O-232 Extracellular vesicles secreted by the maternal endometrium functionally regulate processes related to embryo development and implantation in human blastocysts
Authors: Segura-Benitez M , Carbajo-Garcia M C , Quiñonero A , De M J , Santos Los , Pellicer A , Ferrero H 
Published: June 2023

Abstract

Study question
What is the response of human embryos in a transcriptomic level to the uptake of extracellular vesicles (EVs) secreted by the human endometrium?

Summary answer
EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation.

What is known already
Communication between the maternal endometrium and the embryo is essential for a successful implantation, and EV role in this cross-talk has been recently established. Results of our previous experiments showed that endometrial EVs carry miRNAs and proteins related to endometrial receptivity, embryo implantation process and early embryo development, and that they can be uptaken by human blastocysts. However, there are no studies which address the transcriptomic response of human embryos to the uptake of these EVs, which would demonstrate the functional relevance of this communication system.

Study design, size, duration
Endometrial biopsies were collected from oocyte donors (n = 20) with confirmed fertility, on oocyte retrieval day, and primary human endometrial epithelial cells (pHEECs) were isolated and in vitro cultured. Hormonal treatment was added to mimic secretory phase of menstrual cycle. Conditioned medium was collected and EVs were isolated and characterized. Day 5 human blastocysts (n = 24) were divided into two groups (n = 12/group) and cultured in vitro with or without these EVs. RNA-sequencing of these embryos was performed.

Participants/materials, setting, methods
Conditioned media from pHEECs was pooled into 4 replicates (n = 5 biopsies/replicate) and EVs were isolated by ultracentrifugation. Characterization was performed by western blot, nanoparticle tracking analysis and transmission electron microscopy (TEM). Human blastocysts were devitrified and cultured in vitro for 24h with or without EVs previously isolated. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (FDR&lt;0.05). QIAGEN Ingenuity Pathway Analysis (IPA) was used to perform the functional enrichment analysis.

Main results and the role of chance
EVs presented a size range within 100-300nm, and expression of EV protein markers HSP70, TSG101, CD9 and CD81. EV morphology was corroborated by TEM. RNA-sequencing identified 26610 genes, being significant 519 upregulated and 395 downregulated in EV-embryos compared with control. Among top20 most significant upregulated genes, CSF1R (log2FoldChange [logFC]=3.36) has been associated with formation of blastocyst cavity, and PLOD2 (logFC=2) deficiency leads to early embryonic lethality. Genes related to antioxidant defense, as PDK1 (logFC=2.82), or vesicle trafficking, as SEC24D (logFC=2.17), were also upregulated. Regarding top20 downregulated genes, DAB2IP (logFC=-1.19) negative regulation was found beneficial in early pregnancy, and DUSP6 (logFC=-2.07) was associated with cell differentiation and pluripotency. Functional analysis demonstrated positive regulation of biological processes related to embryonic development, cellular invasion and migration, cell cycle, cellular organization, and cell viability in EV-embryos. Contrary, there was a negative regulation of processes related to cell death, apoptosis, and DNA fragmentation. Some pathways upregulated in EV-embryos were NANOG role in embryonic pluripotency (z-score [z]=1.89), unfolded protein response (z = 3.05), involved in cellular homeostasis, VEGF signaling (z = 1.67), that regulates trophoblast adaptation to hypoxia, and integrin signaling (z = 3.36), which regulates trophoblast adhesion and differentiation; PTEN signaling (z=-1.73) was inhibited in EV-embryos, suppression of which prevents apoptosis.

Limitations, reasons for caution
This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment.

Wider implications of the findings
EVs secreted by the maternal endometrium and uptaken by human blastocysts regulate processes related to embryo development and implantation, suggesting the functional relevance of this endometrial-embryo crosstalk during the implantation process. This study opens further research insight to define potential targets of implantation success and embryo competence.

Trial registration number
Not applicable