Biological Tools to Study the Effects of Environmental Contaminants at the Feto-Maternal Interface.

The human endometrium is a fertility-determining tissue. It consists of various cell types. Epithelial cells and glandular-epithelial cells are located in the lining of the uterine lumen. Stromal, endothelial, and immune cells are mainly located in endometrial stroma. The cell types that reside in the endometrium result to be more sensitive to steroid hormones compared to the cell types of other tissues. 31 , 74 This raises concern about the possible deleterious effects of hormone-mimicking compounds on the cycling and pregnant human endometrium. 16 , 75 Primary cells from human endometrium can be easily cultured and are able to proliferate in vitro. 76 These primary cultures of endometrial cells respond to hormonal stimuli but, unfortunately, tend to dedifferentiate after several passages and therefore cannot be cultured for long periods. 77 , 78 Immortalized and carcinoma-derived cell lines, such as Ishikawa cells (derived from epithelial endometrial cells) and St-T1b (derived from stromal endometrial cells), are also available. 6 , 78 , 79 These cell lines are easily cultured and maintained for long periods, but they often lose responsiveness to hormonal stimuli and this can lead to misleading results. In vitro models of human endometrial epithelium are used to study blastocyst attachment and generally to focus on the very first steps of pregnancy establishment. Cell models of endometrial stroma are a powerful tool to study trophoblast invasion in the maternal uterus. Furthermore, such models are often used to study endometrial pathologies such as endometriosis. 80 Endothelial stromal cells can also be used to investigate the physiological and pathological mechanisms that take place in the human endometrium. Unfortunately, primary endothelial cells from human endometrium tend to lose their hormonal responsiveness after few passages. 74 Nevertheless, endothelial cells of uterine origin are far more sensitive to hormonal stimuli than endothelial cells from other tissues, that is, HUVECs, and thus tissue-specificity should be always considered when selecting an in vitro model. 74 Models of tissue explants from human endometrium have been also used. 81 – 83 These can be obtained from the decidual fragments remaining in the human placenta after elective termination of pregnancy or after delivery at term. More recently, three-dimensional (3D) models of human endometrium have been established and are gaining increasing interest since these models are closer to the in vivo situation. Indeed, endometrial cells show different features when grown in 3D matrices compared to cell monolayers. 76 , 77 , 84

Human pregnancy is a complex and finely regulated process during which 2 genetically different organisms, the mother and the embryo/fetus, establish a very intimate contact. 1 – 3 A huge number of molecules are secreted at the feto–maternal interface by both the mother and the embryo/fetus and act on both sides. 4 Thus, the mother and the embryo interact via specific tissues in a reciprocal exchange of molecules that act as communication signals. Placentation in human pregnancy involves invasion of the blastocyst in maternal endometrium up to the spiral arteries. The trophoblast cells, which surround the blastocyst, first contact the uterine epithelium, then dislodge the endometrial epithelial cells, and invade the maternal endometrium. During its journey, the trophoblast follows 2 differentiation pathways—the villous and the extravillous ones ( Figure 1 ). 5 In the villous pathway, the mononuclear cytotrophoblast cells forming the internal layer of villi fuse and form the multinucleated syncytiotrophoblast, which is the epithelial covering of the chorionic villi (floating villi). In the extravillous pathway, the cytotrophoblast cells move beyond the overlying syncytium and form multilayer cell columns of extravillous trophoblast that fix the villi to the maternal tissues (anchoring villi). These cells then move deeper into the maternal tissues up to the proximal third of the myometrium and endometrial spiral arteries ( Figure 1 ). Villous and extravillous trophoblast in human placentation. Human endometrium has a key role for reproductive efficiency, and its remodeling takes place under the control of steroid hormones. 6 , 7 This tissue undergoes dramatic cyclic changes regarding cell proliferation, secretory functions, regression, and regeneration. Hormonal stimuli lead the maternal tissues to the formation of the decidua at the end of each cycle. If fertilization has occurred, the blastocyst reaches the uterine epithelium and implantation starts. The correct signaling between the decidua and the fetal trophoblast is of paramount importance for blastocyst implantation and successful pregnancy ( Figure 2 ). 2 , 7 The interplay of endocrine and paracrine mediators at the feto–maternal interface. hCG indicates human chorionic gonadotropin; GFs, growth factors; PGs, prostaglandins. In the complex scenario of the feto–maternal interface, endocrine, paracrine, and autocrine factors must be taken into consideration. Hormones, such as estrogens, progesterone, and human chorionic gonadotropin (hCG) are the major players of the endocrine regulation that take place at the feto–maternal interface. Human chorionic gonadotropin, which is the hormone produced by the trophoblast in the very early stages of pregnancy, plays a key role in making sure that the endometrium is ready to receive the embryo implantation. 8 Those molecules which have an autocrine/paracrine action are important factors in the establishment and advancement of pregnancy. 9 Among these are cytokines including the interleukins (IL)s IL-1, IL-4, IL-5, IL-6, IL-8, and IL-10, the macrophage migration inhibitory factor (MIF), colony stimulating factors (CSFs) and the leukemia inhibiting factor, and different growth factors (GFs) such as the epidermal GF and the vascular endothelial GF. All these molecules are potent immunoregulators that play a key role in the feto–maternal tolerance to the semiallogeneic embryo. 10 These molecules are also mediators of cell proliferation/differentiation and apoptosis contributing to fetal growth and expansion in the maternal tissues. 11 , 12 Therefore, their presence at the feto–maternal interface may modulate the maternal immune response and contribute to the expansion of fetal tissues in the maternal uterus. Prostaglandins (PGs) also play a pivotal role in angiogenesis, mitogenesis, cell proliferation, and differentiation and exert an important role during the early stages of pregnancy. 7 , 13 , 14

Because of their ability to interfere with steroid hormones and their widespread distribution in everyday products, many of the chemicals in the environment represent a hazard to the reproductive system of adult women. Much greater concern arises when we take into account prenatal exposure to these bioactive compounds. Prenatal life is indeed the most critical phase of the life cycle, as it is the period in which organs and tissues are formed. 42 Already during the periimplantation period, the intrauterine environment is able to provoke epigenetical changes that will persist later in life. 43 Barker and coworkers initiated the investigation of the developmental origins of many diseases that appear in later life. 44 Then many researchers followed, revealing a dramatic scenario in which alterations in the intrauterine environment can result in pathologies and dysfunctions in adulthood. 45 For many decades, the human placenta has been considered a protective organ. It was assumed that the placenta interdicted the passage of harmful compounds to the fetus. However, the placenta has not been adapted to act as a barrier to the many substances that have been produced by man in the last decades. Therefore, the human placenta might not be able to prevent fetal exposure to these potentially dangerous compounds. Indeed, due to their high lipophilicity, many environmental estrogens are able to elude the placental barrier and potentially harm the fetus. 46 Moreover, the placenta is a highly sensitive tissue to environmental contaminants with estrogenic activity as it expresses both ERα and ERβ. 47 Since the placenta is the fundamental organ to maintain pregnancy and assure fetal development and growth, it seems to be extremely important to estimate the risk of exposure during in utero life by evaluating the effects of these contaminants on the placenta and the uterus. Animal models remain a solid and important shield to study human pregnancy establishment and development, but, unfortunately, they often fail to completely reproduce the complexity of human reproduction. Many efforts have been taken in order to shed light on the physiological, pathological, and toxicological aspects of human pregnancy, and in vitro models represent a valid help for researchers in this field. In this report, we will mainly focus on BPA and p -NP as representative chemicals with estrogen-like activity. Special emphasis will be put on the effects at low doses that are relevant with or are lower than those found in the environment and in human tissues. In fact, it is important to underline that environmental chemicals with estrogenic activity can have different effects at different doses. 6 In particular, some of these compounds have their detrimental effects at very low concentrations while higher concentrations are not effective in the same way. 48 – 50

Coculture models including cells and/or tissues representative of both the fetus and the maternal counterpart have been developed. 76 , 81 – 83 Many of these focus on the mechanisms of blastocyst implantation. Usually, human blastocyst is mimicked by spheroids of placental cells 76 , 77 that are allowed to attach to monolayer cultures of endometrial cells or 3D matrices. Other coculture models allow the study of a wider spectrum of interactions at the feto–maternal interface. 81 – 83 Such approaches mainly focus on the invasion of human placenta inside the maternal decidua. To mimic this situation, ex vivo explants of human placenta and decidua have been utilized. 81 , 82 , 99 These coculture models are a powerful tool as they include all the main cell types involved at the feto–maternal interface. They nevertheless require synchronization of primary cultures from tissues which are sometimes difficult to obtain. In order to overcome these limitations, Mannelli and co-workers 49 recently applied an in vitro system that could be helpful to study the molecular interactions at the feto–maternal interface even in laboratories that do not have availability of fresh tissues. Indeed, in their article, the authors presented an in vitro model in which explants of chorionic villi were exposed to an endometrial stromal cell-conditioned medium collected in another laboratory. 49 Such encouraging approach could widen the possibilities of different research groups across the world, and many similar efforts have been taken in the last years. Indeed, Huppertz and co-workers 100 developed the method of cryogenic preservation of placental explants in order to overcome the paucity of this tissue in other laboratories.

Coculture models of feto–maternal interface could represent a useful end point of toxicological studies, in which the effects of chemicals are tested under more complex and physiological conditions. These models have been largely used to investigate physiological processes of pregnancy 76 , 81 – 83 while only little has been reported for toxicological studies. Using the model of endometrial stromal cell-conditioned medium and placental explants, Mannelli and co-workers 49 demonstrated that the maternal compartment is able to protect the placenta from the effects of BPA. The study indeed showed that direct exposure of placental explant cultures with very low BPA concentrations (0.5-1 nmol/L) triggered the secretion of MIF and β-hCG and that these effects were abolished/diminished in the placental cultures exposed to cell-conditioned medium from endometrial stromal cells pretreated with BPA. Although the exact mechanism/s of maternal protection are not known, the data highlight the importance of in vitro models reproducing the complex interactions between the mother and the fetus to verify the effect of environmental chemicals in pregnancy. What is more, the choice to use very low concentrations of BPA revealed how concentrations in the range of 1 nmol/L are able to trigger a significant physiological unbalance in reproductive tissues. 49

Although not fully respecting the in vivo situation, in vitro models are informative about the effect of chemicals at the maternal–fetal interface and can give fruitful insights about the possible correlations between environmental pollutants and reproductive disorders. However, given the dynamic and complex mechanisms responsible for blastocyst implantation and placenta development in the maternal uterus, it is difficult to classify chemically induced alterations as adverse or nonadverse effects. One can only state that chemicals that are widely distributed in the environment and present in daily used products such as BPA and p -NP have the potential to interfere with the physiological processes of preparation of pregnancy and placentation. What raises great concern is that chemical concentrations lower than those causing cell death can alter fundamental biological processes such as uterine receptivity and placenta development. This might indicate that maternal contamination with these types of chemicals, at concentrations that do not cause termination of pregnancy, may cause dysfunction in pregnancy and fetal development. Moreover, as prenatal life is a critical period of life in which the body is formed and develops, any abnormality in its development will have negative consequences for adult life. This review aimed to give an exhaustive overview of all the in vitro models available and of their current use. Our hope is that more complex models will be applied to studies of environmental toxicology, and that the use of low concentrations of chemicals in toxicological tests will become of common use. Indeed, due to the limitations of in vitro models, it is important to prepare an experimental design that is as close as possible to the in vivo situation. The different dose–response effects and the tissue-specific effects of chemicals should be always considered as well. 6 , 91 In conclusion, the data reported here raise great concern for the environmental risk to pregnancy, indicating the need to develop more accurate tests for the protection of both the prenatal life and the development of the fetus.

There are increasing data that support the adverse effects on human reproductive health caused by environmental contaminants, especially endocrine-disrupting chemicals. These chemicals are man-made or plant-derived compounds that can bind to the receptors of steroid hormones and thus impair hormone-driven physiological functions in female and male reproductive system. 15 , 16 Among these, diethylstilbestrol (DES), a nonsteroidal estrogenic compound, has been commercialized as therapeutic for reproductive disorders. Regrettably, only after its commercialization its deleterious effects became evident. Among industrial compounds is bisphenol A (BPA), a polymer that can be released by polycarbonated plastics and by the linings of metal cans that are used for food and beverages. 17 , 18 Consequently, BPA can be easily absorbed via the food chain, 19 and indeed it can be easily detected in the human body. With regard to pregnancy, levels of BPA have been detected in the placenta, fetal liver, in the blood, and in the follicular fluid in a range of 0.3-40 nmol/L. 18 , 20 , 21 Bisphenol A shares many similarities with the endogenous estrogens 22 and acts on estrogen-responsive organs by binding to estrogen receptor (ER) isoforms, ERα and ERβ. 23 , 24 The estrogenic activity of BPA has been demonstrated in Ishikawa cells, an endometrial cell line. 6 Bisphenol A also binds to the progesterone receptors (PRs) and thus exert antiprogestin activity. 25 , 26 Furthermore, BPA is able to mimic glucocorticoids, another class of steroid hormones fundamental at the feto–maternal interface. Bisphenol A is indeed able to bind with the mineralocorticoid and glucocorticoid receptors (GRs). 27 , 28 Interestingly, it has been demonstrated that environmental concentrations of BPA (10 nmol/L) increased the messenger RNA (mRNA) expression and enzymatic activity of the enzyme 11β-hydroxysteroid dehydrogenase type 1 in the adipose tissue and the visceral adipocytes isolated from children. 29 Furthermore, perinatal exposure to BPA resulted in a decreased expression of GR in the brain of female rats compared to controls. 30 This environmental chemical was listed as reference compound within the European ReProTect Programme because of its well-known reproductive toxicity. 31 , 32 Another environmental chemical derived from the manufacturing industry is para-nonylphenol ( p -NP), an alkylphenol applied as a plasticizer and surfactant, known to have estrogenic activity since 1991. 33 Human exposure to p -NP may occur by cutaneous absorption, ingestion of contaminated food or water, and inhalation. 34 , 35 Estrogenic activity of p -NP has been known since 1991 33 and reported in a number of in vitro 36 and in vivo studies. 37 Interestingly, studies in rats showed that maternal exposure to p -NP resulted in an increase in uterine calbinding-D 9k (CaBP-9k) mRNA and protein expression in maternal and neonatal uteri, suggesting its potential transfer through the placenta. 38 , 39 CaBP-9k is a cytosolic calcium-binding protein expressed in various tissues (eg, intestine, uterus, and placenta) and a marker of estrogenic compounds exposure. 40 Since the number of man-made chemicals released in the environment have been growing exponentially in the last decades, 41 the assessment of the risks that derive from the exposure to chemicals is of paramount importance. 31