- Email: [email protected]
Effect of the immunomodulator leflunomide on the induction of endometriosis in an experimental rat model
CORRESPONDENCE Effect of the immunomodulator leflunomide on the induction of endometriosis in an experimental rat model The effect of immunomodulator leflunomide on the development of an experimental endometriosis model was assessed by surgically transplanting autologous fragments of endometrial tissue onto the inner surface of the abdominal wall and arterial cascades of the small intestines. Leflunomide was found to affect the development of endometriosis negatively and seemed to interfere with the growth and maintenance of the uterine explant in this experimental rat model. (Fertil Steril威 2007;87:699 –702. ©2007 by American Society for Reproductive Medicine.)
Inflammatory cells and their secretory products have been recognized as important mediators of the pathophysiology of endometriosis (1, 2). In women with endometriosis, it has been reported that activated macrophages, endometriotic lesions, and mesothelial cells of the peritoneum secrete cytokines, such as tumor necrosis factor (TNF superfamily member 2, previously known as TNF-␣) and interleukin (IL-1) (3– 6). In turn, these cytokines modulate other cytokines and chemokines, such as IL-8, CCL2 (also known as monocyte chemotactic protein [MCP1]), and CCL5 (also known as RANTES for “regulated on activation, normal T-cell expressed and secreted”) (3, 4, 7). Therefore it can be speculated that modulation of these cytokines might alter the course of the disease. Some investigators have shown that TNF-␣– binding protein can prevent the development of induced endometriosis, and treatment with anti-TNF antibody c5N has been suggested to reduce the extent of induced endometriosis (8). Also, the soluble form of TNF receptor type 1 was found to produce regression of endometriotic implant growth (9). Leflunomide is a novel immunomodulator used mainly in rheumatoid arthritis with antiinflammatory, antipyretic, and analgesic activities (10). Its active metabolite A77 1726 has the ability to suppress IL-1 and TNF-␣ selectively (11). Based on the above considerations, the purpose of this study was to evaluate the effect of an immunomodulator, leflunomide, on the development of an experimental endometriosis model in which survival of autologous transplants of uterine fragments was assessed. Twenty female nonpregnant Wistar albino rats weighing between 210 and 240 g, purchased from Government of Received January 19, 2006; revised and accepted July 19, 2006. Reprint requests: Hakan Aytan, M.D., Bankaci Sok, 19/10, 06640, Kucukesat, Ankara, Turkey (FAX: ⫹90 356 2133179; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1527
Health Ankara Education and Research Hospital experimental animal laboratory, were used as a model for experimental induction of endometriosis. The rats were caged individually in a controlled environment with 12-hour light/dark cycles and were fed ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). The guidelines for the care and use of the animals approved by the local institution were followed. Before the operation, rats were randomly assigned into two groups each consisting of ten rats. The first group was assigned as the control group. A vehicle treatment of 2 mL saline to the rats in group 1 and 35 mg/kg/day leflunomide (Arava; Aventis, Istanbul, Turkey) to the rats in group 2 were orally administered starting 3 days before the operation and continuing for 4 weeks after the operation. Endometriosis was surgically induced in rats by transplanting an autologous fragment of uterine tissue onto the inner surface of the abdominal wall and arterial cascades of the small intestines adjacent to mesenteric blood vessels, as proposed by Vernon and Wilson (12) with modifications by Lebovic et al. (13). Briefly, each rat was anesthetized with an IM injection of ketamine (70 mg/kg Ketalar; Eczacibasi, Istanbul, Turkey). Before surgery, the abdominal skin was shaved and antisepsis was obtained by 10% povidone iodine solution. Using sterile techniques, a 5-cm vertical midline incision was made and both uterine horns were exposed. A distal segment 1 cm in length was resected from the right uterine horn. The segment was placed in phosphate-buffered saline at 37°C, split longitudinally, and cut into two 5 ⫻ 5–mm squares. These pieces of uterine tissue were transplanted without removing the myometrium onto the inner surface of the right abdominal wall and arterial cascades of the small intestines adjacent to mesenteric blood vessels, with the serosal surface apposed and secured with single nonabsorb-
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
699
able 5-0 polypropylene suture at the middle to the abdominal wall. Before closure of the abdominal wall, 2 mL saline was administered into the abdominal cavity to prevent drying and minimize adhesion formation. The abdominal incision was closed in two layers with the use of a simple interrupted 4-0 polyglactin 910 suture for the peritoneum-fascia and for the skin. The operation was limited to 20 minutes for each rat to control the effect of room air tissue drying. All surgeries were performed by the same investigator, who was blinded to the treatment group. The rats were individually caged after the operation and were left for a recovery period of 4 weeks. During this period 35 mg/kg leflunomide was administered to the rats in the study group. During the treatment period, the rats were weighed daily. Vaginal secretion was assessed daily for 5 days before the second operation. Starting 28 days after the operation and lasting 5 days, daily vaginal smears were performed, and all rats that were in estrous state were killed and a second-look laparotomy was performed while the rats were fixed in the supine position. All three dimensions (length ⫻ width ⫻ height in mm) of ectopic uterine tissues were measured in situ using a caliper by the second author, who had no prior knowledge of which group was being evaluated. To minimize any possible bias, rats to be evaluated were randomly chosen and not sequentially brought to the investigator who was evaluating the ectopic endometria. The spherical volume of each ectopic uterine tissue was calculated using the prolate ellipsoid formula: V (mm3) ⫽ 0.52 ⫻ A ⫻ B ⫻ C, where A, B, and C denote width, length, and height, respectively. Tissues were photographed using a digital camera and then excised and weighed (in mg). Then tissue samples were fixed in 10% buffered formalin solution for 24 hours. After fixation, routine tissue processing procedure was performed, and then sampled tissues were embedded in paraffin. Paraffin wax blocks were cut in 4-m thick slices. Prepared sections were stained with hematoxylin-eosin. The histologic diagnosis of endometriosis was based on the morphologic identification of endometrial glandular tissue and stroma; glands and stroma of the endometrial type, with epithelial lining and luminal formation. In microscopic examination, the preservation of endometrial tissue was semiquantitatively evaluated according to a previously published method (14). From each rat, two samples, one from abdominal wall and one from the intestinal arterial cascades, were evaluated. The fourth author evaluated sections from each explant in a blinded fashion. Lilliefors-adjusted Kolmogorov-Smirnov test was used to test whether the variables used in the study were normally distributed. For comparison of volumes and weights, t test was used. For comparison of histopathologic evaluation, Mann Whitney U test was used. A P value of ⬍.05 was assumed to be significant. 700
Aytan et al.
Correspondence
Three rats in the study group and one rat from the control group died the day after surgery owing to complications related to surgery. The standardized surgical procedures and the administration of the protocols were well tolerated by the remaining animals. All laparotomy sites were intact and none of the animals had an incisional hernia. Side effects on the estrous cycle were determined by vaginal smears. Determination of the occurrence of any other side effects was done daily by observation. General appearance of the rats, lacrimation, porphyria, vocalizations, presence of loss of appetite, any changes in weight, and color of urine were observed. Morphologically the implants were cystic (Fig. 1). There was a significant difference (P⬍.0001; 95% CI 97.56 – 157.39) in spherical volume between the control group (166.23 ⫾ 54.95 mm3, range 80 –250 mm3) and the group treated with leflunomide (38.76 ⫾ 29.17 mm3, range 6.8 – 84 mm3). Similarly the two groups were significantly different with respect to weight: mean 188.79 ⫾ 59.95 mg (range 90.86 –283.93 mg) for the control group and 44.01 ⫾ 33.13 mg (range 7.72–95.39 mg) for the leflunomidetreated group (P⬍.0001; 95% CI 110.8 –178.75). The histopathologic finding of endometrial glands and stroma in the surgical site of implantation of endometrial squares allowed the diagnosis of experimental endometriosis. Histologically, epithelia of the cystic implants in the control group were found to be more persistent compared with the leflunomide-treated group (Fig. 1). The median scores were 1.5 (interquartile range 2.25) and 0 (interquartile range 1), respectively, with a P value of .015 (Z ⫺2.43). Findings suggest that an abnormality in monocyte or macrophage function may, at least, be supportive of ectopic endometrial growth and, at most, be causal for it (15). Although it is certain that the elevated cytokine levels or inflammation are a direct result of the disease, it is still unclear whether the secretion of these proinflammation proteins and associated immune cells into the peritoneal microenvironment contributes to the cascade of events that results in the establishment and further progression of endometriosis. In this context, cytokines that have gained particular attention in the pathophysiology of endometriosis are TNF-␣ and IL-1. They have been reported to affect various processes in endometriosis. Based on these data, we postulated that immunomodulator leflunomide with the ability to suppress TNF-␣ and IL-1 may be effective in the prevention of endometriosis development. Leflunomide is the selective inhibitor of the rate-limiting enzyme of de novo pyrimidine synthesis dihydroorotate dehydrogenase (11). At higher doses, its active metabolite A77 1726 has the ability to suppress IL-1 and TNF-␣, and recent evidence suggests that it is a potent inhibitor of nuclear factor (NF-kB)—a transcriptional factor critical to the function of cells in the immune system Vol. 87, No. 3, March 2007
FIGURE 1 Gross morphologic appearance and histology of implanted uterine autografts in the control group (A [note the adhesion] and C [note the preserved epithelial linings] and in the leflunomide-treated group (B and D [note the destroyed epithelia]). Hematoxylin-eosin stain, 200⫻ magnification.
Aytan. Inhibition of endometriosis with leflunomide. Fertil Steril 2007.
and which plays a part in inflammation—activation (11, 16). In addition, A77 1726 inhibits tyrosine kinases responsible for early T cell and B cell signaling in the G0/G1 phase of the cell cycle (11). In the present study, a rat model was used to investigate the efficacy of leflunomide on the development of experimental endometriosis, and leflunomide was found to affect the development of endometriosis negatively and seemed to interfere with the growth and maintenance of the uterine explant. There are some limitations in the present study that must be acknowledged. The semiquantitative evaluation of the preservation of endometrial tissue with a scoring system is subjective, and there may be interobserver variability. Second, although it has been noted that rat endometriotic tissues and cells perform in a similar manner as human endometriotic cells in organ explant culture and isolated cell culture (17–19), there is a disadvantage of extrapolating data across species, and immunologic properties of species are different. Estrogen levels in the rat are much lower than in humans, and rats do not have a menstrual cycle, all of which could be important and limit extrapolation of results from this model to humans. Third, leflunomide is potentially a toxic drug with some side effects (11, 20 –24). Fertility and Sterility姞
In conclusion, because of its immunomodulator properties the effect of leflunomide on the development of endometriosis model in rats was assessed in the present study. Leflunomide was found to affect the development of endometriosis negatively in this experimental rat model and seemed to interfere with the growth and maintenance of the uterine explant. Although there are concerns about the toxicity of this particular compound, novel therapeutic approaches for the important and yet unsolved problem of endometriosis and related morbidities are needed. Leflunomide may be a candidate; however, further studies are required to evaluate its effects. Hakan Aytan, M.D.a Pinar Caglar, M.D.b Dilek Uygur, M.D.b Sema Zergeroglu, M.D.c Sertac Batioglu, M.D.d a Department of Gynecology and Obstetrics, Gaziosmanpasa University, Tokat, Turkey; and Departments of b Gynecology and Obstetrics, c Pathology, and d Reproductive Endocrinology, Zekai Tahir Burak Women’s Health Training and Research Hospital, Ankara, Turkey 701
REFERENCES 1. Garcia-Velasco JA, Arici A, Zreik T, Naftolin F, Mor G. Macrophage derived growth factors modulate Fas ligand expression in cultured endometrial stromal cells: a role in endometriosis. Mol Hum Reprod 1999;5:642–50. 2. Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG, Taylor RN. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1 . Mol Hum Reprod 2000;6:269 –75. 3. Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96. 4. D’Hooghe TM, Nugent NP, Cuneo S, Chai DC, Deer F, Debrock S, et al. Recombinant human TNFRSF1A (r-hTBP1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Biol Reprod 2006;74:131– 6. 5. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. IL-1 beta, TNF-alpha, and IL-2 in peritoneal fluid and macrophageconditioned media of women with endometriosis. Am J Reprod Immunol 1995;34:381–5. 6. Cao X, Yang D, Song M, Murphy A, Parthasarathy S. The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: implications for endometriosis. Fertil Steril 2004;82 Suppl 3:999 –1007. 7. Hornung D, Bentzien F, Wallwiener D, Kiesel L, Taylor RN. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol Hum Reprod 2001;7: 163– 8. 8. Falconer H, Mwenda JM, Chai DC, Wagner C, Cornillie FJ, D’Hooghe TM. Treatment with antiTNF antibody c5N reduces the extent of induced endometriosis in the baboon. Fertil Steril 2004;82 Suppl 2:54 –5. 9. D’Antonio M, Martelli F, Peano S, Papoian R, Borrelli F. Ability of recombinant human TNF binding protein-1 (r-hTBP-1) to inhibit the development of experimentally-induced endometriosis in rats. J Reprod Immunol 2000;48:81–98. 10. Rozman B. Clinical pharmacokinetics of leflunomide. Clin Pharmacokinet 2002;41:421–30. 11. Breedveld FC, Dayer JM. Leflunomide: mode of action in the treatment of rheumatoid arthritis. Ann Rheum Dis 2000;59:841–9. 12. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684 –94.
702
Aytan et al.
Correspondence
13. Lebovic DI, Kir M, Casey CL. Peroxisome proliferator–activated receptor-gamma induces regression of endometrial explants in a rat model of endometriosis. Fertil Steril 2004;82 Suppl 3:1008 –13. 14. 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. 15. Arici A, Oral E, Attar E, Tazuke SI, Olive DL. Monocyte chemotactic protein-1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril 1997;67:1065–72. 16. Murray BE. New aspects of antimicrobial resistance and the resulting therapeutic dilemmas. J Infect Dis 1991;163:1184 –94. 17. Sharpe KL, Zimmer RL, Khan RS, Penney LL. Proliferative and morphogenic changes induced by the coculture of rat uterine and peritoneal cells: a cell culture model for endometriosis. Fertil Steril 1992;58:1220 –9. 18. Sharpe KL, Zimmer RL, Griffin WT, Penney LL. Polypeptides synthesized and released by human endometriosis differ from those of the uterine endometrium in cell and tissue explant culture. Fertil Steril 1993;60:839 –51. 19. Isaacson KB, Xu Q, Lyttle CR. The effect of estradiol on the production and secretion of complement component 3 by the rat uterus and surgically induced endometriotic tissue. Fertil Steril 1991;55: 395– 402. 20. Mladenovic V, Domljan Z, Rozman B, Jajic I, Mihajlovic D, Dordevic J, et al. Safety and effectiveness of leflunomide in the treatment of patients with active rheumatoid arthritis. Results of a randomized, placebo-controlled, phase II study. Arthritis Rheum 1995;38:1595– 603. 21. Strand V, Cohen S, Schiff M, Weaver A, Fleischmann R, Cannon G, et al.; Leflunomide Rheumatoid Arthritis Investigators Group. Treatment of active rheumatoid arthritis with leflunomide compared with placebo and methotrexate. Arch Intern Med 1999;159:2542–50. 22. Liote H. Respiratory complications of new treatments for rheumatoid arthritis. Rev Mal Respir 2004;21:1107–15. 23. Martin K, Bentaberry F, Dumoulin C, Longy-Boursier M, Lifermann F, Haramburu F, et al. Neuropathy associated with leflunomide: a case series. Ann Rheum Dis 2005;64:649 –50. 24. Gensburger D, Kawashima M, Marotte H, Kanitakis J, Miossec P. Lupus erythematosus with leflunomide: induction or reactivation? Ann Rheum Dis 2005;64:153–5.
Vol. 87, No. 3, March 2007
Inflammatory cells and their secretory products have been recognized as important mediators of the pathophysiology of endometriosis (1, 2). In women with endometriosis, it has been reported that activated macrophages, endometriotic lesions, and mesothelial cells of the peritoneum secrete cytokines, such as tumor necrosis factor (TNF superfamily member 2, previously known as TNF-␣) and interleukin (IL-1) (3– 6). In turn, these cytokines modulate other cytokines and chemokines, such as IL-8, CCL2 (also known as monocyte chemotactic protein [MCP1]), and CCL5 (also known as RANTES for “regulated on activation, normal T-cell expressed and secreted”) (3, 4, 7). Therefore it can be speculated that modulation of these cytokines might alter the course of the disease. Some investigators have shown that TNF-␣– binding protein can prevent the development of induced endometriosis, and treatment with anti-TNF antibody c5N has been suggested to reduce the extent of induced endometriosis (8). Also, the soluble form of TNF receptor type 1 was found to produce regression of endometriotic implant growth (9). Leflunomide is a novel immunomodulator used mainly in rheumatoid arthritis with antiinflammatory, antipyretic, and analgesic activities (10). Its active metabolite A77 1726 has the ability to suppress IL-1 and TNF-␣ selectively (11). Based on the above considerations, the purpose of this study was to evaluate the effect of an immunomodulator, leflunomide, on the development of an experimental endometriosis model in which survival of autologous transplants of uterine fragments was assessed. Twenty female nonpregnant Wistar albino rats weighing between 210 and 240 g, purchased from Government of Received January 19, 2006; revised and accepted July 19, 2006. Reprint requests: Hakan Aytan, M.D., Bankaci Sok, 19/10, 06640, Kucukesat, Ankara, Turkey (FAX: ⫹90 356 2133179; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1527
Health Ankara Education and Research Hospital experimental animal laboratory, were used as a model for experimental induction of endometriosis. The rats were caged individually in a controlled environment with 12-hour light/dark cycles and were fed ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). The guidelines for the care and use of the animals approved by the local institution were followed. Before the operation, rats were randomly assigned into two groups each consisting of ten rats. The first group was assigned as the control group. A vehicle treatment of 2 mL saline to the rats in group 1 and 35 mg/kg/day leflunomide (Arava; Aventis, Istanbul, Turkey) to the rats in group 2 were orally administered starting 3 days before the operation and continuing for 4 weeks after the operation. Endometriosis was surgically induced in rats by transplanting an autologous fragment of uterine tissue onto the inner surface of the abdominal wall and arterial cascades of the small intestines adjacent to mesenteric blood vessels, as proposed by Vernon and Wilson (12) with modifications by Lebovic et al. (13). Briefly, each rat was anesthetized with an IM injection of ketamine (70 mg/kg Ketalar; Eczacibasi, Istanbul, Turkey). Before surgery, the abdominal skin was shaved and antisepsis was obtained by 10% povidone iodine solution. Using sterile techniques, a 5-cm vertical midline incision was made and both uterine horns were exposed. A distal segment 1 cm in length was resected from the right uterine horn. The segment was placed in phosphate-buffered saline at 37°C, split longitudinally, and cut into two 5 ⫻ 5–mm squares. These pieces of uterine tissue were transplanted without removing the myometrium onto the inner surface of the right abdominal wall and arterial cascades of the small intestines adjacent to mesenteric blood vessels, with the serosal surface apposed and secured with single nonabsorb-
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
699
able 5-0 polypropylene suture at the middle to the abdominal wall. Before closure of the abdominal wall, 2 mL saline was administered into the abdominal cavity to prevent drying and minimize adhesion formation. The abdominal incision was closed in two layers with the use of a simple interrupted 4-0 polyglactin 910 suture for the peritoneum-fascia and for the skin. The operation was limited to 20 minutes for each rat to control the effect of room air tissue drying. All surgeries were performed by the same investigator, who was blinded to the treatment group. The rats were individually caged after the operation and were left for a recovery period of 4 weeks. During this period 35 mg/kg leflunomide was administered to the rats in the study group. During the treatment period, the rats were weighed daily. Vaginal secretion was assessed daily for 5 days before the second operation. Starting 28 days after the operation and lasting 5 days, daily vaginal smears were performed, and all rats that were in estrous state were killed and a second-look laparotomy was performed while the rats were fixed in the supine position. All three dimensions (length ⫻ width ⫻ height in mm) of ectopic uterine tissues were measured in situ using a caliper by the second author, who had no prior knowledge of which group was being evaluated. To minimize any possible bias, rats to be evaluated were randomly chosen and not sequentially brought to the investigator who was evaluating the ectopic endometria. The spherical volume of each ectopic uterine tissue was calculated using the prolate ellipsoid formula: V (mm3) ⫽ 0.52 ⫻ A ⫻ B ⫻ C, where A, B, and C denote width, length, and height, respectively. Tissues were photographed using a digital camera and then excised and weighed (in mg). Then tissue samples were fixed in 10% buffered formalin solution for 24 hours. After fixation, routine tissue processing procedure was performed, and then sampled tissues were embedded in paraffin. Paraffin wax blocks were cut in 4-m thick slices. Prepared sections were stained with hematoxylin-eosin. The histologic diagnosis of endometriosis was based on the morphologic identification of endometrial glandular tissue and stroma; glands and stroma of the endometrial type, with epithelial lining and luminal formation. In microscopic examination, the preservation of endometrial tissue was semiquantitatively evaluated according to a previously published method (14). From each rat, two samples, one from abdominal wall and one from the intestinal arterial cascades, were evaluated. The fourth author evaluated sections from each explant in a blinded fashion. Lilliefors-adjusted Kolmogorov-Smirnov test was used to test whether the variables used in the study were normally distributed. For comparison of volumes and weights, t test was used. For comparison of histopathologic evaluation, Mann Whitney U test was used. A P value of ⬍.05 was assumed to be significant. 700
Aytan et al.
Correspondence
Three rats in the study group and one rat from the control group died the day after surgery owing to complications related to surgery. The standardized surgical procedures and the administration of the protocols were well tolerated by the remaining animals. All laparotomy sites were intact and none of the animals had an incisional hernia. Side effects on the estrous cycle were determined by vaginal smears. Determination of the occurrence of any other side effects was done daily by observation. General appearance of the rats, lacrimation, porphyria, vocalizations, presence of loss of appetite, any changes in weight, and color of urine were observed. Morphologically the implants were cystic (Fig. 1). There was a significant difference (P⬍.0001; 95% CI 97.56 – 157.39) in spherical volume between the control group (166.23 ⫾ 54.95 mm3, range 80 –250 mm3) and the group treated with leflunomide (38.76 ⫾ 29.17 mm3, range 6.8 – 84 mm3). Similarly the two groups were significantly different with respect to weight: mean 188.79 ⫾ 59.95 mg (range 90.86 –283.93 mg) for the control group and 44.01 ⫾ 33.13 mg (range 7.72–95.39 mg) for the leflunomidetreated group (P⬍.0001; 95% CI 110.8 –178.75). The histopathologic finding of endometrial glands and stroma in the surgical site of implantation of endometrial squares allowed the diagnosis of experimental endometriosis. Histologically, epithelia of the cystic implants in the control group were found to be more persistent compared with the leflunomide-treated group (Fig. 1). The median scores were 1.5 (interquartile range 2.25) and 0 (interquartile range 1), respectively, with a P value of .015 (Z ⫺2.43). Findings suggest that an abnormality in monocyte or macrophage function may, at least, be supportive of ectopic endometrial growth and, at most, be causal for it (15). Although it is certain that the elevated cytokine levels or inflammation are a direct result of the disease, it is still unclear whether the secretion of these proinflammation proteins and associated immune cells into the peritoneal microenvironment contributes to the cascade of events that results in the establishment and further progression of endometriosis. In this context, cytokines that have gained particular attention in the pathophysiology of endometriosis are TNF-␣ and IL-1. They have been reported to affect various processes in endometriosis. Based on these data, we postulated that immunomodulator leflunomide with the ability to suppress TNF-␣ and IL-1 may be effective in the prevention of endometriosis development. Leflunomide is the selective inhibitor of the rate-limiting enzyme of de novo pyrimidine synthesis dihydroorotate dehydrogenase (11). At higher doses, its active metabolite A77 1726 has the ability to suppress IL-1 and TNF-␣, and recent evidence suggests that it is a potent inhibitor of nuclear factor (NF-kB)—a transcriptional factor critical to the function of cells in the immune system Vol. 87, No. 3, March 2007
FIGURE 1 Gross morphologic appearance and histology of implanted uterine autografts in the control group (A [note the adhesion] and C [note the preserved epithelial linings] and in the leflunomide-treated group (B and D [note the destroyed epithelia]). Hematoxylin-eosin stain, 200⫻ magnification.
Aytan. Inhibition of endometriosis with leflunomide. Fertil Steril 2007.
and which plays a part in inflammation—activation (11, 16). In addition, A77 1726 inhibits tyrosine kinases responsible for early T cell and B cell signaling in the G0/G1 phase of the cell cycle (11). In the present study, a rat model was used to investigate the efficacy of leflunomide on the development of experimental endometriosis, and leflunomide was found to affect the development of endometriosis negatively and seemed to interfere with the growth and maintenance of the uterine explant. There are some limitations in the present study that must be acknowledged. The semiquantitative evaluation of the preservation of endometrial tissue with a scoring system is subjective, and there may be interobserver variability. Second, although it has been noted that rat endometriotic tissues and cells perform in a similar manner as human endometriotic cells in organ explant culture and isolated cell culture (17–19), there is a disadvantage of extrapolating data across species, and immunologic properties of species are different. Estrogen levels in the rat are much lower than in humans, and rats do not have a menstrual cycle, all of which could be important and limit extrapolation of results from this model to humans. Third, leflunomide is potentially a toxic drug with some side effects (11, 20 –24). Fertility and Sterility姞
In conclusion, because of its immunomodulator properties the effect of leflunomide on the development of endometriosis model in rats was assessed in the present study. Leflunomide was found to affect the development of endometriosis negatively in this experimental rat model and seemed to interfere with the growth and maintenance of the uterine explant. Although there are concerns about the toxicity of this particular compound, novel therapeutic approaches for the important and yet unsolved problem of endometriosis and related morbidities are needed. Leflunomide may be a candidate; however, further studies are required to evaluate its effects. Hakan Aytan, M.D.a Pinar Caglar, M.D.b Dilek Uygur, M.D.b Sema Zergeroglu, M.D.c Sertac Batioglu, M.D.d a Department of Gynecology and Obstetrics, Gaziosmanpasa University, Tokat, Turkey; and Departments of b Gynecology and Obstetrics, c Pathology, and d Reproductive Endocrinology, Zekai Tahir Burak Women’s Health Training and Research Hospital, Ankara, Turkey 701
REFERENCES 1. Garcia-Velasco JA, Arici A, Zreik T, Naftolin F, Mor G. Macrophage derived growth factors modulate Fas ligand expression in cultured endometrial stromal cells: a role in endometriosis. Mol Hum Reprod 1999;5:642–50. 2. Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG, Taylor RN. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1 . Mol Hum Reprod 2000;6:269 –75. 3. Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285–96. 4. D’Hooghe TM, Nugent NP, Cuneo S, Chai DC, Deer F, Debrock S, et al. Recombinant human TNFRSF1A (r-hTBP1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Biol Reprod 2006;74:131– 6. 5. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. IL-1 beta, TNF-alpha, and IL-2 in peritoneal fluid and macrophageconditioned media of women with endometriosis. Am J Reprod Immunol 1995;34:381–5. 6. Cao X, Yang D, Song M, Murphy A, Parthasarathy S. The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: implications for endometriosis. Fertil Steril 2004;82 Suppl 3:999 –1007. 7. Hornung D, Bentzien F, Wallwiener D, Kiesel L, Taylor RN. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol Hum Reprod 2001;7: 163– 8. 8. Falconer H, Mwenda JM, Chai DC, Wagner C, Cornillie FJ, D’Hooghe TM. Treatment with antiTNF antibody c5N reduces the extent of induced endometriosis in the baboon. Fertil Steril 2004;82 Suppl 2:54 –5. 9. D’Antonio M, Martelli F, Peano S, Papoian R, Borrelli F. Ability of recombinant human TNF binding protein-1 (r-hTBP-1) to inhibit the development of experimentally-induced endometriosis in rats. J Reprod Immunol 2000;48:81–98. 10. Rozman B. Clinical pharmacokinetics of leflunomide. Clin Pharmacokinet 2002;41:421–30. 11. Breedveld FC, Dayer JM. Leflunomide: mode of action in the treatment of rheumatoid arthritis. Ann Rheum Dis 2000;59:841–9. 12. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684 –94.
702
Aytan et al.
Correspondence
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