Porcine Endometrium Responds to Conceptus IFNG and Transitions from a Nidatory Inflammatory State to Anti-Inflammatory State between Days 15 and 20 of the Peri-Implantation Period of Pregnancy

Alexandria Ross¹; Joe W. Cain¹; Fuller W. Bazer²; Gregory A. Johnson¹; Gunter P. Wagner³

¹Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843;

²Department of Animal Science, Texas A&M University, College Station, TX 77843;

³Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520

Abstract Text: Pigs have a true epitheliochorial placenta as the conceptus (embryo and associated placental membranes) trophectoderm (Tr) attaches to the endometrial luminal epithelium (LE) but does not invade. Despite the lack of invasion, there is an inflammatory reaction in the endometrium during the early stages of conceptus attachment, yet there is no pregnancy loss. The Tr cells secrete the pro-inflammatory cytokine, interferon gamma (IFNG), during the period of conceptus elongation, with maximum secretion on Day 15 of pregnancy and recruitment of T cells to the endometrium. Paradoxically, the presence of IFNG null conceptuses leads to uncontrolled endometrial inflammation and pregnancy failure. While an initial inflammatory response seems to be essential to successful attachment, we propose that an inflammatory resolution response is necessary for continuation of gestation. To characterize immune cells within the endometrium, gilts were mated to boars with proven fertility and hysterectomized on either Day 13, 15, 17, 20, or 25 of gestation. Using bulk RNAseq, 35,670 genes were mapped and 15,440 subjected to Spearman Correlation Analysis. Gene expression grouped similarly for endometria from Days 13 and 15 (initial attachment for implantation), and for endometria from Days 20 and 25 (initial placentation). Expression from Day 17 endometria grouped with either group, however there was a clear difference between the groups illustrating a transition in gene expression between Days 15 and 20. From RNA sequencing data of the maternal endometrium, mRNA expression of CD80 and TNF, markers for pro-inflammatory M1 macrophages, was not significantly affected by day of gestation, however, expression of CD86, IL10, and TNF mRNAs, markers for M2b macrophages, increased (p < 0.05) by Day 20 of gestation, and then decreased by Day 25. From data for the M2c macrophage markers, CD206 mRNA expression increased (p < 0.05) between Days 15 and 25 of gestation, CD163 mRNA expression increased between Days 20 and 25, while mRNA expression of toll-like receptor (TLR) 1 increased on Day 20 and decreased by Day 25. Whole uterine sections were collected from gilts on Days 13, 15, 20, and 25 of pregnancy, corresponding to RNA sequencing data. Using a myeloperoxidase (MPO) antibody, neutrophils were localized to the endometrial stroma and vasculature across all gestational days studied. Immunofluorescent staining for TLR4, an M1 macrophage marker, revealed strongest expression in smooth muscle cells, but no clear evidence of M1 presence. Immunofluorescent staining for arginase 1 (ARG1), an M2a macrophage marker, did not detect specific staining, consistent with low ARG1 RNA expression on Day 20 of gestation. Immunohistochemical staining for CD206 revealed localized expression to possible fibroblasts and dendritic cells within the maternal endometrium on Day 25 of pregnancy and a small population of large cells in the fetal compartment, refuting the possibility of M2c polarization. Immunostaining for CD86 revealed high expression to the conceptus Tr on Days 15 and 20 of pregnancy, the endometrial blood vessels on Day 20, and less intense staining in the chorionic epithelium on Day 25. Results of these studies establish for the first time that endometrial gene expression changes dramatically between Days 15 and 20 of the peri-implantation period in pigs, and strongly suggests these changes are in part in response to conceptus secretion of IFNG to resolve the nidatory inflammatory state of the endometrium to prepare for subsequent placentation.