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Blastocyst stage vitrification and re-expansion after warming. Human blastocysts vitrified with the Rapid-i. (a,b) Morphology immediately after warming, (c,d) Morphology two hours later. Both blastocysts now fully expanded and hatching out from the opening in the zona created after warming. Transfer resulted in a singleton pregnancy. Magnification 300 ×.
Source publication
Background
The 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...
Context in source publication
Context 1
... expansion and size of the blastocoel cavity were morphologic features closely linked to successful implantation of vitrified-warmed blastocysts. Blastocysts with high implantation potential appeared completely collapsed when recovered from the vitrification device and expanded by the time of transfer as shown in Figure 4. ...Similar publications
The aim of the present study was to evaluate the post-thaw survival and hatching rates of sheep blastocysts using different cryoprotectants. In Experiment 1, Day 6 sheep embryos were cryopreserved by a slow freezing protocol using 10% ethylene glycol (EG), 10% dimethyl sulfoxide (DMSO) or a mixture of 5% EG and 5% DMSO. Hatching rates were higher i...
Vitrification is the freezing method for animal's germplasms cryopreservation. Cryoprotectants (CPAs) influence the ability of oocytes survives after vitrification. However, a high concentration of single CPAs might induce a toxic effect on oocytes. In this study, three CPAs, ethylene glycol (EG), dimethyl-sulfoxide (DMSO) and propylene glycol (PG)...
The objective of our present experiment was to investigate the effects of closed pulled straw (CPS) vitrification on the viability and development of mouse embryo. The experiment was arranged according to completely randomized design (CRD) consisting of 4 treatments, namely not vitrification (NV) is control, CPS 1 (mCZB Hepes + 20% Bovine Serum Alb...
BACKGROUND: Immature oocytes are more sensitive to cold injury than mature oocytes. OBJECTIVE: The study was to evaluate the post thaw normal oocytes, cleavage and blastocyst rates of ovine cumulus oocyte complexes (COC's) using different cryoprotectants by slow freezing and Open pulled straw OPS vitrification. METHODS: In five replicates, abattoir...
Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number o...
Citations
... More data focusing on the impact of this aspect, analyzing the evidence from a cryobiology perspective, are needed. What is more, the lack of direct contact with liquid nitrogen in closed systems lowers the cooling rate of oocytes, ranging from −522 to −1220 • C per minute, when compared to −15,000 • C per minute in open systems, identifying another focal difference [56,57]. Warming rates are also potentially affected when using a closed device versus an open one. ...
Background/Objectives: Open and closed vitrification systems are commonly employed in oocyte cryopreservation; however, there is limited evidence regarding a comparison of their separate impact on oocyte competence. This study uniquely brings to the literature, data on the effect of open versus closed vitrification systems on laboratory and clinical outcomes, and the effect of cooling and warming rates. Methods: A systematic search of the literature was performed using the databases PubMed/MEDLINE and the Cochrane Central Library, limited to articles published in English up to January 2023. A network meta-analysis was conducted comparing each vitrification system versus fresh oocytes. Results: Twenty-three studies were included. When compared to fresh oocytes, both vitrification devices resulted in lower fertilization rates per MII oocyte retrieved. When comparing the two systems in terms of survival rates, no statistically significant difference was observed. However, interestingly open systems resulted in lower cleavage and blastocyst formation rates per 2 pronuclear (2PN) oocyte compared to fresh controls, while at the same time no statistically significant difference was detected when comparing closed devices with fresh oocytes. Conclusions: In conclusion, closed vitrification systems appear to exert a less detrimental impact on the oocytes’ competence, which is reflected in the blastocyst formation rates. Proof of superiority of one system versus the other may lead to standardization, helping to ultimately determine optimal practice in oocyte vitrification.
... 9 With the popularization of vitrification and freezing technology, vitrification and freezing carriers have been developed and applied, further improving the effect of vitrification and freezing. This method uses many types of commercial [10][11][12] and non-commercial [13][14][15] vitrification carriers. Regardless of whether the embryos are directly in contact with liquid nitrogen when the embryos are stored in a liquid nitrogen tank after freezing, the carriers can be divided into three categories: fully open carriers, 16 open cooling and closed storage carriers, 17 and closed carriers. ...
Objective
This study aimed to compare the effects of two brands of commercial vitrification carriers on pregnancy outcomes in freeze–thaw cycles.
Methods
We included 4871 patients who underwent a “freeze all” strategy using the commercial carriers J.Y. straw and OYASHIPS straw in the Reproductive Center of the First Hospital of Jilin University. The pregnancy outcomes of cleavage-stage embryos and blastocysts were studied separately. Detailed data and the safety of children born from mothers with the two types of carriers were also compared.
Results
Patients who used J.Y. straw had similar clinical pregnancy and live birth rates with one and two cleavage-stage embryo transplantation to those who used OYASHIPS straw. In patients who had blastocyst transplantation, the clinical pregnancy rate of one blastocyst transplanted in those who used OYASHIPS straw was significantly higher than that in those who used J.Y. straw (57.85% vs 47.09%). Among children born from mothers who used J.Y. straw, the congenital disability rate was significantly higher than that in those with OYASHIPS straw.
Conclusion
The OYASHIPS straw carrier is cheap and can achieve clinical pregnancy and live birth outcomes comparable to those of J.Y. straw. Therefore, OYASHIPS straw is a good alternative option.
... In agreement with these findings, we confirm that the same type and concentration of permeating CPA in Kit 1 and Kit 2 could be possible cooperating factors in prior mentioned suboptimal conditions. Furthermore, clinical studies on blastocyst vitrification and warming already showed the independence of permeating CPA composition with low or high osmolarities of nonpermeating CPA [35,36,38,41,45]. The increased CPA exchange with increased working temperature together with the plasticity of blastocyst cells could allow potential perspectives toward a more rapid and simplified method without saccharide gradients for blastocyst warming. ...
This study investigates whether there is an effect on laboratory results and clinical outcome using commercial kits with similar vitrification but different warming procedures for blastocysts vitrified on day 5 or day 6. A single-center retrospective cohort study was performed between 2011 and 2020. A change from a stage-specific kit (Kit 1) to a universal kit (Kit 2) was undertaken in 2017. A total of 1845 untested blastocysts were warmed for single vitrified-warmed blastocyst transfers (SVBT). Eight hundred and twenty-five blastocysts were vitrified with Kit 1 and 1020 with Kit 2. Blastocyst survival was not different (96.1% versus 97.3%). Seven hundred seventy-seven SVBT were performed from Kit 1 and 981 from Kit 2. Overall clinical pregnancy and live birth rates were not different (35.4% versus 34.1% and 30.9% versus 30.5% for Kit 1 and 2, respectively). Subgroup analysis for live birth rates in relation to the day of blastocyst vitrification showed no differences (36.1% and 36.1% for day 5 and 25.4% and 23.5% for day 6 blastocysts, respectively). For both kits, the mean gestational age was not different (38.8 ± 2.5 weeks versus 38.8 ± 2.0 weeks) with a singleton birth weight of 3413 ± 571 g and 3410 ± 528 g for Kit 1 and Kit 2, respectively. Differences in warming procedures do not affect laboratory performance or clinical outcome after blastocyst vitrification. The plasticity of a human blastocyst may allow for further investigation on simplification of blastocyst warming procedures.
... Blastocysts were also cryopreserved by vitrification for future transfer experiments. Methodology for blastocyst vitrification using the Rapid i carrier and warming has been previously described [29]. ...
Abstract Background Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. Materials and Methods Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. Results A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p
... For some time, vitrification has been recognized as a more effective freezing protocol for embryo cryopreservation than slow freezing methods [8,22] . The pregnancy outcomes achieved using different vitrification protocols were excellent [7,23,24] . However, our experience suggests that vitrification does not improve clinical outcomes for certain types of embryos of relatively poor quality. ...
Objective: To evaluate the effect of morphologic factors on survival rate (SR), pregnancy rate (PR), and implantation rate (IR) of human embryo vitrification following frozen embryo transfer (FET) on day 3 post-ovulation. Methods: Women undergoing FET (n = 921) with embryos cryopreserved by vitrification between 2012 and 2013 were enrolled in this retrospective cohort study. Results: Embryos with >9 blastomeres yielded the highest SR of 100%. Lower SR was observed in embryos with 5 to 6 (57.5%) and 4 blastomeres (41.4%). In terms of blastomere symmetry, the SR of embryos with equally sized blastomeres was significantly higher than that of embryos with unequally sized cells (82.5% vs. 64.6%, P < 0.05). As fragmentation increased, SR decreased from 92.1% to 20.6% (P < 0.05). Significant differences were observed among groups when analyzing PR and IR according to the 3 embryonic parameters before vitrification. Embryos with 13 to 16 blastomeres yielded the highest PR (39.5%) and IR (24.1%). The PR and IR of embryos with blastomeres of equal size were significantly higher than those with unequally sized blastomeres (36.5% vs. 21.7%, 23.7% vs. 12.4%, P < 0.05). After warming, embryos with 13 to 16 blastomeres yielded the highest PR and IR (40.9% and 24.2%, respectively). The PR and IR were observed to grow with an increase in the percentage of intact blastomeres (23.2%-38.2%, 14.2%-23.2%). Conclusions: These results show that vitrification methods do not effectively improve survival outcomes for embryos of poor quality and it is needed to develop a comprehensive vitrification protocol that considers all the practical aspects, including the current limitation regarding cleavage-stage embryos of poor quality.
... Only fully expanded blastocysts were included in the randomization of sibling blastocysts for intact transfer and artificial collapsing. The rest of the blastocysts in the earlier stages of blastocoel expansion were never collapsed, because early blastocysts do not benefit from blastocoel shrinkage (Desai et al., 2013). The comparative analysis covered only the first transfers of single vitrified-warmed blastocysts. ...
Research question
Does laser-induced artificial blastocoel collapse result in better blastocyst cryopreservation survival and a higher live birth rate (LBR) in comparison with intact counterparts?
Design
Half of the supernumerary blastocysts from IVF cycles were randomly selected before vitrification for laser-induced artificial collapsing or vitrification in intact form. A matched case-control study of first transfers of single blastocysts artificially collapsed (case) or intact (control) before vitrification was conducted. Controls were matched to cases on a 1:1 ratio by female age, parity, fresh and vitrified cycle protocol, blastocyst age and quality, resulting in 309 case-control pairs.
Results
The two groups were comparable in terms of their characteristics. Survival rates in the case and control groups (97.8% and 95.7%; P = 0.133) were comparable, but the optimal survival rate was higher in the case group (78.2% and 69.3%; P = 0.03). Clinical pregnancy rates (38.2% and 35.3%; P = 0.518), miscarriage rates (15.2% and 22%; P = 0.190), LBR per transfer (32.4% and 27.5%; P = 0.221) and LBR per warmed blastocyst (31.6% and 26.3%; P = 0.137) were not statistically different between the case and control groups. No significant difference in preterm births (11.1% versus 15.7%), birthweights (3333 ± 723 g versus 3304 ± 609 g) or sex ratio (49.3% versus 50.7% boys) was observed between the two groups. No major malformations were detected in the study population.
Conclusions
Compared with vitrification of intact blastocysts, collapsed blastocysts resulted in a significantly higher optimal survival rate, and although they resulted in a 5% higher LBR, this was not significant for the chosen sample size. Neonatal outcomes were comparable in the two groups.
... The principle of this method is the preparation of a sequencing library, where individual samples are marked with specific indices, which is then amplified and sequenced by SBS (MiSeq, Illumina). Biopsied embryos were vitrified 1 h after biopsy (Rapid VitBlast, Vitrolife) [23]. ...
The selection of the best embryo for embryo transfer (ET) is one of the most important steps in IVF (in vitro fertilisation) treatment. Preimplantation genetic testing (PGT) is an invasive method that can greatly facilitate the decision about the best embryo. An alternative way to select the embryo with the greatest implantation potential is by cultivation in a time-lapse system, which can offer several predictive factors. Non-invasive time-lapse monitoring can be used to select quality embryos with high implantation potential under stable culture conditions. The embryo for ET can then be selected based on the determined morphokinetic parameters and morphological features, which according to our results predict a higher implantation potential. This study included a total of 1027 morphologically high-quality embryos (552 normal and 475 abnormal PGT-tested embryos) from 296 patients (01/2016–06/2021). All embryos were cultivated in a time-lapse incubator and PGT biopsy of trophectoderm cells on D5 or D6 was performed. Significant differences were found in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the stage of two-cell and four-cell embryos between the group of genetically normal embryos and abnormal embryos. At the same time, significant differences in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the two-cell and four-cell embryo stage were found between the group of genetically normal embryos that led to clinical pregnancy after ET and the group of abnormal embryos. From the morphokinetic data found in the PGT-A group of normal embryos leading to clinical pregnancy, time intervals were determined based on statistical analysis, which should predict embryos with high implantation potential. Out of a total of 218 euploid embryos, which were transferred into the uterus after thawing (single frozen embryo transfer), clinical pregnancy was confirmed in 119 embryos (54.6%). Our results show that according to the morphokinetic parameters (cc2, t5, tSB) and the occurrence of multinucleations during the first two cell divisions, the best euploid embryo for ET can be selected with high probability.
... Similarly, clinical outcomes in terms of pregnancy rate (33.4% vs 34.6%) and miscarriage rate (24.8% vs 22.2%) after transfer of vitrified-warmed assisted-hatching laser-treated blastocysts were reported (Miwa et al., 2020). Although closed vitrification devices are associated with lower cooling and warming rates, it has been suggested that similar cryosurvival rates for embryos and blastocysts can be achieved when compared to open vitrification carrier systems (Kuwayama et al., 2005;Chen et al., 2013;Desai et al., 2013;Panagiotidis et al., 2013). The present study corroborates this notion on cryosurvival of 2PN oocytes in the context of a semi-automated device with the evidence generated being supportive of only negligible outcome differences (exemplified by the À4.7% lower bound CI of the difference in mean 2PN oocyte survival rate). ...
STUDY QUESTION
What are outcome and procedural differences when using the semi-automated closed Gavi® device versus the manual open Cryotop® method for vitrification of pronuclear (2PN) stage oocytes within an IVF program?
SUMMARY ANSWER
A semi-automated closed vitrification method gives similar clinical results as compared to an exclusively manual, open system but higher procedure duration and less staff convenience.
WHAT IS KNOWN ALREADY
A semi-automated closed vitrification device has been introduced to the market, however, little evaluation of its performance in a clinical setting has been conducted so far.
STUDY DESIGN, SIZE, DURATION
This prospective, randomised, open non-inferiority trial was conducted at three German IVF centers (10/2017–12/2018). Randomization was performed on day of fertilization check, stratified by center and by indication for vitrification (surplus 2PN oocytes in the context of a fresh embryo transfer (ET) cycle or ‘freeze-all’ of 2PN oocytes).
PARTICIPANT/MATERIAL, SETTING, METHODS
The study population included subfertile women, aged 18–40 years, undergoing IVF or ICSI treatment after ovarian stimulation, with 2PN oocytes available for vitrification. The primary outcome was survival rate of 2PN oocytes at first warming procedure in a subsequent cycle and non-inferiority of 2PN survival was to be declared if the lower bound 95% CI of the mean difference in survival rate excluded a difference larger than 9.5%; secondary, descriptive outcomes included embryo development, pregnancy and live birth rate, procedure time and staff convenience.
MAIN RESULTS AND THE ROLE OF CHANCE
The randomised patient population consisted of 149 patients, and the per-protocol population (patients with warming of 2PN oocytes for culture and planned ET) was 118 patients. The survival rate was 94.0% (±13.5) and 96.7% (±9.7) in the Gavi® and the Cryotop® group (weighted mean difference −1.6%, 95% CI −4.7 to 1.4, P = 0.28), respectively, indicating non-inferiority of the Gavi® vitrification/warming method for the primary outcome. Embryo development and the proportion of top-quality embryos was similar in the two groups, as were the pregnancy and live birth rate. Mean total procedure duration (vitrification and warming) was higher in the Gavi® group (81 ± 39 min vs 47 ± 15 min, mean difference 34 min, 95% CI 19 to 48). Staff convenience assessed by eight operators in a questionnaire was lower for the Gavi® system. The majority of respondents preferred the Cryotop® method because of practicality issues.
LIMITATIONS, REASON FOR CAUTION
The study was performed in centers with long experience of manual vitrification, and the relative performance of the Gavi® system as well as the staff convenience may be higher in settings with less experience in the manual procedure. Financial costs of the two procedures were not measured along the trial.
WIDER IMPLICATIONS OF THE FINDINGS
With increasing requirements for standardization of procedures and tissue safety, a semi-automated closed vitrification method may constitute a suitable alternative technology to the established manual open vitrification method given the equivalent clinical outcomes demonstrated herein.
STUDY FUNDING/COMPETING INTERESTS
The trial received no direct financial funding. The Gavi® instrument, Gavi® consumables and staff training were provided for free by the distributor (Merck, Darmstadt, Germany) during the study period. The manufacturer of the Gavi® instrument had no influence on study protocol, study conduct, data analysis, data interpretation or manuscript writing. J.H. has received honoraria and/or non-financial support from Ferring, Merck and Origio. G.G. has received honoraria and/or non-financial support from Abbott, Ferring, Finox, Gedeon Richter, Guerbet, Merck, MSD, ObsEva, PregLem, ReprodWissen GmbH and Theramex. The remaining authors have no competing interests.
TRIAL REGISTRATION NUMBER
ClinicalTrials.gov NCT03287479.
TRIAL REGISTRATION DATE
19 September 2017.
DATE OF FIRST PATIENT’S ENROLMENT
10 October 2017.
... Using CM, vitrified/warmed Blastocysts had a survival rate of 98.3% (n = 60/61), while for HM, it was 98.9% (n=92/93; P=0. 27), survival rate at 6-8 hrs with CM 96.7% (n=59/61), while for HM, it was 97.8% (n=91/93; P=0.48), or expansion for CM 93.4% (n=57/61), while for HM, it was 95.7% (n=92/93; P=0.39) after 6-8 h of culture following thawing (Fig. 2). ...
... The results found that vitrification did not affect blastocyst survival in any of the treatment groups. These findings confirm earlier results, which showed that thawed blastocysts which survived vitrification weren't different from fresh blastocysts, in terms of quality, DNA and chromosome integrity, ultrastructure, and developmental competence [27][28][29] . ...
... Many techniques and approaches have been developed in order to improve vitrification protocols. Rapid I is a newly adapted and promising method that yields high rates of developed embryos after IVF, especially in human embryology (8,16) and there are relatively few studies about using the Cryotop method on bovine species (12,31). Whereas cryopreservation of human embryos has become a routine part of the treatment of infertility and is crucial for a safe and effective treatment, advancement in oocyte cryopreservation is still one of the most sought of in reproductive medicine (17). ...
... Several cryo-devices have been invented and can be classified as either open or closed. Open vitrification devices such as cryoloop or open pulled-straw (OPS) allow direct contact of the embryos with liquid nitrogen, and this is undesirable in terms of potential risk of crosscontamination between specimens (1,8). Cryotop carrier in Rapid-I method is a ''closed'' vitrification device which allows loading of the oocytes or embryos with minimum vitrification solution. ...
... Cryotop carrier in Rapid-I method is a ''closed'' vitrification device which allows loading of the oocytes or embryos with minimum vitrification solution. Cryotop carrier has shown its superiority to other devices (8,12,15,18,31). Interestingly, Marco-Jimenez et al. (20) compared two devices for vitrification, namely, Cryotop and inoculation loop, mainly used by microbiologists to retrieve an inoculum from a Different superscripts demonstrate significant differences: a, b = p<0.05 ...
BACKGROUND: Vitrification is commonly used for cryopreservation of gametes. OBJECTIVE: The study aimed at evaluating viability and developmental competence of bovine oocytes vitrified by Rapid-I method. MATERIALS AND METHODS: Oocytes after collection (group 1) and IVM (group 2) were vitrified using medium containing 18% Ficoll, 40% ethylene glycol, 0.3 M sucrose. To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 0.5 µM ionomycin in TCM 199 combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum. RESULTS: Survival rate in group 1 was 58%, and 88% in the control. In group 2, 63% viable oocytes were found, compared to 82% in the control group. After parthenogenetic activation 27.2% morulas were observed. This percentage was lower than in the non-vitrified group (31%). CONCLUSION: Maturity stage of bovine oocytes has no effect on their survival after vitrification.
