Oxidative stress and peritoneal endometriosis. Anne Van Langendonckt, Ph.D., Françoise Casanas-Roux, Ph.D., and Jacques Donnez, M.D., Ph.D.

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1 FERTILITY AND STERILITY VOL. 77, NO. 5, MAY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MODERN TRENDS Edward E. Wallach, M.D. Associate Editor Oxidative stress and peritoneal endometriosis Anne Van Langendonckt, Ph.D., Françoise Casanas-Roux, Ph.D., and Jacques Donnez, M.D., Ph.D. Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium Objective: To review the literature associating pelvic endometriosis with oxidative stress and to discuss the potential causes and consequences of a pro-oxidant environment in the peritoneal cavity. Design: Literature survey. Result(s): Several studies suggest that oxidative stress is a component of the inflammatory reaction associated with endometriosis. Evidence includes the prevention of endometriosis induction in rabbits by the addition of antioxidants, an increase in reactive oxygen species release by macrophages, increased peritoneal levels of oxidized low-density lipoproteins and their by-products, altered expression of endometrial pro-oxidant and antioxidant enzymes, and consumption of peritoneal fluid vitamin E. Retrograde menstruation is likely to carry highly pro-oxidant factors, such as heme and iron, into the peritoneal cavity, as well as apoptotic endometrial cells, which are well-known inducers of oxidative stress. Reactive oxygen species may be involved in endometriosis-associated infertility and may play a role in the regulation of the expression of genes encoding immunoregulators, cytokines and cell adhesion molecules implicated in the pathogenesis of endometriosis. Conclusion(s): Better understanding of the mechanisms of reactive oxygen species production and detoxification and further investigation of their effect on the peritoneal environment are essential to obtain new insight into this disease and eventually develop new diagnostic and therapeutic strategies. (Fertil Steril 2002; 77: by American Society for Reproductive Medicine.) Key Words: Pelvic endometriosis, hemoglobin, iron, antioxidant, reactive oxygen species, NF- B Received August 29, 2001; revised and accepted November 14, Supported by a grant from the Fonds National de la Recherche Scientifique de Belgique. Dr. Van Langendonckt is a scientific collaborator of the Fonds National de la Recherche Scientifique de Belgique. Reprint requests: Jacques Donnez, M.D., Ph.D., Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires St Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium (FAX: ; donnez@gyne. ucl.ac.be) /02/$22.00 PII S (02)02959-X Endometriosis is characterized by the implantation and growth of endometrial tissue outside the uterine cavity. Most studies of pelvic endometriosis are based on the implantation theory of Sampson (1). According to this theory, desquamated endometrial cells are transported into the peritoneal cavity after retrograde menstruation, and the still-viable cells subsequently implant and grow. Recent studies suggest that menstrual effluent contains factors that induce alterations in the morphology of the peritoneal mesothelium (2), which may create adhesion sites for endometrial cells. Attachment of endometrial cells appears to be enhanced by induction of adhesion molecules and their receptors (3 5) and overexpression of matrix metalloproteinases (6) and plasminogen activators (7), which ensure local destruction of the extracellular matrix in endometriosis. After adhesion, endometrial cells proliferate and gradually invade the peritoneal tissue. Some factors induce vascularization of endometriotic implants, allowing their further development (8 12). It has been suggested that this process leads to the formation of red, flame-like lesions with high vascular and metabolic activity. Cytokines and growth factors, such as transforming growth factor-, interleukin-8, interleukin-1, tumor necrosis factor-, interferon- (13), and vascular endothelial growth factor (11), have been implicated as inducers of attachment, proliferation, and neovascularization. Endometriosis also seems to be associated with increased recruitment and activation of macrophages, which are thought to play an important role in the development of the disease (14). Red lesions regrow constantly after partial shedding. A scarification process is then initiated, and lesions appear as black and, later, white quiescent or latent lesions (8, 9, 15). Endometriosis is a multifactorial disease associated with a general inflammatory response in the peritoneal cavity. Oxidative stress has been proposed as a potential factor involved in 861

2 the pathophysiology of the disease. Reactive oxygen species have been implicated in the pathogenesis of many human diseases and aging (16, 17). We provide an overview of studies associating oxidative stress with peritoneal endometriosis. The possible causes and consequences of an oxidant environment in the peritoneal cavity are discussed in the light of recent advances in this field. REACTIVE OXYGEN SPECIES Many comprehensive reviews have discussed production of reactive oxygen species and cellular response to oxidative stress (16 18); we therefore cover these topics only briefly. Reactive oxygen species are intermediaries produced by normal oxygen metabolism. To protect themselves from the deleterious effects of reactive oxygen species, cells have developed a wide range of antioxidant systems to limit production of reactive oxygen species, inactivate them, and repair cell damage. However, oxidative stress may occur when the balance between reactive oxygen species production and antioxidant defense is disrupted. The increasing number of diseases associated with oxidative stress suggests that oxidative balance may be precarious (17). Univalent reduction in oxygen results in production of the superoxide anion. The dismutation of two superoxide anions, catalyzed by superoxide dismutase, leads to the formation of hydrogen peroxide. Hydrogen peroxide is detoxified by glutathione peroxidase or inactivated to H 2 O and O 2, a reaction catalyzed by catalase. Optimal protection is achieved only when an appropriate balance among superoxide dismutase, glutathione peroxidase, and catalase is maintained (19). Alternatively, in the presence of transition metals such as iron, hydrogen peroxide produces the highly reactive hydroxyl radical. The hydroxyl radical is highly toxic to cells because it reacts instantaneously with all cellular components. Almost all major classes of biomolecules, including lipids, proteins, and nucleic acids, are potential targets for reactive oxygen species. The oxidative destruction of polyunsaturated fatty acids, known as lipid peroxidation, is particularly damaging because it is a self-perpetuating chainreaction that may alter the integrity of cell membranes. These reactions often involve transition-metal ion catalysis (16). The sequestration of iron by iron-binding proteins seems to be crucial in protecting cells from damage by reactive oxygen species and lipid peroxidation. Cells have evolved enzymatic antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase, and nonenzymatic antioxidants, including vitamin E, vitamin C, taurine, and glutathione (18). Nitric oxide (NO), another oxygen-containing radical, is of growing interest in biology and medicine. Nitric oxide synthase is involved in the production of NO. Nitric oxide plays an important role as a mediator of paracrine interactions, as seen in the vascular system (20). However, as a free radical with an unpaired electron, NO also reacts with a wide range of cellular molecules. In pathophysiologic conditions, NO may be overproduced and the highly toxic radical peroxynitrite, which is formed from NO and superoxide, may accumulate. Peroxynitrite is considered to be a strong prooxidant similar to the hydroxyl radical (17). EVIDENCE OF INCREASED OXIDATIVE STRESS IN ENDOMETRIOSIS Generation of Reactive Oxygen Species In 1987, Zeller et al. (21) showed that production of reactive oxygen species by peritoneal fluid mononuclear cells was increased in endometriosis. They concluded that large amounts of reactive oxygen species were released after chronic stimulation of peritoneal fluid macrophages in women with endometriosis. Production of reactive oxygen species is known to increase after activation of immune cells, especially polymorphonuclear leukocytes and macrophages. However, further studies based on direct measurement of reactive oxygen species production failed to show an obvious oxidant or antioxidant imbalance in the peritoneal cavity of patients with endometriosis. Wang et al. (22) found similar levels of reactive oxygen species (detected by a chemiluminescent method) in the peritoneal fluid of patients with endometriosis and disease-free controls. Furthermore, Ho et al. (23) showed that total antioxidant status, as assessed by spectrophotometry, was not increased in endometriosis; Polak et al. (24) recently confirmed this finding. Ota et al. (25) also found evidence that oxidative stress occurs in endometriosis. Expression of xanthine oxidase, an enzyme which produces reactive oxygen species, in ectopic and eutopic endometrium remained high throughout the menstrual cycle in women with endometriosis; in contrast, cyclic variations in its expression were seen in controls. This apparent discrepancy between results may be due to the fact that only persistent markers of oxidative stress, such as enzymes or stable by-products of oxidative reactions, can still be detected when endometriosis is diagnosed. Another possible explanation is that oxidative stress occurs only locally for example, at the site of bleeding and does not result in an increase in total peritoneal fluid concentrations. Generation of the NO. Radical Endothelial nitric oxide synthase, an enzyme that generates NO, is also overexpressed in endometriosis and adenomyosis (26). Three isoforms of nitric oxide synthases have been identified to date: inducible, endothelial, and neuronal. Expression of endothelial and inducible nitric oxide synthases has been demonstrated in the endometrium (20), and increased expression of endothelial nitric oxide synthase was observed throughout the cycle in the endometrium of women with endometriosis and adenomyosis (26, 27). However, no evidence of increased NO metabolism was found in the 862 Van Langendonckt et al. Oxidative stress and endometriosis Vol. 77, No. 5, May 2002

3 peritoneal fluid of women with and without endometriosis (23). Of note, generation of peroxynitrite by ectopic endometrium was recently demonstrated in patients with adenomyosis (28). Expression of endothelial and inducible nitric oxide synthase and peroxynitrite generation were markedly reduced after GnRH agonist therapy, supporting their potential role in the pathophysiology of adenomyosis (28). Animal Models A study by Portz et al. (29) supports the hypothesis that oxidative stress is involved in endometriosis. They found that injection of the antioxidant enzymes superoxide dismutase and catalase into the peritoneal cavity prevented formation of intraperitoneal adhesions at endometriosis sites in the rabbit. Oxidation of Low-Density Lipoproteins In contrast, Murphy et al. (30) found increased oxidation of low-density lipoprotein in patients with pelvic endometriosis and increased concentrations of oxidized low-density lipoproteins in the peritoneal fluid of women in whom the disease was developing (30, 31). Oxidative modification of these molecules involves peroxidation of the lipid component, which leads to release of aldehydes, such as malondialdehyde, and reaction with lysine residues of proteins. However, Arumugam and Yip (32) found no relation between levels of malondialdehyde in peritoneal fluid and severity of endometriosis. Higher levels of lysophosphatidyl choline, another indicator of lipoprotein peroxidation, were found in the peritoneal fluid of patients with endometriosis (30, 33). Murphy et al. (34) demonstrated increased modified lipidprotein complexes at the level of the endometrium. Ectopic endometrial cells were also immunostained with antibodies to oxidatively modified proteins. The occurrence of oxidatively modified lipid-protein complexes was further ascertained by an increase in serum autoantibodies to three markers of oxidative stress: lipid-peroxide modified serum albumin, malondialdehyde-modified low-density lipoprotein, and oxidized low-density lipoprotein (35). The latter two molecules are examples of terminal decomposition of proteins damaged by peroxidized lipids. Antioxidant Enzymes Several recent studies appear to show that in women with endometriosis, the endometrium shows altered expression of enzymes involved in defense against oxidative stress. Ota et al. (36) found that manganese and copper/zinc superoxide dismutase (37) and glutathione peroxidase (38) are overexpressed in the eutopic endometrium of patients with endometriosis or adenomyosis. Expression of both superoxide dismutases remains high throughout the cycle (37), unlike in patients without disease, who have cyclic variations in their levels of expression (37, 39). Expression of manganese superoxide dismutase (40) and glutathione peroxidase (38) has been demonstrated in ectopic endometrium of women with adenomyosis. Expression of these enzymes, which are induced during increased release of reactive oxygen species, can be considered as an indicator of oxidative stress (36). It has been suggested that eutopic endometrium undergoes oxidative stress even in patients who do not develop endometriosis (30), but probably to a lesser extent. Heat-Shock Proteins Abnormal expression of heat-shock proteins 27 and 70 has been reported in the eutopic endometrium of women with endometriosis (13). These stress response proteins are induced by numerous stimuli, including oxidative stress, and can also be considered as indicators of an atypical stress response (13). Vitamin E Vitamin E levels are significantly lower in the peritoneal fluid of women with endometriosis, perhaps because antioxidants in peritoneal fluid are consumed during oxidation reactions (30, 33). Vitamin E plays an important role in protecting biological membranes by preventing peroxidation. It may also play a role in preventing activation of redox-sensitive pathways, which have been implicated in abnormal cell proliferation and inflammatory response. A decrease in antioxidant capacity may explain why low-density lipoproteins in the peritoneal fluid of patients with endometriosis are more readily oxidized than are lowdensity lipoproteins from control patients (33). As far as we know, apart from this study of vitamin E, the antioxidant response of the peritoneal environment has not been examined. Taken together, the data strongly indicate the occurrence of oxidative stress in the peritoneal cavity of women with endometriosis and, to a lesser extent, in those without disease. ORIGIN OF OXIDATIVE STRESS IN THE PERITONEAL CAVITY Several hypotheses have been proposed to explain why oxidative stress is induced in cases of pelvic endometriosis. Erythrocytes (41), apoptotic endometrial tissue and cell debris (30) transplanted into the peritoneal cavity by menstrual reflux and macrophages (30) have been implicated as potential inducers of oxidative stress (Fig. 1). Excessive production of reactive oxygen species may also result from exposure to environmental toxicants and heavy metals, which may disrupt the balance of pro-oxidants and antioxidants. Erythrocytes and Their Pro-oxidant By- Products Erythrocytes are likely to release pro-oxidant and proinflammatory factors, such as hemoglobin and its highly toxic by-products heme and iron, into the peritoneal environment. Iron and heme are essential to living cells (42). However, unless they are appropriately chelated, free iron FERTILITY & STERILITY 863

4 FIGURE 1 Hypotheses explaining oxidative stress in the peritoneal cavity of women with endometriosis. Bold type indicates factors that have been studied specifically in relation to pelvic endometriosis. CO carbon dioxide; HO heme oxygenase; NO nitric oxide; NOS nitric oxide synthase. Van Langendonckt. Oxidative stress and endometriosis. Fertil Steril (16) and, to a lesser extent, heme (43, 44) play a key role in the formation of deleterious reactive oxygen species. Ironinduced oxidative stress has been implicated in numerous pathologic processes (16, 17). However, erythrocytes are found in the peritoneal cavity of 90% of menstruating women (45). Thus, why do some patients develop macroscopically visible peritoneal endometriotic lesions but others do not? One hypothesis is that in some patients, peritoneal protective mechanisms are swamped by menstrual reflux, either because of the abundance of the reflux or because of defective scavenging systems (13). Several studies have suggested that the amount of retrograde menstruation may be related to endometriosis (13). Shorter cycles (46) and heavier and longer menstrual flow have been reported in women with endometriosis (47). Moreover, in patients in whom the disease develops, bleeding from red endometrial lesions may further increase the number of erythrocytes. Data are lacking on how the peritoneal environment copes with the presence of erythrocytes. However, cellular response is probably similar to that observed in most tissues during hemorrhage. Some studies indicate that amounts of hemoglobin and its by-products, as well as proteins involved in their scavenging or catabolism, are increased in patients with endometriosis. Increased levels of hemoglobin have been found in the peritoneal fluid of patients with pelvic endometriosis compared with disease-free controls (48). Studies by Piva and Sharpe-Timms (49) and Sharpe-Timms et al. (50) suggested 864 Van Langendonckt et al. Oxidative stress and endometriosis Vol. 77, No. 5, May 2002

5 that ectopic endometrial cells react by synthesizing and secreting endo-1, a haptoglobin-like protein that is closely related to the hemoglobin scavenger haptoglobin. These investigators recently confirmed that more endo-1 protein is localized in endometriotic lesions and eutopic endometrium of women with endometriosis than in eutopic endometrium of controls (51). The fact that infertile patients with or without endometriosis have reduced serum levels of antibodies against a haptoglobin-like protein corroborates these findings (52). These articles discuss the potential role of haptoglobin as an angiogenic or immunomodulatory factor. Further studies are needed to assess whether endo-1 binds hemoglobin. In contrast, Dunselman et al. (53) found no increase in haptoglobin in the peritoneal fluid of women with endometriosis. Catabolism of hemoglobin involves its degradation into its protein component and nonprotein core, heme. Most cells protect themselves from heme toxicity by rapid expression of scavenger proteins, such as hemopexin, and by induction of heme oxygenase-1, which catalyzes heme degradation (54). All products of heme catabolism are biologically active. These products include iron; carbon monoxide acting as a signal molecule; and biliverdin, which is further converted into bilirubin, both biliverdin and bilirubin acting as antioxidants. Van Langendonckt et al. (55) showed that endometrial cells express constitutive heme oxygenase-2 and inducible heme oxygenase-1 and that endometrial lesions, especially red lesions, strongly express inducible heme oxygenase-1 (48). However, the fact that heme oxygenase-1 was poorly expressed in macrophages and mesothelial cells and that levels of bilirubin, the final by-product of hemoglobin catabolism, in peritoneal fluid were not increased may suggest that detoxifying systems are overwhelmed by excess hemoglobin in endometriosis (48). This idea is in accordance with the findings of Gaulier et al. (56), who reported the presence of bilirubin deposits around endometriotic lesions and an absence of deposits in the mesothelium in a patient with endometriosis. Evidence suggests that iron overload occurs in the peritoneal cavity of patients with peritoneal endometriosis. Higher levels of iron are found in the peritoneal fluid of patients with endometriosis; concentrations are related to the severity of the disease (41, 57). Moreover, iron pigment is more frequently found in endometriotic than in nonendometriotic lesions (58). Most iron is likely to be stored bound to proteins in a soluble nontoxic form. Levels of ferritin, the major iron-storing molecule (59), and transferrin, which ensures iron transport (60), were found to be increased in the peritoneal fluid of women with endometriosis. This is consistent with the finding that patients with endometriosis have higher serum levels of antibodies to transferrin (61). As in most tissue, activated macrophages probably play an important role in the degradation of erythrocytes. The presence of siderophages, known as iron-storing macrophages, is considered to indicate endometriosis (56, 62). Macrophages usually internalize and lyse senescent erythrocytes, releasing hemoglobin and ensuring its degradation by heme oxygenase. The iron released is then returned to the iron transporter, transferrin (42). Martinez-Roman et al. (63) found increased transferrin receptor expression in peritoneal macrophages of patients with endometriosis compared with fertile and infertile controls. This further supports the hypothesis of increased iron metabolism by macrophages in endometriosis. However, Becker et al. (64) found no increase in transferrin receptor expression (CD71) in macrophages of patients with endometriosis. Macrophages, endometriotic lesions, and peritoneal cells of patients with endometriosis show typical features of ironoverloaded tissue; specifically, they are heavily laden with hemosiderin (56, 58, 62). Several authors have suggested that hemosiderin is formed when abundant ferritin cores come into close contact after partial digestion of their protein coat by lysosomal-siderosome enzymes (42, 65). In patients with endometriosis, the presence of tissue iron deposits in the peritoneum appears to be related to the presence of endometriotic lesions, since the rate of deposits encountered was higher in peritoneum close to a lesion than in normallooking peritoneum (59). Taken together, these studies suggest that iron homeostasis in the peritoneal cavity may be disrupted in women with endometriosis. Recruitment and Activation of Macrophages Murphy et al. (30) proposed a hypothesis based on analogies between endometriosis and arteriosclerosis to explain the occurrence of oxidative stress in the peritoneal cavity of patients with endometriosis. Both diseases share common features, including the presence of tissue macrophages that express scavenger receptors, increased levels of oxidized lipoproteins, and the presence of inflammatory cytokines and growth factors. Murphy et al. (30) suggested that senescent erythrocytes and apoptotic endometrial cells may be responsible for recruitment and activation of mononuclear phagocytes in the peritoneal cavity. These investigators also showed that the activation of macrophages in endometriosis patients is associated with an increase in scavenger receptor activity (31). The presence of scavenger receptors seems to be essential for clearance of oxidized low-density lipoproteins. Scavenger receptor activity appears to be induced in adherent macrophages only, as suggested by data showing the uncommon occurrence of such receptors in nonadherent macrophages (66). Nonadherent macrophages would generate oxidative stress; as a consequence, levels of lipid peroxides and oxidatively modified lipoproteins would further increase, triggering a cascade of FERTILITY & STERILITY 865

6 proinflammatory reactions involving recruitment of macrophages and secretion of proinflammatory cytokines (30). This hypothesis is supported by the fact that derivatives of low-density lipoprotein oxidation, such as lysophosphatidylcholine (levels of which are increased in patients with endometriosis), may act as a chemotactant for monocytes and induce secretion of cytokines (30, 33). Exposure to Toxicants Exposure to environmental toxicants, such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), may also promote oxidative stress in patients with developing endometriosis. Inhalation of airborne particles or ingestion of toxicants and heavy metals has been shown to induce oxidative stress and has been suggested as a contributory factor in the pathogenesis of several diseases (17, 67). Studies indicate that toxicants such as TCDD may also contribute to the development of endometriosis (67, 68). The potential role of TCDD in the pathophysiology of endometriosis may be based on its role as a disrupter of steroid action. However, other adverse effects of TCDD, including induction of oxidative stress (69), may also be involved. CONSEQUENCES OF INCREASED OXIDATIVE STRESS IN THE PERITONEAL CAVITY Decreased Fertility Most reports discuss the potential consequences of increased oxidative stress in endometriosis relative to decreased fertility. However, the association between endometriosis and infertility, especially in patients with mild disease, is still a matter of debate (70). Oxidative stress is thought to be one of the factors responsible for impaired fertility in patients with endometriosis (36). The peritoneal fluid of patients with endometriosis is toxic to sperm (57). This effect may be mediated by reactive oxygen species (71). This detrimental effect appears to be associated with concentrations of iron in peritoneal fluid (57). Reactive oxygen species and NO may also play a role in ovulation (72 75). Studies have shown that inhibition of reactive oxygen species (73) and NO production may impair ovulation (74, 75). The effects of oxidative stress on fertilization, preimplantation embryo development (76), and implantation (20) are well documented. Induction of Cellular Damage In other tissues, iron is known to induce oxidative stress, leading to macromolecular oxidative damage, tissue injury, and chronic inflammation (17). Oxidative stress was suggested to be responsible for local destruction of the peritoneal mesothelium, thereby creating adhesion sites for ectopic endometrial cells (41). It may also promote apoptosis (30); some studies indicate that apoptosis and oxidative stress are related (77). However, a recent study appears to show that morphologic changes in mesothelial cells induced by menstrual effluent are not due to necrosis or apoptosis (78). Induction of Adhesion and Growth of Ectopic Endometrial Cells Reactive oxygen species can also alter cell function by regulating protein activity and gene expression. In particular, reactive oxygen species seem to play an essential role in the regulation of the transcription factors NF- B and AP-1, which control genes involved in immunologic response, cellular defense, and expression of cytokines and cell adhesion molecules (79). Of note, many factors that are overexpressed in patients with endometriosis have NF- B binding sites in their genes (30) (Table 1). NF- B is a cytoplasmic protein that, when activated, is translocated into the nucleus, where it binds to B sequences of DNA and activates target genes (Fig. 2). Activation of NF- B involves release of its I B subunit, which is induced by cytokines, such as tumor necrosis factor- and interleukin-1; proinflammatory factors, such as heme; toxicants, such as dioxin; and factors that induce oxidative stress (Table 1). The potential activation of NF- B by oxidative stress is supported by many studies demonstrating that the addition of antioxidant prevents NF- B activation (79). Endometrial cells have been found to express NF- B and its activating kinases (80). Progesterone has been shown to increase levels of I- B and thereby inhibit NF- B activation (81). Thus, altered expression and lower biological activity of the progesterone receptor in ectopic endometrial cells (82 84) may result in NF- B activation. Expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin, in endometrial and peritoneal cells may also be induced by heme (44) and lysophosphatidyl choline, a byproduct of lipoprotein peroxidation (33). Ectopic endometrial cells have been shown to overexpress intercellular adhesion molecule-1 (4). Moreover, heme has been reported to induce activated macrophages of patients with endometriosis to produce cytokines, such as tumor necrosis factor-, interleukin-1, and interleukin-6 (85). Similarly, oxidized lipids and modified proteins might induce interleukin-1 and colony-stimulating factor-1 synthesis by macrophages and promote endometrial cell growth as pointed out by Murphy et al (33). CONCLUSIONS Compelling evidence suggests that oxidative stress is increased in women with pelvic endometriosis. Oxidative stress occurs when there is an imbalance between production of reactive oxygen species, which are intermediaries released by normal oxygen metabolism, and antioxidant systems, which are designed to control production of reactive oxygen species, inactivate reactive oxygen species, and repair cell 866 Van Langendonckt et al. Oxidative stress and endometriosis Vol. 77, No. 5, May 2002

7 TABLE 1 Molecules that are induced in endometriosis and contain an NF- B responsive sequence in their genes or are involved in NF- B activation. Molecule Regulation by NF- B Function Link with endometriosis a RANTES 87 Monocytes chemotactant 88 Monocyte chemoattractant protein 89 Monocytes chemotactant 90 Intercellular adhesion molecule 91 Adhesion 4 Vascular cell adhesion molecule 44 Adhesion 92 Heme oxygenase-1 93 Heme degradation 48 Urokinase plasminogen activator 94 Fibrinolysis 95 Inducible nitric oxide synthase 96 Nitric oxide production 28 Cyclooxygenase-2 97 Prostaglandin synthesis 98 Activators of NF- B Tumor necrosis factor- 88 Cytokine 99 Interleukin Cytokine 101 Integrin 102 Cell adhesion 3 Fibronectin 103 Cell adhesion 3 Dioxin 104 Toxicant 68 a Columns show reference numbers. Van Langendonckt. Oxidative stress and endometriosis. Fertil Steril FIGURE 2 Mechanism of NF- B activation by factors that have been implicated in the pathogenesis of endometriosis and induction of target genes. COX cyclooxygenase; IL interleukin; i-nos inducible nitric oxide synthase; TNF tumor necrosis factor. Van Langendonckt. Oxidative stress and endometriosis. Fertil Steril FERTILITY & STERILITY 867

8 damage. Production of reactive oxygen species appears to be increased in women in whom endometriosis is developing, as indicated by increased release of reactive oxygen species by macrophages; higher levels of oxidized low-density lipoproteins and their by-products in peritoneal fluid; and overexpression by endometrial cells of enzymes that produce reactive oxygen species, such as xanthine oxidase and nitric oxide synthase. Several studies indicate that antioxidant defenses may be altered in endometriosis, as suggested by aberrant expression of endometrial antioxidant enzymes and lower levels of the antioxidant vitamin E in peritoneal fluid. Further studies are warranted to assess the contribution of oxidative stress to the physiopathology of endometriosis. It would be particularly valuable to determine whether antioxidant treatment of endometriosis is effective. Use of mifepristone to treat endometriosis has had promising results (86). This agent has been shown to inhibit endometrial cell growth, an effect which appears to be due to its antioxidant properties. The data do not clearly indicate whether oxidative stress associated with pelvic endometriosis results from increased production of reactive oxygen species, defective antioxidant defense, or both conditions. Information is lacking on how the peritoneal environment, and particularly mesothelial cells, respond to the presence of blood, which repeatedly carried into the peritoneal cavity by retrograde menstruation, ovulation, and bleeding lesions. Expression of antioxidant enzymes by endometrial cells appears to be enhanced in patients with developing endometriosis. Some data seem to indicate that catabolism of hemoglobin by peritoneal macrophages and release of proteins that chelate iron in peritoneal fluid are also increased in endometriosis. However, the occurrence of oxidative stress suggests that despite this increase in antioxidant defense, detoxifying mechanisms might be overloaded in endometriosis. Reactive oxygen species have also been increasingly studied as regulators of protein activity and inducers of gene expression (76). As a potential inducer of NF- B, which activates genes involved in cell adhesion, secretion of inflammatory cytokines, and recruitment of macrophages, oxidative stress may help to trigger the chain of events that leads to the development of endometriotic lesions. Further studies are needed to examine the potential involvement of oxidative stress in the pathophysiology of endometriosis. References 1. Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927;14: Koks CAM, Demir-Weusten AY, Groothuis PG, Dunselman GAJ, de Goeij AFPM, Evers JLH. Menstruum induces changes in mesothelial cell morphology. 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