Study of Complex Mosaicism in Human Embryos: Revealing Extensive Aneuploidy During Early Embryonic Development

Background and Research Objectives

Pre-implantation Genetic Testing for Aneuploidy (PGT-A) is a widely used technology in in vitro fertilization (IVF) aimed at screening out abnormal embryos to improve implantation and live birth rates. However, limited understanding of human embryo genetics means that the use of PGT-A is based on unvalidated assumptions and guidelines, which may lead to the mistaken disposal of embryos with developmental potential. Existing studies show that the incidence of embryo mosaicism ranges from 2% to 90%, but the assessment methods of most studies are based on limited multi-cell samples, which may underestimate the true mosaic status. Furthermore, the dynamic changes of early embryo mosaicism and its impact on embryonic development remain unclear. This study systematically analyzed chromosomal mosaicism in human embryos from the blastocyst stage to the fetal development stage through single-cell whole genome sequencing and single-cell RNA sequencing (scRNA-seq).

Research Source and Publication Information

This study was conducted by the research team led by Fan Zhai, Siming Kong, and Zhiqiang Yan from the Center for Reproductive Medicine of Peking University Third Hospital, and was published in the journal Cell Discovery in 2024. The research team analyzed genetic information from the blastocyst stage to multiple fetal developmental stages using single-cell sequencing technology.

Research Procedures and Methods

Sample Collection and Analytical Design

1. Blastocyst Stage Analysis
   A total of 20 blastocyst samples were collected, and each blastocyst was fully dissociated into single cells, resulting in the collection of 1,133 single cells. Single-cell whole genome sequencing (scWGS) was used to detect chromosomal aneuploidy, and copy number variation (CNV) analysis was performed to confirm mosaicism.

2. Post-implantation Embryo and Fetal Organ Analysis
   The research team further used scRNA-seq data generated in previous studies to analyze chromosomal copy number changes in embryos covering development days 8 to 14 and fetal organs (including cerebral cortex, heart, and kidney) spanning 5 to 26 weeks.

3. Retrospective Analysis of PGT-A Mosaicism
   CNV analysis was performed on 116 backup biopsy samples from embryos previously diagnosed as euploid by PGT-A, which had developed into healthy newborns, to verify the prevalence of mosaicism.

Experimental Techniques and Analytical Methods

• High-quality sequencing technology combined with innovative analysis algorithms was used for comprehensive genome-wide CNV detection.
• In the blastocyst stage, low-quality amplified single-cell data were excluded, and CNV analysis was conducted on the remaining 1,072 single-cell data.
• In the post-implantation stage, chromosomal abnormalities were inferred precisely by combining scRNA-seq data with allelic imbalance analysis.
• In the retrospective analysis, single-cell sequencing results were compared with PGT-A diagnoses to reassess the true chromosomal composition of mosaic embryos.

Main Research Findings

1. Widespread Mosaicism in the Blastocyst Stage

   • All 20 blastocysts showed mosaicism, with an average mosaicism rate of 25%. 70% of blastocysts contained cells with “complementary” chromosomal abnormalities (i.e., some cells showed a gain of a chromosome while others showed a loss of the same chromosome).
   • There was no significant correlation between the mosaic rate and maternal age.

2. Chromosomal Mosaic Characteristics in the Post-implantation Stage and Fetal Organs

   • A total of 4,820 embryonic single cells from the 8–14 day development period were analyzed, and 96% of the embryos showed mosaicism, with the mosaic rate increasing with developmental progression.
   • Significant mosaicism was observed in fetal brain, heart, and kidney, and the distribution of aneuploid cells in different organs indicated differences in organ tolerance to chromosomal abnormalities.

3. Misdiagnosed Mosaic Embryos in PGT-A

   • In 116 backup samples from healthy newborns, 10.34% contained mosaic cells. The findings suggest that embryos previously diagnosed as euploid may actually include mosaic embryos.

Research Significance and Value

1. Scientific Significance
   This study reveals the widespread occurrence of mosaicism from the blastocyst stage to fetal development, challenging the negative evaluation of mosaic embryos by traditional PGT-A and suggesting that mosaic embryos may have developmental potential comparable to euploid embryos.

2. Clinical Application Value
   The study emphasizes the need for more tolerance towards mosaic embryos in PGT-A, especially for couples without available euploid embryos, as mosaic embryos could serve as an alternative option.

3. Technological Innovation
   The study employed a combination of single-cell whole genome sequencing and single-cell RNA sequencing techniques, providing new tools for a comprehensive analysis of embryo mosaicism.

Highlights and Future Directions

• Key Findings
  This study is the first to systematically reveal the complex mosaic phenomenon in human embryos at a single-cell resolution, linking the prevalence of mosaic embryos to healthy newborn outcomes.

• Novel Methodology
  The innovative single-cell analytical techniques and rigorous data filtering standards provide a technical paradigm for future studies.

• Future Directions
  The research team suggests further exploration of the developmental mechanisms of mosaic embryos and clinical screening and management strategies for mosaic embryos.

This study not only expands the understanding of human embryo genetic characteristics in the scientific field but also has far-reaching implications for the clinical practice of reproductive medicine.