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Endometriosis is sustained by tumour necrosis factor-α
Medical Hypotheses (2003) 60(1), 84–88 ª 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-9877(02)00336-5, available online at http://www.idealibrary.com
Endometriosis is sustained by tumour necrosis factor-a D. W. Bullimore Horbury, W. Yorkshire, UK
Summary Endometriosis is a common gynaecological disorder causing pain, infertility, and emotional distress. Evidence presented here suggests that abnormal production of tumour necrosis factor-a (TNF-a) is required for the establishment and maintenance of endometriosis and also is responsible for the associated infertility through its effect on sperm motility and function and oocyte development. Infliximab, which blocks TNF-a function, could be used in the treatment of endometriosis to reverse the above effects. ª 2002 Elsevier Science Ltd. All rights reserved.
HYPOTHESIS Endometriosis is one of the most common gynaecological disorders. It affects 5–10% of the female population of child-bearing age, or about 6 million women in the US (1). In this paper I will argue two main points. First, that in endometriosis tumour necrosis factor-a (TNF-a) plays a pivotal role in the establishment and maintenance of endometrial deposits. Second, that TNF-a also plays a major role in the infertility often seen in women with endometriosis, even when the endometriosis is relatively mild. The consequence of my hypothesis is that inhibitors of TNF-a, such as the hybrid monoclonal antibody infliximab, should provide effective treatment for endometriosis and, in many cases, the associated infertility. I will also briefly outline how my hypothesis can be tested. Early work demonstrated that the level of TNF-a in the peritoneal fluid (PF-TNF) of women with endometriosis undergoing laparoscopy was significantly higher than in controls (P < 0:01) (2). A large number of further studies has confirmed this finding (3–10). Using the
Received 15 March 2002 Accepted 12 July 2002 Correspondence to: Dr. D.W. Bullimore FRCP, Barnsley District General Hospital Trust, Gawber Road, Barnsley S75 2EP, UK. Phone: +44-01226-777879 E-mail: [email protected]
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revised American Fertility Society (rAFS) classification for endometriosis those with severe Stage III/IV disease have higher levels of PF-TNF than those with Stage I/II disease who in turn have higher levels than healthy controls. As an example, in one study median concentration of PF-TNF rose from 3.3 pg/ml in controls to 60.2 pg/ml in a group with Stage I/II disease and to 168 pg/ml in a group with Stage III/IV disease (9). The high levels of TNF-a detected suggest local release from activated peritoneal macrophages. Increased PF-TNF has also been noted in other conditions such as pelvic inflammatory disease where the presence of activated macrophages would be expected (4,10). Interestingly, in some studies raised TNF-a levels were also noted in primary infertility (2,6). TNF-a is produced by activated monocytes and macrophages. In vitro culture of peritoneal macrophages from women with endometriosis produce increased amounts of TNF-a compared with controls suggesting that they are a source of the peritoneal fluid TNF-a (3). Increased TNF-a production from peritoneal macrophages in endometriosis occurs both in the basal state and after activation with lipopolysaccharide (11). The above demonstrates that PF-TNF is elevated in endometriosis and derives in part, at least, from activated peritoneal macrophages. This is of course not sufficient to support the claim that TNF-a is involved in the development and maintenance of endometriosis. We must now consider the evidence that TNF-a, directly or indirectly, promotes the proliferation and adhesion of
Endometriosis is sustained by tumour necrosis factor-a
endometrial cells and the associated angiogenesis seen in endometriosis. As early as 1993, Hammond et al. (12) showed that TNF-a and fibroblast growth factor act synergistically to promote human endometrial stromal cell proliferation in an in vitro cell culture system. In the same year it was shown, again using an in vitro system, that pre-treatment of mesothelial cells with TNF-a increased the adherence of endometrial stromal cells (13). More recently, Sillem et al. (14) studied integrin function and its relation to TNF-a and other cytokines in endometriosis. Integrins are a family of molecules required for cellular adhesion. Endometrial cell cultures from women with or without endometriosis demonstrated functional integrin expression. (Cellular adhesion was blocked by anti-integrin antibodies showing that it was due to functional integrins.) However, in the endometriosis group TNF-a caused a significant increase in adhesion to laminin, fibronectin, and collagen. A variety of cytokines, in addition to TNF-a, is secreted by activated macrophages in endometriosis. These include interleukin-8 (IL-8), a potent angiogenic cytokine. Lesions in endometriosis can be classified as red, black, or white lesions. Red lesions represent an active form of endometriosis with high mitotic activity and vascularisation. TNF-a and IL-8 concentrations correlate with the size and number of active lesions (15). Peritoneal fluid IL-8 levels are increased in endometriosis. (Typically, IL-8 levels are controls 4.8 pg/ml, rAFS Stage I/II 27.5 pg/ml, and rAFS Stage III/IV 530 pg/ml (16).) Cell culture shows that peritoneal mesothelial cells were the source of IL-8 and treatment with TNF-a resulted in higher levels of IL-8. Cultured mesothelial cells expressed TNF-a-inducible IL-8 mRNA and secreted IL-8 protein. This suggests a role for the TNF-regulated expression of IL-8 in endometriosis (7). Recent work by Iwabe et al. (17) showed that TNF-a promoted endometrial stromal cell proliferation in culture and induced IL-8 gene and IL-8 protein expression in a dose-dependent manner. Importantly they demonstrated that the effect of TNF-a on endometrial stromal cell proliferation was abolished by adding anti-IL-8 antibody, showing that TNF-a is acting via its effect on IL-8 production. While most studies have used in vitro systems D’Antonio et al. (18) have used an in vivo rat system to study the proliferation of ectopic endometrial tissue. They transplanted autologous uterine tissue onto the inner surface of the abdominal wall of rats. Some of the rats were then given recombinant human tumour necrosis factorbinding protein-1 (r-hTBP-1), which is the soluble form of tumour necrosis factor-a receptor type-1. This effectively blocks TNF-a. The implants were less well-maintained in treated animals than controls supporting a role for TNF-a in the development of endometriosis (18). (It ª 2002 Elsevier Science Ltd. All rights reserved.
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should be noted that the experimental system used is not one in which you would expect there to be an increased number of activated peritoneal macrophages.) The above evidence supports the role of TNF-a in the development of the cellular adhesion and the angiogenesis present in endometriosis. Outlined below is the further role of TNF-a in recruitment of macrophages into the peritoneal cavity in endometriosis by its effect on chemokine production which provides a positive feedback loop to maintain the endometriosis. Macrophages are recruited into the peritoneal cavity in endometriosis. Local secretion of various chemotactic attractant factors (chemokines) appear to contribute to this process. Monocyte chemotactic protein-1 (MCP-1) is one such chemokine. TNF-a or IL-1b stimulated endometriotic cells synthesise and secrete increased MCP1 in culture (19,20). Peritoneal mesothelial cells produce MCP-1 the production of which is upregulated by both interleukin-1b (IL-1b) and TNF-a (21,22) as judged by production of MCP-1 protein and MCP-1 mRNA. Peritoneal fluid levels of MCP-1 are greatly increased in endometriosis. For example from median values of MCP-1 of 137 pg/ml (controls) to 205 pg/ml (moderate endometriosis) to 1,165 pg/ml (severe endometriosis) (21). RANTES is a further chemokine detected in peritoneal fluid in endometriosis with broad ranging monocyte, T-lymphocyte and eosinophil attractant and activating properties. (RANTES stands for ‘regulated upon activation, normal T-cell expressed and secreted’.) It is released in culture by human endometrial and endometriotic cells (23). Cultures of endometrial cells from Macaca nemestrina monkeys show a low constitute release of RANTES which is increased by up to nearly 500fold by stimulation with a combination of TNF-a and interferon-c. The stimulated RANTES production was nearly all released into the culture fluid. RANTES production in endometriosis could therefore provide a potent attractant and activator for macrophages, T-lymphocytes and eosinophils (23,24). With the important role of TNF-a as a promoter of cell adhesion and angiogenesis this provides a positive feedback loop to provide further establishment and development of endometriotic tissue. Similarly IL-1b upregulates RANTES mRNA 5-fold and RANTES protein concentration 12-fold compared with controls in cultured endometriotic cells to provide further positive feedback loop (25). While the above argument for the development of endometriosis has concentrated on local changes within the peritoneum there is much evidence of systemic immune dysfunction. In endometriosis peripheral blood monocytes also produce increased amounts of TNF-a and other cytokines, such as IL-6 and IL-8, in vitro (3,26,27). The peripheral blood cytokine levels although raised do not show a close correlation with the stage of Medical Hypotheses (2003) 60(1), 84–88
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endometriosis present (27,28). Danazol, used in the treatment of endometriosis significantly reduces the levels of peripheral blood TNF-a (29,30). Thus in some cases the development of a local inflammatory reaction with peritoneal macrophage recruitment following reflux of endometrial cells into the peritoneum might be one explanation for the development of endometriosis. However, in other cases the changes in peripheral blood cytokine levels and activation of peripheral blood monocytes point to disordered systemic immune function which might predispose to the establishment of endometriosis. Of particular interest in this regard is work on the female rhesus monkey exposed to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Exposure in the rhesus monkey is associated with a dose-dependent increase in the incidence of endometriosis (1,31,32). Examination of peripheral blood monocytes 13 years after exposure to TCDD in these animals was ended showed that TCDD exposure correlated with increased peripheral blood monocyte TNF-a secretion in response to T-cell mitogen (31). Related work used the Macaca fascicularis monkey, exposed to a range of TCDD over a year. Autologous transplants of endometrial fragments into the peritoneum showed significantly higher survival rates of implants, and greater diameter of the implants, in monkeys exposed to higher doses of TCDD (33). While the exact mechanism underlying these observations is uncertain work has focused on the role of matrix metalloproteinases (MMPs). This is a group of enzymes important for the control of extracellular matrix turnover (34). MMPs are expressed during menstrual breakdown and oestrogen-mediated growth of endometrial tissue and are normally suppressed by progesterone (35). TCDD, which promotes endometriosis in monkeys, disrupts MMP regulation by progesterone and also allows promotion of ectopic lesions in a mouse model of endometriosis (36). TNF-a and IL-1 cause upregulation of several MMPs which could contribute to increased invasiveness of uterine endometrial fragments in endometriosis patients (37). The expression of tissue inhibitors of MMPs is decreased in endometriosis giving a further proteinase-inhibitor imbalance (38). Thus again in endometriosis we have evidence of a mechanism involving regulation by TNF-a. The OXEGENE study, which aims to identify genes which give increased susceptibility to endometriosis includes genes related to dioxin detoxification as potential candidate genes (39). The conclusion from the above multiple lines of evidence must be that TNF-a plays a pivotal role in the development and maintenance of endometriosis. I will now turn to the evidence related to the second part of my hypothesis that suggests it plays a role in the infertility experienced by many women with endometriosis even when they have only mild to moderate disease. Medical Hypotheses (2003) 60(1), 84–88
TNF-a is cytotoxic to gametes (3,40). Sperm motility is decreased when they are incubated with either peritoneal fluid from infertile women with minimal endometriosis or with recombinant TNF-a. Motility is inhibited in proportion to TNF-a concentration. Either heat inactivation of TNF-a in the peritoneal fluid or neutralisation with anti-TNF-a antibody reverses the inhibitory effect on sperm motility (41). PF-TNF-a concentration is higher in infertile women with endometriosis compared with a group of infertile women without endometriosis or normally fertile women. Donor sperm, when incubated with the peritoneal fluid of infertile women with endometriosis, show less progressive movement, total motility and hypoosmotic swelling, showing a negative correlation with PF-TNF levels. This again supports the view that PF-TNF has a detrimental effect on sperm motility (42). Furthermore, the binding of sperm to the zona pellucida in vitro is significantly impaired when the incubation medium contains conditioned medium from activated or unactivated macrophages as compared to control medium. The inhibition of sperm binding was greatest when conditioned medium from activated macrophages, which produce the greatest amount of cytokine, was used (43). While a number of cytokines were produced by the activated macrophages incubation of the sperm with the individual cytokines in the assay system used identified TNF-a as the key cytokine responsible for the effect. Studies of embryo toxicity, using a mouse two-cell model, show that peritoneal fluid from women with endometriosis is toxic. This fluid contains TNF-a and inhibits cleavage of the two-cell embryo (5). Interestingly, patients who had received Danazol for treatment of endometriosis had near normal PF-TNF levels and showed little embryo toxicity. Granulosa cells from women with endometriosis produce higher levels of TNF-a compared with control granulosa cells obtained from women undergoing in-vitro fertilisation (IVF) for male infertility (44). IL-1b, IL-6, and IL-8 were also increased but only for TNF-a was the increase statistically significant. Work has suggested that the follicular fluid TNF-a levels is associated with poor oocyte quality and outcome in in-vitro fertilisationembryo transfer work (45). Other recent work has shown that women with endometriosis undergoing IVF and oocyte donation from a woman without endometriosis have embryo development and implantation rates similar to women without endometriosis. However, when the donor egg is from a woman with endometriosis and the recipient is a woman without endometriosis then the implantation rate is significantly reduced (46). This again points to lower oocyte quality in some women with endometriosis. There are therefore multiple lines of evidence to support the role of raised TNF-a levels as a cause of ª 2002 Elsevier Science Ltd. All rights reserved.
Endometriosis is sustained by tumour necrosis factor-a
lowered fertility in endometriosis, both by its effect on the sperm and on the oocyte. Both parts of my hypothesis can be tested as there is already a clinical product available which effectively blocks TNF-a. This product, infliximab, is currently used clinically in the management of Crohn’s disease and rheumatoid arthritis. Studies in endometriosis could be on a named patient basis with patients who could understand the arguments for its use and were prepared to accept the side-effects of infliximab therapy. These are well documented and generally minor. If there was evidence of benefit in these preliminary studies then a formal randomised controlled trial could be used. End-points could include pregnancy success rates, regression of endometriotic lesions at ‘second-look’ laparoscopy or change in patient quality of life scores. Pain scores do not correlate well with rAFS severity grading for endometriosis (6) but within-subject changes might show benefit from treatment. Endometriosis is a common and distressing condition. Current treatment options, both medical and surgical, are often unsatisfactory. The evidence presented above in support of the hypothesis suggests that it is time to determine whether TNF-a blockade with infliximab will provide a more satisfactory management option.
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24. Hornung D., Klingel K., Dohrn K., Kandolf R., Wallwiener D., Taylor R. N. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal and endometriotic implants: assessment of autocrine/ paracrine regulation by in situ hybridization. Am J Pathol 2001; 158(6): 1949–1954. 25. Lebovic D. I., Chao V. A., Martini J. F., Taylor R. N. Il-1b induction of RANTES (regulated upon activation, normal T-cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-jb site in the proximal promoter. J Clin Endocrinol Metab 2001; 86(10): 4759–4764. 26. Braun D. P., Gebel H., House R., Rana N., Dmowski N. P. Spontaneous and induced synthesis of cytokines by peripheral blood monocytes in patients with endometriosis. Fertil Steril 1995; 65: 1125–1129. 27. Fang X., Lin Q., Chen Y. Study on in vitro cytokines levels induced from peripheral mononuclear cells in patients with endometriosis. Zhonghua Fu Chan Ke Za Zhi 1999; 34(10): 609–611. 28. D’Hooghe T. M., Xiao L., Hill J. A. Cytokine profiles in autologous peritoneal fluid and peripheral blood of women with deep and superficial endometriosis. Arch Gynecol Obstet 2001; 265(1): 40–44. 29. Metalliotakis I., Neonaki M., Zolindaki A., Hassan E., Georgoulias V., Koumantakis E. Changes in immunologic variables (TNF-a, sCD8, and sCD4) during danazol treatment in patients with endometriosis. Int J Fertil Womens Med 1997; 42: 211–214. 30. Metalliotakis I. M., Neonaki M. A., Panidis D. K., Goumenou A. G., Koumantakis E. E. Three-year follow-up of [CA-125, CA 19-9, CA 15-3, sIL-2R, Il-6, Il-1a, TNF-a, sCD8, and sCD4] levels in a woman with severe endometriosis. Eur J Obstet Gynecol Reprod Biol 2000; 93(2): 127–129. 31. Rier S. E., Coe C. L., Lemieux A. M., Martin D. C., Morris R., Lucier G. W., Clark G. C. Increased tumor necrosis factor-a production by peripheral blood leukocytes from TCDDexposed rhesus monkeys. Toxicol Sci 2001; 60(2): 327–337. 32. Rier S. E., Martin D. C., Bowman R. E., Dmowski W. P., Becker J. L. Endometriosis in the rhesus monkey (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol 1993; 21(4): 433–441. 33. Yang J. Z., Agarwal S. K., Foster W. G. Subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin modulates the pathophysiology of endometriosis in the cynomolgus monkey. Toxicol Sci 2000; 56(2): 374–381. 34. Bruner K. L., Eisenberg E., Gorstein F., Osteen K. G. Progesterone and transforming growth factor-b coordinately
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ª 2002 Elsevier Science Ltd. All rights reserved.
Endometriosis is sustained by tumour necrosis factor-a D. W. Bullimore Horbury, W. Yorkshire, UK
Summary Endometriosis is a common gynaecological disorder causing pain, infertility, and emotional distress. Evidence presented here suggests that abnormal production of tumour necrosis factor-a (TNF-a) is required for the establishment and maintenance of endometriosis and also is responsible for the associated infertility through its effect on sperm motility and function and oocyte development. Infliximab, which blocks TNF-a function, could be used in the treatment of endometriosis to reverse the above effects. ª 2002 Elsevier Science Ltd. All rights reserved.
HYPOTHESIS Endometriosis is one of the most common gynaecological disorders. It affects 5–10% of the female population of child-bearing age, or about 6 million women in the US (1). In this paper I will argue two main points. First, that in endometriosis tumour necrosis factor-a (TNF-a) plays a pivotal role in the establishment and maintenance of endometrial deposits. Second, that TNF-a also plays a major role in the infertility often seen in women with endometriosis, even when the endometriosis is relatively mild. The consequence of my hypothesis is that inhibitors of TNF-a, such as the hybrid monoclonal antibody infliximab, should provide effective treatment for endometriosis and, in many cases, the associated infertility. I will also briefly outline how my hypothesis can be tested. Early work demonstrated that the level of TNF-a in the peritoneal fluid (PF-TNF) of women with endometriosis undergoing laparoscopy was significantly higher than in controls (P < 0:01) (2). A large number of further studies has confirmed this finding (3–10). Using the
Received 15 March 2002 Accepted 12 July 2002 Correspondence to: Dr. D.W. Bullimore FRCP, Barnsley District General Hospital Trust, Gawber Road, Barnsley S75 2EP, UK. Phone: +44-01226-777879 E-mail: [email protected]
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revised American Fertility Society (rAFS) classification for endometriosis those with severe Stage III/IV disease have higher levels of PF-TNF than those with Stage I/II disease who in turn have higher levels than healthy controls. As an example, in one study median concentration of PF-TNF rose from 3.3 pg/ml in controls to 60.2 pg/ml in a group with Stage I/II disease and to 168 pg/ml in a group with Stage III/IV disease (9). The high levels of TNF-a detected suggest local release from activated peritoneal macrophages. Increased PF-TNF has also been noted in other conditions such as pelvic inflammatory disease where the presence of activated macrophages would be expected (4,10). Interestingly, in some studies raised TNF-a levels were also noted in primary infertility (2,6). TNF-a is produced by activated monocytes and macrophages. In vitro culture of peritoneal macrophages from women with endometriosis produce increased amounts of TNF-a compared with controls suggesting that they are a source of the peritoneal fluid TNF-a (3). Increased TNF-a production from peritoneal macrophages in endometriosis occurs both in the basal state and after activation with lipopolysaccharide (11). The above demonstrates that PF-TNF is elevated in endometriosis and derives in part, at least, from activated peritoneal macrophages. This is of course not sufficient to support the claim that TNF-a is involved in the development and maintenance of endometriosis. We must now consider the evidence that TNF-a, directly or indirectly, promotes the proliferation and adhesion of
Endometriosis is sustained by tumour necrosis factor-a
endometrial cells and the associated angiogenesis seen in endometriosis. As early as 1993, Hammond et al. (12) showed that TNF-a and fibroblast growth factor act synergistically to promote human endometrial stromal cell proliferation in an in vitro cell culture system. In the same year it was shown, again using an in vitro system, that pre-treatment of mesothelial cells with TNF-a increased the adherence of endometrial stromal cells (13). More recently, Sillem et al. (14) studied integrin function and its relation to TNF-a and other cytokines in endometriosis. Integrins are a family of molecules required for cellular adhesion. Endometrial cell cultures from women with or without endometriosis demonstrated functional integrin expression. (Cellular adhesion was blocked by anti-integrin antibodies showing that it was due to functional integrins.) However, in the endometriosis group TNF-a caused a significant increase in adhesion to laminin, fibronectin, and collagen. A variety of cytokines, in addition to TNF-a, is secreted by activated macrophages in endometriosis. These include interleukin-8 (IL-8), a potent angiogenic cytokine. Lesions in endometriosis can be classified as red, black, or white lesions. Red lesions represent an active form of endometriosis with high mitotic activity and vascularisation. TNF-a and IL-8 concentrations correlate with the size and number of active lesions (15). Peritoneal fluid IL-8 levels are increased in endometriosis. (Typically, IL-8 levels are controls 4.8 pg/ml, rAFS Stage I/II 27.5 pg/ml, and rAFS Stage III/IV 530 pg/ml (16).) Cell culture shows that peritoneal mesothelial cells were the source of IL-8 and treatment with TNF-a resulted in higher levels of IL-8. Cultured mesothelial cells expressed TNF-a-inducible IL-8 mRNA and secreted IL-8 protein. This suggests a role for the TNF-regulated expression of IL-8 in endometriosis (7). Recent work by Iwabe et al. (17) showed that TNF-a promoted endometrial stromal cell proliferation in culture and induced IL-8 gene and IL-8 protein expression in a dose-dependent manner. Importantly they demonstrated that the effect of TNF-a on endometrial stromal cell proliferation was abolished by adding anti-IL-8 antibody, showing that TNF-a is acting via its effect on IL-8 production. While most studies have used in vitro systems D’Antonio et al. (18) have used an in vivo rat system to study the proliferation of ectopic endometrial tissue. They transplanted autologous uterine tissue onto the inner surface of the abdominal wall of rats. Some of the rats were then given recombinant human tumour necrosis factorbinding protein-1 (r-hTBP-1), which is the soluble form of tumour necrosis factor-a receptor type-1. This effectively blocks TNF-a. The implants were less well-maintained in treated animals than controls supporting a role for TNF-a in the development of endometriosis (18). (It ª 2002 Elsevier Science Ltd. All rights reserved.
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should be noted that the experimental system used is not one in which you would expect there to be an increased number of activated peritoneal macrophages.) The above evidence supports the role of TNF-a in the development of the cellular adhesion and the angiogenesis present in endometriosis. Outlined below is the further role of TNF-a in recruitment of macrophages into the peritoneal cavity in endometriosis by its effect on chemokine production which provides a positive feedback loop to maintain the endometriosis. Macrophages are recruited into the peritoneal cavity in endometriosis. Local secretion of various chemotactic attractant factors (chemokines) appear to contribute to this process. Monocyte chemotactic protein-1 (MCP-1) is one such chemokine. TNF-a or IL-1b stimulated endometriotic cells synthesise and secrete increased MCP1 in culture (19,20). Peritoneal mesothelial cells produce MCP-1 the production of which is upregulated by both interleukin-1b (IL-1b) and TNF-a (21,22) as judged by production of MCP-1 protein and MCP-1 mRNA. Peritoneal fluid levels of MCP-1 are greatly increased in endometriosis. For example from median values of MCP-1 of 137 pg/ml (controls) to 205 pg/ml (moderate endometriosis) to 1,165 pg/ml (severe endometriosis) (21). RANTES is a further chemokine detected in peritoneal fluid in endometriosis with broad ranging monocyte, T-lymphocyte and eosinophil attractant and activating properties. (RANTES stands for ‘regulated upon activation, normal T-cell expressed and secreted’.) It is released in culture by human endometrial and endometriotic cells (23). Cultures of endometrial cells from Macaca nemestrina monkeys show a low constitute release of RANTES which is increased by up to nearly 500fold by stimulation with a combination of TNF-a and interferon-c. The stimulated RANTES production was nearly all released into the culture fluid. RANTES production in endometriosis could therefore provide a potent attractant and activator for macrophages, T-lymphocytes and eosinophils (23,24). With the important role of TNF-a as a promoter of cell adhesion and angiogenesis this provides a positive feedback loop to provide further establishment and development of endometriotic tissue. Similarly IL-1b upregulates RANTES mRNA 5-fold and RANTES protein concentration 12-fold compared with controls in cultured endometriotic cells to provide further positive feedback loop (25). While the above argument for the development of endometriosis has concentrated on local changes within the peritoneum there is much evidence of systemic immune dysfunction. In endometriosis peripheral blood monocytes also produce increased amounts of TNF-a and other cytokines, such as IL-6 and IL-8, in vitro (3,26,27). The peripheral blood cytokine levels although raised do not show a close correlation with the stage of Medical Hypotheses (2003) 60(1), 84–88
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endometriosis present (27,28). Danazol, used in the treatment of endometriosis significantly reduces the levels of peripheral blood TNF-a (29,30). Thus in some cases the development of a local inflammatory reaction with peritoneal macrophage recruitment following reflux of endometrial cells into the peritoneum might be one explanation for the development of endometriosis. However, in other cases the changes in peripheral blood cytokine levels and activation of peripheral blood monocytes point to disordered systemic immune function which might predispose to the establishment of endometriosis. Of particular interest in this regard is work on the female rhesus monkey exposed to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Exposure in the rhesus monkey is associated with a dose-dependent increase in the incidence of endometriosis (1,31,32). Examination of peripheral blood monocytes 13 years after exposure to TCDD in these animals was ended showed that TCDD exposure correlated with increased peripheral blood monocyte TNF-a secretion in response to T-cell mitogen (31). Related work used the Macaca fascicularis monkey, exposed to a range of TCDD over a year. Autologous transplants of endometrial fragments into the peritoneum showed significantly higher survival rates of implants, and greater diameter of the implants, in monkeys exposed to higher doses of TCDD (33). While the exact mechanism underlying these observations is uncertain work has focused on the role of matrix metalloproteinases (MMPs). This is a group of enzymes important for the control of extracellular matrix turnover (34). MMPs are expressed during menstrual breakdown and oestrogen-mediated growth of endometrial tissue and are normally suppressed by progesterone (35). TCDD, which promotes endometriosis in monkeys, disrupts MMP regulation by progesterone and also allows promotion of ectopic lesions in a mouse model of endometriosis (36). TNF-a and IL-1 cause upregulation of several MMPs which could contribute to increased invasiveness of uterine endometrial fragments in endometriosis patients (37). The expression of tissue inhibitors of MMPs is decreased in endometriosis giving a further proteinase-inhibitor imbalance (38). Thus again in endometriosis we have evidence of a mechanism involving regulation by TNF-a. The OXEGENE study, which aims to identify genes which give increased susceptibility to endometriosis includes genes related to dioxin detoxification as potential candidate genes (39). The conclusion from the above multiple lines of evidence must be that TNF-a plays a pivotal role in the development and maintenance of endometriosis. I will now turn to the evidence related to the second part of my hypothesis that suggests it plays a role in the infertility experienced by many women with endometriosis even when they have only mild to moderate disease. Medical Hypotheses (2003) 60(1), 84–88
TNF-a is cytotoxic to gametes (3,40). Sperm motility is decreased when they are incubated with either peritoneal fluid from infertile women with minimal endometriosis or with recombinant TNF-a. Motility is inhibited in proportion to TNF-a concentration. Either heat inactivation of TNF-a in the peritoneal fluid or neutralisation with anti-TNF-a antibody reverses the inhibitory effect on sperm motility (41). PF-TNF-a concentration is higher in infertile women with endometriosis compared with a group of infertile women without endometriosis or normally fertile women. Donor sperm, when incubated with the peritoneal fluid of infertile women with endometriosis, show less progressive movement, total motility and hypoosmotic swelling, showing a negative correlation with PF-TNF levels. This again supports the view that PF-TNF has a detrimental effect on sperm motility (42). Furthermore, the binding of sperm to the zona pellucida in vitro is significantly impaired when the incubation medium contains conditioned medium from activated or unactivated macrophages as compared to control medium. The inhibition of sperm binding was greatest when conditioned medium from activated macrophages, which produce the greatest amount of cytokine, was used (43). While a number of cytokines were produced by the activated macrophages incubation of the sperm with the individual cytokines in the assay system used identified TNF-a as the key cytokine responsible for the effect. Studies of embryo toxicity, using a mouse two-cell model, show that peritoneal fluid from women with endometriosis is toxic. This fluid contains TNF-a and inhibits cleavage of the two-cell embryo (5). Interestingly, patients who had received Danazol for treatment of endometriosis had near normal PF-TNF levels and showed little embryo toxicity. Granulosa cells from women with endometriosis produce higher levels of TNF-a compared with control granulosa cells obtained from women undergoing in-vitro fertilisation (IVF) for male infertility (44). IL-1b, IL-6, and IL-8 were also increased but only for TNF-a was the increase statistically significant. Work has suggested that the follicular fluid TNF-a levels is associated with poor oocyte quality and outcome in in-vitro fertilisationembryo transfer work (45). Other recent work has shown that women with endometriosis undergoing IVF and oocyte donation from a woman without endometriosis have embryo development and implantation rates similar to women without endometriosis. However, when the donor egg is from a woman with endometriosis and the recipient is a woman without endometriosis then the implantation rate is significantly reduced (46). This again points to lower oocyte quality in some women with endometriosis. There are therefore multiple lines of evidence to support the role of raised TNF-a levels as a cause of ª 2002 Elsevier Science Ltd. All rights reserved.
Endometriosis is sustained by tumour necrosis factor-a
lowered fertility in endometriosis, both by its effect on the sperm and on the oocyte. Both parts of my hypothesis can be tested as there is already a clinical product available which effectively blocks TNF-a. This product, infliximab, is currently used clinically in the management of Crohn’s disease and rheumatoid arthritis. Studies in endometriosis could be on a named patient basis with patients who could understand the arguments for its use and were prepared to accept the side-effects of infliximab therapy. These are well documented and generally minor. If there was evidence of benefit in these preliminary studies then a formal randomised controlled trial could be used. End-points could include pregnancy success rates, regression of endometriotic lesions at ‘second-look’ laparoscopy or change in patient quality of life scores. Pain scores do not correlate well with rAFS severity grading for endometriosis (6) but within-subject changes might show benefit from treatment. Endometriosis is a common and distressing condition. Current treatment options, both medical and surgical, are often unsatisfactory. The evidence presented above in support of the hypothesis suggests that it is time to determine whether TNF-a blockade with infliximab will provide a more satisfactory management option.
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