Although human in vitro fertilization (IVF) was introduced over 35 years ago as a means to assist infertile couples in conceiving a child, the average live birth rate per IVF cycle is still only ~30-35% (cdc.gov/art). A primary contributor to IVF failure are whole chromosomal abnormalities (aneuploidy) that occur during pre- implantation embryo development. Using high-resolution genomic techniques, it was recently shown that ~50- 80% of cleavage-stage human embryos have one or more chromosomally abnormal cells. These aneuploid embryos often display phenomena known as cellular fragmentation and micronucleation, which we hypothesize are corrective mechanisms to aid in restoring proper chromosome numbers. To improve our understanding of why these abnormalities occur and uncover solutions to ameliorate their impact on IVF success, animal models with pre-implantation development closely resembling humans are needed. Besides rarely containing micronuclei and cellular fragments, mouse embryos also exhibit considerably less aneuploidy than humans at ~1-15%, and therefore, do not make a good model for studying these processes. Therefore, this proposal seeks to investigate the use of a bovine model, which shares several key aspects of early embryogenesis with humans, to increase our understanding of human embryonic aneuploidy.
The first aim of these studies will determine the exact aneuploidy frequency in cleavage-stage bovine embryos by combining next generation DNA-sequencing of single blastomeres and cellular fragments with time-lapse monitoring (TLM) of pre-implantation development.
In Aim II, fragmented versus non-fragmented bovine embryos will be assessed by RNA-sequencing following TLM to identify gene expression changes resulting from, and contributing to, aneuploidy for functional analysis.
Aim III will investigate the role of BUB1B, a mitotic checkpoint protein important for mediating normal blastomere divisions and nuclear structure in our preliminary data, and/or other genes identified from Aim II via morpholino microinjection. Gene expression changes in BUB1B deficient embryos will be assessed to determine the effect of knockdown on chromosome segregation and maintenance during early mitosis. Completion of the proposed studies will allow us to unravel the precise relationship between embryonic cleavage divisions, cellular fragmentation, micronucleation, and aneuploidy as well as increase our understanding of the molecular pathways mediating normal pre-implantation development. These studies will contribute valuable findings to our long term goal of identifying and avoiding chromosomal abnormalities associated with early embryo loss for increased IVF success.
Despite efforts to increase human IVF success, the average live birth rate remains low at only ~30-35% with chromosomal abnormalities identified as a major contributing factor to early embryo loss. The studies proposed here aim to establish a bovine model to study the mechanisms regulating human embryonic aneuploidy during pre-implantation development. Completion of these studies will provide the knowledge necessary for translational application to human embryos and improved pregnancy success.
