Exploring the Impacts of Senescence on Implantation and Early Embryonic Development Using Totipotent Cell-Derived Blastoids

Academic Background

With the increasing trend of delayed childbearing in modern society, reproductive issues among women of advanced maternal age (AMA) have become increasingly prominent. Studies have shown that the implantation and pregnancy rates of embryos from older women are significantly reduced, but the underlying mechanisms remain unclear. Implantation is a critical process for embryo survival and development, involving complex interactions between the embryo and the endometrium. However, due to the lack of suitable research models and ethical constraints, scientists’ understanding of this process is limited. The mouse model, due to its similarity in gene expression profiles and implantation regulatory pathways with humans, has become an important tool for studying human implantation. In recent years, scientists have developed blastoid models, which resemble natural blastocysts in morphology, cell lineage allocation, and transcriptomic features, providing new insights into early embryonic development and implantation.

However, existing blastoid models have limitations in construction efficiency, cell lineage localization, and developmental potential, making them unable to fully replicate the developmental processes of natural blastocysts. Therefore, developing a blastoid model with enhanced developmental potential is crucial for accurately simulating early embryonic development and implantation processes. This study aims to explore the impact of senescence on implantation ability by constructing a blastoid model using totipotent blastomere-like cells (TBLCs), and to reveal the potential molecular mechanisms behind implantation failure in embryos from older women.

Source of the Paper

This paper was co-authored by Yuxin Luo, Chenrui An, Ke Zhong, and other researchers from multiple institutions, including Peking University Third Hospital and the Third Affiliated Hospital of Guangzhou Medical University. The paper was published online on February 24, 2024, in the Journal of Advanced Research, titled “Exploring the Impacts of Senescence on Implantation and Early Embryonic Development Using Totipotent Cell-Derived Blastoids.” The study was supported by the National Key Research and Development Program of China and the National Natural Science Foundation of China.

Research Workflow

1. Induction and Identification of TBLCs

Researchers first induced mouse pluripotent stem cells (mPSCs) into totipotent blastomere-like cells (TBLCs) by inhibiting the spliceosome. These TBLCs exhibited transcriptomic features similar to 2-cell and 4-cell stage embryos but were distinct from pluripotent stem cells. Through quantitative PCR (qRT-PCR) and immunofluorescence staining, the researchers verified that TBLCs highly expressed totipotency genes (e.g., Zscan4), while pluripotency genes were expressed at lower levels. Additionally, TBLCs exhibited proliferation capabilities similar to totipotent cells but showed slower growth rates in long-term culture.

2. Construction and Characterization of Blastoids

Researchers constructed blastoids (TB-blastoids) using TBLCs and optimized a three-dimensional culture system by adding lysophosphatidic acid (LPA), an inhibitor of the Hippo pathway. During the culture process, the blastoids gradually formed typical blastocyst-like structures, containing three cell lineages: the inner cell mass (ICM), trophectoderm (TE), and primitive endoderm (PE). Immunofluorescence staining revealed that the cell lineage allocation of the blastoids was similar to that of natural blastocysts. Single-cell RNA sequencing (scRNA-seq) further confirmed the high similarity in transcriptomic features between the blastoids and natural blastocysts.

3. Impact of Senescence on Blastoid Development

To simulate the senescence characteristics of embryos from older women, researchers treated TBLCs with hydrogen peroxide (H2O2), an oxidative stress inducer, to establish a senescence-related blastoid model. Through immunofluorescence staining and qRT-PCR, researchers found that senescent TBLCs exhibited reduced cell proliferation capabilities and displayed typical senescence features, such as increased senescence-associated β-galactosidase (SA-β-gal) activity. Subsequently, researchers constructed senescent blastoids using senescent TBLCs and found that their developmental potential was significantly reduced, manifested as decreased implantation rates and the degeneration of embryo-like structures.

4. In Vitro and In Vivo Developmental Potential of Blastoids

Researchers cultured the blastoids in vitro and observed their ability to form structures resembling post-implantation embryos. Additionally, researchers transplanted the blastoids into the uteri of pseudopregnant mice and found that they could successfully implant and induce decidualization. However, the implantation rates and developmental potential of senescent blastoids were significantly lower than those of normal blastoids, consistent with the phenomenon of implantation failure in embryos from older women.

5. Gene Expression and Disease Association Analysis

By comparing the transcriptomic data of senescent and normal blastoids, researchers found that genes related to senescence and apoptosis were upregulated in senescent blastoids, while genes related to pluripotency and oxidative phosphorylation were downregulated. Furthermore, through gene-disease association analysis, researchers identified a series of genes associated with infertility, ovarian failure, and pregnancy complications, which may play important roles in implantation failure in embryos from older women.

Research Results

1. Induction and Identification of TBLCs: Researchers successfully induced TBLCs and verified their totipotency characteristics. TBLCs exhibited transcriptomic features similar to early embryonic cells but were distinct from pluripotent stem cells.

2. Construction and Characterization of Blastoids: TBLCs successfully constructed blastoids, which exhibited morphology and cell lineage allocation highly similar to natural blastocysts. Single-cell RNA sequencing further confirmed the consistency in transcriptomic features between the blastoids and natural blastocysts.

3. Impact of Senescence on Blastoid Development: Blastoids constructed from TBLCs induced by oxidative stress exhibited significantly reduced developmental potential, manifested as decreased implantation rates and the degeneration of embryo-like structures.

4. In Vitro and In Vivo Developmental Potential of Blastoids: Blastoids exhibited developmental potential similar to natural blastocysts in both in vitro culture and in vivo transplantation experiments, but the developmental potential of senescent blastoids was significantly reduced.

5. Gene Expression and Disease Association Analysis: Transcriptomic data from senescent blastoids revealed upregulation of genes related to senescence and apoptosis, as well as changes in gene expression associated with infertility and pregnancy complications.

Conclusion and Significance

This study successfully constructed a blastoid model (TB-blastoids) based on totipotent cells, which exhibited high similarity to natural blastocysts in morphology, cell lineage, and transcriptomic features. By simulating the senescence process, researchers revealed the negative impact of senescence on embryo implantation and early development, and identified a series of genes associated with implantation failure in embryos from older women. These findings provide a new research model for exploring the mechanisms of reproductive issues in older women and offer potential intervention targets for improving pregnancy outcomes in this population.

Research Highlights

1. Novel Blastoid Model: This study is the first to construct a blastoid model using totipotent cells, which exhibits superior developmental potential and transcriptomic features compared to existing models.

2. Exploration of Senescence Mechanisms: By inducing senescence in TBLCs through oxidative stress, researchers successfully simulated the senescence characteristics of embryos from older women and revealed the negative impact of senescence on embryo development.

3. Gene-Disease Association Analysis: Through gene-disease association analysis, researchers identified a series of genes associated with infertility and pregnancy complications, providing new research directions for improving reproductive health in older women.

Other Valuable Information

The blastoid model developed in this study provides an important tool for future research on early embryonic development and implantation, particularly for exploring the mechanisms of reproductive issues in older women. Additionally, this study offers potential targets for developing therapeutic strategies to address fertility problems in older women.