Increased vascular endothelial growth factor

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Aug 15, 2009 - Apgar BS, Brotzman G. Management of cervical cytologic abnormalities. Am Fam Physician. 2004;70:1905-1916. 3. Branca M, Giorgi C, Santini ...

Original Article

Increased Vascular Endothelial Growth Factor Expression, CD3-Positive Cell Infiltration, and Oxidative Stress in Premalignant Lesions of the Cervix ˜ a, MD2; Chiquinquira ´ Silva, MD3; Yenddy Carrero, MSc1; Diana Callejas, MSc1; Freddy Alan 1 4 Raimy Mindiola, MSc ; and Jesu ´ s Mosquera, MD

BACKGROUND: Vascular endothelial growth factor (VEGF) plays an important role in cervical intraepithelial neoplasia (CIN) progression. The occurrence of leukocytes has been documented in CIN; however, their role in VEGF production remains unknown. Oxidative stress has been involved in the progression of malignant neoplasias, but to the authors’ knowledge tissue oxidative stress in CIN has not been documented. The objective of the current study was to investigate the expression of VEGF, leukocyte infiltration, leukocyte VEGF expression, and nitrogen/oxygen metabolism in cervical tissues from patients with CIN. METHODS: Indirect immunofluorescence was used to study the expression of VEGF and leukocyte infiltration in cervical samples from 55 patients with CIN and 7 normal controls. Superoxide anion (O2) expression was determined by a cytochemical method, and tissue and serum nitric oxide by the Griess reaction. Human papillomavirus (HPV) DNA and HPV types were identified by the hybrid capture 2 HPV DNA test. RESULTS: Increased expression of VEGF was observed related to the progression of CIN. A significant increment of CD3 lymphocytes was found in CIN type 3 (CIN 3) and coexpression of CD3/VEGF and monocyte-macrophage/VEGF in CIN 2 and 3. Increased O2-positive cells were found in CIN 2 and 3; however, tissue nitrate-nitrite content remained similar to controls. The incidence of HPV infection was 16% in patients with CIN. No significant differences were observed in the values of HPV-positive or HPV-negative patients. CONCLUSIONS: Different factors leading to cervical neoplasia progression may be involved in the evolution of CIN, and the presence of these factors is most likely not related to the HPV infection status. C 2009 American Cancer Society. Cancer 2009;115:3680–8. V KEY WORDS: intraepithelial cervical neoplasia, vascular endothelial growth factor, lymphocytes, oxidative stress.

Corresponding author: Jesu´s Mosquera, MD, Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette,’’ Facultad de Medicina, Universidad del Zulia, Apartado Postal 23, Maracaibo 4001-A, Zulia, Venezuela; Fax: (011) 58-261-7597247; [email protected] 1 Regional Laboratory of Virological Reference, Faculty of Medicine, Zuila University, Maracaibo, Venezuela; 2Medical Odontology, Faculty of Medicine, Zuila University, Maracaibo, Venezuela; 3Department of Histology, Zuila University, Maracaibo, Venezuela; 4Institute of Clinical Investigation ‘‘Dr. Ame´rico Negrette,’’ Faculty of Medicine, Zuila University, Maracaibo, Venezuela

We thank Dr. Humberto Martinez for critical review of the English. Received: October 29, 2008; Revised: January 7, 2009; Accepted: January 13, 2009 C 2009 American Cancer Society Published online: May 28, 2009 V

DOI: 10.1002/cncr.24411,



August 15, 2009

Cervical Neoplasia and VEGF/Carrero et al

Cervical carcinogenesis has well-defined stages of disease progression, including 3 grades of preinvasive lesions, cervical intraepithelial neoplasia (CIN) types 1 through 3, and invasive cervical cancer.1,2 However, the biologic properties of CIN lesions prone to develop invasive disease are not well defined. Angiogenesis and oxidative stress are relevant parameters in carcinogenesis. Previous observations suggest that early invasive disease spreads to regional lymph nodes in several tumor types, and that vascular endothelial growth factor (VEGF) is involved in lymphangiogenesis related to the progression of CIN lesions to invasive cervical carcinoma.3-5 Local leukocyte infiltration appears to play an important immunologic role in the progression of CIN6,7; however, the role of leukocytes in the production of VEGF during the different grades of CIN remains to be elucidated. Oxidative damage of DNA also appears to be important during cervical carcinogenesis8; however, to our knowledge little information regarding the local oxidative status in CIN has been reported. Therefore, the aim of this study was to determine the expression of VEGF and its coexpression with leukocyte markers and local nitrosative and oxidative status in uterine cervical biopsies from patients with cervical intraepithelial neoplasia. The results of the current study demonstrated increased VEGF production and oxidative stress in advanced intraepithelial neoplasia, and suggest a possible role of leukocytes in angiogenesis by the production of VEGF.

MATERIALS AND METHODS Tissue Specimens We evaluated exocervix samples spanning the spectrum from normal cervical tissue to high-grade squamous intraepithelial lesions. Patients were informed of study procedures, and their consent was obtained before enrollment in the investigation following the ethical committee guidelines of each of the hospitals involved, the Committee of Bioethics and Biosecurity of FONACIT (Caracas, Venezuela), and the Committee of Bioethics of the University of Zulia Medical School (Maracaibo, Venezuela). Each patient had a personal interview, including gynecologic and obstetric history, and underwent a gynecologic examination, Papanicolaou test smear, and a colposcopic examination of the exocervix. This study included patients Cancer

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who were sexually active, were not pregnant, and had cervical lesions, and who had not undergone immunosuppressant treatment or treatment for human papillomavirus (HPV). All individuals in this study were negative for human immunodeficiency virus (HIV). Colposcopic examination was performed under a standard protocol that included conventional visual assessment, application of 5% acetic acid, identification of the squamocolumnar junction and transformation zone, and recognition of suspected CIN lesions for directed cervical biopsies. A direct biopsy was taken when a colposcopic abnormality was found. Uterine cervical samples from CIN patients (n ¼ 55: CIN 1 in 45 patients, CIN 2 in 5 patients, and CIN 3 in 5 patients) and from 7 healthy individuals with normal cervices (controls) were taken. Controls were age matched, and neither patients nor controls had menstrual bleeding. All specimens were fixed in 10% buffered formalin, embedded in paraffin for 6-lm sections, stained with hematoxylin and eosin, and analyzed following the Richart terminology.1 Colposcopic cervical samples were included in OCT compound (Tissue Tek; Miles Inc. Diagnostics Division, Kankakee, Ill), frozen in dried ice and acetone, and kept at 70 C until use for immunofluorescence studies and superoxide anion (02) expression. Immunofluorescence Studies The frozen sections (4 lm) from control and patient tissues were fixed with cold acetone and then reacted with 1 of the following mouse monoclonal antibodies: antihuman CD3, antihuman RIII/C16 b, or antihuman macrophage (R&D Systems, Minneapolis, Minn). Mouse immunoglobulin was detected on tissues with fluorescein isothiocyanate–conjugated F(ab0 )2 rat antimouse immunoglobulin G antibody (Accurate Chemical and Scientific Corporation, Westbury, NY). Thereafter, tissues were reacted with a biotinylated monoclonal antibody against human VEGF (R&D Systems) and phycoerythrin-conjugated streptavidin. Controls included sections subjected to monoclonal antibodies with the same isotype, but against nonrelevant antigens as primary antibodies. Samples were examined using an epifluorescence microscope (Axioskop, Zeiss, Go¨ttingen, Germany). Results were expressed as number of positive cells per microscopy field (400) from 10 randomly selected areas. 3681

Original Article

Detection of HPV DNA in Cervical Specimens HPV DNA was isolated from cervical tissue by a commercial kit following manufacturer indications (HPV DNA Purification Kit; Promega Corporation, Madison, Wis). HPV DNA was analyzed using a Digene Hybrid Capture kit (Qiagen, Valencia, Calif). This kit detects 34 HPV types, including 13 high-risk types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low-risk types (types 6, 11, 42, 43, and 44). Determination of Oxidative Status Superoxide anion production was determined at cellular levels in the cervical samples by a cytochemical method previously described.9 Results were expressed as positive cells per microscopy field of 400. Nitric oxide (NO) formation was detected by nitrite and nitrate accumulation in homogenates from fresh samples of cervix and serum from patients and controls by the Griess reaction10 using a commercial kit (QuantiChrom Nitric Oxide Assay Kit [DINO-250]; BioAssay Systems, Hayward, Calif). Results were expressed as lmol/mg of tissue protein and as lmol (serum). Total protein content was measured in the cervical homogenates by the method of Lowry et al.11 Statistical Analysis Results in the groups are shown as mean  the standard deviation (SD). Comparisons between groups were performed using the Student t test and analysis of variance with the Bonferroni post-test. Two-tailed P < .05 was interpreted as being statistically significant.

RESULTS VEGF Expression and Leukocyte Infiltration Increased expression of epithelial and stromal VEGF was observed in all grades of CIN (Fig. 1). A low number of VEGF-positive cells in the epithelium and the absence of these cells in the stroma were observed in control tissues. When analysis of coexpression of VEGF and leukocyte markers was performed, increased numbers of CD3/ VEGF and macrophage/VEGF cells related to CIN pro3682

FIGURE 1. Vascular endothelial growth factor (VEGF) in cervical intraepithelial neoplasia (CIN) is shown. Increased expression of VEGF was observed in the (A) epithelium and (B) stroma of cervical samples from patients with CIN. Low values in the epithelium and the absence of VEGF-positive cells in the stroma were observed in control tissues. W ¼ 0.

gression were found (Figs. 2 and 3). In general, low numbers of CD3-positive cells expressing VEGF were observed in the epithelium of controls. Coexpression of stromal CD3/VEGF or macrophages expressing VEGF was absent in control tissues. This association was also evident when the total cellular count/number of CIN (1, 2, or 3) patient index was calculated (Fig. 4). Leukocyte populations in cervix tissues were determined by monoclonal antibodies. Increased epithelial and stromal CD3-positive cells were observed in CIN 3. Neutrophils were observed in epithelium and stroma from control tissues; however, absence or low number of neutrophils was observed in Cancer

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Cervical Neoplasia and VEGF/Carrero et al

FIGURE 2. Coexpression of vascular endothelial growth factor (VEGF) and leukocyte markers in cervical intraepithelial neoplasia (CIN) is shown. High numbers of (A and B) CD3/VEGF cells and (C and D) macrophage (Mf)/VEGF cells were observed in the epithelium and stroma of cervical samples from patients with CIN. Only a low number of CD3/VEGF–positive cells was found in the normal epithelium. A lack of stromal CD3/VEGF–positive cells and MU/VEGF was observed in control tissues. W ¼ 0.

CIN 2 and 3. Values of epithelial and stromal macrophages remained at basal numbers (Fig. 5). Oxidative Metabolism Lower values of oxygen and nitrogen oxidative metabolism were observed in controls when compared with patients. An increased number of O2-positive cells in epithelium and stroma was observed in CIN 2 and 3 (Figs. 6 and 7). However, the tissue nitrate-nitrite content in biopsies and serum from CIN patients remained similar to control values (Fig. 8). HPV Infection Sixteen percent of the CIN patients were positive for HPV, as shown by detection of HPV DNA. Normal controls were negative for HPV infection. The incidence of HPV infection was related to the CIN grades (CIN 1, 8.88 %; CIN 2, 40%; and CIN 3, 60%). Fifty percent of high-risk type infection was detected in CIN 2 and 3. There were no significant differences when values from Cancer

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positive or negative HPV infection status were compared, except in stromal O2-positive cells, in which values from HPV-infected patients were higher than those of noninfected patients (P < .001).

DISCUSSION Increased angiogenesis leads to invasion in cervical cancer. VEGF is involved in angiogenesis and progression of CIN.3-5 Accordingly, increased expression of VEGF in epithelium and stroma of uterine cervix in CIN was observed in this study. High expression of VEGF in CIN 1 suggests an early marker for neovascularization and cancer dissemination. In this regard, transcriptional activity of the VEGF gene and its receptors, indicating autocrine mechanism regulation of angiogenic genes, has been demonstrated to be increased with low-grade intraepithelial changes.12 However, to our knowledge, this early expression has not been reported by others.13 Analysis to determine the role of tissue leukocytes in the production of VEGF demonstrated increased expression of this factor in CD3 cells and macrophages in high-risk grades of CIN 3683

Original Article

FIGURE 3. Double staining for vascular endothelial growth factor (VEGF) and CD3 in a cervical sample from a patient with type 3 cervical intraepithelial neoplasia is shown. A CD3positive cell in Panel A (arrow) was also positive for VEGF in Panel B (arrow). En indicates epithelium; St, stroma.

(CIN 2-3), suggesting that infiltrating leukocytes could be in part responsible for progression of CIN. Because a high percentage of cells were only positive for VEGF, cells other than leukocytes are involved in VEGF production in CIN. It has been reported that HIV-positive status induces increased angiogenesis in high- and low-grade cervical lesions,14 and increased VEGF production has been related to high-risk HPV infection in CIN.3 In the current study, only 16% of CIN patients were positive for HPV, and there were no differences noted with regard to the VEGF values when infected and noninfected patients were compared; in addition, patients had HIV-negative status, suggesting no restricted conditions to increase VEGF production during CIN. The local immune response appears to play an important role in the progression of CIN. In the current study, an increased number of CD3-positive cells in the cervical epithelium and stroma related to the grade of 3684

FIGURE 4. Total cell count/number of individuals index in controls and patients with cervical intraepithelial neoplasia (CIN) is shown. Increased index of total vascular endothelial growth factor (VEGF), CD3/VEGF, macrophage (Mf)/VEGF, and non-CD3– or Mf/VEGF–positive cells was observed in the (A) epithelium and (B) stroma related to the progression of CIN. c ¼ 0.

CIN was found. This finding could be involved in a causal role in the progression of CIN. In addition to the production of VEGF by CD3 lymphocytes, the increased number of CD3-positive cells in CIN 3 could represent regulatory T cells, which might be involved in the failure Cancer

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Cervical Neoplasia and VEGF/Carrero et al

FIGURE 5. Leukocyte infiltration in cervical samples from patients with cervical intraepithelial neoplasia (CIN) is shown. An increased number of CD3-positive cells was found in the (A) epithelium and (B) stroma in type 3 CIN (CIN 3). (C and D) Epithelial and stromal neutrophils were observed in control tissues, and a decreased number or the absence of neutrophils was observed in the epithelium and stroma of samples from CIN 2 and CIN 3 patients. (E and F) No significant differences were found with regard to monocyte-macrophage (Mf) infiltration in patients with CIN compared with controls. PMN indicates polymorphonuclear. W ¼ 0.

of the immune response, with further CIN progression.7,15 In this regard, CD3 lymphocytes have been noted to be correlated with CIN recurrence after conization because of CIN 3.6 Monocyte-macrophage infiltration was not statistically increased in cervical tissues, suggesting an inadequate monocyte inflammatory response in this disease. These results agree with previous information indicating similar CD68 (macrophage-specific marker) and ICAM-1 in normal, CIN 2, and CIN 3 lesions, results that did not correlate with HPV infection.16 In addition, intraepitheCancer

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lial and stromal macrophages were found to be reduced in low-grade and high-grade squamous intraepithelial lesions from patients with HIV infection.17 In this study, a low percentage of CIN patients were HPV positive, and all patients were HIV negative, suggesting that this lack of monocyte infiltration could be independent of HPV or HIV infections. Decreased monocyte-macrophage infiltration during CIN could be related to a diminished chemokine production. In this regard, a complete lack of both monocyte-chemoattractant protein-1 expression and CD68 þ cell infiltration has been reported in CIN 3.18 3685

Original Article

FIGURE 7. Superoxide anion (02)-positive cells in a cervical sample from a patient with type 3 cervical intraepithelial neoplasia are shown. Positive cells were observed in the epithelium (crossed arrow) and in the stroma (arrow). Ep indicates epithelium; St, stroma. FIGURE 6. Oxygen oxidative metabolism in cervical intraepithelial neoplasia (CIN) is shown. Increased expression of superoxide anion (02)-positive cells was found in the (A) epithelium and (B) stroma, which was related to CIN progression. Several cells producing 02 were observed in both localizations.

Interestingly, even when tissue macrophage infiltration was not found to be increased, the number of macrophages expressing VEGF was high in CIN, suggesting a contribution of these cells in the lymphangiogenesis and progression of CIN. Neutrophil migration is a key event in inflammatory response of any origin, and neutrophils may be involved in tumor dissemination. Angiogenesis is necessary for solid tumor growth and dissemination, and inflammation can promote tumor angiogenesis.19,20 Neutrophils participate in the angiogenic process by secreting cytokines that may affect endothelial cell functions.19 In addition, neutrophils produce large amounts of reactive oxygen species in inflamed tissue that can pave the way for malignant 3686

tumors.21 In this study, decrease or absence of infiltrating neutrophils was observed in CIN 2 and 3, suggesting an impaired promoting mechanism in CIN. There are no previous reports related to the kinetics of neutrophil infiltration during the progression of CIN; however, increased blood neutrophil counts and decreased neutrophil chemotactic activity have been shown in patients with cervical cancer (stages II-IV), assuming that NO could interfere early in the capacity of neutrophils to migrate, thus impairing host immune response.22 Because blood NO levels and tissue content of NO remained at the normal levels, the chemotactic blocking effect of NO most likely did not play a role in the diminished neutrophil infiltration found in this study. Oxidative stress could have an important role during the progression of neoplasias.19,21 Oxidative status has been reported altered in blood of patients with CIN. Lipid peroxidation, reduced glutathione levels, and catalase activity were shown to be increased in patients with highgrade squamous intraepithelial lesion or invasive cervical Cancer

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Cervical Neoplasia and VEGF/Carrero et al

FIGURE 8. Nitrite-nitrate content in cervical intraepithelial neoplasia (CIN) is shown: (A) cervical tissue values; (B) serum values. Values from both CIN patients and controls were found not to be statistically significant.

cancer.23,24 In this study, an increased number of O2positive cells was found to be related to the progression of CIN; however, no alteration was observed in the nitritenitrate tissue content, suggesting a relevant effect of oxygen oxidative stress. The increased production of O2 in CIN lesions could induce oxidative damage. Supporting this, oxidative DNA damage increases during cervical carcinogenesis at early stages of the process and might help to predict patients at high risk of progression.8 In addition, oxidant compounds may also be involved in angiogenesis, leading to tumor growth and dissemination.20 When HPV-positive and HPV-negative patients were compared, increased production of O2 was observed in the stroma of tissues from HPV-infected patients. Because no differences were observed in the epithelium (neoplastic cells), Cancer

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this finding could represent an inflammatory response to the virus infection by stromal cells. No evidence of increased systemic or local NO production was observed in this study. However, an increased content of nitritesnitrates in endocervical and vaginal secretion samples from patients with CIN 1 through CIN 3 has been reported, suggesting a role for this mediator in the immune response against the tumor.25 In general, the incidence of HVP infection in this study was low; only 16% of individuals were positive for HPV, and extensive HPV infection was not observed. This percentage agrees with prevalence studies reporting that between 5% and 20% of the general population has HPV DNA detectable in cervical samples26 and that, in most cases, infection with HPV is transient and may or may not be associated with cervical abnormalities such as low squamous intraepithelial lesion.27 The low incidence observed in the patients in our population also agrees with other studies in the country, which have reported an incidence of 9.9% to 12.5 % in the occidental Venezuelan population.28,29 However, higher frequency of high-risk HPV infection was observed in patients with CIN 2 and CIN 3 (50%), suggesting an association of this infection with advanced stages in the evolution of CIN. VEGF production, leukocyte infiltration, and oxidative metabolism parameters had similar values in HPV-negative and HVPpositive patients, suggesting that virus infection was not responsible for the findings observed in the course of uterine premalignant lesions. The results of this investigation indicate the existence of factors capable of inducing tumor growth and dissemination during the evolution of CIN. Increased production of VEGF, oxidative stress, and increased Tlymphocyte infiltration in advanced stages of CIN could lead to invasive cervical neoplasia. Because a limited number of controls were analyzed in this study, we cannot rule out the possibility that our results may be changed using a greater number of controls. The changes observed in these factors may be potential molecular targets for studies of cervical cancer prevention and treatment. Further studies are required to determine the evolution of these parameters in invasive cancer. Conflict of Interest Disclosures Supported by FONACIT (S1-2002000531) and Mision Ciencias (2007001088).


Original Article


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Branca M, Giorgi C, Santini D, et al. HPV-Pathogen ISS Study Group. Aberrant expression of VEGF-C is related to grade of cervical intraepithelial neoplasia (CIN) and high risk HPV, but does not predict virus clearance after treatment of CIN or prognosis of cervical cancer. J Clin Pathol. 2006;59:40-47.

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Lee JS, Kim HS, Park JT, Lee MC, Park CS. Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status. Anal Quant Cytol Histol. 2003;25:303-311.


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18. Kleine-Lowinski K, Gillitzer R, Kuhne-Heid R, Rosl F. Monocyte-chemo-attractant-protein-1 (MCP-1)-gene expression in cervical intra-epithelial neoplasias and cervical carcinomas. Int J Cancer. 1999;82:6-11. 19. Naldini A, Carraro F. Role of inflammatory mediators in angiogenesis. Curr Drug Targets Inflamm Allergy. 2005;4:3-8. 20. Noonan DM, Benelli R, Albini A. Angiogenesis and cancer prevention: a vision. Recent Results Cancer Res. 2007;174: 219-224. 21. Roessner A, Kuester D, Malfertheiner P, Schneider-Stock R. Oxidative stress in ulcerative colitis-associated carcinogenesis. Pathol Res Pract. 2008;204:511-524. 22. Fernandes PC Jr, Garcia CB, Micheli DC, Cunha FQ, Murta EF, Tavares-Murta BM. Circulating neutrophils may play a role in the host response in cervical cancer. Int J Gynecol Cancer. 2007;17:1068-1074. 23. Kim YT, Kim JW, Choi JS, Kim SH, Choi EK, Cho NH. Relation between deranged antioxidant system and cervical neoplasia. Int J Gynecol Cancer. 2004;14:889-895. 24. Maldonado PA, Negrini LA, Kaizer RR, et al. Oxidative status in patients submitted to conization and radiation treatments for uterine cervix neoplasia. Clin Chim Acta. 2006;366:174-178. 25. Tavares-Murta BM, de Resende AD, Cunha FQ, Murta EF. Local profile of cytokines and nitric oxide in patients with bacterial vaginosis and cervical intraepithelial neoplasia. Eur J Obstet Gynecol Reprod Biol. 2008;138:93-99. 26. Critchlow CW, Koutsky LA. Epidemiology of human papillomavirus infection. In: Mindel A, ed. Genital Warts: Human Papillomavirus Infection. London, UK: Edward Arnold; 1995:53-81. 27. von Knebel Doeberitz M. The role of papillomaviruses in the etiology of cervix cancer [in German]. Geburtshilfe Frauenheilkd. 1990;50:511-517. 28. Munoz M, Mendoza JA, Tellez L, et al. Deteccion de VPH-16 y 18 en muestras de cervix de mujeres que acuden a centros asistenciales de la ciudad de Merida, Venezuela. [Detection of HPV-16 and 18 in cervical samples from women of Merida, Venezuela.] Rev Biomed. 2003;14:61-68. 29. Mendoza JA, Munoz M, Vielma S, Noguera ME, Lopez M, Toro M. Infeccion cervical por el virus del papiloma humano: diagnostico por citologia y por captura de hibridos del ADN viral. [Human papilloma virus cervix infection: diagnostic by cytology and viral DNA hybrid capture.] Rev Obstet Ginecol Venez. 2000;60:103-107.


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