As well as delivering sperm to fertilise oocytes, seminal fluid induces molecular changes in the female reproductive tract to promote reproductive success. Transforming growth factor-β and other factors in seminal fluid have been identified as signalling agents but do not fully explain the female response. Seminal fluid extracellular vesicles (SFEVs) likely have signalling potential given their suggested roles in immune regulation. This is yet to be assessed in humans.
Aim: To investigate the impact of SFEVs on female reproductive tract signalling using a well-established human ectocervical epithelial (Ect1) cell culture model.
Methods: Human SFEVs were isolated from seminal fluid using sequential density-gradient ultracentrifugation and characterised via immunoblotting, nanoparticle tracking analysis, and electron microscopy. Ect1:SFEV interactions were evaluated by immunofluorescence with biotin-labelled SFEVs (n=3). Gene expression in Ect1 cells 8h after incubation with SFEVs was analysed by transcriptomics (n=4) and qPCR (n=8). Transcriptomic data were analysed using Ingenuity Pathway Analysis.
Results: SFEVs rapidly docked with Ect1 cells and deposit biotinylated cargo within 5-minutes post-incubation. Following 8h co-incubation, SFEVs altered gene expression profiles in Ect1 cells (>1.5FC, 216 genes induced, 211 suppressed, FDR<0.05). Notably, cytokines including IL1A (p≤0.01, 2.6FC) and IL6 (p≤0.01, 2.8FC) were confirmed by qPCR as being induced by SFEVs. Bioinformatic analysis revealed that 55% of activated pathways were immune signalling pathways and identified epidermal growth factor (Z-score=4), and NFkB (Z-score=5.78) as key components of the SFEV cargo putatively responsible for regulating these responses.
Conclusion: These studies provide evidence that SFEVs regulate the female immune environment, thereby promoting reproductive success.