Ram sperm and seminal plasma alters immune transcriptome and cytokine secretion in ovine cervical explants ex vivo

The ovine cervix presents a unique barrier to frozen-thawed ram spermatozoa, reducing pregnancy following cervical artificial insemination (AI). This is hypothesised to be related to a modified sperm molecular profile following seminal plasma (SP) exposure and cryopreservation, altering communication with the female environment. However, the impact of different sperm types on the cervix itself has never been explored, in part due to the difficulty of profiling these in vivo interactions. Here, an ex vivo cell culture model was used to explore changes in the transcriptome of cervical explants following exposure to spermatozoa and SP. Explants were collected from Merino ewes ( n =6) post-mortem and cultured with epididymal, epididymal + SP, frozen and frozen + SP ram spermatozoa, SP only or a media control for 6 hours. Gene expression was profiled by RNA sequencing. Multiplex ELISAs quantified the abundance of cytokines in culture supernatant. Cytokeratin and H&E staining confirmed the presence of cervical epithelial cells in cultured explants. The presence of epididymal sperm caused the differential expression of 18 genes compared to the media only control, while the addition of SP to epididymal sperm altered 781 genes. Differentially expressed genes were involved in pathways related to inflammation, signalling and ATP synthesis. SP exposure to frozen sperm led to significantly increased IL-1a and reduced TGF-b production compared to other treatments. Results show an altered cervical immune response in the presence of ram SP and sperm. Further research is required to determine molecular candidates which facilitate immune tolerance and sperm transit through the cervix. IN BRIEF Cervical explants alter gene expression and cytokine secretion in response to seminal plasma compared to spermatozoa alone. Results validate model to study mechanisms behind sperm-immune tolerance in the ovine cervix ex vivo.