Catherine A. Boyd

Randomized controlled study of human zona pellucida dissection using the Zona Infrared Laser Optical System: evaluation of blastomere damage, embryo development, and subsequent hatching

OBJECTIVE To assess the effect of laser hatching on human embryo damage and subsequent development using the Zona Infrared Laser Optical System (ZILOS). DESIGN Randomized controlled study. SETTING Tertiary care fertility clinic. PATIENT(S) One hundred fourteen donated and discarded frozen human embryos. INTERVENTION(S) Embryos were thawed, cultured with cleavage and morphology evaluated periodically, and randomized into control, partial hatching, or complete hatching groups. The laser hatching procedure was performed by ZILOS. Zona thickness and embryo diameter were recorded. Complete hatching involved the production of a full-thickness defect in the zona and partial hatching, a defect in the outer half of the zona. No laser treatment was administered to the control group. MAIN OUTCOME MEASURE(S) Blastocyst development and completion of hatching process. RESULT(S) No significant difference was noted between the three study groups for their baseline characteristics. There was no significant difference in blastocyst development among the three groups. However, the complete hatching group showed a significant increase in hatching compared to the control group. CONCLUSION(S) Complete laser hatching of human embryos using the ZILOS does not have an adverse effect on subsequent development and increases the rate of completion of hatching.

Effects of sperm viability on fertilization and embryo cleavage following intracytoplasmic sperm injection.

AbstractPurpose: In the human, intracytoplasmic sperm injection is typically performed using “viable” sperm which has been mechanically rendered nonmotile. The purpose of the present study was to determine the ability of nonviable sperm to fertilize human oocytes and the early developmental normalcy of the resulting embryos. Methods: In this study, immature, prophase I oocytes from a total of 27 consenting patients were matured in vitro and then randomized into two groups: injection with a viable human sperm or injection with a sperm rendered nonviable by freeze-thawing in liquid nitrogen. The rates of fertilization and cleavage were compared between the two groups. Results: The results demonstrated a significantly higher two-pronuclear fertilization rate when oocytes were injected with viable sperm (62.2%) compared to when oocytes were injected with nonviable sperm (16.2%). Oocytes injected with viable sperm also demonstrated a higher cleavage rate (91 vs 33%). Conclusions: These findings suggest that while the intracytoplasmic injection of nonviable human sperm can result in normal fertilization, it does so at a much reduced rate compared to viable sperm and may not result in normally cleaving embryos.

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.

The effect of coculture on the postfertilization development of in vitro-matured monkey oocytes *†*Presented at the 50th Annual Meeting of The American Fertility Society, San Antonio, Texas, November 5 to 10, 1995.†Supported in part by the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust, Richmond, Virginia.

OBJECTIVE To determine if the developmental potential of embryos resulting from in vivo- and in vitro-matured monkey oocytes could be increased through the use of a coculture system. DESIGN Randomized prospective comparison of embryos resulting from either in vitro- or in vivo-matured oocytes cocultured with Vero cells or cultured in medium alone (control). SETTING Basic research laboratory. MAIN OUTCOME MEASURES In vitro embryo development to the blastocyst stage and blastocyst hatching. RESULTS No significant difference in development was noted between coculture and control groups with embryos resulting from in vivo-matured oocytes. However, coculture was found to improve significantly the development of monkey embryos resulting from in vitro-matured oocytes. CONCLUSIONS These results demonstrate that primate embryos resulting from in vitro-matured and in vitro-fertilized oocytes differ in their culture requirement when compared with embryos resulting from in vivo-matured oocytes.