Evaluation of Choroidal Vascularity Index, Retinal and Optic Nerve Changes in Erectile Dysfunction

doi:10.21203/rs.3.rs-5955829/v1

Objective: To investigate choroidal alterations through assessment of subfoveal choroidal thickness (SFCT) and choroidal vascularity index (CVI), optic nerve modifications through evaluation of peripapillary retinal nerve fiber layer (RNFL), and macular changes through assessment of ganglion cell layer (GCL) in patients with erectile dysfunction, and to compare these parameters with age-matched healthy controls.

Methods: This cross-sectional study included 30 eyes of 30 patients diagnosed with erectile dysfunction based on the International Index of Erectile Function (IIEF-15) questionnaire. Spectral-domain optical coherence tomography (SD-OCT) was employed to evaluate peripapillary RNFL and GCL thickness. OCT images were acquired and processed using the Image-J software to calculate CVI. All measurements were compared with those obtained from 30 age-matched healthy volunteers.

Results: RNFL thickness values in the inferior, superior and nasal quadrants and CVI values were found to be statistically significantly lower in the erectile dysfunction group compared to the control group (p<0.001, p=0.006, p<0.001, p<0.001). GCL thickness values in all quadrants and SFCT values were observed to be lower in the erectile dysfunction group compared to the control group. However, the difference was not statistically significant. In addition, a moderate positive correlation was found between the CVI and erectile function (r=0.473, p=0.008).

Conclusions: RNFL and CVI may be valuable markers in addition to questionnaires in the diagnosis of erectile dysfunction. Furthermore, due to the rapid, repeatable and non-invasive nature of the SD-OCT device, RNFL and CVI measurements may act as predictive factors in erectile dysfunction.

Choroidal vascularity index

erectile dysfunction

ganglion cell layer

optical coherence tomography

retinal nerve fiber layer

Erectile dysfunction is a condition defined as the inability to achieve or maintain a satisfactory erection for sexual intercourse [1]. The prevalence among men in their 40s to 70s in the United States is approximately 52%, while in Turkey, according to the Turkish Society of Andrology, the prevalence is estimated to be 33% in men older than 40 years [2]. Diagnosis and severity assessment generally require subjective grading such as questionnaires [3]. The International Index of Erectile Function (IIEF-15) is a questionnaire preferred by many clinicians for diagnosing erectile dysfunction, determining its severity, and also for monitoring its response to treatment [3].

The etiology is multifactorial, including organic and psychological reasons, and studies have shown that chronic vascular diseases such as hypertension and coronary artery disease are the main etiological reasons [4]. The hypothesized pathophysiological mechanism involves progressive degenerative alterations leading to endothelial dysfunction and consequential reduction in nitric oxide synthesis and bioavailability [5]. Moreover, the "artery size" hypothesis posits that atherosclerotic processes preferentially initiate in vessels of smaller caliber, thereby simultaneously affecting both penile arteries and the microvasculature of the retina and choroid [6]. This hypothesis elucidates the sequential manifestation of atherosclerotic symptomatology across diverse vascular territories by postulating that vessels of smaller diameter, such as penile arteries (1–2 mm) or retinal/choroidal vessels, exhibit clinical manifestations earlier than larger vessels such as coronary arteries (3–4 mm) when subjected to comparable atherosclerotic burden. Specifically, a 50% stenotic lesion induces substantially greater hemodynamic compromise in smaller caliber arteries compared to larger vessels, thereby explaining the frequent antecedence of erectile dysfunction to coronary symptomatology despite the systemic nature of atherosclerotic progression. This conceptual framework substantiates several clinical observations: erectile dysfunction and coronary arteriopathy represent different manifestations of identical underlying pathology; smaller vessels function as early indicators of vascular integrity; and the temporal sequence of vascular symptomatology across different anatomical locations adheres to a predictable pattern based on vessel diameter [7–9] (Figure-1). Furthermore, the structural and functional similarities between the choroid and corpus cavernosum, characterized by dense vascularity and phosphodiesterase type 5 expression, augment the plausibility of concurrent pathological involvement [10].

For the objective assessment of these vascular alterations, spectral-domain optical coherence tomography (SD-OCT) offers a rapid, reproducible, and non-invasive imaging modality capable of generating high-resolution cross-sectional visualization of retinal and optic nerve head architecture. SD-OCT imaging facilitates precise quantification of retinal layer thickness and identification of specific neurodegenerative changes, including axonal and ganglion cell loss, through measurement of retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) thickness [11]. Additionally, subfoveal choroidal thickness (SFCT) can be accurately and reliably quantified using enhanced depth imaging OCT (EDI-OCT), enabling identification and classification of choroidal vascular alterations [12]. The choroidal vascularity index (CVI) represents an advanced quantitative parameter derivable through specialized analysis of EDI-OCT images. Defined as the ratio of choroidal luminal area to total choroidal area [13], this metric provides a comprehensive assessment of choroidal vascularity and overcomes numerous limitations inherent to isolated measurements of choroidal thickness [14]. Contemporary literature has established CVI as a more reliable and consistent methodology for evaluating choroidal structural integrity compared to choroidal thickness measurements alone [13].

The present study aims to report the choroidal changes by evaluating SFCT and CVI, optic nerve changes by evaluating peripapillary RNFL and macular changes by evaluating GCL in erectile dysfunction patients and compare these findings with age-matched healthy volunteers. In addition, the correlation between IIEF-15 parameters and SD-OCT findings was analyzed. To our knowledge, this is the first study to evaluate RNFL and GCL thickness and CVI in erectile dysfunction patients.

This study was approved by the institutional review board of xxx Training and Research Hospital (date: 28.02.2024, number: 2024/241) and adheres to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject. After approval by the Institutional Review Board, 30 patients who applied to the Urology Department of Buca Seyfi Demirsoy Training and Research Hospital and were diagnosed with erectile dysfunction and 30 age-matched healthy volunteers were enrolled in this study. In the urology department, the diagnosis was made by the same urologist (T.T.) by evaluating the IIEF-15 questionnaire. The questionnaire has 15 questions that assess five domains: erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction. Through the IIEF-15 questionnaire, erectile dysfunction can be graded based on the erectile function domain. An erectile function score in the range of 26–30 is defined as no erectile dysfunction, a score in the range of 22–25 is defined as mild erectile dysfunction, a score in the range of 17–21 is defined as mild to moderate, a score in the range of 11–16 is defined as moderate, and a score in the range of 6–10 is defined as severe erectile dysfunction [3]. All information regarding the domains was recorded. In addition, the patients' testosterone levels were recorded.

To minimize potential confounding factors, stringent exclusion criteria were applied. Patients with established diagnoses of hypertension, diabetes mellitus, or coronary artery disease were excluded from participation. Additionally, individuals with other systemic pathologies potentially affecting retinal or choroidal structures, or conditions that might alter ocular hemodynamics, such as smoking, were excluded. Patients undergoing any topical or systemic pharmacotherapy were similarly excluded. Ocular exclusion criteria encompassed any pathological conditions that could compromise measurement accuracy, including significant cataract, glaucoma, congenital or acquired retinal abnormalities, refractive errors exceeding ± 3 diopters, nystagmus, and corneal opacities. Furthermore, individuals with a history of ocular surgical intervention or trauma were excluded from participation.

The control group consisted of age-matched healthy individuals who presented to our ophthalmology outpatient clinic for routine ophthalmic evaluation during the study period. Potential control subjects underwent consecutive screening for eligibility based on the study's inclusion and exclusion parameters. Those who fulfilled all criteria and provided informed consent were enrolled as controls. Meticulous attention was directed toward ensuring demographic concordance between the control and erectile dysfunction cohorts to minimize potential confounding variables.

All patients incuded in the study, initially were performed a detailed ophthalmological examination including best corrected visual acuity determination with the Snellen chart, intraocular pressure measurement with applanation tonometry and anterior-posterior segment evaluation with slit-lamp biomicroscopy. After the examination, SD-OCT (DRI-OCT Triton, Topcon, Inc, Tokyo, Japan) images were taken to evaluate RNFL thickness values in 4 quadrants and GCL thickness values in 6 quadrants. EDI-OCT mode was preferred to measure SFCT and calculate CVI. To avoid diurnal variations, all EDI-OCT scans were performed at the same time of day, between 10:00 and 12:00, for each patient and the volunteer group. Two blinded physicians (P.K. and O.K.) conducted the SFCT measurements. The mean value of the two measurements was calculated, and the differences between the readings of the blinded physicians were found to be within 10 µm of the mean. In the event that the measurements differed by more than 10 µm, they were repeated. The inter-examiner reproducibility of the subfoveal choroidal thickness (SFCT) measurements was evaluated using the intraclass correlation coefficient (ICC).

To evaluate the RNFL and GCL thickness values in each group, SD-OCT device’s automatic calculation and reporting system was used, and the thickness values of 4 quadrants (superior, temporal, inferior and nasal) for RNFL, and 6 quadrants (superior, superotemporal, superonasal, inferior, inferotemporal, and inferonasal) for GCL were recorded.

For CVI analysis, the SD-OCT images through the fovea were selected for image binarization and segmented according to the protocol described by Agrawal et al [15] using Image J version 1.53 a (National Institutes of Health, Bethesda, MD, USA). The polygon selection tool helped to detect the total choroidal area (TCA) between the retinal pigment epithelium and the choroid-scleral junction for the subfoveal region within a width of 1500 µm (750 µm either side of the fovea). Regions of interest (ROIs) were added to the ROI manager. After the image was converted to 8-bit, a Niblack autolocal thresholding tool was applied. This tool helped to obtain the mean pixel value with standard deviation (SD) for all points. The luminal area (LA) was defined using the color thresholding tool and was also added to the ROI manager. To determine the area of vascularity within the selected polygon, both areas were selected in the ROI manager and merged using an "AND" operation. The CVI value was defined as the ratio of LA to TCA.

In addition, the correlations between the SD-OCT measurements and the IIEF-15 results (erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall sexual satisfaction) and total testosterone levels were analyzed.

Statistical Analysis

IBM The Statistical Package for the Social Sciences version 25 (SPSS Inc., Chicago, IL, USA) was used for statistical purposes. Categorical variables were expressed as frequencies and percentages, and numerical variables were expressed as means and standard deviations. Kolmogorov-Smirnov tests were used to determine whether the data were normally distributed. Independent t-test was used to evaluate differences in normally distributed data. Mann-Whitney U test was used to determine differences in non-normally distributed data. Pearson's correlation test and Spearman's correlation test were used to examine correlations in both normally and non-normally distributed data. A p-value less than 0.05 was considered statistically significant. For statistical purposes, values from the left eyes were analyzed in both the study and control groups. The sample size was calculated based on the findings of a previous study by Kim et al. (6), which observed that the Small Choroidal Vessel Layer (SCVL) thickness in patients with erectile dysfunction (ED) was significantly thinner than in the control group (control: 69.8 ± 24.3 µm vs. ED: 55.1 ± 19.9 µm; p = 0.017). The G*Power program was used for the sample size calculation. A 95% confidence interval and 80% power were targeted to detect a significant difference between the two groups. For 80% power (effect size: 0.5, alpha error probability: 0.05), the minimum required sample size was determined to be 26 patients.

The mean age was 50.9 ± 8.8 years in the erectile dysfunction group and 51.4 ± 8.4 years in the control group. There was no statistically significant difference in age between the groups (p = 0.723). Information on age, IIEF-15 scores and testosterone levels of erectile dysfunction patients are summarized in Table 1. The RNFL, GCL, SCFT and CVI values of the erectile dysfunction and control groups are summarized in Table 2. RNFL thickness values in the inferior, superior and nasal quadrants and CVI values were found to be statistically significantly lower in the erectile dysfunction group compared to the control group (p < 0.001, p = 0.006, p < 0.001, p < 0.001, p < 0.001, respectively). GCL thickness values in all quadrants and SFCT values were observed to be lower in the erectile dysfunction group compared to the control group. However, the difference was not statistically significant. In addition, a moderate positive correlation between CVI and erectile function was observed (r = 0.473, p = 0.008). Correlations between SD-OCT measurements and IIEF-15 and total testosterone levels are summarized in Table 3.

Table 1. Demographic characteristics, International Index of Erectile Function-15 (IIEF-15) domain scores, and serum testosterone levels in patients with erectile dysfunction compared to age-matched controls.

Statistical analysis was performed using the Independent t-test for normally distributed variables and the Mann-Whitney U test for non-parametric data.

Table 2

Table 2. Quantitative comparison of retinal nerve fiber layer (RNFL) thickness, ganglion cell layer (GCL) thickness, and choroidal vascularity index (CVI) between erectile dysfunction cohort and control subjects.
	Study group	Control group	p
Retinal Nerve Fiber Layer Thickness (µm)
Inferior quadrant (mean ± SD)	125.16 ± 22.33	143.80 ± 15.81	> 0.001^a
Superior quadrant (mean ± SD)	127.66 ± 17.60	140.26 ± 16.94	0.006 ^a
Nasal quadrant (mean ± SD)	74.20 ± 14.28	88.70 ± 14.05	> 0.001^a
Temporal quadrant (mean ± SD)	72.26 ± 11.61	77.46 ± 11.25	0.083^a
Ganglion Cell Complex Thickness (µm)
Superior quadrant [median (min-max)]	110.0 (78–123)	112.0 (97–146)	0.214 ^b
Supero-nasal quadrant [median (min-max)]	121.5 (92–132)	120.5 (106–155)	0.325^b
Infero-nasal quadrant (mean ± SD)	118.16 ± 9.89	122.03 ± 10.28	0.143^a
Inferior quadrant (mean ± SD)	107.33 ± 9.16	108.60 ± 8.52	0.581^a
Infero-temporal quadrant [median (min-max)]	101.0 (76–111)	101.50 (89–130)	0.446 ^b
Supero-temporal quadrant [median (min-max)]	97.0 (74–107)	100.0 (85–122)	0.125^b
Choroidal Vascularity Index (%) [median (min-max)]	60.0 (57.6–71)	67.3 (59.8–74.5)	> 0.001 ^b
Subfoveal Choroidal Thickness (µm) (mean ± SD)	312.70 ± 59.51	334.56 ± 60.42	0.163^a
^a = Independent t test; ^b = Mann-Whitney U test.
Data were analyzed using the Independent t-test for parametric variables and the Mann-Whitney U test for non-parametric variables.Abbreviations: SD; standard deviation, min; minimum, max; maximum.

Table 3

Correlation analysis between spectral-domain optical coherence tomography (SD-OCT) parameters and erectile function metrics (IIEF-15 domain scores and total testosterone levels).
	Total Testesteron (pg/mL)	Erectile function	Orgasmic function	Sexual desire	Intercourse satisfaction	Overall sexual satisfaction
RNFL
Inferior quadrant	r = 0.172 p = 0.363	r =-0.205 p = 0.277	r =-0.015 p = 0.938	r =-0.035 p = 0.854	r =-0.100 p = 0.599	r = 0.042 p = 0.825
Superior quadrant	r = 0.039 p = 0.839	r = 0.042 p = 0.824	r =-0.088 p = 0.644	r =-0.404, p = 0.027	r = 0.220 p = 0.243	r = 0.009 p = 0.961
Nasal quadrant	r =-0.328 p = 0.077	r =-0.177 p = 0.349	r = 0.026 p = 0.891	r =-0.145 p = 0.445	r = 0.159 p = 0.402	r = 0.063 p = 0.741
Temporal quadrant	r = 0.073 p = 0.703	r = 0.227 p = 0.227	r = 0.321 p = 0.084	r = 0.270 p = 0.149	r =-0.011 p = 0.955	r = 0.167 p = 0.377
GCL
Superior quadrant	r = 0.040 p = 0.834	r =-0.120 p = 0.258	r =-0.050 p = 0.793	r =-0.198 p = 0.295	r = 0.216 p = 0.251	r = 0.033 p = 0.861
Superonasal quadrant	r =-0.100 p = 0.599	r =-0.124 p = 0.515	r =-0.114 p = 0.547	r =-0.046 p = 0.809	r = 0.105 p = 0.580	r =-0.096 p = 0.615
Inferonasal quadrant	r =-0.098 p = 0.606	r =-0.040 p = 0.835	r = 0.042 p = 0.825	r = 0.01 p = 0.930	r =-0.064 p = 0.738	r = 0.012 p = 0.949
Inferior quadrant	r = 0.087 p = 0.646	r =-0.085 p = 0.653	r = 0.173 p = 0.362	r = 0.174 p = 0.357	r =-0.100 p = 0.599	r = 0.032 p = 0.865
Inferotemporal quadrant	r =-0.159 p = 0.400	r =-0.029 p = 0.881	r =-0.069 p = 0.716	r =-0.012 p = 0.949	r =-0.007 p = 0.969	r =-0.019 p = 0.920
Superotemporal quadrant	r =-0.176 p = 0.351	r =-0.163 p = 0.389	r = 0.127 p = 0.503	r =-0.080 p = 0.674	r = 0.070 p = 0.714	r =-0.118 p = 0.535
CVI	r =-0.104 p = 0.583	r = 0.473 p = 0.008	r =-0.068 p = 0.720	r = 0.234 p = 0.213	r = 0.037 p = 0.845	r = 0.114 p = 0.547
SFCT	r =-0.136 p = 0.475	r = 0.074 p = 0.698	r =-0.261 p = 0.163	r = 0.007 p = 0.971	r =-0.075 p = 0.692	r = 0.360 p = 0.051
Abbreviations: SFCT, subfoveal choroidal thickness; CVI, choroidal vascularity index; RNFL, retinal nerve fiber layer; GCL, ganglion cell layer; IIEF, The International Index of Erectile Function; SD-OCT, Spectral-Domain Optical Coherence Tomography
Associations were evaluated using Pearson's correlation coefficient for normally distributed variables and Spearman's rank correlation coefficient for non-parametric data.

In the present study, RNFL thickness values in the inferior, superior, and nasal quadrants and CVI values were statistically significantly lower in the erectile dysfunction group compared with the control group. GCL thickness values in all quadrants and SFCT values were observed to be lower in the erectile dysfunction group compared to the control group. However, the difference was not statistically significant. Furthermore, a moderate positive correlation was demonstrated between the choroidal vascularity index and erectile function.

To our knowledge, there are no previous studies evaluating CVI, RNFL, and GCL in patients with erectile dysfunction. However, several investigations have examined the small choroidal vascular layer—defined as the choriocapillaris layer and medium-sized choroidal vessels—in this population. In a study by Kim et al. [6], this layer was found to be significantly thinner in patients with erectile dysfunction compared to healthy controls. Additionally, the authors reported that the thickness of this layer decreased proportionally with the severity of erectile dysfunction as determined by IIEF scoring.

Previous research has also explored the effect of phosphodiesterase type 5 inhibitors, the first-line pharmacological intervention for erectile dysfunction, on choroidal thickness. In a study by Aslan et al. [16], an increase in small vessel layer and total choroidal thickness was observed in erectile dysfunction patients receiving tadalafil. Similarly, another investigation demonstrated that sildenafil administration increased choroidal thickness by enhancing blood flow to the short posterior ciliary arteries [17]. These findings align with the results of our study regarding choroidal vascular alterations as assessed by CVI, suggesting that choroidal vasculopathy may represent a significant pathophysiological feature in patients with erectile dysfunction.

Endothelial dysfunction resulting from progressive degenerative changes is widely recognized as a potential underlying mechanism in the development of erectile dysfunction [18]. Metabolic conditions such as cardiovascular disease, hypertension, and diabetes mellitus, which are frequently associated with erectile dysfunction, share a common pro-inflammatory pathophysiological background that can precipitate endothelial dysfunction. This dysfunction is characterized by impaired vasodilation, diminished production and release of nitric oxide, and increased vascular permeability [19]. Importantly, the endothelial dysfunction implicated in erectile pathophysiology is not confined to the penile vasculature. A study by Montorsi et al. [20] demonstrated that erectile dysfunction may serve as an early warning sign of coronary artery disease and a potential marker for systemic vascular pathology. Additional research has suggested that individuals without clinically diagnosed atherosclerosis may nonetheless exhibit impaired endothelial function if they present with erectile dysfunction, thus positioning erectile dysfunction as a potential early indicator for atherosclerotic development [21]. The "artery size" hypothesis provides a compelling explanatory framework for our findings. This hypothesis posits that small-diameter arteries exhibit increased susceptibility to atherosclerotic processes, elucidating the relationship between erectile dysfunction and choroidal vascular anomalies, as both the choroid and corpus cavernosum possess similar microvascular architecture characterized by smaller vessel caliber [6]. Consequently, the correlation between choroidal vascular parameters such as SFCT and CVI with erectile function observed in our study is not unexpected. Several factors contribute to this association, including the shared dense vascular infrastructure, comparable vessel diameter profiles, presence of phosphodiesterase type 5 in both tissues, and similar underlying pathophysiology centered on endothelial dysfunction.

Previous studies have also suggested that chronic low-grade inflammation may contribute significantly to the development of erectile dysfunction in men. Therefore, the reduction in RNFL and GCL thickness in the erectile dysfunction group observed in our study may result from the combined effects of endothelial dysfunction and sustained inflammatory processes [22]. Although autoregulatory mechanisms normally maintain stable ocular blood flow to the retina, choroid, and optic nerve head, research by Güçlü et al. [23] demonstrated that patients with atherosclerosis exhibited impaired autoregulatory capacity, resulting in reduced perfusion and subsequent apoptosis of retinal ganglion cells. Perfusion fluctuations secondary to atherosclerotic changes may induce damage to both ganglion cells and their axons. Thus, compromised blood flow associated with erectile dysfunction represents a plausible explanation for the RNFL and GCL thinning observed in these patients.

The current study has several limitations. The relatively small sample size of 30 patients with ED may restrict the statistical power and generalizability of our findings. This small sample size also limited our ability to perform meaningful multivariable regression analysis, which would require a larger number of subjects per variable analyzed. Future multi-center studies with larger cohorts would help validate our observations and potentially uncover additional associations that may not have been detectable in our current sample, while also enabling multivariable regression models to better control for confounding variables and establish more definitive associations. Psychological factors known to be associated with erectile function were not collected and analyzed. While we excluded major vascular diseases such as hypertension, diabetes, and coronary artery disease, we did not specifically control for all vascular risk factors such as dyslipidemia and obesity, which could potentially influence both erectile function and ocular parameters. Another limitation is the potential for inter- and intra-observer bias during manual choroidal thickness measurements. However, this bias is a common limitation encountered in studies investigating choroidal thickness. The utilization of two independent observers has the potential to mitigate this issue to a certain extent. Additionally, our statistical analysis relied primarily on p-values rather than reporting effect sizes and confidence intervals, which limits a comprehensive understanding of the observed differences between groups.

In conclusion, the anatomical similarities between vascular structures in the choroid and corpus cavernosum, underlying inflammatory processes, and comparable mechanisms of endothelial dysfunction may explain the correlations observed in our study. Our preliminary findings indicate that RNFL thickness and CVI measurements might be associated with erectile dysfunction, though prospective longitudinal studies would be necessary to establish any causal relationship. While our results are promising, we acknowledge that the cross-sectional design of our study precludes definitive conclusions regarding causality or the predictive value of these parameters. Nevertheless, given the rapid, reproducible, and non-invasive nature of spectral-domain optical coherence tomography, further research into the relationship between ocular vascular parameters and erectile dysfunction is warranted. Future prospective studies with larger cohorts and longitudinal follow-up are needed to determine whether these parameters could eventually serve as adjunctive tools in the assessment of patients with erectile dysfunction.

Acknowledgements: None

Author contributions: All authors contributed significantly to the creation of this manuscript; each fulfilled criteria as established by the ICMJE.

Human Ethics and Consent to participate: Both obtained. This study was approved by the institutional review board of Buca Seyfi Demirsoy Training and Research Hospital and adheres to the tenets of the Declaration of Helsinki. (date: 28.02.2024, number: 2024/241) .Authors obtained consent from the patients for publishing the photo.

Consent for publication: Authors obtained consent from the patients for publishing the photo.

Declaration of conflicting interest: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Pelin Kiyat and Omer Karti. The first draft of the manuscript was written by Pelin Kiyat and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Informed consent was obtained from all individual participants included in the study.

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No competing interests reported.

Evaluation of Choroidal Vascularity Index, Retinal and Optic Nerve Changes in Erectile Dysfunction

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Abstract

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Introduction

Materials and Methods

Statistical Analysis

Results

Discussion

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References

Additional Declarations

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