JCI: Using DNA methylation to trace the source of free DNA cells in blastocyst culture fluid

Human pregnancy efficiency is very low, less than 50% of embryos in natural pregnancy can develop to term

On June 15, 2021, Peking University Biomedical Frontier Innovation Center Tang Fuchou/Wen Lu team cooperated with Peking University Third Hospital Qiao Jie /Huang Jin team to publish the title DNA methylome reveals cellular origin of in The Journal of Clinical Investigation .

Figure 1 Schematic diagram of the study of cfDNA methylation profile in embryo culture fluid

1.

The study performed a systematic unsupervised analysis of the methylation of cell-free DNA in the culture medium and the methylation of genomic DNA of preimplantation embryo cells and granular cells, and found that 50/191 SECM and granular cells are grouped together.

In order to further quantify the proportion of DNA from granular cell contamination in SECM, the study identified 769 granular cell-specific hypermethylated differentially methylated regions (C-DMRs).

Figure 2 Contamination of granular cells in embryo culture medium (SECM)

　　2.

　　In order to further explore the accurate source of free DNA in the SECM without granule cell contamination, the study continued to conduct a systematic unsupervised cluster analysis of the methylation of free DNA in the culture medium and the genomic methylation of preimplantation embryo cells.

　　In order to further quantify the proportion of free DNA in polar somatic cells contaminated in SECM, the study identified 548 egg/polar somatic cell-specifically hypermethylated differentially methylated regions (O-DMRs), which are highly methylated in egg cells and female pronuclei.

　　Figure 3 Polar body cell contamination in the embryo culture medium (SECM)

　　3.

　　Using the methylation level data of 769 C-DMRs and 548 O-DMRs, the research team established a calculation method to derive the ratio of granular cell and polar body cell DNA in embryo culture fluid

　　The study then calculated the proportion of each component in the free DNA of the actual culture fluid, and found that 1/3 of the embryo culture fluid free DNA samples contained granular cell contamination, and 22% of the samples had granular cell contamination more than 60%

　　Subsequently, the study defined the sum of the contamination ratios of granular cells and polar body cells as net maternal contamination, and found that only 1/3 of embryo culture fluid free DNA samples did not have obvious maternal pollution, and nearly 1/3 of embryo culture fluid Cell-free DNA samples have serious maternal contamination (Figure 4B)

　　Figure 4 Derivation of maternal DNA contamination rate in embryo culture medium (SECM)

　　4.
Integrated analysis of maternal contamination of free DNA in embryo culture fluid and chromosome aneuploidy

　　DNA methylation sequencing can infer chromosome copy number (CNV)
.
The study evaluated the accuracy of single-cell BS-seq inferred copy number results in cell lines, and found that the copy number obtained by single-cell BS-seq was consistent with the results obtained with MALBAC, and the required starting amount was very low (Figure 5A)
.
The study also explored the effect of maternal contamination of free DNA in embryo culture fluid on the inferred embryonic cell chromosome copy number
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The results showed that the gender inconsistency rate and false negative rate of chromosome copy number increased with the increase of the net contamination rate of granular cells, polar body cells and maternal origin
.
When the percentage of net maternal pollution is higher than 60%, the gender inconsistency rate and false negative rate increase to 100% and 75%, respectively
.
Therefore, when the maternal source is heavily contaminated, the CNV of embryonic cells inferred by SECM becomes inaccurate
.

　　Figure 5 Integration analysis of maternal DNA contamination and chromosome copy number in embryo culture medium (SECM)
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(A) scBS-seq infers CNV; (B) the influence of maternal pollution on SECM infers CNV
.

　　To sum up, this research traces the cell source of free DNA in embryo culture fluid to blastocyst cells, granulosa cells and polar body cells for the first time, and has developed a fast and convenient algorithm to quantify the amount of maternal contamination in embryo culture fluid.
At the same time, through the integrated analysis of maternal contamination and chromosomal aneuploidy, the efficiency of detecting aneuploid embryos using embryo culture fluid is improved
.
This study provides new insights for the characterization of free DNA in embryo culture fluid and provides a new perspective for non-invasive preimplantation embryo aneuploidy genetic testing (PGT-A)
.

　　Chen Yidong and Gao Gao, Ph.
D.
candidates at the Institute of Frontier Interdisciplinary Studies, Peking University, are the co-first authors of this research paper
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Beijing Future Gene Diagnosis Advanced Innovation Center (ICG), Peking University Biomedical Frontier Innovation Center (BIOPIC), Professor Tang Fuchou, School of Life Sciences, Associate Researcher Wen Lu, and Professor Qiao Jie and Deputy Director Huang Jin of Peking University Third Hospital The physician is the co-corresponding author of the paper
.
This research project was supported by the National Natural Science Foundation of China and the Beijing Future Gene Diagnosis Advanced Innovation Center
.

　　Paper link:

　　https://