*2.3. En Face CVI estimation*

Adaptive histogram equalization was employed (using a built in MATLAB v2018b function) in order to increase the contrast between choroidal vessel lumen and the stroma. Blood vessels were then separated using the block-based particle swarm optimization (PSO) thresholding [22,27]. The binarized images were reviewed by two independent observers blinded to each other to assess whether the images were correctly converted by comparing with the original en face OCT images. This process was performed twice for each image by each observer.

CVI was calculated for every en face image separated by 5 μm within the choroid volume. The layer of small choroidal vessels, including choriocapillaris, was defined as a dense network of small vessels just 10 μm beneath Bruch's membrane.

The points of measurements were manually identified in each eye, focusing on major anatomical locations (i.e., Bruch's membrane, choriocapillaris and choroidoscleral interface) and at various depths from RPE–Bruch's membrane complex. The maximum choroidal thickness across the volume cube was divided by three (superficial or inner layer, medium and deep or outer layer) for both eyes. Hence, the mean CVI was calculated for the choriocapillaris, the inner/superficial third, the middle/medium third and the outer/deep third of the choroidal thickness. (Figures 1 and 2).

#### *2.4. Statistical Analysis*

The statistical analysis was conducted with R (version 4.0.0) and RStudio (version 1.2.5042) software. The Kolmogorov–Smirnov test was used to evaluate the normal distribution for each variable. The CVI was compared between CSC eyes and fellow eyes by using paired samples t-test or Wilcoxon test. A repeated measures ANOVA or Friedman test was used to compare the choroidal vascularity of choriocapillaris, superficial, medium and deep third of the choroid. A *p* value < 0.05 was considered statistically significant.

**Figure 1.** *Cont*.

**Figure 1.** Original en face optical coherence tomography (OCT) scans and the software-processed images of an affected eye of a patient with central serous chorioretinopathy. Original en face OCT scan images of the superficial (**a**), medium (**b**) and deep (**c**) choroidal layer; binarized images of the superficial (**d**), medium (**e**) and deep (**f**) choroidal layer; OCT B-scan across the foveal center (**g**).

**Figure 2.** *Cont*.

**Figure 2.** Original en face optical coherence tomography (OCT) scans and the software-processed images of the fellow eye of the same patient of Figure 1. Original en face OCT scan images of the superficial (**a**), medium (**b**) and deep (**c**) choroidal layer; binarized images of the superficial (**d**), medium (**e**) and deep (**f**) choroidal layer; OCT B-scan across the foveal center (**g**).

#### **3. Results**

A total of 20 patients (16 males and 4 females) were included. The average age was 50.7 ± 9.96 years. The average BCVA was 0.28 ± 0.35 logMAR for CSC eyes and 0.03 ± 0.09 logMAR for fellow eyes. Previous treatments included only nonsteroidal anti-inflammatory drugs (10 patients). The average time between the diagnosis and the evaluation was 2.42 ± 2.47 years.

The gender- and age-matched control group included 20 eyes of 10 individuals (eight males and two females) with a mean age of 48.8 ± 3.5 years. The demographic data showed no statistical difference with the study group (all *p* > 0.05).

The choroidal parameters in CSC, fellow and healthy eyes are reported in Table 1.

The subfoveal CT was significantly higher in eyes with CSC compared with fellow eyes (489.8 ± 13.4 vs. 433.7 ± 12.2; *p* = 0.047). The first third segment thickness resulted on average 163.3 ± 44.8 μm and 144.6 ± 41.6 μm for affected eyes and fellow eyes, respectively. Consecutively, these values represented the average thicknesses of the choroidal segments. For the average en face CVI, no significant difference between the CSC and fellow eyes was observed (*p* = 0.681). Similarly, no significant differences in the choriocapillaris, superficial, medium and deep CVI were found (respectively, *p* = 0.940, *p* = 0.685 and *p* = 0.411; *p* = 0.627) (Table 1).


**Table 1.** Choroidal parameters in eyes with CSC, fellow and healthy eyes.

CSC—central serous chorioretinopathy; CT—choroidal thickness; CVI—choroidal vascularity index.

There was a significant difference in subfoveal CT between healthy eyes and both eyes of CSC patients. However, with regard to the CVI layers' comparison, no difference was shown in the layer comparison between healthy and CSC or fellow eyes.

Although a different trend of changes between CSC eyes and fellow eyes, choriocapillaris, superficial, medium and deep CVI did not significantly differ for both (*p* = 0.73; *p* = 0.16). On the contrary, healthy eyes showed a significant difference of CVI among the various choroidal layers (*p* < 0.01).
