P–547 Single-cell RNA sequencing identifies molecular regulations associated with poor maturation performance on rescue in vitro matured oocytes
Authors: Lee W T , Ng K W , Liao J , Luk A C S , Suen H C , Chan T H T , Cheung M Y , Chu D , Zhao M , Chan Y L , Li T C , Lee T L 
Published: August 2021

Abstract

Study question
What is the transcriptome signature associated with rescuein vitro matured (rIVM) oocytes?

Summary answer
GATA–1/CREB1/WNT signaling axis was repressed in rIVM oocytes of poor quality.

What is known already
rIVM aims to produce mature oocytes (MII) for in vitro fertilization (IVF) through IVM of immature oocytes collected from stimulated ovaries. It is less popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress, and the absence of cumulus cell support in oocytes could account for the failure of rIVM.

Study design, size, duration
We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo (IVO) oocytes (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors, followed by studying the maternal age effect and ovarian responses on rIVM oocyte transcriptomes.

Participants/materials, setting, methods
Human oocytes were collected from donors aged 28–41 years with a body mass index of &lt; 30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analyzed. Selected genes in therIVM vs. IVO comparison were validated by quantitative real-time PCR.

Main results and the role of chance
The transcriptome profiles of rIVM/IVO showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least two-fold change and adjusted p &lt; 0.05) were found to be enriched in metabolic processes, biosynthesis, and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signaling in rIVM when compared with IVO oocytes. We found that estradiol level exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio). rIVM oocytes with higher MII ratio showed over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor outcomes of rIVM, we compared oocytes collected from young donors with high MII ratio versus donors of advanced maternal age and revealed CREB1was an important regulator in rIVM. Our study identified GATA–1/CREB1/WNT signaling was repressed in both rIVM condition and rIVM oocytes of low-quality.

Limitations, reasons for caution
In the rIVM oocytes of high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle so polar body extrusion was the only indicator.
Wider implications of the findings: This study showed that GATA–1/CREB1/WNT signaling and antioxidant actions were repressed in rIVM condition and was further downregulated in rIVM oocytes of low-quality, providing us the foundation of subsequent follow-up research on human subjects.

Trial registration number
Not applicable