Impact of seminal plasma on the equine endometrial transcriptome

Establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the mare accepts the semi-allogenic fetus while still being capable of combating uterine pathogens. The first uterine exposure to paternal antigens is during mating when the endometrium and draining lymphatics are introduced to semen. Seminal plasma (SP) plays an important role in preparing the female tract for a suitable immunologic environment in other species. The role of SP has been determined in the immediate inflammatory response to breeding, but its impact on the endometrium at the initial time of exposure to an embryo has not been investigated in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with or without SP. We hypothesized that removal of SP would affect transcripts relating to immunotolerance, antigen presentation, and embryo growth and development. Six (n=6) mares were inseminated over four estrous cycles. Upon the detection of a pre-ovulatory follicle (>35mm), mares were assigned to one of three treatment groups: 1) uterine infusion with 30mL lactated Ringer's solution (LRS; negative control), 2) AI with 500 × 106 progressively motile sperm (PMS) in conjunction with 30mL SP (SP+), and 3) AI with 500 × 106 PMS without SP (reduced via gradient centrifugation), and resuspended in 30mL LRS (SP-). The order of SP+ and SP- wasrandomly assigned in a switch-back design, and separated by a washout cycle in which mares received a uterine infusion with 30mL LRS. hCG was administered to standardize the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilizing Trizol, and RNASeq was performed using Illumina NovaSeq6000, with 97.79% Total Mapping and 40 million read depth. The False Discovery Rate (FDR) was set to <0.05. In total 2100 genes were differentially expressed between all groups. When comparing the SP+ and SP- groups, 241 differentially expressed genes (DEGs) were identified. Biological processes that were impacted by the presence of SP included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Differentially expressed genes associated with these pathways included multiple genes relating to antigen presentation (HLA-DM Beta Chain, HLA-DRB, HLA-DQA, RASGRP1), immune cell signaling (CXCL9, CXCL1, B-defensin, MIP-2B), embryo growth and development (Activin, KLF2, RDH10, LAMA3, SLC34A2), and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2, IGFBP3). In conclusion, the presence of SP had a significant effect on the endometrial transcriptome at the time when an embryo first is exposed to the uterine environment. Further research will evaluate the impact of these alterations on embryo maturation, placental development, and pregnancy outcome.