**2.1 Subjects**

212 Endometriosis - Basic Concepts and Current Research Trends

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).

role in endometriosis development?

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 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.

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

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 analysis.

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

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 215

Laboratories, USA). Flow cytometry analyses were performed on FACScan (Becton Dickinson, USA) using CellQuest Pro software. Data from forward versus side scatter was obtained to analyze the CD45+CD14+ macrophage population and Vimentin+ endometrial

The content of IL-8, MCP-1 and calprotektin in the peritoneal fluid of women with and without endometriosis was assessed by ELISA (enzyme-linked immunosorbent assay) using

The influence of peritoneal fluid and culture media of 24-h cultures of peritoneal macrophages of women with endometriosis upon invasiveness of eutopic endometrial cells of women with endometriosis was estimated in the 2D Matrigel system using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, USA). 50 μl of prepared endometrial stromal cells isolated from eutopic endometrium of women with endometriosis by enzymatic method were placed on top of porous membrane coated with Matrigel. Autologous peritoneal fluid or pooled culture media of 24-h cultures of peritoneal macrophages of women with endometriosis were added to the lower chamber and then incubated overnight. At the end of the incubation period the upper side of the membrane was cleaned with a cotton wool bud. For assessment of the number of invaded cells, the filters were stained with hematoxylin and eosin and mounted on glass microscope slides. Total number of cells that had invaded onto the underside of the filter

Results were presented as the mean ± standard error. All variables were checked for normal distribution with the Kolmogorov-Smirnov test. All the parameters studied showed a normal distribution. Student's t-test was used to compare results from different groups.

**3. Impairment of the apoptosis and invasiveness of endometrial tissue in** 

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

The growing bodies of evidence indicate that the impairment of endometrial cell's apoptosis and the increase of tissue proteolysis supposedly are involved in the pathogenic

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

stromal cell population.

**2.6 Cell invasion assays** 

commercial kits (Bender MedSystems, Austria).

was counted manually at 200x magnification.

Statistical significance was defined as p < 0.05.

**2.5 ELISA** 

**2.7 Statistics** 

**endometriosis** 

mechanisms of endometriosis.

concentration of macrophages in culture media was 2x106 cell/ml. Endometrial explants were cultured at 370 С and 5% СО2 for 24 hours. Samples of endometrium or peritoneal macrophages, cultured in the same conditions only in RPMI 1640, were used as controls. After termination of cultivation, endometrial tissue was washed up in phosphate-buffered saline (PBS) twice and was taken for subsequent RNA isolation or for enzymatic isolation of stromal endometrial cells*.* Peritoneal macrophages were collected from wells, filtered through 6 layers of gauze and analyzed using flow cytometry.
