The retina is one of the few tissues in the human body where the blood circulatory system can be non-invasively observed (1, 2). Retinal vascular characteristics can reflect the status of the microcirculation affected by ocular, cardiovascular, and systemic diseases, such as age-related macular degeneration, diabetic retinopathy, Alzheimer's disease, hypertension, etc. (3–11). Since early vascular alterations induced by diseases typically happened in microvasculature (12, 13), changes of the capillaries in the retinal macula are thought to be more sensitive to the early stage of disease progression and could provide significant information for the early diagnosis and monitoring of pathologic changes of related diseases (14–16).

Retinal blood vessels can be observed by imaging techniques such as fundus photography (17), optical coherence tomography angiography (OCTA) (18), fundus fluorescein angiography (FFA) (19), providing abundant retinal vascular structure and perfusion information. While OCTA and FFA could show the more detailed structure and functional changes of microvasculature, FFA is an invasive examination that may induce anaphylactic reactions (20), OCTA provides vascularity of both superficial and deep plexus but is prone to generate artifacts with a scanning imaging regime and its widespread in clinical use and disease screening is limited by the high cost, high patient cooperation and operation difficulties (18, 21). Currently, fundus photography is still the most widely used imaging technique for retinal examination and disease screening due to its advantages of non-invasiveness, relatively low cost, and ease to use the property.

The use of digital image analysis and machine learning techniques for fundus photography has become increasingly common over the past decades (22, 23), offering sophisticated techniques to acquire minute details and quantify the geometry of retinal vascular network (24, 25). Semiautomatic methods developed with Image J (26) and computer-assisted software such as Interactive Vessel Analysis (IVAN), Singapore I Vessel Assessment (SIVA), and Quantitative Analysis of Retinal Vessel Topology and Size (QUARTZ) (25, 27–29) could offer retinal vascular parameters such as vascular length, diameter, tortuosity, and so on. Clinical applications have been conducted showing these parameters may be important indicators of microvascular diseases that can be used in risk prediction (13, 15, 16). Nevertheless, most of the studies focused only on the larger blood vessels around the optic disc (5, 30).

Our former studies have developed a multiparametric retinal vascular network analysis method with a vessel segmentation algorithm based on dense block generative adversarial network (D-GAN) and automated vascular geometry quantification (14, 31). The method has been applied to explore the macular vascular geometry characteristics of fundus photography of patients with diabetes mellitus, showing microvascular morphological parameters may be indicators of the early retinal vessel changes for diabetic retinopathy (14). Though searching for disease-related potential vascular biomarkers is of great importance, the vascular geometry could be different between gender and changes could also be introduced during the normal aging process. While only a few studies have examined the potential sex and age interactions with retinal vasculatures (32, 33), no detailed research has been conducted to quantify the sex- and age-related changes of the microvascular network with fundus photography. In this study, we demonstrated a cross-sectional study aiming to explore the macular vascular geometry variations between sexes and characterized the age-related morphological changes with our multiparametric fundus photography analysis method, finding sensitive indicators of macular vascular geometry changes. Moreover, normative baseline data of both genders in different age groups was provided that can be used as references in identifying progressive macular vascular changes due to different pathologies.

The general age and gender characteristics of the studied subjects are shown in Table 1. There were no significant differences between the mean age of all the male and female subjects (32.706 ± 10.372 and 33.494 ± 10.620, respectively, p > 0.05) and the mean ages of both sexes in each age group (p > 0.05). Sex ratios were also equally represented in each age group.

Table 2 shows the comparison of macular vascular geometric parameters between all the male and female subjects. Figure 2 shows the mean ETDRS retinal vascular geometric parameters maps with gender differences. The D_f, VAR, and D_m of the inner ring, the D_f of the outer ring, and the D_f and VAR of the whole macular were significantly greater in males than females (Table 2; p < 0.001, p < 0.001, and p < 0.05, respectively), especially in the inferior quadrants (Figure 2; p < 0.001). There was no significant difference of τ_n between males and females (Table 2; Figure 2; p > 0.05).

Table 3 shows the association results between macular vascular geometric parameters and age. The D_f, VAR, and D_m of the whole macular, inner and outer rings all negatively associated with age (Table 3; Figure 3; p < 0.001), whereas the τ_n of the macular did not correlate with age (Table 3; Figure 3; p > 0.05).

The comparison of macular vascular geometric parameters of the whole, the inner and the outer rings among the different age groups are shown in Figure 4. It is resolved that, in the inner ring, G2 showed a significant decrease of the D_f compared with G1 (Figure 4; p < 0.05), while D_f, VAR, and D_m all decreased from G3 compared with the younger groups in the whole, inner and outer rings (Figure 4; p < 0.05). τn also has no significant differences (Figure 4; p > 0.05).

Table 4 shows the baseline values of D_f, VAR, D_m, and τ_n of both sexes in 4 age groups.

This is the first study that quantifies the sex- and age-related macular vascular geometry alterations in healthy populations with fundus photography using our customized automatic multiparametric vascular analysis software. We demonstrated that macular vascular D_f, VAR, and average vessel diameter D_m were all significantly lower in the female than male and all decrease with the increase of age, while vessel tortuosity τ_n showed no statistical difference, revealing the physiological variations of macular vascular geometry induced by sex and age. Normative baseline data of macular vascular geometric parameters for both the genders in different age groups were acquired that can potentially be used as references in identifying progressive macular vascular changes due to different pathologies.

In the analysis of the macular vascular geometric difference between male and female healthy subjects, we found that D_f and VAR in the macular area and D_m in the inner area of the macula were significantly greater in men than in women, indicating that male has more complex retinal vascular structures and larger vessel occupation and diameter in the macular area as compared with female. While no studies have demonstrated macular vascular structure analysis with fundus photography between genders, our results showing more abundant vascular network in men are in consistent with the former studies. Using OCTA, Yu et al. (37) reported lower blood flow index in the macula and larger fovea avascular zone in women, and Wang et al. (38) revealed higher superficial retinal vascular density in men than in women. Using fundus photography, Tapp et al. (39) showed that the retinal vascular diameter around the optic disk was larger in male. Since the retina is a tissue with high-oxygen consumption that need to rely on blood perfusion to maintain its metabolic levels, the retinal vascular structure is mainly influenced by the blood perfusion level (37, 40), which is regulated by blood pressure, intraocular pressure (IOP) (41), body mass index (BMI) (42), and hormone (43). Thus, the proven differences in these factors between genders (44) might be the reasons leading to the macular vascular geometry divergence. There were studies exploring gender differences in the epidemiology of ophthalmic diseases that have shown that macular diseases such as age-related macular degeneration (AMD) and macular holes (45, 46) were more likely to happen in females, which could also potentially be related to the macular vascular geometry differences. The detailed mechanisms of the gender-specific differences of macular vascular geometry remain unclear and require further study.

We have described the association between age and macular vascular geometric parameters showing that D_f, VAR, and D_m of the macular area all decrease with the increase of age. The association between age and D_f, VAR, and D_m were significant while the correlation coefficient is relatively low, indicating small age-related macular vascular geometry changes. This is similar to those reported in other studies (47, 48). One possible reason is that the age range of the samples in our study is not wide enough with the fewer elderly samples. The other reason could be that many factors influence macular vessels such as gender, axial length, refractive error (47, 49, 50), while this study limited in exploring only the age-induced changes. In the comparison of macular vascular geometric parameters of different age groups, we have revealed that statistical variation of vascular D_f happened from 26 to 35 age group while VAR and D_m decrease from 36 to 45 age group. These are in accordance with studies demonstrated in exploring retinal vascular alterations with aging (34, 51). Using fundus photography, Azemin et al. (51) and Liew et al. (34) measured the whole retinal and observed a significant decrease in the D_f with aging. Sun et al. (52) found both arterial and venular caliber decrease in the older groups using IVAN software to analyze peripapillary diameter of 3,019 health participants; Pose-Reino et al. (53) analyzed the retinal blood vessel caliber and found that the reduction of retinal arteriole caliber occurs during aging. While the retinal vascular structure is closely related to blood perfusion, measuring by OCTA, Yu et al. (37) have shown that macular perfusion decreased with increasing age in healthy Chinese eyes, and the parafoveal flow index and vessel area density decrease with aging at a rate of 0.6 and 0.4% per year. You et al. also reported that higher superficial and deep capillary densities were significantly associated with younger age and a general negative association between macular vessel density and age (47). Similar results have been reported in several other studies (48). Similar to other human organs, the physiological function of the eye decreases with aging. Since maintaining normal function of the retina depends on normal metabolism level (54), aging causes tissue loss and a corresponding reduction in oxygen demand, altering vascular system, and declining the perfusion of macular, resulting in a reduction in vascular structure and complexity. Studies have also determined that aging could cause thickening of the vascular wall, reducing vascular elasticity (55), and the rigidity of the vessel wall would lead to the decrease of the vessel diameter (56). The reduction in the macular vascular structure might potentially account for some ocular diseases related to age such as age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and so on (57, 58).

In this study, we also found that the macular vascular tortuosity τ_n has no variation between gender and across aging. Many studies have shown that retinal vascular tortuosity variation is one of the earliest indicators of a number of relevant diseases such as diabetes, cerebrovascular disease, stroke, ischemic heart disease (14, 59, 60). Our observation indicates no statistical difference of macular vascular tortuosity in healthy individuals of different gender and age further enhancing the feasibility of using tortuosity as a sensitive indicator for the early diagnosis of related diseases.

There are several limitations of this study. First, the number of subjects in the elderly age group was relatively small due to the fact that the healthy subjects we have collected tend to be young since older people are more likely to suffer from systemic diseases such as hypertension, heart disease, and diabetes or cloudy refractive interstitium affecting the fundus image quality (6, 9, 14, 34, 50). Future collection of more healthy elderly subjects to balance the sample size of all the age groups would be beneficial. Second, while this study is limited in exploring macular vascular geometry changes with age and gender, there are many other factors that affect the macular vasculature, such as axial length, refractive error, IOP, blood pressure, and so on (34, 41, 49, 50, 61), it is worth further in-depth study considering these factors. Third, since this is a cross-sectional study without temporal information, future prospective investigation with systematic clinical information acquired could provide more detailed support of the vascular alteration mechanisms. Moreover, with the binocular data acquired in this study, analyzing the intereye correlation and difference of the macular vasculature with fundus photography and comparing it to those acquired with OCTA would be interesting (62). Finally, with the baseline data from the healthy subjects gained, the vascular geometric variation induced by different diseases could be further explored. Improvement of our current vascular analysis software to distinguish the retinal arteries and veins would also provide more functional information to study the retinal vascular circulation.

In conclusion, findings from this study reveal the sex- and age-related macular vascular geometry alterations in healthy human subjects and provide the normative baseline data for both genders in different age groups. The application of our automatic retinal vascular analysis system could provide a useful approach for detecting subtle macular vessels change with fundus photography.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Guangzhou, China 2017KYPJ104, Medical Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

JY and PX conceived and planned the research and reviewed and modified the manuscript. ZF, GW, and HX carried out the studies and wrote the first draft of the manuscript. GL and TD conducted the fundus imaging. GW, PX, and ML processed the images and organized the database. ZF and GL performed the statistical analysis. All the authors provided critical feedback and helped to shape the research, analysis, and manuscript.

This study was supported by the Key-Area Research and Development Program of Guangdong Province (No. 2019B010152001), the National Natural Science Foundation of China (81901788), and the Guangzhou Science and Technology Program (202002030412).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.