Open Vitrification System Research Articles

Implantation and survival rates of vitrified/warmed blastocysts using a closed semi-automated vitrification system compared to an open manual vitrification system in a regional IVF clinic

Background and Aim: The success of blastocyst vitrification and warming relies on the control and standardization of laboratory environmental factors such as temperature and air quality as well as competency-based technical factors such as and speed and consistency of the scientist. The aim of this study was to determine if the implementation of a closed, semi-automated vitrification system (Gavi) could improve the survival and implantation rates in frozen embryo transfers (FET's) compared to using an open, manual vitrification system (Cryotop).

P-763 effect of duration of cryo-storage of vitrified embryos on obstetric and perinatal outcomes

Abstract Study question Does the duration of cryo-storage of vitrified embryos affect obstetric and perinatal outcomes? Summary answer Duration of cryo-storage with an open vitrification system did not affect obstetric and perinatal outcomes. What is known already Frozen-thawed blastocyst transfer has been performed widely with improvement of embryo culture conditions and cryopreservation techniques. Although blastocyst vitrification has become an essential method to improve clinical outcomes of IVF, there has been little study into the relationship between a long duration of cryo-storage in liquid nitrogen and obstetric and perinatal outcomes. Study design, size, duration This retrospective study was conducted at Kyono ART Clinic from January 2007 to December 2020. This study includes a total of 1053 singletons derived from 2461 frozen-thawed blastocyst transfers in 2461 patients. Steel’s multiple comparison test was performed for clinical and perinatal outcomes with cases of cryo-storage of less than 3 months as a control group. P < 0.05 was considered statistically significant. Participants/materials, setting, methods The subjects are patients who underwent their first single frozen-thawed blastocyst transfer (FBT) with an open vitrification system. Females >40 years old at cryopreservation and those who underwent preimplantation genetic testing were excluded. According to cryo-storage duration, patients were grouped as follows: group A, 0-3 months (1255 cycles); group B, 3-6 months (1008 cycles); group C, 6-12 months (162 cycles), group D, 12-24 months (36 cycles). Main results and the role of chance Both the mean maternal age at blastocyst cryopreservation (A: 33.7±3.6, B: 34.1±3.5, C: 34.8±3.2, D: 35.1±3.3) and the mean maternal age at FBT (A: 33.8±3.6, B: 34.4±3.5, C: 35.5±3.1, D: 36.4±3.3) in groups B, C, and D were significantly higher compared to those in group A.. There was no significant difference in the survival rate after blastocyst thawing [A: 97.4% (1256/1290), B: 98.3% (1010/1027), C: 99.4% (163/164), D: 97.2% (35/36)]. The pregnancy rate in group C was significantly lower compared to group A [A: 60.0% (752/1254), B: 61.4% (619/1008), C: 48.1% (78/162), D: 63.9% (23/36)] The gestational age of group C was significantly lower compared to group A (A: 39.5±1.8, B: 39.4±1.8, C: 38.4±3.1, D: 39.8±1.6), but there were no significant differences in live birth weight or height. There were no significant differences in congenital abnormality rate [A: 1.3% (7/541), B: 2.5% (11/440), C: 1.8% (1/56), D: 0.0% (0/16)], placental abnormalities such as placenta previa [A: 1.3% (7/541), B: 1.6% (7/440), C: 1.8% (1/56), D: 0.0% (0/16)], perinatal abnormalities such as hypertensive disorders of pregnancy or gestational diabetes [A: 6.1% (33/541), B: 8.6% (38/440), C: 1.8% (1/56), D: 6.3% (1/16)] among the four groups. Limitations, reasons for caution We could not provide sufficient information on confounding factors such as smoking habits, and the sample size was too small for multivariate analysis. The safety of longer storage will need to be verified as this has not been clarified in this study. Wider implications of the findings The pregnancy rate in group C was significantly lower than that in group A, but the effect was small: 0.065, power of 0.78. Our data suggested that the duration of cryopreservation with an open vitrification system in liquid nitrogen did not affect obstetric or perinatal outcomes. Trial registration number not applicable

The effect of different vitrification protocols on cell survival in human ovarian tissue: a pilot study

BackgroundVitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are as yet no standard protocols for its use in ovarian tissue cryopreservation (OTC). Published protocols diverge mainly with regard to the extent of supplementation of dimethyl sulfoxide (DMSO) to the vitrification medium, and to the use of an open or closed vitrification system.We investigated the viability of cells after vitrification/warming, using ovarian tissue of transgender patients, by means of Fluorescence Activated Cells Sorting (FACS), and histomorphological analyses using a DMSO-containing (P1) and a DMSO-free protocol (P2) in an open or closed vitrification setting.ResultsTwelve ovarian samples were donated from female-to-male transgender patients: 6 were vitrified according to protocol 1, the other 6 according to protocol 2. The amount of viable cells was 90.1% (P1) and 88.4% (P2) before vitrification. After vitrification and subsequent warming, viable cells were reduced to 82.9% (P1, p = 0.093) and 72.4% (P2, p = 0.019). When comparing the closed and the open systems, the decline in cell viability from pre- to post-vitrification was significant only for the latter (p = 0.037). Histological examination reveals no significant differences with respect to degenerated follicles before or after vitrification.ConclusionThese results led us to conclude that a protocol containing DMSO results in a higher viability of ovarian cells than a protocol that uses ethylene glycol as cryoprotective agent in vitrification. The use of an open vitrification system led to significant decline in the rate of viable cells.Trial registrationNCT03649087, retrospectively registered 28.08.2018.

High-security closed devices are efficient and safe to protect human oocytes from potential risk of viral contamination during vitrification and storage especially in the COVID-19 pandemic

PurposeThe main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes’ vitrification.MethodsA prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital (Italy) between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups.ResultsNo statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women’s mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection (792 and 594 respectively, P = .304).ConclusionsThe present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples’ contamination during vitrification and storage.

Efficiency and efficacy of vitrification in 35 654 sibling oocytes from donation cycles.

Is oocyte vitrification/warming as efficient and effective as using fresh oocytes in donation cycles? IVF with vitrified donor oocytes is less efficient than using fresh oocytes, but its efficacy remains comparable to that of fresh cycles. Oocyte vitrification is used to preserve the reproductive potential of oocytes. A small number of randomized controlled trials carried out by experienced groups have shown that this technique provides fertilization, pregnancy, implantation and ongoing pregnancy rates comparable to those of fresh oocytes. However, large registry-based analyses have consistently reported lower live birth rates (LBRs) in cycles using vitrified oocytes. It is not clear whether this decrease may be due to the effect of vitrification per se on the oocytes or to the lower efficiency of the technique, as some of the oocytes do not survive after warming. Retrospective cohort analysis of 1844 cycles of oocyte donation (37 520 oocytes), each donor in the study provided enough oocytes for at least one reception cycle with fresh oocytes (2561 cycles) and one reception cycle with vitrified oocytes (2471 cycles) from the same ovarian stimulation (sibling oocytes). Overall, 35 654 oocytes were considered in the analysis. All embryo transfers (n = 5032) were carried out between 2011 and 2017. Differences in reproductive outcomes after the first embryo transfer were evaluated using Pearson's Chi-squared test and regression analysis adjusted for recipient's age, BMI, sperm origin and state, day of embryo transfer, morphological score and number of transferred embryos. We performed two additional sub-analyses, to test whether the efficiency and/or effectiveness of vitrification/warming impacts reproductive results. One analysis included paired cycles where the same number of fresh and vitrified oocytes were available for ICSI (SAME sub-analysis), while the second analysis included those cycles with a 100% survival rate post-warming (SAME100 sub-analysis). Baseline and cycle characteristics of participants were comparable between groups. Overall, fertilization rates and embryo morphological scores were significantly lower (P < 0.001) when using vitrified oocytes; moreover, vitrified oocytes also resulted in lower reproductive outcomes than sibling fresh oocytes using both unadjusted and adjusted analyses: ongoing pregnancy (32.1% versus 37.5%; P < 0.001; OR 0.88, 95% CI 0.77, 1.00) and live birth (32.1% versus 31.9%; P = 0.92; OR 1.16, 95% CI 0.90, 1.49). However, when the efficiency of warming was taken into account, reproductive outcomes in recipients became comparable: ongoing pregnancy (33.5% versus 34.1%; P = 0.82; OR 1.11, 95% CI 0.87, 1.43) and LBR (32.1% versus 32%; P = 0.97; OR 1.15, 95% CI 0.89, 1.48). Moreover, after selecting only cycles that, in addition to having the same number of oocytes available for ICSI, also had 100% post-warming survival rate in the vitrified group, reproductive outcomes were also comparable between fresh and vitrified oocytes: ongoing pregnancy (34.8% versus 32.4%; P = 0.42; OR 1.32, 95% CI 0.98, 1.77) and live birth (32.9% versus 31.0%; P = 0.52; OR 1.27, 95% CI 0.95, 1.71), indicating that reproductive outcomes of these cycles are affected by the efficiency of the vitrification/warming technique performed rather than the oocyte damage due to the fast cooling process to which oocytes are subjected. An open vitrification system was used for all cases, and oocyte vitrification/warming was performed by experienced embryologists with consistently high survival rates; caution must be exerted when extrapolating our results to data obtained using other open vitrification systems, closed vitrification systems or to IVF units with survival rates <90%. This is the largest cohort study comparing reproductive outcomes of vitrified and fresh sibling donor oocytes to date. We found that, when the number of oocytes available after warming is equal to the number of fresh oocytes, reproductive results including live birth are comparable. Consequently, the efficiency of vitrification must be taken into account to achieve the same reproductive outcomes as with fresh oocytes. We recommend implementing strict indicators of vitrification/warming efficiency in clinics and refining vitrification/warming protocols to maximize survival. This work was supported by intramural funding of Clínica EUGIN and by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 048). The authors declare no conflicts of interest. N/A.

Embryons vitrifiés, embryons frais : comparaison des poids de naissance

ObjectiveIt is already known that children born after slow frozen embryo replacement have a significantly higher birth weight compared to children born after fresh embryo transfer. Similar data have been reported related to frozen embryo transfer using an open vitrification system. However, few data relative to birth weight using a complete embryo closed vitrification system has been reported. The purpose of this study was to know if frozen embryo in closed vitrification system is associated with a higher birth weight compared to fresh embryo replacement. DesignThis was a monocentric retrospective cohort study, 371 children were issued from fresh embryo replacement and 127 from vitrified embryo transfer. Materials and MethodsAll singletons born after fresh or vitrified embryo transfer between January 2011 and April 2015 were included. Births from the vitrified or fresh transfers of egg or sperm donation, and preimplantation genetic diagnosis were excluded. In addition, pregnancies with more than one gestational sac at the first ultrasound were excluded. An analysis of covariance (ANCOVA) was used for multivariate analysis. ResultsMean birth weight was 205g higher in the frozen embryo compared with fresh embryos transfer groups (3368g vs. 3163g respectively, P<0.001). This difference remained after multivariate analysis adjusted on confounding factors such as gestational age, maternal age, maternal body mass index (BMI), tobacco exposure, number of embryo transferred and birth order (P<0.001).. ConclusionsEmbryo frozen in closed vitrification system is associated with a higher birth weight compared to fresh embryo replacement. Our findings are consistent with the previous studies related to slow freezing and open vitrification systems data. The effects of controlled ovarian stimulation (COS), ex vivo culture conditions and cryopreservation systems on birth weight of children born should be further explored.

Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified–warmed blastocysts

A retrospective cohort study of 8736 autologous single vitrified-warmed blastocyst transfer cycles was conducted in a single centre to investigate the effect of cryostorage on clinical and neonatal outcomes. Cryostorage duration was classified into three groups: (A) 0-2 months (n = 4702); (B) 2-13 months (n = 2853) and (C) 13-97 months (n = 1181). Blastocysts were vitrified using the Cryotop method. No significant differences were observed in live birth rates: (A) 37.3%; (B) 34.9%; (C) (35.2%). Gestational period was significantly shorter in group C: (A) 38.7 ± 1.8; (B) 38.6 ± 1.6; (C) 38.1 ± 1.7; P < 0.05. This was clinically unimportant as the average gestational age was more than 38 weeks. No significant differences between groups were observed in birth weight: (A) 3060 ± 455 g; (B) 3052 ± 449 g; (C) 2992 ± 445 g, or congenital malformation rates: (A) 2.2%; (B) 1.9%; (C) 1.8%. The limitation of this study was that maximum storage duration was 8 years; most blastocysts were in cryostorage for much shorter periods. Long-term storage of blastocysts that are vitrified using an open device vitrification system has no negative effect on pregnancy and neonatal outcomes.

Cryopreservation of human ovarian tissue using the silver closed vitrification system.

The aim of this study is to evaluate the feasibility of using a hand-made silver container for the cryopreservation of human ovarian cortex. Human ovarian cortex tissues were vitrified using an open vitrification system (OVS) of needle immersed vitrification (NIV) and two closed vitrification systems (CVS) of a plastic vial (plastic CVS) and a silver container (silver CVS). Outcomes of vitrification were evaluated morphologically and histologically by in vitro culture and xenotransplantation. The apoptosis of primordial follicles was assessed by TUNEL staining. The production of E2 and P4 was examined by a chemiluminescent immunoassay. Blood vessels were visualized with CD31 staining. Compared with the fresh ovarian cortex tissue, ovarian cortex tissues that were vitrified using the three different carriers and then warmed showed significantly reduced percentages of normal primordial follicles, viability of primordial follicles, E2 and P4 levels during in vitro culture and decreased amounts of blood vessels. However, much better outcomes were obtained with NIV and silver CVS than with plastic CVS, based on the better morphology and viability of primordial follicles, higher E2 and P4 production during an in vitro culture, and greater numbers of blood vessels after xenografting. Importantly, the outcomes of ovarian cortex cryopreservation with silver CVS were similar and comparable to those with NIV. The hand-made silver container as a CVS is a promising carrier for the cryopreservation of the human ovarian cortex.

Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program

There is a clinical demand for cryopreservation of both in vivo-recovered and in vitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both in vivo-recovered and IVP blastocysts. In vivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10 hours to the laboratory before vitrification. IVP blastocysts (<300 μm in diameter) were produced by intracytoplasmic sperm injection and in vitro embryo culture. All vitrified-warmed embryos were shipped (0.5–12 hours) for transfer to recipient mares. In experiment 1, 47 IVP embryos from our clinical intracytoplasmic sperm injection program were vitrified using the MPT and transferred. The rates of initial pregnancy (59%) and foaling (45%) were equivalent to those for 52 IVP embryos from the same mare aspiration sessions and shipped for the same duration but transferred fresh (75% and 45%, respectively). The pregnancy and foaling rates for in vivo-recovered embryos were 76 and 71%, respectively for 17 small blastocysts (<300 μm in diameter), and 55 and 45%, respectively for 11 large blastocysts (303–608 μm in diameter, collapsed before vitrification; P > 0.1). In experiment 2, the MPT was cut lengthwise to form an open vitrification device, designated “Sujo”. Research IVP blastocysts were vitrified at 1, 2, or 3 embryos per Sujo (n = 34 embryos), or singly on a commercial open device (Cryolock; n = 11). After warming, 97% and 91% of embryos, respectively, grew in culture. Similarly, culture of two in vivo-recovered large blastocysts after collapse and vitrification on Sujos both resulted in embryo growth. However, transfer of four in vivo-recovered expanded blastocysts after collapse, vitrification on Sujos, and warming resulted in only one foal. These data indicate that vitrification of equine IVP embryos and small in vivo-recovered embryos is efficient under clinical conditions. Collapse and vitrification of in vivo-recovered large blastocysts in MPT under our clinical conditions resulted in a 45% foaling rate. While numbers are low, use of an open vitrification system did not appear to improve results for these embryos.

Closed vitrification of human oocytes and blastocysts: outcomes from a series of clinical cases.

High survival rates and clinical outcomes similar to those from fresh oocytes and blastocysts have been observed with open oocyte vitrification systems. It has been suggested that the extremely fast cooling rates that are only achieved with open systems are necessary for human oocyte and blastocyst vitrification. However, there is a potential risk of introducing contamination with open systems. The aim of this study was to assess whether similar survival and subsequent implantation rates could be achieved using a closed vitrification system for human oocytes and blastocysts. Initially, donated immature oocytes that were matured in vitro were vitrified using the cryoprotectants ethylene glycol (EG) + dimethyl sulphoxide (DMSO) + sucrose and either a closed system (Rapid-i®) or an open system (Cryolock). The closed system was subsequently introduced clinically for mature oocyte cryopreservation cases and blastocyst vitrification. Using in vitro matured oocytes, a similar survival was achieved with the open system of 92.4% (73/79) and with the closed system of 89.7% (35/39). For clinical oocyte closed vitrification, high survival rate of 90.5% (374/413) and an implantation rate of 32.7% (18/55) from the transfer of day 2 embryos was achieved, which is similar to fresh day 2 embryo transfers. Blastocysts have also been successfully cryopreserved using the Rapid-i closed vitrification system with 94% of blastocysts having an estimated ≥75% of cells intact and a similar implantation rate (31.5%) to fresh single blastocyst transfers. Closed vitrification can achieve high survival and similar implantation rates to fresh for both oocytes and blastocysts.

Étude prospective comparant l’efficacité de la vitrification ovocytaire en système fermé ou en système ouvert

ObjectivesOocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). MethodsForty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). ResultsThe global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). ConclusionTaking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.

Neonatal outcomes after the implantation of human embryos vitrified using a closed-system device.

Closed vitrification poses a risk of adversely affecting embryo development, while it may minimize the risk of contamination. We assessed the effects of closed-system human embryo vitrification on fetal development after implantation, neonatal outcome, and clinical safety. This was a retrospective cohort study conducted at a private fertility clinic. A total of 875 vitrified-warmed blastocysts that were single-transferred under hormone-replacement cycles between November 2011 and December 2013 were randomly divided into two groups (closed vitrification, n 313; open vitrification, n 562) after receiving the patients' consent forms. Developmental competence after implantation, including gestational age, birth weight, sex, Apgar score, and anomalies of newborns, after the transfer of blastocysts vitrified by closing vitrification was compared with that obtained in the case of open vitrification. There were no significant differences between the use of closed and open vitrification systems in embryo development after implantation, gestational age, birth weight, sex ratio, Apgar score, and congenital anomalies of newborns. Human embryos can be vitrified using a closed vitrification system without impairment of neonatal development.

Closed versus open vitrification systems for human oocytes and embryos. A mini-review

Closed versus open vitrification systems for human oocytes and embryos. A mini-review

Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system

BackgroundIncreasing evidence indicates that closed vitrification has been successfully used in the cryopreservation of human oocytes and embryos. Little information is available regarding the neonatal outcome of closed blastocysts vitrification. The aim of this study was to evaluate the effectiveness and safety of blastocyst vitrification using a high-security closed vitrification system compared with an open vitrification system.MethodsA total of 332 vitrified-warmed blastocyst transfer cycles between April 2010 and May 2012 were analyzed retrospectively. The post-thaw survival rate, implantation rate, clinical pregnancy rate, live birth rate, and neonatal outcome were recorded.ResultsThere were no significant differences between the open vitrification group and the close vitrification group regarding the post-thaw survival rate (98% versus 95.8%), clinical pregnancy rate (47.6% versus 42.2%), implantation rate (42.9% versus 35.6%), and live birth rate (39.8% versus 32.1%). In total, 332 warming cycles produced 131 healthy babies. There were no significant differences in the mean gestational age, the birth weight, and the birth length between the two groups. No adverse neonatal outcomes were observed in the children born after the transfer of closed vitrified blastocysts compared with the transfer of open vitrified blastocysts.ConclusionsThese data suggest that blastocyst vitrification using a closed vitrification device seems safe and effective with results comparable to those obtained through open vitrification.

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.

A closed system supports the developmental competence of human embryos after vitrification : Closed vitrification of human embryos.

Closed-system vitrification may enable the risk of contamination to be minimised. We performed three studies to compare the developmental competence of human embryos vitrified using either a closed vitrification system (CVS; Rapid-i®) or an open vitrification system (OVS; Cryo-top®). The first study was performed in vitro using 66 zygotes previously vitrified at pronuclear stage. These were warmed and randomised 1:1 to revitrification using either the OVS or the CVS. After re-warming, embryo development and blastocyst cell number were assessed. For the second study, also performed in vitro, 60 vitrified-warmed blastocysts were randomised 1:1:1 into three groups (OVS or CVS revitrification, or no revitrification). The proportion of dead cells was assessed by staining. The third study was performed in vivo, using 263 high-grade blastocysts randomly assigned to vitrification using either the CVS (n = 100) or the OVS (n = 163). After warming, single blastocyst transfer was performed. There were no differences between the CVS and the OVS in survival rate (100% vs. 97%), blastulation rate (96h: 50% vs. 50%; 120h: 68% vs. 56%), proportion of good blastocysts (96h: 32% vs. 22%, 120h: 47% vs. 41%), or mean number of cells (137 vs. 138). The proportion of dead cells in blastocysts re-vitrified by CVS (31%) was similar to that for OVS (38%) and non-revitrification (32%). In vivo, the implantation rate for blastocysts vitrified using the CVS (54%) was similar to that with the OVS (53%). Our studies consistently indicate that human embryos may be vitrified using a CVS without impairment of developmental competence.

The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

BackgroundThe Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (−15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an “open” vitrification system routinely used in our laboratory.MethodsHuman embryos were vitrified at either the 8–10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2–3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant.ResultsA total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going.ConclusionsThe Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8–10 cell stage as well as the blastocyst stage. Use of this type of “closed” sealed system that prevents direct contact between the embryos and liquid nitrogen reduces the potential risk of sample cross-contamination or infection. These preliminary data and live birth outcomes have paved the way toward transitioning to a closed vitrification system in our own IVF program.

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.

A closed vitrification system enables a safe and an aseptic vitrification without impairing the developmental competence of human embryos

To avoid the risk of contamination by bacteria, virus and prions, we compared the developmental competence of human embryos vitrified using a closed vitrification system (CVS) with that using an open vitrification system (OVS). Prospective randomized human study. This study was approved by a local IRB of IVF Namba Clinic. In experiment 1 and 2, vitrified embryos donated from patients who completed fertility treatment and gave informed consent were used. In experiment 1, 66 embryos at pronuclear stage were divided randomly into 2 groups after warming: CVS (Rapid-I, Vitrolife) and OVS (Cryotop, Kitazato medical) and were re-vitrified using CVS or OVS. After warming, embryos development and blastocysts cell number were assessed. In experiment 2, 60 vitrified-warmed blastocysts were divided randomly into 3 groups (CVS, OVS, and non-revitrified) and were assessed the proportion of dead cell by staining with Hoechst and Propidium iodide. In experiment 3, 27 high grade blastocysts were randomly divided into two groups and were vitrified using CVS (14) and OVS (13). After warming, single blastocyst transfer was performed. There were no differences between CVS and OVS in the survival rates (100% vs. 100%, respectively), the blastulation rates (Day 5: 50% vs. 50%; Day 6: 68% vs. 56%, respectively), the rate of good blastocyst (Day 5: 32% vs. 22%, Day 6: 47% vs. 41%, respectively), and the mean cell numbers (137 vs. 138) in experiment 1. In experiment 2, the proportion of dead cells in re-vitrified blastocysts were similar at 31%, 38% and 32% in the groups CVS, OVS and non-revitrified respectively. In experiment 3, the implantation rate of blastocysts vitrified using CVS (73%) was comparable to that for the OVS group (54%). The closed vitrification system overcomes the concerns associated with direct liquid nitrogen contact in the open system without impairing the developmental competence of human embryos.

The new rapid i closed vitrification system is technically easy to use and gives excellent outcomes with both blastocyst and cleavage stage embryos

The Rapid i is a new FDA approved closed carrier for embryo vitrification. he cooling rate of -1200 °C/min is far lower than that reported with open vitrification systems such as the cryoloop (-15,000 °C/min). Clinical outcome studies are still needed to determine efficacy.