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Published in final edited form as: Toxicol Lett. 2022 Dec 16;375:1–7. doi: 10.1016/j.toxlet.2022.12.004

High-Throughput Screening of Toxicants that Modulate Extravillous Trophoblast Migration

Cassandra Meakin a, Christine Kim a, Thomas Lampert b, Lauren M Aleksunes a,c,d
PMCID: PMC9877196  NIHMSID: NIHMS1861698  PMID: 36535517

Abstract

Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris™ cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 hours and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.

Keywords: extravillous trophoblast, migration, placenta, cadmium, bisphenol, zearalenone

Graphical Abstract

graphic file with name nihms-1861698-f0005.jpg

1. Introduction

The human placenta is a unique organ that while temporary has a lasting effect on fetal development (Burton and Fowden, 2015; Jansson and Powell, 2007). A major step in proper formation of the placenta is the migration and invasion of extravillous trophoblasts (EVT) into the maternal decidua and remodeling of spiral arteries (Albrecht and Pepe, 2020; Chen et al., 2012). During this process, arteries transition from high resistance to low resistance leading to efficient exchange of nutrients, waste, solutes, and oxygen at the maternal-fetal interface (Moser et al., 2018; Sato et al., 2012). Aberrations in trophoblast migration have been associated with a variety of gestational outcomes including preeclampsia, eclampsia, placenta accreta spectrum, fetal growth restriction, and more (Ball et al., 2006; Kaufmann et al., 2003; Tantbirojn et al., 2008). Therefore, it is essential to understand how perturbations to these processes mediate adverse gestational outcomes.

There are two methods commonly used to investigate trophoblast migration. The first method is the scratch assay (James et al., 2016; Liang et al., 2007). Upon the formation of an EVT monolayer, cells are scratched with a pipette tip or similar device. Images of the cells entering the ‘wound’ area are captured over time (Liang et al., 2007; Riahi et al., 2012). While this method is convenient and relatively inexpensive, some drawbacks include the inconsistency of results from scratch-to-scratch, the initiation of apoptosis upon scratch formation leading to release of cell death signals, and the relatively low number of side-by-side comparisons that can be made (De Ieso and Pei, 2018; Liang et al., 2007). The second commonly utilized method to evaluate EVT migration is the Transwell, or Boyden chamber, assay. Cells are seeded into the upper chamber of a Transwell insert until a monolayer is formed. Conditioned media is then added to the lower chamber of the Transwell system which stimulates cells to migrate through the membrane (Angelova et al., 2013). This membrane can then be stained and imaged at a terminal timepoint (James et al., 2016). While this assay is a robust method to evaluate changes in cell migration, there are several limitations. These include varying results based on how the Transwell membrane is stained and prepared for imaging, reliance upon a chemoattractant to stimulate migration, and the limited number of replicates or chemicals that can be screened (Katt et al., 2016).

Despite these drawbacks, both in vitro trophoblast methods have been widely used to identify chemicals that are known to alter EVT migration. For example, epidermal growth factor (EGF) stimulates migration whereas pertussis toxin and the toxic heavy metal cadmium reduce migration (Alvarez and Chakraborty, 2011; Han et al., 2010; Szilagyi et al., 2020). Exposure to chemicals in the environment is a contributing factor for adverse pregnancy outcomes. In addition to long-standing concerns regarding reproductive toxicity due to persistent chemicals such as pesticides and polyaromatic hydrocarbons, there is increasing scientific concern regarding estrogenic mycotoxins, such as zearalenone (ZEN), and newer chemicals developed for consumer products including bisphenols and organophosphate flame retardants. Early data suggest that in utero exposure to these chemicals is associated with adverse gestational complications including fetal growth restriction, low birthweight, impaired placental vascularization and loss of the fetus, among others (Kunishige et al., 2017; Luo et al., 2020; Müller et al., 2018; Zhang et al., 2014). One of multiple key physiological processes that can be disrupted by chemicals is the establishment of the uteroplacental interface. As a result, there is an urgent need to establish a reliable and high thoughput approach to identify environmental and dietary chemicals that alter EVT migration. In the current study, we sought to develop a new screening method that evaluates the ability of chemicals to inhibit or enhance trophoblast migration. We employed 96-well Oris™ migration plates containing stoppers that create cell-free zones to enable real-time imaging of EVT cell proliferation and movement.

2. Methods

2.1. Chemicals.

All chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri) unless otherwise stated.

2.2. Cell Culture.

Human immortalized HTR8/SVneo EVT cells were generously provided by Dr. Andy Babwah (Rutgers University). Cells were cultured in a 37°C incubator with 5% CO2, and HEPA-filtered air and maintained in phenol red-free RPMI 1640 media (Gibco, Waltham, MA) supplemented with glutamine (Gibco, Waltham, MA), 1% penicillin-streptomycin (Gibco, Waltham, MA), 1mM sodium pyruvate (Gibco, Waltham, MA), 1% non-essential amino acids (Gibco, Waltham, MA), and 10% fetal bovine serum (Atlantic Biologicals, Frederick, MD). Cells were used between passages 112 and 125.

2.3. Immunocytochemistry.

To ensure that the HTR8/SVneo cells used for the assay were not contaminated by fibroblasts (Abou-Kheir et al., 2017), immunocytochemistry was performed. In brief, cells were seeded into a 96-well plate at 8 x103 cells/well with 100 μL media. Cells were washed with PBS (Gibco, Waltham, MA) and fixed in 4% paraformaldehyde before blocking with 5% goat serum (Thermo-Fisher, Waltham, MA) in 0.1% Triton X in PBS. After blocking, primary antibodies against the fibroblast marker vimentin (MA5-11883; 1:100, Thermo Fisher, Hanover Park, IL) and the cytotrophoblast marker cytokeratin-7 (MA5-11986; 1:100, Thermo Fisher, Hanover Park, IL) were applied overnight. Cells were then rinsed and incubated with secondary goat anti-mouse antibody (A11029, 1:100, Thermo Fisher, Hanover Park, IL) for one hour. Cell nuclei were then stained with 5 μM Hoechst 33342 dye and imaged at 100X magnification.

2.4. Cytotoxicity.

Cytotoxicity studies were performed to identify non-toxic concentrations for use in cell migration experiments. HTR8/SVNeo cells were seeded at 0.8 x 104 cells per well and allowed to adhere overnight. On the next day, 100 μL of control or treatment media was added to each well. After 48 hours, cells were treated with 1 μM Hoechst 33342 and 0.5 μM propidium iodide (PI) for 30 mins at 37°C and then imaged on the Cytation (Agilent), SONY IMX 264 camera, using the DAPI filter at ex: 350 nm and em: 461 nm with bandwidths 377/50 and 477/60, respectively.

2.5. Cell Migration.

Oris™ tissue culture treated 96-well plates (Platypus Technologies, Fitchburg, WI) with stoppers were used for this assay. On day 1, cells were seeded (5 x104 cells per well) to achieve a confluent monolayer overnight. On day 2, stoppers were removed, revealing a cell-free detection zone and cells washed with culture medium. Fresh media containing vehicle (0.1% DMSO) or chemicals was then added and incubated for 48 hours at 37°C, with 5% CO2, and 98% relative humidity. Pertussis toxin (Cayman Chemical, Ann Arbor, MI) and EGF (Gibco, Waltham, MA) stocks were diluted to 10 ng/mL using culture medium. Stocks of test chemicals (bisphenols, cadmium chloride, flame retardants, and mycoestrogens) were diluted to non-toxic concentrations (0.1-10 μM) using culture medium and added to the plates. All chemicals were soluble at the concentrations tested.

2.6. Imaging and Quantification of Cell Migration.

Following removal of stoppers, plates were imaged once every 6 hours for two days using a Cytation 5 imager with Gen5 software. A high-contrast bright-field annulus used for these experiments allowed for the nuclei to be clearly identified and counted, generating a quantified cell count without the use of toxic dyes or phototoxicity. Cells were cultured in media with Hoechst for capture of representative images (ex: 350 nm em: 461 nm); however, quantification of EVT migration was performed in the absence of Hoechst dye.

To evaluate the ability of test compounds to alter EVT migration, a relative cell density (RCD) was calculated using an adapted equation from a prior in vitro migration assay (Grada et al., 2017; Johnston et al., 2015). In brief, a percent relative cell density (RCD) for each timepoint was calculated as follows (WT−WO)(CT−WO)×100 where Wt represents the number of cells inside the detection zone at timepoint of interest, WO represents of cells inside of the detection zone at time 0, and CT represents the number of cells outside of the detection zone at the timepoint of interest. To calculate the number of cells inside and outside of the detection zone, the area of each well was determined to be 2500 μm x 2500 μm. The plug area was then inserted and defined as Data Set 1 (DS1). Detection zone area was then defined as (2500 x 2500)-DS1. To determine changes in cell proliferation versus migration, the number of cells on the outside of the detection zone were calculated by subtracting the number of cells outside of the detection zone from timepoint 0 from the number of cells outside of the detection zone at the timepoint of interest.

2.7. Statistical Analysis.

Quantitative results were expressed as mean ± standard deviation (SD) (n = 4-6 replicates per treatment group). Data were analyzed using a one or two-way analysis of variance (factors: time and treatment) with a Dunnett posthoc test using GraphPad Prism 9.1.0 software (GraphPad Software Inc., La Jolla, CA). Asterisks (*) represent a statistical difference between control and chemical-treated groups. Significance was set at p<0.05.

3. Results

3.1. Development of a Trophoblast Migration Method.

Initial studies were designed to evaluate HTR8/SVneo cell migration in response to known modulators including stimulation (10 ng/mL EGF, positive control) and repression (10 ng/mL pertussis toxin, negative control). As shown in Figure 1, the detection cell-free zones were similar across all treatment groups at 0 hours. By 24 and 48 hours, there was greater accumulation of EVT cells in the detection zone for wells containing EGF whereas fewer cells were observed in this region following treatment with pertussis toxin. Similar treatment-related changes in EVT migration in response to EGF and pertussis toxin were also observed in the absence of the Hoechst 33342 dye (data not shown).

Figure 1. Time-dependent migration of human trophoblasts into cell-free detection zones.

Figure 1.

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Migration of HTR8/SVneo cells was analyzed at 0, 24, and 48 hours following treatment with epidermal growth factor (10 ng/ml, positive control) and pertussis toxin (10 ng/mL, negative control). Trophoblast nuclei were stained with Hoescht 33342 dye (100 nM). Images were captured at 10X magnification. White bars represent 1000 μm.

The relative cell density (RCD) inside the detection zone was calculated every 6 hours and used to quantitate migration and detect treatment-related changes. As early as 12 hours, increased migration with EGF and decreased migration with pertussis toxin compared to vehicle-treated control cells was observed (Figure 2A). These differences persisted throughout the entire 48-hour period. Moreover, by 24 hours, concentration-dependent changes in EVT migration were observed with both EGF and pertussis (Figure 2B). These data indicated that EGF (0.1-10 ng/mL) enhanced trophoblast migration up to 65% when compared to control cells. Conversely, pertussis toxin (1-10 ng/mL) inhibited trophoblast migration up to 39%.

Figure 2. Time- and concentration-dependent migration of human trophoblasts following pharmacologic treatments.

Figure 2.

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Migration of HTR8/SVneo cells (n=6 wells/treatment group) into cell-free detection zones was quantified as relative cell density (RCD) following treatment with increasing concentrations (0-10 ng/mL) of epidermal growth factor (positive control) and pertussis (negative control). A. Heatmap of trophoblast migration every 6 hours for 48 hours. The scale denotes low (white) to high (red) trophoblast migration. B. Comparison of trophoblast migration at 24 hours. Data are presented as mean RCD values ± SD. Two-way ANOVA demonstrated time (p<0.001) and treatment (p<0.001) as significant variables. Asterisks (*) represent a statistically significant difference (p<0.05) from control.

3.2. Evaluation of Trophoblast Migration Following Treatment with Environmental Chemicals.

Previous studies have demonstrated repression of EVT migration following treatment with the heavy metal cadmium (Alvarez and Chakraborty, 2011; Brooks and Fry, 2017). To determine whether this assay could similarly detect the ability of cadmium to repress trophoblast migration, HTR8/SVneo cells were treated with CdCl2 (0.01-10 μM) for 48 hours. Notably, these concentrations of CdCl2 did not cause apoptosis or alter cell viability in HTR8-SVneo cells for up to 48 hours as determined by a propidium iodide/Hoechst toxicity assay (Supplemental Figure 2). Similar to prior reports, treatment of trophoblasts with CdCl2 at concentrations of 1 μM or higher significantly reduced migration (Figure 3A and 3B). No change in EVT migration was observed at lower concentrations. The most notable treatment-related differences in migration could be observed at 18 hours and continued through 48 hours (Figure 3A). Interestingly, the impaired migration of EVT cells treated with cadmium chloride was completely rescued by the addition of EGF (Figure 3).

Figure 3. Time-dependent migration of human trophoblasts following cadmium chloride treatment.

Figure 3.

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Migration of HTR8/SVneo cells (n=6 wells/treatment group) into cell-free detection zones was quantified as relative cell density (RCD) following treatment with CdCl2 (0-10 μM) for up to 48 hours. Cells were co-treated with vehicle or EGF (10 ng/mL) A. Heatmap of trophoblast migration every 6 hours for 48 hours. The scale denotes low (white) to high (red) trophoblast migration. B. Comparison of trophoblast migration at 24 hours. Data are presented as mean RCD values ± SD. The dashed line represents control trophoblast migration. Two-way ANOVA demonstrated time (p<0.05) and treatment (p<0.05) as important variables. Asterisks (*) represent a statistically significant difference (p<0.05) from control.

To extend this method to additional chemicals, three classes of environmental contaminants were selected as case studies for screening effects on EVT migration: organophosphate flame retardants (OPFRs), bisphenols, and estrogenic mycotoxins. These chemicals are known to cause reproductive and developmental toxicities in rodents. Interestingly, the most significant changes in EVT migration were observed at 30 hours for every class of compound examined (Figure 4A). Of the three OPFRs tested (TPP, TCP, TDCPP) all significantly increased migration up to 20% compared to controls (Figure 4B). Conversely, bisphenol A, B, F, and S (BPA, BPB, BPF, BPS) all reduced trophoblast migration up to 15% at hour 30 (Figure 4B). The third class of chemicals screened were estrogenic mycotoxins. Zearalenone (ZEN) and its metabolites, α and β-zearalenol (α and β-ZOL), decreased EVT cell migration up to 17% at 24 and 30 hours (Figure 4A) whereas no change was observed with the metabolite and synthetic mycoestrogen zeranol (ZER).

Figure 4. Time- and concentration-dependent migration of human trophoblasts following screening of xenobiotics.

Figure 4.

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A. Migration of HTR8/SVneo cells (n=4 wells/group) into cell-free detection zones was quantified as relative cell density (RCD) following treatment with increasing concentrations (0.1-10 μM) of xenobiotics. Classes of chemicals screened included: organophosphate flame retardants (tricresyl phosphate (TCP), triphenyl phosphate (TPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP)), BBisphenols (bisphenol A (BPA), bisphenol B (BPB), bisphenol S (BPS), or bisphenol F (BPF)), and estrogenic mycotoxins including zearalenone (ZEN), its metabolites (α-ZOL and β-ZOL), and the synthetic form zeranol (ZER). B. Comparison of trophoblast migration at 30 hours. Data are presented as mean RCD values ± SD. The dashed line represents control trophoblast migration. Two-way ANOVA demonstrated time (p<0.001) and treatment (p<0.001) as important variables. Asterisks (*) represent a statistically significant difference (p<0.05) from control.

4. Discussion

Migration of trophoblasts and invasion into the endometrium are essential for anchoring the placenta to the uterus and ensuring maternal delivery of nutrients to the fetus. Currently, there is a need for a high-throughput screening method that reliably evaluates changes in EVT migration following exposure to xenobiotics. Therefore, we have developed a novel, high-throughput screening assay that quantifies changes in migration of human HTR8/SVneo cells in response to environmental and dietary contaminants including heavy metals, OPFRs, bisphenols, and estrogenic mycotoxins. A Z’-factor was calculated to assess reliability of the assay. When calculated, the Z’-factor for RCD was found to be 0.62, which is considered an excellent value as it falls between 0.5-1 (Buchser et al., 2004). Taken together, this real-time imaging screen is a reliable assay to evaluate EVT migration in the presence of environmental and dietary chemicals associated with pregnancy complications.

Several of these findings are consistent with previous literature. Pertussis toxin is a potent G-protein coupled receptor inhibitor and inhibits trophoblast migration by causing a reduction in the expression of several chemokine receptors that play a role in regulating cell motility (Gilder et al., 2016; Martinez-Olmedo and Garcia-Sainz, 1984; Szilagyi et al., 2020). Conversely, EGF enhances migration of EVTs by increasing the expression of genes that regulate cell cycle progression and growth (Malik et al., 2017; Qiu et al., 2004). As a result, pertussis toxin and EGF serve as suitable negative and positive controls, respectively for evaluating trophoblast migration (Han et al., 2010; Qiu et al., 2004; Szilagyi et al., 2020). In this study, HTR8-SVneo cells treated with EGF and pertussis toxin exhibited concentration-dependent responsive changes in migration where higher treatment concentrations resulted in increased and decreased migration, respectively. As has been reported previously in both JEG-3 cells and HTR8/SVneo cells (Alvarez and Chakraborty, 2011; Brooks and Fry, 2017), treatment with the toxic metal cadmium reduced migration of trophoblasts compared to controls. This finding is significant as in utero exposure of humans to cadmium has been associated with a heightened risk for development of adverse gestational outcomes such as preeclampsia, potentially due to shallow invasion of EVTs into the uterus (Goldman-Wohl and Yagel, 2002; Laine et al., 2015). Interestingly, the decreases in migration from cadmium exposure were completely rescued by the addition of 10 ng/mL EGF. EGF activates downstream signaling cascades that are known to increase trophoblast migration including extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling. These kinases affect the expression of genes involved in cell growth, death, and proliferation and may mediate the restorative effect that EGF has on impaired EVT migration following treatment with cadmium (Lin et al., 2022; Thomas et al., 2003).

Moving from cadmium to emerging toxicants of concern, our screening assay indicated that bisphenols, mycoestrogens, and OPFRs impact EVT migration without affecting trophoblast viability or proliferation (Supplemental Figure 3). At 30 hours, BPA, BPB, BPS, and BPF decreased migration up to 15% relative to controls. This is significant as previous studies have shown decreases in EVT migration following treatment with BPA using both the scratch and the transwell assay systems at treatments ranging from 0.01-100 μM suggesting a greater range of toxicity than observed in this assay (Spagnoletti et al., 2015; Wang et al., 2015; Wei et al., 2020). Furthermore, a recent paper demonstrated that BPS (~0.4 - 4.0 μM) inhibited EGF-stimulated migration and signaling in HTR8/SVneo cells, which may underlie observed reductions in migration in this study and others (Ticiani et al., 2022). By comparison, less is known about the developmental toxicity of ZEN and its metabolites. Gestational exposure to zearalenone in rodents is associated with increased fetal loss, infertility, and delays in fetal development and maternal toxicity (Gao et al., 2017; Zhang et al., 2014). We demonstrate for the first time that ZEN, α-ZOL, and β-ZOL decrease EVT cell migration which could lead to improper cellular signaling in the maternal uterus. Additionally, while no studies to date have examined the effects of OPFRs on EVT migration, OPFRs accumulate in the human placental tissues and in utero exposures to OPFRs induce placental cell death in rodent and human cell line models (Ding et al., 2016; Hu et al., 2017; Rock et al., 2018; Rock et al., 2020). Previous studies have similarly observed a stimulation of cell migration and invasion by TPP including glioma cells (Zhang and Song, 2022), colorectal cancer cells (Hong et al., 2022), and hepatocellular cancer cells (Ye et al., 2022). In the case of hepatocellular cancer cells, TPP stimulated expression of migration-related genes including CXCL3, SCUBE3, and MICAL2 (Ye et al., 2022). Whether similar mechanisms are responsible stimulating EVT migration by OPFRs are yet to be determined but future studies should also interrogate potential signaling pathways and whether OPFRs can enhance the depth of EVT invasion into the uterus. Taken together, these data demonstrate the utility of a novel screening assay reveal mechanistic data that could explain the placental toxicity of some environmental chemicals.

While developing this methodology, several factors had to be considered. It has been demonstrated that some stocks of HTR8/SVneo cells are contaminated with fibroblast cells, which would affect data interpretation (Abou-Kheir et al., 2017). HTR8/SVneo cells used for this study were stained with antibodies against cytokeratin-7 (marker of cytotrophoblasts) and vimentin (marker of fibroblasts) to ensure the cells used consisted of a trophoblast-enriched population (Supplemental Figure 1). All HTR8/SVneo cells stained were negative for vimentin and positive for cytokeratin-7, indicating a pure trophoblast population. In order for primary EVTs to maintain their invasive phenotype of interest for migration assays, EVTs must be isolated from first trimester placentas (Aboagye-Mathiesen et al., 1996; Tarrade et al., 2001). For this study, we were unable to isolate primary EVTs due to limited access to first trimester placentas. However, the use of HTR8/SVneo cells to characterize invasive trophoblasts has been well established in the literature (James et al., 2016; Silva and Serakides, 2016). Notably, for some chemicals screened, there was a clear concentration-response relationship where higher concentrations caused greater decline in EVT migration (e.g., EGF, pertussis toxin, CdCl2). However, for other chemicals, there were similar changes across concentrations (e.g., TCP, TPP, ZEN) which may be due to complex signaling changes and non-monotonic responses and/or a limitation of this method. As a result, this assay may serve best in a screening capacity for hazard identification with consideration of traditional or alternative models for full characterization of the concentration response. For example, this model is currently a 2D system, which may not be as representative of a “real-world” in vivo setting as a 3D model. To address this, future studies aim to expand our screening platform to evaluate EVT invasion into a simulated uterine environment. Recently, the Huh laboratory engineered a 3D system using co-cultures of first trimester HTR8/SVneo cells, immune, and uterine cells (Park et al., 2022). This model represents an advanced in vitro platform that recapitulates the human maternal and fetal interface formed during implantation/invasion and remodeling of uterine vessels by trophoblasts. Using a tiered testing approach, a screening assay using Oris™ plates could prioritize chemicals for additional mechanistic and functional evaluation in a 3D model.

One of the greatest advantages of this assay is the ability to scale up to screen hundreds of chemicals. The Biospa (Agilent) incubator allows up to eight 96-well microtiter plates to be loaded and incubated at one time allowing for the user to screen a myriad of substances simultaneously. Future studies using this platform will involve the screening of endocrine disrupting compounds, heavy metals, drugs, and others to evaluate their impacts on EVT migration. The findings from the study can be used to inform a larger body of literature that aims to elucidate the mechanisms by which exposure to environmental chemicals, such as cadmium, are associated with adverse gestational outcomes including preeclampsia and accrete spectrum disorders. EVT migration is a highly regulated process that requires proper signaling of proteins, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) (Zhu et al., 2012). Future studies should characterize the expression of signaling proteins (MMPs and TIMPs) that regulate EVT motility in cells treated with chemicals that significantly affected migration, such as ZEN, OPFRs, and bisphenols. Furthermore, the migration platform in this study may be used for kinetic high-throughput screening assays to assess whether toxicants alter EVT migration.

Supplementary Material

Suppl Fig 1

Supplemental Figure 1. Immunofluorescent Characterization of HTR8/SVneo cells. Indirect immunofluorescent staining (green) against cytokeratin-7, a trophoblast marker, and vimentin, a fibroblast marker, was performed on fixed HTR8/SVneo cells. Nuclei were stained with Hoechst 33342 dye (blue). Images show no presence of fibroblasts indicating an enriched trophoblast population. Images were captured at 100X magnification. White bars represent 1000 μm.

Suppl Fig 2

Supplemental Figure 2. Concentration-dependent cytotoxicity in human trophoblasts following cadmium chloride treatment. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1-10 μM) of cadmium chloride for 48 hours. Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p<0.05) from vehicle control.

Suppl Fig 3

Supplemental Figure 3. Concentration-dependent cytotoxicity in human trophoblasts following treatment with environmental chemicals. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1-10 μM) of screening chemicals for 48 hours. Environmental chemicals included organophosphate flame retardants (tricresyl phosphate (TCP), triphenyl phosphate (TPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP)), bisphenols (bisphenol A (BPA), bisphenol B (BPB), bisphenol S (BPS), bisphenol F (BPF)), and estrogenic mycotoxins including zearalenone (ZEN), its metabolites (α-ZOL and β-ZOL), and the synthetic form zeranol (ZER). Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p<0.05) from vehicle control.

Highlights.

  • Trophoblast entry into a cell-free zone can be monitored with real-time imaging.

  • This migration assay can be used to screen potential human placenta toxicants.

  • Cadmium chloride, bisphenols, and mycoestrogens reduce trophoblast migration.

  • Organophosphate flame retardants increase trophoblast migration.

Acknowledgements

This work was supported by the National Institute of Environmental Health Sciences [Grants R01ES029275, T32ES007148, and P30ES005022] and the National Center for Advancing Translational Sciences [Grant UL1TR003017].

Abbreviations:

BPA

bisphenol A

BPB

bisphenol B

BPS

bisphenol S

CdCl2

cadmium chloride

EGF

epidermal growth factor

EVT

extravillous trophoblast

PI

propidium iodide

RCD

relative cell density

TCP

tricresyl phosphate

TPP

triphenyl phosphate

TDCPP

tris(1,3-dichloro-2-propyl) phosphate

ZEN

zearalenone

ZER

zeranol

α-ZOL

α-zearalenol

β-ZOL

β-zearalenol

Footnotes

Conflicts of Interest

The authors do not have conflicts of interest to declare.

Data Statement

The authors are able to make data available upon request.

References

  1. Aboagye-Mathiesen G, Laugesen J, Zdravkovic M, Ebbesen P, 1996. Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi. Clin Diagn Lab Immunol 3, 14–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abou-Kheir W, Barrak J, Hadadeh O, Daoud G, 2017. HTR-8/SVneo cell line contains a mixed population of cells. Placenta 50, 1–7. [DOI] [PubMed] [Google Scholar]
  3. Albrecht ED, Pepe GJ, 2020. Regulation of uterine spiral artery remodeling: a review. Reprod Sci 27, 1932–1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alvarez MM, Chakraborty C, 2011. Cadmium inhibits motility factor-dependent migration of human trophoblast cells. Toxicol In Vitro 25, 1926–1933. [DOI] [PubMed] [Google Scholar]
  5. Angelova M, Machado HL, Swan KF, Morris C, Sullivan DE, 2013. Extravillous Trophoblast Migration and Invasion Assay. Bio Protoc 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ball E, Bulmer JN, Ayis S, Lyall F, Robson SC, 2006. Late sporadic miscarriage is associated with abnormalities in spiral artery transformation and trophoblast invasion. J Pathol 208, 535–542. [DOI] [PubMed] [Google Scholar]
  7. Brooks SA, Fry RC, 2017. Cadmium inhibits placental trophoblast cell migration via miRNA regulation of the transforming growth factor beta (TGF-beta) pathway. Food Chem Toxicol 109, 721–726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buchser W, Collins M, Garyantes T, Guha R, Haney S, Lemmon V, Li Z, Trask OJ, 2004. Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging. In: Markossian S, Sittampalam GS, Grossman A, Brimacombe K, Arkin M, Auld D, Austin CP, Baell J, Caaveiro JMM, Chung TDY, Coussens NP, Dahlin JL, Devanaryan V, Foley TL, Glicksman M, Hall MD, Haas JV, Hoare SRJ, Inglese J, Iversen PW, Kahl SD, Kales SC, Kirshner S, Lal-Nag M, Li Z, McGee J, McManus O, Riss T, Saradjian P, Trask OJ Jr., Weidner JR, Wildey MJ, Xia M, Xu X (Eds.) Assay Guidance Manual, Bethesda (MD). [Google Scholar]
  9. Burton GJ, Fowden AL, 2015. The placenta: a multifaceted, transient organ. Philos Trans R Soc Lond B Biol Sci 370, 20140066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen JZ, Sheehan PM, Brennecke SP, Keogh RJ, 2012. Vessel remodelling, pregnancy hormones and extravillous trophoblast function. Mol Cell Endocrinol 349, 138–144. [DOI] [PubMed] [Google Scholar]
  11. De Ieso ML, Pei JV, 2018. An accurate and cost-effective alternative method for measuring cell migration with the circular wound closure assay. Biosci Rep 38 (5):BSR20180698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ding J, Xu Z, Huang W, Feng L, Yang F, 2016. Organophosphate ester flame retardants and plasticizers in human placenta in Eastern China. Sci Total Environ 554-555, 211–217. [DOI] [PubMed] [Google Scholar]
  13. Gao X, Sun L, Zhang N, Li C, Zhang J, Xiao Z, Qi D, 2017. Gestational Zearalenone exposure causes reproductive and developmental toxicity in pregnant rats and female offspring. Toxins (Basel) 9(1):21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilder AS, Wang L, Natali L, Karimi-Mostowfi N, Brifault C, Gonias SL, 2016. Pertussis toxin is a robust and selective inhibitor of high grade glioma cell migration and invasion. PLoS One 11, e0168418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldman-Wohl D, Yagel S, 2002. Regulation of trophoblast invasion: from normal implantation to pre-eclampsia. Mol Cell Endocrinol 187, 233–238. [DOI] [PubMed] [Google Scholar]
  16. Grada A, Otero-Vinas M, Prieto-Castrillo F, Obagi Z, Falanga V, 2017. Research techniques made simple: analysis of collective cell migration using the wound healing assay. J Invest Dermatol 137, e11–e16. [DOI] [PubMed] [Google Scholar]
  17. Han J, Li L, Hu J, Yu L, Zheng Y, Guo J, Zheng X, Yi P, Zhou Y, 2010. Epidermal growth factor stimulates human trophoblast cell migration through Rho A and Rho C activation. Endocrinology 151, 1732–1742. [DOI] [PubMed] [Google Scholar]
  18. Hong Z, Li Y, Deng X, Chen M, Pan J, Chen Z, Zhang X, Wang C, Qiu C, 2022. Comprehensive analysis of triphenyl phosphate: An environmental explanation of colorectal cancer progression. Ecotoxicol Environ Saf 241, 113778. [DOI] [PubMed] [Google Scholar]
  19. Hu W, Gao F, Zhang H, Hiromori Y, Arakawa S, Nagase H, Nakanishi T, Hu J, 2017. Activation of peroxisome proliferator-activated receptor gamma and disruption of progesterone synthesis of 2-ethylhexyl diphenyl phosphate in human placental choriocarcinoma cells: comparison with triphenyl phosphate. Environ Sci Technol 51, 4061–4068. [DOI] [PubMed] [Google Scholar]
  20. James J, Tun W, Clark A, 2016. Quantifying trophoblast migration: In vitro approaches to address in vivo situations. Cell Adh Migr 10, 77–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jansson T, Powell TL, 2007. Role of the placenta in fetal programming: underlying mechanisms and potential interventional approaches. Clin Sci (Lond) 113, 1–13. [DOI] [PubMed] [Google Scholar]
  22. Johnston ST, Shah ET, Chopin LK, Sean McElwain DL, Simpson MJ, 2015. Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM assay data using the Fisher-Kolmogorov model. BMC Syst Biol 9, 38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC, 2016. In Vitro tumor models: advantages, disadvantages, variables, and selecting the right platform. Front Bioeng Biotechnol 4, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaufmann P, Black S, Huppertz B, 2003. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 69, 1–7. [DOI] [PubMed] [Google Scholar]
  25. Kunishige K, Kawate N, Inaba T, Tamada H, 2017. Exposure to zearalenone during early pregnancy causes estrogenic multitoxic effects in mice. Reprod Sci 24, 421–427. [DOI] [PubMed] [Google Scholar]
  26. Laine JE, Ray P, Bodnar W, Cable PH, Boggess K, Offenbacher S, Fry RC, 2015. Placental cadmium levels are associated with increased preeclampsia risk. PLoS One 10, e0139341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liang CC, Park AY, Guan JL, 2007. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329–333. [DOI] [PubMed] [Google Scholar]
  28. Lin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, Aoki K, Iwamoto R, Terai K, Matsuda M, 2022. Redundant roles of EGFR ligands in the ERK activation waves during collective cell migration. Life Sci Alliance 5(1):e202101206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Luo D, Liu W, Tao Y, Wang L, Yu M, Hu L, Zhou A, Covaci A, Xia W, Li Y, Xu S, Mei S, 2020. Prenatal Exposure to organophosphate flame retardants and the risk of low birth weight: a nested case-control study in China. Environ Sci Technol 54, 3375–3385. [DOI] [PubMed] [Google Scholar]
  30. Malik A, Pal R, Gupta SK, 2017. Interdependence of JAK-STAT and MAPK signaling pathways during EGF-mediated HTR-8/SVneo cell invasion. PLoS One 12, e0178269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martinez-Olmedo MA, Garcia-Sainz JA, 1984. Direct action of pertussis toxin in isolated hamster fat cells. Eur J Pharmacol 99, 115–118. [DOI] [PubMed] [Google Scholar]
  32. Moser G, Windsperger K, Pollheimer J, de Sousa Lopes SC, Huppertz B, 2018. Human trophoblast invasion: new and unexpected routes and functions. Histochem Cell Biol 150, 361–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Müller JE, Meyer N, Santamaria CG, Schumacher A, Luque EH, Zenclussen ML, Rodriguez HA, Zenclussen AC, 2018. Bisphenol A exposure during early pregnancy impairs uterine spiral artery remodeling and provokes intrauterine growth restriction in mice. Scientific Reports 8, 9196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Park JY, Mani S, Clair G, Olson HM, Paurus VL, Ansong CK, Blundell C, Young R, Kanter J, Gordon S, Yi AY, Mainigi M, Huh DD, 2022. A microphysiological model of human trophoblast invasion during implantation. Nature Communications 13, 1252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Qiu Q, Yang M, Tsang BK, Gruslin A, 2004. Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration. Mol Hum Reprod 10, 677–684. [DOI] [PubMed] [Google Scholar]
  36. Riahi R, Yang Y, Zhang DD, Wong PK, 2012. Advances in wound-healing assays for probing collective cell migration. J Lab Autom 17, 59–65. [DOI] [PubMed] [Google Scholar]
  37. Rock KD, Horman B, Phillips AL, McRitchie SL, Watson S, Deese-Spruill J, Jima D, Sumner S, Stapleton HM, Patisaul HB, 2018. EDC IMPACT: Molecular effects of developmental FM 550 exposure in Wistar rat placenta and fetal forebrain. Endocr Connect 7, 305–324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rock KD, St Armour G, Horman B, Phillips A, Ruis M, Stewart AK, Jima D, Muddiman DC, Stapleton HM, Patisaul HB, 2020. Effects of prenatal exposure to a mixture of organophosphate flame retardants on placental gene expression and serotonergic innervation in the fetal rat brain. Toxicol Sci 176, 203–223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sato Y, Fujiwara H, Konishi I, 2012. Mechanism of maternal vascular remodeling during human pregnancy. Reprod Med Biol 11, 27–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Silva JF, Serakides R, 2016. Intrauterine trophoblast migration: A comparative view of humans and rodents. Cell Adh Migr 10, 88–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Spagnoletti A, Paulesu L, Mannelli C, Ermini L, Romagnoli R, Cintorino M, Ietta F, 2015. Low concentrations of Bisphenol A and para-nonylphenol affect extravillous pathway of human trophoblast cells. Mol Cell Endocrinol 412, 56–64. [DOI] [PubMed] [Google Scholar]
  42. Szilagyi JT, Freedman AN, Kepper SL, Keshava AM, Bangma JT, Fry RC, 2020. Per- and Polyfluoroalkyl substances differentially inhibit placental trophoblast migration and invasion in vitro. Toxicol Sci 175, 210–219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tantbirojn P, Crum CP, Parast MM, 2008. Pathophysiology of placenta creta: the role of decidua and extravillous trophoblast. Placenta 29, 639–645. [DOI] [PubMed] [Google Scholar]
  44. Tarrade A, Lai Kuen R, Malassine A, Tricottet V, Blain P, Vidaud M, Evain-Brion D, 2001. Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta. Lab Invest 81, 1199–1211. [DOI] [PubMed] [Google Scholar]
  45. Thomas CY, Chouinard M, Cox M, Parsons S, Stallings-Mann M, Garcia R, Jove R, Wharen R, 2003. Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells. Int J Cancer 104, 19–27. [DOI] [PubMed] [Google Scholar]
  46. Ticiani E, Pu Y, Gingrich J, Veiga-Lopez A, 2022. Bisphenol S impairs invasion and proliferation of extravillous trophoblasts cells by interfering with epidermal growth factor receptor signaling. Int J Mol Sci 23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang ZY, Lu J, Zhang YZ, Zhang M, Liu T, Qu XL, 2015. Effect of Bisphenol A on invasion ability of human trophoblastic cell line BeWo. Int J Clin Exp Pathol 8, 14355–14364. [PMC free article] [PubMed] [Google Scholar]
  48. Wei P, Ru D, Li X, Shi D, Zhang M, Xu Q, Zhou H, Wen S, 2020. Exposure to environmental bisphenol A inhibits HTR-8/SVneo cell migration and invasion. J Biomed Res 34, 369–378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ye L, Zhang X, Wang P, Zhang Y, He S, Li Y, Li S, Liang K, Liao S, Gao Y, Zhou S, Peng Q, 2022. Low concentration triphenyl phosphate fuels proliferation and migration of hepatocellular carcinoma cells. Environ Toxicol 37, 2445–2459. [DOI] [PubMed] [Google Scholar]
  50. Zhang W, Song G, 2022. A comprehensive analysis-based study of triphenyl phosphate-environmental explanation of glioma progression. Ecotoxicol Environ Saf 248, 114346. [DOI] [PubMed] [Google Scholar]
  51. Zhang Y, Jia Z, Yin S, Shan A, Gao R, Qu Z, Liu M, Nie S, 2014. Toxic effects of maternal zearalenone exposure on uterine capacity and fetal development in gestation rats. Reprod Sci 21, 743–753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhu JY, Pang ZJ, Yu YH, 2012. Regulation of trophoblast invasion: the role of matrix metalloproteinases. Rev Obstet Gynecol 5, e137–143. [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl Fig 1

Supplemental Figure 1. Immunofluorescent Characterization of HTR8/SVneo cells. Indirect immunofluorescent staining (green) against cytokeratin-7, a trophoblast marker, and vimentin, a fibroblast marker, was performed on fixed HTR8/SVneo cells. Nuclei were stained with Hoechst 33342 dye (blue). Images show no presence of fibroblasts indicating an enriched trophoblast population. Images were captured at 100X magnification. White bars represent 1000 μm.

NIHMS1861698-supplement-Suppl_Fig_1.tiff (19.8MB, tiff)
Suppl Fig 2

Supplemental Figure 2. Concentration-dependent cytotoxicity in human trophoblasts following cadmium chloride treatment. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1-10 μM) of cadmium chloride for 48 hours. Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p<0.05) from vehicle control.

NIHMS1861698-supplement-Suppl_Fig_2.tiff (14.8MB, tiff)
Suppl Fig 3

Supplemental Figure 3. Concentration-dependent cytotoxicity in human trophoblasts following treatment with environmental chemicals. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1-10 μM) of screening chemicals for 48 hours. Environmental chemicals included organophosphate flame retardants (tricresyl phosphate (TCP), triphenyl phosphate (TPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP)), bisphenols (bisphenol A (BPA), bisphenol B (BPB), bisphenol S (BPS), bisphenol F (BPF)), and estrogenic mycotoxins including zearalenone (ZEN), its metabolites (α-ZOL and β-ZOL), and the synthetic form zeranol (ZER). Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p<0.05) from vehicle control.

NIHMS1861698-supplement-Suppl_Fig_3.tiff (22.5MB, tiff)

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