Evaluation of a New Vitrification Protocol for Oocyte Cryopreservation (original) (raw)
Vitrification of human oocyte using cryoloop
2005
Background: The cryopreservation of human oocyte would make a significant contribution to infertility treatment, such as using it for oocyte donation and for patients a bout to lose ovarian function due to surgery or chemotherapy. Despite of using standard freezing straws and cryovials or even open pulled straws, only a few successful pregnancies have been arisen from cryopreserved human oocytes. This situation has been primarily attributed to poor survival, fertilization and development of cryopreserved oocytes. Objective: The aim of this study was to evaluate the novel cryoloop vitrification method for cryopreservation of human oocytes. Materials and Methods: Nine infertile couples participated in this study. In all women proper regulation and desensitization was done using GnRH agonist during luteal phase. Mature oocytes allocated into two groups randomly. In group I, 34 oocytes were vitrified in conventional straws, while in group II, 33 oocytes were vitrified in cryoloop. After a store time of 1-6 months the oocytes were thawed, incubated for 2 hours and subsequently the ICSI was done on survived oocytes. To verify normal fertilization of vitrified oocytes the number of pronuclei in the cytoplasm was counted 16-18 hours after ICSI and good morphological quality embryos were transferred on day 2 or 3 after sperm injection. Pregnancy was identified by the serum ß HCG level, checked 14 days after embryo transfer. Results: The present study shows that the rate of survival of vitrified human oocytes in two groups has no significant difference (52.94% in group I versus 63.63% in group II) but the fertilization rate of vitrified oocytes by cryoloop was greater than vitrified oocytes by conventional straws (73.7% versus 55.55% respectively). One of the embryo transfers achieved clinical pregnancy and resulted in the delivery of healthy baby. Conclusion: Vitrification by using cryoloop can improved the fertilization rate and developmental capacity of vitrified thawed oocyte.
Canadian Journal of Animal Science, 2011
Habibi, A., Hosseini, A., Farrokhi, N., Amidi, F., Carvalhais, I., Chaveiro, A. and Moreira da Silva, F. 2011. Short Communication: Successful vitrification of mouse oocytes using the cryotop method with moderate cryoprotectant concentrations. Can. J. Anim. Sci. 91: 385–388. The response of vitrified mouse MII oocytes in the presence of two concentrations of cryoprotectants [vit1 (15%: 7.5% dimethyl sulfoxide (DMSO)+7.5% ethylene glycol (EG) and vit2 (30%: 15% DMSO+15% EG)] was analyzed to investigate whether reducing cryoprotectant concentrations can affect oocyte survival after cryopreservation by the cryotop method. After thawing the survival, fertilization, cleavage and blastocyst rates were compared with unfrozen oocytes. It can be concluded that 15% cryoprotectant (7.5% DMSO+7.5% EG), instead of the commonly used 30% (15% DMSO+15% EG), could be helpful by moderating the probable toxic effects of vitrification solution in mouse oocyte during vitrification by cryotop.
Reproduction, Fertility and Development, 2013
Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24 h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified ...
Highly efficient vitrification method for cryopreservation of human oocytes
Reproductive BioMedicine Online, 2005
Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.
Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations
Reproductive BioMedicine Online, 2010
The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000°C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l propane-1,2-diol and 0.5 mol/l trehalose and achieve survival rates of 92.5%. Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.
Asian-Australasian Journal of Animal Sciences, 2002
Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to 10°C, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to 10°C or to the process of vitrification, regardless of the cryoprotectant used.
83 Cryopreservation of in Vitro Porcine Oocytes by Solid Surface Vitrification (SSV)
Reproduction, Fertility and Development
The effects of the cytosceletal inhibitor cytochalasin in solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) were investigated on in vitro matured (IVM) porcine oocytes. Cumulus-free IVM oocytes were subjected to one of the following: SSV (equilibration in 4% ethylene glycol (EG) for 10 min followed by vitrification in 35% EG, 5% polyvinyl pirrolidone, and 0.3 M trehalose on a cold (about −150°C) surface; warming in 0.4 M trehalose at 37°C), SSV pre-treatment with 5 μg/mL cytochalasin B (SSV + CB), or the steps of SSV without cooling, i.e. toxicity control (TC). Non-lysed oocytes together with the non-treated controls were subjected to parthenogenetic activation and then in vitro cultured (IVC) for six days. The proportion of non-lysed oocytes was higher when pre-treatment with CB was performed compared to SSV. However, both results were significantly lower than that of the TC. After parthenogenetic activation via a combination of a direct current elect...
Vitrification of human immature oocytes before and after in vitro maturation: a review
Journal of Assisted Reproduction and Genetics
The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.
Technical aspects of oocyte cryopreservation
Molecular and Cellular Endocrinology, 2000
Since the successful development in the mouse, the oocyte cryopreservation has been applied with varying success to a number of different species including the human. The recently reported successes in terms of pregnancies obtained by human oocyte cryopreservation are encouraging. Several studies typically reported different rates of survival (20-80%), fertilization (30-60%) and cleavage (32-100%). This variability of results throws some doubts on the usefulness of oocyte cryopreservation in IVF treatment cycles. It remains to be determined whether the relatively different success rates reported in literature, mainly in terms of survival rate, are due to methodological differences. We tried to investigate the effect of some factors on the oocyte survival rate after thawing: the presence or absence of cumulus oophorus and the exposure time of the oocytes to cryoprotectant. We suggest that a combination of several factors including both morphological and biophisical ones can affect the oocyte survival rate.
Cryobiology, 2013
As survival of mouse oocytes subjected to vitrification depends far more on the warming rate than on the cooling rate, we wished to determine whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The morphological and functional survival of the oocytes showed little or no relation to whether or not the EAFS medium vitrified or froze. We studied if the droplet size and the elapsed time (between placing the droplet on the Cryotop and the start of cooling) affects the result through modification of the cooling rate and solute concentration. Dehydration was rapid; consequently, the time between the placing the droplets into a Cryotop and cooling must be held to a minimum. The size of the EAFS droplet that is being cooled does not seem to affect vitrification. Finally, the degree to which samples of EAFS vitrify is firmly dependent on both its solute concentration and the cooling rate.