Cannabidiol (CBD) but not tetrahydrocannabinol (THC) dysregulate in vitro decidualization of human endometrial stromal cells by disruption of estrogen signaling

Highlights

• THC does not affect differentiation process and presents poor anti-aromatase activity.

• CBD impairs endometrial stromal cells differentiation.

• CBD is a good aromatase inhibitor.

• CBD prevents the increase of CYP19A1 transcripts and of estradiol levels in differentiating cells.

• CBD impairs decidualization that may be associated with pregnancy-related disorders and infertility.

Abstract

Decidualization, which comprises proliferation and differentiation of endometrial stromal cells (ESCs), is essential for the establishment of a receptive endometrium and pregnancy to occur. A deregulation of decidualization has been associated with miscarriage, infertility and other pregnancy-related disorders. The role of estradiol (E₂) on decidualization has already been shown, since it regulates proliferation of ESCs and expression of progesterone receptor. In this study, we investigated the effects of phytocannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD), in proliferation and differentiation of ESCs, as well as, in E₂ metabolism/signaling. We found that CBD, but not THC, inhibits ESCs differentiation. We also show that CBD prevents the increase on transcript levels of CYP19A1 gene and the elevation of E₂ levels that are observed in differentiating ESCs. Moreover, we found that CBD presents anti-aromatase activity. In overall, we highlight a novel effect of CBD on human endometrial differentiation, which may lead to infertility problems.

Introduction

In each menstrual cycle, human endometrial stromal cells (hESCs) proliferate and differentiate into specialized decidual cells, a process named decidualization [1]. The decidualized endometrium provides not only a receptive microenvironment for blastocyst implantation and pregnancy progression but is also considered an active gatekeeper to implantation [2]. Therefore, deregulation of the decidualization process is associated with endometrial and pregnancy complications, including unexplained infertility, recurrent spontaneous abortion, intrauterine growth retardation, preeclampsia and other clinical gynecological diseases, such as endometriosis and endometrial cancer [3].

Classical biochemical markers of decidualizing hESCs are, among others, forkhead box transcription factor (FOXO1), prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) [1,4]. This process is tightly regulated by the ovarian steroids, estradiol (E₂) and progesterone (P), as well as, by other molecules such as, cytokines and cell cycle regulators [2]. The aromatase enzyme, encoded by CYP19A1 gene, is involved in the biosynthesis of estradiol. This hormone regulates cellular proliferation and modulates the expression of the progesterone receptor during the secretory phase of the menstrual cycle, allowing hESCs to respond to progesterone [[5], [6], [7]]. Endometrium has already been described as a steroidogenic tissue, in which, aromatase expression and the production of E2 are increased on human endometrial stromal cells undergoing differentiation [8,9].

During the last few years, the endocannabinoid system has emerged as a critical modulator of female reproductive processes, particularly, in cellular differentiation and turnover [10]. We have previously demonstrated that the main endogenous cannabinoid, anandamide (AEA), inhibits hESCs proliferation and differentiation through the activation of cannabinoid receptor 1 (CB₁) and down-regulation of cyclooxygenase-2 enzyme expression [11,12]. Additionally, we recently reported that AEA inhibits aromatase activity and affects E₂ signaling in hESCs, mechanisms that may be implicated in endometrial dysfunction and, consequently, in fertility disorders [13]. It is known that in rat, the abundance of AEA in the uterine lumen disrupts the decidualization process and has an impact on N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH) expression [14]. Thus, the consumption of cannabis, which contains phytocannabinoids as tetrahydrocannabinol (THC) and cannabidiol (CBD) that act on the same receptors as the endocannabinoids, may induce a deregulation on the homeostasis of ECS and, in that way, affect the decidualization process. For instance, it has been reported that CBD may enhance endogenous AEA signaling by inhibiting FAAH, which is responsible for the intracellular degradation of AEA [15], and/or by inhibiting the cellular uptake of AEA [16].

There are only few studies regarding the potential (anti)-estrogenic effects of the two major phytocannabinoids, THC and CBD and, yet, the results presented are not consensual [17]. In 1997, it was reported that, in MCF-7 breast cancer cells, neither THC nor CBD activate estrogen receptor (ER) [18]. However, in rat uterus, it was shown that, although THC did not bind to ER, it could lead to an anti-estrogenic effect [19], whereas in another study, it was found that THC exhibited a weak estrogenic activity [20]. More recently, it was shown that marijuana induced an anti-estrogenic effect via the ER-mediated pathway, while per se, THC, CBD or cannabinol (CBN) did not induce any anti-estrogenic activity in human breast cancer cells [17]. In addition, Cannabis consumption has been associated with infertility, gestational disorders and increased risk of miscarriage. Nevertheless, the effects of phytocannabinoids in human decidualization have never been addressed.

Overall, in this work we investigated the effect of THC and CBD on hESCs differentiation and in E₂ metabolism and signaling. For this purpose, it was used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) isolated from term placenta.