P-266 Study on the mechanisms of ovol1/2 during the morula-to-blastocyst transition
Authors: Zhu Y , Xueqin C , Zhiqi Z , Qinwei Z , Lizhi L , Lin G 
Published: June 2023

Abstract

Study question
Screen the key gene groups that determine the development of blastocyst. Explore the effect of ovol1/2 gene on early embryonic development.

Summary answer
We founded that there were insufficient activation of the Major ZGA genes in the AE group,and focused on candidate gene ovol1/2.

What is known already
At present, the clinical success rate of assisted reproductive technology (ART) is less than 50%. A large number of embryos cultured in vitro can not develop to blastocyst, and there are cleavage or morula stage arrest. The developmental arrest of preimplantation embryos is an important reason for treatment failure of infertile patients. However, the cause of early embryonic development arrest is not clear, and there is also a lack of effective methods to improve embryo quality.

Study design, size, duration
In this study, fertilized embryos were obtained through ICSI and a single cell from eight-cell embryo was biopsied through micromanipulation for single-cell RNA sequencing (scRNA-seq). Subsequently, we tracked the developmental potential of remaining cells and divided them into high-quality blastocyst(Bla) group and high-quality eight-cell failed blastocyst formation(arrest embryo, AE) group. In addition,we employed a RNAi approach to study the function of candidate gene ovol1/2, by injecting ovol1/2-targeting small interfering RNAs (siovol1/2) into wild-type (WT) zygotes.

Participants/materials, setting, methods
The study collected 21 human blastocysts and 5 arrest embryos. Human immature oocytes from ICSI treatments were clinically discarded and donated by women after signing informed consent by donors. C57B6 background mouse strains were used in this study. Mice were maintained under specific pathogen free conditions in a controlled environment of 20–22 °C,with a 12/12 h light/dark cycle, 50–70% humidity, and food and water provided.

Main results and the role of chance
The results showed that the gene expression of embryos in AE group was significantly different from that of Bla group: 79.5% of differential expression genes were down-regulated, including 48.3% of the Major ZGA gene. Then we focused on the candidate gene ovol1/2. When ovol1/2 were knocked down alone, there was no significant difference in the development rate of embryos from 2-cell to blastocyst between knockdown group and normal control group. When ovol1/2 were knocked down at the same time, there was no significant difference in the development rate of embryos among three groups before the morula stage. But in the morula developed to the blastocyst stage, there were significant differences among three groups of embryos: about 73% of embryos in the normal control group formed blastocyst; about 39% of the embryos in the low-dose knockdown group; the high-dose knockdown group embryos were all arrested in the morula stage. Further investigation revealed that the number of inner cell mass(ICM) cells in low-dose knockdown group did not change significantly, while the number of trophoblastic ectoderm(TE) cells significantly decreased. In addition, the TE functions of low-dose knockdown group such as blastocyst cavity size, paved area, hatching and adhesion rate were damaged.

Limitations, reasons for caution
Further research of the mechanisms is needed.

Wider implications of the findings
This is significant to understand molecular mechanisms of early embryonic development, and will provide important theoretical basis for clinical diagnosis and intervention of embryonic development arrest. Our study provides clues for the causes of abnormal blastocyst formation and new ideas for possible clinical improvements in the success rate of ART.

Trial registration number
not applicable