- Email: [email protected]
Is resveratrol a potential substitute for leuprolide acetate in experimental endometriosis?
European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Is resveratrol a potential substitute for leuprolide acetate in experimental endometriosis? Yesim Bayoglu Tekin a,*, Suleyman Guven b, Aynur Kirbas c, Yıldıray Kalkan d, Levent Tumkaya d, Emine Seda Guvendag Guven a a
Departments of Gynecology and Obstetrics, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey Department of Obstetrics and Gynecology, School of Medicine, Black Sea Technical University, 61080 Trabzon, Turkey Departments of Biochemistry, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey d Departments of Histology, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 August 2014 Received in revised form 13 October 2014 Accepted 22 October 2014
Objective: Resveratrol, a phytoalexin polyphenol, has anti-angiogenic, antioxidant, anti-inflammatory properties. We aimed to compare the anti-inflammatory and anti-angiogenic effects of resveratrol and leuprolide acetate (LA) in an experimental endometriosis model. Study design: A prospective experimental study was conducted in a University Surgical Research Center. Thirty-three non-pregnant female Sprague-Dawley rats, in which experimental model of endometriosis were surgically induced were randomly divided into four groups. Group 1 was administered 30 mg/kg resveratrol i.m. for 14 days, group 2 was given 1 mg/kg s.c. single dose LA, group 3 was administered both resveratrol and LA, and group 4 had no medication. After two weeks medication rats were sacrificed and size, histopathology and immunreactivity to matrix metalloproteinase (mmp)2, mmp9, vascular endothelial growth factor (VEGF) of the endometriotic implants were evaluated. Plasma and peritoneal fluid levels of interleukin (IL)-6, IL-8, and tumor necrosis factor-a (TNF-a) were analyzed. Results: The endometriotic implant volumes, histopathological grade and immunreactivity to mmp2, mmp9 and VEGF were significantly reduced (p < 0.001), and plasma and peritoneal fluid levels of IL-6, IL8 and TNF-a were significantly decreased in group 1 and group 2 in comparison to group 3 and group 4 (p < 0.001). Conclusion: Resveratrol alone is a potential agent for the treatment of endometriosis and may be an alternative to LA. In contrast, the combination of LA and resveratrol decreased the anti-inflammatory and anti-angiogenic effects of each agent. Since resveratrol is widely used as an alternative therapy for a variety of conditions, it can undermine the effectiveness of LA. Therefore, caution should be exercised when used in combination with other agents. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometriosis Rat model Resveratrol Leuprolide acetate Treatment
Introduction Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside of the uterine cavity [1]. Endometriosis is a common disease of reproductive-age women and associated with chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility [2]. Although endometriosis is one the most investigated disorders of gynecology, it’s pathogenesis remains unclear and has been based on two theories: retrograde menstruation and coelomic metaplasia [3,4]. Further investigations have demonstrated a role
* Corresponding author. Tel.: +90 5055171973; fax: +90 4642123015. E-mail address: [email protected] (Y. Bayoglu Tekin). http://dx.doi.org/10.1016/j.ejogrb.2014.10.041 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
for immune dysregulation in the microenvironment of the peritoneal fluid for the development of endometriosis, including elevated levels of cytokines and activated macrophages with reduced phagocytic activity in the peritoneal fluid [5]. The development and maintenance of the endometrial implants depend on the implantation, differentiation, invasion of endometrial cells, and their neovascularization. Medical treatments have been aimed at maintaining a hypo-estrogenic environment by suppressing the hypothalamic–pituitary axis with gonadotropin releasing hormone (GnRH) analogues, but the side effects of these therapies limit their long-term usage. Resveratrol, a phytoalexin polyphenol, is a compound found in red wine, grapes, and berries. The anti-angiogenic, antioxidant, anti-inflammatory properties of resveratrol have been well established. It has been found to be beneficial for cardiovascular
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Y. Bayoglu Tekin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
diseases, cancer, type-2 diabetes mellitus, and neurodegenerative diseases [6,7]. The aim of this study was to evaluate the therapeutic effect of resveratrol and to compare this effect with a GnRH analogue, leuprolide acetate (LA). To our knowledge, no studies comparing the effects of resveratrol with LA on surgically-induced endometriosis in a rat model have been conducted. Materials and methods The Black Sea Technical University Committee on the Use and Care of Animals approved the experimental procedure and all investigations were performed in compliance with international guidelines on the ethical use of animals. The study was performed at the Surgical Research Center of Black Sea Technical University, Trabzon, Turkey. Forty mature, non-pregnant female Sprague-Dawley rats weighting 200–250 g were used for the induction of the experimental endometriosis model. The guidelines for care and use of animals that were approved by the institutional review board were followed. Before the surgical induction of endometriosis, the rats underwent daily vaginal lavages between 8:00 a.m. and 10:00 a.m. to detect the estrus cycle. Vaginal secretions were examined under a light microscope to identify the estrus cycle by the dominancy of the anucleate cornified cells [8].
polypropylene 4–0 suture. Before the enclosure of the abdominal wall, 2 mL of saline was put into the abdominal cavity to prevent drying of the serosal surfaces and to minimize adhesions. An uninterrupted 3–0 polyglactin 910 suture was used for the closure of the peritoneum and fascia and a simple interrupted 2–0 silk was used for the skin. After the surgery, all rats were caged individually and their body weight and wound healing were observed without any medication. Step 2: Measurement of endometrial implants At the second exploratory laparotomy, ectopic endometrial tissues were identified and three dimensional measurements were performed (length x width x height) using a caliper. The prolate ellipsoid formula was used for calculation of the spherical volume of each ectopic uterine tissue: V (mm3) = 0.52 length width height. All implants were photographed and their sizes and volumes were recorded (Fig. 1a). After the second laparotomy, two rats died because of anesthesia complications, and no implant was detected in five of the remaining animals. The remaining 33 rats were randomly divided into four groups. Group 1 was given 30 mg/kg resveratrol i.m. (R5010, Sigma-Aldrich TM Co, Saint Louis, MO, USA) for 14 days, group 2 was given 1 mg/kg s.c. single dose LA, group 3 was administered both resveratrol and LA, and group 4 had no medication. All rats were observed for 14 days as it was shown that LA begins to exert its effect earlier than two weeks [11].
Surgical procedure Step 1: Establishment of the endometriosis model For anesthetizing the rats, ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) 50 mg/kg and xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey) 7 mg/kg were administered intraperitoneally. Using sterile surgical techniques, a 4–5 cm vertical midline incision was made to expose the bilateral uterine horns. The experimental endometrial model was induced by transplanting autologous fragments of endometrial tissue onto the inner surface of the abdominal wall as described by Vernon and Wilson [9] with the modifications of Lebovic et al. [10]. A 1 cm segment was removed from the right uterine horn after ligation with a polypropylene 4–0 suture. The excised fragment was immersed in sterile phosphate-buffered saline (PBS) solution and opened longitudinally from the anti-mesenteric side. The endometrial tissue was trimmed to 5 5 mm without removing any myometrial tissue. The fragment was transplanted onto the inner surface of the right abdominal wall and secured with non-absorbable
Step 3: Evaluation of the outcomes At the end of 14 days, the rats were euthanized with ketamine, and a laparotomy was performed. Before excising the endometrial implants, peritoneal lavage with 2 mL saline was performed to assess the inflammatory markers in the peritoneal fluid. The sizes of the implants (Fig. 1b) were measured, and their volumes were calculated. For the histopathological examination, the implants were fixed in 10% formalin. A blood sample was obtained by cardiac puncture using 5 mL syringe for the detection of the cytokine levels of the plasma. Biochemical analysis Blood and peritoneal fluid samples were centrifuged at 3000 rpm for 10 min at room temperature and stored at 20 8C before the biochemical assay. Serum and peritoneal fluid levels of rat TNF-a and IL-6 were quantified by enzyme-linked immunosorbent assay (ELISA) using commercially available matched antibodies (for TNF-a and IL-6:
Fig. 1. The macroscopic appearance of endometriotic implants: two pre-treatment hypertrophic cystic implants in the abdominal wall (a), and the same implants after therapy (b). Arrows denote atrophic implants.
Y. Bayoglu Tekin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
Invitrogen, Camarillo, CA, USA and for IL-8: SunRed, Shanghai, China). According to the supplier of the kits, the intra-assay and inter-assay coefficients of variation (CV) for TNF-a were 6% and 8%, respectively, for IL-6, were 4.5% and 6.5%, and for IL-8 were 8% and 10%, respectively. The sensitivity was calculated to be <4 pg/mL for TNF-a, <5 pg/mL for IL-6, and 1.772 ng/L for IL-8. Histopathological examination Endometriotic implants were embedded in paraffin blocks after formalin fixation. 5 mm thick tissue sections were obtained, stained in hematoxilen eosin, and evaluated with light microscopy (BX-51, Olympus). The epithelial lining of the endometrial implants was evaluated semi-quantitatively as follows: grade 3: well-preserved epithelial lining, grade 2: moderately preserved epithelium with leukocyte infiltrate, grade 1: poorly preserved epithelium (occasional epithelial cells only), and grade 0: no epithelium. This histologic evaluation was made blindly by the same histologist in accordance with a previous rat endometriosis study [12] and photographed. For immunohistochemical staining, 3–5 mm thick sections of the endometriotic implants were cut and left to stand in xylene for 2 10 min, before the application of an alcohol series (50–100%) (10 min each), and then left to stand for 30 min in an H2O2 solution. After being rinsed twice in PBS, these sections were heated in a citrate buffer solution at 800 W for 4–5 min, four times and allowed to stand in a secondary blocking substance for 30 min. Each slide was incubated for 60 min with different dilutions of the following primary antibodies from Abcam Plc, Cambridge, UK: anti-mmp-9 (EP1254, code: ab76003) (1/100 dilution), anti-mmp-2 antibody (CA-4001/CA719E3C, code: ab3158) (1/100 dilution) and antiVEGF(VG-1code: ab1316) (1/200 dilution) before being stained by anti-mmp9, anti-mmp2, and anti-VEGF. A diaminobenzidine solution was used as a chromogen, and Mayer’s hematoxylin was used as a counterstain, for 3–5 min. PBS was used as a negative control. The immunohistochemical staining was evaluated by the same histologist blindly by a semi-quantitative method using the H-score. For each section, positive areas were scored at X400 magnification from 0 to 3+ with no staining (0), weak (1+), moderate (2+), and strong (3+). H-score was calculated as H = S Pi (I + 1). ‘Pi’ represents the density of immunhistochemical staining and ‘I’ is the percentage of the staining cells. Statistical analysis SPSS (Statistical Package for the Social Sciences) version 17.0 was used to record and statistically analyze the data. Variables were tested with the Kolmogorov–Smirnov test with Lilliefor’s significance correction for normal distribution. Before analysis, the data were normalized by log-transformation and ANOVA was performed with Bonferroni correction for the comparison of the pretreatment and post-treatment implant volumes, cytokine levels, histopathological and immunohistochemical results. Kruskal–Wallis test was used for comparison of the nonparametric data. The value of p < 0.05 was accepted as statistically significant.
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Results The surgical procedures were well tolerated by the animals, and no adverse effects of the medications were observed in the treatment groups. Group 1 had nine rats while the other groups had eight. During the second surgery, the volume of the cystic structured endometriotic implants was measured. There was no significant difference in the sizes of the pre-treatment volumes (p > 0.05) (Table 1). The post-treatment volumes of the endometriotic implants in groups 1, 2, and 3 were less than those of group 4, and this difference was statistically significant (p < 0.001) except for group 3. In addition, the histopathological scores of the implants were significantly lower in groups 1 and 2 than those of groups 3 and 4 (p < 0.001) (Table 1). The histopathological scores were evaluated and compared between the groups (Table 1). Groups 1 and 2 had significantly lower histopathological scores than those of groups 3 and 4 (p < 0.001) (Table 1, Fig. 2). Levels of IL-6, IL-8 and TNFa in plasma and peritoneal fluid are given in Fig. 3a, b and c, respectively. Both plasma and peritoneal fluid levels of all three cytokines were statistically significantly lower in groups 1 and 2 than those of groups 3 and 4. Immunohistochemical staining Mmp2 expression was significantly lower in groups 1 and 2 (0.67 0.16 and 1 0.19) in comparison to groups 3 and 4 (3.13 0.22 and 3.13 0.29) (p < 0.001) (Fig. 4a to d). Mmp9 expression was lower in groups 1 and 2 (0.89 0.2 and 0.75 0.16) compared to groups 3 and 4 (2.13 0.22 and 3 0.26) (p < 0.001) (Fig. 4e–h). Similarly, VEGF expression was lower in groups 1 and 2 (0.78 0.22 and 0.75 0.25) than those of groups 3 and 4 (2.63 0.25 and 3 0.26) (p < 0.001) (Fig. 4i–l). Comments To our knowledge, this is the first study evaluating both the anti-inflammatory effects of resveratrol in the plasma and peritoneal fluids, and the anti-angiogenic effects of resveratrol in endometriotic implants in comparison to LA. We found that resveratrol is as effective as LA in endometriosis treatment when used alone. Interestingly, combination of resveratrol and LA resulted in the lack of complementary/additive therapeutic effect. In recent years, natural therapies have been employed for the treatment of various inflammatory and degenerative diseases and cancers. Resveratrol, a non-flavonoid polyphenol has antiinflammatory effect which is elicited by down-regulation of proinflammatory cytokines including IL-1b, IL-6, IL-8, and TNF-a [13]. Furthermore, resveratrol inhibits tumorogenesis by the induction of apoptosis and promotion of cellular proliferation and differentiation [14]. Resveratrol is reported to be a pleiotropic agent and effective in several steps of neovascularization [15]. Resveratrol suppresses the microvessel activity by having a direct impact on the proliferation and migration of endothelial and vascular smooth muscle cells. Furthermore, resveratrol decreases mmp2 and mmp9
Table 1 Comparison of the implant sizes (mm3) and histopathological grades of the study groups. Parametre
Group 1
Group 2
Group 3
Group 4
p Value
Pretreatment Posttreatment Histopathological scores
241 72 27 13b 0.67 0.23c
147 50 41 58b 0.63 0.18c
83 14 65 31 2.25 0.25
169 88 297 149 2.5 0.53
0.373a 0.049a <0.001a
a b c
Kruskal–Wallis test. p < 0.05 Compared with group 4 (ANOVA test. Refer to materials and methods for further details). p <0.001 Compared with groups 3 and 4 (ANOVA test).
Y. Bayoglu Tekin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
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Fig. 2. Histopathological appearance of endometrial implants (HEx10). Endometrial implants with grade 0 lesions: intense neutrophil and macrophage infiltration with no epithelial lining in groups 1 and 2 ((a) and (b), respectively), and endometrial implants with grade 3 lesions: well-preserved epithelial lining in groups 3 and 4 ((c) and (d), respectively).
plasma periton
80 IL-8 (ng/ml)
120 IL-6 (pg/ml)
b 100
160
80 40
c 40
plasma periton
30 TNFα (pg/ml)
a
60 40 20
0
0 Res
LA
LA-Res Control
Res
LA
LA-Res Control
plasma periton
20 10 0 Res
LA
LA-Res Control
Fig. 3. Cytokine levels in the plasma and peritoneal fluids of treatment groups and controls (Res: resveratrol, LA: leuporlide acetate, LA-Res: leuprolide acetate and resveratrol).
Fig. 4. Immunohistochemical staining of endometriotic implants with mmp2, 9, and VEGF (20). Few scattered mmp2 positive stromal cells around the neovascularization areas in groups 1 and 2 (a and b), and extensive and severe staining of mmp2 in the stroma of the endometriotic implant in groups 3 and 4 (c and d), very small numbers of mmp9 positive stromal cells in groups 1 and 2 (e and f), intense staining of mmp9 in the stroma and epithelial lining of endometrial implants in groups 3 and 4 (g and h), scattered weak staining of VEGF in the stromal cells of endometriotic implants in groups 1 and 2 (i and j), intense epithelial and stromal staining with VEGF in groups 3 an 4 (k and l).
Y. Bayoglu Tekin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
activities, which participate in the degradation of extracellular components and vascular formation [16]. Retrograde menstruation and coelomic epithelial metaplasia, the proposed mechanisms for the development and maintenance of endometriosis, remain insufficient to explain this condition. A growing amount of evidence supports the adjuvant effect of inflammatory activity in the peritoneal fluid on the development of endometriosis. The increased numbers of macrophages play a key role in the elevation of pro-inflammatory cytokine levels, such as Il-6, IL-8 and TNF-a in the peritoneal fluid [17]. Implantation of endometrial grafts depends on the breakdown of the basal membrane, degradation of the extracellular matrix, and neovascularization. Many factors work in this invasion procedure, such as matrix metalloproteinases [18,19] and VEGF [20]. It is likely that resveratrol inhibits endometriosis. Brunner-Tran et al. first reported the therapeutic effect of resveratrol in an experimental endometriosis model by showing that it decreased endometrial cell proliferation and increased apoptosis [21]. Ricci et al. [22] demonstrated both an increase in cell death and the antiangiogenic effects of resveratrol in an endometriosis model and human endometrial cell culture. Rudzitis-Auth et al. reported that resveratrol reduced the proliferation of endothelial cells in the ectopic implants, which was shown by significantly decreased micro-vessel density [23]. Finally, Ergenoglu et al. demonstrated a significant decrease in VEGF, monocyte chemotactic protein 1 (MICP-1), and TNF-a levels in peritoneal fluid and decreased expression of VEGF in endometriotic implants [24]. The systemic effect of LA was related to the decrease of endogenous estrogen levels, and the local effect was demonstrated by the reduction of macrophage infiltration, suppression of cytokine levels, and decreased levels of chemotactic agents [25]. Our results showed that depot LA had sufficient effect on the establishment of a hypo-estrogenic milieu and caused atrophy of the endometriotic implants in 14 days. Furthermore, depot LA decreased cytokine levels in the plasma and peritoneal fluid and also suppressed angiogenic activity by inhibiting VEGF, mmp2 and mmp9. The unexpected effect of resveratrol and LA combination can be explained by the molecular structure of resveratrol. Resveratrol is a partial agonist for estrogen receptor and has agonistic effect at low estrogen concentrations and antagonistic effect at high estrogen concentrations in cell culture [26]. Moreover, the transcriptional activity of resveratrol increases when it binds to ER-b receptor but has antagonistic activity with ER-a receptor in a mammary tumor model [27]. Consumption of resveratrol with LA could bestow a superagonistic effect to resveratrol on the estrogen receptor and stimulate expression of estrogen-regulated genes. This agonistic effect could block the anti-estrogenic effect of LA and decrease the therapeutic effects of each agent in the treatment of endometriosis. In other words, the LA mediated hypo-estrogenic environment may have triggered the agonistic effect of resveratrol. Also the bioavailability of resveratrol is low because of poor solubility in water and isomerization. In our study, we dissolved resveratrol in saline and administered it by intramuscular injection, which could have provided higher plasma levels of resveratrol compared to oral administration. Higher dose of resveratrol could have alterations in estrogen receptor expression and facilitate the agonistic activity in GnRH-managed hypoestrogenic milieu. There are several limitations in our study. First, this is an experimental model with a small number of animals because of ethical concerns. Second, the histological and immunohistochemical scoring were semi-quantitative. A computer based image— analysis system would be a more objective and accurate method for evaluating and scoring the endometriotic implants. Another
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limitation was the inability to evaluate the plasma and peritoneal fluid levels of resveratrol to determine the therapeutic dose for endometriosis. Future research is necessary for the evaluation of the bioavailability and adverse effects of resveratrol in combination with other medications for endometriosis. Conclusion Our results indicate that resveratrol inhibits inflammation and angiogenesis in endometriosis. Further, resveratrol reduces proinflammatory cytokines levels of IL-6, IL-8 and TNF-a in the peritoneal fluid and expression of matrix-metalloproteinases and VEGF in endometriotic implants. Since resveratrol is widely used as an alternative therapy for a variety of conditions, it can undermine the effectiveness of other agents like LA. Therefore, caution should be exercised when used in combination with other medications. Condensation Resveratrol has a remarkable effect on the reduction of endometrial implant growth; however combination with leuprolide acetate decreases effectiveness of both agents. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgement This study was funded by Recep Tayyip Erdogan University, Scientific Research Project Unit (Project number: 2012.106.02.3). References [1] Galle PC. Clinical presentation and diagnosis of endometriosis. Obstet Gynecol Clin North Am 1989;16:29–42. [2] Nothnick WB, Zhang X. Future targets in endometriosis treatment, targeting the endometriotic implant. Mini Rev Med Chem 2009;9:324–8. [3] Sampson J. Peritoneal endometriosis due to the menstrual dissemination of endothelial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927;14: 422–69. [4] Nap AW, Groothuis PG, Demir AY, Evers JL, Dunselman GA. Pathogenesis of endometriosis. Best Pract Res Clin Obstet Gynaecol 2004;18:233–44. [5] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. [6] Yu W, Fu YC, Wang W. Cellular and molecular effects of resveratrol in health and disease. J Cell Biochem 2012;113:752–9. [7] Markus MA, Morris BJ. Resveratrol in prevention and treatment of common clinical conditions of aging. Clin Interv Aging 2008;3:331–9. [8] Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 2002;62:609–14. [9] Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684–94. [10] Lebovic DI, Kir M, Casey CL. Peroxisome proliferator—activated receptorgamma induces regression of endometrial explants in a rat model of endometriosis. Fertil Steril 2004;82:1008–13. [11] Zanagnolo VL, Beck R, Schlaff WD, Damewood MD, Bobbie D, Rock JA. Timerelated effects of gonadotropin-releasing hormone analog treatment in experimentally induced endometriosis in the rat. Fertil Steril 1991;55:411–5. [12] Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999;72:135–41. [13] Bergman M, Levin GS, Bessler H, Djaldetti M, Salman H. Resveratrol affects the cross talk between immune and colon cancer cells. Biomed Pharmacother 2013;61:43–7. [14] Nobili S, Lippi D, Witort E, et al. Natural compounds for cancer treatment and prevention. Pharmacol Res 2009;59:365–78. [15] Chen Y, Tseng SH. Review. Pro- and anti-angiogenesis effects of resveratrol. In Vivo 2007;21:365–70. [16] Kaneko H, Anzai T, Morisawa M, et al. Resveratrol prevents the development of abdominal aortic aneurysm through attenuation of inflammation, oxidative stress, and neovascularization. Atherosclerosis 2011;217:350–7. [17] Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96.
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[18] Chung HW, Wen Y, Chun SH, Nezhat C, Woo BH, Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness. Fertil Steril 2001;75:152–9. [19] Huang HF, Hong LH, Tan Y, Sheng JZ. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertil Steril 2004;81:1235–9. [20] Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998;13:1686–90. [21] Bruner-Tran KL, Osteen KG, Taylor HS, Sokalska A, Haines K, Duleba AJ. Resveratrol inhibits development of experimental endometriosis in vivo and reduces endometrial stromal cell invasiveness in vitro. Biol Reprod 2011;84:106–12. [22] Ricci AG, Olivares CN, Bilotas MA, et al. Natural therapies assessment for the treatment of endometriosis. Hum Reprod 2013;28:178–88.
[23] Rudzitis-Auth J, Menger MD, Laschke MW. Resveratrol is a potent inhibitor of vascularization and cell proliferation in experimental endometriosis. Hum Reprod 2013;28:1339–47. ¨ , Erbas¸ O, et al. Regression of endometrial implants by [24] Ergenoglu AM, Yeniel AO resveratrol in an experimentally induced endometriosis model in rats. Reprod Sci 2013;20:1230–6. [25] Khan KN, Kitajima M, Hiraki K, et al. Changes in tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma after GnRH agonist therapy. Hum Reprod 2010;25:642–53. [26] Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 2000;141:3657–67. [27] Bhat KP, Lantvit D, Christov K, Mehta RG, Moon RC, Pezzuto JM. Estrogenic and antiestrogenic properties of resveratrol in mammary. Cancer Res 2001;61:7456–63.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Is resveratrol a potential substitute for leuprolide acetate in experimental endometriosis? Yesim Bayoglu Tekin a,*, Suleyman Guven b, Aynur Kirbas c, Yıldıray Kalkan d, Levent Tumkaya d, Emine Seda Guvendag Guven a a
Departments of Gynecology and Obstetrics, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey Department of Obstetrics and Gynecology, School of Medicine, Black Sea Technical University, 61080 Trabzon, Turkey Departments of Biochemistry, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey d Departments of Histology, Recep Tayyip Erdogan University, School of Medicine, 53020 Rize, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 August 2014 Received in revised form 13 October 2014 Accepted 22 October 2014
Objective: Resveratrol, a phytoalexin polyphenol, has anti-angiogenic, antioxidant, anti-inflammatory properties. We aimed to compare the anti-inflammatory and anti-angiogenic effects of resveratrol and leuprolide acetate (LA) in an experimental endometriosis model. Study design: A prospective experimental study was conducted in a University Surgical Research Center. Thirty-three non-pregnant female Sprague-Dawley rats, in which experimental model of endometriosis were surgically induced were randomly divided into four groups. Group 1 was administered 30 mg/kg resveratrol i.m. for 14 days, group 2 was given 1 mg/kg s.c. single dose LA, group 3 was administered both resveratrol and LA, and group 4 had no medication. After two weeks medication rats were sacrificed and size, histopathology and immunreactivity to matrix metalloproteinase (mmp)2, mmp9, vascular endothelial growth factor (VEGF) of the endometriotic implants were evaluated. Plasma and peritoneal fluid levels of interleukin (IL)-6, IL-8, and tumor necrosis factor-a (TNF-a) were analyzed. Results: The endometriotic implant volumes, histopathological grade and immunreactivity to mmp2, mmp9 and VEGF were significantly reduced (p < 0.001), and plasma and peritoneal fluid levels of IL-6, IL8 and TNF-a were significantly decreased in group 1 and group 2 in comparison to group 3 and group 4 (p < 0.001). Conclusion: Resveratrol alone is a potential agent for the treatment of endometriosis and may be an alternative to LA. In contrast, the combination of LA and resveratrol decreased the anti-inflammatory and anti-angiogenic effects of each agent. Since resveratrol is widely used as an alternative therapy for a variety of conditions, it can undermine the effectiveness of LA. Therefore, caution should be exercised when used in combination with other agents. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometriosis Rat model Resveratrol Leuprolide acetate Treatment
Introduction Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside of the uterine cavity [1]. Endometriosis is a common disease of reproductive-age women and associated with chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility [2]. Although endometriosis is one the most investigated disorders of gynecology, it’s pathogenesis remains unclear and has been based on two theories: retrograde menstruation and coelomic metaplasia [3,4]. Further investigations have demonstrated a role
* Corresponding author. Tel.: +90 5055171973; fax: +90 4642123015. E-mail address: [email protected] (Y. Bayoglu Tekin). http://dx.doi.org/10.1016/j.ejogrb.2014.10.041 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
for immune dysregulation in the microenvironment of the peritoneal fluid for the development of endometriosis, including elevated levels of cytokines and activated macrophages with reduced phagocytic activity in the peritoneal fluid [5]. The development and maintenance of the endometrial implants depend on the implantation, differentiation, invasion of endometrial cells, and their neovascularization. Medical treatments have been aimed at maintaining a hypo-estrogenic environment by suppressing the hypothalamic–pituitary axis with gonadotropin releasing hormone (GnRH) analogues, but the side effects of these therapies limit their long-term usage. Resveratrol, a phytoalexin polyphenol, is a compound found in red wine, grapes, and berries. The anti-angiogenic, antioxidant, anti-inflammatory properties of resveratrol have been well established. It has been found to be beneficial for cardiovascular
2
Y. Bayoglu Tekin et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 184 (2015) 1–6
diseases, cancer, type-2 diabetes mellitus, and neurodegenerative diseases [6,7]. The aim of this study was to evaluate the therapeutic effect of resveratrol and to compare this effect with a GnRH analogue, leuprolide acetate (LA). To our knowledge, no studies comparing the effects of resveratrol with LA on surgically-induced endometriosis in a rat model have been conducted. Materials and methods The Black Sea Technical University Committee on the Use and Care of Animals approved the experimental procedure and all investigations were performed in compliance with international guidelines on the ethical use of animals. The study was performed at the Surgical Research Center of Black Sea Technical University, Trabzon, Turkey. Forty mature, non-pregnant female Sprague-Dawley rats weighting 200–250 g were used for the induction of the experimental endometriosis model. The guidelines for care and use of animals that were approved by the institutional review board were followed. Before the surgical induction of endometriosis, the rats underwent daily vaginal lavages between 8:00 a.m. and 10:00 a.m. to detect the estrus cycle. Vaginal secretions were examined under a light microscope to identify the estrus cycle by the dominancy of the anucleate cornified cells [8].
polypropylene 4–0 suture. Before the enclosure of the abdominal wall, 2 mL of saline was put into the abdominal cavity to prevent drying of the serosal surfaces and to minimize adhesions. An uninterrupted 3–0 polyglactin 910 suture was used for the closure of the peritoneum and fascia and a simple interrupted 2–0 silk was used for the skin. After the surgery, all rats were caged individually and their body weight and wound healing were observed without any medication. Step 2: Measurement of endometrial implants At the second exploratory laparotomy, ectopic endometrial tissues were identified and three dimensional measurements were performed (length x width x height) using a caliper. The prolate ellipsoid formula was used for calculation of the spherical volume of each ectopic uterine tissue: V (mm3) = 0.52 length width height. All implants were photographed and their sizes and volumes were recorded (Fig. 1a). After the second laparotomy, two rats died because of anesthesia complications, and no implant was detected in five of the remaining animals. The remaining 33 rats were randomly divided into four groups. Group 1 was given 30 mg/kg resveratrol i.m. (R5010, Sigma-Aldrich TM Co, Saint Louis, MO, USA) for 14 days, group 2 was given 1 mg/kg s.c. single dose LA, group 3 was administered both resveratrol and LA, and group 4 had no medication. All rats were observed for 14 days as it was shown that LA begins to exert its effect earlier than two weeks [11].
Surgical procedure Step 1: Establishment of the endometriosis model For anesthetizing the rats, ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) 50 mg/kg and xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey) 7 mg/kg were administered intraperitoneally. Using sterile surgical techniques, a 4–5 cm vertical midline incision was made to expose the bilateral uterine horns. The experimental endometrial model was induced by transplanting autologous fragments of endometrial tissue onto the inner surface of the abdominal wall as described by Vernon and Wilson [9] with the modifications of Lebovic et al. [10]. A 1 cm segment was removed from the right uterine horn after ligation with a polypropylene 4–0 suture. The excised fragment was immersed in sterile phosphate-buffered saline (PBS) solution and opened longitudinally from the anti-mesenteric side. The endometrial tissue was trimmed to 5 5 mm without removing any myometrial tissue. The fragment was transplanted onto the inner surface of the right abdominal wall and secured with non-absorbable
Step 3: Evaluation of the outcomes At the end of 14 days, the rats were euthanized with ketamine, and a laparotomy was performed. Before excising the endometrial implants, peritoneal lavage with 2 mL saline was performed to assess the inflammatory markers in the peritoneal fluid. The sizes of the implants (Fig. 1b) were measured, and their volumes were calculated. For the histopathological examination, the implants were fixed in 10% formalin. A blood sample was obtained by cardiac puncture using 5 mL syringe for the detection of the cytokine levels of the plasma. Biochemical analysis Blood and peritoneal fluid samples were centrifuged at 3000 rpm for 10 min at room temperature and stored at 20 8C before the biochemical assay. Serum and peritoneal fluid levels of rat TNF-a and IL-6 were quantified by enzyme-linked immunosorbent assay (ELISA) using commercially available matched antibodies (for TNF-a and IL-6:
Fig. 1. The macroscopic appearance of endometriotic implants: two pre-treatment hypertrophic cystic implants in the abdominal wall (a), and the same implants after therapy (b). Arrows denote atrophic implants.
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Invitrogen, Camarillo, CA, USA and for IL-8: SunRed, Shanghai, China). According to the supplier of the kits, the intra-assay and inter-assay coefficients of variation (CV) for TNF-a were 6% and 8%, respectively, for IL-6, were 4.5% and 6.5%, and for IL-8 were 8% and 10%, respectively. The sensitivity was calculated to be <4 pg/mL for TNF-a, <5 pg/mL for IL-6, and 1.772 ng/L for IL-8. Histopathological examination Endometriotic implants were embedded in paraffin blocks after formalin fixation. 5 mm thick tissue sections were obtained, stained in hematoxilen eosin, and evaluated with light microscopy (BX-51, Olympus). The epithelial lining of the endometrial implants was evaluated semi-quantitatively as follows: grade 3: well-preserved epithelial lining, grade 2: moderately preserved epithelium with leukocyte infiltrate, grade 1: poorly preserved epithelium (occasional epithelial cells only), and grade 0: no epithelium. This histologic evaluation was made blindly by the same histologist in accordance with a previous rat endometriosis study [12] and photographed. For immunohistochemical staining, 3–5 mm thick sections of the endometriotic implants were cut and left to stand in xylene for 2 10 min, before the application of an alcohol series (50–100%) (10 min each), and then left to stand for 30 min in an H2O2 solution. After being rinsed twice in PBS, these sections were heated in a citrate buffer solution at 800 W for 4–5 min, four times and allowed to stand in a secondary blocking substance for 30 min. Each slide was incubated for 60 min with different dilutions of the following primary antibodies from Abcam Plc, Cambridge, UK: anti-mmp-9 (EP1254, code: ab76003) (1/100 dilution), anti-mmp-2 antibody (CA-4001/CA719E3C, code: ab3158) (1/100 dilution) and antiVEGF(VG-1code: ab1316) (1/200 dilution) before being stained by anti-mmp9, anti-mmp2, and anti-VEGF. A diaminobenzidine solution was used as a chromogen, and Mayer’s hematoxylin was used as a counterstain, for 3–5 min. PBS was used as a negative control. The immunohistochemical staining was evaluated by the same histologist blindly by a semi-quantitative method using the H-score. For each section, positive areas were scored at X400 magnification from 0 to 3+ with no staining (0), weak (1+), moderate (2+), and strong (3+). H-score was calculated as H = S Pi (I + 1). ‘Pi’ represents the density of immunhistochemical staining and ‘I’ is the percentage of the staining cells. Statistical analysis SPSS (Statistical Package for the Social Sciences) version 17.0 was used to record and statistically analyze the data. Variables were tested with the Kolmogorov–Smirnov test with Lilliefor’s significance correction for normal distribution. Before analysis, the data were normalized by log-transformation and ANOVA was performed with Bonferroni correction for the comparison of the pretreatment and post-treatment implant volumes, cytokine levels, histopathological and immunohistochemical results. Kruskal–Wallis test was used for comparison of the nonparametric data. The value of p < 0.05 was accepted as statistically significant.
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Results The surgical procedures were well tolerated by the animals, and no adverse effects of the medications were observed in the treatment groups. Group 1 had nine rats while the other groups had eight. During the second surgery, the volume of the cystic structured endometriotic implants was measured. There was no significant difference in the sizes of the pre-treatment volumes (p > 0.05) (Table 1). The post-treatment volumes of the endometriotic implants in groups 1, 2, and 3 were less than those of group 4, and this difference was statistically significant (p < 0.001) except for group 3. In addition, the histopathological scores of the implants were significantly lower in groups 1 and 2 than those of groups 3 and 4 (p < 0.001) (Table 1). The histopathological scores were evaluated and compared between the groups (Table 1). Groups 1 and 2 had significantly lower histopathological scores than those of groups 3 and 4 (p < 0.001) (Table 1, Fig. 2). Levels of IL-6, IL-8 and TNFa in plasma and peritoneal fluid are given in Fig. 3a, b and c, respectively. Both plasma and peritoneal fluid levels of all three cytokines were statistically significantly lower in groups 1 and 2 than those of groups 3 and 4. Immunohistochemical staining Mmp2 expression was significantly lower in groups 1 and 2 (0.67 0.16 and 1 0.19) in comparison to groups 3 and 4 (3.13 0.22 and 3.13 0.29) (p < 0.001) (Fig. 4a to d). Mmp9 expression was lower in groups 1 and 2 (0.89 0.2 and 0.75 0.16) compared to groups 3 and 4 (2.13 0.22 and 3 0.26) (p < 0.001) (Fig. 4e–h). Similarly, VEGF expression was lower in groups 1 and 2 (0.78 0.22 and 0.75 0.25) than those of groups 3 and 4 (2.63 0.25 and 3 0.26) (p < 0.001) (Fig. 4i–l). Comments To our knowledge, this is the first study evaluating both the anti-inflammatory effects of resveratrol in the plasma and peritoneal fluids, and the anti-angiogenic effects of resveratrol in endometriotic implants in comparison to LA. We found that resveratrol is as effective as LA in endometriosis treatment when used alone. Interestingly, combination of resveratrol and LA resulted in the lack of complementary/additive therapeutic effect. In recent years, natural therapies have been employed for the treatment of various inflammatory and degenerative diseases and cancers. Resveratrol, a non-flavonoid polyphenol has antiinflammatory effect which is elicited by down-regulation of proinflammatory cytokines including IL-1b, IL-6, IL-8, and TNF-a [13]. Furthermore, resveratrol inhibits tumorogenesis by the induction of apoptosis and promotion of cellular proliferation and differentiation [14]. Resveratrol is reported to be a pleiotropic agent and effective in several steps of neovascularization [15]. Resveratrol suppresses the microvessel activity by having a direct impact on the proliferation and migration of endothelial and vascular smooth muscle cells. Furthermore, resveratrol decreases mmp2 and mmp9
Table 1 Comparison of the implant sizes (mm3) and histopathological grades of the study groups. Parametre
Group 1
Group 2
Group 3
Group 4
p Value
Pretreatment Posttreatment Histopathological scores
241 72 27 13b 0.67 0.23c
147 50 41 58b 0.63 0.18c
83 14 65 31 2.25 0.25
169 88 297 149 2.5 0.53
0.373a 0.049a <0.001a
a b c
Kruskal–Wallis test. p < 0.05 Compared with group 4 (ANOVA test. Refer to materials and methods for further details). p <0.001 Compared with groups 3 and 4 (ANOVA test).
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Fig. 2. Histopathological appearance of endometrial implants (HEx10). Endometrial implants with grade 0 lesions: intense neutrophil and macrophage infiltration with no epithelial lining in groups 1 and 2 ((a) and (b), respectively), and endometrial implants with grade 3 lesions: well-preserved epithelial lining in groups 3 and 4 ((c) and (d), respectively).
plasma periton
80 IL-8 (ng/ml)
120 IL-6 (pg/ml)
b 100
160
80 40
c 40
plasma periton
30 TNFα (pg/ml)
a
60 40 20
0
0 Res
LA
LA-Res Control
Res
LA
LA-Res Control
plasma periton
20 10 0 Res
LA
LA-Res Control
Fig. 3. Cytokine levels in the plasma and peritoneal fluids of treatment groups and controls (Res: resveratrol, LA: leuporlide acetate, LA-Res: leuprolide acetate and resveratrol).
Fig. 4. Immunohistochemical staining of endometriotic implants with mmp2, 9, and VEGF (20). Few scattered mmp2 positive stromal cells around the neovascularization areas in groups 1 and 2 (a and b), and extensive and severe staining of mmp2 in the stroma of the endometriotic implant in groups 3 and 4 (c and d), very small numbers of mmp9 positive stromal cells in groups 1 and 2 (e and f), intense staining of mmp9 in the stroma and epithelial lining of endometrial implants in groups 3 and 4 (g and h), scattered weak staining of VEGF in the stromal cells of endometriotic implants in groups 1 and 2 (i and j), intense epithelial and stromal staining with VEGF in groups 3 an 4 (k and l).
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activities, which participate in the degradation of extracellular components and vascular formation [16]. Retrograde menstruation and coelomic epithelial metaplasia, the proposed mechanisms for the development and maintenance of endometriosis, remain insufficient to explain this condition. A growing amount of evidence supports the adjuvant effect of inflammatory activity in the peritoneal fluid on the development of endometriosis. The increased numbers of macrophages play a key role in the elevation of pro-inflammatory cytokine levels, such as Il-6, IL-8 and TNF-a in the peritoneal fluid [17]. Implantation of endometrial grafts depends on the breakdown of the basal membrane, degradation of the extracellular matrix, and neovascularization. Many factors work in this invasion procedure, such as matrix metalloproteinases [18,19] and VEGF [20]. It is likely that resveratrol inhibits endometriosis. Brunner-Tran et al. first reported the therapeutic effect of resveratrol in an experimental endometriosis model by showing that it decreased endometrial cell proliferation and increased apoptosis [21]. Ricci et al. [22] demonstrated both an increase in cell death and the antiangiogenic effects of resveratrol in an endometriosis model and human endometrial cell culture. Rudzitis-Auth et al. reported that resveratrol reduced the proliferation of endothelial cells in the ectopic implants, which was shown by significantly decreased micro-vessel density [23]. Finally, Ergenoglu et al. demonstrated a significant decrease in VEGF, monocyte chemotactic protein 1 (MICP-1), and TNF-a levels in peritoneal fluid and decreased expression of VEGF in endometriotic implants [24]. The systemic effect of LA was related to the decrease of endogenous estrogen levels, and the local effect was demonstrated by the reduction of macrophage infiltration, suppression of cytokine levels, and decreased levels of chemotactic agents [25]. Our results showed that depot LA had sufficient effect on the establishment of a hypo-estrogenic milieu and caused atrophy of the endometriotic implants in 14 days. Furthermore, depot LA decreased cytokine levels in the plasma and peritoneal fluid and also suppressed angiogenic activity by inhibiting VEGF, mmp2 and mmp9. The unexpected effect of resveratrol and LA combination can be explained by the molecular structure of resveratrol. Resveratrol is a partial agonist for estrogen receptor and has agonistic effect at low estrogen concentrations and antagonistic effect at high estrogen concentrations in cell culture [26]. Moreover, the transcriptional activity of resveratrol increases when it binds to ER-b receptor but has antagonistic activity with ER-a receptor in a mammary tumor model [27]. Consumption of resveratrol with LA could bestow a superagonistic effect to resveratrol on the estrogen receptor and stimulate expression of estrogen-regulated genes. This agonistic effect could block the anti-estrogenic effect of LA and decrease the therapeutic effects of each agent in the treatment of endometriosis. In other words, the LA mediated hypo-estrogenic environment may have triggered the agonistic effect of resveratrol. Also the bioavailability of resveratrol is low because of poor solubility in water and isomerization. In our study, we dissolved resveratrol in saline and administered it by intramuscular injection, which could have provided higher plasma levels of resveratrol compared to oral administration. Higher dose of resveratrol could have alterations in estrogen receptor expression and facilitate the agonistic activity in GnRH-managed hypoestrogenic milieu. There are several limitations in our study. First, this is an experimental model with a small number of animals because of ethical concerns. Second, the histological and immunohistochemical scoring were semi-quantitative. A computer based image— analysis system would be a more objective and accurate method for evaluating and scoring the endometriotic implants. Another
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limitation was the inability to evaluate the plasma and peritoneal fluid levels of resveratrol to determine the therapeutic dose for endometriosis. Future research is necessary for the evaluation of the bioavailability and adverse effects of resveratrol in combination with other medications for endometriosis. Conclusion Our results indicate that resveratrol inhibits inflammation and angiogenesis in endometriosis. Further, resveratrol reduces proinflammatory cytokines levels of IL-6, IL-8 and TNF-a in the peritoneal fluid and expression of matrix-metalloproteinases and VEGF in endometriotic implants. Since resveratrol is widely used as an alternative therapy for a variety of conditions, it can undermine the effectiveness of other agents like LA. Therefore, caution should be exercised when used in combination with other medications. Condensation Resveratrol has a remarkable effect on the reduction of endometrial implant growth; however combination with leuprolide acetate decreases effectiveness of both agents. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgement This study was funded by Recep Tayyip Erdogan University, Scientific Research Project Unit (Project number: 2012.106.02.3). References [1] Galle PC. Clinical presentation and diagnosis of endometriosis. Obstet Gynecol Clin North Am 1989;16:29–42. [2] Nothnick WB, Zhang X. Future targets in endometriosis treatment, targeting the endometriotic implant. Mini Rev Med Chem 2009;9:324–8. [3] Sampson J. Peritoneal endometriosis due to the menstrual dissemination of endothelial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927;14: 422–69. [4] Nap AW, Groothuis PG, Demir AY, Evers JL, Dunselman GA. Pathogenesis of endometriosis. Best Pract Res Clin Obstet Gynaecol 2004;18:233–44. [5] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. [6] Yu W, Fu YC, Wang W. Cellular and molecular effects of resveratrol in health and disease. J Cell Biochem 2012;113:752–9. [7] Markus MA, Morris BJ. Resveratrol in prevention and treatment of common clinical conditions of aging. Clin Interv Aging 2008;3:331–9. [8] Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 2002;62:609–14. [9] Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684–94. [10] Lebovic DI, Kir M, Casey CL. Peroxisome proliferator—activated receptorgamma induces regression of endometrial explants in a rat model of endometriosis. Fertil Steril 2004;82:1008–13. [11] Zanagnolo VL, Beck R, Schlaff WD, Damewood MD, Bobbie D, Rock JA. Timerelated effects of gonadotropin-releasing hormone analog treatment in experimentally induced endometriosis in the rat. Fertil Steril 1991;55:411–5. [12] Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999;72:135–41. [13] Bergman M, Levin GS, Bessler H, Djaldetti M, Salman H. Resveratrol affects the cross talk between immune and colon cancer cells. Biomed Pharmacother 2013;61:43–7. [14] Nobili S, Lippi D, Witort E, et al. Natural compounds for cancer treatment and prevention. Pharmacol Res 2009;59:365–78. [15] Chen Y, Tseng SH. Review. Pro- and anti-angiogenesis effects of resveratrol. In Vivo 2007;21:365–70. [16] Kaneko H, Anzai T, Morisawa M, et al. Resveratrol prevents the development of abdominal aortic aneurysm through attenuation of inflammation, oxidative stress, and neovascularization. Atherosclerosis 2011;217:350–7. [17] Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96.
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[18] Chung HW, Wen Y, Chun SH, Nezhat C, Woo BH, Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness. Fertil Steril 2001;75:152–9. [19] Huang HF, Hong LH, Tan Y, Sheng JZ. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertil Steril 2004;81:1235–9. [20] Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998;13:1686–90. [21] Bruner-Tran KL, Osteen KG, Taylor HS, Sokalska A, Haines K, Duleba AJ. Resveratrol inhibits development of experimental endometriosis in vivo and reduces endometrial stromal cell invasiveness in vitro. Biol Reprod 2011;84:106–12. [22] Ricci AG, Olivares CN, Bilotas MA, et al. Natural therapies assessment for the treatment of endometriosis. Hum Reprod 2013;28:178–88.
[23] Rudzitis-Auth J, Menger MD, Laschke MW. Resveratrol is a potent inhibitor of vascularization and cell proliferation in experimental endometriosis. Hum Reprod 2013;28:1339–47. ¨ , Erbas¸ O, et al. Regression of endometrial implants by [24] Ergenoglu AM, Yeniel AO resveratrol in an experimentally induced endometriosis model in rats. Reprod Sci 2013;20:1230–6. [25] Khan KN, Kitajima M, Hiraki K, et al. Changes in tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma after GnRH agonist therapy. Hum Reprod 2010;25:642–53. [26] Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 2000;141:3657–67. [27] Bhat KP, Lantvit D, Christov K, Mehta RG, Moon RC, Pezzuto JM. Estrogenic and antiestrogenic properties of resveratrol in mammary. Cancer Res 2001;61:7456–63.