114 LIVE-CELL IMAGING FOR THE QUALITY ASSESSMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO IN CYNOMOLGUS MONKEY

Abstract

We have developed a live-cell imaging technique to assess measures of embryo quality such as epigenetic status and chromosome integrity during the early cleavage stages of pre-implantation development in the mouse. The advantages of this method are that the procedure is safe for the embryo and pups are not transgenic even after the imaging (Yamagata et al. 2009 Hum. Reprod. 24, 2490–2499). One of the valuable indexes in using this imaging technique is chromosome segregation (CS) during first mitosis; the embryos showing normal CS (NCS) result in normal offspring, whereas abnormal CS (ACS) embryos do not. In this study, we established a live-cell imaging technique for cynomolgus monkey intracytoplasmic sperm injection (ICSI) embryos and we succeeded in obtaining a normal offspring from NCS embryos after the assessment of live-cell imaging. Ovarian stimulation was carried out as previously described by Torii et al. (2001 Exp. Anim. 50, 259). Oocytes were collected by follicular aspiration using laparoscopy and ICSI was performed to metaphase II oocytes. After the ICSI, a mixture of mRNA encoding fluorescent labelled tubulin and histone was injected into ICSI embryos for the evaluation. Live-cell imaging was initiated 4 h after injection by laser confocal microscopy and 2-cell embryos were classified as NCS or ACS the next day. After embryo culture, embryo transfer (ET) was carried out to recipient donors (NCS embryos: 13, ACS embryos: 2) and pregnancy was diagnosed by ultrasonography at 4 weeks after ET. In another experiment, we tried to assess the 2-cell embryos with a snapshot image taken by a conventional fluorescent microscope as a simplified method. A total 121 embryos from 15 monkeys were analysed and embryos were classified as NCS or ACS. Live-cell imaging revealed that the NCS rate was 43.3% and the ACS rate was 56.7%. Pregnancy was confirmed in 2 NCS embryos from 13 ET (15.4%; 2/13); however, no pregnancy was observed in the ACS group (0%, 0/2). Furthermore, one normal offspring was achieved from ET of 2 NCS embryos that were diagnosed by live-cell imaging. In addition, we could also assess the status of chromosome and nuclei in the 2-cell embryos even by fluorescent microscopy and in this case, the NCS rate was 69.2% and the ACS rate was 30.8%. In conclusion, live-cell imaging can be used to evaluate the status of chromosome segregation of ICSI embryos in the cynomolgus monkey under laser confocal and fluorescent microcopy. The results indicate that ACS would be a detrimental factor in the embryonic development in the monkey, similar to in the mouse. Moreover, a normal offspring was born after the imaging and therefore this new technique could be applicable to assessment of embryo quality in human assisted reproductive technology.