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An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.

Objective: To evaluate whether previously enucleated mouse oocytes that are vitrified are usable for the analysis of human sperm chromosomes., Design: Prospective, comparative laboratory study., Setting: Animal breeding and reproduction department in a rural Japan university., Patient(s): Sperm samples were obtained from a fertile donor., Intervention(s): Human sperm were injected into previously enucleated-vitrified mouse oocytes., Main Outcome Measure(s): The rates of appearance of the human sperm chromosomes that were injected into previously enucleated-vitrified mouse oocytes., Result(s): The rates of appearance of human sperm chromosomes were 71%-72% in enucleated-vitrified mouse oocytes., Conclusion(s): The use of enucleated-vitrified mouse oocytes allowed visualization of human sperm chromosomes.

Background: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes., Methods: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI., Results: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017)., Conclusions: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.