**2.3 Immunological markers**

422 Endometriosis - Basic Concepts and Current Research Trends

suggest its assessment in women with suspected endometriosis, in association with laparoscopy and biopsy. In addition, in literature was reported that measurement of serum CA125 levels might be useful in identifying patients with infertility that may have severe

CA125 levels were assessed in the PF of patients with and without endometriosis. Although levels of CA125 in the PF were almost 10 times higher than serum levels, no differences were found between women with and without endometriosis. In addition, CA125 levels are measured also in other body fluids, such as menstrual discharge and the uterine fluid, but

CA19-9 is a high-molecular-weight glycoprotein that was initially thought to be an oncofetal antigen. Serum CA19-9 levels were elevated in patients with some malignant tumour, such as gastrointestinal adenocarcinoma, pancreatic carcinoma, or lung carcinoma; thus, the measurement of serum CA19-9 levels is useful in the diagnosis of these tumours. In gynaecology, the serum CA19-9 levels are elevated in patients with malignant and benign ovarian tumours. Furthermore, a case of an ovarian chocolate cyst with a markedly elevated serum CA19-9 level has been reported. In addition, it has been reported that serum CA19-9 levels in women with endometriosis are significantly reduced during therapy compared with the basal levels before treatment. Serum CA19-9 levels in patients with endometriosis are significantly higher than those in patients without endometriosis and that serum CA19-9 levels increase in accordance with the advancement of the clinical stage of endometriosis. CA19-9 was also detected in the endometrial glandular epithelium in ovarian chocolate cysts by immunohistochemistry. These results reveal that the measurement of the serum CA19-9 levels, as well as the serum CA125 level, may prove to be a valuable tool for predicting the severity of endometriosis as

The endometrium in endometriosis behaves like tumorous tissue and the growth factors involved in tumour proliferation, angiogenesis and invasiveness have been investigated for their expression in endometriosis. Indeed, the degree of endometriosis is positively correlated with the concentration in peritoneal fluid and serum of *Hepatocyte Growth Factor/Scatter Factor (HGF/SF*), a multifunctional polypeptide that has been implicated in embryo development, tissue repair, and cancer growth, produced mainly by mesenchymal cells with activity mediated through the c-met receptor found principally on epithelial and

Inflammatory macrophages and the inflammatory mediators they release could be related to the ectopic implantation of endometriosis: *Fibroblast Growth Factor (FGF), Epidermal Growth* 

α

stimulate in vitro proliferation of endometrial stromal cells, suggesting that they could

α

*), Transforming Growth Factor-beta* 

*).* It is been shown that these growth factors

endometriosis and could benefit from early surgical treatment.

are not useful in clinical practice.

diagnosed by laparoscopy.

endothelial cells (Zong et al., 2003).

*Factor (EGF), Transforming Growth Factor-alpha (TGF-*

*) and Tumor Necrosis Factor-alpha (TNF-* 

improve the implantation of endometrial cells.

**2.2 Growth factors** 

*(TGF*β

**2.1.2 CA19-9** 

The immune system plays an important role in the pathogenesis of endometriosis, which begins, therefore, to be treated as an autoimmune disease. T-helper, T-suppressor and natural killer (NK) cells concentrations are altered in serum and peritoneal fluid of patients with endometriosis (Lebovic et al., 2001; Nothnick, 2001). In addition, IgG and IgA antiendometrial antibodies have been detected in the sera and vaginal and cervical secretions of endometriosis patients. The presence of anti-phospholipids and anti-histones antibodies has been documented by some authors and questioned by others. These observations would believe that markers of immune reactivity, particularly cytokines, might be used as a diagnostic aid for endometriosis.

#### **2.3.1 Cytokines**

Macrophages are a major source of many cytokines involved in immune response, haematopoiesis, inflammation and many other homeostatic processes. Upon stimulation by microorganisms, microbial products or endogenous factors including cytokines, macrophages can *de novo* synthesize and release a large variety of cytokines (i.e. IL-1, IL-1ra, IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-α, IFN-γ, MCP-1, MCP-3, MIF, M-CSF, G-CSF, GM-CSF, MIP-1, MIP-2, LIF, OSM, TGF-β). Some cytokines can up-regulate the production of cytokines by macrophages (IL-3, GM-CSF, IFN-γ) while others can inhibit it (IL-4, IL-10, IL-13, TGF-β). In addition, these cytokines can modulate most of the macrophage functions and cell surface marker expression. Other cytokines (chemokines such as MCP-1, 2, 3, MIP-1,2 and RANTES) contribute to the recruitment of circulating monocytes within tissues.

T lymphocytes are important regulatory cells that secrete several cytokines and participate actively in this inflammatory response. According to the pattern of cytokines secreted, the immune response is classified as cytotoxic or type 1 (IFN-γ, IL-2, IL-12) and humoral or type 2 (IL-4, IL-5, IL-10 and IL-13) *(*Barcelò et al., 2006).

Current Insights and Future Advances in Endometriosis Diagnostics 425

patients. In contrast, serum levels of IL-6 were significantly higher in women with endometriosis than in controls and the highest levels were found in women with chocolate

Endometriosis is supposed to be an autoimmune disorder and many autoantibodies have been proposed as a diagnostic test. A variety of autoantibodies have been detected in endometriosis patients (thyroid peroxidase antibody, IgG anti-laminin-1 antibodies, antiphospholipid antibodies and the novel anti-PDIK1L antibodies). The most commonly reported types are antiendometrial antibodies, autoantibodies against the oxidative-stressinduced, antigens to malondialdehyde-modified low-density lipoprotein (LDL) and

Some investigators have hypothesized that antiendometrial antibodies may cause infertility in some women with endometriosis by preventing the fertilized embryo from implanting in the uterus. In addition, increasing evidence suggests that oxidative stress occurs in the PF of women with endometriosis and oxidatively modified lipoproteins

Serum and PF hormones levels vary in patients affected with endometriosis. Luteinizing hormone (LH) levels are significantly higher in both serum and PF in patients with endometriosis than in normal controls (Illera et al., 2001). However, levels of prolactin, thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH) in the serum

Recently it was reported that serum concentrations of leptin are increased in patients with endometriosis. This increase may play an anti-apoptotic role in activated endometrial stromal cells into the peritoneal cavity, stimulating endometrial cell implantation and cause infertility (Tanaka et al., 2003). Furthermore another study measured the serum concentration of leptin using a radioimmunoassay method, showing a significant association between leptin concentrations and stage of endometriosis (Viganò et al., 2002).

The physiological changes in endometriosis involve multiple steps of matrix remodelling. Endometriosis associated to abnormal matrix remodelling is affected by several molecular factors including proteolytic enzymes and their inhibitors, which mediate tissue turnover, and ovarian steroids, which normally regulate reconstruction of endometrium in the

The extracellular matrix (ECM) constitutes a well-organized network structure that surrounds the cells. The tissue remodelling involving ECM turnover is regulated by the pooled action of proteolytic enzymes, matrix metalloproteinases (MMP) and tissue inhibitors for MMP (TIMP). The inappropriate expression of MMP and TIMP is associated

with tumorigenesis and metastasis, as well as with endometriosis.

cysts (Wieser et al., 2003; Iwabe et al., 2003).

oxidized low-density lipoprotein (Ox-LDL).

were no different between the different groups.

**2.5 Proteolytic enzymes and their inhibitors** 

**2.3.2 Autoantibodies** 

were found in the PF.

**2.4 Hormones** 

menstrual cycle.

The role of cytokines and growth factors in the pathophysiology of endometriosis is evident. They are probably responsible for the proliferation of endometrial cells and implantation of endometrial cells or tissue. In addition, cytokines increase the tissue remodelling through their influence on matrix metalloproteinases. Probably the most important effect of cytokines on ectopic endometrial tissue is an increase in angiogenesis of ectopic endometrial tissue and neovascularisation of the affected region. Therefore, cytokines play an important role in the initiation, propagation and regulation of immune and inflammation responses. The activation of immune cells results in a burst and cascade of inflammatory cytokines.

ELISA kits are available to assess the cytokines in the serum and peritoneal fluid (PF) of endometriosis patients. PF is rich with variable cellular components including macrophages, mesothelial cells, lymphocytes, eosinophils and mastcells. Approximately 85% of PF leukocytes are macrophages. It has been hypothesized that peritoneal macrophage activation is a key step in disease initiation and progression. Activated macrophages in the peritoneal cavity of women with endometriosis are potent producers of cytokines (Bedaiwy et al., 2002). Thus, PF contains a rich mixture of cytokines. Cytokines, such as TNF-α, IL-1, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1 and IFN-γ, are elevated in the PF of women with endometriosis, suggesting that they are involved in the progression of the disease. The level of IL-1 in PF is positively correlated with the progression of endometriosis, but the serum level of IL-1 seems to have no correlation with endometriosis.

*The Tumor Necrosis Factors (TNF)* superfamily of cytokines represents a multifunctional group proinflammatory cytokines, which activate signalling pathways for cell survival, apoptosis, inflammatory responses, and cellular differentiation. Induction of cellular responses to TNF occurs through two receptors, TNFR1 (TNF Receptor-1 or CD120a) and TNFR2 (TNF Receptor-2 or CD120b). TNFR1 is activated in most human tissues by the binding of TNFα. On the other hand, TNFR2 is primarily expressed in immune cells and is activated by both TNFα and TNFβ (Kawasaki et al., 2002).

The main TNF is TNF-α, which is produced by neutrophils, activated lymphocytes, macrophages, NK cells and several non-hematopoietic cells. The TNF-α is involved in the normal physiology of the endometrial proliferation in the human endometrium. TNF-α is expressed predominantly in epithelial cells, especially in the secretory phase. The stromal cells stain for TNF-α mostly in the proliferative phase of the menstrual cycle. These data suggest that hormones influence the role of this cytokine.

Some reports found that concentrations of TNF-α in both serum and PF were very high at the early stage of the disease and decreased with the severity of the endometriosis. Moreover, the assessment of TNF-α levels in the PF can be used as a basis for non-surgical diagnosis of endometriosis.

The role of IL-6 in the pathogenesis of endometriosis has been widely studied. IL-6 is a regulator of inflammation and immunity, which may represent a physiological link between the endocrine and immune systems. IL-6 also modulates the secretion of other cytokines, promotes T-cell activation, differentiation of B cells and inhibits the growth of several human cell lines (Nothnick, 2001).

The data about IL-6 levels in the PF of patients with endometriosis are controversial. In fact, no statistically significant differences are reported between controls and endometriosis patients. In contrast, serum levels of IL-6 were significantly higher in women with endometriosis than in controls and the highest levels were found in women with chocolate cysts (Wieser et al., 2003; Iwabe et al., 2003).
