Scientific Updates

Cell: Genome Analyses of Single Human Oocytes

Many parents concern their children's health, even when their children are still an embryo, and now scientists have pushed the diagnosis limit to as early as just after fertilization. As described in Dec.19 paper in Cell, a united team of researchers, led by Professors Xiaoliang Sunney Xie and Fuchou Tang of Biodynamic Optical Imaging Center at Peking University and Prof. Jie Qiao at PKU’s affiliated Third hospital, realized single human oocyte's genome sequencing and offers the proof of principle for pre-implantation genetic screening (PGS) in in vitro fertilization (IVF).
During human egg formation, homologous recombination occurs between the paternal and maternal genomes. Then after meiosis process, two dispensable product cells containing copies of the genome, namely polar bodies, are relegated to the outer edge leaving a female pronucleus in egg fertilized with the sperm. "These two polar bodies can be harmlessly removed, and their genomes can be amplified and sequenced" said Fuchou Tang, a professor in BIOPIC, Peking University.
"In vitro fertilization (IVF) is a powerful technology to help infertile couples to get their own babies. Although the IVF technology has been successfully used for more than 35 years, the success rate is still relatively low, around 30 to 40%. Many miscarriages and diseases are associated with aneuploidies or single base mutations, but the current techniques array CGH and SNP array cannot detect them genome wide simultaneously, "said Jie Qiao, director of the fertility center at the Third Hospital of Peking University."
"The newly emerging single cell whole genome amplification technique multiple annealing looping based amplification cycle (MALBAC) can achieve 93% uniform genome coverage with little sequencing-based bias, which shows that counting 0,1, 2, 3 or 4 copies in a single cell is possible." said Xiaoliang Sunney Xie, the Mallinckrodt Professor of Chemistry and Chemical Biolog, Harvard University.
They successfully amplified and sequenced the genomes 183 single cells from 70 human oocytes. They constructed the crossover map for each single human oocyte cell and demonstrated the grandmother and grandfather contributions.
"Each person has two sets of genomes which are difficult to separate," Tang said, "But with the second polar body, which has only one set of them, we can easily phase them genome wide for each female."
The total number of genome sets in two polar bodies and female pronucleus of an oocyte should be four, two sets of which are maternal's and the other two are paternal's," Tang said, "By subtracting the two polar bodies from the total, we can deduce which set is left in the egg for embryo growing."
"Determining one inherited point mutation in 3 billion base pairs in human genome is like looking for a needle in a haystack, which needs very high accuracy of amplification and very high sequencing depth for each sample" Tang said, "But thanks to the separated set of genome, we can deduce each mutation according to which set of genome it located in."
To confirm the deduced results, they also sequenced the female pronucleus of each egg under donors' consents. The deduced results are highly consistent with the confirmed results, which proves the feasibility for PGS in IVF.
Obtaining three progeny cells of an oocyte enables researchers to accomplish a comprehensive analysis of recombination properties, like crossover interference and chromatid interference, during human oocyte meiosis.
"By comparison, polar body sequencing offers the whole genome information of the female pronucleus for aneuploidy screening and point mutation screening simultaneously."
"Polar body sequencing technique has its limitation when the genomic abnormalities are inherited from the father or arises during egg's mitosis," Qiao said, "Considering these situations, we also have tried to applied single cell genome amplification and sequencing to aneuploidy screening in blastocyst-stage biopsy. We obtained higher resolution than array CGH."
"We need to develop new methods to drive improvements in this field" said Sunney Xie, "I am very excited about our successful collaboration with Jie Qiao and Fuchou Tang on IVF. I am delighted to see an example of single molecule technology allows for the probing, understanding and bettering of life at a single molecule level."
Related readings:
Original Paper
Yu Hou, Wei Fan, Liying Yan, Rong Li, Ying Lian, Jin Huang, Jinsen Li, Liya Xu, Fuchou Tang, X. Sunney Xie, Jie Qiao, Genome Analyses of Single Human Oocytes, Cell, Volume 155, Issue 7, 19 December 2013, Pages 1492-1506, ISSN 0092-8674,
Perspective on Nature