Effect of method cryopreservation

In recent years, many studies have revealed the superiority of vitrification over slow freeze protocols in reducing chilling damage to oocytes during cryopreservation (Saragusty and Arav, Reference Saragusty and Arav2011). Fadini et al. (Reference Fadini, Brambillasca, Renzini, Merola, Comi, De Ponti and Dal Canto2009) reported higher pregnancy rates for vitrification (18.2 vs. 7.6%). In addition, vitrification has been proposed as a cheaper and more reasonable strategy for the cryopreservation of human and animal oocytes instead of the slow freezing technique using a higher concentration of cryoprotectants and a rapid cooling rate to prevent the formation of intracellular ice crystals (Antinori et al., Reference Antinori, Licata, Dani, Cerusico, Versaci and Antinori2007). Furthermore, the vitrification method has also been applied to cryopreserve oocytes or embryos produced from IVM programmes (Mogas, Reference Mogas2019). Unlike the slow freezing method, vitrification presents the capacity to control solute penetration, the dehydration rate, and the maintenance of physiological temperature during the equilibration process. Breaking the zona pellucida and formation of growing ice crystals are possible during the slow freezing process, but not with vitrification (Alcolak et al., Reference Alcolak, Abu Marar, Mytas, Chalvatzas, Palapelas, Schöpper, Diedrich and Al-Hasani2011).

Results showed that the addition of melatonin to the freezing medium can effectively improve the development ability of frozen human oocytes by increasing mitochondrial function, reducing oxidative damage (by expressing proteins related to the phosphorylation pathway), maintaining reactive oxygen species (ROS)/GSH homeostasis and, as a result, normal ATP production and the ROS/GSH ratio were improved (Zhu et al., Reference Zhu, Ding, Yang, Zou, Yang, Wang, Zhang, Chen, Ji, Hao, Xue, Xu, Wang, Wang, Yan, Cao, Zou and Zhang2022). Limited numbers of studies on oocytes have provided controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE), and aneuploidy. A study of human embryos has shown that vitrification affects DNA integrity much less than slow freezing. Animal studies have shown increased mitochondrial DNA mutations in embryos after cryopreservation (Kopeika et al., Reference Kopeika, Thornhill and Khalaf2015). Studies have shown that slow freezing and vitrification alter the distribution of mitochondria in mouse embryos. Also, in the vitrification method, ROS are higher in the morula stages. Therefore, in addition to the freezing method, the embryonic stage also determines the changes in the cells caused by freezing (Somoskoi et al., Reference Somoskoi, Martino, Cardone, Lacalandra, Dell’Aquila and Cseh2015; Hayashi et al., Reference Hayashi, Kansaku, Abe, Ueda and Iwata2019). A study on rabbit embryos showed that there was no significant difference in the expression of genes (SCGB1A1, EMP1, C1QTNF1, ANXA3, EGFLAM and TNFAIP6) between embryos frozen with the two techniques (slow freezing and vitrification; Saenz-de-Juano et al., Reference Saenz-de-Juano, Marco-Jimenez, Viudes-de-Castro, Lavara and Vicente2014). A comparison between the transcriptional profiles of two vitrified oocyte techniques (semi-automated and manual vitrification) using single-cell RNA sequencing showed that the transcriptional changes between the two techniques were very low and not significant, but specific adverse effects of oocyte freezing on genomic expression were prominent compared with fresh oocytes. Given that fertility preservation through egg freezing will be developed worldwide in the coming years, More studies in this field are needed for the health of children born from frozen oocytes (Barberet et al., Reference Barberet, Ducreux, Bruno, Guilleman, Simonot, Lieury, Guilloteau, Bourc’his and Fauque2022).

Type of CPA and miRNAs

Results of the assessment of IVM bovine oocytes submitted to vitrification showed that DMSO had a significant effect on the incidence of abnormal microtubule distribution when compared with fresh oocytes (Verheijen et al., Reference Verheijen, Lienhard, Schrooders, Clayton, Nudischer, Boerno, Timmermann, Selevsek, Schlapbach, Gmuender, Gotta, Geraedts, Herwig, Kleinjans and Caiment2019). Frequencies found when comparing the DMSO group to glycerol were similar (P > 0.05). Additionally, no differences were found when comparing fresh oocytes to those submitted to vitrification using the combination of ethylene glycol (EG) and glycerol (P > 0.05). No differences between oocytes vitrified using EG and glycerol and fresh oocytes were found. Conversely, oocytes vitrified using the combination of EG and glycerol presented a higher incidence of normal microtubule configuration. It has been widely suggested that the incubation process allows the reorganization of microtubules. The chromosomal arrangement was not affected by vitrification and the incubation process did not affect it (Verheijen et al., Reference Verheijen, Lienhard, Schrooders, Clayton, Nudischer, Boerno, Timmermann, Selevsek, Schlapbach, Gmuender, Gotta, Geraedts, Herwig, Kleinjans and Caiment2019). In a recent study, DMSO effects were assessed with complete transcriptome analysis (using ribodepleted total RNA sequencing and microRNA sequencing), whole-genome methylation profiling (using MeDIP-seq) and proteomics analysis (using mass spectrometry). The results demonstrated that DMSO could not be considered biologically inert, but induced significant changes in miRNAs and the epigenetic landscape in cells in culture even at very low concentrations, especially in the maturing cardiac model. Zhang et al. (Reference Zhang, Mu, Ding, Zou, Li, Chen, Leung, Chang, Zhu, Wang, Xue, Xu, Zou, Zhou, Wei and Cao2021) evaluated the effects of EG as a cryoprotectant in open vitrification medium in human oocytes. Global gene expression studies based on fragments per kilobase million (FPKM) cluster analyses showed that 3740 genes were upregulated and 956 genes were downregulated in vitrified donor oocytes vs. fresh donor oocytes.

Stage of oocyte cryopreservation

Despite the proven superiority of the vitrification method over slow freezing, there is still debate about the vitrification stage of the oocyte (before and after in vitro maturation). Some studies in species such as pigs (Martin and Zhang, Reference Martin and Zhang2007) and humans (Ma et al., Reference Ma, Pan, Montgomery, Olson and Schultz2012; Gao et al., Reference Gao, Jia, Li, Ma, Huang, Zhu, Hou and Fu2017) have reported that vitrified oocytes at the GV stage have a lower survival rate and efficiency than at the metaphase II stage (MII). Conversely, one study reported that vitrification of mouse oocytes in the MII stage was more suitable for transcription (Gao et al., Reference Gao, Jia, Li, Ma, Huang, Zhu, Hou and Fu2017). Also, a study using the same cohort of oocytes (oocytes from the same donors) reported that vitrified oocytes did not show any statistically significant differences in fertilization, embryo quality, or clinical outcome compared with fresh oocytes (Solé et al., Reference Solé, Santaló, Boada, Clua, Rodríguez, Martínez, Coroleu, Barri and Veiga2013). A study using RNA-seq showed that vitrification of in vitro-matured bovine oocytes caused changes in the expression of some genes (Wang et al., Reference Wang, Li, Zhu, Hao, Wang, Yan, Zhao, Du, Wang, Liu, Pang and Zhao2017a, Reference Wang, Zhang, Zhao, Kuang and Xue2018). Song et al. (Reference Song, Peng, Chen, Jin, Yao, Shi, Yang, Zhang and Sun2016) also used GV-stage and GVBD (MI)-stage oocytes generated from IVF cycles. A comparison between matured oocytes in vivo without vitrification with groups of vitrified oocytes before and after maturation in vitro showed that, after thawing, the cleavage rate in vitrified oocytes at the MII stage after IVM was higher than in vitrified immature oocytes before IVM.

One study evaluated the expression of DNMT1, apoptosis-related (Bcl-2 and Bax) and stress-related (Sod1 and HSP70) genes in fresh IVM oocytes, vitrified GV (vGV) and vitrification of in vitro-matured oocytes (vIVM). The results showed that the expression of Bcl-2, Bax, Sod1, and HSP70 genes in the vIVM group was significantly increased, and also the DNMT1 gene in both groups [vitrification before (vGV) and after (vIVM) maturation]. But the rate of expression was reduced in vitrified IVM (vIVM) oocytes (Shahedi et al., Reference Shahedi, Hosseini, Ali Khalili and Yeganeh2017). Therefore, vitrification after maturation caused more stress on the oocytes, and higher expression of stress-related genes in the oocyte. The major problem associated with freezing mature oocytes is meiotic spindle sensitivity to low temperatures and cryoprotectants. To solve this problem, GV oocytes were used instead of mature oocytes. However, poor maturation, fertilization, and embryo development were the main disadvantages associated with the cryostorage of immature oocytes (Walls et al., Reference Walls, Junk, Ryan and Hart2012).

One study found that vitrification acted as a stressor and altered the expression of these genes, thereby reducing the developmental competence of mouse zygotes (Borini and Bianchi, Reference Borini and Bianchi2010). In addition, it should be noted that various factors in the freezing process, such as high concentrations of cryoprotectants, freezing, and osmotic stress, changed the pattern of gene expression as well as the onset of apoptosis, which threatened the viability and development of oocytes (Men et al., Reference Men, Monson, Parrish and Rutledge2003). Men et al. (Reference Men, Monson, Parrish and Rutledge2003) showed that the degeneration mechanism of cryopreserved oocytes was apoptosis. The results showed that the percentage of oocyte maturation in the oocytes matured in vitro before vitrification was significantly higher than in immature oocytes, which were vitrified, and then matured in vitro. Cao et al. (Reference Cao, Xing, Zhang, Wei, Zhou and Cong2009) similarly announced that the oocyte maturation rate in oocytes that were vitrified at the GV stage and afterwards matured in vitro, compared with oocytes that were first matured in vitro and followed by vitrification, was significantly reduced.

Epigenetic effects of cryopreservation on oocytes (such as non-coding RNAs; miRNAs)

Vitrification of porcine oocytes prompted changes in histone H4 acetylation and histone H3 methylation of lysine 9 (Spinaci et al., Reference Spinaci, Vallorani, Bucci, Tamanini, Porcu and Galeati2012). Also, vitrification significantly reduced the expression of DNMT1 mRNA in mouse metaphase II oocytes (Zhao et al., Reference Zhao, Ren, Du, Hao, Wang, Qin, Liu and Zhu2013; Cheng et al., Reference Cheng, Fu, Zhang, Jia, Hou and Zhu2014). The results of experimental studies showed that the expression level of DNMT1 decreased in the vitrified IVM group. Decreased DNMT1 gene expression usually alters DNA methylation patterns, leading to impaired gene expression, genomic printing, and genome stabilization, ultimately leading to cell death. Various studies have shown that, following the freezing and thawing of oocytes, H3K9 methylation and H4K5 acetylation increased sharply. Usually, no H3K14 acetylation occurs in fresh or frozen MII oocytes. Vitrified oocytes are highly sensitive to changes in H3K9 and H4K5 (Yan et al., Reference Yan, Yan, Qiao, Zhao and Liu2010; Rasti and Vaquero, Reference Rasti and Vaquero2018). Histone methylation involves arginine and lysine residues and plays an important role in regulating transcription, genome integrity and epigenetic inheritance. Overall, the results showed that cryopreservation may be associated with changes in physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives.

According to some reports, vitrification of oocytes did not affect zygote cleavage rates, but reduced the blastocyst rate. Chen et al. (Reference Chen, Zhang, Deng, Zou, Wang, Quan and Zhang2016) reported that the degrees of DNA methylation and H3K9me3 in vitrified oocytes and early cleavage embryos were lower than in the control group (non-vitrified group), albeit in the vitrified group the level of acH3K9 increased in the early cleavage stages. These changes in methylation may be due to changes in the expression of micoRNAs in vitrified oocytes. The expression levels of putative-imprinted genes (PEG10, XIST, and KCNQ1O1T) were upregulated in blastocysts. These epigenetic abnormalities may be partially explained by the altered expression of genes associated with epigenetic regulations. DNA methylation and H3K9 modification suggested that oocyte vitrification may excessively relax the chromosomes of oocytes and early cleavage embryos. Consequently, these epigenetic indexes can be used as damage markers of oocyte vitrification during early embryonic development (Chen et al., Reference Chen, Zhang, Deng, Zou, Wang, Quan and Zhang2016). Overall, methylation in bovine MII oocytes has been reported to decrease significantly after vitrification with EG and DMSO as cryoprotectants and 0.25-ml straws as a container (Hu et al., Reference Hu, Marchesi, Qiao and Feng2012). In mice, DNA methylation was also significantly decreased in vitrified MII oocytes and in 2-cell- to 8-cell-stage embryos compared with the control. The data indicated that vitrification of the oocyte seemed to always results in the reduction or loss of DNA methylation in the oocyte or embryo, which may lead to impaired embryonic development and qualities in later stages (Hu et al., Reference Hu, Marchesi, Qiao and Feng2012).

Specific characteristics of miRNAs in oocytes (oocyte maturation)

miRNAs are an important class of small non-coding RNAs of ∼22 nucleoids that act as effectors of post-transcriptional gene silencing, and also act as a potent regulator of gene expression in most biological systems and cellular contexts. In eukaryotic organisms, miRNA genes are highly conserved, affect specific biological processes such as amplification, differentiation, and signal transduction, and are widely involved in development, physiology, and pathology (Flynt and Lai, Reference Flynt and Lai2008; Sun and Lai, Reference Sun and Lai2013). miRNAs typically suppress the expression of ‘target’ genes by anti-sense base-pairing interactions with the 3′ untranslated region (3′UTR). An active miRNA molecule binds to the ‘target’ mRNA through Watson–Crick base pairs using an essential seven-nucleotide seed sequence (Sun and Lai, Reference Sun and Lai2013). Due to this short pairing, a miRNA has the potential to bind to hundreds of 3′UTR targets. Hundreds of miRNAs have been examined based on in silico analysis; miRNAs were initially reported to regulate ∼30% of the expressed mammalian genes (Sevignani et al., Reference Sevignani, Calin, Siracusa and Croce2006), which increased to more than 60% (Friedman et al., Reference Friedman, Farh, Burge and Bartel2009). Given the potential for these small molecules to dramatically alter cellular behaviour and function, it continues to be crucial to understand how miRNAs are themselves regulated. miRNA biosynthesis is a sequential process that provides ample opportunity for complex regulation.

General and specific studies of miRNA profiles have shown that changes in the expression pattern of biologically relevant miRNAs occur during the growth and development of mammalian oocytes (Tesfaye et al., Reference Tesfaye, Worku, Rings, Phatsara, Tholen, Schellander and Hoelker2009; Xu et al., Reference Xu, Wang, Ding, Li, Gu and Zhou2011). For example, miR-21 is a miRNA that potentially targets genes involved in fatty acid metabolism and fatty acid biosynthesis, and is expressed in different patterns during oocyte maturation (Song et al., Reference Song, Peng, Chen, Jin, Yao, Shi, Yang, Zhang and Sun2016). Inhibition of miR-21 by an anti-miR21 peptide nucleic acid leads to a reduced ratio of porcine oocytes to the MII stage and further embryo development (Wright et al., Reference Wright, Hale, Yang, Njoka and Ross2016). Similar results by inhibiting the activity of let-7c, miR-27a and miR-322 in mouse oocytes (Kim et al., Reference Kim, Ku, Kim, Liu, Chi, Kim, Choi, Kim and Moon2013), miR-15/16 in Xenopus oocytes (Wilczynska et al., Reference Wilczynska, Git, Argasinska, Belloc and Standart2016) and miR-378 in the porcine oocyte (Pan et al., Reference Pan, Toms, Shen and Li2015) have been reported. Conversely, increasing the expression of miR-224 in cumulus cells reduced the rate of oocyte maturation and blastocyst growth by regulating PTX3 expression (Li et al., Reference Li, Wang, Sheng and Wang2017).

The results showed that specific changes in miRNA expression were associated with significant changes in the function and morphology of aged oocytes. Experimental studies on the function of key proteins predicted by KEGG analysis and injection of miR-98 mimics or inhibitors have confirmed that miRNAs have stimulatory/inhibitory roles in post-ovulatory oocyte ageing (Wang et al., Reference Wang, Zhang, Zhu, Lian, Yuan, Gao, Luo and Tan2017b). Whereas miR-21 acts as an anti-apoptotic, miR-29b, miR-15a and miR-16, miR-128, and miR-98 act as pro-apoptotics (Xia et al., Reference Xia, Lu, Wang, Yang, Zhou and Huang2018). However, all these six apoptosis-related miRNAs significantly increased their expression from 18 to 24 h after hCG injection. The study showed that miR-98 upregulated the expression of caspase-3, which increased the release of Ca²⁺ from the endoplasmic reticulum into the oocyte, leading to increased spontaneous ovulation activity (SA). Therefore, the results suggested that increased oocytes in SA were associated with increased cytoplasmic Ca²⁺ (Zhang et al., Reference Zhang, Cui, Zhang, Zhang, Wang, Zhu, Jiao and Tan2014). Experimental results showed that suppressed expression of miR-224 in cumulus cells (CC) was essential for oocyte maturation. Overexpression of miR-224 stopped or delayed the progression of oocyte maturation, whereas inhibition of miR-224 induced oocyte development. As a result, overexpression of miRNA-224 led to the decreased expression of genes and proteins associated with oocyte maturation such as GDF9, BMP15, CX37, and ZP3 in CC and oocytes (Li et al., Reference Li, Wang, Sheng and Wang2017).

Oocyte cryopreservation and miRNAs

In general, growth and development in oocytes occurred as a result of various cellular and molecular changes during ovarian follicular development. These changes are controlled by endocrine and paracrine factors coordinated by the oocyte and surrounding somatic cells. These processes are regulated by the precise expression and interaction of many genes in different parts of the ovary (oocytes, granulosa, and theca cells) to achieve oocyte development (Bonnet et al., Reference Bonnet, Tran and Sirard2007) and are possibly regulated by miRNAs. Studies on miRNAs have shown that miRNAs are involved in the regulation of oocyte cross-communication and CC (Assou et al., Reference Assou, Al-Edani, Haouzi, Philippe, Lecellier, Piquemal, Commes, Aït-Ahmed, Dechaud and Hamamah2013). Therefore, altering the expression of miRNAs at different stages of oocyte development can have a negative effect on the maturation and competence of the oocyte and the resulting embryo.

In most mammals, the process of meiotic division in oocytes arrests at the stage of the dictate of prophase I until the resumption of meiosis. Despite the extensive transcript turnover in the oocyte, it is almost accepted that the meiotic maturity of the oocyte is transcriptionally quiescent. Therefore, the fertilization and early development of the embryo depend on the maternal transcripts that have accumulated during the development of the oocyte. This mother-to-zygotic transition occurs in different species at different times in the embryonic stage. Recent studies have detailed conflicting reports on the occurrence of transcription following the resumption of oocyte meiotic maturation. Increased expression of some transcripts during oocyte maturation has been reported between immature (GV) and in vivo matured oocytes (MII) in mice, cattle, and humans (Assou et al., Reference Assou, Anahory, Pantesco, Le Carrour, Pellestor, Klein, Reyftmann, Dechaud, De Vos and Hamamah2006). The comparison of results between GV and MII oocytes showed an increased expression of four miRNAs (hsa-miR-193a-5p, hsa-miR-297, hsa-miR-625, and hsa-miR-602), and decreased expression of 11 miRNAs (hsa-miR-888, hsa-miR-212, hsa-miR-662, hsa-miR-299–5p, hsa-miR-339–5p, hsa-miR-20a, hsa-miR-486–5p, hsa-miR-141, hsa-miR-768–5p, hsa-miR-376a, and hsa-miR-15a; Xu et al., Reference Xu, Wang, Ding, Li, Gu and Zhou2011). Expression of specific miRNAs in human oocytes showed dynamic changes during meiosis. High concentrations of FSH in IVM medium had the opposite effect on the expression of hsa-miR-15a and hsa-miR-20a. A study to confirm miRNA dynamic changes in bovine GV, MII oocytes, and presumptive zygotes (PZ) showed that bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a had progressive changes between stages from GV to PZ. pri-miR-155 and pri-miR-222 are not normally detected in GV oocytes, but pri-miR-155 is present in MII oocytes, indicating transcription during maturation. miR-148a was found in large amounts with stable expression at all stages (Gilchrist et al., Reference Gilchrist, Tscherner, Nalpathamkalam, Merico and LaMarre2016).

However, in the maturing oocyte, several significant pathways have the strong potential for repression by miRNAs that are expressed, including phosphatidyl inositol phosphate binding (miR-222), transcriptional regulatory region DNA binding (miR-21), and regulation of the protein kinase cascade activity (let-7d). Studies of miRNAs in other cells, such as cancer tissue, have shown that p53 is targeted by the let-7 family of miRNAs. p53 is known to be widely involved in cell cycle checkpoints and is present in early bovine embryos (Tomek and Smiljakovic, Reference Tomek and Smiljakovic2005). In addition, miR-155 targets the mRNA of inositol 5-phosphatase 1 (INPP5D) and reduces inositol 5-phosphate 1 (O’Connell et al., Reference O’Connell, Chaudhuri, Rao and Baltimore2009). A decrease in INPP5D has been shown to increase AKT activity, a pathway involved in bovine oocyte maturation (Yamanaka et al., Reference Yamanaka, Tagawa, Takahashi, Watanabe, Guo, Iwamoto, Yamashita, Saitoh, Kameoka, Shimizu, Ichinohasama and Sawada2009). The data showed that let-7 (Takahashi et al., Reference Takahashi, Nakaoka and Yamashita2012) and miR-27a (Kim et al., Reference Kim, Ku, Kim, Liu, Chi, Kim, Choi, Kim and Moon2013) regulate cell cycle signalling pathways. Studies have shown that miR-322 modulates ovarian development, folliculogenesis, and cell differentiation (Leem et al., Reference Leem, Han, Lee, Ha, Kwon, Ho, Kim, Tran, Bae and Kang2011; Kim et al., Reference Kim, Ku, Kim, Liu, Chi, Kim, Choi, Kim and Moon2013).

The results showed that several miRNAs were expressed in human oocytes and blastocysts, targeting key genes involved in DNA repair and cell cycle checkpoints. Most miRNAs are generally expressed at lower levels in the blastocyst than in the oocyte. The results of the correlation analysis showed that there was an inverse and direct relationship between miRNAs and their target mRNAs (Tulay et al., Reference Tulay, Naja, Cascales-Roman, Doshi, Serhal and SenGupta2015). This study investigated the expression of 10 mRNAs and 20 miRNAs in human oocytes and blastocysts. The results showed that all mRNAs studied and 11 miRNAs were expressed in both oocytes and blastocysts. However, mRNA expression levels in oocytes were higher relative to blastocysts. This result appeared normal because the oocyte must transfer enough mRNAs to support the resulting zygote’s early development and maintain survival and development until embryonic genome activation. The results of the molecular analysis showed that miRNA can be involved in various processes of oocyte maturation. The importance of miRNA in oocyte maturation has initially been demonstrated in knockdown or knockout studies, and it has become clear that Dicer is required for meiotic spindle integrity and completion of meiosis I (Murchison et al., Reference Murchison, Stein, Xuan, Pan, Zhang, Schultz and Hannon2007). Many factors, such as oocyte maturation (in vitro or in vivo), cryopreservation methods and cryopreservation procedures (slow freezing or vitrification), oocyte vitrification stages (GV or MII oocyte), maternal age etc., can adversely cause changes in the expression pattern of miRNAs in the oocyte (Monzo et al., Reference Monzo, Haouzi, Roman, Assou, Dechaud and Hamamah2012; Wang et al., Reference Wang, Li, Zhu, Hao, Wang, Yan, Zhao, Du, Wang, Liu, Pang and Zhao2017a).

According to some studies, ∼520 miRNAs have been detected in oocytes, of which only 22 miRNAs were significantly expressed in vitrified oocytes compared with fresh oocytes (Li et al., Reference Li, Yang, Liu, Song and Liu2019). Changes in miRNA expression are dynamic during oocyte vitrification. The data showed that miR-21–3p expression increased after cryopreservation. It has been identified that miR-21–3p target genes are involved in physiological and pathological processes (Yan et al., Reference Yan, Chen, Gong, Yin, Zhou, Chaugai and Wang2015; Xia et al., Reference Xia, Lu, Wang, Yang, Zhou and Huang2018). This suggested that miR-21–3p suppressed PTEN gene expression in umbilical cord blood plasma exosomes (Hu et al., Reference Hu, Rao, Wang, Cao, Tan, Luo, Li, Zhang, Chen and Xie2018). It also proved that miR-21–3p reduced PTEN gene expression by targeting its 3′UTR. PTEN always acts towards the PI3K/AKT pathway, important signalling in regulating cell apoptosis through the regulation of oxidative stress (Matsuda et al., Reference Matsuda, Nakagawa, Kitagishi, Nakanishi and Murai2018). Studies have shown that decreased PTEN expression is associated with increased levels of ROS (Li et al., Reference Li, Mao, Zheng, Dong and Ling2013; Noh et al., Reference Noh, Park, Song, Kim, Lee, Song, Hong, Whang, Han, Kwon, Kim and Lee2016), which acts as a factor in reducing the developmental potential of vitrified oocytes (Pan et al., Reference Pan, Toms, Shen and Li2015; Wang et al., Reference Wang, Zhang, Zhao, Kuang and Xue2018). In the study, ultrastructural malformations such as increased vacuolation, aberrant dynamic variations in mitochondria–smooth endoplasmic reticulum and mitochondria–vesicle complexes, and scarce cortical granules were observed in vitrified oocytes (Nottola et al., Reference Nottola, Albani, Coticchio, Palmerini, Lorenzo, Scaravelli, Borini, Levi-Setti and Macchiarelli2016). In addition, disturbed mitochondrial localization has been reported in vitrified mouse oocytes (Yan et al., Reference Yan, Yan, Qiao, Zhao and Liu2010, Reference Yan, Chen, Gong, Yin, Zhou, Chaugai and Wang2015). Also, vitrification causes damage to microtubules, actin filaments, and chromosome integrity in the oocytes (Wen et al., Reference Wen, Zhao, Chao, Yu, Song, Shen, Chen and Deng2014).

Studies have shown that storage duration did not affect the gene expression pattern in frozen oocytes and did not alter their expression (Goldman et al., Reference Goldman, Kramer, Hodes-Wertz, Noyes, McCaffrey and Grifo2015; Stigliani et al., Reference Stigliani, Moretti, Anserini, Casciano, Venturini and Scaruffi2015). Oocyte developmental competence has been shown to depend on the accumulation of maternal RNAs and proteins during oogenesis. Conversely, reduced developmental competence in the oocyte is one of the main reasons for IVF failure. Therefore, this proves that the developmental potential of frozen/thawed oocytes is not related to their retention time in liquid nitrogen. Therefore, the possible damage to frozen/thawed oocytes is only due to the method and procedure of cryopreservation, which seems to be more important than the storage time. In support of this, clinical studies have been previously performed on controlled-rate cryopreserved and vitrified human oocytes (Goldman et al., Reference Goldman, Kramer, Hodes-Wertz, Noyes, McCaffrey and Grifo2015).

Under in vitro conditions, the dynamic miRNA profile changes are partly attributed to the in vitro maturation environment or ingredients used under in vivo conditions. The miRNA profiles could be affected by physiological conditions such as the animal’s age. For example, in humans, treatment of metaphase I (MI) human oocytes with insulin-like growth factor 1 activated the expression of miR-133a, miR-205–5p and 145 miRNAs and suppressed 200 others, including miR-152 and miR-142–5p (Xiao et al., Reference Xiao, Xia, Yang, Liu, Du, Kang, Lin, Guan, Yan and Tang2014). Conversely, altered expression of 12 miRNAs (including let-7b-5p and let-7e-5p) in oocytes derived from older women compared with young women suggested that the in vivo miRNA profile in the oocytes was affected by oocyte ageing (Battaglia et al., Reference Battaglia, Vento, Ragusa, Barbagallo, La Ferlita, Di Emidio, Borzí, Artini, Scollo, Tatone, Purrello and Di Pietro2016).

A study in mice showed that, although the expression pattern of the miRNA biogenesis pathway varied between 8-cell embryos and blastocysts, vitrification did not affect the expression level of these genes in preimplantation embryos. Expression levels of miR-21 and let-7a were significantly reduced in vitrified 8-cell embryos and fresh blastocysts compared with fresh 8-cell embryos. STAT-3 expression in blastocysts decreased significantly after vitrification (Azizi et al., Reference Azizi, Ghaffari Novin, Naji, Amidi, Hosseinirad and Shams Mofarahe2021). However, changes in the expression pattern of miRNAs in the oocyte and multicellular embryos were compensated in the blastocyst stage. These changes were very small in vitrified and fresh embryos in the blastocyst stage. The results indicated that re-vitrification of embryos changed the expression of miR-16, miR-let-7a and their target genes. These changes caused increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively (Daneshvar et al., Reference Daneshvar, Movahedin, Salehi and Noruzinia2021).

Method of preservation (slow freezing or vitrification) and miRNAs

Vitrification is relatively simple, does not require expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. In addition, cryopreservation of oocytes using vitrification has been suggested to maintain female fertility by servicing and freezing their oocytes at optimal times. Experimental data showed that both cryopreservation methods (slow freezing and glass vitrification) had a negative effect on miRNAs and the pattern and gene expression of human MII oocytes compared with non-frozen MII oocytes, but the effects of vitrification on oocyte physiology (developmental competence of oocyte) was less in slow freezing (Monzo et al., Reference Monzo, Haouzi, Roman, Assou, Dechaud and Hamamah2012; Quan et al., Reference Quan, Wu and Hong2017). However, slow-frozen and vitrified MI oocytes showed signatures for specific gene expression. Slow freezing with downregulation of genes that maintain chromosomal structure (KIF2C and KIF3A) and cell cycle regulation (CHEK2 and CDKN1B) may reduce oocyte developmental competence. In vitrified oocytes, many genes of the ubiquitination pathway are downregulated (including members of the ubiquitin-specific peptidase family and subunits of the 26S proteasome; Monzo et al., Reference Monzo, Haouzi, Roman, Assou, Dechaud and Hamamah2012). The controlled-rate cryopreservation method (slow freezing) seriously alters specific transcript levels, leading to loss of transcript content (Chamayou et al., Reference Chamayou, Bonaventura, Alecci, Tibullo, Di Raimondo, Guglielmino and Barcellona2011; Monzo et al., Reference Monzo, Haouzi, Roman, Assou, Dechaud and Hamamah2012; Stigliani et al., Reference Stigliani, Moretti, Anserini, Casciano, Venturini and Scaruffi2015).

Experimental data showed that the expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 downregulated and 29 upregulated unique transcripts. Gene Ontology analysis using the DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development (such as chromosome organization, RNA splicing and processing, cell cycle, cellular response to DNA damage and to stress, DNA repair, calcium ion binding, malate dehydrogenase activity, and mitochondrial activity; Stigliani et al., Reference Stigliani, Moretti, Anserini, Casciano, Venturini and Scaruffi2015). A study on mice indicated that the influence of vitrification on the transcriptome of oocytes was negligible as no differentially expressed genes were found between vitrified and fresh oocytes. The MII stage is more suitable for oocyte vitrification with respect to the transcriptome (Gao et al., Reference Gao, Jia, Li, Ma, Huang, Zhu, Hou and Fu2017).

Comparison of IVF results from slow-frozen and vitrified oocytes showed that vitrification improved survival, fertilization, and pregnancy rates (Cao et al., Reference Cao, Xing, Zhang, Wei, Zhou and Cong2009; Fadini et al., Reference Fadini, Brambillasca, Renzini, Merola, Comi, De Ponti and Dal Canto2009), although only Fadini et al. (Reference Fadini, Brambillasca, Renzini, Merola, Comi, De Ponti and Dal Canto2009) reported significantly higher pregnancy rates (18.2 vs. 7.6%). Increasing evidence on the effectiveness of IVF with vitrified oocytes showed that it could achieve similar results to IVF using fresh oocytes, with an oocyte survival rate of more than 84% (Rienzi et al., Reference Rienzi, Cobo, Paffoni, Scarduelli, Capalbo, Vajta, Remohí, Ragni and Ubaldi2012). A meta-analysis of five studies showed that the fertilization rate, embryo cleavage, high-quality embryos and ongoing pregnancy progress did not differ between the vitrification and fresh oocyte groups (Cobo and Diaz, Reference Cobo and Diaz2011). Some IVF programmes now use vitrification to cryopreserve oocytes (Brison et al., Reference Brison, Cutting, Clarke and Wood2012; Glujovsky et al., Reference Glujovsky, Riestra, Sueldo, Fiszbajn, Repping, Nodar, Papier and Ciapponi2014).

Applications of miRNAs in assisted reproduction technology

Due to the important role of miRNAs in the suppression or expression of post-translational mRNAs, in addition to treating some diseases, they can be used to improve the maturity of oocytes, in vitro-produced embryos and determine the non-invasive quality of oocytes. Data showed that increased expression of HDAC6 by miR-762 suppression may improve the current protocol for oocyte vitrification. Overexpression of HDAC6 or reduction of miR-762 improved the survival rate, cleavage rate, and blastocyst rate of oocytes after oocyte vitrification (Wang et al., Reference Wang, Zhang, Zhao, Kuang and Xue2018). The results showed that fresh ovine embryos expressed the HDAC1 gene at a higher level than their vitrified counterpart embryos (Bahr et al., Reference Bahr, Robey, Luchenko, Basseville, Chakraborty, Kozlowski, Pauly, Patel, Schneider, Gottesman and Bates2016). Histone acetylation is critical for proper cellular functions, including chromosome condensation, DNA double-stranded breakage repair, and mRNA transcription, but there is no evidence for the effect of HDAC6 on in vivo development of the blastocysts from cryopreserved oocytes. The interpretation of this issue will depend on future works.