Vitrification Cryopreservation Research Articles

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3mprotein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. N/A.

Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts, which is fundamentally important to ligament reconstruction. However, conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents (CPAs). Here, we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation. Comprehensive optimization was conducted on the methods for vitrification and rewarming, as well as the loading and unloading conditions, based on the critical cooling rate (CCR), critical warming rate (CWR), and permeation properties of the cryoprotectant. Using biomechanical and histological level tests, we demonstrate the superior performance of our method in ligament cryopreservation. After 30 days of vitrification cryopreservation, parameters such as the Young's modulus, tensile stress, denaturation temperature, and glycosaminoglycans (GAGs) content of the ligament remained essentially unchanged. This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament, providing valuable insights for future technique development.

Conservation of Green and White Ash Germplasm Using the Cryopreservation of Embryogenic Cultures

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2–3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (p < 0.05) between experiments and with genotype (p < 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (p > 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and restoration.

Comparative Cryopreservation of Indian Wild Orange (Citrus Indica Tanaka) Embryonic Axes

BACKGROUND: Indian Wild Orange (Citrus indica Tanaka) is an endangered and endemic species from northeast India for which effective ex situ conservation strategies, including embryo cryopreservation, are urgently needed. MATERIALS AND METHODS: Desiccation tolerance and cryopreservation ability for embryonic axes of Citrus indica was determined using three techniques (air desiccation-freezing, PVS2 vitrification-freezing and encapsulation-dehydration-freezing). Success was assessed as survival and recovery in vitro. RESULTS: Successful cryopreservation of embryonic axes was achieved using all three methods, with the highest survival achieved when using air desiccationfreezing (90%) followed by encapsulation-dehydration (85%) and PVS2 vitrification cryopreservation (80%). Regeneration levels were lower than survival levels for all three proceedures. Post-cryo regeneration success was: encapsulation-dehydration (64%) > air desiccation-freezing (55%) > PVS2 vitrification (52%). CONCLUSION: Although there was relatively high post-cryopreservation recovery growth obtained using all the three techniques, the air desiccation-freezing technique is preferred, as it is a simple, practical and reproducible technique for the long-term cryobanking of this important wild species.

New Cryoprotectant Loading Method for Cell Droplet Vitrification with Continuous Evaporation.

Droplet-based vitrification is considered to be a promising cryopreservation method, which achieves high cell viability through high cooling rates and low concentrations of cryoprotective agents (CPAs). However, the droplet vitrification cryopreservation process needs in-depth research, such as the balance of the CPA concentration and the cooling rate, the CPA loading process, and the droplet encapsulation method. Here, we developed a chip with a high cooling rate for vitrification droplet encapsulation and provided a new method for continuous loading of low-concentration CPA droplets by evaporation. The results showed that the CPA droplet volume decreased exponentially with the evaporation time, and the larger the initial droplet size, the longer the evaporation time to achieve the critical vitrification concentration. There was no significant difference in the viability of MSCs, NHEK, and A549 cells between the evaporation loading vitrification method and the traditional slow freezing method, but the former was easier to operate and can balance the cooling rate and concentration by controlling the evaporation time. Moreover, a theoretical model was proposed to predict the CPA concentration inside the microdroplets dependent on the evaporation time. The current work provides a potential method to load low-concentration CPAs for cell vitrification preservation, which is more beneficial for cell therapy and other regenerative medicine applications.

P-220 Ice crystals propagation velocity is related to the type and concentration of cryoprotectants and impacts cells and tissue vitrification success

Abstract Study question How different cryoprotectants at various concentrations affect ice crystals formation and propagation during vitrification? Summary answer The velocity of ice crystal propagation is inversely related to concentration and type of cryoprotectants and is responsible for downstream cell damage during vitrification. What is known already Vitrification has replaced slow freezing for oocyte and embryo cryopreservation and is now being considered also for gonadal tissue. However, the results in terms of both vitrification and rewarming survival rates are still not uniform. Understanding the complex physical events controlling ice crystals formation, propagation and expansion are key for improving cryoprotectant solutions and overcoming challenges. Study design, size, duration Basic science research. Ice crystal propagation velocity was measured in solutions of four cryoprotectants: dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG) and glycerol (Gly), at various concentrations in supercooled temperatures. Each experiment used five 0.25ml and five 2ml straws (volume 0.1 ml and 1 ml) containing different cryoprotectant combinations (DMSO:EG, EG:PG, DSMO:PG, DMSO:Gly, EG:Gly, PG:Gly and EG+PG vs EG+DMSO), immersed in alcohol baths prepared at 8-10 °C below the freezing points of each solution. Participants/materials, setting, methods Alcohol baths were set as follows: (1) between -10 °C and -12 °C for straws with 0% and 10% (v/v) DMSO, EG, PG and Gly; (2) -15 °C to -19 °C for the 20%; (3) -19.5 to -24 °C for the 30%, and for the mixture of 15% EG and 15% PG and 15% EG and 15% DMSO (most used vitrification solutions); and (4) -25 to -36 °C for the 40%. A 50µm thermocouple measured supercooling. Student’s T-test analyzed differences. Main results and the role of chance Seeding was done by touching the straw end with LN- precooled forceps until visualizing a small ice crystal. Following 10 to 60s the distance to which the ice crystals grew inside the straws was evaluated using a fine caliber. Ice crystal propagation was inversely correlated to type and cryoprotectant concentrations. Propylene glycol showed statistically significant lower ice crystals growth velocity up to concentrations of 30% (v/v), compared to DMSO and EG at the same concentrations (p < 0.05). Moreover, the ice crystal velocity of PG was also lower than Gly at these same concentrations. The combination of EG with PG decreased ice crystal propagation better than EG with DMSO. At concentrations of 40%, there were no differences. These results suggest that current cryopreservation vitrification solutions are not optimized and combining PG with other CPs has advantages. Using a vitrification solution of 40% DMSO or EG, the velocity of crystal growth was in the range of 0.1mm/sec (6mm/min). This means that to vitrify large tissues, a cooling rate of over 1000 °C/min from RT to a temperature of -196 °C (ΔT of 220 °C in 13 seconds) is necessary to avoid a single ice nucleus to grow to a size of 1.3mm during cooling or warming. Limitations, reasons for caution Assessing the velocity of ice propagation is a novel concept to perfect vitrification/warming solutions. Wider implications of the findings These results pave the way to develop protocols and platforms able to minimize the deleterious impact of cryoprotectant concentrations on both cells and tissues survival after vitrification. Trial registration number not applicable

First live birth in Greece after blastocyst trophectoderm biopsy and preimplantation genetic testing for hereditary angioedema

Preimplantation genetic testing for monogenic/single-gene defects (PGT-M) is a well established tool in assisted reproduction. A couple, where the male was carrier of the c.550G>A (p.Gly184Arg) mutation of the SERPING 1 gene causing hereditary angioedema (HAE) attended our clinic for PGT-M. Employing a strategy of preserving embryos after blastocyst trophectoderm biopsy by vitrification cryopreservation, we managed to screen 10 embryos collected from a single IVF cycle. Three embryos were found unaffected after preimplantation genetic analysis for the paternal mutation. The transfer of one normal blastocyst post-thaw resulted in a healthy and uneventful pregnancy and in the live birth of a male neonate on the 38th week of pregnancy. This is the first report of a live birth in Greece after blastocyst trophectoderm biopsy and preimplantation genetic analysis for hereditary angioedema.

Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents

BackgroundVitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6–8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size.ResultsIn this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of size-controlled 3-D spheroids for cryopreservation of organoid with high survivability.ConclusionsOur results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.

A simplified method for high recovery of kiwifruit (Actinidia spp.) shoot tips after droplet vitrification cryopreservation suitable for long-term conservation

Kiwifruit (Actinidia spp., Family Actinidiaceae) is a recently domesticated crop with highly endangered genetic resources in field genebanks and in the centre of diversity. Improved genotypes of several species in the genus have been commercialised and several other species are used in breeding programmes. Cryopreservation is considered a safe and cost-effective option for ex-situ conservation of clonal crops. In the current research we tested if the currently available method of droplet vitrification (DV) cryopreservation could be simplified whilst improving plant regeneration. Plant regeneration in the nine accessions belonging to A. chinensis var. chinensis, and A. chinensis var. deliciosa, A. arguta, A. macrosperma and A. polygama after cryopreservation ranged from 59 to 88% with the improved method where cold acclimation and antioxidant treatment steps were eliminated while sucrose pretreatment of shoot tips has been reduced to two steps in two days. Use of smaller (0.5–1 mm compared to 2 mm) in vitro shoot tips from younger (two weeks after subculture), fast-growing shoots and the use of liquid sucrose for shoot tip pretreatment are considered to be the reasons for this improved response. We also introduced the use of sodium dichloroisocyanurate as an alternative to household bleach as a sterilant for kiwifruit tissue culture establishment. The improved DV protocol is being used to cryopreserve valuable kiwifruit genetic resources with greater efficiency. Use of small shoot tips (0.5–1mm) from young shoots pretreated in liquid sucrose media in the dark significantly improved plant regeneration in five Actinidia species after droplet vitrification cryopreservation

Clinical application of vitrification freezing of oocytes

Objective: To evaluate the effect of oocyte vitrification on embryo quality and developmental potential. Methods: From January 2014 to December 2017, 30 cases of oocytes vitrification in the reproductive center of Sir Run Run Shaw hospital were collected as the frozen group, due to failure of sperm extraction or inability to obtain sufficient sperm at the ovum pick-up-day because of oligoospermia or azoospermia. 220 cases of fresh oocytes from the same period were selected as the fresh group. The fertilization rate, embryo rate, pregnancy rate, implantation rate and live birth rate of the two groups were compared retrospectively. Results: The survival rate of oocyte resuscitation was 91.4% (180/197). In the frozen group, 24 cases were transferred with 14 pregnancies, while in the fresh group, 31 cases were transferred with 18 pregnancies. The number of 2PN fertilized eggs, 2PN embryos and 2PN high-quality embryos in the frozen group was significantly lower than that in the fresh group (3.7±2.5 vs 7.3±4.8), (3.3±2.5 vs 7.2±4.8), and (1.2±1.8 vs 2.9±2.7) (all P<0.05). The fertilization rate of the frozen group was 77.2% (115/149) and the high-quality embryo rate was 36.6% (37/101), lower than that of the fresh group 77.6% (1 637/2 109) and 40.9% (651/1 591) (P>0.05).The pregnancy rate of the frozen group was 58.3% (14/24) higher than that of the fresh group 58.1% (18/31), the implantation rate of each mature oocyte and the live birth rate of each mature oocyte were 10.8% (15/138) and 10.8% (15/138), both higher than that of the fresh group 9.6% (21/218) and 8.7% (19/218) (all P>0.05). Conclusions: Vitrification cryopreservation of oocytes may lead to a decrease in embryo quality, but embryo development potential is still considerable. Higher pregnancy rate, implantation rate and live birth rate may be obtained.

Cryopreservation of Pleione bulbocodioides (Franch.) Rolfe protocorm-like bodies by vitrification

In this study, a vitrification cryopreservation system for the protocorm-like bodies of monolingual orchids was successfully established and the factors involved in cryopreservation by vitrification, including preculture, loading, cryoprotection, cooling, rewarming, and recovery, were investigated. Histological observation, biochemical and genetic analyses were carried out in every step. The results revealed a high survival rate of 93.33% when the protocorm-like bodies were precultured in 0.4 M sucrose for 2 days, then loaded in 50% plant vitrification solution 2 (15% glycerin, 7.5% ethylene glycol, 7.5% dimethyl sulfoxide, 7.5% sucrose and 62.5% liquid MS) at 25 °C for 40 min, and subsequently treated with modified PVS (25% glycerin, 15% ethylene glycol, 2% polyethylene glycol, 13% dimethyl sulfoxide, 15% sucrose and 30% liquid MS) at 0 °C for 50 min, which improved the vitrification efficiency. The samples were rewarmed directly in a water bath at 60 °C for 2 min after storage in liquid nitrogen. The PLBs were then transferred to a solid medium with 1.0 mg L⁻¹ 6-benzyl adenine, 0.2 mg L⁻¹α-naphthylacetic acid, and 0.5 g L⁻¹ activated charcoal. The PLBs were maintained in the dark for 3 weeks before growth in the light in a culture room. Biochemical analyses revealed that the content of soluble protein and sugar increased during the early stage but decreased during cryopreservation. Histological analyses displayed visible nuclei, few damaged cells, and denser cytoplasm in the cryopreserved PLBs. Inter-simple sequence repeat molecular marker analysis revealed no genetic changes in vitrified PLBs, validating the use of this protocol as an efficient long-term conservation option for this species.

Causes of oocyte vitrification and its value in assisted reproductive technology

To explore the causes of oocyte vitrification and its application in assisted reproduction. We retrospectively analyzed the data of 26 patients with 27 cycles of oocyte vitrification cryopreservation undergoing intracytoplasmic sperm injection (ICSI) and embryo transfer between January, 2008 and October, 2018. The causes of oocyte vitrification and the outcomes of ICSI and clinical pregnancy were analyzed. The causes of oocytes vitrification included mainly azoospermia or severe spermatogenesis disorder of the husband, failure to obtain sperms from the husband, failure of the husband to be present on the day of oocyte retrieval and acute diseases of the husband to not allow sperm collection. A total of 274 oocytes were frozen in 27 oocyte retrieval cycles, and 217 eggs were thawed in 19 cycles with a survival rate of 81.11% (176/217). The normal fertilization rate, cleavage rate and high-quality embryo rate was 74.81% (98/131), 89.80% (88/98) and 36.73% (36/98), respectively. Fifteen patients underwent embryo transfer, and the clinical pregnancy rate and live birth rate was 53.33% (8/15) and 33.33% (5/15), respectively. Compared with patients below 35 years of age, the patients aged above 35 years had significantly lower oocyte survival rate after thawing (82.76% vs 74.42%, P=0.211), clinical pregnancy rate (77.78% vs 16.67%, P=0.041) and live birth rate (55.56% vs 0, P=0.044). Oocytes vitrification can be used as a remedy for infertile couples who fail to provide sperms due to male factors on the day of oocyte retrieval. Vitrification of the oocytes does not significantly affect the fertilization rate or the clinical pregnancy rate. The survival rate of the thawed oocytes is related to the age of the wife, and an age younger than 35 years can be optimal for achieving favorable clinical pregnancy outcomes after oocyte vitrification.

Comparison between Slow Freezing and Vitrification for Human Ovarian Tissue Cryopreservation and Xenotransplantation.

Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.

Vitrified Wharton’s jelly tissue as a biomaterial for multiple tissue engineering applications

Wharton’s Jelly (WJ) tissue is a promising biomaterial, for tissue engineering applications. However, its preservation over a long period in order to be readily available needs further optimization. A possible solution could be the vitrification and storage of WJ tissue at low temperatures. The aim of this study is to evaluate the effect of low temperature in the WJ tissue, which was stored at −196 °C, either with the vitrification or conventional cryopreservation methods. WJ tissues were isolated from human umbilical cords, cryopreserved with the above methods and remained for 1 year at −196 °C. Histological analysis of tissue’s extracellular matrix (ECM), isolation, and characterization of mesenchymal stromal cells (MSCs) were performed. Histological analysis revealed the presence of ECM components such as collagen, sulfated glycosaminoglycans (sGAGs), and the presence of cell nuclei only in vitrified samples. Furthermore, MSCs were isolated and expanded successfully from vitrified WJ tissues, whereas a few viable cells were obtained from conventionally cryopreserved tissues that were not further expanded. In conclusion, this study indicated the proper preservation of vitrified WJ tissues after 1 year of storage, which eventually could be used in tissue engineering and regenerative medicine approaches.

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.

Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis

ObjectiveThe objective is to systematically review and synthesize the literature on the efficacy with two different cryopreservation methods used for human spermatozoa and evaluate whether vitrification protocol and quality of sperm influence effect estimates. DesignThe following electronic databases were searched up to September 2017: Pubmed, Embase and Web of Science. The search strategy used the following the relevant medical subject heading (MeSH) terms, keywords, and word variants for: sperm parameters, conventional freezing, and vitrification. Queries were limited to those involving humans. Randomized controlled trials (RCTs) that published in English languages were considered eligible. Studies and references were included if they reported total motility, progressive motility, morphology, or DNA fragmentation index (DFI) for vitrified or conventional cryopreserved human spermatozoa. Patients recruited in RCTs considering sperm vitrification as one of the experimental arms and conventional freezing (including slow freezing or vapor fast freezing) sperm control as the other. Studies that had high risks of allocation concealment were excluded when performing sensitivity analysis. We specified 2 subgroup variables, including vitrification protocol and quality of spermatozoa cryopreserved, to investigate sources of heterogeneity. A meta-analysis was performed using a random effects (I2 > 50%) or fixed effects (I2 < 50%) model to calculate weighted mean differences (MD) and 95% CI. Result(s)The search yielded a total of 2428 articles and 13 RCTs were included for analysis. They involved 486 vitrified and 486 conventional cryopreserved sperm samples. Four sperm parameters were reported as mean differences and based on adjusted estimates in all included studies. Meta-analysis of these studies showed significantly higher total motility [weighted mean differences (WMD) 6.98; 95% confidence interval (CI) 2.94; 11.02; P < 0.0001] and progressive motility [WMD 4.59; 95% CI 0.78; 8.39; P = 0.02] of past-thawed sperm following vitrification compared with conventional freezing methods. However, DNA fragmentation index (DFI) [WMD -1.18; 95% CI -2.81; 0.45; P = 0.16] and morphology [WMD 0.11; 95% CI -0.42; 0.63; P = 0.69] of past-thawed sperm are similar between two freezing groups. Subgroup analysis shown that the vitrification protocol and quality of spermatozoa are potential risk factors for the efficacy of vitrification. Higher past-thawed sperm parameters following the cryoprotectants-free (CPAs-free) vitrification were observed, as well as a lower past-thawed sperm parameters with the cryoprotectants-presence (CPAs-presence) vitrification, which could reflect the CPAs related cytotoxicity. Meanwhile, vitrification had higher ability in preservation of high quality of spermatozoa compared with vitrification of low quality spermatozoa. Conclusion(s)According to the results of present meta-analysis, vitrification is superior to conventional freezing methods in preservation of spermatozoa, regarding total and progressive motility. However, the efficacy of vitrification is influence by using different vitrification protocol and cryopreservation of different quality spermatozoa. It is must emphasized that the results of present meta-analysis is limited by the small number of studies of variable vitrification protocol. Further well conducted studies are required to confirm the efficacy of vitrification in cryopreservation of spermatozoa, in addition, allow the examination of the two cryopreservation methods in terms of pregnancy achievement and determination of the role of clinical variable on efficacy of vitrification.

A case report of clinical pregnancy in which oocyte retrieval was carried out in the early follicular phase of the menstrual cycle

To report a clinical pregnancy after frozen embryo transfer (FET) for which oocyte was picked up in the early follicular phase of the menstrual cycle. A 40-year-old woman with a two-year history of primary infertility was recruited. After in vitro fertilization (IVF), the embryo underwent vitrification cryopreservation process and FET was carried out five months later. The patient successfully conceived after FET. Follicle recruitment and selection cannot only be performed during a female patient's interovulatory interval (IOI), but oocytes can also be picked up in the early follicular phase of the menstrual cycle conception through IVF, embryo vitrification cryopreservation, and FET. Oocyte retrieval in the early follicular phase of the menstrual cycle increases the chances of an infertile female to conceive, especially in cancer patients and in women with poor ovarian response (POR).

Oxidative damage and antioxidative indicators in 48h germinated rice embryos during the vitrification-cryopreservation procedure.

Cu/Zn SOD and other genes may be critical indicators of a stress response to reactive oxygen species (ROS) accumulation in 48 h germinated rice embryos subjected to vitrification cryopreservation. In the current study, reactive oxygen species (ROS) accumulation was investigated in 48h germinated rice embryos during the vitrification-cryopreservation process. We found that vitrification-cryopreservation significantly affected ROS levels, especially superoxide anion levels, in 48h germinated rice embryos. Malonaldehyde content in the apical meristems of germinated embryos was significantly positively correlated with the rate of superoxide anion generation and the highest levels of malonaldehyde content were reached after vitrification treatment. Cell viability in 48h germinated embryos was significantly negatively correlated with the rate of superoxide anion generation, malonaldehyde content, and electrolyte leakage. Spatial and temporal patterns in ROS accumulation in these embryos existed during the vitrification procedure. Among the vitrification-cryopreservation treatments we assessed, the preculture treatment was found to stimulate superoxide anion generation and to activate the response system in the apical meristems of germinated embryos. Loading treatments motivated the catalase and ascorbate peroxidase activities. During the vitrification-dehydration treatment, oxidative stress reached the highest levels causing an antioxidative response. This response involved antioxidant enzymes promoting detoxification of ROS. Based on a comprehensive correlation analysis involving ROS accumulation, cell viability, the activities of antioxidant enzymes, and gene expression profiles, Cu/Zn SOD, CAT1, APX7, GR2, GR3, MDHAR1, and DHAR1 may be critical indicators of oxidative stress affected by the vitrification-cryopreservation treatments. The investigation of these antioxidative responses in 48h germinated rice embryos may, therefore, provide useful information with respect to plant vitrification-cryopreservation.

Indications for fertility preservation in women from malignant diseases to benign conditions to age-related fertility decline.

Oocyte vitrification and ovarian tissue cryopreservation are two common fertility preservation options. Oocyte vitrification provides the highest yield for women with benign diseases or those seeking fertility preservation for age-related fertility decline. This technique is also indicated for cancer women if cancer treatment can be postponed. Ovarian tissue cryopreservation has some specific indications. There is indeed no other alternative for fertility preservation in prepubertal girls, and in cancer women in whom cancer treatment cannot be postponed. This review focuses on the indications for fertility preservation from malignant diseases to benign conditions to age-related fertility decline.

An Ultrafast Vitrification Method for Cell Cryopreservation

Ultrafast cooling is the key to successful cell vitrification cryopreservation of lower concentration cryoprotective solution. This research develops a cell cryopreservation methodology which utilizes thin film evaporation and achieves vitrification of relatively low concentration cryoprotectant with an ultrafast cooling rate. Experimental results show that the average cooling rate of dimethylsulfoxide (DMSO) cryoprotective solution reaches 150,000 °C/min in a temperature range from 10 °C to −180 °C. The ultrafast cooling rate can remarkably improve the vitrification tendencies of the cryoprotective solution. This methodology opens the possibility for more successful cell vitrification cryopreservation.