**2.5.1 Subject selection**

30 women with endometriosis and 20 without the disease were included in the study. Presence/absence of endometriosis was confirmed by diagnostic laparoscopy. It was ensured that these women had not received any kind of medical or hormonal treatment during the past three months. Women with history of chocolate cyst removal, previous history of any surgery, with other possible causes of pain or pelvic pathology including pelvic tuberculosis were excluded.

#### **2.5.2 Sample collection**

Blood samples collected from patients were allowed to clot and the serum separated by centrifugation at 3,000 rpm for 5 min at 4°C. Serum samples were stored at -20°C until further use. Endometrial biopsy was performed on the 7th day after confirmation of ovulation. The collected tissue was washed in phosphate buffer saline (PBS) and divided into three parts: one part was used for stromal and epithelial cells isolation for flow cytometric analysis of different molecular repertoires of the endometrium, the other part was fixed for immunohistochemistry (IHC) and scanning electron microscopy of these receptivity markers. From the third part, RNA was isolated immediately.

Alteration in Endometrial Remodeling: A Cause for Implantation Failure in Endometriosis? 331

sequence data. Total RNA was isolated from tissue by RNA isolation kit (Trizol Reagent, Invitrogen, Carlsbad, California, USA) and 10 μl of total RNA isolated was subjected to reverse transcription for cDNA synthesis with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Carlsbad, California, USA), according to the manufacturer's instructions. After synthesis, 5 μl of cDNA was used for the RT-PCR mixed with syber green. At the end of each reaction, Cycle threshold (Ct) was manually set at the level that reflected the best kinetic PCR parameters, and melting curves were acquired and analyzed. Relative quantification was used to measure gene expression by relating the PCR signal.

The endometrial tissue was homogenized in tissue lysis buffer. The tissue lysate was then centrifuged at 15,000 g for 15 min and the protein concentration of the homogenates was determined by the GeNei™ Protein Estimation Kits (Bangalore Genei, India). 30 µg of homogenate protein were separated by SDS-polyacrylamide gel electrophoresis (SDS- PAGE). The separated proteins were electroblotted onto a Hybond PVDF membrane (GE Healthcare) at 30 volt for 13 hrs. After blocking the non-specific binding sites with non-fat dry milk in TBST buffer for 1 hr at room temperature, the blots were incubated overnight at 4°C with rabbit polyclonal antibody against COX-2, mouse monoclonal antibody against MMP-2,-9, TIMP-1 and -2 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), rabbit polyclonal antibody against VEGF, VEGFR1+VEGFR2 (Abcam, Cambridge, UK). The blots were then washed three times with TBST buffer, incubated for 1 hr at room temperature with horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). After further

washing, the immunoreactive proteins were revealed using the DAB as substrate.

this purpose. Statistical significance was defined as p≤0.05.

Data were compared using independent two sample't' test and chi-square test, as applicable. Ky Plot version 2.0 beta 13 software and Graphpad Prism Software were used for

The clinical characteristics such as age, BMI, endometrial thickness, serum estrogen and

**Endometrial thickness (cm)** 9.25 ± 0.25 8.25 ± 0.41 P>0.05 **Serum estrogen level (pg/ml)** 258.5 ± 13.83 193.6 ± 14.66 P≤0.05 **Serum progesterone level (ng/ml)** 12.39 ± 1.28 26.43 ± 2 P≤0.05

Low levels of immunoreactivity of the endometrial receptivity markers including αvβ<sup>3</sup> integrin, LIF, L-selectin ligands (MECA-79) and Muc-1 were observed in women with

**Parameters Endometriosis Control P value Age** 29.5±0.61 29.32±0.83 P>0.05 **BMI** 28.18 ± 0.7 26.51 ± 0.6 P>0.05

progesterone levels of women participating in this study are summarized in Table I.

**2.5.7 Western blotting** 

**2.5.8 Statistical analysis** 

**3. Result** 

(Mean ± SEM) Table 1.
