# Exposure of the human placental primary cells to nanoplastics induces cytotoxic effects, an inflammatory response and endocrine disruption

Léa Poinsignon, Bertrand Lefrère, Amani Ben Azzouz, Audrey Chissey, Juliette Colombel, Raja Djelidi, Ioana Ferecatu, Thierry Fournier, Jean-Louis Beaudeux, Gaetane Lespes, et al.

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## To cite this version:

Léa Poinsignon, Bertrand Lefrère, Amani Ben Azzouz, Audrey Chissey, Juliette Colombel, et al. Exposure of the human placental primary cells to nanoplastics induces cytotoxic effects, an inflammatory response and endocrine disruption. Journal of Hazardous Materials, 2025, 490, pp.137713. 10.1016/j.jhazmat.2025.137713. hal-04985127

HAL Id: hal-04985127

https://univ-pau.hal.science/hal-04985127v1

Submitted on 10 Mar 2025

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# Exposure of the human placental primary cells to nanoplastics induces cytotoxic effects, an inflammatory response and endocrine disruption

Léa Poinsignon<sup>a</sup>, Bertrand Lefrère<sup>a,b</sup>, Amani Ben Azzouz<sup>c</sup>, Audrey Chissey<sup>a</sup>,
Juliette Colombel<sup>a</sup>, Raja Djelidi<sup>a</sup>, Ioana Ferecatu<sup>a</sup>, Thierry Fournier<sup>a</sup>,
Jean-Louis Beaudieux<sup>a,b,c</sup>, Gaëtane Lespes<sup>c</sup>, Amal Zerrad-Saadi<sup>a,*</sup>

## HIGHLIGHTS

* Nanopolystyrene (PS-NP) exposure is evaluated on primary trophoblastic cells.

* PS-NP translocate in chorionic villi, mainly in trophoblastic cells.

* Lower trophoblastic viability is observed at environmental concentrations of PS-NP.

* Cellular defenses are enhanced after PS-NP of 100 nm exposure.

* PS-NP induce inflammatory response and endocrine disruption (β-hCG decrease).

## GRAPHICAL ABSTRACT

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**Abbreviation:** ARE, Antioxidant Response Element; Bcl2, B-cell lymphoma 2; BPA-F-S, Bisphenol A-F-S; CK7, cytokeratine 7; CPP, Comité de protection des personnes; DEHP, Bis 2-ethylhexyl phthalate; DOHaD, Developemental Origins of Health and Disease; DMEM, Dulbecco modified Eagle's medium; DMSO, dimethylsulfoxide; DRP1, Dynamine-1-like protein; GA, Glutaraldehyde; GPx1, Gluathion peroxidase 1; GSH, Reduced Glutathion; GSSG, Oxidized Glutathion; HBSS, Hank's balanced salt solution; HCG, human chorionic hormone; HO-1, Heme-oxygenase 1; IF, Immunofluorescence; IL-6, Interleukin 6; IL-1β, Interleukin 1β; IL-10, Interleukin 10; IR, Infra-red; LC3, Microtubule-associated protein 1 light chain 3; LIR, LC3- interacting region; MP/NP, Micro/nanoplastics; MP, microplastics; MFN1, Mitofusine 1; MFN2, Mitofusine 2; Nf-kB, nuclear factor-kappa B; NIST, Nationale Institute of standards and Technology; NLRP3, NOD-like receptor family pyrin domain containing 3; NP, nanoplastics; Nrf2, Nuclear factor erythroid-2-related factor 2; OPA-1, Optic atrophy type 1; PARP-1, poly ADP-ribose polymerase 1; PBS, Phosphate buffer saline; PCB, Polychlorinated biphenyl; PCR, Polymerase Chain Reaction; PE, polyethylene; PET, polyethylene terephtalate; PFA, Paraformaldehyde; PFOA, Perfluoro octanoic acid; Pg, Progesterone; PlGF, placental growth factor; PP, Polypropylene; Prx-SO3, Peroxiredoxin-SO3; PS, Polystyrene; PS-NP, Polystyrene nanoparticles; PU, polyurethane; qPCR, quantitative PCR; RNA, Ribonucleic acid; ROS, Reactive Oxygen Species; RPLP0, Ribosomal Protein Lateral Stalk Subunit P0; RPL13, Ribosomal Protein L13; SDHA, Succinate dehydrogenase; SOD1, Superoxide 1; SOD2, Superoxide 2; ST, Syncytiotrophoblast; t-BHP, Tert-butyl hydroperoxide; TEM, Transmission Electronic Microscopy; TNF-α, Tumor Necrosis Factor; UBA, Ubiquitin associated domain; VCT, villous cytotrophoblast; ZrO₂, Dioxide Zirconium.

<footnote><sup>a</sup> Université Paris-Cité, INSERM U1139 (FPRM), Faculté de Pharmacie, FHU Prem'impact, Paris 75006, France</footnote>

<footnote><sup>b</sup> Service de Biochimie, AP-HP, Hôpital Necker Enfants Malades, Paris F-75006, France</footnote>

<footnote><sup>c</sup> IPREM, CNRS, Université de Pau et des pays d'Adour, Pau 64000, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: amal.zerrad-saadi@u-paris.fr (A. Zerrad-Saadi).</footnote>
ARTICLE INFO

Keywords:

Nanoplastics

Nanopolystyrenes

Nanotoxicology

Placenta

Trophoblast

Pregnancy

Endocrine disruptor

ABSTRACT

Humans are inevitably exposed to micro- and nanoplastics (MP/NP). These particles are able to cross the bio-
logical barriers and enter the bloodstream with levels close to 1.6 µg mL⁻¹; MP/NP have been detected in
placentas and meconium of newborns. However, the consequences of this exposure on the integrity, development
and functions of the human placenta are not documented. In this study, trophoblasts purified from human
placentas at term were exposed for 48 h, to two different sizes of polystyrene nanoparticles (PS-NP) of 20 nm (PS-
NP₂₀) and 100 nm (PS-NP₁₀₀), at environmental and supra-environmental concentrations (0.01–100 µg mL⁻¹).
Cell viability, oxidative stress, mitochondrial dynamics, lysosomal degradation processes, autophagy, inflam-
mation/oxidative responses and consequences for placental endocrine and angiogenic functions were assessed.
PS-NP size determines their internalization rate and their behavior in trophoblasts. Indeed, PS-NP₂₀ are more
rapidly translocated, and accumulated in lysosomes as shown by confocal and TEM imaging. They induce higher
cytotoxicity than PS-NP₁₀₀, as early as 1 µg mL⁻¹ (p < 0.05). In addition, they induce a pro-inflammatory cy-
tokines response: IL-1β is induced from 0.01 µg mL⁻¹ for the both nanoparticle sizes; IL-6, and TNF-α are
overexpressed at 100 µg mL⁻¹ only for PS-NP₂₀ (p < 0.05). For the first time, we report that PS-NP disrupt
endocrine function, as observed by a decreased hCG release at concentrations found in human blood. This work,
provides an in-depth *in vitro* assessment of the effects of PS-NP on the human placenta.

1. Introduction

Environmental pollution by plastics has been steadily increasing since 1950. Its emergence corresponds to the industrial era and the development of petrochemistry. Plastics are used for their low cost, but above all for their properties of lightness, formability, stability and their excellent sustainability [1]. This last characteristic means that plastics do not degrade quickly. Their degradation involves a process of constant fragmentation by UV-oxidation, erosion or digestion by microorganisms. Particles generated are defined as micro or nanoplastics (MP/NP) depending on their size: MP < 5 mm and NP < 100 nm [2,3]. These particles – mainly polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS) and polyurethane (PU) – contaminate all environmental matrices: soil, air and water [3-5]. Concentrations of MP/NP in environment and quantification of human exposure are not clearly defined. Important variations are observed between studies, especially according to the geographical areas, where these works are conducted [6]. Leslie *and al* have suggested that concentrations of MP in blood of healthy volunteers average 1.6 µg mL⁻¹, with some exceeding 10 µg mL⁻¹ [7]. Exposure during pregnancy is therefore inevitable. Indeed, MP have recently been identified in the human placenta at the end of pregnancy [8-15].

Placenta is a temporal and transitional organ that mediates nutrient and waste exchanges to enable in utero fetal existence. Placental tissue is composed of chorionic villi that constitute the structural and functional unit. It undergoes major reorganization throughout pregnancy, according to a well-orchestrated differentiation process that involves the fusion of villous cytotrophoblast (VCT) into syncytiotrophoblast (ST). It is a specialized layer of epithelial cells that totally covers the surface of the villi and is in direct contact with maternal blood [16,17]. The ST assures various functions including immune-tolerance and endocrine function such as the synthesis and release of human chorionic gonadotropin (hCG) hormone and placental growth factor (PIGF). All these functions contribute to maintain the pregnancy by inhibiting uterine contractions and inducing vasculogenesis at the fetomaternal interface [18-20]. The ST is a polarized epithelium that constitute a selective and protective biological barrier to xenobiotics. Nevertheless, some pollutants such as MP are able to cross the placental barrier. MP have been discovered in term human placentas, in newborn's meconium and now constitute an entire part of the prenatal exposome [8,9,21,22]. In this context, and with the emergence of "Developmental Origins of Health and Disease" (DOHaD) concept [23,24], MP/NP exposure requires important investigations, particularly on placental development, pregnancy outcome and fetal health [25].

The presence of MP in the placenta, also called *plasticentas*, was established for the first time by Ragusa *and al*, in 2021 in term placentas of Italian mothers [13]. Later, MP were detected in Iranian, Chinese and

Haitian placentas [10,12,15]. In these reports, particles were various in
terms of polymer type, chemical composition, size and shape: most of
them were PP, PET and PS fibers, subspherical and of indefinite geo-
metric shape. *Plasticentas* were localized from basal plate to the fetal
side. In trophoblastic cells, plastics were found free in cytosol or local-
ized into organelles (vesicular bodies, lysosomes, peroxisomes). Some
organelles, such as mitochondria and the endoplasmic reticulum,
showed ultrastructural morphological changes such as swelling indi-
cating possible alterations in their functions [26]. Consequences of the
*plasticentas* on pregnancy outcomes are still unexplored and knowledge
on MP/NP toxicity on human placental models is still limited. Some
studies conducted on trophoblastic cell lines (BeWo b30, ACH-3P, JAR,
JEG-3, 3A-subE and SWAN-71) indicate a lower viability for doses of
MP/NP above 25–100 µg mL⁻¹ [27–31] that was associated with reac-
tive oxygen species production [29,30], inflammation and alterations of
migration/invasion properties [29]. A recent *in vitro* study has shown
that NP could induce apoptosis at high doses [33]. Until now, the *in vitro*
studies, which provide first insights into the internalization and toxicity
of MP/NP on placental tissue, were carried out on immortalized
trophoblastic cell lines- more resistant to stress and cell death than
primary villous trophoblast [20,31]. This does not constitute a repre-
sentative model for studying the effects of realistic exposure to MP/NP.
Other studies have evaluated MP/NP exposure on gestational mouse
model and suggest an increased fetal resorption rate, a lower birth
weight associated to fetoplacental hypoperfusion and fetal growth re-
striction [30,32,33] as well as alterations in offspring metabolism and
cerebral development [34–37]. In humans, MP-NP are suspected to be
involved in the occurrence of intra-uterine growth retardation [12].

The aim of our work was to assess the consequences of polystyrene nanoparticles (PS-NP) exposure at concentrations compatibles with values found in human blood (0.01–100 µg mL⁻¹), on the human placenta at term using particles of two different sizes (20 and 100 nm). Given that NP quantification is complex and largely perfectible, and that they are likely to accumulate in biological tissues, the choice of a wide range of concentrations (0.01–100 µg mL⁻¹), focused on the concentrations actually found in human blood (1–10 µg mL⁻¹), makes it possible to assess the consequences of infra-, normo- and supra-environmental exposure to PS-NP. PS-NP translocation/accumulation was assessed on *ex vivo* chorionic villus explants and *in vitro* primary VCT were used to explore toxicity effects. These biological models make it possible to study the placental exposure of PS-NP on non-transformed human tissues. In addition, the VCT primary culture model allows to evaluate the consequences of this exposure on the main functions of the placental barrier mediated by ST (endocrine, immune functions), since VCT isolated from human placentas spontaneously differentiate (by fusion) into ST after 24 h of culture.
## 2. Results

Before studying the consequences of exposure to PS-NP on the human placenta, a physico chemical characterization of the particles in the culture medium was carried out (concentration, size, surface charge, stability), as these characteristics modulate the reactivity of NP with living organisms and their toxicological profile.

Next, the translocation of particles across the human placenta was assessed through chorionic villi (explants) and trophoblastic cells (primary culture of VCT). The cytotoxicity of PS-NP on VCT was assessed and the defense mechanisms developed by the trophoblasts were studied (type of cell death induced, activation of antioxidant pathways, inflammatory response, etc.). Finally, the functional repercussions for VCT were analyzed by studying endocrine and angiogenic functions as well as the capacity of the VCT to fuse into ST (Fig. 1).

### 2.1. Physicochemical characterization of PS-NP

Polystyrene is a low-density plastic ($1.06 g.cm^{-3}$) composed of one aromatic ring. PS-NP used in the study are spherical nanobeads commercialized in milliQ water (Fig. 2.A). Concentrations used are expressed in mass concentration (µg mL$^{-1}$); correspondences between PS-NP$_{20}$ and PS-NP$_{100}$ mass concentration to particle concentration were calculated (Fig. 2. B). At high concentration ($100 \mu g mL^{-1}$), PS-NP$_{20}$ mainly formed agglomerates with a mean diameter of $110 \pm 7$ nm whereas PS-NP$_{100}$ slightly agglomerates with a mean diameter close to $130 \pm 6$ nm (Fig. 2.C). After 24 h in culture medium, NP$_{20}$-PS size distribution was polydisperse – ranging from isolated particle of 20 nm to aggregates over 400 nm in diameter- while the NP$_{100}$-PS solution remained homogeneous (Fig. 2.D). Zeta potential was slightly negative for both sizes of particles, $-10 \pm 1$ mV and $-7 \pm 1$ mV for PS-NP$_{20}$ and PS-NP$_{100}$ respectively (Fig. 2.C). The agglomerated state of both particles was generally stable in the culture media until 24 h and the particles quantitatively remained in suspension (Fig. 2.C-D-E). Finally, particles were observed by TEM before and after incubation with VCT, at the lower and upper limits to the concentration range ($0.01$ and $100 \mu g mL^{-1}$) (Fig. 2.F) as well at $1$ and $10 \mu g mL^{-1}$ (Supplementary data 1). Prior to exposure, PS-NP$_{20}$ appeared less agglomerated at low ($0.01 \mu g mL^{-1}$) than at high concentrations. At $100 \mu g mL^{-1}$, particles

were polydisperse with a mixture of isolated PS-NP$_{20}$ and agglomerated-particles around 150 nm diameter confirming DLS measurements. PS-NP$_{100}$ did not appear agglomerated at any concentration. After 24 h incubation with trophoblastic cells, no further aggregation was observed, regardless of particle size (Fig. 2.F).

### 2.2. PS-NP translocation in chorionic villi and VCT

Chorionic villi explants were exposed to fluorescent nanospheres PS-NP$_{20}$ and PS-NP$_{100}$ at high concentration ($100 \mu g mL^{-1}$). Confocal microscopy images, after labelling of trophoblastic cytoskeleton with cytokeratin7 (CK7) (Fig. 3.A-B), showed that after 2 h of incubation, PS-NP$_{20}$ crossed trophoblastic membranes and were mainly localized in VCT and ST, while no labeling was observed for PS-NP$_{100}$. After 24 h, a strong green labeling was observed in the mesenchyme and trophoblastic cells for both particle sizes. Quantification of fluorescence corresponding to PS-NP specifically localized into VCT and ST was performed (Fig. 3.C) and confirms that PS-NP accumulate over time with a faster translocation of PS-NP$_{20}$ than PS-NP$_{100}$. PS-NP$_{100}$ translocation was also confirmed by TEM observations, on primary culture of human VCT after a 24 h incubation (Fig. 3.D). PS-NP$_{100}$ were localized in single membrane vesicles nearly the apical side of VCT, probably in endosomes. Particles were singly or in pairs and never free in the cytosol (Fig. 3.D). Unfortunately, the small size of the PS-NP$_{20}$ and the lack of sensitivity made it impossible to distinguish them with certainty within the VCT. Nevertheless, aggregates/agglomerates of PS-NP$_{20}$ over $200-400$ nm were theoretically examinable by TEM. Their absence in VCT observed by TEM (Supplementary data 2) implies that they do not internalize, or only to a limited extent, into the cells. The PS-NP$_{20}$ observed in chorionic villi by confocal microscopy were therefore probably small particles ($< 100$ nm). These results suggest that the transfer of PS-NP into trophoblastic cells is dependent on the size and/or number of particles and also on the time required for internalization.

### 2.3. PS-NP cytotoxicity and ROS production

The viability of VCT exposed to PS-NP was studied using the WST1 assay, which measures mitochondrial dehydrogenase activity and reflects the metabolic activity of the cells. Firstly, the absence of

<figure><img src="image_5.png" /></figure>

Using Biorender

<caption>Fig. 1. Analysis strategy created by BioRender.com</caption>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Physicochemical characterization of PS-NP A/Polystyrene chemical structure and PS-NP in water (stock solution) observed by TEM. Scale bar = 200 nm. B/ Correspondence between mass concentrations and particle concentrations used in this study. C/ Diameter and zeta-potential, respectively measured by DLS and zettameter, in water and culture medium (DMEM with serum) at 100 µg mL⁻¹. D/Size distribution of PS-NP after 24 h in culture medium (DMEM with serum) at 100 µg mL⁻¹. E/ Suspension concentration measured by UV-vis in culture medium until 72 h at 100 µg mL⁻¹. F/ PS-NP are diluted in culture medium and observed by TEM before and after 24 h incubation with VCT. Scale bar = 200 nm. Legend: → : PS-NP.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** PS-NP translocation in chorionic villi and VCT.A/ Chorionic villi from term placentas are exposed to PS-NP~20~ or PS-NP~100~ for 2 h to 24 h at 100 µg mL~⁻¹~, then fixed in 4% PFA and included in agarose. Immunostaining of CK7 is revealed with Alexafluor 555 (red); PS-NP are revealed by Alexafluor 488 (green) fluorochrome and nuclei are marked with TOPRO-3 (blue). Scale bar = 20 µm. B/ Schema of chorionic villi. Legend: VCT: Villous cytotrophoblast, ST: Syncytiotrophoblast, nucleus are represented in blue. C/ Quantification of PS-NP translocation in VCT and ST using ImageJ software. Experiments are represented as mean + /- SEM (N = 4). Statistical analysis was performed using paired t-test. \* p < 0.05. D/ VCT are exposed 24 h to PS-NP~100~ (100 µg mL~⁻¹~) and fixed for TEM observations. Scale bar = 500 nm. Legend: → PS-NP, N: Nucleus, M: Mitochondria, LD: Lipid droplet.</figcaption></figure>

cytotoxicity possibly induced by the surfactants used to resuspended and avoid particle aggregation, was confirmed (supplementary data 3). At high concentration (100 µg mL⁻¹), cell viability was decreased around 15 % and 25 % after 4 h and 48 h of PS-NP~100~ exposure respectively

(p < 0.05) (Fig. 4.A). Cell viability was reduced, compared to the control, from 24 h (- 12 %) and 48 h (- 30 %) incubation with PS-NP~20~. No cytotoxicity was observed at a lower concentration of PS-NP~100~, whereas a decrease in cell viability of around 20 % after 24 h of exposure was
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** PS-NP cytotoxicity and ROS production. A/ VCT are exposed to PS-NP~20~ or PS-NP~100~ or t-BHP (200 µM) for 4 h to 48 h in 48-well plate. VCT metabolic activity is measured by WST1 assay. Each condition is reported to control activity (VCT not exposed). Experiments are represented as SEM (N = 7). Statistical analysis was performed using Wilcoxon-test. \* p < 0.05 (compared to control), \# p < 0.05 (between experimental conditions). B/ VCT are exposed to PS-NP~20~ or PS-NP~100~ or t-BHP (200 µM) for 4 h to 48 h in 96-well plate. ROS production is measured by CM-H~2~DCFDA assay. ROS oxidize the probe whose emits fluorescence wich is measured. Each condition is reported to control (VCT not exposed). Experiments are represented as mean + /- SEM (N = 6). Statistical analysis was performed using Wilcoxon-test. \* p < 0.05 (compared to control), \# p < 0.05 (between experimental conditions). C/ Immunoblot using PARP1 and Caspase 3 antibodies is performed on VCT exposed 24 h to PS-NP~20~ or PS-NP~100~. Exposure 3 h to staurosporine (500 nM) is used as positive control. Actin is used as loading control. Graphs represent the total amount of protein relative to actin level determined by quantification of immunoblot using ImageStudiolight from LICOR. Results are represented as mean + /- SEM (N = 7 for PARP1 and N = 5 for Caspase 3). Statistical analysis was performed using paired *t*-test.</figcaption></figure>

shown from 1 µg mL⁻¹ for PS-NP~20~ (p < 0.05) (Fig. 4.A). Oxidative stress was investigated by measuring ROS production using CM-H~2~DCFDA probe over a shorter exposure time. Cell treatment with PS-NP~100~ showed no ROS production whereas exposure to PS-NP~20~ revealed a significant increase in ROS levels after 2 h and 4 h at 1 and 100 µg mL⁻¹ (p < 0.05) (Fig. 4.B). The overoxidation level of peroxiredoxin enzyme (Prx) reflects intracellular oxidative status and Prx-SO₃ represents the over oxidized form under H₂O₂ treatment. After 24 h exposure, protein level of Prx-SO₃, the overoxidized form of Prx, remained unchanged with PS-NP~20~ while PS-NP~100~ treatment tended to increase Prx-SO₃ level at 100 µg mL⁻¹ (p = 0.07) (Supplementary data 4). Apoptosis process was evaluated by quantifying protein level of

PARP1, Caspase 3 and their cleavage products. Cleaved-Caspase 3 is the effector of both intrinsic and extrinsic apoptosis pathways, and PARP1 is a substrate of Caspase, which is cleaved by Caspase-3 and Caspase-7 into PARP1 fragments (24 kDa N-terminal and 89 kDa C-terminal). Indeed, after 3 h incubation of VCT with staurosporine (an apoptosis inducer), cleaved-PARP1, the ratio of cleaved-PARP1/PARP1, cleaved-Caspase 3 and the ratio of cleaved-Caspase 3/Caspase 3 were highly increased (p < 0.05). However, no significant differences in the expressions of cleaved-PARP1 and cleaved-Caspase 3 proteins were observed after 24 h of exposure to PS-NP, irrespective of particle size (Fig. 4. C).
## 2.4. Antioxidant response to PS-NP exposure

Antioxidant response was explored by analyzing Nrf2/HO-1 pathway and antioxidant enzymes expression after 24 h of exposure to PS-NP. Nrf2 immunofluorescence showed basal expression of the protein under control condition, both in nuclear and cytoplasmic compartments

(Fig. 5.A). After 24 h of t-BHP treatment (a positive control for Nrf2 pathway activation), Nrf2 was highly expressed exclusively in the nucleus. When VCT were exposed to PS-NP (both sizes, all doses), Nrf2 was also translocated to the nucleus; no labeling was observed in the cytoplasm (Fig. 5.A and supplementary data 5.A). These results thus suggest the induction of Nrf2 pathway after PS-NP exposure. Comparing the

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Antioxidant response to PS-NP exposure. A/ VCT are exposed to PS-NP<sub>20</sub> or PS-NP<sub>100</sub> for 24 h. Cells are then fixed in 4% PFA. Immunostaining of Nrf2 is revealed with Alexafluor 488 (green) and nuclei are marked with TOPRO-3 (blue). Non-specific IgG is performed as negative control. Experiments are reproduced 3 times per condition. Scale bar = 10 µm. B/ Level of Nrf2 in VCT exposed 24 h to PS-NP or t-BHP (50 µM) measured by q.PCR, normalized to the geometric mean of SDHA, RPLPO, RPL13 and Actin transcripts. Statistical analysis is performed using Wilcoxon and paired *t*-test depending on conditions (N = 6). Results are represented as mean + /- SEM. \* *p* < 0.05. C/ Immunoblot using HO-1 antibody is performed on VCT exposed 24 h to PS-NP<sub>20</sub> or PS-NP<sub>100</sub> or t-BHP (50 µM). Actin is used as loading control. Graphs represent the total amount of protein relative to actin level determined by quantification of immunoblot using ImageStudiolight from LICOR. Results are represented as mean + /- SEM (N = 7). Statistical analysis was performed using paired *t*-test. \* *p* < 0.05, \* \* *p* < 0.01. D/ Level of SOD1, SOD2 and Catalase in VCT exposed 24 h to PS-NP or t-BHP (50 µM) measured by q.PCR, normalized to the geometric mean of SDHA, RPLPO, RPL13 and Actin transcripts. Statistical analysis is performed using Wilcoxon and paired *t*-test depending on conditions (N = 6). Results are represented as mean + /- SEM. \* *p* < 0.05, \* \* *p* < 0.01, \* \* \*p < 0.0001.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 5. (continued).</figcaption></figure>

expression of the transcriptional factor Nrf2 and the expression of the protein HO-1, its transcriptional target (by q.PCR and Western-Blot, respectively), allows us to understand the kinetic of activation of the Nrf2/HO-1 pathway. Indeed, Nrf2 transcriptional factor expression was not significantly increased after t-BHP exposure (Fig. 5.B), while HO-1 protein expression, its transcriptional target, was raised around 50 % (p < 0.01) (Fig. 5.B), suggesting a rapid antioxidant response. Nrf2 transcripts increased by almost 30–40 % after PS-NP₁₀₀ at 1 µg mL⁻¹ and 10 µg mL⁻¹ (p < 0.05), and was not significant at 100 µg mL⁻¹ (p = 0.09) (Fig. 5.B). Exploration of HO-1 protein expression, in response to Nrf2 activation, showed that PS-NP₁₀₀ induced an increase in HO-1 expression at low concentrations (0.01 µg mL⁻¹) (p < 0.05) (Fig. 5.C). PS-NP₂₀, which internalize more rapidly than PS-NP₁₀₀, induced earlier response of the Nrf2/HO-1 pathway, with a surprising U-shaped dose effect. Indeed, the HO-1 expression increased at 0.01 and 100 µg mL⁻¹ while Nrf2 transcript expression was unchanged (Fig. 5.B and C). Regard antioxidant enzymes responses, SOD1/2 transcriptional expressions were significantly increased or tended to increase (+50–100 %) at high concentration (100 µg mL⁻¹) for both particle sizes (p < 0.001 and 0.01 for PS-NP₂₀ and p = 0.06 for PS-NP₁₀₀) (Fig. 5.D). At 10 µg mL⁻¹, SOD1/2 expression increased significantly (+25 %, p < 0.05) after PS-NP₁₀₀ exposure. At 1 µg mL⁻¹ of PS-NP₁₀₀, SOD1 expression was significantly enhanced (+55 %, p < 0.01) and SOD2 expression tended to increase (+30 %, p = 0.06). Catalase expression was also increased after PS-NP exposure from 1 µg mL⁻¹ for PS-NP₁₀₀ and 10 µg mL⁻¹ for PS-NP₂₀ (p < 0.01) (Fig. 5.D). No modulation of GPx1 expression was observed after PS-NP exposure (Supplementary data 5.B). Protein expression of antioxidant enzymes was also studied and showed a tendency to increase for exposure to PS-NP₁₀₀ above 10 µg mL⁻¹ but in general we observed a high degree of inter-placental variability (Supplementary data 5. C).

## 2.5. Mitochondria network and dynamics

In order to study potential changes in mitochondria caused by PS-NP exposure, mitochondrial network and transmembrane potential were analyzed by Mitotracker probe, and mitochondrial dynamic fusion-fission processes, by Western-Blot. In control conditions, mitochondria were abundant around the nucleus and the network was reticulated (Fig. 6.A). When exposed to PS-NP above 10 µg mL⁻¹, no major differences in mitochondria network were observed despite a visually weaker mitochondrial labelling at 100 µg mL⁻¹ for PS-NP₁₀₀ (Fig. 6.A). Mitochondrial potential was also conserved even to high concentrations of PS-NP (Fig. 6.A). Furthermore, fusion proteins MFN1, MFN2 and OPA-1, were overexpressed after PS-NP₁₀₀ exposure at the highest dose; + 20 % expression at 100 µg mL⁻¹ (p < 0.05) and same tendency was observed for MFN1 and OPA-1 at 10 µg mL⁻¹ (p = 0.09 and p = 0.06 respectively) (Fig. 6.B). Finally, there is no increase observed on fission proteins expression (FIS1 and DRP1) for both sizes of particles (Fig. 6. B).

## 2.6. Autophagy process

Activation of autophagy was explored by analyzing the expression of LC3, a protein involved in autophagosome formation. LC3 expression investigating by immunofluorescence (IF) staining showed a basal expression in VCT primary culture in control condition (Fig. 7.A). This result suggests a basal activation of the autophagy process at term of pregnancy, which must be taken into consideration in the interpretation of our data. Rapamycin treatment, an autophagy inductor increased more LC3 expression as observed by IF. LC3 labeling intensity was unmodified after PS-NP₂₀ exposure but was enhanced with PS-NP₁₀₀ despite the fact that no colocalization observed between LC3 and PS-NP (Fig. 7.A smaller squares). Exposure of VCT to 100 µg mL⁻¹ of PS-NP₁₀₀
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Mitochondria network and dynamic after PS-NP exposure. A/ VCT are exposed to PS-NP~20~ or PS-NP~100~ for 24 h and then exposed to CMX-ROS probe (50 nM). Cells are fixed in 4% PFA. Mitochondria is revealed with CMX-ROS probe (red) and nuclei are marked with TOPRO-3. Mitochondria network is analyzed using ImageJ software. Experiments are reproduced 3 times per condition. Scale bar = 10 µm. B/ Immunoblot using MFN1, MFN2, OPA1, DRP1, FIS1 antibodies is performed on VCT exposed 24 h to PS-NP~20~ or PS-NP~100~. Actin is used as loading control. Graphs represent the total amount of protein relative to actin level determined by quantification of immunoblot using ImageStudiolight from LICOR. Results are represented as mean + /- SEM (N = 7). Statistical analysis was performed using Wilcoxon and paired t-test depending on conditions. \* p < 0.05.</figcaption></figure>

during 24 h followed by TEM observation, showed that PS-NP~100~ accumulated extensively in double-membrane vesicles similar to autophagosomes and were systematically localized in perinuclear zone (Fig. 7. B). PS-NP~20~ could not be observed using TEM due to a lack of sensitivity.

Autophagy process was more precisely analyzed by the study of protein expressions of P62, Beclin-1 and LC3 using Western-Blot (Fig. 7.C). P62, a cargo protein that recognizes and initiates degradation processes was significantly increased at high concentrations of PS-NP: + 50 %
<figure><img src="image_12.png" /><figcaption>**Fig. 7. Autophagy process.** A/ VCT are exposed to PS-NP~20~ or PS-NP~100~ for 24 h. Rapamycin (500 nM) for 24 h is used as positive control. Cells are then fixed in -20°C methanol. Immunostaining of LC3 is revealed with Alexafluor 555 (red); PS-NP are revealed by Alexafluor 488 (green) fluorochrome and nuclei are marked with TOPRO-3 (blue). Non-specific IgG is performed as negative control. Experiments are reproduced 3 times per condition. Scale bar = 30 µm. On the right of the panel, magnification was done. Scale bar = 10 µm. B/ VCT are exposed 24 h to PS-NP~100~ (100 µg mL⁻¹) and fixed for TEM observations. Experiments are reproduced 3 times per condition. Scale bars = 1 µm and 500 nm. Legend: → PS-NP, N: Nucleus, M: Mitochondria, A: Autophagosome. C/ Immunoblot using P62, Beclin and LC3 antibodies is performed on VCT exposed 24 h to PS-NP~20~ or PS-NP~100~. Actin is used as loading control. Graphs represent the total amount of protein relative to actin level determined by quantification of immunoblot using ImageStudiolight from LICOR. Results are represented as mean ± SEM (N = 7). Statistical analysis was performed using Wilcoxon and paired t-test depending on conditions. * p < 0.05.</figcaption></figure>

expression at 100 µg mL⁻¹ for PS-NP~20~ and around +30% above 10 µg mL⁻¹ for PS-NP~100~. Beclin, autophagy inducer acting on m-TOR pathway, was increased at 10 µg mL⁻¹ and 100 µg mL⁻¹ after PS-NP~100~ exposure (+20%, p = 0.1 and +30%, p < 0.05 respectively). No effect was observed for PS-NP~20~ exposure. Finally, LC3-II/LC3-I ratio reflecting autophagosomes formation (modification of LC3 I to II by conjugation to phosphatidyl-ethanolamine, recruited to the autophagosome membrane), was unchanged between control condition and cells exposed to

PS-NP (Fig. 7C). PS-NP~100~ initiated the autophagy process, but the autophagic flux mechanism was blocked, preventing the continuous renewal of autophagosomes. In parallel, the induction of autophagy was evaluated with zirconium dioxide nanoparticles of around 100 nm diameter (particle known to be a neutral bioceramic metal compound [38]) (Supplementary data 6.A-B). These neutral particles were used to distinguish the impact of the particle's chemical composition on the autophagy induction. Zirconium dioxide did not alter P62 and Beclin-1
C

<figure><img src="image_13.png" /><figcaption>Fig. 7. (continued).</figcaption></figure>

expression after 24 h of exposure but the ratio LC3-II/LC3-I increased significantly (Supplementary data 7.A). Also, TEM showed accumulation of Zirconium dioxide in large vesicles similar to autophagosomes (Supplementary data 7.B).

## 2.7. Lysosomal response

The autophagic flux requires autophagosomes and lysosomes fusion, for this reason lysosomal response was analyzed with lysotracker by confocal microscopy and also analyzed by TEM. After 48 h of culture without PS-NP, lysosomes were more abundant (red labeling) than after 24 h of culture (Fig. 8.A) confirming the senescent state of the trophoblast at term of pregnancy. When exposed to PS-NP during 24–48 h, PS-NP (green) were accumulated in trophoblasts and were colocalized with lysosomes (merge in yellow) (Fig. 8.A). This colocalization was quantified by Pearson correlation coefficient, which increased after PS-NP exposure (Fig. 8.B). Similarly, the proportion of PS-NP localized in lysosomes was estimated by M2 coefficient on ImageJ-JACoP BIOP software. Around 60 % and 40 % of PS-NP are included in lysosomes after

24 h and 48 h of exposure respectively, with no significant difference between particle sizes (Fig. 8.B). We can also mention the weaker marking of the lysotracker after 48 h of culture with PS-NP compared to control condition. Lysotracker integrates exclusively into intact lysosomes, so their membranes have probably been damaged by the PS-NP (Fig. 8.A). Finally, lysosomes observed by TEM revealed an accumulation of particles. Particle accumulation in lysosomes was observed at an intermediate concentration of PS-NP₁₀₀ (10 µg mL⁻¹) for which, lysosomal membranes appeared intact (Fig. 8. C). Lysosomes exposed to PS-NP₁₀₀ at 100 µg mL⁻¹ were very dense and membranes were difficult to discern (Fig. 8. C). In contrast, exposure to high concentrations of PS-NP₁₀₀ induced particle accumulation in lysosomes and led to late-stage vesicles.

## 2.8. Inflammatory response

Inflammatory response following the PS-NP exposure was evaluated by the measure of Nf-kB and cytokines mRNA expressions (Fig. 9). After 24 h of exposure, Nf-kB expression was enhanced around 20 % for
<figure><img src="image_14.png" /><figcaption>**Fig. 8.** PS-NP accumulation in lysosomal compartments. A/ VCT are exposed to PS-NP<sub>20</sub> or PS-NP<sub>100</sub> for 24 h to 48 h and then exposed to lysotracker (100 nM). Lysosomes are revealed with lysotracker (red) on live cells, PS-NP are revealed by Alexafluor 488 (green) fluorochrome and nuclei are marked with Hoechst (blue). Scale bar = 10 µm. B/ Colocalization of PS-NP and lysosomes (Pearson coefficient) and proportion of PS-NP included in lysosomes are analyzed using ImageJ- JACOPIOP software. Experiments are reproduced 3 times per condition. C/ VCT are exposed 24 h to PS-NP<sub>100</sub> (10 or 100 µg mL<sup>-1</sup>) and fixed for TEM observations. Scale bar = 2 µm. Magnification of PS-NP accumulated in lysosomes is done. Experiments are reproduced 3 times per condition. Scale bar = 500 nm. Legend: → PS-NP, N: Nucleus, M: Mitochondria, L: Lysosome.</figcaption></figure>
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Fig. 9.** Inflammatory response to PS-NP exposure. Level of Nf-kB, TNF-α, IL-6, IL-1β and IL-10 in VCT exposed 24 h to PS-NP or t-BHP (50 µM) measured by q-PCR, normalized to the geometric mean of SDHA, RPLPO, RPL13 and Actin transcripts. Statistical analysis is performed using Wilcoxon and paired t-test depending on conditions (N = 6). Results are represented as mean +/- SEM. \* p < 0.05, \*\* p < 0.01.</figcaption></figure>

0.01 µg mL⁻¹ (p < 0.05) and 100 µg mL⁻¹ (p = 0.07) for PS-NP₂₀ and around 30%-40% at 1 µg mL⁻¹ and 100 µg mL⁻¹ for PS-NP₁₀₀ (p < 0.05). Pro-inflammatory cytokines - TNF-α, IL-6 and IL-1β - were strong enhanced after 24 h of treatment with t-BHP (p < 0.05 for TNFα, IL-6, and p = 0.06 for IL-1β). PS-NP₂₀ at high concentration (100 µg mL⁻¹) induced an increase on TNF-α and IL-6 expressions (+60%, p < 0.01 and +40%, p = 0.06 respectively), whereas no increase was observed for other experimental conditions. IL-1β was enhanced after exposure to PS-NP₂₀ at 0.01 (+50%, p < 0.05) and tended to increase at 100 µg mL⁻¹ (+20%, p = 0.08 respectively). After PS-NP₁₀₀ exposure, IL-1β expression increased in a dose-dependent manner from 0.01 µg mL⁻¹ (+10%, p = 0.06) to 100 µg mL⁻¹ (+100%, p < 0.01). No effect on the expression of the regulatory cytokine IL-10 was observed for either PS-NP size.

## 2.9. Placental functions

The impact of PS-NP exposure on placental endocrine function was studied by assessing secretion levels of the hormone hCG. Prior to hCG secretion analysis, interferences of PS-NP with potentially adsorbed

hormones discarded from samples by centrifugation was performed (Supplementary data 8.A) as described in the "Materials and methods (MM)" section. Overloading with PS-NP₁₀₀ showed no interferences between hCG and PS-NP₁₀₀ at any of the concentrations tested (1 µg mL⁻¹ to 100 µg mL⁻¹) (Supplementary data 8.B). However, a decrease in hCG concentration was measured after PS-NP₂₀ above 50 µg mL⁻¹ indicating adsorption of hCG on PS-NP₂₀ at high concentration (n = 3). We therefore concluded that the PS-NP₂₀ coating reduced hCG by around 30% above 50 µg mL⁻¹ (Supplementary data 8.B). After 24 h of exposure to PS-NP₂₀, a decrease of around 20% in hCG secretion was observed only for 0.01 µg mL⁻¹ (p < 0.05) (Fig. 10.A), while higher concentrations had no effect. Exposure for 24 h to PS-NP₁₀₀ induced a dose-dependent decrease in hCG secretion marked by a 20% drop at 1 and 10 µg mL⁻¹ and up to 70% at 100 µg mL⁻¹ (Fig. 10.A). The effects observed for the two particle sizes specifically concerned the secretion of hCG, since RNA transcript levels remained unchanged (Supplementary data 9. A). The impact of PS-NP on VCT fusion into ST was also investigated, by calculating the fusion index as explained in section MM. Fusion was considered to occur when 2 or more nuclei were nested in a same cell (syncytium). As observed on confocal images, ST
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

Control

IgG

C

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

D

**Fig. 10. Placental functions after PS-NP exposure.** A/ Total hCG secretion after 24 h exposure to PS-NP~20~ and PS-NP~100~. Statistical analysis is performed using paired *t*-test (N = 6–7). Results are represented as mean + /- SEM. \* p < 0.05, \*\* p < 0.01. B/ VCT are exposed to PS-NP~20~ or PS-NP~100~ for 24 h. Cells are then fixed in 4 % PFA. Immunostaining of E-cadherin is revealed with Alexafluor 555 (red) and nuclei are marked with TOPRO-3 (blue). Non-specific IgG is performed as negative control. Fusion of VCT into ST is analyzed using ImageJ software. Scale bar = 20 µm. C/ Fusion index is defined as: IF = (number of nuclei in ST) / (number of total nuclei) × 100; ST is defined as two or more nuclei include in a same membrane. D/ PlGF secretion after 24 h exposure to PS-NP~20~ and PS-NP~100~. Statistical analysis is performed using paired *t*-test (N = 6). Results are represented as mean + /- SEM.
with large number of nuclei was observed for all concentrations and for both PS-NP sizes (Fig. 10.B). Quantification confirmed the stability of the fusion index for all PS-NP concentrations (Fig. 10.C), and syn-cytin1–2 gene expressions remained unchanged after 24 h exposure (Supplementary data 9.B). Thus, VCT exposed to PS-NP conserved their ability to fuse into ST, and hCG secretion appears to be independent of the cell fusion process. Finally, PlGF secretion, a reflection of angiogenic function was unchanged after PS-NP~20~ exposure but tended to decrease

after PS-NP~100~ at 100 µg mL⁻¹ (-25 %, p = 0.6) (Fig. 10.D). As with hCG, interference assays revealed the presence of a PlGF coating on PS-NP~20~ above 50 µg mL⁻¹ (Supplementary data 8.C).

### 3. Summary

Primary VCT were exposed to PS-NP of two sizes, 20 nm and 100 nm, from 0.01 to 100 µg mL⁻¹. Both particles were weakly negatively

<figure><img src="image_21.png" /><figcaption>**Fig. 11.** PS-NP exposure consequences on human trophoblast A/ Molecular and cellular pathways induced by PS-NP exposure (created with BioRender.com). B/ Physicochemical characteristics of PS-NP: particle size and particle concentration- dependent effects.</figcaption></figure>
charged in culture medium (around −10 mV) but interacted differently with it. PS-NP~20~ aggregated in a dose-dependent manner, while PS-NP~100~ remained alone or in pairs in the culture medium.

PS-NP~20~ quickly translocated and rapidly induced the production of ROS (from 30 min) from low concentrations (1 µg mL⁻¹). Due to the lack of TEM sensitivity, PS-NP~20~ couldn't be observed in VCTs and we were therefore unable to conclude on their state of aggregation. Given the high and rapid pro-oxidant potential of these particles, we can assume that a fraction of them end up free in the cytosol before accumulating in the lysosomes, and that their high surface reactivity has contributed to the oxidation of intracellular components. The cell then establishes antioxidant defense systems (Nrf2 pathway, Catalase and SOD expressions), nevertheless a pro-inflammatory response appeared (particularly at high concentrations) mediated by the Nf-kB pathway and the expression of IL-6 and TNF-α. VCT were probably overwhelmed and cell death pathways were triggered, leading to a drop in viability with dose-dependent effects, observed as early as 24 h of exposure and at low doses (from 1 µg mL⁻¹) (Fig. 11.A).

PS-NP~100~ translocated into the VCT and were never free in the cytosol. They were systematically surrounded by endosomes-like membranes and did not induce an increase in ROS until 4 h of exposure. After 24 h, PS-NP~100~ were mostly accumulated in lysosomes, stimulated by P62 with dose-dependent effect. During the translocation process, PS-NP~100~ also induced autophagy initiation via Beclin-1 expression. Nevertheless, the autophagic flux was blocked due to the lysosomal overload and a moderate pro-oxidant response was enhanced. Antioxidant pathway was then mediated (Nrf2/HO-1) and antioxidant enzymes were overexpressed (Cat, SOD). Lysosomal overload probably led to membrane-lysosomal permeability and the release of their contents contributing to the IL1-β-mediated pro-inflammatory response. The dose-dependent increase in IL1-β could thus explain the dose-dependent decrease of VCT viability. The lysosomal accumulation of NP-PS~100~ and the cellular stress generated ultimately led to endocrine disruption through a decrease in β-hCG levels in the extracellular compartment (Fig. 11.A).

## 4. Discussion

Our work reports for the first time the consequences of a PS-NP exposure on primary human placental cells. Primary trophoblast isolation is demanding because of the organization required for sample recovery and the technical difficulty of trophoblast purification. Nevertheless, this allows us to take account of inter-individual variability, which depends on intrinsic predispositions and the specific individual response to pollutants exposure. These factors make our results particularly robust. PS is one of the most abundant plastic in our environment after PE, PET and PP [4,7,39]. PS-NP are largely recovered in placental tissues and meconium from neonates [8–11,14,15,22,26,40] but to date, their consequences on placental/fetal development are not understood. This study provides data on the consequences of acute placental exposure to PS-NP and the importance of particle-size on induced toxicity. Because of the limited lifespan of VCT in primary culture, exposure to PS-NP was limited to 48 h. The entry of trophoblasts into senescence, after ST fusion, remains a limiting step in this model [41] and consequences of chronic exposure have therefore not been evaluated. Also, intrinsic contamination of MP/NP of the recovered placentas could exist and may constitute a slight background for the PS-NP concentrations studied. Nevertheless, to take account of this minor bias, the PS-NP exposure conditions were systematically analyzed and compared with the “unexposed” condition. This paired dataset allows us to study the cellular response following induced exposure to PS-NP. This study is the first to explore the effects of PS-NP exposure on primary trophoblastic cells from a very low (0.01 µg mL⁻¹) to supra-environmental concentrations (100 µg mL⁻¹) through concentrations currently detected in human blood and accepted as reference according to Leslie et al., between approximately 1 and 12 µg mL⁻¹ [7].

According to physico chemical properties, PS-NP~20~ and PS-NP~100~ are distinguished by their different abilities to agglomerate in the culture medium. Indeed, PS-NP~100~ are weakly agglomerated whatever their concentration whereas PS-NP~20~ tend to agglomerate in a dose-dependent manner. This dose-dependent agglomeration has already been mentioned for other nanomaterials and is related to the greater surface reactivity of small particles compared to larger ones. [42]. This property led to inhomogeneous PS-NP~20~ suspension in term of particle size, mixing single particle (20 nm) with agglomerates of around 200 nm. The agglomeration phenomenon was observed immediately after incubation PS-NP~20~ in the culture medium and could probably be observed when nanoparticles are in direct contact with human samples. High surface reactivity frequently favors the formation of a protein corona around the particle [42] as observed in this study with hCG coating on PS-NP~20~ (around 30 % at 100 µg mL⁻¹), partially desorbed by treating the supernatants with Sodium dodecyl sulfate (data not shown). Under real conditions, the formation of a protein corona around NP can influence the protein bioavailability and, in case of hCG, might contribute to pregnancy disorders. The corona can also adsorb other pollutants (plasticizers, aromatic hydrocarbon etc), which could facilitate internalization of particles in tissues and thus promote cocktail exposure [43].

Consequences of placental exposure to PS-NP on trophoblastic cells were firstly explored in term of translocation, viability, ROS production and antioxidant response. In the first part of this work, we demonstrated PS-NP internalization in trophoblastic cells (CTV and ST). Internalization is faster for 20 nm particles, with accumulation of particles in the chorionic villi up to 48 h of exposure. These results are in agreement with those obtained with the perfused cotyledon model [44,45] and complement them with longer exposure times (6 h for the perfused cotyledon versus 48 h here). PS-NP~100~ could be identified by TEM and are systematically surrounded by endosomes-like membranes close to the apical pole. The mechanisms of PS-NP~100~ internalization likely involve clathrin-dependent endocytosis, as observed in other murine and human cell types [46–48] but how do PS-NP~20~ internalize: by endocytosis or by passive diffusion, or are they free in the cytoplasm?

Concerning viability, previous studies on choriocarcinoma-derived trophoblasts (Bewo b30, Jeg-3, JAR, ACH-3P) [28] assessed PS-NP toxicity and showed no significant decrease in cell viability. However, ROS production has sometimes been reported [29,30]. A recent study evaluating the cytotoxicity of PS-NP 50 nm at high concentrations (> 50 µg mL⁻¹) on a transformed cell line derived from primary trophoblast (Swan 71) showed induction of apoptosis linked to increased cleaved-caspase 3 and Bcl2 expression [30]. Our study showed a decrease in VCT viability without evidence of apoptosis. Primary culture of trophoblasts purified from human placentas appear thus to be more sensitive to PS-NP than trophoblastic cell lines, whose resistance to apoptosis and cell death makes them less representative of the physiological response to stress caused by exposure to pollutants [20]. The decrease in cell viability after 24 h exposure to PS-NP~20~ at the environmental concentration was preceded with ROS production, an effect not observed for PS-NP~100~. Nevertheless, oxidative stress through oxidation of Peroxiredoxine (Prx) was reported after 24 h exposure to PS-NP~100~. These results showed that both PS-NP sizes induce oxidative stress, more rapidly for PS-NP~20~ than PS-NP~100~, which is probably explained by translocation kinetic. Antioxidant response is clearly triggered after exposure to both sizes PS-NP. Indeed, the Nrf2/HO-1 antioxidant pathway was increased, again with different activation kinetics: PS-NP~20~ translocate and promote HO-1 protein expression rapidly, while PS-NP~100~ lead to an increase in Nrf2 transcript expression later. The transcriptional factor Nrf2 is linked to Keap1, a sensor in the cytosol. When oxidative stress occurs, Keap1 is oxidized and releases Nrf2 that, translocates to the nucleus and leads to the induction of HO-1, SOD1-2 and catalase via an antioxidant response element (ARE) [49]. The expressions of SOD1-2 and catalase were also enhanced after exposure to high concentrations (10–100 µg mL⁻¹) of PS-NP~20~, while the increase
appears at relatively low concentrations for PS-NP₁₀₀ (from 1 µg mL⁻¹) (Fig. 11). Thus, for a same order of particle concentration (10⁹ particles mL⁻¹), SOD and Catalase expression tended to be higher when exposed to 100 nm particles than to 20 nm particles. This suggests that the antioxidant response is particularly dependent on particle size. Antioxidant response elements were mainly analyzed by q.PCR because protein expression measured by Western Blot was extremely variable, possibly due to the lower sensitivity of Western Blot compared with q.PCR. Investigation of enzymatic activities would be highly instructive as they can be directly modulated, with a size-dependent effect, by MP/NP [50]. Indeed, *in vitro* studies have demonstrated SOD activation through changes of secondary structure following adsorption to the particle surface. The conformational change appears to be greater after exposure to PS-NP₁₀₀ (increased dismutase activity) than with PS-NP₂₀ [50]. Measurement of GSH-GSSG content would inform on the antioxidant reserve still available. As mentioned below, PS-NP exposure led to a decrease in metabolic activity without involving apoptosis process. This could be due to a too short exposure duration to observe modulations in apoptosis markers such as PARP1 and Caspase3, or due to the involvement of another type of cell death (pyroptosis, ferroptosis) or also a metabolic slowdown without cell death by mitochondrial damage and/or intracellular pro-oxidant imbalance (NAD⁺ >> NAD(P)H), then limiting the availability of NAD(P)H required for WST-1 assay [51].

Further research has shown that particle size has an impact on the intracellular response. Both sizes induced P62 protein expression, a cargo protein involved in the orientation towards degradation by the proteasome. However, P62 structure is complex, with 6 interactions domains that give it a central role in the cellular response to pollutants [52]. Following P62-Ser349 phosphorylation, KIR motif can interact with Keap1, resulting in the release of Nrf2 and the activation of the antioxidant pathway activation. P62 could therefore contribute to the activation Nrf2/HO-1 as described above, but it could also induce the Nf-kB inflammation pathway by interacting with the ZZ or TB motifs, or promote autophagy by acting via the LIR motif and the UBA motif [52]. In our study, PS-NP₁₀₀, from 10 to 100 µg mL⁻¹, increased Beclin-1 expression and autophagosomes accumulating a number of particles were observed by TEM. However, LC3-II, the membrane protein of autophagosomes, was not increased by Western-Blot. PS-NP₁₀₀ initiate autophagy but the process appears to be blocked without autophagosome turnover [53,54] (Fig. 11). PS-NP₂₀ did not initiate the autophagy process, showing that the effects depend on particle size. This size-effect was also demonstrated in a HUVEC model exposed to 100 and 500 nm PS-NP, in which PS-NP₅₀₀ did not induce autophagy unlike PS-NP₁₀₀ [55]. Autophagosomes fuse with acidic compartments, the lysosomes, to recycle their content into autophagolysosomes [54]. In this work, exposure to PS-NP (20 and 100 nm) led to an accumulation of particles in the lysosomes, revealed by lysotracker and observed by confocal microscopy. In addition, TEM showed very dense lysosomes with damages to membrane integrity after PS-NP₁₀₀ exposure at high concentrations. We can therefore hypothesize that PS-NP induce a lysosomal overload that prevents the fusion of lysosomal with autophagosomes (Fig. 11). Lysosomal overload has already been explored in other tissues following exposure to NP/MP and silica nanoparticles [53,56,57]. This overload can lead to permeabilization of lysosomal membrane, release of cathepsin B and cell death by pyroptosis; a programmed cell death associated with inflammation [58–61]. Pyroptosis involves the inflammasome pathway by NLRP3-caspase1 activation and the release of the specific IL1-β cytokine [58,59,62,63]. Our work showed a strong increase in IL-1β expression (+ 20 %) from very low concentrations (0.01 µg mL⁻¹) of PS-NP₁₀₀ which was not observed for PS-NP₂₀. A possible link between IL-1β release, lysosomal pathways and pyroptosis pathways would be extremely interesting to explore and could, in part, explained the cytotoxicity observed.

The consequences of PS-NP exposure on trophoblast functions were assessed at three levels: inflammation, endocrine secretion and fusion of VCT into ST. Once again, the inflammatory profile showed that PS-NP₂₀

and PS-NP₁₀₀ exposure did not have the same effect on trophoblastic cells: PS-NP₂₀ enhanced the pro-inflammatory pathway Nf-kB and the expression of cytokines (TNF-α and IL-6) at high concentration (100 µg mL⁻¹), whereas PS-NP₁₀₀ did not affect the expression of these markers but, as previously described, induced a high expression of the pro-inflammatory IL-1β from low concentrations (1 µg mL⁻¹) (Fig. 11). Such results are worrying, as they suggest an inflammatory phenomenon triggered at concentrations currently considered as sub environmental. An increase in IL-1β has also been reported in the placenta, correlating with premature rupture of membranes and inflammatory placental pathologies such as chronic villitis [64]. The induction of IL-6 and TNF-α, following 24 h exposure to high concentrations of PS-NP₂₀, should not be overlooked and questions should be asked about the consequences of repeated or chronic exposure to these particles, which may lead to a low-noise inflammatory response favoring the development of placental pathologies such as preeclampsia [65].

The consequences of PS-NP exposure on endocrine function were also explored. hCG, commonly known as “the pregnancy hormone” plays an essential role in placentation by taking over from the corpus luteum to increase progesterone secretion and prevent uterine contractions, and by promoting angiogenesis and placental growth throughout pregnancy [16,18,19]. In addition to the risk of hormonal adsorption of nanoparticles already mentioned above, our work has shown a decrease in hCG release after exposure to PS-NP at environmental concentrations: 0.01 µg mL⁻¹ for PS-NP20 and a dose-dependent response of 1–100 µg mL⁻¹ for PS-NP100. This decrease was not observed at the transcriptional level and was not associated with reduced VCT fusion into ST. Since the ST mainly express hCG compared with the VCT, reduced fusion could have contributed to a reduction in hCG secretion. We can assume PS-NP₂₀ induce more rapid and severe cell stress, resulting in a greater cell mortality but also allowing selection of a cell phenotype that is more resistant to stress and whose endocrine function is less affected. In contrast, cells exposed to PS-NP₁₀₀ undergo less stress at low doses (low mortality) but nevertheless alter their cellular functions, resulting in a greater drop in hCG secretion. Two main mechanisms of this drop in β-hCG release could be emit. The first is linked to the volume occupied by particles accumulated in lysosomes (in particular PS-NP₁₀₀) that could lead to a reduction of β-hCG exocytosis. Indeed, in the absence of cellular stress, motile endosomes are involved in a variety of cellular processes to promote cell growth and development. Some endosomes recycle cargo, others are engaged in plasma membrane repair, protrusion formation, mTORC1 signaling, or exosomes secretion [66]. During PS-NP exposure, endosomes and lysosomes are filled with particles that could saturate the endomembrane system, possibly preventing the renewal of vesicles and the availability of β-hCG exocytosis vesicles. The second hypothesis is that β-hCG stability could be compromised. Indeed, β-hCG stability in extracellular compartments depends of its high degree of glycosylation [19] which takes place in endoplasmic reticulum (ER). ER stress, notably caused by oxidative stress, could thereby alter the degree of glycosylation and thus the half-life of β-hCG [67]. ER stress has already been shown in response to nanoparticles exposure, particularly NP-PS, on other cell types [68,69]. This hypothesis is all the more interesting as we observed the same endocrine disorder profile when VCT were exposed to t-BHP-induced moderate oxidative stress (Supplementary data 10). Exposure to PS-NP could thus contributes to ER stress, via oxidative stress but also via the endomembrane imbalance mentioned just before, and in consequences to a modification of glycosylation levels of β-hCG. It would be interesting to explore the levels of glycosylation of β-hCG as well as the status of ER stress, especially as hyperglycosylation of β-hCG (hCG-H) is a clinical marker of pathological pregnancy [70]. Indeed, a high level of hCG-H at the early second trimester of pregnancy is correlated with a higher risk of Down syndrome while a lower level of hCG-H during the first trimester (8–13 GW) is associated with a higher risk of early preeclampsia [70]. Endocrine disrupting is poorly documented in relation to MP/NP exposure in human tissues, but some works have shown alterations in thyroid
hormones and steroid secretion (testosterone) in rodent models [71,72]. To our knowledge, our work is the first study to demonstrate an endocrine disrupting effect of plastic particles on human tissues. Impaired endocrine function during pregnancy, in particular hCG secretion, can have major repercussions on pregnancy outcome, leading to miscarriage, premature birth and IUGR, pathologies that have already been associated with exposure to environmental pollutants during pregnancy (air pollution, pesticides) [73–75].

In the course of this work, we demonstrated size-dependent molecular and cellular effects of NP-PS (Fig. 11.B). Indeed, at identical particle concentration (2.10¹¹ particles mL⁻¹, i.e. 1 µg mL⁻¹ and 100 µg mL⁻¹ for PS-NP₂₀ and PS-NP₁₀₀ respectively), exposure to PS-NP₁₀₀ induced: (i) a higher expression of the antioxidant system, (ii) an activation of the mitochondrial fusion process, (iii) an initiation of autophagy, (iv) a dose-dependent reduction in β-hCG secretion (no effect observed with PS-NP₂₀). These results therefore suggest that particle size, and hence the volume occupied by the particles, is a key factor in the cellular stress generated and the cellular defense responses developed by trophoblasts. In parallel, at identical mass concentration of particles (1 µg mL⁻¹) - and thus volume occupied-, there was (i) a significant drop in viability and (ii) an induction of ROS production after PS-NP₂₀ exposure (no effect with PS-NP₁₀₀). These results suggest the importance of particle number and surface occupied on induced cytotoxicity probably linked to a rapid oxidation of cellular components [42]. This work highlights a size effect of PS-NP whose trophoblastic consequences would depend on two main factors: the specific surface area of the particles (i.e. surface reactivity) and the volume occupied by NP (Fig. 11.B). Cellular defense and adaptive responses - antioxidant, mitochondrial, autophagic, lysosomal - are thus triggered in response to NP-PS exposure, distinctly according to their size, and it remains to be understood by what mechanisms one signaling pathway would be privileged over another.

Cellular effects are clearly concentration-dependent for PS-NP₁₀₀: cell death, mitochondrial fusion, initiation of autophagy, pro-inflammatory state, reduced β-hCG secretion. In contrast, dose effects are more complex to analyze with PS-NP₂₀, marked by a dose-dependent response to cell death and ROS production, but a surprising “U-inverted” dose-effect of Nrf2 and NfkB expressions, and β-hCG secretion. This behavior can probably be explained by an increasing aggregation state

of PS-NP₂₀ as a function of mass concentration in the culture medium. The state of aggregation modifies not only the diameter of the particle but also all the physicochemical characteristics that depend on it (surface reactivity, shape, corona) and therefore makes it more difficult to assess the effect of increasing particle doses, since the toxicological profile is closely linked to the physicochemical profile of the particle [76].

Our study provides first insights into the placental consequences of acute exposure to PS-NP during pregnancy and proposes a toxicological mechanism for these particles in the human placenta (Fig. 12): once internalized, NP generate oxidative stress through their surface reactivity and accumulate in lysosomes. The alterations caused by oxidative stress and saturation of the endomembrane system lead to cellular inflammation, resulting in endocrine dysfunction and trophoblastic cell death. Endocrine disruption associated with premature cell death could then be responsible for adverse pregnancy outcomes such as recurrent miscarriages, premature birth or intrauterine growth retardation (Fig. 12). Nevertheless, many questions remain unanswered and analytical limitations need to be resolved, in particular the detection and quantification of nanoparticles smaller than 50-nm. In our study, PS-NP₂₀ could not be observed by TEM, which makes understanding of the cellular impacts more complex than with PS-NP₁₀₀. Although an increasing number of studies have recently identified the presence of MP in placentas by IR-laser and Raman microscopy, the size detection limit (around 2 µm) and the sensitivity of quantification methods do not allow detection or quantification of NP in placentas. Exposure to MP/NP is complex because of the number of plastic polymers to which we are exposed (PE, PET, PP, PS, PU mainly) as well as their different sizes, shapes (spherical, fragments or fibers) and incorporated plasticizers (Bisphenol-A-F-S, phthalates (DEHP), perfluoroalkyl, Polychlorinated biphenyls (PCB)) [43,77,78]. This cocktail effect should therefore be studied, particularly following the discovery of the endocrine disrupting effect of PS-NP at low concentrations. This effect could be synergic when combined with substances classified and recognized as endocrine disruptor, such as DEHP or BPA [79,80]. Finally, our work did not explore the consequences of repeated exposure of MP/NP which can, over time, potentiate cell damages as observed during chronic diseases. Exploring the consequences of MP/NP exposure on during placental development

<figure><img src="image_22.png" /><figcaption>Fig. 12. Proposed toxicological mechanism of PS-NP on human placenta (created with BioRender.com).</figcaption></figure>
and understanding the impact on post-natal development through
cohort studies is essential in the future.

**5. Materials and methods (MM)**

**5.1. Materials**

Hank's balanced salt solution (HBSS, # 14175-053), phosphate buffer saline (PBS, # 14190-094), Triton X-100 (ref: 11891445) and the protein assay kit Micro BCATM (#23235) were from Thermo Fisher Scientific (Waltham, MA, USA). DMEM (#11880-028) and Trypsine (#27250-018) for the cell culture were from Gibco. DNase type 4 (# SLBV9316) was from Sigma and PercollTM (# 17544501) from Cytiva. Tissue protein extraction reagent, RIPA Buffer™ (# 9806) was purchased from Cell Signaling Technology. Protease inhibitor cocktail (#539131) and phosphatase inhibitor cocktail (# 524629) were from VWR (Radnor, PA, USA). For PCR and q.PCR, Fast start HIFI PCR system DNT pack 125 U was purchased from Merck (# 04738284001), ARN-PicoPureTM (#KIT0204), PowerTrack SYBR Green TM Master Mix (# 10459604) was from Fisher Scientific. For immunolocalization studies, human IgG was from Jackson ImmunoResearch (Ely, UK), (# 009-000-003), and fluorescence mounting medium (#S3025), was from Dako (Trappes, France). Bovine serum albumin IgG-free (BSA, # 011-000-162) was from Interchim (San Diego, CA, USA), TO-PRO-3 (#3605) and Hoechst 33342 (#H3570) were from Thermofisher Scientific. Cell proliferation reagent WST1 (#11644807001) was from Roche; CM-H2DCFHDA (#C6827), Orange CM-H2TMRos (#M7512) and LysoTrackerTM deep-red (#L12492) were purchased from Thermofisher Scientific. Anti-CK7 mouse antibody (#M7018) was from Dako, anti-E-cadherin mouse antibody (#ab40772), anti-PrxSO3 rabbit polyclonal antibody (#ab16830) and anti-caspase 3 (#13585) were from Abcam. Anti-HO-1 rabbit polyclonal antibody (#ADI-SPA-895-D) was from Enzo, anti-PARP1 rabbit antibody (#9542), anti-MFN1 rabbit monoclonal antibody (#D6E2S), anti-DRP1 rabbit polyclonal antibody (#5391), anti-SOD1 mouse monoclonal antibody (#4266), anti-SOD2 rabbit monoclonal (#13141) and anti-catalase rabbit monoclonal antibody (#12980) were from Cell Signaling. Anti-MFN2 mouse polyclonal antibody (#56889) and anti-OPA1 (#42364) were from Abcam, anti-P62 rabbit polyclonal antibody (# 18420-1-AP), anti-Beclin1 rabbit polyclonal antibody (#11306-1-AP), anti-LC3 rabbit polyclonal antibody (# 14600-1-AP) were from Proteintech. Anti-β-actin mouse monoclonal antibody (A5441, 2.6 µg mL⁻¹) and anti-FIS1 rabbit polyclonal (#HPA017430) were from Simga-aldrich and Nrf2 (#A-10) was from Santa Cruz. DyLight 800-labeled anti-mouse (#35521) and DyLight 680-labeled anti-rabbit (#35568) were from Thermo Fisher Scientific. NanosphereTM size standards National Institute of standards and Technology (NIST) traceable mean diameter (#3200 A and #3100 A) and fluorescent microspheres Fluoro-Max™ G100 (0.100 µm) and G25 (0.026 µm) were from Thermofisher scientific. Dioxide zirconium nanoparticles (#643122) and Staurosporine (#S4400) and Tert-butyl hydroperoxide (t-BHP, Luperox t-BHP70X, #458139) were from Sigma-Aldrich. PS-NP particles of 20-nm and 100-nm (Nanospheres size standards), suspended in water (1 % solids), were certified by NIST and provided from Thermofisher scientific. Modified PS-NP (Fluorescent nanospheres) used for confocal observations were purchased from Thermoscientific. Particles of 22-nm and 100-nm diameter, suspended in water (1 % solids), have an Alexafuor488 in the core of the particle (λexcitation = 468 nm et λemission = 508 nm).

**5.2. Methods**

**5.2.1. Ethics statement**

The study respected the Declaration of Helsinki. Placental tissues
used were obtained after written informed consent from patients of
Cochin Port-Royal maternity units, Anthony Hospital, Diaconesses ma-
ternity, Mutualist Institute (Paris, France). The protocol was approved

by the institutional review board "Comité de Protection des Personnes
(CPP) 2015-mai-13909". Placentas and patient details were anonymized
by a serial number.

Placentas were recovered from singleton pregnancy after caesarian
(third trimester, 37-39 gestational weeks (GW)). Placentas issued from
an assisted reproduction procedure, in a context of fetal abnormalities or
genetic diseases were excluded.

**5.2.2. Placental explants**

Chorionic villi were taken and washed within HBSS at 37°C. Basal
membrane was eliminated and placental explants were cultured in 48-
wells plate during 16 h (37°C, 5% CO₂), with Dulbecco's modified Ea-
gle's medium (DMEM) containing 1 g/L glucose, without red phenol and
supplemented with 10% fetal calf serum (FCS).

**5.2.3. Cytotrophoblast purification**

Villous cytotrophoblasts (VCTs) were purified from human term
placentas as previously described [81]. Briefly, basal layer was elimi-
nated, then pieces of cotyledons were isolated and rinsed in Ca²⁺ and
Mg²⁺-free HBSS. Placental dissection was performed within 30 min to
prevent tissue degradation. After dissection, chorionic villi were incu-
bated for 30 min at 37°C in digestion buffer containing 1 % trypsin
(5 mL/g), 100 mmol/L MgSO₄, 100 mmol/L CaCl₂, 25 IU DNase, and
7 % semi skimmed milk. The following digestions of 10 min were
monitored by observation under an optical microscope until the disso-
ciated cells emerged. For each digestion, the trypsin buffer was renewed.
Finally, the chorionic villi were washed with HBSS and the supernatant
was collected, filtered (cut-off: 40 µm), and incubated with 10 % of FCS
(v/v) to stop trypsin activity. Next, all cell suspensions were cen-
trifugated (200 g for 10 min) at room temperature. Cell pellets were
suspended in DMEM, pooled, layered on the top of a preformed Percoll®
gradient (5-70 %), and centrifuged at 840 g at room temperature
without braking during 20 min. The layer between the 45 % and 35 % of
Percoll® containing trophoblast cells was collected, suspended in
DMEM supplemented with 10 % FCS, and centrifuged at 200 g for
10 min. The cell pellet was re-suspended in complete DMEM and the
cells were counted using a TC20 automated cell counter from Biorad.
The cells were seeded at a density of 125,000 cells/cm² on plates and
then incubated in 5 % CO₂ at 37°C for 16 h.

**5.2.4. PS-NP particle characterization**

PS-NP<sub>20</sub> and PS-NP<sub>100</sub> morphology was observed by transmission electron microscope 120 keV 1400 Flash (Jeol) (TEM). Size of particles and agglomerates were analyzed in water and culture medium, by dynamic light scattering (DLS), using a Dyna Pro NanoStar analyzer (Wyatt Technology) with a temperature set at 25 °C. The zeta-potential of the PS-NP particles was measured by a zetasizer pro (Malvern Instruments). The level of sedimentation was assessed by UV spectroscopy at 254 nm using Dionex UltiMate 3000 Variable Wavelength Detector (Thermo Scientific).

**5.2.5. PS-NP preparation and cells treatments**

PS-NP were diluted in complete culture medium at 0.01; 1; 10; 100 µg mL⁻¹ and sonicated for 3 min in ice (70 % amplitude, 450 W and 50/60 Hz) to avoid agglomerations/aggregations. Cells were rinsed with HBSS and treatments were applied for 30 min to 48 h depending on experiments. t-BHP (tert-butylhydroperoxide), an oxidative stress inducer, was used at 50 µM or 200 µM from 30 min to 48 h, depending on experiments. Staurosporine, an apoptosis inducer, was resuspended in DMSO at 2 mM, diluted in culture medium at 500 nM, and incubated for 3 h. Rapamycin, an autophagy inducer, was resuspended in DMSO (250 µM) and then diluted in culture medium (500 nM) and incubated for 24 h.

**5.2.6. PS-NP treatments with chorionic villi and immunofluorescence**

The fluorescent PS-NP nanospheres, having the same physico-
chemistry features of PS-NP nanoparticles, were used to visualize nanoparticle translocation within placental tissue. They were diluted in complete culture medium and sonicated for 3 min in ice (450 W and 50/ 60 Hz) before being brought into contact with chorionic villi. PS-NP were incubated with chorionic villi for 2–48 h under agitation at 37°C, 5 % CO₂. They were then rinsed with PBS and fixed with 4 % PFA for 24 h (4°C). Explants were included in 4 % agarose (weight/vol) and sections (80 µm of thickness) were made using vibratome. Tissues were incubated 2 h at room temperature under agitation with saturation solution (3 % BSA/0.1 % X100 Triton/ 5 % donkey serum). Trophoblast membrane was visualized by anti-CK7 (1 µg mL⁻¹) added, 1 h at room temperature, and coupled to Alexa fluor 555 (1:500, in 1 % BSA). TOPRO-3, was used for nuclei staining, and deposited for 20 min at room temperature (1:500). Blades were prepared with fluorescent mounting medium and observed with SP8 confocal microscope (Leica).

### 5.2.7. Metabolic activity- WST1 assay

Viability was assessed using the WST-1 assay (Sigma-Aldrich #11644807001). This assay measures the metabolic activity of cells studying the cleavage of the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo phenyl)-2H-tetrazolium (WST-1) into formazan by dehydrogenases. According to the manufacturer's instructions, VCT were treated for 4 h to 48 h with PS-NP. At the end of the exposure time, cells were rinsed with culture medium and WST-1 reagent was added (1:100). After 3 h incubation at 37 °C, absorbance was measured using a microplate reader (EnSpire 2300 Multilabel reader, PerkinElmer, Villebon-sur-Yvette, France) at 440 nm. Nonspecific emission at 600 nm was measured and omitted from the initial value.

### 5.2.8. CM-H₂DCFDA detection of intracellular reactive oxygen species (ROS)

ROS production was assessed using the CM-H₂DCFDA probe according to the manufacturer's instructions. This probe penetrates passively the cells before being esterified by intracellular esterase produced ROS oxidize the probe, which emits a detectable fluorescent signal. CM-H₂DCFDA probe was suspended in DMSO and diluted in HBSS at 5 µM. VCT were incubated with the probe for 1 h (37°C, 5 % CO₂). Probe was then removed and cells were treated with t-BHP or PS-NP for 30 min, 2 h and 4 h. For each time, fluorescent signal was measured using microplate reader (λexcitation = 492 nm and λemission = 520 nm). Control wells without cells or without probe were used as negative controls. The lack of interferences of PS-NP and the probe (without cells) was also checked.

### 5.2.9. Western-blot analysis

Protein was extracted from VCT using extraction buffer (RIPA®) containing 1/50 of a protease inhibitor cocktail and 1/100 of a phosphatase inhibitor cocktail and then was sonicated for 2 min (450 W and 50/60 Hz). The homogenate was centrifuged for 10 min at 13 000 g at 4°C and the supernatant was collected and stored at -80°C.

Protein concentration was determined using the Micro BCA Assay Kit following the manufacturer's instructions, and absorbance was measured with the spectrophotometer Enspire® (λ= 562 nm).

Protein extracts were heated for 1 h at 60°C, and then resolved by 10 % SDS-PAGE. Immunoblotting was performed with anti-PARP1, anti-HO-1, anti-MFN1, anti-MFN2, anti-OPA1, anti-DRP1, anti-FIS, anti-LC3, anti-Actin, anti-SOD1, anti-SOD2, anti-catalase (1:1000), and anti-P62, anti-Beclin1, anti-PrxSO₃ (1: 2000) antibodies. After incubation with an appropriate Alexa Fluor® conjugated secondary antibody (680 or 800 conjugate), blots were revealed by using an Odyssey® Infrared Imaging System, then analyzed using ImageStudioLight from LICOR®.

### 5.2.10. Primer design, RNA extraction, reverse transcription and PCR

Primers were designed and amplification products ranged between 100 and 200 bp to an adequate qPCR. [Table 1]

Total RNA from VCT was extracted following instructions of

<table><caption>**Table 1**
Primer sequences.</caption><thead><tr><th></th><th>Primer sequences</th></tr></thead><tbody><tr><td>Actin</td><td>5'-GTGGGGCGCCCCAGGCACCA-3'<br>5'-CTCCTTAATGTCACGACGATTTC-3'</td></tr><tr><td>Catalase</td><td>5'- TTCATCCAGAAGAAAGCGGTCAA- 3'<br>5'- TGGATGTGGCTCCCGTAGTCA- 3'</td></tr><tr><td>IL-1β</td><td>5'-AGCTACGAATCTCCGACCAC-3'<br>5'-CGTTATCCCATGTGTCGAAGAA-3'</td></tr><tr><td>IL-6</td><td>5'-ACTCACCTCTTCAGAACGAATTG-3'<br>5'-CCATCTTTGGAAGGTTCAGGTTG-3'</td></tr><tr><td>IL-10</td><td>5'-GTGATGCCCCAAGCTGAGA-3'<br>5'-CACGGCCTTGCTCTTGTTTT-3'</td></tr><tr><td>Nf-kB</td><td>5'- GTGAAGACCACCTCTCAGGC- 3'<br>5'- AAGCTGAGTTTGCGGAAGGA- 3'</td></tr><tr><td>Nrf2</td><td>5'-AACCAGTGGATCTGCCAACTACTC-3'<br>5'-CTGCGCCAAAAGCTGCAT-3'</td></tr><tr><td>RPLPO</td><td>5'- AACATCTCCCCCTTCTCCT- 3'<br>5'- ACTCGTTTGTACCCGTTGAT- 3'</td></tr><tr><td>RPL13</td><td>5'- AAGGTCGTGCGTCTGAAG- 3'<br>5'- GAGTCCGTGGGTCTTGAG- 3'</td></tr><tr><td>SDHA</td><td>5'- TGGAACAACAGGGCATCTG- 3'<br>5'- CCACCACTGCATCAAATTCATG- 3'</td></tr><tr><td>SOD1</td><td>5'- CTGAAGGCCTGCATGGATTC- 3'<br>5'- CCAAGTCTCCAACATGCCTCTC- 3'</td></tr><tr><td>SOD2</td><td>5'- CGTGACTTTGGTTCCTTTGACA- 3'<br>5'- AAGTGTCCCCCGTTCCTTATTGA- 3'</td></tr><tr><td>TNF-α</td><td>5'- GCTCCCCAAGAAGACAGG- 3'<br>5'- GCCAGAGGGCTGATTAGA- 3'</td></tr></tbody></table>

PicoPure™ kit and then quantified with a NanoDrop spectrophotometer. Reverse transcription was performed on 0.5–1 µg of RNA (depending on samples) using the SuperScript III First Strand Synthesis System (Invitrogen #18080044, Villebon-sur-Yvette, France) and random primers (Invitrogen # 48190011). The cDNA obtained was stored at -20°C. To verify the specificity of primers, PCR was performed and amplicons were detected under UV light (Minibis, DNR Bio-imaging systems, Dublin, Ireland) before being sequenced by Sanger (Eurofins Genomics). PCR reaction volume containing 0.5 µM of each primer, 300 µM of dNTPs, 3.5 mM MgCl₂, 1.25 U of Taq polymerase high fidelity and 100 ng of cDNA. Forty cycles were performed using the following steps: Taq polymerase activation (15 min, 95°C), denaturation (30 s, 94°C), annealing (30 s, 55°C), extension (30 s, 72 °C), and final extension (10 min, 72°C). Amplification products were migrated by electrophoresis on 1 % agarose gel containing 1/10,000 ethidium bromide. Then, amplicons sequences were analyzed using 4 peaks® software and were blasted using NCBI's Primer-Blast.

### 5.2.11. Nrf2 by immunofluorescence

Trophoblastic cells were fixed in 4 % PFA for 20 min at room temperature. Cells were washed 3 times in PBS for 5 min and then treated with NH₄Cl 50 mM for 10 min at room temperature. Cells were permeabilized in 3 % BSA- 0.1 % Triton for 30 min and then saturated with 3 % BSA - 5 % donkey serum for 2 h. VCT were incubated overnight with specific antibody against Nrf2 (3 µg mL⁻¹) in PBS-1 % BSA – 0.1 % triton and non-specific rabbit IgG was made as control. Cells were then washed and incubated 1 h with AlexaFluor 488 (1/500) at room temperature. Nuclei were marked with TOPRO-3 (1/500, 15 min). Blades were observed using SP8 confocal microscope (Leica).

### 5.2.12. qPCR

Quantitative real-time PCR was performed in a 10 µL reaction volume containing 100 ng of cDNA, 0.5 µM of each primer, and SYBR™ MasterMix (SYBR™ Rox SYBR® MasterMix dTTP Blue, Eurogentec, Angers, France), using an ABI Prism 7900 Sequence Detector System (Applied Biosystems). qPCR was performed on 40 cycles of amplification using the following steps: SYBR activation (3 min, 95°C), denaturation (10 s, 95°C), annealing and extension (1 min, 60°C). The number of cycles required to cross the threshold was corresponded to the Ct
(threshold cycle). mRNA levels were normalized by geometric averaging of housekeeping genes: SDHA, RPLPO, RPL13 and Actin. The relative amounts of mRNA were measured using the ΔCt method and expressed as 2⁻ΔCt.

### 5.2.13. Mitotracker - CM-H2TMRos

Cells were washed with HBSS and treated with Orange CM-H2TMRos following manufacturer's instructions. CM-H2TMRos probe was diluted in complete culture medium at 50 nM and incubated with cells for 30 min at 37°C. Probe was then removed and cells washed in PBS. Fixation was performed using 4% PFA, 20 min at room temperature before adding TOPRO-3 (1:500) for 10 min. Finally, blades were mounted with fluorescent mounting medium and observed with SP8 confocal microscope (Leica).

### 5.2.14. LC3 by immunofluorescence

Trophoblastic cells were fixed in methanol at -20°C for 20 min. Cells were washed 3 times in PBS for 5 min and saturated with 5% donkey serum, 12.5 µg mL⁻¹ human IgG and then incubated for 1 h at room temperature. Immunostaining was performed with specific antibody against LC3 (2.5 µg mL⁻¹) in PBS-1% BSA and non-specific rabbit IgG was made as control. Cells were incubated 1 h with AlexaFluor 555 and nuclei were marked with TOPRO-3 (1/500, 15 min). Blades were mounted in Dako Faramount aqueous mounting medium and observed using SP8 confocal microscope (Leica).

### 5.2.15. Lysotracker

Cells were washed with PBS and treated with LysoTracker deep-red following manufacturer's instructions. The probe was diluted in culture medium without serum at 100 nM with Hoechst (5 µM) and incubated with cells for 10 min at room temperature. Probe was then removed and cells were washed in PBS. Blades were observed on live cells with SP8 confocal microscope (Leica).

### 5.2.16. Transmission electronic microscopy (TEM)

After treatments with PS-NP, cells were rinsed and then incubated successively with 3 fixation solutions: glutaraldehyde-PFA (1.5%/1.5%) diluted in Caco buffer; 1% osmium oxide (OsO4)/1% Potassium ferrocyanide; and 1% uranyl acetate in H₂O. Dehydration using gradual ethanol concentration was practiced and cells were included in 100% Epon, overnight at room temperature. Observations were done using Transmission electron microscope 120 keV 1400 Flash (Jeol).

### 5.2.17. Total hCG assay

After centrifugation (200 g, 10 min) of culture supernatants with no more than one congelation cycle, assays were performed for total hCG using a chemiluminescent microparticle immunoassay (CMIA), with an Alinity reagent kit and analyzer (Abbott) held by an accredited (Cofrac) clinical chemistry laboratory. Samples were assayed by series as regards to the low inter-run variability coefficient.

### 5.2.18. Fusion Index (FI)

Cells were incubated for 24 h with PS-NP at 37°C, 5% CO₂. They were then rinsed with HBSS, fixed with 4% PFA, 20 min at room temperature, and permeated 30 min with 0.5% X100-Triton-PBS. Then, cells were saturated, 1 h at room temperature with 5% BSA, PBS-0.1% tween20. The primary antibody, anti-E-cadherin, diluted at 1 µg mL⁻¹, was incubated with the sample for 1 h, followed by the secondary antibody for 1 h: Alexa fluor 555 (1:400), then TOPRO-3, a DNA-labelling intercalant, for 10 min (1:500). Washing steps were carried out between the different incubations and the entire procedure was performed at room temperature. Blades were prepared with fluorescent mounting medium and observed with SP8 confocal microscope (Leica). Fusion index was calculated as: $IF = \frac{\text{number of nuclei in ST}}{\text{number of total nuclei}} \times 100$. The cell was considered as ST when two or more nuclei were included in the same

membrane.

### 5.2.19. PlGF assay

After centrifugation (200 g, 10 min) of culture supernatants with no more than one congelation cycle, assays were performed for PlGF using a commercial kit validated as a tool for screening of pre-eclampsia (DELFIA Xpress PLGF 1–2–3 assay, Perkin Elmer, two-site fluoroimmunometric assay). Variation coefficient was inferior to the variation observed.

### 5.2.20. Statistical analysis

Data were analyzed with GraphPad Prism software (La Jolla, CA, USA). The normality of the samples was verified by a Shapiro-test followed by a Fisher-test. Paired t-test when parametric or Wilcoxon-test when non-parametric were applied for comparison of two appaired groups. A p-value lower than 0.05 was considered statistically significant, with p < 0.05, p < 0.01 or p < 0.001 represented as *, ** or *** respectively. Graphical representations show experimental results with mean ± SEM. Western-blotting and q.PCR were performed respectively on 7 and 6 different placentas (N = 7 and N = 6).

## Environmental implication

Nanoplastics cross the biological barriers and enter the bloodstream (levels around 1.6 µg mL⁻¹). The evidence of human and fetal placental exposure has been established, but the consequences on the human placental development are not documented.

For a realistic exposure, we used primary cells that constitute a physiologic model unlike trophoblastic cell lines currently used in the literature. We evaluated nanoplastics exposure on trophoblasts purified from human term placentas, exposed for 24 h to polystyrene nanoparticles at environmental doses (0.01–10 µg mL⁻¹). We report for the first time an endocrine disruption from 1 µg mL⁻¹ on primary cells.

## Funding sources

This work is supported by The French National Research Program for Environmental and Occupational Health of Anses (ANSES-21-EST-121).

## CRediT authorship contribution statement

**Zerrad-Saadi Amal:** Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.
**Lespes Gaëtane:** Writing – review & editing, Supervision, Methodology, Funding acquisition.
**Beaudeux Jean-Louis:** Writing – review & editing, Supervision, Methodology.
**Fournier Thierry:** Writing – review & editing, Funding acquisition.
**Ferecatu Ioana:** Writing – review & editing, Methodology.
**Djelidi Raja:** Investigation.
**Colombel Juliette:** Investigation.
**Chissey Audrey:** Investigation.
**Ben Azzouz Amani:** Writing – review & editing, Investigation.
**Lefrère Bertrand:** Writing – review & editing, Investigation.
**POINSIGNON Léa:** Writing – original draft, Methodology, Investigation.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Amal Zerrad-Saadi reports financial support was provided by ANSES. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

## Acknowledgements

The authors would like to thank the platform “PICMO”, Faculty of Pharmacy of Paris, Paris Cité University, France, for access to a Leica
confocal microscope and in particular C. Izabelle and B. Saubame for TEM (Jeol) tissue observations. A special thanks for Sonja Boland for her invaluable advice and help with nanotoxicology. The following are acknowledged for providing placentas: Hópital Cochin-Port Royal, maternité privée des Diaconesses, Institut Mutualiste Montsouris and Hôpital privé d'Antony. The diagrams were created with BioRender.com <http://BioRender.com>.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2025.137713.

## Data availability

Data will be made available on request.

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