{"paper_id":"e5800236-2636-4838-a93f-0d60f92c65d7","body_text":"Over the past forty years, ART has been steadily on the rise, allowing millions of infertile couples to conceive. Currently, it is estimated that over nine million children have been conceived using ART [ 1 , 2 ]. While the number of IVF cycles varies widely worldwide, approximately 5% of births are secondary to the use of ART in some European countries [ 2 ]. The main driver of IVF utilization is individuals being affected by infertility (approximately 15% of couples). However, there is a continuous rise in the number of individuals who freeze their eggs or embryos for future use [ 2 , 3 , 4 , 5 , 6 , 7 ]. For example, nearly 310,000 frozen embryo transfer (FET) cycles were performed in Europe in 2018 [ 2 ].\nCurrent evidence indicates that ART is safe; however, an association between ART and an increased incidence of low birth weight, birth defects, altered growth, and metabolic disorders has been reported [ 8 , 9 ]. These findings might be secondary to epigenetic dysregulation of gametes and embryos [ 10 , 11 , 12 ]. Given the continuous rise in the number of cycles that involve oocytes and embryo cryopreservation, it is critical to understand whether cryopreservation is harmful to the future health of children. In this manuscript, we describe the impact that vitrification has on potential epigenetic modifications and consequences for future offspring health.\n\nThe advancements in oocyte cryopreservation found a perfect application for fertility preservation for social reasons or in patients affected by cancer. Indeed, societal changes have resulted in the postponement of the age of the first pregnancy [ 1 , 2 ], at the time when diminished ovarian reserve significantly reduces the chance of success. Therefore, multiple patients aim to freeze their eggs for future use. In the UK, elective egg freezing is the fastest growing fertility treatment, with an increase of 10% per year [ 13 ]. In Spain, egg freezing cycles increased from 4% of total vitrification procedures to 22% in 10 years [ 14 ]. In the USA, fertility preservation cycles increased from 9607 in 2017 to 13,275 in 2018; similar trends have been observed in other countries [ 15 , 16 ].\nEgg freezing is also used by young cancer patients, since treatment for malignancies might negatively affect future fertility [ 17 , 18 ]. According to the International Agency for Research on Cancer, in 2020, there were an estimated 19.3 million new cancer cases, with nearly 10 million cancer deaths. Female breast cancer has surpassed lung cancer as the most commonly diagnosed cancer, with more than two million new cases per year [ 17 ].\nAnother important application of oocyte cryopreservation is in egg donor programs [ 19 ]. Since the description by Trounson of the first successful pregnancy following oocyte donation in Australia [ 20 ], the number of oocyte donation cycles has doubled in the last decade. For example, in the USA, the number of cycles increased from 10,801 in 2000 to 24,300 in 2016 and 49,193 in 2017 [ 21 ]. In 2017, 17,099 donors underwent an average of 2.4 oocyte collections [ 22 ]. The need for finding a large number of egg donors has resulted in the creation of multiple oocyte banks. In particular, an oocyte bank performs the egg retrieval and cryopreservation of oocytes, which are later transported to the receiving clinic. Then, the imported oocytes, in the IVF laboratory of the recipient center, are warmed, fertilized with the ICSI technique using fresh or frozen sperm, cultured, and transferred to the recipient’s uterus or possibly biopsied for PGT procedure and frozen again [ 23 , 24 , 25 , 26 ].\nSeveral studies have analyzed the efficiency of oocyte vitrification. Importantly, egg donor vitrification provides high survival rates after warming and a similar pregnancy rate compared to cycles performed using fresh donor oocytes ( Figure 1 ) [ 25 , 26 , 27 , 28 ].\nIn addition to egg freezing cycles, the ART field has assisted in a significant increase in embryo freeze-all cycles. It has been estimated that 600,000 embryos were stored from 2004 to 2013 in the USA alone, and 309,475 FET were completed in 2018 in Europe ( Figure 2 ) [ 2 ]. Reasons for embryo cryopreservation are multiple and include storage of surplus embryos following a fresh transfer [ 29 , 30 ], fertility preservation for cancer patients, and pre-implantation genetic testing ( Table 1 ) [ 31 , 32 ]. Additional reasons include abnormalities of the stimulation cycle, including elevated progesterone at the time of trigger (which has been reported to have a negative impact on pregnancy outcomes [ 33 ]) or prevention of ovarian hyperstimulation syndrome, a potentially life-threatening complication [ 34 , 35 , 36 ].\nIt is important to note that FET is associated with a higher birth weight compared to fresh embryo transfer and no embryo freezing [ 37 , 38 , 39 ]. A meta-analysis of 26 studies reported that singletons born following freezing and thawing had higher birth weights, were large for gestational age, and the pregnancy had an increased risk of hypertensive disorders [ 40 ]. An increased birthweight in ART babies conceived following FET has been reported by several authors [ 41 , 42 , 43 , 44 ]. At present, it is unclear whether the vitrification procedure itself, the use of cryoprotective agents (CPAs), the drugs used for endometrial preparation, or parental infertility are responsible for the higher birthweight in offspring. However, since no difference in birth weight has been observed when embryos are transferred in a natural cycle, it is possible that the drugs applied for endometrial preparation might be responsible for that condition [ 45 ].\n\nCryopreservation enables the long-term preservation of tissue or cells at ultra-low temperatures (stored in liquid nitrogen at −196 °C) in a state of suspended animation. This process interrupts all biological activities and maintains cell viability and physiological competency for future use. The first report of a live birth following the transfer of a cryopreserved and thawed embryo was recorded in Australia by Trounson and Mohr in 1983 by the “slow freezing” procedure [ 46 ]. Later, in the 1990s, a great advancement in the field was achieved with the introduction of the “vitrification” protocol in Japan and Australia [ 4 , 5 , 47 ]. Vitrification was rapidly adopted since it achieved better outcomes in terms of gamete and embryo survival and higher pregnancy rates, compared to slow freezing [ 29 , 30 , 48 ]. Vitrification is performed using a high concentration of CPAs. These agents increase viscosity and inhibit ice crystal formation, inducing the solution to enter a “glassy state” [ 7 ]. The success of vitrification is correlated with several factors, such as the temperature in the vitrification and warming steps, which depends on the choice of carrier used (open or closed vitrification) and, most importantly, the concentration and type of CPAs used ( Table 2 ). Regarding the temperature, it has been clearly shown that the warming rate is as important as the cooling rate. Seki and Mazur reported that cryo-damage might also be induced by re-crystallization in the warming step. They examined the relationship between cooling versus warming rates in a mouse model and concluded that a warming rate of at least 3000 °C/min is imperative to obtain an acceptable survival rate above 80% [ 49 ]. CPAs play a critical role in the success of cryopreservation and are classified into two categories: Permeating and non-permeating agents. The first group includes small molecular weight compounds (less than 400 Da) that can cross cell membranes and, once inside, protect the cell from cryo-induced damage. Permeating agents include ethylene glycol (EG), dimethyl sulfoxide (DMSO; an amphipathic molecule), propylene glycol or 1,2 propanediol (PG), glycerol (GLY), formamide (FMD), methanol (METH), and butanediol (BD; 2,3-butanediol). DMSO and glycerol are the two most used ( Table 3 ). Non-penetrating CPAs are non-diffusible, normally have a higher molecular weight, and therefore cannot cross the cell membrane. Examples are trehalose, sucrose, glucose, mannitol, galactose, and polyvinylpyrrolidone (PVP). These molecules induce an osmotic gradient that removes water from inside to outside the cell (dehydration), reducing the temperature at which ice starts to form and thus preserving membranes and intracellular structures [ 50 , 51 ].\n\nThe principal problem that can occur with cryopreservation is the formation of ice crystals. Human embryos and oocytes contain a high content of water, which might be converted into ice, causing irreversible damage and cellular death. This concern was elegantly described by Mazur in 1963 [ 56 ]. The sharp reduction of temperature might lead to cold-shock harm and impair the function of several sensible structures located in the oocyte cytoplasm, including membrane permeability, cytoskeleton architecture, and, importantly, the meiotic spindle apparatus [ 57 , 58 ]. The meiotic spindle is a cytoskeletal structure, formed of microtubules and associated proteins [ 59 ]. It is considered an indicator of oocyte health; its stability is linked with normal fertilization and is directly responsible for the correct segregation of chromosomes, avoiding errors in chromatin division, accountable for aneuploidies and miscarriage [ 60 ]. It is well established that temperature changes can debilitate meiotic spindle stability [ 61 ]. At a temperature of 33 °C or lower, the meiotic spindle starts to depolymerize, and only a few minutes of exposure to non-physiologic pH or temperature is sufficient to induce disassembly of the spindle [ 62 ]. Several studies on both animals and humans have demonstrated a negative association between temperature, as well as osmolality on normal microtubule disassembly, and spindle alterations [ 59 , 60 , 61 , 62 , 63 ]. Additional impairment following cooling and warming includes premature hardening of the zona pellucida (ZP), which is essential at the time when sperm fertilizes the oocyte. These facts indicate the use of ICSI to fertilize oocytes. However, questions remain concerning the impact of ZP hardening and implantation of the embryo [ 64 ]. It is also possible to observe cryo-damage to intracellular organelles, as well as an increased risk of parthenogenetic activation of the oocytes [ 65 ]. Oocyte exposure to CPAs might cause ultrastructural modification of the mitochondria and smooth endoplasmic reticulum [ 66 , 67 ]. Animal studies have suggested that oocyte cryopreservation, particularly vitrification, might be associated with increased levels of reactive oxygen species (ROS) and apoptotic events [ 68 , 69 , 70 ], which might alter the epigenetic mechanisms associated with oocyte competence and future embryo development and viability [ 70 , 71 ]. In particular, DMSO is a known radical scavenger and, as an antioxidant, helps to protect cells from the damage caused by free radicals. However, at normal or decreased levels of ROS, it may restrict cell metabolism by scavenging the electrons needed for ATP production. Therefore, a decrease in DMSO-induced ATP might cause downstream effects that may disrupt cellular function, fetal development, and implantation potential [ 72 , 73 , 74 , 75 ]. Finally, over the past few years, several reports have shown the detrimental effects of cryopreservation programs on the epigenetic makeup of the embryo, protein expression, and DNA integrity [ 76 , 77 , 78 , 79 , 80 ], as well as alteration of such genes involved in critical biological processes [ 79 , 80 , 81 ], inducing an increase in free radical production and apoptosis [ 81 , 82 , 83 , 84 , 85 ].\n\nIn 1942, Conrad Waddington, a biologist at Edinburgh University, was the first to emphasize the importance of environmentally directed changes during the early stages of mammalian embryo development and introduced the term “Epigenetics”. Epigenetics is a gene-regulatory mechanism that leads to heritable changes in gene function that are not associated with changes in DNA sequence [ 86 ]. The importance of epigenetics in the ART field is secondary to the fact that epigenetic changes can be caused by different environmental agents and that important epigenetic changes occur during embryo development. There are two epigenetic reprogramming phases. The first resets DNA methylation marks in primordial germ cells (PGCs) when they migrate to the fetal gonadal ridge. The second wave of DNA methylation changes occurs during the early stage of embryo development, following fertilization; the parental genome is actively demethylated, while the maternal genome is passively demethylated with a wave of re-methylation at the blastocyst stage ( Figure 3 ) [ 87 , 88 , 89 ]. In summary, the epigenome of the preimplantation embryo is highly susceptible to external and internal modifications.\nDNA methylation is the most investigated epigenetic process and involves the addition of a methyl group at the 5′ carbon position of the cytosine pyrimidine ring in the context of CG dinucleotide (CpG sites). Those epigenetic modifications are maintained by daughter cells throughout cell divisions by DNA methyltransferases (DNMTs). To date, five different types of DNMTs have been identified: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, and Dnmt3L [ 90 , 91 ]. DNA methylation is generally correlated with gene silencing, but it is also involved in other regulatory mechanisms such as imprinting or X-chromosome inactivation and silencing of centromeric sequences [ 90 , 91 , 92 ]. Additional epigenetic regulations comprise post-translational histone modifications, including acetylation, methylation, phosphorylation, and glycosylation ubiquitination [ 6 ]. Histone lysine acetylation is particularly important, since it plays a role in cellular differentiation and might be associated with disease processes [ 93 ]. This histone modification is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) and is generally associated with transcriptionally active regions of the genome, as it relaxes the chromatin structure, allowing for increased accessibility of the DNA to transcription factors and other regulatory proteins [ 92 , 93 , 94 ]. Acetylation leads to open chromatin configuration, enhances transcriptional activity, and encourages transcription factor binding to DNA. On the contrary, deacetylation is correlated with transcriptional inactivation and gene silencing [ 94 ]. SUMOylation and de-SUMOylation marks indicate the addition and removal of SUMO (small ubiquitin-related modifier) polypeptides on lysine residues [ 95 ], which are essential for the occurrence of oocyte maturation, meiotic resumption, and spindle formation [ 95 , 96 , 97 ]. Finally, another newly identified epigenetic modification is lactylation, affected by cellular lactate levels, which directly stimulates gene transcription [ 98 ].\nAn important subgroup of genes affected by epigenetic regulation are imprinted genes [ 99 , 100 ]. Currently, around 150 genes have been identified in mice, and less than 100 in humans [ 100 ]. A list of the current mammalian imprinted genes is available online at [ https://www.otago.ac.nz/biochemistry/research/facilities/otago652955.html , accessed on 1 January 2023]. These genes are characterized by a monoallelic expression that is dependent on the parental origin of the allele. The parental imprint is linked to differential epigenetic labeling of parental alleles, and importantly is established during gametogenesis and maintained during the early stage of preimplantation embryo development [ 101 , 102 , 103 ]. The correct expression of those imprinted genes depicts a critical role in growth and development and are prevalently located in the placenta and brain [ 104 , 105 , 106 , 107 ]. Examples include loss of imprinted DNA methylation at the Kvdmr icr, found in ART-conceived children with Beckwith–Wiedemann syndrome (BWS) [ 108 ] or gain of methylation because of maternal uniparental disomy on chromosome 7 at the Mest icr in approximately 10% of Silver–Russell Syndrome (SRS) cases, as well as Angelman syndrome (AS) and Prader–Willi syndrome [ 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ]. While epigenetic changes can affect the individual, new evidence suggests that there could be a transgenerational transmission of epigenetic information [ 114 ]. It is therefore possible that the presence of chemical compounds such as cryoprotective agents could alter the reprogramming machinery and cause long-term risk of disease, as postulated by the Developmental Origin of Health and Disease [ 115 , 116 , 117 , 118 ].\n\nIn the past few years, several research groups have investigated the relationship between vitrification and epigenetic disruption in early embryo development [ 119 ]. The most studied molecule and the one most widely used is DMSO. DMSO may impact cellular functions, metabolism, enzyme activities, cell growth, and apoptosis, as well as might induce alterations in microRNAs (miRNA) and epigenetic changes [ 120 , 121 ]. Studies have shown that DMSO has temperature-, time-, and concentration-dependent toxic effects [ 73 , 74 ]. Studies focusing on the effect of DMSO and epigenetic changes have reported that DMSO interferes with the activity of the enzyme DNMT3a, even though the specific mechanism is unknown [ 119 , 120 , 121 ]. Studies on animal models have shown that following vitrification-warming of mouse oocytes, the expression of the imprinted gene  Kcnq1ot1  decreased significantly [ 122 ]. Chen and collaborators reported that following vitrification of mature bovine oocytes, the expression of imprinted genes  Peg10 ,  Kcnq1ot1 , and  Xist  in blastocysts obtained by ICSI increased abnormally [ 123 ]. The same group in a subsequent publication found that vitrification of mouse MII oocytes affected the expression of the maternally imprinted genes  Peg3 ,  Peg10 , and  Igf2r  in oocytes, and maternally imprinted genes  Peg3  and  Peg10  and paternal imprinted gene  Gtl2  in cleavage stage embryos [ 124 ]. Another study found that methylation of imprinted genes  H19 ,  Peg3 , and  Snrpn  decreased in mouse blastocysts obtained from vitrified mouse oocytes [ 125 ]. Comparable results have been reported by other authors, showing a reduction in the overall DNA methylation level in oocytes and early embryos following the vitrification process [ 126 , 127 ]. In summary, animal models suggest that vitrification may affect the normal expression of imprinted genes by changing the DNA methylation level, affecting the regulatory region of those genes ( Table 4 ).\nHuman studies are limited. A study on the effects of DMSO on the DNA methylation profile in human cardiac microtissues found dysregulation of DNA methylation pathways. Methyltransferase DNMT1, a key factor for the maintenance of DNA methylation, as well as DNMT3A, essential for both de novo and maintenance of DNA methylation, were upregulated, while  TET1 , which plays an important role in active de-methylation, was downregulated [ 121 ]. Overall, no or limited changes in DNA methylation and imprinted gene expression were found in human oocytes or embryos following vitrification ( Table 4 ). The imprinted genes  H19  and  Kcnq1ot1  showed no differences in DNA methylation in vitrified oocytes. In this study immature oocytes were donated after egg retrieval, and after vitrification warming were in-vitro matured to MII stage [ 130 ]. Liu and colleagues estimated the effects of vitrification on nuclear configuration and global DNA methylation in GV-stage oocytes after vitrification warming and in-vitro maturation to MII stage. They found no significant differences in the distribution of mitochondria and global DNA methylation patterns between the groups. However, the authors reported a significantly higher abnormal configuration of the spindle following vitrification [ 129 ]. De Munck reported no significant change in the overall DNA methylation level of in-vitro cultured eight-cell embryos derived from vitrified oocytes [ 128 ]. Huo and colleagues, using 16 donated human MII oocytes, observed that a total of 1987 genes were differentially expressed following oocyte vitrification warming compared to fresh mature oocytes and found that about 82% of these genes were downregulated, while 18% were upregulated [ 136 ]. Those genes involved in several critical biological processes, such as two meiosis-related genes,  Ncapd2  and  Tubgcp5 , were significantly downregulated following oocyte vitrification. In addition, cryopreservation might induce histone changes in oocytes and preimplantation embryos. Suo and colleagues found that the acetylation status of histone H4 at lysine K12 in mouse oocytes was significantly increased in cryopreserved compared to fresh oocytes [ 137 ]. Another study evaluated the consequences of mouse embryo vitrification at two cell stages on specific histone marks (H3K9 methylation and H3K9 acetylation) for the genes  Igf2  and  Oct4.  The authors found no significant difference in the expression level of these genes and their histone marks in vitrified and non-vitrified embryos, while only embryo culture induced changes on these loci [ 138 ]. Other pathways that were altered following vitrification included several physiological processes, such as oogenesis, cellular response to heat, microtubule-based processes, methylation, ubiquinone biosynthetic processes, sister chromatid migration, DNA repair, oxidative phosphorylation, and ATP metabolic processes [ 139 , 140 , 141 , 142 ]. The authors also investigated the time of storage of vitrified oocytes in nitrogen and found no alteration in gene expression, suggesting that overall, the potential damage resulting from oocyte vitrification might be associated with the cryopreservation process itself rather than the storage [ 136 ]. This finding was confirmed by Stigliani and collaborators, who analyzed the gene expression status between surviving warmed oocytes after three and six years of storage in liquid nitrogen and found no differently expressed genes [ 143 ]. The effects of the length of freezing embryos in liquid nitrogen on thawing survival, blastocyst viability, and implantation were recently investigated by Yan and colleagues, who evaluated pregnancy outcomes following different lengths of storage (from less than three years up to 10 years). The authors found a reduced survival rate for blastocysts that were stored for longer than six years. Similarly, clinical pregnancy and live birth rates were significantly decreased in blastocysts stored for more than six years compared with the group frozen for less than three years. No difference was reported in the rates of miscarriage and ectopic pregnancy [ 144 ]. In summary, while epigenetic changes in oocytes and embryos following cryopreservation exist, their significance and clinical consequences remain to be fully elucidated [ 145 , 146 , 147 , 148 ]. Future studies are needed to clarify this important issue [ 148 ].\n\nSperm cryopreservation is an essential component of ART that has wide clinical applications while being critical for cancer patients to protect their fertility before receiving chemotherapy or radiotherapy [ 149 , 150 , 151 ]. Cryopreservation of human sperm has been practiced for more than 50 years [ 151 ]. In the past decade, sperm vitrification has been shown to achieve a higher survival rate and reduced sperm DNA damage compared to slow-freezing protocols [ 151 , 152 , 153 , 154 ]. Several studies have investigated the impact of sperm cryopreservation on epigenetic markers, including DNA methylation, histone modification, and non-coding RNA molecules [ 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 ]. De Mello and co-authors investigated the effect of CPA, methanol, ethyl glycol, and glycerol dimethylsulfoxide on DNA methylation of  Colossoma macropomum  sperm and embryo evolution and found that the cryoprotectants investigated induced an overall reduction in DNA methylation levels in spermatozoa, and also caused a significant delay in embryonic development [ 163 ]. In contrast, a study by Depince and collaborators reported that DNA methylation of zebrafish spermatozoa significantly increased after cryopreservation with methanol [ 164 ]. Salehi and colleagues studied DNA methylation and histone modification, as well as cellular features, including membrane integrity, mitochondria activity and apoptosis, and fertility potential, of rooster semen before and after cryopreservation. The results showed that cryopreservation leads to significantly reduced values of the parameters examined when correlated with fresh samples. Furthermore, there was a significant reduction in H3K9 acetylation and H3K4 methylation compared to the fresh samples [ 165 ]. Another study showed that cryoprotectant and freezing–thawing protocols significantly increased global DNA methylation levels in ram spermatozoa [ 166 ]. Additionally, a study on humans by Khosravizadeh and co-authors investigated the effects of cryopreservation on DNA methylation in promoter regions of the SNURF–SNRPN and UBE3A imprinted genes, PWS-ICR, and AS-ICR in the chromosome 15q11–q13 region [ 167 ]. The authors reported the cryopreservation method to be safe concerning DNA methylation in the chromosome 15q11–q13 region. They found that exposure to cryoprotectants had no significant effect on ROS levels and DNA fragmentation. Neither cryopreservation nor exposure to cryoprotectant significantly affected DNA methylation of the selected gene regions. However, DNA fragmentation had a positive correlation with DNA methylation of AS-ICR [ 167 ]. Different mechanisms could lead to epigenetic changes following cryopreservation. First, cryoprotectant agents could be responsible. For example, CPA, a widely used agent for sperm cryopreservation, is cytotoxic and can harm sperm cells, causing osmotic injury and physiological alterations and potentially influencing the epigenetic state of sperm cells indirectly [ 149 , 154 , 155 , 156 , 157 ]. Second, raising the level of ROS during the freezing–thawing process [ 156 , 159 , 160 , 161 ] might induce site-specific hypermethylation through either the upregulation of DNA methyltransferases (DNMTs) or the formation of new DNMT-including complexes [ 158 , 159 ]. It is important to emphasize that sperm epigenetic changes could be secondary to additional factors, including sperm manipulation alone or patient characteristics [ 166 , 167 , 168 , 169 , 170 ]. For example, it is well known that oligospermic men have more epigenetic changes than normospermic men [ 160 , 161 , 162 ]. However, the number of studies currently available on the topic is still limited. Given the relatively low number of studies conducted using human spermatozoa, additional multicenter studies utilizing the same cryopreservation protocols and DNA methylation analysis are needed to clarify the issue.\n\nIn the last decade, advancements made in the field of cryobiology have contributed to the increased success of ART. However, concerns about the association between cryopreservation and alteration in epigenetic reprogramming exist. This is relevant, given the association between epigenetic changes and future offspring health. Unfortunately, evidence is lacking, and the number of published reports is limited. Future studies and utilization of novel technologies (such as single-cell sequencing and epigenomics) are needed to fully assess the potential epigenetic aberration that occurs at the time of oocytes or embryo cryopreservation, in order to improve its safety and efficacy in ART.","source_license":"CC-BY-4.0","license_restricted":false}