Oral Presentation ESA-SRB-ANZOS 2025 in conjunction with ENSA

Seminal extracellular vesicle mediated nfκb signalling in the female reproductive tract (128667)

Pipper Miller 1 2 , Shanmathi Parameswaran 1 2 , Cottrell T Tamessar 1 2 3 , Elizabeth Torres-Arce 1 2 , Judith Weidenhofer 4 5 , Huiming Zhang 6 , Elizabeth G Bromfield 1 2 7 , Sarah Robertson 8 , Brett Nixon 1 2 , David Sharkey 8 , John E Schjenken 1 2
  1. Infertility and Reproduction Research Program. Hunter Medical Research Institute, Hunter Medical Research Institute and School of Environmental and Life Sciences. University of Newcastle, Australia, New Lambton Heights, New South Wales, Australia
  2. Centre for Reproductive Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, Australia
  3. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde, Sweden
  4. School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia
  5. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  6. Central Analytical Facility, Research and Innovation Division, University of Newcastle, Callaghan, NSW, Australia
  7. Bio21 Institute, School of BioSciences, University of Melbourne, Melbourne , VIC, Australia
  8. The Robinson Research Institute and School of Biomedicine, University of Adelaide, Adelaide, SA, Australia

Seminal fluid exposure at coitus activates immune responses in the epithelial cells lining the female reproductive tract, promoting molecular and cellular changes that support successful pregnancy. In addition to soluble signalling mediators present in the fluid portion of the ejaculate, the seminal plasma, and seminal extracellular vesicles (SEVs) may also contribute. However, the identity of SEV signalling mediators remains unknown. In this study, we used bioinformatics analysis of transcriptomic data from SEV co-cultured with ectocervical cells (Ect1 cells) to predict SEV signalling mediators. Immunoblotting and qPCR were then employed to provide evidence of the role of these mediators in SEV signalling. Prominent among the upstream regulators predicted by bioinformatics analysis was the Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) complex (Z-score=5.78, p<0.05) with other NFκB associated molecules including Interleukin-1A (Z-score=5.14, p<0.05), RELA proto-oncogene, NF-kB subunit (Z-score=4.79, p<0.05), Toll-Like Receptor 4 (Z-score=4.61, p<0.05) and Tumour Necrosis Factor (TNF, Z-score=7.15, p<0.05). Immunoblotting further characterised NFkB complex molecules in SEVs showing the presence of NFκB inhibitor epsilon (NFκBIE) and nuclear factor kappa B subunit 2 (NFκB2). However, these subunits were also expressed in resting Ect1 cells, and both NFκB2 and NFκBIE were upregulated at the gene expression level following SEV co-incubation, compared to untreated media alone controls. These findings suggest NFκB signalling is activated in female reproductive tract cervical epithelial cells in response to SEV exposure. We are currently exploring whether paternal SEV-derived NFκB signals are driving these inflammatory gene expression changes or whether other SEV signals may be responsible. Overall, this suggest that SEVs may deliver specific NFκB mediators to epithelial cells in the female reproductive tract, potentially priming the local immune environment to support a healthy pregnancy.