barrier in diabetic patients is correlated with a dysfunction of the blood-aqueous barrier. In fact, several researches showed that flare levels change after photocoagulation and anti-VEGF therapy in patients with DME. However, although interesting, there are no reports showing significant correlation between flare levels and choroidal thickness in DME.

In this study, the flare level of DME patients was not investigated, and its relationship could not be examined.

This will be an issue for a further study.

Reviewer #2:

1) Although the authors found -in each DM group- that there was no significant difference in systolic blood pressure, this latter was significantly higher in DM group than in control group (P<0.01). Do they think that this could have influenced their results? Please make a comment on it.

Thank you so much for your comments.

It has been pointed out that there is a systemic confounding factors in the choroidal thickness. Therefore, high systolic blood pressure and serum lipids in the DM group may have influenced the results. Therefore, in relation to reply to the reviewer 1’s comment, the revised version added the following in the discussion section.

Page 19, 2th Paragraph: Fifth, this study was able to adjust the local confounding factor that could affect the choroid structure. However, systolic blood pressure and serum lipids were significantly higher in the DM group than normal controls. These systemic confounding factors can affect the acquisition of data regarding choroidal structure.

2) Authors indicated in the methods that data from the total choroid layer, inner layer (choriocapillaris + medium vascular layer), and outer layer was manually identified based on findings of EDI-OCT horizontal scan through the fovea, as reported previously [22]. Since patients with DME usually do not have foveal depression, which scan did the authors include to measure choroidal layers?

Thank you so much for your comments.

The position where the foveal bulge was confirmed, or the position where the vertical height of the DME was the highest were defined as the central retinal thickness, and then the choroid thickness was measured beneath the positions.

Therefore, the following was added to the method section.

Page 7: To identify the measurement position of the choroid thickness, the position where the foveal bulge was confirmed, or the position where the vertical height of the DME was the highest were defined as the central retinal thickness, and then the choroid thickness was measured beneath the positions.

3) I know they considered excluded images with signal strength less than 6. Nevertheless, I believe that in this kind of study (manual measurements) it is important to statistically correct all the OCT images for signal strength index. Also, what is about the presence of significant motion artifacts?

Thank you so much for your comments.

It is a well-known fact that low-quality OCT images affect analysis. In this study, obtained images with a signal strength of 6 or more were included. Motion artifacts were resolved by using a fundus tracking system. Therefore, the following was added to the method section.

Page 7: Therefore, images with a signal strength of less than 6 were excluded from this study. In addition, motion artifacts were removed by the fundus tracking system.

4) I would suggest the authors to include just one eye per patient, as they correlated several parameters referred to a subject (for example: duration of diabetes, age, blood pressure and so on) to retinal parameters that are measured separately in a single eye. Although the numbers will be more limited, it would be more correct to use a single eye in the statistical analyses.

Thank you so much for your comments.

In the previous reports, there are many reports analyzing choroidal thicknesses using both eyes on the topic of DME (1. Unsal E et al. Choroidal thickness in patients with diabetic retinopathy. Clin Ophthalmol. 2014. 2. Kim JT et al. Changes in choroidal thickness in relation to the severity of retinopathy and macular edema in type 2 diabetic patients. Invest Ophthalmol Vis Sci. 2013. 3. Rewbury R et al. Subfoveal choroidal thickness in patients with diabetic retinopathy and diabetic macular oedema. Eye (Lond). 2016.).

In this study, the proportion of binocular DME cases with different forms of edema in each eye was 20%. This fact indicates that the DME even in the same case may have different pathology and different severity in each eye. In addition, this is a retrospective study, and if we selected and examined the one eye in binocular cases, bias may occur in DME showing different conditions in both eyes. Thus, prospective studies may be more appropriate for unilateral studies. Therefore, we think that the current study design including both eyes is meaningful.

5) The found that in stage of DR, inner CCT layer was significantly thicker in severe non-proliferative diabetic retinopathy compared to the control group (P < 0.05). How many patients with DME in this group?

Thank you so much for your comments.

The sNPDR-DME group included 17 eyes from 14 patients.

Therefore, the following was added to the discussion section.

Page 18: However, the mechanisms underlying thickened inner choroidal layers in sNPDR-DME+ group compared to the controls are unknown. In fact, the major histopathological findings in the inner layer of DM eyes were occlusion of choriocapillaris [3]. Therefore, in order to validate the clinicopathological correlation, it is necessary to investigate morphological changes in choroidal vessels and/or stromal components using recent imaging techniques.

6) Did they include patients with Type 2 DM? Type 2 and type 1 DM? These latter usually are younger and have fewer confounding factors or systemic comorbidities.

Thank you so much for your comments.

All diabetic patients examined in this study were type 2 DM. Hence, subjects of type 1 DM patients may be examined for future investigations. The revised version added the following statements in the method section.

Page 6, 1st paragraph: This retrospective observation study was approved by the institutional review committee of Teine Keijinkai hospital, and informed consent was obtained from all subjects (patients diagnosed with type 2 diabetes and healthy controls).

7) Based on their finding, the authors stated that systemic DM treatment alone is not an independent factor involving the choroidal thickness. Also, they speculated that one of the mechanisms underlying significantly thickened outer layer in DME＋DT-group might be correlated to chronic hyperglycemic condition caused by untreated DM.

One could argue why patients with DME- DT- (97 patients and second largest group) do not present any choroidal statistically significant difference as an early sign of the deteriorated choroidal microcirculatory system.

Please make a comment on it.

Thank you so much for your comments.

The choroid thickness may be regulated by VEGF produced in the eye. In fact, it has been reported that the intraocular VEGF protein concentration is higher than the serum concentration in DME eyes (Funatsu, H., et al., Increased levels of vascular endothelial growth factor and interleukin-6 in the aqueous humor of diabetics with macular edema. Am J Ophthalmol, 2002). Therefore, from the standpoint that both ocular local and systemic factors are related to CCT, it is considered that the single factor DT alone did not show a significant change, as we showed no significant changes between DME+DT- and DME+DT-.

Minor:

Page 5, please delete the double references #19

Thank you so much for your comments.

The corresponding part was corrected.

Thank you

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Dec 2019

Relationship between diabetic macular edema and choroidal layer thickness

PONE-D-19-26407R1

Dear Dr. Kase,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Demetrios G. Vavvas

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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 #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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 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 sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space 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 #1: (No Response)

Reviewer #2: Afeter the revision, authors have addressed all my comments and the manuscript quality has been improved.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

10 Dec 2019

PONE-D-19-26407R1

Relationship between diabetic macular edema and choroidal layer thickness

Dear Dr. Kase:

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 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Demetrios G. Vavvas

Academic Editor

PLOS ONE