**4. Experimental designs**

Oxidative stress, proinflammatory responses and angiogenesis of endometriosis are important during early development of endometriosis. We postulate that the refluxed endometrial tissues in peritoneal cavity could stimulate oxidative stress and proinflammatory cytokines which promote the endometriotic adhesion, angiogenesis and implantation (Fig. 3). The methods we used are complementary to each other's insufficiency (Table 2).

Fig. 3. Summary of early pathogenesis in endometriosis


Table 2. Comparison of available study methods

Oxidative stress, proinflammatory responses and angiogenesis of endometriosis are important during early development of endometriosis. We postulate that the refluxed endometrial tissues in peritoneal cavity could stimulate oxidative stress and proinflammatory cytokines which promote the endometriotic adhesion, angiogenesis and implantation (Fig. 3). The methods we used are complementary to each other's insufficiency

Method Advantage Disadvantage

Expensive

expensive,

superficial vessels

expensive, nonlongitudinal

expensive, nonlongitudinal

non-longitudinal

stable

*in vitro*, quantitative, sensitive non-longitudinal, not

*in vitro*, quantitative non-longitudinal

IVIS *in vivo*, longitudinal, semiquantitative

quantitative

quantitative

Multiplex small sample, selected cytokines, quantitative

ELISA large sample, specific cytokines,

quantitative, sensitive

**4. Experimental designs** 

Fig. 3. Summary of early pathogenesis in endometriosis

Angiogenesis Cellvizio, IVIS *in vivo*, longitudinal, semi-

Cytokines Antibody Array small sample, all cytokines, semi-

ROS or RNS markers

markers

Table 2. Comparison of available study methods

Angiogenic

(Table 2).

Oxidative stress

## **4.1 Oxidative stress in early endometriosis**

In order to monitor the oxidative stress response in early development of endometriosis, an experimental endometriosis model in C57 mice was established by subcutaneous injection of mouse endometrium fragments. A chemiluminescent probe, L-012 (25mg/kg s.c.), was injected to the mice for the noninvasive *in vivo* oxidative stress imaging. L-012 is a new luminol derivative and sensitive chemiluminescence probe reacting with various types of ROS. ROS and reactive nitrogen species (RNS) production in the transplanted lesion can be monitored longitudinally by Xenogen IVIS 200 Imaging System. The results showed that *In vivo* imaging demonstrated significant increased bioluminescence signals for ROS/RNS from the transplanted lesions at the first hour interval. The signal reached a peak after 4 hours of transplantation. Then, the signal gradually decreased and maintained at minimal intensity in the rest of experiment. Immunohistochemistry showed positive lag correlation for the stained Hypoxia-inducible factors (HIF-1) in glandular epithelial cells and stromal tissue from the isolated lesions across the later time after transplantation. For angiogenesis, CD34, VEGF and Von Willebrand factor (vWF) signals were increased in parallel with HIF expression at 1 week thereafter. The non-invasive *in vivo* imaging method provides a valuable tool for monitoring oxidative stress in endometriosis and to understand its role in the early development and growth of endometriosis. The study indicated oxidative stress preceded HIF activation and VEGF angiogenesis in the pathogenesis of early endometriosis.

#### **4.2 Cytokine profiling in early endometriosis**

Both donor and recipient BALB/c mice at 7 weeks old were subjected to ovariectomy (OVX) and then were supplemented with 100ug/kg estradiol. Uterine horns from the donor mice were removed into F12 medium. Endometrium was punched into endometrial fragments after peeling off the serosa and myometrium under microscope. Fragments suspended in 0.3ml PBS were injected into peritoneal cavity of recipient mice. Peritoneal fluid was collected at experiment time intervals after transplantation. Cytokines profiles in peritoneal fluid were detected simultaneously. Differentially expressed cytokines were confirmed by ELISA quantification.

The results showed that the levels of CD30, CD36/SR-B3, Dickkopf-related protein (Dkk-1), epidermal growth factor (EGF), Eotaxin, IL-1 receptor antagonist (IL-1ra), IL-6 and Vascular cell adhesion protein 1 (VCAM-1) were significantly increased with the first hour of transplantation. This is the first report to analyze the peritoneal fluid cytokines profiles in experimental endometriosis in mice. The change pattern of cytokines could provide insights in understanding the early development of endometriosis. From the results, we can see that the oxidative stress and abnormal cytokine profiles might contribute to the early development of endometriosis.

#### **4.3 Angiogenesis in early endometriosis**

Mice were randomly treated with epigallocatchin-3-gallate (EGCG) extracted from green tea, Vitamin E (antioxidant controls) or vehicle (negative controls) for *in vivo* and *in vitro*  microvessel imaging at the end of intervention. Microvascular networks in the endometriotic lesions *in vivo* were imaged by Cellvizio LAB LSU-488 with ProFlex Microprobe S1500. Microvessel length and area were measured using Cellvizio LAB Vessel Detection software and averaged from 4 perpendicular regions of the lesion in replicate.

Pathophysiological Changes in Early Endometriosis 469

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

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

Fig. 4. Hypotheses and clinical potentials of endometriosis

**5.2 Study limitations** 

as below:

to approve.

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 supplementation attenuated the inhibitory effects by EGCG.
