Role of cytokines in endometriosis

FERTILITY AND STERILITY威 VOL. 76, NO. 1, JULY 2001 Copyright ©2001 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

Role of cytokines in endometriosis Tasuku Harada, M.D., Tomio Iwabe, M.D., and Naoki Terakawa, M.D. Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago, Japan

Objective: To review the literature on the role of cytokines in the pathogenesis of endometriosis and endometriosis-associated infertility. Design: Pertinent studies were identified by a computer search of MEDLINE. References of selected articles were hand-searched for additional citations. Result(s): Recent studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages that secrete various local products, such as growth factors and cytokines. Levels of several cytokines were reported to be elevated in the peritoneal fluid of women with endometriosis. Because the peritoneal environment may be controlled by locally regulated factors, cytokines are believed to play a role in the development and progression of endometriosis and endometriosis-associated infertility. A possible pathogenic mechanism links cytokines with endometriosis. Conclusion(s): Cytokines, which are produced by many cell types including endometriotic tissues, play diverse roles in the pathogenesis of endometriosis and endometriosis-associated infertility. More studies about the specific role of these cells and soluble factors are needed to improve understanding of endometriosis and to develop novel therapies. (Fertil Steril威 2001;76:1–10. ©2001 by American Society for Reproductive Medicine.) Key Words: Cytokine, macrophage, endometriosis, peritoneal fluid

Received December 3, 2000; revised and accepted January 29, 2001. Reprint requests: Tasuku Harada, M.D., Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 6838504, Japan (FAX: 81-85834-8089). 0015-0282/01/$20.00 PII S0015-0282(01)01816-7

Endometriosis, a common disease among women of reproductive age, is characterized by the presence and growth of endometrial tissue (glands and stroma) outside the uterus. Dysmenorrhea and infertility, which are common symptoms of endometriosis, compromise quality of life. Despite a long history of clinical experience and experimental research, endometriosis remains an enigma and its pathogenesis is still controversial.

Studies have reported elevated levels of several cytokines in the peritoneal fluid of women with endometriosis, thus implicating these cytokines in the development and progression of endometriosis and endometriosis-associated infertility. We review the current understanding of the role of cytokines in the pathogenesis of endometriosis and endometriosis-associated infertility.

Endometriosis is associated with infertility even among affected women who ovulate and have anatomically patent fallopian tubes. The exact mechanism by which endometriosis interferes with fertility is not known, but data suggest that an aberrant immunologic mechanism is involved in its pathophysiology. An important general concept is that endometriosis is a local pelvic inflammatory process with altered function of immune-related cells in the peritoneal environment. Supporting this concept are recent studies suggesting that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages that secrete various local products, such as growth factors and cytokines.

Peritoneal fluid containing immune-related cells is often seen in the vesicouterine cavity or the pouch of Douglas during gynecologic surgery and bathes the pelvic cavity, uterus, fallopian tubes, and ovaries. It may be a major factor controlling the peritoneal microenvironment that influences the development and progression of endometriosis and endometriosisassociated infertility.

Peritoneal Fluid

Fluid Volume and Cellular Components Peritoneal fluid volume and its components depend on follicular activity, corpus luteum vascularity, and hormonal production. The volume of peritoneal fluid in the peritoneal cavity 1

varies during the menstrual cycle, reaching a peak of 20 mL at the time of ovulation (1). Changes in fluid volume and the presence of various cells, hormones, and other compounds during normal menstrual cycles and in pathologic conditions have been described (2). Syrop and Halme (3) analyzed peritoneal fluid volume in 426 patients and found that women with endometriosis had a greater peritoneal fluid volume than fertile controls, patients with adhesive disease, or those with unexplained infertility. The volume of peritoneal fluid in women with unexplained infertility was also higher than that in controls. Therefore, an increased volume of peritoneal fluid may be commonly associated not only with endometriosis but also with long-term unexplained infertility. Peritoneal fluid contains various free-floating cells, including macrophages, mesothelial cells, lymphocytes, eosinophils, and mast cells. Normally, peritoneal fluid contains leukocytes in concentrations of 0.5 to 2.0 ⫻ 106/mL, of which approximately 85% are macrophages (2). Halme et al. (4) postulated that peritoneal macrophage activation may be a central contributor to the pathogenesis of endometriosis. Activated macrophages in the peritoneal cavity of women with endometriosis (5) are potent producers of cytokines (6 – 8). Thus, peritoneal fluid contains a rich cocktail of cytokines.

Cytokines in the Peritoneal Fluid Cytokines play a major role in the initiation, propagation, and regulation of immune and inflammatory responses. Immune cell activation results in a burst and cascade of inflammatory cytokines. These cytokines have pleiotropic and redundant activities that culminate in recruitment of numerous cell types to the site of inflammation. A highly sensitive enzyme-linked immunosorbent assay has made it possible to measure several cytokines in the peritoneal fluid of women with endometriosis. These cytokines include interleukin (IL)-1 (9); IL-4 (10); IL-5 (11); IL-6 (12–16); IL-8 (17–19); IL-10 (15, 20); IL-12 (21, 22); IL-13 (23); interferon-␥ (14); tumor necrosis factor (TNF)-␣ (9, 16); regulated on activation, normal T cell expressed and secreted (RANTES) (24); monocyte chemotactic protein-1 (MCP-1) (25–27); macrophage colony stimulating factor (MCSF) (28); transforming growth factor (TGF)-␤ (29); and vascular endothelial growth factor (VEGF) (30, 31). Several studies have reported that the level of many cytokines is increased in the peritoneal fluid of women with endometriosis. Cytokines may regulate the actions of leukocytes in the peritoneal fluid or may act directly on ectopic endometrium, where they may play various roles in the pathogenesis and pathophysiology of endometriosis.

Sources of Cytokines Increased levels of cytokines in the peritoneal fluid of women with endometriosis may reflect increased synthesis of cytokines by peritoneal macrophages, lymphocytes, ec2

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Endometriosis and cytokines

topic endometrial implants, or mesothelial cells of the peritoneum, all of which can produce cytokines (32, 33). The main source of cytokines is thought to be the macrophages, which originate in bone marrow, circulate as monocytes, and migrate to various body cavities. Chemoattractants, including MCP-1, RANTES, and IL-8, facilitate recruitment of macrophages into the peritoneal cavity. T lymphocytes are also implicated in the pathogenesis of endometriosis. T-helper cells can be classified into two subsets: type 1 (Th1) and type 2 (Th2). Th1 cells produce IL-2, IL-12, and interferon-␥, which are potent inducers of cellmediated immunity. Th2 cells produce mainly IL-4, IL-5, IL-10, and IL-13, which are involved in suppression of cell-mediated immunity. Hsu et al. (10) investigated the expression of Th1 (IL-2 and interferon-␥) and Th2 (IL-4 and IL-10) cytokines in peripheral blood monocytes and peritoneal fluid from patients with endometriosis. They found that cytokine secretion by Th1 and Th2 cells is altered in women with endometriosis. The shift in the balance of Th1 and Th2 cells toward the Th2 arm may contribute to the derangement of an immunologic defense mechanism in endometriosis. Recent studies suggest that endometriotic implants also produce cytokines (34 –36). Interleukin-6 is secreted from many types of cells, including endometrial epithelial cells (37), macrophages (the main source of IL-6 in peritoneal fluid), and other leukocytes (14, 38). Recently, Tsudo et al. (36) demonstrated that endometriotic cells constitutively express IL-6 messenger RNA and produce IL-6 protein and that adding TNF-␣ stimulated IL-6 gene and protein expression in a dose-dependent manner. When they compared IL-6 production by macrophages and endometriotic stromal cells in patients with endometriosis, they found that similar levels of IL-6 were produced in stromal cells derived from an endometrioma and by macrophages under basal- and TNF␣–stimulated conditions. These findings suggest that endometriotic tissue may be another important source of this cytokine (36).

PATHOGENESIS Plausible Hypothesis The pathogenesis of endometriosis is still in question, despite extensive research efforts since Sampson’s landmark article in 1927 (39). Sampson’s theory of retrograde menstruation, which describes endometrial cells that may attach, implant, and grow, seems plausible because peritoneal lesions are most frequently found in the ovaries and the posterior cul-de-sac, where regurgitated menstrual material pools (40). Furthermore, retrograde menstruation is a common physiologic condition in women with patent tubes (41), and viable endometrial cells can be recovered from the peritoneal fluid during menstruation (42, 43), suggesting an obvious route by which endometrial cells are introduced into the peritoneal cavity. Vol. 76, No. 1, July 2001

FIGURE 1 Factors involved in the pathogenesis of endometriosis.

Those investigators postulated that metaplasia of the invaginated coelomic epithelium caused ovarian endometriosis. The rectovaginal endometriotic nodule is an adenomyotic nodule whose histogenesis is related to the metaplasia of mu¨llerian remnants located in the rectovaginal septum.

The Role of Cytokines in Pathogenesis Implantation Although retrograde menstruation occurs in about 90% of cycling women, endometriosis is diagnosed in only about 10% of women. This discrepancy raises questions about features of refluxed eutopic endometrium in patients with endometriosis, which may have distinct features for adherence or proliferation at the ectopic sites. Recent studies have shown increased IL-6 production by endometriotic cells in both basal and cytokine-stimulated conditions compared with normal conditions (34 –36). Tseng et al. (35) examined eutopic endometrium from patients with endometriosis and found an increased basal- and IL-1␤stimulated production of IL-6 compared with patients without endometriosis. This is an important avenue of future investigation because it suggests that the endometrial cells of women who develop endometriosis may function differently from those in women who do not develop this condition.

Harada. Role of cytokines in edometriosis. Fertil Steril 2001.

Two studies (44, 45) demonstrated that endometriosis can be induced by exposing the pelvis to large amounts of menstrual discharge. Another study showed that endometrium can attach to the mesothelial surface of the peritoneum in vitro (46). The investigators observed that in all cases of adhesion to intact mesothelium, the endometrium was attached through stromal cells. Another theory, the metaplasia theory, is also attractive because it explains some rare cases of endometriosis, such as in patients with no menstruation (the Rokitansky-Kuster-Hauser syndrome) (47). A recent in vitro study supports the metaplasia theory (48). Unfortunately, neither theory can explain all cases of endometriosis (Fig. 1). A recent study proposed that three types of endometriotic lesions—peritoneal, ovarian, and rectovaginal—should be considered separate entities, each with a different pathogenesis (49). The implantation theory can explain peritoneal endometriosis, in which the different aspects (black, red, and white) represent distinct steps in the evolutionary process. FERTILITY & STERILITY威

To implant and grow, endometrial cells must establish cell– cell or cell– extracellular matrix interactions with the peritoneal lining. In this regard, cell adhesion molecules are of great importance (50). A recent report clearly showed that endometrial stromal cells are the critical cells in endometrial attachment to the mesothelial surface of the peritoneum and that endometrial epithelial cells fail to attach to the mesothelium (45). Most of these interactions between endometrial cells and extracellular matrix are mediated by the integrin family of cell surface receptors, which can transduce intracellular signals. It has also been suggested that cellular adhesion itself stimulates chemokine expression (51). Garcia-Velasco and Arici (52) showed that increasing the dose of IL-8 stimulates the ability of endometrial stromal cells to adhere to an extracellular matrix protein, fibronectin. They also showed that the adhesion of endometrial stromal cells to different extracellular matrix proteins induces variable levels of IL-8 gene expression and protein secretion and that this event is integrin-mediated (53). Interleukin-8 may play a role in the attachment of endometrial implants in the pathogenesis of endometriosis. According to Sampson’s theory of retrograde menstruation, deficient cellular immunity, in particular impaired natural killer (NK) cell function, is one of the etiologic factors that may contribute to survival and implantation of refluxed endometrial cells. Several investigators have found decreased NK cell activity in the peritoneal fluid of women with endometriosis compared with women without endometriosis (54 –56). This observation suggests that the clearing 3

mechanism of retrograde menstruated endometrial cells may be impaired in women with endometriosis because of a defect in the local immune defense system. Oosterlynck et al. (29) found increased TGF-␤ activity in the peritoneal fluid of women with endometriosis. Transforming growth factor-␤ may be a cytokine that inhibits NK activity in the peritoneal fluid of women with endometriosis (29). Intercellular adhesion molecule (ICAM)-1–mediated cell– cell adhesion is essential for various immunologic functions, including NK cell–mediated cytotoxicity against endometrium. Recently Somigliana et al. (57) reported that soluble ICAM-1 was constitutively shed from the surface of endometrial stromal cells obtained from patients with endometriosis into the culture medium. The enhanced release of soluble ICAM may allow the endometrial stromal cells of patients with endometriosis to escape immunosurveillance and, therefore, to implant in ectopic sites. More interesting, soluble ICAM-1 production from the macrophages of patients with endometriosis was up-regulated by interferon-␥ and IL-6 (58). Interleukin-12 acts on T and NK cells, inducing cytokine production (primarily interferon-␥), enhancing NK cell cytotoxic activity, and favoring generation of Th1 cell response (59, 60). Concentrations of IL-12 in the peritoneal fluid are low, but detectable, regardless of the presence or absence of endometriosis (21). Administration of IL-12 significantly prevented ectopic endometrial implantation in a murine model of endometriosis (61). A direct growth inhibitory effect on endometrial cells seems unlikely because endometrial cells do not express receptors for IL-12. A potential explanation for these results is that IL-12 enhances the growth and augments the cytolytic activity of both NK and T cells. These data support the idea that manipulation of cytokine activity in the peritoneal fluid is a novel management approach to controlling the establishment of endometriosis.

Angiogenesis, which is the process of generating new capillary blood vessels, occurs in a variety of normal and pathologic processes. In angiogenesis, the basement membrane is dissolved by protease derived from vascular endothelial cells, endothelial cells migrate and proliferate, and the capillary tube forms (62). Each of these steps is regulated by various angiogenic factors. Neovascularization is probably required for the implant to grow larger than 2 to 3 mm3 during tumor growth (63). An angiogenic mechanism may be involved in the pathogenesis of endometriosis. We can postulate that further outgrowth of ectopic endometrial implants will depend on new capillary growth, on the basis of several studies indicating that tumors are angiogenesis-dependent (64). Vascular endothelial growth factor (VEGF) is a heparinbinding growth factor of 30 – 46 kDa that is active as a Harada et al.

McLaren et al. (31) demonstrated that peritoneal fluid macrophages are the principal source of the angiogenic growth factor VEGF and that anti-VEGF antibody abolished the enhanced endothelial cell proliferation induced by conditioned medium from macrophages isolated from the peritoneal cavity of women with endometriosis. These findings suggest that activated macrophages are a major source of VEGF in endometriosis and that estradiol and progesterone directly regulate this expression (31). Since endometriosis is characterized by pronounced vascularization in and around the ectopic tissue, elevated levels of the potent angiogenic growth factor VEGF in the peritoneal fluid and the presence of VEGF-positive macrophages in the ectopic tissue are clinically important in this disease. Vascular endothelial growth factor–induced angiogenesis may therefore be a critical aspect of the pathophysiology of endometriosis. Interleukin-8, a chemoattractant for neutrophils and an angiogenic agent, induces proliferation of human melanoma and glioma cells (68, 69). Arici et al. reported that IL-8 is produced in the human endometrium in vivo, mainly in glandular cells (70) and that IL-8 induces proliferation of endometrial stromal cell as a potential autocrine growth factor (71). Iwabe et al. (19, 72) demonstrated that IL-8 exerts its growth-promoting actions in endometriotic as well as in normal endometrial cells. Tumor necrosis factor-␣, a secretory product of activated macrophages and a potent inducer of new blood vessel growth (73), also stimulates proliferation of endometriotic stromal cells (72). These angiogenic cytokines may play a role in the angiogenesis of endometriosis. Progression and Infiltration

Angiogenesis

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disulphide-linked homodimer (65) and is a potent mitogen, morphogen, and chemoattractant for endothelial cells (65, 66). The angiogenic activity of peritoneal fluid, as well as levels of VEGF in peritoneal fluid, are elevated in women with endometriosis (30, 67).

Endometriosis and cytokines

Surrey and Halme (74) demonstrated a direct stimulatory effect of the cell-free fraction of peritoneal fluid samples from patients with endometriosis on the proliferation of normal uterine endometrial cells in short-term culture, indicating that factors within the peritoneal fluid are involved in the progression of endometriosis. Several cytokines, such as IL-8 and TNF-␣, have growth-promoting effects on endometrial and endometriotic cells (19, 71, 72). These findings suggest that an elevated peritoneal fluid level of cytokine promotes the progression and spread of endometriotic implants in the peritoneal cavity. Iwabe et al. (19) found that peritoneal fluid levels of IL-8 significantly enhanced proliferation of stromal cells derived from ovarian endometriomas. Expression of IL-8 receptor type A messenger RNA was detected in endometriotic stromal cells (Fig. 2). These results suggest that IL-8 may promote the progression of endometriosis. Iwabe et al. (72) Vol. 76, No. 1, July 2001

FIGURE 2 Reverse transcriptase polymerase chain reaction determination of interleukin (IL)-8 receptor messenger RNA expression in endometrial and endometriotic stromal cells. (A), Ethidium bromide-stained agarose gel analysis of the amplified products. Lanes 1 and 3 are endometrial stromal cells; lanes 2 and 4 are endometriotic stromal cells. HaeIII-digested X174 size markers were used in the left lane (lane M). (B), The specificity of polymerase chain reaction products was confirmed by hybridization of oligonucleotide probe. Lane 1 is endometrial stromal cells, and lane 2 is endometriotic stromal cells. Glycerol-3-phosphate dehydrogenase (G3PDH) was used as a positive control.

Harada. Role of cytokines in edometriosis. Fertil Steril 2001.

also tested the hypothesis that elevated levels of TNF-␣ in peritoneal fluid of patients with endometriosis may contribute to the progression of endometriosis by inducing production of IL-8. Gene and protein expression of IL-8 in the stromal cells of endometriotic tissues are up-regulated by TNF-␣ (72), and TNF-␣ stimulated the proliferation of the endometriotic stromal cells. This stimulatory effect of TNF-␣ was abolished by adding anti-TNF-␣ antibody or anti-IL-8 antibody. Therefore, TNF-␣ may act on stromal cells by mediating the proliferative effects of IL-8. Expression of type I and type II receptors for TNF-␣ was observed in endometriotic stromal cells (Fig. 3). This evidence suggests that TNF-␣ action mediated by IL-8 may not only be an initiating factor that facilitates adhesion of endometrial cells to the peritoneum but may also contribute to development and progression of endometriosis. Harada et al. (16) and Iwabe et al. (19) found that the extent of superficial red endometriotic lesions was related to increased levels of IL-6, IL-8, and TNF-␣ in the peritoneal fluid. Red lesions, such as red flame-like lesions, gland-like lesions, and red vesicles, were classified as active lesions of endometriosis because angiogenesis is more pronounced in red lesions than in black or white lesions (75) and because early red lesions invade extracellular matrix (76). Thus, cytokines may play a role in the early stage of endometriosis. P-450 aromatase is an enzyme complex that catalyzes the formation of estrogens from C-19 androgenic steroids. Analysis using a sensitive RT-PCR technique revealed that eutopic endometrium and normal pelvic peritoneum of patients without endometriosis did not express P-450 transcripts. In FERTILITY & STERILITY威

contrast, the eutopic endometrium and peritoneal implants of patients with endometriosis did express the transcripts (77). The results suggest that localized estrogen production, acting in an autocrine or paracrine fashion, may promote the growth of the implants. More interesting, IL-6 family cytokines, such as IL-6, IL-11, leukemia inhibitory factor, and oncosta-

FIGURE 3 Reverse transcriptase polymerase chain reaction revealed tumor necrosis factor receptor (TNFR)-I and -II messenger RNA expression in endometriotic cells. The U937 cell lines (lymphoma) were used as a positive control for tumor necrosis factor receptors. Lane 1 is U937 cells, lane 2 is endometriotic stromal cells, and lane 3 is reverse transcriptase (⫺). Glycerol-3-phosphate dehydrogenase (G3PDH) was used as a positive control (data not shown).

Harada. Role of cytokines in edometriosis. Fertil Steril 2001.

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TABLE 1 Proposed mechanism of endometriosis-associated infertility. 1. Impaired function of ovary and fallopian tube Anatomical distortion and tubal obstruction Ovulation failure Impaired follicle development Luteinized unruptured follicle Hyperprolactinemia Luteal phase defect 2. Immunologic defect Autoimmunity Antiendometrial antibody 3. Altered characteristics of peritoneal fluid Activated macrophages Prostaglandins Cytokines Harada. Role of cytokines in edometriosis. Fertil Steril 2001.

tin M, were shown to be potent stimulators of aromatase expression in adipose stromal cells in culture (78). Recently, Yoshioka et al. (79) reported that IL-6 inhibits proliferation of endometrial stromal cells derived from the secretory phase but not from the proliferative phase. In contrast, negative regulation by IL-6 was not observed in the stromal cells of endometriotic tissues, suggesting that the biological characteristics of endometriotic cells differ from those of eutopic endometrial cells.

INFERTILITY Pelvic endometriosis is frequently associated with infertility, even if affected women ovulate and have functional, patent tubes. A conservative estimate suggests that about 20% to 25% of infertile women have endometriosis, compared with 2% to 5% of women undergoing tubal ligation (most of whom have proven fertility) (80). Even in its early stages, before adhesion or anatomic distortion take place, endometriosis has been associated with infertility. The exact mechanism by which endometriosis interferes with fertility is not fully understood, although many possible causes have been suggested (Table 1). Several studies have suggested that ovulatory dysfunction contributes to infertility in women with minimal endometriosis (81). The association between the luteinized unruptured follicle syndrome and endometriosis was described in women (82– 84) and primates (85). In women with the luteinized unruptured follicle syndrome, steroid hormone concentrations in peritoneal fluid are much lower after the ovulatory cycle (86). It was suggested that this lower steroid environment may facilitate development of endometriosis. Hyperprolactinemia and luteal phase defects have also been proposed as a possible cause of subfertility in endometriosis (87, 88), but the issue remains controversial. 6

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Endometriosis and cytokines

Several lines of clinical evidence from IVF and oocyte donation programs have led to the hypothesis that follicular development may be impaired in patients with endometriosis (89 –91). One recent study suggested that patients with endometriosis have a defect in granulosa cell steroidogenesis, which could affect oocyte function and explain reduced rates of fertilization in endometriosis (89). At least two recent studies have demonstrated reduced pregnancy rates in IVF cycles in women with minimal and mild endometriosis (90, 91). The reasons proposed for this reduction were impaired oocyte quality (90) and defects in implantation (91). One study aimed to determine the possibility of subtle oocyte or sperm dysfunction contributing to the reduced fertilization rates associated with minimal and mild endometriosis in couples undergoing donor insemination (92). The results suggest that the reduction in natural fertility associated with endometriosis appears to be due at least in part to reduced potential for fertilization of the oocyte. Gleicher et al. (93) proposed that endometriosis is associated with autoimmune disease. They noted that a significant proportion (40% to 60%) of women with endometriosis has elevated autoantibody titers when tested against a panel of common autoantibodies (phospholipid, ribonucleoprotein, and double-strand DNA). Autoantibodies have been reported to interfere with various processes during human reproduction, including sperm function, fertilization, and normal progress of pregnancy (93). These lines of evidence may all be partially involved in subfertility in endometriosis. Cytokines are now believed to be related to infertility in women with endometriosis. Since the ovaries and fallopian tubes are bathed in peritoneal fluid, substances present in peritoneal fluid may greatly affect reproductive function by altering tubal motility, ovum pick-up, or ovulation. Oocytes are exposed to the peritoneal environment even after they are captured by the fimbriae, and spermatozoa are present in the peritoneal fluid after intercourse. Therefore, gametes and early embryos are exposed to peritoneal fluid, which may influence early reproductive process.

Peritoneal Fluid and Infertility Peritoneal fluid contains many types of cells. Macrophages are primarily responsible for phagocytosis of cellular debris, including sperm, in the pelvis. Muscato et al. (94) demonstrated that peritoneal macrophages phagocytized sperm in vitro and that macrophages from women with endometriosis were more active than those from women without the disease. Peritoneal fluid diffusing into the tubal and endometrial environment may affect sperm and their interaction with the oocyte. Studies showed that the peritoneal fluid of patients with endometriosis has detrimental effects on sperm function. Sperm motility (95, 96), acrosome reaction (97), gamete interaction (98), and ovum capture by tubal fimbriae (99) have been studied. Aeby et al. (100), using a hamster peneVol. 76, No. 1, July 2001

tration assay, recently showed that peritoneal fluid from patients with endometriosis impaired gamete interaction. In their study, the mean number of eggs penetrated by sperm mixed with peritoneal fluid from patients with endometriosis was significantly less than that observed in controls. These data suggest that substances in the peritoneal fluid of patients with endometriosis contribute to infertility by impairing sperm function. The effects of peritoneal fluid on development of the preimplantation embryo have also been examined in a murine model. Although several investigators showed that peritoneal fluid obtained from patients with endometriosis had adverse effects on mouse embryonic development in vitro (9, 101–103), others have found that peritoneal fluid had no adverse effects (104). The reason for these contradictory findings is unclear, but peritoneal fluid from patients with endometriosis has frequently been shown to be toxic to the preimplantation embryo. A study demonstrated that medical treatment of endometriosis eliminated the embryotoxicity of the peritoneal fluid (9). In this study, the levels of IL-1 and TNF-␣ were markedly reduced in the peritoneal fluid of women who had undergone medical treatment (danazol or intranasal buserelin for 4 to 6 months) of endometriosis. This finding supports the hypothesis that increased levels of cytokines in peritoneal fluid may be involved in the pathogenesis of endometriosis-associated infertility.

FIGURE 4 The role of peritoneal fluid and cytokines in the pathophysiology of endometriosis-associated infertility. Accumulated data suggested that substances found in the peritoneal fluid of patients with endometriosis contribute to infertility by impairing embryo development, egg penetration by sperm, and sperm function.

Harada. Role of cytokines in edometriosis. Fertil Steril 2001.

Cytokines and Infertility It is speculated that a substance or substances from endometriotic tissues enter the peritoneal fluid and interfere with the reproductive process. However, few data have demonstrated any direct effect of specific elements of the peritoneal fluid on reproductive function. One study demonstrated that the addition of human recombinant IL-6 to culture medium suppressed the rate of blastocyst formation of mouse embryos (105), suggesting that increased IL-6 in the peritoneal fluid of endometriosis patients may contribute to infertility by adversely affecting embryonic development.

plants, which can produce various cytokines, may contribute to reduced fecundity in patients with endometriosis. The role of peritoneal fluid and cytokines in the pathophysiology of endometriosis-associated infertility is shown in Figure 4. However, data on cytokines and their role in infertility are still incomplete, and future investigation that can reveal the critical roles of cytokines is still needed.

Levels of cytokines, such as TNF-␣, IL-6, and IL-8, have been shown to be increased in the peritoneal fluid of women with endometriosis (16, 19). Levels of these cytokines are also correlated with the extent of red lesions. Cytokine production by these early lesions has not yet been evaluated, but IL-6 is known to be produced by endometriotic stromal cells derived from ovarian chocolate cysts (36). Early lesions are likely to be producers of these cytokines.

Cytokines, which are produced by many cell types in peritoneal fluid, play a diverse role in constructing the peritoneal environment that induces the development and progression of endometriosis and endometriosis-associated infertility. Intense basic research into the specific role of these cells and soluble factors may improve our understanding of endometriosis and result in novel therapies for endometriosis.

The causal link between endometriotic lesions and infertility is debatable, as is the value of resecting or ablating them to treat infertility. Convincing evidence emerged from a recent randomized clinical trial of 341 infertile patients with minimal or mild endometriosis in which diagnostic laparoscopy alone was compared with resection or ablation of visible lesions (106). Surgery was found to enhance fecundity. These findings suggest that endometriotic im-

Acknowledgement: The authors thank Professor Yasunori Yoshimura, M.D., Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, for critical reading of this manuscript and helpful comments.

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CONCLUSIONS

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