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. 2024 Nov 2;18(45):31244–31260. doi: 10.1021/acsnano.4c09734

Copper Oxide Nanoparticles Impair Mouse Preimplantation Embryonic Development through Disruption of Mitophagy-Mediated Metabolism

Yunyao Luo †,‡, Xi Zeng †,‡, Xue Dai †,‡, Yin Tian †,‡, Jie Li §, Qi Zhang †,‡, Qiang Dong †,‡, Lifeng Qin †,‡, Guoning Huang †,‡,*, Qi Gu ∥,*, Jianyu Wang §,*, Jingyu Li †,‡,*
PMCID: PMC11562798  PMID: 39487804

Abstract

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Copper oxide nanoparticles (CuONPs) have been widely applied, posing potential risks to human health. Although the toxicity of CuONPs on the liver and spleen has been reported, their effects on reproductive health remain unexplored. In this study, we investigate the effects of CuONPs on embryonic development and their potential mechanisms. Our results demonstrate that CuONPs exposure impairs mouse preimplantation embryonic development, particularly affecting the morula-to-blastocyst transition. Additionally, CuONPs were found to reduce the pluripotency of the inner cell mass (ICM) and mouse embryonic stem cells (mESCs). Mechanistically, CuONPs block autophagic flux and impair mitophagy, leading to the accumulation of damaged mitochondria. This mitochondrial dysfunction leads to reduced tricarboxylic acid (TCA) cycle activity and decreased α-ketoglutarate (α-KG) production. Insufficient α-KG induces the failure of DNA demethylation, reducing corresponding chromatin accessibility and consequently inhibiting ICM-specific genes expressions. Similar reduced development and inhibitions of pluripotency gene expression were observed in CuONPs-treated human blastocysts. Moreover, in women undergoing assisted reproductive technology (ART), a negative correlation was found between urinary Cu ion concentrations and clinical outcomes. Collectively, our study elucidates the mitophagy-mediated metabolic mechanisms of CuONPs embryotoxicity, improving our understanding of the potential reproductive toxicity associated with it.

Keywords: CuONPs, preimplantation embryonic development, mitophagy, α-KG, DNA demethylation

Copper oxide nanoparticles (CuONPs) have been widely used in various fields, such as food packaging, antibacterial products, and agriculture. Due to the physicochemical properties, CuONPs can easily penetrate physiological barriers, and induce associated toxic effects.1,2 Several studies have shown that CuONPs can induce pro-inflammatory response, trigger oxidative stress, leading to hepatotoxicity, neurotoxicity, and even reproductive toxicity.3 Embryos are more vulnerable to chemical and foreign particle invasion compared to other cell types.4 The quality of embryos is a critical factor influencing the success rate of assisted reproductive technology (ART). The embryotoxicity of CuONPs has been reported in earthworms and zebrafish;5,6 however, its effects on mammalian embryonic development remain unclear.

Recent studies suggest that the toxicity induced by CuONPs is correlated to autophagy dysfunction. For instance, Zhang et al. found that CuONPs perturb autophagy in endothelial cells, lung epithelial cells, and tumor cells.7−9 Autophagy, a lysosome-mediated pathway essential for cytoplasmic degradation, is essential for maintaining cellular homeostasis.10−12 Additionally, autophagy plays a significant role in preimplantation embryonic development.13,14 Oocyte-specific Atg5 (autophagy-related protein 5) knockout mice are unable to develop beyond the 4- and 8-cell stages when fertilized with Atg5-deficient sperm;15−17 similarly, knockdown of Atg3 led to developmental arrest at the 4-cell stage.18 Therefore, it is hypothesized that CuONPs may affect preimplantation embryonic development by influencing autophagy.

Besides autophagy, nanoparticles can affect cellular functions through epigenetic modifications. After fertilization, epigenetic modifications undergo drastic and precise changes that are essential for the normal development of preimplantation embryos. For instance, H3K4me3 in the promoter region experiences reorganization during early cleavage, which is associated with the zygotic genome activation.19 H3K9me3 has been shown to play a role in lineage differentiation. In the blastocysts, de novo established H3K9me3 suppresses the expression of lineage-specific genes, thus promoting lineage differentiation of the inner cell mass (ICM) and trophectoderm (TE).20−23 The genome-wide DNA methylation undergoes reprogramming during preimplantation embryonic development, exhibiting a dynamic balance between global demethylation and significant remethylation. The DNA methylation levels drop to the lowest points during the blastocyst stage.24,25 Epigenetic modifications are known to be influenced by environment and behavioral factors such as nutrition, smoking, and air pollution, which may affect preimplantation embryonic development.26,27 Previous studies have shown that TiO2 NPs and AuNPs can induce DNA hypermethylation, while AgNPs reduce H3 methylation in erythroid MEL cells.28−30 Additionally, CuONPs exposure significantly alters the expression of DNA methyltransferase in the RAW264.7 cell line.31 In Caenorhabditis elegans, CuONPs affect the expression of histone methyltransferase (MET-2) and H3K4me2 demethylase.32 However, the effects of CuONPs on preimplantation embryonic development, and whether they exert toxicity through epigenetic modifications, require further investigation.

The preimplantation embryonic development process experiences first lineage differentiation and the blastocyst consists of two cell types: the ICM, which forms the fetus, and the TE, which forms the placenta.33 Embryonic stem cells (ESCs) can be produced by the ICM cultured in vitro. Because ESCs are pluripotent and capable of self-renewal, they are frequently employed in developmental toxicity studies.34−36 In this study, we observed that exposure to CuONPs reduces the development of preimplantation embryos. Mechanistically, CuONPs impair mitochondrial function by disrupting mitophagy, which results in decreased α-ketoglutarate (α-KG) production within the tricarboxylic acid (TCA) cycle. The reduction in α-KG disrupts DNA demethylation processes, leading to an increased methylation level that silences the expression of essential developmental genes, thereby affecting the developmental potential of preimplantation embryos. Furthermore, our findings indicate that CuONPs exposure could adversely impact the clinical outcomes of women undergoing ART. Therefore, this study aims to provide insights into the reproductive toxicity of CuONPs exposure through metabolic mechanisms.

Results

CuONPs Exposure Affect the Preimplantation Embryonic Development in Mice

First, the characterization of CuONPs was confirmed through transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses, and the images showed that CuONPs exhibited spherical or cubical with 50 nm diameter on average (Figure 1A,B). Moreover, energy-dispersive spectroscopy (EDS) substantiated the presence of special peaks that correspond to copper (Cu) and oxygen (O) elements (Figure 1C). The averages of hydrodynamic radius and zeta potential in ultrapure water were 453.8 ± 9.0 nm and −10.39 ± 1.24 mV, respectively; in culture medium, they were 274.2 ± 7.6 nm and −19.70 ± 2.07 mV, respectively (Figure 1D).

Figure 1.

Figure 1

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Physical characteristics of CuONPs. (A) Representative TEM image of CuONPs. Scale bar: 50 nm. (B) Representative SEM image of CuONPs. (C) Chemical elemental composition of CuONPs. Scale bar: 5 μm. (D) Hydrodynamic diameters, PDI, and zeta potentials of CuONPs in water or medium were measured using dynamic light scattering (DLS).

To determine the effects of CuONPs on embryos, zygotes were incubated with CuONPs in a KSOM medium to monitor preimplantation embryonic development and count the birth rates after blastocyst transplantation (Figure 2A). First, we found that embryos exposed to 10 μg/mL CuONPs displayed a 50 percent reduction in blastocyst formation rates compared to the control group, with most embryos arresting at the morula stage (Figure 2B). This concentration was used in the subsequent experiments. A time-dependent manner was observed for the effects of CuONPs on embryonic development (Figure 2C). To further explore the effects of CuONPs on the preimplantation embryonic development, only those embryos that reached the blastocyst stage following CuONPs treatment were included in the analysis. The results demonstrated that embryonic development was delayed in the CuONPs group (Figure S1A), which was accompanied by a reduction in the blastocyst hatching rate (Figure S1B). Then, the blastocyst cells were counted using immunostaining against the ICM marker Sox2, and the TE marker Cdx2. Exposure to CuONPs significantly reduced the number of blastocyst cells, especially the ICM (Figure 2D). Then, morphologically normal blastocysts were transferred into pseudopregnant mice. CuONPs exposure at 10 μg/mL significantly decreased the birth rate of pups (Figure 2E). No significant differences were observed in the sex ratio or body weight of the pups (Figure S1C–E). These results suggest that CuONPs exposure adversely affects preimplantation embryonic development, especially impacting the morula-to-blastocyst transition.

Figure 2.

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CuONPs impair mouse preimplantation embryonic development. (A) Schematic diagram of the experimental procedures. (B) Representative images (left) and development rates (right) of mouse embryos at 2-cell, morula, and blastocyst stages after treatment with different concentrations of CuONPs (0, 1, 5, 10, 20 μg/mL) (n = 23, 24, 23, 24, 22, respectively). Scale bar: 100 μm. The 2-cell rate is calculated as the ratio of 2-cell embryos to 2PN embryos, and the morula or blastocyst rate represents the proportion of morula or blastocyst to 2-cell embryos, respectively. (C) Representative images (left) and blastocyst rate (right) post-treatment with 10 μg/mL CuONPs at different time durations (0, 24, 48, 72, 96 h) (n = 22, 23, 25, 26, 28, respectively). (D) Immunofluorescence images (left) show Cdx2 (TE marker, red), Sox2 (ICM marker, green), and nuclei (blue), along with statistical analysis (right) of control and CuONPs-treated blastocyst cells, and ICM ratio (ICM/TE) in control and CuONPs-treated (n = 16 in each group) blastocysts. Scale bar: 20 μm. (E) Representative images (left) and live birth rate (right) after blastocysts transfer in control and CuONPs-treated groups (n = 12 in each group). Data in (B), (C), and (D) represent means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

CuONPs Decrease the Pluripotency of ICM by Inhibiting Development Related Gene Expression

The observed reduction in the number of live births in the CuONPs group suggests its potential effects on the ICM. H2B-mcherry mRNA and CuONPs were coinjected into a blastomere of two-cell embryo (Figure 3A), and it was found that CuONPs inhibited the contribution of the injected blastomere to ICM formation (Figure 3B,C). Outgrowth incubation experiment showed that CuONPs exposure reduced the outgrowth rate and inhibited ICM differentiation, while the TE area was comparable between the control and CuONPs groups (Figure S2A). These findings indicate that CuONPs exposure may impair the pluripotency of the ICM. In addition, the effects of CuONPs on pluripotency were further evaluated using mESCs. CuONPs were observed to reduce clone formation and cell proliferation of mESCs in a dose-dependent manner, with significant reductions at 2 μg/mL (Figure S2B). Additionally, CuONPs significantly reduce the stemness of mESCs, as evidenced by a decrease in alkaline phosphatase (AKP) positive clones (Figure S2C). CuONPs-treated mESCs were then injected into nude mice, showing that CuONPs weaken the ability of mESC to form teratoma and differentiate into three germ layers (Figure 3D,E). To further evaluate pluripotency, CuONPs-treated mESCs were injected into the blastocyst and then transferred to the female body of the pseudo-pregnancy receptor. The results indicated that CuONPs significantly reduced the number of chimeric pups, as evidenced by their coat color (Figure 3F,G).

Figure 3.

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The effects of CuONPs exposure on pluripotency and gene expression patterns of the ICM. (A) Schematic diagram of 2-cell embryos microinjection. (B) Immunofluorescence images of Sox2 (ICM marker, green) and Cdx2 (TE marker, purple) in blastocyst. Scale bar: 20 μm. Merge 1&2: blastocyst cell development from the injected blastomere; Merge 2&3: ICM development from the injected blastomere (yellow); Merge 2&4: TE development from the injected blastomere (purplish red). (C) Statistical analysis of blastocyst cells and the ICM ratio (ICM/TE) in injected and uninjected blastomere in control (n = 25) and CuONPs (n = 27) groups. (D) Representative images of teratomas in the control and CuONPs groups. (E) H&E staining of a teratoma showing three germ layers. Scale bar: 30 μm. (F) Representative images of chimeric pups and (G) the efficiency of chimerism in control and CuONPs (2 μg/mL) groups. (H) Volcano plots display the DEGs (downregulated, blue, fold change < −1, P < 0.05; upregulated, red, fold change >1, P < 0.05; ICM-specific, orange; nonsense, gray) in CuONPs-treated ICM compared to the controls. (I) Gene-set enrichment analysis (GSEA) reveals the ICM-specific genes in CuONPs-treated ICM compared to the controls. (J) GO enrichment analysis was conducted on the 1302 down-regulated genes in CuONPs-treated ICM compared to the controls. (K) Expression of genes were revealed by real-time PCR in the control and CuONPs-treated ICM. Data in (C) and (K) are means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

RNA sequencing (RNA-seq) was performed on the ICM to explore the mechanism by which CuONPs affect preimplantation embryonic development. A total of 3015 differentially expressed genes (DEGs) (P <0.05, fold change >1.0), with 1302 down-regulated and 1713 up-regulated genes in the CuONPs-treated group (Figure 3H, S2D). A list of ICM-specific genes was identified using the data published in a previous study20 (Figure S2E). Strikingly, GSEA data showed that CuONPs decreased the ICM specific gene expressions, including several key pluripotency genes such as Sox2, Klf2, Klf4, and Esrrb (Figure 3H,I). Among the down-regulated genes, 17.82% (232/1302) were ICM-specific genes (Figure S2F). Gene Ontology (GO) data indicated that down-regulated genes related to CuONPs exposure were primarily related to three germ layer differentiation (Figure 3J). Real-time PCR further confirmed the down-regulated ICM-specific genes (Figure 3K). Collectively, these observations indicate that CuONPs may inhibit the expression of development related genes, thereby reducing the pluripotency of the ICM.

CuONPs Induce Accumulation of Damaged Mitochondria by Blocking Autophagic Flux

Previous studies have reported a link between autophagy and CuONPs exposure,9,37 leading us to investigate its role in the toxic effects of CuONPs on embryos (Figure 4A). Immunofluorescence (IF) staining and Western blot analysis showed increased LC3B and p62 levels, autophagy markers, in CuONPs-treated embryos (Figure 4B–D). The accumulation of autophagosomes and the increase in the number of autophagic substrates may be due to the induction of autophagy or impaired autophagic flux. To elucidate the mechanism behind LC3B accumulation, CuONPs-treated embryos were incubated with or without BafA1, an inhibitor of autophagosome-lysosome fusion. The levels of LC3B and p62 were comparable between the two groups (Figure 4B–D,F), indicating that CuONPs exposure impairs the autophagic flux rather than activating autophagy. Furthermore, a fluorescence-based method was used to visualize the autophagic activity in live embryos (Figure 4E). IF analysis showed significant accumulation of GFP-LC3 puncta in the CuONPs-treated embryos (Figure 4F), further indicating an impaired autophagic flux. Lysosomes play an important role in autophagic degradation. TEM images showed that CuONPs entered the cytoplasm, accumulated in lysosomes, and impaired their structure, displaying swelling, rupture, and loss of the complete membrane structure (Figure 4G). Subsequent assessments of lysosomal function showed that CuONPs disrupted the degradation ability of lysosomes, leading to an aberrant accumulation of lysosomes (Figure 4H,I). Altogether, these results indicate that CuONPs impair autophagic flux by destroying the lysosomal function in embryos.

Figure 4.

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CuONPs block autophagic flux by disrupting lysosomal function. (A) Schematic diagram of cellular autophagic flux. (B) Immunofluorescence images (left) and statistical analysis (right) of LC3B foci in 4-cell embryos (n = 17). Scale bar: 30 μm. (C) Immunofluorescence images (left) and statistical analysis (right) of p62 foci in 4-cell embryos (n = 15). Scale bar: 30 μm. (D) Western blotting images (left) and statistical analysis (right) of LC3B and p62 protein levels in 4-cell embryos (n = 50). (E) Schematic diagram of mRNA microinjection. (F) Representative images (left) and statistical analysis (right) showing the total fluorescence intensities of GFP-LC3 and control RFP in 4-cell embryos (n = 28). Scale bar: 100 μm. (G) TEM images of lysosomes in control and CuONPs-treated 4-cell embryos. Red arrow, the destroyed lysosomes. Scale bar: 5 μm. (H) Immunofluorescence images (left) and statistical analysis (right) of Lysosensor in control (n = 20) and CuONPs-treated (n = 23) 4-cell embryos. Scale bar: 100 μm. (I) Immunofluorescence images (left) and statistical analysis (right) of LysoTracker in control (n = 22) and CuONPs-treated (n = 23) 4-cell embryos. Scale bar: 100 μm. Data in (B), (C), (D), (F), (H), and (I) are means ± SD from at least three independent experiments. *P < 0.05, ** P < 0.01, **** P < 0.0001.

Previous studies have illustrated that CuONPs deposition can release toxic copper ions (Cu ions) into the surrounding media.37 Therefore, we warranted investigating whether the embryotoxicity of CuONPs is due to the release of Cu ions. The Cu ions chelator ammonium tetrathiomolybdate (TTM) was added in an embryo medium, showing TTM treatment partially mitigated the abnormal embryonic development induced by CuONPs (Figure S3A–D). This finding was further confirmed by the concentration of Cu ions (Figure S3E). Next, we cultured the embryos with CuCl2 (Cu ion donor); the exposed concentration was close to the Cu ion level in the CuONPs-treated culture medium. We observed that although Cu ions could reduce the blastocyst rate by 10% (Figure S3F), it has no effects on lysosomal functions and autophagic flux (Figure S3G–I). TTM supplementation could not rescue the abnormal lysosomal function and autophagic flux induced by CuONPs (Figure S3J–M). These observations indicate that the destruction of autophagy by CuONPs is not mediated by Cu ions release.

Mitophagy, a selective form of autophagy, degrades dysfunctional mitochondria to maintain cellular homeostasis and is essential for various physiological processes.38 Impairment of autophagic flux can also affect mitophagy. In this study, increased expressions of mitophagy markers such as Pink1, Parkin, and Tom20 in CuONPs-treated embryos suggest that the mitophagy flux is blocked (Figure 5A). CuONPs exposure results in extensive mitochondrial damage and accumulation in embryos (Figure 5B–D). High levels of colocalization of Lysotracker with Tom20 indicated decreased clearance of damaged mitochondria in lysosomes (Figure 5E). Furthermore, exposure to CuONPs impaired mitochondrial function, as evidenced by a weaker JC-1 fluorescence intensity (red/green ratio) in treated embryos (Figure 5F). Reactive oxygen species (ROS) were associated with mitochondrial function, and plays important roles in embryonic development. We found excessive ROS production in blastocysts treated with CuONPs (Figure S4A). We used the ROS inhibitor NAC, which effectively reduced ROS levels but did not improve embryonic development (Figure S4B–D), indicating that excessive ROS is not the primary cause of abnormal embryonic development induced by CuONPs. These findings suggest that the CuONPs induce mitochondrial dysfunction in blastocysts primarily through affecting mitophagy.

Figure 5.

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CuONPs impair mitophagy and result in mitochondrial dysfunction. (A) Western blot analysis depicting levels of mitophagy-related proteins in blastocysts. (B) TEM images of mitochondria (left) and statistical analysis (right) in control (n = 52) and CuONPs-treated (n = 50) blastocysts. Black arrow: normal mitochondria; White arrow: abnormal mitochondria. Scale bar: 500 nm. (C) Immunofluorescence images (left) and statistical analysis (right) of MitoTracker in control and CuONPs-treated (n = 14 in each group) blastocyst. Scale bar: 20 μm. (D) The mtDNA copy number in the control and CuONPs (n = 100 in each group) groups. mtDNA, mitochondrial DNA. (E) Immunofluorescence images of LysoTracker (red), Tom20 (green), and nuclei (blue) (left) and statistical analysis (right) of Tom20 fluorescence intensity in control and CuONPs-treated (n = 10 in each group) blastocyst. Scale bar: 20 μm. (F) Representative images of mitochondrial membrane potential (ΔΨm, staining with JC-1; red, high ΔΨm; green, low ΔΨm) (left), and statistical analysis (right) of JC-1 (red-to-green ratio) in control (n = 9) and CuONPs-treated (n = 11) blastocyst. Scale bar: 100 μm. Data in (B), (C), (D), (E), and (F) are means ± SD from at least three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001.

CuONPs Impair TCA Cycle and Reduce α-KG Production Inducing Embryonic Developmental Toxicity

Mitochondrial function is closely associated with the TCA cycle.39 In our study, RNA-seq analysis indicated that exposure to CuONPs inhibited the expression of TCA cycle genes, such as Aco2, Idh1, and Idh2 (Figure 6A,B), which was confirmed by real-time PCR (Figure S5A). IF and Western blotting analyses further demonstrated decreased expression of Aco2 and Idh1 in CuONPs-treated embryos (Figure 6C–E, S5B). To investigate the specific effects of CuONPs on the TCA cycle, targeted metabolomics analysis of energy metabolism in blastocysts was performed. Of note, we identified a significant reduction of α-ketoglutarate (α-KG) levels in the CuONPs-treated group (Figure 6F). This reduction in α-KG was confirmed in both CuONPs-treated blastocysts and mESCs (Figure 6G). To further explore whether the reduction of α-KG mediates the embryotoxicity effect of CuONPs, α-KG was supplemented in the culture medium. This supplementation significantly restored the CuONPs-induced reductions in blastocysts rate (Figure 6H), live birth rate (Figure 6I), ICM number (Figure S5C), AKP positive clone numbers (Figure S5D), and teratoma formation (Figure 6J,K). RNA-Seq analysis showed that 76.57% (997/1302) of the downregulated genes in the CuONPs group were rescued by α-KG supplementation (Figure 6L, S5E), which are involved in the development of the three germ layers (Figure S5F). Among these rescued genes, 19.26% were ICM-specific (Figure S5G), and this restoration of gene expression was validated by real-time PCR (Figure S5H). These findings indicate that CuONPs impair the TCA cycle activity, subsequently reducing α-KG production, which adversely affects preimplantation embryonic development.

Figure 6.

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CuONPs impair TCA cycle and reduce α-KG production. (A) Schematic diagram of the TCA cycle. Cs, Citrate Synthase; Aco, aconitase; Idh, isocitrate dehydrogenase. (B) Heatmap displaying the expression levels of TCA cycle-related genes in control and CuONPs-treated blastocyst from our RNA-Seq data. (C) Immunofluorescence images (left) and statistical analysis (right) of Aco2 in control and CuONPs-treated (n = 15 in each group) blastocyst. Scale bar: 20 μm. (D) Immunofluorescence images (left) and statistical analysis (right) of Idh1 in control (n = 14) and CuONPs-treated (n = 15) blastocyst. Scale bar: 20 μm. (E) Western blotting showing the protein levels of Aco2 and Idh1 in control and CuONPs-treated blastocyst. (F) Heatmap displaying the relative abundance of metabolites in control and CuONPs-treated blastocyst. For each group, n = 5 biological replicates. Each biological replicate collected 120 blastocysts. (G) The concentration of α-KG in blastocysts (left) and mESCs (right) (n = 120 in each group). (H) Representative images (left) and blastocyst rate (right) in control (n = 32), CuONPs (n = 54), and CuONPs+α-KG (n = 52) groups. Scale bar: 100 μm. (I) Representative images (left) and live birth rate (right) after blastocyst transplantation in CuONPs and CuONPs+α-KG (n = 12 in each group) groups. (J) Representative images of teratoma in CuONPs and CuONPs+α-KG groups. (K) H&E staining of teratoma showing three germ layers in CuONPs and CuONPs+α-KG groups. (L) Heatmap displaying downregulated genes in CuONPs-treated blastocyst (compared to the control) and rescued by α-KG supplementation. Data in (C), (D), (G) and (H) are means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Reduction of α-KG Induced by CuONPs Impairs DNA Demethylation and Subsequently Inhibits Gene Expression

α-KG acts as a substrate for DNA demethylase enzymes, facilitating the conversion of 5-methylated cytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Therefore, we hypothesized that CuONPs might disrupt preimplantation embryonic development by influencing the α-KG-mediated DNA demethylation process. As expected, CuONPs increased 5mC levels in both blastocyst and mESCs, an effect that could be reversed by α-KG supplementation (Figure 7A, S6A). Inhibition of DNA demethylation by Bob339 (inhibitor of demethylase enzyme) led to a decreased blastocyst rate (Figure S6B, S6C), suggesting that the failure of α-KG-mediated DNA demethylation may be the potential mechanism by which CuONPs affect preimplantation embryonic development. To verify this hypothesis, we used 5-azacytidine (5AZAC), a DNA methylation inhibitor, which successfully rescued abnormal embryonic development induced by CuONPs (Figure 7B, S6D).

Figure 7.

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Reduced α-KG result in DNA demethylation failure and inhibition of gene expression. (A) Immunofluorescence images (left) and statistical analysis (right) of 5mC in control (n = 15), CuONPs (n = 17) and CuONPs+α-KG (n = 15) groups. Scale bars: 25 μm. (B) Representative images (left) and the blastocyst rate (right) in the control (n = 24), CuONPs (n = 32), and CuONPs+5azac (n = 30) groups. Scale bars: 100 μm. (C) The averaged CpG DNA methylation levels of the control, CuONPs, and CuONPs+α-KG groups, along the gene bodies and 2 kb upstream of the transcription start sites (TSS) and 2 kb downstream of the transcription end sites (TES) of all RefSeq genes. (D) Volcano plots showing the DMRs in CuONPs compared to control embryos (Hypo, blue; Hyper, orange). (E) Venn plots showing the overlap between hypermethylated DMRs in CuONPs vs Control (gray) and hypomethylated DMRs in CuONPs+α-KG vs CuONPs (orange). (F) The percentage of ATAC-seq peaks assigned to promoters, introns, exons, and intergenic regions. (G) Metaplot showing the ATAC-seq enrichment at the promoters (Z-score normalized). (H) TFs-binding motif enrichment analysis of differential peaks in ATAC-seq between control and CuONPs-treated ICM. TFs, transcription factors. (I) Heatmap illustrating the relative gene expression, DNA methylation level, and chromatin accessibility around the TSS regions (z-score of the TPM values) of α-KG-rescued genes. (J) Genome browser view of RNA-seq, WGBS, and ATAC-seq signals at the Klf2 locus in the control, CuONPs, and CuONPs+α-KG groups. Data in (A) and (B) are means ± SD from at least three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.

We further analyzed the DNA methylation level of the ICM using whole-genome bisulfite sequencing (WGBS). In CuONPs-treated embryos, the overall DNA methylation was significantly increased, which could be reversed by α-KG supplementation (Figure 7C, S6E). We identified a total of 33,718 high-confidence differentially methylated regions (DMRs), comprising 20,548 hypermethylated and 13,170 hypomethylated DMRs in the CuONPs group (Figure 7D). Notably, 40.01% (8,252/20,548) hypermethylated DMRs induced by CuONPs could be restored by α-KG supplementation (Figure 7E). Given that DNA methylation can lead to chromatin inaccessibility and repress gene expression,40,41 Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) was performed in the ICM. Hierarchical clustering distinguished the control, CuONPs and CuONPs+α-KG groups, confirming the data quality (Figure S6F). CuONPs significantly reduced the chromatin accessibility, particularly in promoter regions, an effect restored by α-KG supplementation (Figure 7F, 7G). In addition, the IF analysis of RNA polymerase II (Pol II) displayed similar trends (Figure S6G). Of note, many decreased ATAC peaks in the CuONPs group harbored motifs of pluripotent transcription factors (TFs) such as Esrrb, Klf4, Sox2, and Nr5a2, highlighting the toxic effects of CuONPs on pluripotency of the ICM (Figure 7H). By integrating RNA-seq, WGBS and ATAC data, we found that the expression of down-regulated genes caused by CuONPs was negatively correlated with DNA methylation levels and is accompanied by reduced chromatin accessibility at promoters (Figure 7I), such as that of Klf2 (Figure 7J). These changes could be restored by α-KG supplementation (Figure 7I, 7J). These findings demonstrate that CuONPs impair α-KG-mediated DNA demethylation, resulting in reduced chromatin accessibility and subsequent inhibition of normal gene expression in the ICM.

Association between Cu Ions Concentration and ART Outcome

Previous studies, as well as our findings, indicated that CuONPs can increase Cu ions concentration. In addition, we demonstrated that the destruction of autophagy by CuONPs is not mediated by Cu ions release. In clinical practice, direct detection of CuONPs concentration is not feasible; therefore, we use Cu ion concentration as an indicator to infer CuONPs exposure in patients. To investigate the clinical embryotoxicity of CuONPs, we measured urinary Cu ions concentration in a cohort of 60 patients undergoing ART. The schematic diagram for patient recruitment in our study is shown in Figure 8A. Among these patients, five were removed due to undetectable Cu ions levels, and two were excluded due to loss of follow-up, leaving data from 53 patients available for analysis (Figure 8A). The baseline parameters are detailed in Supplementary Table S3. Urine Cu ion concentration was quantified using the ICP-MS method (Figure 8B). Patients were divided into low (<4.4 μg/L) and high (≥4.4 μg/L) groups based on the median urinary Cu ions concentration. The results showed that patients who had a positive clinical pregnancy after IVF had significantly lower Cu ion levels (Figure 8C, 8D). A Receiver Operating Characteristic (ROC) curve was created to assess the predictive value of Cu ions levels for pregnancy outcomes, with an Area Under the Curve (AUC) of 0.67 (P < 0.05) (Figure 8E). Furthermore, to determine whether the effects of CuONPs were conserved between humans and mice, we exposed human embryos to CuONPs from the zygote to blastocyst. Similar to the mouse embryos, we found that CuONPs reduced blastocyst formation (Figure 8F, 8G), and inhibited the expression of pluripotent genes in the ICM of human blastocysts (Figure 8H-8K).

Figure 8.

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The association between urinary cooper ions concentration and clinical pregnancy in IVF women. (A) Flow diagram of data extraction. (B) Schematic diagram of urine copper ions (Cu ions) detection in women. (C) and (D) Association between the concentration of Cu ions in urine and clinical pregnancy rate in IVF women (n = 53). (E) ROC curves for pregnancy rate and Cu ions concentration in urine. (F) Stacked bar plots showing fraction of human embryos at the different developmental stages in control (n = 21) and CuONPs (n = 20) groups. (G) Representative images of the development of control human embryos. Scale bar: 20 μm. (H) - (K) Representative images of digital PCR (left) and statistical analysis (right) of pluripotent genes (Sox2, Klf2, Klf4, Nanog) in control and CuONPs-treated human blastocyst. Data in (C), (D), (H), (I), (J), and (K) are means ± SD at least three independent experiments. *P < 0.05, **P < 0.01.

Discussion

Due to their physicochemical characteristics, CuONPs have been widely utilized in various fields. Their extensive use has sparked worries about their toxicity, leading to a number of studies evaluating their safety. The success of preimplantation embryonic development is crucial for achieving clinical pregnancy and ensuring offspring health. However, our understanding of the impacts and underlying mechanisms of CuONPs on preimplantation embryonic development in mammals is still limited. In this study, we demonstrate the toxic effects of CuONPs on mouse preimplantation embryonic development. Our findings indicate that CuONPs impair autophagic flux and damage mitophagy, resulting in mitochondrial dysfunction, thereby reducing α-KG production in the TCA cycle. Insufficient α-KG induces the failure of DNA demethylation, increasing corresponding heterochromatin and subsequently suppressing the expression of developmental related genes (Figure 9).

Figure 9.

Figure 9

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Schematic diagram of the mechanism of the CuONPs effects on preimplantation embryos. CuONPs enter the cellular lysosome, disrupt its function, and impair autophagic flux. Mitophagy, a critical pathway for maintaining mitochondrial homeostasis, is further impaired, resulting in the accumulation of damaged mitochondria. The mitochondrial dysfunction reduced tricarboxylic acid (TCA) cycle activity and decreased α-ketoglutarate (α-KG) production. Insufficient α-KG resulted in a failure of DNA demethylation, leading to DNA hypermethylation and consequent inhibition of developmental and pluripotency-related gene expression. The embryonic developmental toxicity of CuONPs results in a reduction in the number of ICM and subsequently leading to arrested embryonic development.

Previous studies have shown that CuONPs in the environment can be toxic to various organisms, including fish, algae, bacteria, fungi, and protozoa.42 CuONPs typically have an embryotoxicity concentration of 5–60 μg/mL in zebrafish,43,44 which is comparable to those found in other cultured cells.45−47 However, there is currently no information available on CuONPs’ impact on mammalian embryos. In this study, CuONPs at a concentration of 10 μg/mL reduced the blastocyst rate by half, a concentration that is only 1/5 to 1/25 of those observed in zebrafish embryos and cultured cells. Additionally, exposure to CuONPs at 80 μg/mL decreased the cell viability of human intestinal cells.2 Intriguingly, in this study, CuONPs at 2 μg/mL can reduce both cell viability and pluripotency of mESCs. These findings suggest that preimplantation mammalian embryos are more vulnerable to the harmful effects of exposure to CuONPs. In addition to impaired embryonic development, we are interested in determining the developmental potential of embryos that successfully reach the blastocyst stage after CuONPs exposure. Our results indicate that CuONPs exposure reduced the live birth rate after blastocyst transplantation. Furthermore, we found that the ICM formation was affected by CuONPs through two-cell embryo injection and an outgrowth experiment. The ICM of a blastocyst undergoes differentiation into embryonic lineages, which give rise to various of developing fetal tissues.48,49 Therefore, we speculate that the embryotoxicity of CuONPs may be mediated through effects on the ICM. Considering that mESCs and the ICM share characteristics including pluripotency regulatory networks,50 we used mESCs to investigate the toxicity of CuONPs on embryonic developmental. We discovered that exposure to CuONPs reduced the pluripotency of mESCs using teratoma and diploid chimera tests. In first fate determination, the inner cells differentiate into the ICM, and express specific pluripotency genes.51−53 Our RNA-seq data showed that CuONPs reduced the ICM-specific gene expressions, especially key pluripotency genes such as Sox2, Klf2, and Nanog. Similarly, in humans, the inhibition of pluripotency genes in a blastocyst was also observed.

Autophagy, the catabolic pathway mediated by lysosomes, has been preserved throughout evolution and is crucial for the recycling of macromolecules and organelles, playing crucial roles in preimplantation embryonic development. Satoshi et al. suggested that autophagy is triggered by fertilization and intensifies from the 2-cell to 4-cell stage.54 Inhibition of autophagy reduced protein synthesis and led to the developmental arrest at the 4-cell and 8-cell stages. Previous studies have suggested that CuONPs can cause lysosomal dysfunction and autophagic stress, leading to toxicological consequences.37 In this study, CuONPs accumulated in lysosomes and disrupted their function, leading to blocked autophagic flow and not impaired autophagy activation. Our results are consistent with previous studies indicating CuONPs treatment induce Cu ions released.37 Cu ions are one of the essential components for preserving homeostasis and can be harmful by oxidative stress and DNA damage. In this study, treatment with Cu ions has no effects on lysosomal function and autophagic flux, indicating the toxicity of CuONPs is not mediated by the release of Cu ions. Mitophagy is a type of selective autophagy that targets and breaks down old and damaged mitochondria,55,56 was further impaired due to the disrupted autophagic flux, resulting in the accumulation of damaged mitochondria in blastocyst. Mitochondria, the vital cellular energy factories, are essential for various cellular processes and preimplantation embryonic development. In CuONPs-treated blastocysts, impaired mitochondrial function was evidenced by the reduced membrane potential and increased ROS levels and TEM analysis. However, reducing ROS levels did not rescue development, indicating that ROS might result from other molecular alterations rather than being the direct cause of abnormal embryonic development induced by CuONPs. Therefore, these results indicate that CuONPs blocked autophagic flux and damaged mitophagy, leading to the mitochondrial dysfunction in the preimplantation embryos.

Cellular metabolism displays dynamic patterns during preimplantation embryonic development. Initially, embryos utilize pyruvate and lactate and later switch to glucose. In this study, the blocked mitophagy induced by CuONPs impaired mitochondrial function and further disrupted energy metabolism, displaying a poor TCA cycle in CuONPs-treated embryos. Among the metabolites of the TCA cycle, α-KG is particularly critical. Zhao et al. suggested that the blastocyst has an active TCA cycle and abundant α-KG. Insufficient α-KG can affect the formation and hatching of blastocysts,57 indicating that α-KG plays an important role in embryonic development. Consistent with the decreased TCA activity, we observed a significant reduction in α-KG levels in CuONPs-treated embryos. Notably, α-KG supplementation was able to alleviate the toxicity induced by CuONPs in preimplantation embryos and mouse embryonic stem cells (mESCs). These results suggest that reduced α-KG levels may be a crucial factor in the embryonic development toxicity associated with CuONPs. Changes in metabolites are linked to the epigenetic remodeling, which plays a significant role in mammalian preimplantation development. Our previous studies revealed that metabolism regulates preimplantation embryonic development in mammals through epigenetic modifications, such as NAD+ mediated erasure of H3K27ac, causing the embryo to exit the minor zygotic genome activation (ZGA) and maintain its continued development; lactic acid regulates major ZGA activation in mammalian embryos through H3K18lac.58,59 α-KG is recognized as an essential cofactor for DNA demethylases, which dynamically regulate DNA demethylation to control gene expression.60,61 Genome-wide DNA methylation plays a crucial role in the epigenetic regulation of mammalian embryonic development and experiences reprogramming, including demethylation and remethylation. Disruption of this dynamic balance can lead to embryonic development arrest.25 Using WGBS, we observed a global increase in DNA methylation in the ICM following the CuONPs exposure. CuONPs-induced downregulated genes are associated with increased DNA methylation and decreased chromatin accessibility at promoters, indicating that CuONPs suppress ICM-specific gene expression through promoting DNA methylation-dependent heterochromatinization. In addition, these regions of reduced chromatin accessibility are enriched with pluripotent TF motifs. Notably, these toxic effects of CuONPs can be reversed by α-KG supplementation. Thus, we speculate that reduced α-KG production caused by CuONPs disrupts gene expression patterns in the ICM by affecting DNA demethylation.

The long-term health effects of offspring exposed to CuONPs require careful consideration. Previous studies have reported toxic effects from nanoparticles exposure during pregnancy, including reduced birth rates and birth weight.62 In this study, CuONPs exposure has no obvious effect on the sex ratio, birth weight, or subsequent weight gain of pups (Figure S1C–E). CuONPs-exposed embryos were transferred to recipient female mice not exposed during pregnancy. We speculate that CuONPs may reduce ICM pluripotency and embryonic developmental potential, resulting in early miscarriage, thus sparing surviving offspring from further impact. These finding align with our previous study showing that ZnONPs exposure during preimplantation had no effect on offspring weight.63 Although CuONPs affected DNA demethylation in embryos, further research is required to determine whether these epigenetic modifications persist in the offspring.

Although CuONPs exposure can release Cu ions, our results indicated that the impaired autophagy by CuONPs is not mediated by Cu ions. In clinical patients, it is not practical to directly test the concentration of CuONPs; therefore, we employed Cu ion concentrations as an indicator to infer CuONP exposure. Our results demonstrate a negative correlation between Cu ion concentration and clinical pregnancy rates, indicating CuONPs exposure may adversely affect outcomes in ART. However, there is a limitation that whether the increase in Cu ion concentration is directly due to CuONPs exposure, warranting further research. To mitigate the potential adverse effects of nanoparticles on reproductive health, raising public awareness regarding nanoparticle exposure is crucial, and a washout period before the ART cycle is recommended for exposed individuals. Additionally, the incorporation of α-KG into the embryo culture medium may offer benefits for couples exposed to CuONPs.

Conclusions

Our findings indicate the embryotoxicity of CuONPs from the standpoint of autophagy influencing metabolism-mediated DNA methylation. This study provides an assessment of reproductive health hazards associated with CuONPs exposure.

Experimental Section

Ethics Statement

This study was reviewed and approved by the Chongqing Health Center for Women and Children (Approval No.: 2024010). All embryos used from patients in this research were not suitable for clinical use, and written consent was provided by the patients. Animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing Health Center for Women and Children (Approval No.: 2021013).

Characterization of CuONPs

The CuONPs (<50 nm particle size) were purchased from Sigma-Aldrich (544868). The particle morphology and agglomeration degree of CuONPs were observed using transmission electron microscopy (TEM) (JEOL JEM F200, Tokyo, Japan) and scanning electron microscopy (HITACHI SU8010). Additionally, the chemical elemental composition of CuONPs was analyzed using energy-dispersive spectroscopy (Oxford X-MAN 50) equipped with a field emission scanning electron microscope (FE-SEM/EDS). Furthermore, dynamic light scattering (DLS) measurements of the size and zeta potential of CuONPs in water and KSOM were performed using a Zetasizer Nano-ZS (Malvern, Nano, ZS90, UK) (MR-121-D; Merck).

Mice

In this study, all mice were purchased from Beijing SPF Biotechnology Co., Ltd. (ICR mice, 6–8 weeks old). These mice were kept in a specific pathogen-free (SPF) environment with controlled temperatures of 20–23 °C, humidity ranging from 50% to 70%, and a standard 12 h light/dark cycle. They were ensured free access to food and water.

Mice Embryo Collection

ICR female mice were first administered with 6.5 IU pregnant mare serum gonadotropin (PMSG) for 46–48 h, then received a 5 IU human chorionic gonadotropin (hCG) injection for superovulation, and then mated with ICR males. Embryos were collected 18 h later. The embryos were cultured in KSOM medium under mineral oil in an incubator at 37 °C with 5% CO2.

Culture of Mouse Embryonic Stem Cells (mESCs)

mESCs were cultured in KnockOut DMEM (Gibco, 10829018). The mESCs are cultured on culture dishes coated with gelatin (Solarbio, G0040) and seeded with feeder cells isolated from embryos of ICR mice at day 13.5 of gestation (mouse embryonic fibroblasts, MEF).

Treatment of Embryos and mESCs

CuONPs was stored at a concentration of 1 mg/mL as a stock solution and ultrasonically treated at 100 W for 20 min before dilution using an ultrasonic instrument (Science-IID, Ningbo Keke Biological Technology Co., Ltd., China). Subsequently, CuONPs were diluted to the desired concentration using the corresponding embryo or mESC culture medium. For inhibitor treatments, Bafilomycin A1 (S1413, Selleck), DM-αKG (349631, Sigma-Aldrich), 5-Azacytidine (HY-10586, MCE), and Bobcat339 (HY-111558, MCE) solutions were prepared in DMSO. NAC (N-acetylcysteine, A0737, Sigma-Aldrich) and TTM (3,3′,5,5′-tetramethylbenzidine, 323446, Sigma-Aldrich) solutions were prepared in water. These were cultured together with embryos or mESCs for subsequent experiments.

Time-Lapse Monitoring of Embryo Morphokinetics

The Vitrolife Embryoscope system was used for time-lapse imaging. T0 represents the moment when two protons fade (tPNf). The times t2, t3, t4, t5, t8, tM, and tBL respectively represent the time points of division into 2-cell, 3-cell, 4-cell, 5-cell and 8-cell embryos, morula stage, and blastocyst stage duration.

Blastocyst Transfer

Select ICR female mice in the estrus and mate them with vasectomized males. After vaginal plugs were confirmed, these females are considered pseudopregnant recipient mice. The pseudopregnant recipient mice at 2.5 days postcoitum are anesthetized with an intraperitoneal injection of 1.25% Avertin at a dose of 0.02 mL/g. Following disinfection with 75% alcohol, six blastocyst stage embryos are transferred into each uterine horn using a Pasteur pipet. Subsequently, the surgical site is sutured, and the mice are placed in an SPF environment to ensure their health and recovery.

Transmission Electron Microscopy (TEM)

Embryos were fixed with 2.5% glutaraldehyde solution at room temperature for 2 h. Then, they were stained with eosin (BC-DL-043, Bio-Channel) and embedded in agarose (1110GR100, BIOFROXX). The agarose gel containing the embryos was then cut into small pieces and placed in 2.5% glutaraldehyde at 4 °C overnight. After fixation with 1.0% osmium tetroxide, dehydration, and embedding, the embryo samples were mounted on slides with a layer of bone collagen membrane and stained with uranyl acetate and lead citrate. Finally, the ultrastructure of embryonic organelles was observed using transmission electron microscopy (TEM, JEOL JEM-1400Flash).

Alkaline Phosphatase (ALP) Staining

After fixing with ice-cold methanol for 30 min, the mESCs were washed with PBS, followed by staining with an alkaline phosphatase (AKP) staining solution (P0321M, Beyotime) for 60 min. Finally, the mESCs were observed under a microscope.

Teratoma Formation

Inject mESC (1 × 106) was subcutaneously injected into the thigh region of NOD-SCID mice. Three weeks later, we collected the tumors formed by mESC and fixed them with 4% paraformaldehyde. Subsequently, embed the tumors in paraffin to process them into sections and observe after staining with hematoxylin and eosin.

Production of Chimeras

Approximately 12–16 cells were injected into the blastocoel cavity. The blastocysts transferred into the uterus of pseudopregnant ICR mice 2.5 days after intercourse. The successful development of chimeras was verified by examining the coat color patterns of the pups.

In Vitro Transcription of LC3 mRNA and Microinjection

To prepare mRNA for microinjection, the pcDNA3.1-EGFP-LC3 and pcDNA3.1-RFP vectors (GenePharma, Shanghai, China) were linearized, extracted using the phenol chloroform method, and then precipitated with ethanol. Then, the mMESSAGE mMMACHINE kit (AM1344, Invitrogen) was used to transcribe linearized DNA in vitro. The obtained mRNA was polyadenylated using the mMESSAGE mMACHINE kit (AM1350, Invitrogen) and separated by a lithium chloride precipitation. Then, the mRNA was collected and injected into embryos.

Immunofluorescence Assay

The mouse embryos were fixed in 4% paraformaldehyde (FB002; Invitrogen) for 30 min. Afterward, the embryos were permeabilized in a 0.5% Triton X-100 solution for 15 min. Next, the embryos were blocked with 1% BSA (A1933, Sigma), and then were incubated with primary antibodies targeting LC3B (2775s, Cell Signaling Technology), p62 (D5L7G, Cell Signaling Technology), ACO2 (ab110321, Abcam), IDH1 (sc-515396, Santa Cruz), or 5mc (ab214727, Abcam) in 1% BSA. Next, the embryos were incubated with the second antibody, using Alexa Fluor 555-conjugated goat antirabbit IgG (A-11008, Invitrogen) or Alexa Fluor 488-conjugated goat antimouse IgG (A-11001, Invitrogen). Then, the embryos were stained with Hoechst 33342 (14533, Sigma-Aldrich) for 15 min. Finally, the embryos were placed on glass slides and examined under a laser scanning confocal microscope (TCS SP8; Leica, Wetzlar, Germany). LysoTracker Red DND-99 (L7528, Invitrogen) and LysoSensor Green DND-189 (L7535, Invitrogen) were used to detect lysosomal function, while MitoTracker Red CMXRos (M7512, Invitrogen), JC-1 (C2006, Beyotime), and DCFH (S0063, Beyotime) were applied to detect mitochondrial function. After incubation at 37 °C, the embryos were placed in an M2 medium-filled confocal dish for observation under a microscope. After the regions of interest (ROIs) were selected, ImageJ software (NIH, Bethesda, MD, USA) was utilized to calculate the fluorescence intensity.

Western Blotting

First, we used a RIPA buffer (containing PMSF) to lyse the embryos and then added a 5-fold loading buffer (G2075, Servicebio) to the embryo lysis buffer, and then boiled the mixture for 5 min. Subsequently, electrophoresis separation was performed and transferred onto a PVDF membrane (03010040001, Roche). After blocking, the membrane was incubated with primary antibodies targeting LC3B, p62, β-actin (4970, Cell Signaling Technology, China), Tom20 (ab186735, Abcam), Pink (Novus Biologicals, BC100-494, Novus Biologicals), Perkin (SC32282, Santa Cruz), ACO2, and IDH1 at 4 °C overnight. Then, the secondary antibodies (7076S, antimouse IgG, Cell Signaling Technology; 7074S, antirabbit IgG, Cell Signal Technology) were incubated. Finally, ECL Plus (G2020, Servicebio) was used for chemiluminescence, and the signal was captured using the Protein Simple imaging system.

Copper Ions Concentration Measurement and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Analysis

The concentration of copper ions released by mouse embryos in the culture medium was detected and quantified using the Quanti-Chrom Copper Assay Kit (BioAssay Systems, USA). Additionally, the concentration of copper ions in the urine of female patients was detected and quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (PerkinElmer NexIONTM 300X, Norwalk, Connecticut.

Collection of Inner Cell Mass

Incubating the blastocyst with antimouse rabbit serum diluted in medium. After washing PBS–PVA five times, the blastocysts were further incubated with guinea pig serum total complement (CH50) in medium (37 °C, 30 min). Then, a narrow glass pipet was used to separate the inner cell mass (ICM) from the lysed trophectoderm.

RT-qPCR

First, total mRNA was extracted from 50 ICM cells using an RNA Isolation Kit (Thermo Fisher). Subsequently, cDNA was synthesized by a reverse transcription kit (RR036A, Takara). Then, amplification was performed using TB Green Premix (RR420A, Takara). Each sample was measured three times. The data calculation was performed using the 2-ΔΔCt value after normalized by the Hrpt gene. All primers used in this section are listed in Supplementary Table S1.

Droplet Digital PCR (ddPCR) Assay

The ddPCR primers are listed in Supplementary Table S2, and the analysis was performed using the Qx200 ddPCR EvaGreen Supermix (1864033, Bio-Rad). The sample wells were filled with 20 μL of a mixture of reactants containing primers, cDNA samples, premix (2×), and H2O, and then the addition of 70 μL of QX200 droplets for EvaGreen to the oil well to generate oil (1864005, Bio Rad). Subsequently, the DG8 cartridge was placed into the QX200 droplet generator (1864002, Bio Rad) to generate droplets. The droplet lotion was transferred to the PCR plate for amplification. Finally, we used the QX200 droplet reader for automatic measurements and data analysis (QuantaSoft, v. 1.7.4.0917).

Metabolomics

Four replicate samples were collected for each group and sent to Applied Protein Technology in Shanghai, China for the targeted metabolomics analysis of energy metabolites. Blastocysts (n = 120) were added to a solvent containing methanol/acetonitrile and ultrapure water, and the samples were treated with ultrasounication. The supernatant was collected and stored after centrifugation. Subsequently, the sample was dissolved in a solvent composed of acetonitrile and water (1:1). After vigorous shaking, the supernatant was centrifuged and collected and analyzed using UHPLC (Agilent Technologies, 1290 Infinity LC) combined with QTRAP (AB Sciex 5500).

RNA-Sequencing (RNA-seq) and Data Processing

RNA-seq libraries were prepared according to the previous description.58 Each sample was prepared using three ICM cells from blastocysts, with two replicates per group. We prepared the library using Phusion Hot Start II High-Fidelity PCR Master Mix (F-565S, Thermo Fisher) and NEBNext Ultra II DNA Library Preparation Kit (E7805S, New England Biolabs). The library sequencing was performed on the NovaSeq 6000 platform (Illumina). After obtaining the raw data, we used Fastp v0.23.2 to remove low-quality reads. Then, the quality-controlled data (clean data) were aligned to the mouse genome (GRCm38) in paired-end mode using HISAT2 v2.1.0. Finally, the gene level was quantitatively analyzed using the featureCounts software to obtain the gene expression matrix. R package DESeq2 v1.30.0 was used for differential expression analysis. Genes with log fold change >1 and P-value <0.05 are considered differential expression genes (DEGs). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the default parameters of metescape v 3.5.

Identification of ICM-Specific Genes

The RNA-seq data for the ICM and TE were sourced from the GSE98150 data set. The method for processing the raw sequencing data is the same as we that used for our RNA-seq data.20 R package DESeq2 (version 1.30.0) was used to differential expression analysis. The differentially expressed genes were the absolute fold change value >1.5 and the P-value <0.05. ICM-specific genes were the genes upregulated in the ICM group.

ATAC-seq and Data Analysis

ICM (n = 20) was dissolved in a lysis buffer for 10 min on ice. Next, the sample was incubated with Tn5 transposase at 37 °C for 1 h. Afterward, TS buffer was added followed by incubation at room temperature for 5 min to stop the reaction. Subsequently, library amplification and purification were performed. Then, the library was sequenced. For data analysis, Fastp (version 0.23.2) was used to trim the original sequence containing adapters and low-quality bases. The BWA-MEM (version 0.7.17) command was used to align the filtered sequence with the mouse reference genome (mm10). Picard was used to remove duplicate items. Samtools was used to merge biological replication samples from different groups. Subsequently, the peaks were named and annotated using MACS2 (version 2.1.1) and Homer. Deeptools were used to visualize chromatin accessibility upstream and downstream of the transcription start site (TSS) by 1000 nucleotides.

Whole Genome Bisulfite Sequencing (WGBS) and Data Analysis

The ICMs (n = 30, three replicates per group) were washed with PBS and snap-frozen in liquid nitrogen. Samples were sent to Shenzhen BGI Genomics Co., Ltd. for WGBS library construction and sequencing. The construction of the WGBS library followed standard protocol 65. After obtaining the raw sequencing data, we used fastp v0.23.2 to filter low-quality bases and adapters. Then, the quality-controlled data (clean data) were aligned to the mouse genome (GRCm38) in paired-end mode using Bismark v2.5.1, followed by extracting methylation information from the aligned BAM files using the Bismark methylation extractor function. Subsequently, DNA methylation levels at cytosine sites were determined by the ratio of supporting methylation reads (C) to total reads (methylated and unmethylated). The methylation levels of genomic regions were calculated by calculating the average methylation levels of all CpG sites covered in these regions. “DNA methylation level” refers to the methylation level of CpG sites in a specific region. DNA methylation region analysis included only informative 5000 bp tiles; tiles with at least 10 CpG sites covered in each sample were included in the subsequent analyses. We utilized the methylKit v1.28.0 R package to detect differentially methylated regions (DMRs) with a difference of ≥25, q-value of ≤0.01, win.size = 5000, and step.size = 5000. The identified DMRs were annotated using Homer (version 1.28.0).

Collection of Human Triploid Embryos and Urine

Human triploid embryos and urine samples were donated by women using assisted reproductive technology at the Chongqing Health Center for Women and Health from February to March 2024. All samples were collected with written informed consent.

Statistical Analyses

Analysis was performed using GraphPad Prism 8.0. The difference between the two sets of continuous variables (mean ± standard deviation) was assessed using the student t test, and the categorical variables n (%) were assessed using the chi-square test. Binary logistic regression was used to investigate the relationship between each factor and clinical outcome. P-values <0.05 were considered statistically significant.

Acknowledgments

This work was supported by CQMU Program for Youth Innovation in Future Medicine (W0207), the Joint project of Chongqing Health Commission and Science and Technology Bureau (2023MSXM003), Natural Science Foundation of Chongqing (CSTC2021JCYJ-MSXMX0785, CSTB2022NSCQ-MSX0875), National Reserve Program for Excellent Young Scholars in the Health Field of Chongqing (HBRC2024015), and National Nature Science Foundation of China (No. 82201724).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c09734.

  • Data on embryonic development and offspring; CuONPs reduce the pluripotency of ICM and mESCs; release of Cu ions from CuONPs has no effects on autophagic flux; effects of CuONPs on ROS; effects of CuONPs on TCA cycle activity and α-KG production; insufficient α-KG induced by CuONPs result in a failure of DNA demethylation; summary of the primers used for real time PCR; summary of the primers used for digital PCR; baseline parameters of women undergoing IVF (PDF)

Author Contributions

# Y.L., X.Z., X.D., and Y.T. contributed equally to this work. J.L., G.H., and J.W. designed the study. Y.L., X.Z, X.D., and Y.T. performed most of the experiments and prepared figures. Jie.L., Q.Z., and Q.D. performed some of the experiments. X.D., X.Z. and L.Q. analyzed the data. Y.L., Q.G., and J.L. wrote the manuscript. All authors have read and approved the final version of this manuscript.

The authors declare no competing financial interest.

Notes

The research was reviewed and approved by the Medical Ethics Committee of the Chongqing Health Center for Women and Children (2021013, 2024010).

Notes

The manuscript does not contain any personal information in any form.

Supplementary Material

nn4c09734_si_001.pdf (1.3MB, pdf)

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