84 women with endometriosis who underwent surgery in the Gynecology Department of Nanhai District Maternal and Child Health Hospital of Foshan City from November 2016 to October 2018 (Table 1) were provided informed consent and the study was approved by the Ethics Committee of the hospital. The patients ranged from 26 to 49 years (average, 34 ± 6.9 years). All patients underwent laparoscopic ovarian endometriosis cystectomy.
The control group comprised 32 patients, aged 22–48 years (average, 39.2 ± 7.0
years) from the same hospital who underwent endometrial curettage or total hysterectomy during the same time period because of tubal infertility and uterine fibroids. All of the study patients underwent surgery in the proliferative phase of the menstrual cycle, following 3–7 days after menstruation. None of the patients took steroids, were pregnant, or in lactation 6 months before any of the surgeries
All subjects were asked for a detailed medical history, which included information
on vaccinations, menstruation, marriages, and family. They received a detailed physical examination, laboratory and functional examinations, including hematuria, coagulation, vaginal secretions, liver and kidney function, thyroid function, electrocardiogram, detailed pelvic abdominal B ultrasound, and chest X-ray, to rule out the presence of any other endocrine diseases.
Paraffin-embedded tissues were archived. Each block was serially sectioned at 4-µm thickness for immunohistochemical staining. Baked at 60°C for 2 hours, deparaffinized and rehydrated for high-pressure, heat-mediated antigen retrieval for 5 minutes. The sheets were blocked endogenous peroxidase activity by using 3% hydrogen peroxide in 10 minutes, followed by washes (5 minutes each) with 0.01 M phosphate-buffered saline (PBS), and then blocked with the same serum as the secondary antibody source for 20 minutes at room temperature. The working solution for the primary detection antibodies, rabbit anti-Beclin-1(Abcam, ab55878, Cambridge, UK) and rabbit anti-LC3A/B (Abcam, ab48394, Cambridge, UK, was prepared in accordance with the primary antibody specification. The primary detection antibodies and the tablets were placed in a wet box and transferred to a 4°C refrigerator overnight, then washed three times with 0.01 M PBS (5 minutes each). Using the instructions for the horse radish peroxidase-labeled secondary antibody, the concentration of the secondary antibody was determined, and incubated with the homologous primary antibody in the wet box for 1 hour at room temperature. The sheets were washed three times with 0.01 M PBS, incubated with streptavidin-biotin complex in a wet box for 30 minutes at room temperature, and washed another three times with 0.01 M PBS (5 minutes each). Immunolabeling was visualized by dropwise addition of 3,3′-diaminobenzidine color staining solution for 5 to 10 minutes. The sheets were washed with water, and the tablets were stained with hematoxylin, then dehydrated and finally sealed with a neutral gum.
The sections were independently reviewed by two pathologists using a double-blind method. Beclin-1 was mainly expressed in the membrane and cytoplasm. LC3 was mainly expressed in the cytoplasm. Some nuclei and stroma were visible when strongly positive.
We observed five 40 × 10 high power fields at random; 100 cells were counted, and the color depth of the cells was observed. Ultimately, the ratio of cells and the depth of staining were assessed comprehensively. Beclin-1 and LC3 have the same scoring criteria.
1)Positive staining cell score: 1, positively stained cells accounting for ≤ 10% of the total cells; 2, positively stained cells accounting for 11–50% of the total cells; and 3, positively stained cells accounting for 51–75% of the total cells.
2)Dyeing depth score: 0, no staining; 1, light yellow; 2, yellow; and 3, dark yellow to brown.
3)Total score: positive staining cell ratio score × staining intensity score; ≤ 3 is negative, > 3 is positive.
Grading And Staging Of Endometriosis
The original records and surgical images of the endometriosis group were reviewed. Using the records of the location, number, size and adhesion of the endometriotic lesions, the American Society for Reproductive Medicine classification was used to correct the endometriosis staging method .
Statistical analysis was performed using SPSS 22.0 software. Expression of Beclin-1 and LC3 protein in both groups, and in subgroups at different stages of endometriosis, were analyzed by the chi-square test. Spearman correlation analysis was used to analyze the correlation. P < 0.05 was used to indicate a statistically significant difference.