Establishment Of Endometrial Receptivity Research Articles

Activated THP-1 Macrophage-Derived Factors Increase the Cytokine, Fractalkine, and EGF Secretions, the Invasion-Related MMP Production, and Antioxidant Activity of HEC-1A Endometrium Cells.

Endometrium receptivity is a multifactor-regulated process involving progesterone receptor-regulated signaling, cytokines and chemokines, and additional growth regulatory factors. In the female reproductive system, macrophages have distinct roles in the regulation of receptivity, embryo implantation, immune tolerance, and angiogenesis or oxidative stress. In the present study, we investigated the effects of PMA-activated THP-1 macrophages on the receptivity-related genes, cytokines and chemokines, growth regulators, and oxidative stress-related molecules of HEC-1A endometrium cells. We established a non-contact co-culture in which the culture medium of the PMA-activated macrophages exhibiting the pro-inflammatory phenotype was used for the treatment of the endometrial cells. In the endometrium cells, the expression of the growth-related factors activin and bone morphogenetic protein 2, the growth hormone EGF, and the activation of the downstream signaling molecules pERK1/2 and pAkt were analyzed by ELISA and Western blot. The secretions of cytokines and chemokines, which are involved in the establishment of endometrial receptivity, and the expression of matrix metalloproteinases implicated in invasion were also determined. Based on the results, the PMA-activated THP-1 macrophages exhibiting a pro-inflammatory phenotype may play a role in the regulation of HEC-1A endometrium cells. They alter the secretion of cytokines and chemokines, as well as the protein level of MMPs of HEC-1A cells. Moreover, activated THP-1 macrophages may elevate oxidative stress protection of HEC-1A endometrium cells. All these suggest that pro-inflammatory macrophages have a special role in the regulation of receptivity-related and implantation-related factors of HEC-1A cells.

Excessive proliferating cell nuclear antigen attenuates endometrial adhesive capacity and decidualization in patients with recurrent implantation failure.

Does the expression of proliferating cell nuclear antigen (PCNA) in the endometrium regulate endometrial receptivity in patients with recurrent implantation failure (RIF)? A high abundance of PCNA attenuates endometrial adhesive capacity and decidualization in patients with RIF. Aberrant expression of PCNA has been discovered in multiple infertility-related disorders. However, the expression pattern and role of PCNA in the establishment of endometrial receptivity and endometrial decidualization in patients with RIF remain unclear. We analysed the expression of PCNA in mid-secretory endometrial tissues from 24 patients with RIF and 24 healthy women. Additionally, PCNA expression levels were measured in proliferative and mid-secretory phase endometrial tissue samples from women with regular menstrual cycles and in decidual tissue samples taken from ten women during normal early pregnancy (n = 10 per phase for each group). The function and regulatory mechanisms of PCNA in endometrial adhesive capacity and endometrial decidualization were investigated using BeWo spheroids, Ishikawa cells, and human endometrial stromal cells (HESCs). The expression of PCNA in mid-secretory endometrial tissues of patients with RIF and women with normal endometrium and in endometrial tissue at different stages of the menstrual cycle and in decidualized tissues was analysed by RT-qPCR, western blot, and immunohistochemistry staining (IHC). Furthermore, the number of BeWo spheroids directly attached to the Ishikawa cell monolayers, and the potential molecular mechanisms involved, were compared between cells overexpressing PCNA and a control group. Additionally, the effect and regulatory mechanisms of PCNA on the decidualization of HESCs in vitro were investigated. Our findings indicated that the abundance of PCNA was dramatically greater in mid-secretory endometrial tissues from patients with RIF than in those from women with healthy endometrium. The expression of PCNA increased in the proliferative phase of the menstrual cycle but decreased gradually in the mid-secretory phase and in decidual tissues. Interestingly, PCNA was expressed in both human endometrial epithelial cells (HEECs) and HESCs. In Ishikawa cells, PCNA overexpression dramatically reduced the endometrial adhesive capacity by inhibiting the expression of adhesion molecules (E-cadherin and integrin β3) and activating the FAK/paxillin signalling pathway. Furthermore, in HESCs, PCNA overexpression attenuated endometrial decidualization by activating the AKT/β-catenin signalling pathway and increasing tight junctions between cells by upregulating ZO-1 and occludin expression. In addition, PCNA-ELAVL1 interactions were confirmed by coimmunoprecipitation in decidualized HESCs. N/A. The functional analysis of PCNA was limited by the number of human endometrial tissues. A larger sample size is required to further explore the potential roles of PCNA during embryo implantation. Moreover, the present results should be taken with caution, as only a few of the embryos that were transferred in RIF patients population underwent preimplantation genetic testing for embryonic chromosome aneuploidies (PGT-A), despite embryo ploidy testing being significant in the diagnosis of unexplained RIF. High PCNA expression attenuates endometrial adhesive capacity and decidualization in patients with RIF. These findings provide new insights into the potential mechanisms underlying the occurrence of implantation failure. This work was supported by the National Natural Science Foundation of China (82101698), Shandong Provincial Natural Science Foundation (ZR2021MH012), and the Science and Technology Plan of Yantai (2023YD021 and 2022YD031). The authors have no conflicts of interest to disclose. N/A.

Loss of KLF15 impairs endometrial receptivity by inhibiting EMT in endometriosis.

The impaired endometrial receptivity is a major factor contributing to infertility in patients with endometriosis (EM), but the underlying mechanism remains unclear. Our study aimed to investigate the role of Kruppel-like factor 15 (KLF15) in endometrial receptivity and its regulation in EM. We observed a significant decrease in KLF15 expression in the mid-secretory epithelial endometrial cells of EM patients compared to normal females without EM. To confirm the role of KLF15 in endometrial receptivity, we found a significantly reduced KLF15 expression and a significant decrease in embryo implantation number in the rat model via uterine horn infection with siRNA. This highlights the importance of KLF15 as a regulator receptivity. Furthermore, through ChIP-qPCR, we discovered that the progesterone receptor (PR) directly binds to KLF15 promoter regions, indicating that progesterone resistance may mediate the decrease in KLF15 expression in EM patients. Additionally, we found that the mid-secretory endometrium of EM patients exhibited impaired epithelial-mesenchymal transition (EMT). Knockdown of KLF15 upregulated E-cadherin and downregulated vimentin expression, leading to inhibited invasiveness and migration of Ishikawa cells. Overexpression KLF15 promotes EMT, invasiveness, and migration ability, and increases the attachment rate of JAR cells to Ishikawa cells. Through RNA-seq analysis, we identified TWIST2 as a downstream gene of KLF15. We confirmed that KLF15 directly binds to the promoter region of TWIST2 via ChIP-qPCR, promoting epithelial cell EMT during the establishment of endometrial receptivity. Our study reveals the involvement of KLF15 in the regulation of endometrial receptivity and its downstream effects on EMT. These findings provide valuable insights into potential therapeutic approaches for treating non-receptive endometrium in patients with EM.

IFNT-induced IRF1 enhances bovine endometrial receptivity by transactivating LIFR

Interferon-τ (IFN-τ) participates in the establishment of endometrial receptivity in ruminants. However, the precise mechanisms by which IFN-τ establishes bovine endometrial receptivity remain largely unknown. Interferon regulatory factor 1 (IRF1) is a classical interferon-stimulated gene (ISG) induced by type I interferon, including IFN-τ. Leukemia inhibitory factor receptor (LIFR) is a transmembrane receptor for leukemia inhibitory factor (LIF), which is a key factor in regulating embryo implantation in mammals. This study aimed to investigate the roles of IRF1 and LIFR in the regulation of bovine endometrial receptivity by IFN-τ. In vivo, we found IRF1 and LIFR were upregulated in the bovine endometrial luminal epithelium on Day 18 of pregnancy compared to Day 18 of the estrous cycle. In vitro, IFN-τ could upregulate IRF1, LIFR, and endometrial receptivity markers (LIF, HOXA10, ITGAV, and ITGB3) expression, downregulate E-cadherin expression and reduce the quantity of microvilli of bovine endometrial epithelial cells (bEECs). Overexpression of IRF1 had similar effects to IFN-τ on endometrial receptivity, and interference of LIFR could block these effects, suggesting the positive effects of IRF1 on endometrial receptivity were mediated by LIFR. Dual luciferase reporter assay verified that IRF1 could transactivate LIFR transcription by binding to its promoter. In conclusion, IFN-τ can induce IRF1 expression in bovine endometrial epithelial cells, and IRF1 upregulates LIFR expression by binding to LIFR promoter, contributing to the enhancement of bovine endometrial receptivity.

ITRAQ-based Proteomic Analysis Unveils ACSL4 as a Novel Potential Regulator of Human Endometrial Receptivity.

Competent endometrial receptivity is a prerequisite for successful embryo implantation. Identification of novel key molecules involved in endometrial receptivity is essential to better interpret human implantation and improve pregnancy rates in assisted reproduction treatment. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to profile the proteomes of the prereceptive (luteinizing hormone [LH] + 2, n = 4) and receptive (LH + 7, n = 4) endometrial tissues. A total of 173 differentially expressed proteins (DEPs) between LH + 2 and LH + 7 endometrial samples were identified. Integrated analysis of the proteomic data and published transcriptomic data was performed to identify the concordant DEPs with differential expression at both the messenger RNA and protein levels. Protein-protein interaction (PPI) network analysis was performed on concordant DEPs. We first identified 63 novel concordant DEPs and 5 hub proteins (ACSL4, ACSL5, COL1A1, PTGS1, and PLA2G4F) between LH + 2 and LH + 7 endometrial samples. ACSL4 was predominantly expressed in endometrial epithelial cells and its expression was significantly upregulated by progesterone in the LH + 7 endometrium and significantly downregulated in repeated implantation failure patients. Knockdown of ACSL4 in endometrial epithelial cells induced the downregulation of endometrial receptivity markers (HOXA10, COX2, and LIF) and the significant decrease of implantation rate during in vitro implantation analysis. This study provides the first gel-independent quantitative proteomes of the LH + 2 and LH + 7 human endometrium using iTRAQ technology. The identified concordant DEPs and hub proteins open a new avenue for future studies aimed at elucidating the underlying mechanisms governing endometrial receptivity. ACSL4 was identified as a novel regulatory molecule in the establishment of endometrial receptivity and might play important roles during implantation.

Coupling miR/isomiR and mRNA Expression Signatures Unveils New Molecular Layers of Endometrial Receptivity.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants—isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5′3′-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

Attenuated monoamine oxidase a impairs endometrial receptivity in women with adenomyosis via downregulation of FOXO1†.

The establishment of endometrial receptivity is a prerequisite for successful pregnancy. Women with adenomyosis possess a lower chance of clinical pregnancy after assisted reproductive technology, which is partially due to impaired endometrial receptivity. The establishment of endometrial receptivity requires the participation of multiple processes, and proper endometrial epithelial cell (EEC) proliferation is indispensable. Monoamine oxidase A (MAOA) is a key molecule that regulates neurotransmitter metabolism in the nervous system. In the present study, we demonstrated a novel role for MAOA in the establishment of endometrial receptivity in women with adenomyosis and in an adenomyotic mouse model. Attenuated MAOA impairs endometrial receptivity by promoting inappropriate proliferation of EECs via the downregulation of FOXO1 during the window of implantation. These results revealed that MAOA plays a vital role in endometrial receptivity in female reproduction.

Identification of HOXA10 target genes in human endometrial stromal cells by RNA-seq analysis.

Homeobox A10 (HOXA10) is a transcription factor belonging to the homeobox gene family. It is highly expressed in endometrial stromal cells (ESCs) and plays essential roles in the proliferation and differentiation of endometrium, the establishment of endometrial receptivity and embryo implantation. However, little is known about the target genes and signaling pathways regulated by HOXA10 in ESCs. In this study, we identified 1830 transcripts regulated by HOXA10 in ESCs by RNA interference (RNAi) and RNA-sequencing (RNA-seq) analysis, of which 980 were positively regulated by HOXA10 and 850 were negatively regulated by HOXA10. Interestingly, matrix metallopeptidase-11 was downregulated by HOXA10 in stromal cells verified by quantitative real-time polymerase chain reaction and western blot analysis. Pathway analysis demonstrated that the target genes were enriched in various pathways, including cellular metabolism, DNA replication and repair, cell junction, and lysosome and signal transduction. The results of the present study provide novel insights into the mechanism underlying HOXA10 regulation in ESCs and may identify novel targets for the diagnosis and treatment of endometrium-related infertility.

Decay-accelerating factor promotes endometrial cells proliferation and motility under ovarian hormone stimulation

The intent of the study was to explore the elevating expression of decay-accelerating factor(DAF) exerts influence on biological behaviors of endometrial stromal cells except in classical immunology on the basis of bioinformatic statistics and clinical miscarriages findings suggesting its potential role in the establishment of endometrial receptivity. We confirmed that DAF locates on the cellular surface of endometrial epithelium and stroma. By using plasmid transfection to down-regulate DAF expression in primary endometrial stromal cells(ESCs), we discovered that DAF expression in ESCs increases in response to estradiol and progesterone stimulation in dose- and time-dependent manners; moreover, tamoxifen and RU486 stimulations to block estrogen receptors(ERs) and progesterone receptors(PRs) respectively result in reduced DAF mRNA and protein, and it is more obvious to block PRs. Meanwhile, knocked-down DAF in ESCs weakens the proliferation, migration and invasion of endometrial cells. Cell cycle analysis showed knocked-down DAF accumulates cells in S phase and diminishes cells in G0/G1 phase, which substantiates DAF mediates endometrial cells proliferation. In conclusion, DAF is a potential molecule involving in endometrial cellular proliferation and motility to verify up-expressed DAF during the WOI may facilitate endometrial physiobiological behavior changes, which shed light on DAF function and potential role in the endometrial receptivity establishment.

Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation.

Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

EPAC2-mediated calreticulin regulates LIF and COX2 expression in human endometrial glandular cells.

The proper production of the implantation-related factors, leukemia inhibitory factor (LIF), cyclooxygenase 2 (COX2, PTGS2), and prostaglandin E2 (PGE2) in the uterine glands is essential for embryo implantation and the establishment of endometrial receptivity. It has been shown that cAMP-mediated protein kinase A (PKA) signaling regulates the production of these factors. We have previously reported that exchange protein directly activated by cAMP 2 (EPAC2, RAPGEF4), another cAMP mediator, is involved in the differentiation of endometrial stromal cells through the regulation of the expression of calreticulin (CALR). To address whether EPAC2-CALR signaling is involved in the expression of implantation-related factors, we examined the effect of EPAC2 and CALR knockdown on their expression in cultured human endometrial glandular epithelial EM1 cells, treated with forskolin, an adenylyl cyclase activator, an EPAC-selective cAMP analog (8-(4-chlorophenylthio)-2'-O-methyl cAMP (CPT)), or a PKA-selective cAMP analog (N(6)-phenyl-cAMP (Phe)). In addition, the status of cell senescence was examined. EPAC2 knockdown suppressed the expression of CALR protein and mRNA in EM1 cells. Forskolin- or Phe-, but not CPT-, induced expression of LIF or PTGS2 and secretion of PGE2 was inhibited in EPAC2- or CALR-silenced EM1 cells. In addition, knockdown of EPAC2 or CALR increased senescence-associated beta galactosidase activity and expression of p21 but decreased expression of p53. These findings indicate that expression of CALR regulated by EPAC2 in endometrial glandular epithelial cells is critical for the expression of LIF and PTGS2-mediated production of PGE2 through cAMP signaling. Furthermore, EPAC2 and CALR could play a role in the maintenance of gland function.

Combined Analysis of MicroRNome and 3′-UTRome Reveals a Species-specific Regulation of Progesterone Receptor Expression in the Endometrium of Rhesus Monkey

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.

Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

166. EMBRYO IMPLANTATION IS ASSOCIATED WITH SPECIFIC EXPRESSION OF PROPROTEIN CONVERTASE 6 IN THE RABBIT UTERUS

Proprotein convertase 5/6 (PC6) is a member of the proprotein convertase family that endoproteolytically cleave latent precursor proteins into their biologically active state. We have previously demonstrated that endometrial PC6 is critical for embryo implantation in mice and primates, including human. PC6 regulates the endometrial physiology specifically at implantation in association with epithelial differentiation during the establishment of endometrial receptivity (in human and monkey) and stromal cell decidualization (in the mouse, human and monkey). PC6 was further confirmed to be a unique PC member that is tightly regulated in the endometrium in relation to implantation. Our further studies (unpublished) suggest that PC6 regulates adhesion molecules in the endometrial epithelium for implantation in women. It is known that between the mouse and human, the endometrial stroma-mediated responses are similar whereas the epithelial cells behave differently. Because PC6 regulates primarily the stromal component (decidualization) in the mouse, in vivo mouse models are critical to investigate the roles of PC6 in decidualization. To address the function of PC6 in endometrial epithelium, non-mouse models relevant to human implantation are required. The rabbit is regarded as an excellent model to study the molecular events of embryo adhesion and attachment. The current study aimed to determine the expression pattern and localisation of PC6 in the rabbit uterus during early pregnancy. Quantitative RT-PCR analysis showed that PC6 mRNA expression was dynamically up-regulated in the rabbit uterus immediately prior to implantation. Western blotting and immunohistochemical analyses demonstrated that PC6 protein was predominantly localised to the basal glands throughout pregnancy, and up-regulated specifically in the epithelium at the embryo attachment site. These findings suggest that PC6 may play an essential role in rabbit implantation, and that the rabbit is a useful animal model to investigate the function of PC6 during embryo attachment.

164. INTEGRIN CLEAVAGE IS MEDIATED BY PROPROTEIN CONVERTASE 6 IN HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR ENDOMETRIAL RECEPTIVITY AND IMPLANTATION

Integrins are transmembrane glycoproteins composed of non-covalently associated α and β chains. Integrins participates in cell–cell interaction and binding to components of the extracellular matrix. Integrin expression changes during the establishment of receptivity for implantation. Integrins α1β1, α4β1 and αvβ3 are expressed in the endometrium during the window of implantation [1, 2] and deficiency in integrin αvβ3 is associated with idiopathic infertility and delayed endometrial maturation [1, 3, 4]. Proprotein convertases (PCs) cleave proproteins at the basic amino acids consensus motif (K/R)-(X)n-(K/R)↓ (where n = 0, 2, 4 or 6 and X is any aa) for activation. Integrins require post-translational cleavage for activation and are known to be cleaved by PCs. PC6 plays a critical role in the establishment of endometrial receptivity. We hypothesized that PC6 cleaves integrins for its activation in the endometrial epithelium for implantation. The aim of this study was to investigate the functional importance of PC6 in integrin cleavage, using a stable PC6-knockdown in HEC1A cells (HEC1A-HP) by siRNA technology.Cells transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. To determine the amount of functional integrins on the cell surface, HEC1A-PC6 and HEC1A-control were subjected to an integrin monoclonal antibody array. The amount of functional integrins α1, α3, α5, αv, αvβ3, β1, β3, β4 and α5β1 on the cell surfacewas much less in HEC1A-HP than HEC1A-control cells. Western blot analysis confirmed that cleavage of pro-integrin α5 into disulfide-linked heavy chain (110kDa) and light chain (35kDa) was greater in HEC1A-control compared to HEC1A-HP cells, suggesting that knockdown of PC6 affects integrin cleavage. Our studies imply that integrin cleavage is mediated by PC6 in endometrial epithelial cells for the establishment of receptivity for embryo implantation. (1) Lessey BA, et al., Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest, 1992. 90(1): 188–95.(2) Tabibzadeh S, Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod, 1992. 7(6): 876–82.(3) Lessey BA, et al., Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril, 1994. 62(3): 497–506.(4) Lessey BA, et al., Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab, 1994. 79(2): 643–9.

156. IDENTIFICATION OF SCAFFOLDING PROTEINS AS PC6 SUBSTRATES IN THE HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR EMBRYO IMPLANTATION

Successful embryo implantation is an important step in establishing pregnancy, which requires a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, however the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms, they are thus regarded as critical ‘master switch’ molecules. The aim of this study was to identify target proteins of PC6 in the endometrial epithelial cells important for implantation. We used a HEC1A cell line in which PC6 was stably knocked down by siRNA approach (HEC1A-PC6). HEC1A cells that were similarly transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. Previous study confirmed that HEC1A-PC6 cells had much reduced capacity to adhere to blastocyst. A proteomic comparison between HEC1A-PC6 treated with or without human recombinant PC6 identified ezrin as a potential PC6 substrate. Ezrin is a cytoplasmic protein which is known to bind to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) thereby translocating to the plasma membrane.This complex has been associated with cytoskeletal re-organisation and changes in cell polarity. Co-immunoprecipitation of ezrin and EBP50 showed that knockdown of PC6 allowed the binding of ezrin to the C-terminus of EBP50 in HEC1A-PC6, whereas PC6 cleavage of EBP50 in HEC1A-control prevented the binding. This was also confirmed by immunofluorescence showing that ezrin and EBP50 were co-localized to the plasma membrane in HEC1A-PC6. This study thus identified that PC6 regulates scaffolding proteins such as EBP50 and ezrin in the endometrium for embryo implantation.

529. PROPROTEIN CONVERTASE 6 PLAYS A CRITICAL ROLE IN MODULATING THE HUMAN ENDOMETRIAL EPITHELIUM FOR RECEPTIVITY AND IMPLANTATION

Successful embryo implantation is an important step in establishing pregnancy, requiring a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, but the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation, in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical “master switch” molecules. The present study aimed to determine whether PC6 is a critical regulator in the endometrial epithelium for receptivity and implantation. We examined whether endometrial epithelial PC6 dys-regulation is associated with implantation failure in women and whether knockdown of PC6 by siRNA in human endometrial epithelial cells affects embryo adhesion in a cell culture model. Endometrial PC6 expression was assessed by immunohistochemistry in the mid-secretory phase of the menstrual cycle (receptive phase) in two unique clinical cohorts comprising women of known fertility and infertility (with no obvious gynecological disorders, and with fertile males). Endometrial epithelial PC6 levels were significantly lower in infertile vs fertile women in both cohorts. To further establish that PC6 is important for receptivity, a cell model relevant to human implantation was used involving co-culture of uterine epithelial cells with mouse embryos. The epithelial cells were stably transfected with PC6 siRNA and PC6 knock down was confirmed at the levels of mRNA, protein, and activity by real-time RT-PCR, Western blotting and activity assay respectively. Embryos readily adhered to normal epithelial cells, but the adhesion was significantly reduced in the PC6 knockdown epithelial cells. We are currently using proteomics technology to identify the pathways affected by PC6 knockdown. These results strongly suggest that PC6 plays a critical role in modulating the human endometrial epithelium for receptivity and implantation.

Effects of galectin-3 inhibition on endometrial cell cycle and adhesion

Galectin-3 (gal-3) and its ligands have been implicated in cell transformation and cancer metastasis. Gal-3 protein has been found in uterine epithelial cells adjacent to implanting blastocysts in different cell types. In order to investigate the role of gal-3 in the establishment of human endometrial receptivity, the expression of gal-3 in human endometrial cell line RL95-2 was silenced by RNA interference technology using gal-3 specific small RNA. The expression of gal-3 was detected by the reverse transcriptase-polymerase chain reaction and Western blot analysis. After the suppression of gal-3, cell cycle changes and the expression of integrin β1 were detected by flow cytometry. The adhesive ability of RL95-2 cells was analyzed by the adhesion test. Gal-3 siRNA transfection efficiency reached 70%-90%. The expression of gal-3 mRNA and protein in RL95-2 cells was strongly inhibited by 70%-90% after RNA interference. Inhibition of gal-3 expression decreased S-phase but increased G1 phase cells. Integrin β1 expression was down-regulated, and the adhesive ability of RL95-2 cells to fibronectin (FN) was significantly reduced. Gal-3 may be involved in the establishment of endometrial receptivity by regulating the proliferation and adhesion of endometrial cells. The influence on adhesion may be related with the integrin modulation.

Macrophage migration inhibitory factor up-regulates alpha(v)beta(3) integrin and vascular endothelial growth factor expression in endometrial adenocarcinoma cell line Ishikawa

Human endometrium undergoes a series of dynamic physiological changes during the menstrual cycle of reproductive age women. Many factors, including hormones, cytokines, growth factors, matrix metalloproteinases and integrins, are essential for the success of embryonic implantation into endometrial tissue. Herein, we used a well-differentiated endometrial adenocarcinoma cell line, Ishikawa, to investigate in vitro the role played by macrophage migration inhibitory factor (MIF) in the regulation of endometrial receptivity markers. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that MIF induced a slight increase in alpha(v) (αv) mRNA integrin subunit expression during the first 12 h, but reached a significant difference after 24 h MIF treatment compared to control, whereas beta(3) (β3) integrin subunit displayed significant increase in mRNA 2 h following treatment. Immunocytofluorescence showed strong αv and β3 immunostaining at 25 ng/ml MIF, and Western blotting clearly indicated increased αv and β3 protein expression. MIF treatment significantly stimulated vascular endothelial growth factor (VEGF) mRNA expression in a dose- and time-dependent manner after 24 h treatment. Moreover, immunocytofluorescence revealed positive VEGF immunostaining compared to control, and analysis by ELISA of VEGF release in culture supernatants demonstrated that MIF (25 ng/ml) significantly induced VEGF secretion at 12 and 24 h. In conclusion, this study provides evidence that MIF directly up-regulates αvβ3 integrin and VEGF expression in human endometrial Ishikawa cells and may advance our understanding of factors involved in the establishment of endometrial receptivity and successful implantation.

Endometrial receptivity: Clinical assessment in relation to fertility, infertility, and antifertility

Fertility in humans and other mammalian species depends absolutely on synchronous events that render the developing blastocyst and the receiving uterus competent for implantation. Endometrial receptivity is defined as the period during which the endometrial epithelium acquires functional, but transient, ovarian steroid-dependent status supportive to blastocyst acceptance and implantation. Once inside the uterus, the blastocyst is surrounded by an intact luminal epithelium, which is considered to act as barrier to its attachment, except for this short period of high endometrial receptivity to blastocyst signal(s). Its transport and permeability properties, in conjunction with cellular action of the endometrium and the embryo, have been suggested to influence creation and maintenance of informational and nutritional status of uterine luminal milieu. This period, also termed as the 'window of implantation,' is limited to days 20-24 of menstrual cycle in humans. However, establishment of endometrial receptivity is still a biological mystery that remains unsolved despite marked advances in our understanding of endometrial physiology following extensive research associated with its development and function. This review deals with various structural, biochemical, and molecular events in the endometrium coordinated within the implantation window that constitute essential elements in the repertoire that signifies endometrial receptivity and is aimed to achieve a better understanding of its relationship to fertility, infertility, and for the development of targeted antifertility agents for human use and welfare.