Replication Stress Impairs Chromosome Segregation and Preimplantation Development in Human Embryos

Spontaneous DNA damage, such as double-strand breaks (DSBs), affects cell-cycle progression and contributes to embryo attrition. There is a strong correlation among mitotic cell-cycle progression, ploidy, the quality of the blastocyst formed, and successful implantation from in vitro fertilization. The mechanism by which spontaneous chromosomal breaks occur and are repaired in the human embryo, as well as the location of these breaks, has not been investigated through controlled experimentation. First, it was identified that foci indicating DNA damage and repair were present shortly after fertilization and pronucleus formation, suggesting they were formed de novo after the first S phase. Next, experiments in 1-cell embryos showed that DNA damage foci observed on day 1 in human zygotes are in G2 phase cells, and DNA synthesis by replicative DNA polymerase continues into the G2 phase while incompletely replicated DNA delays entry into mitosis. Assessing DNA replication forks progression showed slow speed at the 1-cell stage that increased through the blastocyst stage and was associated with frequent asymmetry in diverging sister forks, particularly at the zygote stage, indicating fork stalling and reflecting DNA replication stress. Spontaneous DSBs were found to result in a net loss of DNA, both segmental and whole-chromosome losses, in cleavage stage embryos. Mapping chromosomal break sites in blastomeres of day-3 embryos showed mirroring, or reciprocal breaks tended to locate to gene-poor regions and common fragile sites. A total of 154 break sites (55 different genomic locations) were observed in the same genomic region in different embryos. Correlating these regions to published gene expression data sets showed that the expression of neither long nor short transcripts correlated with chromosome fragility in the cleavage stage embryo. The results of this study show that human embryos acquire DNA damage during embryonic DNA replication characterized by replication fork stalling, DNA synthesis in G2 phase, incomplete replication at mitosis, chromosome breakage, micronucleation, and aneuploidy. Future research should study limiting factors involved in DNA fork stability and DSB repair, as well as the molecular causes of DNA replication stress in the early embryo.