Steen Willadsen

Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs

readers with a two-factor (blinding status and meta-analysis topic), weighted analysis of variance model with weights equal to the inverse of the variance of the in odds ratio for each meta-analysis performed by each team of readers.

Mitochondrial DNA heteroplasmy after human ooplasmic transplantation

OBJECTIVE To determine the patterns of mitochondrial inheritance in embryos, fetuses, and infants after ooplasmic transplantation using the technique of mitochondrial DNA (mtDNA) fingerprinting. DESIGN Prospective clinical study. SETTING The IVF program at Saint Barnabas Medical Center, a nonprofit community hospital. PATIENT(S) In a total of 23 cases with recurrent implantation failure after IVF ooplasmic transplantation was performed. Thirteen embryos from two patients and amniotic cells from four patients were investigated for heteroplasmy. Placenta and fetal cord blood cells from four newborn babies/infants were also investigated. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) mtDNA fingerprinting, polymerase chain reaction, and DNA sequencing analysis. RESULT(S) In addition to the recipient maternal mitochondrial DNA, a small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (n = 6/13), amniocytes (n = 1/4), placenta (n = 2/4), and fetal cord blood (n = 2/4). Fingerprinting showed that nuclear DNA was not inherited from the donor in placenta or fetal cord blood of the babies. CONCLUSION(S) Ooplasmic transfer can result in sustained mtDNA heteroplasmy representing both donor and recipient. This was shown by mtDNA fingerprinting of embryos, amniocytes, fetal placenta, and cord blood. These results show that the donor-derived mitochondrial population persists after ooplasmic transfer and may be replicated during fetal development.

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.

Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos

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.

Highly effective method of human oocyte activation.

Fresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium, magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.