Diane L. Wright

Human embryos derived from in vitro and in vivo matured oocytes: analysis for chromosomal abnormalities and nuclear morphology.

AbstractPurpose: To determine whether embryos resulting fromoocytes matured in vitro have a higher incidence of nuclearand/or genetic abnormalities compared to embryos resultingfrom oocytes matured in vivo. Methods: Fluorescence in situ hybridization analysis forchromosomes X, Y, and 18 was used to compare the ratesof aneuploidy, mosaicism, and nuclear abnormalities inembryos derived from oocytes that were prophase I ataspiration (immature group) to that observed in embryos resultingfrom oocytes that were metaphase I or II at aspiration(mature group). Results: Based on nuclear morphology, significantly moreembryos in the mature group (23percnt;) were classified as normalcompared to embryos in the immature group (3percnt;). Nodifference was found in the rate of aneuploidy or in the incidenceof mosaicism involving these chromosomes. Conclusions: These findings suggest that few embryosderived from prophase I oocytes collected following ovarianstimulation are morphologically normal.

PHYSIOLOGY: Evaluation of the Meiotic Spindle Apparatus in Metaphase II Human Oocytes Following Cytoplasmic Donation

Purpose: To determine if the removal of cytoplasm from metaphase II human donor oocytes damages the meiotic spindle apparatus.Materials and Methods: Cryopreservation of metaphase II human oocytes was performed using a fast-freeze, fast-thaw protocol. Upon thaw, oocytes were incubated for 3–4 h and then used for cytoplasmic donation (test oocytes). Oocytes thawed but not used for donation served as controls. Test and control oocytes were fixed using a microtubule-stabilizing buffer. Tubulin was localized using antitubulin monoclonal antibody. Chromosomes were identified by counterstaining with DAPI.Results: Forty-four oocytes had cytoplasm removed (test group) while 12 were not used for the procedure (controls). Twenty-three oocytes survived the donation procedure. Rates of normal spindle structure for the control and test groups were 21/23 (91.3%) and 12/12 (100%), respectively.Conclusion: The removal of cytoplasm from a metaphase II human donor oocyte does not appear to significantly increase the damage to chromosome alignment or to the spindle structure.

Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines

OBJECTIVE To investigate the use of donated gametes for the production of human embryonic stem cell lines. DESIGN Basic research study. SETTING Assisted Reproductive Technology (ART) program at an academic institution. PATIENT(S) Consenting oocyte and sperm donors. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Oocytes were aspirated from oocyte donors (n = 12) and inseminated with frozen-thawed donor (n = 2) sperm followed by culture of embryos to day 5 or 6 in sequential media. The inner cell masses of expanded blastocysts were isolated using immunosurgery and cultured for 4-11 days on irradiated primary mouse embryonic fibroblasts (PMEFs). Viable cell colonies were passed every 7-10 days onto fresh PMEFs in the presence of leukemia inhibitory factor (0.1 microg/mL) and evaluated for appropriate cell surface markers. RESULT(S) Immunosurgery of 40 blastocysts resulted in the culture of 18 inner cell masses, which have produced three cell lines. One of these cell lines has been shown to stain positive for alkaline phosphatase and stage-specific embryonic antigen (SSEA)-4 and negative for SSEA-1, express telomerase activity, and produce hCG when allowed to differentiate. CONCLUSION(S) These findings demonstrate that the future production of human embryonic stem cell lines for therapeutic use is possible with the use of donated gametes. Many ethical issues were considered before the initiation of this study, and it was our goal to ensure that both oocyte and sperm donors understood the nature and purpose of the research before their participating in the study.

Characterization of Telomerase Activity in the Human Oocyte and Preimplantation Embryo

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.