**2. Development of endometriosis**

#### **2.1 Sampson's implantation theory**

Endometriosis is one of most common gynecological disorder, but poorly understood condition. As early as in 1860, von Rokitansky (Rokitansky, 1860) is the first one to describe

Pathophysiological Changes in Early Endometriosis 461

peritoneal fluid of women with endometriosis was significantly higher than women without this disease, the function of increased immune cells was decreased (Berkkanoglu *et al.*, 2003). Meanwhile, these defected immune cells may secrete some cytokines and growth factors, such as interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) etc., which may help the endometrial cells escape from immune surveillance to adhere to the peritoneum, establish microvessels and finally grow under the stimulation of estrogen cycle (Kyama *et al.*, 2003). However, the reasons causing impaired immune cells are not known. When and what kind of cells as well as molecules taking part in the process from survival to steady growth are still not clear.

Endometrial cells have to contact with immune cells, peritoneal lining and vascular endothelial before final growth and maintenance in ectopic location. Cellular immune response is responsible for implantation of the retrograde and vital endometrial cells. The molecules secreted by immune cells would effect the reaction between endometrial cells and

Macrophages are the most abundant immune cells in peritoneal fluid and their main role is to phagocytose cellular debris and pathogens. They can also promote lymphocytes and other immune cells to respond to pathogens (Tariverdian *et al.*, 2009; van Furth *et al.*, 1979). It has been reported that the number and activity of macrophages in peritoneal fluid significantly increased. However they cannot clear the ectopic endometrial cells and inhibit the development of endometriosis. Modulators of activated macrophages for both immune and non-immune cells promote growth and maintenance of ectopic lesion (Lebovic *et al.*, 2001). There are several evidence to support the change of receptors expression on macrophages leads to impaired scavenger function, which might be caused by abnormal cytokines and growth factors in the peritoneal fluid of women with endometriosis

The two main types of lymphocytes are: B cells accounting for humoral acquired response by secreting soluble antibodies into the body's fluids for eliminating foreign antigens, and T cells responsible for cellular responses. Both of which recognize specific antigen targets. T cells are mainly differentiated into helper T cells promoting antibody production secreted by B cells, regulatory T cells controlling immune response, and cytotoxic/suppressor T cells killing infected cells and cancer cells in the thymus. In endometriosis, it was reported that the proliferation and cytotoxic activity of lymphocytes in peripheral blood is decreased (Dmowski *et al.*, 1981; Steele *et al.*, 1984). Increased T cells, both helper and suppressor T cells, in peritoneal fluid and ectopic endometriotic tissue was observed in women with endometriosis (Dmowski *et al.*, 1994; Hill *et al.*, 1988; Mettler *et al.*, 1996). However, the changes are not consistent. Besides, the function and activity of peripheral T cells might be different from those in peritoneal fluid. In all, it's controversial if the alteration of lymphocytes in peripheral and peritoneal fluid play a role in the development of

**2.3 Cellular communications** 

other cells.

**2.3.1 Macrophages** 

(Berkkanoglu *et al.*, 2003).

**2.3.2 Lymphocytes** 

endometriosis.

this disease in detail. Since then, several postulated theories explaining the pathogenesis of endometriosis were raised. The most popular theory is Sampson's classical implantation theory in 1921 (Sampson, 1921). He proposed that the endometrial fragments of uterine endometrium during menstruation can regurgitate through the fallopian tubes and survive in the peritoneal cavity, developing to endometriosis.

There have been numerous studies in human and primate support the implantation theory (Bartosik et al., 1986; Halme et al., 1984). However, this hypothesis cannot explain why only about 10% women suffer from endometriosis, but the incidence of retrograde menstruation should be much higher. What's more, the endometriotic lesion sometimes is present out of peritoneal cavity, such as lungs, brain and heart, instead of peritoneal cavity only (Felson et al., 1960; Joseph et al., 1994; Thibodeau et al., 1987). Besides, genetic, immunological factors and vascular and lymphatic spread are also essential for endometriosis development. Therefore, endometriosis is multifactorial and complicated condition. More studies are needed to explicitly understand the pathogenesis of endometriosis.

#### **2.2 Pathophysiology**

With numerous clinical and basic researches on endometriosis, especially peritoneal endometriosis, based on Sampson's retrograde menstruation theory, it's well accepted that the appearance of vital endometrial cells is the first step. Then immune escape, adhesion, implantation, angiogenesis and proliferation are all very important during the development of endometriosis (Fig. 1).

Fig. 1. Summary of the development of endometriosis

In healthy women, macrophages, lymphocytes, natural killer cells and leukocytes eliminate ectopic endometrial cells (Braun *et al.*, 1996). Though the number of immune cells in peritoneal fluid of women with endometriosis was significantly higher than women without this disease, the function of increased immune cells was decreased (Berkkanoglu *et al.*, 2003). Meanwhile, these defected immune cells may secrete some cytokines and growth factors, such as interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) etc., which may help the endometrial cells escape from immune surveillance to adhere to the peritoneum, establish microvessels and finally grow under the stimulation of estrogen cycle (Kyama *et al.*, 2003). However, the reasons causing impaired immune cells are not known. When and what kind of cells as well as molecules taking part in the process from survival to steady growth are still not clear.

#### **2.3 Cellular communications**

Endometrial cells have to contact with immune cells, peritoneal lining and vascular endothelial before final growth and maintenance in ectopic location. Cellular immune response is responsible for implantation of the retrograde and vital endometrial cells. The molecules secreted by immune cells would effect the reaction between endometrial cells and other cells.

#### **2.3.1 Macrophages**

460 Endometriosis - Basic Concepts and Current Research Trends

this disease in detail. Since then, several postulated theories explaining the pathogenesis of endometriosis were raised. The most popular theory is Sampson's classical implantation theory in 1921 (Sampson, 1921). He proposed that the endometrial fragments of uterine endometrium during menstruation can regurgitate through the fallopian tubes and survive

There have been numerous studies in human and primate support the implantation theory (Bartosik et al., 1986; Halme et al., 1984). However, this hypothesis cannot explain why only about 10% women suffer from endometriosis, but the incidence of retrograde menstruation should be much higher. What's more, the endometriotic lesion sometimes is present out of peritoneal cavity, such as lungs, brain and heart, instead of peritoneal cavity only (Felson et al., 1960; Joseph et al., 1994; Thibodeau et al., 1987). Besides, genetic, immunological factors and vascular and lymphatic spread are also essential for endometriosis development. Therefore, endometriosis is multifactorial and complicated condition. More studies are needed to explicitly understand the pathogenesis of

With numerous clinical and basic researches on endometriosis, especially peritoneal endometriosis, based on Sampson's retrograde menstruation theory, it's well accepted that the appearance of vital endometrial cells is the first step. Then immune escape, adhesion, implantation, angiogenesis and proliferation are all very important during the development

In healthy women, macrophages, lymphocytes, natural killer cells and leukocytes eliminate ectopic endometrial cells (Braun *et al.*, 1996). Though the number of immune cells in

in the peritoneal cavity, developing to endometriosis.

Fig. 1. Summary of the development of endometriosis

endometriosis.

**2.2 Pathophysiology** 

of endometriosis (Fig. 1).

Macrophages are the most abundant immune cells in peritoneal fluid and their main role is to phagocytose cellular debris and pathogens. They can also promote lymphocytes and other immune cells to respond to pathogens (Tariverdian *et al.*, 2009; van Furth *et al.*, 1979). It has been reported that the number and activity of macrophages in peritoneal fluid significantly increased. However they cannot clear the ectopic endometrial cells and inhibit the development of endometriosis. Modulators of activated macrophages for both immune and non-immune cells promote growth and maintenance of ectopic lesion (Lebovic *et al.*, 2001). There are several evidence to support the change of receptors expression on macrophages leads to impaired scavenger function, which might be caused by abnormal cytokines and growth factors in the peritoneal fluid of women with endometriosis (Berkkanoglu *et al.*, 2003).

#### **2.3.2 Lymphocytes**

The two main types of lymphocytes are: B cells accounting for humoral acquired response by secreting soluble antibodies into the body's fluids for eliminating foreign antigens, and T cells responsible for cellular responses. Both of which recognize specific antigen targets. T cells are mainly differentiated into helper T cells promoting antibody production secreted by B cells, regulatory T cells controlling immune response, and cytotoxic/suppressor T cells killing infected cells and cancer cells in the thymus. In endometriosis, it was reported that the proliferation and cytotoxic activity of lymphocytes in peripheral blood is decreased (Dmowski *et al.*, 1981; Steele *et al.*, 1984). Increased T cells, both helper and suppressor T cells, in peritoneal fluid and ectopic endometriotic tissue was observed in women with endometriosis (Dmowski *et al.*, 1994; Hill *et al.*, 1988; Mettler *et al.*, 1996). However, the changes are not consistent. Besides, the function and activity of peripheral T cells might be different from those in peritoneal fluid. In all, it's controversial if the alteration of lymphocytes in peripheral and peritoneal fluid play a role in the development of endometriosis.

Pathophysiological Changes in Early Endometriosis 463

Cellular communications in endometriosis mediated by inflammatory cytokines and growth factors is mainly regulated by nuclear factor-κB (NF-κB) signaling pathway (Fig. 2) (Gonzalez-Ramos *et al.*). NF-κB mediated gene transcription promoting inflammation, invasion, angiogenesis, and cell proliferation and inhibiting apoptosis of endometriotic cells through p50/p65 dimers and NF-κB inhibitor IκBα has been found *in vitro* and *in vivo* studies (Gonzalez-Ramos *et al.*, 2010). Constitutive activation of NF-κB has been demonstrated in endometriotic lesions and peritoneal macrophages of patients with endometriosis (Laird *et al.*, 2000). Some drugs such as GnRH blocking NF-κB have been proven efficient at reducing endometriosis-associated symptoms in women (Han *et al.*, 2003). Overload iron produced by erythrocytes from menstruation shedding and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) as well as oxidative stress stimulate NF-κB activation in macrophages and ectopic endometrial cells, which stimulates synthesis of proinflammatory cytokines, sending a positive feedback loop to the NF-κB signaling pathway. NF-κB activation enhances factors of anti-apoptosis, growth, invasion and angiogenesis as well as proinflammatory cytokines such as cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), which promote the development of endometriosis. Intercellular adhesion molecule-1 (ICAM-1) and RANTES up-regulated by

NF-κB activity could attract more macrophages to sites of inflammation.

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

Fig. 2. NF-κB signaling pathway in endometriosis

**3. Early pathogenesis** 

**3.1 Oxidative stress** 

**2.4 Molecular modulations** 

### **2.3.3 Natural killer cells**

Natural killer cells (NK cells) constitute a major component of the innate immune system. They have two ways to take part in host defense by expressing different receptors binding to target cells. One receptor type binds immunoglobulin G (IgG). The other includes killeractivating receptors promoting cytotoxic activity and killer-inhibitory receptors (KIR) suppressing cytotoxic activity (Moretta *et al.*, 1995). Oosterlynck and Wilson have found that the cytotoxic activity of peripheral and peritoneal fluid NK cells from women with endometriosis was obviously decreased with the severity of endometriosis (Oosterlynck *et al.*, 1991; Wilson *et al.*, 1994). The decreased NK-mediated cytotoxicity in the peritoneal fluid might contribute to the establishment of endometriosis. The mechanisms that cause aberrant NK cell cytotoxicity are unclear, but seem to be involved in KIR expression (Wu *et al.*, 2000). In a recent study, Maeda et al. reported KIR2DL1 as the subclass of KIR overexpressed on NK cells in peripheral and peritoneal fluid of patients with endometriosis.

#### **2.3.4 Peritoneal cells**

Several adhesion moleculars were found to be expressed in endometrium, which mediate the adhesion and invasion of endometrial cells to peritoneum. Koks found that endometrium preferentially adhere to the extracellular matrix (ECM) of the peritoneum mediated by integrin (Koks *et al.*, 2000). Endometrium expresses various integrins during menstruation shedding and the adhesion can be disrupted by blocking integrin. Integrins are cell-surface glycoproteins acting as receptors for ECM proteins. In normal eutopic endometrium, integrins are important in the interaction between glandular epithelial and stromal cells, and essential for implantation (Lessey *et al.*, 1992). After adherence of endometrial cells to the peritoneum, local degradation of the ECM is required for invasion and implantation. Metalloproteinases (MMPs) causing ECM breakdown, tissue collapse and menstruation was up-regulated in late secretory phase (Salamonsen *et al.*, 1996). This implies that the vital endometrial cells in peritoneal cavity during menstruation shedding already have the potential to invade into peritoneum. What's more, MMPs are present independent of the cycle phase in peritoneal and ovarian endometriosis (Salamonsen *et al.*, 1996), which promotes endometriotic cells to infiltrate into peritoneum further although endometriosis has been established.
