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Immunohistochemical analysis of vascular endothelial growth factor cellular expression in ovarian endometriomata
FERTILITY AND STERILITY威 VOL. 81, NO. 6, JUNE 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Immunohistochemical analysis of vascular endothelial growth factor cellular expression in ovarian endometriomata Gaia Goteri, M.D.,a Guendalina Lucarini, Ph.D.,b Alessandra Filosa, M.D.,a Alessandra Pierantoni, M.D.,c Nina Montik, M.D.,c Graziella Biagini, M.D.,b Guidalberto Fabris, M.D.,a and Andrea Ciavattini, M.D.c School of Medicine, Universita` Politecnica delle Marche, Ancona, Italy
Received June 16, 2003; revised and accepted November 14, 2003. Reprint requests: Gaia Goteri, M.D., Anatomia Patologica-Dipartimento di Neuroscienze-Universita` Politecnica delle Marche, Ospedale Torrette, Via Conca 60020, Ancona, Italy (FAX: 39-071-889985; E-mail: [email protected]). a Sezione di Anatomia Patologica, Dipartimento di Neuroscienze, Ospedale Torrette, Ancona, Italy. b Istituto di Istologia, Polo Didattico Torrette, Ancona, Italy. c Clinica di Ginecologia ed Ostetricia, Ospedale G. Salesi, Universita` Politecnica delle Marche, Ancona, Italy. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 11.035
1528
Objective: To evaluate the expression of vascular endothelial growth factor (VEGF) in the cell populations of ovarian endometriomata cyst layers. Design: Experimental retrospective study. Setting: University hospital. Patient(s): Twenty-eight patients with ovarian endometriomata. Intervention(s): Surgical excision of 32 ovarian cysts. Main Outcome Measure(s): Histologic and VEGF immunohistochemical analysis of cyst layers. Result(s): Though the least represented cell types, macrophages exhibited the highest frequency of strong immunoreactivity, followed by capsular vessel endothelial and subepithelial stromal cells and by epithelial cells and capsular fibroblasts. Endothelia of the subepithelial stroma were the least immunoreactive cells. Diffuse VEGF expression in epithelial cells was associated with cyst diameters greater than 5.4 cm, and high VEGF expression in capsular fibroblasts was associated with bilateral cysts. Conclusion(s): Angiogenesis plays an active role in ovarian endometriosis, especially in the presence of large and bilateral cysts. Expression of VEGF in epithelial cells, capsular fibroblasts, and vessels was found to be related, suggesting that neoangiogenesis might especially affect the outer cyst wall, thus contributing to the fibrosing process of adhesion formation during cyst growth. (Fertil Steril威 2004;81:1528 –33. ©2004 by American Society for Reproductive Medicine.) Key Words: Ovarian endometriosis, angiogenesis, VEGF
Endometriosis is a relatively frequent disease affecting women of reproductive age. It is characterized by the presence of ectopic endometrium outside the uterus, with pelvic pain and infertility as the major complaints. The two main sites of disease location are the pelvic peritoneum, where active lesions appear as small, red maculae and old, inactive ones as black pigmented plaques, and the ovaries, where lesions become cystic and rather larger. The presence of large amounts of steroid hormones in the ovary has been invoked to explain the larger diameter of endometriomata compared with extraovarian implants. Several pathogenic mechanisms have been suggested for the two different locations (1). Peritoneal endometriosis can reasonably be explained by the implantation of endometrial tissue coming
from the uterine cavity through a retrograde reflux, whereas in the ovary endometrial tissue might colonize a surface inclusion or a functional follicular cyst; it might form an adherence with the peritoneal wall or the broad ligament and grow inside the ovary due to cortical invagination, or could derive from metaplasia of the celomatic lining (2, 3). As survival of ectopic endometrium and development of endometriosis could be enormously facilitated by the formation of new vessels, angiogenesis has been studied extensively in this connection (4 –15). One key mediator of angiogenesis is vascular endothelial growth factor (VEGF), a secreted homodimeric glycoprotein that stimulates proliferation and migration of endothelial cells and enhances vascular permeability (16). There are at least
FIGURE 1 (a) High power view of an ovarian endometrioma with cuboidal epithelium and absent specialized stroma; fibrous tissue with some vessels and macrophages below the epithelium (H&E). (b) The same field immunostained with anti-VEGF antibody, showing that epithelial cells are strongly positive (score ⫹⫹⫹) together with some macrophages and vessel endothelial cells. (c, d) Score ⫹⫹⫹ immunostaining of capsular fibroblasts and vessels (c) and macrophages (d).
TABLE 1 Distribution of VEGF expression scores in the various cell populations. VEGF expression score
Cyst layer Internal layer Epithelial cells Stromal cells Endothelial cells Macrophages Outer layer Fibroblasts Endothelial cells Macrophages
⫹ (no. cases)
⫹⫹ (no. cases)
⫹⫹⫹ (no. cases)
16 8 22 0
7 9 4 4
5 7 2 5
19 13 2
8 13 2
4 6 3
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
(8, 9, 15), but it was not demonstrated in ovarian cysts by Deguchi et al. (13). Recently, Tan et al (15) detected greater VEGF expression in peritoneal lesions than in ovarian cysts. The aim of our study was to further investigate the immunohistochemical expression of VEGF in the various cell populations of ovarian endometriotic cysts. Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
MATERIALS AND METHODS five isoforms of VEGF, resulting from alternative splicing of a single gene. The larger isoforms, VEGF-189 and VEGF206, remain cell-bound, whereas the smaller subtypes, VEGF-121 and VEGF-165, are efficiently secreted after production (17). Although VEGF is normally produced by various tissues in the female genital tract, such as endometrium, ovary, and placenta, VEGF receptors are found exclusively in endothelial cells. Vascular endothelial growth factor is involved in normal endometrial turnover during the menstrual cycle and is under the control of ovarian steroids (18). As in malignant tumors, where VEGF overexpression has been reported to correlate with tumor invasiveness and metastatic potential (19), VEGF might be overexpressed in endometriotic lesions, resulting in formation of the vessel network required for implant survival. The involvement of tissue hypoxia in the up-regulation of VEGF expression occurring in the phase of implantation and growth of ectopic endometrial tissue has also been hypothesized (20). The role of VEGF and of other angiogenic factors has been investigated in both conditions, with conflicting results (5, 6, 8, 9, 13, 15). High concentrations of VEGF have been reported in peritoneal liquid in pelvic endometriosis (5, 7, 11) and in cystic liquid content of ovarian endometriomata (12). In tissues, VEGF expression has been detected in macrophages (6) and in glandular epithelial and stromal cells FERTILITY & STERILITY威
Clinical Data We reviewed the clinical records of 28 patients consecutively referred to the Department of Gynecology and Obstetrics of G. Salesi Hospital, Ancona, Italy, between 1999 and 2000 for diagnosis and surgical removal of ovarian endometriotic cysts. The following clinical data were collected: age, presence and intensity of pelvic pain (absent to mild vs. severe), cycle characteristics (regular vs. irregular), cycle phase at the time of surgery (preovulatory or postovulatory), and hormone therapy before surgery. Cyst diameter, bilateral cysts, and adhesions to the pelvic wall or to other pelvic viscera were also recorded. The Revised American Fertility Society (AFSR) scores for endometriosis were calculated for each patient (21).
Pathologic Examination Surgical samples were sent fresh or in formalin to the Institute of Pathology of Polytechnic University of Marche, Ancona. Pathologic description of cysts and histologic slides were retrieved from the archives to evaluate morphologic parameters. Similarities were sought between epithelial lining (columnar or cuboidal) and subepithelial specialized stroma (small and spindle-shaped or plump and polygonal cells) of ectopic endometrium and the glandular and stromal components of eutopic endometrium in its proliferative or secretive phases. We also assessed cytologic atypia, hyperplasia, and frank malignant proliferation of the epithelial 1529
FIGURE 2 The correlation between epithelial and capsular fibroblastic VEGF immunoreactivity is statistically significant. Purple ⫽ fibroblastic VEGF score ⫹⫹⫹. Green ⫽ fibroblastic VEGF score ⫹⫹. Light green ⫽ fibroblastic VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
component; presence of macrophages with foamy or pigmented cytoplasm; morphologic evidence of an outer fibrous capsule with activated fibroblasts and small vessels; and preservation of an invaginated ovarian cortical layer between cyst and medulla (4). Representative areas of the cysts were selected for immunohistochemical investigations. Multiple cysts were considered individually.
Immunohistochemistry
Immunohistochemical analysis was performed on 5-m paraffin-embedded tissue sections on poly-L-lysine-coated glass slides. After heat-drying, sections were deparaffinized in xylene and sequentially rehydrated in gradients of ethanol, and treated with TUF solution (Histo-line Laboratories, Milan, Italy) at 90°C for 10 minutes better to unmask antigenic sites; after three washes with H2O, they were incubated overnight at 4°C with anti-VEGF-165 monoclonal antibody (dil. 1:200, Santa Cruz Biotechnology, Santa Cruz, CA). The reaction was revealed using the streptavidin-biotin-peroxidase technique: sections were incubated with 3,3⬘-diaminobenzidine (0.05 diaminobenzidine in 0.05 M Tris buffer, pH 7.6 and 0.01% hydrogen peroxide) and counterstained with Mayer’s hematoxylin. Positive controls were paraffin-em1530 Goteri et al.
VEGF expression in ovarian endometriosis
bedded sections from gastric carcinomas. Negative controls were performed by substituting the primary antibody with nonimmune serum. Semiquantitative analysis of VEGF immunostaining was carried out by light microscopy in epithelium; specialized cells, vessel endothelia, and macrophages of the subepithelial stroma; and fibroblasts, vessel endothelia, and macrophages of the fibrous capsule. A rising score was attributed based on the proportion of VEGF-positive cells observed in each of these populations: ⫹ (up to 25%), ⫹⫹ (⬎25% and ⬍50%), and ⫹⫹⫹ (⬎50%). Sections were separately examined twice by two independent observers; interobserver and intraobserver variability was evaluated, and concordance was ⬎80%. Discordant cases were discussed until complete agreement.
Statistical Analysis Clinical and pathologic data were entered into StatView 4.5 (SAS Institute, Cary, NC) on a Macintosh computer. Cases were analyzed based on the clinical and pathologic parameters (pain intensity, cycle characteristics, cyst diameter, bilaterality, adhesions to pelvic wall and viscera, type of Vol. 81, No. 6, June 2004
FIGURE 3 The correlation between epithelial and capsular endothelial VEGF immunoreactivity is statistically significant. Purple ⫽ capsular vessel VEGF score ⫹⫹⫹. Green ⫽ capsular vessel VEGF score ⫹⫹. Light green ⫽ capsular vessel VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
epithelial and stromal cells, and cellular atypia). Differences in the distribution of VEGF expression among groups and cell populations were compared using the chi-square test. Differences in mean age and AFSR scores among the three groups of patients with different VEGF expression scores were analyzed using the nonparametric Kruskal-Wallis test. Pⱕ.05 was considered statistically significant.
RESULTS Clinical Data The age of the 28 patients ranged from 21 to 43 years (mean ⫾ SD, 31.46 ⫾ 5.99). Only three women had irregular cycles. Pain was cited by 24 patients: mild in 8 and severe in 16. The AFSR scores ranged from 20 to 84 (mean ⫾ SD, 39.43 ⫾ 15.07). At the time of surgery, 21 patients were in the preovulatory and 7 in the postovulatory phase. There were 32 cysts: 24 single cysts, 3 bilateral cysts, and 2 cysts in the same ovary. Adhesions were present in 25 cases. None of the patients was receiving hormonal therapy at the time of surgery; 11 patients had discontinued anticontraceptive hormonal treatment more than 12 months before surgery (range: 13 to 96 months). FERTILITY & STERILITY威
Pathologic Findings The diameter of the 32 cysts ranged from 2 to 10 cm (mean ⫾ SD, 5.49 ⫾ 2.1). Histologically, they exhibited an inner layer of ectopic endometrium and an outer fibrosclerotic wall recognizable in all cases, with numerous small vessels and activated fibroblasts (Fig. 1a). A third, external, layer of ovarian cortex with oocytes, follicles, functional cysts, and corpora albicantes was also noted in 25 cases. The thickness of each layer varied considerably from cyst to cyst. The epithelial lining was absent in four cysts and the specialized subepithelial stroma in nine. The epithelial cells forming the lining and the glands were columnar, with basal nuclei resembling those of the estrogenic phase of normal endometrium in 17 cases; and cuboidal, resembling those of the progestinic phase, in 11. Regressive atypia was found in eight cysts. No hyperplastic or carcinomatous foci were detected. The endometrial-type stroma below the epithelium and around the glands showed variable thickness and numerous extravascular erythrocytes. Endometrial-type stromal cells appeared small and spindle-shaped, as in the proliferative phase, in 17 cases, and plump and even pseudo-decidualized, as in the secretory 1531
FIGURE 4 The correlation between capsular fibroblastic and endothelial VEGF immunoreactivity is statistically significant. Purple ⫽ fibroblastic VEGF score ⫹⫹⫹. Green ⫽ fibroblastic VEGF score ⫹⫹. Light green ⫽ fibroblastic VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
phase, in 6 cases. Small vessels were recognized in the stroma in 28 cysts. Large aggregates of pigmented or foamy macrophages were present in nine cysts.
VEGF Immunostaining The VEGF immunoreactivity was variable in the different cyst layers, as shown in Table 1. Although macrophages were detected in a minority of cysts, they exhibited the highest frequency of diffuse immunoreactivity, followed by capsular vessel and subepithelial stromal cells, and by epithelial cells and capsular fibroblasts (see Fig. 1). The least immunoreactive cells were the endothelia of the subepithelial stroma. Epithelial and subepithelial stromal immunoreactivity was more diffuse, albeit not significantly, in cysts with features of secretory-type endometrial lining. Diffuse epithelial VEGF expression was associated with cyst diameters greater than 5.4 cm (P⬍.05). Bilateral cysts were related to diffuse fibroblastic VEGF expression (P⬍.05) and tended to be associated with stronger VEGF immunoreactivity in the vessels of the outer fibrous capsule (P⫽.06). Epithelial staining for VEGF correlated with positivity of fibrous wall fibroblasts (P⫽.01; Fig. 2) and endothelial cells (P⫽.01; Fig. 3). The VEGF expression in fibroblasts correlated highly 1532 Goteri et al.
VEGF expression in ovarian endometriosis
with staining in fibrous wall endothelial cells (P⬍.0001; Fig. 4). None of the clinical parameters correlated with VEGF expression scores in the different cell populations.
DISCUSSION The present study shows that VEGF is expressed in the cellular constituents of ovarian endometriomata. Some researchers from our group reported absent or low VEGF expression in epithelial and endothelial cells of benign inclusion cysts and serous cystadenomas, more intense immunoreactivity in borderline tumors, and still stronger staining in invasive cystadenocarcinomas of the ovary (22). Thus, VEGF expression in endometriotic cysts seems to be comparable to that of malignant ovarian neoplasms rather than of benign nonendometriotic cysts. In line with the literature (9), our data implicate neoangiogenesis in ovarian endometriosis as being similar to neoplastic conditions. The present study was also directed at seeking correlations among VEGF cellular expression, clinical findings, and cellular composition of the ovarian cysts. None of the clinical parameters evaluated correlated with VEGF expression. However, the more interesting findings came from pathologic analysis of the Vol. 81, No. 6, June 2004
histology of immunoreactive cell types and of gross parameters (cyst diameter and bilaterality). In line with McLaren et al. (6), who demonstrated that activated macrophages are a major source of VEGF in endometriosis, we found that the cells with the highest frequency of diffuse VEGF expression were macrophages, detected in a minority of cysts, followed by capsular vessel and subepithelial stromal cells, and by epithelial cells and capsular fibroblasts. The least immunoreactive cells were the endothelia of the subepithelial stroma. A correlation was observed between VEGF expression in epithelial cells and capsular fibroblasts and vessels, suggesting that these cell types might form a cellular network sharing regulation by the stimulatory signals promoting angiogenesis. Because E2 induces VEGF expression in cultured endometrial cells (7), endometrial cancer cell lines (23), and macrophages (6), and hypoxia up-regulates VEGF expression (16, 20), further research is needed to evaluate the role of these factors in endometriosis. Although we failed to find a correlation between VEGF expression and the histologic appearance of epithelial or stromal cells, there was a strong, nearly statistically significant trend for greater VEGF immunoreactivity in cysts with secretory compared with proliferative features. An intriguing finding was the association between strong VEGF expression in epithelial cells and a cyst diameter greater than 5.4 cm. Only two studies have analyzed the relationship between expression of angiogenic factors and cyst size (4, 12). Like us, Di Blasio et al. (4) showed a similar relationship between greater cyst diameter and higher basic fibroblastic growth factor (bFGF) mRNA levels, whereas Fasciani et al. (12) found an inverse correlation between VEGF concentration in cystic fluid and cyst diameter. Although the latter data may appear to contrast with ours, VEGF concentrations in fluid might in fact be inversely related to VEGF expression in the epithelial lining of cysts and in other cell compartments. In our study, bilateral cysts were associated with diffuse VEGF expression in the cells of the outer fibrous capsule. The present results are consistent with a pathogenic mechanism that could combine the relationships of VEGF expression in epithelium and outer fibrous capsular wall and with cyst diameter and bilaterality. Based on this mechanism, angiogenesis in ovarian cysts would involve the external wall more than the other layer(s), contributing to cyst growth, to the fibrosing process in the capsule, and to formation of adhesions. References 1. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997;68:585–96.
FERTILITY & STERILITY威
2. Brosens I, Puttemans P, Deprest J. The endoscopic localization of endometrial implants in the ovarian chocolate cyst. Fertil Steril 1994; 61:1034 –8. 3. Scurry J, Whitehead J, Healey M. Classification of ovarian endometriotic cysts. Int J Gynecol Pathol 2001;20:147–54. 4. Di Blasio AM, Centinaio G, Carniti C, Somigliana E, Vigano P, Vignali M. Basic fibroblast growth factor messenger ribonucleic acid levels in eutopic and ectopic human endometrial stromal cells as assessed by competitive polymerase chain reaction amplification. Mol Cell Endocrinol 1995;115:169 –75. 5. McLaren J, Prentice A, Charnock-Jones DS, Smith SK. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod 1996;11:220 –3. 6. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. 7. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, et al. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112–8. 8. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13:1686 –90. 9. Fujishita A, Hasuo A, Khan KN, Masuzaki H, Nakashima H, Ishimaru T. Immunohistochemical study of angiogenic factors in endometrium and endometriosis. Gynecol Obstet Invest 1999;48(Suppl 1):36 –44. 10. Fujimoto J, Sakaguchi H, Hirose R, Wen H, Tamaya T. Angiogenesis in endometriosis and angiogenic factors. Gynecol Obstet Invest 1999; 48(Suppl 1):14 –20. 11. Mahnke JL, Dawood MY, Huang JC. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 2000;73:166 –70. 12. Fasciani A, D’Ambrogio G, Bocci G, Monti M, Genazzani AR, Artini PG. High concentrations of the vascular endothelial growth factor and interleukin-8 in ovarian endometriomata. Mol Hum Reprod 2000;6:50 –4. 13. Deguchi M, Ishiko O, Sumi T, Yoshida H, Yamamoto K, Ogita S. Expression of angiogenic factors in extrapelvic endometriosis. Oncol Rep 2001;8:1317–9. 14. Kressin P, Wolber EM, Wodrich H, Meyhofer-Malik A, Buchweitz O, Diedrich K, et al. Vascular endothelial growth factor mRNA in eutopic and ectopic endometrium. Fertil Steril 2001;76:1220 –4. 15. Tan XJ, Lang JH, Liu DY, Shen K, Leng JH, Zhu L. Expression of vascular endothelial growth factor and thrombospondin-1 mRNA in patients with endometriosis. Fertil Steril 2002;78:148 –53. 16. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4 –25. 17. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1992;13:18 –32. 18. Greb RR, Heikinheimo O, Williams RF, Hodgen GD, Goodman AL. Vascular endothelial growth factor in primate endometrium is regulated by oestrogen-receptor and progesterone-receptor ligands in vivo. Hum Reprod 1997;12:1280 –92. 19. Itakura J, Ishiwata T, Shen B, Kornmann M, Korc M. Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer. Int J Cancer 2000;85:27–34. 20. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359:843–5. 21. American Fertility Society. Revised American Fertility Society Classification of Endometriosis, 1985. Fertil Steril 1985;43:351–2. 22. Garzetti GG, Ciavattini A, Lucarini G, Pugnaloni A, DeNictolis M, Amati S, et al. Expression of VEGF related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors. Cancer 1999;85:2219 –25. 23. Charnock-Jones DS, Sharkey AM, Rajput-Williams J, Burch D, Schofield JP, Fountain SA, et al. Identification and localization of alternately spliced mRNAs for VEGF in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol Reprod 1993;48: 1120 –8.
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Immunohistochemical analysis of vascular endothelial growth factor cellular expression in ovarian endometriomata Gaia Goteri, M.D.,a Guendalina Lucarini, Ph.D.,b Alessandra Filosa, M.D.,a Alessandra Pierantoni, M.D.,c Nina Montik, M.D.,c Graziella Biagini, M.D.,b Guidalberto Fabris, M.D.,a and Andrea Ciavattini, M.D.c School of Medicine, Universita` Politecnica delle Marche, Ancona, Italy
Received June 16, 2003; revised and accepted November 14, 2003. Reprint requests: Gaia Goteri, M.D., Anatomia Patologica-Dipartimento di Neuroscienze-Universita` Politecnica delle Marche, Ospedale Torrette, Via Conca 60020, Ancona, Italy (FAX: 39-071-889985; E-mail: [email protected]). a Sezione di Anatomia Patologica, Dipartimento di Neuroscienze, Ospedale Torrette, Ancona, Italy. b Istituto di Istologia, Polo Didattico Torrette, Ancona, Italy. c Clinica di Ginecologia ed Ostetricia, Ospedale G. Salesi, Universita` Politecnica delle Marche, Ancona, Italy. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 11.035
1528
Objective: To evaluate the expression of vascular endothelial growth factor (VEGF) in the cell populations of ovarian endometriomata cyst layers. Design: Experimental retrospective study. Setting: University hospital. Patient(s): Twenty-eight patients with ovarian endometriomata. Intervention(s): Surgical excision of 32 ovarian cysts. Main Outcome Measure(s): Histologic and VEGF immunohistochemical analysis of cyst layers. Result(s): Though the least represented cell types, macrophages exhibited the highest frequency of strong immunoreactivity, followed by capsular vessel endothelial and subepithelial stromal cells and by epithelial cells and capsular fibroblasts. Endothelia of the subepithelial stroma were the least immunoreactive cells. Diffuse VEGF expression in epithelial cells was associated with cyst diameters greater than 5.4 cm, and high VEGF expression in capsular fibroblasts was associated with bilateral cysts. Conclusion(s): Angiogenesis plays an active role in ovarian endometriosis, especially in the presence of large and bilateral cysts. Expression of VEGF in epithelial cells, capsular fibroblasts, and vessels was found to be related, suggesting that neoangiogenesis might especially affect the outer cyst wall, thus contributing to the fibrosing process of adhesion formation during cyst growth. (Fertil Steril威 2004;81:1528 –33. ©2004 by American Society for Reproductive Medicine.) Key Words: Ovarian endometriosis, angiogenesis, VEGF
Endometriosis is a relatively frequent disease affecting women of reproductive age. It is characterized by the presence of ectopic endometrium outside the uterus, with pelvic pain and infertility as the major complaints. The two main sites of disease location are the pelvic peritoneum, where active lesions appear as small, red maculae and old, inactive ones as black pigmented plaques, and the ovaries, where lesions become cystic and rather larger. The presence of large amounts of steroid hormones in the ovary has been invoked to explain the larger diameter of endometriomata compared with extraovarian implants. Several pathogenic mechanisms have been suggested for the two different locations (1). Peritoneal endometriosis can reasonably be explained by the implantation of endometrial tissue coming
from the uterine cavity through a retrograde reflux, whereas in the ovary endometrial tissue might colonize a surface inclusion or a functional follicular cyst; it might form an adherence with the peritoneal wall or the broad ligament and grow inside the ovary due to cortical invagination, or could derive from metaplasia of the celomatic lining (2, 3). As survival of ectopic endometrium and development of endometriosis could be enormously facilitated by the formation of new vessels, angiogenesis has been studied extensively in this connection (4 –15). One key mediator of angiogenesis is vascular endothelial growth factor (VEGF), a secreted homodimeric glycoprotein that stimulates proliferation and migration of endothelial cells and enhances vascular permeability (16). There are at least
FIGURE 1 (a) High power view of an ovarian endometrioma with cuboidal epithelium and absent specialized stroma; fibrous tissue with some vessels and macrophages below the epithelium (H&E). (b) The same field immunostained with anti-VEGF antibody, showing that epithelial cells are strongly positive (score ⫹⫹⫹) together with some macrophages and vessel endothelial cells. (c, d) Score ⫹⫹⫹ immunostaining of capsular fibroblasts and vessels (c) and macrophages (d).
TABLE 1 Distribution of VEGF expression scores in the various cell populations. VEGF expression score
Cyst layer Internal layer Epithelial cells Stromal cells Endothelial cells Macrophages Outer layer Fibroblasts Endothelial cells Macrophages
⫹ (no. cases)
⫹⫹ (no. cases)
⫹⫹⫹ (no. cases)
16 8 22 0
7 9 4 4
5 7 2 5
19 13 2
8 13 2
4 6 3
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
(8, 9, 15), but it was not demonstrated in ovarian cysts by Deguchi et al. (13). Recently, Tan et al (15) detected greater VEGF expression in peritoneal lesions than in ovarian cysts. The aim of our study was to further investigate the immunohistochemical expression of VEGF in the various cell populations of ovarian endometriotic cysts. Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
MATERIALS AND METHODS five isoforms of VEGF, resulting from alternative splicing of a single gene. The larger isoforms, VEGF-189 and VEGF206, remain cell-bound, whereas the smaller subtypes, VEGF-121 and VEGF-165, are efficiently secreted after production (17). Although VEGF is normally produced by various tissues in the female genital tract, such as endometrium, ovary, and placenta, VEGF receptors are found exclusively in endothelial cells. Vascular endothelial growth factor is involved in normal endometrial turnover during the menstrual cycle and is under the control of ovarian steroids (18). As in malignant tumors, where VEGF overexpression has been reported to correlate with tumor invasiveness and metastatic potential (19), VEGF might be overexpressed in endometriotic lesions, resulting in formation of the vessel network required for implant survival. The involvement of tissue hypoxia in the up-regulation of VEGF expression occurring in the phase of implantation and growth of ectopic endometrial tissue has also been hypothesized (20). The role of VEGF and of other angiogenic factors has been investigated in both conditions, with conflicting results (5, 6, 8, 9, 13, 15). High concentrations of VEGF have been reported in peritoneal liquid in pelvic endometriosis (5, 7, 11) and in cystic liquid content of ovarian endometriomata (12). In tissues, VEGF expression has been detected in macrophages (6) and in glandular epithelial and stromal cells FERTILITY & STERILITY威
Clinical Data We reviewed the clinical records of 28 patients consecutively referred to the Department of Gynecology and Obstetrics of G. Salesi Hospital, Ancona, Italy, between 1999 and 2000 for diagnosis and surgical removal of ovarian endometriotic cysts. The following clinical data were collected: age, presence and intensity of pelvic pain (absent to mild vs. severe), cycle characteristics (regular vs. irregular), cycle phase at the time of surgery (preovulatory or postovulatory), and hormone therapy before surgery. Cyst diameter, bilateral cysts, and adhesions to the pelvic wall or to other pelvic viscera were also recorded. The Revised American Fertility Society (AFSR) scores for endometriosis were calculated for each patient (21).
Pathologic Examination Surgical samples were sent fresh or in formalin to the Institute of Pathology of Polytechnic University of Marche, Ancona. Pathologic description of cysts and histologic slides were retrieved from the archives to evaluate morphologic parameters. Similarities were sought between epithelial lining (columnar or cuboidal) and subepithelial specialized stroma (small and spindle-shaped or plump and polygonal cells) of ectopic endometrium and the glandular and stromal components of eutopic endometrium in its proliferative or secretive phases. We also assessed cytologic atypia, hyperplasia, and frank malignant proliferation of the epithelial 1529
FIGURE 2 The correlation between epithelial and capsular fibroblastic VEGF immunoreactivity is statistically significant. Purple ⫽ fibroblastic VEGF score ⫹⫹⫹. Green ⫽ fibroblastic VEGF score ⫹⫹. Light green ⫽ fibroblastic VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
component; presence of macrophages with foamy or pigmented cytoplasm; morphologic evidence of an outer fibrous capsule with activated fibroblasts and small vessels; and preservation of an invaginated ovarian cortical layer between cyst and medulla (4). Representative areas of the cysts were selected for immunohistochemical investigations. Multiple cysts were considered individually.
Immunohistochemistry
Immunohistochemical analysis was performed on 5-m paraffin-embedded tissue sections on poly-L-lysine-coated glass slides. After heat-drying, sections were deparaffinized in xylene and sequentially rehydrated in gradients of ethanol, and treated with TUF solution (Histo-line Laboratories, Milan, Italy) at 90°C for 10 minutes better to unmask antigenic sites; after three washes with H2O, they were incubated overnight at 4°C with anti-VEGF-165 monoclonal antibody (dil. 1:200, Santa Cruz Biotechnology, Santa Cruz, CA). The reaction was revealed using the streptavidin-biotin-peroxidase technique: sections were incubated with 3,3⬘-diaminobenzidine (0.05 diaminobenzidine in 0.05 M Tris buffer, pH 7.6 and 0.01% hydrogen peroxide) and counterstained with Mayer’s hematoxylin. Positive controls were paraffin-em1530 Goteri et al.
VEGF expression in ovarian endometriosis
bedded sections from gastric carcinomas. Negative controls were performed by substituting the primary antibody with nonimmune serum. Semiquantitative analysis of VEGF immunostaining was carried out by light microscopy in epithelium; specialized cells, vessel endothelia, and macrophages of the subepithelial stroma; and fibroblasts, vessel endothelia, and macrophages of the fibrous capsule. A rising score was attributed based on the proportion of VEGF-positive cells observed in each of these populations: ⫹ (up to 25%), ⫹⫹ (⬎25% and ⬍50%), and ⫹⫹⫹ (⬎50%). Sections were separately examined twice by two independent observers; interobserver and intraobserver variability was evaluated, and concordance was ⬎80%. Discordant cases were discussed until complete agreement.
Statistical Analysis Clinical and pathologic data were entered into StatView 4.5 (SAS Institute, Cary, NC) on a Macintosh computer. Cases were analyzed based on the clinical and pathologic parameters (pain intensity, cycle characteristics, cyst diameter, bilaterality, adhesions to pelvic wall and viscera, type of Vol. 81, No. 6, June 2004
FIGURE 3 The correlation between epithelial and capsular endothelial VEGF immunoreactivity is statistically significant. Purple ⫽ capsular vessel VEGF score ⫹⫹⫹. Green ⫽ capsular vessel VEGF score ⫹⫹. Light green ⫽ capsular vessel VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
epithelial and stromal cells, and cellular atypia). Differences in the distribution of VEGF expression among groups and cell populations were compared using the chi-square test. Differences in mean age and AFSR scores among the three groups of patients with different VEGF expression scores were analyzed using the nonparametric Kruskal-Wallis test. Pⱕ.05 was considered statistically significant.
RESULTS Clinical Data The age of the 28 patients ranged from 21 to 43 years (mean ⫾ SD, 31.46 ⫾ 5.99). Only three women had irregular cycles. Pain was cited by 24 patients: mild in 8 and severe in 16. The AFSR scores ranged from 20 to 84 (mean ⫾ SD, 39.43 ⫾ 15.07). At the time of surgery, 21 patients were in the preovulatory and 7 in the postovulatory phase. There were 32 cysts: 24 single cysts, 3 bilateral cysts, and 2 cysts in the same ovary. Adhesions were present in 25 cases. None of the patients was receiving hormonal therapy at the time of surgery; 11 patients had discontinued anticontraceptive hormonal treatment more than 12 months before surgery (range: 13 to 96 months). FERTILITY & STERILITY威
Pathologic Findings The diameter of the 32 cysts ranged from 2 to 10 cm (mean ⫾ SD, 5.49 ⫾ 2.1). Histologically, they exhibited an inner layer of ectopic endometrium and an outer fibrosclerotic wall recognizable in all cases, with numerous small vessels and activated fibroblasts (Fig. 1a). A third, external, layer of ovarian cortex with oocytes, follicles, functional cysts, and corpora albicantes was also noted in 25 cases. The thickness of each layer varied considerably from cyst to cyst. The epithelial lining was absent in four cysts and the specialized subepithelial stroma in nine. The epithelial cells forming the lining and the glands were columnar, with basal nuclei resembling those of the estrogenic phase of normal endometrium in 17 cases; and cuboidal, resembling those of the progestinic phase, in 11. Regressive atypia was found in eight cysts. No hyperplastic or carcinomatous foci were detected. The endometrial-type stroma below the epithelium and around the glands showed variable thickness and numerous extravascular erythrocytes. Endometrial-type stromal cells appeared small and spindle-shaped, as in the proliferative phase, in 17 cases, and plump and even pseudo-decidualized, as in the secretory 1531
FIGURE 4 The correlation between capsular fibroblastic and endothelial VEGF immunoreactivity is statistically significant. Purple ⫽ fibroblastic VEGF score ⫹⫹⫹. Green ⫽ fibroblastic VEGF score ⫹⫹. Light green ⫽ fibroblastic VEGF score ⫹.
Goteri. VEGF expression in ovarian endometriosis. Fertil Steril 2004.
phase, in 6 cases. Small vessels were recognized in the stroma in 28 cysts. Large aggregates of pigmented or foamy macrophages were present in nine cysts.
VEGF Immunostaining The VEGF immunoreactivity was variable in the different cyst layers, as shown in Table 1. Although macrophages were detected in a minority of cysts, they exhibited the highest frequency of diffuse immunoreactivity, followed by capsular vessel and subepithelial stromal cells, and by epithelial cells and capsular fibroblasts (see Fig. 1). The least immunoreactive cells were the endothelia of the subepithelial stroma. Epithelial and subepithelial stromal immunoreactivity was more diffuse, albeit not significantly, in cysts with features of secretory-type endometrial lining. Diffuse epithelial VEGF expression was associated with cyst diameters greater than 5.4 cm (P⬍.05). Bilateral cysts were related to diffuse fibroblastic VEGF expression (P⬍.05) and tended to be associated with stronger VEGF immunoreactivity in the vessels of the outer fibrous capsule (P⫽.06). Epithelial staining for VEGF correlated with positivity of fibrous wall fibroblasts (P⫽.01; Fig. 2) and endothelial cells (P⫽.01; Fig. 3). The VEGF expression in fibroblasts correlated highly 1532 Goteri et al.
VEGF expression in ovarian endometriosis
with staining in fibrous wall endothelial cells (P⬍.0001; Fig. 4). None of the clinical parameters correlated with VEGF expression scores in the different cell populations.
DISCUSSION The present study shows that VEGF is expressed in the cellular constituents of ovarian endometriomata. Some researchers from our group reported absent or low VEGF expression in epithelial and endothelial cells of benign inclusion cysts and serous cystadenomas, more intense immunoreactivity in borderline tumors, and still stronger staining in invasive cystadenocarcinomas of the ovary (22). Thus, VEGF expression in endometriotic cysts seems to be comparable to that of malignant ovarian neoplasms rather than of benign nonendometriotic cysts. In line with the literature (9), our data implicate neoangiogenesis in ovarian endometriosis as being similar to neoplastic conditions. The present study was also directed at seeking correlations among VEGF cellular expression, clinical findings, and cellular composition of the ovarian cysts. None of the clinical parameters evaluated correlated with VEGF expression. However, the more interesting findings came from pathologic analysis of the Vol. 81, No. 6, June 2004
histology of immunoreactive cell types and of gross parameters (cyst diameter and bilaterality). In line with McLaren et al. (6), who demonstrated that activated macrophages are a major source of VEGF in endometriosis, we found that the cells with the highest frequency of diffuse VEGF expression were macrophages, detected in a minority of cysts, followed by capsular vessel and subepithelial stromal cells, and by epithelial cells and capsular fibroblasts. The least immunoreactive cells were the endothelia of the subepithelial stroma. A correlation was observed between VEGF expression in epithelial cells and capsular fibroblasts and vessels, suggesting that these cell types might form a cellular network sharing regulation by the stimulatory signals promoting angiogenesis. Because E2 induces VEGF expression in cultured endometrial cells (7), endometrial cancer cell lines (23), and macrophages (6), and hypoxia up-regulates VEGF expression (16, 20), further research is needed to evaluate the role of these factors in endometriosis. Although we failed to find a correlation between VEGF expression and the histologic appearance of epithelial or stromal cells, there was a strong, nearly statistically significant trend for greater VEGF immunoreactivity in cysts with secretory compared with proliferative features. An intriguing finding was the association between strong VEGF expression in epithelial cells and a cyst diameter greater than 5.4 cm. Only two studies have analyzed the relationship between expression of angiogenic factors and cyst size (4, 12). Like us, Di Blasio et al. (4) showed a similar relationship between greater cyst diameter and higher basic fibroblastic growth factor (bFGF) mRNA levels, whereas Fasciani et al. (12) found an inverse correlation between VEGF concentration in cystic fluid and cyst diameter. Although the latter data may appear to contrast with ours, VEGF concentrations in fluid might in fact be inversely related to VEGF expression in the epithelial lining of cysts and in other cell compartments. In our study, bilateral cysts were associated with diffuse VEGF expression in the cells of the outer fibrous capsule. The present results are consistent with a pathogenic mechanism that could combine the relationships of VEGF expression in epithelium and outer fibrous capsular wall and with cyst diameter and bilaterality. Based on this mechanism, angiogenesis in ovarian cysts would involve the external wall more than the other layer(s), contributing to cyst growth, to the fibrosing process in the capsule, and to formation of adhesions. References 1. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997;68:585–96.
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2. Brosens I, Puttemans P, Deprest J. The endoscopic localization of endometrial implants in the ovarian chocolate cyst. Fertil Steril 1994; 61:1034 –8. 3. Scurry J, Whitehead J, Healey M. Classification of ovarian endometriotic cysts. Int J Gynecol Pathol 2001;20:147–54. 4. Di Blasio AM, Centinaio G, Carniti C, Somigliana E, Vigano P, Vignali M. Basic fibroblast growth factor messenger ribonucleic acid levels in eutopic and ectopic human endometrial stromal cells as assessed by competitive polymerase chain reaction amplification. Mol Cell Endocrinol 1995;115:169 –75. 5. McLaren J, Prentice A, Charnock-Jones DS, Smith SK. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod 1996;11:220 –3. 6. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. 7. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, et al. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112–8. 8. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13:1686 –90. 9. Fujishita A, Hasuo A, Khan KN, Masuzaki H, Nakashima H, Ishimaru T. Immunohistochemical study of angiogenic factors in endometrium and endometriosis. Gynecol Obstet Invest 1999;48(Suppl 1):36 –44. 10. Fujimoto J, Sakaguchi H, Hirose R, Wen H, Tamaya T. Angiogenesis in endometriosis and angiogenic factors. Gynecol Obstet Invest 1999; 48(Suppl 1):14 –20. 11. Mahnke JL, Dawood MY, Huang JC. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 2000;73:166 –70. 12. Fasciani A, D’Ambrogio G, Bocci G, Monti M, Genazzani AR, Artini PG. High concentrations of the vascular endothelial growth factor and interleukin-8 in ovarian endometriomata. Mol Hum Reprod 2000;6:50 –4. 13. Deguchi M, Ishiko O, Sumi T, Yoshida H, Yamamoto K, Ogita S. Expression of angiogenic factors in extrapelvic endometriosis. Oncol Rep 2001;8:1317–9. 14. Kressin P, Wolber EM, Wodrich H, Meyhofer-Malik A, Buchweitz O, Diedrich K, et al. Vascular endothelial growth factor mRNA in eutopic and ectopic endometrium. Fertil Steril 2001;76:1220 –4. 15. Tan XJ, Lang JH, Liu DY, Shen K, Leng JH, Zhu L. Expression of vascular endothelial growth factor and thrombospondin-1 mRNA in patients with endometriosis. Fertil Steril 2002;78:148 –53. 16. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4 –25. 17. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1992;13:18 –32. 18. Greb RR, Heikinheimo O, Williams RF, Hodgen GD, Goodman AL. Vascular endothelial growth factor in primate endometrium is regulated by oestrogen-receptor and progesterone-receptor ligands in vivo. Hum Reprod 1997;12:1280 –92. 19. Itakura J, Ishiwata T, Shen B, Kornmann M, Korc M. Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer. Int J Cancer 2000;85:27–34. 20. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359:843–5. 21. American Fertility Society. Revised American Fertility Society Classification of Endometriosis, 1985. Fertil Steril 1985;43:351–2. 22. Garzetti GG, Ciavattini A, Lucarini G, Pugnaloni A, DeNictolis M, Amati S, et al. Expression of VEGF related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors. Cancer 1999;85:2219 –25. 23. Charnock-Jones DS, Sharkey AM, Rajput-Williams J, Burch D, Schofield JP, Fountain SA, et al. Identification and localization of alternately spliced mRNAs for VEGF in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol Reprod 1993;48: 1120 –8.
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