Hypoxia is a critical factor influencing sperm viability by compromising their structural integrity, functional activity, and fertilizing capacity. In reproductive biology and cryobiology, both hypoxia and cryopreservation are known to independently induce cellular stress; however, their combined effects on sperm remain inadequately explored. Here we systematize current knowledge on the effects of hypoxia on sperm cryoresistance, elucidate the molecular mechanisms underlying these effects and evaluate modern strategies for mitigating the combined damage caused by hypoxia and cryopreservation on sperm structure and function. We conducted a comprehensive literature review by analyzing peer-reviewed studies focused on the molecular and cellular responses of mammalian spermatozoa to hypoxia and cryopreservation. Key mechanisms investigated included oxidative stress pathways, mitochondrial function, membrane homeostasis, ion transport and genetic and epigenetic changes. The review also considered current experimental approaches and therapeutic interventions targeting these mechanisms. The analysis revealed that hypoxia disrupts cellular energy metabolism and enhances oxidative stress, leading to reduced sperm survival following cryopreservation. Additionally, cryopreservation itself causes further damage through membrane destabilization, osmotic imbalance and impaired intracellular signaling. These cumulative effects intensify structural and functional deterioration in sperm cells. Emerging mitigation strategies, including antioxidant supplementation, ion regulation, alterations to cryopreservation protocols and the use of hypoxic adaptogens demonstrate potential for improving post-thaw sperm viability. The combined impact of hypoxia and cryopreservation significantly impairs sperm integrity and function, primarily through oxidative and metabolic stress mechanisms. Targeted interventions aimed at counteracting these effects hold promise for enhancing sperm cryoresistance. Finally, this work highlights important avenues for further research and practical applications in reproductive medicine, particularly in fertility preservation and assisted reproductive technologies.
BACKGROUND: Jurkat cells, a human T lymphocyte line widely used in immunological studies, require reliable cryostorage to maintain post-thaw quality. Dimethyl sulfoxide (Me 2 SO) is the standard cryoprotectant, but its cytotoxicity may impair cell performance after thawing. OBJECTIVE: To develop a Me 2 SO-free multicomponent cryoprotectant agent (CPA) for Jurkat cell cryopreservation. MATERIALS AND METHODS: A Me 2 SO-free CPA composed of ethylene glycol (EG), disaccharides (trehalose, sucrose, glucose), and amino acids (histidine, isoleucine, tetrahydropyrimidine) was formulated. The effects of CPA composition and concentration on Jurkat cell cryopreservation were evaluated. Post-thaw viability and proliferation capacity were measured to identify optimal formulations. Cryomicroscopy was used to observe ice crystal growth and recrystallization under different CPA conditions. RESULTS: Disaccharides at 0.1 M improved post-thaw viability, whereas higher concentrations caused a marked decline in viability, indicating concentration-dependent cryoinjury. Among the disaccharides tested, trehalose showed the most consistent protection. Adding 20 mM histidine to 12.5% EG + 0.1 M trehalose increased viability from 87.8% to 93.8% (p < 0.05). Reducing EG to 10% while maintaining trehalose and histidine preserved viability above 91%, comparable to 10% Me 2 SO. Proliferation assays confirmed sustained recovery, with 40 mM histidine producing the highest proliferation. Cryomicroscopy showed that trehalose and histidine suppressed ice crystal growth and recrystallization. CONCLUSION: The optimized Me 2 SO-free, EG-based multicomponent CPA provides effective cryoprotection for Jurkat cells with reduced cytotoxicity and represents a practical alternative to Me 2 SO-based protocols.
This review recounts the advances in cryopreservation technologies of red blood cells (RBC). The limitations of traditional glycerol-based methods and the latest developments in cryopreservation with no or less glycerol are elaborated. Although the use of glycerol at high-concentrations effectively prevents ice crystal formation, the associated osmotic stress damages RBC. Moreover, the cumbersome and time-consuming deglycerolization process not only increases operational costs, but also poses the risks of contamination and additonal cell damage, thereby limiting the application of frozen blood for emergency situations. To overcome these challenges, novel strategies have focused on rapid freezing with no or less glycerol, new permeable cryoprotectants and the synergistic use of non-permeable macromolecular/saccharide protectants (such as trehalose and hydroxyethyl starch). These approaches aim to achieve more efficient and safer preservation through multi-faceted protective mechanisms via the reduction of ice crystal damage, osmotic stress and oxidative damage. New strategies demonstrate significant advantages in streamlining processes (eliminating or simplifying deglycerolization steps), improving cell recovery and quality (low hemolysis) and enhancing in vivo efficacy. However, challenges such as long-term storage stability, scale-up production costs, standardization and regulatory approval remain critical issues to be addressed before clinical translation can be realized. Future research needs to focus on optimizing cryoprotectant formulations, elucidating molecular mechanisms, establishing a standardized quality control system and clinical validation, thereby revolutionizing blood inventory management.
BACKGROUND: Cryopreservation is integral to artificial insemination and germplasm conservation in goats, yet post-thaw sperm survival remains inconsistent due to structural and oxidative damage. Age, a key intrinsic factor, critically modulates sperm functional competence and antioxidative capacity, making it a potential predictor of cryotolerance. Understanding age-associated functional and oxidative biomarkers can refine buck selection strategies to enhance fertility outcomes. OBJECTIVE: To systematically evaluate the influence of age on semen quality, oxidative stress markers, and functional competence of spermatozoa in both fresh and cryopreserved semen of Barbari bucks, with the aim of identifying age-associated functional and oxidative biomarkers predictive of sperm cryotolerance and post-thaw survival. MATERIALS AND METHODS: Eighteen breeding bucks were categorized into three groups: Group I (15 to 25-month young adults), Group II (26 to 40-month mid adults), and Group III (41-to 55-month old adults). Fresh and post-thaw semen was analyzed using CASA, flow cytometry, and biochemical assays for motility, viability, acrosomal and plasma membrane integrity, mitochondrial activity, DNA fragmentation, capacitation status, and antioxidative enzyme activity. RESULTS: Mid-aged bucks (26-40 months) showed significantly higher acrosomal integrity, antioxidant enzyme activity and genomic stability with better cryotolerance compared with younger and older bucks. Younger bucks exhibited incomplete functional maturation, while older bucks displayed oxidative deterioration, higher DNA fragmentation, and reduced antioxidant defense. CONCLUSION: The optimal age window for semen collection in bucks is mid-aged bucks (26 to 40-month old). Functional and oxidative biomarkers provide better predictors of cryotolerance than conventional semen traits, offering actionable insights for AI programs and germplasm conservation.
BACKGROUND: Post-thaw quality of frozen buck sperm is strongly influenced by the choice of diluent and antioxidant supplementation. L-carnitine, sodium pyruvate and casein enhance sperm cryopreservation by providing antioxidant protection, stabilizing membranes, supporting mitochondrial energy metabolism and preserving DNA integrity during the freeze-thaw process. OBJECTIVE: This study aimed to assess the combined effect of L-carnitine, casein and sodium pyruvate addition in semen extenders on cryopreserved buck semen survivability, in vitro sperm parameters and oxidative stress at various months and seasons in a tropical area. MATERIALS AND METHODS: A total of 12 adult Barbari bucks were used in this study. Semen was cryopreserved using an extender supplemented with L-carnitine (2 mM), sodium pyruvate (1 mM), and casein (0.068 g). Post-thaw semen was evaluated for volume, sperm concentration, mass motility, progressive motility, viability, plasma membrane integrity (HOST), DNA integrity, capacitation and apoptosis status, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and superoxide dismutase (SOD) activity. RESULTS: Supplementation significantly improved post-thaw sperm motility, viability, plasma and acrosomal membrane integrity, mitochondrial function, and DNA stability while reducing apoptosis and cryocapacitation compared to the control group. Seasonal variations were observed, with semen collected during September-November and the rainy season showing superior parameters, followed by the transition from rainy to winter. The antioxidants effectively mitigated oxidative stress and preserved mitochondrial and DNA integrity throughout the freeze-thaw process. CONCLUSION: The inclusion of L-carnitine, sodium pyruvate, and casein in extenders significantly improved post-thaw buck sperm quality by preserving motility, membrane integrity, mitochondrial function, and DNA stability while reducing oxidative stress and apoptotic-like changes. These findings highlight the potential of this formulation to enhance the fertilizing capacity of cryopreserved semen across seasonal variations.
Hypoxia is a critical factor influencing sperm viability by compromising their structural integrity, functional activity, and fertilizing capacity. In reproductive biology and cryobiology, both hypoxia and cryopreservation are known to independently induce cellular stress; however, their combined effects on sperm remain inadequately explored. Here we systematize current knowledge on the effects of hypoxia on sperm cryoresistance, elucidate the molecular mechanisms underlying these effects and evaluate modern strategies for mitigating the combined damage caused by hypoxia and cryopreservation on sperm structure and function. We conducted a comprehensive literature review by analyzing peer-reviewed studies focused on the molecular and cellular responses of mammalian spermatozoa to hypoxia and cryopreservation. Key mechanisms investigated included oxidative stress pathways, mitochondrial function, membrane homeostasis, ion transport and genetic and epigenetic changes. The review also considered current experimental approaches and therapeutic interventions targeting these mechanisms. The analysis revealed that hypoxia disrupts cellular energy metabolism and enhances oxidative stress, leading to reduced sperm survival following cryopreservation. Additionally, cryopreservation itself causes further damage through membrane destabilization, osmotic imbalance and impaired intracellular signaling. These cumulative effects intensify structural and functional deterioration in sperm cells. Emerging mitigation strategies, including antioxidant supplementation, ion regulation, alterations to cryopreservation protocols and the use of hypoxic adaptogens demonstrate potential for improving post-thaw sperm viability. The combined impact of hypoxia and cryopreservation significantly impairs sperm integrity and function, primarily through oxidative and metabolic stress mechanisms. Targeted interventions aimed at counteracting these effects hold promise for enhancing sperm cryoresistance. Finally, this work highlights important avenues for further research and practical applications in reproductive medicine, particularly in fertility preservation and assisted reproductive technologies.
BACKGROUND: Dynamic plate cryolipolysis (DPC) has emerged as a non-invasive alternative technique for treating localized adiposity, particularly in anatomically challenging areas. OBJECTIVE: To evaluate the effects of DPC on the cutaneous and subcutaneous tissues of the abdominal region through histological and immunohistochemical analyses. METHODS: Four volunteers underwent distinct protocols of DPC (10, 20 or 30 min) or static cryolipolysis (20 min). DPC protocols were performed at-5 degrees C, followed by 3 min of reperfusion and manual massage. The right infraumbilical region was treated, while the contralateral side served as control. Samples were collected during elective abdominoplasty and analyzed for biomarkers of apoptosis, inflammation, fibroblast activity, collagen remodeling, heat stress response, and metabolic or hormonal modulation. RESULTS: DPC induced adipocyte apoptosis, confirmed by detection of caspase-3, COX-2, and macrophage (CD68 and CD163) expression, fibroblast activation (FGF2 and FGFR1), and a predominance of type I collagen deposition. Additionally, modulation of metabolic and hormonal markers was observed, including reduced PPAR-gamma and aromatase expression, and partial UCP1 expression, suggesting metabolic modulation with potential thermogenic effects. CONCLUSIONS: DPC induced significant and controlled cellular responses, supporting its safety and therapeutic potential. Beyond aesthetic applications, these findings point to broader clinical implications. Larger-scale studies are required to validate and expand these findings.
BACKGROUND: Cryopreservation offers a robust approach for the long-term conservation of sugarcane (Saccharum spp.) germplasm, particularly for maintaining the viability of true seeds used in hybridization programs. According to conservation biology, any plant part capable of regenerating a whole plant can be conserved. However, the effectiveness of cryogenic treatments in preserving sugarcane true seeds requires systematic evaluation. OBJECTIVE: To evaluate the survival dynamics of five sugarcane genotypes under different cryogenic treatments and to determine the efficacy of silica gel dehydration combined with liquid nitrogen exposure in enhancing cryo-tolerance. MATERIALS AND METHODS: Five sugarcane genotypes (Co 1148, 1148-13-11-2-255, CoSe 92423, CoM 9217, and LG 07590) were subjected to four storage treatments: (i) silica gel dehydration followed by liquid nitrogen (Si+LN+), (ii) direct liquid nitrogen storage without dehydration (Si-LN+), (iii) low-temperature storage at -23 degrees C, and (iv) non-cryogenic control. Survival probabilities over 21 days were analyzed using the Kaplan-Meier method with NCSS software. Survival functions were compared across treatments using logrank tests. RESULTS: Across genotypes, Si+LN+ consistently showed the highest survival probabilities over a short-term poststorage assessment duration. Survival curves under Si+LN+ significantly differed from controls and low-temperature treatments (p < 0.001). Among the genotypes, Co 1148 and 1148-13-11-2-255 exhibited the greatest cryo-tolerance, maintaining survival >40% after 21 days, whereas CoM 9217 was the most sensitive. Incorporation of silica gel dehydration effectively mitigated cryo-injury during liquid nitrogen exposure. CONCLUSION: Kaplan-Meier survival analysis provided detailed temporal insights into treatment-genotype interactions, offering more robust statistical evaluation than endpoint germination alone. The findings emphasize the utility of silica gel dehydration in enhancing cryogenic survival and highlight the Si+LN+ protocol as optimal for conserving sugarcane reproductive viability in germplasm conservation programs. The controls maintained higher survival only over the short experimental timeframe, whereas cryopreserved seeds are intended for indefinite storage without genetic or physiological deterioration.
BACKGROUND: Carboxymethyl cellulose (CMC), a cellulose derivative, is osmotically inert and has low viscosity compared with other non-permeating cryoprotectants. OBJECTIVE: To assess whether a combination of CMC with glycerol can improve the efficiency of freezing extender for dog sperm cryopreservation. MATERIALS AND METHODS: Dog spermatozoa were diluted and frozen in freezing extender supplemented with 0% (control), 0.1%, 0.25%, 0.5%, or 0.75% (w/v) CMC. Post-thaw, the sperm kinematic parameters (CASA), plasma membrane integrity (SYBR14/PI), and acrosome integrity (FITC-PSA) were evaluated. The expression of genes related to apoptosis (BCL2, BAX, and ANAXA), motility (BACTIN), cold shock proteins (YBX3), and ROS (CAT, ROMO1, and SMOX) was evaluated using qPCR. RESULTS: The total motility was higher in the 0.1% CMC group compared to the other groups ( P <0.05). In addition, the expression of the antiapoptotic gene BCL2 was upregulated in the 0.1% CMC group compared to the control, whereas the gene expression of BAX and SMOX exhibited lower levels in the 0.1% and 0.25% CMC groups than the control group ( P <0.05). The expression of YBX3 genes was significantly decreased with 0.25% CMC supplementation. CONCLUSION: The addition of CMC to a freezing extender improved sperm motility and alleviated cryostress by regulating the gene expression of BCL2, BAX, SMOX, and YBX3.
Over the past 35 years, encapsulation technologies have played an important role in the development and diversification of cryopreservation techniques for cells and organized structures from numerous plant species. Encapsulation with calcium alginate was initially developed as a technological approach for the production of synthetic seeds. However, the coating of biological material with a hydrogel matrix subsequently promoted new applications that provided additional benefits to in vitro multiplication and preservation techniques. Alginate coatings can be complemented with growth regulators, antioxidants, and nanoparticles. These compounds have helped mitigate the toxic effects of cryoprotectants by regulating their penetration rate and have enabled the implementation of drastic desiccation treatments that would have otherwise been lethal. Furthermore, encapsulation facilitates the simultaneous handling of large quantities of samples, simplifying the ongoing handling required. Cryopreservation protocols based on encapsulation include Encapsulation-Dehydration, Encapsulation-Vitrification, V- and D-cryoplate methods. The objective of this review is to provide information on the impact of encapsulation of plant material on the advancement of cryogenic procedures and, consequently, on the understanding of tolerance of plant tissues to cryopreservation. In view of the advantages of encapsulation, it is considered that it will facilitate the long-term conservation of a greater number of plant species.
BACKGROUND: Arundina graminifolia is a traditional medicinal plant of the Dai people in China. To enhance its ex situ conservation greater knowledge is needed on the species' seed storage behavior, particularly the effects of developmental age, moisture content and temperature and storage time on seed survival (germination). OBJECTIVE: To investigate whether A. graminifolia seeds can be preserved using conventional seed banking and/or cryopreservation in relation to seed developmental age and lipid thermal fingerprinting. MATERIALS AND METHODS: Naturally pollinated and artificially pollinated capsules at three maturity stages were collected (i.e., six seed lots), equilibrated to 15%-75% relative humidity (RH), and stored at 4 degrees C,-20 degrees C and in liquid nitrogen (LN). Seed quality, including after 3 and 6 months of storage, were evaluated using asymbiotic germination. Seed water and lipid thermal properties were determined by differential scanning calorimetry. RESULTS: Seed maturity state was confirmed by the moisture content decrease of fresh seeds as well as the lipid transition enthalpy increase (from c. 10 to 20 J g(-1) DW) and melting peak temperatures of dry seeds. Immature seeds (either as green capsules [GC] or 60 days after pollination [DAP60]) had some level of desiccation sensitivity (up to germination halving) and significant losses of germination during storage. Mature seeds (from capsules just before dehiscence [JBD], fully dehisced [FD], DAP90 and DAP120]) retained high germination levels under the vast majority of RH x Temperature x Time combinations. DSC analysis of the enthalpy of transitions during cooling and warming revealed that seeds equilibrated to <= 75% RH were below the unfrozen moisture content. CONCLUSION: Mature seeds of A. graminifolia seem to have orthodox seed storage behaviour, and both cold storage and cryopreservation appear feasible for the long-term ex situ conservation of this species.
Modern biological and medical technologies require low-temperature storage of cells, tissues, and tissue-engineered constructs to facilitate their transfer and to prolong their usability prior to application. Many industries, including biological research, medicine, and agriculture, depends on low-temperature preservation to preserve the integrity and viability of cells, tissues, and organs for long periods of time. Nanotechnology has had an influence on low-temperature preservation in recent years, providing advanced solutions that greatly enhance biological sample storage. Development of metal oxide and inorganic metal nanoparticles (MNPs) has drawn interest in several biotechnological and medicinal domains, including cryopreservation. The integration of MNPs into reproductive biology protocols represents a novel and emerging research area. Although AI has long been used in medicine, the recent rise of deep learning is a major factor in the growth of this field. Sperm morphology categorization has been automated using deep learning frameworks that focused on different sperm cell components. ROS generation is significantly elevated during sperm cryopreservation. Low, medium, and high ROS levels in cells cause apoptosis, auto-phagocytosis, and necrosis, respectively. Antioxidants can be added to semen extenders to reduce the elevated ROS levels during cryopreservation.
BACKGROUND: Sperm cryopreservation is essential for assisted reproductive technologies, yet current methods face challenges regarding cryoprotectant toxicity and protocol standardization. OBJECTIVE: To develop an optimized cryopreservation system by investigating cryoprotective mechanisms and establishing standardized freezing protocols without using any animal-derived material. MATERIALS AND METHODS: Thermodynamic analysis was performed to evaluate the cryoprotectant efficacy of glycerol (i.e., its ice crystal inhibition properties). An optimized formulation combining 10% glycerol with recombinant human serum albumin (rHSA) was developed to replace traditional egg yolk components. A novel freezing protocol incorporating gradient cooling and precise nucleation at-8.5 degrees C was implemented using a custom-designed automated cryopreservation device (+/- 1 degrees C precision). RESULTS: The optimized formulation significantly reduced the melting enthalpy while it maintained post-thaw sperm motility of 75.4 +/- 3.2% and DNA fragmentation index <15%. The freezing protocol achieved 82.6 +/- 4.1% recovery rate and reduced ice crystal formation by 62.3%. Preclinical study demonstrated significant improvements versus conventional methods (increased motility by 27.8% and enhanced DNA integrity by 34.6%, both p < 0.01). CONCLUSION: The study established a standardized, pathogen-free cryopreservation system with demonstrated efficacy in preserving sperm quality. The findings provide both technical solutions for clinical practice and fundamental insights into cryoprotective mechanisms.
BACKGROUND: Spermatogonia is sensitive to the toxicity of chemotherapy and/or radiotherapy agents. Cryopreservation of testis tissue may offer fertility restoration for pre-pubertal male cancer survivors. OBJECTIVE: To investigate the effects of PHD-inhibitor GSK360A on the function of testis graft following cryopreservation. Besides, the underlying mechanism is explored. MATERIALS AND METHODS: Wister rats were randomly divided into three groups: fresh control group (neither freezing nor autograft), vehicle group (freezing + autograft + vehicle), and GSK360A group (freezing + autograft + GSK360A). The rate of apoptotic Leydig cells and expression of RIPK1 and caspase-8 were spermatogonia, as well as microvessel density of graft were measured. RESULTS: GSK360A statistically increased the number of spermatogonia per round tubule, redued the rate of apoptotic Leydig cells, and enhanced the microvessel density of the graft. Furthermore, diminished protein expression of RIPK1 and enhanced caspase-8 expression were found in the GSK360A group than in vehicle group. CONCLUSION: GSK360A exhibits protective effects on testicular frozen graft by increasing the number of spermatogonia and reducing the rate of apoptotic Leydig cells. Its mechanism may be related to attenuating ischaemic injury and switching cellular death mode from necroptosis to apoptosis in testis grafts.
Sperm cryopreservation is a key technology in reproductive medicine, providing patients the possibility to retain viability before medical interventions or age-related decline. Despite its clinical significance, current cryopreservation procedures suffer substantial limits due to cryoinjury, most notably from intracellular ice formation, osmotic imbalance, membrane instability, and oxidative damage. These conditions significantly affect sperm motility, viability, and genetic integrity post-thaw. To overcome these problems, recent breakthroughs have focused on merging nanotechnology and smart biomaterial science to produce next generation cryoprotectants and preservation systems. Nanoengineered cryoprotectants comprising customized nanomaterials such as liposomes, polymeric nanoparticles, and biologically derived exosomes have shown improved membrane protection, effective antioxidant delivery, and reduction of ice nucleation compared to traditional agents. Early preclinical tests reveal that these alterations considerably enhance post-thaw sperm sustainability, minimize DNA fragmentation, and sustain functional ability for fertilization. Moreover, the combination of individualized cryopreservation protocols leveraging microfluidic technology and embedded biosensors allows unprecedented control and real-time monitoring of cryopreservation quality suited to unique patient demands. Despite these gains, further study into nanotoxicity, long-term safety, and regulatory standards is necessary before widespread clinical adoption. Collectively, nanoengineered cryoprotectants and smart biomaterials constitute a promising new frontier, seeking to enhance male fertility preservation with higher efficiency, safety, and tailored solutions.
BACKGROUND: The scientific advance in cryobiology allows the cryostorage of human semen without the use of toxic permeable cryoprotectants. To do this, the cellular suspensions are supplemented with non-permeable cryoprotective agents that possess mild cytotoxicity. Alternatively, the freezing is executed in vitrification solutions and preservation extenders, with preliminary selected spermatozoa via density-gradient centrifugation and swim-up techniques, although these procedures sometimes cause sperm DNA fragmentation. OBJECTIVE: To introduce unique results concerning the cryopreservation of larger volumes native human semen (above 20-30 & micro;L) on water-repellent soot-coated surfaces, eliminating the involvement of any freezing extenders and formulations. RESULTS: The natural biomolecular matrix of seminal plasma combined with the icephobicity of rapeseed oil soot promoted the survival of over 5,500,000 forward-moving spermatozoa in 1 mL liquefied ejaculate. CONCLUSION: The future improvement of this technology may facilitate the assisted human reproduction, helping to solve the demographic problem in Europe.
BACKGROUND: Vitrification freezing has been shown to reduce mammalian oocyte developmental competence, and this has been strongly associated with abnormal mRNA expression in oocytes thawed by vitrification freezing. However the effect of vitrification freezing on the transcriptional machinery of oocytes examined by RNA sequencing has rarely been reported. OBJECTIVE: To study differentially expressed genes in mouse oocytes as a result of vitrification freezing. MATERIALS & METHODS: Differential gene expression was determined using DEseq2 (p-value <0.05, minimum multiplicity set at 1.5), and the transcriptomes of mouse stage MII oocytes from the Fresh and Vitrification groups were analyzed by employing the Smart-seq technique. The differentially expressed mRNAs were retrieved according to the Gene Ontology (GO) and KEGG databases, and finally, a real-time fluorescence quantitative PCR was used to characterize the reliability of the transcriptome data. RESULTS: Among them, 545 genes were detected to be up-regulated and 274 genes were up-regulated. Ten genes related to oxidative phosphorylation were screened to be down-regulated, and 44 genes were down-regulated with genes related to the HIF-signaling pathway, ribosomes and apoptosis.GO enrichment analysis showed that these genes were mainly concentrated in the cytoplasm, organelle membranes, mitochondrial membranes, and the Golgi apparatus. The four randomly selected candidate genes' mRNA expression levels were consistent with the results of RNAseq. CONCLUSION: Our results suggest that vitrification freezing affects mouse M II stage oocytes mainly through down-regulation of transcription, which leads to a decrease in the developmental capacity of vitrified oocytes. Our findings will help to identify ways to improve the vitrification efficiency of oocytes.
