Vitrification Warming Research Articles

Effects of Cryoprotectant Concentration and Exposure Time during Vitrification of Immature Pre-Pubertal Lamb Cumulus-Oocyte Complexes on Nuclear and Cytoplasmic Maturation.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Maternal age and its impact on implantation and clinical pregnancy rates in patients undergoing day 3 versus day 5 embryo transfer in Indian population

Objective: The present study compared the reproductive outcomes (implantation rate, pregnancy rate) between day 3 and day 5 transfers in two different age groups of patients (aged <36 years vs. aged ≥36 years). Materials and methods: The present study was conducted in the RIDGE IVF Centre, Gouri Hospital, New Delhi (India), after obtaining institutional ethical committee clearance. For frozen embryo transfers (FETs), day 3 embryos were thawed using Origio vitrification warming kit. The outcome parameters [anti mullerian hormone (AMH), antral follicle count (AFC), follicle-stimulating hormone, E2, mean number of oocytes retrieved, embryo grades A, B, and C, beta human chorionic gonadotropin (HCG), number of sacs, miscarriages, and live birth rate] were compared between age <36 versus >36 years, day 3 versus day 5 embryo, and fresh versus frozen embryo. The unpaired t test was used for comparing quantitative variables and Chi-squared test was used for comparing qualitative variables. Results: The mean age of the study population was 31.84 ± 3.74 years. The mean AMH, AFC, and oocytes retrieved were significantly more among <36 years compared to ≥36 years age group. Beta HCG and number of sacs did not differ between <36 years and ≥36 years age groups. The number of sacs formed and live birth were significantly more among frozen embryo. Day 5 embryo had significantly more positive rate of beta HCG, more number of sac formations, and live birth. Conclusion: The present study dictates that in vitro fertilization rate showed better success with women <36 years of age than >36 years old. Day 5 blastocysts had good implantation rates along with higher resulting pregnancy rates. The FET had better live birth rate.

Validation testing of alternative blastocyst vitrification warming protocols to facilitate international shipments

Validation testing of alternative blastocyst vitrification warming protocols to facilitate international shipments

Vitrification negatively affects the Ca2+-releasing and activation potential of mouse oocytes, but vitrified oocytes are potentially useful for diagnostic purposes.

To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca2+ dynamics in response to SrCl2 or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl2 exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. Vitrified-warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified-warmed human oocytes is required.

Successful application of a single warming protocol for embryos cryopreserved by either slow freezing or vitrification techniques

ABSTRACTThe aim of this study was to evaluate the feasibility and efficiency of using a sucrose gradient-based warming protocol as a universal warming approach on human cleavage stage embryos. Between January 2013 and November 2014, a total of 118 warming cycles were performed on 705 embryos which had previously been cryopreserved/thawed by slow freezing protocols or cryopreserved by slow freezing and warmed by vitrification thaw solution. Clinical outcomes have been retrospectively analyzed depending on cryopreservation and warming techniques used, embryo viability, day of cryopreservation, clinical pregnancy, implantation, and live birth rate. Results indicate that, the use of the vitrification warming protocol for warming after slow freezing results in comparable post-warming survival (71.6% and 71.1%; p = 0.890). Higher clinical pregnancy, implantation, and live birth rates were obtained in the cryopreserved embryos by slow freezing and warmed by vitrification group in comparison to the cryopreserved/thawed by slow freezing protocols group but the results did not show statistically significant differences between groups (p > 0.05). These results indicate that such an approach can eliminate the need to search for a brand-dependent product, as well as case-dependent hands-on planning. Further research that evaluates the effectiveness of this approach on a larger case series is underway.Abbreviations: CPA: concentrated cryoprotective agent; COH: controlled ovarian stimulation; FET: frozen embryo transfer; HSG: hysterosalpingogram; mHTF: modified human tubal medium; SSM: single step media; SSS: synthetic serum substitute; TV-USG: transvaginal ultrasound.

Effect of vitrification procedures on the subsequent development of invitro matured swamp buffalo oocytes following invitro fertilization.

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after invitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after invitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3±5.84%) of vitrified oocytes than that combined with VB solution (69.8±4.19%-75.8±4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1±0.07%-5.2±0.24%) compared with that of untreated oocytes (18.0±1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.

Bovine oviductal and uterine fluid support in vitro embryo development.

In order to mimic the maternal oviductal environment, we evaluated the effect of oviductal fluid (OF) and/or uterine fluid (UF) supplementation on in vitro embryo development and quality. In vitro-produced zygotes were cultured with 1.25% OF from Day 1 to Day 4 after insemination (OF group), 1.25% OF from Day 1 to Day 4 followed by 1.25% UF from Day 4 to Day 9 (OF+UF group) or 1.25% UF only from Day 4 to Day 9 (UF group). Control groups were cultured in the presence of synthetic oviduct fluid (SOF) supplemented with 3mgmL-1 bovine serum albumin (BSA) or 5% fetal calf serum (FCS). Supplementation of the culture medium with OF and/or UF (both at 1.25%) supported embryo development (Day 9 blastocyst rate 28.2-30.6%). At 72h after vitrification-warming, the survival of blastocysts from the OF and OF+UF groups was similar to that of blastocysts in the SOF+BSA group (61.0±5.7% and 62.8±6.4% vs 64.8±6.4% respectively), but significantly higher than that of blastocysts from the SOF+FCS group (31.6±4.9%; P<0.001). Blastocysts from the OF group exhibited upregulation of epigenetic genes (i.e. DNA methyltransferase 3α (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R)), compared with expression in the SOF+FCS group (P<0.05). Whereas those from OF+UF and UF groups exhibited downregulation of oxidative stress genes compared to SOF+BSA and OF groups for glutathione peroxidase (GPX1) and to SOF+FCS, SOF+BSA and OF groups for chloride intracellular channel 1 (CLIC1) (P<0.05). In addition, accumulation of reactive oxygen species was lower in blastocysts from the OF, OF+UF and UF groups. In conclusion, the use of low concentrations of OF and UF in in vitro serum-free culture supports embryo development, with OF providing a better control of embryo methylation, whereas UF may have antioxidant activity.

Impact of prolonged oocyte incubation time before vitrification on oocyte survival, embryo formation, and embryo quality in mice.

Oocyte incubation time before freezing is one of the factors affecting oocyte vitrification. In the assisted reproductive technology (ART) clinics, it is sometimes decided to perform oocyte vitrification after a long period of incubation time due to various conditions, such as inability to collect semen samples, unsuccessful urological interventions (PESA, TESE, etc.), or unexpected conditions. A time factor of up to 6h has been studied in the available reports. Therefore, this study was designed to evaluate oocyte incubation time before freezing at 0, 6, 12, 18, and 24h after retrieval. Metaphase II (MII) oocytes were obtained from NMRI female mice after being randomly divided into the five groups of 0, 6, 12, 18, and 24h of freezing via hormonal stimulation following retrieval and entered into the vitrification-warming process. The thawed oocytes were evaluated according to the survival criteria and then inseminated with the sperms of male mice for in vitro fertilization. The next day, the embryo formation rate and embryo quality were assessed. Our results demonstrated that even after 24h of incubation, the survival rate of oocytes was 51.35% with the embryo formation rate of 73.21%. However, the survival and embryo formation rates significantly decreased within 12, 18, and 24h after retrieval compared to the groups vitrified at 0h. The embryo quality was significantly reduced by vitrification at 0 to 24h after retrieval. According to our data, although a prolonged incubation time before freezing reduced the survival rate, there was still a chance for oocytes to stay alive with acceptable embryo formation and quality rates after vitrification warming of oocytes.

Appendix F: Quinn's Advantage Embryo Freeze Kit.

Despite a large focus on the use of vitrification to cryopreserve embryos in recent years, there are still arguments for the use of slow freezing for the cleavage-stage embryo. Having said this, there are lessons to be learned from the process of vitrification that could be applied to slow freezing to improve post-thaw survival and ultimately clinical pregnancy rates. Specifically, increasing the concentration of sucrose in the freezing solution from 0.1 to 0.2M and subsequently increasing the sucrose concentrations in thawing solutions could prove beneficial. The use of vitrification warming solutions in the thawing of slow-frozen embryos may also be an option that not only improves survival but also streamlines product purchasing and protocols within the laboratory.

Effective vitrification and warming of porcine embryos using a pH-stable, chemically defined medium.

The use of pH-stable media would simplify embryo vitrification and the warming of porcine embryos and might facilitate the application of embryo transfer in practice. In this work, we investigated whether a pH-stable basal medium constituted of Tyrode’s lactate medium, polyvinyl alcohol, and HEPES for buffering was suitable for porcine embryo vitrification warming in place of the conventional gas-equilibrated media. A high percentage (>90%) of embryos survived vitrification and warming in this medium, achieving in vitro survival rates similar to embryos vitrified-warmed using the conventional protocol and their fresh counterparts. The pH-stable medium did not affect the in vivo developmental competence of the vitrified-warmed embryos. A farrowing rate of 71.4% (5/7) with 10.4 ± 3.1 piglets born was obtained for the embryos vitrified and warmed in this medium and transferred to selected recipients. This medium will enable the use of simple, safe and standardized protocols for the vitrification and warming of porcine embryos for optimal embryo survival and quality when applied under field conditions. This study opens new possibilities for the widespread use of embryo transfer in pigs.

Effect of Exogenous Anti-Müllerian Hormone Treatment on Cryopreserved and Transplanted Mouse Ovaries.

Follicle loss occurs after ovary cryopreservation and transplantation. To preserve the follicle pool of cryopreserved or grafted ovaries, anti-Müllerian hormone (AMH), which inhibits ovarian follicle recruitment, was used in a mouse model. In experiment 1, ovaries were vitrified warmed with different doses of AMH (0, 5, 15, or 45 μg/mL) supplementation. In experiment 2, AMH (0, 50, 250, and 1250 μg/mL) was injected into mice before and/or after cryopreserved ovary autotransplantation, and the recipients remained for 7 or 28 days after grafting. Ovaries were evaluated by follicle morphology, density, and apoptosis ratio. Additionally, serum follicle-stimulating hormone was measured in experiment 2. Significantly decreased follicle apoptosis were detected in AMH-treated groups when compared to the control ovaries in experiment 1, meanwhile no positive effect of exogenous AMH was found in experiment 2. Thus, we suggest AMH supplementation during ovary vitrification warming has beneficial effect on reducing follicle apoptosis.

Co-culture with granulosa cells improve the in vitro maturation ability of porcine immature oocytes vitrified with cryolock

This study was designed to evaluate the efficiency of two oocyte vitrification–warming procedures using two different devices: Superfine Open Pulled Straws (SOPS) and Cryolock, as well as the effect of the co-culture of vitrified immature oocytes with fresh granulosa cells to improve in vitro maturation (IVM). Immature oocytes were vitrified with two procedures: A) Oocytes were exposed to an increasing concentration of ethylene glycol (EG) from 4% to 35% with 0.5M trehalose. They then, were loaded in SOPS or Cryolock. For warming, oocytes were exposed to decreasing concentrations of trehalose 0.3, 0.2 and 0.1M for IVM. B) Oocytes were exposed to two mixtures of EG and dimethylsulfoxide (Me2SO), at 7.5% and 16%, both with 0.4M of sucrose and then loaded in SOPS or Cryolock and stored in liquid nitrogen. For warming, oocytes were exposed to a single concentration of sucrose 0.5M. After warming, viability was determined; and after 44h of IVM both viability and meiotic stages were evaluated. The results indicate no significant differences between procedures A and B with SOPS in all maturation stages, reaching a maximum maturation rate of 21%. As to Cryolock, significant differences were observed between both procedures, being procedure B, more efficient with a yield of 38% in MII stage and increased to 49% due to the co-culture with fresh granulosa cells. In conclusion, viability and maturation rates were improved with Cryolock and procedure B with the co-culture system in vitrified immature oocytes.

Effects of varying tissue sizes on the efficiency of baboon ovarian tissue vitrification

ObjectiveThe aim of this study was to detect the effects of varying tissue sizes on the efficiency of baboon ovarian tissue vitrification. Study designThe percentages of morphologically normal primordial follicles and the follicles expressing bax protein in ovarian tissues after vitrification–warming were measured. Besides, the 17-β estradiol levels in the culture supernatants were measured. ResultsThe percentages of morphologically normal primordial follicles in vitrified–warmed ovarian tissues slicing in 0.5–1.5mm in length and wide, and 1.0mm in thickness were significantly higher than those slicing in 2.0mm in length and wide, and 1.0mm in thickness. Moreover, the follicles expressing bax protein in vitrified–warmed ovarian tissues slicing in 0.5–1.5mm in length and wide, and 1.0mm in thickness were significantly lower than those slicing in 2.0mm in length and wide, and 1.0mm in thickness. The 17-β estradiol levels in the culture supernatants slicing in 1.0–1.5mm in length and wide, and 1.0mm in thickness were significantly higher than those slicing in 0.5mm or 2.0mm in length and wide, and 1.0mm in thickness. ConclusionsCortex piece slicing in 1.0–1.5mm in length and wide, and 1.0mm in thickness is suitable for baboon ovarian vitrification.

Effects of supra-zero storage on human ovarian cortex prior to vitrification–warming

This study investigated the effects of supra-zero storage on ovarian cortex of 12 premenopausal patients before cryopreservation. Fresh ovarian tissue (control) was either vitrified immediately or stored at 4°C for 24 h or 48 h before vitrification. This study assessed malondialdehyde release during storage and the capacity to synthesize oestradiol in culture, follicle morphology and the proportion of apoptotic follicles subsequent to vitrification–warming. The malondialdehyde concentration in the storage medium increased significantly in a time-dependent manner (P = 0.029). Oestradiol release during tissue culture decreased statistically significantly in both storage groups compared with the immediate vitrification group (P = 0.043). The proportion of high-quality follicles was significantly different between the fresh group and the two storage groups (24 h, P < 0.01; 48 h, P < 0.001). The proportion of apoptotic follicles was significantly different between the fresh and immediate vitrification groups (P < 0.05). The 24-h and 48-h groups were not different compared with the other groups. This study provides evidence that storage at supra-zero conditions has detrimental effects on the human ovarian cortex. Storage duration should therefore be limited to a minimum to prevent potential damage.

Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol?

Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. The aim of this study was to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification. This was a prospective study on 216 sibling oocytes randomized for either conventional rapid thawing or rapid warming with vitrification warming solution. The primary endpoint was morphological assessment of survival at 2h. Surviving oocytes were divided into two subgroups: (i) parthenogenetically activated; and (ii) fixed and observed for spindle/chromosome configuration. Secondary endpoints were parthenogenetic development and spindle/metaphase configuration. Survival rate with rapid warming was higher (92/102, 90.2%) than with rapid thawing (85/114, 74.6%; P=0.005), and after 3d of culture the rapidly warmed parthenotes had more blastomeres compared with those rapidly thawed (P=0.042). Meiotic spindle and chromosomal configuration were not significantly influenced by rapid warming or rapid thawing. The finding of this study allows IVF centres to increase the efficiency of oocyte slow freezing, enabling survival rates comparable to vitrification protocols, and potentially to optimize costs by using the same warming protocol for both slow-frozen and vitrified reproductive cells.Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. Due to the high survival rate guaranteed by vitrification, this procedure is increasingly applied worldwide. Nevertheless, to date, perhaps millions of slow-frozen oocytes/embryos have already been stored in IVF cryobanks. The aim of this study is to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification in order to optimize the slow freezing survival rate and reduce costs by using the same solutions for both slow freezing and vitrification warming. Between December 2012 and January 2013, 216 slow-frozen oocytes donated for research were randomized for the rapid thawing conventionally used for slow-frozen oocytes, or rapid warming as for vitrification protocols. We observed that rapid warming significantly increased the survival rate of slow-frozen oocytes; furthermore, slow-frozen oocytes warmed via rapid warming had better developmental competence than their sibling counterparts thawed with conventional rapid thawing. The application of this warming protocol allows us to increase the efficiency of oocyte slow freezing procedure, enabling survival rates comparable with that reported in previous vitrification studies. This finding redefines the scenario of slow-frozen and vitrification, confirming the pivotal role of warming and indicating that the results obtained until now with slow-frozen oocytes/embryos can be improved; this could be the biggest breakthrough in human oocyte cryopreservation since the introduction of vitrification. Furthermore, this finding allows IVF centres to reduce costs by using the same ‘universal warming protocol’ for both slow-frozen and vitrifed reproductive cells.

Vitrification of day 3 cleavage-stage embryos yields better clinical outcome in comparison with vitrification of day 2 cleavage-stage embryos

The objective of this retrospective study was to determine an optimal time point for vitrification of cleavage-stage human embryos. This study included patients who were undergoing day 2 or day 3 vitrified-warmed cleavage-stage embryo transfer at the In Vitro Fertilization (IVF) Programme of the Shanghai First Maternity and Infant Hospital, China, affiliated to the Tongji University School of Medicine, from April 2010 to March 2012. Intervention was made for the entire cohort of vitrified embryos for poor responder patients so as to avoid severe ovarian hyperstimulation syndrome. Embryo survival rate (SR) after vitrification-warming, implantation rate (IR), and clinical pregnancy rate (CPR) were the main outcome measurements. In total, 380 vitrified-warmed cleavage-stage embryo transfer (VWT) cycles were included. We found that the SR after vitrification and warming for day 2 embryos and day 3 embryos were 92.7% and 92.8%, respectively. For poor ovarian responders, the IR of day 2 and day 3 vitrified-warmed embryos was 6.4% and 13.2%, respectively (P = 0.186). The CPR for day 3 vitrified-warmed embryos was significantly higher than that of day 2 vitrified-warmed embryos (17.6 vs. 4.0% per transfer cycle, P = 0.036). For patients who had their entire cohort of embryos vitrified to prevent severe ovarian hyperstimulation syndrome (OHSS), the IR and CPR were not significantly different for day 2 and day 3 vitrified-warmed embryo transfer. In conclusion, for vitrified-warmed embryo transfer, cryopreservation of the entire cohort of embryos on day 3 resulted in better clinical outcomes compared with cryopreservation on day 2. Therefore, it is highly recommended that cleavage-stage embryos should be vitrified on day 3, but not on day 2, particularly for poor ovarian responder patients.

Comparative effects of adding β-mercaptoethanol or L-ascorbic acid to culture or vitrification–warming media on IVF porcine embryos

The aims of the present study were to; (1) determine the effects of supplementation with two antioxidants during in vitro culture (IVC) on embryo development and quality; and (2) test the effects of adding the antioxidants to vitrification-warming media on the cryotolerance of in vitro-produced (IVP) porcine blastocysts. In Experiment 1, presumptive zygotes were cultured without antioxidants, with 50 µM β-mercaptoethanol (β-ME) or with 100 µM L-ascorbic acid (AC). After culture, blastocyst yield, quality and cryotolerance were evaluated in each treatment group. In Experiment 2, survival rates (3 and 24 h), total cell number, apoptosis index and the formation of reactive oxygen species (ROS) in blastocysts vitrified-warmed with 100 µM AC or 50 µM β-ME or without antioxidants added to the vitrification medium were compared. Antioxidant addition during IVC had no effect on embryo development, total cell number or the apoptosis index, and culturing embryos in the presence of β-ME had no effects on cryotolerance. In contrast, ROS levels and survival rates after vitrification-warming were significantly improved in embryos cultured with AC. Furthermore, addition of AC into vitrification-warming media enhanced embryo survival and embryo quality after warming. In conclusion, our results suggest that supplementing culture or vitrification media with 100 µM AC improves the quality and cryosurvival of IVP porcine blastocysts.

Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study

Vitrification has been successfully applied in the cryopreservation of oocytes and embryos. It can be achieved either by direct (open system) or indirect (closed system) contact with liquid nitrogen. Unlike embryo vitrification, few reports have been published regarding oocyte vitrification in closed systems. In order to validate the effectiveness of a closed and aseptic vitrification approach for oocyte cryopreservation, a prospective, randomized study was performed. Sibling oocytes donated from the same donor were randomly and equally assigned into closed or open vitrification groups. A total of 75 vitrification–warming cycles were performed in each group. Apart from the survival rate (82.9% versus 91.0%, P<0.05), no statistically significant differences were observed in pregnancy (β-human chorionic gonadotrophin positive) (42.7% versus 33.3%), clinical pregnancy (36.0% versus 28.0%), implantation (13.8% versus 10.1%), ongoing pregnancy (33.3% versus 24.0%) and live birth (36.0% versus 24.0%) rates between the closed and open groups, and 27 and 18 healthy babies were born, respectively. This study shows that the replacement of the open vitrification system by a closed system has no impact on clinical pregnancy and implantation rates. Therefore, the closed vitrification system provides an aseptic alternative to the open method for oocyte vitrification.Recently, the need for oocyte cryopreservation has increased due to a number of medical and social reasons. Vitrification is the most efficient technique for cryopreserving oocytes. A main characteristic of this successful technique is the direct contact of the oocytes with liquid nitrogen, which increases cooling rates. Since there are many concerns of contamination hazards due to this direct contact, the need for finding an appropriate aseptic way for vitrifying oocytes has arisen. In aseptic vitrification methods, the direct contact with liquid nitrogen is avoided but the cooling rates are decreased. Therefore the efficiency of this technique can be questioned. In order to validate the effectiveness of an aseptic vitrification approach for oocyte cryopreservation, a prospective randomized study was performed. Oocytes donated from the same donor were randomly and equally assigned to two groups. In one group, oocytes were vitrified in an aseptic way (closed system) and in the other group, oocytes were vitrified in an non-aseptic way (open system). A total of 75 vitrification–warming cycles were performed in each group. The biological and clinical results in the two groups were similar, and 27 and 18 healthy babies were born in the closed and open groups. This study shows that the replacement of the open vitrification system by a closed system has no impact on the pregnancy and implantation rates. Therefore, the closed vitrification system may be an alternative for oocyte vitrification in an aseptic way.

Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification–warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency.Vitrification is defined as the solidification of a liquid without the formation of ice crystals. The basic principle lies in finding the correct balance between the intracellular amount of cryoprotectants and the cooling rate, without inducing a toxic effect. Before plunging in liquid nitrogen, the embryos are deposited on a carrier device that can be described as open or closed. The use of open carriers have raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to analyse the outcome of vitrification of blastocysts randomly allocated to either open vitrification (group I) or aseptic vitrification (group II) using the VitriSafe as the embryo carrier in both groups. In group I, blastocysts were exposed to two cryoprotectant solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were initially exposed to an additional solution of lower concentration (5%/5%). A total of 208 and 224 vitrification–warming cycles were initiated for group I and group II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. There was no statistically significant difference between the two groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). Ultra-rapid vitrification can be replaced by aseptic vitrification without affecting cryopreservation efficiency.