Magosaburo Kasai

Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique

OBJECTIVE Clinical application of vitrification for the cryopreservation of human blastocysts. DESIGN Clinical trial of vitrification of human blastocysts. SETTING Private assisted reproductive technology clinic. PATIENT(S) Supernumerary blastocysts after fresh blastocyst transfer were vitrified for subsequent transfer. INTERVENTION(S) Culture of pronuclear embryos to the blastocyst stage in sequential media and subsequent vitrification of supernumerary blastocysts using a cryoloop technique. MAIN OUTCOME MEASURE(S) Clinical outcome after transfer of vitrified blastocysts. RESULT(S) A total of 60 vitrified blastocysts from 21 patients were warmed, and the survival rate at 2 hours after warming was 63%. Six clinical pregnancies were achieved after 19 transfers. One healthy baby was born, four pregnancies are ongoing, and one ended in miscarriage. CONCLUSION(S) Human blastocysts can be successfully vitrified by suspension on a small nylon loop and a direct plunge into liquid nitrogen. A delivery and ongoing pregnancies prove the safety of this method. This report documents the first successful pregnancy and delivery achieved by blastocyst vitrification using the cryoloop containerless technique.

Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method.

Mouse oocytes and embryos at various developmental stages were exposed directly to an ethylene glycol-based vitrification solution (EFS) for 2 or 5 minutes at 20 degrees C. They were then vitrified at -196 degrees C and were warmed rapidly. At the germinal vesicle stage, the proportion of morphologically normal oocytes was 36 to 39% if they had cumulus cells, whereas in cumulus-removed immature oocytes and in ovulated oocytes it was only 2 to 4%. This low survival was attributed to the harmful action of ethylene glycol. After fertilization, on the other hand, the post-warming survival rate of 1-cell zygotes, as assessed by cleavage to the 2-cell stage, increased markedly (62%). As the developmental stage proceeded, higher proportions of vitrified embryos developed to expanded blastocysts; the rates increased up to 77 and 80% in 2-cell and 4-cell embryos, respectively. For embryos at the 8-cell, morula and early blastocyst stages, the proportion of embryos developed after vitrification (90 to 95%) was not significantly different from that of the untreated embryos (95 to 100%) when the period of exposure to EFS solution was 2 minutes. As the blastocoel began to enlarge, however, survival began to decrease again, with rates of 79 and 57% in blastocysts and expanded blastocysts, respectively. After the cryopreserved 2-cell, 4-cell and 8-cell embryos as well as morulae and blastocysts were transferred to recipients, 43 to 57% of the recipients became pregnant, and 48 to 60% of these various stage embryos developed into live young.

Artificial Expression of Aquaporin-3 Improves the Survival of Mouse Oocytes after Cryopreservation

Abstract Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (LP) and glycerol permeability (PGLY) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25°C. Mean ± SD values of LP and PGLY of cRNA-injected oocytes (3.09 ± 1.22 μm min−1 atm−1 and 3.69 ± 1.47 × 10−3 cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 ± 0.02 μm min−1 atm−1 and 0.07 ± 0.02 × 10−3 cm/min, respectively; n = 13) and water-injected oocytes (0.87 ± 0.10 μm min−1 atm−1 and 0.08 ± 0.02 × 10−3 cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve.

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.

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.

Simple and efficient methods for vitrification of mammalian embryos

Abstract Vitrification greatly simplifies the cooling process for embryo cryopreservation, and eliminates any injuries caused by extracellular ice. However, embryos may be injured by various factors, such as the toxicity of the solution, intracellular ice, fracture damage and osmotic swelling. By minimising the effect of each of these factors, refined procedures for simple and efficient vitrification have been developed for mouse embryos.

Cryopreservation of mammalian embryos

As an innovative method for embryo cryopreservation, vitrification not only reduced the cooling stage duration to a minimum, but also eliminated any injuries cased by extracellular ice, which is a major cause of cell injury. Therefore, if embryos are treated adequately, high survival can be obtained. As a component of a vitrification solution, a permeating cryoprotective agent is essential, and additional inclusion of a macromolecule and a small saccharide makes the solution more effective. The author’s group composed a solution, designated EFS40, with ethylene glycol. Ficoll, and sucrose. This solution proved effective for the cryopreservation of various stages of embryos in many species. In this article, the author describes the detailed procedure for the vitrification of mouse morulae; related information is also described.

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.

Development of a reliable in vitro maturation system for zebrafish oocytes

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50-100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5-9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16-29%; hatching rate, 8-21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes.

The Role of Aquaporin 3 in the Movement of Water and Cryoprotectants in Mouse Morulae

Abstract The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.