**1. Introduction**

Embryo implantation, the process of attachment and invasion of the uterus endometrium by the conceptus, is a complex physiological process tightly regulated by multiple biological molecules. Implantation requires a well orchestrated interaction between maternal and foetal tissues and consists of a fine balanced cross talk of cytokines, hormones and chemokines [1,2].

Chemokines play important key roles in several physiologic and pathologic aspects of human reproductive system, among them, the menstrual cycle, ovulation, implantation, cervical ripening, preterm labour and endometriosis [3].

The chemokine fractalkine (FKN) is synthesized as a 373 amino-acid transmembrane molecule. It is the only CX3C-chemokine which has been described [4,5]. FKN exists as both membrane-anchored and soluble forms. As an individual chemokine, its function is not merely chemoattraction, but it also acts as an adhesion molecule and is capable of regulating the immune response via CX3CR1 corresponding receptor interaction [5–7].

CX3CR1 belongs to a family of G protein-coupled receptors [7], expressed on cytotoxic effector lymphocytes, including NK cells and cytotoxic T lymphocytes [8]. The FKN-CX3CR1 axis is a crucial regulator of the microglia, the immune cells of the central nervous system (CNS) [9,10].

FKN and its receptor are expressed in numerous reproductive tissues, comprising testis, uterus, ovaries and Fallopian tubes [11,12]. According to previous studies, fractalkine might have an important involvement in the feto–maternal communication during gestation since the trophoblast cells express CX3CR1 and the endometrium cells produce FKN [13,14].

CX3CR1 controls three major signalling pathways, the PLC-PKC pathway, the PI3K/AKT/NFκB pathway and the Ras-mitogen-activated protein kinases (MAPK) pathways (p38, ERK1/2 and JNK) [15]. MAPKs are one of the oldest known signal transduction pathways [16,17] regulating proliferation, growth, migration and apoptosis [18]. PLC-PKC pathway can trigger the MAPK pathway and the secretion of various hormones [15]. The PI3K/AKT signalling pathway is activated via growth factors and plays a critical role in regulating diverse cellular functions including cell growth, metabolism, proliferation, survival, transcription, protein synthesis and mitogenesis [19,20].

Cell migration and invasion are pivotal processes for endometrial cells during implantation, cell trafficking and embryonal morphogenesis [21]. The endometrium undergoes different highly regulated physiological changes during implantation [22,23]. The main supporters of migration are protein kinases, including extracellular signal-regulated protein kinase (ERK)1/2, the phosphatidylinositol 3 kinase (PI3K), the focal adhesion kinase (FAK) and other kinases that can be activated by different cytokines, growth factors and the extracellular matrix [24]. The JNK and p38 pathways are activated in mouse preimplantation development [25].

Progesterone receptor (PR) is involved in the proliferation and differentiation processes by regulating the transcription of specific genes (c-myc, p21, EGFR etc.). The human progesterone receptor has two classical isoforms—A and B—but non-classical intracellular progesterone receptor variants have also been detected (PRC, PRM, PRS and PRT) [26]. PRA and PRB are transcribed from the same gene by using two distinct promoters. The PR can be phosphorylated at basal phosphorylation sites, as well as in response to progesterone. Different protein kinases, including mitogen-activated protein kinases (MAPK), are known to phosphorylate PR at specific serine amino acid residues and modify PR's activity [27].

Trophoblast invasion into the uterus is an essential step at implantation of the human blastocyst. The action is facilitated by the degradation of the extracellular matrix of the endometrium/decidua by various proteinases, including the matrix metalloproteinases (MMPs) [28]. The tissue inhibitors of matrix metalloproteinases (TIMPs) are key regulators of the metalloproteinases. They inhibit the activity of the MMPs by binding to the highly conserved zinc-binding site of active MMP [29]. It is suggested that MMPs and their regulators control many aspects of reproductive function. The expression of MMP2 and MMP9 depends on the activation of the aforementioned MAPK pathways [30,31] that are regulated by CX3CR1, as well. MMPs have been localized most strongly to the placental bed in early pregnancy suggesting that these proteins are involved in the invasion of trophoblasts [32,33]. MMP expression is also regulated by other factors, e.g., activin signalling and BMP2 signalling pathways [34–36].

Activins secreted by endometrial cells belong to the pleiotropic family of the transforming growth factor beta (TGFβ) superfamily of cytokines [37] and are potential factors for maternal–embryo interactions [38]. Activin/Nodal signalling through activin receptors plays an important role at implantation [34,38]. Activin is antagonized by another secreted molecule, called follistatin, which is a key regulator of the biological actions of activin [37]. Follistatins are able to bind directly and irreversibly to activins, neutralizing the ligand [39]. In early development, many processes are contingent on BMPs signalling for cell growth and differentiation [40]. Some follistatin isoforms can bind bone morphogenetic proteins (BMPs) and hinder their biological activities [41].

A bilaminar co-culture system is used to study cell–cell interactions and the technique can be modified for co-culturing any variety of adherent cell types. In this system, the two cell types can connect to each other physically. Therefore, they are able to communicate with each other not only via their released signalling molecules, but also through different cell surface molecules. This way, we can get a better view of action/reaction both of the examined cells [42–45]. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 19 via their released signalling molecules, but also through different cell surface molecules. This way,

In this study, we focused on the molecular mechanisms governing FKN supporting HEC-1A/JEG-3 interaction and influencing the expression of implantation-related genes in an in vitro co-culture model. Understanding how these mechanisms contribute to implantation might open new targeted medical therapies to give reassurance to women suffering from its failure. we can get a better view of action/reaction both of the examined cells [42–45]. In this study, we focused on the molecular mechanisms governing FKN supporting HEC-1A/JEG-3 interaction and influencing the expression of implantation-related genes in an in vitro coculture model. Understanding how these mechanisms contribute to implantation might open new
