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Numerous environmental and endogenous factors affect the level of genetic diversity in natural populations. Genetic variability is the cornerstone of evolution and adaptation of species. However, currently, more and more plant species and local varieties (landraces) are on the brink of extinction due to anthropopression and climate change. Their preservation is imperative for the sake of future breeding programs. Gene banks have been created worldwide to conserve different plant species of cultural and economic importance. Many of them apply cryopreservation, a conservation method in which ultra-low temperatures (−135 °C to −196 °C) are used for long-term storage of tissue samples, with little risk of variation occurrence. Cells can be successfully cryopreserved in liquid nitrogen (LN) when the adverse effect of ice crystal formation and growth is mitigated by the removal of water and the formation of the so-called biological glass (vitrification). This state can be achieved in several ways. The involvement of key cold-regulated genes and proteins in the acquisition of cold tolerance in plant tissues may additionally improve the survival of LN-stored explants. The present review explains the importance of cryostorage in agronomy and presents an overview of the recent works accomplished with this strategy. The most widely used cryopreservation techniques, classic and modern cryoprotective agents, and some protocols applied in crops are considered to understand which parameters provide the establishment of high quality and broadly applicable cryopreservation. Attention is also focused on the issues of genetic integrity and functional genomics in plant cryobiology.

Vitrification, is an ultra-rapid, manual cooling process that produces glass-like (ice crystal-free) solidification. Water is prevented from forming intercellular and intracellular ice crystals during cooling as a result of oocyte dehydration and the use of highly concentrated cryoprotectant. Though oocytes can be cryopreserved without ice crystal formation through vitrification, it is still not clear whether the process of vitrification causes any negative impact (temperature change/chilling effect, osmotic stress, cryoprotectant toxicity, and/or phase transitions) on oocyte quality, which translates to diminished embryo developmental potential or subsequent clinical outcomes. In this review, we attempt to assess the technique's potential effects and the consequence of these effects on outcomes. Summary Sentence: The osmolarity changes and water movement during oocyte vitrification and warming procedures are illustrated. Graphical Abstract

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Genetic improvements in plant breeding are dependent upon having access to novel plant genetic resources that are available in plant genebanks. Many crops that are vegetatively-propagated are maintained as plants in the field or greenhouse, making them vulnerable to biotic and abiotic threats. Increasingly, plant genebanks are using cryopreservation technologies to secure vegetatively propagated collections at secondary locations. Droplet vitrification and cryo-plate cryopreservation methods have been used to successfully cryopreserve the shoot tips of many plant species. New propagule types, including small leaf square-bearing adventitious buds, stem disc-bearing adventitious buds, microtubers and rhizome buds are alternative explants for use in cryopreservation. This review describes new technologies for in-vitro based cryopreservation systems that have advanced the field of plant cryopreservation. Future advances will allow even more diverse germplasm to be successfully preserved in cryobanks.Key messageNew technologies for in-vitro based cryopreservation systems have advanced the field of plant cryopreservation since the twenty first century. Further advances will certainly facilitate even more diverse germplasm to be successfully preserved in cryobanks.

In this review, we provide a perspective on the science and technology of vitrification of waste. First, we provide a background on the general classes of wastes for which vitrification is currently used for immobilization or is proposed, including nuclear and industrial hazardous wastes. Next, we summarize the issues surrounding solubility of waste ions and resulting uncontrolled crystallization or phase separation. Some newer waste form designs propose a controlled crystallization, resulting in a glass-ceramic. A summary of glass systems and glass-ceramic systems is given, with the focus on immobilizing waste components at high waste loading. Throughout, design and processing considerations are given, and the difference between uncontrolled undesirable and controlled desirable crystallization is offered.

Vitrification is a highly efficient technique for the cryopreservation of the human embryo. The effect of delayed blastulation may be responsible for implantation failures and negatively affects in vitro fertilization (IVF) outcomes. The current literature displays discordant results; some studies have announced higher pregnancy rates after day 5 (D5) transfer compared with day 6 (D6) transfer, while others have shown equivalent outcomes. In the present study an investigation into the clinical implications of delayed blastulation (D5 versus D6) was carried out. We performed a retrospective study comparing clinical pregnancies and implantation rates following warmed single blastocyst transfer (WSBT). All patients coming for a programmed warmed transfer at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, were included in this study and divided in two groups according to the day of blastocyst vitrification: D5 (n = 1563) and D6 (n = 517). The overall survival rate was 95.0% (1976/2080) with no significant difference between the D5 and D6 groups: 95.3% (1489/1563) and 94.2% (487/517) respectively. WSBT of D6 blastocysts resulted in a lower implantation and clinical pregnancy compared with D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.4% and 42.6% for the D5 and 37.4% and 32.2% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.32% (1.14% for D5 vs 1.84% for D6). Although the transfer of D6 vitrified-warmed blastocyst remains a reasonable option, priority to a D5 embryo would reduce the time to successful pregnancy.

PurposeTo investigate the clinical efficacy of a “Universal Warming” protocol, based on subsequent steps with 1 M and 0.5 M concentration of extracellular cryoprotectant (ECCP), on shipped oocytes. Oocytes are vitrified using different brands of ready-to-use kits which recommend that the use of their own warming kit and combining different vitrification/warming kits may have legal consequences for assisted reproductive (AR) centers, until this practice has been validated with clinical studies.MethodsRetrospective multi-center transnational observational study. Number of oocytes warmed 1.898. Vitrification performed with vitrification kit (Kitazato, Japan); warming carried out randomly with two different kits: Kitazato warming kit and Vit Kit®-Thaw (FujiFilm Irvine, USA). Warmed oocytes were assigned to 2 groups: KK (Kitazato/Kitazato) 939, and KI (Kitazato/Irvine) 959. Primary endpoint: survival rate. Secondary endpoints: fertilization rate; blastulation rate; implantation rate; live birth rate.ResultsSurvival was comparable between the groups: 84.6% (795/939) in group KK vs 82.1% (787/959) in group KI. Fertilization rate was lower (P = 0.027) in group KK (75.7%—602/795) than in group KI (80.4%—633/787). Blastulation and implantation and live birth rates were all statistically comparable between the study groups: blastulation rate was 58.5% (352/602) vs 57.8% (366/633); implantation rate was 41.5% (80/193) vs 45.9% (84/183); live birth rate was 52.5% (62/118) in KK and 45.0% (54/120) in KI.ConclusionThe use of this “Universal Warming” protocol simplifies vitrified oocyte exchange between AR centers in different countries, and overcomes potential regulatory/commercial/availability differences affecting clinical practice.

We compared post-cryopreserved outcomes of normozoospermic semen samples after cryopreservation by vitrification and liquid nitrogen vapor freezing, using the original and the simplified preservation media. Forty normozoospermic semen samples were used in the study. Post-prepared semen samples were divided into five aliquots: one served as non-cryopreserved control; two were vitrified using in-house (In-house-V) or sucrose media (Simp-V); and the last two aliquots were frozen in liquid nitrogen vapor, using commercial (Com-L) or sucrose media (Simp-L). Sperm after cryopreservation regardless of the media and method used, significantly decreased in motility, viability, and increased ROS level without changes in sperm morphology and DNA fragmentation. Simplified sucrose freezing medium significantly increases post-thawed motility (57.6% (53.2-68.9) vs. 34.5% (27.3-43.6) in Simp-L and Com-L) and viability (61.0% (52.0-67.8) vs. 37.0% (29.3-46.0) in Simp-L and Com-L) in vapor freezing but significantly decrease post-thawed motility (58.8% (51.4-63.3) vs. 77.8% (70.8-81.3) in Simp-V and In-house-V) and viability (59.0% (53.2-66.0) vs. 75.5% (68.0-83.0) in Simp-V and In-house-V) in vitrification. A simplified medium does not affect sperm morphology, ROS level, and DNA fragmentation. In liquid nitrogen vapor freezing, a simplified medium significantly improved sperm motility and viability compared with commercial medium. In vitrification, the simplified medium gave inferior results on sperm motility and viability compared to the original preservation medium.

Vitrification could enable long-term organ preservation, but only after loading high-concentration, potentially toxic cryoprotective agents (CPAs) by perfusion. In this paper, we combine a two-compartment Krogh cylinder model with a toxicity cost function to theoretically optimize the loading of CPA (VMP) in rat kidneys as a model system. First, based on kidney perfusion experiments, we systematically derived the parameters for a CPA transport loading model, including the following: Vb = 86.0% (ra = 3.86 μm), Lp = 1.5 × 10–14 m3/(N·s), ω = 7.0 × 10–13 mol/(N·s), σ = 0.10. Next, we measured the toxicity cost function model parameters as α = 3.12 and β = 9.39 × 10–6. Combining these models, we developed an improved kidney-loading protocol predicted to achieve vitrification while minimizing toxicity. The optimized protocol resulted in shorter exposure (25 min or 18.5% less) than the gold standard kidney-loading protocol for VMP, which had been developed based on decades of empirical practice. After testing both protocols on rat kidneys, we found comparable physical and biological outcomes. While we did not dramatically reduce toxicity, we did reduce the time. As our approach is now validated, it can be used on other organs lacking defined toxicity data to reduce CPA exposure time and provide a rapid path toward developing CPA perfusion protocols for other organs and CPAs.

Sucrose and trehalose are commonly used as non-permeating cryoprotectants in cryopreservation, primarily due to their ability to increase extracellular osmolality, which promotes cellular dehydration and minimizes intracellular ice formation. While the effects of these cryoprotectants on clinical outcomes in human oocytes and sperm have been extensively studied, their roles in the vitrification of human embryo remain underexplored. The optimization of cryoprotectants is crucial for improving pregnancy outcomes in assisted reproductive technology (ART), particularly in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, where surplus embryos or embryos unsuitable for fresh transfer are cryopreserved due to clinical considerations. This study aimed to compare the efficacy of sucrose-based and trehalose-based vitrification solutions for human blastocyst cryopreservation and to evaluate their impact on embryological and clinical outcomes. A retrospective cohort study was performed on 616 patients who underwent frozen embryo transfer (FET) cycles at a single reproductive center from January 2021 to December 2023. The participants were categorized into two groups based on the cryoprotectant used for blastocyst vitrification: sucrose or trehalose. Key outcomes, including implantation rates, proportions of good-quality and poor-quality blastocysts, and clinical pregnancy outcomes, were compared between the groups. A comparative analysis of the sucrose-based and trehalose-based groups yielded the following outcomes: Trehalose-based vitrification solution was associated with improved implantation rates and better post-warming blastocyst quality compared to sucrose-based one. However, since the trehalose-based solution was commercially available, while the sucrose-based solution was laboratory-prepared, the observed differences may not solely be attributed to the type of sugar used. Future studies should aim to clarify the independent effects of trehalose by comparing it with sucrose under standardized conditions within the same type of vitrification solution.