Morphokinetic analysis of early embryos subjected to double stranded DNA damage provides evidence for cell cycle checkpoint activity

Abstract Study question Is there evidence of cell cycle checkpoint activation in preimplantation embryos with double stranded breaks (DSBs) in their DNA, induced using CRISPR? Summary answer Cells with persistent double strand DNA damage exhibit longer timeframes to reach key morphological milestones than those with successful repair, consistent with checkpoint activation. What is known already In most cell types, DNA damage leads to activation of cell cycle checkpoints that halt the cycle while DNA repair is attempted. It has been hypothesised that early embryos lack (or have relaxed) checkpoints, and that this may be necessary to allow the embryo to divide rapidly and synchronously during the first few mitoses. It is thought that maternal (oocyte derived) inhibitors may be responsible for suppressing checkpoints until activation of the embryonic genome. Inadequate checkpoint control may be responsible for the genetic instability and sensitivity to DNA damage seen during early preimplantation stages, which is of clinical importance. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods Precise induction of DNA damage involved injection of CRISPR-Cas9 ribonuclear complex (RNP), along with the sperm at the time of fertilization (ICSI). All embryos were disaggregated on day-3 and their cells subjected to whole genome amplification. Amplified products underwent low-pass next generation sequencing to detect segmental aneuploidy related to failure of DNA repair, while the site targeted using CRISPR-Cas9 was PCR amplified and sequenced to confirm whether it had successfully undergone repair. Main results and the role of chance Embryos that successfully repaired the induced DNA damage showed timings of pronuclei appearance/disappearance and cell divisions that were indistinguishable from control embryos. Rates of embryo arrest prior to completion of the second mitotic division were also similar between the two groups (25% versus 24%). In contrast, embryos with unresolved DNA damage, as evidenced by the detection of chromosomal fragments involving a breakpoint at the site targeted using CRISPR, were delayed in reaching the same developmental milestones (p < 0.0001 for time to first cleavage division) and displayed a much higher incidence of arrest (63% before the second mitotic division; p = 0.0002). The delay in mitotic progression in blastomeres with DNA damage represents strong evidence that a checkpoint is active in the cells of cleavage stage embryos, sensing double strand DNA breaks and slowing the cell cycle. However, the fact that 36% of embryos with unresolved DNA damage continued to progress, albeit at a slower rate, is consistent with the notion that checkpoint control is less stringent in early human embryos. This relaxation of control is likely to contribute to the genetic instability seen at the cleavage stage, including a risk of unresolved DNA damage and a high frequency of chromosomal malsegregation (causing mosaicism). Limitations, reasons for caution While altered cell cycle timings are consistent with checkpoint activation, other factors could conceivably influence the duration of key morphokinetic events. Complimentary experiments to test the functionality of specific checkpoints and/or the activity of their individual components will be required for definitive proof that embryonic checkpoints are active but weakened. Wider implications of the findings Segmental aneuploidy, micronucleation and mosaicism are frequently seen in human preimplantation embryos and are associated with reduced likelihood of ongoing pregnancy. Such abnormalities are symptomatic of genetic instability associated with excessively permissive checkpoints. These results highlight the need for culture systems that protect embryos from cellular stressors, especially DNA damage. Trial registration number N.A.