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ABSTRACT: 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.
Reproductive biomedicine online 09/2006; 13(2):222-7. · 2.04 Impact Factor
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ABSTRACT: The timing of cytoplasmic fragmentation in relation to the cell cycle was studied in mature oocytes and early cleavage stages using mouse oocytes and embryos as experimental models. The central approach was to remove the nuclear apparatus, in whole or in part, from non-activated and activated oocytes and early embryos, and follow their response during subsequent culture in vitro. Oocytes arrested in metaphase of the second meiotic division did not fragment following complete removal of the meiotic apparatus, provided they were not subsequently activated. Exposure of spindle-chromosome-complex-depleted oocytes to activation conditions immediately after enucleation led to fragmentation, although not until control embryos entered first mitosis. Delaying activation until 24 h post-enucleation led to earlier fragmentation. Enucleation of normally fertilized or artificially activated oocytes after emission of the second polar body also led to fragmentation coinciding with the first mitosis in nucleated control embryos. However, if artificially activated oocytes were prevented from completing second meiosis, by exposure to cytochalasin, and then enucleated, this universal wave of fragmentation was preceded in some cytoplasts by limited fragmentation after just a few hours in culture, and coinciding with completion of meiosis II in nucleated oocytes. Fragmentation also occurred in the second mitotic cell cycle, but it was limited to blastomeres of fertilized oocytes that were enucleated in late interphase. These results indicate that fragmentation in oocytes and early embryos, though seemingly uncoordinated, is a precisely timed event that occurs only in mitotically active cells, during the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to altered cytoskeletal organization.
Molecular Human Reproduction 06/2005; 11(5):335-44. · 3.85 Impact Factor
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ABSTRACT: 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.
Cryobiology 03/2002; 44(1):5-13. · 2.06 Impact Factor
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ABSTRACT: This study examined the developmental capacity of aggregates of surviving mono-nucleated cells isolated from several non-viable human embryos on day 3 or day 4 after fertilization. The results clearly demonstrate that some blastomeres from non-viable embryos do indeed maintain their developmental potential and regulatory capacity to the extent of being able to contribute to a normally organized blastocyst, with as many as 90% diploid cells. Although the chimaeric nature of such blastocysts excludes them from use in therapeutic IVF, they are of particular relevance to the discussion of embryonic and trophectodermal stem cell line production.
Reproductive biomedicine online 5(1):56-8. · 2.04 Impact Factor
