Comprehensive chromosomal and mitochondrial copy number profiling in human IVF embryos

Abstract

Single cell whole genome sequencing helps to decipher the genome heterogeneity within a cell population and facilitates the analysis of trace amounts of genetic material, such as is found in human embryos. The mitochondrial genome, although an important part of the genetic composition of eukaryotic cells, is often neglected in single cell genome analysis. A recently developed single cell whole genome amplification method was used, known as multiple annealing and looping based amplification cycles (MALBAC-NGS), for simultaneous analysis of chromosomal and mitochondrial genomes at the single cell level. The platform was validated by a series of technical and biological replicates and used for chromosomal and mitochondrial copy number analysis in 399 in-vitro fertilized embryos from 81 couples. A positive correlation of maternal age with increased mitochondria quantity (β = 0.176, P = 0.001) was observed after adjusting for the impact of cell type. Lower numbers of mitochondria were detected in successfully implanted embryos, although the difference was not significant. It is proposed that MALBAC-NGS could potentially be used for an advanced pre-implantation genetic screening procedure with both chromosomal constitution and mitochondrial copy number being evaluated.

Mitochondrial DNA and genomic DNA ratio in embryo culture medium is not a reliable predictor for in vitro fertilization outcome

“…Another study chose Alu, a multiple copies gene, as a detection fragment 44 , which was not technically superior to that of a single copy gene, and the frequency and composition of the Alu sequence are significantly different in population 45 . multiple annealing and looping-based amplification cycles (MALBAC) and NGS has been successfully used in various studies of DNA at single cell level, such as detecting single nucleotide variation (SNV) and copy number variation (CNV), analyzing meiotic recombination 46,47 , and performing PGS of in-vitro fertilized embryos 47,48 . Studies have also shown that NGS is sensitive enough to detect small differences between subculture populations from the same cell line and that MALBAC-NGS is also suitable to study mtDNA copy number in single cells with good reproducibility 49 .…”

“…2500 platform for a total of approximately 1.5 million reads. The NGS data was analyzed and the chromosomal copy number variation was investigated as described in previous reports 48,52,53 . The high quality read numbers were counted along the whole genome with a bin size of 1.5~2 Mb and normalized by the GC content and a reference dataset.…”

Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening

“…As discussed above, the universal MARSALA strategy we reported in this study eliminates lengthy preliminary work-ups on linkage analysis of specific mutations and SNP markers and the need of additional information from relevant family members is. Based on the NGS platform, this approach also possesses some advantages over other technologies: (a) it detects the mutation sites directly allowing the discovery of de novo mutations; (b) the detection of chromosomal mosaicism in the blastocysts can be achieved with greater sensitivity and at a finer resolution than array-based tests [31]; (c) the analyses on mitochondrion genome (for instance copy number or mutation site) are also achievable [32].…”

Mitochondrial reactive oxygen species regulate mitochondrial biogenesis in porcine embryos

“…However, the relationship between mitochondrial number and reproductive potential in blastocysts is not completely clear. Accumulating evidence supports the quiet embryo hypothesis [12], which states that blastocysts with higher mitochondrial DNA copy numbers have low quality or low reproductive ability [13][14][15][16].…”