It has been suggested that bovine IVF embryos have a higher frequency of occurrence of chromosomal abnormalities than invivo-fertilised embryos, which may explain low pregnancy success, but the details have not been clarified (Yao et al. 2018 Sci. Rep. 8, 7460). In this study, chromosomal aneuploidy in blastocysts of bovine IVF and invivo-fertilised was analysed by copy number variations (CNVs) based on next-generation sequencing. The IVF bovine embryos were cultured in well of-the-well culture dishes (LinKID micro25: Dai Nippon Printing) containing 125µL of CR1aa supplemented with 5% calf serum at 38.5°C in 5% O2 and 5% CO2 for 8 days after insemination. Invitro development of embryos was monitored using time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970-78). Invivo embryos were produced by collection of a superstimulated Japanese Black cow. Embryos that reached the blastocyst stage were divided into inner cell mass (ICM) and trophectoderm (TE) fractions by a micromanipulator with a blade. The TE and ICM samples were biopsied individually from 10 IVF and 4 invivo-derived embryos, and extracted DNA was amplified using the SurePlex DNA amplification System (Illumina). The whole-genome amplified DNA libraries were sequenced using MiSeq (Illumina). The sequencing reads were mapped onto the Bos taurus reference genome ARS-UCD1.2, obtained from the National Center for Biotechnology Information. In all 29 autosomal chromosomes and the X chromosome, CNV analysis was performed by CNV-seq (Xie and Tammi 2009 BMC Bioinformatics 10, 80). Male or female Japanese Black cattle DNA sequence was used for the reference genome. The parameter of CNV-seq was run with P-value=0.001, log2=0.6, and window size=1M. Four IVF embryos showed chromosomal duplications or deletions in either ICM- or TE-cell samples (4/10, 40%). The CNV loci between ICM and TE cells were relatively similar in each embryo. One of them was a code 1-expanded blastocyst with normal cleavage. Interestingly, CNV was not identified in another code 1-expanded blastocyst that underwent direct cleavage from 1 cell to 3 or more cells. In invivo embryos, only one embryo had a CNV (1/4, 25%). Observed CNVs in both IVF and invivo embryos were segmental duplication or deletion in each chromosome. Hence, to improve pregnancy success in bovine IVF embryos, cytogenetic evaluation may be useful for quality evaluation of embryos that are prone to chromosomal abnormalities, as well as morphological scoring.
Reliability of next generation sequencing (NGS) diagnosis of aneuploidy or mosaicism by re-biopsy and repeat NGS of inner cell mass (ICM) and trophectoderm (TE) from human embryos
