Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes\' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Assisted reproduction technologies (ARTs) are generally considered safe; however, emerging evidence highlights the need to evaluate potential risks in adulthood to improve safety further. ART procedures like rederivation of embryos by vitrification differ from natural conditions, causing significant disparities between in vitro and in vivo embryos, affecting foetal physiology and postnatal life. This study aims to investigate whether hepatic transcriptome and metabolome changes observed postnatally are already present in foetal livers at the end of gestation. This study compared fresh and vitrified rabbit embryos, finding differences between foetuses obtained by the transfer of fresh and vitrified embryos at 24 days of gestation. Rederived embryos had reduced foetal and liver weights and crown-rump length. However, the offspring of vitrified embryos tended to be born with higher weight, showing compensatory growth in the final week of gestation (59.2 vs. 49.8 g). RNA-Seq analysis revealed 43 differentially expressed genes (DEGs) in the foetal liver of vitrified embryos compared to the fresh group. Notably, downregulated genes included BRAT1, CYP4A7, CYP2B4, RPL23, RPL22L1, PPILAL1, A1BG, IFGGC1, LRRC57, DIPP2, UGT2B14, IRGM1, NUTF2, MPST, and PPP1R1B, while upregulated genes included ACOT8, ERICH3, UBXN2A, METTL9, ALDH3A2, DERPC-like, NR5A2-like, AP-1, COG8, INHBE, and PLA2G4C. Overall, a functional annotation of these DEGs indicated an involvement in lipid metabolism and the stress and inflammatory process or immune response. Thus, our results suggest that vitrification and embryo transfer manipulation induce an adaptive response that can be observed in the liver during the last week of gestation.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

Vitrification is the most promising method for cryopreservation of complex structures such as organs and tissue constructs. However, this method requires multimolar concentrations of cell-permeant cryoprotective agents (CPAs), which can be toxic at such elevated levels. The selection of CPAs for organ vitrification has been limited to a few chemicals; however, there are numerous chemicals with properties similar to commonly used CPAs. In this study, we developed a high-throughput method that significantly increases the speed of cell membrane permeability measurement, enabling ~100 times faster permeability measurement than previous methods. The method also allows assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 °C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation.

Plasma membrane damage in vitrified oocytes is closely linked to mitochondrial dysfunction. However, the mechanism underlying mitochondria-regulated membrane stability is not elucidated. A growing body of evidence indicates that mitochondrial activity plays a pivotal role in cell adaptation. Since mitochondria work at a higher temperature than the constant external temperature of the cell, we hypothesize that suppressing mitochondrial activity would protect oocytes from extreme stimuli during vitrification. Here we show that metformin suppresses mitochondrial activity by reducing mitochondrial temperature. In addition, metformin affects the developmental potential of oocytes and improves the survival rate after vitrification. Transmission electron microscopy results show that mitochondrial abnormalities are markedly reduced in vitrified oocytes pretreated with metformin. Moreover, we find that metformin transiently inhibits mitochondrial activity. Interestingly, metformin pretreatment decreases cell membrane fluidity after vitrification. Furthermore, transcriptome results demonstrate that metformin pretreatment modulates the expression levels of genes involved in fatty acid elongation process, which is further verified by the increased long-chain saturated fatty acid contents in metformin-pretreated vitrified oocytes by lipidomic profile analysis. In summary, our study indicates that metformin alleviates cryoinjuries by reducing membrane fluidity via mitochondrial activity regulation.

UNASSIGNED: Vitrification is a recently introduced yet widely applied assisted reproduction technique. So far, the effects of the chemicals and devices in vitrification on sperm motility and DNA integrity are still unclear.
UNASSIGNED: This study aimed to examine sperm quality, as determined by semen analysis and sperm DNA integrity when vitrified with or without cryoprotectant agents (CPAs) using pulled-glass capillaries.
UNASSIGNED: Between February and June 2020, 50 infertile men from the Hue Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy, Vietnam, were enrolled. Sperm samples, prepared using the swim-up technique, were divided into 2 groups: vitrification with CPAs (group 1) and without CPAs (group 2). Vitrified sperm samples were preserved in 10 µL pulled-glass capillaries. Motility, sperm membrane integrity, and the DNA fragmentation index were tested.
UNASSIGNED: Sperm motility in vitrified media with CPAs (54.4 ± 11%) was statistically higher than in media without CPAs (51.14 ± 10.6%, p < 0.05). CPAs did not affect sperm membrane integrity or large halo ratio (71.34 ± 8.47 vs. 70.38 ± 8.11 and 50.84 ± 18.92 vs. 51.98 ± 19.44, respectively). Group 2 exhibited a lower DNA fragmentation index than group 1 after vitrification (14.2 ± 8.47 vs. 12.60 ± 9.03, p = 0.021).
UNASSIGNED: Using a pulled-glass capillary for sperm vitrification, the presence of CPAs in the vitrification medium resulted in higher progressive motility and lower DNA fragmentation index than the medium without CPAs.

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1\'s efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

Female fertility preservation is a rapidly growing field in medicine. Oocyte cryopreservation and assisted reproductive technique with vitrified-warmed oocytes have been successful with in vivo matured oocytes after conventional ovarian stimulation protocols. The use of in vitro matured oocytes after vitrification and warming has been limited. Capacitation in vitro maturation (CAPA-IVM) represents the latest refinement of IVM protocols and provides in vitro matured oocytes with improved competence. This case report describes the first successful live birth following oocyte vitrification from a CAPA-IVM cycle. This milestone achievement holds a significant promise to expand fertility preservation options and improve accessibility for women wishing to cryopreserve their eggs for future use.