James J Stachecki

An overview of oocyte cryopreservation

The ability to cryopreserve human oocytes and store them indefinitely would be beneficial for cancer patients at risk of becoming sterile after therapy, allow women to delay reproduction, and alleviate religious concerns associated with embryo storage. In 1986, Chen was the first to report a pregnancy originating from a frozen-thawed human oocyte. Although over 100 babies have been born from oocyte storage since then, pregnancy rates remain unacceptably low. Adapting embryo cryopreservation techniques to oocyte storage has had limited success and new reproducible methods are needed. Problem areas other than intracellular ice formation and osmotic effects need to be identified. A broad approach of critical analysis should be conducted regarding the entire cryopreservation process from pre-equilibration and cooling, to thawing and stepout. All established facets deserve reanalysis in order to assess which aspects can be optimized or changed so that cellular demise can be avoided and cellular viability enhanced. New methods, including the use of choline-based media and vitrification have proven useful in increasing survival and pregnancy rates in some clinics. Other methods yet untested, such as injection of complex carbohydrates into the oocyte, deserve further studies. Vitrification research has led to the formulation of new ideas and has demonstrated the flexibility of cells to survive cryopreservation. Although successful, vitrification protocols are potentially harmful and technically challenging, due to elevated cryoprotectant concentrations and rapid cooling rates. Bovine embryo vitrification methods have been used to store human oocytes and embryos, particularly blastocysts with some success. Vitrification solutions containing high molecular weight polymers have also proved beneficial by reducing solution toxicity. In general, further advances are needed to improve human oocyte storage before widespread routine clinical use.

Spindle organization after cryopreservation of mouse, human, and bovine oocytes.

Oocyte cryopreservation would alleviate a number of ethical, social, and religious problems associated with human embryo storage. One potential problem is the effect of cryopreservation on the metaphase II spindle and chromosomes. The microtubules that make up the spindle tend to depolymerize at sub-physiological temperatures. Although there are numerous reports in the literature on this topic, discrepancies as to whether the spindle can or cannot reform persist. One of the confounding factors may be the low cryosurvival rates (around 50%) for mammalian oocytes. In recent years, a cryopreservation medium and protocol have been developed that allow oocytes of several species to be cryopreserved with high survival rates (>85%). Bovine, mouse, and human oocytes consistently reformed a morphologically normal spindle with chromosomes aligned along the metaphase plate (70% or higher) after first surviving cryopreservation (>87% survival for all species tested). Normal chromosome numbers were found in every second polar body tested by FISH (second polar bodies n = 4). It is concluded that the mammalian spindle, although depolymerized during cryopreservation, has the ability to reform, and in the mouse has been shown to function normally. Therefore, spindle reformation may not be a major cause for concern when storing mammalian MII oocytes.

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium.

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.

A new safe, simple and successful vitrification method for bovine and human blastocysts

This study examined a new method for vitrification of blastocysts that is safe, simple and easy to learn and use. Current vitrification techniques have shortcomings that include the use of dimethyl sulphoxide, one of the more toxic cryoprotectants, and minute containers that are difficult to handle and are usually open to contamination. Cell handling and loading times are very short, which allows no room for user-associated errors and increases the difficulty of the procedure. This study describes a method of vitrification without these shortcomings. Human and bovine blastocysts were exposed to a series of three cryoprotectant solutions and loaded into a 0.25 ml sterile straw, heat sealed at both ends and vitrified. This technique allowed sufficient time for cryoprotectant exposure, loading, sealing and vitrification. Research blastocysts were thawed, cultured for 24 h, and stained for cell viability. The majority survived and on average had few lysed cells. In clinical studies from three different centres, 81.4% of vitrified blastocysts were intact after thawing. Out of 43 transfers with 76 blastocysts replaced, 44.7% implanted, 43.4% yielded a fetal heart beat, and a total of 32 babies have been delivered or are ongoing. The overall clinical pregnancy per transfer rate was 60.4%. The high survival rates and clinical pregnancy rates obtained with this new, safe and easy-to-use vitrification procedure are encouraging.

Cryopreservation of unfertilized human oocytes

Previous investigations revealed that choline-based freezing media developed in our laboratory were superior to conventional sodium-based media for storing mouse oocytes. This paper examines the ability of the choline-based medium CJ2 and a modified form of this medium, CJ3, to cryopreserve unfertilized human oocytes. Oocytes that were consented for research and matured overnight, as well as freshly collected, donor, mature metaphase II (MII) oocytes, were cryopreserved using choline-based media and an optimized slow-cooling protocol. The results showed higher survival and fertilization rates when CJ3 supplemented with 0.2 mmol/l sucrose was used as compared with CJ2 supplemented with either 0.1 mmol/l or 0.2 mmol/l sucrose. Freshly collected oocytes were more difficult to cryopreserve than those matured in vitro. Modification of the base medium proved to be one of the key factors in obtaining survival rates over 90%. Fertilization rates, embryo development, and genetic analysis of embryos resulting from control and frozen-thawed oocytes are provided. There appears to be a high correlation between chromosomal anomalies and abnormal morphology in embryos from thawed oocytes.

Cryopreservation of biopsied cleavage stage human embryos.

The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.