O-122 Transcriptomic landscape of human oocytes and trophectoderm cells following ultra-rapid or standard vitrification

Abstract

Abstract Study question Can ultra-rapid vitrification and thawing preserve the transcriptomic profiles of oocyte and trophectoderm cells better than standard vitrification procedures? Summary answer Compared to conventional vitrification, ultra-rapid vitrification alters 97.5% fewer genes in oocytes and has a similar magnitude of transcriptomic change in trophectoderm cells. What is known already While traditional vitrification avoids intracellular ice crystal formation compared to cryopreservation techniques, the elevated concentrations of cryoprotectants used may be cytotoxic. Vitrification protocols for human oocytes and embryos have evolved to improve safety and efficiency, however, there remain concerns about the downstream risks of micromanipulation during 8–15 minutes. Ultra-rapid vitrification is a promising approach that minimizes micromanipulations to under two minutes, preserving oocyte viability and embryo quality as confirmed by secretome and metabolic biomarkers. Given the novelty of the technique for human oocytes, there have been no comprehensive analyses of how the transcriptomic landscape is affected. Study design, size, duration This prospective study included 48 donor oocytes collected between January 2022 and November . Sibling oocytes were match-paired for standard or ultra-rapid vitrification and thawing. Surviving oocytes were fertilized by intracytoplasmic sperm injection and the resulting embryos were cultured to the blastocyst stage (day 5/6). Good-quality blastocysts (grade A/B trophectoderm according to ESHRE criteria) were biopsied for transcriptomic analysis. Participants/materials, setting, methods Oocytes and trophectoderm cells from fresh cycles, conventional vitrification, or ultra-rapid vitrification (n = 8 of each cell type/protocol) underwent scRNA-Seq. Once transcriptomic data was pre-processed and visualized, statistical differences in differentially expressed genes (DEGs; |log2FC|>1 and adjusted p-value <0.05) were evaluated using the Shapiro-Wilk test followed by the Student’s t-test (parametric variables) or the Mann-Whitney U-Test (non-parametric variables). The number of cells are limited while RNA library is very complicated and expensive process. Main results and the role of chance scRNA-Seq generated an average of 35 million >Q20 paired-end reads per sample. Oocyte and trophectoderm samples were clearly distinguished by principal component analysis and clustered heatmap analysis. The average Euclidean distance between samples of the same cell type was 160, whereas this distance increases to an average of 570 between samples of different cell types. Differential expression analysis revealed that conventional oocyte vitrification significantly altered the expression of 16,016 genes, resulting in 53.18% upregulated and 46.81% downregulated DEGs with respect to fresh oocytes. Ultra-rapid oocyte vitrification was associated with 97.56% fewer DEGs than the conventional oocyte vitrification group (390 vs. 16,016 DEGs, respectively). Between vitrification protocols there were 8,246 (51.20%) upregulated and 7,877 (48.85%) downregulated DEGs. On the other hand, conventional vitrification was associated with 15,692 DEGs in trophectoderm cells, of which 7,814 were upregulated and 7,878 were downregulated compared to the fresh group. Similarly, ultra-rapid vitrification was associated with 16,010 DEGs, being 7,806 upregulated and 8,204 downregulated compared to the fresh group. Comparing ultra-rapid vitrification and conventional vitrification profiles, 68.16% (623/914) of the DEGs were found to be downregulated. Limitations, reasons for caution Trophectoderm cells appear to be more susceptible to ultra-rapid vitrification-related transcriptomic changes than oocytes. Functional analyses are needed to understand whether these molecular changes can affect cell function and the embryo’s implantation potential. The main limitation is creation of RNA library for more number of cells. Wider implications of the findings This study begins to elucidate how ultra-rapid vitrification affects the transcriptomic landscape of human oocytes and embryos. Coupling the most significant DEGs with clinical data of reproductive outcomes may reveal new biomarkers for oocyte and embryo competence.This is the first transcriptoic study in the field. Trial registration number Yes