Molecular Biomarkers of Endometrial Function and Receptivity in Natural and Stimulated Assisted Reproductive Technology (ART) Cycles

Abstract

The success of embryo implantation and pregnancy depends on a complex interaction between the trophoblast and the endometrial environment, where endometrial receptivity plays a crucial role in this process. Assisted reproductive technologies (ARTs) are essential in overcoming biological barriers and enabling implantation in women with fertility issues. However, one of the main challenges in ART is ensuring that the endometrium is receptive at the time of implantation. Therefore, identifying endometrial receptivity biomarkers is essential to optimize ART treatments, improving success rates. A comprehensive literature review was conducted by searching peer-reviewed articles published in PubMed, Scopus, and Web of Science databases. The search included studies focusing on molecular and cellular mechanisms underlying endometrial receptivity in both natural and stimulated cycles. Various experimental methods, including proteomic and microRNA studies, have identified key biomarkers involved in endometrial receptivity, such as adhesion molecules, growth factors, and others. However, ovarian stimulation in fertility treatments can alter endometrial receptivity, making approaches like frozen embryo transfer necessary. Despite advancements, many questions persist regarding the endometrial receptivity and implantation mechanisms in both natural and stimulated cycles. This article reviews the main molecules involved in endometrial receptivity in natural and stimulated cycles, highlighting their potential role as biomarkers for embryo implantation.

1. Introduction

Assisted reproductive technology (ART) represents a significant advancement in the treatment of human infertility, enabling the management of many causes that can hinder natural conception. These procedures are particularly effective in addressing issues such as ovulation disorders, which impair the release of mature oocytes, fertilization failure, or even cases of Fallopian tube obstruction, which can impede fertilization [1]. Despite advancements in ART, approximately 30–40% of women fail to conceive after embryo transfer [2]. The need to better understand and redefine the concept of recurrent implantation failure (RIF) arises in consideration of new technologies and advances in reproductive biology, especially when failures occur despite the transfer of genetically normal embryos, which should ensure successful implantation. This highlights the importance of more accurately identifying whether the causes are related to embryonic issues or other factors, such as endometrial receptivity. In fertile women, the receptive phase lasts about 5 days and occurs in a period that corresponds to the interval from the seventh to the eleventh day of the secretory phase (LH +7 up to LH +11) in an endometrial regular cycle, which corresponds to the Window of Implantation (WOI) [3,4,5]. Transcriptomic studies have shown that the WOI is not fixed but varies among individuals. This variability can lead to asynchrony between the endometrium and embryonic development, which is a common cause of in vitro fertilization (IVF) failure. Furthermore, ovarian stimulation can further alter endometrial timing, causing the WOI to close earlier before embryo transfer [6]. After ovulation, if fertilization occurs, the endometrium provides a binding site and nutrients for the embryo until the placenta develops [7,8,9]. Successful implantation depends on a receptive endometrium, a functional embryo at the blastocyst stage, and a coordinated molecular interaction between maternal and embryonic tissues [4,8,10,11,12].

In ART, embryo development to the blastocyst stage (days 5–6) enables the selection of the most viable embryos, with the highest likelihood of implantation. This stage reflects the embryo’s ability to interact with the endometrium and sustain its own development. At this stage, the blastocyst must complete the process of hatching from the zona pellucida to make direct contact with the endometrium and thus initiate the process of implantation in the uterus [13].

However, factors related to endometrial receptivity, such as the quality of the uterine environment, immune responses, and hormonal variations, can pose a challenge even when the embryo is of good quality and has reached the blastocyst stage. In this context, despite significant advances in assisted reproduction techniques, the interaction between the embryo and the endometrium remains a complex process, strongly influenced by hormonal balance. In this regard, estrogens and growth factors play a crucial role in regulating the proliferation and differentiation of endometrial cells, processes essential for successful implantation. In particular, the expression of specific receptors for these hormones and factors allows for the creation of an optimal microenvironment for the initiation of pregnancy. Consequently, any alteration in the regulation of these mechanisms can compromise endometrial preparation, leading to implantation failure. On the other hand, pathological conditions such as adenomyosis, endometriosis, endometritis, insulin resistance, obesity, and polycystic ovary syndrome (PCOS) can also interfere with endometrial receptivity through hormonal imbalances, immune dysfunction, inflammatory processes, and changes in gene expression, ultimately contributing to implantation failure [14].

For that reason, assessing endometrial receptivity is crucial for identifying the optimal time for implantation and improving the chances of success in assisted reproductive technologies. To this end, several experimental methods have been developed, including thin-layer chromatography, immunohistochemistry, inverse polymerase chain reaction (PCR), DNA microarrays, microRNA studies, and proteomic analysis, which have allowed the identification of key factors regulating the interaction between progesterone and the endometrium, cell proliferation, chemotaxis, and epidermal growth [15,16,17,18,19,20,21,22,23,24,25,26]. Furthermore, recent advances in the Endometrial Receptivity Array (ERA) test have further improved the assessment of the optimal implantation window by analyzing gene expression related to these factors [27].

Concomitantly, in clinical practice, ovarian hyperstimulation can alter endometrial receptivity, making the use of frozen embryos and hormonal preparation of the endometrium a strategic approach [28].

However, in spite of the efforts and research, a high number of questions remain with no answer, such as the accurate mechanisms regulating the implantation and endometrial receptivity in natural and stimulated cycles that could improve the ability to treat infertility, recurrent miscarriages, and develop effective strategies that positively impact the clinical outcomes of ART.

The aim of this evidence-based study was to describe the most important molecules that are known to have a role in endometrial function and receptivity during the natural and stimulated cycles and their possible function as biomarkers to summarize the current understanding of their regulation and changes in both physiological and pathological conditions. The originality of the present work lies in presenting an integrated and comparative analysis of the molecular mechanisms regulating endometrial receptivity in both natural and stimulated cycles, linking these mechanisms to clinical outcomes in ART. This review synthesizes hormonal, molecular, and pathological factors, highlighting their interplay and potential utility as biomarkers. This comprehensive perspective aims to clarify why implantation may fail, thus providing a complete picture of current knowledge regarding endometrial receptivity and the potential clinical implications for ART.

2. Endometrial Receptivity and Implantation

The human endometrium is composed of both glandular and luminal epithelium, as well as stroma, which is made up of mesenchymal cells, leukocytes, endothelium, and smooth vascular muscle. These components undergo biochemical and morphological changes through the menstrual cycle that facilitate the development of pregnancy. The implantation process requires complex cell signaling to lead to the endometrium being receptive in a short period; however, the disruption of this time negatively impacts pregnancy success, increasing the spontaneous abortion rate [29].

Implantation begins with the molecular interaction between the blastocyst and the receptive endometrium, mediated by hormones and growth factors [30]. This process involves the apposition of the blastocyst to the luminal epithelium, followed by adhesive contact and invasion of maternal tissues [31] (Figure 1). During the apposition phase, trophoblast cells adhere to endometrial luminal receptors via pinopods on the uterine surface [32,33], stabilizing the adhesion between the blastocyst, basal endometrium, and stromal extracellular matrix. This event triggers a signaling cascade critical for pregnancy establishment, involving cell adhesion factors, cytokines, growth factors, lipids, and other molecules [4,12,26]. In women, the reaction occurs between days 20 and 21 of the cycle, coinciding with increased stromal vascular permeability at the binding site [34].

The final phase of implantation involves the invasion of the blastocyst into the endometrial stroma, which is primarily regulated by trophoblast cells, while the decidua limits the depth of this invasion [35]. This process depends on interactions between cells and the extracellular matrix (ECM) [36] and is mediated by the combined action of cell adhesion molecules and metalloproteinases (MMPs), which degrade the ECM and initiate the cascade necessary for implantation and early embryonic development [37]. Human cytotrophoblastic (CTB) cells exhibit invasiveness through their ability to secrete MMPs [38].

In response to trophoblastic invasion and progesterone distribution, endometrial stromal cells and the extracellular matrix undergo decidualization, with remodeling of uterine glands and vessels and the influx of specialized uterine natural killer (NK) cells [39]. After implantation, the trophoblast differentiates into extraembryonic tissues, assuming key functions such as hormone secretion and the exchange of nutrients and wastes between the fetus and mother through the fetoplacental circulation. Hormone production is carried out by the multinucleated syncytiotrophoblast, while uterine invasion is facilitated by extravillous cytotrophoblast (EVCT) cells [40].

3. Molecular Dynamics of Endometrial Function in Normal and Stimulated Conditions

Endocrine and paracrine mechanisms play a crucial role in the synchronization between the embryo and the endometrium [14], creating a complex system in which sex hormones and growth factors interact to ensure the proper function of the endometrium. These mechanisms are crucial for regulating the proliferation and differentiation of endometrial cells, thereby preparing them for the potential implantation of an embryo.

Estrogens induce the proliferation of both epithelial and stromal cells by regulating the synthesis and secretion of growth factors. In the early stages of endometrial regeneration, epithelial cells migrate from glandular remnants to the surface within 48 h of tissue removal, despite low circulating estrogen levels and the absence of estrogen receptor α (ERα) in epithelial cells [41]. Subsequently, estrogens stimulate epithelial and stromal cell proliferation by regulating the synthesis and secretion of transforming growth factor beta (TGFβ), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) [42]. These growth factors also contribute to the decidualization of stromal cells [43].

In particular, in decidualized stromal cells, the IGF-binding proteins, IGFBP-1 and IGFBP-3, limit the mitogenic effects of IGF-1 during endometrial differentiation [44]. Estrogens also enhance the expression of EGF receptors [45], which mediate the proliferative actions of EGF and transforming growth factor-alpha (TGF-α) [42,46].

Furthermore, hepatocyte growth factor (HGF), produced by endometrial stromal cells, stimulates epithelial cell proliferation through paracrine signaling, independently of sex steroid hormones [43,47]. Other factors, such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), promote stromal cell proliferation through autocrine interactions with their respective receptors, Fibroblast Growth Factor Receptor 1 (FGF-R1) and Platelet-Derived Growth Factor Receptor beta (PDGF-Rβ), during the proliferative phase [48,49,50].

In parallel, endometrial epithelial cells synthesize and secrete several factors, including EGF, TGFα, and IGF-I. During the phase of rapid epithelial proliferation, the expression of receptors for TGFα, IGF-I, bFGF, epidermal growth factor receptor (EGFR), and PDGFβ increases. HGF, leukaemia inhibitory factor (LIF), and stem cell factor (SCF) are also thought to play key roles in endometrial cell proliferation, angiogenesis, implantation, and potentially regeneration. In addition, these growth factors may further modulate the effects of estrogen and progesterone by regulating the expression of their respective receptors [50,51]. The final purpose of the differentiation and maturation of these components during the menstrual cycle is to ensure the appropriate conditions for implantation and establishment of pregnancy [50,52].

During ovarian stimulation protocols for IVF, it has been shown that the molecular profile of the endometrium is altered, affecting the levels of cytokines and enzymes crucial for endometrial receptivity. For instance, studies have shown that certain mediators, such as interleukin(IL)-1β, IL-5, IL-10, and tumour necrosis factor (TNF)-α, exhibit significantly higher concentrations in endometrial secretions during stimulated cycles compared to natural cycles. Conversely, lower levels of monocyte chemoattractant protein-1 (MCP-1) have been associated with higher implantation rates, as it attracts fewer uterine NK cells, whose excessive accumulation can hinder fertility. Similarly, vascular endothelial growth factor (VEGF) has been detected at lower levels during ovarian stimulation, suggesting a potential negative impact on uterine angiogenesis [53] (Figure 2). Similar alterations were also observed at the gene level in endometrial biopsies from fertile donors after gonadotropin-releasing hormone (GnRH) agonist stimulation, affecting genes related to cytokines, growth factors, and the immune response. Furthermore, changes in NK cell signaling and extracellular matrix degradation were detected, consistent with the functional changes observed in endometrial secretions [54]. A study suggested that ovarian stimulation may impair the endometrium’s ability to support embryo implantation properly. Specifically, the analysis of endometrial gene expression profiles during the window of implantation showed that after ovarian stimulation, many genes involved in endometrial receptivity were abnormally expressed. These changes in gene expression make the endometrium less receptive, hindering and delaying its development [55]. However, administration of a single dose of GnRH a during the luteal phase has also been reported to improve the implantation rate in assisted reproduction cycles [56], likely due to significant changes in the expression of genes targeting the ECM pathway and adhesion molecule genes [57].

4. Biomarkers of Endometrial Receptivity

The description of molecular biomarkers of endometrial receptivity has opened an extensive field of investigation that aims to improve the implantation rates in natural or stimulated cycles for ART techniques or even promote a new model of contraception. The criteria for selecting biological markers are that they should be present in the receptive phase at the implantation site of the endometrium and disappear after the receptive window. The problem is that the number of candidates is constantly increasing.

5. Evolution in Receptivity Evaluation Methods

5.1. Histology and Noyes Criteria

The histology dating of the human endometrium has been used as a relevant criterion during the evaluation of an infertile couple [58] to determine if ovulation has been achieved, with the formation of the corpus luteum in the ovary and the progesterone rise effects in the glands [59,60]. However, it was shown in 49% of fertile women an ‘out of phase’ endometrium based on Noyes’ criteria [61], highlighting how the precision required for accurate dating is difficult to achieve [62]. For this reason, many markers help in the histologic criteria evaluation for clinical and research purposes. In a previous study, it has been demonstrated that endometrial dating based on Noyes’ criteria did not show differences between untreated women and those stimulated with CC and recombinant follicle-stimulating hormone (rFSH) [33,63].

5.2. Pinopods or Uterodomes

Another important feature during the endometrial receptivity period is the appearance of transient membrane projections called pinopods, visible between days 20 and 21 of the regular menstrual cycle and dependent on progesterone [64]. These anchor-like structures facilitate the molecular interactions required to retain the blastocyst, likely by removing uterine fluid and preventing cilia from penetrating the blastocyst [65]. Human studies suggest that, as they do not perform a pinocytic function, pinopods are more correctly termed uterodomes [66,67]. Their appearance is limited to less than 48 h per cycle and correlates with serum progesterone concentrations and downregulation of the progesterone receptor B [60,68]. In contrast, in hyperstimulated ovarian cycles, formation occurs earlier, between days 18 and 20 [69].

The observation that pinopods are clinically relevant is based on the fact that their appearance coincides with the loss of steroid receptors and with the maximal expression of integrin α(v)β(3), osteopontin, LIF, and its receptor, indicating a correlation with other markers of endometrial receptivity [69]. A high score of pinopods (>85%) is associated with greater pregnancy success, while their absence or paucity is associated with lower implantation rates and increased risk of spontaneous abortion. However, their reliability as a biomarker remains controversial due to the variability in observation and classification methods [64].

6. Molecular Factors

6.1. Growth Factors and Cytokines

The study of endometrial physiology has revealed crucial insights into the mechanisms regulating implantation and pregnancy. Morphological and biochemical mechanisms of action for several growth factors and cytokines have recently been elucidated, primarily during the process of decidualization. For instance, LIF, a member of the IL-6 cytokine family, which also includes oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin 1 (CT-1) [70], binds to its specific receptors and induces dimerization with glycoprotein (gp) 130. This forms a high-affinity receptor complex that activates several signaling pathways, including the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, the Src homology 2 domain-containing tyrosine phosphatase (SHP-2)/Ras/extracellular signal-regulated kinase (ERK) pathway, and the phosphatidylinositol-3-kinase (PI3K)/Akt pathway [71].

Some authors have observed that LIF mRNA is highly expressed in the cervical epithelium during the implantation phase in both mice and humans [72]. Studies on mouse models have demonstrated that wild-type embryos fail to implant in the endometrium of females homozygous for LIF gene deficiency. However, implantation could be restored through LIF supplementation [73,74]. In fertile women, LIF mRNA and protein are present in glandular and luminal epithelium, between days 18 and 28 of a 28-day menstrual cycle. Similarly, LIF receptor mRNA and protein are expressed in the epithelial compartments during the secretory phase, correlating with the formation of pinopods [75,76,77,78].

During the first trimester, LIF is expressed in the chorionic villi, decidual leukocytes, and uterine NK cells. It also regulates trophoblast invasiveness and influences immune tolerance by modulating the expression of human leukocyte antigen-G (HLA-G) in invasive cytotrophoblast cells during implantation [71,79,80]. A microarray study on 43 endometrial genes in women in the mid-secretory phase, who received intracytoplasmic sperm injection (ICSI), demonstrated a positive correlation with 6 genes and pregnancy success, including LIF [81,82]. In fertile and infertile women treated with the selective estrogen modulator (SERM) ormeloxifene, LIF immunostaining was reported to be normal, although the expression of estrogen and progesterone receptors was altered in endometrial tissue [83].

A study investigating the expression of several key markers—progesterone receptor-B (PGR-B), LIF, glycodelin/progestagen-associated endometrial protein (PAEP), homeobox A10 (HOXA10), heparin-binding EGF-like growth factor (HB-EGF), calcitonin, and chemokine ligand 14 (CXCL14)—in fertile and infertile women revealed a significant cyclic and tissue-specific regulation of each protein, with marked dysregulation in infertile subjects. Correlation analyses also suggested potential interactions among these proteins, which appeared disrupted in infertility [84]. Further in vitro evidence supports the role of these growth factors in endometrial receptivity and pregnancy. Experiments assessing the expansion of spheroids stimulated with EGF, HB-EGF, IL-1β, and LIF showed a stronger attachment to undifferentiated endometrial stromal cells (ESCs) than to standard culture surfaces. Moreover, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an adhesion molecule involved in trophoblast invasion, was upregulated by conditioned medium from decidualized ESC and by the combined action of HB-EGF, IL-1β, and LIF. These findings suggest that decidualized ESC promotes trophoblast invasion through paracrine signaling mechanisms that may involve HB-EGF, IL-1β, and LIF [84,85].

6.2. Vascular Endothelial Growth Factor (VEGF)

Among angiogenic factors, VEGF is considered crucial for physiological neovascularization, such as cyclic-dependent angiogenesis in the endometrium, and acts as a stimulator of microvascular permeability. The presence of VEGF in the human endometrium has been confirmed by the detection of its mRNA, protein, and receptors within endometrial tissue. In both fertile and infertile women, the expression of VEGF and its receptors is cyclically modulated by ovarian steroids during the secretory and proliferative phases [86]. Several studies have examined the link between VEGF levels and embryo implantation failure. While some have found elevated serum VEGF levels in women with RIF compared to fertile women, others have reported a decrease in VEGF expression in the endometrium during embryo implantation. Despite these discrepancies, evidence suggests that estrogen and progesterone enhance VEGF expression in endometrial stromal and vascular endothelial cells during early pregnancy, supporting the hypothesis that VEGF is a key mediator in the implantation process [87].

Thus, an endometrium with good vascularization seems to be crucial for proper receptivity and the success of embryo implantation. Women with unexplained infertility tend to have lower VEGF expression compared to fertile women, who, in contrast, present higher VEGF levels. This suggests that increased angiogenesis in the endometrium could promote better implantation and improved pregnancy outcomes, and that VEGF could be used as a marker of endometrial receptivity, providing valuable information to predict treatment success and personalize therapies for patients with unexplained infertility [88].

6.3. Cell Adhesion Molecules (CAMs)

During the WOI, the expression of CAMs in the endometrium and embryo is elevated, and this contributes to blastocyst attachment to the uterine mucosa. The CAM family comprises integrins, cadherins, selectins, and immunoglobulins, which are surface ligands and glycoproteins that mediate cell-to-cell adhesion [89].

Integrins are a family of transmembrane glycoproteins with two non-covalent α and β subunits [90]. In the human endometrium, three cycle-specific integrins—α1β1, α4β1, and αVβ3—are co-expressed; however, only the β3 mRNA subunit exhibits a marked increase after day 19 of the menstrual cycle, being undetectable before this stage [60]. αVβ3 integrin and its ligand, osteopontin, were positively detected by immunohistochemistry on the luminal epithelial surface of the endometrium, where it first interacts with the trophoblast [91] and has been suggested as a potential receptor for embryonic attachment [92]. The cycle-specific pattern of integrin expression in the endometrium suggests that it is hormonally regulated. The expression of αVβ3 integrin in the human endometrium is enhanced by growth factors such as EGF and HBEGF, while it is reduced by 17β-estradiol [93]. During the proliferative phase, elevated estrogen levels acting through the ERα suppress integrin expression. In the subsequent luteal phase, progesterone reduces the number of estrogen receptors, thereby counteracting this inhibitory effect of estrogen (E2) on integrins. Progesterone may also enhance the production of paracrine stromal factors, such as EGF and HB-EGF, which stimulate epithelial β3 integrin expression—considered the rate-limiting step in αVβ3 formation [92]. Furthermore, the homeobox gene HOXA10 plays a role in regulating β3 subunit expression, as treatment of cultured endometrial cells with HOXA10 markedly increases β3 expression [94].

6.4. Cadherin

Cadherins are a group of calcium-dependent glycoproteins that mediate cell-to-cell adhesion through a homophilic mechanism, comprising E-, P-, and N-cadherins, each with distinct tissue distributions. In the human endometrium, E-cadherin is the most extensively studied and plays a key role in maintaining epithelial cell junctions [95,96]. Studies in mice have shown that mutations in the E-cadherin gene cause defects in preimplantation development [97]. Its mRNA levels increase during the secretory phase [98], although protein expression remains relatively constant throughout the cycle [99].

Intracellular calcium levels also influence epithelial adhesion and polarity by regulating the distribution of CAMs. In Ishikawa cells, calcitonin-induced calcium elevation suppresses E-cadherin expression [100]. Since calcitonin is upregulated by progesterone during the mid-secretory phase [101,102,103], progesterone may indirectly regulate E-cadherin expression via calcium signaling.

Beta-catenin, a cytoplasmic protein bound to E-cadherin, strengthens cell adhesion and participates in Wnt/Wingless signaling. Mutations in β-catenin or adenomatous polyposis coli (APC) genes cause their nuclear accumulation, leading to the activation of transcription of proliferation-related genes, such as cellular myelocytomatosis oncogene (c-Myc) and cyclin D1 [104]. Interestingly, endometrial samples from infertile women with endometriosis or unexplained infertility show significantly higher E-cadherin and β-catenin expression than fertile controls, suggesting altered regulation of these adhesion molecules during the WOI [105].

6.5. Mucin 1 (MUC1)

MUC1 is responsible for the cell surface’s resistance to enzymatic actions and the cell and cell matrix adhesion [106]. MUC-1 is a highly expressed apical epithelium glycoprotein in the endometrium and has a different pattern of expression during the menstrual cycle [107]. It may also play a role in preventing infections in the upper genital tract [108]. In mice (and most mammals), MUC1 is proposed as a barrier to implantation, disappearing at the time of implantation and downregulated by the blastocyst. In humans, MUC1 is expressed throughout the window of implantation phase, with unique glycosylation patterns that likely explain why MUC1 is involved in endometrial receptivity [109]. The uterine flushings from normal fertile women showed a notable increase in secreted MUC1 concentration after the Luteinizing Hormone (LH) surge [110], and the high concentrations persist in the flushings until day 13 after the LH peak. However, the function of the secreted form of MUC1 remains largely unknown.

6.6. CD44 Protein

CD44 is a transmembrane glycoprotein that participates in cell migration and adhesion [111]. CD44 is present in the blastocyst stage of human preimplantation development; however, it is absent from the first-trimester trophoblast [112], which suggests an involvement in peri-implantation interactions. CD44 can recognize polyanionic glycans, including hyaluronan and chondroitin sulfate [113]; sulfated and sialylated oligosaccharides are abundant on the endometrial apical epithelium [114,115,116], and could act as ligands. Peritoneal cells secrete hyaluronan (HA), a glycosaminoglycan whose primary receptor is CD44. In the human endometrium, CD44 is expressed in both epithelial and stromal cells, with the epithelium exhibiting large variant isoforms generated by alternative splicing [117]. CD44 also binds osteopontin [118], potentially bridging to αVβ integrins. Recent studies indicate that inhibiting N- and O-linked glycosylation of CD44 reduces endometrial cell attachment to peritoneal mesothelial cells, likely because glycosylation-induced conformational changes affect HA binding, highlighting CD44’s key role in cell adhesion [111]. In another study, the endometrial expression of cadherins and CD44 was evaluated in infertile women with hydrosalpinx, and it was found that these patients had lower expression of E-cadherin and CD44 [119].

6.7. Selectin

Selectins are glycoproteins that play a crucial role in cellular adhesion mechanisms and are involved in fundamental immune and inflammatory responses. These molecules participate in a series of interactions between circulating leukocytes and endothelial cells in blood vessels, a phenomenon known as the adhesion cascade [120]. Studies have shown that L-selectin, expressed by the trophoblast, binds to oligosaccharide ligands on the maternal endometrium, promoting adhesion and enhancing the connection between the blastocyst and the endometrium [121]. Although further studies are needed to clarify the role of the L-selectin ligand (LSL) system in endometrial receptivity, five peptide components—podocalyxin, endomucin, nepmucin, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and cluster of differentiation 34 (CD34)—have been identified in the human endometrium during both natural menstrual cycles and menopause. Previous studies have also shown that L-selectin ligands are expressed during the implantation window in both natural and stimulated cycles, with reduced expression in stimulated cycles, suggesting that ovarian hyperstimulation may negatively impact endometrial receptivity [122].

This suggests that LSLs may act as crucial adhesion molecules and serve as a variable biomarker between fertile and non-fertile endometria. Although a study has found a reduced expression of L-selectin ligands in the luminal epithelium following ovarian stimulation, it has not been determined whether this reduction is directly caused by the stimulation or whether it negatively affects the implantation rate [123]. In fact, a recent literature review concluded that, although proteins such as L-selectin are differentially expressed in the human endometrium, further studies are still needed to clarify their actual role [124].

6.8. Immunoglobulin

The immunoglobulin (Ig) superfamily is the largest among CAMs. A key member, intercellular CAM-1 (ICAM-1, CD54), is a transmembrane glycoprotein expressed on endothelial and epithelial cells, fibroblasts, and leukocytes, and acts as a ligand for β2 integrins, mediating leukocyte transendothelial migration and immune functions. In the human endometrium, leukocytes (including macrophages, T lymphocytes, and granulocytes) contribute to decidualization, menstruation, and parturition [125,126,127,128,129]. Intercellular adhesion molecule 1 (ICAM-1) localizes to the apical surface of glandular and luminal epithelium and stroma, with upregulation observed during menstruation [130].

Activated leukocyte CAM (ALCAM, CD166) is a type I transmembrane protein and ligand for CD6 on T lymphocytes. In the endometrium, ALCAM is expressed on luminal and glandular epithelial cells during proliferative and secretory phases, but not in the stroma. mRNA analyses confirm epithelial-specific expression, and flow cytometry validates its presence on the surface of epithelial cells. Notably, ALCAM mRNA was found to be present in human blastocysts but absent in earlier stages, such as the 8-cell and morula stages. Transcriptome studies further show that several cytokines, growth factors (platelet-derived growth factor—PDGFA), placenta growth factor, insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), (IGF2BP3), and ECM proteins, mean corpuscular haemoglobin (MCAM), integrin alpha E, platelet endothelial cell adhesion molecule 1, laminin subunit alpha 1 (LAMA1), are upregulated in human trophectoderm cells; other proteins, including receptors platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), PECAM1, integrin subunit beta 8 (ITGB8), and LAMA2 are restricted to the receptive endometrium [131]. These observations enhance our understanding of the mechanisms underlying the early communication between the blastocyst and the endometrium during implantation, highlighting the significant role of ALCAM in this process. A summary of the main key molecules involved in endometrial receptivity and embryo implantation is described in

Table 1. Key molecules involved in endometrial receptivity and embryo implantation.

Molecule	Family	Function	Expression Pattern	Reference(s)
LIF	Cytokine	Promotes embryo adhesion and trophoblast differentiation	Peaks during the WOI	[71,72,73,74]
VEGF	Growth factor	Promotes angiogenesis, vascularization, embryo implantation	Peaks in mid-luteal phase	[86,87,88]
EGF/HBEGF	Growth factor	Regulates epithelial cell growth and endometrial remodeling	Increases during mid-luteal phase	[92,93]
Integrin αvβ3	Integrin (CAM)	Mediates adhesion between trophoblast and endometrial epithelium	Reduced during proliferative phase	[91,92,93]
Selectin L	Selection (CAM)	Facilitates initial tethering of blastocyst to endometrium	Expressed on trophoblast	[120,121]
CD44	Hyaluronan receptor (CAM)	Aids in cell adhesion	Expressed in epithelium and in stromal cells	[111,117,118]
E-cadherin	Cadherin (CAM)	Maintains epithelial integrity for embryo attachment	Downregulated locally at implantation site	[105]
	Ig superfamily	Facilitates immune cell adhesion, support embryo- endometrium contact	Upregulated during The secretory phase	[125,126,127,128,129,130]
ICAM-1	(CAM)
	Glycopritein	Prevents adhesion	Downregulated at site of embryo attachment	[107,108,109]
MUC1	(anti-adhesive)
	Ig superfamily	Supports interaction between blastocyst and the endometrium	Expressed in the proliferative and secretory phase and in blastocysts	[131]
ALCAM	(CAM)

.

7. Modulators of Trophoblast Invasion and Decidualization

The importance of the trophoblast invasion has been extensively described, and it is known that an insufficient invasion of the extravillous trophoblast (EVT) into the uterine endometrium leads to pregnancy-related complications such as spontaneous abortion, fetal growth restriction and pregnancy-induced hypertension [85].

7.1. Glycodelin

Glycodelin plays a significant role in trophoblast invasion. This secretory glycoprotein is found in the reproductive systems of primates and exists in four glycosylated variants: glycodelin (Gd)-S, GdA, GdF, and GdC. These isoforms are synthesized in distinct regions of the reproductive tract [132,133]. GdS is the only isoform known in males, while the other isoforms are found in females. The isoforms differ only in their N-glycan profile [134,135,136]. GdA is abundantly secreted from the endometrial glands and the decidual glandular epithelium in response to progesterone, human chorionic gonadotropin (hCG), and relaxin [137,138].

Early studies have demonstrated that GdA inhibits leukocyte proliferation in vitro [136]. The peak concentrations of GdA in serum and amniotic fluid at 10 and 16 weeks of gestation, respectively, suggest a role in early pregnancy [139]. GdA is believed to contribute to immune protection at the foeto-maternal interface [132], although its mechanisms are not fully understood. Recent research suggests that GdA affects multiple leukocyte types and regulates trophoblast function [140,141]. The functional properties of glycodelin isoforms depend on glycosylation, as demonstrated in sperm studies, where deglycosylation abolishes their effects [132,133,135,139]. Similarly, GdA activity in immune and trophoblast cells relies on its glycosylation.

The receptors mediating GdA interactions with T cells remain unclear. In 2003, GdA was proposed to act as a calcium-dependent lectin targeting CD45, with binding influenced by its sialylation state [142,143]. In contrast, in 2009, a calcium-independent lectin function recognizing N-glycans on CD7 to trigger T cell death was suggested [144]. Other potential GdA receptors include fucosyltransferase-5 [145], E-selectin [146], CD22 [134], and dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) [147], although definitive evidence is lacking. Further studies are required to clarify the binding mechanisms, intracellular signaling, and biological effects of GdA interactions [69,148].

7.2. Stem Cell Factor (SCF)

During embryonic development, SCF and its receptor, c-kit, play key roles in supporting the survival, proliferation, and migration of germ cells. In the postnatal ovary, c-kit inhibition prevents the initiation of primary follicles. In the human endometrium, SCF is significantly higher in decidual tissue than in non-pregnant tissue, while c-kit is present in placental tissue during pregnancy, suggesting a role at the foeto-maternal interface in supporting embryo development, trophoblast function, and placental growth [149]. During implantation, SCF acts via both paracrine and autocrine mechanisms on trophoblast cells through the c-kit receptor. c-kit mRNA is expressed in both spreading mouse blastocysts and endometrial cells, and immunohistochemistry confirms protein expression. SCF stimulation enhances the surface area expansion of spreading blastocysts [150].

Another study investigated the role of c-kit and its ligand, SCF, in human placental development, examining their expression in first-trimester and term placental tissues, as well as in pregnant and non-pregnant endometrium, using immunocytochemistry and flow cytometry. In the non-pregnant endometrium, SCF expression was absent. However, in the decidua of the first trimester, SCF was strongly detected in the arterial media of maternal blood vessels. SCF was expressed throughout pregnancy by invasive fetal extravillous trophoblast [151]. A recent study assessed the protein profile of implanted and non-implanted human blastocysts using protein microarray technology and observed that SCF was decreased in non-implanted embryos [152].

7.3. CD34 Protein

CD34 is a single-pass transmembrane sialomucin protein expressed in early hematopoietic and vascular tissues, functioning as an adhesion molecule that facilitates T cell entry into lymph nodes. During the first trimester, decidual NK cells populate the maternal decidua, contributing to immune regulation, tissue remodeling, and neovascularization. Recent evidence suggests that decidual CD34-positive (dCD34) cells differ from cord or peripheral blood CD34 precursors, as they express the IL-5/IL-2 receptor beta chain, the IL-7 receptor alpha chain, and mRNA for E4BP4 and ID2. In vitro, exposure to growth factors or co-culture with decidual stromal cells drives dCD34 differentiation into functional CD56 (bright) CD16 (−) KIR (+/−) NK cells, suggesting that dNK cells may arise locally from CD34+ precursors via interactions with the decidual microenvironment.

7.4. Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) and IGF 1 Receptor (IGF-1R)

In the establishment of the endometrial receptivity and maintenance of pregnancy, the decidualization process is fundamental. Decidualization is defined as the differentiation of endometrial fibroblast-like mesenchymal cells into decidual cells, which are morphologically and biochemically distinct [153]. Biochemically, the cells express new proteins, such as prolactin and members of the IGF family, including IGFBP-1 and IGF-1R.

The IGF family, particularly IGF-1, promotes the migration and invasion of EVTs, supporting placental development. The expression of insulin-like growth factor binding protein 1 (IGFBP-1) is regulated by various hormones, including insulin, glucocorticoids, progesterone, cytokines, and hypoxia. Studies show that elevated progesterone upregulates members of the IGF family, including IGF-1, IGF1R, and various IGFBPs [154]. In the human decidua, progesterone stimulates IGFBP-1 production, possibly via a glucocorticoid response element [155], and may promote its polymerization, enhancing IGF-I activity and trophoblast invasion at the foeto-maternal interface [156].

IGFBP-1, secreted by decidual cells, contains an RGD motif that binds α5β1 integrin, facilitating trophoblast–fibronectin interactions and migration [157]. Its expression is regulated by FOXO1, a transcription factor upregulated in the secretory and early pregnancy phases of the endometrium. FOXO1 activates the IGFBP-1 promoter and interacts with nuclear factors, including estrogen, retinoic acid, and thyroid hormone receptors, as well as HOXA10, thereby further enhancing IGFBP-1 activity [158].

7.5. Homeobox Genes

Homeobox genes play a crucial role in regulating development and cell differentiation. An in vitro study revealed that IGFBP1, prolactin, HOXA10, IL-11, and IL-15 are co-regulated during steroid hormone-driven decidualization of human endometrial stromal cells. However, the study also showed that transient suppression of HOXA10 expression in predecidual cells did not impair their ability to undergo decidualization, indicating that hormone-induced decidual transformation and cytokine production in vitro can occur independently of HOXA10 [159]. Another study evaluated the modulation of HOXA10, VEGF, and GdA by hCG in an endometrial explant model and found increased gene expression in response to augmented hCG concentrations. Additionally, the association between age and HOXA10 expression was evaluated, and it was found to be positive, providing novel evidence of the role of uterine age in molecular endometrial responses [160]. A summary of the main molecules involved in trophoblast invasion and decidualization, along with their roles in the embryo implantation process, is presented in

Table 2. Molecules involved in trophoblast invasion and decidualization categorized by their family and primary functions.

Molecule	Family	Function(s)	Reference(s)
Glycodelin (GdA)	Glycoprotein isoform	Modulates trophoblast invasion, maternal immune response	[132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148]
Stem cell factor (SCF)	Cytokine	Promotes trophoblast growth, embryo expansion, via interaction with c-kit	[149,150,151,152]
CD34	Transmembrane sialomucin	Supports the origin of decidual NK cells, immune modulation, and angiogenesis	[153,154,155]
IGFBP-1 and IGF-1R	IGF family	Regulates trophoblast invasion, placental function, enhances IGF-1 activity under progesterone and FOXO1 stimulation	[154,155,156,157,158]
Homeobox genes (HOXA 10)	Transcription factor	Regulates decidualization markers and molecular responses of endometrium to hormonal stimulus	[159,160]

.

8. Signaling Pathways in Embryo Implantation

Wnt/β-catenin signaling is essential for the correct implantation and invasion of trophoblast cells into the endometrium and myometrium, playing a crucial role in placental formation and in the development and function of the female reproductive tract. Indeed, dysregulated Wnt/β-catenin signaling may contribute to the development of various uterine disorders [161]. During the development of the female reproductive tract, this signaling pathway regulates several processes: Wnt4 is indispensable for the initiation of the Müllerian duct, Wnt7A for its differentiation, and Wnt5A for posterior growth [162,163]. Furthermore, WNT4 has been shown to act downstream of BMP2 and function through the β-catenin pathway to regulate the differentiation of human endometrial stromal cells [164]. In the adult endometrium, the Wnt/β-catenin pathway plays a crucial role in the normal menstrual cycle, with nuclear accumulation of β-catenin during the proliferative phase and its absence during the secretory phase [165,166]. Inhibition of Wnt/β-catenin signaling, for example, by the Wnt/β-catenin inhibitor SFRP2, suppresses E2-induced endometrial proliferation, highlighting the importance of precise regulation for uterine function [167,168]. Furthermore, progesterone counteracts the proliferative activity of E2 by inducing Dickkopf-related protein 1 (DKK1) and forkhead transcription factor 1(FOXO1), which inhibit the Wnt pathway, a mechanism essential for endometrial homeostasis and potentially relevant in tumour pathogenesis [169].

The invasive properties of trophoblasts are finely regulated by several cytokines, including LIF, IL-6, HGF, and granulocyte-macrophage colony-stimulating factors (GM-CSFs), which modulate trophoblast invasion through the activation of specific receptors on trophoblast cells and, consequently, intracellular signaling pathways. Among these, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, in particular phosphorylated STAT3, plays a key role in enhancing the invasiveness of both tumour cells and trophoblastic cells, where it is mainly activated by LIF [170]. Indeed, transcriptomic analyses of the endometrium of fertile women and patients with RIF have highlighted a higher expression of genes related to IL-6/JAK/STAT3 signaling and allograft rejection in healthy endometrium, suggesting that LIF is crucial for endometrial preparation for embryo implantation and may represent a potential therapeutic target to improve endometrial receptivity and promote implantation [171].

On the other hand, the TGFβ superfamily, including TGFβ, activins, bone morphogenetic proteins (BMPs), and growth differentiation factors (GDFs), regulates essential processes such as differentiation, proliferation, apoptosis, and tissue remodeling, which are essential for various reproductive processes [172]. TGFβ isoforms (TGFβ1, TGFβ2 and TGFβ3), expressed in the endometrial epithelium and stroma of both rodents and humans, contribute to maternal immunotolerance during implantation and modulate key implantation molecules, such as VEGF, MMP-9, insulin-like growth factor-binding protein 1 (IGFBP-1) and LIF in vitro and considering that TGFβ knockout mouse embryos die in utero or soon after birth [173], it was impossible to draw conclusions on the potential role of TGFβ in implantation using this model [174]. Furthermore, a study on secretory human endometrium assessed the expression of several members of the TGFβ superfamily, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, and TGFβ and Nodal, demonstrating that all members except Nodal were expressed in secretory endometrium, with only BMP2, GDF5, and TGFβ1 being localized in decidual cells [175]. Furthermore, in human endometrial stromal cell cultures, all ligands except Nodal were expressed, and it was seen that during the decidualization process of human embryonic stem cells, activin inhibition reduced decidualization, while BMP2 and TGFβ1 secretion increased during this process; their exogenous administration further enhanced their efficiency, confirming the crucial role of these proteins in the regulation of decidualization [175].

Some authors evaluated the expression of the mRNAs for the TGF-β receptor types I and II and their signal transducers, Smad2 and Smad4, in the rat endometrium during the cycle and early pregnancy and found that the TGF-β signals are spatially and temporally related to uterine receptivity and a decrease in trophoblast invasion [174,176]. In human endometrial stromal cells, TGF-β plays a crucial role in regulating the expression of matrix metalloproteinases (MMPs). In particular, a study found that TGF-β1 increased the expression and activity of MMP2 and MMP11, while progesterone inhibited TGF-β1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGF-β1 also reduced the expression of progesterone receptor (PR)-A and PR-B in HESCs, with a more evident effect on PR-A [177]. TGF-β1 effects are known to be mediated primarily by the suppressor of mothers against decapentaplegic (SMAD) pathway, with specific actions on decidual markers, including prolactin (PRL) and IGFBP1. The resistance of decidual stromal cells (DSCs) to TGF-β1 during early pregnancy appears crucial for tissue homeostasis [178].

In recent years, endometrial bleeding-associated factor (EBAF/LEFTY B) has been recognized as a soluble cytokine within the TGF-β superfamily, known to promote tissue remodeling by inducing collagenolysis via matrix metalloproteinases. Its expression in the human endometrium is low during the receptive period and peaks during the perimenstrual and menstrual phases. Studies have shown that transgenic mice with induced overexpression of EBAF/LEFTY B exhibit impaired implantation. EBAF/LEFTY B inhibits the expression of crucial decidual proteins, such as IGFBP-1 and PRL, through the regulation of the transcription factors FOXO1 and ETS1. In infertile patients, however, EBAF/LEFTY B expression was prematurely elevated during the implantation window, confirming the role of LEFTY as a molecular switch regulating both the induction and maintenance of decidual differentiation associated with the expression of IGFBP1 and PRL. However, because decidual cells express high levels of LEFTY, its action on decidualization may occur through an autocrine mechanism, inhibiting the differentiation of uterine stromal cells [179,180].

An additional critical mediator of decidualized stromal cell survival is serum/glucocorticoid-inducible kinase 1 (SGK1), a kinase involved in epithelial ion transport and survival, which can be stimulated in some contexts by TGF-β. Studies in human endometrium have shown that SGK1 is upregulated in women with unexplained infertility, primarily in the luminal epithelium, but downregulated in women with recurrent pregnancy loss, resulting in increased oxidative stress and decidualized stromal cell death [181]. These findings highlight the interconnection between TGF-β signaling, the regulation of intracellular factors such as SGK1, and proper decidual function required for implantation and pregnancy maintenance. A summary of the signaling pathways involved in the regulation of decidualization, immunotolerance, and tissue remodeling processes in embryo implantation can be found in

Table 3. Wnt/β-catenin Signaling and other Molecules in Endometrial and Trophoblast Functions.

Molecule	Pathway/Nature	Function(s)	Reference(s)
Wnt/β-catenin	Wnt signaling pathway	Regulates implantation, trophoblast invasion, and dysregulation leading to uterine disorders	[161,162,163,164,165,166,167,168,169]
Wnt 4 Wnt 5A Wnt 7A	Wnt signaling pathway	Mullerian duct initiation	[162,163]
		Posterior outgrowth during female tract development Mullerian duct differentiation
DKK1	Wnt signaling molecule	Inhibits Wnt signaling, maintains endometrial homeostasis	[168]
LIF	Cytokine	Enhances endometrial receptivity, regulates trophoblast invasion via JAK/STAT3 pathway	[170,171]
TGFβ (1,2,3)	TGFβ superfamily	Modulates immune tolerance, regulates implantation-related molecules, and supports decidualization	[172,173,174,175]
BMP (2,4,7)	Bone morphogenetic proteins of TGFβ superfamily	Promote decidualization and regulate endometrial remodeling	[175]
LEFTY (EBAF)	Endometrial bleeding- associated factor of TGFβ superfamily	Inhibits decidual proteins, impairs implantation when overexpressed, regulates decidual differentiation	[179,180]
SGK1	Kinase	Involved in epithelial ion transport, dysregulation linked to recurrent pregnancy loss via oxidative stress	[181]

.

9. Advancements in Embryo Receptivity Diagnostics

Innovative developments in reproductive medicine provide a more precise assessment of the WOI, which includes ERA. It is a customized microarray test that analyzes the transcriptomic signature of the endometrium to assess its receptivity for implantation. By examining 238 genes known to be involved in the hormonal implantation process, the test compares the genetic profile of samples taken from natural cycle controls with those subjected to hormonal stimulation. Using a bioinformatics predictor, the sample is then classified as receptive or non-receptive [6]. However, while the ERA test represents a significant advancement in endometrial receptivity diagnostics, it still has some limitations that require further investigation. The ERA test helps identify the WOI, but its reliability can be influenced by factors such as progesterone levels, body mass index (BMI), and the presence of chronic endometritis, making early diagnosis of the latter necessary [182]. Although some studies highlight its benefits, there is no scientific consensus on its real effectiveness. Therefore, further research is needed to assess its impact on implantation and pregnancy rates, especially in cases of RIF. Nonetheless, tests like ERA also use machine learning to analyze gene expression and personalized embryo transfer timing, representing the way forward.

Research on non-coding RNAs could also open new opportunities to improve the diagnosis and treatment of endometrial receptivity. miRNAs are promising diagnostic biomarkers for evaluating the WOI and RIF. Recent studies have identified specific miRNAs in plasma, biological fluids, and the endometrium as indicators of RIF [183]. Among the miRNAs involved in the implantation process, miR-31 has recently been identified as a promising biomarker for monitoring endometrial receptivity and predicting implantation failure. It is a small RNA that regulates gene expression, with significantly elevated levels during the secretory phase of the endometrium, which corresponds to the WOI. In patients with RIF, an abnormal increase in miR-31 has been observed during the proliferative phase of the menstrual cycle. This suggests a key role of miR-31 in endometrial receptivity and highlights its potential diagnostic value in predicting implantation failure cases [184]. Another potential microRNA biomarker is let-7a. It has been observed that the let-7 family influences both cell proliferation by suppressing the Wnt/β-catenin signaling pathway and the presence of physical barriers, such as mucin. Therefore, its expression on the fourth day of embryonic development would promote implantation [185]. miR-182-5p also regulates endometrial receptivity and influences embryo attachment to the endometrium. Specifically, in fertile women, miR-182-5p levels were lower during the secretory phase compared to the proliferative phase, whereas in infertile women, they were higher. Moreover, an induced increase in miR-182-5p also leads to a reduction in E-cadherin expression, which, being a key protein for cell adhesion, is essential for embryo implantation [186]. As well, miR-135a-5p has been identified as a key regulator of endometrial receptivity, with its overexpression inhibiting the formation of pinopods and decidualization. The study demonstrated that miR-135a-5p suppresses endometrial receptivity by inhibiting the expression of genes such as HOXA10 and bone morphogenetic protein receptor 2 (BMPR2) in ESCs, thereby reducing decidualization and trophoblast invasion, which are crucial for implantation [187].

A recent study analyzed serum samples collected from patients who underwent the ERA test, dividing them into two groups: receptive and non-receptive. Using high-throughput sequencing, they examined the expression profile of sncRNAs in the blood to identify potential non-invasive biomarkers for endometrial receptivity. Among the various categories of sncRNAs analyzed, in addition to miRNAs and piRNAs, particular attention was given to transfer RNA-derived small RNAs (tsRNAs). A total of 286 of these small RNA fragments, derived from tRNA fragmentation, showed differential expression between the receptive and non-receptive groups. These tsRNAs regulate the expression of genes involved in key signaling pathways crucial for endometrial receptivity, including the mitogen-activated protein kinase (MAPK) pathway, essential for hormonal regulation and the preparation of the endometrium for implantation, the FOXO pathway, involved in decidualization, the Ras-associated protein 1 (Rap1) pathway, critical for as adhesion, proliferation and migration, and the circadian rhythm, which appears to play a role in the regulation of endometrial decidualization. The authors suggest that tsRNAs could serve as novel non-invasive biomarkers for assessing endometrial receptivity and, in the future, could be used to prevent implantation failure or treat infertility related to receptivity issues [188].

10. Summary and Clinical Implications

The process of embryonic implantation requires the participation of both embryo and endometrium, culminating in the attachment of the blastocyst to the maternal endometrium during the window of implantation. The interaction between the embryo and the endometrium is characterized by a molecular dialogue, with the participation of cytokines, growth factors, hormones and cell adhesion molecules, which may function as biochemical markers of endometrial receptivity. The assessment of endometrial receptivity has historically relied on histological dating, such as Noyes criteria, but these have shown limitations in accuracy, with a significant percentage of fertile women displaying “out-of-phase” endometrial dating. In response to this, new biomarkers have been developed for a more accurate evaluation of receptivity. Pinopods, temporary structures that appear on the surface of endometrial epithelial cells during the implantation window, are historically the most studied biomarkers. Their formation appears to be crucial for embryo adhesion; however, although the presence of pinopods has been associated with higher pregnancy success rates, their reliability as a biomarker remains controversial due to variability in classification methods. Similarly, various molecular factors, such as growth factors and cytokines, play a crucial role in enhancing endometrial receptivity. VEGF, for example, stimulates angiogenesis and endometrial vascularization, improving implantation chances. Additionally, cell adhesion molecules such as integrins, cadherins, and selectins are involved in blastocyst implantation, as well as Immunoglobulin superfamily molecules, such as ICAM-1, also play a significant role in cell adhesion and immune responses during embryo implantation, with increasing evidence suggesting that lower ICAM-1 levels are associated with recurrent pregnancy loss. Similarly, molecules like IGFBP-1 and HOXA10, which regulate decidualization and trophoblast invasion modulation through glycoproteins and factors like SCF, are essential in creating a favourable environment for embryo implantation. Finally, understanding the molecular mechanisms involved, including Wnt/β-catenin and TGF-β signaling pathways, is not only crucial for improving ART outcomes but also provides insights into managing pregnancy complications.

Despite the evidence, identifying reliable biomarkers for endometrial receptivity remains a challenge. The main limitations stem from the short temporal window of expression, individual variability, the influence of hormonal treatments, and complex molecular regulation. Another significant obstacle is the difficulty in standardizing biomarker identification and quantification methods. Differences in analytical protocols and the impact of hormonal conditions often make it difficult to reproduce results. Furthermore, in ovarian stimulation cycles, the expression of these biomarkers undergoes considerable variations. Pinopods, for example, appear earlier than in natural cycles, while VEGF, a key molecule for endometrial vascularization, may be altered. Integrins and other adhesion molecules also undergo modifications, which limit their reliability as indicators of receptivity. To further complicate the issue, the presence of certain biomarkers does not necessarily guarantee successful embryo implantation. Conflicting studies on molecules such as E-cadherin and β-catenin demonstrate that the mechanisms regulating implantation are not yet fully understood. Additionally, many of these biomarkers have been studied mainly in animal or in vitro models. These limitations suggest that, although the described biomarkers offer great potential for improving pregnancy success in ART, further studies are needed to understand the action of these molecules on endometrial receptivity. While the ERA test and emerging biomarkers such as tsRNAs offer promising advances in the diagnosis and treatment of endometrial receptivity, further research and validation are essential to fully harness their potential in improving implantation success and fertility outcomes after ovarian stimulation treatments.