and then transferring them to recipient mice is challenging, and information other than the category will be lost, making detailed analysis difficult. Combining live-cell imaging and single embryo transfer could overcome this problem 13 , and we could directly link the relationship between the type/severity of the result of transplantation. Further, previous studies on the relationship between embryo ploidy and developmental potential used biopsy of blastocysts and subsequent chromosome analysis 14-16 ; in this study, ploidy of blastomeres of 2-cell embryos was investigated by single-cell genome sequencing after live-cell imaging of 1 st mitosis to link the imaging data of chromosome segregation and ploidy of embryo. Through live-cell imaging, single embryo transfer, and genome sequencing at single-cell resolution, we demonstrated that early chromosomal segregation error resulting in aneuploidy in mouse pre-implantation embryos is a developmental risk to the blastocyst, but some blastocysts retain their developmental potential. Methods Animals. This study conformed to the Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Animal Care and Use Committee at the Research Institute for Kindai University (permit number: KABT-31-016). ICR or B6D2F1 (BDF1) strain mice (12-16 weeks old) were obtained from Japan SLC, Inc. (Shizuoka, Japan). Room conditions were standardized, with the temperature maintained at 23 °C, relative humidity at 50%, and a 12-h/12-h light-dark cycle. Animals had free access to water and commercial food pellets. Mice used for experiments were sacrificed by cervical dislocation.

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In cases where GnRHa fails to trigger oocyte maturation, a rescue protocol entailing re-triggering with HCG can produce successful outcomes.

To culture preimplantation embryos in vitro, water-jacketed CO2 incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O2 was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.

Klinefelter's syndrome and spinal cord injury are major causes of male infertility. Intracytoplasmic sperm injection (ICSI) is a relatively new method of assisted reproduction. A testicular biopsy was obtained from a patient with the double complications of non-mosaic 47,XXY Klinefelter's syndrome and spinal cord damage, and motile spermatozoa were collected. ICSI was then performed. Of the four sperm-injected oocytes, three became fertilized and cleaved. Two embryos were implanted, resulting in a single pregnancy with visible evidence of a heartbeat appearing at 6 weeks gestation. The pregnancy is now entering its 20th week. To the best of our knowledge, this is the first case of a pregnancy resulting from the sperm of a patient with double complications.