Vitrification Method Research Articles

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P<0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P<0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

Development of ovarian tissue vitrification method by using closed device

Development of ovarian tissue vitrification method by using closed device

Advances in basic research related to immunogenicity of allogeneic tendon

Tendon transplantation is one of the most commonly used procedures for patients with injured tendons. The materials used for tendon transplantation include the tendon autograft, tendon allograft, xenogenic tendon and artificial ligament.Of these, the allogeneic tendon has become more and more widely accepted because of the abundant donor source, absence of complications at donor sites and reduced operation time. However, it would meanwhile increase the risks of immunological rejection, disease transmission and delayed tendon-bone integration. A lot of studies have reported many processing technics to solve thementioned drawbacks of tendon allograft. Regarding to the immunological rejection, many approaches have been proposed including physical freezing (such as deep freezing, freeze drying and cryopreservation using the vitrification method) and chemical decellularization (such as deoxyguanosine culture solution, trinbutyl phosphate, chloroform/methyl alcohol, sodium dodecyl sulfate, 95% ethanol and Triton X-100). Among the physical freezing methods, freeze-drying could remove the immunogenicity of tendon more effectively, but it also tends to cause damage to the mechanics, structure and histology of the tendon. And it is more likely to cause damage to tendon especially when the method is used in combination with irradiation sterilization. Deep freezing has less damage to the mechanical and histological characteristics of tendon, and this method is currently the mostcommonly used in clinical and scientific research. The vitrification preservation method has unique advantages in protecting thetendon mechanical properties and cell activity. It retains similar mechanical properties to fresh tendons. However, the complicated preparation procedures involved in this method, the high cost and the cytotoxicity of the cryoprotectant have always restrictedits application in actual production. Different chemical decellularization methods have their own advantages and disadvantages when used to reduce the immunogenicity of tendons. In order to achieve more thorough removal of cellular components in tendonswhile maximally retaining the structural and mechanical integrity of tendons, strict control on the concentration of the decellularizing agent and the treatment time is often required.

Cryopreserved potato shoot tips showed genotype-specific response to sucrose concentration in rewarming solution (RS)

Cryopreservation protocols have been successfully developed for hundreds of species and thousands of genotypes in laboratories around the world. In many of the protocols, the rewarming process occurs in a rewarming solution (RS) with a high concentration of sucrose (0.8–1.2 M). Warming rate and associated conditions influence cell rehydration, loss of accumulated solutes (e.g. cryoprotectants) and recrystallization of small ice crystals in the nuclei. The need for, and effect of, high sucrose concentrations in the RS post-thawing regrowth rate after liquid nitrogen exposure was assessed in the range of 0.0-1.2 M sucrose with a set of 16 potato landraces cryopreserved with the PVS2-droplet vitrification method. The results showed no significant difference for the average recovery rate (81–87%) between sucrose concentrations of 0.3 M to 1.2 M. Fourteen of 16 accessions had their highest recovery rate with sucrose concentrations between 0.3 and 0.9 M. The experimental results were subsequently extended to a genetically diverse set of 85 potato accessions (nine taxa), which demonstrated significantly higher recovery rates of 55–61% with RS sucrose concentrations of 0.3–0.9 M, compared to the sucrose concentrations of 0.0 M (37%) and 1.2 M (44%). Only one of 85 accessions showed its highest recovery rate with the routinely used RS sucrose concentration of 1.2 M. Of all the concentrations tested, 0.6 M sucrose appeared to be the best bet in terms of recovery rates across the genotypes; therefore, our routine protocol has been changed from 1.2 M sucrose to 0.6 M. The specific response to low (0.0 M) and high RS sucrose concentrations (1.2 M) was highly variable within species/subspecies and appears to be genotype specific. Thus, caution should be taken in generalizing experimental cryopreservation results obtained with a limited number of accessions to larger germplasm collections.

The profiling of pre- and post-warming DNA in mouse embryos with microsatellite method

Aims:This research aimed to identify the deoxyribonucleic acid (DNA) profile and changes of post-warming embryo after being frozen with vitrification method using microsatellite method.Materials and Methods:This research examined the mouse embryo blastocysts that were divided into four groups: Post-warming living blastocyst, post-warming living blastocyst with half fragmented cell, post-warming dead blastocyst, and pre-freezing living blastocyst. The isolation sample applied phenol-chloroform method. After obtaining polymerase chain reaction results, all the samples of pre-freezing fresh embryo, post-warming living embryo, dead embryo, and degenerated embryo were then examined by single-strand conformation polymorphism (SSCP).Results:The amplification with D18mit14 primer was 100 bp and 150bp with D18mit87 primer, 150bp with D7mit22, and 300bp with D7mit25. The result of SSCP with D18mit14 primer showed that the blastocysts were fragmented and dead after warming process and formed into two DNA strand fragments, while the fresh embryos which passed freezing process did not form any fragment. D18mit87 primer SSCP indicated different fragments for each treatment. The result of SSCP using D7mit22 formed two different fragments for each treatment. While using D7mit25, the SSCP result formed some different fragments for each sample. Post-warming living embryo had similar ribbon to pre-freezing fresh embryo.Conclusion:D7mit222, D7mit25, and D18mit87 primers could be used as the aneuploidy marker on mouse embryos that were induced by post-warming process. The profile of living blastocyst, dead blastocyst, and post-warming fragmented blastocyst had different DNA tapes.

The CARD Method for Simple Vitrification of Mouse Oocytes: Advantages and Applications.

A light modification on the mouse embryos CARD simple vitrification protocol makes it possible the oocyte freezing. This rapid and handy method for cryopreservation of mouse oocytes has many advantages and multiple applications like banking the female gamete for future use, controlling the IVF timing, and facilitating the transgenic production. In this chapter we described the protocol for cryopreserving oocytes by simple vitrification method and the in-vitro fertilization carried out using fresh or frozen sperm. With this method, nearly all of the cryopreserved oocytes were recovered, resulting in more than 90% morphologically normal. From those oocytes used for in vitro fertilization, 89% of the oocytes were fertilized.

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage.

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo-flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2-8mm in diameter, washed three times in TCM-199 and then examined under stereo-microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40%+DMSO 40%. The obtained results revealed that addition of EG 40%+DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG+40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro-produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.

The Unusual Properties of Polytetrafluoroethylene Enable Massive-Volume Vitrification of Stem Cells with Low-Concentration Cryoprotectants.

Injectable stem cell-hydrogel constructs hold great potential for regenerative medicine and cell-based therapies. However, their clinical application is still challenging due to their short shelf-life at ambient temperature and the time-consuming fabrication procedure. Banking the constructs at cryogenic temperature may offer the possibility of "off-the-shelf" availability to end-users. However, ice formation during the cryopreservation process may compromise the construct quality and cell viability. Vitrification, cooling biological samples without apparent ice formation, has been explored to resolve the challenge. However, contemporary vitrification methods are limited to very small volume (up to ~0.25 ml) and/or need highly toxic and high concentration (up to ~8 M) of permeable cryoprotectants (pCPAs). Here, we show that polytetrafluoroethylene (PTFE, best known as Teflon for making non-stick cookware) capillary is flexible and unusually stable at a cryogenic temperature. By using the PTFE capillary as a flexible cryopreservation vessel together with alginate hydrogel microencapsulation and Fe3O4 nanoparticle-mediated nanowarming to suppress ice formation, massive-volume (10 ml) vitrification of cell-alginate hydrogel constructs with a low concentration (~2.5 M) of pCPA can be achieved. This may greatly facilitate the use of stem cell-based constructs for tissue regeneration and cell based therapies in the clinic.

Genetic analysis of embryos, obtained from vitrificated oocytes in donor’s programs of artificial reproductive technologies

The factors of cryopreservation can disrupt molecular mechanisms of normal chromosome’s segregation during meiosis and to increase the number of aneuploidy in embryos. The aim of the paper was to determine the frequency of aneuploid embryos obtained from the thawed oocytes in comparison with the fresh embryo cycles. Preimplantation genetic testing for aneuploidy was performed using fluorescence in situ hybridization with the study of such chromosomes as 13, 16, 18, 21, 22, X, Y. Statistical hypotheses were checked using criteria t, c2 at levels of significance p<0.05, p<0.01, p<0.001. The results have shown that the embryos obtained from vitrificated and fresh oocytes had identical frequency of chromosomal nondisjunctions. However, there was marked the increasing of the mosaicism frequency in the embryos get from thawed oocytes. Aneuploidies were rarer determine among the embryos obtained from vitrificated oocytes, which may indicate a less viability of oocytes with violations in the number of certain chromosomes. This research is the fragment of SRW IPCandC NASU 2.2.6.58 “Study of influence of cryopreservation factors by the vitrification method on morphologically functional descriptions of reproductive cells and embryos”, number of state registration 0116u003498.

Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.

Development of a vitrification method for preserving human myoblast cell sheets for myocardial regeneration therapy

BackgroundTissue-engineered cardiac constructs have potential in the functional recovery of heart failure; however, the preservation of these constructs is crucial for the development and widespread application of this treatment. We hypothesized that tissue-engineered skeletal myoblast (SMB) constructs may be preserved by vitrification to conserve biological function and structure.ResultsScaffold-free cardiac cell-sheet constructs were prepared from SMBs and immersed in a vitrification solution containing ethylene glycol, sucrose, and carboxyl poly-l-lysine. The cell sheet was wrapped in a thin film and frozen rapidly above liquid nitrogen to achieve vitrification (vitrification group, n = 8); fresh, untreated SMB sheets (fresh group, n = 8) were used as the control. The cryopreserved SMB sheets were thawed at 2 days, 1 week, 1 month, and 3 months after cryopreservation for assessment. Thawed, cryopreserved SMB sheets were transplanted into rat hearts in a myocardial infarction nude rat model, and their effects on cardiac function were evaluated. Cell viability in the cardiac constructs of the vitrification group was comparable to that of the fresh group, independent of the period of cryopreservation (p > 0.05). The structures of the cell-sheet constructs, including cell-cell junctions such as desmosomes, extracellular matrix, and cell membranes, were maintained in the vitrification group for 3 months. The expression of cytokine genes and extracellular matrix proteins (fibronectin, collagen I, N-cadherin, and integrin α5) showed similar levels in the vitrification and fresh groups. Moreover, in an in vivo experiment, the ejection fraction was significantly improved in animals treated with the fresh or cryopreserved constructs as compared to that in the sham-treated group (p < 0.05).ConclusionsOverall, these results show that the vitrification method proposed here preserves the functionality and structure of scaffold-free cardiac cell-sheet constructs using human SMBs after thawing, suggesting the potential clinical application of this method in cell-sheet therapy.

Radionuclide disposal using the pyrochlore supergroup of minerals as a host matrix-A review.

Since the first large scale commercial nuclear power plant became operational in 1958, the nuclear power industry has been faced with the growing problem of disposal of radionuclides produced from nuclear fission. The current global production of high level nuclear waste is approximately 10,000 metric tons p.a., consisting predominantly of uranium, plutonium, actinides and other minor radionuclides. Developing a safe and cost-effective method for long term storage and disposal of nuclear waste is a key issue of concern to the nuclear power industry. A promising approach to radionuclide disposal is incorporation of the nuclear waste into refractory oxide host minerals or mineral matrices. This technique offers lower leaching rates when compared to the commonly used glass-based vitrification approaches. The refractory pyrochlore supergroup of minerals are particularly attractive for this purpose as they can incorporate considerable amounts of the radionuclides: 93Zr, 133Ba, 135Cs, Th, U, 238Pu, and 244Cm, while demonstrating very low leachability. This review examines the structural, compositional and chemical properties of radionuclide-containing pyrochlore supergroup minerals. Compiled leaching data for radionuclides hosted in pyrochlores demonstrates that these materials offer a high degree of aqueous durability making them strong candidates for radionuclide disposal, offering a viable storage alternative to traditional vitrification methods.

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring.

BackgroundThe open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.MethodsOvine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.ResultsNo significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.ConclusionsThe OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

Innovative multi-protectoral approach increases survival rate after vitrification of ovarian tissue and isolated follicles with improved results in comparison with conventional method

BackgroundIn recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation.ResultsThe results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity.ConclusionsThe multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.

Minimum volume Spatula MVD vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos

This study was conducted to compare the effect of minimum volume Spatula MVD vitrification (VIT) versus traditional slow freezing (SLF) of mouse embryos. A total of 2,617 8-cell in vivo derived and 2-cell in vitro produced B6D2 mouse embryos were subjected to freezing/thawing or vitrification/warming, while fresh embryos were used as control group. Embryo recovery, survival and development rate, pregnancy rate and offspring production were analyzed. In Experiment 1, 8-cell in vivo derived embryos were subjected to in vitro culture, resulting in greater survival and development rates at 3.5 days post coitum stage in VIT than in SLF group (P < 0.05). Although both methods reached an acceptable hatching rate (41.0% and 49.7% for VIT and SLF, respectively; P=NS), it was significantly lower respect to the control group (67.8%, P < 0.01). In Experiment 2, 2-cell in vitro produced mouse embryos showed a similar recovery rate from the device after freezing/thawing or vitrification/warming (∼84%), however survival rate was significantly higher for vitrified/warmed (94.7%) than frozen/thawed embryos (85.1%; P < 0.01). Vitrified/warmed and control fresh embryos were transferred to surrogate mothers, revealing no differences both in pregnancy and offspring production rates. Our data demonstrate that minimum volume Spatula MVD method is a simple home-made useful technique for vitrification of 2-cell and 8-cell mouse embryos produced either in vitro or in vivo.

Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential

Abstract Background There are difficulties associated with testicular sperm freezing. Different methods of sperm cryopreservation developed. Not enough detailed studies about the real efficacy of these techniques exist. For sperm morphologic assessment, motile sperm organelle morphology examination (MSOME) is able to identify not only conventional morphological sperm alterations but also sperm head vacuoles. Objective To assess the effect of cryopreservation on testicular spermatozoa vacuoles by MSOME and its correlation to DNA fragmentation and mitochondrial membrane potential. Materials and methods After preparation, testicular sperm extraction samples of 15 azoospermic men, aged 20–40 years old were divided into three groups. Group 1 was assessed freshly. Group 2 was cryopreserved with vitrification method and Group 3 with cooling in liquid nitrogen vapor using droplet. Pre and post warming assessment in terms of spermatozoa head vacuoles by MSOME, DNA fragmentation, and mitochondrial membrane potential were performed. Results The number of spermatozoa with no vacuoles significantly decreased after two cryopreservation techniques (P 0.05). DNA fragmentation and mitochondrial membrane potential increased after cryopreservation (P Conclusion Cryopreservation affects spermatozoa vacuolization, DNA structure, and mitochondrial membrane potential. Using MSOME in the selection of post-thaw morphologically normal testicular spermatozoa for ICSI procedure will be of particular value.

Recent Advances in Single Particle Cryo-electron Microscopy and Cryo-electron Tomography to Determine the Structures of Biological Macromolecules

A detailed three-dimensional structure of macromolecular assemblies is necessary to understand their function which in turn helps to understand life. Cryo-electron microscopy (cryo-EM) is a powerful method for structural studies of a wide range of different sizes biological macromolecules and their complexes. Cryo-EM has three different imaging modalities based on specimen and imaging condition: single particle analysis (SPA), cryo-electron tomography (cryo-ET) plus sub-tomogram averaging (STA)/sub-volume averaging (SVA) and electron diffraction. Richard Henderson and Nigel Unwin revealed the structure of the first membrane protein bacteriorhodopsin from electron diffraction data. This led to the beginning of understanding molecular structures of biomolecules in three-dimension. Soon after that, a unique vitrification method of biomolecules has been successfully developed by Jacques Dubochet more than two decades ago. Ordered 2D array or biomolecules with internal symmetry have long been considered for structure determination to achieve better resolution. But structure calculation by electron microscopy was at that time known as blobology to others due to low resolution Images with small number of pixels and less information and less detail. (image with less information) compared to X-ray. Since then imaging and software technologies have steadily improved and after 2013, with the development and success of direct detectors, the world witnessed a resolution revolution in cryo-EM. Now cryo-EM more specifically single particle analysis has achieved the resolution at which protein complex can be studied at near-atomic level. This once a highly skilled and difficult technique has now become a widely accepted biophysical technique in structural biology. Here the two methods of cryo-EM (SPA and cryo-ET) and recent studies are reviewed.

GDF9-β promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs

Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-β expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-β can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue. Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-β to the grafted tissue. After 5days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software. Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-β cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-β was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-β cultured group. GDF9-β, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.