BDNF and TrKB expression levels in patients with endometriosis and their associations with dysmenorrhoea

This study included patients diagnosed with endometriomas and ovarian benign tumours who were referred to our centre from May 2017 to July 2018. The study was approved by the local research and ethics committee of Beijing Obstetrics and Gynaecology Hospital (No. 2016-KY-01). Written informed consent was obtained from each patient before sampling. The study group included forty-six patients who underwent laparoscopic surgery for ovarian endometrioma and received a histopathological diagnosis at Beijing Obstetrics and Gynaecology Hospital. Endometriosis was surgically and histologically diagnosed as stage I, II, III, or IV according to the revised American Fertility Society (r-AFS) classification scheme [14]. The demographic and clinical characteristics of the patients were as follows: 6 had stage II endometriosis, 3 in the proliferative phase and 3 in the secretory phase, and the mean age was 33.70 ± 5.80 years; 19 had stage III endometriosis, 9 in the proliferative phase and 10 in the secretory phase, with a mean age of 33.20 ± 6.20 years; and 21 had stage IV endometriosis, 9 in the proliferative phase and 12 in the secretory phase, with a mean age of 32.50 ± 6.40 years. The differences in age among these patient groups were not statistically significant (P > 0.05). The exclusion criteria were as follows: patients with no history of a sexual life, with hormonal medicine use within 3 months before surgery, or with adenomyosis, polycystic ovary syndrome (PCOS), pelvic inflammatory disease, endometrial lesions, genital dysplasia, malignant tumours of any organ, pregnancy or neurological diseases. In patients undergoing laparoscopic surgery, the cyst was completely detached under the laparoscope, the contents were aspirated, and a small amount of the cyst wall was then cut under strict aseptic conditions; this sample was used to isolate and culture primary ectopic endometrial cells, and parts of the sample were paraffin-embedded and cryopreserved.

Sixteen patients of reproductive age with laparoscopic surgery for benign ovarian tumours and who were diagnosed by histopathology or who had no endometriosis were recruited as the control group. Eight were in the proliferative phase, the other 8 were in the secretary phase, and the mean age was 33.40 ± 7.00 years. The difference in age between this group and the endometriosis group was not statistically significant (P > 0.05). The exclusion criteria were the same as those for the endometriosis group. The basic characteristics of the two groups of patients are shown in Table 1. Eutopic endometrial and ovarian endometriotic lesions were taken from the endometriosis group, and normal endometrium of approximately 1 cm × 1 cm × 0.5 cm in size was obtained from each subject in the control group.

Was used before surgery to evaluate. The presurgical severity of dysmenorrhoea in patients with endometriosis was evaluated by means of the VAS (0–10 score, 0 = no pain and 10 = maximum pain). The VAS is considered the ‘gold standard’ for pain measurement and can be used for multiple types of pain, including dysmenorrhoea, dyspareunia, dyschezia and chronic pelvic pain [15]. VAS scores were grouped into three levels: mild (ranging from 0 to 3), moderate (ranging from 4 to 6), and severe (ranging from 7 to 10).

All samples were fixed with 10 mM PBS-buffered 10% formalin, embedded in paraffin, and cut into sections of 4-μm thickness. For heat-induced epitope retrieval, deparaffinized sections in 0.01 mmol/L citrate buffer were heated for 20 min at 95 °C using a microwave oven. Immunohistochemical staining was carried out according to the avidin–biotin immunoperoxidase method using the UltraSensitiveTM S-P kit (Maixin Corporation, China). Endogenous peroxidase activity was blocked for 10 min with 50 μl peroxidase blocker from the UltraSensitive™ S-P kit, followed by washing with PBS 3 times for 2 min each time. The sections were incubated at 4 °C overnight with the following primary antibodies: anti-BDNF (1:50 dilution, ab108319; Abcam, Cambridge, MA, USA) and anti-TrKB (1:100 dilution, ab108319; Abcam, Cambridge, MA, USA). The sections were then rinsed and incubated with biotinylated secondary antibodies for 10 min. After washing with PBS, the sections were further incubated with horseradish peroxidase-conjugated streptavidin for 5 min and finally treated with diaminobenzidine in 0.01% H₂O₂ for 5 min. The slides were counterstained with Mayer’s haematoxylin, and the stained sections were observed under a microscope (Axio Imager 2, Zeiss, Oberkochen, Germany). The immunoreactive intensity of the BDNF- and TrKB-stained cells was quantified by a modified method of histogram scoring (H-score) as described previously [16]. The staining intensity was graded as follows: 0, no staining; 1, weak; 2, moderate; or 3, strong. A total percentage score (% of cells with staining intensity ≥ 1, namely, the sum of the percentage of cells with intensities of 1, 2, and 3) was used to semiquantitatively evaluate the tissue expression levels of BDNF and TrKB. The H-score was calculated using the following formula: H-score = [(% at 0) × 0] + [(% at 1 +) × 1] + [(% at 2 +) × 2] + [(% at 3 +) × 3].

BDNF and TrKB were stained in normal endometrium, eutopic endometrium, and ectopic endometrium. In both proliferative and secretory phases, BDNF had the highest protein expression level in ectopic endometrial tissue, followed by eutopic and normal endometrium (P < 0.05). The same was true for the expression of TrKB (P < 0.05). As shown in Fig. 1D and 2D. However, when comparing the expression of BDNF and TrKB between the proliferative and secretory phases, there was no significant difference in either ectopic or normal endometrium (P > 0.05).

Afterwards, the expression of BDNF in eutopic and ectopic endometria at each stage was analysed. In patients with stage II endometriosis, the expression of BDNF in the ectopic endometrium was significantly higher than that in the eutopic endometrium (P < 0.05). In patients with endometriosis stages III and IV, the expression of BDNF in the ectopic endometrium was also higher than that in the eutopic endometrium (P < 0.05). As shown in Fig. 2.

Furthermore, the analysis of the expression of BDNF in each stage of the ectopic endometrium showed that the expression of BDNF in stage IV was significantly higher than that in stage III and II. The expression in phase III was also significantly higher than that in phase II (P < 0.05). However, the expression of BDNF in phase III was not significantly different from that in phase II (P > 0.05). Meanwhile, in the analysis of BDNF expression in the eutopic endometrium in each stage, it was found that as the stage increased, its expression gradually increased (P < 0.05).

Similarly, in the analysis of TrKB expression in each stage, we found the same pattern. In patients with stage II, stage III and stage IV endometriosis, TrKB expression was found to be higher in ectopic endometrium than in eutopic endometrium (P < 0.05), as shown in Fig. 2. In eutopic and ectopic endometria of patients with endometriosis, TrKB expression increased with increasing stage. There were significant differences in TrKB expression in each phase of the eutopic endometrium (P < 0.05). In the analysis of TrKB expression in ectopic endometrium, there were significant differences between each stage (P < 0.05) but not between stage III and stage II (P > 0.05).

To analyse the correlations among BDNF, TrKB and dysmenorrhoea, the mRNA expression levels of BDNF and TrKB in the endometriosis group were assessed, as shown in the Fig. 3. The Spearman rank correlation coefficient for the association between BDNF mRNA expression in the eutopic endometrium of the endometriosis group and the dysmenorrhoea VAS score was r = 0.52, demonstrating that there was a moderate positive association between BDNF expression in the eutopic endometrium and dysmenorrhoea VAS score (P < 0.05); however, there was no association between BDNF expression in ovarian endometriotic lesions and the dysmenorrhoea VAS score (P > 0.05).

The Spearman rank correlation coefficient of the association between TrKB expression in the eutopic endometrium of the endometriosis group and the dysmenorrhoea VAS score was r = 0.56, meaning there was a moderate positive association between TrKB expression in the eutopic endometrium and the dysmenorrhoea VAS score (P < 0.05); however, there was no association between TrKB expression in ovarian endometriotic lesions and the dysmenorrhoea VAS score (P > 0.05).

Pearson analysis coefficients for the associations between BDNF and TrKB in both eutopic and ectopic endometrium were r = 0.82 and r = 0.66, respectively (P < 0.05).