Embryo-maternal cross-talk: key players in successful implantation and live birth rates.

Prior to implantation, the human embryo undergoes a series of cellular divisions, ultimately resulting in the formation of a blastocyst that comprises a distinctly inner cell mass (ICM) and TE [ 123 ]. In a process known as blastocyst activation, the ovarian hormones, specifically progesterone and estrogen, typically regulate the blastocyst to a state of competence for implantation [ 124 ]. Human embryos undergo programming to generate secretory markers and surface markers, which enable the embryos to appropriately respond to maternal factors and regulate the endometrium environment [ 4 ]. As a result, establishing effective mutual communication between the embryo and the endometrium facilitates the implantation process. Blastocysts express receptors and adhesion molecules on their surface prior to implantation. These receptors bind to secretory ligands that originate from maternal cells (paracrine signaling), as well as ligands released by the embryo (autocrine signaling). Thouas et al. have thoroughly reviewed the surface receptors, which include cytokine receptors, growth factor receptors, chemokine receptors, and hormone receptors [ 4 ]. Furthermore, the expression of adhesion molecules enables blastocysts to establish physical interactions with endometrial components during the process of implantation. As previously mentioned, integrins are one of the adhesion molecules expressed in both the endometrium and the trophoblast [ 113 ]. Embryos have variable integrin expression at different stages of the implantation process. For example, the TE mainly expresses αⱱβ3, α5β1, αⱱβ6, and αⱱβ5 in early adhesion [ 125 ], whereas α6β1, α1β1, and α7β1 are likely to be more prominent in embryo invasion [ 126 ]. Interactions between integrin αvβ3 and its ligand, glycoprotein osteopontin (OPN), may contribute to the stable attachment of the trophoblast cells to the endometrial tissue [ 127 ]. In the feto-maternal interaction, the selectin adherence mechanism is firmly established. The entire embryo’s surface has displayed L-selectin molecules, and in the endometrium, the expression of their ligands increases during the implantation period [ 61 , 128 ]. CD44 is another structure with an adhesion effect that is present on the surface of human blastocysts’ TE. For the first time, Berneau et al. [ 129 ] have shown the role of this molecule in the initial phases of the embryo’s attachment to the uterus with the help of an in vitro model. During implantation, the blastocyst expresses and releases several factors that have been shown to have both paracrine and autocrine effects [ 130 ]. These factors, known as the embryo secretome, include cytokines, growth factors, hormones, various proteins, and microRNAs, which mediate interactions with the endometrial tissue, supporting adherence, invasion, and immunological regulation [ 4 , 130 ]. Assessing these molecules and analyzing their mechanisms of activity could enhance our insight into the embryo’s behavior in the implantation process, thereby contributing to the adoption of new, practical diagnostic and therapeutic approaches for women with infertility [ 5 ]. Cytokines are critical immunological components in the interaction between the embryo and the mother [ 4 , 131 ]. Some studies demonstrated the embryonic synthesis of cytokines, including IL1B [ 132 ], TNF [ 59 ], LIF [ 133 ], TGFA and TGFB1 [ 4 ], CXCL8 [ 134 ], IL6 [ 134 ], IL10 [ 135 ], IFN-β and IFN-α [ 4 ], and CCL4 [ 135 ]. The evidence confirms that these cytokines play a role in improving blastocyst hatching rates [ 136 ]. Cytokines such as IL1B, TGFB1, and CSF1 regulate trophoblastic MMPs [ 4 , 61 ], so the invasive capacity of trophoblasts is attributed to the synthesis of MMPs 9 and 2 [ 137 ]. he blastocyst-secreted factors, in addition to their autocrine function, have a paracrine effect on the endometrium to improve endometrial receptivity. For example, IL1 can trigger the elevation of endometrial αⱱβ3 in the specific region where the embryo implants [ 138 , 139 ], and TNF removes MUC1, which acts as an anti-adhesion structure [ 108 ]. However, increased levels of embryo-derived TNF through TNF receptor activation correlate with implantation failure [ 140 ]. The interaction between trophoblast cells and endometrial immune cells includes three distinct stages. Initially, trophoblasts release chemokines to attract immune cells to the embryo implantation site. Subsequently, during the education stage, they control the differentiation of immune cells by releasing regulatory cytokines. Finally, over the response stage, immune cells generate factors that facilitate the normal development and function of the placenta [ 141 ]. In this regard, it has been demonstrated that hCG (a blastocyst-derived prominent paracrine regulatory hormone in effective implantation) might improve the recruitment of endometrial Tregs by stimulating the chemokine CCL2, and the signals generated by this hormone, along with insulin like growth factor 1 (IGF1) as well as IGF2, increase LIF production by endometrial cells [ 142 – 144 ]. MicroRNAs (miRNAs) are non-coding small RNAs that operate as regulatory agents in gene expression, and existing evidence reflects their effects on developmental processes in embryos, endometrial activities, and embryo-maternal interactions [ 145 ]. In addition to their maternal origin, these molecules have an embryonic origin and are produced both intracellularly and extracellularly [ 146 ]. Several studies investigating embryo-derived miRNAs in culture medium have shown an association between their upregulation and downregulation with embryo quality as well as implantation outcomes [ 147 , 148 ]. The extensive examination of miRNAs in a study indicated that miR-30c and miR-20a are highly expressed in spent blastocyst culture medium (SBCM) from implanted blastocysts compared to non-implanted ones. Although this hypothesis was not confirmed in this research, it is suggested that these miRNAs, which are involved in the proliferation of endometrium through their effects on genes such as NRAS , PTEN , APC , MAPK1 , PIK3CD , KRAS , and SOS1 , act as uterine regulators in implantation [ 146 , 149 ]. Berkhout et al. [ 150 ] analyzed 752 miRNAs and detected hsa-miR-320a as the sole miRNA specifically released by good-quality embryos. This miRNA not only differentiates good-quality embryos from poor-quality ones but also appears to support the migration of decidualized endometrial stromal cells to achieve effective implantation [ 150 ]. Furthermore, a study demonstrated that both miR-29b and miR-145 affect critical activities, including cell attachment and invasion, by regulating the expression of MMP9 and ITGB3 [ 151 ]. Several miRNAs, such as miR-519d-3p, miR-191, miR-661, miR-142-3p, and miR-372, have been discovered to be significantly expressed in the SBCM from non-implanted blastocysts and, through different signaling pathways, result in implantation failure [ 152 – 154 ]. For example, miR-372 and miR-191 affect the MAP3K (mitogen-activated protein kinase kinase kinase), CDK6 (cyclin-dependent kinase 6), and apoptotic pathways, while miR-661 suppresses the expression of MTA1 , MTA2 , PVRL1 , and EPHB2 in endometrium epithelial cells [ 145 ]. Blastocyst synthesizes several other secretory molecules, such as ubiquitin [ 155 ], apolipoprotein A1 (APOA1) [ 156 ], and preimplantation factor (PIF) [ 157 ]. PIF, initially identified by Dr. Eytan Barnea, is a 15-amino acid protein released by viable human embryos from the 4-cell stage and reaches its highest levels at the blastocyst stage [ 158 ]. By examining the serum of patients with RPL, the specificity of PIF secretion from viable embryos was confirmed. In patients who were β-hCG positive but had miscarriages, no PIF activity was detected [ 159 ]. This protein carries out several activities to facilitate a successful pregnancy, of which we will discuss the most important ones. Given that successful implantation requires a strongly regulated invasion of EVT cells into the endometrium, several in vitro studies using synthetic PIF (sPIF) have investigated the role of PIF in the process of decidualization and trophoblast invasion. Esther Dos Santos et al. [ 160 ] demonstrated that PIF had positive effects on decidualization in human endometrial stromal cells (HESCs) by significantly improving prolactin release and upregulating the mRNA expression of connexin 43 ( CX43 ) and insulin-like growth factor binding protein 1 ( IGFBP1 ) [ 161 ] (Fig. 3 e). Fig. 3 Supportive effects of PIF during implantation. PIF is released by viable human embryos from the 4-cell stage and peaks at the blastocyst stage. This protein supports implantation through autocrine and paracrine effects. a  PIF increases MMP9 and decreases TIMP1 in EVT cells, promoting invasion. b  It also induces environmental tolerance in favor of the embryo by increasing the expression of HLA-E, HLA-C, and HLA-G molecules. c  This embryo-derived protein reduces trophoblast apoptosis by increasing BCL2 expression, lowering BAK and BAX mRNA expression, and decreasing p53 phosphorylation at serine (Ser)−15. PIF modifies many cell populations to promote embryo implantation via paracrine effects. d  This protein controls the production of integrins, including α2β3 integrin, in endometrial epithelial cells, which are essential markers for facilitating implantation. e  In stromal cells, PIF works in several ways. By upregulating inflammatory mediators such as IL6, CXCL8, IL1B, ICAM1, CXCL1, and CCL2, it prepares the uterus for implantation. Through a reduction in MMP9 activity, it suppresses an excessive invasion of trophoblasts. PIF contributed to decidualization by increasing the release of prolactin and the expression of CX43 and IGFBP1. f  Immune cells are another group of PIF target cells. PIF promotes a Th2 predominance while also sustaining an effective anti-pathogenic Th1 response and reducing NK cell toxicity by downregulating CD69 expression. PIF: Preimplantation factor, BCL2: B-cell lymphoma 2, BAK: BCL2 antagonist/killer, BAX: BCL2-associated X protein, CX43: Connexin 43, IGFBP1: Insulin like growth factor binding protein 1, NK: Natural killer cells, EVT: Extravillous trophoblast, HLA-: Human leukocyte antigen-, MMP9: Matrix metalloproteinase 9, TIMP1: Tissue inhibitors of metalloproteinase1, ECM: Extracellular matrix, ICAM1: intercellular adhesion molecule 1, IL: Interleukin. (Created by biorender) Supportive effects of PIF during implantation. PIF is released by viable human embryos from the 4-cell stage and peaks at the blastocyst stage. This protein supports implantation through autocrine and paracrine effects. a  PIF increases MMP9 and decreases TIMP1 in EVT cells, promoting invasion. b  It also induces environmental tolerance in favor of the embryo by increasing the expression of HLA-E, HLA-C, and HLA-G molecules. c  This embryo-derived protein reduces trophoblast apoptosis by increasing BCL2 expression, lowering BAK and BAX mRNA expression, and decreasing p53 phosphorylation at serine (Ser)−15. PIF modifies many cell populations to promote embryo implantation via paracrine effects. d  This protein controls the production of integrins, including α2β3 integrin, in endometrial epithelial cells, which are essential markers for facilitating implantation. e  In stromal cells, PIF works in several ways. By upregulating inflammatory mediators such as IL6, CXCL8, IL1B, ICAM1, CXCL1, and CCL2, it prepares the uterus for implantation. Through a reduction in MMP9 activity, it suppresses an excessive invasion of trophoblasts. PIF contributed to decidualization by increasing the release of prolactin and the expression of CX43 and IGFBP1. f  Immune cells are another group of PIF target cells. PIF promotes a Th2 predominance while also sustaining an effective anti-pathogenic Th1 response and reducing NK cell toxicity by downregulating CD69 expression. PIF: Preimplantation factor, BCL2: B-cell lymphoma 2, BAK: BCL2 antagonist/killer, BAX: BCL2-associated X protein, CX43: Connexin 43, IGFBP1: Insulin like growth factor binding protein 1, NK: Natural killer cells, EVT: Extravillous trophoblast, HLA-: Human leukocyte antigen-, MMP9: Matrix metalloproteinase 9, TIMP1: Tissue inhibitors of metalloproteinase1, ECM: Extracellular matrix, ICAM1: intercellular adhesion molecule 1, IL: Interleukin. (Created by biorender) In the endometrial epithelial cells, PIF regulates the expression of integrins, such as α2β3 integrin, which are important biomarkers for inducing implantation (Fig. 3 d). It also increases the expression and release of several inflammatory mediators in stromal cells associated with uterine preparation for implantation. These inflammatory mediators include IL6, CXCL8, IL1B, intercellular adhesion molecule 1 (ICAM1), CXCL1, and CCL2 (Fig. 3 e). Additionally, an upregulation in the expression of epiregulin and amphiregulin genes, which play a role in enhancing decidua development, has been shown [ 162 ]. Another critical determinant of an effective invasion is the maintenance of balance among MMPs and tissue inhibitors of metalloproteinases (TIMPs) [ 163 ]. PIF maintains this balance by exerting different effects on two distinct target cells. By affecting EVT cells, PIF increases MMP9 activity, reduces mRNA expression of TIMP1, and ultimately enhances invasion [ 164 ] (Fig. 3 a); conversely, by affecting endometrial cells, it decreases MMP9 activity and thus controls excessive invasion [ 160 ] (Fig. 3 e). YANG et al. [ 165 ] employed the isobaric tags for relative and absolute quantification (iTRAQ) proteomics technique to identify a protein profile that interacts with PIF throughout invasion. The analysis provides evidence that interacting between myosin heavy chain 10 (MYH10) and PIF greatly improved the invasion and migratory abilities of HTR-8 trophoblast cells [ 165 ]. In addition to its previously mentioned effects, PIF exerts crucial roles in immunological regulation during pregnancy. To identify the effect of PIF on signaling between the mother and preimplantation embryo, human peripheral blood mononuclear cells (PBMCs) isolated from non-pregnant patients were used, and PIF binding to immune cells was investigated. The binding characteristics of PIF change in conditions of immunological activation and unstimulated immunity [ 166 ]. PIF creates a Th2 predominance while maintaining an efficient anti-pathogenic Th1 reaction [ 167 ], as well as directly regulating NK cell activity [ 166 , 168 ]. Low-dose PIF effectively diminishes NK cell toxicity through the downregulation of CD69 expression [ 168 ] (Fig. 3 f). Beyond exerting PIF paracrine effects on the endometrium and immune cells, the embryo demonstrates PIF autotrophic activities. Animal studies have confirmed the self-protection of embryos by demonstrating the capability of blastocysts to uptake PIF [ 169 ]. Moreover, the administration of anti-PIF antibodies to the culture media of murine embryos resulted in growth retardation and mortality, indicating the crucial role of PIF in proper embryonic development. In contrast, adding PIF to the culture media of bovine embryos substantially enhanced their development up to the blastocyst stage [ 169 ]. It has also been shown that PIF reduces the deaths of mouse embryos exposed to toxic serum derived from RPL patients; therefore, using diagnostic and therapeutic strategies based on PIF may be effective in diminishing recurrent miscarriage [ 170 ]. Lindsay F. Goodale et al. demonstrated that PIF improves this self-protection by targeting protein disulfide isomerase/thioredoxin in the embryo and diminishing reactive oxygen species (ROS) [ 171 ]. Furthermore, in JEG-3 cells as a model for studying trophoblasts, PIF can create a tolerance environment by improving both intracellular and surface expression of specific HLA class-I molecules, including HLA-E, HLA-C, and HLA-G, in time- and dose-dependent pathways [ 172 ] (Fig. 3 b). The advantage of using the JEG-3 cell line over other cell lines is the ability to express HLA-G and HLA-C, similar to EVT, making it an appropriate model for trophoblast research [ 173 ]. PIF exerts an anti-apoptotic effect on trophoblasts by modulating the p53 signaling pathways. In more detail, this embryo-derived protein increases the expression of B-cell lymphoma 2 (BCL2), decreases mRNA expression for BCL2 antagonist/killer 1 (BAK) and BCL2-associated X protein (BAX), and reduces the phosphorylation level of p53 at serine (Ser)−15 [ 161 ] (Fig. 3 c).

Based on the evidence presented in this review, studying the embryo-endometrial cell interactions is crucial for comprehending implantation physiology and developing approaches to manage reproductive disorders and female infertility. Nonetheless, the intricate mechanisms governing this interaction remain unknown because of ethical and technological limitations in acquiring human biological specimens. Thus, employing in vitro models that imitate implantation provides ethical and efficient alternatives. Most existing developed models of the endometrium comprise a single cell type and Matrigel, which is probably not identical to the endometrium ECM [ 122 ]. In contrast, models representing the endometrium have to incorporate several endometrial cellular types and authentic endometrium matrices, alongside trophoblastic cultures for modeling early pregnancy. Human in vitro studies have moved forward considerably by the establishment of organoids, which are 3D models derived from pluripotent or tissue-specific stem cells. In culture, these cells exhibit significant expandability while effectively preserving their phenotype [ 174 ]. In addition to endometrial organoids, trophoblastic organoids and embryonic organoids have also been established, enabling the examination of the implantation process and early stages of development [ 175 , 176 ]. However, the anatomical characteristics of conventional endometrium organoids restrict their use in investigating endometrial interactions with embryonic models during implantation. Consequently, Shibata et al. [ 177 ] have bridged this existing gap by establishing a modified endometrium organoid that, when co-cultured with embryonic blastoids, mimics the key stages of implantation, such as apposition, attachment, and invasion. Generally, in research focused on examining variables influencing embryo-endometrial interactions, the more similar the models are to biological conditions in the human body, the stronger and more valid the outcomes become. The intricate interactions among immune cells at the embryo-maternal interface are incredibly dynamic, with immunological composition quickly altering in reaction to semen exposure, hormones, embryo signals, microbiota, and systemic inflammation. Most studies provide just a static picture, failing to account for dynamic interactions; thus, the time-based and spatial control of these activities in humans is unknown. The discovery of embryo-derived proteins is another element that might improve our knowledge about the implantation process, since the embryo, functioning as a semi-allogeneic entity, potentially undergoes modifications in itself and the endometrium that may facilitate successful implantation. For example, PIF, by exhibiting both autocrine and paracrine effects, facilitates interactions between the embryo and the endometrium. Moreover, identifying these proteins will enable the employment of an appropriate marker to assess embryo quality in patients undergoing IVF procedures, thereby enhancing IVF outcomes.

The two unique biological components that make up the uterine endometrium are the epithelium cells and stromal cells. The increased levels of progesterone during the WOI induce the fibroblast-like stromal cells within the endometrium to undergo cellular alterations and become bigger and more rounded decidual cells [ 7 ]. This process, known as decidualization, occurs in humans independently of embryonic existence; however, if implantation occurs, high progesterone levels perpetuate these cellular modifications to preserve the pregnancy [ 8 ]. Changes in the levels of various immune cells, cytokines, chemokines, growth factors, and adhesion molecules accompany the enhancement of secretory glandules and the formation of pinopodes and microvilli on the luminal epithelium. Thus, these expression changes contribute to the overall growth and development of the reproductive system, facilitating the necessary physiological processes for successful reproduction [ 9 ]. The immune cells of the uterus undergo fundamental variations during a normal menstrual cycle, and the functions of these leukocytes include immune protection of the uterus mucosa, the process of decidualization, and embryo implantation [ 10 ]. Various hypotheses regarding the source of endometrial leukocytes have been proposed. One hypothesis is that circulating blood transports leukocytes to the endometrial site in response to the synthesis of chemoattractants. For example, the chemokines CX3CL1, CXCL10, and CCL2, which are regulated by sex hormones, are involved in the recruitment of peripheral blood natural killer (pbNK) cells [ 11 ]. Another theory suggests the local expansion of resident immune cells, such that undifferentiated cells in the endometrium, with the ability for proliferation and self-renewal, develop into immune cells in response to cytokines. For instance, progesterone induces the synthesis of interleukin 15 (IL15) from endometrial cells, and IL15 can potentially stimulate the in situ expansion of endometrial NK cells [ 12 ]. Furthermore, it has been suggested that hematopoietic progenitors are recruited and undergo differentiation within the endometrium [ 13 ]. Before menstruation begins, in the absence of pregnancy, there is a drop in the levels of estrogen and progesterone, which leads to an increase in immune cells like mast cells, granular lymphocytes, eosinophils, neutrophils, B and T lymphocytes, and granular lymphocytes. These leukocytes upregulated the expression of inflammatory mediators, specifically CXCL8 and CCL2 [ 14 ]. Throughout the menstrual phase, which is an inflammatory state with the shedding of the endometrial wall, dendritic cells (DCs), along with other antigen-presenting cells (APCs), facilitate the cleaning of the uterine cavity [ 10 ]. Endometrial tissue debris may also infiltrate the uterine-draining lymph nodes (LNs) via the lymphatic system and be eliminated by immune cells. A study published by Marina Berbic et al. [ 15 ] confirmed this hypothesis by showing an increase in the number of CD10⁺ endometrial stromal cells, CD79⁺, CD68⁺, CD20⁺, FoxP3⁺, DC-Sign⁺, CD4⁺, and CD3⁺ cells present in the LN during menstruation. However, given insufficient sample numbers, this trend didn’t become significant in some populations [ 15 ]. The maternal immune system undergoes three stages during a normal pregnancy. The first stage is a pro-inflammatory reaction in which embryo implantation occurs. Then, in the second stage, anti-inflammatory conditions cause rapid growth and development of the fetus. Finally, parturition is an inflammatory phase that is accompanied by the invasion of immune cells into the myometrium [ 16 ]. During pregnancy, the cross-talk between trophoblast and decidual immune cells results in various outcomes, for example, its involvement in immune tolerance to implanted allogeneic embryos. In this way, human leukocyte antigen (HLA)-G expressed on the surface of trophoblast cells interacts with immunoglobulin-like transcription inhibitory receptors (ILT) that are differentially expressed on T, B, natural killer (NK) cells, and phagocytes, ultimately leading to suppression of the immune system [ 17 ]. In addition to HLA-G, progestagen-associated endometrial protein (PAEP) and indoleamine 2, 3-dioxygenase (IDO) are two factors that play a role in immune suppression and cause regulated chemotaxis of immune cells to the fetal-maternal interface [ 18 , 19 ]. Embryo implantation is another outcome of mutual dialogue between endometrial immune cells and embryonic trophoblasts. In the following, we will discuss the role of the innate and adaptive immune system in more detail during implantation. Within the population of innate immune cells, NK cells are the most critical decidual cells in establishing pregnancy. Uterine natural killer (uNK) cells and pbNK cells exhibit distinct phenotypes. CD56 dim CD16 + NK cells make up 90–95% of pbNK cells, while CD56 bright CD16 − NK cells make up the majority of uNK cells [ 20 ]. Once pregnancy has occurred, the population of uNK cells expands, making up 60%–90% of the immune cells in the decidual [ 21 ]. Studies have shown that human uNK cells express some activating receptors, such as KLRK1 (killer cell lectin-like receptor K1), NCR3 (natural cytotoxicity triggering receptor 3), NCR1, CD244, and NCR2. However, they are not as effective at cytotoxic activity as NK cells in peripheral blood [ 22 ]. Despite their limited cytotoxicity, uNK cells can spontaneously release a diverse range of cytokines and chemokines, indicating that they undergo activation within the decidua [ 23 ]. Ulrike von Rango et al. [ 24 ] conducted a study that examined the number and distribution of specific types of leukocytes in intrauterine and fallopian tube implantation sites. The examination of leukocytes revealed a significant difference in CD56 + NK cells between fallopian tube mucosa and endometrial decidua. Therefore, NK cells may play an essential role in regulating trophoblast invasion because it is thought that the lack of these cells at the tubal implantation site may cause the abnormal invasion of trophoblasts in this pathological condition [ 24 ]. Furthermore, in another study using trophoblast organoids, it was shown that NK-derived cytokines and chemokines such as colony stimulating factor 1 (CSF1), CSF2, XCL1, and CCL5 facilitate trophoblast differentiation during the last step of the invasion [ 25 ]. Some researchers suppose that uNK cells do not directly participate in the implantation; however, they assist in the process by remodeling the uterine spiral artery. In this regard, it has been shown that in both mice and humans, uNK cells can produce angiogenic factors like vascular endothelial growth factor (VEGF), angiopoietin 2 (ANGPT2), and placental growth factor (PGF) [ 23 , 26 ] (Fig. 1 a). Fig. 1 Immune cell distribution during implantation. a  uNK cells are the most common cells in the secretory phase. By secreting angiogenic factors, they play a role in spiral artery remodeling. Additionally, these cells, in cooperation with T cells, prevent excessive invasion by induction of apoptosis. b  Mφs and uNK cells destroy ECM by producing MMPs; as a result, trophoblast invasion into blood vessels is facilitated. c  Mφs-derived IL-1β and LIF cytokines increase the α1,2-fucosyltransferases (FUT1, FUT2) expression and fucosylated structures, increasing the embryo’s adhesion. d  Cytokines cause monocyte migration to the uterus and differentiate into uDC, which regulates angiogenesis and Treg development. e , f  Mast cells play a role in the regulation of implantation by secreting VEGF and histamine. Tr: Trophoblast, ICM: Inner cell mass, IL-: Interleukin-, LIF: Leukemia inhibitory factor, dMQ: Decidual macrophage, uMC: Uterine mast cell, VEGF: Vascular endothelial growth factor, ECM: Extracellular matrix, MMP: Matrix metallopeptidase, uNK: Uterine natural killer cell, ANGPT2: Angiopoietin 2, PGF: Placental growth factor, Treg: Regulatory T cell, TGFB1: transforming growth factor beta 1, CSF: Colony stimulating factor, uDC: Uterine dendritic cell, sFLT1: Soluble FMS-like tyrosine kinase 1, MUC1: Mucin 1 (created by biorender) Immune cell distribution during implantation. a  uNK cells are the most common cells in the secretory phase. By secreting angiogenic factors, they play a role in spiral artery remodeling. Additionally, these cells, in cooperation with T cells, prevent excessive invasion by induction of apoptosis. b  Mφs and uNK cells destroy ECM by producing MMPs; as a result, trophoblast invasion into blood vessels is facilitated. c  Mφs-derived IL-1β and LIF cytokines increase the α1,2-fucosyltransferases (FUT1, FUT2) expression and fucosylated structures, increasing the embryo’s adhesion. d  Cytokines cause monocyte migration to the uterus and differentiate into uDC, which regulates angiogenesis and Treg development. e , f  Mast cells play a role in the regulation of implantation by secreting VEGF and histamine. Tr: Trophoblast, ICM: Inner cell mass, IL-: Interleukin-, LIF: Leukemia inhibitory factor, dMQ: Decidual macrophage, uMC: Uterine mast cell, VEGF: Vascular endothelial growth factor, ECM: Extracellular matrix, MMP: Matrix metallopeptidase, uNK: Uterine natural killer cell, ANGPT2: Angiopoietin 2, PGF: Placental growth factor, Treg: Regulatory T cell, TGFB1: transforming growth factor beta 1, CSF: Colony stimulating factor, uDC: Uterine dendritic cell, sFLT1: Soluble FMS-like tyrosine kinase 1, MUC1: Mucin 1 (created by biorender) After uNK cells, macrophages are the predominant immune cells in the decidua, constituting around 20–25% of all leukocytes in this tissue. These cells are major APCs in the decidua and become attracted to the endometrium in reaction to seminal fluid and initial signals through the preimplantation phase [ 27 , 28 ]. For example, a study has shown that the interaction between RANK + decidual macrophages (dMφs) and RANKL on decidual stromal cells (DSCs) stimulates the production of dMφs adherence molecules, facilitating their accumulation throughout the initial stages of pregnancy [ 29 ]. Based on the level of CD11c marker expression, Brandy L. Houser et al. [ 30 ] discovered two different subsets of dMφs in human endometrium. They termed these types CD11c LO dMφs and CD11c HI dMφs, and both populations release pro-inflammatory cytokines (tumor necrosis factor (TNF) and IL1B (interleukin 1 beta)) and anti-inflammatory cytokines (IL10 and TGFB1). They also have different gene signatures, making it impossible to classify them as either M1 or M2 macrophages. Instead, these cells are macrophages that reside in decidual tissue and play specific roles at the interface between the mother and fetus. It is believed that human dMφs, which are found in proximity to invasive trophoblasts, have a role in the invasion of trophoblasts and the development of the placenta [ 31 ]. These leukocytes significantly produced matrix metallopeptidase 7 (MMP7) and MMP9, suggesting their role in breaking down the extracellular matrix (ECM) of surrounding tissues. This process creates a favorable environment for trophoblast cells to invade the blood vessels [ 32 ] (Fig. 1 b). Furthermore, local macrophages help the expression of fucosyltransferases in the epithelial cell by releasing specific cytokines, such as IL1B and leukemia inhibitory factor (LIF). This, in turn, leads to an increase in fucosylated structures on the cell surface, and as a result, the embryo’s adhesion to the uterine wall increases [ 33 , 34 ] (Fig. 1 c). More research demonstrated that not all CD11c + cell populations were macrophages; thus, the CCR2 marker was also suggested alongside CD11c to categorize dMQs. Consequently, macrophages at the embryo-maternal interface have been classified into three subgroups for the first time: CCR2 + CD11c HI , CCR2 − CD11c HI , and CCR2 − CD11c LO , which reside in distinct locations on the decidua [ 35 ]. In CCR2 − dMQs, the expression of M2 macrophage-related genes was considerably higher in comparison with M1-related genes. Consequently, they may assume an anti-inflammatory effect in the decidua. In contrast, CCR2 + dMQs exhibited much higher expression of M1 macrophage genes, which led to increased pro-inflammatory properties [ 36 ]. Like dMφs and uNK cells, uterine dendritic cells (uDCs) have different phenotypic characteristics from DCs in other tissues. The phenotype of DCs is regulated by a mutually beneficial connection between them and the milieu of the uterus [ 20 ]. Cytokines, including CSF1, IL4, and CSF2, prompt the migration of monocytes into the uterus, where they undergo differentiation into tolerogenic cells known as uDCs. UDCs exert a local effect and control angiogenesis in the endometrium by the production and release of soluble FMS-like tyrosine kinase 1 (sFLT1) and TGFB1. They also contribute to the development of regulatory T (Treg) cells [ 37 ] (Fig. 1 d). Another type of immune cell present in both human and mouse decidua tissue is mast cells (MCs) [ 38 ]. Uterine mast cells (uMCs) are also a distinct subset compared to MCs that exist in diverse tissues and refer to a heterogeneous group that consists of connective tissue-type mast cells, mucosal mast cells, and a transitional type that exhibits characteristics of both mast cell types. CD117 and FcεRIα are expressed by a majority of these cells, whereas only a minority of them express Mcpt5 and Mcpt8 [ 39 ]. It is hypothesized that histamine produced by MCs has a beneficial effect during implantation because it aids tissue remodeling [ 40 ]. The findings from Rodent’s research utilizing disodium cromoglycate to inhibit MC degranulation suggest that VEGF derived from these cells plays a role in regulating implantation as a pro-angiogenic component [ 41 ]. In a study, a model for how human MCs function in the establishment of pregnancy through Killer Cell Ig-Like Receptor 2DL4 (KIR2DL4/CD158D) was presented [ 42 ]. Trophoblasts express HLA-G, which activates KIR2DL4 receptors on decidual MCs, and this activation triggers the release of LIF and serine proteases, such as MMP9. LIF promotes STAT3 activation and trophoblast migration, whereas serine proteases break down protease-activated receptors (PARs) and trigger trophoblast tube development. In vitro, developing a cellular tube network on Matrigel is the outcome of necessary biological processes, such as cell proliferation and migration. Thus, the production of trophoblast tubes represents an increase in trophoblast invasion in response to mast cells [ 42 , 43 ]. Prior to pregnancy, T cells make up 50–60% of all lymphocytes in the endometrial tissue [ 44 ]. However, during early pregnancy, T cells comprise around 5–20% of total CD45 + decidual lymphocytes, and this percentage increases to 40–80% with gestational age [ 45 ]. The T cells in the decidua constitute a heterogeneous subtype of cells that exhibit significant differences in comparison to the T cells seen in peripheral blood circulation. Intense activation of CD8 + T lymphocytes with cytotoxic effects takes place throughout the early to middle proliferative stage of the menstrual cycle, which is critical to maintaining immune surveillance [ 10 ]. Also, the increase in cytotoxic activity may play an essential role in protecting the uterine environment from pathogens before embryo implantation [ 46 ]. As preovulatory estrogen concentrations rise, cytotoxic T-cell activity suddenly decreases, and this reduction is thought to facilitate effective embryo implantation. At this stage of the cycle, the increase in Foxp3 + Tregs is believed to be involved in reducing this cytotoxicity [ 47 ]. The adaptive immune system responses play a crucial role in establishing tolerance for pregnancy, and the lack of balance between Treg lymphocytes and effector T lymphocytes is a significant contributing factor to infertility and prevalent obstetric diseases [ 48 ]. The rate of decidual T helper 1 (Th1) cells has somewhat increased, although Th17 and Th2 cell populations are generally not abundant, reflecting a moderate inflammatory state regulated by Tregs [ 49 , 50 ]. The research in mice provides convincing evidence for the importance of Treg cells in the processes of embryo implantation and placenta establishment. Aluvihare et al. [ 51 ] initially proved the necessity of Treg cells by transferring either complete T lymphocyte populations or populations lacking CD4 + CD25 + Treg lymphocytes into pregnant mice deficient in T cells. When Treg cells are absent, fetuses from parents with different major histocompatibility complex ( MHC ) genes are always rejected, whereas fetuses from parents with the same MHC genes typically survive [ 51 ]. Cytokines, chemokines, and factors such as TGFB1 and prostaglandin (PG) that exist in semen, as well as IL10 and IFN-γ secreted by uNK cells, CSF2, and chemokines released by uterine epithelial cells, contribute to macrophages and DCs acquiring tolerogenic DCs (tDCs) and M2 phenotypes [ 52 – 54 ] (Fig. 2 a). Subsequently, the tDCs induce the differentiation of Th0 cells into peripheral Tregs (pTregs) by presenting paternal antigens [ 48 ]. These cells, along with thymus-derived Tregs (tTregs) cells in response to epithelial cell-derived chemokines such as CCL19, CCL4, CCL3, and CCL5, migrate to the uterus pre- and during implantation [ 48 , 55 , 56 ] (Fig. 2 a). Decidual Treg cells release IL10 and TGFB1 as well as possess CD25, CD274, and cytotoxic T-lymphocyte associated protein 4 (CTLA4), which are all key factors involved in Treg-mediated suppression. These factors likely have a role in limiting the activity of effector T cells (Teff) during the early stages of pregnancy [ 48 , 49 ]. Fig. 2 Role of cytokine and chemokine networks in implantation. a  MQs and DCs transform into M2 and tDCs phenotypes in response to cytokines secreted by uNK and semen. Then tDCs differentiate TH0 into pTregs, which, together with tTregs, migrate to the uterus in response to chemokines produced by epithelial cells. b  In addition to recruiting immune cells to the uterus, chemokines can also be directly involved in implantation; for example, CCL3/CCL5, CXCL12, and CXCL16 modulate invasion via interacting with their receptors on the trophoblast cells. c  Also, trophoblast-derived CXCL12 facilitates the accumulation of uNK cells. uNK: Uterine natural killer cells, MQ: Macrophage, DCs: Dendritic cells, tDCs: Tolerogenic dendritic cells, M2: M2 macrophages, TH0: T helper, pTregs: Peripheral regulatory T cells, tTreg: Thymus-derived regulatory T cell, TGFB1: transforming growth factor beta 1, IL-10: Interleukin-10, IFN-ᵞ: Interferon-ᵞ, Tr: Trophoblast. (Created by biorender) Role of cytokine and chemokine networks in implantation. a  MQs and DCs transform into M2 and tDCs phenotypes in response to cytokines secreted by uNK and semen. Then tDCs differentiate TH0 into pTregs, which, together with tTregs, migrate to the uterus in response to chemokines produced by epithelial cells. b  In addition to recruiting immune cells to the uterus, chemokines can also be directly involved in implantation; for example, CCL3/CCL5, CXCL12, and CXCL16 modulate invasion via interacting with their receptors on the trophoblast cells. c  Also, trophoblast-derived CXCL12 facilitates the accumulation of uNK cells. uNK: Uterine natural killer cells, MQ: Macrophage, DCs: Dendritic cells, tDCs: Tolerogenic dendritic cells, M2: M2 macrophages, TH0: T helper, pTregs: Peripheral regulatory T cells, tTreg: Thymus-derived regulatory T cell, TGFB1: transforming growth factor beta 1, IL-10: Interleukin-10, IFN-ᵞ: Interferon-ᵞ, Tr: Trophoblast. (Created by biorender) Regulation of embryo-endometrium communication is primarily achieved via the effects of various cytokines and chemokines [ 57 ]. Parallel with the immunological adaptations necessary for implantation, the embryo experiences division and differentiation to reach the stage of blastocyst. After the zona pellucida dissolves, the blastocyst attaches itself to the receptive endometrium. The maternal immunological system also contributes to these processes through cytokine signaling, which regulates embryonic development [ 58 , 59 ]. Optimal implantation requires the presence of both inflammatory and anti-inflammatory cytokines, and maintaining an appropriate balance between them is critical [ 57 ]. Inflammatory cytokines appear to trigger trophoblast invasion and remodeling of spiral arteries by establishing an inflammatory milieu. Among these cytokines, IL-1β is one of the most critical mediators, which, with secretion from trophoblast cells, decidual cells, as well as other leukocytes such as T cells, macrophages, and NK cells, plays an essential role in the process of decidualization and successful implantation [ 60 , 61 ]. One of the vital factors in the decidualization process is cyclic adenosine monophosphate (cAMP), so IL1B increases the level of cAMP in stromal cells through the induction of cyclooxygenase 2 (COX2) expression and consequently the production of prostaglandin E2 (PGE2) [ 62 ]. Studies have demonstrated that IL1B can additionally stimulate the production of MMP9, a crucial enzyme that mediates trophoblast invasion [ 63 ]. In a recent study, Zhi Ma et al. [ 64 ] discovered that during implantation, IL1B raises the level of endometrial sialyl Lewis X (sLeX) that binds to L-selectin on the surface of trophoblast cells and facilitates the embryo-endometrial interaction. Another endometrial receptivity marker upregulated by IL1B is the integrin β3 [ 63 ]. Additional inflammatory cytokines involved in successful implantation are TNF, IFN-γ, IL17A, and IL22. A model of the inflammatory network necessary for implantation by TNF and IL17A has been proposed. TNF stimulates IL17A production in stromal cells, and subsequently IL17A increases the expression of CXCL1, CXCL8, IL6, CSF2, CD274, MMP9, and MMP3 in trophoblast cells. This process improves trophoblast migratory and invasion capabilities [ 65 ]. Thus, IL17A deficiency could be associated with implantation failure, while other research indicates a link between extensive high IL17A and miscarriage [ 66 , 67 ]. The simultaneous production of IL17A and IL4 appears to affect pregnancy outcome. In successful pregnancies, IL17A coexists with IL4, resulting in a balanced immune response, but in cases of unexplained recurrent miscarriage, IL17A is mostly produced without IL4 [ 68 ]. Similar to IL17A, IL22 might be another interleukin essential for pregnancy maintenance if it is generated concurrently with IL4 [ 69 ]. IL22 is a cytokine with reparative properties in epithelial cells [ 70 ]. Its receptor (IL-22R1) has been identified on the surface of trophoblast cells, and evidence suggests that reduced expression of this receptor is associated with the occurrence of spontaneous miscarriage. Accordingly, IL22 plays an important role in the success of implantation and pregnancy continuation by regulating the survival and proliferation of trophoblasts [ 71 ]. Conversely, IFN-γ, by a process that induces trophoblast cell apoptosis, preserves maternal tissue against excess invasion [ 72 ]. Cytokines with anti-inflammatory effects have vital roles in several processes, such as decidualization and placentation, embryonic development, and establishing tolerance between the mother and fetus, which are required for a successful pregnancy. In addition, these mediators facilitate essential events at all implantation phases, including apposition, adhesion, invasion, and migration of trophoblast cells inside the decidua [ 57 , 73 ]. Cytokines that are classified under the IL6 family category, especially LIF, IL6, and IL11, effectively support the processes as mentioned above [ 59 , 74 ], and the unique property of them is their dependence on gp130 for signaling to exert their function [ 75 , 76 ]. LIF is the most important IL6 family cytokine in the implantation process [ 77 ]. Through activating its receptor and the transcription factor STAT3 signaling pathway, LIF is recognized for supporting molecular and cellular interaction at the embryo-maternal interface [ 78 ]. Considering that the receptors of this cytokine are expressed both in the preimplantation blastocyst and in the endometrium, LIF could interact with embryonic as well as uterine tissues at the time of implantation and is also responsible for the infiltration of specific subgroups of immune cells, especially macrophages and NK cells, which play a role in an inflammatory reaction during implantation [ 79 ]. In a study by inducing LIF receptor deficit in uterine epithelial cells of mice, researchers demonstrated that LIF is crucial in forming the implantation chambers and, consequently, the effective attachment of blastocyst [ 80 ]. IL6, another member of the IL6 family, has activities similar to LIF during implantation [ 81 ]. In humans, this cytokine exhibits low expression throughout the proliferative phase but reaches its maximum expression level in the mid-luteal phase [ 82 ]. Microarray investigation of the mid-luteal phase endometrium in women with repeated implantation failure (RIF) reveals reduced IL6 expression compared to normal control groups [ 83 ]. Like the LIF receptor, the IL6 receptor is present in both the endometrium and the trophoblast. As a result, IL6 triggers trophoblast invasion by upregulating the expression of β1, α5, and α1 integrins in trophoblasts, as well as the stimulation of MMP9 and MMP2 [ 82 ]. In addition to the cytokines already noted, there exist other anti-inflammatory cytokines such as IL10, IL4, IL11, and TGFB1 that regulate establishing tolerance between the mother and embryo in implantation [ 58 ]. Similar to the cytokines that were discussed, chemokines, along with their receptors, are required for the processes associated with successful implantation and are widely expressed by decidual stromal cells, trophoblast cells, and decidual immune cells at the embryo-maternal interface [ 84 , 85 ]. Sexual steroid hormones can directly as well as indirectly influence the expression of various chemokines throughout the menstrual cycle. For example, progesterone increases the expression levels of CXCL8 and CCL2, as well as CXCL11 (ITAC) and CXCL10 (IP10) [ 58 ]. Chemokines, such as CCL3/CCL5 [ 86 ], CXCL12 [ 87 ], and CXCL16, regulate invasion via interacting with their receptors on the trophoblast surface [ 85 ] (Fig. 2 b). Extravillous trophoblasts (EVTs) then express dipeptidyl peptidase IV (DPPIV), which metabolizes CCL5, to prevent excessive invasion [ 88 ]. CXCL12 and CXCL16, in addition to their role in invasion, are involved in the function and regulation of decidual immune cells. For example, Tao Y et al. [ 89 ] found that CXCL12/CXCR4 is essential for the attraction of CD25⁺ decidual NK (dNK) cells and facilitates the CD3 ¯ CD56bright CD25⁺dNK cell accumulation at the maternal-embryo interface (Fig. 2 c). Additional chemokines involved in NK cell chemotaxis, produced by decidualized stromal cells during the mid-late secretory phase, include CCL22, CCL7, CX3CL1, CCL21, and CCL4 [ 90 ]. CXCR6/CXCL16 can attract and facilitate the migration of T cells to the decidua, contributing to the immunological regulation in pregnancy [ 85 ]. Also, CCL19 and CCL5 are expressed during the mid-secretory phase, which is essential for attracting Treg cells [ 91 ]. During the process of preparing the uterus for embryo implantation, the endometrium undergoes a receptive phenotype, which is characterized by changes in gene expression as well as diverse structures. Increasing evidence suggests that these adaptations play a crucial role in controlling embryo invasion and endometrial angiogenesis [ 92 ]. Inappropriate changes in endometrial gene expression can result in unsuccessful implantation. Table 1 demonstrates the significance of some critical genes and their associated expression products regarding successful implantation and infertility disorders. Table 1 Some genes and their associated expression products regarding endometrial receptivity, their effect on successful implantation, and infertility Gene/expression product Effect on implantation Infertility Ref. PAEP /GdA Suppression of maternal immunity, shifts the Th1/Th2 ratio toward a Th2-dominated ratio, proliferation of the endometrium Lower expression in patients with RIF, RPL, and endometriosis [ 93 – 98 ] SPP1 /OPN Mediation of endometrial-trophoblast interactions in implantation Association of abnormal expression with idiopathic infertility, PCOS, and subfertility in patients with endometriosis [ 95 , 99 , 100 ] HOXA10 / HOXA10 expression of αVβ3 integrin and E-cadherin downregulation in patients with RIF, Endometriosis, and RM [ 95 , 101 , 102 ] LPAR3 /LPAR3 Stimulation of COX2 and PG signaling Reduced expression in endometriosis [ 95 ] CD55 /DAF Protection of the embryo from maternal complement and preventing epithelium destruction Decreased in RPL patients with APS, and PCOS women [ 103 , 104 ] MIR30D /miR-30d-5p upregulation throughout the achievement of endometrial receptivity Reduced expression in RIF patients [ 105 ] PTGS2/ COX2 Production of PGs and the decidualization process Multiple mutations in this gene result in RIF [ 106 ] IL15 /IL15 Proliferation of uNK and regulation of invasion Association of IL15 deficiency with reduced uNK cells in women with RIF [ 103 ] Abbreviations: PAEP Progestagen-associated endometrial protein, GdA Glycodelin-A, RIF Recurrent implantation failure, RPL Recurrent pregnancy loss, SPP1 Secreted phosphoprotein 1, OPN Osteopontin, PCOS Polycystic ovarian syndrome, HOXA Homeobox A, RM  Recurrent miscarriage, LPAR3 Lysophosphatidic acid receptor 3, COX Cyclooxygenase, PG Prostaglandin, DAF Decay accelerating factor, APS Antiphospholipid syndrome, miR MicroRNA, PTGS2 Prostaglandin-endoperoxide synthase 2, IL  Interleukin, uNK Uterine NK cell Some genes and their associated expression products regarding endometrial receptivity, their effect on successful implantation, and infertility HOXA10 / HOXA10 Decreased in RPL patients with APS, and PCOS women Reduced expression in RIF patients PTGS2/ COX2 Abbreviations: PAEP Progestagen-associated endometrial protein, GdA Glycodelin-A, RIF Recurrent implantation failure, RPL Recurrent pregnancy loss, SPP1 Secreted phosphoprotein 1, OPN Osteopontin, PCOS Polycystic ovarian syndrome, HOXA Homeobox A, RM  Recurrent miscarriage, LPAR3 Lysophosphatidic acid receptor 3, COX Cyclooxygenase, PG Prostaglandin, DAF Decay accelerating factor, APS Antiphospholipid syndrome, miR MicroRNA, PTGS2 Prostaglandin-endoperoxide synthase 2, IL  Interleukin, uNK Uterine NK cell During the WOI, the endometrium expresses structures called adhesion molecules, such as mucins and integrins, that facilitate the effective attachment of the embryo to the uterine surface. MUC1 is a common glycoprotein that is expressed throughout both the proliferative and secretory phases of the menstrual cycle. However, the presence of MUC1 hinders the process of implantation, and reducing its levels could potentially enhance the ability of the endometrium to receive an embryo. Researchers have proposed that humans require a mechanism that acts locally to remove the MUC1 barrier to embryo implantation [ 107 , 108 ]. The repellent activity of MUC1 may have an essential function in directing the blastocyst to the most suitable location for implantation [ 108 ]. Francis et al. [ 109 ] demonstrated that MUC1, while possessing anti-adhesion characteristics, can have adhesive ability when it is significantly glycosylated and that its interaction with L-selectin improves the attachment of the blastocyst to the endometrium. In a study of patients experiencing RIF, it was found that MUC1 significantly diminished in both the glandular and luminal epithelium [ 110 ]. Additionally, women diagnosed with recurrent pregnancy loss (RPL), endometriosis, and PCOS have been shown to have lower levels of endometrial MUC1 [ 111 ]. Integrins are another type of adhesion molecule developed by endometrial cells, with hormonal regulation governing their expression throughout the menstrual cycle. Three specific integrins, including αVβ3, α1β1, and α4β1, exhibit distinct expression patterns in the endometrium, and due to the existence of αVβ3 ligands in the embryo and endometrium, this integrin is of particular importance in the embryo-endometrium interaction and implantation process [ 112 , 113 ]. A study showed that when LIF binds to its target receptor, it increases the expression of αVβ5 and αVβ3 in endometrial cells, so this association facilitates the attachment of the trophoblast to the endometrium during blastocyst implantation [ 114 ]. The aberrant expression of αVβ3 has been linked to disorders such as PCOS [ 115 ], endometriosis [ 116 , 117 ], and unexplained infertility [ 118 ]. Given that αVβ3 is recognized as a biomarker for implantation, assessing the mRNA level of integrin β3 on day 21 can be valuable in estimating the successful outcome of in vitro fertilization (IVF) [ 119 ]. According to a study by Sarah G. Paule et al. [ 120 ], podocalyxin (PCX) was introduced as a novel adhesion-related structure in the human endometrium that is downregulated by the presence of progesterone during the mid-secretory stage, when the endometrium is preparing for implantation. Furthermore, the findings suggest that PCX inhibits the adherence and penetration of trophoblastic spheroids [ 120 ]. Trophoblastic spheroids are three-dimensional (3D) cultures used to investigate trophoblast invasion in vitro, since they precisely imitate the properties of their origin tissue and the in vivo invasion process [ 121 ]. Overall, based on the review of studies presented in this section, abnormal endometrial cell differentiation and immune cell distribution contribute to an improper uterine environment that adversely affects fertility. Challenges in the assessment of human endometrial activity and preliminary pregnancy in vivo have resulted in the establishment of reproductive models. Mice are one of the animal models employed to study several aspects of human reproduction [ 26 , 51 , 80 ]. But mice and humans are not exactly alike. For example, in mice, decidualization is a process that depends on embryonic signals, while in humans, the onset of this process is independent of the embryo’s presence and occurs spontaneously in each menstrual cycle [ 8 ]. Numerous studies on endometrial modifications rely on monolayer endometrial cell culture models. However, a significant drawback of these models is their limitation to a singular endometrium cell type, ignoring the epithelial-stromal communication present in the in vivo milieu, along with the contributions of local immunological and endothelial cells [ 122 ].

Pregnancy is an intricate and permanent process that involves several stages, such as implantation of embryos, decidualization, placentation, and ultimately the birth of infants by parturition [ 1 ]. The interaction between a receptive uterus and an appropriate blastocyst can only happen within a specific time, referred to as the “window of implantation” (WOI), and the rate of spontaneous abortion is significantly increased when implantation occurs outside this time [ 2 ]. In humans, the menstrual cycle consists of proliferative (follicular) and luteal (secretory) phases. In the follicular phase, the increase in estrogen level leads to the proliferation of the epithelium, vascular endothelium, and stroma with the aim of endometrial regeneration, which is followed by the thickening of the endometrium and the secretion of progesterone in the luteal phase to prepare the uterus for implantation [ 3 ]. Implantation consists of three distinct stages, making it a multifaceted process. In the initial stage, known as apposition, the embryo does not make direct physical contact with the endometrium; however, there is a mutual cross-talk facilitated by soluble molecules [ 4 ]. During the subsequent phase, termed adhesion, the human blastocyst comes into direct contact with the epithelial cells of the endometrium through interactions between ligands and receptors, resulting in the formation of a stronger and more stable connection compared to the preceding stage [ 5 ]. Finally, after reaching the invasion stage, the trophectoderm (TE) cells initiate a process of differentiation, stimulating the embryonic trophoblast to invade the epithelium; as a consequence, the maternal spiral arteries are remolded, and the small vessels with high resistance are replaced by larger vessels with lower resistance [ 6 ]. The embryo and the endometrium are key players in implantation, both of them establishing effective embryo-maternal cross-talk through necessary adaptations. The focus of this review is the investigation of the alterations in the immune system and gene expression within the endometrium, along with exploring the embryonic surface and secretory markers crucial for successful implantation.