OBJECTIVE: Does vitrification cryopreservation of embryos for more than 5 years affect the pregnancy outcomes after frozen embryo transfer (FET)?
CONCLUSIONS: Vitrification cryopreservation of good-quality blastocysts for more than 5 years is associated with a decrease in the implantation rate (IR) and live birth rate (LBR).
BACKGROUND: Previous studies have predominantly focused on embryos cryopreserved for relatively short durations (less than 5 years), yet the impact of extended cryopreservation duration on pregnancy outcomes remains a controversial issue. There is a relative scarcity of data regarding the efficacy and safety of storing embryos for 5 years or longer.
METHODS: This retrospective study involved 36 665 eligible vitrified-thawed embryo transfer cycles from 1 January 2016 to 31 December 2022, at a single fertility center in China.
METHODS: Patients were divided into three groups according to embryo storage time: Group 1 consisted of 31 565 cycles, with storage time of 0-2 years; Group 2 consisted of 4458 cycles, with a storage time of 2-5 years; and Group 3 included 642 cycles, with storage time exceeding 5 years. The main outcome measures were IR and LBR. Secondary outcome variables included rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage, as well as neonatal outcomes. Reproductive outcomes were analyzed as binary variables. Multivariate logistic regression analysis was used to explore the effect of preservation time on pregnancy outcomes after correcting for confounding factors. In addition, we also assessed neonatal outcomes, such as large for gestational age (LGA) and small for gestational age (SGA).
RESULTS: IRs in the three groups (0-2, 2-5, and >5 years) were 37.37%, 39.03%, and 35.78%, respectively (P = 0.017), and LBRs in the three groups were 37.29%, 39.09%, and 34.91%, respectively (P = 0.028). After adjustment for potential confounding factors, compared with the 0-2 years storage group, prolonged embryo vitrification preservation time (2-5 years or >5 years) did not affect secondary outcomes such as rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage (P > 0.05). But cryopreservation of embryos for more than 5 years reduced the IR (adjusted odds ratio (aOR) 0.82, 95% CI 0.69-0.97, P = 0.020) and LBR (aOR 0.76, 95% CI 0.64-0.91, P = 0.002). Multivariate stratified analysis also showed that prolonging the cryopreservation time of blastocysts (>5 years) reduced the IR (aOR 0.78, 95% CI 0.62-0.98, P = 0.033) and LBR (aOR 0.68, 95% CI 0.53-0.87, P = 0.002). However, no effect on cleavage embryos was observed (P > 0.05). We further conducted stratified analyses based on the number and quality of frozen blastocysts transferred, and the results showed that the FET results after transfers of good-quality blastocysts in the >5 years storage group were negatively affected. However, the storage time of non-good-quality blastocysts was not significantly associated with pregnancy outcomes. Regarding the neonatal outcomes (of singletons), embryo vitrification preservation time had no effect on preterm birth rates, fetal birth weight, or neonatal sex ratios. However, as the storage time increased, rates of SGA (5.60%, 4.10%, and 1.18%) decreased, while rates of LGA (5.22%, 6.75%, and 9.47%) increased (P < 0.05). After adjusting for confounding factors, the increase in LGA and the decrease in SGA were significantly correlated with the duration of storage time.
CONCLUSIONS: This was a retrospective study using data from a single fertility center, even though the data had been adjusted, our findings still need to be validated in further studies.
CONCLUSIONS: With the full implementation of the two-child policy in China, there may be more patients whose embryos have been frozen for a longer time in the future. Patients should be aware that the IR and LBR of blastocysts are negatively affected when the cryopreservation time is longer than 5 years. Couples may therefore consider shortening the time until FET treatment.
BACKGROUND: This study was supported by the National Nature Science Foundation of China (No. 82101672), Science and Technology Projects in Guangzhou (No. 2024A03J0180), General Guidance Program for Western Medicine of Guangzhou Municipal Health Commission (No. 20231A011096), and the Medical Key Discipline of Guangzhou (2021-2023). None of the authors have any conflicts of interest to declare.
BACKGROUND: N/A.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4-6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139-3p, miR-214 and miR-885-3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-β signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-β (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testis was vitrified with the solution. The post-vitrification survival (72.8±3.5%) was markedly improved compared with that achieved using the sucrose-containing solution (44.7±4.2%). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg\'s repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17×105) and conventional slow freezing (10.80×105), compared to the DNA breakpoints of fresh semen samples (5.19×105). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

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UNASSIGNED: Female fecundity decreases significantly after the age of 32, and rapidly so after age 37. There is no treatment to prevent this decline. Furthermore, globally, women are getting married later and the age at which they have their first child is increasing. As of July 2023, elective egg freezing (EEF) or oocyte cryopreservation (OC) for age-related fertility decline, commenced in Singapore. With medical advancements in OC, EEF is no longer considered experimental. The aim of this review is to examine the existing literature around EEF with regard to reproductive outcomes and its safety, to better guide clinicians in counselling young single women.
UNASSIGNED: Published studies were examined to increase understanding on optimal age for EEF, ideal number of oocytes for a live birth, recommended OC protocols, cryopreservation techniques affecting thaw survival or fertilisation, oocyte storage and pregnancy risks.
UNASSIGNED: Models predict that EEF should be performed at age <37 years and to achieve a 70% chance of live birth, women would need 14, 15 and 26 mature oocytes at ages 30-34, 35-37 and >38 years, respec-tively. An antagonist stimulation protocol with an agonist trigger would minimise ovarian hyper-stimulation syndrome and duration of stimulation without affecting outcomes. Oocyte vitrification in comparison to slow freezing increases thaw survival, fertilisation and clinical pregnancy rates. No increased risks exist for the woman, future pregnancy or child when compared with conventional IVF.
UNASSIGNED: EEF is a viable option for single women desiring fertility preservation. Financial costs are significant, but returns are worthwhile if oocytes are utilised.

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte.

This study aimed to evaluate whether the addition of vitamins E and C as two conventional antioxidants improves the cryotolerance of preantral follicles enclosed in ovine ovarian tissue slices. For this purpose, ovarian slices were obtained from abattoired juvenile lambs and randomly distributed to the following groups: fresh, toxicity, vitrified (control), and three treatment groups in two experiments. Vitamin E, vitamin C, or vitamin E + C was added to the vitrification media alone in the first experiment and added to all vitrification, warming, and culture media in the second experiment. Finally, the treated tissues were cultured in vitro for 12 hours. The histological analysis showed that single or combined use of vitamins E and C increases intact preantral follicles in comparison to the control in two experiments (p < 0.05), and simultaneous use of vitamins E and C had a synergistic effect on increasing the percentage of normal preantral follicles in experiment 2 (p < 0.05). Due to the better results in Experiment 2, stromal cell density, antioxidant activity, and molecular evaluation were followed only in this experiment. The vitamin E + C group had higher stromal cell density compared with control group (p < 0.05). Vitamin E strengthened antioxidant capacity compared with the control and vitamin C groups (p < 0.05). This effect was exacerbated when used in combination with vitamin C (p < 0.05). The expression of all evaluated genes (BMP4, BMP15, GDF9, and KITLG) was significantly increased in ovarian tissue treated with vitamin E + C compared with the control group (p < 0.05). This increase was also observed in BMP4, GDF9, and KITLG genes compared with the vitamin C group (p < 0.05). In conclusion, this study revealed the positive effects of vitamins E and C on preantral follicle viability and to some extent a synergistic action of vitamin C on the protective effects of vitamin E against preantral follicle degeneration and increasing antioxidant capacity and development of preantral follicles after ovine ovarian tissue vitrification.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.