**3.1 Regulation of apoptosis in the endometrium of women with endometriosis**

Apoptosis is a fundamental process responsible for maintaining homeostasis in multicellular organisms (Mei et al., 2010). In contrast to necrotic cell death, which is usually a result of trauma, programmed cell death is a physiological process. The mechanisms of apoptosis are highly complex and sophisticated, involving an energy dependent cascade of molecular events. Two major apoptotic routes exist in mammalian cells: the intrinsic or mitochondrial pathway and extrinsic or death receptor pathway. The intrinsic pathway is

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 217

apoptotic factor Bcl-2 and decrease expression of pro-apoptotic factor Bax were found in proliferative eutopic endometrium from women with endometriosis (Meresman et al., 2000). These differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and development of endometriosis (Taniguchi 2011). In ectopic endometrium of women with endometriosis the level of Bcl-2 expression was significantly increased in stromal cells (Harada et al., 2004). In endometriotic lesions the alterations in expression of different apoptosis-associated genes, such as PTEN, p52 and Bcl-2 were also demonstrated (Nezha et al., 2008). Authors thought that these changes are analogous in tumor tissue and evidence about possible malignant transformation of endometriotic tissue. At the same time there are some data about absence of significant changes of apoptotsis both in eutopic and in ectopic endometrium in women with endometriosis (Hassa et al., 2009). So, the study of the mechanisms, regulating apoptosis in endometriosis, need to be

We also have checked out the expression of mRNA of some genes, possessed both pro- and anti-apoptotic action, in eutopic and ectopic endometrium of women with endometriosis. It was found that the expression of genes, regulating apoptosis, in endometrial tissue of women with endometriosis significantly differed from that in healthy women (Table 1).

(n=15)

Table 1. The level of the expression of mRNAs genes, regulating apoptosis, in the eutopic and ectopic endometrium of women with endometriosis (\* – given in comparison to the endometrium of healthy women, \*- p<0.05; x - given in comparison to the eutopic

In eutopic endometrium of women with endometriosis the expression of anti-apoptotic factor XIAP (X-linked inhibitor of apoptosis) mRNAs were statistically increased compared

In ectopic endometrial tissue the high levels of caspase-3 and heat shock protein 27 (HSP27) mRNAs expression were seen. Both in eutopic and ectopic endometrium of endometriosis women the low level of metallothionein-1 (MT-1) mRNA expression was found compared to

Thus, the profile of genes, regulating apoptosis, was different in eutopic and ectopic endometrium in endometriosis except the expression of MT-1 mRNA which was decreased both in eutopic endometrium and in endometriotic lesions. It is known that metalloteoneins (MTs) are a group of ubiquitous low-molecular-weight proteins essential for the protection of cells against heavy metal ion toxicity. These molecules also are directly involved in regulation of cell growth, differentiation and apoptosis in different pathological conditions and in tumour as well (Inoue et al., 2009). Earlier the low level of MT-1 protein was demonstrated in ovarian endometriomas (Wicherek et al., 2006).

mRNA MT-1 361.89±124.74 46.56±23.76 \* 42.36±17.42\* mRNA XIAP 0.38±0.18 22.51±9.62\* 2.90±1.59<sup>х</sup> mRNA caspase-3 0.72±0.33 0.38±0.09 3.80±1.25\*хх mRNA HSP27 0.23±0.05 0.22±0.08 1.75±0.55\*<sup>х</sup>

Eutopic endometrium,

Ectopic endometrium,

endometriosis

(n=15)

endometriosis

Endometrium, control (n=7)

endometrium of women with endometriosis, x – p<0.05, xx- p<0.01)

to that in the endometrium of women from the control group.

that in the endometrium of women without endometriosis.

continued.

Parameter, copies number/μl

characterized by the permeabilization of the outer mitochondrial membrane and the release of several pro-apoptotic factors into the cytosol. These include cytochrome c, Smac/Diablo, AIF (apoptosis-inducing factor), and serine protease HtrA2/Omi (Elmore 2007). These proteins activate the caspase dependent mitochondrial pathway. Cytochrome *c* binds and activates Apaf-1 (the apoptotic protease activating factor-1) as well as procaspase-9, forming an "apoptosome" (Hill et al., 2004). The clustering of procaspase-9 in this manner leads to caspase-9 activation. Different molecules from IAP family (inhibitors of apoptosis proteins) can inhibit activity of Smac/DIABLO and HtrA2/Omi on this stage of apoptosis (Schimmer et al., 2006). The control and regulation of these apoptotic mitochondrial events occurs through members of HSP (heat shock proteins) family, acting as inhibitors of apoptosis. Proteins from Bcl-2 family also regulate the mitochondrial stage of apoptosis and these factors can be either pro-apoptotic or antiapoptotic. Till date, a total of 25 genes have been identified in the Bcl-2 family. Some of the anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG, and some of the pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk. These proteins have special significance since they can determine if the cell is committed to apoptosis or else abort the process. The tumor suppressor protein p53 has a critical role in regulation of the Bcl-2 family of proteins (Elmore 2007). The extrinsic pathway is activated by engaging death receptors such as Fas and Tumor Necrosis Factor Receptor (TNFR) with their cognate ligands. This leads to the formation of the membrane-bound death inducing signaling complex (DISC), which recruits the initiator procaspase-8 by the adaptor protein FADD. The intrinsic pathway responds to "intracellular" signals such as DNA damage, oncogene activation, nutrient deprivation, and lineage information (Elmore 2007). The final common end point of both pathways is initiation of execution stage of apoptosis and result in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, crosslinking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells (Elmore 2007). Caspase-3 is considered to be the most important of the executioner caspases and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 specifically activates the endonuclease CAD and CAD then degrades chromosomal DNA within the nuclei and causes chromatin condensation. Caspase-3 also induces cytoskeletal reorganization and disintegration of the cell into apoptotic bodies (Elmore 2007). So, the apoptosis process is very complex and regulated numerous pro- and anti-apoptotic factors.

The role of apoptosis in endometriosis development is studied very intensively at last time. It was demonstrated that apoptosis aids in maintaining cellular homeostasis during the menstrual cycle by eliminating aging cells from the functional layer of the uterine endometrium (Agic et al., 2009). In the normal endometrium apoptosis was detected in the glandular epithelium of late secretory and menstruating endometrium, while very little apoptosis was detected during the proliferative or at the beginning of the secretory phase (Harada et al., 2004). Eutopic endometrium from women with endometriosis has some differences in apoptosis compared with normal endometrium. These differences could contribute to the survival of the regurgitating endometrial cells into the peritoneal cavity and the endometriosis development. It was found, that the percentage of apoptosis in sloughed endometrial cells was greatly reduced among women with endometriosis, implying that the number of surviving cells that enter the peritoneal cavity is greater in women who develop endometriosis (Harada et al., 2004). An increased expression of anti-

characterized by the permeabilization of the outer mitochondrial membrane and the release of several pro-apoptotic factors into the cytosol. These include cytochrome c, Smac/Diablo, AIF (apoptosis-inducing factor), and serine protease HtrA2/Omi (Elmore 2007). These proteins activate the caspase dependent mitochondrial pathway. Cytochrome *c* binds and activates Apaf-1 (the apoptotic protease activating factor-1) as well as procaspase-9, forming an "apoptosome" (Hill et al., 2004). The clustering of procaspase-9 in this manner leads to caspase-9 activation. Different molecules from IAP family (inhibitors of apoptosis proteins) can inhibit activity of Smac/DIABLO and HtrA2/Omi on this stage of apoptosis (Schimmer et al., 2006). The control and regulation of these apoptotic mitochondrial events occurs through members of HSP (heat shock proteins) family, acting as inhibitors of apoptosis. Proteins from Bcl-2 family also regulate the mitochondrial stage of apoptosis and these factors can be either pro-apoptotic or antiapoptotic. Till date, a total of 25 genes have been identified in the Bcl-2 family. Some of the anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG, and some of the pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk. These proteins have special significance since they can determine if the cell is committed to apoptosis or else abort the process. The tumor suppressor protein p53 has a critical role in regulation of the Bcl-2 family of proteins (Elmore 2007). The extrinsic pathway is activated by engaging death receptors such as Fas and Tumor Necrosis Factor Receptor (TNFR) with their cognate ligands. This leads to the formation of the membrane-bound death inducing signaling complex (DISC), which recruits the initiator procaspase-8 by the adaptor protein FADD. The intrinsic pathway responds to "intracellular" signals such as DNA damage, oncogene activation, nutrient deprivation, and lineage information (Elmore 2007). The final common end point of both pathways is initiation of execution stage of apoptosis and result in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, crosslinking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells (Elmore 2007). Caspase-3 is considered to be the most important of the executioner caspases and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 specifically activates the endonuclease CAD and CAD then degrades chromosomal DNA within the nuclei and causes chromatin condensation. Caspase-3 also induces cytoskeletal reorganization and disintegration of the cell into apoptotic bodies (Elmore 2007). So, the apoptosis process is very complex and regulated numerous pro- and

The role of apoptosis in endometriosis development is studied very intensively at last time. It was demonstrated that apoptosis aids in maintaining cellular homeostasis during the menstrual cycle by eliminating aging cells from the functional layer of the uterine endometrium (Agic et al., 2009). In the normal endometrium apoptosis was detected in the glandular epithelium of late secretory and menstruating endometrium, while very little apoptosis was detected during the proliferative or at the beginning of the secretory phase (Harada et al., 2004). Eutopic endometrium from women with endometriosis has some differences in apoptosis compared with normal endometrium. These differences could contribute to the survival of the regurgitating endometrial cells into the peritoneal cavity and the endometriosis development. It was found, that the percentage of apoptosis in sloughed endometrial cells was greatly reduced among women with endometriosis, implying that the number of surviving cells that enter the peritoneal cavity is greater in women who develop endometriosis (Harada et al., 2004). An increased expression of anti-

anti-apoptotic factors.

apoptotic factor Bcl-2 and decrease expression of pro-apoptotic factor Bax were found in proliferative eutopic endometrium from women with endometriosis (Meresman et al., 2000). These differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and development of endometriosis (Taniguchi 2011). In ectopic endometrium of women with endometriosis the level of Bcl-2 expression was significantly increased in stromal cells (Harada et al., 2004). In endometriotic lesions the alterations in expression of different apoptosis-associated genes, such as PTEN, p52 and Bcl-2 were also demonstrated (Nezha et al., 2008). Authors thought that these changes are analogous in tumor tissue and evidence about possible malignant transformation of endometriotic tissue. At the same time there are some data about absence of significant changes of apoptotsis both in eutopic and in ectopic endometrium in women with endometriosis (Hassa et al., 2009). So, the study of the mechanisms, regulating apoptosis in endometriosis, need to be continued.

We also have checked out the expression of mRNA of some genes, possessed both pro- and anti-apoptotic action, in eutopic and ectopic endometrium of women with endometriosis. It was found that the expression of genes, regulating apoptosis, in endometrial tissue of women with endometriosis significantly differed from that in healthy women (Table 1).


Table 1. The level of the expression of mRNAs genes, regulating apoptosis, in the eutopic and ectopic endometrium of women with endometriosis (\* – given in comparison to the endometrium of healthy women, \*- p<0.05; x - given in comparison to the eutopic endometrium of women with endometriosis, x – p<0.05, xx- p<0.01)

In eutopic endometrium of women with endometriosis the expression of anti-apoptotic factor XIAP (X-linked inhibitor of apoptosis) mRNAs were statistically increased compared to that in the endometrium of women from the control group.

In ectopic endometrial tissue the high levels of caspase-3 and heat shock protein 27 (HSP27) mRNAs expression were seen. Both in eutopic and ectopic endometrium of endometriosis women the low level of metallothionein-1 (MT-1) mRNA expression was found compared to that in the endometrium of women without endometriosis.

Thus, the profile of genes, regulating apoptosis, was different in eutopic and ectopic endometrium in endometriosis except the expression of MT-1 mRNA which was decreased both in eutopic endometrium and in endometriotic lesions. It is known that metalloteoneins (MTs) are a group of ubiquitous low-molecular-weight proteins essential for the protection of cells against heavy metal ion toxicity. These molecules also are directly involved in regulation of cell growth, differentiation and apoptosis in different pathological conditions and in tumour as well (Inoue et al., 2009). Earlier the low level of MT-1 protein was demonstrated in ovarian endometriomas (Wicherek et al., 2006).

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 219

It has been shown that early lesions formation is an invasion event that requires breakdown of the extracellular matrix (ECM) proteins (Nap et al., 2004). The ECM consists of collagens, proteoglycans and glycoproteins, including fibronectin and laminin (Curry & Osteen, 2003). The ECM has become recognized as a key regulatory component in cellular physiology, providing an environment for cell migration, division, differentiation, anchorage, and, in some cases, an ultimate fate between cell survival and cell death. Additionally, the ECM is important in metabolic processes, influencing cellular proliferation, differentiation and apoptosis and it serves as a repository for biologically active growth factors (Nap et al., 2004). The highly regulated control of ECM turnover and homeostasis occurs, in part, by the action of a specific class of proteolytic enzymes. Supposedly, enzymes which belong to the system of plasminogen and matrix metalloproteinase family play the important role in

Plasminogen, a ubiquitous protein secreted by liver, is activated into a protease plasmin by two types of activators. These are tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which bind to a specific cellular receptor (uPA-R). uPA initiates pericellular proteolysis of the ECM. This system is perfectly controlled by powerful inhibitors, some at the level of the plasminogen activators (type 1 and type 2 Plasminogen Activator Inhibitor, PAI-1 and PAI-2, respectively), and others at the level of the plasmin. uPA plays an important role in uterine physiology and in menstruation (Vinatier et al., 2001). Some results evidence about involvement of plasminogen system in mechanisms of endometriosis development. It was found that concentrations of uPA and PAI-1 were strongly increased both in endometriotic lesions and in matched eutopic endometrium of women with endometriosis compared to that in healthy women (Bruse et al., 1998). But there are some works which didn't find any differences in the expression of the proteins from plasminogen system in endometrial tissue in endometriosis (Vinatier et al., 2001). So,

In last years the role of proteases from matrix metalloproteinase (MMPs) family in mechanisms of endometriosis establishment is intensively investigated. It was found that the MMP system is actively involved in the control of different aspects of reproductive function. In the ovary and uterus, MMP system regulates the dynamic structural changes that occur throughout the menstrual cycle (Curry & Osteen, 2003). The MMPs family consists of several structurally related Zn2+-dependent secreted endopeptidases. Currently, it is recognized more that 26 endopeptidases that include four broad classes: the collagenases, gelatinases, stromelysins, and membrane type enzymes (MT-MMPs). These proteinases exhibit numerous structural and functional similarities. All of them have conserved domain structures and specific domains related to substrate specificity and recognition of other proteins (Amalinie et al., 2010). Common features of the MMPs family include: 1) the presence of zinc in the active site of the catalytic domain, 2) synthesis of the MMPs as preproenzymes that are secreted in an inactive form, 3) activation of the latent zymogen in the extracellular space, 4) recognition and cleavage of the ECM by the catalytic domain of the enzyme, and 5) inhibition of enzyme action by both serum-borne and tissuederived metalloproteinase inhibitors or TIMPs in the extracellular environment (Curry & Osteen, 2003). Although similarities exist in the structure of the MMPs, there are also distinct differences in the recognition and specificity for components of the ECM. For

**3.2 Changes of the invasiveness of endometrium in endometriosis** 

ECM remodeling during endometriotic lesions formation (Vinatier et al., 2001).

the role of these enzymes in endometriosis is not clear yet.

Likely, diminishment of the MT-1 synthesis in endometrium of women with endometriosis might lead to the impairment of apoptosis control during this pathology. Another important apoptosis regulator, XIAP, is one of the members of IAP (inhibitory apoptosis proteins) family (Mufti et al., 2007). IAPs proteins are selectively bind and inhibit caspases-3, -7 and -9. XIAP is the only member of this family able to directly inhibit both the initiation and execution phase of the caspase cascade. XIAP is frequently overexpressed in malignant cells and is associated with poor clinical outcome (Schimmer et al., 2006). According to our data XIAP synthesis was significantly increased in eutopic endometrium in endometriosis, but in endometriotic lesions the level of XIAP mRNA expression didn't differ from that in normal endometrium. The high level of XIAP mRNA expression in eutopic endometrium possibly can lead to the decrease of spontaneous apoptosis in endometrium of women with endometriosis and in menstrual endometrium as well. This phenomenon might be responsible for the elevation of viability of endometrial cells in peritoneal cavity and play an important role at the initial stages of endometriotic lesions formation. But the absence of the elevation of XIAP synthesis in ectopic endometrium evidence in favor of the benign character of endometriotic lesions growth. Likely, the growth of already formed endometriotic tissue isn't under the XIAP control. Evidently, mechanisms, regulating endometrial XIAP synthesis, are different in eutopic and ectopic endometrium. The same was noted for the caspase-3 and HSP27. Only in ectopic endometrium we have seen the high level of the caspase-3 and HSP27 synthesis compared to that in endometrium of healthy women. From one side, these results are controversial, because caspase-3 is one of the main proapoptotic factors, a central effector caspase involved in numerous apoptotic pathways (Voss et al., 2007). On the contrary, HSP27 is well known inhibitor of apoptosis because the most heat shock proteins and HSP27 as well have strong cytoprotective effects and behave as molecular chaperone for other cellular proteins (Schmitt et al., 2007). HSP27 can bind to pro-caspase-3 to prevent its cleavage and activation by caspase-9 (Pandey et al., 2000). It does so by directly sequestering cytochrome c when released from the mitochondria into the cytosol (Schmitt et al., 2007). But as it is known HSP27 can interact with different partners implicated in the apoptotic process. For example, under stress conditions HSP27 increases IκBα ubiquitination/degradation, which results in an increase in NFκ-B activity and increased survival (Parcellier et al., 2003). So, the high level of HSP27 synthesis in endometriotic lesions can protect cells form apoptosis in caspase-independent pathway. Possibly the thin balance between mechanisms inducing and inhibiting apoptosis exists in endometriosis tissue. The growth of the endometriotic tissue was accompanied by the high level of synthesis of anti-apoptotic molecules HSP27. These changes are similar to that in the cancer cells and might be responsible for the invasion of endometrium into the peritoneum in the process of lesions formation. But in contrast to tumor cells the high level of caspase-3 mRNA expression in endometriosis lesions might provide the benign character of its growth.

Thus, both literature data and our own results evidence the impairment of apoptosis regulation in endometriosis. It must be special noted, that mechanisms of regulation of apoptosis in eutopic and ectopic endometrium are different. In eutopic endometrium the expression profile of apoptosis-related genes possibly contribute to the increase of viability of endometrial cells. But in endometriotic lesions we found the simultaneously elevated expression of both pro- and anti-apoptotic genes. Likely, this phenomenon might be responsible for benign type of ectopic growth of endometrium in endometriosis.

#### **3.2 Changes of the invasiveness of endometrium in endometriosis**

218 Endometriosis - Basic Concepts and Current Research Trends

Likely, diminishment of the MT-1 synthesis in endometrium of women with endometriosis might lead to the impairment of apoptosis control during this pathology. Another important apoptosis regulator, XIAP, is one of the members of IAP (inhibitory apoptosis proteins) family (Mufti et al., 2007). IAPs proteins are selectively bind and inhibit caspases-3, -7 and -9. XIAP is the only member of this family able to directly inhibit both the initiation and execution phase of the caspase cascade. XIAP is frequently overexpressed in malignant cells and is associated with poor clinical outcome (Schimmer et al., 2006). According to our data XIAP synthesis was significantly increased in eutopic endometrium in endometriosis, but in endometriotic lesions the level of XIAP mRNA expression didn't differ from that in normal endometrium. The high level of XIAP mRNA expression in eutopic endometrium possibly can lead to the decrease of spontaneous apoptosis in endometrium of women with endometriosis and in menstrual endometrium as well. This phenomenon might be responsible for the elevation of viability of endometrial cells in peritoneal cavity and play an important role at the initial stages of endometriotic lesions formation. But the absence of the elevation of XIAP synthesis in ectopic endometrium evidence in favor of the benign character of endometriotic lesions growth. Likely, the growth of already formed endometriotic tissue isn't under the XIAP control. Evidently, mechanisms, regulating endometrial XIAP synthesis, are different in eutopic and ectopic endometrium. The same was noted for the caspase-3 and HSP27. Only in ectopic endometrium we have seen the high level of the caspase-3 and HSP27 synthesis compared to that in endometrium of healthy women. From one side, these results are controversial, because caspase-3 is one of the main proapoptotic factors, a central effector caspase involved in numerous apoptotic pathways (Voss et al., 2007). On the contrary, HSP27 is well known inhibitor of apoptosis because the most heat shock proteins and HSP27 as well have strong cytoprotective effects and behave as molecular chaperone for other cellular proteins (Schmitt et al., 2007). HSP27 can bind to pro-caspase-3 to prevent its cleavage and activation by caspase-9 (Pandey et al., 2000). It does so by directly sequestering cytochrome c when released from the mitochondria into the cytosol (Schmitt et al., 2007). But as it is known HSP27 can interact with different partners implicated in the apoptotic process. For example, under stress conditions HSP27 increases IκBα ubiquitination/degradation, which results in an increase in NFκ-B activity and increased survival (Parcellier et al., 2003). So, the high level of HSP27 synthesis in endometriotic lesions can protect cells form apoptosis in caspase-independent pathway. Possibly the thin balance between mechanisms inducing and inhibiting apoptosis exists in endometriosis tissue. The growth of the endometriotic tissue was accompanied by the high level of synthesis of anti-apoptotic molecules HSP27. These changes are similar to that in the cancer cells and might be responsible for the invasion of endometrium into the peritoneum in the process of lesions formation. But in contrast to tumor cells the high level of caspase-3 mRNA expression in endometriosis lesions might provide the benign

Thus, both literature data and our own results evidence the impairment of apoptosis regulation in endometriosis. It must be special noted, that mechanisms of regulation of apoptosis in eutopic and ectopic endometrium are different. In eutopic endometrium the expression profile of apoptosis-related genes possibly contribute to the increase of viability of endometrial cells. But in endometriotic lesions we found the simultaneously elevated expression of both pro- and anti-apoptotic genes. Likely, this phenomenon might be

responsible for benign type of ectopic growth of endometrium in endometriosis.

character of its growth.

It has been shown that early lesions formation is an invasion event that requires breakdown of the extracellular matrix (ECM) proteins (Nap et al., 2004). The ECM consists of collagens, proteoglycans and glycoproteins, including fibronectin and laminin (Curry & Osteen, 2003). The ECM has become recognized as a key regulatory component in cellular physiology, providing an environment for cell migration, division, differentiation, anchorage, and, in some cases, an ultimate fate between cell survival and cell death. Additionally, the ECM is important in metabolic processes, influencing cellular proliferation, differentiation and apoptosis and it serves as a repository for biologically active growth factors (Nap et al., 2004). The highly regulated control of ECM turnover and homeostasis occurs, in part, by the action of a specific class of proteolytic enzymes. Supposedly, enzymes which belong to the system of plasminogen and matrix metalloproteinase family play the important role in ECM remodeling during endometriotic lesions formation (Vinatier et al., 2001).

Plasminogen, a ubiquitous protein secreted by liver, is activated into a protease plasmin by two types of activators. These are tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which bind to a specific cellular receptor (uPA-R). uPA initiates pericellular proteolysis of the ECM. This system is perfectly controlled by powerful inhibitors, some at the level of the plasminogen activators (type 1 and type 2 Plasminogen Activator Inhibitor, PAI-1 and PAI-2, respectively), and others at the level of the plasmin. uPA plays an important role in uterine physiology and in menstruation (Vinatier et al., 2001). Some results evidence about involvement of plasminogen system in mechanisms of endometriosis development. It was found that concentrations of uPA and PAI-1 were strongly increased both in endometriotic lesions and in matched eutopic endometrium of women with endometriosis compared to that in healthy women (Bruse et al., 1998). But there are some works which didn't find any differences in the expression of the proteins from plasminogen system in endometrial tissue in endometriosis (Vinatier et al., 2001). So, the role of these enzymes in endometriosis is not clear yet.

In last years the role of proteases from matrix metalloproteinase (MMPs) family in mechanisms of endometriosis establishment is intensively investigated. It was found that the MMP system is actively involved in the control of different aspects of reproductive function. In the ovary and uterus, MMP system regulates the dynamic structural changes that occur throughout the menstrual cycle (Curry & Osteen, 2003). The MMPs family consists of several structurally related Zn2+-dependent secreted endopeptidases. Currently, it is recognized more that 26 endopeptidases that include four broad classes: the collagenases, gelatinases, stromelysins, and membrane type enzymes (MT-MMPs). These proteinases exhibit numerous structural and functional similarities. All of them have conserved domain structures and specific domains related to substrate specificity and recognition of other proteins (Amalinie et al., 2010). Common features of the MMPs family include: 1) the presence of zinc in the active site of the catalytic domain, 2) synthesis of the MMPs as preproenzymes that are secreted in an inactive form, 3) activation of the latent zymogen in the extracellular space, 4) recognition and cleavage of the ECM by the catalytic domain of the enzyme, and 5) inhibition of enzyme action by both serum-borne and tissuederived metalloproteinase inhibitors or TIMPs in the extracellular environment (Curry & Osteen, 2003). Although similarities exist in the structure of the MMPs, there are also distinct differences in the recognition and specificity for components of the ECM. For

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 221

the prominent changes of MMPs production in animals with experimental endometriosis have been demonstrated. The level of MMP-3 and MMP-9 was significantly higher in endometriotic lesions in rats with experimental endometriosis than in matched eutopic endometrium (Machado et al., 2010; Cox et al., 2001). Suppression of MMP-2 and MMP-9 activity in mice led to the inhibition of endometriosis progression in animals with the decrease of endometriotic lesions weight (Chen et al., 2010). Investigation of the role of MMPs in human endometrial explants in a chicken chorioallantoic membrane model (CAM) of endometriosis allowed to establish that endometrium both of healthy women and patients with endometriosis showed a statistically significant increase of MMP-1 mRNA expression 24, 48, 72, and 96 hours after transfer to the CAM and no statistically significant difference regarding the MMPs mRNA expression was shown for endometrium of healthy women and endometriosis patients (Juhasz-Boss et al., 2010). Earlier we have also studied the synthesis of mRNA of MMP-2 and TIMP-2 in rat ectopic endometrium at different stages of experimental endometriosis development. We found that the dynamic of MMP-2 mRNA expression in ectopic experimental lesions was characterized by its significant increase at 7th day after transplantation comparing to that in native uterine endometrium with subsequent declining to normal values at 14th and 21st days after surgery. The level of TIMP-2 mRNA expression on the contrary was sharply decreased at early and middle stages of endometriotic lesions formation and was elevated at the late stage of experimental endometriosis comparing to that in intact uterine endometrium (Sotnikova et al., 2010). So, during the first 7 days of experimental endometriosis lesions development, the balance of pro- and anti-proteolytic activity in endometrial tissue was shifted towards MMP-2 prevalence. It is known that during this period the maximal level of endometrial tissue invasion and cell proliferation are seen in rat endometriosis model. Our results evidence about the coincidence of high level of MMP-2 expression and high tissue's invasiveness in early experimental endometriosis lesions and allow us to suggest the direct involvement of MMP-2 in invasion of

endometrial cells at the initial stage of endometriosis development.

al., 2010).

The role of MMP-2 and MMP-9 in endometriosis development has a special interest. It was demonstrated that these two gelatinases actively participate in tumor invasion and progression (Amalinei et al., 2010). Initially, MMP-9 or gelatinase-B, was considered a key MMP in the invasion and metastasis, overexpressed by cancer cells and induced by several cytokines, growth factors and oncogene products (Okada et al., 2001), and its inhibition resulting in loss of metastatic potential. Subsequent studies demonstrated that the activation ratios of pro-MMP-2, not of pro-MMP-9, correlate with lymph node metastasis in breast, lung, thyroid and digestive tract carcinomas (Amalinei et al., 2010). MMP-2 has been shown to play a key role in promotion of invasiveness both of normal and neoplastic cells. Cellular localization in many tumor tissues indicates that MMP-2 mRNA appears to be localized to the stromal fibroblast adjacent to the sites of tumor invasion (Amalinei et al., 2010). It was also shown that MMP-2 and MMP-9 expression are closely associated with the parameters of tumor aggressiveness (Karahan et al., 2007) and tumor growth is reduced by the absence of MMP-2, and the metastatic processes is reduced by the lack of MMP-9 ( Egeblad & Werb, 2002). But MMP-2 and MMP-9 are not tumor specific and are involved in ECM remodeling in a wide range of non-neoplastic processes, including embryonic development, trophoblastic invasion, angiogenesis, T-cell transmigration and wound healing (Amalinei et

example, collagenases (MMP-1, MMP-8, and MMP-13) degrade native fibrillar collagen of types I, II, III, V, and XI. Gelatin, another important protein of ECM, is susceptible to a wide range of tissue proteinases, including the gelatinases MMP-2 and MMP-9. The stromelysin enzymes (MMP-3, MMP-7, MMP-10, and MMP-11) act on a broad and diverse array of ECM substrates. Both gelatinases and stromelysins are capable of degrading major constituents of basement membranes, including type IV collagen, laminin, and fibronectin. In addition to degrading the ECM, the MMPs exhibit activity toward other MMPs, growth factors, and cytokines such as IGF binding proteins, epidermal growth factor (EGF), TNF-α (Curry & Osteen, 2003).

Involvement of MMPs in endometriosis development was suspected after collagen breakdown products were found in the peritoneal fluid of patients with endometriosis (Spuijbroek et al., 1992) and since that time many studies have demonstrated that the pattern of MMPs expression in endometrium and peritoneal fluid of women with endometriosis significantly differ from that in healthy women (Nap et al, 2004). Increased levels of MMP-1, MMP-2, MMP-7 and MMP-9 were detected in peritoneal fluid of patients with endometriosis (Amalinie et al., 2010). At the same time the amount of the MMP-13 in peritoneal fluid of patients was significantly decreased in comparison to that in healthy women (Laudanski et al., 2005). In eutopic endometrium of women with endometriosis the elevation of the expression of MMP-1, MMP-2, MMP-3, MMP-9 was found (Di Carlo et al., 2009; Shaco-Levy et al., 2008). Diminishment of the expression of MMPs inhibitors TIMP-1 and TIMP-2 in endometrium of endometriosis patients was also noted (Colett et al., 2004; Uzan et al., 2004). The study of the character of MMPs production at the level of ectopic endometrium had shown that MMP-2 and MMP-3 overexpression were related to the infiltrative nature of endometriotic lesion (Uzan et al., 2004). It was also shown that circulating mRNA for MMP-3 was significantly higher in peripheral blood of patients with endometriosis than in control patients, regardless of the degree of endometriosis severity (De Sanctis et al., 2011). In a prospective, blinded, longitudinal study MMP-2 and MMP-9 were more likely to be detected in the urine of patients with endometriosis than in controls (Becker et al., 2010). Thus, practically all received data evidence about the increased production of MMPs both at the systemic and local level in endometriosis. But there are some reports that contradict these results. For example, Colett with coworkers didn't find any difference in the MMP-2 production in endometrial tissue of women with endometriosis in comparison with healthy women (Colett et al., 2004). Very low production of MMP-9 by endometrial cells was seen in endometriosis women (Sillme et al., 2001). So, the role of MMPs in endometriosis pathogenesis is still unclear.

The majority of works are only descriptive and functional involvement of MMPs in the development or progression of endometriosis has not been proven. It is very difficult to study the ectopic endometrium in humans as controlled experiments are limited, because it is not possible to monitor the disease progression without repeated laparoscopies which is difficult on many grounds. In such cases animal models are an extremely important tool in elucidating the pathogenesis of the disease. Animals with experimental endometriosis let us to evaluate the different stages of ectopic lesion formation. Both nonprimate and primate models have been used to study endometriosis for many years. Nonprimates, including rodents, do not undergo spontaneous disease, but it can be induced using either autologous uterine tissue or human endometrium (Story and Kennedy, 2004). Up to now

example, collagenases (MMP-1, MMP-8, and MMP-13) degrade native fibrillar collagen of types I, II, III, V, and XI. Gelatin, another important protein of ECM, is susceptible to a wide range of tissue proteinases, including the gelatinases MMP-2 and MMP-9. The stromelysin enzymes (MMP-3, MMP-7, MMP-10, and MMP-11) act on a broad and diverse array of ECM substrates. Both gelatinases and stromelysins are capable of degrading major constituents of basement membranes, including type IV collagen, laminin, and fibronectin. In addition to degrading the ECM, the MMPs exhibit activity toward other MMPs, growth factors, and cytokines such as IGF binding proteins, epidermal growth factor (EGF), TNF-α (Curry &

Involvement of MMPs in endometriosis development was suspected after collagen breakdown products were found in the peritoneal fluid of patients with endometriosis (Spuijbroek et al., 1992) and since that time many studies have demonstrated that the pattern of MMPs expression in endometrium and peritoneal fluid of women with endometriosis significantly differ from that in healthy women (Nap et al, 2004). Increased levels of MMP-1, MMP-2, MMP-7 and MMP-9 were detected in peritoneal fluid of patients with endometriosis (Amalinie et al., 2010). At the same time the amount of the MMP-13 in peritoneal fluid of patients was significantly decreased in comparison to that in healthy women (Laudanski et al., 2005). In eutopic endometrium of women with endometriosis the elevation of the expression of MMP-1, MMP-2, MMP-3, MMP-9 was found (Di Carlo et al., 2009; Shaco-Levy et al., 2008). Diminishment of the expression of MMPs inhibitors TIMP-1 and TIMP-2 in endometrium of endometriosis patients was also noted (Colett et al., 2004; Uzan et al., 2004). The study of the character of MMPs production at the level of ectopic endometrium had shown that MMP-2 and MMP-3 overexpression were related to the infiltrative nature of endometriotic lesion (Uzan et al., 2004). It was also shown that circulating mRNA for MMP-3 was significantly higher in peripheral blood of patients with endometriosis than in control patients, regardless of the degree of endometriosis severity (De Sanctis et al., 2011). In a prospective, blinded, longitudinal study MMP-2 and MMP-9 were more likely to be detected in the urine of patients with endometriosis than in controls (Becker et al., 2010). Thus, practically all received data evidence about the increased production of MMPs both at the systemic and local level in endometriosis. But there are some reports that contradict these results. For example, Colett with coworkers didn't find any difference in the MMP-2 production in endometrial tissue of women with endometriosis in comparison with healthy women (Colett et al., 2004). Very low production of MMP-9 by endometrial cells was seen in endometriosis women (Sillme et al., 2001). So, the role of

The majority of works are only descriptive and functional involvement of MMPs in the development or progression of endometriosis has not been proven. It is very difficult to study the ectopic endometrium in humans as controlled experiments are limited, because it is not possible to monitor the disease progression without repeated laparoscopies which is difficult on many grounds. In such cases animal models are an extremely important tool in elucidating the pathogenesis of the disease. Animals with experimental endometriosis let us to evaluate the different stages of ectopic lesion formation. Both nonprimate and primate models have been used to study endometriosis for many years. Nonprimates, including rodents, do not undergo spontaneous disease, but it can be induced using either autologous uterine tissue or human endometrium (Story and Kennedy, 2004). Up to now

MMPs in endometriosis pathogenesis is still unclear.

Osteen, 2003).

the prominent changes of MMPs production in animals with experimental endometriosis have been demonstrated. The level of MMP-3 and MMP-9 was significantly higher in endometriotic lesions in rats with experimental endometriosis than in matched eutopic endometrium (Machado et al., 2010; Cox et al., 2001). Suppression of MMP-2 and MMP-9 activity in mice led to the inhibition of endometriosis progression in animals with the decrease of endometriotic lesions weight (Chen et al., 2010). Investigation of the role of MMPs in human endometrial explants in a chicken chorioallantoic membrane model (CAM) of endometriosis allowed to establish that endometrium both of healthy women and patients with endometriosis showed a statistically significant increase of MMP-1 mRNA expression 24, 48, 72, and 96 hours after transfer to the CAM and no statistically significant difference regarding the MMPs mRNA expression was shown for endometrium of healthy women and endometriosis patients (Juhasz-Boss et al., 2010). Earlier we have also studied the synthesis of mRNA of MMP-2 and TIMP-2 in rat ectopic endometrium at different stages of experimental endometriosis development. We found that the dynamic of MMP-2 mRNA expression in ectopic experimental lesions was characterized by its significant increase at 7th day after transplantation comparing to that in native uterine endometrium with subsequent declining to normal values at 14th and 21st days after surgery. The level of TIMP-2 mRNA expression on the contrary was sharply decreased at early and middle stages of endometriotic lesions formation and was elevated at the late stage of experimental endometriosis comparing to that in intact uterine endometrium (Sotnikova et al., 2010). So, during the first 7 days of experimental endometriosis lesions development, the balance of pro- and anti-proteolytic activity in endometrial tissue was shifted towards MMP-2 prevalence. It is known that during this period the maximal level of endometrial tissue invasion and cell proliferation are seen in rat endometriosis model. Our results evidence about the coincidence of high level of MMP-2 expression and high tissue's invasiveness in early experimental endometriosis lesions and allow us to suggest the direct involvement of MMP-2 in invasion of endometrial cells at the initial stage of endometriosis development.

The role of MMP-2 and MMP-9 in endometriosis development has a special interest. It was demonstrated that these two gelatinases actively participate in tumor invasion and progression (Amalinei et al., 2010). Initially, MMP-9 or gelatinase-B, was considered a key MMP in the invasion and metastasis, overexpressed by cancer cells and induced by several cytokines, growth factors and oncogene products (Okada et al., 2001), and its inhibition resulting in loss of metastatic potential. Subsequent studies demonstrated that the activation ratios of pro-MMP-2, not of pro-MMP-9, correlate with lymph node metastasis in breast, lung, thyroid and digestive tract carcinomas (Amalinei et al., 2010). MMP-2 has been shown to play a key role in promotion of invasiveness both of normal and neoplastic cells. Cellular localization in many tumor tissues indicates that MMP-2 mRNA appears to be localized to the stromal fibroblast adjacent to the sites of tumor invasion (Amalinei et al., 2010). It was also shown that MMP-2 and MMP-9 expression are closely associated with the parameters of tumor aggressiveness (Karahan et al., 2007) and tumor growth is reduced by the absence of MMP-2, and the metastatic processes is reduced by the lack of MMP-9 ( Egeblad & Werb, 2002). But MMP-2 and MMP-9 are not tumor specific and are involved in ECM remodeling in a wide range of non-neoplastic processes, including embryonic development, trophoblastic invasion, angiogenesis, T-cell transmigration and wound healing (Amalinei et al., 2010).

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 223

growth of ectopic lesion. But from another side, it was shown that excessive levels of TIMP-1 protein such as those secreted by endometriotic lesions into the peritoneal cavity negatively affects the reproduction function of rats with experimental endometriosis (Stilley et al., 2010). It was also shown that the rat embryo treated in vitro with endometriotic peritoneal fluid concentrations of TIMP-1 developed abnormally, and rats treated with a TIMP-1 function-blocking antibody had normal zygote, follicle and embryo quality (Stilley et al., 2010). Summarizing their results authors hypothesize that excessive TIMP-1 was deleterious to ovulation and embryo development because endometriotic lesion-secreted TIMP-1 can translocate to ovary/or oviduct and cause poor preimplantation embryo quality, developmental arrest and the subsequent embryo loss found in endometriosis (Stilley et al., 2010). Thus, the high level of TIMP-1 synthesis in ectopic endometrium might be associated

with mechanisms of infertility development in women with endometriosis.

Fig. 1. Comparative characteristic of the synthesis of the enzymes with pro- and antiproteolytic activity in eutopic and ectopic endometrium of women with endometriosis

factors influence upon apoptosis and invasiveness of endometrial cells.

**endometriosis** 

**4. Influence of peritoneal macrophages and peritoneal fluid upon** 

**mechanisms, regulating apoptosis and invasiveness of endometrium in** 

The mechanisms by which regurgitated endometrial cells are cleared from the peritoneal cavity in the majority of women are poorly understood yet. However, it has been suggested that a peritoneal microenvironment might subserve this role (Vigano et al., 2004). There are

Thus, endometriotic lesion establishment and growth are accompanied by the impairment of regulation of apoptosis and invasion in endometrial tissue. These changes at the level of eutopic endometrium possibly are associated with the increase of the viability of endometrial cells and participate in early lesions formation events. At the level of already formed ectopic endometrium we found the significant increase of invasive capacity and elevated synthesis of both pro- and anti-apoptotic factors which might be responsible for the benign type of ectopic lesions growth. Earlier it was suggested that many differences observed between eutopic and ectopic tissue of a patients with endometriosis can be explained as the direct consequence of the different environment of peritoneal fluid (Hara et al., 2004). So, the next step of our investigation was to study the possible peritoneal fluid

Endometriosis often is defined as benign tumor. Taking into account literature data about action of MMP-2 and MMP-9 during tumor progression we checked out synthesis on these MMPs and its inhibitors TIMP-2 and TIMP-1 in eutopic and ectopic endometrium of healthy women and women with endometriosis. We found, that in eutopic endometrium of women with endometriosis the expression of MMP-2, TIMP-2 and TIMP-1 mRNAs were statistically increased compared to that in the endometrium of women from control group. In ectopic endometrial tissue the high levels of MMP-2, MMP-9 and TIMP-1 mRNAs expression were seen (Table 2). So, the distinct characteristic of endometriotic lesion is the high level of MMP-2 and MMP-9 synthesis which might provide invasive character of ectopic endometrium growth. But the eutopic endometrium doesn't posses such invasive activity. Evidently, some factors may influence upon MMPs synthesis in endometrial tissue of women with endometriosis elevating the mRNA expression of MMP-2 and MMP-9.


Table 2. The level of the expression of mRNAs for genes, regulating invasiveness, in the eutopic and ectopic endometrium of women with endometriosis (\* – given in comparison to the endometrium of healthy women, \*- p<0.05; x – given in comparison to the eutopic endometrium of women with endometriosis, x – p<0.05)

It must be noted that the balance of pro- and anti-proteolytic enzymes synthesis was different in eutopic end ectopic endometrium (Fig.1). In eutopic endometrium we have seen the significant prevalence of the mRNA expression of specific inhibitors of MMPs - TIMP-2 and TIMP-1 upon the activity of MMP-2 and MMP-9, respectively. But in ectopic endometrium the level of mRNA expression of MMP-2 and TIMP-2 was practically the same (Fig.1). This situation allows us to compare the growth of ectopic endometrium with tumor growth. It is well known that in urothelial cancer patients the mean MMP-2/TIMP-2 ratio in patients with recurrence is significantly higher than that in patients without recurrence and the disease-free survival of patients with high MMP-2/TIMP-2 ratio is extremely poor compared with that of patients with lower ratios (Gohji et al., 1996). It was demonstrated also that evaluation of MMP-2: TIMP-2 mRNA balance may constitute an early prognostic indicator of human cancer (Onisto et al., 1995). Evidently, the elevation of the balance of MMP-2 and TIMP-2 in endometriotic tissue results in increase of invasive potential of ectopic endometrium. But mechanisms regulating such changes need to be further elucidated. It can be supposed that peritoneal fluid factors can directly influence upon endometrial invasiveness. But in our case we didn't observe such changes in the balance of MMP-9 and TIMP-1. Moreover, the synthesis of TIMP-1 in ectopic endometrium was very high. From one side these changes can be connected with the specific action of TIMP-1 during tumor progression. It was shown that in certain situations TIMP-1 can act as growth promoting factor and/or antiapoptotic factor to the cancer cells (Mannello & Gazzanelli, 2001). And high level of TIMP-1 synthesis might be attributive to enhance the invasive

Endometriosis often is defined as benign tumor. Taking into account literature data about action of MMP-2 and MMP-9 during tumor progression we checked out synthesis on these MMPs and its inhibitors TIMP-2 and TIMP-1 in eutopic and ectopic endometrium of healthy women and women with endometriosis. We found, that in eutopic endometrium of women with endometriosis the expression of MMP-2, TIMP-2 and TIMP-1 mRNAs were statistically increased compared to that in the endometrium of women from control group. In ectopic endometrial tissue the high levels of MMP-2, MMP-9 and TIMP-1 mRNAs expression were seen (Table 2). So, the distinct characteristic of endometriotic lesion is the high level of MMP-2 and MMP-9 synthesis which might provide invasive character of ectopic endometrium growth. But the eutopic endometrium doesn't posses such invasive activity. Evidently, some factors may influence upon MMPs synthesis in endometrial tissue of

women with endometriosis elevating the mRNA expression of MMP-2 and MMP-9.

mRNA MMP-2 0.89±0.27 2.40±0.63\* 5.89±2.29\* mRNA TIMP-2 4.09±0.74 13.53±3.94\* 6.44±1.59 mRNA MMP-9 0.27±0.16 1.37±0.62 13.78±5.04\*x mRNA TIMP-1 2.72±1.33 24.44±9.02\* 87.73± 31.19\*

Eutopic endometrium,

Ectopic endometrium,

endometriosis

(n=18)

endometriosis

(n=17)

Table 2. The level of the expression of mRNAs for genes, regulating invasiveness, in the eutopic and ectopic endometrium of women with endometriosis (\* – given in comparison to the endometrium of healthy women, \*- p<0.05; x – given in comparison to the eutopic

It must be noted that the balance of pro- and anti-proteolytic enzymes synthesis was different in eutopic end ectopic endometrium (Fig.1). In eutopic endometrium we have seen the significant prevalence of the mRNA expression of specific inhibitors of MMPs - TIMP-2 and TIMP-1 upon the activity of MMP-2 and MMP-9, respectively. But in ectopic endometrium the level of mRNA expression of MMP-2 and TIMP-2 was practically the same (Fig.1). This situation allows us to compare the growth of ectopic endometrium with tumor growth. It is well known that in urothelial cancer patients the mean MMP-2/TIMP-2 ratio in patients with recurrence is significantly higher than that in patients without recurrence and the disease-free survival of patients with high MMP-2/TIMP-2 ratio is extremely poor compared with that of patients with lower ratios (Gohji et al., 1996). It was demonstrated also that evaluation of MMP-2: TIMP-2 mRNA balance may constitute an early prognostic indicator of human cancer (Onisto et al., 1995). Evidently, the elevation of the balance of MMP-2 and TIMP-2 in endometriotic tissue results in increase of invasive potential of ectopic endometrium. But mechanisms regulating such changes need to be further elucidated. It can be supposed that peritoneal fluid factors can directly influence upon endometrial invasiveness. But in our case we didn't observe such changes in the balance of MMP-9 and TIMP-1. Moreover, the synthesis of TIMP-1 in ectopic endometrium was very high. From one side these changes can be connected with the specific action of TIMP-1 during tumor progression. It was shown that in certain situations TIMP-1 can act as growth promoting factor and/or antiapoptotic factor to the cancer cells (Mannello & Gazzanelli, 2001). And high level of TIMP-1 synthesis might be attributive to enhance the invasive

Endometrium, control (n=5)

endometrium of women with endometriosis, x – p<0.05)

Parameter, copies number/μl growth of ectopic lesion. But from another side, it was shown that excessive levels of TIMP-1 protein such as those secreted by endometriotic lesions into the peritoneal cavity negatively affects the reproduction function of rats with experimental endometriosis (Stilley et al., 2010). It was also shown that the rat embryo treated in vitro with endometriotic peritoneal fluid concentrations of TIMP-1 developed abnormally, and rats treated with a TIMP-1 function-blocking antibody had normal zygote, follicle and embryo quality (Stilley et al., 2010). Summarizing their results authors hypothesize that excessive TIMP-1 was deleterious to ovulation and embryo development because endometriotic lesion-secreted TIMP-1 can translocate to ovary/or oviduct and cause poor preimplantation embryo quality, developmental arrest and the subsequent embryo loss found in endometriosis (Stilley et al., 2010). Thus, the high level of TIMP-1 synthesis in ectopic endometrium might be associated with mechanisms of infertility development in women with endometriosis.

Fig. 1. Comparative characteristic of the synthesis of the enzymes with pro- and antiproteolytic activity in eutopic and ectopic endometrium of women with endometriosis

Thus, endometriotic lesion establishment and growth are accompanied by the impairment of regulation of apoptosis and invasion in endometrial tissue. These changes at the level of eutopic endometrium possibly are associated with the increase of the viability of endometrial cells and participate in early lesions formation events. At the level of already formed ectopic endometrium we found the significant increase of invasive capacity and elevated synthesis of both pro- and anti-apoptotic factors which might be responsible for the benign type of ectopic lesions growth. Earlier it was suggested that many differences observed between eutopic and ectopic tissue of a patients with endometriosis can be explained as the direct consequence of the different environment of peritoneal fluid (Hara et al., 2004). So, the next step of our investigation was to study the possible peritoneal fluid factors influence upon apoptosis and invasiveness of endometrial cells.

#### **4. Influence of peritoneal macrophages and peritoneal fluid upon mechanisms, regulating apoptosis and invasiveness of endometrium in endometriosis**

The mechanisms by which regurgitated endometrial cells are cleared from the peritoneal cavity in the majority of women are poorly understood yet. However, it has been suggested that a peritoneal microenvironment might subserve this role (Vigano et al., 2004). There are

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 225

macrophages and peritoneal fluid of healthy women increased the amount of apoptotic Annexin V+ endometrial cells (Table 4). The elevation of the amount of apoptotic cells was noted predominantly in population of endometrial stromal cells, entered in the

Annexin V+ 63.36±2.86 78.14±4.21\* 81.90±1.24\*\*\* Annexin V+Pi- 25.62±2.96 29.54±2.24 27.55±2.35 Annexin V+Pi+ 37.74±4.12 53.23±4.25\* 54.35±2.25\*\* Table 4. The influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrium stromal cells in healthy women (\* – given in comparison to the

So, our results let us to conclude that in physiological conditions the endometrial cells are effectively eliminated from the peritoneal cavity due to induction of apoptosis by peritoneal macrophages. Peritoneal fluid, according to our data, is less important for induction of apoptosis in endometrium of healthy women. Only macrophages of women without endometriosis are capable to increase the synthesis one of the most important pro-apoptotic factor - caspase-3 in autologous endometrial tissue. Earlier it was shown that the control of tissue homeostasis by induction of apoptosis in aging and transformed cells is one of the important macrophages function (Gordon & Freedman, 2006). It is also known that apoptotic cells are rapidly engulfed by phagocytes in a process akin to macro pinocytosis. Normally, the uptake of apoptotic cells is accompanies by the induction of antiinflammatory cytokine synthesis of phagocytes (Erwig & Henson, 2007). Our results have shown that MT-1 synthesis significant elevated in endometrium of healthy women after its incubation with peritoneal macrophages. It is known that metallothioneins demonstrated strong antioxidant properties and able to scavenge a wide range of reactive oxygen species thus serving as anti-inflammatory mediators (Inoue et al., 2009). So, the increase of MT-1 synthesis in endometrial cells, undergoing apoptosis, might be one of the factors, participating in the development of anti-inflammatory environment in the process of endometrial apoptotic cells uptake by phagocytes. Summarizing our data about the influence of peritoneal fluid factors upon apoptosis of endometrial cells in healthy women it can be concluded that the increase of the amount of endometrial stromal cells at the late stages of apoptosis after its incubation with autologous peritoneal macrophages or with peritoneal fluid is physiological process that prevent the survival and growth of menstrual

In women with endometriosis the response of endometrial cells upon the influence of peritoneal fluid factors significantly differed from that in healthy women. In vitro incubation of endometrial explants of women with endometriosis with the autologous peritoneal macrophages had led to the decrease of MT-1, XIAP, and caspase-3 mRNAs expression (Table 5). In these conditions the peritoneal fluid of endometriosis women significantly increased the level of HSP27 mRNA expression (Table 5) and diminished the

Endometrium+ autologous macrophages

Endometrium + autologous Peritoneal fluid

(n=4)

(n=5)

late/irreversible apoptosis.

Parameter, % Endometrium +RPMI

control, \*- p<0.05, \*\*- p<0.01, \*\*\*- p<0.001)

endometrial cells in peritoneal cavity.

amount of early apoptotic endometrial stromal cells (Table 6).

(n=5)

1640 (control)

two main factors which can contribute to the mechanisms regulating endometrial apoptosis and invasion – soluble biologically active factors of peritoneal fluid, including numerous cytokines, growth factors, metabolites of arachidonic acid, reactive oxygen species, etc. and peritoneal macrophages, the most abundant leukocytes population in peritoneal fluid. To estimate the possible influence both of humoral and cellular components of peritoneal fluid upon the apoptosis and invasiveness of endometrial cells which entered the peritoneal cavity due to the menstrual reflux, we had incubated explants of eutopic endometrial tissue in the presence of autologous peritoneal macrophages or in the presence of autologous peritoneal fluid. After termination of incubation we assessed the level of expression of genes regulating apoptosis and invasiveness in endometrial tissue.

#### **4.1 Influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrial tissue in healthy women and in women with endometriosis**

Accumulation evidences suggest that apoptosis helps to maintain cellular homeostasis during the menstrual cycle through the elimination of senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phases of the cycle (Vigano et al., 2004). It was proposed that in healthy women the majority of menstrual reflux cells undergo programmed cells death and do not survive in the peritoneal fluid (Gebel et al., 1998; Nasu et al., 2009). On the contrary in women with endometriosis the percentage of menstruated endometrial cells undergoing apoptosis is greatly reduced, increasing the number of surviving cells that could continue to exhibit physiological activity (Vigano et al., 2004). In our work we have checked out the influence of peritoneal fluid factors upon apoptosis endometrial cells both in healthy women and in women with endometriosis to elucidate the mechanisms responsible for elimination of menstrual endometrial cells from peritoneal cavity in women with and without endometriosis.

It was found that in women without endometriosis incubation of the endometrial explants with macrophages had led to the significant increase of the caspase-3 and MT-1 mRNAs expression in endometrial tissue (Table 3). Peritoneal fluid in this case didn't influence upon apoptosis-related gene expression in endometrial tissue.


Table 3. The influence of peritoneal macrophages and peritoneal fluid upon the expression of mRNAs of genes, regulating apoptosis, in endometrium of healthy women (\* – given in comparison to the control, \*- p<0.05)

We also estimated the amount of apoptotic endometrial stromal cells in endometrium of healthy women after its incubation with macrophages or with peritoneal fluid in standard test with Annexin V and propidium iodide (Pi) staining. It was established that both

two main factors which can contribute to the mechanisms regulating endometrial apoptosis and invasion – soluble biologically active factors of peritoneal fluid, including numerous cytokines, growth factors, metabolites of arachidonic acid, reactive oxygen species, etc. and peritoneal macrophages, the most abundant leukocytes population in peritoneal fluid. To estimate the possible influence both of humoral and cellular components of peritoneal fluid upon the apoptosis and invasiveness of endometrial cells which entered the peritoneal cavity due to the menstrual reflux, we had incubated explants of eutopic endometrial tissue in the presence of autologous peritoneal macrophages or in the presence of autologous peritoneal fluid. After termination of incubation we assessed the level of expression of genes

**4.1 Influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrial tissue in healthy women and in women with endometriosis** 

endometrial cells from peritoneal cavity in women with and without endometriosis.

mRNA MT-1 21.51±2.59 54.19±13.90\* 68.79±21.68 mRNA XIAP 0.73±0.15 0.73±0.16 1.26±0.62 mRNA caspase-3 15.69±5.91 39.51±9.64\* 131.21±73.54 mRNA HSP27 5.28±3.02 8.57±0.45 13.27±2.45

Table 3. The influence of peritoneal macrophages and peritoneal fluid upon the expression of mRNAs of genes, regulating apoptosis, in endometrium of healthy women (\* – given in

We also estimated the amount of apoptotic endometrial stromal cells in endometrium of healthy women after its incubation with macrophages or with peritoneal fluid in standard test with Annexin V and propidium iodide (Pi) staining. It was established that both

It was found that in women without endometriosis incubation of the endometrial explants with macrophages had led to the significant increase of the caspase-3 and MT-1 mRNAs expression in endometrial tissue (Table 3). Peritoneal fluid in this case didn't influence upon

> Endometrium+ autologous macrophages (n=10)

Endometrium + autologous peritoneal fluid

(n=10)

Accumulation evidences suggest that apoptosis helps to maintain cellular homeostasis during the menstrual cycle through the elimination of senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phases of the cycle (Vigano et al., 2004). It was proposed that in healthy women the majority of menstrual reflux cells undergo programmed cells death and do not survive in the peritoneal fluid (Gebel et al., 1998; Nasu et al., 2009). On the contrary in women with endometriosis the percentage of menstruated endometrial cells undergoing apoptosis is greatly reduced, increasing the number of surviving cells that could continue to exhibit physiological activity (Vigano et al., 2004). In our work we have checked out the influence of peritoneal fluid factors upon apoptosis endometrial cells both in healthy women and in women with endometriosis to elucidate the mechanisms responsible for elimination of menstrual

regulating apoptosis and invasiveness in endometrial tissue.

apoptosis-related gene expression in endometrial tissue.

comparison to the control, \*- p<0.05)

Endometrium +RPMI

1640 (control) (n=10)

Parameter, copies number/μl macrophages and peritoneal fluid of healthy women increased the amount of apoptotic Annexin V+ endometrial cells (Table 4). The elevation of the amount of apoptotic cells was noted predominantly in population of endometrial stromal cells, entered in the late/irreversible apoptosis.


Table 4. The influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrium stromal cells in healthy women (\* – given in comparison to the control, \*- p<0.05, \*\*- p<0.01, \*\*\*- p<0.001)

So, our results let us to conclude that in physiological conditions the endometrial cells are effectively eliminated from the peritoneal cavity due to induction of apoptosis by peritoneal macrophages. Peritoneal fluid, according to our data, is less important for induction of apoptosis in endometrium of healthy women. Only macrophages of women without endometriosis are capable to increase the synthesis one of the most important pro-apoptotic factor - caspase-3 in autologous endometrial tissue. Earlier it was shown that the control of tissue homeostasis by induction of apoptosis in aging and transformed cells is one of the important macrophages function (Gordon & Freedman, 2006). It is also known that apoptotic cells are rapidly engulfed by phagocytes in a process akin to macro pinocytosis. Normally, the uptake of apoptotic cells is accompanies by the induction of antiinflammatory cytokine synthesis of phagocytes (Erwig & Henson, 2007). Our results have shown that MT-1 synthesis significant elevated in endometrium of healthy women after its incubation with peritoneal macrophages. It is known that metallothioneins demonstrated strong antioxidant properties and able to scavenge a wide range of reactive oxygen species thus serving as anti-inflammatory mediators (Inoue et al., 2009). So, the increase of MT-1 synthesis in endometrial cells, undergoing apoptosis, might be one of the factors, participating in the development of anti-inflammatory environment in the process of endometrial apoptotic cells uptake by phagocytes. Summarizing our data about the influence of peritoneal fluid factors upon apoptosis of endometrial cells in healthy women it can be concluded that the increase of the amount of endometrial stromal cells at the late stages of apoptosis after its incubation with autologous peritoneal macrophages or with peritoneal fluid is physiological process that prevent the survival and growth of menstrual endometrial cells in peritoneal cavity.

In women with endometriosis the response of endometrial cells upon the influence of peritoneal fluid factors significantly differed from that in healthy women. In vitro incubation of endometrial explants of women with endometriosis with the autologous peritoneal macrophages had led to the decrease of MT-1, XIAP, and caspase-3 mRNAs expression (Table 5). In these conditions the peritoneal fluid of endometriosis women significantly increased the level of HSP27 mRNA expression (Table 5) and diminished the amount of early apoptotic endometrial stromal cells (Table 6).

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 227

We also established that peritoneal fluid factors directly influenced upon invasive capacity of endometrial cells of women with endometriosis. In vitro incubation of endometrial explants of women with endometriosis with the autologous peritoneal macrophages had led to the decrease of TIMP-1 and TIMP-2 mRNAs expression and the peritoneal fluid of endometriosis women significantly increased the level of MMP-2 mRNA expression by

> Endometrium+ autologous macrophages

Endometrium + autologous peritoneal fluid

(n=6)

(n=6)

**4.2 The influence of peritoneal macrophages and peritoneal fluid upon the invasiveness of eutopic endometrial tissue in endometriosis women** 

Endometrium +RPMI

mRNA MMP-2 4.83±2.40 2.97±0.89 20.44±6.19\* mRNA TIMP-2 10.36±2.60 1.76±0.64\* 8.25±2.57 mRNA MMP-9 0.18±0.09 0.17±0.13 0.98±0.68 mRNA TIMP-1 10.12±3.91 0.52±0.25\* 15.00±6.98

Table 7. The influence of peritoneal macrophages and peritoneal fluid upon the expression of mRNAs of genes, regulating invasiveness, in eutopic endometrium of women with

So, in endometriosis both soluble factors of peritoneal fluid and macrophages evidently increased the invasiveness of eutopic endometrial cells. The literature data about stimulatory action of peritoneal fluid from women with endometriosis upon uPA protein expression in endometrial cell culture from women with and without endometriosis are in a

Results of our experiments, received after estimation of the invasiveness of endometrial stromal cells of women with endometriosis in matrigel system also have shown the prominent stimulatory effect of soluble factors of peritoneal fluid of women with endometriosis on endometrial cells invasiveness. We found the enhanced invasion of endometrial stromal cells in matrigel in the presence of autologous peritoneal fluid or in the presence of supernatants of 24-hours cultures of peritoneal macrophages of women with endometriosis (Fig.2). In every case we estimated the action of autologous peritoneal fluid, and the action of pooled supernatants of peritoneal macrophages cultures. When endometrial cells migrated through membrane pores with matrigel only in presence of culture media RPMI 1640 we have seen the only few migrated cells (coefficient of spontaneous invasion – 53.52±23.33%). In the cases of endometrial stromal cells invasion in the presence of autologous peritoneal fluid, coefficient of invasion was 71.85±2.15%, and addition of supernatants of peritoneal macrophages cultures in culture media elevated the

Thus, summarizing our results about the influence of peritoneal fluid factors on apoptosis and invasiveness of endometrial cells in endometriosis, it might be concluded that immunosurveillance in the peritoneal cavity of women with endometriosis promotes conditions favorable for apoptosis inhibition and invasiveness facilitation of endometrial cells. Incapability of macrophages of women with endometriosis to eliminate the ectopic

1640 (control)

endometriosis (\* – given in comparison to the control, \*- p<0.05).

good accordance with our results (Cosin et al., 2010).

coefficient of invasion to 76.07±1.43% (p<0.001 in both cases).

(n=6)

eutopic endometrial cells (Table 7).

Parameter, copies number/μl


Table 5. The influence of peritoneal macrophages and peritoneal fluid upon the expression of mRNAs of genes, regulating apoptosis, in eutopic endometrium of women with endometriosis (\* – given in comparison to the control, \*- p<0.05)


Table 6. The influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrium stromal cells in women with endometriosis (\* – given in comparison to the control, \*- p<0.05).

Thus, the influence of both peritoneal macrophages and peritoneal fluid of women with endometriosis upon apoptosis of autologous eutopic endometrium was opposite to that in healthy women. Peritoneal macrophages decreased the expression of pro-apoptotic genes, and peritoneal fluid increased the synthesis of anti-apoptotic protein HSP27, diminishing the early apoptosis of endometrial stromal cells. We have seen only one exception from the anti-apoptotic action of peritoneal fluid factors in endometriosis. Peritoneal macrophages of women with endometriosis decreased the level of mRNA expression of apoptosis inhibitor factor XIAP. We thought that this effect might be responsible for the phenomenon of low expression of XIAP gene in ectopic endometrium in vivo. But in the general the action of peritoneal factors of women with endometriosis was directed to the inhibition of apoptosis of autologous endometrial cells. Evidently, this action of peritoneal fluid factors might serve as one of the fundamental mechanisms leading to survival of endometrial cells in peritoneal cavity and promotion of ectopic lesions formation and growth. Our results are in a good accordance to the literature data. Earlier it was shown that addition of plasma or peritoneal fluid of women with endometriosis to in vitro cultures of neutrophils from healthy donors reduced the percents of apoptotic cells (Kwak et al., 2002). Decrease of the sensitivity of endometrial cells of women with endometriosis to cytolysis by peritoneal macrophages was also noted (Dmowski et al., 1998). But all these works didn't light the mechanisms of apoptosis-inhibitory action of peritoneal fluid and peritoneal macrophages of women with endometriosis.

Endometrium+ autologous macrophages (n=10)

Endometrium+ autologous macrophages

(n=8)

Endometrium + autologous peritoneal fluid

(n=10)

Endometrium + autologous peritoneal fluid

(n=5)

Endometrium +RPMI

mRNA MT-1 43.95±7.88 13.02±4.03\*\* 26.82±7.12 mRNA XIAP 0.47±0.19 0.04±0.01\* 0.19±0.06 mRNA caspase 3 18.52±8.20 0.42±0.22\* 2.81±1.09 mRNA HSP27 14.52±6.20 12.86±5.52 70.32±26.19\*

of mRNAs of genes, regulating apoptosis, in eutopic endometrium of women with

Annexin V+ 59.96±4.67 54.76±3.68 45.35±3.38\* Annexin V+Pi- 24.59±1.88 20.35±1.54 17.38±2.46\* Annexin V+Pi+ 35.00±5.28 34.79±3.57 28.00±3.11

Table 6. The influence of peritoneal macrophages and peritoneal fluid upon apoptosis of eutopic endometrium stromal cells in women with endometriosis (\* – given in comparison

Thus, the influence of both peritoneal macrophages and peritoneal fluid of women with endometriosis upon apoptosis of autologous eutopic endometrium was opposite to that in healthy women. Peritoneal macrophages decreased the expression of pro-apoptotic genes, and peritoneal fluid increased the synthesis of anti-apoptotic protein HSP27, diminishing the early apoptosis of endometrial stromal cells. We have seen only one exception from the anti-apoptotic action of peritoneal fluid factors in endometriosis. Peritoneal macrophages of women with endometriosis decreased the level of mRNA expression of apoptosis inhibitor factor XIAP. We thought that this effect might be responsible for the phenomenon of low expression of XIAP gene in ectopic endometrium in vivo. But in the general the action of peritoneal factors of women with endometriosis was directed to the inhibition of apoptosis of autologous endometrial cells. Evidently, this action of peritoneal fluid factors might serve as one of the fundamental mechanisms leading to survival of endometrial cells in peritoneal cavity and promotion of ectopic lesions formation and growth. Our results are in a good accordance to the literature data. Earlier it was shown that addition of plasma or peritoneal fluid of women with endometriosis to in vitro cultures of neutrophils from healthy donors reduced the percents of apoptotic cells (Kwak et al., 2002). Decrease of the sensitivity of endometrial cells of women with endometriosis to cytolysis by peritoneal macrophages was also noted (Dmowski et al., 1998). But all these works didn't light the mechanisms of apoptosis-inhibitory action of peritoneal fluid and peritoneal macrophages of women with

Table 5. The influence of peritoneal macrophages and peritoneal fluid upon the expression

1640 (control) (n=10)

endometriosis (\* – given in comparison to the control, \*- p<0.05)

1640 (control)

Parameter, % Endometrium +RPMI

to the control, \*- p<0.05).

endometriosis.

(n=8)

Parameter, copies number/μl

#### **4.2 The influence of peritoneal macrophages and peritoneal fluid upon the invasiveness of eutopic endometrial tissue in endometriosis women**

We also established that peritoneal fluid factors directly influenced upon invasive capacity of endometrial cells of women with endometriosis. In vitro incubation of endometrial explants of women with endometriosis with the autologous peritoneal macrophages had led to the decrease of TIMP-1 and TIMP-2 mRNAs expression and the peritoneal fluid of endometriosis women significantly increased the level of MMP-2 mRNA expression by eutopic endometrial cells (Table 7).


Table 7. The influence of peritoneal macrophages and peritoneal fluid upon the expression of mRNAs of genes, regulating invasiveness, in eutopic endometrium of women with endometriosis (\* – given in comparison to the control, \*- p<0.05).

So, in endometriosis both soluble factors of peritoneal fluid and macrophages evidently increased the invasiveness of eutopic endometrial cells. The literature data about stimulatory action of peritoneal fluid from women with endometriosis upon uPA protein expression in endometrial cell culture from women with and without endometriosis are in a good accordance with our results (Cosin et al., 2010).

Results of our experiments, received after estimation of the invasiveness of endometrial stromal cells of women with endometriosis in matrigel system also have shown the prominent stimulatory effect of soluble factors of peritoneal fluid of women with endometriosis on endometrial cells invasiveness. We found the enhanced invasion of endometrial stromal cells in matrigel in the presence of autologous peritoneal fluid or in the presence of supernatants of 24-hours cultures of peritoneal macrophages of women with endometriosis (Fig.2). In every case we estimated the action of autologous peritoneal fluid, and the action of pooled supernatants of peritoneal macrophages cultures. When endometrial cells migrated through membrane pores with matrigel only in presence of culture media RPMI 1640 we have seen the only few migrated cells (coefficient of spontaneous invasion – 53.52±23.33%). In the cases of endometrial stromal cells invasion in the presence of autologous peritoneal fluid, coefficient of invasion was 71.85±2.15%, and addition of supernatants of peritoneal macrophages cultures in culture media elevated the coefficient of invasion to 76.07±1.43% (p<0.001 in both cases).

Thus, summarizing our results about the influence of peritoneal fluid factors on apoptosis and invasiveness of endometrial cells in endometriosis, it might be concluded that immunosurveillance in the peritoneal cavity of women with endometriosis promotes conditions favorable for apoptosis inhibition and invasiveness facilitation of endometrial cells. Incapability of macrophages of women with endometriosis to eliminate the ectopic

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 229

as phagocytes when activated. Within tissues, differentiation of monocytes into macrophages occurs in response to the surrounding microenvironmental context, which directs the acquisition of tissue-specific phenotype. Within most organs macrophages are involved in tissue homeostasis via their ability to execute diverse functional activity, including (i) phagocytosis and degradation of foreign antigens, (ii) matrix dissolution and tissue remodeling, and (iii) production and secretion of growth factors, cytokines and chemokines (Wu et al., 2004). These effector functions allow macrophages to regulate local

Macrophages are identified in tissues by their expression of specific proteins markers which are predominantly cell surface receptors. The proteins considered most exclusively restricted to macrophages are CD68 molecules, class II MHC antigens, receptors, that are involved in phagocytosis, including Fc receptors, complement receptors, integrins, mannose receptor, sialoadhesin and scavenger receptors (Wu et al., 2004). Macrophages are considered "professional phagocytes" and can internalize particles much more rapidly and efficiently then other cells due to their expression of specific cell surface receptors, which initiates actin polymerization and internalization of the foreign molecule or organism into a phagosome. Macrophages phagocytose endogenous and exogenous substances, such as cell debris, bacteria and viruses. Macrophages in vivo recognize and internalize apoptotic and necrotic cells (Wu et al., 2004). Macrophages sequestered in the peritoneal cavity remove red blood cells, damaged tissue fragments, apoptotic cells, and probably endometrial cells that gain access to the peritoneal cavity through the fallopian tubes (Dmowski & Braun, 2004). In endometriotic peritoneal fluid the concentration and number of peritoneal macrophages are significantly increased as compared to healthy controls (Tariverdian et al., 2007). These are large, activated macrophages that produce high levels of smooth-muscle-contracting prostaglandins, such as PGE2 and PGF2α (Gazvani & Templeton, 2002; Dmowski & Braun, 2004). Elevated PGE2 in the peritoneal fluid of endometriosis patients due to macrophages activation have been proposed to subsequently aggravate endometriosis-associated pain by altering uterine and tubal contractility and cause infertility due to a delayed ovum transport (Tariverdian et al., 2007). But despite of activated status macrophages evidently are not capable to effectively control the growth of endometrial tissue in peritoneal cavity of women with endometriosis. Molecular basis for this phenomenon isn't completely elucidated yet. One of the mechanisms by which abnormally functioning macrophages could contribute to the growth of ectopic endometrial cells is through defective scavenging activity (Sidell et al., 2002). Important function of macrophages in the face of an invading "foreign" material or when encountering cellular debris and apoptotic cells is the scavenger function (Sidell et al., 2002). A family of specific scavenger receptors (SRs) is involved in this activity. This family was aptly named because these receptors have been found to bind and "scavenge" a broad array of modified self and nonself ligands, including apoptotic cells, anionic phospholipids and amyloid and pathogen components (Moore & Freeman, 2006). The SRs are believed to be members of the group of pattern recognition receptors (PRR) that mediate the innate immune host response through recognition of highly conserved pathogen-associated molecular patterns (PAMP). This evolutionarily ancient but highly effective system of host defense enables the immune system to discriminate between "noninfectious self" and "infectious nonself". However, there is a growing body of evidence to suggest that SRs may recognize endogenous neoantigens and apoptotic cells through molecular mimicry of

immune and inflammatory responses as well as influence normal tissue function.

cells from the peritoneal cavity and the stimulatory effect of humoral peritoneal factors on the synthesis of proteolytic enzymes by endometrial cells might be considered as the important pathogenetic mechanisms of endometriotic tissue formation and growth.

Fig. 2. Invasion of eutopic endometrium stromal cells of women with endometriosis. Holes represent the 8-μm pore in the filter. A- spontaneous invasion of endometrial stromal cells in RPMI 1640 medium; B - invasion of endometrial stromal cells in the presence of the supernatants of 24-h culture of macrophages; C - invasion of endometrial stromal cells in the presence of the autologous peritoneal fluid. Arrows indicate cells. (Original magnification x200)
