This study was approved by the Institutional Review Board at Pusan National University Hospital (PNUH IRB 2010144; Yangsan, Gyeongnam, South Korea) and all experiments involving animals were approved by the Animal Care and Use Committee of Pusan National University (PNU-2012-0120). Females with (n=15; endometriosis) and without (n=15; control) histologically confirmed endometriosis were enrolled. All the recruited subjects signed an informed consent form prior to their participation in the study. Females in the control group (aged, 25–48) were undergoing laparoscopy or laparotomy for gynecological problems other than endometriosis, i.e., ovarian cysts (mature cystic teratoma, functional cysts and hemorrhagic cysts). The endometriosis group consisted of females (aged, 27–40) who were diagnosed with endometriosis following laparoscopy or laparotomy. These patients complained of abdominal/pelvic pain, dysmenorrhea and/or subfertility. The patients underwent ultrasonography, pelvic CT and/or pelvic MRI in an outpatient clinic. Two patients were classified with minimal to mild disease (stages I and II) and 13 patients with moderate to severe disease (stages III and IV). Blood samples were collected during the proliferative phase from recruited patients. Blood was centrifuged at 400 × g for 15 min at 4°C and then the supernatant was collected and stored at −80°C until assayed.

Seven-week-old female BALB/C mice (n=16) were obtained from Koa Tech, Ltd. (Pyeongtaek, Gyeonggi, South Korea). The mice were housed in cages in environmentally controlled rooms (ambient temperature, 22±2°C; relative humidity, 50±10%) under a 12/12 h light-dark cycle (lights on 8:00 am-8:00 pm). Food and water were available ad libitum.

The endometriosis mouse model was established according to the methods previously described (19). Briefly, donor mice were subcutaneously injected with β-estradiol in corn oil (100 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). One week later, donor mice were anesthetized by intraperitoneal injection of Zoletil (weight, 15 mg/kg; Virbac, Carros, France) and 2% xylazine (Rompun; 15 mg/kg body weight; Bayer, Leverkusen, Germany). The entire uterus was then removed and transferred to a petri dish containing saline. Isolated uterine horns were reduced to small fragments with scissors, which were then resuspended in saline. Half of the preparation was injected into the pertitoneum of two anesthetized recipient mice with a syringe (day 0). Then, the incision was closed with running 4-0 prolene muscle and skin sutures. Following surgery, recipient mice (n=8; endometriosis) were injected with β-estradiol in corn oil (5 mg/kg; Sigma-Aldrich) every week, whereas control mice (n=4) were treated with corn oil only.

Two weeks later, all mice were sacrificed by CO₂ asphyxiation. Blood samples were collected into tubes coated with heparin and the plasma was obtained following centrifugation at 400 × g for 15 min. Aliquots of plasma samples were frozen (−80°C) until analyzed. In addition, the uterine horns and endometriotic lesions were collected and fixed in formalin for three days for the following histological evaluation. Subsequent to fixation, mice endometrial lesions and uterine horns were imbedded in paraffin wax and sectioned into 5-μm thick slices. The sections were stained with hematoxylin and eosin (Sigma-Aldrich). Stained sections were observed by light microscopy (CX31, Olympus, Tokyo, Japan).

Albumin and IgGs were removed from the human plasma using a Multiple Affinity Removal System Spin Cartridge HAS/IgG (Agilent Technologies, Wilmington, DE, USA). Following this, the samples were concentrated using a Spin Concentrator (Agilent Technologies) according to the manufacturer’s instructions. The total protein concentrations of the samples were determined using a bicinchoninic acid assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Electrophoretic separation of the proteins was performed as previously described (20). Briefly, 100 μg proteins diluted in isoelectric focusing buffer was loaded onto pH 3–10 NL Immobiline DryStrip gels (18 cm; GE Healthcare, Piscataway, NJ, USA). The strips were then equilibrated for 15 min in an equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol and 0.002% (w/v) bromophenol blue] containing 1% dithiothreitol or 135 mM iodoacetamide, separately. The equilibrated strips were inserted into 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels (18 cm) in an Ettan DALT 2-D gel system (GE Healthcare) and the gels were stained using a PlusOne Silver Staining kit (GE Healthcare). The spots were analyzed by ProteomWeaver software 2.2 (Definiens AG, Munich, Germany). Spots exhibiting differences in staining intensity of at least 2-fold were selected for electrospray ionization-quadrupole-time of flight/mass spectrometer (ESI-Q-TOF/MS) analysis. The details of ESI-Q-TOF/MS analysis have been described in a previous study (21).

Each human or mouse (10 or 80 μg) sample was separated by 8 or 10% SDS-PAGE and then transferred onto nitrocellulose membranes using a Trans-blot® SD Semi-dry Transfer Cell (Bio-Rad, Hercules, CA, USA) for 30 min. The nitrocellulose membrane (Whatman, Dassel, Germany) was immediately blocked with a 5% non-fat milk solution at room temperature for 1 h. The membrane was then incubated overnight at 4°C with the appropriate primary antibodies: Anti-α-2macroglobin (1:1000; Abcam, Cambridge, MA, USA), anti-apolipoprotein E (1:1000; Abcam), anti-apolipoprotein L1 (1:1000; Abcam), anti-C4A (1:5000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-haptoglobin (HP) protein (1:250000; Abcam) and anti-leucine-rich α-2-glycoprotein precursor (LRG; 1:1000; Abcam). Following this, membranes were washed three times with 1X Tris-buffered saline with Tween-20 and incubated (for 20 min at room temperature) with the following secondary antibodies: Anti-rabbit IgG-horseradish peroxidase (HRP) (1:5000 or 1:1000, Thermo Fisher Scientific, Inc., Rockford, USA) and anti-mouse IgG-HRP (1:5000; Abcam). The proteins on the membrane were visualized using an enhanced chemiluminescence detection kit (Surmodics, Eden Prairie, MN, USA). Bands were quantified using Image J 1.43 software (http://rsb.info.nih.gov/ij/download.html) and protein levels were normalized to those of β-actin on the same membrane.

All results were expressed as the mean ± standard error of the mean. A comparison between the two groups was performed using Student’s t-test and Tukey’s HSD Kramer comparison test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using JMP 7.0.1 (SAS Institute Inc., Cary, NC, USA).

Plasma samples from endometriosis and control patients were pooled into two groups and the differences in spot density within 2-DE maps were determined using ProtemoWeaver software (Definiens AG). A total of 220 protein spots in the endometriosis group (n=15) and 203 spots in the control group (n=15) were observed, of which 25 of these spots were identified to have differences in spot density of at least 2-fold. Twenty spots in the endometriosis samples had lower densities, whereas five spots were detected only in the control. Seven spots were then selected after the densities comparison followed the clinical stage (stage I=1, stage II=1, stage III=3, stage IV=10) of endometriosis. (Fig. 1). Finally, they were identified as Hp, LRG, C4A protein, apolipoprotein E precursor (Apo-E), apolipoprotein L1 precursor (ApoL-1) and alpha-2-macroglobulin precursor (α-2M) by ESI-Q-TOF/MS (Table I).

Western blotting was performed on a total of 13 samples including from females with endometriosis (early stage=2, advanced stage=7) and the control group (n=4). Quantification of protein densities was conducted by Image J software (Fig. 2).

As demonstrated in Fig. 2B, the relative densities of Hp, ApoL-1 and LRG in the endometriosis group were decreased compared with those in the the control group. In particular, quantitation of the Hp expression was reduced 3.05-fold compared with that of the control (P<0.05). By contrast, there was no change in Apo-E expression in females with endometriosis compared with those without. C4A and α-2M protein expression in females with endometriosis generally increased compared with that in the control group. LRG expression gradually decreased from the early to advanced stages.

Fig. 2C demonstrates the results of early (stages I and II) and advanced (stages III and IV) stage groups when compared with the control, in patients with and without endometriosis. The relative densities of Hp were reduced in early and advanced stages compared with the control (P<0.05). The expression of ApoL-1 generally decreased in the early and advanced stage groups compared with the control group. However, the expression of ApoL-1 in the advanced stage group increased compared with the early stage. The relative density of Apo-E was not observed to be altered in each group. The levels of α-2M and C4A increased in the early and advanced stages compared with the controls.

Histological examination revealed that uterine tissue samples at 14 days following transplantation into the peritoneal cavity had developed into endometriotic lesions with a typical histological appearance (Fig. 3). As illustrated in Fig. 4, the plasma Hp level was significantly downregulated in the surgically induced mouse model, as compared with the control mice (P<0.01).