Next Generation Sequencing-Based Comprehensive Chromosome Screening in Mouse Polar Bodies, Oocytes, and Embryos

Abstract

Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond.

Keywords: aging; aneuploidy; embryo; oocyte; whole-genome sequencing.

Fig. 1

NGS can be used to identify the karyotype of mouse cells of known ploidy. A) Sequencing results are shown of genomic DNA isolated from pooled mouse embryonic fibroblasts engineered to have stable Trisomy 16 (copy number of 3). This cell line is of female origin. B) Average yield of DNA isolated from individual fibroblasts, polar bodies, oocytes, or blastocysts following WGA. Representative sequencing plots are shown from a single Trisomy 16 female MEF (C) and a single euploid male MEF (D) following WGA and NGS. *Autosomes that have copy numbers that deviate from 2, which indicates aneuploidy.

Fig. 2

Use of WGA and NGS for chromosome assessment in individual matched polar bodies and chromosomes is shown. A) MII oocytes with visible polar bodies were used for sequencing analysis. A representative image is shown in the left panel (*polar body). The oocyte and polar body were separated from each other following removal of the zona pellucida. The matched oocyte and polar body (inset) are shown in the right panel following separation. Bar = 50 μm. B) Schematic shows the experimental workflow. The zona pellucida was removed from each MII oocyte using acidic Tyrode solution, the matched polar body and oocyte were gently separated by aspiration and WGA, and NGS was performed in each cell. Representative sequencing plots of a euploid oocyte (C) and its matched polar body (D) are shown. Note, all autosomes and the X chromosome have a copy number of 2, whereas the Y chromosome has a copy number of 0.

Fig. 3

Detection of reciprocal aneuploidies in metaphase II oocytes and matched polar bodies. Sequencing plots from matched polar bodies (A, C, and E) and oocytes (B, D, and F) that reveal reciprocal aneuploidies, including premature separation of sister chromatids (loss/gain of 1 copy of chromosome 15 [A and B]) non-disjunction of homologs (loss/gain of 2 copies of chromosome 9 [C and D]), and complex aneuploidy (loss/gain of 1 copy of chromosomes 11 and 12 [E and F]). *Chromosomes with an abnormal copy number indicative of aneuploidy.

Fig. 4

Frequency of chromosome-specific aneuploidies in oocytes and embryos from reproductively aged mice. Histograms show frequency of segregation errors for each specific chromosome observed in aneuploid oocytes (A), IVO-derived embryos (B), and IVM-derived embryos (C).

Fig. 5

Use of WGA and NGS for chromosome assessment in individual blastocysts originating from young and reproductively aged female. A) Blastocysts derived after IVM and IVF were used for sequencing. A representative blastocyst is shown. Representative sequencing plots of euploid (B) and aneuploid blastocysts (C and D) are shown. Aneuploidy is due to a gain of 1 copy of chromosome 13 (C) and a gain of 1 copy of chromosome 8 (D). *Chromosomes with an abnormal copy number indicative of aneuploidy.