During embryogenesis, cells repeatedly divide and dynamically change their positions in three-dimensional (3D) space. A robust and accurate algorithm to acquire the 3D positions of the cells would help to reveal the mechanisms of embryogenesis. To acquire quantitative criteria of embryogenesis from time-series 3D microscopic images, image processing algorithms such as segmentation have been applied. Because the cells in embryos are considerably crowded, an algorithm to segment individual cells in detail and accurately is needed. To quantify the nuclear region of every cell from a time-series 3D fluorescence microscopic image of living cells, we developed QCANet, a convolutional neural network-based segmentation algorithm for 3D fluorescence bioimages. We demonstrated that QCANet outperformed 3D Mask R-CNN, which is currently considered as the best algorithm of instance segmentation. We showed that QCANet can be applied not only to developing mouse embryos but also to developing embryos of two other model species. Using QCANet, we were able to extract several quantitative criteria of embryogenesis from 11 early mouse embryos. We showed that the extracted criteria could be used to evaluate the differences between individual embryos. This study contributes to the development of fundamental approaches for assessing embryogenesis on the basis of extracted quantitative criteria.

Highlights d Filamentous actin is a hallmark of pronuclei in mouse zygotes d Polymerized nuclear actin is important for embryonic development d Zygotic nuclear F-actin is required for efficient DNA damage repair d The timely disassembly of nuclear F-actin is key to subsequent development

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.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4–8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

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