Evaluation of a New Vitrification Protocol for Oocyte Cryopreservation (original) (raw)
Related papers
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.
The present study was design to establish the optimal vitrification protocol for the immature swine oocytes using different concentration of ethylene glycol (40, 45 and 50%) in the vitrification media and different exposure time (30, 40, 50 and 60 seconds). Vitrification was performed by the SSV method in a stepwise manner. After thawing morphological and viability assessments were done. The results shows that, once the concentration of the EG and the exposure time are increased, the percentage of morphologically normal oocytes after freezing/thawing is decreasing. Meanwhile the rate of viable oocytes is increasing once the concentration of EG reaches 45% in the VM and no longer exposing the eggs then 40 seconds. It is not justified the increasing of it in the VM over 45%. At 50%, the cryoprotectant has no longer a beneficial effect in the vitrification process. High concentrations lead to cell toxicity and cell death. An acceptable vitrification protocol can be obtained only with repeated exploratory regarding to both cryoprotectant concentration and exposure time.
2010
Objective: To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Design: Prospective randomized. Setting: Academically affiliated, private fertility center. Patient(s): Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Intervention(s): Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Main Outcome Measure(s): Oocyte survival, fertilization, embryo development, and clinical pregnancy. Result(s): Patient use has resulted in 30 thaws and 48 warmings. Women's age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/ thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/ thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. Conclusion(s): Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy.
Fertility & Reproduction
Background: Oocyte Cryopreservation has become an important part of infertility treatment for various reasons such as fertility preservation in women going for oncological treatment; in oocyte donation cycles; in eliminating several religious, ethical, and legal concerns of embryo freezing and in women who wish to delay childbirth. The newer ”vitrification” technique for freezing has further improved the success rates for actual conception than the earlier method of slow freezing. A successful oocyte freezing program can help in establishment of oocyte banks, which would help to provide compatible oocytes immediately, thus would eliminate the several problems of fresh donor cycles. Methods: In this retrospective observational study, total 60 oocyte donation cycles were included (38 were fresh and 22 were vitrified oocytes cycle, respectively). After a thorough screening, controlled ovarian hyperstimulation for donors was performed using flexible antagonist protocol. All mature oocyt...
Cryobiology, 2010
This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84±5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol+0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol+15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV+MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p=0.01) and vitrification (p<0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p<0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.
Fertility and Sterility, 2008
Objective: To evaluate the outcome of oocyte vitrification using the Cryotop method, observed in an egg donation program by simultaneously evaluating embryos derived from vitrified and fresh oocytes coming from the same stimulated cycle. Design: Cohort prospective randomized study. Setting: Instituto Valenciano de Infertilidad (IVI) Valencia, Spain. Patient(s): Thirty oocyte donors and 30 recipients with informed consents. Intervention(s): Vitrification by the Cryotop method. Warming 1 hour after vitrification. Microinjection of surviving MII and fresh oocytes, evaluation of fertilization, embryo development, and clinical results. Main Outcome Measure(s): Survival, fertilization, and cleavage rate. Embryo quality, pregnancy rate (PR), and implantation rate. Result(s): Survival rate observed was 96.7%. There was no difference in fertilization rates (76.3% and 82.2%), day 2 cleavage (94.2% and 97.8%), day 3 cleavage (80.8% and 80.5%), and blastocyst formation (48.7% and 47.5%) for vitrified and fresh oocytes, respectively. Embryo quality on day 3 and on day 5-6 were similar for vitrification and fresh oocyte group (80.8% vs. 80.5% and 81.1% vs. 70%, respectively). A total of 23 embryo transfers were carried out in the vitrification group. Pregnancy rates, implantation rates, miscarriage rates, and ongoing PR were 65.2%, 40.8%, 20%, and 47.8%, respectively. Conclusion(s): The Cryotop method preserves the potential of vitrified oocytes to fertilize and further develop, which is similar, when evaluated simultaneously, to fresh counterparts. Excellent clinical outcome indicates the possible use of this technology for egg donation programs, as well as a high potential for establishing oocyte banking. (Fertil Steril Ò 2007;-:---.