Extravillous Trophoblast Migration and Invasion: Impact of Environmental Chemicals and Pharmaceuticals

Abstract

During pregnancy, the migration and invasion of extravillous trophoblasts (EVTs) into the maternal uterus is essential for proper development of the placenta and fetus. During the first trimester, EVTs engraft and remodel maternal spiral arteries allowing for efficient blood flow and the transfer of essential nutrients and oxygen to the fetus. Aberrant migration of EVTs leading to either shallow or deep invasion into the uterus has been implicated in a number of gestational pathologies including preeclampsia, fetal growth restriction, and placenta accreta spectrum. The migration and invasion of EVTs is well-coordinated to ensure proper placentation. However, recent data point to the ability of xenobiotics to disrupt EVT migration. These xenobiotics include heavy metals, endocrine disrupting chemicals, and organic contaminants and have often been associated with adverse pregnancy outcomes. In most instances, xenobiotics appear to reduce EVT migration; however, there are select examples of enhanced motility after chemical exposure. In this review, we provide an overview of the 1) current experimental approaches used to evaluate EVT migration and invasion in vitro, 2) ability of environmental chemicals and pharmaceuticals to enhance or retard EVT motility, and 3) signaling pathways responsible for altered EVT migration that are sensitive to disruption by xenobiotics.

1.1. Introduction.

A successful pregnancy requires a healthy and properly functioning placenta to provide nutrition and protect the fetus throughout gestation [1]. The placenta produces a number of hormones and cytokines required to maintain pregnancy, ensure appropriate fetal development, and initiate parturition and lactation [2,3]. In addition, the placenta supports the nutritional demands of the growing fetus by exchanging oxygen, carbon dioxide, waste and nutrients at the fetal-maternal interface [4,5]. Arguably one of the most important roles of the placenta is its role as a semi-permeable barrier during development that selectively regulates exposure of the fetus to exogenous and endogenous compounds [4]. Nonetheless, even chemicals that do not cross the placenta can have direct effects on key physiological functions that can otherwise adversely impact fetal health.

Trophoblasts are among the first cells that give rise to the formation of the placenta. Trophoblasts differentiate to form several subtypes, each with specialized functions (Figure 1). Following fusion of the sperm and egg, the zygote becomes a blastocyst [6]. During this time, a trophectoderm forms around the blastocyst and invades the maternal uterine wall giving rise to cytotrophoblasts (CTBs). CTBs in contact with the uterus establish the maternal-fetal junctional zone. Upon this stage, CTBs are then able to differentiate into multinucleated synctiotrophoblasts (STBs) or form anchoring villi cell columns that attach to the uterine wall. STBs are bathed in maternal blood and are the major site of placental-fetal exchange of nutrients, oxygen, waste, and potential endogenous or exogenous exposures [7]. Furthermore, STBs are the main site of production of hormones, including human chorionic gonadotropin (hCG) and progesterone, that support pregnancy and fetal development [2].

Figure 1. Origin and Differentiation of Trophoblast Subpopulations within the Placenta.

The earliest epithelial cells arise from the trophectoderm within the blastocyst. Cytotrophoblasts are then able to differentiate into cell populations required for invasion into the uterus (extravillous, endovascular, and interstitial trophoblasts) as well as form villous structures that allow for nutrient, gas, and chemical exchange, metabolism, and hormone production (syncytiotrophoblasts). Created using Biorender and Smart.servier.

Extravillous trophoblasts (EVTs) are similarly derived from the differentiation of CTBs but instead provide different functions. EVTs detach from cellular columns of the anchoring villi and migrate into the maternal decidua (Figure 2). During this developmental stage, EVTs include both endovascular and interstitial trophoblasts. Both cell types invade the myometrium and remodel the uterine spiral arteries utilizing epithelial-mesenchymal transition [8]. The process of epithelial-mesenchymal transition is regulated by a myriad of proteins including transforming growth factor β (TGFβ), epidermal growth factor (EGF), Raf and Rho-associated kinase (ROCK), hypoxia-inducible transcription factor (HIF-1α), epithelial-cadherin (E-cadherin), and E-cadherin transcriptional repressors, Snail (SNA1) and Slug (SNAI2), among others [8,9].

Figure 2. Migration and Invasion of Trophoblasts into the Maternal Uterus During Early Placenta Development.

Early in the first trimester, extravillous trophoblasts migrate from the column of cytotrophoblasts and form plugs within the spiral arteries of the endometrium. Endovascular trophoblasts will insert within the endothelium of spiral arteries and interstitial trophoblasts will migrate into the endometrial tissue. Created using Biorender and Smart.servier.

This complex process involves the interaction of several molecular signaling cascades to ensure proper EVT migration and invasion [10,11] into the maternal endometrium leading to healthy placentation [12,13]. Around day 15 of human pregnancy, EVTs begin to detach from cellular columns and penetrate the maternal decidua basalis and the underlying maternal myometrium [14,15]. EVTs further differentiate into two subtypes: interstitial trophoblasts and endoarterial trophoblasts [12]. Interstitial trophoblasts penetrate the uterine interstitium and further differentiate into endoarterial trophoblasts that invade into the maternal spiral arteries, one of the most important steps in placentation [15,16]. Recruitment of EVTs to invade and remodel the spiral artery is accomplished by uterine natural killer cells and macrophages [17,18]. EVTs that invade into the lumen of maternal spiral arteries transform the artery to a low resistance vessel, which allows for transport of oxygen and nutrients to the developing fetus [10,12]. At a molecular level, EVT invasion and migration are regulated, in part, by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [19]. MMPs degrade the extracellular matrix and enable EVT invasion into the uterine interstitial tissue or spiral arteries. As their name suggests, TIMPs counteract this process and function as inhibitors of MMPs [19]. Moreover, a myriad of growth factors that coordinate signals through the ERK and P13K pathways regulate EVT invasion, migration, and proliferation [9]. Aberrations in molecular signaling of these pathways, through environmental exposures or pathologies, is thought to mediate a number of gestational hypertensive disorders.

Mounting evidence points to aberrant EVT invasion as a contributor to poor placentation and pathological pregnancies that can have detrimental effects on maternal health, fetal growth, maintenance of pregnancy, and the timing of parturition [15]. The purpose of this review is to 1) provide a brief overview of the role of EVT dysfunction in placental pathologies and 2) examine the ability of drugs and toxicants to either enhance or impair EVT migration thereby contributing to poor pregnancy outcomes.

1.2. Pathologic Disorders Associated with Altered Trophoblast Invasion and Migration.

A healthy pregnancy requires a properly formed placenta with remodeled maternal spiral arteries that regulate the fetal environment and ensure the fetus is carried to term [1]. Aberrations in EVT invasion are of concern as improper EVT migration is associated with poor outcomes such as preeclampsia, placenta previa or accreta, fetal growth restriction, and early pregnancy loss [16,20–22]. Given the fine-tuned relationship between EVT invasion and pregnancy success, it is essential to understand the exact mechanisms by which EVTs mediate the development of gestational disorders.

Hypertensive disorders during pregnancy, such as preeclampsia, are a major public health concern. Preeclampsia was first documented in 1637 by Francois Mauriceau, and while the field of preeclampsia research has grown substantially over the years, the exact etiology of preeclampsia still remains unclear. Preeclampsia is diagnosed when a mother exhibits the onset of hypertension with proteinuria or other end-organ damage after 20 weeks gestation [23]. Critically, preeclampsia is a major public health concern affecting 2 to 8% of all pregnancies worldwide [24] and it is a major contributor to maternal and fetal morbidity and mortality, including preterm birth [25]. It has been posited that preeclampsia arises in part from the shallow invasion on EVTs into spiral arteries and insufficient remodeling [16,26]. As a result, the spiral arteries exhibit high resistance to vascular flow, ultimately hindering maternal-fetal circulation and resulting in dangerously high maternal blood pressure [27]. In terms of molecular markers associated with preeclampsia, the expression of placental growth factor (PlGF) and placental protein 13 (PP-13), among others, are decreased in the serum of women with preeclampsia [28–30]. Conversely, levels of soluble FLT-1 (sFLT-1), inhibin A, and activin A are typically increased in the serum of women with preeclampsia [31,32]. In addition, insulin resistance and abnormal lipid profiles are often observed in women with preeclampsia [33]. Currently, the primary treatment for preeclampsia is bed rest and delivery of the fetus [23]. In terms of later life health outcomes, mothers who previously experienced preeclampsia are more predisposed to developing type 2 diabetes and a number of cardiovascular outcomes such as myocardial infarction and heart failure [34–37]. Future research is aimed at elucidating the mechanisms and underlying causes of preeclampsia with the goal of being able to alleviate the detrimental effects of the disease on both the mother and the fetus.

Excessive invasion of the uterine wall by EVT trophoblasts can also adversely affect placental development leading to placenta accreta spectrum (PAS). PAS was first observed in 1937, and is defined a pathologic condition where trophoblasts invade deep into the myometrium and beyond [38]. PAS is classified as: 1) accreta, when the placenta adheres to the myometrium; 2) increta, with invasion deep into the myometrium; and 3) percreta, where invasion reaches the uterine serosa [39]. Interestingly, in the Unites States alone, the incidence of PAS has increased 10- to 15-fold over several decades [40]. While the exact mechanism(s) responsible for developing PAS are unknown, two hypotheses currently exist. The first is that trophoblast function is impaired leading to an extra invasive phenotype of EVTs [41,42]. The second, more current theory, is that the decidua itself has failed to form in the area of the uterine scar allowing the trophoblasts to invade through thin decidual tissue [22]. Biomarkers of PAS include low Pregnancy-Associated Plasma Protein A (PAPP-A) levels in maternal serum, reduced levels of hCG, elevated levels of cell-free Fetal DNA (cffDNA), and circulating cell-free placental mRNA [43–46]. Treatment of PAS often involves a caesarian section with peripartum hysterectomy. With the placenta anchored securely to the uterus, PAS is a major cause of maternal morbidity due to hemorrhagic shock during delivery and uterine rupture before the onset of labor (usually early in pregnancy) [47,48].

Increased trophoblast invasion is also a common occurrence with placenta previa [49]. This condition occurs when the placenta adheres to the bottom of the uterus and partially or fully eclipses the cervix, preventing the delivery of the fetus [49]. As with many gestational disorders, further research needs to be conducted to determine why PAS develops and how to combat the detrimental effects of this disorder from a pharmacological perspective.

Other gestational disorders that may involve aberrations in EVT invasion (in at least some cases) include fetal growth restriction, miscarriage, and gestational trophoblastic disease [16,20,50,51]. Fetal growth restriction is thought to originate similarly to preeclampsia. Shallow invasion of EVTs into the myometrium limits blood flow from the mother to the fetus leading to less delivery of key nutrients at the maternal-fetal interface [16,21]. Fetal growth restriction imparts lasting effects on postnatal growth and health including increased susceptibility to the development of hypertension, obesity, diabetes, or other endocrine complications later in life [52]. It has been estimated that nearly 2/3 of all early pregnancy loss is due to improper trophoblast invasion [53]. In terms of trophoblast invasion, it has been demonstrated that a lack of invasion of EVTs into myometrial spiral arteries was observed in miscarriages compared to healthy, successful pregnancies in humans [20,54]. Gestational trophoblastic disease (GTD) occurs when benign or malignant tumors grow from malformed pregnancies [51]. GTD includes choriocarcinoma, invasive mole, hydatidiform mole (partial and complete), epithelioid trophoblastic tumor, and placental site trophoblastic tumor [55]. This condition arises from abnormal trophoblast growth following conception [51]. Treatment for GTD hinges on the specific type of tumor that develops and can potentially be fatal if left untreated [51]. However, the mechanism that causes these EVTs to continue to grow post-pregnancy remains to be elucidated. Taken together, much evidence exists that implicates proper EVT invasion in the regulation of successful pregnancy. However, more studies need to be conducted to delineate the exact cellular mechanisms underpinning abnormal EVT invasion and the pathogenesis of gestational disorders.

1.3. Impact of Chemicals and Drugs on Trophoblast Migration or Invasion.

Given the importance of proper trophoblast migration and invasion in placentation, there exists a clear need to investigate the effects of in utero exposure to xenobiotics on functional changes in trophoblasts. The majority of migration studies detailed below employ one of two methods, the scratch (wound healing assay) or the transwell (Boyden chamber) assay. Both assays quantify invasion by the addition of a Matrigel and involve time-lapse microscopy to analyze images using data quantification software, such as ImageJ, and quantify results [56]. In addition, the most common human cell types used in vitro to investigate placental cell invasion and migration are the placental cytotrophoblast cell line, the JEG3, and the immortalized extravillous cell line, the HTR-8/SvNEO. Currently, several studies have examined the effects of various drugs and environmental contaminants on trophoblast migration and/or invasion as shown in Tables 1–4.

Table 1.

Effect of Metals on the In Vitro Migration and Invasion of Extravillous Trophoblasts.

Chemical	Concentrations	Experimental Model	Migration or Invasion	Direction of Change	Assay Type	Ref
Cadmium	0.5–1μM	HTR-8/SVneo cells	Migration	Reduced	Transwell	[64]
Cadmium	1, 10, 25μM	JEG3 cells	Migration	Reduced	Transwell	[65]
Methylmercury	0.047, 4.7μM	HTR-8/SVneo cells	Migration	Increased	Gap closure assay (Cell Biolabs, Inc)	[77]
Sodium arsenite	0.625, 1.25, 2.5μM	HTR-8/SVneo cells	Migration and invasion	Reduced; no change	Invasion assay kit (ECM 555 QCM™ from Chemicon) and a migration assay kit (ECM 510 QCM™ Chemotaxis from Chemicon)	[76]
Zinc	10μM	HTR-8/SVneo cells	Both	Increased	Transwell	[80]

Table 4.

Effect of Pharmaceuticals, Phytochemicals, and Other Chemicals on the In Vitro Migration and Invasion of Extravillous Trophoblasts.

Chemical	Concentrations	Experimental Model	Migration or Invasion	Direction of Change	Assay Type	Ref
Valproate	100–400μM	HTR-8/SVneo cells	Migration	Reduced	Wound healing	[110]
Thalidomide	100–400μM	HTR-8/SVneo cells	Migration	Reduced	Wound healing	[110]
Dexamethasone	100–10,000nM	HTR-8/SVneo cells	Invasion	Reduced	Spheroid invasion	[118]
Δ 9 -Tetrahydrocannabinol	30μM	HTR-8/SVneo cells	Invasion	Reduced	Spheroid invasion	[118]
Ethyl Alcohol	25mM; 100mM	HTR-8/SVneo cells	Migration	Reduced; Increased	Scratch wound	[110]
PM2.5 1	120μg/ml	HTR-8/SVneo cells	Both	Reduced	Transwell	[114]

¹PM: Particulate matter less than 2.5 μm.

1.3.1. Heavy Metals.

Metals exist in two major classes: toxic and essential metals [57]. Exposure to metals is ubiquitous, persistent, and potentially detrimental to proper placental and fetal development [58]. However, the exact mechanisms that underpin these observed adverse health effects remain largely unknown. To date, several studies have investigated how metals, both toxic and essential, affect the motility of placental trophoblasts as shown in Table 1. Cadmium exposure is a major public health concern as individuals are exposed through food products, contaminated water or air, and cigarette smoke [59]. Cadmium is of particular importance to trophoblast invasion as exposure to this metal has been associated with hypertensive disorders during pregnancy as well fetal growth restriction [60–62]. Because of this association, it has been postulated that cadmium may reduce the ability of extravillous trophoblasts to invade the uterine wall, resulting in shallow invasion of the spiral artery, thereby leading to maternal hypertension and reduced fetal growth [63]. In HTR-8/SVneo cells, environmentally-relevant nontoxic concentrations of cadmium chloride (0.5–1 μM) reduced the extent of trophoblast migration [64]. In a second placental cell line, the JEG-3 cells, cadmium chloride treatment (1, 10, 25 μM) also decreased the migration of trophoblasts by altering expression of the transforming growth factor beta (TGF-β) pathway [65]. This is significant as TGF-β regulates cell growth, cell cycle progression, differentiation as well as extracellular matrix signaling, which all impact cell invasion [66]. Furthermore, expression of TGF-β genes has been shown to be down-regulated in preeclamptic placentas [67–69]. Taken together, these studies are significant and provide initial mechanistic insight into how cadmium exposure in utero can alter EVT functionality, potentially leading to hypertensive disorders, such as preeclampsia.

Exposure to sodium arsenite, another ubiquitous, toxic metal, during pregnancy is also of concern as millions of individuals across the world may be exposure to harmful levels of arsenic through contaminated drinking water [70]. Sodium arsenite (NaAsO₂) is the sodium salt of arsenous acid that contains inorganic arsenic and is often found in an aqueous form in the environment [71]. Exposure to sodium arsenite has been associated with low infant birthweight, increased incidence of infection, and preterm birth, among others [72–75]. Interestingly, treatment with sodium arsenite (at 0.625, 1.25, 2.5 μM) reduced migration but not invasion of HTR-8/SVneo cells [76]. The results from this study highlight the potential role that non-toxic environmentally relevant doses of sodium arsenite play in impacting trophoblast migration, which may underlie observed associations with adverse gestational outcomes and in utero arsenic exposure.

Methylmercury (MeHg), a potent toxic metal found naturally occurring in the earth’s crust and at high levels in some seafood, was shown to increase the migration of HTR-8/SVneo cells following exposure to nanomolar to micromolar concentrations [77]. MeHg exposures in utero are associated with extreme gestational outcomes such as fetal death, which may be mediated by modifying the invasive phenotype of EVTs, resulting in shallow placentation and ultimately fetal death [78]. Zinc is an essential metal that regulates cellular growth, division, and maturation during development [79]. Using the HTR-8/SVneo cell line and a transwell assay, 10μM zinc increased both migration and invasion of EVTs [80]. Collectively, these studies contribute to a larger body of literature that seeks to elucidate the cellular mechanisms that underlie metal exposure during pregnancy to better inform adverse placental pathologies observed in response to exposure.

1.3.2. Organic Contaminants.

Persistent organic pollutants (POPs) are of concern to toxicologists due to their ability to accumulate in the environment and persist in humans as a result of their long half-lives. POPs bioaccumulate in and can cross the human placenta [81]. In utero exposure to POPs has been associated with the development of gestational diabetes, hypertension, and fetal growth restriction, which have long-term impacts on fetal/child development [81,82]. One such POP, per- and polyfluoroalkyl substances (PFAS), are a class of synthetic chemicals used in a wide array of common household items [83]. While legacy chemicals, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) have been studied extensively and are being phased out of manufacturing; newer replacement chemicals, such as GenX, are less characterized [84]. Using a scratch assay, Szilagyi et. al demonstrated that PFAS, PFOA, and GenX at 1000ng/mL all decreased EVT migration of HTR-8/SVneo cells whereas only GenX caused a significant reduction in invasion [85]. This study is among the first to investigate how PFAS alters EVT migration/invasion and future studies should be employed as exposure to these compounds is ubiquitous and their role in EVT motility is largely understudied. Another emerging contaminant of interest is perfluorobutane sulfonate (PFBS), which is an alternate to PFAS and is thought to be more easily transferred through the environment compared to PFAS due to its shorter carbon chain structure [86,87]. High concentrations of PFBS (10 and 100 μM) reduced both migration and invasion, while low concentrations of PFBS (0.1 μM) enhanced cell invasion [87]. This observed biphasic phenotype was attributed to altered HIF-1α signaling, which plays an important role in trophoblast functions [87]. HIF-1α is a nuclear transcription factor that is induced in low oxygen environments to increase glucose transport and release growth factors important for angiogenesis. It has also been shown to up-regulate trophoblast migration and proliferation [88,89].

Benzo(a)pyrene (BaP) is a well-known polycyclic aromatic hydrocarbon (PAH) generated from the incomplete combustion of organic matter and is found at concentrations in the environment that can be detrimental to human health. Once metabolized, BaP metabolites can damage DNA or RNA. In terms of pregnancy outcomes, BaP metabolite exposure in utero is associated with the formation of DNA adducts in both maternal and fetal cord blood, which may lead to potentially life threatening birth defects or loss of the fetus [90,91]. Results demonstrated that concentrations of BaP ranging between 1 and 10 μM significantly reduced both the invasion and migration of HTR-8/SVneo cells as determined by a transwell assay [90]. This may be a result of BaP activating extracellular signal- regulated kinases (ERKs) ERK1/2 and c- Jun N- terminal kinase (JNK) signaling [90]. JNKs are activated by inflammatory stimuli whereas ERKs are stimulated by growth hormones. During the process of migration, both of these proteins are activated, subsequently increasing protein levels of MMP- 2, MMP- 9, and E- cadherin [90]. By increasing the levels of MMPs that break down extracellular matrix, trophoblast migration is enhanced.

Older, legacy POPs, such as flame retardants and pesticides, have also been well studied as reproductive and developmental toxicants. One such class of POP are the flame retardants polybrominated diphenyl ethers (PBDEs) utilized in many consumer products. There are over 209 different analogs of PBDEs, but BDE-47 is of most concern as it is found at high levels in both the fetus and the mother and in utero exposures are thought to be detrimental to neurodevelopment [92,93]. Using the HTR-8/SVneo cell line, Park et. al demonstrated that 20 μM BDE-47 stimulated trophoblast migration while reducing invasion [94]. This surprising discrepancy was attributed to altered MMP signaling, however further studies need to be conducted. Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), is a metabolite of BaP. While exposure to BaP has been associated with adverse pregnancy outcomes, the effects of its metabolite, BPDE, on placenta are understudied [95]. One study by Wang et. al was able to demonstrate that BPDE reduced both migration and invasion of HTR-8/SVneo cells as a result of reduced MMP2 activity [95]. Given the ubiquitous nature of these exposures, these studies provide further vital insight as to how exposure effects gestational health and EVT function. In addition, Robinson and colleagues were able to isolate primary CTBs from second trimester human placental tissue and evaluate the effects of BDE-47 on both migration and invasion. This study is unique as primary cells have been infrequently used to investigate changes in invasion but provide great insight as they are not transformed or immortalized like the HTR-8/SVNeo and JEG3 cell lines. Similar to the results from HTR-8/SVneo cells, BDE-47 reduced both migration and invasion in primary CTBs [96]. While cell lines are convenient and robust tools to evaluate trophoblast invasion, validation in primary CTBs offers confirmation of the relevance of toxicity responses to human placental cells.

1.3.3. Endocrine Disrupting Compounds (EDCs).

Due to the placenta’s role as a critical endocrine organ during pregnancy, disruption of placental endocrine function by exposure to EDCs is a public health concern. Endocrine disruption during pregnancy can lead to inflammation, fetal growth restriction, and impaired neurological function [97,98]. Bisphenol A (BPA) is one of the most widely studied EDCs in relation to in utero toxicity and trophoblast function. This is due to the fact that BPA has been used in the production of plastics and other commercial products for many years [99]. Exposure occurs on a large scale through consumer care plastics including water bottles and the lining of aluminum cans as well as other food packaging materials and receipts [99]. Treatment of human choriocarcinoma BeWo cells with BPA reduced migration in a transwell assay, which was a result of a down-regulation in MMP-2/MMP-9 expression, and a corresponding up-regulation of TIMP-1/TIMP-2 [100]. Two similar studies that used HTR-8/SVneo cells determined that BPA reduced both invasion and migration of trophoblasts by the similar mechanism of the down-regulation MMP-2 and MMP-9, and the up-regulation of TIMP-1 and TIMP-2. [101,102]. Conversely, a fourth study using HTR-8/SVneo cells in a scratch-based assay observed opposing results. Instead, BPA at concentrations between 0.1 and 50 μM enhanced trophoblast migration due to an increase in the expression of MMP-2 and MMP-9 [103]. The discrepancy between this study and the others could be due to the difference in methodology of scratch versus transwell assay and should be addressed in future investigations.

An additional EDC is p-Nonylphenol (p-NP). p-NP is a metabolite of alkyl phenol ethoxylates that are used for cleaning and is widely found in the environment [104]. Results from transwell assays revealed that p-NP treatment significantly reduced HTR-8/SVneo cell migration and invasion without altering proliferation [101]. The metabolism of di(2-ethylhexyl) phthalate (DEHP) to mono-2-ethylhexyl phthalate (MEHP) is also of concern as exposures to phthalates during pregnancy are associated with fetal loss and may be due to high levels of MEHP [105]. Treatment with MEHP in HTR-8/SVneo cells inhibited the activity of MMP-9 and resulted in a reduction of trophoblast invasion as determined in transwell studies [106]. Pesticides and fungicides are also major environmental sources of endocrine disruption. Within the class of pesticides, are triazole fungicides that are highly consumed worldwide and are shown to result in adverse pregnancy complication in rodents [107]. To investigate the effects of triazole fungicide on human trophoblast migration and invasion, treatment of HTR-8/SVneo cells with tebuconazole for 24 hours impaired both the invasion and migration of trophoblasts due to down-regulation of MMP-2 and MMP-9, and an up-regulation of TIMP-1 and TIMP-2 [108]. While a growing body of literature exists that investigates the role of EDCs on trophoblast motility, further studies are needed to how their metabolism and signaling affect EVT function.

1.3.4. Other Chemicals.

Alcohol readily crosses the placenta leading to fetal alcohol syndrome. While alcohol can induce cellular proliferation and growth of trophoblasts, the role that ethanol plays in altering trophoblast migration is a relatively understudied area [109]. One investigation utilized HTR-8/SVneo cells and found that ethanol treatment favored trophoblast migration in monolayer wounds and impaired migration in multilayer wounds [110] though more research is needed to identify key cellular mechanisms involved.

Global increases in environmental pollution have led to greater exposure to atmospheric pollutants during pregnancy. Fine particulate matter, less than 2.5 μm in aerodynamic diameter (PM2.5), is a pollutant of high concern. Epidemiological studies have reported relationships between high exposure to PM2.5 and fetal growth restriction, stillbirth, and spontaneous abortion, among a number of other adverse outcomes [111–113]. Using a transwell assay, researchers demonstrated that PM2.5 treatment of HTR-8/SVneo cells reduced migration, which may underlie observed associations between in utero PM2.5 exposure and adverse pregnancy outcomes in humans [114]. With over 85,000 chemicals in commercial use, more studies need to be conducted to investigate how EDC exposure during pregnancy affects EVT migration and invasion.

1.3.5. Drugs.

Certain drugs, such as dexamethasone, are prescribed during pregnancy for women experiencing complications resulting from inflammation, illness, or preterm birth [115]. Historically, the field of toxicology has recognized the ability of pharmaceuticals such as thalidomide and valproic acid to cause structural birth defects [116,117]. Valproic acid and thalidomide can notably reduce HTR-8/SVneo cell migration in a wound healing assay [110]. Other xenobiotics have also been evaluated for their effects on migration and invasion. The corticosteroid dexamethasone and the active ingredient of marijuana Δ⁹-tetrahydrocannabinol reduce the invasion of HTR-8/SVneo cells when grown in an innovative 3D EVT spheroid invasion model [118]. With recent increases in state-by-state legalization of medicinal marijuana and recreational use of marijuana among pregnant women, there exists a clear need to explore the effects of cannabis on trophoblast function. Additionally, the effects of many drugs on trophoblast invasion or migration represents a gap in the literature that should be addressed by future studies.

1.4. Perspectives and Future Directions.

Many of the current in vitro assays utilize transwell or wound healing (scratch-based) assays). While these assays are a cost-effective way to gain insight as to how exposures to various compounds alter EVT motility, they are relatively low throughput and data analysis can be tedious [56]. Additionally, scratch-based assays that do not use automated scratching methods may introduce considerable variability when interpreting results as scratch may differ in thickness from researcher-to- researcher. A second form of variation with both scratch and transwell assays is the establishment of a confluent cell monolayer as this is essential to reproducibility [56]. A non-confluent monolayer is known to considerably alter data interpretation and experimental results. Transwell invasion assays often requires the removal of non-invading cells with a cotton swab, which can create variability and affect reproducibility [119]. Even though they are not high throughput, these assays provide researchers with a wealth of foundational knowledge regarding trophoblast motility. One method that seeks to address the issue of the translatability of data from transwell studies and other 2D systems, is the 3D placenta spheroid model. This model aims to recapitulate changes and interactions between cell types seen in a real-world setting by assembling HTR-8/SVneo cells into a 3D sphere to more closely model an in vivo environment [118,120]. An alternative 3D placental cell model utilizes a microfluidics system with EVTs cultured in matrigel to recapitulate a 3D extracellular matrix found in vitro [121]. While these two 3D systems function differently, they both aim to create an assay design that closely recapitulates human placental development when compared to 2D modeling. However, several issues still exist with the adaptation and application of 3D assays including co-culturing of relevant cell types, sheer stress involved in microfluidics systems, and the consistent formation of spheroids. Future studies should address the differences between 2D and 3D placental models to determine the most accurate way to model in utero exposures and changes in trophoblast motility.

A majority of current in vitro placental migration and invasion utilize the HTR-8/SVneo cell line. This cell line provides many advantages including the fact that it did not arise from a choriocarcinoma (as is the case for most other human placental cell lines), is able to secrete placental hormones such as hCG, progesterone, and estrogen, and they are able to maintain the invasive properties of EVTs [122]. Recently, it has been shown that laboratory stocks of HTR-8/SVneo HTR-8/SVneo cell stocks for non-trophoblast cell markers including vimentin. Furthermore, there exist many differences between cell lines and primary EVTs. One stark difference is that cell lines will continue to proliferate in culture but primary EVTs differentiate and stop proliferating, particularly from term placentas [9]. This may affect the applicability of findings from cell lines to human populations. However, acquiring primary EVTs poses a challenge to researchers and may not be feasible for some due to specimen availability, particularly to first and second trimester placentas.

Current studies often examine the effects of a single contaminant on EVT migration and invasion. This is significant as exposures in a “real world” setting would exceed a single contaminant at a time, and co-exposures may potentially alleviate or exacerbate observed findings, further informing the field. For example, cadmium has been shown to reduce EVT invasion and migration, but zinc has the opposite effect [62,64,80]. A co-treatment or pretreatment with zinc may be able to rescue the observed less invasive phenotype. There also exists a significant gap in the literature regarding whether exposure to drugs in vitro alters EVT functionality. There is a lack of information on drug exposure during pregnancy including HIV, antidepressant, antiepileptic, or anti-inflammatory drugs in addition to other corticosteroids beyond dexamethasone. Investigating migratory invasion phenotypes of EVTs and elucidating molecular mechanisms is fundamental for the field of reproductive biology and toxicology. Current in vitro assays provide many advantages including that they are user-friendly, findings are reproducible, and are fast and cost effective. However, these in vitro assays lack the ability to screen compounds in a high-throughput manner. Future studies should aim to develop novel in vitro time-lapse microscopy techniques to increase throughput and the body of knowledge regarding exposures and EVT motility.

1.5. Conclusion.

The placenta is a key organ that is necessary for proper fetal development [1]. Placentation is regulated by trophoblast migration and invasion that are essential for a healthy pregnancy. EVT differentiation and motility are highly complex processes involving interrelationships between many molecular signaling cascades [8,15]. EVT migration and invasion is crucial to pregnancy as this phenomenon allows for proper transport of nutrients and oxygen to the developing fetus. Any aberrations in these processes can lead to shallow or extra invasive EVTs, which are associated with a myriad of gestational complications including growth restriction, preterm birth, placenta accreta spectrum, preeclampsia, and pregnancy loss. While the current literature describes several mechanisms by which exposures alter trophoblast migration and invasion, several aspects of current in vitro invasion and migration methodologies must be considered.

Table 2.

Effect of Organic Contaminants on the In Vitro Migration and Invasion of Extravillous Trophoblasts.

Chemical	Concentrations	Experimental Model	Migration or Invasion	Direction of Change	Assay Type	Ref
GenX	2.89 μM	HTR-8/SVneo cells	Both	Reduced	Scratch assay	[85]
Perfluorooctane sulfonic acid	2μM	HTR-8/SVneo cells	Migration	Reduced	Scratch assay	[85]
Perfluorooctanoic acid	2.415μM	HTR-8/SVneo cells	Migration	Reduced	Scratch assay	[85]
Perfluorobutane sulfonate	10, 100μM	HTR-8/SVneo cells	Both	Reduced; 0.1 μM increased cell invasion only	Scratch wound for migration; Transwell for invasion	[87]
Benzo(a)pyrene	1–10μM	HTR-8/SVneo cells	Both	Reduced	Transwell	[90]
Benzo[a]pyrene-7,8-diol-9,10-	0.25–1μM	HTR-8/SVneo cells	Both	Reduced	Scratch wound for migration; Transwell for invasion	[95]
Brominated diphenyl ether-47	20μM	HTR-8/SVneo cells	Invasion; Migration	Reduced; Increased	Scratch wound for migration; Transwell for invasion	[94]
Brominated diphenyl ether-47	1 and 5 μM	Second trimester CTBs	Both	Reduced	Minimum distance between nuclei for migration; Transwell for invasion	[96]

Table 3.

Effect of Endocrine Disrupting Chemicals on the In Vitro Migration and Invasion of Extravillous Trophoblasts.

Chemical	Concentrations	Experimental Model	Migration or Invasion	Direction of Change	Assay Type	Ref
Bisphenol A	0.01μM-100μM	BeWo	Invasion	Reduced	Transwell	[100]
Bisphenol A	1μM-1μM	HTR-8/SVneo cells	Both	Reduced	Transwell	[101]
Bisphenol A	0.0001μM-1μM	HTR-8/SVneo cells	Both	Reduced	Transwell	[102]
Bisphenol A	0.1μM-50μM	HTR-8/SVneo cells	Migration	Increased	Scratch wound	[103]
Para-nonylphenol	1μM-1μM	HTR-8/SVneo cells	Both	Reduced	Transwell	[101]
Mono-2-ethylhexyl phthalate	10, 100, 200μM	HTR-8/SVneo cells	Invasion	Reduced	Transwell	[106]
Tebuconazole	10–80μM	HTR-8/SVneo cells	Both	Reduced	Transwell	[108]

Highlights:

• Gestational complications are often associated with improper EVT invasion.

• Signaling of MMPs, TMPs, TGF-β, and JNK signaling are involved in EVT invasion.

• Many in vitro EVT studies use the traditional scratch assay or a transwell assay.

• Environmental chemicals and xenobiotics typically reduce the migration of EVTs.

• Reliable, high throughput methods are needed to evaluate EVT motility.