**3. Cytokynes (IL-1, IL-6, IL-8, TNF- a)**

Cytokines are small cell-signaling protein molecules that are secreted by several cells types and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin (Gilman et al., 2001). Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages (many near the interface with the external environment) are potent producers of IL-1, IL-6, and TNF-α (Boyle, 2005) .Studies have reported elevated levels of several cytokines in the peritoneal fluid of women with endometriosis, thus implicating these proteins in the development and progression of endometriosis and endometriosis-associated infertility (Koninckx et al.,1998); (Harada et al.,2001); (Bedaiwy et al., 2003); (Kalu et al., 2007). Peritoneal fluid is derived from plasma transudate and ovarian exudates and in a small part from secretions of the mesothelial surface and tubal luminal fluid. Some studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages and other immune cells that secrete various local products, such as growth factors and cytokines, which exert a paracrine action on endometriotic cells. (Harada et al., 2001)

endometriosis cells. The virus induces apoptosis in endometriotic cells in vitro (Rein DT. Et al, 2010). Ad VEGFE1 allowed specific replication and efficient killing of endometriotic cells. Another approach, which has been recently been published (Essam –Eldin R et al, 2008) is the transfection of endometriotic cells by dominant negative estrogen receptor gene via Ad vector. Dominant negative mutants of the estrogen receptor are altered estrogen receptors forms that are unable to activate transcription of estrogen-responsive genes when estradiol

Human endocrine gland derived vascular endothelial growth factor is a secreted angiogenetic mitogen growth factor expressed in the steroidogenic glands, ovary, testis, adrenal and placenta. It induces proliferation, migration and fenestration (formation of membrane discontinue) in capillary endothelial cells derived from endocrine glands. Human EG-VEGF is a 9.6 kDa protein consisting of 86 amino acid residues. Endocrine gland-derived VEGF (EG\_VEGF) belongs to the prokineticin family. It is also known as prokineticn 1 (PK1). Although EG- VEGF is structurally distinct from VEGF, they induce similar angiogenic response in the ovary. The EG- VEGF acts through G-protein couplesd

EG-VEGF was found to be expressed in non-endocrine tissues including endometrium: in human, it is highly expressed during the secretory phase of the menstrual cycle, when angiogenesis occurs. Lee et al evaluated the expression of EG-VEGF and its receptors in eutopic and ectopic endometrial tissues. A significant increase in molecule expression was found in the stromal cells of ectopic endometrium. It is possible that the stromal cells may synthesize EG-VEGF or that it is synthesize in the epithelial cells but is accumulated in the extracellular matrix of stroma. The endocrine gland-vascular endothelial growth factor, through its heparin- binding domain can, as VEGF, accumulated in the extracellular matrix

Cytokines are small cell-signaling protein molecules that are secreted by several cells types and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin (Gilman et al., 2001). Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages (many near the interface with the external environment) are potent producers of IL-1, IL-6, and TNF-α (Boyle, 2005) .Studies have reported elevated levels of several cytokines in the peritoneal fluid of women with endometriosis, thus implicating these proteins in the development and progression of endometriosis and endometriosis-associated infertility (Koninckx et al.,1998); (Harada et al.,2001); (Bedaiwy et al., 2003); (Kalu et al., 2007). Peritoneal fluid is derived from plasma transudate and ovarian exudates and in a small part from secretions of the mesothelial surface and tubal luminal fluid. Some studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages and other immune cells that secrete various local products, such as growth factors and cytokines,

which exert a paracrine action on endometriotic cells. (Harada et al., 2001)

binds them, resulting in decreased cell proliferation and increased apoptosis.

receptors, pkr1.

(Lee K et al, 2010).

**3. Cytokynes (IL-1, IL-6, IL-8, TNF- a)** 

**2.2 Endocrine Gland derived Vascular Endothelial Growth Factor (EG-VEGF)** 

#### **3.1 Cytokines and pathogenesis of endometriosis**

Studies have shown the role of some cytokines in the implantation of ectopic endometrial tissue, and its progression and infiltration. In the implantation and growth of ectopic tissue, a primary role was attributed to several cytokines contained in the peritoneal fluid including interleukin IL-1,IL-6, IL-8, IL-12 and tumor necrosis factor-a (TNF-a) (Arici et al.,1996); (Iwabe et al., 1998); (Ho et al.,1997) Normally, peritoneal fluid contains leukocytes in concentrations of 0.5 to 2.0 3 106/mL, of which approximately 85% are macrophages (Syrop et al., 1987). Halme et al., (Halme et al.,1984) postulated that peritoneal macrophage activation may be a central contributor to the pathogenesis of endometriosis and activated macrophages in the peritoneal cavity of women with endometriosis (Vinatier et al., 1996) are potent producers of cytokines (Halme, 1989) ;( Fakih et al., 1987); (Rana et al., 1996). Thus, peritoneal fluid contains a rich cocktail of cytokines. 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 (Harada et al, 2001). More, 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. Increased levels of cytokines in the peritoneal fluid of women with endometriosis may reflect increased synthesis of cytokines by peritoneal macrophages, lymphocytes, ectopic endometrial implants, or mesothelial cells of the peritoneum, all of which can produce cytokines (Tabibzadeh et al., 1989) ; (Betjes et al., 1993). 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. It seems that the cytokines playing the most important role in the endometriosis are : IL-1, IL-6, IL-8,IL-12, TNF- α.

**• Interleukin-1 (IL1)** is one of the major proinflammatory cytokine found in the peritoneal fluid of women with endometriosis (Mori et al., 1991); (Taketani et al., 1992); (Fakih et al., 1987). This multifunctional cytokine was shown to stimulate the production of angiogenic factors by ectopic endometrial cells and therefore play a role in ectopic endometrial cell growth (Lebovic et al., 2000). Other studies pointed to a possible role for IL1 in endometriosis-associated infertility (Fakih et al.,1987); (Sueldo et al.,1990). Both ectopic and eutopic endometrial cells of women with endometriosis display an increased sensitivity to IL1, which results in an enhanced production of angiogenic, growth, and proinflammatory factors (Lebovic et al., 2000); (Akoum et al., 1995); (Akoum et al.,2002).Some previous studies showed that the increased endometrial and endometriotic cell responsiveness to IL1 may in part be due to a deficiency in the expression of interleukin-1 receptor type II (IL1R2) revealed in eutopic and ectopic endometrial tissues (Akoum et al., 2001);(Kharfi et al.,2002). The soluble IL1R2 levels were found to be reduced in the peripheral blood of women with endometriosis, which may account for the activation of peripheral blood monocytes in them (Kharfi et al., 2002). More, the IL1R2 has no signaling properties in contrast to the functional signaling IL1R1, which mediates cell activation by IL1 (Bossu et al., 1995); (Dinarello 2004); (Colotta et al.,1993). However, the membrane form of this receptor and the soluble form, which is shed by proteolysis from the cell surface (Cui et al.,2003); (Orlando et al.,1997), bind to IL1 and with higher affinity to IL1b, which is the circulating and the preferential ligand for IL1R2, in particular for its soluble form (Bossu et al., 1995). This inhibits the interaction of

Endometriosis and Angiogenic Factors 191

**• Interleukin-8 (IL-8)** is a potent angiogenic cytokine produced by mesothelial cells, macrophages, and endometrial and other cells. Its concentration in the peritoneal fluid of patients with red endometriosis is found to increase as the size and number of active lesions increase (Iwabe et al.,1998). It stimulates adhesion of endometrial stromal cells to extracellular matrix proteins, matrix metalloproteinase activity and endometrial stromal cell proliferation in a dose-dependent manner, all of which can help to promote the implantation and growth of ectopic endometrium (Garcia-Velasco et al.,1999); (Ryan et al.,1995); (American Society for Reproductive Medicine, 1997); (Harada et al.,1997); (Punnonen et al.,1996); (Jolicoeur et al., 1998); (Akoum et al.,2000); (Pizzo et al., 2002) ; (Arci et al., 1998). • Arici et al. reported that IL-8 is produced in the human endometrium in vivo, mainly in glandular cells (Arici A, et al.,.) and that this interleukin induces proliferation of endometrial stromal cell as a potential autocrine growth factor (Arici et al., 1998). Iwabe et al. (Iwabe et al.,1998) also 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 These results suggest that IL-8

**• Tumor necrosis factor** α (**TNF-** α is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. The primary role of TNF is in the regulation of immune cells. TNF is able to induce apoptotic cell death, to induce inflammation and to inhibit tumorigenesis and viral replication. It is considered the most representative cytokine involved in the pathogenesis of endometriosis: a clear positive association between the content of TNF- α in peritoneal fluid and the severity of endometriosis has been demonstrated (Overton et al.,1996). Numerous studies indicate that both activated macrophages and the ectopic endometrium itself are responsible for the abnormal production of TNF- α in the peritoneal fluid and that this cytokine is involved in the proliferation of endometriotic stromal cells (Iwabe et al., 2000). It acts through both direct and indirect mechanisms, and by mediating the proliferative effect of IL-8 (Sakamoto Y et al., 2003 ). Moreover, gene and protein expression of IL-8 in the stromal cells of endometriotic tissues are up-regulated by TNF- α (Iwabe et al., 2000), and TNF- α stimulates the proliferation of the endometriotic stromal cells. This stimulatory effect of TNF- α was abolished by adding anti-TNF- α antibody or anti-IL-8 antibody. Therefore, TNF- α may act on stromal cells by mediating the proliferative effects of IL-8. Expression of type I and type II receptors for TNF- α was observed in endometriotic stromal cells. This evidence suggests that TNF- α 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. Thus, the differential response of endometrial cells to TNF- α in women with and without endometriosis may reflect differential regulation of TNF-receptor expression or signaling by this cytokine. Braun et al. (Braun et al., 2002) published data suggesting that in women without endometriosis, endometrial cells do not implant in ectopic locations because normal apoptotic mechanisms are activated by TNF- α through the TNFR1 receptor and because the proliferation enhancing effects of TNF- α are inhibited by down-regulation of the TNFR2 receptor. Disruption or dysregulation of the normal, cyclical expression of these two TNF- α receptors on endometrium from women with endometriosis could create cells that can grow in the presence of high concentrations of TNF- α a possibility for which evidence is avalaible (Ding et al.,2000). Harada et al. (Harada et al., 1997) and Iwabe et al. (Iwabe et al.,1998) found that the extent of superficial red

may promote the progression of endometriosis.

IL1 with its functional receptor type I and, consequently, IL1-mediated cell activation (Bossu et al., 1995); (Colotta et al.,1993); (Subramaniam et al., 2004);(Symons et al, 1995). Akoum et al. (Akoum et al., 2008) showed an imbalance in IL1/soluble IL1R2 levels in women with endometriosis suffering from infertility and pelvic pain and a relationship with endometriosis initial stages and infertility. This is in keeping with other findings showing a reduced expression of IL1R2 in the eutopic endometrial tissue of women with endometriosis, particularly in those who were infertile, and provide evidence for a deficiency in the regulation of IL1 actions at the local peritoneal level in initial endometriosis stages, which may result in increased cell reactivity and contribute to endometriosis development and the manifestation of its clinical symptoms.

**• Interleukin-6 (IL-6)** is a pleiotropic cytokine that is produced by a variety of cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells, and mesangial cells. It is said to mediate numerous physiological and pathogenic processes and acts on a wide variety of cells. IL-6 may also have important functions in reproductive physiology, including the regulation of ovarian steroid production, folliculogenesis and early events related to implantation. (Jacobs et al.,1992); (Akoum et al., 1996). Both eutopic and ectopic endometrium are known to produce IL-6 (Harada et al., 1997). Infact IL-6 belongs to the group of cytokines produced in increased amount by endometriotic cells both in basal and cytokine-stimulated conditions (Akoum et al., 1996), (Tsudo et al., 2000). IL-6 in turn is able to increase the secretion of several other cytokines and promotes the activation of immune cells (Iwabe et al., 2002).Examining eutopic endometrium from patients with endometriosis , it was found an increased basal- and IL-1b stimulated production of IL-6 compared with patients without endometriosis. This suggests that the endometrial cells of women who develop endometriosis may function differently from those in women who do not develop this condition. Endometrial stromal cells were considered the critical cells in endometrial attachment to the mesothelial surface of the peritoneum and that endometrial epithelial cells fail to attach to the mesothelium (Scott et al., 1953). It has also been suggested that cellular adhesion itself stimulates chemokine expression (Smith et al.,1997). More interestingly IL-6 family cytokines, such as IL-6, IL-11, leukemia inhibitory factor, and oncostatin M, were shown to be potent stimulators of aromatase expression in adipose stromal cells in culture (Zhao et al.,1995). Yoshioka et al. (Yoshioka et al.,1999) 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. Bedaiwy et al. (Bedaiwy et al., 2002) reported that serum IL-6 and peritoneal fluid TNF- α, could be used to discriminate between patients with and without endometriosis with a high degree of sensitivity and specificity. On the other hand, other authors, such as Kalu (Kalu et al, 2007) have failed to confirm this. They did not find any significant differences in the concentration of IL-6 in the sera of women in the two groups and more, they try to explain this result, considering that the measurement of cytokine concentrations is complicated by the fact that they have very short half-lives and are never produced in isolation, but as a mixture which may have similar or opposing effects. So, it is possible that the circulating IL-6, IL-1 b, and TNF- α found by some investigators may be non-functional or antagonized by anti-inflammatory cytokines or cytokine inhibitors. While this may also be true of peritoneal fluid cytokines, the presence of activated macrophages does suggest that at least some of the cytokines are functionally active.

IL1 with its functional receptor type I and, consequently, IL1-mediated cell activation (Bossu et al., 1995); (Colotta et al.,1993); (Subramaniam et al., 2004);(Symons et al, 1995). Akoum et al. (Akoum et al., 2008) showed an imbalance in IL1/soluble IL1R2 levels in women with endometriosis suffering from infertility and pelvic pain and a relationship with endometriosis initial stages and infertility. This is in keeping with other findings showing a reduced expression of IL1R2 in the eutopic endometrial tissue of women with endometriosis, particularly in those who were infertile, and provide evidence for a deficiency in the regulation of IL1 actions at the local peritoneal level in initial endometriosis stages, which may result in increased cell reactivity and contribute to endometriosis

**• Interleukin-6 (IL-6)** is a pleiotropic cytokine that is produced by a variety of cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells, and mesangial cells. It is said to mediate numerous physiological and pathogenic processes and acts on a wide variety of cells. IL-6 may also have important functions in reproductive physiology, including the regulation of ovarian steroid production, folliculogenesis and early events related to implantation. (Jacobs et al.,1992); (Akoum et al., 1996). Both eutopic and ectopic endometrium are known to produce IL-6 (Harada et al., 1997). Infact IL-6 belongs to the group of cytokines produced in increased amount by endometriotic cells both in basal and cytokine-stimulated conditions (Akoum et al., 1996), (Tsudo et al., 2000). IL-6 in turn is able to increase the secretion of several other cytokines and promotes the activation of immune cells (Iwabe et al., 2002).Examining eutopic endometrium from patients with endometriosis , it was found an increased basal- and IL-1b stimulated production of IL-6 compared with patients without endometriosis. This suggests that the endometrial cells of women who develop endometriosis may function differently from those in women who do not develop this condition. Endometrial stromal cells were considered the critical cells in endometrial attachment to the mesothelial surface of the peritoneum and that endometrial epithelial cells fail to attach to the mesothelium (Scott et al., 1953). It has also been suggested that cellular adhesion itself stimulates chemokine expression (Smith et al.,1997). More interestingly IL-6 family cytokines, such as IL-6, IL-11, leukemia inhibitory factor, and oncostatin M, were shown to be potent stimulators of aromatase expression in adipose stromal cells in culture (Zhao et al.,1995). Yoshioka et al. (Yoshioka et al.,1999) 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. Bedaiwy et al. (Bedaiwy et al., 2002) reported that serum IL-6 and peritoneal fluid TNF- α, could be used to discriminate between patients with and without endometriosis with a high degree of sensitivity and specificity. On the other hand, other authors, such as Kalu (Kalu et al, 2007) have failed to confirm this. They did not find any significant differences in the concentration of IL-6 in the sera of women in the two groups and more, they try to explain this result, considering that the measurement of cytokine concentrations is complicated by the fact that they have very short half-lives and are never produced in isolation, but as a mixture which may have similar or opposing effects. So, it is possible that the circulating IL-6, IL-1 b, and TNF- α found by some investigators may be non-functional or antagonized by anti-inflammatory cytokines or cytokine inhibitors. While this may also be true of peritoneal fluid cytokines, the presence of activated macrophages does suggest that at least some of the cytokines are functionally

development and the manifestation of its clinical symptoms.

active.

**• Interleukin-8 (IL-8)** is a potent angiogenic cytokine produced by mesothelial cells, macrophages, and endometrial and other cells. Its concentration in the peritoneal fluid of patients with red endometriosis is found to increase as the size and number of active lesions increase (Iwabe et al.,1998). It stimulates adhesion of endometrial stromal cells to extracellular matrix proteins, matrix metalloproteinase activity and endometrial stromal cell proliferation in a dose-dependent manner, all of which can help to promote the implantation and growth of ectopic endometrium (Garcia-Velasco et al.,1999); (Ryan et al.,1995); (American Society for Reproductive Medicine, 1997); (Harada et al.,1997); (Punnonen et al.,1996); (Jolicoeur et al., 1998); (Akoum et al.,2000); (Pizzo et al., 2002) ; (Arci et al., 1998).

• Arici et al. reported that IL-8 is produced in the human endometrium in vivo, mainly in glandular cells (Arici A, et al.,.) and that this interleukin induces proliferation of endometrial stromal cell as a potential autocrine growth factor (Arici et al., 1998). Iwabe et al. (Iwabe et al.,1998) also 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 These results suggest that IL-8 may promote the progression of endometriosis.

**• Tumor necrosis factor** α (**TNF-** α is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. The primary role of TNF is in the regulation of immune cells. TNF is able to induce apoptotic cell death, to induce inflammation and to inhibit tumorigenesis and viral replication. It is considered the most representative cytokine involved in the pathogenesis of endometriosis: a clear positive association between the content of TNF- α in peritoneal fluid and the severity of endometriosis has been demonstrated (Overton et al.,1996). Numerous studies indicate that both activated macrophages and the ectopic endometrium itself are responsible for the abnormal production of TNF- α in the peritoneal fluid and that this cytokine is involved in the proliferation of endometriotic stromal cells (Iwabe et al., 2000). It acts through both direct and indirect mechanisms, and by mediating the proliferative effect of IL-8 (Sakamoto Y et al., 2003 ). Moreover, gene and protein expression of IL-8 in the stromal cells of endometriotic tissues are up-regulated by TNF- α (Iwabe et al., 2000), and TNF- α stimulates the proliferation of the endometriotic stromal cells. This stimulatory effect of TNF- α was abolished by adding anti-TNF- α antibody or anti-IL-8 antibody. Therefore, TNF- α may act on stromal cells by mediating the proliferative effects of IL-8. Expression of type I and type II receptors for TNF- α was observed in endometriotic stromal cells. This evidence suggests that TNF- α 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. Thus, the differential response of endometrial cells to TNF- α in women with and without endometriosis may reflect differential regulation of TNF-receptor expression or signaling by this cytokine. Braun et al. (Braun et al., 2002) published data suggesting that in women without endometriosis, endometrial cells do not implant in ectopic locations because normal apoptotic mechanisms are activated by TNF- α through the TNFR1 receptor and because the proliferation enhancing effects of TNF- α are inhibited by down-regulation of the TNFR2 receptor. Disruption or dysregulation of the normal, cyclical expression of these two TNF- α receptors on endometrium from women with endometriosis could create cells that can grow in the presence of high concentrations of TNF- α a possibility for which evidence is avalaible (Ding et al.,2000). Harada et al. (Harada et al., 1997) and Iwabe et al. (Iwabe et al.,1998) found that the extent of superficial red

Endometriosis and Angiogenic Factors 193

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. 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 (Keenan et al.,1995). In this study, the levels of IL-1 and TNF- α 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 endometriosisassociated infertility. One study demonstrated that the addition of human recombinant IL-6 to culture medium suppressed the rate of blastocyst formation of mouse embryos (Harada et al., 1997), suggesting that increased IL-6 in the peritoneal fluid of endometriosis patients may contribute to infertility by adversely affecting embryonic development. Moreover, other authors (Minici et al., 2008) showed that in endometriosis, the milieu surrounding the uterine cavity may be involved in impaired eutopic endometrial stromal cell decidualization, partially due to increased peritoneal levels of TNF- α. So they concluded that in endometriosis either intrinsic defects of endometrial stromal cell differentiation or the biochemical environment of the uterine cavity could concur to compromise the normal

In conclusion, 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

Intense basic research into the specific role of these cells and soluble factors may improve

Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular differentiation and other functions in most cells. TGF-β is a secreted protein that exists in at least five isoforms called TGF- ß 1, TGF- ß 2 and TGF- ß 3. It was also the original name for TGF- ß 1, which was the founding member of this family. TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. Some cells that secrete TGF- ß also have receptors for TGF- ß. This is known as autocrine signalling. Cancerous cells increase their production of TGF-β, which also acts on surrounding cells.Oosterlynck et al. (Oosterlynck et al.,1994) found increased TGF-b activity in the peritoneal fluid of women with endometriosis. Transforming growth factor-b may be a cytokine that inhibits NK activity in the peritoneal fluid of women with endometriosis (Oosterlynck et al.,1994). It may play a major role in the biological processes leading to establishment and maintenance of endometriosis., in fact TGF- ß is implicated in the gene expression, cell motility, proliferation, apoptosis, differentiation, immune responses and tumorigenesis (Derynck et al., 2001). TGF-b is abundantly and differentially expressed in the endometrium and is secreted by endometrial cells and macrophages into the uterine fluid where interaction with the preimplantation embryo is suspected (Jones et al., 2006).

our understanding of endometriosis and result in novel therapies for endometriosis.

decidualization required for optimal implantation.

**4. Transforming growth factor beta (TGF-ß)** 

progression of endometriosis and endometriosis-associated infertility.

endometriotic lesions was related to increased levels of IL-6, IL-8, and TNF- α 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 (Wiegerick et al.,1993) and because early red lesions invade extracellular matrix (Spuijbroek et al.,1992). Braun et al. (Braun et al., 2002) also suggested that ectopic growth of endometrial cells and the physiological consequences of that growth in women with endometriosis may be retarded by agents that block the effects of TNF- α. Presumably, this could be achieved by blocking TNF- α production (e.g., administration of pentoxifylline or ciprofloxacin) or by blocking the effects of TNF- α on target tissues (e.g., administration of etanercept). The attenuation of the proliferation-enhancing activity in peritoneal fluid from women with endometriosis by etanercept that the athors observed, and results in an animal model of endometriosis using a recombinant human TNF-binding protein (D'Antonio et al., 2000;), support this idea. Blocking the effects of TNF- α on target tissues might be especially appropriate in patients with extensive or intractable disease and might be useful in the postsurgical adjuvant setting to reduce the likelihood of recurrence. Given the potential of TNF- α to play a prominent role in both the etiology and the pathogenicity of endometriosis, studies of such treatment are warranted

**• Interleukin-12 (IL-12**) is naturally produced by dendritic cells ( Kaliński Pet al., 1997), macrophages and human B-lymphoblastoid cells in response to antigenic stimulation. It is known as a T cell-stimulating factor, which can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFNγ. It acts on T and NK cells, inducing cytokine production, enhancing NK cell cytotoxic activity, and finally favoring generation of Th1 cell response, (Kobayashi et al., 1989) ;(Wysocka et al., 1995) so it seems to play an important role, in the controll of the endometriosis. Harada et al., (Harada et al.,2001)demonstrated that the concentrations of IL-12 in the peritoneal fluid are low, but detectable, regardless of the presence or absence of endometriosis (Zeyneloglu et al., 1998). Administration of IL-12 significantly prevented ectopic endometrial implantation in a murine model of endometriosis (Somigliana et al.,1999). 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.

#### **3.2 Cytokines and endometriosis related to infertility**

Muscato et al. (Muscato et al., 1982) 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 (Curtis et al., 1993) ;(Drudy et al., 1994), acrosome reaction (Arumugam et al.,1994), gamete interaction (Coddington et al, 1992.), and ovum capture by tubal fimbriae (Suginami et al., 1988) have been studied. Aeby et al. (Aeby et al.,1996), using a penetration assay, showed that peritoneal fluid from patients with

endometriotic lesions was related to increased levels of IL-6, IL-8, and TNF- α 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 (Wiegerick et al.,1993) and because early red lesions invade extracellular matrix (Spuijbroek et al.,1992). Braun et al. (Braun et al., 2002) also suggested that ectopic growth of endometrial cells and the physiological consequences of that growth in women with endometriosis may be retarded by agents that block the effects of TNF- α. Presumably, this could be achieved by blocking TNF- α production (e.g., administration of pentoxifylline or ciprofloxacin) or by blocking the effects of TNF- α on target tissues (e.g., administration of etanercept). The attenuation of the proliferation-enhancing activity in peritoneal fluid from women with endometriosis by etanercept that the athors observed, and results in an animal model of endometriosis using a recombinant human TNF-binding protein (D'Antonio et al., 2000;), support this idea. Blocking the effects of TNF- α on target tissues might be especially appropriate in patients with extensive or intractable disease and might be useful in the postsurgical adjuvant setting to reduce the likelihood of recurrence. Given the potential of TNF- α to play a prominent role in both the etiology and the pathogenicity of endometriosis, studies of such treatment

**• Interleukin-12 (IL-12**) is naturally produced by dendritic cells ( Kaliński Pet al., 1997), macrophages and human B-lymphoblastoid cells in response to antigenic stimulation. It is known as a T cell-stimulating factor, which can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFNγ. It acts on T and NK cells, inducing cytokine production, enhancing NK cell cytotoxic activity, and finally favoring generation of Th1 cell response, (Kobayashi et al., 1989) ;(Wysocka et al., 1995) so it seems to play an important role, in the controll of the endometriosis. Harada et al., (Harada et al.,2001)demonstrated that the concentrations of IL-12 in the peritoneal fluid are low, but detectable, regardless of the presence or absence of endometriosis (Zeyneloglu et al., 1998). Administration of IL-12 significantly prevented ectopic endometrial implantation in a murine model of endometriosis (Somigliana et al.,1999). 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.

Muscato et al. (Muscato et al., 1982) 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 (Curtis et al., 1993) ;(Drudy et al., 1994), acrosome reaction (Arumugam et al.,1994), gamete interaction (Coddington et al, 1992.), and ovum capture by tubal fimbriae (Suginami et al., 1988) have been studied. Aeby et al. (Aeby et al.,1996), using a penetration assay, showed that peritoneal fluid from patients with

**3.2 Cytokines and endometriosis related to infertility** 

are warranted

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. 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 (Keenan et al.,1995). In this study, the levels of IL-1 and TNF- α 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 endometriosisassociated infertility. One study demonstrated that the addition of human recombinant IL-6 to culture medium suppressed the rate of blastocyst formation of mouse embryos (Harada et al., 1997), suggesting that increased IL-6 in the peritoneal fluid of endometriosis patients may contribute to infertility by adversely affecting embryonic development. Moreover, other authors (Minici et al., 2008) showed that in endometriosis, the milieu surrounding the uterine cavity may be involved in impaired eutopic endometrial stromal cell decidualization, partially due to increased peritoneal levels of TNF- α. So they concluded that in endometriosis either intrinsic defects of endometrial stromal cell differentiation or the biochemical environment of the uterine cavity could concur to compromise the normal decidualization required for optimal implantation.

In conclusion, 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.
