**3. Early pathogenesis**

#### **3.1 Oxidative stress**

Oxidative stress has been proposed as a potential factor in the pathogenesis of endometriosis (Van Langendonckt *et al.*, 2002). Oxidative stress may occur when the balance of reactive oxygen species (ROS) and antioxidant is disturbed. Several studies have demonstrated that the oxidative stress is involved in endometriosis with increased concentration of ROS, enzymes producing ROS and lower concentration of antioxidant in peritoneal fluid and in the eutopic and ectopic endometrium of women with endometriosis (Ota *et al.*, 2001; Zeller *et al.*, 1987). It's postulated that oxidative stress is stimulated by erythrocytes (Brosens, 1994), apoptotic

Pathophysiological Changes in Early Endometriosis 465

Differentiate T cells (Giudice, 1994) Stimulate angiogenesis (Lin *et al.*, 2006)

Differentiates B cells (Lebovic *et al.*, 2000) Increase IL-6, sICAM-1, IL-8 & VEGF (Arici *et al.*, 1993)

Recruit neutrophils and lymphocytes (Garcia-Velasco *et al.*,

Inhibit NK cells cytotoxicity (Koninckx *et al.*, 1998)

Stimulate endometrial cell proliferation (Arici *et al.*, 1997)

(Iwabe *et al.*, 1998)

(Arici *et al.*, 1998)

(Laird *et al.*, 1996)

(Zhang *et al.*, 1993)

1999)

Cytokines Functions References

IL-1 Activates T-lymphocytes (Vigano *et al.*, 1998)

IL-6 Stimulate B cell activity (Le *et al.*, 1989)

MCP-1 Activate macrophages (Oral *et al.*, 1996)

Inhibit T and B lymphocytes and NK cell activity

Table 1. Functions of cytokines and growth factors involved in endometriosis

therapy possible for prevention of reoccurrence after surgical treatment.

RANTES Attract macrophages and lymphcytes (Khorram *et al.*, 1993) ICAM-1 Mediate cell adhesion (Oral *et al.*, 1996)

VEGF Stimulate angiogenesis; Attract monocytes (McLaren *et al.*, 1996)

The establishment of new blood vessels is essential in growth and survival of endometriosis. Increased angiogenic activity has been demonstrated in peritoneal fluid of women with endometriosis and strong expression of angiogenic factors has been shown in active lesions (Donnez *et al.*, 1998; Nisolle *et al.*, 1993). Moreover, inhibition of endometrial implants by anti-angiogenic agents or VEGF receptors (VEGFR) blocker was observed in animal studies (Dabrosin *et al.*, 2002; Nap *et al.*, 2004). Many anti-angiogenic compounds are studied extensively in animal models of endometriosis. Vlahos stated that pentoxifylline used in the treatment of peripheral vascular disease for many years may cause suppression of endometriotic tissue by inhibiting angiogenesis through VEGF-C and VEGFR-2 expression in rat model (Vlahos *et al.*). Besides, progestins already used in the treatment of endometriosis inhibit human ectopic endometrial lesions in a mouse model by regulating cysteine-rich angiogenic inducer (CYR61), basic fibroblast growth factor (bFGF) and VEGFA (Monckedieck *et al.*, 2009). Endostain, a potent endogenous inhibitor of blood vessel growth, suppress angiogenesis by inhibiting endothelial migration without effecting normal estrous cycles (Becker *et al.*, 2006). What's more, either selective cyclooxygenase-2 (COX-2) inhibitor or immunoconjugate molecule (Icon) suppress angiogenesis in animal models by microvessels density assessment (Krikun *et al.*). However, there is no study to investigate the early process of angiogenesis in endometriosis, which makes new anti-angiogenesis

TGF-β Attract monocytes (Oosterlynck *et al.*, 1994)

IL-8 Promote proliferation of endometrial and endometriotic stromal cells

TNF-α Initiate the cascade of cytokines and inflammatory response

endometrial stromal cells

**3.3 Angiogenesis** 

Increase the adherence of cultured

fibronectin

Stimulate adhesion of endometrial cells to

endometrial tissue, cell debris and macrophages (Murphy *et al.*, 1998). These inducers may cause activation and recruitment of mononuclear phagocytes which induce oxidative stress. Oxidative stress might lead to a localized pelvic inflammatory reaction with increased proinflammatory mediators, cytokines and growth factors (Gupta *et al.*, 2006). These cytokines and growth factors have been widely accepted to promote the immune modulation, adhesion, invasion and angiogenesis of endometriosis. Therefore, understanding of oxidative stress could give a light in the initiation and process of angiogenesis and inflammation during the development of endometriosis. However, two other studies could not find the imbalance between ROS and antioxidant in the peritoneal fluid of women with endometriosis (Ho *et al.*, 1997; Wang *et al.*, 1997). This discrepancy might be due to the use of markers of oxidative stress. Thus, further studies are needed to identify when and how oxidative stress play a role in the pathophysiology of endometriosis in particular during early development.

#### **3.2 Proinflammatory responses**

It's widely accepted that endometriosis is a pelvic inflammatory process with defected function of immune system and increased level of abnormal cytokines, chemokines and growth factors in the peritoneal fluid modulating the growth and inflammation of endometriosis. The proinflammatory cytokines and chemokines involved in development of endometriosis include IL-1, IL-6, IL-8, MCP-1 and RANTES (Table 1). These cytokines are mostly secreted by activated immune cells and endometrial cells. They act as paracrine and autocrine messengers in cellular communication. On the one hand, some of these cytokines mediate the adhesion of endometrial cells to peritoneum, such as ICAM-1 and TNF-α and promote proliferation of endometrial cells, such as IL8, as well as stimulate angiogenesis such as VEGF. On the other hand, some cytokines modulate immune cells function: transforming growth factor beta (TGF-β) inhibiting T and B lymphocytes and NK cells which may cause immune tolerance; MCP-1 activating macrophages. The imbalance and abnormal distribution of peritoneal fluid cytokines and their functions imply that inflammation plays a key role in the development of endometriosis.

Whether pelvic inflammation cause endometriosis or endometriosis results in pelvic inflammation is still not well defined. Due to the unsatisfactory diagnostic methods and the limitation of human researches, we are not able to answer this question in human because most patients have had endometriosis for an unknown disease course at the time of diagnosis. Studying endometriosis using animal models complement the understanding endometriosis in human. Chen et al. have found that the endometrial cells in the peritoneal fluid induced the production of IL-1β, TNF-α, VEGF and MCP-1 at 24 hours in the peritoneal fluid of mice. (Chen *et al.*). Similarly, IL-2, IL-4, IL-6, IL-10 and MCP-1, eosinophil chemotactic protein (eotaxin), macrophage inflammatory protein and RANTES as well as CC chemokine receptor (CCR1) were found remarkably expressed in endometriotic lesions on the 4th day in rat model by autologous transplantation of endometrial epithelial fragment to peritoneum (Umezawa *et al.*, 2008). These inflammation cytokines found in early endometriosis is consistent with those found in peritoneal fluid of women with endometriosis. Based on this finding, it can be supposed that endometrial cells in peritoneal cavity might cause inflammation mediating and promoting the development of endometriosis. More studies about early endometriosis are necessary to confirm this hypothesis.

endometrial tissue, cell debris and macrophages (Murphy *et al.*, 1998). These inducers may cause activation and recruitment of mononuclear phagocytes which induce oxidative stress. Oxidative stress might lead to a localized pelvic inflammatory reaction with increased proinflammatory mediators, cytokines and growth factors (Gupta *et al.*, 2006). These cytokines and growth factors have been widely accepted to promote the immune modulation, adhesion, invasion and angiogenesis of endometriosis. Therefore, understanding of oxidative stress could give a light in the initiation and process of angiogenesis and inflammation during the development of endometriosis. However, two other studies could not find the imbalance between ROS and antioxidant in the peritoneal fluid of women with endometriosis (Ho *et al.*, 1997; Wang *et al.*, 1997). This discrepancy might be due to the use of markers of oxidative stress. Thus, further studies are needed to identify when and how oxidative stress play a role

It's widely accepted that endometriosis is a pelvic inflammatory process with defected function of immune system and increased level of abnormal cytokines, chemokines and growth factors in the peritoneal fluid modulating the growth and inflammation of endometriosis. The proinflammatory cytokines and chemokines involved in development of endometriosis include IL-1, IL-6, IL-8, MCP-1 and RANTES (Table 1). These cytokines are mostly secreted by activated immune cells and endometrial cells. They act as paracrine and autocrine messengers in cellular communication. On the one hand, some of these cytokines mediate the adhesion of endometrial cells to peritoneum, such as ICAM-1 and TNF-α and promote proliferation of endometrial cells, such as IL8, as well as stimulate angiogenesis such as VEGF. On the other hand, some cytokines modulate immune cells function: transforming growth factor beta (TGF-β) inhibiting T and B lymphocytes and NK cells which may cause immune tolerance; MCP-1 activating macrophages. The imbalance and abnormal distribution of peritoneal fluid cytokines and their functions imply that

Whether pelvic inflammation cause endometriosis or endometriosis results in pelvic inflammation is still not well defined. Due to the unsatisfactory diagnostic methods and the limitation of human researches, we are not able to answer this question in human because most patients have had endometriosis for an unknown disease course at the time of diagnosis. Studying endometriosis using animal models complement the understanding endometriosis in human. Chen et al. have found that the endometrial cells in the peritoneal fluid induced the production of IL-1β, TNF-α, VEGF and MCP-1 at 24 hours in the peritoneal fluid of mice. (Chen *et al.*). Similarly, IL-2, IL-4, IL-6, IL-10 and MCP-1, eosinophil chemotactic protein (eotaxin), macrophage inflammatory protein and RANTES as well as CC chemokine receptor (CCR1) were found remarkably expressed in endometriotic lesions on the 4th day in rat model by autologous transplantation of endometrial epithelial fragment to peritoneum (Umezawa *et al.*, 2008). These inflammation cytokines found in early endometriosis is consistent with those found in peritoneal fluid of women with endometriosis. Based on this finding, it can be supposed that endometrial cells in peritoneal cavity might cause inflammation mediating and promoting the development of endometriosis. More studies about early endometriosis are necessary to confirm this

in the pathophysiology of endometriosis in particular during early development.

inflammation plays a key role in the development of endometriosis.

**3.2 Proinflammatory responses** 

hypothesis.


Table 1. Functions of cytokines and growth factors involved in endometriosis
