Vascular endothelial growth factor (VEGF)-C and its receptors, soluble VEGFR-2 and VEGFR-3, in polycystic ovary syndrome

Introduction

Polycystic ovary syndrome (PCOS) is generally accompanied by a combination of endocrine and reproductive disorders with a wide range of clinical signs, abnormal menstruation cycles, hyperandrogenism, and polycystic ovary morphology [1]. Although the etiopathogenesis of the disease has not yet been thoroughly defined, the condition of low-grade inflammation such as obesity, insulin resistance, hyperinsulinemia, and dyslipidemia is frequently observed in PCOS and is believed to play a pivotal role in its development [2]. Moreover, many studies have found a higher risk of pregnancy-related complications in women with PCOS [3]. The diagnosis of PCOS is received by two of these criteria such as ovulation dysfunction, hyperandrogenism, and polycystic ovaries on ultrasonography [2]. However, there is still no specific biomarker that can be an effective method for the diagnosis of the disease. Treatment options for PCOS require lifestyle changes and medical management to limit the metabolic features of the disease [4].

The disruption of the angiogenesis process in the ovary, follicular fluid, and serum plays a significant role in the pathophysiology of PCOS [5]. A key component of angiogenesis, vascular endothelial growth factor (VEGF) promotes the proliferation and migration of endothelial cells [6]. Several studies have shown high VEGF levels in the serum and follicular fluid of PCOS patients and VEGF gene expression in PCOS ovaries [7], [8], [9]. VEGF gene upregulation in the ovarian stroma observed in PCOS has also been linked to ovulation disorders and subfertility [9]. VEGF-C is a family member of VEGF that is involved in angiogenesis and lymphangiogenesis [10]. The decreased VEGF-C protein level in serum and decreased VEGF-C gene and protein expression in granulosa cells of PCOS patients have been reported previously [11, 12]. VEGF-C functions via VEGF receptor (VEGFR)-2 and VEGFR-3 [10]. A soluble form of VEGFR-2 (sVEGFR-2) can decrease VEGF-mediated angiogenic activity by binding the VEGF before reaching its membranous receptors or directly binding its membrane receptor [13]. Subjects with obesity and the metabolic syndrome demonstrated higher serum levels of sVEGFR-2 [14, 15]. VEGFR-3 is mainly involved in lymphangiogenesis as well as angiogenesis [16]. A study by Zheng et al. demonstrated a decrease in serum VEGFR-3 protein level in PCOS patients according to protein array analysis [11, 12].

In the current research, we focused on evaluating the differences in the concentration of serum VEGF-C, sVEGFR-2, and VEGFR-3 between subjects with PCOS and healthy controls and determining the association between these proteins and hormonal and biochemical parameters to identify the value of serum VEGF-C and its receptors as potential diagnostic circulating markers among patients with PCOS. Furthermore, this is the first study evaluating the levels of circulating sVEGFR-2 and its relationship with serum levels of VEGF-C and VEGFR-3 in PCOS.

Materials and methods

Results

ROC analysis of serum sVEGFR-2 levels for PCOS diagnosis

The cutoff value was 4.24 ng/mL, with an appropriate sensitivity (68 %) and specificity (64 %) to distinguish patients with PCOS from controls (area under the curve [AUC]=0.772, 95 % confidence interval [CI]=0.650–0.893, p<0.001) (Figure 1).

Figure 1:

ROC curve analysis of sVEGFR-2 levels for PCOS. AUC: 0.772 (95 % CI: 0.650–0.893, p<0.001).

Discussion

Growth factors are thought to be involved in the etiology of PCOS, and according to previous studies, serum VEGF levels in the serum are noticeably greater in those with PCOS [7, 8, 17]. Furthermore, it has been reported that elevated serum VEGF levels in PCOS may be related to increased ovarian vascularization [17], [18], [19]. Thus, the evaluation of antiangiogenic sVEGFR-2 together with VEGF-C and VEGFR-3 in serum is also valuable. Although a study by Zheng et al. demonstrated a decrease in serum VEGF-C and VEGFR-3 protein levels in patients with PCOS based on protein array results [11], the changes in serum levels of these markers were not examined by specific ELISA kits. Thus, this study could be a continuation of this previous study in terms of serum VEGF-C and VEGFR-3 levels with different samples, sizes, and methods. According to our data, there were no appreciable variations in the serum levels of VEGF-C and VEGFR-3 among the groups, which was inconsistent with previous findings [11]. However, the correlation analysis revealed that the serum level of VEGF-C was positively correlated with VEGFR-3 in the PCOS group, which could support the involvement of angiogenesis mechanisms in PCOS. As far as we know, there have been no previous reports on the serum levels of sVEGFR-2 in women with PCOS. The findings demonstrated that sVEGFR-2 levels were considerably increased and correlated significantly with serum VEGF-C and VEGFR-3 levels in participants diagnosed with PCOS, indicating a possible relationship between PCOS, angiogenesis and the presence of elevated sVEGFR-2 levels. It can be suggested that serum sVEGFR-2 has a relationship with these two VEGF-related molecules in PCOS.

PCOS is defined as a low-grade inflammation state [20]. Insulin resistance and obesity could be inducers of the inflammatory state in PCOS [20]. Elevated CRP levels in women with PCOS were found in previous studies [20, 21], which is similar to our finding. Compared with women without PCOS, our findings demonstrated lower HDL-C levels in the PCOS patients, which was consistent with a study by Rudnicka et al. [20]. In our data, LH and total testosterone levels were higher in individuals with PCOS than in controls, in line with other investigations [1, 20]. Testosterone can modulate the actions of ovarian granulosa cells by increasing AMH expression [22]. Previous studies have also reported significant relationships between higher AMH levels and elevated LH and testosterone concentrations in patients with PCOS [23, 24]. Moreover, it has been known that polycystic ovaries lead to increased synthesis and secretion of AMH [25]. Similarly, our results showed that serum AMH levels in the PCOS group were higher than those in controls, as reported in earlier studies [26, 27]. In addition, our results showed increased SBP and DBP, but the differences were statistically non-significant, probably due to the limited number of participants in the current study. These data also supported the results of Liu et al. [1] for SBP and DBP.

VEGF is the major mediator of sVEGFR-2 levels as circulating fragments of VEGFR-2 [28], [29], [30]. The availability of VEGF in endothelial cells is assumed to be negatively regulated by the soluble receptors via downregulating angiogenic activity [13, 31]. sVEGFR-2 inhibits VEGF-mediated angiogenesis in a strong and selective manner [30] and lymphatic vessel growth [32]. In the current study, we could not measure serum VEGF levels. However, increased serum VEGF levels in PCOS women have been reported in many previous reports [7, 8, 18], thus, according to previous studies and our findings, it could be deduced that an increased serum sVEGFR-2 (angiogenesis inhibitor) concentration in PCOS could be a compensatory mechanism to counteract the angiogenesis and block the action of VEGF. Furthermore, it has been reported that sVEGFR-2 plays a role as an endogenous VEGF-C antagonist [32]. The reason for unchanged serum VEGF-C levels in women with PCOS could be related to increased sVEGFR-2 serum levels. Similar to our results, serum sVEGFR-2 levels have also been reported to be significantly high in subjects with obesity and metabolic syndrome [14, 15]. Furthermore, diminished soluble VEGFR-1 (sVEGFR-1) and a rise in the bioavailability of VEGF in the serum have been shown in PCOS women undergoing controlled ovarian stimulation [33]. Furthermore, in our study, serum levels of sVEGFR-2 were positively correlated with VEGF-C, and VEGFR-3 indicated that the growing concentrations of sVEGFR-2 responded to higher VEGF-C and VEGFR-3 levels, and serum levels of VEGF-C and VEGFR-3 could affect the expression of sVEGFR-2. In addition, sVEGFR-2 was correlated with DBP and total cholesterol among clinical variables. Likewise, in a previous clinical trial, serum sVEGFR-2 levels indicated positive correlations with DBP and total cholesterol in women with metabolic syndrome [15]. We can suggest that circulating sVEGFR-2 levels are associated with some metabolic parameters, such as DBP and total cholesterol in PCOS. Moreover, serum sVEGFR-2 was positively correlated with AMH levels. Tal et al. reported that AMH was positively correlated with a VEGF family member, placental growth factor levels in follicular fluid in women with PCOS [34]. In another study by Wu et al., serum AMH levels were negatively correlated with follicular fluid VEGF levels in patients receiving in vitro fertilization treatment [35]. The role of angiogenic factors and AMH and their related relationship have also been reported previously in the pathophysiology of follicular development in PCOS [36]. These findings strengthen the possible link between AMH and angiogenesis, which is consistent with our finding. In addition, it seems that serum sVEGFR-2 levels could be an acceptable predictor to identify the likelihood of PCOS.

In the current study, no difference was noted regarding serum levels of VEGF-C and its receptor, VEGFR-3 between the groups. Firstly, the sample size and the subset of PCOS patients may be the reasons for these results. A study by Liu et al. demonstrated that serum VEGF levels were significantly higher in PCOS patients with normal androgen levels than in PCOS women who fulfilled all PCOS diagnostic criteria [37]. Moreover, Liu et al. also found a negative correlation between VEGF and testosterone in PCOS patients, which could be an explanation for the increased VEGF levels in PCOS patients with normal androgen levels compared to PCOS patients with hyperandrogenemia [37]. Based on this, it can be suggested that the hormonal profile of patients with PCOS could affect the levels of angiogenic factors. In the present study, PCOS patients were not stratified into subsets according to PCOS inclusion criteria. If we had grouped PCOS patients according to PCOS phenotypes, we could have achieved different results regarding the levels of studied biomarkers. In addition, previous studies have demonstrated that the VEGF-C/VEGFR-3 signaling system is involved in regulating both angiogenesis and mostly lymphangiogenesis [38, 39]. Thus, no alteration in the levels of VEGF-C and VEGFR-3 in PCOS might be related to their predominant function in lymphangiogenesis besides angiogenesis. VEGF-C, which is the ligand for VEGFR-3, can bind and induce the activation of VEGFR-3 [40]. A positive correlation between VEGF-C and its receptor, VEGFR-3 may be related to their receptor-ligand interaction to elicit biological responses. Moreover, the hormonal regulation of VEGF-C gene levels in cultured human granulosa-luteal cells has been previously demonstrated [41]. Based on this information, serum VEGF-C levels could also be affected by hormone levels in subjects with PCOS. In previous studies, decreased protein levels of serum VEGF-C and VEGFR-3 in patients with PCOS and decreased VEGF-C gene and protein expression in granulosa cells of PCOS patients were reported [11, 12]. A study by Wei et al. [12] suggested that the elevated apoptosis in ovarian granulosa cells might lead to the dysregulation of folliculogenesis in PCOS, and this might be related to decreased VEGF-C expression. A previous study demonstrated high VEGF-C serum levels in overweight and obese individuals [14]. In a study by Zhang et al., treatment with VEGF-C-stimulated adipose-derived stem cells decreased chronic intestinal inflammation in mice by activating VEGF-C/VEGFR-3 signaling [42]. These results suggest that VEGF-C could be associated with inflammatory mechanisms in PCOS. Moreover, Wada et al. [43] indicated that circulating levels of VEGF-C were significantly related to dyslipidemia in human subjects, whereas we observed a positive correlation between serum VEGF-C and HDL-C levels in the PCOS group, possibly demonstrating that increasing VEGF-C levels might not be linked to dyslipidemia in PCOS subjects.

The limited sample size of this study is its principal weakness, which may impair statistical power. Secondly, this study only measured serum levels of VEGF-C and its receptors. However, the effect of elevated serum levels of sVEGFR-2 on the ovary tissue angiogenesis was not evaluated. In addition, angiogenesis mechanisms could be mediated by a large number of angiogenic factors and concurrently antiangiogenic factors [14]. Our study was limited to the analysis of only one angiogenic molecule, VEGF-C, and its two receptors, sVEGFR-2 and VEGFR-3. Therefore, the lack of data evaluating the ratio of angiogenic VEGF to its antiangiogenic soluble receptor, sVEGFR-2, could be another limitation of the study. Thus, this aspect makes it difficult to evaluate the relationship and imbalance between the studied biomarkers and other angiogenesis-related factors in patients with PCOS.

Conclusions

Since this study is the first to evaluate the serum levels of sVEGFR-2 and its relationship with VEGF-C and VEGFR-3 in women with PCOS, it could be a pioneering study on this subject. Circulating sVEGFR-2 levels were significantly elevated and positively correlated with VEGF-C, VEGFR-3, and AMH serum levels in women with PCOS. VEGF-C/VEGFRs axis appears effective in PCOS. More research is needed to understand the function of VEGF-C and its receptors in the pathophysiology of PCOS and to validate the utility of these markers as prospective treatment targets.