The abnormal expression of oxytocin receptors in the uterine junctional zone in women with endometriosis

The study was approved by the Ethics Committee of the First Affiliated Hospital of Medical University of Anhui (reference number: PJ20160409). There were two groups of patients with and without endometriosis. The mean age of patients in the endometriosis and control groups was 42.3 ± 3.9 and 41.5 ± 4.1 years old, respectively. The inclusion criteria for women with endometriosis were who had regular menstrual cycles (23–35 days), histologically confirmed endometriosis, experienced dysmenorrhea or not, received no hormone therapy or used an intrauterine device ≥ six months before hysterectomy. The exclusion criteria for study group were menopausal, adenomyosis, pelvic inflammatory disease and pregnancy 3 months before hysterectomy. Endometriosis and adenomyosis came to be seen as distinct entities. MRI is the method of choice for imaging and evaluation of JZ as an important diagnostic marker in the diagnosis of adenomyosis [19]. Because adenomyosis can be confidently diagnosed using MRI when the altered junctional zone thickness is greater than 12 mm and the expression pattern of oxytocin receptor in the junctional zone in women with adenomyosis had been studied by Zhang, women with evidence of adenomyosis were excluded from the scope of this study [20, 21]. The strict inclusion and exclusion criteria can insure the validity of causal relationship of the findings with the endometriosis against the probably confounder factors exist in our study. The control group included women with regular menstrual cycles who underwent hysterectomy due to cervical intraepithelial neoplasia III (CIN III) or stage I cervical cancer, but with no history of primary dysmenorrhea, no evidence of endometriosis and adenomyosis through gynecological and sonographic examination before surgery, no hormone therapy or use of an intrauterine device ≥ six months before surgery. There were no evidences of endometriosis and adenomyoisis through surgical examination and histology after surgery. According with the principle of ethics, all samples containing endometrium and junctional zone must from the excised uterus. There was no evidence that OTR expression in smooth muscle cells of CIN III or stage I cervical cancer changed comparing with normal uterus. In many studies about OTR expression in smooth muscle cells, CIN III or stage I cervical cancer patients were listed as control groups [13, 20]. Therefore, we incline to the view that OTR expression in JZ of CIN III or stage I cervical cancer patients have no changed compare with normal uterus. Based on the endometrial histology, there were 21 women with endometriosis, 15 women in the proliferative phase and 6 women in the secretory phase, as well as 48 women without endometriosis, with 26 women in the proliferative phase and 22 women in the secretory phase. In the control group, 9 patients suffered from secondary dysmenorrhea due to uterine leiomyoma. Dysmenorrhea is one of the most prevalent symptoms of uterine leiomyoma. High expression of myostatin and matrix- metalloproteinases-14 in uterine leiomyoma correlate with the presence of svere dysmenorrhea [22]. In many studies about OTR expression in smooth muscle cells, uterine leiomyoma patients were listed as control groups [13, 23]. Staging of endometriosis was performed according to the revised classification of the American Society of Reproductive Medicine (revised ASRM: I = 4; II = 2; III = 8, IV = 7). Seventeen ovarian endometriosis (OEM) and four deep infiltrating endometriosis (DIE) patients were in the study group. The severity of the recent dysmenorrhea in the endometriosis group was evaluated by a 10-cm Visual Analog Scale (VAS) before the surgery. The characteristics of the recruited patients of the case and control groups are listed in Table 1.

The uterine junctional zone was defined as the inner third of the myometrium [24]. All samples containing endometrium and junctional zone were collected from the mid-fundal and isthmic areas of the uterine anterior wall. The uterus was opened along the sagittal plane after surgery. Multiple 1 × 1 × 1 cm³ samples were collected and fixed in buffered formaldehyde and processed routinely for paraffin embedding. Serial 4 μm sections were prepared from each paraffin-embedded tissue block and handled for immunohistochemical staining. All uterine samples were examined by optical microscope to confirm presence of endometriosis, absence of adenomyosis and respective phases of the menstrual cycle. Under low light microscopy magnification, the uterine junctional zone was located just 3 mm below the endometrium [20].

After routine de-paraffinization and rehydration procedures, the slides were heated in a microwave oven (700 W) in citrate buffer saline (9.0) for twelve min and cooled at room temperature for antigen retrieval. Every sections were incubated with a drop of 3% H₂O₂ deionized water (PV-6000, Wuxi, China) for 20 min at 37 °C temperature. After 2 washes with phosphate-buffered saline (PBS), the slides were hatched with polyclonal rabbit anti-OTR (1:100 dilution, bs-1314R, Bioss, Beijing, China) overnight at 4 °C refrigerator. After 3 washes with phosphate-buffered saline (PBS), the sections were incubated with biotinylated anti-rabbit immunoglobulin G (1:400) for 30 min at room temperature. The bound antibody complexes were stained for 3 mins with diaminobenzidine. The slides were then washed, counterstained with hematoxylin, dried and mounted. Negative control sections were processed by omitting the primary antibody. Myometrium of pregnant uterus were used as positive controls. Immunoreactivity staining was characterized quantitatively by digital image analysis on the Image Pro-Plus 6.0 (Nikon, Japan). Images were obtained with a microscope fitted with a digital camera. A series of 4 random images on several sections were taken for each immunostained parameter to obtain a mean value. Staining was defined by color intensity, and a color mask was made. The mask was then applied equally to all images, and measurements were obtained. Immunohistochemical parameters were assessed in the area detected by total optical density and mean optical density, which is equivalent to the intensity of staining in the positive cells.

The results were presented as mean ± standard error of the mean. Statistical Program for Social Sciences (SPSS) for windows version 16.0 (IBM Corp, Armonk, NY, USA) was used to perform statistical analysis. Statistical comparison of data was carried out by Student’s t-test for non-paired samples. The normality tests showed that all data were in normal distribution. To evaluate possible effect of OTR expression levels on VAS score, a linear regression model was used. P-value less than <0.05 (p < 0.05) were considered statistically significant.