MetrioGene BioSciences (a subsidiary of PROCREA BioSciences), Montréal, Canada, H4P 2R2
1 To whom correspondence should be addresssed. e-mail: dgosselin{at}metriogene.com
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Abstract |
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Key words: angiogenesis/endometriosis/serum/VEGF/VEGF serum levels
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Introduction |
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Although the pathogenesis of endometriosis remains unclear, the presence of endometrial cells in the peritoneal cavity is explained by retrograde menstruations (Sampson, 1927). 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., 1999
). 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, 2000
; Taylor et al., 2002
).
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, 2001; Taylor et al., 2002
). VEGF is a 2345 kDa heparin-binding glycoprotein with potent endothelial cell-specific mitogenic and vascular permeability activities (Taylor et al., 2002
). 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., 2002
).
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., 1998; Tan et al., 2002
). 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., 1996
; Donnez et al., 1998
; Smith, 2001
). 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., 1998
; Tan et al., 2002
). 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., 1993; McLaren et al., 1996
; Taylor et al., 1997
). 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. (1998
) reported no significant difference in VEGF serum levels of patients with endometriosis compared with control individuals. In contrast, Matalliotakis et al. (2003
) 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.
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Materials and methods |
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The stage of endometriosis was determined according to the classification of the American Society of Reproductive Medicine (1997). Cases were defined as subjects with minimal/mild endometriosis (stages III) or moderate/severe endometriosis (stages IIIIV) 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 (1953). 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 manufacturers 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 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 Students 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.
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Results |
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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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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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Discussion |
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In the present study, we have undertaken an extensive casecontrol 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., 1996; Hyder et al., 2000
; reviewed in Taylor et al., 2002
), 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., 2003). 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, 2002
). 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., 1993
; Cramer and Missmer, 2002
).
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, 2002). 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., 2001
).
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., 2001; Shaarawy and El-Sharkawy, 2001
). 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.
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Acknowledgements |
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References |
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Submitted on December 21, 2002; accepted on April 30, 2003.