Pregnancy is a miracle of immunology, as well as of development. During pregnancy, a developing fetus is a semi-allogeneic graft that expresses paternally derived antigens. Yet, it is nurtured for several months without being rejected by the maternal immune system. During implantation, extravillous trophoblasts (EVT) arise from the tips of anchoring villi and invade the decidua, defining the boundary between mother and fetus: the maternal-fetal interface (Leonardo et al., (2017)). EVT evade maternal immune surveillance while inducing immune tolerance, expressing a unique subset of major histocompatibility complex (MHC) molecules: classical highly polymorphic HLA-C and nonclassical low polymorphic HLA-G (Apps et al., 2009, Tilburgs et al., 2015), as well as HLA-E at a low level.

Progesterone is critical for the establishment and maintenance of pregnancy. In early pregnancy, progesterone is produced by the corpus luteum during pregnancy and promotes endometrial chemotaxis, providing suitable conditions for embryo implantation. Progesterone also induces immune protective mechanisms to avoid embryo rejection by the mother, and is responsible for preventing preterm labor by maintaining the myometrium in a quiescent state (Shah et al. 2019). The biological effects of progesterone are thought to be primarily mediated through the classical progesterone signaling pathway via ligand binding to cytoplasmic and nuclear progesterone receptors (PRs). Ligand-bound PRs traffic from the cytoplasm to the nucleus and subsequently bind to gene regulatory regions, including progesterone response elements (PREs), to initiate transcription (Jacobsen and Horwitz 2012). Interestingly, while progesterone has been shown to upregulate HLA-G in EVT and contribute to immune tolerance (Moreau et al. 2009), several studies using both microarray (Tilburgs et al. 2015) and RNA-seq data (Ferreira et al. 2016) have shown that EVTs lack expression of PRs, raising questions as to how progesterone mediates this important effect.

In addition to the classical pathway of progesterone signaling, there is also a non-classical pathway. A variety of second messengers and signaling pathways play an important role, exerting rapid hormonal effects through non-classical progesterone signaling (Mani and Oyola 2012). Recently, a growing body of evidence has implicated two families of novel membrane proteins unrelated to classical PRs, progesterone membrane receptor component (PGRMC) and progestin and adiponectin receptor (PAQR), in progesterone signaling in several tissues. Non-classical receptors that are expressed in placental tissue, specifically in trophoblasts, include PGRMC1, PGRMC2, PAQR3, PAQR4, PAQR7, and PAQR9 (Fernandes et al., 2005, Zhang et al., 2008, Zachariades et al., 2011, Zachariades et al., 2012). PGRMC1 was found to be the most highly expressed of these genes in both microarray and RNA-seq data (Tilburgs et al., 2015, Ferreira et al., 2016). These clues focused our attention on PGRMC1 as a candidate factor for the action of progesterone at the maternal-fetal interface.

In a separate line of investigation, it has been shown that the surface expression of HLA-G is modulated by progesterone (Yie et al., 2006, Sheshgiri et al., 2008). In order to circumvent the difficulties associated with long-term culture of primary trophoblasts and genetically modifying patient EVT, the trophoblast model cell line JEG3 was employed for the studies reported here. JEG3 is generally considered to be a suitable but imperfect model of EVT (Hannan et al. 2010). Of special relevance for this project is their specific expression profile: JEG3 express HLA-C, HLA-G, PGRMC1, and lack expression of PRs. This pattern closely emulates the most relevant gene expression characteristics of EVT.