P–549 What trophectoderm cells from mosaic embryos tell us about embryonic competence at the transcriptional level
Authors: Martin A , Mercader A , Insua F , Escrich L , Grau N , Tejera A , Mifsud A , Pellicer A , lo. Santos M.J. D 
Published: August 2021

Abstract

Study question
Does transcriptome of remaining trophectoderm (TE) reflect the developmental potential of mosaic blastocysts after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer: TE from low-degree mosaic (Low-mos) and high-degree mosaic (High-mos) blastocysts are transcriptionally equivalent, standing between euploid and aneuploid categories and displaying key deregulated developmental processes.

What is known already
Blastocysts classified as mosaic by PGT-A are associated with lower implantation and higher miscarriage rates than those classified as euploid, yet they still lead to healthy babies. Unveiling the true developmental identity of these embryos faces a dilemma: understanding to which extent they represent technical artefacts or whether they hold own potential to implant and give rise to normal pregnancies. Current RNA sequencing (RNA-seq) techniques allow for the determination of whole transcriptomic profiles even from single cells, which paves the way for the identification of new molecular keys of embryonic competence.

Study design, size, duration
Prospective study comparing RNA-seq data of remaining TE from blastocysts classified as euploid (n = 4), Low-mos (n = 5), High-mos (n = 4) and aneuploid (n = 6) by PGT-A. Participants were recruited between October 2018 and November 2019 at IVI-RMA Valencia.

Participants/materials, setting, methods
Chromosomal mosaicism was defined in the range 30%-&lt;50% (Low-mos) and 50%-&lt;70% (High-mos) using a next-generation sequencing (NGS) validated algorithm. Whole TE fractions were separately collected and processed for RNA-seq. Differentially expressed genes (DEGs) were calculated with DESeq2 package [Benjamini-Hochberg (BH)-adjusted p &lt; 0.01 & abs(log2FoldChange)&gt;2 significant]. Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms (BH-adjusted p &lt; 0.01 significant).

Main results and the role of chance
For comparisons, TE from euploid blastocysts were used as control. At the gene level, 15 DEGs were found in Low-mos, 20 DEGs in High-mos, and 64 DEGs in aneuploid blastocysts. To address the functional implications of these differences, pathways significantly deregulated according to KEGG and GO categories were identified. TE from aneuploid blastocysts displayed significant downregulation in up to 115 KEGG and GO processes directly involved in processing and integrity maintenance of nuclear and mitochondrial genomes, a reflection of their aberrant chromosomal identity. In addition, TE from High-mos and Low-mos were transcriptionally equivalent (0 DEGs between both groups), with 23 overlapping KEGG and GO processes significantly downregulated compared with control. Importantly, main significantly-affected processes included mitotic sister chromatid segregation, NIK NF-kB activity, regulation of apoptosis, and pathways related to the biosynthesis and metabolism of proteins, fatty acids, carbohydrates and steroid hormones. These findings indicate that mosaic embryos comprise a unique developmental entity, which swims between the euploid and aneuploid waterfronts and may regulate survival by diverse mechanisms, including cell proliferation and apoptosis.

Limitations, reasons for caution
This is a descriptive, single-center study with limited sample size. TE fractions were obtained by micromanipulation, which may have led to potential cross-contamination with the inner cell mass.
Wider implications of the findings: Transcriptomic equivalence between Low-mos and High-mos TE fractions questions the biological significance of inferring mosaicism degrees from single biopsies. Deregulated processes in these embryos support their reduced developmental and live birth potential, pointing to mechanisms that may mediate survival in the presence of aneuploid cells, as shown in the mouse.

Trial registration number
Not applicable