Bo Peng1, Fahad T Alotaibi1, Sadaf Sediqi1, Mohamed A Bedaiwy1, Paul J Yong1. 1. Department of Obstetrics and Gynaecology, University of British Columbia, BC Children's Hospital Research Institute and Women's Health Research Institute, Vancouver, British Columbia, Canada, V6H3N1.
Abstract
STUDY QUESTION: Does interleukin-1β (IL-1β) play a role in promoting nerve growth factor expression, neurogenesis and deep dyspareunia in endometriosis? SUMMARY ANSWER: IL-1β directly stimulates nerve growth factor (NGF) expression in endometriosis and is associated with local neurogenesis around endometriosis and more severe deep dyspareunia. WHAT IS KNOWN ALREADY: Local nerve density around endometriosis (using the pan-neuronal marker PGP9.5) is associated with deep dyspareunia in endometriosis, mediated in part by NGF expression. STUDY DESIGN, SIZE, DURATION: This in vitro study included endometriotic tissue samples from 45 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a university hospital affiliated research institute and included 45 women with surgically excised deep uterosacral/rectovaginal endometriosis (DIE, n = 12), ovarian endometriomas (OMA, n = 14) or superficial peritoneal uterosacral/cul-de-sac endometriosis (SUP, n = 19). Immunolocalisation of IL-1β, IL-1 receptor type 1 (IL-1R1), NGF and PGP9.5 in endometriotic tissues was examined by immunohistochemistry (IHC), and the intensity of IHC staining in the endometriotic epithelium and stroma was semi-quantitatively evaluated using the Histoscore method (H-score). For each case, deep dyspareunia was pre-operatively rated by the patient on an 11-point numeric rating scale (0-10). In addition, primary endometriosis stromal cells were isolated and cultured from surgically excised endometriosis. These cells were treated with IL-1β alone or in combination of Anakinra (an inhibitor of IL-1R1), small inference RNA (siRNA) against IL-1R1, siRNA against c-FOS or NGF neutralising antibody. The mRNA and protein levels of target genes (NGF and c-FOS) were assessed by reverse-transcription qPCR and western blot/ELISA, respectively. Furthermore, immunofluorescent microscopy was used to examine the neurite growth of rat pheochromocytoma PC-12 cells, as an in vitro model of neurogenesis. MAIN RESULTS AND THE ROLE OF CHANCE: For IHC, IL-1β expression in the endometriosis epithelium was significantly associated with more severe deep dyspareunia (r = 0.37, P = 0.02), higher nerve fibre bundle density around endometriosis (r = 0.42, P = 0.01) and greater NGF expression by the endometriosis epithelium (r = 0.42, P = 0.01) and stroma (r = 0.45, P = 0.01). In primary endometriosis stromal cells, treatment with exogenous IL-1β significantly increased the mRNA and protein levels of NGF and c-FOS. Pre-treatment with Anakinra, siRNA against IL-1R1, or siRNA against c-FOS, each attenuated IL-1 β-induced increases of NGF expression. In addition, supernatants from IL-1β treated endometriosis stromal cells significantly stimulated PC-12 neurite growth compared to controls, and these effects could be attenuated by pre-treatment with NGF neutralising antibody or Anakinra. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We did not have data from cultures of endometriosis glandular epithelium, due to the known difficulties with primary cultures of this cell type. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed a mechanism for deep dyspareunia in endometriosis, whereby IL-1β stimulates NGF expression, promoting local neurogenesis around endometriosis, which in turn leads to tender pelvic anatomic sites and thus deep-hitting dyspareunia. There may also be potential for drug targeting of IL-1β and/or NGF in the management of endometriosis-associated pain. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from the Canadian Institutes of Health Research (MOP-142273 and PJT-156084). P.Y. is also supported by a Health Professional Investigator Award from the Michael Smith Foundation for Health Research. MB has financial affiliations with Abbvie and Allergan. Otherwise, there are no conflicts of interest to declare.
STUDY QUESTION: Does interleukin-1β (IL-1β) play a role in promoting nerve growth factor expression, neurogenesis and deep dyspareunia in endometriosis? SUMMARY ANSWER: IL-1β directly stimulates nerve growth factor (NGF) expression in endometriosis and is associated with local neurogenesis around endometriosis and more severe deep dyspareunia. WHAT IS KNOWN ALREADY: Local nerve density around endometriosis (using the pan-neuronal marker PGP9.5) is associated with deep dyspareunia in endometriosis, mediated in part by NGF expression. STUDY DESIGN, SIZE, DURATION: This in vitro study included endometriotic tissue samples from 45 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a university hospital affiliated research institute and included 45 women with surgically excised deep uterosacral/rectovaginal endometriosis (DIE, n = 12), ovarian endometriomas (OMA, n = 14) or superficial peritoneal uterosacral/cul-de-sac endometriosis (SUP, n = 19). Immunolocalisation of IL-1β, IL-1 receptor type 1 (IL-1R1), NGF and PGP9.5 in endometriotic tissues was examined by immunohistochemistry (IHC), and the intensity of IHC staining in the endometriotic epithelium and stroma was semi-quantitatively evaluated using the Histoscore method (H-score). For each case, deep dyspareunia was pre-operatively rated by the patient on an 11-point numeric rating scale (0-10). In addition, primary endometriosis stromal cells were isolated and cultured from surgically excised endometriosis. These cells were treated with IL-1β alone or in combination of Anakinra (an inhibitor of IL-1R1), small inference RNA (siRNA) against IL-1R1, siRNA against c-FOS or NGF neutralising antibody. The mRNA and protein levels of target genes (NGF and c-FOS) were assessed by reverse-transcription qPCR and western blot/ELISA, respectively. Furthermore, immunofluorescent microscopy was used to examine the neurite growth of rat pheochromocytoma PC-12 cells, as an in vitro model of neurogenesis. MAIN RESULTS AND THE ROLE OF CHANCE: For IHC, IL-1β expression in the endometriosis epithelium was significantly associated with more severe deep dyspareunia (r = 0.37, P = 0.02), higher nerve fibre bundle density around endometriosis (r = 0.42, P = 0.01) and greater NGF expression by the endometriosis epithelium (r = 0.42, P = 0.01) and stroma (r = 0.45, P = 0.01). In primary endometriosis stromal cells, treatment with exogenous IL-1β significantly increased the mRNA and protein levels of NGF and c-FOS. Pre-treatment with Anakinra, siRNA against IL-1R1, or siRNA against c-FOS, each attenuated IL-1 β-induced increases of NGF expression. In addition, supernatants from IL-1β treated endometriosis stromal cells significantly stimulated PC-12 neurite growth compared to controls, and these effects could be attenuated by pre-treatment with NGF neutralising antibody or Anakinra. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We did not have data from cultures of endometriosis glandular epithelium, due to the known difficulties with primary cultures of this cell type. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed a mechanism for deep dyspareunia in endometriosis, whereby IL-1β stimulates NGF expression, promoting local neurogenesis around endometriosis, which in turn leads to tender pelvic anatomic sites and thus deep-hitting dyspareunia. There may also be potential for drug targeting of IL-1β and/or NGF in the management of endometriosis-associated pain. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from the Canadian Institutes of Health Research (MOP-142273 and PJT-156084). P.Y. is also supported by a Health Professional Investigator Award from the Michael Smith Foundation for Health Research. MB has financial affiliations with Abbvie and Allergan. Otherwise, there are no conflicts of interest to declare.
Authors: Bo Peng; Hong Zhan; Fahad Alotaibi; Ghadeer M Alkusayer; Mohamed A Bedaiwy; Paul J Yong Journal: Reprod Sci Date: 2017-07-04 Impact factor: 3.060