P–189 The transcriptional profile of arrested cleavage stage human embryos
Authors: Pérez M , Domínguez F , Quiñonero A , Beltrán D , Arantza D , Mercader A , Martín Á , Pellicer A , Santos M.J. D. lo 
Published: August 2021

Abstract

Study question
What are the molecular pathways overactivated in arrested cleavage stage human embryos?

Summary answer
There is an upregulation of mitochondrial activity and cellular stress-related pathways in arrested cleavage embryos, which is in agreement with the “quiet hypothesis”.

What is known already
mtDNA content decreases during embryo development, however there is a high increase in mtDNA content in arrested cleavage embryos that may correspond to a response to intrinsic or extrinsic factor creating stress. This reasoning would be in agreement with stablished hypothesis showing that a basal metabolism with a moderate-low energy consumption is actually a sign of embryo health, so a viable embryo does not need to use an extra energy to accommodate to the environment (Leese, 2012). The study of the transcriptional profile during human embryo development will give further information about key molecular process involved in in-vitro embryonic competence.

Study design, size, duration
A prospective cohort study was performed with 11 MII oocytes (average age= 22,9 years), 10 non-arrested cleavage embryos (average age= 29,9 years), 5 arrested cleavage embryos (average age= 38,8 years) and 8 blastocysts (average age= 39,1 years). All specimens were warmed and sampled in PCR tubes with 2 μl of suitable reaction buffer for the RNA sequencing protocol.

Participants/materials, setting, methods
Specimens were analyzed by single-cell RNA sequencing (scRNA-seq). Correlation studies, principal component and differential expression analysis were performed with DESeq2 package. Differential gene expression analyses were done using the parametric Wald test, with Benjamini-Hochberg multiple test correction (padj). Finally, Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene ontology (GO) terms.

Main results and the role of chance
We do not observe mitochondria-related activity pathways significantly (P&gt;.05) deregulated between MII oocytes and non-arrested cleavage embryos considering GO and KEGG categories.
When comparing non-arrested cleavage embryos versus blastocysts, we observe several ATP production/consumption and cristae formation-related pathways significantly (P&lt;.05) upregulated in blastocysts compared to non-arrested cleavage embryos considering GO and KEGG categories. This change in activity coincides with the metabolic activation event that occurs in the blastocyst stage.
However, when we analyze arrested cleavage embryos versus non-arrested cleavage embryos, we observe several ATP production related-pathways and mitochondria-related apoptosis pathways significantly (P&lt;.05) upregulated in arrested cleavage embryos compared to non-arrested cleavage embryos considering GO categories. With KEGG categories, we notice a significant (P&lt;.05) upregulation of oxidative phosphorylation in arrested cleavage embryos. On the other hand, when we analyze the differences between arrested cleavage embryos and blastocysts, taking into account the differences related to the change of embryo stage, we do not observe ATP production or consumption-related pathways significantly (P&gt;.05) deregulated considering GO and KEGG categories. Then, human arrested embryos in parallel with the increase in mtDNA content, display an upregulation of mitochondrial activity and cellular stress which is in line with the expected overactive metabolism of non-viable embryos.

Limitations, reasons for caution
All analyzed blastocyst were aneuploid, so we are unable to determine what the results would be with euploid blastocysts. Also, although age can it be ruled out, no differences were observed between mean age from cleavage stage embryos (arrested and non-arrested ones) and blastocyst.
Wider implications of the findings: This study evidences the relation between extreme mtDNA content increase and the identification of the pathways involved in active metabolism and apoptosis in arrested cleavage stage human embryos.

Trial registration number
Not applicable