Secretory Proteomic Responses of Endometrial Epithelial Cells to Trophoblast-Derived Extracellular Vesicles

Abstract

Synchronized crosstalk between the embryo and endometrium during the periconception period is integral to pregnancy establishment. Increasing evidence suggests that the exchange of extracellular vesicles (EVs) of both embryonic and endometrial origin is a critical component of embryo–maternal communication during peri-implantation. Here, we investigated whether embryonic signals in the form of EVs can modulate the endometrial epithelial cell secretome. Receptive endometrial analog RL95-2 cells were supplemented with trophoblast analog JAr cell-derived EVs, and the secretory protein changes occurring in the RL95-2 cells were analyzed using mass spectrometry. EVs of non-trophoblastic origin (HEK 293 cells) were used as the control EV source to supplement endometrial cells. Trophoblast cell-derived EVs enriched endometrial epithelial cell secretions with proteins that support embryo development, attachment, or implantation, whereas control EVs were unable to induce the same effect. The present study suggests that embryonic signals in the form of EVs may prime receptive endometrial epithelial cells to enrich their secretory proteome with critical proteomic molecules with functional importance for periconception milieu formation.

Do extracellular vesicles have specific target cells?; Extracellular vesicle mediated embryo maternal communication

“…EV isolation and characterization from JAr and HEK 293 cells were carried out as described in our previous publications ( Midekessa et al, 2020 ; Godakumara et al, 2021 ; Muhandiram et al, 2023 ). Briefly, JAr and HEK cells were grown until it reached 80% confluency and the cell culture-conditioned media were replaced with EV-depleted FBS containing media.…”

The evolving roles of extracellular vesicles in embryo-maternal communication

“…Implantation, a pivotal event in early pregnancy, exhibits highly species-specific mechanisms, yet common features exist in successful implantation across species 3 , 4 , 30 – 33 . Notably, bidirectional communication between the embryo and endometrium is a shared characteristic, with emerging evidence implicating EVs in this dynamic process.…”

Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency

“…This reciprocal communication between conceptus and endometrium is now widely believed to be facilitated by paracrine signalling ( Guzeloglu-Kayisli et al, 2009 ; Yockey and Iwasaki, 2018 ). While soluble factors such as cytokines and growth factors have been critical in this interplay, the role of EVs is also starting to come to light ( Godakumara et al, 2021 ; Hart et al, 2023 ; Muhandiram et al, 2023 ). EVs are a heterogeneous group of nano-sized particles (30–5,000 nm in size) secreted by almost all cell types into the extracellular environment.…”

“…The outer most layer of the pre-implanting embryo (blastocyst) consists with a layer of trophectoderm cells (responsible for blastocyst hatching, endometrial attachment and placentation) and the quality of these cells are known to correlate highly with the pregnancy progression in humans ( Garcia-Belda et al, 2024 ). The EVs derived from trophoblast cells have shown to support endometrial receptivity and embryo attachment ( Es-Haghi et al, 2019 ; Godakumara et al, 2021 ; Muhandiram et al, 2023 ). Trophoblastic EVs also play a role in facilitating epithelial-to-mesenchymal transition of endometrial cells and modulating inflammation during the pre-implantation period (by interacting with immune cells to mitigate maternal rejection of the embryo) ( Shi et al, 2021 ).…”

“…However, there are no proper indicators to evaluate the functionality of trophoblastic EVs in embryo implantation. In a previous study, we demonstrated that increased secretion of Milk fat globule-EGF factor 8 protein (MFGE8 or human lactadherin) serves as a cellular response of EECs to trophoblast-derived EVs ( Muhandiram et al, 2023 ), thereby functioning as a marker for EV signalling during embryo implantation. MFGE8 is a glycoprotein rich in cysteine ( Wang, 2014 ).…”