Concentrations of the endocannabinoid N-arachidonoylethanolamine in the follicular fluid of women with endometriosis: the role of M1 polarised macrophages

Abstract

Although N-arachidonoylethanolamine (AEA; also known as anandamide) is present in human follicular fluid (FF), its regulation remains unknown. Therefore, the aims of the present study were to: (1) investigate the relationships between FF AEA concentrations in women undergoing assisted reproductive technology and their age, body mass index, ART characteristics and fertility treatment outcomes; and (2) assess how different inflammatory patterns may trigger AEA production by human granulosa cells (hGCs). FF AEA concentrations were higher in women undergoing IVF than in those undergoing intracytoplasmic sperm injection group. FF AEA median concentrations were lower in women undergoing ART because of male factor infertility than in women with endometriosis (1.6 vs 2.5nM respectively), but not women with tubal, hormonal or unexplained infertility (1.6, 2.4 and 1.9nM respectively). To evaluate the effects of macrophages on AEA production by hGCs, hGCs were cocultured with monocyte-derived macrophages. The conditioned medium from M1 polarised macrophages increased AEA production by hGCs. This was accompanied by an increase in AEA-metabolising enzymes, particularly N-acyl phosphatidylethanolamine-specific phospholipase D. The results of the present study show that high FF AEA concentrations in patients with endometriosis may be associated with the recruitment of inflammatory chemokines within the ovary, which together may contribute to the decreased reproductive potential of women with endometriosis. Collectively, these findings add a new player to the hormone and cytokine networks that regulate fertility in women.

Molecular Mechanisms Underlying the Association between Endometriosis and Ectopic Pregnancy

“…To date, little is known about the role of ECS in the pathogenesis of endometriosis; however, cannabinoids may contribute to the inflammation closely related to endometriosis [ 92 ]. It has been demonstrated that M1 macrophages stimulated AEA production by granular cells, which may explain the increased level of AEA in the follicular fluid in the course of endometriosis [ 93 ]. Moreover, similarly to EP, increased levels of systemic AEA were shown along with decreased expression of CB1 in the secretory phase as compared to the control.…”

The role of macrophages phenotypes in the activation of resolution pathways within human granulosa cells

“…The differentiation protocol used in this study was based on the one previously published by our research group. We initiated THP-1 differentiation by exposing the cells to PMA (25 ng/mL) for 48 h, followed by a 24 h resting period [18]. Differentiation of monocytes into resting M0 was confirmed by observation that macrophage-like cells adhered to tissue culture flask, adopting a stellate 2 Effects conditioned media obtained from culture of M1 and M2 macrophages in hGCs and COV434 cells viability.…”

“…THP-1-derived macrophages were obtained following a previous procedure described by Fonseca et al [ 18 ], with some modifications. Firstly, THP-1 cells were seeded at a density of 5.0 × 10 5 cells/well in a 24-well plate and stimulated with PMA (25 ng/mL) for 48 h. After PMA exposure, the medium was discarded and the cells were washed with PBS, followed by a further incubation in fresh medium for 24 h as a resting period.…”

“…To evaluate how macrophage phenotypes found in the ovaries affect the GCs functions, an interaction model was established between hGCs and COV434 cells and conditioned media (CMs) obtained from culture of M0, M1 and M2 macrophages [ 18 ]. hGCs were seeded in a 6-well plate (1.5 × 10 6 cells/well) and 24-well plate (7.0 × 10 5 cells/well) and COV434 cells were also seeded in a 6-well plate (8.0 × 10 5 cells/well) and 24-well plate (3.5 × 10 5 cells/well) in DMEM/F12 10% FBS, and incubated for 24 h for cell adhesion.…”