O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos
Authors: Ono Y , Shirasawa H , Takahashi K , Goto M , Ono T , Sakaguchi T , Iwasawa T , Fukunaga N , Asada Y , Kumazawa Y , Terada Y 
Published: July 2024

Abstract

Study question
How multinucleation and fragmentation occur in the first mitosis in human embryos?

Summary answer
The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation.

What is known already
Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling.

Study design, size, duration
Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin.

Participants/materials, setting, methods
Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ.

Main results and the role of chance
We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p &lt; 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p &lt; 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p &lt; 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p &lt; 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development.

Limitations, reasons for caution
Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics.

Wider implications of the findings
Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans.

Trial registration number
not applicable