Triclosan and bisphenol a affect decidualization of human endometrial stromal cells

Highlights

• Low TCL and BPA concentrations affect human ESCs physiology.

• Progesterone induced decidualization is enhanced by TCL and BPA.

• TCL shows more effects compared to BPA on ESCs.

• TCL and BPA induce ESCs migration.

• Exposure to EDCs may be a risk factor for fertility.

Abstract

In recent years, impaired fertility and endometrium related diseases are increased. Many evidences suggest that environmental pollution might be considered a risk factor for endometrial physiopathology. Among environmental pollutants, endocrine disrupting chemicals (EDCs) act on endocrine system, causing hormonal imbalance which, in turn, leads to female and male reproductive dysfunctions. In this work, we studied the effects of triclosan (TCL) and bisphenol A (BPA), two widespread EDCs, on human endometrial stromal cells (ESCs), derived from endometrial biopsies from woman not affected by endometriosis. Cell proliferation, cell cycle, migration and decidualization mechanisms were investigated. Treatments have been performed with both the EDCs separately or in presence and in absence of progesterone used as decidualization stimulus. Both TCL and BPA did not affect cell proliferation, but they arrested ESCs at G2/M phase of cell cycle enhancing cell migration. TCL and BPA also increased gene expression and protein levels of some decidualization markers, such as insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL), amplifying the effect of progesterone alone. All together, our data strongly suggest that TCL and BPA might alter human endometrium physiology so affecting fertility and pregnancy outcome.

Introduction

Human endometrium undergoes to morphological and functional changes required for a physiological reproduction. Most of these modifications are regulated by the cyclic equilibrium of estrogen and progesterone levels (Martin, 1980, Clarke and Sutherland, 1990). Briefly, when estrogen levels increase endometrial cells begin to proliferate (proliferative phase) (Gellersen and Brosens, 2003, Huhtinen et al., 2012) while when progesterone levels are predominant, stromal endometrial cells differentiate into decidual cells (secretive phase) (Johansson and Wide, 1969, Guerrero et al., 1975, Zhang et al., 1994). The term “decidualization” refers to the specific transformation of the stromal compartment of endometrium necessary to accommodate pregnancy (Gellersen and Brosens, 2014, Paule et al., 2010). Differentiation of stromal cells, indeed, represents the complex initial stage involved in the establishment of a successful pregnancy. So, impaired decidualization causes infertility because the uterus is not able to allow blastocyst implantation and decreases the endometrial receptivity of high quality embryos (Salker et al., 2010, Salker et al., 2012, Gellersen and Brosens, 2014). As a consequence, it is related to implantation and pregnancy failures (Strowitzki et al., 2006, Salker et al., 2011, Weimar et al., 2012).

Age, genetics, diet and environmental pollutants are considered the main risk factors for the onset of endometrial diseases such as endometriosis, endometrial cancer or altered decidualization and abnormal menstrual cycle (Modugno et al., 2005, Vercellini et al., 2014, Signorile et al., 2010). In this regard, many evidences suggest that the class of environmental pollutants known as endocrine disruptor chemicals (EDCs) may be responsible for endometrial-linked diseases (Cobellis et al., 2003, Kabbarah et al., 2005, Missmer et al., 2004). EDCs act both on reproductive and non-reproductive systems by mimicking the endogenous hormones essential for the correct physiology of the organism (Diamanti-Kandarakis et al., 2009). Bisphenol A (BPA) and Triclosan (TCL) are well known EDCs, widely used in plastic additives, food industry and personal care products (Rochester, 2013, Rodricks et al., 2010). They accumulate as contaminants in rivers, lakes, seas, groundwater and soil and have been found also in drinking water and food (Fang et al., 2010, Kang et al., 2006, Errico et al., 2014). Furthermore, EDCs concentration in human tissues results increased due to their bioaccumulation through the food chain (Muncke, 2009, Dann and Hontela, 2011). In fact, elevated concentrations of BPA or TCL have been reported in human breast milk, amniotic liquid, blood and urine (Calafat et al., 2008, Dayan, 2007, Henry and Fair, 2013, Nicolucci et al., 2013). Epidemiological studies demonstrated a relation between plasmatic and urine levels of EDCs and the onset of menarche, endometriosis, miscarriages and endometrial cancer (Akesson et al., 2008, Hiroi et al., 2004, Sugiura-Ogasawara et al., 2005). In rats, pre-natal exposure to BPA caused endometriosis-like phenotype in F1 generation (Signorile et al., 2010). Moreover, BPA has been found to affect in mice the placenta patho-physiology and the age of puberty and to stimulate proliferation of endometrial cancer cells (Gertz et al., 2012, Lee et al., 2005, Yang et al., 2014). Clinical studies reported that BPA is associated with miscarriages, decreased blastocyst formation and implantation failure (Sugiura-Ogasawara et al., 2005, Ehrlich et al., 2012a, Ehrlich et al., 2012b). Recently, our group demonstrated in human children, that BPA could also affect metabolic pathways, lipogenesis and insulin production (Menale et al., 2015). On the contrary, to date no findings have been reported in literature regarding the relation between TCL and endometrial pathophysiology. However, it has been demonstrated that TCL exerts both estrogen and anti-estrogen activity in vitro and it may be considered as a risk factor for estrogen-related cancer (Ahn et al., 2008, Gee et al., 2008). On the basis of these considerations, in this study we analyzed the effects of TCL and BPA on primary human stromal endometrial cells (ESCs), investigating on cellular proliferation, cell migration and decidualization, three mechanisms strictly linked each others (Petrie et al., 2009, Weimar et al., 2013). ESCs represent a useful cellular model for the analysis of placenta formation (Zhu et al., 2014), since they are able to differentiate in vitro under decidualization stimuli, such as progestins and cyclic adenosine monophosphate (cAMP) analogs (Telgmann et al., 1997, Hess et al., 2007). Here we investigated the ability of TCL and BPA to alter the physiological program of endometrium. To this aim in vitro experiments have been carried out using different concentrations of TCL or BPA on ESCs both undifferentiated or undergone to decidualization.