Alterations of mitotic checkpoint genes play a role in embryonic chromosome mosaicism

Abstract

OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts.DESIGN: Experimental study.MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (≥Grade 3BB) (n=21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR.RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n=8 euploid, Group B, n=8 uniform aneuploidy (meiotic error) and Group C, n=5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes.CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome. OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts. DESIGN: Experimental study. MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (≥Grade 3BB) (n=21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR. RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n=8 euploid, Group B, n=8 uniform aneuploidy (meiotic error) and Group C, n=5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes. CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome.