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Vitrification affects the post-implantation development of mouse embryos by inducing DNA damage and epigenetic modifications

Clinical Epigenetics volume 17, Article number: 20 (2025) Cite this article

Abstract

Vitrification is widely used in assisted reproductive technology (ART) for female infertility, but the long-term effect on the embryo of vitrification has not been comprehensively studied. The study aimed to investigate the effect of vitrification on long-term development of mouse embryos. The warmed embryos which were frozen at 8-cell stage were cultured in vitro until the blastocyst stage and were transferred into recipients. Immunofluorescence staining was performed to evaluate the reactive oxygen species (ROS) level, mitochondrial function, cell apoptosis, DNA damage and histone epigenetic modification in blastocysts. Transmission electron microscopy (TEM) analysis was performed to exam the mitochondrial ultrastructure in blastocysts. The related gene expression and transcriptome profiles were investigated by RT-qPCR and RNA-seq, respectively. Blastocyst and implantation frequencies were not significantly affected by vitrification. However, vitrification significantly reduced blastocyst cell number and the live pup frequency. Vitrification induced ROS accumulation, DNA damage, and apoptosis in mouse blastocysts. The homologous recombination (NHEJ) is the major DNA repair pathway for vitrified embryos. Vitrification elevated H3K4me2/3, H4K12ac, and H4K16ac levels and reduced m6A modification in blastocysts. Moreover, vitrification significantly altered transcriptome profiles of mice placentas and brains at embryonic day 18.5 (E18.5). Thus, vitrification exhibited a long-term effect on mouse embryo viability by increasing ROS levels, DNA damage, altering the epigenetic modifications and transcriptome profiles.

Introduction

Assisted reproductive technologies (ARTs) are widely used in the treatment of human infertility, and more than 10 million children are born by ART worldwide [1]. In a fresh embryo transfer cycle, the best-developing embryos will be selected for fresh embryo transfer (ET) at 2–6 days after the in vitro fertilization (IVF) of oocyte retrieval in healthy women [2]. However, many patients are suffering from polycystic ovary syndrome (PCOS) [3], high risk of ovarian hyperstimulation syndrome (OHSS) [4], hydrosalpinx [5] and high progesterone levels, and frozen-warmed embryo transfer (FET) is more appropriate for these women [6]. Frozen embryo transfer could provide sufficient time for the recovery of endometrial receptivity (ER) from the ovarian stimulation [7]. In addition, the "freeze-all" policy has been increasingly performed in pre-implantation genetic testing (PGT) in recent years [8, 9].

Although FET may be associated with better clinical outcomes in these certain patient populations, several side effects of FET, including an increased risk of large gestational age (LGA) [10,11,12,13], hypertensive disorders, and pre-eclampsia [14,15,16]. And whether embryo vitrification is related to these complications needs further research. More importantly, recent retrospective studies have demonstrated that live birth frequencies of FET were significantly lower than that of fresh ET when using fresh donor oocytes to achieve pregnancy [17, 18]. Moreover, donor oocytes can minimize the effects of the supraphysiologic endometrial environment that occurs in fresh ET cycles using autologous oocytes. Therefore, the freeze-warm process may decrease the long-term developmental potential of embryos.

Vitrification is widely used in human ART, and it is much more efficient than slow freezing due to the fact that vitrification can quickly freeze embryos to avoid the formation of larger ice crystals which is the major cause of embryo damage during freezing [19]. However, several studies have found increased reactive oxygen species (ROS) in vitrified embryos and oocytes. Moreover, several negative effects of vitrification on embryos and oocytes could be alleviated by supplementing the culture medium with antioxidants [20,21,22]. The accumulation of ROS underlies oxidative injury to the cells, including DNA damage, lipid peroxidation, and mitochondrial altered distribution and dysfunction [23]. Overproduction of ROS may induce cell apoptosis and even change the gene expression profile associated with cell differentiation of blastocysts [24]. More importantly, the mitochondrial apoptotic pathway may participate in the apoptosis of vitrified pig blastocysts [25]. Furthermore, vitrification may induce DNA damage, but no report about the role of DNA repair in the development of vitrified embryos was found [20].

Furthermore, vitrification was used for the preservation of embryos at pre-implantation stage which is an epigenetic sensitive period of embryos [26]. Genome-wide reprogramming occurs during this stage, like DNA (de)methylation, RNA (de)methylation, and histone modification, which could affect the implantation and fetus development [27, 28]. Vitrification is associated with decreased global DNA methylation and altered histone modification in oocytes and blastocysts [21, 29, 30]. In addition, some groups have reported that vitrification affects the gene expression and epigenetic modification in both the placenta and fetus [31,32,33,34].

In this study, using mouse model, we investigated the effect of vitrification on long-term development of mouse embryo and explored the underlying mechanisms such as oxidative stress responses, DNA damage, epigenetic modification, and transcriptome profiles of blastocysts, and E18.5 fetuses and placentas. Our results demonstrated that vitrification-induced oxidative stress and epigenetic changes contributed to the compromised implantation outcomes. Therefore, our results emphasized detrimental effects of vitrification on embryonic development and provided important evidence and references for guiding the improvement of this technology.

Methods

Zygote collection and embryo culture

Male and female ICR mice were purchased from Charles River Laboratory (Beijing, China) and housed in specific pathogen-free (SPF) conditions with a 12-h light/dark cycle under controlled temperature and humidity. Female mice (6–8 weeks old) were intraperitoneally injected with 10 IU pregnant mare serum gonadotrophin (PMSG) (Ningbo Sansheng Pharmaceutical Co, China), followed by human chorionic gonadotropin (hCG) (Ningbo Sansheng Pharmaceutical Co, China) at intervals of 48 h. Females were then mated with male ICR mice (8–10 weeks old). Approximately, 50 female mice were used for zygote collection in vitrified and control group, respectively. And 10 males were used for mating with donors. Mating was confirmed for the presence of a copulatory plug at the following morning. Zygotes were collected approximately 18 h post-hCG injection and washed in M2 medium (M7157, Sigma) containing 1 mg/ml hyaluronidase to remove cumulus cells [35]. After washing in M2 medium for 3 times, the zygotes with two pronuclei (2PN) were transferred to 20 ul drops of KSOMaa medium (IVL08, Caisson Labs) under paraffin oil (Sigma) and cultured in groups of 10 at 37 ℃ with 5% CO2 for further study. All experiments involving animals were reviewed and approved by Institutional Animal Care and Use Committee of First Hospital, Jilin University (Approval no. SYXK 2019–0012) and all experiments involving animals performed according to Guide for the Care and Use of Laboratory Animals.

Embryo vitrification and warming

The embryos at the 8-cell stage were divided into two groups. In group I, embryos were cultured to blastocysts stage without any treatment. In group II, embryos were vitrified and warmed, then cultured to the blastocyst stage. The cryotop, vitrification, and warming media were purchased from Kitazato (Tokyo, Japan). Ten embryos were placed in an equilibration solution for 8 min and then transferred to a vitrification solution for 30–60 s. Subsequently, embryos were placed on a cryotop and immediately plunged into liquid nitrogen. For warming, the cryotop removed from liquid nitrogen was quickly exposed to the warming solution for less than 1 min and then to the diluent solution for 3 min. Embryos were transferred to washing solution I for 5 min and then to washing solution II for 3 min. All the warming steps were performed at room temperature. The time interval between vitrification and warming was one month. The survival rate of vitrified embryos was evaluated at 2–4 h culture in KSOMaa. The embryo with at least 50% surviving blastomeres was considered as a survivor [36].

N-acetylcysteine treatment

The vitrified 8-cell stage embryos were warmed in cryopreservation solutions with or without 1 μM N-acetylcysteine (NAC) (Selleck, US). After warming, those survival embryos were cultured in KSOMaa with (Vit + NAC group) or without (Vit group) 1 μM NAC until the blastocyst stage (E4.5) in 37 ℃, 5% CO2 incubator.

Inhibition of DNA repair pathway

To investigate the effects of homologous recombination (HR) pathway on vitrified embryo development, embryos were treated with RAD51 inhibitor B02 (Selleck, US). Survival embryos of the vitrified group and control embryos were cultured in KSOMaa with three concentrations (0, 10, 50 uM) of B02 until the blastocyst stage (E4.5). To investigate the effects of NHEJ pathway on vitrified embryo development, survival embryos were treated with DNA-PK inhibitor KU57788 (Selleck, USA). Survival embryos of the vitrified group and control embryos were cultured in KSOMaa with three different concentrations (0, 1, 10 uM) of KU57788 until the blastocyst stage (E4.5).

ROS measurement

Embryos were incubated in KSOMaa with 10uM DCFH-DA (Beyotime Biotechnology, Jiangsu, China) at 37 ℃ for 30 min. Embryos were then mounted on the glass slides after three washing in PBS with 0.1% PVP. Images were captured by a fluorescent microscope (Nikon, Tokyo, Japan) or a confocal microscope (Zeiss LSM880) and analyzed using Image J software.

Mitochondrial activity measurement

Embryos were incubated in KSOMaa with 500 nM/L Mito Tracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ℃ for 30 min. After three washing in PBS with 0.1% PVP, embryos were then fixed in 4% paraformaldehyde (PFA) for 30 min and mounted on the glass slides. Images were captured by a fluorescent microscope (Nikon, Tokyo, Japan) or a confocal microscope (Zeiss LSM880) and analyzed using Image J software.

Mitochondrial membrane potential measurement

Mitochondrial membrane potential was detected using JC-1 staining kit (Beyotime Biotechnology, Jiangsu, China). Embryos were incubated in JC-1 staining working solution at 37 ℃ for 20 min according to the manufacturer’s instructions. After three washing in KSOMaa, embryos were then mounted on the glass slides and observed under a fluorescent microscope (Nikon, Tokyo, Japan). Detected fluorescence signals were analyzed using Image J software.

Mitochondrial permeability transition pore (mPTP) measurement

Mitochondrial permeability transition pore was detected using mitochondrial permeability transition pore assay kit (Beyotime Biotechnology, Jiangsu, China). Embryos were incubated in Calcein AM + CoCl2 staining working solution at 37 ℃ for 30 min according to the manufacturer’s instructions. After three washing in KSOMaa, embryos were placed in a drop of KSOMaa media on a dish and observed under a fluorescent microscope (Nikon, Tokyo, Japan). Detected fluorescence signals were analyzed using Image J software.

Real-time PCR for mitochondrial DNA (mtDNA) copy number

Total DNA was extracted from indicated embryos using AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). Real-time PCR (quantitative PCR, qPCR) was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences used were: for Nd1, Forward, 5′-CAGCCACCCGAGATTGAGCA-3′, reverse, 5′-TAGTAGCGACGGGCGGTGTG-3′; for 18 s rRNA, Forward, 5′-CTAACAACTATTATCTTCCTAGGAC-3′, reverse, 5′-GATGTATAAGTTGATCGTAACGG-3′. The Nd1/18 s rRNA ratio was used as an estimate for the relative mtDNA copy number.

Comet assay

The glass microscope slides were coated with 1% normal melting point (NMP) agarose. Embryos were transferred to 30uL 1% low-melting point agarose (LMP) and then added to the slides. Then the slides were incubated with freshly prepared lysing solution for 2 h at 4 ℃. Subsequently, the slides were placed into alkaline electrophoresis solution for 20 min at 4 ℃. Then the slides were transferred to a horizontal gel electrophoresis tank. Electrophoresis was run for 20 min at an electric field strength of E = 1 V/cm. After electrophoresis, the slides were stained with 5 μg/mL EB for 30 min in the dark. Finally, the slides were observed under a fluorescent microscope (Nikon, Tokyo, Japan).

TUNEL assay

Embryos were washed 3 times in PBS with 0.1% PVP and fixed in 4% PFA for 30 min. Following three washing in 0.1% PVP-PBS, embryos were permeabilized in PBS with 1% Triton X-100 for 20 min. TUNEL assay was performed using One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Jiangsu, China) for 1 h at 37 ℃ according to the instruction. DNA was stained by Hoechst 33,342 (10ug/mL). Images were captured by a fluorescent microscope (Nikon, Tokyo, Japan) or a confocal microscope (Zeiss LSM880) and analyzed using Image J software.

Immunofluorescent staining and blastomere counting

Immunofluorescent staining was performed as previously described[37]. Then embryos were incubated with anti-γH2A.X rabbit monoclonal antibody (1:200 dilution, 2577, Cell Signaling Technology), anti-H3K9me3 rabbit polyclonal antibody (1:200 dilution, ab8898, Abcam), anti-H3K4me3 rabbit polyclonal antibody (1:200 dilution, ab8580, Abcam), anti-H3K4me2 rabbit polyclonal antibody (1:200 dilution, ab7766, Abcam), anti-H4K12ac (1:200 dilution, ab46983, Abcam), anti-H4K16ac rabbit monoclonal antibody (1:200 dilution, ab109463, Abcam), anti-OCT4 mouse monoclonal antibody (1:200 dilution, sc-5279, Santa Cruz Biotechnology) or m6A rabbit polyclonal antibody (1:1000 dilution, 202003, Synaptic Systems) at 4 ℃ for overnight. Following three washing, embryos were incubated with a secondary antibody labeled with Alexa488 goat anti-rabbit (1:500 dilution, A11008, Invitrogen) or Alexa594 goat anti-rabbit (1:500 dilution, A11005, Invitrogen). DNA was stained by Hoechst 33342 (10ug/mL). Finally, images were captured by a fluorescent microscope (Nikon, Tokyo, Japan) or a confocal microscope (Zeiss LSM880) and analyzed using Image J software. The cell number of inner cell mass (ICM) or trophectoderm (TE) was obtained by counting the numbers of OCT4 positive nuclei (ICM) and negative nuclei (TE) in Hoechst 33342-stained blastocysts.

Electron microscopy

Briefly, the embryos were fixed in 2.5% glutaraldehyde for 2 h at 4 °C. After being rinsed in PBS, post-fixed was performed with 1% osmium tetroxide. Then samples were rinsed in PBS again. Then embryos were embedded in small blocks of 1.5% agar, dehydrated in graded series of ethanol, immersed in 1:1 epon–acetone, and finally embedded into pure epoxy resin. The ultrathin Sects. (60–70 nm) sectioned using a diamond knife were mounted on nickel and copper grids. After poststained with 1% aqueous uranyl acetate and 3% lead citrate, sections were analyzed by transmission electron microscopy (Hitachi H-7650).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from indicated embryos and reversed to cDNA using SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen, MA, USA). qPCR was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). All primers are listed in Table 1.

Table 1 Primer sequences of genes for quantitative RT-PCR
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Embryo transfer and post-implantation analysis

Pseudopregnant ICR females are generated through mating with vasectomized males on the day of collecting zygotes. A total of 47 pseudopregnant female mice were used, 22 in control group and 25 in vitrified group. And 15 vasectomized males were used for mating with recipients. Mating was confirmed by the presence of a copulatory plug the following morning. Ten expanded blastocysts were transferred into one uteri of pseudopregnant females on postcoital day 3.5. The fetal and placental tissues were collected and weighted at E18.5. The brain of the fetus and placenta were collected and stored at - 80 ℃.

RNA-seq and analysis

Total RNA was isolated from placentas and brains using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After quantification and qualification, the purified RNA libraries were sequenced using the Illumina NovaSeq6000 platform with 150 bp paired-end sequencing at the Berry Genomics Corporation (Beijing, China). Paired-end clean reads were aligned to the reference genome using Hisat2. Feature count was used to count the reads numbers mapped to each gene. Differential expression analysis was performed using the EdgeR package. The resulting p values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with | log2 (Fold Change) | > 1 and adjusted p value < 0.05 were considered as differentially expressed. GO, KEGG and GSEA analyses of the differentially expressed genes (DEGs) were performed using the clusterProfiler R package. The terms with adjusted p value < 0.05 were considered significantly enriched.

Western blot

Protein was extracted from 50–100 blastocysts using RIPA Lysis Buffer (Solarbio, Beijing, China). Protein was separated using 10% SDS-PAGE gel, and transferred to the Immobilon-p transfer membrane (Millipore, MA, USA). The membrane was blocked with 5% nonfat milk for 1 h at room temperature and then was incubated with anti-BAX antibody (1:500 dilution, sc-7480, Santa Cruz Biotechnology), anti-BCL2 antibody (1:500 dilution, ab59348, Abcam) for overnight at 4 °C. After 3 washings with PBST, the membrane was incubated with the corresponding secondary antibody for 1 h at room temperature. The membrane was washed three times with PBST after incubation.

Statistical analysis

Statistical analysis was performed by t-test for Binary comparisons and two-way ANOVA for multiple comparisons using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA), p < 0.05 was considered statistically significant. Fisher's exact test was used to compare pregnancy frequencies. The data were presented as the mean ± SEM, and each experiment was performed at least 3 times.

Results

Vitrification decreased the blastocyst cell number live pup frequency

Firstly, we investigated the effect of vitrification on in vitro development of mice embryos. Notably, although there was no significant difference in blastocyst frequency between two groups, the percentage of high-quality blastocysts (undergoing both dilatation and hatching) in vitrified group was lower than that in control group (vitrified vs control, 65.15 ± 3.52 vs 77.62 ± 2.84, p < 0.05) (Fig. 1A). We found that the total cell number of blastocysts derived from the vitrified embryos was fewer than that of the control group (vitrified vs control, 73.93 ± 2.38 vs 86.33 ± 2.70, p < 0.01) (Fig. 1B). And both the cell number of inner cell mass and trophectoderm were decreased. Furthermore, the effects of vitrification on the in vivo development of embryos in terms of fetuses and placentas were evaluated at embryonic days 7.5 and 18.5 (E7.5 and E18.5). Interestingly, we found that vitrification did not significantly affect the implantation frequency of the embryos at E7.5 (Fig. 1D). Moreover, a similar pregnancy frequency was found between the two groups (Fig. 1C). However, in line with the decreased cell number and low proportion of high-quality blastocyst, the live pup frequency of the vitrified group was lower than that of the control group (vitrified vs control, 28.00% ± 4.05% vs 41.67% ± 4.58%, p < 0.05) (Fig. 1E). Although studies in humans with FET have demonstrated that vitrification was associated with large gestational age (LGA) infants [12], we did not find abnormalities in fetuses in the vitrified group compared with the control group (Fig. 1F). While the weight of placenta in the vitrified group was significantly higher than that in the control group at E18.5 (Fig. 1G). And vitrification impaired the placental efficiency (the ratio of fetal weight and placental weight) which is a common method to evaluate placental function (Fig. 1H).

Fig. 1
figure 1

Vitrification impaired pre- and post-implantation development of mouse embryos. (A) The development of E4.5 blastocysts of control and vitrified group. The red arrows denote low-quality blastocysts (n = 4 for Con, n = 4 for Vit). Scale bar, 200 μm. (B) Cell number of E4.5 blastocysts (for total cell number: n = 21 for Con, n = 28 for Vit; for ICM or TE cell number: n = 14 for Con, n = 14 for Vit). (C) The pregnancy frequency at E18.5. (D) The developmental frequency of the decidua at E7.5 (n = 9 for Con, n = 10 for Vit). (E) The live pup frequency at E18.5 (n = 12 for Con, n = 15 for Vit). (F) Fetal weight at E18.5 (n = 33 for Con, n = 41 for Vit). (G) Placental weights at E18.5 (n = 33 for Con, n = 41 for Vit). (H) Placental efficiency at E18.5 (n = 33 for Con, n = 41 for Vit). *p < 0.05, **p < 0.01. Con represents control sample; Vit represents vitrified sample

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Vitrification-induced oxidative stress and mitochondria-mediated apoptosis in blastocysts

Next, we sought to examine the effect of vitrification on oxidative stress which might induce apoptosis in cells. As shown in Fig. 2A, vitrification increased ROS levels in blastocysts (p < 0.001), and mRNA levels of antioxidant genes (Sod1, Sod2, Gpx1) were significantly down-regulated in the vitrified group embryos (p < 0.001) (Fig. 2B). Mitochondrial dysfunction is one of the inducers of abnormal ROS generation [38]. The mitochondrial activity was measured by MitoTracker and a significant decrease in fluorescence density could be seen in blastocysts of the vitrified group (p < 0.05) (Fig. 2C). Furthermore, to check the effect of vitrification on mitochondria ultrastructure of embryos, we performed transmission electron microscopy (TEM) analysis on blastocysts from control and vitrified groups. The results showed mitochondrial swelling, vacuolation, and disappearance of mitochondrial crest in the vitrified blastocysts, which was not detected in the control group embryos (Fig. 2D). The blastocysts of the vitrified group exhibited markedly decreased mitochondrial membrane potential evaluated by JC-1 assay (p < 0.001) (Supplementary file 1: Figure S1A, B). However, there was no difference in the opening of mPTP or mtDNA copy number in the blastocysts between the two groups (Supplementary file 1: Figure S1C, D, E).

Fig. 2
figure 2

Vitrification-induced oxidative stress and mitochondria-mediated apoptosis. (A) Representative images and the fluorescence intensity of ROS in blastocysts (n = 14 for Con, n = 15 for Vit). Scale bar, 50 μm. (B) Relative mRNA abundance of the antioxidant genes Sod1, Sod2 and Gpx1. (C) Representative images and the fluorescence intensity of mitochondrial activity in blastocysts (n = 25 for Con, n = 37 for Vit). Scale bar, 50 μm. (D) Effects of vitrification on mitochondrial ultrastructure. White arrows indicate mitochondrial swelling, vacuolation, and disappearance of mitochondrial crest damage. Black arrow indicates autophagosome. Dotted arrow indicated knife markers. Scale bar, 1 μm. (E) Representative images and the fluorescence intensity of γH2A.X in blastocysts (n = 31 for Con, n = 22 for Vit). Scale bar, 50 μm. (F) Representative images and the percentage of TUNEL-positive cells (green) in blastocysts (n = 27 for Con, n = 24 for Vit). Scale bar, 50 μm. (G) Relative abundance of cell apoptosis related genes Bax and Bcl2 and the ratio of Bax/Bcl2. *p < 0.05, ***p < 0.001. Con represents control sample; Vit represents vitrified sample

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It was reported that the high level of ROS might induce DNA damage [38]. Unsurprisingly, vitrification significantly increased the intensity of γH2A.X of blastocysts (p < 0.05) (Fig. 2E). And the results of the comet assay also indicated that vitrification could induce DNA damage in blastocysts of the vitrified group (Supplementary file 1: Figure S2A, B). To further confirm the hypothesis that ROS might lead to the accumulation of DNA damage, we treated the vitrified embryos with 1 μM NAC in cryopreservation solutions and KSOMaa to inhibit the ROS generated during vitrification. Notably, the blastocysts treated with NAC showed a reduced ROS level (p < 0.001) (Fig. 3A, B). Subsequently, the IF results indicated that NAC treatment can also attenuate DNA damage induced by vitrification (p < 0.05) (Fig. 3D, E). Furthermore, NAC treatment increased the total cell number of vitrified blastocysts (vitrification vs control, 69.00 ± 3.57 vs 78.83 ± 2.47, p < 0.05) (Fig. 3C).

Fig. 3
figure 3

NAC supplement reduced the level of DNA damage induced by vitrification. (A) Representative images of ROS staining in blastocysts treated with 1 μM NAC. Scale bar, 100 μm. (B) The fluorescence intensity of ROS in blastocysts treated with 1 μM NAC (n = 10 for Vit, n = 13 for Vit + NAC). ***p < 0.001. (C) Total cell number of E4.5 blastocysts treated with 1 μM NAC (n = 8 for Vit, n = 12 for Vit + NAC). (D) Representative images of γH2A.X staining in blastocysts treated with 1 μM NAC. Scale bar, 50 μm. (E) The fluorescence intensity of γH2A.X in blastocysts treated with 1 μM NAC (n = 5 for Vit, n = 6 for Vit + NAC). *p < 0.05. Con represents control sample; Vit represents vitrified sample

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Additionally, the results of the TUNEL assay further demonstrated a higher cellular apoptosis rate in blastocysts derived from vitrified 8-cell embryos (p < 0.05) (Fig. 2F). Importantly, the expression levels of genes correlated with mitochondrial-dependent apoptosis such as the Bax and Bax/Bcl2 ratio were markedly increased by vitrification (p < 0.001) (Fig. 2G). Consistently, the western blot results also showed that BAX protein level was increased upon vitrification (Supplementary file 1: Figure S3). Taken together, these results suggest that apoptosis in embryos of the vitrified group may be linked to mitochondrial dysfunction.

Inhibition of NHEJ DNA repair pathway hampered the development of vitrified embryos

Given that vitrification could lead to increased intensity of γH2A.X, a marker of DNA double-strand breaks, the DNA repair may play an important role in the development of vitrified embryos. To investigate which DNA repair pathway mainly contributes to the vitrification-induced DNA damage, HR inhibitor B02 was used to treat vitrified and control embryos. We found that HR inhibition did not affect the development of control or vitrified embryos (Fig. 4A). By contrast, inhibition of NHEJ pathway with its inhibitor KU57788 did not affect the development of control embryos, but significantly decreased the blastocyst frequency of vitrified embryos (0 uM vs 10 uM, 91.54 ± 3.19 vs 59.60 ± 6.01, p < 0.01) (Fig. 4B). Furthermore, NHEJ inhibition with 10 uM KU57788 significantly increased the intensity of γH2A.X and decreased the total cell number of blastocysts (vitrification vs control, 49.40 ± 1.72 vs 60.92 ± 2.39, p < 0.05) of the vitrified group (Fig. 4C, D). Therefore, the NHEJ pathway might be the major pathway responsible for the DNA repair of vitrified embryos.

Fig. 4
figure 4

DNA repair inhibition was necessary for the development of vitrified embryos. (A) Blastocyst frequencies of embryos in control and vitrified group that were exposed to 0, 10, 50 μM B02. Scale bar, 200 μm. (B) Blastocyst frequencies of embryos in control and vitrified group that were exposed to 0, 1, 10 μM KU57788. Scale bar, 200 μm. (C) Representative images and the fluorescence intensity of γH2A.X in the blastocysts treated with 10 μM KU57788 (n = 23 for Con, n = 23 for Vit). Scale bar, 50 μm. (D) Total cell number of the blastocysts treated with 10 μM KU57788 (n = 13 for Con, n = 15 for Vit). *p < 0.05. Con represents control sample; Vit represents vitrified sample

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Vitrification altered histone methylation and acetylation of blastocysts

It is well known that the histone epigenetic modifications including the methylation and acetylation, are critical for normal embryo development [39]. Therefore, we used the immunofluorescence staining (IF) to measure the status of histone methylation (and H3K4me2, H3K4me3 and H3K9me3) and histone acetylation (H4K12ac and H4K16ac) which are reported to be important for the normal embryos development [40,41,42]. Surprisingly, no significant change of H3K9me3 between the vitrified group and normal group was found (Supplementary file 1: Figure S4). However, the level of H3K4me2 (p < 0.01) (Fig. 5A, E) and H3K4me3 (p < 0.05) of vitrification blastocysts was increased (Fig. 5B, F). Furthermore, we found that both H4K12ac and H4K16ac intensity of the vitrified blastocysts was stronger than that in the control blastocysts (p < 0.05) (Fig. 5C, D, G, H), which indicated that vitrification induced an elevation of H4K12ac and H4K16ac. We further analyzed the signal in ICM and TE respectively. The results showed that the intensity of H3K4me2/3 and H4K12ac in both ICM and TE was higher in vitrified group than that in control group (p < 0.05). While the intensity of H4K16ac in ICM was similar between two groups (Supplementary file 1: Figure S5).

Fig. 5
figure 5

Vitrification affected histone methylation and acetylation. (A-D) Representative images of blastocysts stained for H3K4me2, H3K4me3, H4K12ac and H4K16ac, respectively. (E–H) The fluorescence intensity of H3K4me2 (n = 30 for Con, n = 30 for Vit), H3K4me3 (n = 13 for Con, n = 14 for Vit), H4K12ac (n = 16 for Con, n = 21 for Vit) and H4K16ac (n = 19 for Con, n = 19 for Vit) in the blastocysts, respectively. Scale bar, 50 μm. *p < 0.05, **p < 0.01, *** p < 0.001. Con represents control sample; Vit represents vitrified sample

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Vitrification decreased RNA N6-methyladenosine of blastocysts

Recent studies indicated that RNA m6A modification plays an important role in mammalian embryo development [43,44,45]. Therefore, we sought to examine whether the vitrification induced alteration of RNA m6A modification in mouse vitrified embryos. The IF results indicated that m6A signal is mainly localized at the cytoplasm (Fig. 6A) and the m6A level was dramatically decreased in the vitrified group than in the control group (p < 0.05) (Fig. 6A, B). And the m6A signal was weaker in both ICM and TE in the vitrified group than those in control group (p < 0.05) (Supplementary file 1: Figure S6A). We also analyzed the expression of genes correlated to RNA m6A modification by qPCR. The results showed that mRNA levels of Mettl3 and Mettl14 (RNA m6A writer) were down-regulated in the vitrified embryos. Moreover, vitrification increased the mRNA levels of Ytdhc1 (RNA m6A reader) (Supplementary file 1: Figure S6B).

Fig. 6
figure 6

Vitrification decreased the level of RNA N6-methyladenosine. (A) Representative images of blastocysts stained for m6A. (B) The fluorescence intensity of m6A in blastocysts (n = 28 for Con, n = 22 for Vit). Scale bar, 50 μm. *p < 0.05. Con represents control sample; Vit represents vitrified sample

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Vitrification altered transcriptome profile of mouse and human blastocysts

To identify the effect of vitrification on the gene expression profiles of embryos, we downloaded two RNA-Seq data from SRA, SRP355713 of human control and vitrified blastocysts [46] and SRP511002 of mouse control and vitrified blastocysts [47]. There were 2514 DEGs (| log2 (Fold Change) | > 0.585 and p < 0.05) between mouse control and vitrified blastocysts, and 1381 DEGs between human control and vitrified blastocysts (Fig. 7A). The co-DEGs of mice and humans could be more valuable for studying the disturbances of vitrification on embryos, so we compared the DEGs between two species and found that there were totally 167 co-DEGs, including 61 co-up-regulated genes, 46 co-down-regulated genes and 60 specific genes (Fig. 7A, B). Next, we analyzed the role of co-DEGs by GO and KEGG. GO analysis indicated that the genes enriched in several pathways, including “Methyltransferase complex” (GO:0034708), “RNA modification” (GO:0009451), and “Regulation of lipid biosynthetic process” (GO:0046890) (p < 0.05) (Fig. 7C). And the KEGG data showed that “Purine metabolism” (mmu00230) and “Necroptosis” (mmu04217) were the significantly changed pathways (p < 0.05) (Fig. 7D). More interestingly, to further investigate the expression pattern of co-DEGs enriched in “Methyltransferase complex,” we found that the Kmt2a was up-regulated, and the Mettl14 was down-regulated in both human and mouse vitrified blastocysts (Fig. 7E, F). This result was consistent with the IF results of H3K4me2/3 and m6A.

Fig. 7
figure 7

Vitrification altered transcriptome profiles of human and mouse blastocysts. (A) Veen plot shows the overlap of DEGs between human and mouse. (B) Veen plot shows the overlap of co-DEGs between human and mouse. (C) Bubble plot of GO analysis of co-DEGs. (D) Bubble plot of KEGG analysis of co-DEGs. (E) Heatmap of co-DEGs enriched in “methyltransferase complex” in mouse samples. (F) Heatmap of co-DEGs enriched in “methyltransferase complex” in human samples

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Vitrification altered transcriptome profiles of E18.5 placenta

Placenta provides the nutrition for the fetus during pregnancy, and abnormality of placenta is detrimental to embryo development. Gene transcription profile which might be affected by vitrification could provide signs of abnormality [48, 49]. Then RNA sequencing was performed to investigate the alteration of transcriptome profile of placenta derived from the embryos with or without vitrification. Differential mRNA expression was determined when | log2 (Fold Change) | > 1 and p < 0.05. In the placentas, 714 genes were identified with different expression in control and vitrified groups, and there were 428 down-regulated genes and 286 up-regulated genes. The DEGs detected were shown in the volcano plot (Fig. 8A).

We further analyzed all differentially expressed genes via GO and KEGG analyses to obtain insights into their potential functional roles. Functional analysis revealed that the up-regulated DEGs were mainly associated with GO terms for pyroptosis (GO: 0070269), cytolysis (GO: 0019835), and granzyme-mediated apoptotic signaling pathway (GO: 0140507) (Fig. 8B), while the down-regulated DEGs were significantly enriched for cell adhesion molecule binding (GO: 0050839) (Fig. 8C). KEGG analysis revealed that the down-regulated DEGs are mainly involved in the cell adhesion molecules pathway (mmu04514) (Fig. 8C). GSEA analysis revealed that the DEGs are associated with paracetamol adme and blood clotting cascade (Supplementary file 1: Figure S7). Notably, pyroptosis is reported to be associated with pre-eclampsia in human [50,51,52]. To confirm the expression of DEGs correlated with pyroptosis, RT-qPCR was performed to verify the RNA-seq results. The expression patterns of all five genes matched those of high-throughput sequencing data (Fig. 8D).

Given that the imprinted genes play an important role in the normal development of placenta and fetus during pregnancy. RNA-Seq results indicated that Ins2 and Ctnna3 were considerably down-regulated in the vitrified placentas.

Fig. 8
figure 8

Vitrification altered transcriptome profiles of placenta at E18.5. (A) Volcano plot of DEGs. (B) Chordal graph of GO/KEGG analysis of up-regulated genes. (C) Chordal graph of GO/KEGG analysis of down-regulated genes. (D) Relative abundance of pyroptosis-related genes Gsdmc2, Gsdma3, Gzmd, Gzmc, and Gzmg. *p < 0.05, ***p < 0.001. Con represents control sample; Vit represents vitrified sample

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Vitrification altered transcriptome profiles of E18.5 fetus

ART may affect human embryonic brain development [53]. Whether vitrification has an impact on the fetal brain is still unknown. Thus, we applied RNA sequencing to address the effects of vitrification on gene expression of the fetal brain. In the brains, there were 434 genes showing differential expression between control and vitrified groups, among them, 304 genes were up-regulated and 130 genes were down-regulated. The DEGs detected were represented in the volcano plot (Fig. 9A). GO function and KEGG pathway enrichment analyses showed that the up-regulated DEGs were mainly associated with GO terms for serine hydrolase activity (GO: 0017171), while the down-regulated DEGs were enriched for regulation of viral life cycle (GO: 1,903,900) (Fig. 9B, C). GSEA analysis revealed that the DEGs are associated with NABA matrisome (Supplementary file 1: Figure S8). RNA-Seq results indicated that Trpm5 and Slc22a2 were up-regulated, whereas Cdh15 was down-regulated in vitrified brains.

Fig. 9
figure 9

Vitrification altered transcriptome profiles of fetus at E18.5. (A) Volcano plot of the differentially expressed genes. (B) Chordal graph of GO/KEGG analysis of up-regulated genes. (C) Chordal graph of GO/KEGG analysis of down-regulated genes

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Discussion

For reasons of PGT, OHSS avoidance, or desirability, the implementation of embryo cryopreservation in human IVF has dramatically increased in recent years [9]. Compared with slow freezing, vitrification is a simple protocol, that eliminates intracellular ice crystal formation, and provides a significantly higher survival rate [54]. But it was reported that vitrification is still associated with oxidative stress, DNA damage, and epigenetic changes in embryos [55]. Interestingly, we found that vitrification reduced the total cell number of blastocysts and decreased the live pup frequency measured at E18.5. We further detected the alteration of oxidative stress, DNA damage, epigenetic modifications in vitrified embryos and the transcriptome profiles of placentas and fetuses at E18.5.

ROS are by-products of embryo metabolism intracellularly. During the ART procedure, there are multiple external sources for the generation of excess ROS, including in vitro manipulation, exposure to oxygen, temperature, humidity, pH and light changes, culture media additives, and also embryo cryopreservation process [56]. During vitrification, the cryoprotectant agents are subjected to rapid cooling and warming, which may increase ROS in the embryos [57]. The physiological level of ROS is critical for embryogenesis, embryo development, and embryo implantation [58]. However, extensive ROS and the resulting oxidative stress may have detrimental effects on the quality of embryos and the outcomes of ART [59]. Abnormal levels of ROS are associated with DNA damage, lipid peroxidation, enzymatic function alteration, and cell apoptosis [60]. We found that vitrification significantly increased the ROS level and decreased the expression of antioxidant mRNA, which might be the underlying mechanism for the cryo-induced ROS induced injury to embryos. ROS are generated mainly as a by-product of mitochondrial respiration in aerobic cell and excessive ROS may induce mitochondrial genomic instability, respiratory dysfunction, and dynamics [61]. Vitrification significantly impaired the mitochondrial function and increased the Bax mRNA level. Furthermore, an increased level of DNA damage and apoptosis was observed in vitrified blastocysts. Notably, the NAC treatment could reduce the ROS level, attenuate DNA damage induced by vitrification and increase the total cell number of blastocysts. These results implied that the excessive ROS induced by vitrification might be the reason for higher mitochondrial-dependent apoptosis in embryos. Consistently, a recent research found that vitrified embryos a underwent dramatic change of mtDNA after warming, which showed a rapid decline of mtDNA after warming and recovery after 4-5 h in vitro culture. And these changes may be the response to cellular stress and DNA damage [62]. Therefore, the removal of reactive oxygen species and the restoration of mitochondrial function may be the key to the improvement of vitrification technology. Consistent with our results, previous studies also showed that the addition of antioxidants such as A3 (combination of acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic) and L-Glutathione improved the development of mouse embryos [21, 22]. Based on the results described above, we postulated that antioxidant supplementation may improve the development and viability of vitrified embryos.

The maintenance of genome stability and integrity is important for normal embryo development [63]. Our result was consistent with that vitrification contributes to increased γH2A.X signal, which is considered a maker of DNA double-strand breaks (DSBs) [20]. Both NHEJ and HR pathways are responsible for the DSBs repair [64]. NHEJ mainly occurs when no nearby sister chromatid and/or DSB occurs during interphase. And the DSBs that occurred during S phases are mainly repaired by HR, which uses the sister chromatid as a template [65]. It was reported that the HR pathway seems more important than the NHEJ pathway for DSB repair in early swine embryos [66, 67]. Interestingly, using chemical inhibitors for the two pathways, we found that inhibition of NHEJ, but not the HR pathway, significantly reduces the blastocyst frequency of embryos and increases γH2A.X level in the vitrified group. These results suggested that NHEJ might be more important than the HR pathway for the DSB repair induced by vitrification in mouse embryos. In line with this notion, it has been reported that the rapid phase of repair (such as the repairment of most ionizing radiation-induced DSBs) requires NHEJ genes rather than HR [68]. And simple DSBs are predominantly repaired by NHEJ [69, 70]. Thus, the repairmen of vitrification-induced DSBs might be a rapid phase.

Epigenetic reprogramming is related to totipotency, embryonic gene expression, and early lineage development in embryos [71]. A recent study indicated that H3K4me2 plays an important role in activating genes associated with the recovery following DNA damage [72]. Abnormal H3K4me2 modification induced by vitrification may be critical for DNA damage repair. Some studies demonstrated that vitrification alters the acetylation levels of histones (H3/H4) in oocytes [40,41,42]. We found that vitrification also increases both H4K12ac and H4K16ac levels in blastocysts. Previous studies indicated that oxidative stress in oocytes increased intracellular mRNA abundance of acetyltransferase 1 (Hat1) and increased H4K12ac [73, 74]. We hypothesized that the increased H4K12 and H4K16 levels might also be attributed to increased levels of ROS in vitrified embryos. Unlike the increased signal of H3K4me2 and H4K12ac/H4K16ac, the fluorescence intensity of H3K9me3 was not dramatically affected by vitrification. Besides histone modification, RNA m6A also plays a crucial role in embryo development [75]. It has been reported that Cycloleucine exposure increased the m6A level and resulted in severe DNA damage in embryos [76]. Therefore, the reduced RNA m6A signal may contribute to the elevated γH2A.X signal in vitrified embryos. More interestingly, the RNA-seq data showed that the Mettl14 was down-regulated in both mouse and human blastocysts, which indicated that vitrification may affect the embryo development of different species by regulating of RNA m6A level. Further studies are needed to explore the underlying mechanisms and long-term impact of vitrification-induced epigenetic modification changes on embryos.

Recent studies have shown that vitrification altered placental transcriptome in mice and rabbits [48, 49]. We hypothesized that the low live pup frequency of the vitrified group might be attributed to placental dysfunction. In our study, we found that vitrification altered the expression of several genes in mouse placentas. The up-regulated genes were associated with biological processes correlated with pyroptosis. And the qPCR results were consistent with the sequencing results. Therefore, the process of vitrification may induce cell pyroptosis in the placenta. Some studies have reported that pyroptosis is related to pre-eclampsia in humans. Caspase-1, GSDMD, IL-1β, and IL-18 are highly expressed in the placenta of early onset pre-eclampsia. Importantly, several studies have found a high risk of pre-eclampsia in women receiving FET [11, 12]. Consistent with this result, we also observed many DEGs in the placenta of the vitrified group, such as Itgb2, Cd44, and Kcnq, which are influenced by pre-eclampsia [77,78,79,80]. Thus, vitrification may induce placental pyroptosis, which increases the risk for pre-eclampsia and adverse pregnancy outcomes.

In summary, our work revealed that vitrification increased ROS accumulation which may lead to mitochondria-dependent apoptosis and vitrification induces DNA damage which is mainly repaired by the NHEJ pathway. Moreover, the epigenetic modifications such as the H3K4me2/3, H4K12ac/H4K16ac, and m6A were also altered by vitrification in blastocysts which might partially lead to a lower live pup frequency, and transcription profile alterations of placental and fetus. Our study emphasized some safety issues of fresh autologous oocyte ET vitrification technology. The situation that embryo vitrification has been widely used in ART warrants further in-depth investigation into the effects of vitrification on the long-term development of fetuses and optimization of vitrification methods for ART in human.

Data availability statement

The datasets presented in this study can be found in the online repository. Sequencing data were deposited into Genome Sequence Archive (GSA) database with accession numbers CRA012038.

Competing interest

The authors declare no competing interests.

Ethics statement

All experiments involving animals were reviewed and approved by Institutional Animal Care and Use Committee of First Hospital, Jilin University (Approval no. SYXK 2019–0012), and all experiments involving animals performed according to Guide for the Care and Use of Laboratory Animals.

References

  1. Pinborg A, Wennerholm U-B, Bergh C. Long-term outcomes for children conceived by assisted reproductive technology. Fertil Steril. 2023;120:449–56.

    Article  PubMed  Google Scholar 

  2. Glujovsky D, Quinteiro Retamar AM, Alvarez Sedo CR, Ciapponi A, Cornelisse S, Blake D. Cleavage-stage versus blastocyst-stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev. 2022;5:CD002118.

  3. Wei D, Shi Y, Li J, Wang Z, Zhang L, Sun Y, et al. Effect of pretreatment with oral contraceptives and progestins on IVF outcomes in women with polycystic ovary syndrome. Hum Reprod. 2017;32:354–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles. Hum Reprod Update. 2013;19:433–57.

    Article  CAS  PubMed  Google Scholar 

  5. Hammadieh N, Coomarasamy A, Ola B, Papaioannou S, Afnan M, Sharif K. Ultrasound-guided hydrosalpinx aspiration during oocyte collection improves pregnancy outcome in IVF: a randomized controlled trial. Hum Reprod. 2008;23:1113–7.

    Article  PubMed  Google Scholar 

  6. Santos-Ribeiro S, Polyzos NP, Haentjens P, Smitz J, Camus M, Tournaye H, et al. Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration. Hum Reprod. 2014;29:1698–705.

    Article  CAS  PubMed  Google Scholar 

  7. Blockeel C, Campbell A, Coticchio G, Esler J, Garcia-Velasco JA, Santulli P, et al. Should we still perform fresh embryo transfers in ART? Hum Reprod. 2019;34:2319–29.

    Article  CAS  PubMed  Google Scholar 

  8. Papaleo E, Pagliardini L, Vanni VS, Delprato D, Rubino P, Candiani M, et al. A direct healthcare cost analysis of the cryopreserved versus fresh transfer policy at the blastocyst stage. Reprod Biomed Online. 2017;34:19–26.

    Article  PubMed  Google Scholar 

  9. Bosch E, De Vos M, Humaidan P. The future of cryopreservation in assisted reproductive technologies. Front Endocrinol. 2020;11:67.

    Article  Google Scholar 

  10. Pelkonen S, Koivunen R, Gissler M, Nuojua-Huttunen S, Suikkari A-M, Hydén-Granskog C, et al. Perinatal outcome of children born after frozen and fresh embryo transfer: the Finnish cohort study 1995–2006. Hum Reprod. 2010;25:914–23.

    Article  CAS  PubMed  Google Scholar 

  11. Sazonova A, Källen K, Thurin-Kjellberg A, Wennerholm U-B, Bergh C. Obstetric outcome in singletons after in vitro fertilization with cryopreserved/thawed embryos. Hum Reprod. 2012;27:1343–50.

    Article  PubMed  Google Scholar 

  12. Maheshwari A, Raja EA, Bhattacharya S. Obstetric and perinatal outcomes after either fresh or thawed frozen embryo transfer: an analysis of 112,432 singleton pregnancies recorded in the Human Fertilisation and Embryology Authority anonymized dataset. Fertil Steril. 2016;106:1703–8.

    Article  PubMed  Google Scholar 

  13. Ginström Ernstad E, Wennerholm U-B, Khatibi A, Petzold M, Bergh C. Neonatal and maternal outcome after frozen embryo transfer: Increased risks in programmed cycles. Am J Obstet Gynecol. 2019;221:126.e1-126.e18.

    Article  PubMed  Google Scholar 

  14. Maheshwari A, Pandey S, Amalraj Raja E, Shetty A, Hamilton M, Bhattacharya S. Is frozen embryo transfer better for mothers and babies? Can cumulative meta-analysis provide a definitive answer? Hum Reprod Update. 2018;24:35–58.

    Article  PubMed  Google Scholar 

  15. Wei D, Liu J-Y, Sun Y, Shi Y, Zhang B, Liu J-Q, et al. Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial. Lancet. 2019;393:1310–8.

    Article  PubMed  Google Scholar 

  16. Roque M, Haahr T, Geber S, Esteves SC, Humaidan P. Fresh versus elective frozen embryo transfer in IVF/ICSI cycles: a systematic review and meta-analysis of reproductive outcomes. Hum Reprod Update. 2019;25:2–14.

    Article  PubMed  Google Scholar 

  17. Roeca C, Johnson RL, Truong T, Carlson NE, Polotsky AJ. Birth outcomes are superior after transfer of fresh versus frozen embryos for donor oocyte recipients. Hum Reprod. 2020;35:2850–9.

    Article  PubMed  Google Scholar 

  18. Insogna IG, Lanes A, Lee MS, Ginsburg ES, Fox JH. Association of fresh embryo transfers compared with cryopreserved-thawed embryo transfers with live birth rate among women undergoing assisted reproduction using freshly retrieved donor oocytes. JAMA. 2021;325:156–63.

    Article  PubMed  Google Scholar 

  19. Nagy ZP, Shapiro D, Chang C-C. Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment. Fertil Steril. 2020;113:241–7.

    Article  CAS  PubMed  Google Scholar 

  20. Chang H, Chen H, Zhang L, Wang Y, Xie X, Zhang Y, et al. Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos. Mol Reprod Dev. 2019;86:1603–14.

    Article  CAS  PubMed  Google Scholar 

  21. Truong TT, Gardner DK. Antioxidants increase blastocyst cryosurvival and viability post-vitrification. Hum Reprod. 2020;35:12–23.

    Article  CAS  PubMed  Google Scholar 

  22. Abdul Rahman N-S, Mohamed Noor Khan N-A, Eshak Z, Sarbandi M-S, Mohammad Kamal A-A, Abd Malek M, et al. Exogenous L-glutathione improves vitrification outcomes in murine preimplantation embryos. Antioxidants (Basel). 2022;11:2100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang L, Tang J, Wang L, Tan F, Song H, Zhou J, et al. Oxidative stress in oocyte aging and female reproduction. J Cell Physiol. 2021;236:7966–83.

    Article  CAS  PubMed  Google Scholar 

  24. Luo Z, Xu X, Sho T, Zhang J, Xu W, Yao J, et al. ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation. Am J Physiol Cell Physiol. 2019;316:C198-209.

    Article  CAS  PubMed  Google Scholar 

  25. Chen Y-N, Dai J-J, Wu C-F, Zhang S-S, Sun L-W, Zhang D-F. Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts. Anim Reprod Sci. 2018;198:137–44.

    Article  PubMed  Google Scholar 

  26. Jiang Z, Wang Y, Lin J, Xu J, Ding G, Huang H. Genetic and epigenetic risks of assisted reproduction. Best Pract Res Clin Obstet Gynaecol. 2017;44:90–104.

    Article  PubMed  Google Scholar 

  27. Du Z, Zhang K, Xie W. Epigenetic reprogramming in early animal development. Cold Spring Harb Perspect Biol. 2022;14: a039677.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chen C, Gao Y, Liu W, Gao S. Epigenetic regulation of cell fate transition: learning from early embryo development and somatic cell reprogramming†. Biol Reprod. 2022;107:183–95.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Chen H, Zhang L, Deng T, Zou P, Wang Y, Quan F, et al. Effects of oocyte vitrification on epigenetic status in early bovine embryos. Theriogenology. 2016;86:868–78.

    Article  CAS  PubMed  Google Scholar 

  30. Yao J, Geng L, Huang R, Peng W, Chen X, Jiang X, et al. Effect of vitrification on in vitro development and imprinted gene Grb10 in mouse embryos. Reproduction. 2017;154:97–105.

    Article  PubMed  Google Scholar 

  31. Wang Z, Xu L, He F. Embryo vitrification affects the methylation of the H19/Igf2 differentially methylated domain and the expression of H19 and Igf2. Fertil Steril. 2010;93:2729–33.

    Article  CAS  PubMed  Google Scholar 

  32. Hiura H, Hattori H, Kobayashi N, Okae H, Chiba H, Miyauchi N, et al. Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer. Clin Epigenet. 2017;9:79.

    Article  Google Scholar 

  33. Ma Y, Ma Y, Wen L, Lei H, Chen S, Wang X. Changes in DNA methylation and imprinting disorders in E9.5 mouse fetuses and placentas derived from vitrified eight-cell embryos. Mol Reprod Dev. 2019;86:404–15.

    Article  CAS  PubMed  Google Scholar 

  34. Wu Y-T, Dong Z-H, Li C, Zhou D-Z, Zhang J-Y, Wu Y, et al. The effect of blastomere loss during frozen embryo transfer on the transcriptome of offspring’s umbilical cord blood. Mol Biol Rep. 2020;47:8407–17.

    Article  CAS  PubMed  Google Scholar 

  35. Li Y, Tang J, Ji X, Hua M-M, Liu M, Chang L, et al. Regulation of the mammalian maternal-to-embryonic transition by eukaryotic translation initiation factor 4E. Development. 2021;148:190793.

    Article  Google Scholar 

  36. Edgar DH, Karani J, Gook DA. Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival. Reprod Biomed Online. 2009;19:521–5.

    Article  PubMed  Google Scholar 

  37. Bui H-T, Yamaoka E, Miyano T. Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation without Cdc2 kinase and mitogen-activated protein kinase activation in pig oocytes. Biol Reprod. 2004;70:1843–51.

    Article  CAS  PubMed  Google Scholar 

  38. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005;3:28.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Ladstätter S, Tachibana K. Genomic insights into chromatin reprogramming to totipotency in embryos. J Cell Biol. 2019;218:70–82.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Spinaci M, Vallorani C, Bucci D, Tamanini C, Porcu E, Galeati G. Vitrification of pig oocytes induces changes in histone H4 acetylation and histone H3 lysine 9 methylation (H3K9). Vet Res Commun. 2012;36:165–71.

    Article  CAS  PubMed  Google Scholar 

  41. Bakhtari A, Rahmani H-R, Bonakdar E, Jafarpour F, Asgari V, Hosseini S-M, et al. The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst. Cryobiology. 2014;69:419–27.

    Article  CAS  PubMed  Google Scholar 

  42. Barberet J, Barry F, Choux C, Guilleman M, Karoui S, Simonot R, et al. What impact does oocyte vitrification have on epigenetics and gene expression? Clin Epigenetics. 2020;12:121.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Mendel M, Chen K-M, Homolka D, Gos P, Pandey RR, McCarthy AA, et al. Methylation of structured RNA by the m6A Writer METTL16 Is essential for mouse embryonic development. Mol Cell. 2018;71:986-1000.e11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kwon J, Jo Y-J, Namgoong S, Kim N-H. Functional roles of hnRNPA2/B1 regulated by METTL3 in mammalian embryonic development. Sci Rep. 2019;9:8640.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Chen C, Liu W, Guo J, Liu Y, Liu X, Liu J, et al. Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos. Protein Cell. 2021;12:455–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li J, Zhu L, Huang J, Liu W, Han W, Huang G. Long-term storage does not affect the expression profiles of mRNA and long non-coding RNA in vitrified-warmed human embryos. Front Genet. 2021;12: 751467.

    Article  CAS  PubMed  Google Scholar 

  47. Lee C-Y, Tsai H-N, Cheng E-H, Lee T-H, Lin P-Y, Lee M-S, et al. Transcriptomic analysis of vitrified-warmed vs fresh mouse blastocysts: cryo-induced physiological mechanisms and implantation impact. Int J Mol Sci. 2024;25:8658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Saenz-de-Juano MD, Marco-Jimenez F, Schmaltz-Panneau B, Jimenez-Trigos E, Viudes-de-Castro MP, Penaranda DS, et al. Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level. Reproduction. 2014;147:789–801.

    Article  CAS  PubMed  Google Scholar 

  49. Truong T, Harvey AJ, Gardner DK. Antioxidant supplementation of mouse embryo culture or vitrification media support more in-vivo-like gene expression post-transfer. Reprod Biomed Online. 2022;44:393–410.

    Article  CAS  PubMed  Google Scholar 

  50. Cheng S-B, Nakashima A, Huber WJ, Davis S, Banerjee S, Huang Z, et al. Pyroptosis is a critical inflammatory pathway in the placenta from early onset preeclampsia and in human trophoblasts exposed to hypoxia and endoplasmic reticulum stressors. Cell Death Dis. 2019;10:927.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang Y, Liu W, Zhong Y, Li Q, Wu M, Yang L, et al. Metformin corrects glucose metabolism reprogramming and NLRP3 inflammasome-induced pyroptosis via inhibiting the TLR4/NF-κB/PFKFB3 signaling in trophoblasts: implication for a potential therapy of preeclampsia. Oxid Med Cell Longev. 2021;2021:1806344.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Pan Y-J, Zhang M-Z, He L-H, Feng J, Zhang A-H. Expression of urotensin II is positively correlated with pyroptosis-related molecules in patients with severe preeclampsia. Clin Exp Hypertens. 2021;43:295–304.

    Article  CAS  PubMed  Google Scholar 

  53. Husen SC, Koning IV, Go ATJI, Groenenberg IAL, Willemsen SP, Rousian M, et al. IVF with or without ICSI and the impact on human embryonic brain development: the Rotterdam Periconceptional Cohort. Hum Reprod. 2021;36:596–604.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chang C-C, Shapiro DB, Nagy ZP. The effects of vitrification on oocyte quality. Biol Reprod. 2022;106:316–27.

    Article  PubMed  Google Scholar 

  55. Estudillo E, Jiménez A, Bustamante-Nieves PE, Palacios-Reyes C, Velasco I, López-Ornelas A. Cryopreservation of gametes and embryos and their molecular changes. Int J Mol Sci. 2021;22:10864.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Agarwal A, Maldonado Rosas I, Anagnostopoulou C, Cannarella R, Boitrelle F, Munoz LV, et al. Oxidative stress and assisted reproduction: a comprehensive review of its pathophysiological role and strategies for optimizing embryo culture environment. Antioxidants. 2022;11:477.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gupta MK, Uhm SJ, Lee HT. Effect of vitrification and beta-mercaptoethanol on reactive oxygen species activity and in vitro development of oocytes vitrified before or after in vitro fertilization. Fertil Steril. 2010;93:2602–7.

    Article  PubMed  Google Scholar 

  58. Dennery PA. Effects of oxidative stress on embryonic development. Birth Defects Res C Embryo Today. 2007;81:155–62.

    Article  CAS  PubMed  Google Scholar 

  59. Jana SK, K NB, Chattopadhyay R, Chakravarty B, Chaudhury K. Upper control limit of reactive oxygen species in follicular fluid beyond which viable embryo formation is not favorable. Reprod Toxicol. 2010;29:447–51.

  60. Lettieri G, D’Agostino G, Mele E, Cardito C, Esposito R, Cimmino A, et al. Discovery of the involvement in DNA oxidative damage of human sperm nuclear basic proteins of healthy young men living in polluted areas. Int J Mol Sci. 2020;21:4198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Orrenius S, Gogvadze V, Zhivotovsky B. Calcium and mitochondria in the regulation of cell death. Biochem Biophys Res Commun. 2015;460:72–81.

    Article  CAS  PubMed  Google Scholar 

  62. Pérez-Sánchez M, Pardiñas ML, Díez-Juan A, Quiñonero A, Domínguez F, Martin A, et al. The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles. J Assist Reprod Genet. 2023;

  63. Juan H-C, Lin Y, Chen H-R, Fann M-J. Cdk12 is essential for embryonic development and the maintenance of genomic stability. Cell Death Differ. 2016;23:1038–48.

    Article  CAS  PubMed  Google Scholar 

  64. Chapman JR, Taylor MRG, Boulton SJ. Playing the end game: DNA double-strand break repair pathway choice. Mol Cell. 2012;47:497–510.

    Article  CAS  PubMed  Google Scholar 

  65. Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem. 2010;79:181–211.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bohrer RC, Dicks N, Gutierrez K, Duggavathi R, Bordignon V. Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos. FASEB J. 2018;32:1818–29.

    Article  CAS  PubMed  Google Scholar 

  67. Jin Z-L, Shen X-H, Shuang L, Kwon J, Seong M-J, Kim N-H. Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development. Reproduction. 2019;157:223–34.

    Article  CAS  PubMed  Google Scholar 

  68. Beucher A, Birraux J, Tchouandong L, Barton O, Shibata A, Conrad S, et al. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J. 2009;28:3413–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Zhao B, Rothenberg E, Ramsden DA, Lieber MR. The molecular basis and disease relevance of non-homologous DNA end joining. Nat Rev Mol Cell Biol. 2020;21:765–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Shibata A, Conrad S, Birraux J, Geuting V, Barton O, Ismail A, et al. Factors determining DNA double-strand break repair pathway choice in G2 phase. EMBO J. 2011;30:1079–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Xu R, Li C, Liu X, Gao S. Insights into epigenetic patterns in mammalian early embryos. Protein Cell. 2021;12:7–28.

    Article  PubMed  Google Scholar 

  72. Wang S, Meyer DH, Schumacher B. H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage. Nat Struct Mol Biol. 2020;27:1165–77.

    Article  CAS  PubMed  Google Scholar 

  73. Cui M-S, Wang X-L, Tang D-W, Zhang J, Liu Y, Zeng S-M. Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species. Theriogenology. 2011;75:638–46.

    Article  CAS  PubMed  Google Scholar 

  74. Keshavarzi S, Salehi M, Farifteh-Nobijari F, Hosseini T, Hosseini S, Ghazifard A, et al. Melatonin modifies histone acetylation during in vitro maturation of mouse oocytes. Cell J. 2018;20:244–9.

    PubMed  PubMed Central  Google Scholar 

  75. Zhang M, Zhai Y, Zhang S, Dai X, Li Z. Roles of N6-Methyladenosine (m6A) in stem cell fate decisions and early embryonic development in mammals. Front Cell Dev Biol. 2020;8:782.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Zhang M, Zhang S, Zhai Y, Han Y, Huang R, An X, et al. Cycloleucine negatively regulates porcine oocyte maturation and embryo development by modulating N6-methyladenosine and histone modifications. Theriogenology. 2022;179:128–40.

    Article  CAS  PubMed  Google Scholar 

  77. Trifonova EA, Gabidulina TV, Ershov NI, Serebrova VN, Vorozhishcheva AY, Stepanov VA. Analysis of the placental tissue transcriptome of normal and preeclampsia complicated pregnancies. Acta Naturae. 2014;6:71–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Wei X, Zhang Y, Yin B, Wen J, Cheng J, Fu X. The expression and function of KCNQ potassium channels in human chorionic plate arteries from women with normal pregnancies and pre-eclampsia. PLoS ONE. 2018;13: e0192122.

    Article  PubMed  PubMed Central  Google Scholar 

  79. Todd N, McNally R, Alqudah A, Jerotic D, Suvakov S, Obradovic D, et al. Role of a novel angiogenesis FKBPL-CD44 pathway in preeclampsia risk stratification and mesenchymal stem cell treatment. J Clin Endocrinol Metab. 2021;106:26–41.

    Article  PubMed  Google Scholar 

  80. Li X, Liu L, Whitehead C, Li J, Thierry B, Le TD, et al. Identifying preeclampsia-associated genes using a control theory method. Brief Funct Genomics. 2022;21:296–309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was partly supported by the National Natural Science Foundation of China (No: 82273137), Natural Science Foundation of Jilin Province (No: 20210204165YY, No: YDZJ202301ZYTS461), the "Startup funding of First Hospital, JLU."

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Author notes

  1. Yurong Chen, Haibo Zhu, and Fucheng Guo contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital of Jilin University, Changchun, China

    Yurong Chen, Haibo Zhu, Fucheng Guo, Luyao Wang, Xiaoling Zhang & Xiangpeng Dai

  2. National-Local Joint Engineering Laboratory of Animal Models for Human Disease, First Hospital of Jilin University, Changchun, China

    Yurong Chen, Haibo Zhu, Fucheng Guo, Luyao Wang, Xiaoling Zhang & Xiangpeng Dai

  3. Reproductive Medicine Center, Xiamen University Affiliated Chenggong Hospital, Xiamen, Fujian, China

    Yurong Chen

  4. Center of Reproductive Medicine and Center of Prenatal Diagnosis, First Hospital of Jilin University, Changchun, China

    Haibo Zhu & Ruizhi Liu

  5. Comprehensive Testing and Analytical Center of North China University of Science and Technology, Tangshan, Hebei, China

    Wenli Zhang

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  1. Yurong Chen
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Contributions

Yurong Chen contributed to investigation, data curation, writing – original draft. Haibo Zhu contributed to conceptualization, writing – review & editing, revision. Fucheng Guo contributed to writing – review & editing, revision. Luyao Wang contributed to writing – review & editing. Wenli Zhang contributed to revision. Ruizhi Liu contributed to writing – review & editing, supervision, funding acquisition. Xiaoling Zhang contributed to writing – review & editing, supervision, revision. Xiangpeng Dai contributed to conceptualization, writing – review & editing, supervision, funding acquisition.

Corresponding authors

Correspondence to Xiaoling Zhang or Xiangpeng Dai.

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Chen, Y., Zhu, H., Guo, F. et al. Vitrification affects the post-implantation development of mouse embryos by inducing DNA damage and epigenetic modifications. Clin Epigenet 17, 20 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13148-025-01826-y

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13148-025-01826-y

Keywords

Clinical Epigenetics

ISSN: 1868-7083

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