Levels of vascular endothelial growth factor (VEGF) in serum of patients with endometriosis

D. Gagné, M. Pagé, G. Robitaille, P. Hugo and D. Gosselin1

MetrioGene BioSciences (a subsidiary of PROCREA BioSciences), Montréal, Canada, H4P 2R2

1 To whom correspondence should be addresssed. e-mail: dgosselin{at}metriogene.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Elevated concentrations of vascular endothelial growth factor (VEGF) have been detected in the peritoneal fluid of patients with endometriosis. Furthermore, it was postulated that VEGF is involved in the development of endometriotic lesions. The present study is aimed at determining whether high levels of VEGF could also be found in the serum of patients with endometriosis. METHODS: VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum from 131 subjects with surgically confirmed endometriosis and 146 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. Parameters such as demographics, personal habits, menstrual characteristics and clinical profile were collected from each subject included in this study. RESULTS: The mean VEGF levels were not significantly modulated in serum samples of cases compared with controls in a crude general linear model and in a model adjusted for possible confounders. VEGF serum levels did not correlate with the score, stage of endometriosis or the presence of benign gynaecological disorders. However, a correlation was found between circulating concentrations of VEGF and body mass index. CONCLUSION: Although VEGF seems to play a pivotal role locally in the implantation and development of endometriotic lesions, the disease is not associated with a significant modulation in the levels of circulating VEGF.

Key words: angiogenesis/endometriosis/serum/VEGF/VEGF serum levels


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometriosis is one of the most common gynaecological diseases, affecting ~10% of women of reproductive age (Eskenazi and Warner, 1997Go). This condition is characterized by the abnormal presence of endometrial cells in the peritoneal cavity, leading to pelvic pain, dysmenorrhoea, dyspareunia and infertility. The formal diagnosis currently requires direct visualization of the lesions under surgery. However, this procedure has several limitations that clearly establish the need for the development of a less invasive, rapid and reliable diagnostic test for endometriosis.

Although the pathogenesis of endometriosis remains unclear, the presence of endometrial cells in the peritoneal cavity is explained by retrograde menstruations (Sampson, 1927Go). Because this phenomenon is prevalent in most women, several other fundamental processes must contribute to the implantation of endometrial cells and their subsequent development into endometriotic lesions. Indeed, retrogradely seeded endometrial cells must escape apoptosis, adhere to the mesothelium, proliferate and establish a new blood supply (reviewed in Gosselin et al., 1999Go). In fact, there is compelling evidence that angiogenesis plays a key role in the ectopic implantation of endometrial tissue and its development into endometriotic lesions (McLaren, 2000Go; Taylor et al., 2002Go).

Angiogenesis, defined by the formation of new capillaries from pre-existing blood vessels, involves the interaction of a number of tightly regulated molecules including vascular endothelial growth factor (VEGF), which is recognized as a pivotal angiogenic factor (Smith, 2001Go; Taylor et al., 2002Go). VEGF is a 23–45 kDa heparin-binding glycoprotein with potent endothelial cell-specific mitogenic and vascular permeability activities (Taylor et al., 2002Go). Binding of VEGF to one of several tyrosine kinase receptors triggers their autophosphorylation, resulting in the activation of mitogen-activated protein kinases (Taylor et al., 2002Go).

VEGF mRNA and protein expression have been detected in endometriotic lesions and were reported to be greater in red lesions (active) than in black lesions (senescent), a finding associated with higher vascularization in active red lesions (Donnez et al., 1998Go; Tan et al., 2002Go). VEGF mRNA and protein expression have also been detected in the eutopic endometrium and are known to play an important role in blood vessel formation during the menstrual cycle (Shifren et al., 1996Go; Donnez et al., 1998Go; Smith, 2001Go). When comparing eutopic endometrium of subjects suffering from endometriosis with that of normal individuals, increased VEGF mRNA and protein expression have been observed in the luteal phase of the menstrual cycle in diseased females (Donnez et al., 1998Go; Tan et al., 2002Go). These results thus suggest that before retrograde menstruation occurs, eutopic endometrial tissue already has the capacity to promote its implantation at ectopic sites, through the secretion of VEGF.

Furthermore, several studies have reported an elevated angiogenic activity, as well as an increased VEGF concentration in the peritoneal fluid of patients with endometriosis (Oosterlynck et al., 1993Go; McLaren et al., 1996Go; Taylor et al., 1997Go). Increased levels of VEGF in the peritoneal fluid in diseased individuals could indicate that VEGF is also modulated in the serum of these patients. However, whether circulating VEGF is also modulated in endometriosis remains poorly defined. Pellicer et al. (1998Go) reported no significant difference in VEGF serum levels of patients with endometriosis compared with control individuals. In contrast, Matalliotakis et al. (2003Go) reported a slight but significant increase of serum VEGF in diseased females. Such apparent inconsistencies could be explained by the low number of samples tested, by poorly defined study populations and by the absence of adjustment for confounding factors.

The detection of increased levels of VEGF in the serum of patients with endometriosis could have a clear application in the diagnosis, as well as providing a better understanding of the aetiology of this disease. We have, therefore, undertaken an extensive study in which VEGF levels were compared in the serum of 277 individuals with or without endometriosis, taking into account several important parameters such as demographics, gravidity, phase of the menstrual cycle, clinical profile and the presence of benign reproductive disorders.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Subjects were recruited from women who were scheduled to undergo laparoscopy or laparotomy at one of the eight clinical institutions of our clinical network. A total of 35 gynaecologists experienced in the management of endometriosis participated in this study. To be admitted to the study, patients had to be of premenopausal age, not currently menstruating, have regular menstrual cycles (between 21 and 35 days), have no acute salpingitis, have not been pregnant for the last 3 months, and have not been under hormonal treatment nor using an intrauterine device for the last 3 months. Only subjects who were in the luteal phase of the menstrual cycle, based on histopathological dating, were included in the study. Subjects who agreed to participate in this study signed a consent form approved by the Ethics Review Board of PROCREA and that of each of the collaborating institutions. The level of participation among eligible subjects was >90%. For each subject enrolled in the study, the clinical investigators completed a questionnaire in which clinical profile as well as information concerning personal habits, menstrual characteristics, gross evaluation of the intensity of pain, contraception and parity were recorded.

The stage of endometriosis was determined according to the classification of the American Society of Reproductive Medicine (1997Go). Cases were defined as subjects with minimal/mild endometriosis (stages I–II) or moderate/severe endometriosis (stages III–IV) confirmed at the time of surgical examination. Endometriosis was detected by surgeons skilled in the detection and identification of all forms of endometriotic lesions. The control group comprised healthy subjects who underwent surgery for several indications. These subjects had no clinical evidence of endometriosis or detectable endometriotic lesions at the time of surgical examination. A total of 131 patients with endometriosis and 146 normal controls were included in the present study.

Blood collection and serum preparation
Blood samples were collected before anaesthesia by venipuncture of individuals who had abstained from food for at least 8 h. Blood samples were collected into 10 ml sterile tubes containing no additives and were kept at room temperature until centrifugation at 216 g for 10 min. Less than 5 h was allowed between blood collection and processing. Serum aliquots were then frozen at –80°C until measurement of VEGF serum levels.

Histological dating
Endometrial tissue specimens were collected with a suction curette when the patient was under anaesthesia. The phase of the menstrual cycle and the dating were determined by histopathology from a section of formalin-fixed endometrial tissue according to the method of Noyes and Hama (1953Go). Only samples that were confirmed to be in the luteal phase of the menstrual cycle were included in the study.

Measurement of VEGF serum levels
Concentration of VEGF serum levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. Briefly, serum samples and standards were incubated for 2 h in a microplate pre-coated with murine anti-VEGF monoclonal antibodies. After washing three times, enzyme-linked anti-VEGF polyclonal antibodies were added. Unbound antibodies were removed by washing. The intensity of the reaction was then revealed with tetramethylbenzidine and optical density was measured at 450 nm using a Vmax microplate reader and Soft MAX Pro software (Molecular Devices, Menlo Park, CA, USA). All samples were run in duplicate, and a standard curve was established for each assay. The sensitivity of the assay was <9.0 pg/ml. Inter- and intra-assay variations were <10%.

Statistical analysis
Statistical analysis was performed using the SPSS software. Comparisons of cases and controls with respect to several categorical parameters (demographics, personal habits, clinical profile) were done with Pearson {chi}2-tests. The effect of these parameters on VEGF levels was examined with one-way analyses of variance (ANOVA) or by bivariate correlations using a two-tailed test significance and Pearson or Spearman coefficient correlation. The mean serum level of VEGF was compared in cases and controls using the Student’s t-test for independent samples. In another series of analyses, the mean VEGF serum level was adjusted for potential confounders [indication of surgery, gravidity, body mass index (BMI), pelvic pain, infertility and length of menses] using a univariate general linear model. Results were considered statistically different when the P-value was <=0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Before VEGF levels can be compared in the serum of patients with endometriosis and normal controls, it was first important to verify whether some confounders could be identified in the present study. This was achieved by determining whether several parameters among demographics, personal habits and clinical profile were comparable in cases and controls. Any variables that were significantly modulated in cases and controls and/or those shown to correlate strongly with the serum levels of VEGF were considered as potential confounders in the present study and were, therefore, taken into account in the comparison of VEGF serum levels in cases and controls.

Comparison of demographic variables, personal habits and clinical data among cases and controls
The great majority of the subjects enrolled in this study were Caucasians (data not shown). The proportion of subjects among cases and controls within each category of demographic or personal habit variables is presented in Table I. No significant difference was observed between cases and controls regarding age, BMI, smoking and alcohol intake (Table I). Among menstrual characteristics, the length of menses was different in patients with endometriosis and controls (Table I). The proportion of women with menses lasting more than 6 days was significantly greater among cases than controls, whereas no significant modulation was found regarding the length of the cycle (Table I).


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Table I. Distribution of demographics, personal habitsa and clinical profile by study status
 
Subjects included in the present study underwent surgery for several different indications. As expected, the proportion of subjects who underwent surgery for tubal ligation, diagnostic laparoscopy and hysterectomy was significantly different between cases and controls (Table I). Also, the proportion of subjects with at least one pregnancy was dramatically lower in cases compared with controls (Table I). Previous use of oral contraceptives, allergies or recent acute infections were found not to be different between study groups (Table I). The proportion of patients experiencing symptoms associated with endometriosis such as pelvic pain, including dysmenorrhoea and dyspareunia, as well as infertility was significantly higher in cases compared with the control group (Table I).

Influence of demographic variables, personal habits and clinical profile on VEGF serum levels
Among the above-mentioned parameters, BMI was shown to affect the levels of VEGF in the serum (Table II, Figure 1). The present study was restricted to serum samples collected in the luteal phase of the cycle. Results presented in Figure 2 show that VEGF serum levels did not correlate with the histological day of the luteal phase. Moreover, the proportion of subjects within the early, middle and late phase of the menstrual cycle was comparable in cases and controls (Table I). These results thus indicate that it is not necessary to adjust for this variable in the comparison of VEGF levels between cases and controls.


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Table II. VEGF serum levels according to demographics, personal habitsa and clinical profile
 


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Figure 1. Distribution of VEGF serum levels according to body mass index. The level of VEGF was measured in the serum samples by ELISA as described in the Materials and methods section. Spearman’s correlation coefficient: 0.179 (P = 0.004).

 


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Figure 2. Distribution of VEGF serum levels according to the histological day of the menstrual cycle. The level of VEGF was measured in the serum of all subjects (controls and cases) included in the study by ELISA as described in the Materials and methods section.

 
Comparison of VEGF concentration in the serum of cases and controls
Results presented in Table III show no significant difference in mean VEGF serum levels when 131 patients with minimal to severe endometriosis (stages I–IV) are compared with 146 normal controls. In order to verify whether this observation could have been biased by the lack of control for several possible confounders, the mean VEGF serum levels were adjusted with respect to surgery, pelvic pain, gravidity, length of menses, infertility and BMI in a univariate general linear model. Using this model, no significant difference was observed in mean VEGF serum levels between cases and controls (Table III). Furthermore, the concentration of serum VEGF measured in patients with endometriosis did not correlate with the stage or the score of the disease (data not shown).


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Table III. Comparison of VEGF serum levels between cases and controls in crude and adjusted models
 
As subjects included in the present study underwent surgery for several different indications, they reflect a wide variety of clinical profiles. The presence of some benign gynaecological disorders other than endometriosis (i.e. cysts, fibroids, pelvic inflammation, myoma, polyps, tubal obstruction, pelvic varicose) could modulate the levels of VEGF in the serum and alleviate any potential difference that would exist between cases and controls. Results obtained in the present study indicate that VEGF serum levels were not associated with the presence of adhesions, fibroids, cysts or a history of genital infections (data not shown). In addition, to discriminate between the impact of endometriosis and other benign gynaecological disorders on VEGF serum levels, patients with endometriosis and controls were stratified according to the presence or absence of benign gynaecological disorders. VEGF serum levels were compared in cases and controls among subjects with or without gynaecological disorders and, again, no difference was found between cases and controls (Figure 3).



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Figure 3. VEGF serum levels in cases and controls according to the presence of benign gynaecological disorders. Boxes and vertical lines represent 50% and 90% of the samples, respectively. The horizontal bars in the boxes represent the mean. VEGF serum levels were measured in cases (stages I–IV) (n = 62) and control (n = 48) subjects with benign gynaecological disorders (A). VEGF serum levels were also measured in cases (stages I–IV) (n = 69) and control (n = 98) subjects without benign gynaecological disorders (B).

 
Taken together, these results clearly indicate that even under carefully controlled conditions, no difference could be found when VEGF serum levels were compared in patients with endometriosis and normal controls.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several lines of evidence suggest that VEGF plays a determinant role in the development of endometriotic lesions (reviewed in Taylor et al., 2002Go). Endometriosis was shown previously to be associated with some immune systemic manifestations (Sinaii et al., 2002Go; Gagné et al., 2003Go), and many cytokines found in the peritoneal fluid of females with endometriosis were also found to be elevated in their serum (Sharpe-Timms et al., 1998Go; Gurgan et al., 1999Go; Pizzo et al., 2002Go). It is conceivable, therefore, that modulated levels of VEGF found in the peritoneal cavity (McLaren et al., 1996Go; Küpker et al., 1998Go; Mahnke et al., 2000Go) could be similarly altered in the serum of patients with endometriosis. This issue remains controversial, however, as preliminary studies have reported contradictory results (Pellicer et al., 1998Go; Matalliotakis et al., 2003Go).

In the present study, we have undertaken an extensive case–control study in order to establish whether VEGF levels are indeed modulated in the serum of patients with endometriosis. As VEGF levels are under steroid control and peak in the luteal phase of the menstrual cycle (Shifren et al., 1996Go; Hyder et al., 2000Go; reviewed in Taylor et al., 2002Go), only serum samples that were collected in the luteal phase of the cycle were included in this study. Indeed, this approach eliminates possible variation due to the phase of the cycle that could hamper the potential modulation in VEGF serum levels. We have studied 131 cases and 146 normal controls. As expected, the proportion of subjects enrolled in the present study who underwent surgery for tubal ligation, hysterectomy or diagnostic laparoscopy was different among cases and controls. Hence, asymptomatic patients who underwent surgery for tubal ligation were expected to have lower odds of suffering from endometriosis than subjects who underwent surgery for diagnostic laparoscopy for symptoms related to endometriosis.

Our previous work in immunophenotyping blood leukocytes had underlined the importance of controlling for possible confounders in studies involving blood measurements (Gagné et al., 2003Go). A similar approach was taken to determine whether several parameters among demographics, personal habits and clinical profile were comparable in cases and controls. Hence, results presented herein show that the proportion of subjects with at least one pregnancy was dramatically reduced in cases compared with controls. This observation was shown to correlate with infertility, a condition well known to be associated with endometriosis (Cramer and Missmer, 2002Go). Furthermore, the length of menses was shown to correlate positively with an increased risk of endometriosis. In fact, longer menses is a well-documented risk factor for endometriosis consistent with the theory of retrograde menstruation (Darrow et al., 1993Go; Cramer and Missmer, 2002Go).

In this study, we demonstrate for the first time that VEGF serum levels increase with BMI. The mechanisms underlying this effect are unknown, but may be related to the previous observation that adipocytes have the capacity to secrete VEGF (Mora and Pessin, 2002Go). Moreover, it was reported previously that leptin, a well-characterized adipocyte secretory protein, triggers angiogenesis in vivo and in vitro. Leptin can also induce, in a dose-dependent manner, VEGF secretion in vitro by human umbilical vein endothelial cells (HUVECs) (Park et al., 2001Go).

Data reported in the present study thus suggest that the mean VEGF serum levels measured in cases and controls should be adjusted with the following variables: indication for surgery, infertility, BMI, gravida, pelvic pain and length of menses. Results obtained in crude as well as adjusted models have clearly established that no significant modulation in VEGF serum levels was observed between patients with endometriosis and normal controls.

It remains possible, however, that the lack of difference in VEGF serum levels between cases and controls could be explained by the fact that all stages of endometriosis were included in the endometriosis group. Our results indicate that this possibility can be ruled out, as no correlation was found between VEGF serum levels and the stage or the score of the disease.

VEGF has been associated in the past with several benign disorders of the female reproductive tract such as endometrial hyperplasia, abnormal uterine bleeding and ovarian cysts (Fasciani et al., 2001Go; Shaarawy and El-Sharkawy, 2001Go). Given that subjects included in the present study underwent surgery for several different indications, they reflect a wide variety of clinical profiles, other than endometriosis, that could modulate the levels of VEGF in the serum and alleviate any potential differences that would exist between cases and controls. It was thus important to take into account the presence of these disorders among the subjects included in this study. Results reported herein show no correlation between the levels of VEGF and the presence of fibroids, pelvic adhesion, cysts or genital infections. In addition, no significant modulations could be attributed to endometriosis when VEGF serum levels were compared between cases and controls among subjects with or without benign gynaecological disorders.

Together these results clearly indicate that even under carefully controlled conditions, no difference could be found in VEGF serum levels between patients with endometriosis and controls. The absence of modulation in VEGF serum levels in endometriosis cannot, therefore, be attributed to a lack of control of possible confounders, to the histological day of the luteal phase, or to the presence of benign gynaecological disorders. It thus can be concluded that although VEGF seems to play a key role locally in the development of endometriotic lesions, the disease is not associated with a significant modulation in the levels of circulating VEGF.


    Acknowledgements
 
The authors would like to thank Annick Rioux, Julie Poliquin-Fleury and all the team of technicians for their excellent technical assistance, and Dr Fabianne Philippoussis for critical review of the manuscript. The authors are also grateful to all the clinicians for their precious collaboration in sample and data collection. This study would not have been possible without the generous contribution of all women who have participated in this study.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 21, 2002; accepted on April 30, 2003.