Embryo implantation in the mare occurs just over one month after fertilization, coinciding with the production of chorionic gonadotropin. The factors that regulate this late implantation in the mare, and whether they are unique to horses or shared with more invasive embryo implantation in other species, remain poorly understood. This study aimed to determine and compare the transcriptome and subpopulations of endometrial cells before and after embryo implantation in the horse. Single-cell RNA sequencing was used to characterize the transcriptome of nearly 97,000 endometrial cells collected from biopsies of the endometrium at the beginning (day 33 of gestation) and after embryo implantation (day 42 of gestation) in mares. Sixteen immune and 24 non-immune cell clusters were identified, representing known major cell populations as well as novel subpopulations of horse immune cells such as resident innate lymphoid cells and mucosal-associated invariant T cells. Contrary to current knowledge, endometrial natural killer (eNK) cells were the most abundant endometrial leukocyte population during implantation in horses. Moreover, eNK cells not only expressed genes that may interact with fetal MHC I, such as LY49F, but also exert immunoregulatory functions independent of MHC I expression, such as CD96/TIGIT. Analogous to other species studied, upregulation of CXCR4 was found in several subpopulations of immune cells. Our results suggest that despite distinctive and later placentation compared with humans, horses share some key similarities in the mechanisms of embryo implantation.

Summary Sentence

Endometrial NK cells are the most abundant and antigen-presenting cells are the most responsive populations of immune cells in the equine endometrium across implantation. Additional key contributor cells include other innate lymphoid and mucosal-associated invariant T cells, which were defined for the first time in the horse.

Graphical Abstract