Ultra-rapid Vitrification Research Articles

Artificial shrinkage (AS) of blastocoele prior to cooling step of vitrification improves the survival rate of vitrified human blastocysts

Artificial shrinkage (AS) of blastocoele prior to cooling step of vitrification improves the survival rate of vitrified human blastocysts

Cryopreservation of animal and human embryos by vitrification

Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For embryo cryopreservation, the vitrification method has advantages over the slow freezing method. For example, injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short period without a programmed freezer. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulphoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and/or a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conventional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation.

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.

82COMPARISON OF THE SURVIVAL OF IN VITRO-DERIVED PORCINE OOCYTES AND EMBRYOS VITRIFIED BY OPEN PULLED STRAW, ELECTRON MICROSCOPE GRID, AND NYLON LOOP SYSTEM

Various sample containers have been developed for ultra-rapid vitrification of oocytes and embryos to minimize chilling and other injuries. In this study, the survival rates of in vitro-derived porcine oocytes were compared after cryopreservation using open pulled straw (OPS), electron microscope grid (EMG), and nylon loop system (NLS). In addition, the post-thaw survival of porcine morulae and blastocysts was assessed after vitrification by the OPS method. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al., 1994 Theriogenology 41, 1425–1433). Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5mM sodium pyruvate, 0.5mM sodium lactate and 4mgmL−1 bovine serum albumin for 2 days at 39°C; 10% fetal bovine serum was added to the culture medium thereafter. Oocytes and embryos were treated with 7.5μgmL−1 cytochalasin B for 30min, centrifuged at 13,000g for 13min and then exposed sequentially to an ethylene glycol (EG) vitrification solution (0M for 1min, 2M for 5min and 8M plus 7% polyvinylpyrrolidone for 1min). Oocytes (n=100per treatment group) were placed in or on sample containers and plunged into liquid nitrogen. Porcine morulae and blastocysts (n=137) were similarly treated, aspirated into OPSs, and plunged into liquid nitrogen. Oocytes and embryos were thawed rapidly by transferring the sample container into 1M EG for 2min, and then the embryos were serially diluted by transfer into 0.5M for 2min, and medium for 5min. Post-thaw survival of vitrified oocytes was assessed by development after IVF and culture to morulae or blastocysts. Post-thaw survival of vitrified morulae and blastocysts was assessed by their ability to continue development to, respectively, blastocysts and expanded blastocysts. After oocytes were thawed, fertilized and cultured in vitro, the rates of development to the morula- or blastocyst-stage were 8%, 0% and 6% for, respectively, the OPS, EMG and NLS groups. The rate of development of non-cryopreserved control oocytes was 38% (38/100). Although significantly fewer oocytes developed after vitrification compared to the control (P<0.05), OPS and NLS containers were superior to EMG containers (P<0.05). Morula- or blastocyst-stage embryos vitrified using OPS yielded a significantly higher rate of survival than did oocytes (48%, 66/137; P<0.05). This study indicates that very few in vitro-matured porcine oocytes survive vitrification using OPS, EMG and NLS methods. Considerably higher rates of survival were obtained by in vitro-produced porcine morulae and blastocysts following vitrification by the OPS method. Additional research is needed to identify and develop methods to overcome the factors limiting the cryopreservation of porcine oocytes for practical uses. Supported by KOSEF (R05-2002-000-00388-0) grant.

116DEVELOPMENT INTO BLASTOCYSTS OF SWAMP BUFFALO OOCYTES AFTER VITRIFICATION AND NUCLEAR TRANSFER

Oocyte cryopreservation in the domestic species is still at the experimental stage, but recent studies indicated that vitrification characterized by ultra-rapid cooling rate is promising for cryopreservation of bovine oocytes. In the present study, denuded buffalo oocytes were vitrified by minimum volume cooling procedure (Kuwayama and Kato, 2000, J Assist Reprod Genet 17, 477) after IVM or after IVM and enucleation, and developmental potential into blastocysts of the post-warm oocytes after somatic cell nuclear transplantation was examined. Cumulus-oocyte complexes were matured, denuded, and enucleated as described previously (Parnpai et al., 1999, Buffalo J 3, 371–384). The presumptive metaphase-II (M-II) oocytes before and after enucleation were first equilibrated in 7.5% DMSO+7.5% ethylene glycol+20% FCS in TCM199 for 10 min, and then exposed to 15% DMSO+15% ethylene glycol+0.5M sucrose+20% FCS in TCM199 for 1min. Five oocytes were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 5min, directly transferred into TCM199+20% FCS, and kept at room temperature for 1h before being used for a cloning experiment. The post-warm oocytes were fused with ear skin fibroblasts by two DC pulses (26V, 17μs) and activated with 7% ethanol for 5min and then 10μg/mL cycloheximide and 1.25μg/mL cytochalasin-D for 5h. The reconstructed embryos were cultured in mSOFaa+0.2% BSA+0.1% linoleic acid albumin for 2 days, and then co-cultured with bovine oviductal epithelial cells for an additional 5 days. Post-warm morphological survival of M-II oocytes (80%, 187/235) was similar to that of enucleated oocytes (75%, 158/212). Vitrified M-II oocytes were successfully enucleated (96%, 136/142) as were fresh control oocytes (88%, 143/162). Fusion rates of M-II oocytes vitrified before and after enucleation (81%, 94/116 and 78%, 106/136, respectively) were also similar to those of fresh oocytes (81%, 100/123). Percentages of reconstructed embryos developing into hatching blastocysts on Day 7 were 5% (5/91), 6% (6/103), and 8% (8/99) in the groups of oocytes vitrified before and after enucleation, and of fresh control oocytes, respectively (ANOVA tests were not significant different). These results indicate that swamp buffalo oocytes cryopreserved by ultra-rapid vitrification procedure can be used successfully for subsequent somatic cell nuclear transplantation. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology)

Assisted hatching at the time of warming improves the implantation rate of vitrified human blastocysts (BLs)

Objective: To improve pregnancy outcomes of BL transfer, it is essential to establish a simple, easy and reliable procedure for the cryopreservation of BLs. We have reported the successful application of ultra-rapid vitrification using a cryoloop technique for human BLs (Mukaida T, et al. Hum.Reprod. 2003). However, cryopreservation including exposure to cryoprotectant is proven to cause the hardening of zona pellucida. Therefore, it will be possible to improve the implantation rate with assisted hatching (AHA) at the time of warming of vitrified human BLs before transfer.

Comparison of slow freezing in the cleavage stage and ultra-rapid vitrification of blastocysts

Objective: Freezing technology for blastocysts is an absolute necessity at facilities that perform ART. Ultra-rapid vitrification of blastocysts, which can be conveniently performed in a short period of time, is actively performed at our clinic. The objective of the current research has been comparison of results of slow freezing in the cleavage stage and ultra-rapid vitrification of blastocysts. Design: Retrospective. Materials and Methods: From August 2001 to December 2002, 9 cycles of freeze/thaw ET were performed with slow freezing on Day3 and 38 cycles of freeze/thaw ET were performed with ultra-rapid vitrification of blastocysts on Day 5 and 6 at our clinic. Day 3 Embryos were frozen using PROH and sucrose as cryoprotectants. For thawing of the embryos, the cryoprotectants were then removed by stepwise dilutions with thawing solution (5min or 10min each step). The blastocysts are suspended in solution I, which is HEPES-buffered HTF medium containing 5 mg/ml of HSA(base medium) containing 7.5%(v/v) DMSO and 7.5%(v/v) EG at about 37°C. A cryoloop is dipped into cryoprotectant solution II, which is a base medium containing 15%(v/v) DMSO, 15%(v/v) EG, 10mg/ml Ficoll 70, and 0.65M sucrose, at about 37°C to create a filmy layer of the solution on the cryoloop. 1min and 45sec after suspending the blastocysts in solution I, the blastocysts are washed in solution II and transferred onto the filmy layer on the cryoloop. Within 30 sec of suspension in solution II, the cryoloop is plunged into liquid nitrogen. For thawing of the blastocysts, the loop containing blastocysts is removed from the liquid nitrogen and placed quickly and directly into a well of the base medium containing 0.33M sucrose at 37°C. After 2 min, embryos are transferred to the base medium containing 0.2M sucrose. After an additional 3min, embryos are suspended in base medium for 5min. Results: The survival rate was 81.8% (63/77) with slow freezing and 81.6% (93/114) with vitrification; no significant difference was found. ET was possible in 9 of 10 cycles with slow freezing and in 38 of 45 cycles with vitrification. The mean embryo transfer number was 2.6±0.9 with slow freezing and 1.8±0.7 with vitrification; the mean ET number with vitrification was significantly lower. The implantation rate was 21.7% (5/23) with slow freezing and 26.9% (18/67) with vitrification; significance was not noted. The clinical pregnancy rate (FHM) was 33.3% (3/9) with slow freezing and 36.8% (14/38) with vitrification; no significant difference was found. Conclusion: The fact that a high pregnancy rate and implantation rate can be obtained with freeze/thaw ET via vitrification has been demonstrated. Because vitrification can be conveniently performed in a short period of time, it is also linked to a reduction in laboratory work, which grows in complexity each day. However, the safety of vitrification in aspects like the toxicity of the cryoprotectant has yet to be established, so further improvements are needed.

Successful production of blastocysts following ultrarapid vitrification of germinal vesicle oocytes in mice by step-wise equilibrium

Objective: The cryopreservation of oocytes would be important for storage of oocytes from patients who risk losing ovarian function because of extirpative therapy, chemotherapy, or radiation. Cryopreservation of germinal vesicle (GV) oocytes has been reported, but with limited success. The aim of this study was to evaluate viability and subsequent developmental ability in mouse GV oocytes ultrarapidly vitrified by step-wise exposure.

Successful production of blastocysts following ultrarapid vitrification with step-wise equilibriation of germinal vesicle-stage mouse oocytes.

The purpose of this study was to evaluate the viability and subsequent developmental ability of murine germinal vesicle (GV) oocytes ultrarapidly vitrified after step-wise exposure to cryoprotectants (CPAs). Oocytes were transferred to a vitrification solution composed of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 M sucrose in a direct manner (non-preequilibrium) or in a step-wise manner (single-, two-, or ten-step preequilibrium). After ultrarapid vitrification and storage in liquid nitrogen, the oocytes were thawed, washed by diluting the CPAs in five steps, and then subjected to in vitro maturation, fertilization and culture. In the non-preequilibrium group, the rates of post-thawed oocytes surviving, maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 91.8, 87.1, 15.9 and 2.3%, respectively. In the single- and two-step groups, the corresponding rates were 97.0-98.2%, 92.2-95.0%, 22.0-29.4% and 8.8-15.6%, whereas in the ten-step group they were 98.2, 91.8, 38.6 and 22.8%, respectively. In the non-vitrified control group, the rates of oocytes maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 90.2, 75.2 and 51.5%, respectively. The present study shows that the ultrarapid vitrification of murine GV oocytes by a step-wise manner involving 10 steps preequilibrium may have an advantage in maintaining the viability and subsequent production of blastocysts.

Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique

Human embryos have been cryopreserved mainly by slow freezing, but vitrification has also proven effective for embryos at early cleavage stages. However, clinical results on blastocyst cryopreservation have not been consistent. A feasible option appears to be ultrarapid vitrification, in which embryos are vitrified with a reduced amount of solution to achieve extremely high rates of cooling and warming. The cryoloop is a tiny nylon loop connected to the lid by a small metal tube; a metal insert on the lid enables the use of a stainless steel handling rod with a small magnet, and the loop can be stored in the cryovial. In the HART Clinic group, of 444 supernumerary human blastocysts that were vitrified by cryoloops 79% survived after warming, and of 126 recipients 36% became pregnant. The outline of ultrarapid vitrification using cryoloops is described.

Clinical outcome after transfer of human blastocysts (BLs) vitrified on day 5 and on day 6 with a cryoloop technique

Objective: To optimize clinical outcomes of BL transfer, it is essential to establish a simple, fast and reliable procedure for the cryopreservation of BLs. We have reported the effectiveness of ultra-rapid vitrification using a cryoloop technique for human BLs (1). However, the timing of vitrification of BLs might affect the outcome. This study examined the post-warming survival and clinical outcome of human BLs vitrified on day 5 and on day 6. Design: Retrospective study on the vitrification human BLs. Materials/Methods: A total of 129 patients who had supernumerary BLs between Oct 1999 and Nov 2001 entered this study. A sequential culture system was used to obtain BLs. When embryos reached the BL stage 5 or 6 days after oocyte retrieval and patients agreed to cryopreserve the embryos by ultra-rapid vitrification, supernumerary BLs were cryopreserved using a cryoloop technique (1). Blastocysts were mounted on a minute nylon loop, which creates a thin, filmy layer of the solution by surface tension, and vitrified with the use of a two-step loading process with cryoprotectants at 37C. Initially, BLs were placed in cryoprotectant solution I, which is the base medium (Hepes-buffered modified HTF) containing 7.5% DMSO and 7.5% ethylene glycol (EG), for 2 min before being suspended in cryoprotectant solution II, which is the base medium containing 15% DMSO, 15% EG, 1% Ficoll 70 and 0.65 M sucrose, for 30 sec. Blastocysts were warmed and diluted in two steps with 0.33 M sucrose for 2 min and 0.2 M sucrose for 3 min. Blastocysts with morphologically intact inner cell mass and trophectoderm and a re-expanding blastocoel were judged to have survived and were transferred to the patient 5 days after progesterone administration, regardless of the timing of vitrification. Results: Of 301 day-5 BLs collected from 92 cycles, 274 (91%) survived after warming. Of 143 day-6 BLs collected from 45 cycles, however, only 77 (54%) survived (p <0.01). Pregnancy rates with day-5 and day-6 BLs were 39% (35/92) and 28% (10/45), respectively, and implantation rates with day-5 and day-6 BLs were 21% and 17%, respectively, both rates being not significantly different (p >0.05). Conclusions: Blastocysts on day 5 are more resistant to vitrification than those on day 6. Fast-developing embryos might be more viable than slow-developing ones. In addition, since more day-6 BLs had a larger blastocoel, BLs having a smaller blastocoelic cavity may be suitable for vitrification, probably with less risk of intracellular ice formation. However, once BLs survived vitrification, they seem to have similar ability to develop after transfer. Reference: (1) Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K (2001) Successful birth after transfer of vitrified human blastocysts using a cryoloop container-less technique. Fertil Steril 76: 618–620. Supported by: none.

Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultrarapid vitrification.

The cryopreservation of embryos has become a powerful tool in assisted reproduction in several mammalian species. Embryos are cryopreserved by slow freezing or by vitrification. However, consistently high survival has not been obtained in most oocytes and in some embryos. The main reasons for the low survival would be sensitivity to low temperatures, which leads to chilling injury, and low permeability of the cell membrane, which leads to the formation of intracellular ice. As a strategy aiming to overcome these injuries, modified vitrification methods have been devised in which the cooling and warming rate is markedly increased by minimizing the volume of the solution and the container. The modified methods use electron microscope grids, open-pulled straws, cryoloops, or container-less microdrops. In this article, recent developments in the ultrarapid vitrification of mammalian oocytes and embryos are reviewed based on the understanding of the mechanisms of cell injury in cryopreservation. (Reprod Med Biol 2002; 1: 1-9).

Vitrification of mouse oocytes using a nylon loop.

Cryopreservation of mouse oocytes was improved by the use of ultra-rapid vitrification using a nylon loop of 0.5 mm diameter. Oocytes that were vitrified using the loop survived at high rates and were fertilized following a small hole being made in the zona pellucida (69.8%) and developed to the blastocyst stage in culture (67.4%) at similar rates to that of oocytes that were not cryopreserved. Blastocysts resulting from oocytes vitrified using the nylon loop had similar development of the inner cell mass and trophectoderm as blastocysts from non-cryopreserved oocytes. In contrast, oocytes that were cryopreserved using a slow-freezing protocol where most of the Na+ is replaced with choline had lower rates of fertilization (39.5%), reduced development to the blastocyst stage (25.7%), and blastocysts had reduced development of the inner cell mass. Blastocysts derived from oocytes that were vitrified with the nylon loop were able to implant (88.0%) and develop into fetuses (56.5%) at significantly higher rates compared to blastocysts derived from oocytes that were slow-frozen (52.4 and 26.2%, respectively). Vitrification of mouse oocytes using the nylon loop results in the retention of viability of the oocytes and subsequent embryos.