O2184: Fetal Growth Restricted Placental Organoid-Derived Extracellular Vesicles Regulate Extravillous Trophoblast Invasion and Gene Expression via microRNA Signaling in an In Vitro model

Background: Fetal growth restriction (FGR) affects 5-10% of pregnancies and accounts for 50% of stillbirths. Impaired placentation, specifically reduced extravillous trophoblast invasion into the maternal uterine decidua, is a hallmark pathologic finding in FGR. Epigenetic regulators, specifically microRNAs (miRNAs), are implicated in FGR through their ability to modulate gene expression post-transcriptionally. These miRNAs can be transported via extracellular vesicles (EVs), facilitating cell-to-cell communication and influencing trophoblast function. Advancements in prenatal therapeutics have been limited by failure of in vivo and in vitro models to recapitulate disease pathology. Placental organoids are a novel tool to study placentation and only recently have we been able to generate them from term placental tissue. Given this innovation, we generated organoids from placentas associated with FGR to study the epigenetic regulation of failed extravillous trophoblast invasion. We hypothesize that EVs released from FGR-derived placental organoids carry miRNAs that impair invasion of extravillous trophoblasts, contributing to the shallow placentation characteristics of FGR.

Methods: Placentas from healthy and FGR-affected pregnancies were collected at birth, villous tissue was dissected and then enzymatically digested and cultured in Matrigel domes. Conditioned media was exchanged every other day to support the formation of multicellular structures, known as placental organoids. Trophoblast organoids were stained for cell surface antibodies to confirm cellular identity.
Culture media extract was collected 48 hours after exchange and EVs were isolated. RNA was extracted from the EVs and sequenced to analyze miRNA content. In addition, EVs were also exposed to cultured extravillous trophoblasts for 48 hours. Intracellular RNA was then sequenced to determine the effect FGR EVs have on molecular pathways critical to trophoblast invasion.
Differential gene expression analysis was performed, and Ingenuity Pathway Analysis (IPA) was conducted to identify significantly up- and downregulated genes in extravillous trophoblasts in response to FGR-derived EVs.

Results: Trophoblast organoids secreted beta-hCG and stained positive for cytokeratin 7 and Syndecan-1, confirming trophoblast identity (Figure 1). Compared to control-derived EVs, FGR-derived EVs had increased miR-141-3p and miR-503-5p expression, two microRNAs that that target genes involved in angiogenesis, Wnt signaling, and extracellular matrix remodeling. Extravillous trophoblasts exposed to EVs derived from FGR organoids exhibited reduced expression of genes associated with invasion, extracellular remodeling, cell communication, and Wnt signaling (Figure2).

Conclusion: Organoids generated from placentas associated with FGR release EVs containing higher levels of miR-141-3p and miR-503-5p compared to health control pregnancies. Furthermore, when these EVs are exposed to extravillous trophoblasts, they downregulate gene expression of key molecular pathways critical to trophoblast invasion. Further studies will investigate the role of miR-141-3p and miR-503-5p contributing to upstream regulation of these gene expression pathways. Term placental organoids are a novel tool to study epigenetic regulation of failed trophoblast invasion associated with FGR.