Melatonin activity and receptor expression in endometrial tissue and endometriosis

Abstract

Study question: Are melatonin receptors (melatonin receptor 1A (MR1A) and melatonin receptor 1B (MR1B)) expressed in human endometrium and endometriotic tissue, and does melatonin affect endometrial cell proliferation?

Summary answer: Melatonin receptors are expressed in human eutopic endometrium, endometriomas and peritoneal lesions, although to different extents, and melatonin treatment attenuated estradiol-induced endometrial epithelial cell proliferation in culture.

What is known already: Melatonin decreased endometriotic lesion volume in a rat model of endometriosis. Melatonin treatment reduced pain scores in and analgesic use by women with endometriosis.

Study design, size, duration: Basic science study using human endometrial tissue and an endometrial epithelial cell line.

Participants/materials, setting, methods: Measurement of melatonin receptor expression (mRNA and protein) in women with surgically confirmed endometriosis (endometrioma (n = 20) or peritoneal lesion (n = 11) alone) and women without surgical evidence of endometriosis (control, n = 15). Collection of endometrial and endometriotic tissue samples, gynecologic history and demographic information. Quantification of estradiol (1.0 nM) and melatonin (0.1 nM-1.0 μM) ± estradiol-induced endometrial epithelial cell proliferation in cultures of endometrial epithelial cells (CRL-1671) following 24 and 48 hours of culture.

Main results and the role of chance: MR1A and MR1B were localized by immunohistochemistry in glandular epithelial cells of endometrial biopsies from women with and without endometriosis. Both receptors were expressed in eutopic and ectopic endometrial tissue. mRNA expression of MR1A and MR1B was significantly greater in peritoneal lesions than in either endometriomas or eutopic endometrium. However, protein expression of MR1A was decreased in peritoneal lesions compared to control eutopic endometrium, whereas MR1B expression did not differ between the groups. Melatonin (0.1 nM-1.0 μM) treatment inhibited estradiol (1.0 nM)-induced endometrial epithelial cell proliferation at 48 hours but not 24 hours of culture.

Limitations, reasons for caution: Beneficial effects of melatonin seen in culture have yet to be comprehensively evaluated in women with endometriosis.

Wider implications of the findings: Our data suggest that melatonin may be useful as an adjunct to current endometriosis treatments.

Study funding/competing interest(s): This study was supported by the Canadian Institutes of Health Research (grant MOP142230 to W.G.F.). A.A.M. is supported by a resident research grant through the Physicians Services Incorporated Foundation. The authors have no conflicts of interest.

Keywords: CRL1671 cells; MR1A; MR1B; endometriosis; endometrium; epithelial; melatonin; melatonin receptors.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Figures

Figure 1

MR1A immunohistochemical localization in endometrium across the menstrual cycle. Representative photomicrographs of MR1A expression at medium (×100) and high (×200) power magnification during the proliferative (A) and secretory (B) phases along with negative (without primary antibody) controls.

Figure 2

Immunohistochemical localization of MR1B in the endometrium across the menstrual cycle. Representative photomicrographs of MR1B expression at medium (×100) and high (×200) power magnification during the proliferative (A) and secretory (B) phases along with negative (without primary antibody) controls.

Figure 3

MR1A and MR1B expression in control endometrium, eutopic case endometrium, ectopic endometriomas (E) and ectopic peritoneal (P) lesions. MR1A mRNA expression (A) was significantly elevated in ectopic peritoneal lesions (n = 11) relative to control endometrium (n = 14), eutopic case endometrium (n = 23) and ectopic endometriomas (n = 19). MR1B mRNA expression (B) was also significantly higher in ectopic peritoneal lesions (n = 11) relative to control endometrium (n = 15), eutopic case endometrium (n = 28) and ectopic endometriomas (n = 20). mRNA fold expression was relative to the housekeeping mRNA (YWHaz) used. MR1A protein expression (C, representative bands; E, densitometry) was significantly decreased in ectopic peritoneal lesions (n = 6) relative to control endometrium (n = 7) (eutopic case endometrium, n = 14; ectopic endometriomas, n = 10). MR1B protein expression (D, representative bands; F, densitometry) did not significantly differ between groups (control endometrium, n = 7; eutopic case endometrium, n = 14; ectopic endometriomas, n = 10; ectopic peritoneal lesions, n = 6). Statistically significantly different groups based on post hoc tests are denoted with different letters above a particular group. All data are presented as box plots, where the box represents 25th and 75th percentiles, and within which is shown the 50th percentile (the median). The whiskers represent the 5th and 95th percentiles.

Figure 4

Increasing concentrations of melatonin (0.1 nM–1.0 μM) alone and in the presence of estradiol (1.0 nM) attenuated endometrial epithelial cell proliferation at 48 hours of culture. Cell viability in the control wells was set to 1, and data are expressed on a log scale. Endometrial epithelial cells (CRL1671) were incubated in phenol red-free DMEM/F12 media supplemented with 10% charcoal-stripped FBS and 0.1% ITS. Results are the mean ± SEM of three separate experiments with eight replicates/concentration in each experiment. Data were analyzed by two-way ANOVA and Duncan’s multiple range test. A P < 0.05 was considered significant.