Fragmentation of embryos is associated with both necrosis and apoptosis.

OBJECTIVE: To explore the association between embryo fragmentation and necrosis and apoptosis. DESIGN: A prospective study. SETTING: Mizmedi Hospital. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Staining with annexin V (a marker of apoptosis) and propidium iodide (PI, a marker of necrosis), DNA integrity and mitochondrial distribution, and a beneficial effect of fragment removal in human fragmented embryos. RESULT(S): Most of the mouse and human fragmented embryos were stained with PI but not with annexin V. The comet assay revealed severe DNA fragmentation of the fragmented human embryos but not of the unfragmented embryos. Fewer mitochondria were observed in the fragmented compared with the normal blastomeres, indicating a rapid depletion of ATP in the fragmented embryos. Microsurgical fragment removal from the embryos had a beneficial effect on their subsequent development. CONCLUSION(S): Fragments of human embryos exhibited various characteristics of necrosis, such as staining with PI, DNA fragmentation, rapid depletion of ATP, and harmful effects on neighboring blastomeres. We suggest that the fragmentation of embryos is closely associated with both necrosis and apoptosis. Whether this fragmentation is associated with primary or secondary necrosis remains to be elucidated.

Patient-specific embryonic stem cells derived from human SCNT blastocysts.

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.

Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles.

OBJECTIVE: To investigate the effect of calcium ionophore on the fertilization rate of a patient with normozoospermia who nonetheless exhibited a low fertilization rate in intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: In vitro fertilization center. PATIENT(S): A male patient whose sperm, though diagnosed as normal by semen analysis, exhibited a severely low fertilization rate in ICSI cycles. INTERVENTION(S): Oocytes were activated by calcium ionophore after ICSI. MAIN OUTCOME MEASURE(S): Fertilization rate after oocyte activation; ultrastructure and protein expression of the patient's sperm. RESULT(S): The fertilization rate of oocytes activated with calcium ionophore (12 of 15, 80.0%) was higher than that of the nonactivated oocytes (4 of 16, 25.0%). Four embryos derived from the activated oocytes were transferred, resulting in a twin pregnancy. Further investigation revealed abnormalities in the patient's sperm: many nuclear vacuoles were observed and the expression of some proteins was absent. CONCLUSION(S): Oocyte activation with calcium ionophore was effective at increasing the fertilization rate of dysfunctional sperm characterized by ultrastructural and protein expression anomalies.

Cryopreservation of human embryos using ethylene glycol in controlled slow freezing.

BACKGROUND: Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian formula embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). This study was carried out to evaluate the permeation and toxicity of EG and to investigate the effects of its use in a slow-freezing protocol on post-thaw development of mouse embryos and on pregnancy outcome of frozen human embryos. METHODS: Spare human embryos after embryo transfer were cryopreserved using 1.5 mol/l EG or PROH using a slow-freezing protocol which had been tested previously in mouse experiments. RESULTS: The post-thaw survival rate of human embryos in the EG group (80.6%) was significantly higher than that in the PROH group (65.2%, P < 0.05). The implantation and clinical pregnancy rates of human embryos in the EG group (20.3 and 46.9%) were significantly higher than those in the PROH group (7.5 and 24.6%, P < 0.05). CONCLUSIONS: Ethylene glycol may be a good substitute for PROH to cryopreserve human embryos using a slow-freezing protocol.