{"paper_id":"079f2357-e0d7-4bf2-8c33-36073222c18f","body_text":"Angiogenesis is identified as new capillaries being\nformed from pre-existing blood vessels. This\nphenomenon involves the interaction of some of the\nclosely synchronized molecules including vascular\nendothelial growth factor (VEGF) ( 1 ,  2 ). VEGF, as a\nkind of mitogen, is a major promoter of angiogenesis\nin pathological and physiological settings, and is also\nconsidered as a survival factor for endothelial cells ( 3 ).\nFurthermore, through vascular leakage and mobilizing\nleukocytes, VEGF, as a strong vascular permeability\nfactor, promotes inflammatory process ( 4 ,  5 ).\nEndometriosis is known as one of the most common\ndisease in women of reproductive age. Despite\nthe current controversy regarding the pathophysiology\nof this disease ( 6 ), Sampson’s theory\nexplains the existence of endometrial cells in the\nperitoneal cavity by retrograde menstruation. Several factors, such as increased of inflammatory activity\nin the peritoneal fluid (PF), angiogenesis and\nup-regulating of pro-inflammatory cytokines may\nfacilitate the pathogenesis of endometriosis, which\nis assumed to be a complex process ( 7 ,  8 ).\nThere is a controversy among the literatures about\nthe possible variation of VEGF in serum and in PF\nof endometriosis patients ( 9 - 20 ). Some of the published\ndata indicated the increased level of VEGF\nin serum ( 9 ,  13 ,  19 ) and in PF ( 10 ,  11 ,  13 ) of endometriosis\npatients. However, several others studies\nreported no change of VEGF level in serum ( 15 - 18 )\nand in PF ( 12 ,  15 ) of endometriosis patients. On the\nother hand, there have been many theories explaining\nthe etiology, but the most common accepted\none is shedding of the endometrium following retrograde\nmenstruation. In endometriosis, neo-vascularization\nis essential for the successful implantation\nof endometrial cells in ectopic sites ( 2 ). VEGF\nis part of a heparin-binding protein family ( 21 ), and\nthe induction of endometrial cell proliferation is\nfunctioned by VEGF ( 4 ,  22 ), so VEGF was considered\nessential factor in uterine angiogenesis ( 23 ).\nMost of published data related to VEGF variation\nin endometriosis were obtained from small\nsample size patients, and accordingly, we attempted\nto consider this inconsistency in a large number\nof patients referred to a clinic during three years.\nTherefore, we measured the level of VEGF in serum\nand in PF of endometriosis patients and compared\nwith normal subjects.\n\nThis descriptive study was approved by Ethical\nCommittee of Isfahan University of Medical Sciences.\nDuring years 2009-2011, a total of 392 patients\nsubjected to laparoscopy for the evaluation of infertility\nor pelvic pain at the Isfahan Fertility and Infertility\nCenter were considered. The medical record of each\npatient was exactly reviewed by an expert gynecologist\nin order to exclude those patients with hypertension,\ncoronary arterial diseases, diabetes, renal diseases,\nactive pelvic inflammatory disease or polycystic\novarian syndrome. Finally, 179 patients were assigned\nfor this study. This sample size was selected based\non our pilot study. After laparoscopy, the patients\nwere allocated into the following two groups: group\nI: different types of endometriosis patients (n=90) and\ngroup II: non-endometriosis patients (n=89). The official\ninformed consent was obtained for all subjects.\nThe venous blood samples were obtained from\nall patients before induction of anesthesia for laparoscopy\nprocedure. The blood samples were centrifuged,\nand the serums were stored at -20˚C until\nmeasurement. In addition, the PF samples were\ncollected from pelvis before any manipulation.\nThe bloody fluids were excluded. The PF samples\nwere also centrifuged and the supernatant were\nstored at -20˚C until measurement.\nThe serum and peritoneal levels of VEGF were\nmeasured using enzyme immunoassay kit (Immuno-\nBiological Laboratory Co., Japan). Briefly, the kit is a\nsolid phase sandwich enzyme linked immunosorbent\nassay (ELISA) using specific polyclonal and monoclonal\nantibodies, while the coloring agent was tetra\nmethyl benzidine (TMB). The sample (100 μl) was\nput in pre coated plate (Anti-Human VEGF (16F1)\nMouse IgG M0Ab Affinity Purify (Immuno-Biological\nLaboratories Co., Ltd, Japan), then washed after\nincubating for 60 minutes in 37˚C. Afterwards, the labeled\nantibody (HRP conjugated Anti-Human VEGF\nRabbit IgG Fab’ Affinity Purify (Immuno-Biological\nLaboratories Co., Ltd, Japan), was added, incubated\nand washed again. Finally, coloring agent was added,\nfollowed by stop solution, whereas the absorbance\nwas determined at 450 nm by plate reader. The VEGF\nconcentration was determined using standard curve.\nData was expressed as mean ± SEM. Unpaired\nt test was applied to compare the parameters between\nthe groups, while paired t test was applied to\ncompare the parameters within the groups. The value\nof p<0.05 was considered statistically significant.\n\nThe average age of groups I and II are 28.9 (range:\n19-44) and 30.2 (range: 24-42), respectively, while\nthere was no statistically significant difference between\ntwo groups. In this study, 166 women were in\nproliferative phase, and 13 women were in secretory\nphase. The obtained data for VEGF level in serum\nand in PF from group I (the patients with endometriosis)\nand group 2 (control) are demonstrated in figure 1. The VEGF level in serum was significantly less\nthan that in PF in both groups (p=0.00). However no\nsignificant difference in serum level of VEGF was detected\nbetween the two groups, but the result indicates\nthat group I had significantly higher level of VEGF in\nperitoneal fluid in comparison to group II (p=0.043).\nThe VEGF level in serum and peritoneal fluid from\nthe patients with endometriosis (group I) and control subjects\n(group II).\n\nEndometriosis, as a gynecologic disease, has been\nsubjected to many research in order to find the exact\ncause of infertility related to this disorder ( 2 ,  6 - 8 ,  11 ,\n 14 ,  18 ,  19 ,  24 ,  25 ). The main objective of this study\nwas to compare the serum and peritoneal levels of\nVEGF in endometriosis patients and control subjects.\nWe found that the level of VEGF in PF were higher\nthan that in serum in both groups. In addition, the peritoneal\nlevel of VEGF in endometriosis was increased\nsignificantly when compared with control group.\nIn some studies, the serum level of VEGF in endometriosis\nwas reported differently compared to our\nobtained result. Their findings showed higher levels\nof VEGF in the serum of patients with endometriosis\ncompared to specific control group ( 9 ,  10 ); in addition,\nno significant difference was detected in endometriosis\npatients in the luteal phase (which may\naffect the variation of VEGF) with different control\ngroup undergoing surgery for several different indications\n(tubal ligation, hysterectomy or diagnostic\nlaparoscopy) ( 16 ). In other study, the VEGF level\nin patients in different phases of the menstrual cycle\nwas compared with false-positive cases (patients\nwho were suspected to have endometriosis), and no\nstatistically difference was detected even when the\nphase of the cycle was taken into account ( 12 ). One\nof the advantages of our data was the specific control\ngroup consisting of infertile women with no evidence\nof endometriosis in their laparoscopic examination.\nIt seems that this control group was more reliable to\ncompare with endometriosis patients. Similar to the\nother study ( 12 ,  16 ), we also included all stages of endometriosis\nin our study group (group I).\nEndometriosis is a chronic disease associated with\na general inflammatory response in peritoneal cavity.\nEvidence shows that immunological factors ( 26 ) and\nangiogenesis play a decisive role in the pathogenesis\nof endometriosis ( 13 ,  27 ), and there is an increase\nin number, activity and secretion of peritoneal macrophages\nof endometriosis cases ( 28 ,  29 ). In women\nwith endometriosis, the function of peritoneal macrophages,\nkiller cells and lymphocytes are so considerable.\nMoreover, growth factors and inflammatory\nmediators in the peritoneal fluid are mainly produced\nby peritoneal macrophages, and peritoneal leukocytes,\nare modified in endometriosis ( 13 ). It seems\nthe peritoneal macrophages activate ( 29 ) endometrial\ncells ( 30 ), and all neutrophils ( 31 ) are also able to synthesize\nand secret VEGF ( 10 ,  27 ). VEGF promotes\ninflammatory process through vascular leakage and\nleukocytes accumulation ( 32 ). Therefore, a local increase\nof the VEGF in the PF (not in serum) might be\ndue to an increase of the macrophage secretary products,\nwhich is found to be a major source of VEGF in\nendometriosis ( 10 ).\nAlso, VEGF is one of those molecules attributed to\nthe growth and maintenance of angiogenesis ( 4 ,  12 ,\n 22 ,  26 ,  33 ) and vascular permeability ( 10 ,  12 ,  33 ).\nAccordingly, it can be a main factor in physiologic angiogenesis\nin endometrium ( 9 ,  28 ). It has been shown\nhigher angiogenic activity of PF in woman suffering\nfrom endometriosis in comparison to those not suffering\nfrom this disease ( 11 ). It was observed that there\nis an increase in angiogenesis around the peritoneal\nexplants, followed by an increase in angiogenic activity\nin the PF of endometriosis women ( 11 ,  34 ). Vascularization\nwithin and around the tissue have been\npronounced by active endometriotic explants, caused\nby the process of angiogenesis ( 35 ). Surprisingly,\nboth peritoneal macrophages and deep endometriotic\nlesions produce VEGF in large amounts in PF ( 36 ).\nTherefore, the ability of endometriosis lesions in producing\nVEGF is another reason for an increase in PF\nlevel in endometriosis women ( 30 ).\n\nAccording to our data, it seems that angiogenic activity\nmay increase by the elevated level of VEGF in\nthe PF of endometriosis patients. This elevated level\nof VEGF possibly promotes neovascularization within the peritoneal environment. It seems, this disease\nis only associated with pelvic inflammation, while is\nnot related to the change of VEGF level in circulation.","source_license":"CC0","license_restricted":false}