Background

With the increasing number of infertile couples, the importance of assisted reproductive technologies (ART), including in vitro fertilization (IVF), is steadily growing. However, a significant proportion of fertilized eggs fail to develop beyond the blastocyst stage, before implantation in the uterus. In fact, studies have shown that only about half of the primate embryos cultured in vitro successfully reach the blastocyst stage, with the other half undergoing cell death in vitro. This has been a long-standing cause of stagnant success rates in infertility treatments.

Existing research has primarily attributed the causes of embryo developmental failure to genetic defects or chromosomal abnormalities associated with aging in the oocytes. The molecular-level physical errors that occur during the process of embryo self-division after fertilization have remained largely unexplored. Tracking the dynamics within living embryos in real-time has been technically challenging. Most studies have relied on indirect methods, such as staining and analyzing fixed embryo cells, making it nearly impossible to identify precise deviations during the moment of division.

Key Findings

A joint research team led by Dr. Chun So and Dr. Ge Lin at the National Institute of Biological Sciences (NIBS) used state-of-the-art live-cell fluorescence microscopy to observe the early 120 hours of human and cynomolgus monkey embryo development in real-time. The observation focused on over 2,000 blastomere division events. The analysis revealed that the causes of embryo developmental arrest differ significantly depending on the stage.

Early developmental arrest, occurring between 1 and 3 days after fertilization, is primarily caused by centrosome errors. Frequent centrosome errors were observed during the second division of the two-cell stage. This resulted in stochastic centriole overduplication, leading to the formation of multipolar spindles instead of normal bipolar spindles. Multipolar spindles cause aberrant segregation of genetic material, leading to chromosomal segregation errors and micronucleus formation, ultimately causing early embryo death.

The researchers targeted the centrosome assembly-driving kinase, 'PLK4 (Polo-like kinase 4)'. They then conducted experiments in which a low-molecular-weight compound, 'centrinone', which inhibits PLK4, was transiently added to the culture medium at the two-cell stage. The effects were immediate. In the treated embryos, centrosome dysfunction was significantly blocked. The proportion of embryos with a normal number of centrosomes increased from 40% to 80%.

In contrast, late developmental arrest, occurring at the morula stage around 4 days after fertilization, is caused by a different factor. It was not related to chromosomal segregation or spindle errors. At this stage, embryo cells exhibit excessive activation of the endoplasmic reticulum (ER) stress response, even though the chromosomes are normal, leading to growth arrest. Disruption of protein production led to decreased expression of cell adhesion and cell polarity proteins essential for blastocyst formation. As a result, the cells collapsed without completing their structure.

Significance and Prospects

This study is the first to demonstrate that the pathways leading to human pre-implantation embryo developmental failure are completely independent depending on the stage. In the early embryo, stabilization of the centrosome, which aids in genome segregation, is essential. In contrast, in the later morula stage, management of ER stress in the protein production factory is critical. This provides a specific direction for targeted treatment by segmenting the causes by stage. Few studies have demonstrated the possibility of targeted treatment by segmenting the causes by stage.

The assisted reproductive medicine community anticipates that the PLK4 regulation mechanism will provide a new clue to improve the success rate of infertility treatment. Of course, there are still challenges to be addressed before actual clinical application. It is not yet known whether short-term centrosome normalization ensures the long-term stability of the fetal genome. Thorough safety verification is essential. In addition, further research is needed to identify the maternal environmental factors that induce ER stress in the morula stage and to find compounds that can control them.