Saturday, January 18, 2014

Research pathogenesis of endometriosis

Current Concepts and Research in the Pathogenesis of Endometriosis

Erkut Attar, M.D.
Istanbul University, Istanbul Medical School, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility
Istanbul, Turkey
Tel/Fax : +90-216-3273245
Keywords: Endometriosis, Etiology, Pathogenesis, Research
Objective: To review the recent basic research and literature from different disciplines as genetics, environmental science, cancer biology and immunology for better understanding the current status of endometriosis.
Abstract: Endometriosis is a complex gynecologic disorder that affects nearly 1 in 7 women of reproductive age. Ectopic dissemination of endometrial cells and their subsequent implantation are the mechanisms involved in the development of endometriosis. Despite extensive investigation the pathogenesis of endometriosis is poorly understood. Research on the pathogenesis of endometriosis currently interfaces with four areas of basic research: 1) Genetics; 2) Environmental Science; 3) Cancer Biology; 4) Immunology. In this article current research of these disciplines and their pertinence to endometriosis research were reviewed. Data from these disciplines relevant to endometriosis were summarized and conjunctions were highlighted to provide a better understanding of the pathogenesis
Endometriosis is a chronic disease that affects nearly 1 in 7 women of reproductive age (1). Sampson first described the disease formally in 1921. Then he proposed the hypothesis that the origin of peritoneal endometrial implants was tissue delivered by the retrograde menstruation (2). Retrograde menstruation is a nearly universal phenomenon among cycling women (3,4), but it is not clear why endometrial tissue will implant and grow in the peritoneal cavity of only a subgroup of women. These endometrial cells can respond to ovarian hormones and, therefore undergo cyclic menstrual changes with periodic bleedings. Despite extensive investigation the pathogenesis of endometriosis is poorly understood. Research on the pathogenesis of endometriosis currently interfaces with four areas of basic research:        1) Genetics; 2) Environmental Science; 3) Cancer Biology; 4) Immunology. In this article current research of these disciplines and their relevance to endometriosis research are reviewed.
1. Genetics:
1.1 Oxford Endometriosis Gene Study (OXEGENE)
There is increasing evidence that endometriosis has genetic basis. Endometriosis is probably a complex trait, like diabetes mellitus, hypertension and asthma, which implies that the disease is caused by an interaction between multiple genes and environment. Such conditions do not have a clear Mendelian pattern of inheritance and multiple gene loci conferring susceptibility to the condition interact with each other and the environment to produce the phenotype. Oxford Endometriosis Gene Study (OXEGENE) is designed to discover whether there is a genetic cause for endometriosis, and to identify susceptibility loci involved in the development of endometriosis using the linkage analysis. DNA from sisters with surgically confirmed r-AFS stage II-IV disease and their parents are being collected to perform a genome-wide screen. There were 571 Families, 886 patients and 65 collaborators involving this study until the time that this manuscript was in preparation.
1.2 Candidate Genes
Nevertheless, the use of linkage analysis alone is often insufficient to localize susceptibility genes in complex traits because of the limited resolution of the method for fine mapping. As a complementary approach, candidate genes need to be investigated in association studies by comparing the frequency of marker alleles in affected cases and normal controls. There are two genetic associations with endometriosis have been reported: 1) Polymorphism in galactose-1-phosphate uridyl transferase (GALT) (5);  2) Null mutation in Glutathione S-transferase M1 (GSTM1)(6,7). Enzymes belonging to the glutathione S-transferase family are involved in the two stage of detoxification of2,3,7,8-Tetraachlorodibenzo-p-Dioxin (Dioxin) which is a potential pollutant for endometriosis development.
1.3  Familial Clustering in Rhesus Monkeys
Oxford Group recently made some publications using the epidemiological data that they have collected. Familial tendency of disease supporting the hypothesis that endometriosis has a genetic basis was deserved in these studies(8,9). However, defining the familial tendency more thoroughly in whole population is difficult because surgery is required to establish the diagnosis. The clinical features appearance at surgery and histological characteristics of the disease in the rhesus monkey mimic those in human (10). Therefore, the clearer understanding of the epidemiology and inheritability of the disease may emerge from studying spontaneous endometriosis in rhesus monkey colonies (Macaca Mulatta). Oxford Group is collaborating with California Regional Primate Research Center (CRPRC) and Wisconsin Regional Primate Research Center (WRPRC) to study the epidemiology and inheritability of endometriosis. They have identified 121 (8.3%) affected rhesus monkeys among the autopsy records of the 1459 female animals that they died, aged 4 years of more, in the colony between 1982-1996 at CRPRC. They are trying to determine the familial tendency in these affected animals by analyzing the entire colony records over 9000 females from 1965-1977  (11). Hadfield et al studied the autopsy records of 399 rhesus monkeys that died in the WRPRC colony between 1980 and 1995 and reported a prevalence rate of 20% in animals aged 4 years or older at death and 29% in animals aged 10 years or older at death (12).
2.      Environmental Science
2.1 Dioxin
Dioxin is a potent chemical toxicant, which serves as the reference compound for a large class of halogenated aromatic hydrocarbons. The dioxin connection to endometriosis was discovered almost by accident. The aim of the original study was to investigate the long-term reproductive effects of  exposure to dioxin in the rhesus monkeys. Twelve years after the initiation of this work, in 1989 endometriosis was discovered at the autopsy of a dioxin exposed animal.
The major source of dioxin in the environment (95%) comes from incinerators burning chlorinated wastes. Dioxin pollution is also affiliated with paper mills, which use chlorine bleaching in their process and with the production of Polyvinyl Chloride (PVC) plastics.
The major sources of dioxin are in diet. Since dioxin is fat-soluble, it bioaccumulates up the food chain and it is mainly (97.5%) found in meat and dairy products. Men have no ways to get rid of dioxin other than letting it break down according to its chemical half-lives. Women, on the other hand, have two ways which it can exit their bodies: It crosses the placenta into the growing infant causing recurrent pregnancy loss and it is present in the fatty breast milk, which is also a route of exposure which doses the infant.
2.2 Experimental endometriosis in Rhesus Monkeys
Now new thinking about endometriosis has been stimulated by research linking dioxin exposure to the disease in rhesus monkeys. In rhesus monkeys the disease develops spontaneously and resembles the human disease both anatomically and clinically. Scientists who have been studying the disease for two decades or more, unsuccessfully seeking a cause consider the recognition of dioxin as a contributor to the disease in rhesus monkeys a exciting breakthrough. It seems that there is a dose response relationship between the dioxin and severity of endometriois(13) Reproductivity of these monkeys was also affected when they exposed to high levels of dioxin. Fetotoxicity was also reported in rhesus monkeys exposed to dioxin (14)
2.3 Genetics and Dioxin
Target genes for the action of dioxin include cytochrome p450 and growth regulatory genes involved in both inflammation and differentiation, including plasminogen  activator inhibitor-2 and interleukin-1b. (15,16). As it was previously stated in this chapter, glutathione S-transferase M1 (GSTM1) is responsible for detoxification of dioxin that is a candidate gene for endometriosis development. 
2.4  Reproductive System and Dioxin
Dioxin is also modulates various hormone receptor systems which play a role in uterine function, including estrogenreceptor, progesterone receptor, epidermal growth factor  receptor and prolactin receptor (17,18) . Moreover this toxin alters the action of estrogen in reproductive organs in a manner which is both age-dependent and target organ specific (18,19). It modulates steroid receptor expression resulting in altered tissue specific responses to hormone(18) .
2.5 Immune System and Dioxin
In addition to effects on the reproductive system, dioxin is also adversely effect immuncompetence(20,21,22,23,24) Dioxin shows immunosuppressive activities and is a potent inhibitor of T-lymphocyte function(22,23,24).
3. Cancer Biology
While it is not a malignant disorder, endometriosis exhibits cellular proliferation and invasion. Invasive phenotype in endometriosis shares aspects with tumor metastasis (25). T cell mediated invasion may be similar to that which occurs with metastatic neoplasia, where in immune surveillance systems are inadequate or unable to respond the seeding tissue (26,27). Accumulation of various growth factors and the occurrence of angiogenesis to produce a self contained blood supply are the features that implicate the relationship of cancer biology and ectopic endometriotic tissue development.
3.1 Angiogenesis
Endometrial tissue develops its own vascular supply and becomes an independent, growing mass. In ways similar to the spread of neoplasm, a piece of implanted tissue may subsequently break off from the primary site and travel elsewhere in the peritoneal cavity, setting up a peritoneal location or may enter a blood or lymph vessel and disseminate to distant body sites. As the free floating pieces of endometrial tissue themselves implant, grow, and develop their own blood supply, the process repeats itself.
Endometriosis is one of the family of angiogenic diseases and excessive endometrial angiogenesis is proposed as an important mechanism in the pathogenesis of endometriosis. Evidence is reviewed for the hypothesis that the endometrium of  women with endometriosis has an increased capacity to proliferate, implant and grow in the peritoneal cavity. Data is summarized indicating that the endometrium of patients with endometriosis shows enhanced endothelial cell proliferation. Results are also reviewed indicating that the cell adhesion molecule integrin alpha(v)betha3 is expressed in more blood vessels in the endometrium of women with endometriosis when compared with normal women (28). Taken together, these results provide evidence for increased endometrial angiogenesis in women with endometriosis when compared with normal subjects (29). Endometrium is a rich source of growth factors which promote angiogenesis including the fibroblast growth factors, FGF1 and FGF2 and the vascular endometrial growth factor (VEGF) (30,31,32).
Dysmenorrhoea is associated with increased plasma levels of vasopresssin (33,34). Vasopressin induced uterine contractions, but also increases contractility of the uterine resistance artery (35). In addition other peptides including oxytocin, endothelin and noradrenalin contribute to this action on blood levels. The consequence is a large reduction in blood flow in the uterus particularly to the endometrium during uterine contractions. Hypoxia is a critical activator of genes, especially for VEGF and results increased translation for VEGF protein in glandular and stromal cells of endometrium (36) . Thus, the greater hypoxia in the uteri of women with severe dysmenorrhoea results in increased production of VEGF, facilitating angiogenesis at the implantation site of desquamated endometrium. (fig 1)
figure 1
3.2 Extracellular Matrix Remodeling
Essential features of endometrial physiology involve extracellular matrix (ECM). In the pathogenesis of endometriosis interactions of endometriosis cells with ECM can be postulated. Indeed, it has been shown that extracellular matrix (ECM) turnover in the patients with endometriosis is altered (37). A group of enzymes, matrix mettaloproteinases (MMP) are responsible for extracellular matrix and endometrium remodeling. Tumor promotors, growth factors, cytokines, steroids and oncogenes regulate them. Suppression of MMP inhibits establishment of ectopic lesions by human endometrium (38). It has been shown that MMP-1 expression is correlated with the activity of endometriotic tissue suggesting its involvement in tissue remodeling and reimplantation of endometriotic lesion (39) and endometriotic implants express the protease enabling the invasion of surrounding tissue (40). Adhesion to peritoneum has to occur with subsequent implantation and proliferation. Therefore, specific cell adhesion receptors and their extracellular matrix ligands are being investigated to understand the invasive features of endometriosis. Cell adhesion molecules fall into 4 major groups including cadherins, selectins, members of immunoglobulin superfamily and integrins that each may potentially play a role in the development and progression of disease(41,42).
3.3 Apoptosis
Programmed cell death (apoptosis) has been implicated in the pathogenesis of endometrium. Several investigations have revealed that uterine endometrium in mammals can be regulated by apoptosis. It has been suggested that decreased susceptibility of endometrial tissue to apoptosis contributes to the etiology or pathogenesis of endometriosis (43). The increased proportion of Bcl-2 positive macrophages found in women with endometriosis may predispose these cells to resist apoptosis (44) . The continued survival of these active cells could have important consequences for the survival and proliferation of the ectopic tissue. Decreased apoptosis in endometrial cells of  the patients with endometriosis may lead to acquiring the capacity to utilize the products of an activated immune system to establish ectopic foci of disease.
4.  Immunology
There are many immune and inflammatory changes relevant to endometriosis that can be considered as a cause and/or result of the disease development. Genetic characteristics and environmental (dioxin) factors could be responsible for the altered immune functions and disease development in patients with endometriosis. Because the ectopic implants of endometrial tissue are destroyed by a variety of immune and inflammatory reactions, research on cancer biology is a part of the  full understanding of the disease development as it was emphasized in section 3, previously. Nonetheless, altered macrophage functions itself and the role of peritoneal fluid have a special impact on the current endometriosis research.
4.1 Altered Macrophage Functions
Unlike the situation seen in women without endometriosis, in women with endometriosis the macrophages do not appear to be as active in the phagocytosis ad cellular debris. They do, however secrete high concentration of substances as growth factors that restrict natural killer activity, increase angiogenesis and fibrosis and induce endometrial cell proliferation in vitro. As a cause of infertility in the patients with endometriosis, these changes in peritoneal milieu could also be responsible for the failure of fertilization, embryo development and implantation.
4.2 Paracrine Changes in the peritoneal environment of women with endometriosis
Macrophage derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In paricular growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis (fig.2).
figure 2
Interleukin 8 (IL-8) is a chemoattractant and activating factor for human neutrophils and a potent angiogenic agent. IL-8 concentrations in peritoneal fluid show higher levels in patients with endometriosis according to the stage of the disease compared to the control. However potential source of IL-8 in peritoneal fluid are not only the macrophages but also mesothelial cells of the peritoneum and endometrium itself. We found that cultured mesothelial cells constitutively express IL-8 mRNA and secrete IL-8 protein, and the expression of IL-8 from mesothelial cells is modulated by other cytokines such as  IL-1, TNFa . These latter cytokines appear to play some role in the constitutive secretion of IL-8 as well as being capable of greatly stimulating further production and secretion (45). Peritoneal macrophages therefore play an important role in the initiation of the pathogenic cascade as a sources of IL-1 and TNF-a.  
Monocyte Chemotatctic Protein-1 (MCP-1) is also an active participant in the pathogenesis of endometriosis.  The level of the MCP-1 levels was found significantly higher in patients with severe disease. We have observed that the elevated MCP-1 levels in peritoneal fluid of the patients with endometriosis  may play a role in growth and maintenance of ectopic endometrial tissue by directly stimulating endometrial cell proliferation (46).
VEGF is a growth factor related to angiogenesis and released in response to hypoxia. The role of VEGF on endometriosis was discussed previously in this article. Thus, it may reflect a connection between the retrograde menstruation and /or dysmenorrhoea and changes in peritoneal fluid. Activated macrophages in the peritoneal cavity produce large amount of VEGF. (47)
5. Conclusion
It is likely that endometriosis is a common multifactorial disease, caused by an interaction between multiple gene loci and environment. Causes of immune or inflammatory deficiency may be related to the effects of stress on immune functioning, or may be genetically determined. Environmental factors such as Dioxin can be responsible for immunosuppressive activities in patients with endometriosis. In addition this toxin modulates steroid receptor expression resulting in altered tissue specific responses to hormones. Chronic immunosuppression in combination with hormonal regulation may have facilitated the aberrant growth of endometrial tissue with in the peritoneum. Basic research on the field of cancer biology currently interfaces with the pathogenesis of endometriosis and this may lead to a better understanding of disease etiology. It seems that the genetical, environmental, immunological and hormonal (Autocrine and Paracrine) factors interfere each other and, implicating that a circle occurred could be responsible for the development and progression of endometriosis. However, the mechanism appears to require endometrium and retrograde menstruation in most cases of disease. In conclusion, we have just started to bring together the more obvious clinical observations as retrograde menstruation and/or dysmenorrhoea and results of the basic research on genetics, cancer biology and immunology to explain the  disease (fig. 3). 
figure 3
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