Vitrification Protocol Research Articles

Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle.In stimulated IVF cycles, high doses of hormones are given to stimulate multifollicular growth. One drawback of the hormonal substitution is that the uterine environment is not at the same time optimally prepared for embryo implantation. A solution, which is increasingly under discussion, is to cryopreserve the embryos obtained in the stimulated cycle and to transfer them back into the optimal uterine environment in a subsequent cryo-cycle. This procedure requires highly secure and safe cryopreservation protocols in order to ensure benefits for both pregnancy and birth rates. We have established a protocol for the vitrification of zygote-stage embryos in aseptic devices, which minimize the potential risk of contamination during cooling and storage. The vitrified zygotes showed the same blastocyst development as compared with sibling zygotes in fresh culture. A clinical study comprising 173 cryo-cycles with transfer of blastocysts originating from vitrified zygotes shows an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification conditions contributes to a change in transfer strategy and encourages us to increase the cryo-embryo transfer rate for an optimal uterine environment.

Vitrification and xenografting of human ovarian tissue

To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. Pilot study. Gynecology research unit in a university hospital. Ovarian biopsies were obtained from seven women aged 30-41 years. Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.

Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions

Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10–104K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.

Vitrification of ovarian tissue from primates and domestic ruminants: an overview.

In the last decade, vitrification protocols to preserve human ovarian tissue have been regularly reported, even more often than the protocols developed for large mammals, such as ruminants and nonhuman primates. In order to facilitate the use of domestic ruminants (cows, goats, and sheep) and nonhuman primates as animal models, application of similar protocols as used for human material is performed. Next to it, the addition of indispensable or exclusion of avoidable compounds in the vitrification of human ovarian tissue should be tested in such experiments with animal models. The objective of this mini-review is to summarize the current protocols used for the vitrification of ovarian tissue and to evaluate the vitrification methods in humans, nonhuman primates, and domestic ruminants.

Vitreous preservation of articular cartilage from cryoinjury in rabbits

Frozen osteoarticular grafts treated with liquid nitrogen are utilized for joint reconstruction after tumor resection, but the joints may subsequently develop osteoarthritic changes. To preserve articular cartilage from cryoinjury, we modified a vitrification method utilized for embryo cryopreservation and demonstrated in vitro that our vitrification protocol was effective for protecting cartilage from cryoinjury. In this study, we investigated in vivo whether this vitrification method could protect against osteoarthritic changes in articular cartilage. Osteochondral plugs were obtained from the distal femur of rabbits. These grafts were divided into 3 groups: Fresh group (F-group), non-vitrification group (N-group), and vitrification group (V-group). After treatment, the plugs were re-implanted as autografts. Histological findings, chondrocyte viability, and ultrastructural examinations were examined 6, 12, and 24weeks after implantation. Histological findings of chondrocytes for the V-group showed no significant difference from those of the F-group at any time point except at 24weeks postimplantation at the non-weight bearing site (p<0.05). Viability of chondrocyte showed no significant difference from those of the F-group except at 12weeks postimplantation at the bearing site (p<0.05). In contrast, viable cells disappeared from the N-group and histology and viability significantly differed between the N-group and the V-group. Transmission electron microscopy demonstrated preservation of chondrocyte structure in the V-group and the F-group, but chondrocytes of the N-group were abnormally electron dense. Our vitrification method was effective in protecting chondrocytes from cryoinjury that might lead to cartilage degeneration. Reconstructing joints with osteoarticular grafts containing living cartilage may help to avert osteoarthritic changes. Our vitrification method could prove useful for reconstruction with frozen tumor-containing autografts and for long-term storage of living cartilage for allografts.

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus–Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus-oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm(3)) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

Cryopreservation of human failed-matured oocytes followed by in vitro maturation: vitrification is superior to the slow freezing method

BackgroundOocyte cryopreservation is an important method used in a number of human fertility circumstances. Here, we compared the survival, in vitro maturation, fertilization, and early embryonic development rates of frozen-thawed human immature oocytes using two different cryopreservation methods.MethodsA total of 454 failed-matured oocytes [germinal vesicle (GV) and metaphase I (MI) stages] were collected from 135 patients (mean age 33.84 +/- 5.0 y) who underwent intracytoplasmic sperm injection (ICSI) cycles between February 2009 and December 2009 and randomly divided into a slow freezing group [1.5 mol/L-1, 2-propanediol (PROH) + 0.2 mol/l sucrose] and vitrification group [20% PROH + 20% ethylene glycol (EG) + 0.5 mol/l sucrose].ResultsThe vitrification protocol yielded a better survival rate than the slow freezing protocol at each maturation stage assessed. Regardless of the maturation stage (GV + MI), the slow freezing protocol had a significantly lower survival rate than the vitrification protocol (p < 0.001). In addition, a significant difference was found in the survival rates between GV and MI oocytes regardless of the protocol used (90.1 vs. 64.7%, respectively; p < 0.01). We also found that the maturation rates of GV and MI oocytes from the slow freezing and vitrification groups were 16.7 vs. 24.4% and 50.8 vs. 55.4%, respectively. Regardless of the protocol used, the GV oocytes had significantly lower viability than MI oocytes after 36 h of in vitro maturation (21.2 vs. 54.0%, respectively; p < 0.01). In addition, the GV and MI oocytes from the slow freezing group had a markedly lower maturation rate than those from the vitrification group (33.6 vs. 43.1%, respectively), but no statistical difference was found between the two groups (P > 0.05). For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, while a 19.0% (4/21) fertilization rate was observed in the vitrification group. For the MI-matured oocytes, fertilization rates for the slow freezing and vitrified groups were 36% and 61.1%, respectively, but no significant difference was found between the two groups (PIn the Methods section in the MS, all procedures were compliant with ethical guidelines, i.e. approved by the Ethical Committee of our university and Informed Consent signed by each patient. > 0.05). In the GV vitrification group, no embryo formed; however, in the MI slow freezing group, 12 oocytes were fertilized, but only two achieved cleavage and were subsequently blocked at the 2-cell stage. In the MI vitrification group, a total of 22 embryos were obtained, five of which developed to the blastocyst stage.ConclusionsVitrification is superior to the slow freezing method in terms of the survival and developmental rates for the cryopreservation of human failed-matured oocytes. In addition, GV oocytes appeared to be more resistant than MI oocytes to the low temperature and cryoprotectant used during cryopreservation.

CRYOPRESERVATION OF STRAWBERRY GENETIC RESOURCES IN GERMANY

The National German Strawberry Genebank has 369 cultivars and 318 accessions of wild Fragaria species maintained in a field collection at Dresden-Pillnitz. Conservation of clonal crops requires safety duplication. A calculation of the efforts required to establish and maintain a safety backup of the whole collection with in vitro cold storage indicated that it is too labour intensive to maintain the collection in such form. The development of an effective method for cryopreservation is required for a cost effective long-term storage. Four distinct cryopreservation protocols were tested: two protocols using Plant Vitrification Solution 2 (PVS2), encapsulation dehydration and controlled rate cooling. All four methods were equally effective for the cultivars tested, however the Fragaria wild species had significantly better regrowth with the PVS2 vitrification protocol that included 14 d alternating-temperature cold acclimation (16 h at -1°C and 8 h at 22°C). The PVS2 vitrification protocol with cold acclimation will be applied as the standard method for cryopreservation of Fragaria cultivars and wild species. Currently, the Dresden-Pillnitz Institute has 23 different Fragaria accessions in cryopreservation.

Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates

Occasionally, clinical scenarios arise where embryos, previously cryostored and warmed, need to be recryopreserved. The outcome of 30 such transfer cycles from 25 women where embryos were recryopreserved is detailed. In 16 cases, embryos were initially cryopreserved by slow freezing and in 14 cases by vitrification. The cryopreservation stages were the pronuclear stage (n=16), day-3 cleavage stage (n=12), blastocyst (n=1) and oocytes (n=1). All recryopreservation was by Cryotop-based vitrification. From this mixed source, 30/31 twice-cryopreserved embryos survived warming and were transferred, resulting in 13 pregnancies, 11 deliveries with normal gestational age and birthweight, one pre-term birth at 33 weeks and two miscarriages. There were no malformations reported for the live births. Recryopreservation using vitrification by CryoTop has been used in a variety of clinical scenarios to preserve surplus cryopreserved embryos. The current study, although limited in numbers, resulted in high survival rates, clinical pregnancy rates similar to once-cryopreserved embryos and healthy live births independently of the initial stage and cryopreservation method. The technique may increasingly be applicable to elective single-embryo transfer and blastocyst transfer to maximize the pregnancy rate while minimizing the number of cryopreserved embryo transfers.Freezing of embryos has significantly influenced the likelihood of pregnancy after an IVF stimulation cycle. Since it is often unclear which embryos have the potential to continue developing, more embryos may be thawed than transferred. If, in such cases, the surplus embryos remain viable, the ability to refreeze them is an important laboratory tool. In this study, we report on 30 cycles from 25 women where, for a variety of clinical scenarios, surplus embryos previously cryopreserved by either slow freezing or more recently by vitrification were recryopreserved using the Cryotop vitrification protocol. The report details 13 pregnancies, including 11 live births. The study group included one case where oocytes rather than embryos were initially vitrified. When these recryopreserved embryos were subsequently warmed, they exhibited a high survival rate and, after embryo transfer, a pregnancy rate similar to once-cryopreserved embryos. While recryopreservation has to date demonstrated that the embryos retain viability, leading to healthy children, and is, as such, an additional tool for embryologists, its application should proceed with caution.

Is there any difference in vitrification and slow freezing protocol for oocyte cryopreservation?

ObjectiveThis study was an observational retrospective cohort, with the objective to compare oocyte cryopreservation efficiency between slow freezing using choline chloride based medium, and vitrification protocol using cryotop.DesignA total of 101 cycles (slow freezing and vitrification techniques) were performed for those patients that decided to thaw their oocytes from 2009 to 2011.Materials and MethodsAll patients with at least 10 oocytes and not willing to freeze embryos were offered the possibility to cryopreserve oocytes. The oocytes cryopreserved by slow freezing followed Stachecki's (1998) protocol, while the oocytes vitrified followed Kuwayama's. (2005) protocol. The endometrium was prepared using Estradiol Benzoate with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Two days before the replacement of the embryos, the oocytes were thawed. After thawing, the oocytes were kept in a incubator during 2 hours before inseminating by ICSI. The survival, fertilization, cleavage, average embryo transfer and pregnancy rates were compared through the chi-square test (P<0.05).ResultsTable 1Outcome results from slow freezing versus vitrification protocol.Slow freezing (%)Vitrification (%)Cycles5645Oocytes Thawed523399Oocytes Survived*P=0.0085289 (55.2)255 (63.9)Oocytes Fertilized200 (69.2)177 (69.4)Embryos Cleaved185 (92.5)156 (88.1)Embryos Transferred121 (65.4)100 (64.1)Average Embryo Transfer2.22.2Implantation15 (12.4)16 (16.0)Pregnancies16 (28.6)15 (33.3)Clinical Pregnancies14 (25.0)13 (28.9)* P=0.0085 Open table in a new tab ConclusionAlthough the survival rate was statistically higher for the vitrification technique, this did not result in a statistically higher implantation and/or pregnancy rate. Therefore both techniques are reliable to cryopreserve oocytes. ObjectiveThis study was an observational retrospective cohort, with the objective to compare oocyte cryopreservation efficiency between slow freezing using choline chloride based medium, and vitrification protocol using cryotop. This study was an observational retrospective cohort, with the objective to compare oocyte cryopreservation efficiency between slow freezing using choline chloride based medium, and vitrification protocol using cryotop. DesignA total of 101 cycles (slow freezing and vitrification techniques) were performed for those patients that decided to thaw their oocytes from 2009 to 2011. A total of 101 cycles (slow freezing and vitrification techniques) were performed for those patients that decided to thaw their oocytes from 2009 to 2011. Materials and MethodsAll patients with at least 10 oocytes and not willing to freeze embryos were offered the possibility to cryopreserve oocytes. The oocytes cryopreserved by slow freezing followed Stachecki's (1998) protocol, while the oocytes vitrified followed Kuwayama's. (2005) protocol. The endometrium was prepared using Estradiol Benzoate with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Two days before the replacement of the embryos, the oocytes were thawed. After thawing, the oocytes were kept in a incubator during 2 hours before inseminating by ICSI. The survival, fertilization, cleavage, average embryo transfer and pregnancy rates were compared through the chi-square test (P<0.05). All patients with at least 10 oocytes and not willing to freeze embryos were offered the possibility to cryopreserve oocytes. The oocytes cryopreserved by slow freezing followed Stachecki's (1998) protocol, while the oocytes vitrified followed Kuwayama's. (2005) protocol. The endometrium was prepared using Estradiol Benzoate with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Two days before the replacement of the embryos, the oocytes were thawed. After thawing, the oocytes were kept in a incubator during 2 hours before inseminating by ICSI. The survival, fertilization, cleavage, average embryo transfer and pregnancy rates were compared through the chi-square test (P<0.05). ResultsTable 1Outcome results from slow freezing versus vitrification protocol.Slow freezing (%)Vitrification (%)Cycles5645Oocytes Thawed523399Oocytes Survived*P=0.0085289 (55.2)255 (63.9)Oocytes Fertilized200 (69.2)177 (69.4)Embryos Cleaved185 (92.5)156 (88.1)Embryos Transferred121 (65.4)100 (64.1)Average Embryo Transfer2.22.2Implantation15 (12.4)16 (16.0)Pregnancies16 (28.6)15 (33.3)Clinical Pregnancies14 (25.0)13 (28.9)* P=0.0085 Open table in a new tab ConclusionAlthough the survival rate was statistically higher for the vitrification technique, this did not result in a statistically higher implantation and/or pregnancy rate. Therefore both techniques are reliable to cryopreserve oocytes. Although the survival rate was statistically higher for the vitrification technique, this did not result in a statistically higher implantation and/or pregnancy rate. Therefore both techniques are reliable to cryopreserve oocytes.

Rapid vitrification to eliminate abnormal sperm with nuclear vacuoles

The biggest problem in freezing cells is ice formation that destroys cells. Taking advantage of this, the study examined freezing to eliminate abnormal sperm with vacuoles. The objectives were: (a) to compare 2 vitrification protocols and (b) to determine freezing effects on vacuolated sperm.

Slow freezing vs vitrification of oocytes: a comprehensive meta-analysis

To compare the success of 2 oocyte cryopreservation techniques: Slow Freezing vs. Vitrification. Meta-analysis done after systematic review of literature. All the published articles between 1985 and March 2010 that deal with oocyte freezing by either utilizing a slow freezing or vitrification protocols were studied. Only those studies were included which used mature oocytes, used ICSI for fertilization and eventually had a successful pregnancy outcome. The main outcomes measured were the differences in survival rate, fertilization rate, implantation rate and pregnancy rate in both these protocols. The data was analyzed using the Random effects model in Comprehensive Meta analysis software. 240 articles were scrutinized, 120 were selected on inclusion criterion. Total number of oocytes used in the meta analysis study: Vitrification study: 3424 thawed eggs Slow Freezing: 18020 thawed eggs Table 1.Slow Freezing MethodVitrificationP valueSurvival Rate/Oocyte thawed71.9% [67.44, 75.89] 95% CI 11309/ 1802078.6% [70, 85.18] 95% CI. 2682/ 34240.0000001Fertilization Rate/Oocyte thawed51.2% (2245) [42.2, 60.1] 95% CI.55.96% (1710) [47.4, 67.1] 95% CI.0.0000001Implantation Rate/Oocyte thawed7% (420) [4.3, 11.2] 95% CI7.7% (207) [5.35, 11] 95% CI0.0000001Pregnancy Rate/Oocyte thawed4.2% (324) [3.08, 5.56] 95% CI7.6%(154) [5, 11.4] 95% CI0.7 Open table in a new tab

Is there any difference in vitrification and slow freezing protocol for embryo cryopreservation?

This study was an observational retrospective cohort, with the objective to compare the cryopreservation efficiency between slow freezing and vitrification technique using cryotop. A total of 181 cycles were performed for those patients that decided to thaw their embryos from 2009 to 2011. Considering that for the past three years, the embryos have being vitrified in our clinic, we have had the opportunity to thaw embryos either vitrified or frozen by the slow freezing protocol. The embryos cryopreserved by slow freezing followed the traditional protocol with 1.5M of PROH and 0.1M of Sucrose. The embryos cryopreserved by vitrification followed Kuwayama's (2005) protocol. The endometrium was prepared using Estradiol Benzoate with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Three days before the embryo transfer, the patient started using 90mg progesterone gel daily. After thawing, the embryos were kept in a 5% CO2 incubator, humidified in air, at 37°C during 2 hours before transferring. The embryo transfers were performed with a catheter, under ultrasound control. The survival, average embryo transfer, implantation and pregnancy rates were compared through the chi-square test (P<0.05). The results are as follow:Table 1Outcome results from slow freezing versus vitrification protocol.Slow freezing (%)Vitrification (%)Cycles9883Embryos Thawed288226Embryos Survived∗P< 0.0001218 (75.7)206 (91.1)Embryos Transferred213 (97.7)199 (96.6)Average Embryo Transfer2.22.4Implantation22 (10.3)30 (15.1)Pregnancies28 (28.6)29 (34.9)Clinical Pregnancies19 (19.4)25 (30.2)* P< 0.0001 Open table in a new tab Although the survival rate was statistically higher for the vitrification technique, this did not result in a statistically higher implantation and pregnancy rates.

Comparison of the vitrification and slow freezing protocol for day 3 embryo cryopreservation

Comparison of the vitrification and slow freezing protocol for day 3 embryo cryopreservation

Comparison Between Slow Freezing and Vitrification in Blastocyst Cryopreservation

Comparison Between Slow Freezing and Vitrification in Blastocyst Cryopreservation

Dehydration status of ABA-treated and cold-acclimated Hypericum perforatum L. shoot tips subjected to cryopreservation

To improve the vitrification protocol for Hypericum perforatum shoot tips, calorimetric measurements of the intensity of thermal transitions in the samples during warming using differential scanning calorimetry were assessed. Comparison of ABA treatment and cold-acclimation revealed higher content of crystallizable water in the latter. Regeneration capacity of cryopreserved H. perforatum shoot tips increased progressively with the exposure time in PVS3 cryoprotection mixture reaching the maximum between 59.3 and 71.3% after 180min when the proportion of freezable water ranged between 0.0 and 0.4% of total water content respectively. Glass transitions were observed in all samples dehydrated with PVS3 when freezable water was present. However, the decreasing recovery of H. perforatum shoot tips after prolonged PVS3 dehydration (without LN storage) indicated some injuries of meristematic tissue induced by PVS3 dehydration rather than ice nucleation and resulted from genotype-dependent tolerance to abiotic stresses accompanying the entire process.

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.

Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation

To investigate the efficacy of vitrification, rapid freezing, and slow freezing in preserving testicular tissue for subsequent isolation of spermatogonial stem cells. Experimental study. University-based laboratory. Immature mouse testicular tissue. The tunica of the testis was manipulated before cryopreservation. The tunica was either breached with a fine needle or completely removed, or the testis was sectioned longitudinally into halves. Cell viability by Trypan blue exclusion test and flow cytometry analysis of live-dead cytotoxicity test, measurement of hormonal production, enrichment of spermatogonial stem cells with use of magnetic-activated cell sorting technology. Samples with tunica minimally penetrated with a needle point gave the highest cell viability after freezing and thawing. Vitrification protocol with use of an ethylene glycol-sucrose-based vitrification solution (40% vol/vol ethylene glycol-0.6 mol/L sucrose) was able to maintain postwarming cell viability and functions similar to those of noncryopreserved controls and significantly better than both conventional slow and rapid freezing protocols. Primitive spermatogonial stem cells were enriched successfully from vitrified tissue via magnetic-activated cell sorting. Vitrification of testicular tissue is a time- and cost-efficient strategy to preserve spermatogonial stem cells for potential transplantation procedure.

Vitrification of human single pronuclear oocytes following two approaches to polar body biopsy

The purpose of the present study was to investigate whether the size of the opening in the zona pellucida (ZP) of human single pronuclear (1PN) oocytes made by laser and partial zona dissection (PZD) techniques might interfere with the survival and subsequent development to blastocyst stage upon vitrification and warming. Moreover, the viability of these blastocysts was evaluated by comparing their total cell number (TCN) to the TCN of blastocysts developed from control non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using laser-assisted and PZD techniques, respectively. The size of ZP opening made by laser and PZD techniques did not interfere with survival (94.8% and 95.4%) or development to the blastocyst stage (27.8% and 26.1%). However, the TCN of laser-derived blastocysts was significantly lower than the TCN of blastocysts developed from non-vitrified control 1PN oocytes (48.7 ± 3.4 versus 70.8 ± 7.1, P < 0.028). The vitrification protocol used here is thus revealed to be an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the viability of blastocysts developed from laser-treated 1PN oocytes seems to be negatively affected by this method of biopsy. The purpose of the present study was to investigate whether the size of the opening made in the outer membrane (zona pellucida) of human single pronuclear (1PN) oocytes by mechanical dissection and laser techniques followed by polar body removal could hamper survival and development to the blastocyst stage upon vitrification and warming. Vitrification is a simple method of cryopreservation in which the intra- and extracellular solutions transform during cooling into a solid, glass-like substance. Moreover, in order to predict the viability of blastocysts developed from vitrified 1PN oocytes, their total cell number (TCN) was recorded and compared with the TCN of blastocysts developed from non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using the laser-assisted and mechanical method of dissection, respectively. Vitrification was also applied to 61 zona-intact 1PN oocytes. Following vitrification, no disparity in survival or further development to blastocyst stage was observed between either of the two biopsied groups and zona-inact 1PN oocytes, revealing that the vitrification protocol used here is an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the TCN of blastocysts developed from laser-treated 1PN oocytes was considerably lower than the TCN of blastocysts developed from non-vitrified zona-intact 1PN oocytes ( n = 46) suggesting that the viability of blastocysts developed from laser-treated 1PN oocytes was negatively influenced by this method of biopsy.