**5. Prospective and proposal**

#### **5.1 Clinical significance and potential applications**

Up to date, the only way to diagnose endometriosis is laparoscopy which is minimal invasive but expensive. Non-invasive and cheap diagnostic methods are urgent to be developed. CA-125 is a widely used serum marker for the diagnosis and evaluation of recurrent endometriosis or the success of a surgical treatment. A recent meta-analysis including twenty three studies and assessing the diagnostic performance of serum CA125 has shown that it's a poor diagnostic method with 90% specificity and 28% sensitivity (Mol *et al.*, 1998). One study has found serum IL-6 and peritoneal fluid TNF-α were able to discriminate between patients with and without endometriosis (Bedaiwy *et al.*, 2002). They stated the sensitivity and specificity of serum IL-6 reached 80% and 87% respectively, which is significantly higher than that of CA-125. It indicates that no inflammatory cytokine make a potential biomarker for diagnosis of endometriosis. Its predictive values in early endometriosis require further investigation.

In addition, the recognition of pathogenesis is able to provide new concept to the current unsatisfactory treatments. Hormone medicine is a commonly used drug for prevention of occurrence after surgical treatment. However, lots of patients are not sensitive to these medicines or cannot endure the side effects, such as vomiting and weight gain. New therapy with less adverse effects and more effective function needs to be developed. The study of oxidative stress, inflammation and angiogenesis in the pathogenesis of early endometriosis make it possible. For example, antioxidant therapy with 1200 IU of vitamin E and 1 g of vitamin C for a period of two months lead to a decrease in the inflammation cytokines such as MCP-1, RANTES and IL-6 in peritoneal fluid (Nalini Santanam, 2003). Another drug mifepristone with antioxidant effects was also found to exert an inhibitory effect on endometrial cell growth (Guo *et al.*). As angiogenesis, the VEGF inhibitor and anti-VEGF antibody have been demonstrated to be effective to control and inhibit implant lesion in mice model (Hull *et al.*, 2003). As process of endometriosis contains several steps, the ideal medicine has the potential to control the development of progression of endometrium in early pathophysiological stage (Fig. 4).

Fig. 4. Hypotheses and clinical potentials of endometriosis

#### **5.2 Study limitations**

468 Endometriosis - Basic Concepts and Current Research Trends

Endometriotic implants were collected for angiogenesis microarray and pathway analysis after microvessel assessments *in vivo*. Differentially expressed angiogenesis molecules CD34, VEGFA, VEGFB, VEGFC, VEGFD, VEGFR1, VEGFR2 and VEGFR3 were confirmed by quantitative PCR, Western blot and immunhistochemistry. Effects of EGCG on angiogenesis signal transduction were further characterised in human endothelial cell line. Microvessel parameters and angiogenesis VEGFC/VEGFR2 signaling pathway including Jun protooncogene (cJUN), interferon-gamma (IFNG), matrix metallopeptidase-9 (MMP9) and chemokine (C-X-C motif) ligand-3 (CXCL3) in endometriotic implants and endothelial cells were studied. The results showed that EGCG, but not Vitamin E, inhibited microvessels in endometriotic implants. EGCG selectively suppressed VEGFC and tyrosine kinase receptor VEGFR2 expression. EGCG down regulated VEGFC/VEGFR2 signaling through cJUN, IFNG and MMP9/CXCL3 pathways for endothelial proliferation, inflammatory response and mobility. EGCG also suppressed VEGFC expression and reduced VEGFR2 and extracellular signal-regulated kinases (ERK) activation in endothelial cells. VEGFC

Up to date, the only way to diagnose endometriosis is laparoscopy which is minimal invasive but expensive. Non-invasive and cheap diagnostic methods are urgent to be developed. CA-125 is a widely used serum marker for the diagnosis and evaluation of recurrent endometriosis or the success of a surgical treatment. A recent meta-analysis including twenty three studies and assessing the diagnostic performance of serum CA125 has shown that it's a poor diagnostic method with 90% specificity and 28% sensitivity (Mol *et al.*, 1998). One study has found serum IL-6 and peritoneal fluid TNF-α were able to discriminate between patients with and without endometriosis (Bedaiwy *et al.*, 2002). They stated the sensitivity and specificity of serum IL-6 reached 80% and 87% respectively, which is significantly higher than that of CA-125. It indicates that no inflammatory cytokine make a potential biomarker for diagnosis of endometriosis. Its predictive values in early

In addition, the recognition of pathogenesis is able to provide new concept to the current unsatisfactory treatments. Hormone medicine is a commonly used drug for prevention of occurrence after surgical treatment. However, lots of patients are not sensitive to these medicines or cannot endure the side effects, such as vomiting and weight gain. New therapy with less adverse effects and more effective function needs to be developed. The study of oxidative stress, inflammation and angiogenesis in the pathogenesis of early endometriosis make it possible. For example, antioxidant therapy with 1200 IU of vitamin E and 1 g of vitamin C for a period of two months lead to a decrease in the inflammation cytokines such as MCP-1, RANTES and IL-6 in peritoneal fluid (Nalini Santanam, 2003). Another drug mifepristone with antioxidant effects was also found to exert an inhibitory effect on endometrial cell growth (Guo *et al.*). As angiogenesis, the VEGF inhibitor and anti-VEGF antibody have been demonstrated to be effective to control and inhibit implant lesion in mice model (Hull *et al.*, 2003). As process of endometriosis contains several steps, the ideal medicine has the potential to control the development of progression of endometrium in

supplementation attenuated the inhibitory effects by EGCG.

**5.1 Clinical significance and potential applications** 

endometriosis require further investigation.

early pathophysiological stage (Fig. 4).

**5. Prospective and proposal** 

Due to unavailable systematic studies, the understanding about this disease is still limited although endometriosis has been studied for many years. The limitations and difficulties are as below:

First, at the time of diagnosis endometriosis has already been established with unknown history. Hence, it's impossible to undertake clinical research from the onset to maintenance, which mainly makes the etiology still unknown. Second, it's difficult to have adequate control group. The control women involved in most studies are patients without endometriosis, which means they might have other disease, such as uterine myoma and benign ovarian tumor. The difference between normal eutopic and ectopic endometrium as well as normal pelvic environment and that with endometriosis is impossible to study in human. Third, we have already known genetic, immune system and peritoneal microenviroment are associated with endometriosis, which is supported by different curative effect with the same treatment for different patients and diverse symptoms. Therefore, the clinical treatment is individual and personal which increase the difficulty to observe and compare the different treatments. Fourth, only human and primates suffer from spontaneous endometriosis. The reproductive anatomy, physiology and estrogen cycle characteristics of monkeys are similar to human. Therefore, monkeys are the best animal model to do studies which can not carry out in humans. However, only few centers have the capacity to maintain this expensive animal. The most commonly used animal model is rodents, but there is an enormous phylogenetic gap between these animals and human. Hence, the question is how much data from these rodent models can be extrapolated to human situation. Fifth, primary endometrial and endometriotic epithelial cells cannot be passaged and fall into senescence within 2 weeks, but a stable cell line is necessary for the mechanism study. Until now, there are no stable and commercial normal endometrial and endometriotic glandular and stromal cells available for studying. Several researchers immortalized these cells by using human telomerase reverse transcriptase (hTERT) (Kyo *et al.*, 2003) or transfecting SV40 T-antigen vector (Zeitvogel *et al.*, 2001). However, how much characteristics these immortalized cells are similar to primary endometrial cells are needed to approve.

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