Association Between Spindle Assembly Checkpoint Gene Expression and Maternal Age in Human Oocytes

Abstract

Objective: Maternal aging remains the overwhelming factor in the aetiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at metaphase. MAD2 and BUB1 genes encode conserved kinetochore-associated proteins believed to be major components of this regulatory pathway. A failure in this surveillance system could lead to genomic instability, which may underlie the increased incidence of aneuploidy in oocytes of older women.Design: The concentrations of MAD2 and BUB1 transcripts in human oocytes at GV, MI and MII stages of maturation were determined by real-time rapid cycle fluorescent RT-PCR. The incidence of aneuploidy was established by multi-probe FISH analysis.Materials and Methods: Nonviable human mature and immature oocytes (n=68) were obtained from patients undergoing IVF. Rapid fluorescence monitored cycling was used to examine expression levels of the checkpoint and housekeeping genes in individual human oocytes. Unknown concentrations were extrapolated from standards co-amplified producing a standard curve. Polar body biopsy, fixation and multi-probe FISH analysis was performed to determine chromosomal status of MII oocytes.Results: Regression analysis confirmed that a significant linear correlation exists between maternal age and MAD2 transcript concentration for GV, MI and MII stages of maturation. Likewise, a similar trend emerged when the BUB1 transcript copy numbers were plotted versus maternal age but only for GV and MI stages. No association was apparent between maternal age and the concentration of β-actin mRNA. Experiments are in progress comparing the incidence of aneuploidy and checkpoint transcript concentrations. However, preliminary results suggest that an association may exist between aneuploidy status and MAD2 transcript concentrations.Conclusions: The observations reported here suggest that the MAD2 and BUB1 transcripts may degrade as the oocyte ages. Since the concentration of housekeeping transcripts remained constant throughout each stage of maturation examined, it appears that this phenomenon is not universal of all transcripts in oocytes from older women. Potentially, the degradation of these messages may impair spindle checkpoint function in older oocytes and be a contributing factor in age-related aneuploidy. Understanding the checkpoint defects associated with chromosomal abnormalities may have profound therapeutic consequences for older women undergoing assisted reproduction. Objective: Maternal aging remains the overwhelming factor in the aetiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at metaphase. MAD2 and BUB1 genes encode conserved kinetochore-associated proteins believed to be major components of this regulatory pathway. A failure in this surveillance system could lead to genomic instability, which may underlie the increased incidence of aneuploidy in oocytes of older women. Design: The concentrations of MAD2 and BUB1 transcripts in human oocytes at GV, MI and MII stages of maturation were determined by real-time rapid cycle fluorescent RT-PCR. The incidence of aneuploidy was established by multi-probe FISH analysis. Materials and Methods: Nonviable human mature and immature oocytes (n=68) were obtained from patients undergoing IVF. Rapid fluorescence monitored cycling was used to examine expression levels of the checkpoint and housekeeping genes in individual human oocytes. Unknown concentrations were extrapolated from standards co-amplified producing a standard curve. Polar body biopsy, fixation and multi-probe FISH analysis was performed to determine chromosomal status of MII oocytes. Results: Regression analysis confirmed that a significant linear correlation exists between maternal age and MAD2 transcript concentration for GV, MI and MII stages of maturation. Likewise, a similar trend emerged when the BUB1 transcript copy numbers were plotted versus maternal age but only for GV and MI stages. No association was apparent between maternal age and the concentration of β-actin mRNA. Experiments are in progress comparing the incidence of aneuploidy and checkpoint transcript concentrations. However, preliminary results suggest that an association may exist between aneuploidy status and MAD2 transcript concentrations. Conclusions: The observations reported here suggest that the MAD2 and BUB1 transcripts may degrade as the oocyte ages. Since the concentration of housekeeping transcripts remained constant throughout each stage of maturation examined, it appears that this phenomenon is not universal of all transcripts in oocytes from older women. Potentially, the degradation of these messages may impair spindle checkpoint function in older oocytes and be a contributing factor in age-related aneuploidy. Understanding the checkpoint defects associated with chromosomal abnormalities may have profound therapeutic consequences for older women undergoing assisted reproduction.