Endometrial and uterine extracellular vesicles gene expression on day 8 after ovulation in pregnant and nonpregnant mares susceptible or resistant to persistent mating-induced endometritis

Persistent breeding-induced endometritis (PBIE) is the leading cause of subfertility in mares. So far, it is not clear at what time after insemination PBIE is interfering with establishment of pregnancy. Therefore, the first objective of this study was to determine if there is a differential response to the presence of a Day 8 embryo. Furthermore, we analyzed if mares susceptible to PBIE showed differences in endometrial gene expression and in uterine extracellular vesicles (uEVs) RNA cargo on Day 8 post-ovulation (p.o.). First, 20 mares were screened for susceptibility to PBIE and classified as susceptible (n=6) or resistant (n=6) to PBIE or intermediate (n=8). Selected mares (n=12) were inseminated with sperm from one fertile stallion. On Day 8 p.o., low-volume uterine lavage, followed by embryo flushing and collection of endometrial biopsies was performed. Embryos were collected from two susceptible and three resistant mares (Day 8 pregnant, n=5). In 7 mares, no embryo could be found (Day 8 nonpregnant, n=7). Endometrial luminal (LE) and glandular epithelium (GE) were isolated by laser capture microdissection and analyzed by low-input RNA-sequencing. Uterine EVs were isolated from uterine lavage, and their RNA content was analyzed by total RNA-seq. The comparison between Day 8 pregnant and nonpregnant mares revealed 305 differentially expressed genes (DEGs) for LE (FDR 5%) and 50 for GE (FDR 20%, P<0.001), indicating that the main response is present in the LE. The analysis of uEVs revealed 442 differentially abundant (DA) RNAs (FDR 5%) between Day 8 pregnant and nonpregnant mares. At the miRNA level, miR-500-5p and miR-1246 were found in higher concentrations in Day 8 pregnant mares. Furthermore, the comparison between susceptible and resistant mares resulted in 80 DEGs for GE (FDR 20%, P<0.001) and 65 for LE (FDR 28%, P<0.001). Functional annotation analysis revealed a number of genes known as involved in establishment of pregnancy. The analysis of uEVs revealed 7 DA RNAs (FDR 20%) and one miRNA (eca-miR-181a-5p) between susceptible and resistant mares. In conclusion, this is the first study showing a transcriptional response to the presence of an embryo on Day 8 p.o. in the endometrium and uEVs cargo and revealing differences between mares susceptible or resistant to PBIE.