**1. Introduction**

210 Endometriosis - Basic Concepts and Current Research Trends

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in human adipose tissue: role of a Jak/STAT pathway in regulation of the adipose-

growth factor gene and the risk of familial endometriosis. *Molecular human* 

According to common view, endometriosis is characterized by the presence and growth of the endometrium-like glandular tissue and stroma outside the uterus. So, endometriosis would represent an autotransplant in which tissue is transplanted to an ectopic location in an organism. It is well known that the immune system plays the main role in regulation of tissue homeostasis in an organism. Thus, immunologists have a special interest in this disease as insights in the pathogenesis of endometriosis would help not only to understand how ectopic lesions grow but also allow searching new approaches to the medical treatment of this disease.

Numerous theories have been proposed to explain the endometriosis pathogenesis till date. The most widely accepted Sampson's transplantation theory proposes that, during menstruation, there is reflux of endometrial tissue via fallopian tubes into abdominal cavity where endometrium could attach to peritoneal surfaces, proliferate, invade, and become the disease known as endometriosis. However, this theory does not account for the fact that most of women of reproductive age exhibit some degree of reflux of endometrial debris, but only some patients develop endometriosis (Harada et al., 2004; Vinatier et al., 2001). There are two main suggestions explaining this contradiction. The first explanation propose that there are some changes in the endometrium of women with endometriosis and these changes can promote the resistance of endometrial cells to normal peritoneal cleaning (Vinatier et al., 2001). The second theory suggests that impaired immune recognition in peritoneal cavity due to abnormalities of the cellular and humoral immunity can promote the endometriosis development (Kyama et al., 2003).

The suggestion that the primary defect in endometriosis is located in the eutopic endometrium, was proposed many years ago and supported now by many investigators (Vinatier et al., 2001; Kyama et al., 2003; Harada et al., 2004), though the eutopic endometrium of women with and without endometriosis is histologically similar. This hypothesis is confirmed by different studies revealing that there are many fundamental differences between these two tissues. Invasive properties, decreased apoptosis, and increased steroid hormones and cytokine production have been identified in eutopic

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 213

To concretize the role of cellular and humoral immune components of peritoneal fluid in their interaction with endometrial tissue in endometriosis and also to elucidate the local immune mechanisms participating in endometriotic lesions formation we attempted: a) to define the regulatory mechanisms of apoptosis and invasiveness of eutopic and ectopic endometrium in women with endometriosis, b) to elucidate the influence of autologous peritoneal macrophages and humoral factors of peritoneal fluid upon the parameters of eutopic endometrial tissue apoptosis and invasiveness in endometriosis women, c) to establish the changes in the functional state of peritoneal macrophages of women with endometriosis and d) to estimate the character of macrophages activity in response to

The study group consisted of 80 women of reproductive age undergoing diagnostic laparoscopies for infertility or pelvic pain. The presence and extent of the disease were determined laparoscopically and staging was performed according to the revised American Fertility Society classification (1985). Mild endometriosis (stage 1-2) was diagnosed in 51 women (64%), severe endometriosis (stage 3-4) was noted in 29 patients (36%). Laparoscopy was performed prior to the initiation of any treatment. Samples of peritoneal fluid and paired eutopic and ectopic endometrial biopsies were obtained from women with endometriosis. Endometrial tissue and peritoneal fluid from 30 control women without endometriosis, who underwent surgical sterilization were also collected. Informed consent was given by each woman participating in our study, according to local Ethical Committee protocol. Samples of peritoneal fluid from the Douglas pouch were aspirated into sterile tubes. Samples of ectopic and matched eutopic endometrium were collected into sterile

flasks with isotonic saline solution and were immediately recruited into the study.

To evaluate the possible effect of peritoneal environment on the apoptosis and invasive capacity of endometrial tissue, the samples of eutopic endometrium of 10 women without endometriosis and 10 women with endometriosis were cultivated in the presence of autologous peritoneal fluid or autologous peritoneal macrophages. Samples of peritoneal fluid were centrifuged at 2000 g for 10 minutes to remove cellular component. Bloody samples of peritoneal fluid were excluded from our study. Enriched population of peritoneal macrophages was obtained from peritoneal fluid using standard Ficoll-Verografin gradient centrifugation (d-1.078) with subsequent removing of lymphocytes using standard procedure of macrophages adherence to plastic. The percentage of CD14+ macrophages in received fraction was 93-95% as it was established by flow cytometry

Endometrial tissue was minced by scissors into pieces of 1-2 mm in diameter. Approximately 40-50 mg of endometrial tissue were placed in 24 well plate, in 2 ml of whole RPMI 1640 medium with 2 mM of glutamine, 5% fetal calf serum and antibiotics supplemented with autologous peritoneal fluid or with peritoneal macrophages. Ratio of peritoneal fluid and whole RPMI 1640 in medium for cultivation was 1:1. The final

**2.2 Co-cultivation of endometrial tissue and peritoneal macrophages** 

autologous stimulation of endometrial cells.

**2. Methods of investigation** 

**2.1 Subjects** 

analysis.

endometrium of women with endometriosis compared to that in women without disease (Ulukus 2006). Impairment of some genes expression also was found in women with endometriosis. Aberrant expression of several genes such as matrix metalloproteinases, Hox genes, integrins, anti-apoptotic genes Bcl-2 was demonstrated in endometrium of women with endometriosis (Bondza et al., 2009). The significant increase of aromatase expression was shown in eutopic endometrium from endometriosis patients (Weiss et al., 2009). A constitutional or acquired anomaly in the nature of antigens, expressed by endometrium, such as transplantation antigens HLA-DR and HLA-A, B, C antigens, can explain the resistance of endometrium of women with endometriosis to the cytotoxicity of Tlymphocytes (Vinatier et al., 2001). Increased proliferative activity of endometrial cells due to altered expression of c-myc, TGF-β1 and bax genes was also found in patients with endometriosis (Jonson et al., 2005). Eutopic endometrium from endometriosis patients showed increased expression of midkine and pleiotropin mRNA expression compared with endometrium from normal women (Chung et al., 2002). It is known that midkine and pleiotropin function as tumor growth factors positively regulating tumor angiogenesis and metastasis of solid tumors. So these results evidence that eutopic endometrium from endometriosis patients may be more invasive and prone to implantation than that from women without endometriosis (Chung et al., 2002). All these findings let us to propose that cells and tissue elements derived from such an altered eutopic endometrium shed into peritoneal cavity would have a higher potential of implantation and growth on peritoneal surfaces. And many differences, observed between eutopic endometrium and ectopic tissue of patients with endometriosis, can be explained as the direct consequence of the specific environment of peritoneal fluid (Vinatier et al., 2001; Harada et al., 2004).

The local environment that surrounds the endometriotic implants in the peritoneal cavity is a dynamic one. Histologically, the peritoneum consists of a thin layer of loose connective tissue covered by a layer of mesothelium and is the most extensive serous membrane in the body, with a rich supply of subperitoneal blood vessels and lymphatics (Gazvani & Templeton, 2002). The peritoneal cavity is normally empty except for a thin film of fluid that keeps surfaces moist. The peritoneal fluid arises primarily from two sources: plasma transudate and ovarian exudate (Koninckx et al., 1998; Gazvani & Templeton, 2002). Peritoneal fluid contains a variety of free-floating immune cells, including macrophages, natural killer (NK) cells, lymphocytes, eosinophils and mast cells (Gazvani & Templeton, 2002). Macrophages are the most abundant cells among the peritoneal leukocytes. It is well known that macrophages are the main source of different cytokines, growth factors and other biologically active substances in the peritoneal fluid. Recent studies have also suggested that peritoneal fluid of women with endometriosis contain an increased number of activated macrophages and other immune cells and high amount of proinflammatory cytokines and growth factors, which exert a paracrine action on endometrial cells (Minici et al., 2007). So, endometriosis can be considered to be an inflammatory disease (Vigano et al., 2004). Possibly, this peritoneal inflammation might facilitate the implantation and growth of ectopic endometrial tissues. But we don't know exactly yet, whether endometriosis is caused by peritoneal inflammation? Or does endometriosis lead to inflammation caused by inappropriate immune response to endometrial debris? Can the inappropriate peritoneal environment directly influence lesions formation, or may be the intrinsic dysfunctions of endometrial cells play the main role in endometriosis development?

To concretize the role of cellular and humoral immune components of peritoneal fluid in their interaction with endometrial tissue in endometriosis and also to elucidate the local immune mechanisms participating in endometriotic lesions formation we attempted: a) to define the regulatory mechanisms of apoptosis and invasiveness of eutopic and ectopic endometrium in women with endometriosis, b) to elucidate the influence of autologous peritoneal macrophages and humoral factors of peritoneal fluid upon the parameters of eutopic endometrial tissue apoptosis and invasiveness in endometriosis women, c) to establish the changes in the functional state of peritoneal macrophages of women with endometriosis and d) to estimate the character of macrophages activity in response to autologous stimulation of endometrial cells.
