Reproductive endocrinologist Irene Su and her collaborator Sheng Zhong, a biostatistician at the University of California, San Diego, routinely perform liquid biopsies on recovered cancer patients to track cancer regression. In 2019, using minuscule amounts of serum, they identified specific signatures that could identify patients with or without recurrent breast cancer. A few years later, they realized that they could try a similar method to distinguish viable human embryos from abnormal ones after in vitro fertilization (IVF). 

In a recent study published in Cell Genomics, their research team reported a non-invasive method for inspecting human embryo quality using up to 40 µl of embryo culture medium (2). Their technique complements existing technologies for assessing embryo quality and may one day improve the predictions that an embryo will result in a live birth. “Being able to make meaningful measurements from a small amount of input material opens the door for better diagnostics for IVF in the future,” said Rajiv McCoy a biologist at Johns Hopkins University who was not involved in the study. 

Currently, doctors rely on embryo morphology assessments and preimplantation genetic testing to predict which embryos are most likely to develop to term, but these methods are imperfect. “The live birth rate, even after preimplantation genetic testing, is somewhere between 70-75 percent,” said Su. “That's awesome, but we can’t explain why we don’t have a live birth in 25-30 percent of cases.” This uncertainty raises the question of whether analyzing spent media, which is typically discarded, could provide additional insights about embryo development.

As human IVF embryos develop in culture, they release part of their RNA into the extracellular space. The researchers wanted to identify specific extracellular RNA signatures that correlated with known measures of human embryo quality. They began by collecting small media samples from the cultured IVF embryos of 29 participants and performed extracellular RNA sequencing to construct sequencing libraries. They detected around 4,000 RNA molecules at each stage of embryonic development, including the oocyte, zygote, 8-cell, morula, and blastocyst stages.

To correlate extracellular RNA profiles with embryo quality, an embryologist assessed IVF embryos at 120- and 160-hours post-fertilization and categorized them as either developing normally or developmentally arrested. The team then analyzed the culture media from these and found that the developmentally arrested embryos had a distinct extracellular RNA profile characterized by lower overall extracellular RNA levels, but higher levels of specific genes known to negatively regulate cell-cycle progression. 

The live birth rate, even after preimplantation genetic testing, is somewhere between 70- 75 percent. That's awesome, but we can’t explain why we don’t have a live birth in 25-35 percent of cases. 
– Irene Su, University of California, San Diego

Lastly, an embryologist graded embryos based on their morphology, with blastocysts receiving ratings of good, fair, or poor. Initial analysis showed that no single gene could predict embryo quality when considered independently, but a machine-learning model showed a strong correlation between predicted and observed quality scores and identified genes associated with embryo quality. These genes were related to GTPase activity regulation and spindle structure, which are both important for asymmetric cell division.

The researchers used spent media from cocultured embryos, which is considered best practice in IVF. This prevented the association of specific findings with individual embryos. “The practical value of this study is limited at this time, but it's a nice proof of principle that you can get RNA sequencing data out of a small amount of material, and that it's correlated with measurements of embryo quality,” said McCoy.

In future experiments, Su and Zhong plan to correlate the extracellular RNA signatures with preimplantation genetic testing results and test whether this information adds to live birth predictions. “This study draws attention to the idea of coming up with noninvasive ways to improve predictions about pregnancy outcomes,” said McCoy. “It’s a small step toward that goal.”

References

1. Zhou, Z. et al. Extracellular RNA in a single droplet of human serum reflects physiologic and disease states. Proc Natl Acad Sci  116, 19200–19208 (2019).
2. Wu, Q. et al. A temporal extracellular transcriptome atlas of human pre-implantation development. Cell Genomics  4, 100464 (2024).