P-780 Oocyte proteome and cumulus cell transcriptome analysis reveal oocytes live in a hypoxic microenvironment underlie the arrest of human early embryos in vitro culture
Authors: Zhang J , Zhang N , Lin G 
Published: June 2023

Abstract

Study question
Can cumulus cell transcriptome analysis combined with oocyte single-cell proteomics uncover the cause of human early embryos arrest during assisted reproductive treatment?

Summary answer
Oocytes living in a hypoxic microenvironment underlie the arrest of human early embryos at cleavage stage in vitro culture.

What is known already
Preimplantation embryo developmental potential largely depends on gamete quality in assisted reproduction treatment. Human embryonic genome major activation has occurred by the eight-cell stage, up to 3 days after fertilization. Therefore, to a large extent, the development competence of embryo at cleavage stage relies on oocytes quality, which acquire during follicle development. Cumulus cell (CC) plays an essential role in transmitting signals within the ovary and supporting oocyte growth and maturation. We used whole transcriptome analysis of cumulus cells (CC) and single-cell proteomics of oocyte to gain insights into the molecular mechanisms of embryo development arrest at cleavage stage.

Study design, size, duration
Fifty CCs samples and eleven oocyte samples were obtained from fifty-eight couples who underwent IVF/intracytoplasmic sperm injection (ICSI) treatment. The total CCs average age was 30.18±4.51(control group) and 32.59±3.43 (DA group). The total oocyte average age was 33.13±4.83. Donated CCs were recruited between January 2018 and December 2019. Donated oocytes were recruited between September 2022 and October 2022.

Participants/materials, setting, methods
CCs samples were obtained from 33 patients whose rate of good-quality embryos is above 90% (as control) and 17 patients with more than 90% embryos fail to reach six-cell stage (Grade II) on day 3 (as development arrest, DA). Oocytes at GV and MI stage respectively were collected for proteomics. Differentially expressed genes were analyzed using the R package DESeq2 or DEP (filtered with q-value≤0.05 and Foldchange≥1.5). EGSEA package was used to perform pathway analysis.

Main results and the role of chance
RNA sequencing identified 18458 genes expressed in CCs of both groups, 31 of which were differentially expressed. CCs from patients whose embryos development arrested, contains 36 differentially expressed genes (DEGs) up-regulated and 4 DEGs down regulated. Gene ontology showed that up-regulated genes were significantly enriched with annotations related to oxidoreductase activity, mitochondrial respiratory chain complex IV and response to hypoxia. TF enrichment analysis found out that up-regulated DEGs was enriched in hypoxia-inducible factor 1 (HIF-1) induced transcription. It has been reported that HIF-1-mediated gene expression can be elicited by hypoxia, which could restrict mitochondrial biogenesis and ATP-dependent cellular processes. So, we supposed that oocytes live in an ultra-hypoxic microenvironment underlie human early embryos arrest. In consideration of the difficulty in detection of the oxygen level in antral follicles cavity, the protein level changes of oocytes in DA group were used to reflect oocyte status. There were 47 proteins up-regulated and 105 proteins down-regulated at GV stage; 55 gene up-regulated and 48 genes down-regulated at MI stage. Among these, overlap with up-regulated genes in both stages included tuberous sclerosis complex1(TSC1) and Neutral amino acid transporter A (ASCT1), which play key roles in cellular response to oxygen-glucose and hypoxia stress response.

Limitations, reasons for caution
Because of the lack of credible data reflecting accurately the dissolved O2 concentration adjacent to an oocyte. Determination of dissolved O2 within antral follicles is fraught with technical difficulties. We can just verify our hypothesis by single-cell proteomics of oocyte and Ultra-Low-oxygen treatment in vitro.

Wider implications of the findings
We provide a new hypothesis that hypoxic follicular harmed oocyte quality, causing human early embryos arrest. The DEGs we detected in CCs and oocyte could be the biomarker candidates for embryo developmental potential. Ovarian stimulation protocols for embryo development arrested patients could consider diminishing oocyte exposure time in hypoxic follicles.

Trial registration number
Not applicable