BackgroundThe endometrial luminal epithelium is the first point of attachment of embryos during implantation. Failure of embryos to firmly adhere results in implantation failure and infertility. A receptive endometrial luminal epithelium is achieved through the expression of adhesion molecules in the mid-secretory phase and is a requirement for implantation. Cadherin 6 (CDH6) is an adhesion molecule localizing to the endometrial luminal epithelial cell surface in the mid-secretory/receptive phase and knockdown of CDH6 in the Ishikawa cells (receptive endometrial epithelial cell line) compromises cell integrity. However, there are no studies investigating the role of CDH6 on receptivity and infertility. This study aimed to investigate whether CDH6 is dysregulated in the endometrium of women with infertility during the receptive window and the effect of CDH6 on endometrial adhesion and receptivity.MethodsThe expression and the localization of CDH6 in the human endometrium were determined by immunohistochemistry. Ishikawa cells were used to investigate the functional consequences of CDH6 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids in vitro. CDH6 knockdown was assessed by qPCR and immunoblotting. After CDH6 knockdown, the expression of type II cadherin family members and CDH6 functional partners were assessed by qPCR. Two-tailed unpaired student’s t-test or one-way ANOVA as appropriate were used for statistical analysis with a significance threshold of P < 0.05.ResultsA significant reduction of CDH6 immunolocalization was recorded in the luminal and glandular epithelium of endometrium from women with infertility (P < 0.05) compared to fertile group respective cellular compartments in the mid-secretory phase. Functional analysis using Ishikawa cells demonstrated that knockdown of CDH6 (treated with 50 nM CDH6 siRNA) significantly reduced epithelial adhesive capacity (P < 0.05) to HTR8/SVneo spheroids compared to control and other type II cadherin family members likely failed to compensate for the loss of CDH6. The expression levels of CDH6 functional partners, catenin family members were not changed after CDH6 knockdown in Ishikawa cells.ConclusionTogether, our data revealed that CDH6 was dysregulated in the endometrium from women with infertility and altered Ishikawa cell adhesive capacity. Our study supports a role for CDH6 in regulating endometrial adhesion and implantation.

The human endometrium undergoes cycle-dependent changes and is only receptive to an implanting blastocyst within a narrow window of 2-4days in the mid-secretory phase. Such functional changes require delicate interplay between a diversity of factors including cytokines and signaling pathways. The Notch signaling pathway members are expressed in human endometrium. We have previously demonstrated that Notch ligand Jagged1 (JAG1) localizes in the endometrial luminal epithelium (LE) and is abnormally reduced in infertile women during receptivity. However, the functional consequences of reduced JAG1 production on endometrial receptivity to implantation of the blastocyst are unknown. This study aimed to determine the role of JAG1 in regulating endometrial receptivity in humans and mice. Knockdown of JAG1 in both primary human endometrial epithelial cells and Ishikawa cells significantly reduced their adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. We confirmed that in human endometrial epithelial cells, JAG1 interacted with Notch Receptor 3 (NOTCH3) and knockdown of JAG1 significantly reduced the expression of Notch signaling downstream target HEY1 and classical receptivity markers. Knockdown of Jag1 in mouse LE significantly impaired blastocyst implantation. We identified ten genes (related to tight junction, infertility, and cell adhesion) that were differentially expressed by Jag1 knockdown in LE in mice. Further analysis of the tight junction family members in both species revealed that JAG1 altered the expression of tight junction components only in mice. Together, our data demonstrated that JAG1 altered endometrial epithelial cell adhesive capacity and regulated endometrial receptivity in both humans and mice likely via different mechanisms.

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short ‘receptive window’ that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-β, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

Cyclooxygenase-2 network as predictive molecular marker for clinical pregnancy in in vitro fertilization

How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. NA.

Effects of variations in serum estradiol concentrations on secretory endometrial development and function in experimentally induced cycles in normal women

Endometrial receptivity is crucial for successful embryo implantation during early pregnancy. The human endometrium undergoes remodeling within each menstrual cycle to prepare or become receptive to an implanting blastocyst in the mid-secretory phase. However, the mechanisms behind these changes are not fully understood. Recently, using hormone-treated endometrial organoids to model receptivity, we identified that the transcriptional regulator WD-repeat-containing protein-61 (WDR61) was reduced in organoids derived from infertile women. In this study, we aimed to determine the role of WDR61 in endometrial receptivity. Here, we demonstrated that WDR61 immunolocalizes in the nuclei and cytosol of endometrial glandular epithelium, luminal epithelium, and stroma. The staining intensity of WDR61 was significantly higher during the receptive mid-secretory phase compared to the non-receptive proliferative phase in fertile women. In a functional experiment to model blastocyst adhesion to the endometrial epithelium, we found that adhesion of cytotrophoblast progenitor spheroids was blocked when siRNA was used to knockdown WDR61 in primary endometrial epithelial cells. Similarly, in Ishikawa cells (a receptive human endometrial epithelial cell line), siRNA knockdown of WDR61 significantly reduced the cell adhesive and proliferative capacities. qPCR revealed that WDR61 knockdown reduced expression of key genes involved in receptivity including HOXD10, MMP2, and CD44. Chromatin immunoprecipitation sequencing demonstrated that WDR61 directly targeted 2022 genes in Ishikawa cells, with functions including focal adhesion, intracellular signaling and epithelial-mesenchymal transition. Overall, these findings suggest that WDR61 promotes endometrial receptivity by modulating epithelial cell focal adhesions, proliferation, and epithelial-mesenchymal transition.

miR-23b-3p expression is increased in fertile endometrium during receptivity. This study investigates the function of miR-23b-3p on endometrial adhesion and its downstream targets. The human endometrium undergoes dramatic remodeling throughout the menstrual cycle that is essential for successful blastocyst attachment and implantation in the mid-secretory (receptive) phase. microRNA (miR) plays a role in the preparation of endometrial receptivity. miR-23b-3p expression is increased in fertile endometrium during receptivity. Here, we aimed to investigate miR-23b-3p function during receptivity. qPCR and in situ hybridization were used to investigate the expression and localization of miR-23b-3p in human endometrium, respectively. Ishikawa cells (endometrial epithelial cell line) and endometrial organoid-derived epithelial cells were transfected with miR-23b-3p mimic, and trophoblast progenitor spheroid (blastocyst surrogate) adhesion assay was used to determine effects on blastocyst adhesion to endometrial cells. We demonstrated that miR-23b-3p was significantly upregulated in the fertile endometrium of the receptive phase compared to the non-receptive, proliferative phase. No difference was identified for the expression of miR-23b-3p between fertile and infertile mid-secretory phase endometrium. miR-23b-3p localized to the epithelium and stroma in the mid-secretory phase but was undetectable in the proliferative phase of fertile endometrium. Functionally, miR-23-3p overexpression in Ishikawa cells and fertile endometrial organoid-derived epithelial cells significantly improved their adhesive capacity to trophoblast progenitor spheroids. miR-23b-3p overexpression in infertile endometrial organoid-derived epithelial cells did not improve adhesion. Among 10 miR-predicted gene targets examined, miR-23b-3p overexpression in Ishikawa cells significantly reduced the expression of MET, secreted frizzled-related protein 4 (SFRP4) and acyl-CoA dehydrogenase short/branched chain (ACADSB) compared to control. The reduction of SFRP4 after miR23b-3p overexpression was confirmed by immunoblotting in fertile organoid-derived epithelial cells. SFRP4 expression in fertile endometrium exhibited an inverse expression pattern compared to miR-23b-3p and was higher in the proliferative phase compared to the mid-secretory phase. Overall, miR-23b-3p is likely a critical regulator of endometrial epithelial adhesion and receptivity.

The objectives were to investigate the presence, distribution and sex steroid hormone regulation of ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), in human endometrium in relation to endometrial receptivity and fertility. Endometrial biopsies were obtained from women with unexplained infertility and healthy fertile volunteers. Ishikawa cells were used to mimic the action of ghrelin in endometrium. Immunostaining of GHSR was strong in luminal epithelium and stroma during mid-secretory phase. Ghrelin and GHSR expression is less intense in mid-secretory endometrium of infertile women compared to fertile controls. Treatment with estrogen and/or progesterone or their antagonists did not significantly change the relative expression of GHSR in Ishikawa and stromal cells. Ghrelin was present in and secreted from human blastocysts, which suggest that the communication between human blastocyst and endometrium might involve ghrelin. Low levels of GHSR in endometrium from women with unexplained infertility may in part explain the infertility.

Proenkephalin A and the γ-aminobutyric acid A receptor π subunit: expression, localization, and dynamic changes in human secretory endometrium

Prostaglandins (PG) are known to be involved in the process of human implantation. In several animal species treatment with prostaglandin synthesis inhibitors will prevent implantation. Cyclo-oxygenase (COX) is the rate-limiting enzyme in the synthesis of PGs and exists in two isoforms, COX-1 and COX-2. Defective implantation in COX-2-deficient mice has been demonstrated recently. In the present study, the expression of COX-1 and COX-2 was studied during the implantation period in healthy fertile women before and after treatment with the antiprogesterone, mifepristone. The study consisted of one control cycle and one treatment cycle. The subjects served as their own control. During the treatment cycle the subjects received 200 mg of mifepristone 2 days after the luteinizing hormone (LH) surge. Endometrial biopsies were obtained in the mid-luteal phase (LH + 6 to LH + 8) in both cycles. Using polyclonal antibodies against COX-1 and COX-2, immunostaining for COX-1 was found mainly in the glandular and luminal epithelium and for COX-2 mainly in the luminal epithelium and the perivascular cells. After treatment with mifepristone, expression of COX-1 in glandular epithelium and COX-2 in luminal epithelium significantly decreased whilst the immunostaining for COX-2 in the perivascular cells remained strong. This study shows the expression of both COX-1 and COX-2 during the implantation period and also indicates that treatment with mifepristone in early luteal phase impairs glandular epithelial function and endometrial receptivity.

Frozen embryo transfer: evidence-based markers for successful endometrial preparation

Profiling the gene signature of endometrial receptivity: clinical results

Endometrial estrogen and progesterone receptors mediate gene expression during the menstrual cycle in all mammalian species. At the time of implantation, differential patterns of gene expression occur on both glandular and luminal (surface) epithelium. Glandular secretions appear to be essential for embryonic development and viability, whereas luminal epithelium may provide a site for attachment and subsequent invasion of the nascent embryo. We have characterized previously integrins and other extracellular matrix (ECM) molecules as differentiated markers of endometrial epithelium. Both luminal and glandular epithelium express constitutive α3βl and α6β4 integrins and hormonally regulated α1βl, α4βl, α9βl, and αvβ3. The glandular epithelium expresses hormonally regulated ECM glycoproteins, such as osteopontin. Luminal cells exhibit cycle-dependent patterns of other α2βl and selectively express cytokeratin 13. Endometrial cancer cell lines have provided an excellent model to study normal endometrial epithelium. Of the many available cell lines, Ishikawa and ECC-1 cells maintain normal hormonal responsiveness to estrogen and progesterone, as well as to androgens. In this chapter, we review the potential of Ishikawa and ECC-1 cells as surrogate cells for normal endometrial epithelium and discuss their characteristics of glandular and luminal epithelia, respectively. Further characterization of these two cell lines may provide new insight into the mechanism of embryo implantation in humans and shed light on possible defects in endometrial receptivity.

Impaired endometrial receptivity is one of the major causes of recurrent implantation failure (RIF), although the underlying molecular mechanism has not been fully elucidated. In the present study, we demonstrated that chromodomain Y like (CDYL) was highly expressed in the endometrium at mid-secretory phase during the normal menstrual cycles. However, the expression of CDYL was downregulated in the endometrial tissues obtained from women with RIF, consistently with the protein level of LIF, which is a marker of endometrial receptivity. In CDYL-knockdown human endometrial Ishikawa cells, we identified 1738 differentially expressed genes (DEGs). Importantly, the catenin beta 1 (CTNNB1) expression was dramatically reduced responding to the CDYL inhibition, both in Ishikawa cells as well as the primary endometrial epithelial and stromal cells. In addition, the expression of CTNNB1was decreased in the endometrium from RIF patients as well. These results suggested that the expression of CTNNB1 was regulated by CDYL in endometrium. The cell migration was impaired by CDYL-knockdown in Ishikawa cells and primary endometrial stromal cells (ESCs), which could be rescued by CDYL or CTNNB1 overexpression. Collectively, our findings indicated that the decreased expression of CDYL may suppress endometrial cell migration capability by affecting CTNNB1 expression, which would contribute to poor endometrial receptivity in women with RIF.