# Blastocyst exposure to plastic during mice in vitro fertilization impacts placental development

Jean-Philippe Wolf, Yasmine Boumerdassi, Claude Saint-Ruf, Franck

Kouakou, Florence Eustache, Daniel Vaiman, Francisco Miralles

► To cite this version:

Jean-Philippe Wolf, Yasmine Boumerdassi, Claude Saint-Ruf, Franck Kouakou, Florence Eustache, et al.. Blastocyst exposure to plastic during mice in vitro fertilization impacts placental development. Reproductive Toxicology, 2025, 132, pp.108856. 10.1016/j.reprotox.2025.108856. hal-05360444

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# Blastocyst exposure to plastic during mice in vitro fertilization impacts placental development

Claude Saint-Ruf, Yasmine Boumerdassi, Franck Kouakou, Jean-Philippe Wolf, Florence Eustache, Daniel Vaiman, Francisco Miralles*

Institut Cochin U1016 INSERM, UMR 8134 CNRS, Université de Paris, France

## ARTICLE INFO

Handling Editor: Dr. Bal-Price Anna

**Keywords:**

Assisted reproductive technologies
Placenta
Transcriptomics
Epigenetics
Deconvolution
MicroRNAs
Plasticware

## ABSTRACT

**Introduction:** Pregnancies from Assisted Reproductive Technologies (ARTs) are associated with a significant prevalence of maternal, neonatal and long-term adverse health issues. These anomalies are generally attributed to the *in vitro* manipulations involved in these procedures. Concerns have been raised on the quality of the culture media, however the potential influence of the chemical composition of the devices used in the *in vitro* fertilization (IVF) has been poorly analysed. By comparing the transcriptomes of placentas from mouse blastocysts obtained by IVF on plasticware, glassware and naturally conceived, we have previously established that plasticware profoundly impacts placental development.

**Methods:** Transcriptomics, transcriptome deconvolution analysis, Gene Set Enrichment Analysis.

**Results:** Plasticware alters placental gene expression mostly in the trophoblast compartment, and alters cell composition favouring Glycogen Cells. These modifications correlate with alterations of epigenetic mechanisms (alterations of imprinted genes, microRNAs expression, methylation alterations). Also, sex-stratified analysis reveals that these effects are more drastic in female than male placentas. The effect of glassware on the transcriptome and cellular composition of the placenta is milder, and in particular has lower impact on the imprinted gene or microRNAs expression.

**Conclusion:** In vitro culture in plasticware during IVF procedures sex-specifically alters gene expression and/or cell composition in the placenta, possibly through factors released by the plasticware having an action on epigenetic actors (imprinted genes, miRNAs and DNA methylation).

## 1. Introduction

Six percent of births in Europe results from Assisted Reproductive Technologies (ART) use [1,2]. Growing evidence suggests that individuals born using these technologies are at increased risk of intrauterine growth restriction, perinatal complications [3], and to develop metabolic or cardiovascular disease later [4-6]. Maternal adverse conditions are also associated with ART. These include preeclampsia (PE), HELLP syndrome and placenta accreta [7,8]. ART pregnancies are also associated with increased risk of rare imprinting disorders, mainly Beckwith-Wiedemann, Silver-Russell and Angelman syndromes [9-11].

ART includes a large variety of techniques, among these, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are commonly used. IVF involves ovarian stimulation, eggs retrieval, sperm fertilization, culture and embryo transfer into the uterus. These manipulations could affect normal embryonic development and be

responsible for the adverse outcomes associated with ART. Studies using mainly the mouse model, indicate that the placenta is particularly impacted by ART: foetal/placental ratios, placental Junctional Zone overgrowth, loss of genomic imprinting, and altered transcriptome have been observed [12-14]. One aspect that has been little explored is the potential release of harmful chemicals by the culture devices (i.e. poly-carbonate strippers, plastic culture dishes). Plastics could release molecules such as Bisphenol A (BPA), Di(2-ethylhexyl) phthalate (DEHP), Diethylstilbestrol (DES) known to impact placental development [15, 16]. For instance, BPA exposed placentas present loss of imprinting correlating with altered global expression and DNA methylation at ICRs [17,18].

In a recent study we compared the placental transcriptome of mouse conceived either on plasticware, glassware or in vivo [19]. We discovered that compared to the in vivo condition, plasticware induced substantial transcriptome modifications, while glassware induced more

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: francisco.miralles@inserm.fr (F. Miralles).</footnote>
restricted transcriptional alterations. In addition, we observed that these
transcriptional differences in the placenta depended upon the sex of the
concepti. However, bulk transcriptomic analysis of heterogeneous tis-
sues does not allow to determine if modifications in gene expression are
due to altered gene expression or the consequence of altered tissue
composition. This problem can be in part leveraged by the recently
developed techniques of Cell-type deconvolution analysis based on
available profiles of cell-type-specific marker genes [20]. The mouse
placenta is structured into three regions, the decidua (DC), the Junc-
tional Zone (JZ) and the labyrinth (La). The JZ is situated beneath the
DC and is composed of three cell types, the parietal trophoblast giant
cells (PTGC), the spongiotrophoblast (SpT) and the glycogen cells (GC).
The La contains sinusoidal trophoblast giant cells (STGC),
syncytiotrophoblast-I cells (SynTI) syncytiotrophoblast-II (SynTII), and
endothelial cells (EC). The cells of the JZ are involved in hormone and
metabolism functions, while the La cells ensure the gas and nutrients
exchange between the maternal and foetal blood. Given that
high-resolution single-cell/nuclei RNA-seq atlas of the mouse placenta
are now available [21,22], we have extended in the present article our
previous study by analysing how the transcriptional changes induced by
plastic relate to the different layers and cell types of the placenta. In
addition, we have stratified this study by sex. Finally, we present here
for the first-time evidences that gene expression alterations mediated by
factors released by the plasticware, could be epigenetically regulated.

## 2. Methods

### 2.1. Experimental design, oocytes collection, insemination and blastocysts culture

Here we further explored microarray data from our previously
published study [19]. In brief, B6/CBAF1 females and males were used
for this study. The female mice were divided in three experimental
groups. Groups one and two were used to obtain blastocysts grown in
vitro after IVF. The blastocysts from group one were grown in glassware
and those of group two in plasticware. The females of group three
(control group) were used for in vivo fertilization and subsequent
development until blastocyst stage. For females of groups one and two,
the oocytes were recovered after previous superovulation with 5 IU
(0.1 ml) human Chorionic Gonadotropin (hCG). The oocytes were
inseminated 15 h post-hCG with 10⁶ spermatozoa per ml. The fertilized
eggs (23 h post-hCG) were identified by the presence of two pronuclei,
and transferred to 30 µl drops of fresh medium (M16 supplemented with
7 mg/ml BSA) and covered with paraffin oil. Then they were grown
(either in glass or plastic) until the blastocyst stage under a controlled
temperature of 37°C and 5.5 % CO2. In the control group the females
were superovulated in identical conditions as the two other groups. The
blastocysts were collected after in vivo fertilization and development.
They were obtained by flushing the uterus at 3.5 dpc. For the three
groups we ensured to use blastocysts at the same stage of development
prior to transfer to pseudopregnant C57BL6/CBA females on pseudo-
pregnant at day 3.5 following standard procedures. On day 16.5 the
females were sacrificed and the fetus immediately recovered. After
removing the yolk sak the foetuses and the placentas were disposed
separately in Trizol reagent (Invitrogen Life Technology) snap-frozen
and stored at -80°C for further RNA extraction.

### 2.2. Preparation of RNA samples and microarray analysis

The RNAs were extracted from individual placentas using a kit
(Invitrogen Life Technology) and the foetuses sexed. Then 18 RNAs were
selected for each condition (Glass, Plastic and in vivo). These RNAs were
randomly taken to smoothen individual effects. The RNAs were pooled
by sex (three RNAs per pool) in order to generate for each condition 6
samples (3 male and 3 female pools). The microarray analysis of these
samples was performed on mouse Affymetrix 430.2.0 GeneChips

(Affymetrix Inc., Santa Clara, CA, USA) following manufacturer's protocol.

### 2.3. Wet experiments of RT qPCR

To validate the deconvolution analysis, we performed a gene tar-
geted analysis from 46 additional placentas (16 controls, 16 cultivated
in plastic, 14 on glass), from which mRNA were prepared and Reverse
transcription was performed using MMLV (Invitrogen) and following the
manufacturer's instructions. The tested genes with their primers were
Pcdh12 5' GAT-CCC-TGT-CTG-GTG-TCA-TTT-G 3', 5' TTA-GGA-CCC-
GTG-TCT-TGG-TCA 3', Pla2g4 5' AGG-TGG-AGT-TCC-TAC-TCG-AAG 3'
5' TGT-TCT-CGA-AGG-AGT-CTG-TCA 3', Car2 5' TCC-CAC-CAC-TGG-
GGA-TAC-AG 3' 5' CTC-TTG-GAC-GCA-GCT-TTA-TCA-TA 3', Ada 5'
GTC-ACC-CCT-GAT-GAC-GTT-GTG 3', 5' CAG-AAT-GGA-CCG-GAC-CTT-
GAT 3', cd83 5' AGC-TAC-AGA-GTC-AAC-TTT-CAG-GA 3' 5' GCT-TGT-
TCC-GTA-CCA-GGT-TTA-G 3', Pitm15' GAG-AAA-ATC-CAC-GGG-TTC-
ACT 3' 5' CTT-GCC-AGG-TGC-AGG-TAT-CTG 3', Tgfb3 5' GGA-CTT-
CGG-CCA-CAT-CAA-GAA 3' 5' TAG-GGG-ACG-TGG-GTC-ATC-AC 3'
and Gpc15' GGA-GAG-CGC-ACT-CCA-TGA-C 3' 5' CTC-AGC-ATA-TAG-
GTC-CCG-GAA 3', using Sdha as a reference gene to normalize the effi-
ciency of the reverse transcription/mRNA quality. The qPCR was per-
formed on a LightCycler 480 in 96 wells plates, using short cycles and an
annealing temperature fixed at 58°C for all the genes. The quality of the
qPCR was ascertained by analysis of the melting curve that revealed a
major peak for each system, after derivation.

### 2.4. Differential expression analysis

To analyse the expression data from the Affymetrix microarray we
used the Limma Bioconductor R 3.20 package for normalization, log
transformation and to identify statistically significant differentially
expressed genes (DEGs) and miRNAs (DEmiRs). The DEGs and DEmiRs
with statistical significance between the placentas issued of IVF blasto-
cyst on plastic (or glass) versus the in vivo fertilization were selected
using a 1.5 fold variation in either direction and a P value ≤ 0.05.

### 2.5. Gene ontology analysis

The ontology analysis was performed using the Gene Set Enrichment
Analysis (GSEA) tool from the Broad Institute (https://www.gsea-msig
db.org/gsea/index.jsp). Over Representation Analysis (ORA) was con-
ducted using the Webgestalt platform (https://www.webgestalt.org/).

### 2.6. Deconvolution analysis

The deconvolution analysis of the microarray data was carried out
with dtangle [23]. We fed dtangle with a single nuclei RNA-seq
(snRNA-seq) experiment from mice placenta [21]. The algorithm used
this reference to identify specific placenta cell markers and the pro-
portion of the different major cell types: trophoblasts (TC), endothelial
cells (EC), decidual stromal cells (DC), mesenchyme fetal cells (MeFe)
and blood cells (Bl).

### 2.7. miRNA-mRNA regulatory interactions

The miRWalk V2.0 database [24] was used to identify targets of the
miRNAs identified as DEmiRs. The lists of DEmiRs were submitted to the
Target Mining tool using miRBaseIDs as names.

### 2.8. Statistical tests

Statistical tests were based on ANOVA, followed by post-hoc tests, as
presented in each relevant figure, using GraphPad Prism10™ and the
Statist xl add-on on Excel. For the GSEA (Gene Set Enrichment Analysis)
experiments the FDR were calculated after running 1000 Monte-Carlo
permutations of the expression datasets (default value used by the algorithm) and a threshold of 0.05 was set for significance, which is considered highly stringent for this kind of analysis. The wet experiments were analysed using GraphPad Prism 10.4. Against the analysis was performed using two ways ANOVA with the variables sex and treatment, followed by post-hoc Dunnett test using the control samples as the reference. Statistical significance was fixed at $p < 0.05$.

# 3. Results

## 3.1. Increased trophoblasts proportions in E16.5 placentas derived from blastocysts grown on plastic

To more closely investigate the effects of IVF on the placenta, we applied the dtangle package to our microarray data. This software allows to estimate the cell composition from bulk. The reference was a single-nuclei RNA-seq (snRNA-seq) dataset of mouse placenta at E14.5. dtangle used the gene expression signatures of the annotated cell types (trophoblast, endothelial, decidual, fetal mesenchyme, and blood cells) from the snRNA-seq to estimate cell composition. The proportion of trophoblast cells was slightly but significantly increased in the placentas developed from blastocysts grown on plastic. Moreover, this was independent of the sex. Both male or female placentas presented an increased proportion of trophoblasts (Fig. 1A and B). There was nevertheless a minor effect of sex with systematically more trophoblasts in the male than in the female placentas.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Deconvolution analysis. (A) deconvolution of placental microarray data for cell-type specific composition. Each bar represents a pool of three placentas with absolute values of the proportional cell type composition. (B) Differences in trophoblasts cell composition among the placentas issued from blastocysts grown on plasticware, glassware or naturally conceived. Red and blue dots represent respectively pools of placentas from female or male concepti. Comparisons were made using a two-ways ANOVA (Culture effect, $p = 0.0014$; Sex effect, $p = 0.0174$, Interaction effect: not significant; post-hoc Tukey test significant for Plastic vs in vivo, $p = 0.0010$).</figcaption></figure>

## 3.2. DEGs are enriched for genes involved in placental development

We performed Gene Set Enrichment Analysis (GSEA) to identify biological processes associated with genes dysregulated in the E16.5 placentas derived from blastocysts grown on plastic compared to those developed in vivo.

In both female and male placentas GSEA identified many biological processes (GOBP) enriched in the placentas derived from embryos grown in plastic. Top 10 non-redundant GOBPs enriched in females and males are shown (Supplementary tables 1A&B). Many of these are common to female and male placentas including processes involved in ribosome biogenesis, regulation of lactation, mitochondrial gene expression, and epigenetic regulation of gene expression.

A common GOBP highly enriched in both male and female placentas corresponds to Embryonic placenta development (Males: NES=2.30, $p = 0.000$, FDR $q-val=9.2410^{-5}$; Females NES=2.48, $p = 0.000$, FDR $q-val=0.000$). Genes involved in placental development and significantly deregulated in both males and females include: *Gcm1*, *Syna*, *Plac1*, *Sox15*, *Cdx2*, and *Tgfb3* (Fig. 2). The same analysis comparing the placentas derived from blastocyst grown on glass with those developed in vivo shows also an enrichment of genes involved in placental development, on a milder mode (Males: NES=1.49, $p = 0.0069$, FDR $q-val=0.0069$; Females NES=1.85, $p = 0.000$, FDR $q-val=0.000$).

## 3.3. Trophoblast alterations of gene expression are the driver of the whole placenta modifications in gene expression due to plastic culture

To investigate whether plastic culture of IVF blastocysts altered gene expression in specific cell types of the placenta we compared the lists of DEGs in female and male placentas with the same snRNA-seq data used in the deconvolution analysis (using a different level of clusterization that allowed to discriminate the different types of trophoblasts: SpT, GC, STGC, SynTI and SynTII). Also, additional specific cell type gene expression was obtained from single-cell RNA-seq data available from the mouse cell atlas (https://bis.zju.edu.cn/MCA/) [25]. Concerning the female placentas, of the 1166 DEGs identified, 654 are annotated. Of these 108 genes were preferentially expressed in the JZ and 99 in the La. Only 8 DEGs were preferentially expressed in the DC. The remaining DEGs distribute as follows: 407 were either expressed uniformly in more than one cell type, or could not be assigned to specific cell type (not represented in the sc/sn-RNA-seq data), 21 DEGs were detected preferentially in blood immune cells (Bl), 8 DEGs in the foetal mesenchyme (FeMe), 3 DEGs in ECs. In the male placentas we detected 528 DEGs, 385 of which were annotated. Thirty-seven were preferentially expressed in the JZ, 39 in the La, 4 in the DC, 13 in immune cells of the Bl. Also, 13 DEGs correspond to FeMe and 2 were preferentially expressed in EC. The remaining 277 DEGs were either expressed in more than one cell type or cannot be assigned to any cell type. In sum, gene dysregulation in the female and male placentas developed from plastic-cultured blastocysts affects mostly trophoblasts cells located both in the JZ and La regions (Fig. 3).

Some of the DEGs identified in female and male placentas were specific of a particular trophoblast cell type. *Aldh1a3*, *Hsd17b7*, *Pla2g4e*, *Pla2g4d*, *Plac8*, *Pcdh12* specific markers off GC were significantly up-regulated in the placentas developed from plastic-cultured blastocysts. *Gcm1* a marker of SynTII, *Cds1* and *Syna* markers of SynTI and *Slc6a2*, marker of STGC were upregulated in the plastic condition (Fig. 4).

To attempt validating the microarray data, we used a different set of mRNAs from placentas originating from embryos cultivated either on plasticware, or on glassware, or directly transferred (control in vivo), as described in [19] and analysed a subset of genes. We also carried a qPCR analysis of the placentas using Xist that allowed to a posteriori identify male and female placentas. The results are presented as Fig. 5. Overall, the individual variability was higher than in the microarray, presumably due to the fact that individual placentas were used and not pools. Basically, it was clear that the effects due to glass/plastic culture were
Female placentas

Enrichment plot:
GOBP_EMBRYONIC_PLACENTA_DEVELOPMENT

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

<figure><img src="image_6.png" /></figure>

Male placentas

Enrichment plot:
GOBP_EMBRYONIC_PLACENTA_DEVELOPMENT

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Fig. 2.** Enriched expression of genes involved in placenta development. Data analysis using Gene Set Enrichment Analysis (GSEA) reveals that female and male placentas issued from blastocyst grown on plasticware had an enriched expression of genes involved in embryonic placenta development compared with those naturally conceived. The Normalized Enrichment Score represents the enrichment quality above random distributions. The FDR was calculated after 1000 Monte-Carlo permutations.
<figure><img src="image_9.png" /><figcaption>Cell proportions from dTangle deconvolution analysis (external circle = 🟩; internal cycle = 🟥)</figcaption></figure>

**Fig. 3.** Spatial distribution of differentially expressed genes (DEGs) in the placentas issued from blastocysts grown on plasticware. The circle plots summarizes the spatial expression of DEGs in female (654) and male (528) placentas. Genes spatial expression was obtained from single-nuclei RNA-seq data recently published [21] and from single-cell RNA-seq data available from the mouse cell atlas (https://bis.zju.edu.cn/MCA/)[25]. External circle = ♀, inner circle cycle = ♂.

higher on male placentas than on female placentas, which, while the trends was not different between sexes, makes that the statistical significance was attained only in males (not shown). The two markers of glycogen cells (GC), Pcdh12 and Pla2g4 are increased in glass and plastic. The same profile is observed for Gpc1 and Pitm1 (albeit not significant for this last gene), and a similar observation is seen for Tgfb3. Overall, we can say that individual tests confirm most of what the microarray pinpointed.

In addition to a reduced number of DEGs in male compared to female placentas, we observed differences in the expression of some specific cell markers. In male placentas there was a strong downregulation of *Abcb1b* and *Abcc4*, two specific markers of SynTII encoding ATP-dependent transporters that extrude physiological compounds and xenobiotics from cells. Also, *Sgk2*, *Slc22a3* and *Slc27a6* specific markers respectively of SynTI, SynTII and SpT were upregulated only in female placentas.

### 3.4. The gene expression signature of the placentas developed from blastocysts grown in vitro is indicative of placental overgrowth

Placental overgrowth (placentomegaly) is a common feature of IVF and cloning in mice. Placental hyperplasia is also observed in interspecies hybridization (Mus spretus (Ms)-Mus musculus (Mm) matings results in enlarged placentas [26–29]). Hyperplastic placentas show increased spongiotrophoblast expansion and increased differentiation of glycogen cells. Several studies have identified a set of common genes overexpressed in late-gestation hyperplastic placentas [30–32]. Many of these genes are also overexpressed in our dataset following in vitro culture. These include *Pitrm1*, *Ada*, *Car2*, *Cd83*, *Gpc1*, *Plac8*, *Mmp15*, *Adcy7*, *Sgk2*, *Dtx1*, *Anxa4*, *Actn1*, *Prl3c1*, and *Vnn1*. Most of these genes were preferentially expressed in the JZ, either in GCs and/or in the SpT.

### 3.5. Imprinted genes

A role of imprinted genes in the development of hyperplasia by the placentas derived from blastocysts grown in vitro has been largely documented. Thus, we performed GSEA to identify imprinted genes deregulated in both female and male placentas issued from blastocysts grown in plastic versus those issued from blastocysts developed in vivo. This analysis resulted in a considerable enrichment in both female and male placentas (Females: NES=1.69, p = 0.000, FDR q-val=0.000;

Males NES=1.96, p = 0.000, FDR q-val=0.000). In the female placentas the leading edge contained 51 upregulated genes and in males 50 upregulated genes. Of these, 37 genes were common (supplementary table 2). This include among others *Ascl2*, *Sfmbt2*, *Smoc1*, *Slc22a3*, *Slc22a18*, *Gpr1*, *Slc38a2*, *Slc38a1*, *Kcnq1ot1* and *Cdkn1c*. Imprinted genes with modified expression only in female placentas include *Slc38a4*, *Htra3*, *Xlr3b*, *Phlda2*, *Dlk1* and *Pdgfb*. Imprinted genes overexpressed only in male placentas include *Arid1b*, *Jade1*, *Ndn*, *Nnat*, and *Tnfrsf22*. Over representation analysis (ORA) of the list of the upregulated imprinted genes in females using the Mammalian Phenotype Ontology indicate an enrichment in terms such as abnormal trophoblast layer morphology (MP:0005031, FDR = 0.00047838), abnormal placenta labyrinth morphology (MP:0001716, FDR= 0.00088704) or abnormal placenta size (MP:0004258, FDR= 0.0047379). The genes related to these enrichments include: *Ascl2*, *Cdkn1c*, *Dlk1*, *Gab1*, *Kcnq1ot1*, *Pdgfb*, *Phlda2*, *Tle3* and *Rtl1*. The same ORA using the list of deregulated imprinted genes in males returns also the terms abnormal placenta morphology (MP:0001711, FDR = 0.075214) and abnormal trophoblast layer morphology (MP:0005031, FDR = 0.16608) but with less significant values. The genes related to these enrichments are *Ascl2*, *Cdkn1c*, *Gab1*, *Kcnq1ot1*, *Tle3*, and *Rtl1*. GSEA of the placentas grown on glassware failed to detect significant enrichment of imprinted genes either in female or male placentas.

### 3.6. Global placental DNA methylation

Mouse and human embryos generated by IVF have a significant loss of DNA methylation at ICRs [33]. A recent study has shown that loss of global placental DNA methylation is associated with time in culture [33]. Thus, embryos cultured to the blastocyst stage prior to implantation display increased loss of DNA methylation compared to those who were cultured only until morula stage. In this study the authors identified sets of DMRs in intronic or promoters in male and female E18.5 placentas from cultured blastocyst versus naturally conceived. We compared our lists of DEGs in male and female placentas with this dataset. We found that 50 DEGs in our female placentas have a detected DMR while only 10 DEGs present a DMR in males placentas issued of blastocyst cultured on plastic. In females most DMRs were Hypomethylated. Only 7 DEGs were associated with hypermethylated DMRs (*Plod3*, *St6gal1*, *Fbxw8*, *Rassf4*, *Dusp9*, *Apobec1*, *Smoc1*). The 10 DEGs in
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Expression of trophoblasts cell-type specific markers. Expression of cell-type specific markers as identified by the microarray analysis. Spongiotrophoblast cells (SpT), glycogen cells (GC), sinusoidal trophoblast giant cells (S-TGC), syncytiotrophoblast-I cells (SynTI) and syncytiotrophoblast-II (SynTII). Red and black dots represent respectively pools of placentas from female or male concepti. Values are displayed as mean±SEM. Statistical comparisons were made using a two-factors ANOVA followed by a Student-Neuman Keuls post-hoc T-test. ANOVA values are: for Pcdh12 (Condition p < 0.0001, for Sex p < 0.0001); for Pla2g4e (Condition p < 0.0001, for Sex p < 0.001); for Prl8a1 (Condition p = 0.012); for Syna (Condition p = 0.008); for Gcm1 (Condition p = 0.0044); for Slc6a2 (Condition p = 0.0006; for Sex p < 0.0079), (***indicates P < 0.0001; **indicates P < 0.001; *indicates P < 0.01).</figcaption></figure>

male placentas were all associated with hypomethylated DMRs (supplementary table 3A&B).

### 3.7. MicroRNAs (miRNAs) expression is significantly modified in placentas issued of blastocyst grown on plastic

The comparison of the transcriptomes of the placentas from blastocyst grown on plastic with those developed in vivo revealed a significant number of differentially expressed miRNAs (DEmiRs). In females we identified 98 DEmiRs, accounting for 8 % of the total DEGs. Of these 6 where upregulated and 92 downregulated in the placentas of the plastic

group. To investigate the potential impact of these DEmiRs on the transcriptome we used the miRWalk database. This resulted in the identification of 24 of the DEmiRs as possible regulators of 290 DEGs (Fig. 6, supplementary table 4). Most of these DEGs were upregulated (262). Functional enrichment analysis of the 290 DEGs targeted by the 24 DEmiRs reveals significant enrichment for BPs related to glycoprotein metabolic process (GO:0009100, FDR = 0.0005), Cell chemotaxis (GO:0060326, FDR = 0.0009), organic biosynthetic process (GO:0016053, FDR = 0.007) and acute inflammatory process (GO:0002526, FDR = 0.0043211). A subset of 80 DEGs was found to be enriched in terms such embryonic development (GO:0048568, FDR =
<figure><img src="image_11.png" /><figcaption>Fig. 5. qPCR validation of a selection of placental genes. Genes expression levels are presented using Sdha as a reference gene. The sexes were determined a posteriori from Xist expression levels measured by qRT-PCR. \*\*\* indicates P < 0.0001; \*\* indicates P < 0.001; \* indicates P < 0.01, and \* is p < 0.05.</figcaption></figure>

0.02), abnormal extraembryonic tissue morphology (MP:0002086, FDR = 0.001), perinatal lethality (MP:0002081, FDR=0.0001), or abnormal placenta morphology (MP:0001711, FDR= 0.003). Comparation with the snRNA-seq/scRNA-seq data show that the DEGs targeted by DEMiRs are preferentially expressed in trophoblast cells: 33 (GCs), 38 (SpT), 45 (SynTI/ SynTII), 27 (STGCs) and 8(JZP). For the remaining genes spatial information was not available or were expressed by many cell types including BL, DC and FeMe.

The analysis of the chromosomal distribution of the 98 DEMiRs identified in the placentas originated from blastocyst grown on plastic ware (supplementary table 4), shows that 24 of them localize in the C2MC (also known as Sfmbt2 miRNA cluster). These DEmiRs potentially target 163 DEGs, among them genes involved in trophoblast differentiation (Tfap2c, Gcm1, E2f3 or Tgfb3) and genes involved in trophoblasts metabolic functions (Prl4a1, Aldh1a3, Ada, Star, Pla2g4e,

Hsd17b7).

In the placentas of male concepti we identified only 38 DEmiRs, accounting for 7% of the total DEGs. Of these 5 were upregulated and the remaining downregulated in the placentas of the plastic group. Screening potential targets resulted in the identification of 13 DEmiRs as potential regulators of 128 DEGs (Fig. 7, supplementary table 5). Functional analysis identifies hormone metabolic process (GO:0042445, FDR = 0.002) as principal enriched GO term. As for the female placentas the DEGs targeted by DEmiRs are preferentially expressed in trophoblast cells: 23 (GCs/SpT), 9(SynTP1/ SynTP2), 9 (STGCs), 2(JZP) 3 (FeMe), 3 and 3(DC). For the remaining genes spatial information was not available or were expressed by many cell types. The analysis of the chromosomal distribution of the 38 DEmiRs identified in the male placentas originated from blastocyst grown on plastic ware (Fig. 7B), failed to identify any consistent cluster.
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** Differentially Expressed MicroRNAs (DEmiRs) and their targeted Differentially Expressed Genes (DEGS) in female placentas. Volcano plots showing DEmiRs (A) and DEGs (B) identified in placentas of female concepti issued of blastocysts grown on plastic ware. In the DEmiRs volcano plot the color scale represent the number of identified targets among the DEGS. In the DEGs volcano plot the blue dots show DEGs involved placenta development. The cutoff to identify differential expression was placed at an absolute fold change (FC) > 1.5 and P ≤ 0.05.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 7.** Differentially Expressed MicroRNAs (DEmiRs) and their targeted Differentially Expressed Genes (DEGS) in male placentas. Volcano plots showing DEmiRs (A) and DEGs (B) identified in placentas of male concepti issued of blastocysts grown on plastic ware. In the DEmiRs volcano plot the color scale represent the number of identified targets among the DEGS. In the DEGs volcano plot the blue dots show DEGs involved placenta development. The cutoff to identify differential expression was placed at an absolute fold change (FC) > 1.5 and P ≤ 0.05.</figcaption></figure>

Comparison between female and male placentas identified 19 common DEmiRs, but only 8 of these target DEGs (176 DEGs in females, 105 DEGs in males, 40 DEGs common to both sexes). The genes targeted by these DEmiRs include *Aldh1a3*, *Hsd17b7*, *Ada*, *Car2*, *Tph1*, *Sesn2*, *Gbe1*, *Fads2* and *Anxa4*. All these genes are preferentially expressed by the JZ cells either GC or SpT.

The comparison of placentas from blastocyst grown on glass with those developed in vivo reveals a small number of DEmiRs in both female (13 DEmiRs) and males (3 DEmiRs), none shared in common between female and male placentas. These DEmiRs target limited number of DEGs: in female placentas they are *Aopep*, *B4galnt2*, *Desi2*, *Pex19*, *Slc25a48*, *Slc6a2*, *Ubac2*, and in male placentas they are *Hsd17b7*, *Hsd11b1*, *Rsad2*, *Add2*, *Dolk*, *Ptgfrn*, *Slc18a2*, *Slc31a2*.

## 4. Discussion

Assisted reproductive technologies (ART) are not exempt of risks for

perinatal and offspring health. There is compelling evidence that even the blastocyst transfer may affect placentation and foetal growth [12, 26]. In a previous study we discovered that plasticware used in IVF can be harmful for the placenta in mice. The placentas from blastocysts grown in vitro on plastic or glass dishes showed substantial transcriptome alterations compared to those developed in vivo, especially on plastic [19]. Applying deconvolution analysis, we investigate how these transcriptomic alterations refer to the different regions and cell types of the placenta using dtangle, a dedicated software [23]. We took advantage of a recently published snRNA-seq atlas of mouse placental development who served as reference [21]. This snRNA-seq study focalised on mouse placenta development until E14.5 which differs from the E16.5 placentas analysed in our study, but constitutes a valid reference to identify specific cell markers because by E14.5 the mouse is structurally mature.

The placentas from blastocyst grown on plastic dishes have a significant increase in the proportion of trophoblast cells compared with
those developed in vivo. The placentas from blastocyst grown on glass dishes, show a smaller increase. In both cases, this effect was more significant in the placentas from females than male concepti. Consistently, GSEA reveals an enrichment in genes involved in trophoblast differentiation in those placentas derived from blastocyst grown on plastic. This result is significant in both sexes, but the enrichment is higher in female than in male placentas. Again, this enrichment was present (albeit milder) in placentas derived from blastocyst grown on glass. The snRNA-seq reference and the Mouse Cell Atlas provided a list of specific cell markers and preferentially expressed genes to contrast with the list of DEGs identified in our transcriptomic analysis. This analysis shows that from plastic-grown embryos, specific markers of almost all trophoblast cell types were increased. However, we noticed that among the highly specific cell markers the most significantly dysregulated in both female and males was Pcdh12, a marker of GC. Also, other genes such as Aldh1a3, Pla2g4e, Pla2g4d which are preferentially expressed in GC are increased in the placentas from IVF conducted on plasticware. We found DEGs shared among the GC and SpT, but no genes exclusively expressed in the SpT. These results are consistent with previously published studies showing that ART induce placental overgrowth [27,34–36]. In addition, a recent study has investigated the effect of different ARTs on eliciting placental anomalies [26]. The analysis, conducted at different developmental stages (E12.5, E14.5 and E18.5), revealed that naturally conceived blastocysts developed in vivo, without hormone stimulation prior to transfer have impaired early placentation and foetal growth, that normalize by term. In contrast, embryos cultured in vitro before transfer do not exhibit this compensation and display JZ placental overgrowth, reduced foetal weight, and reduced placental DNA methylation. Although the majority of the JZ comprised SpT in all ART groups, IVF placentas displayed a disproportionate increase in GCs at E18.5. In IVF placentas, 26–30 % of the JZ comprised GCs compared with only 13 % in controls.

ART such as IVF induce biallelic expression of maternally and paternally imprinted genes which are normally monoallelic expressed [12,26,34,36]. We revealed by GSEA a significant increase in the expression of imprinted genes in the placentas developed from IVF on plastic, especially in female placentas. Interestingly, glass culture did not significantly differ from in vivo conditions to this respect. Loss of DNA methylation at imprinting Control Regions (ICRs) has been reported in association with overexpression of imprinted genes in placentas from embryo culture [2]. Placental overgrowth was associated to two distinct windows of preimplantation embryo culture, one prior to morula stage and the other from morula to blastocyst [33]. Placental abnormalities and reduced ICR methylation were only associated with culture prior to morula. Extended culture to blastocyst stage induced additional placenta global DNA methylation modifications (mainly hypomethylation), with a higher loss of DNA methylation in female than in males placentas. An important role for imprinted genes located in the Kcnq10t1 in the development of the JZ has been reported [37,38]. In particular the genes *Ascl2*, *Cdkn1c*, *Igf2* and *Phlda2* could be involved in the regulation of the GCs lineage [39]. Thus, our observations are similar to those reported by the above cited studies, and suggest that embryo culture on plastic dishes has a more profound effect than culture on glass on the expression of the imprinted genes.

Another epigenetic mechanism that we have found altered in the placentas from IVF concerns the expression of miRNAs. Placentas from blastocysts grown on plastic dishes had a significant number of differentially expressed miRNAs (DEmiRs) compared with those cultured on glass dishes or the in vivo controls. The majority of these DEmiRs are downregulated with strong differences between female and male placentas. In particular we found a group of DEmiRs targeting DEGs (mainly up-regulated) involved in placenta development. These DEmiRs represent another possible mechanism contributing to the modifications observed in the development of the IVF placentas. Twenty-four DEmiRs identified in the female placentas belong to the paternally imprinted cluster C2MC (Sfmbt2 miRNA cluster). These are predicted to target 163

DEGs in placentas. Targeted deletion of a 53 kb region encompassing the entire miRNA cluster (without affecting Sfmbt2 expression) showed that conceptus reaching term have lower weight and placental defects [40]. The placenta is smaller and the SpT layer thinner. Also, loss of H3K27me3 imprinting in the *Sfmbt2* miRNA cluster causes placental hyperplasia in cloned mice [41]. Interestingly, our GSEA identified *Sfmbt2* as one of the imprinted genes with upregulated expression in both female and male placentas developed from blastocyst grown on plastic. Few studies have addressed the role of miRNAs in the developing placenta in the context of ARTs [42,43]. Nevertheless, owing the recognized role of miRNAs as effectors of placental mediated complications during pregnancy [44], our work highlights the need to more specifically address the question of their altered expression in the context of ART.

Altogether our study indicates that placentas from blastocysts grown on plastic dishes present, compared to those grown on glass or in vivo, transcriptomic modifications evoking a placental overgrowth of the JZ. This differential gene expression is correlated with loss of imprinting, global methylation, and microRNA modifications. Since the alterations were much milder in placentas that from blastocyst cultured on glassware, it is tempting to associate these differences to chemicals released by the plasticware.

Epigenetic modifications could be also related to the observed sex biased effect on the placenta development caused by IVF. Several studies, have reported that IVF has sex-specific effects that can affect embryonic and placental development, as well as postnatal metabolism and growth [45,46]. Our study confirms these results. The recently reported differences among female and male placentas from IVF procedures in the methylation status of the ICRs, and global DNA methylation, compared to those naturally conceived [33] suggests that these epigenetic mechanisms could provide a molecular basis for the IVF sex-biased effects. Gene expression and DNA methylation show sexual dimorphism [47–49], thus, these differences could be at the root of the sex-specific response to environmental exposures such as molecules released by plasticware.

### CRediT authorship contribution statement

**Saint-Ruf Claude:** Formal analysis, Data curation, Conceptualization. **Boumerdassi Yasmine:** Formal analysis, Data curation, Conceptualization. **Kouakou Franck:** Methodology, Investigation. **Wolf Jean-Philippe:** Conceptualization. **Eustache Florence:** Supervision, Methodology, Formal analysis. **Vaiman Daniel:** Writing – review & editing, Writing – original draft, Methodology, Data curation. **Miralles Francisco:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Formal analysis, Data curation, Conceptualization.

### Declaration of Competing Interest

No competing interests to declare.

### Acknowledgements

This paper is exclusively based upon published datasets, and no specific funding was obtained for this study.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.reprotox.2025.108856.

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