Temporal regulation of uterine luminal extracellular vesicle-associated microRNAs during porcine preimplantation embryonic development

Authors: SCHWEIGER, BRITTANY - University Of Nebraska; VYZOUREK, BENJAMIN - University Of Nebraska; Wijesena, Hiruni; Snider, Alexandria; Rempel, Lea; Miles, Jeremy; PANNIER, ANGELA - University Of Nebraska

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/3/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: During early pregnancy in pigs, developing embryos undergo a rapid morphological transition from spherical to filamentous structures over ~3 days. Elongation is required for successful uterine implantation, however, deficiencies in this process are a major contributor to embryonic loss, resulting in reduced litter sizes and decreased sow productivity. Despite its importance, the mechanisms that initiate elongation remain poorly defined, partly due to the lack of physiologically relevant in vitro systems that replicate the mechanical and molecular features of the uterine environment. Furthermore, most previous studies have focused on the role of soluble factors regulating conceptus development, including cytokines, growth factors, and hormones. Recently, extracellular vesicles (EVs) within the uterine environment have been shown to facilitate the transfer of regulatory cargo, including microRNAs (miRNAs) between maternal and conceptus tissues. However, temporal expression patterns of EV-derived miRNAs and their roles in the biological processes driving conceptus elongation remain incompletely characterized. Therefore, the objective of this study was to characterize the temporal shift in EV-associated miRNAs within porcine uterine luminal fluid (ULF) during elongation and identify predicted gene targets and biological processes. Building upon these findings, ongoing studies are utilizing a three-dimensional alginate hydrogel culture system developed to support porcine embryo survival and initiation of elongation in vitro. Embryos encapsulated within hydrogels can be used to isolate embryo-derived EV-miRNAs directly from conditioned media and compare secretion profiles between embryos that successfully elongate and those that fail to elongate, to better understand mechanisms that drive successful embryonic development, to improve sow reproductive outcomes. Uterine luminal fluid was collected from pregnant gilts on gestational days 8-12 following artificial insemination. Uterine horns were flushed with HEPES-buffered RPMI-1640 medium and recovered embryos were morphologically characterized to confirm gestational grouping. ULF samples were centrifuged to remove cellular debris followed by 0.22 µm filtration to remove apoptotic bodies. EVs were isolated using ultracentrifugation at 32,000 RPM for 120 minutes at 4°C and EV pellets were resuspended in sterile PBS. EV concentration, size distribution, and presence of a lipid membrane were characterized using the Flow NanoAnalyzer (NanoFCM, Nottingham, UK) system. Total RNA was isolated from EVs using an exosomal RNA isolation kit and RNA quality was assessed using the Agilent Tapestation assay. Small RNA libraries were prepared using the NEXTFLEX Small RNA-Seq Kit V4 and sequenced using the Illumina NextSeq 200 platform. Raw sequences were quality filtered, adapter trimmed, and aligned to the Sus scrofa reference genome using miRDeep2 software. Differential expression analysis of identified miRNAs was performed using DESeq2 with a significance threshold of FDR-adjusted p-value (p /= 1). Validated and predicted miRNA-target interactions were identified using miRTarBase and miRNet databases, respectively. Gene ontology enrichment analysis of miRNA targets was performed using the g:Profiler platform with the Sus scrofa reference organism and a g:SCS multiple testing correction statistical significance threshold of 0.05. ULF EVs were successfully isolated from all gestational days and displayed characteristic EV size distributions (~66–71 nm), with high purity of lipid-positive particles (>/= 89%) (Figure 1). EV concentrations and EV-associated miRNA abundance varied temporally across gestation, with progressive reductions from gestational days 8 to 11, followed by a distinct increase on gestational day 12 (Figure 1). Differential express