This study was approved by the human Ethics Committee of the Pusan National University Hospital (Institutional Review Board: 2008072) and all of the females recruited for the study signed a consent form prior to participation. Two independent study populations were recruited: the first for identification of biomarkers (n=12) and the second for biomarker validation (n=6). The patients included individuals with a laparoscopic diagnosis of endometriosis aged between 25 and 40 years. Half the females had advanced endometriosis and the other half did not have endometriosis, adenomyosis or leiomyoma.

The eutopic endometrial cells were isolated from ~1 ml menstrual blood collected with a suction catheter on days 2–4 of the menstrual cycle. Menstrual blood samples were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 with 5% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA) and 0.2% collagenase (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C with 5% CO₂ for 1 h. To isolate eutopic endometrial cells, the media were centrifuged at 300 × g for 10 min. The cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin (v/v) and 1% amphotericin B (v/v) (Sigma-Aldrich, St. Louis, MO, USA) for 2 days at 37°C under 5% CO₂. The cells were then washed three times with phosphate-buffered saline (PBS) and resuspended in DMEM containing 10% FBS. After 2 weeks, the eutopic endometrial cells were collected following detachment with trypsin (Lonza Verviers, Belgium) and then washed twice with PBS.

Total RNA was extracted from the eutopic endometrial cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. An aliquot of total RNA (3 μg) was used as a template for single-stranded cDNA synthesis by incubating the RNA with reverse transcriptase (Invitrogen Life Technologies) at 37°C for 1 h. PCR amplification of the selected genes was performed using the appropriate forward and reverse primers (Table I). Real-time PCR was performed in 20 μl reactions in 96-well plates using a MyiQ™ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reaction conditions were optimized using an iQ™ SYBR^®-Green Supermix Kit (Bio-Rad). Gene expression was analyzed using the 2^−ΔΔCT method (22).

Total proteins were extracted from endometrial cells with 8 M urea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% (w/v) ethylenediaminetetraacetic acid (EDTA), 10 mM Tris/HCl (pH 8.3) and protease inhibitor cocktail (GE Healthcare, Piscataway, NJ, USA). Briefly, cells were incubated with the above cell lysis buffer for 30 min at room temperature and stirred every 5 min. The protein samples were then centrifuged at 18,000 × g for 15 min at 20°C and the supernatant was collected. Protein concentrations of all the samples were determined using a PlusOne 2-D Quant Kit (GE Healthcare). Aliquots of the protein samples were stored at −80°C until analysis.

Two separate 2-DE analyses were performed in order to analyse the difference of these two independent study populations, separately. Electrophoretic separation of total cell proteins was performed using a previously described method (23). Briefly, the protein samples of eutopic endometrial cells were diluted in isoelectric focusing (IEF) buffer containing 9 M urea, 2 M thiourea, 4% CHAPS, 500 mM EDTA, 0.002% (w/v) bromophenol blue, 75 mM dithiothreitol (DTT) and 1% (v/v) pharmalyte (pH 3–10 NL). A total volume of 350 μl containing 100 μg pooled proteins was then loaded onto a pH 3–10 NL immobilized pH gradient strip (GE Healthcare; 18 cm). After 12 h rehydration at 20°C, IEF was performed with an IEF electrophoresis unit (GE Healthcare) under the following conditions: a linear ramp from 500 to 1,000 V for 1 h and a constant voltage of 8,000 V for 6 h to deliver a total of 56,000 Vh. Following IEF, the strips were equilibrated twice for 15 min each in equilibration buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol and 0.002% (w/v) bromophenol blue. For the first equilibration, the buffer contained 1% DTT and for the second equilibration the buffer contained 135 mM iodoacetamide. An Ettan DALT 2-D gel system (GE Healthcare) was used for electrophoresis in the second dimension. The equilibrated strips were inserted into the top of the 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The gels were then stained using a PlusOne Silver Staining Kit (GE Healthcare). Spot detection, pair matching and normalization were performed using ProteomWeaver software (Definiens, Munich, Germany). Ratios of spot intensities for the control and endometriosis patients were calculated. Spots with intensities showing a change of >2-fold were selected for ESI-Q-TOF/MS analysis. The details of ESI-Q-TOF/MS analysis have been described previously (24).

Eutopic endometrial proteins (25 μg) were loaded onto a 10% (w/v) polyacrylamide gel and separated with electrophoresis. Proteins in the gels were transferred onto nitrocellulose membranes using a Trans-Blot^® SD Semi-Dry Transfer Cell (Bio-Rad). The membranes were blocked overnight at 4°C with 5% non-fat dried milk (BD Biosciences, Franklin Lakes, NJ, USA) in Tris-buffered saline with Tween-20 [TBST; 20 mM Tris-HCl (pH 7.6), 137 mM NaCl and 0.01% Tween-20]. The membranes were then incubated with an anti-collapsin response mediator protein 2 (CRMP2) antibody (ab62661, 1:10000 dilution; Abcam, Cambridge, MA, USA) for 3 h at 4°C. The membranes were washed three times with TBST and incubated with polyclonal goat anti rabbit IgG-horseradish peroxidase (HRP; ab6721, 1:3000 dilution; Abcam). Immunoreactive proteins on the membrane were visualized by enhanced chemiluminescence using an ECL-Plus Detection Kit (GE Healthcare) and exposure to X-ray film (Fujifilm Corporation, Tokyo, Japan) for 1–5 min. The film was then scanned and the bands were quantified using ImageJ 1.43 software (http://rsb.info.nih.gov/ij/download.html). Protein expression levels were normalized to those of β-actin on the same membrane.

Values are expressed as mean ± standard error of the mean (SEM). Student's t-test was used to compare results from the two groups of study subjects. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of undifferentiated stem cell markers (Oct-4 and CXCR4) in eutopic endometrial cells from endometriosis patients and healthy controls were compared using real-time PCR. Compared to the controls, mRNA expression of Oct-4 and CXCR4 was 21.36- and 23.5-fold higher, respectively, in the endometriosis patients (Fig. 1). These results confirm that the mRNA expression of undifferentiated stem cell markers was higher in the endometriosis patients.

2-DE analysis was performed to characterize differences of protein expression in eutopic endometrial cells from endometriosis patients and normal controls. Proteins corresponding to three selected spots on the 2-DE gel were identified using ESI-Q-TOF/MS. These proteins had a lower (≥3-fold) expression in endometriosis patients compared to that of the controls. Using this technique, we identified three differentially expressed proteins as CRMP2, UCH-L1 and MYL9 (Fig. 2; Table II).

The mRNA expression of four undifferentiated stem cell markers, including Oct-4, CXCR4, SOX2 and mesenchymal-epithelial transition factor (MET) in the second set of collected samples were analyzed using real-time PCR. Partially consistent with the results from the first set of samples, mRNA expression levels of Oct-4, CXCR4, SOX2 and MET were 16.9-, 22.36-, 4.88- and 16.95-fold higher, respectively, in the endometriosis patients compared to those in the controls (Fig. 3). However, these differences were not statistically significant. Increased mRNA expression of undifferentiated stem cell markers in the first and second sets of samples nevertheless suggests that there is a relevant connection between undifferentiated stem cells and endometriosis.

A second 2-DE analysis was performed to confirm the candidate biomarker proteins identified by the first 2-DE analysis. Among the three candidate proteins (CRMP2, UCH-L1 and MYL9) identified by 2-DE analysis of the first set of samples, only CRMP2 expression demonstrated a change of >2-fold (Fig. 4B). Therefore, CRMP2 was selected for further western blot analysis of eutopic endometrial cells in menstrual blood collected from three controls and three patients with advanced endometriosis. Western blot results demonstrated that CRMP2 protein levels were 2.5-fold higher in the controls compared to that of the endometriosis patients (Fig. 4C and D).