Introduction: Uterine Natural Killer (NK) cells are the predominant immune cells within the decidua during early pregnancy. These cells are thought to regulate aspects of decidualization and placental development, but their functions remain poorly characterized, especially in species with deeply invading trophoblasts such as humans and rats. Interleukin-15 (IL-15) is a cytokine required for NK cell development and survival. IL-15 mutant (IL15Δ/Δ) rats lack NK cells and exhibit altered placental development with precocious trophoblast invasion. In this study, we profiled gene expression differences between wild-type and IL15Δ/Δ implantation sites to reveal candidate factors produced by uterine NK cells that may regulate placentation and trophoblast invasion.Methods: Clariom S gene expression profiling was performed using implantation sites collected from pregnant wild-type and IL15Δ/Δ rats on gestational day 9.5. Levels and localization of perforin and osteopontin in implantation sites from wild-type and IL15Δ/Δ rats were further analyzed. The effect of osteopontin on the invasive capacity of rat trophoblasts was evaluated using Matrigel-based Transwell assays.Results: There were 257 genes differentially expressed between wild-type and IL15Δ/Δ implantation sites on gestational day 9.5, including decreased expression of various NK cell markers in IL15Δ/Δ rats, as well as Spp1, which encodes osteopontin. In wild-type rats, osteopontin was present within the decidua basalis and adjacent to the primitive placenta, and osteopontin colocalized with the NK cell marker perforin. Osteopontin was also detectable in uterine glands. Conversely, in IL15Δ/Δ rats, osteopontin and perforin were not readily detectable in the decidua despite robust osteopontin levels in uterine glands. Neutralization of osteopontin in media conditioned by cells isolated from the decidua decreased invasion of rat trophoblasts, suggesting that reduced levels of osteopontin are unlikely to account for the precocious trophoblast invasion in IL15Δ/Δ rats.Conclusion: Osteopontin is expressed by NK cells at the maternal-fetal interface in rats and may contribute to modulation of trophoblast invasion.

Introduction Placenta accreta (PA) is a potentially life‐threatening pregnancy complication in which the placenta invades too deeply into the uterus. The underlying causes of PA remain unknown, but abnormal function of uterine natural killer (uNK) cells may be involved. uNK cells are the most prevalent immune cells within the uterus during early pregnancy. They secrete cytokines and growth factors that are thought to control the depth of placental invasion, yet the function of uNK cells remains poorly characterized. We recently generated rats devoid of uNK cells using targeted genomic editing of the Interleukin‐15 (IL15) locus. Il15 encodes a cytokine needed for uNK cell survival. During pregnancy, Il15‐deficient dams do not have uNK cells, and they exhibit an over invasive placenta. We therefore hypothesize that uNK cells produce factors during early pregnancy that control the depth of placental invasion. Objective: The goal of this study isto profile gene expression differences in the decidua between wild‐type (WT) and IL15‐deficient (IL15Δ/Δ) rats during early pregnancy, in order to deduce which genes are expressed by rat uNK cells. Methods: Pregnant IL‐15Δ/Δ and WT Sprague‐Dawley Holtzman rats (n=3 dams/group) were sacrificed on gestational day (GD) 9.5, conceptuses dissected, mRNA isolated, and global transcriptional changes assessed using Clariom S gene expression array (n=3 conceptuses/dam). Expression of select transcripts was determined in a separate cohort of conceptuses (n=13) using quantitative RT‐PCR. To determine localization of perforin (PRF) and osteopontin (OPN), GD 7.5, 9.5, and 11.5 conceptuses were sectioned, and immunohistochemistry (IHC) was performed. Data was analyzed using Student's t‐test or Analysis of Variance and P≤0.05 was considered statistically significant, unless indicated otherwise. Results: On GD 9.5, 230 genes were differentially expressed between WT and IL‐15Δ/Δ rats (P≤0.01). Gene ontology analysis revealed pathways associated with immune responses, cell migration, and cell adhesion to be significantly altered in IL‐15Δ/Δ conceptuses. Notably, we detected a pronounced decrease in Prf, a uNK cell marker (p≤0.0001), and Spp1 (p=0.0031), which encodes OPN. IHC staining of GD 9.5 conceptuses showed intracellular OPN staining in the uterine glands, decidua, and primitive placenta of WT, but not IL‐15Δ/Δ rats. OPN and PRF staining was co‐localized, suggesting that OPN is produced by uNK cells. Conclusion: IL‐15Δ/Δ rats exhibited impaired immune responses and cellular adhesion and migration pathways in the decidua during early pregnancy. OPN expression was significantly decreased in IL‐15Δ/Δ rats at mRNA and protein levels. We believe that reduced OPN in the decidua of IL‐15Δ/Δ rats may contribute to the increased placental invasion that is apparent in these rats. Significance: This study provides new insights into factors produced by uNK cells that may contribute to the regulation of placental invasion during pregnancy. We are currently determining whether OPN is directly involved...