The Effect of Seminal Plasma on the Equine Endometrial Transcriptome

The establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the female needs to accept a foreign antigen (the semi-allogenic fetus) while still being able to combat pathogens from the uterus. In the horse, the first uterine exposure to paternal antigens is during mating when sperm is introduced to the tissue and draining lymphatics of the uterus. Additionally, it has been suggested that seminal plasma and its proteins within it play an essential role in preparing the female tract for a suitable immunologic environment but this has not been confirmed in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with seminal plasma or with reduced seminal plasma. We hypothesised that reduced seminal plasma would alter the endometrial transcriptome and affect transcripts relating to immunotolerance, antigen presentation and embryo growth and development. To do so, six (n = 6) mares were inseminated in a randomised switch-back design over the course of four oestrous cycles. Mares were rectally palpated and scanned via ultrasonography for the detection of a pre-ovulatory follicle (>35 mm) alongside increasing uterine oedema and relaxed cervix, and then treated with one of four treatment groups including (1) 30 mL lactated Ringers solution (LRS; NegCon), (2) 500 × 10⁶ spermatozoa in conjunction with 30 mL seminal plasma (SP+), (3) 30 mL lactated Ringers solution (LRS; wash out) and (4) 500 × 10⁶ spermatozoa with seminal plasma reduced via gradient centrifugation and resuspended in 30 mL LRS (SP-). Human chorionic gonadotropin (hCG) was administered to standardise the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilising Trizol, and RNA-Seq was performed by Novogene, with 97.79% total mapping and 40 million read depth. p value was set to <0.05. When comparing SP+ to SP-, 158 differentially expressed genes (DEGs) were identified. Biological processes impacted included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Gene ontology (GO) enrichment analysis using DAVID v6.8 revealed that many of these DEGs were involved in biological process such as antigen presentation (HLA-DM beta chain, HLA-DRB, HLA-DQA and RASGRP1), immune cell signalling (CXCL9, CXCL1, DEFB1 and MIP-2B), embryo growth and development (INHA, KLF2, RDH10, LAMA3 and SLC34A2) and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2 and IGFBP3). Overall, reduction of seminal plasma from the insemination dose impacted the endometrial transcriptome at the time of early embryonic exposure to the uterine environment. Further work is justified to evaluate these alterations impact on embryo maturation, placental development, pregnancy outcome and development of offspring.