Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

Abstract

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

Keywords: Dysmenorrhoea; endometriosis; pain; transient receptor potential ankyrin 1; transient receptor potential vanilloid 1.

Figure 1.

Relative gene expressions of TRPA1 (a) and TRPV1 (b) receptors. Columns represent the relative gene expression ratios normalised to RPL29 reference gene with qRT-PCR in the healthy control endometrium (n = 6), compared to autologous eutopic endometrium as autocontrol (n = 6), intact autologous rectosigmoid wall (n = 15), rectosigmoid DIE nodule (n = 15) and dysmenorrhoeic endometrium (n = 7) of women without endometriosis. Data are presented as mean ± SEM. (**P < 0.005, ***P < 0.001, Mann-Whitney U test). TRPA1: transient receptor potential ankyrin 1; TRPV1: transient receptor potential vanilloid 1; RPL29: ribosomal protein L29; qRT-PCR: quantitative real-time polymerase chain reaction; CTRL: healthy control endometrium; Auto CTRL: autologous eutopic endometrium; DIE: deep infiltrating endometriosis.

Figure 2.

Immunohistochemical staining of the TRPA1 receptor in healthy eutopic endometrium and in rectosigmoid DIE nodule. (a) Negative control using tris-buffered saline instead of the primary antibody in normal endometrial tissue. (b) Rectal myenteric ganglia, serving as positive control for TRPA1 expression. (c) Healthy eutopic endometrial tissue. (d) Rectosigmoid DIE nodule. (e) Rectosigmoid DIE nodule, glandular component. (f) Rectosigmoid DIE nodule, stromal component. (d) and (f) Sections shown on panels were taken from the same DIE patient who experienced severe, endometriosis-associated pain. Background staining was performed with haematoxylin and eosin to reveal the tissue structure. Black arrow heads denote TRPA1 receptor labelling. Magnification is X400, except panel (d) where it is X100. Scale bars: 50 µm, except panel (d) where it is 200 µm. TRPA1: transient receptor potential ankyrin 1; DIE: deep infiltrating endometriosis.

Figure 3.

Immunohistochemical staining of TRPV1 receptor in healthy eutopic endometrium and in rectosigmoid DIE nodules. (a) Negative control using tris-buffered saline instead of the primary antibody in normal endometrial tissue. (b) Rectal myenteric ganglia, serving as positive control for TRPA1 expression. (c) Healthy eutopic endometrial tissue. (d) Rectosigmoid DIE nodule. (e) Rectosigmoid DIE nodule, glandular component. (f) Rectosigmoid DIE nodule, stromal component. (d) and (f) Sections shown on panels were taken from the same DIE patient who experienced severe, endometriosis associated pain. Background staining was performed with hematoxylin and eosin to reveal the tissue structure. Black arrow heads denote TRPV1 receptor labelling. Magnification is X400, except panel (d) where it is X100. Scale bars: 50 µm, except panel (d) where it is 200 µm. TRPV1: transient receptor potential vanilloid 1; DIE: deep infiltrating endometriosis.

Figure 4.

Histology scores of TRPA1 (a) and TRPV1 (b) receptors in healthy control endometrium (n = 6) and rectosigmoid DIE nodule (n = 6) epithelium and stroma. Box plots with the whiskers represent the medians ± 25–75 percentiles of the histology score values (*P < 0.05, ***P < 0.001, one-way ANOVA, Bonferroni’s multiple comparison test). TRPA1: transient receptor potential ankyrin 1; TRPV1: transient receptor potential vanilloid 1; DIE: deep infiltrating endometriosis.