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Background: Extravillous trophoblast cell (EVT) differentiation and its communication with maternal decidua especially the leading immune cell type natural killer (NK) cell are critical events for placentation. However, appropriate in vitro modelling system and regulatory programs of these two events are still lacking. Recent trophoblast organoid (TO) has advanced the molecular and mechanistic research in placentation. Here, we firstly generated the self-renewing TO from human placental villous and differentiated it into EVTs (EVT-TO) for investigating the differentiation events. We then co-cultured EVT-TO with freshly isolated decidual NKs for further study of cell communication. TO modelling of EVT differentiation as well as EVT interaction with dNK might cast new aspect for placentation research., Results: Single-cell RNA sequencing (scRNA-seq) was applied for comprehensive characterization and molecular exploration of TOs modelling of EVT differentiation and interaction with dNKs. Multiple distinct trophoblast states and dNK subpopulations were identified, representing CTB, STB, EVT, dNK1/2/3 and dNKp. Lineage trajectory and Seurat mapping analysis identified the close resemblance of TO and EVT-TO with the human placenta characteristic. Transcription factors regulatory network analysis revealed the cell-type specific essential TFs for controlling EVT differentiation. CellphoneDB analysis predicted the ligand-receptor complexes in dNK-EVT-TO co-cultures, which relate to cytokines, immunomodulation and angiogenesis. EVT was known to affect the immune properties of dNK. Our study found out that on the other way around, dNKs could exert effects on EVT causing expression changes which are functionally important., Conclusion: Our study documented a single-cell atlas for TO and its applications on EVT differentiation and communications with dNKs, and thus provide methodology and novel research cues for future study of human placentation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Successful placentation requires delicate communication between the endometrium and trophoblasts. The invasion and integration of trophoblasts into the endometrium during early pregnancy are crucial to placentation. Dysregulation of these functions is associated with various pregnancy complications, such as miscarriage and preeclampsia. The endometrial microenvironment has an important influence on trophoblast cell functions. The precise effect of the endometrial gland secretome on trophoblast functions remains uncertain. We hypothesized that the hormonal environment regulates the miRNA profile and secretome of the human endometrial gland, which subsequently modulates trophoblast functions during early pregnancy. Human endometrial tissues were obtained from endometrial biopsies with written consent. Endometrial organoids were established in matrix gel under defined culture conditions. They were treated with hormones mimicking the environment of the proliferative phase (Estrogen, E2), secretory phase (E2+Progesterone, P4), and early pregnancy (E2+P4+Human Chorionic Gonadotropin, hCG). miRNA-seq was performed on the treated organoids. Organoid secretions were also collected for mass spectrometric analysis. The viability and invasion/migration of the trophoblasts after treatment with the organoid secretome were determined by cytotoxicity assay and transwell assay, respectively. Endometrial organoids with the ability to respond to sex steroid hormones were successfully developed from human endometrial glands. By establishing the first secretome profiles and miRNA atlas of these endometrial organoids to the hormonal changes followed by trophoblast functional assays, we demonstrated that sex steroid hormones modulate aquaporin (AQP)1/9 and S100A9 secretions through miR-3194 activation in endometrial epithelial cells, which in turn enhanced trophoblast migration and invasion during early pregnancy. By using a human endometrial organoid model, we demonstrated for the first time that the hormonal regulation of the endometrial gland secretome is crucial to regulating the functions of human trophoblasts during early pregnancy. The study provides the basis for understanding the regulation of early placental development in humans., Competing Interests: Conflict of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The human placenta is a unique temporary organ with a mysterious immune tolerance. The formation of trophoblast organoids has advanced the study of placental development. HLA-G is uniquely expressed in the extravillous trophoblast (EVT) and has been linked to placental disorders. With older experimental methodologies, the role of HLA-G in trophoblast function beyond immunomodulation is still contested, as is its role during trophoblast differentiation. Organoid models incorporating CRISPR/Cas9 technology were used to examine the role of HLA-G in trophoblast function and differentiation. JEG-3 trophoblast organoids (JEG-3-ORGs) were established that highly expressed trophoblast representative markers and had the capacity to differentiate into EVT. CRISPR/Cas9 based on HLA-G knockout (KO) significantly altered the trophoblast immunomodulatory effect on the cytotoxicity of natural killer cells, as well as the trophoblast regulatory effect on HUVEC angiogenesis, but had no effect on the proliferation and invasion of JEG-3 cells and the formation of TB-ORGs. RNA-sequencing analysis further demonstrated that JEG-3 KO cells followed similar biological pathways as their wild-type counterparts during the formation of TB-ORGs. In addition, neither HLA-G KO nor the exogenous addition of HLA-G protein during EVT differentiation from JEG-3-ORGs altered the temporal expression of the known EVT marker genes. Based on the JEG-3 KO (disruption of exons 2 and 3) cell line and the TB-ORGs model, it was determined that HLA-G has a negligible effect on trophoblast invasion and differentiation. Despite this, JEG-3-ORG remains a valuable model for studying trophoblast differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhuang, Cao, Liu, Wang, Lin, Duan, Lee, Chiu, Yeung and Yao.)

During implantation, a symphony of interaction between the trophoblast originated from the trophectoderm of the implanting blastocyst and the endometrium leads to a successful pregnancy. Defective interaction between the trophoblast and endometrium often results in implantation failure, pregnancy loss, and a number of pregnancy complications. Owing to ethical concerns of using in vivo approaches to study human embryo implantation, various in vitro culture models of endometrium were established in the past decade ranging from two-dimensional cell-based to three-dimensional extracellular matrix (ECM)/tissue-based culture systems. Advanced organoid systems have also been established for recapitulation of different cellular components of the maternal-fetal interface, including the endometrial glandular organoids, trophoblast organoids and blastoids. However, there is no single ideal model to study the whole implantation process leaving more research to be done pursuing the establishment of a comprehensive in vitro model that can recapitulate the biology of trophoblast-endometrium interaction during early pregnancy. This would allow us to have better understanding of the physiological and pathological process of trophoblast-endometrium interaction during implantation., (© 2022. The Author(s).)

Bisphenol A (BPA) is commonly found in epoxy resins used in the manufacture of plastic coatings in food packaging and beverage cans. There is a growing concern about BPA as a weak estrogenic compound that can affect human endocrine function. Chemicals structurally similar to BPA, such as bisphenol F (BPF) and bisphenol S (BPS), have been developed as substitutes in the manufacturing industry. Whether these bisphenol substitutes have adverse effects on human endocrine and reproductive systems remains largely unknown. This study investigated the effects of BPA, BPF, and BPS on regulating the function of decidualized human primary endometrial stromal cells on trophoblast outgrowth and invasion by indirect and direct co-culture models. All three bisphenols did not affect the stromal cell decidualization process. However, BPA- and BPF-treated decidualized stromal cells stimulated trophoblastic spheroid invasion in the indirect coculture model. The BPA-treated decidualized stromal cells had upregulated expressions of several invasion-related molecules including leukemia inhibitory factor (LIF), whereas both BPA- and BPF-treated decidualized stromal cells had downregulated expressions of anti-invasion molecules including plasminogen activator inhibitor type 1 (PAI-1) and tumor necrosis factor (TNFα) . Taken together, BPA and BPF altered the expression of invasive and anti-invasive molecules in decidualized stromal cells modulating its function on trophoblast outgrowth and invasion, which could affect the implantation process and subsequent pregnancy outcome., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Successful implantation happens only when the development of a competent blastocyst synchronized with the differentiation of a receptive uterus. The exact mechanism affecting embryo implantation competency is still unclear. Previous data from our laboratory showed that several members of the let-7 family were up-regulated in the implanting dormant blastocysts and prohibited embryo activation through down-regulation integrin-β3. However, how the mir-let-7 family is regulated is still a question. In this study, the in vitro co-culture model was applied to imitate implantation. Human embryo surrogate Jeg-3 spheroids and endometrium epithelial cells Ishikawa were used. The following views were demonstrated. Firstly，Wnt/β-catenin signaling is essential for Jeg-3 spheroids implantation. Secondly, mir-let-7a is repressed by Wnt signaling, and low let-7a is beneficial for spheroids attachment and outgrowth. Third, in contrast with let-7a, lin28a is up-regulated by Wnt and promotes attachment and outgrowth. Lastly, the function of Wnt in embryo surrogate spheroids in implantation is mediated through lin28a/let-7a axis. In summary, our findings suggest Wnt/β-catenin signaling strength human embryo surrogate spheroids implanting potential through regulation lin28a/let-7a axis., Competing Interests: Declaration of competing interest No potential conflicts of interest were disclosed., (Copyright © 2019. Published by Elsevier Inc.)

Successful pregnancy depends largely on adequate placentation and maternal tolerance of the fetus. Glycodelin-A is a glycoprotein abundant in the decidua during early pregnancy. It plays an important role in placental development and fetomaternal defense. Glycodelin-A interacts by its unique carbohydrate side chains with the cell surface of various cell types in the human fetomaternal interface, particularly the trophoblasts and the immune cells, and modulates their functions and differentiation to permit successful pregnancy. Abnormal levels of glycodelin-A in the endometrium, uterine flushings, and/or maternal serum correlate with unexplained infertility, early pregnancy loss, and recurrent miscarriage. This review integrates recent studies on the role of glycodelin-A in placental development and fetomaternal tolerance in early pregnancy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Study Question: Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation?, Summary Answer: We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation., What Is Known Already: Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research., Study Design, Size, Duration: Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast., Participants/materials, Setting, Methods: Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-β inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells., Main Results and the Role of Chance: After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture., Limitations, Reasons for Caution: The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles., Wider Implications of the Findings: BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro., Design: Experimental study., Setting: Reproductive biology laboratory., Patient(s): Healthy nonpregnant women., Intervention(s): No patient interventions., Main Outcome Measure(s): Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay., Result(s): Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression., Conclusion(s): Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Study Question: Does CD147 regulate trophoblast functions in vitro?, Summary Answer: CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast., What Is Known Already: CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear., Study Design, Size, Duration: A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts., Participants/materials, Setting Methods: CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs., Main Results and the Role of Chance: CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA., Limitations, Reasons for Caution: Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast., Wider Implications of the Findings: This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation., Study Funding/competing Interest(s): This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

Both paracrine and autocrine factors are involved in the regulation of trophoblast invasion. One of these factors is human chorionic gonadotropin (hCG), which stimulates trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) that is expressed in early pregnancy. We compared the stimulatory activities of different forms of hCG and its free β-subunit (hCGβ) on trophoblast invasion. hCG, hCG-h, hCGβ, and its hyperglycosylated form (hCGβ-h) stimulated the invasion of JEG-3 choriocarcinoma cells. The stimulatory effect of hCGβ was also confirmed with primary human trophoblasts. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGβ and hCG on cell invasion. Increased invasion was associated with increased levels of MMP-2, MMP-9 and activity of uPA. Our findings suggest that hCG, hCGβ and their hyperglycosylated forms stimulate the invasion of trophoblast cells independent of the classical LH/hCG-receptor., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM(22-52) (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P < 0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.

Glycodelin contains four isoforms with diverse biological functions. Glycodelin-A is potentially a diagnostic marker for cancer patients and receptivity marker of the secretory endometrium. Yet, direct evidence for the role of glycodelin in the regulation of endometrial epithelial cell migration, invasion and attachment of trophoblastic spheroids (blastocyst surrogate) is lacking. In this study, the human glycodelin gene was stably transfected into human endometrial (HEC1-B) cells. Forced expression of glycodelin in HEC1-B cells did not affect cell proliferation, cell viability or cell-cycle progression, but significantly reduced migration and invasion of the stably transfected cells (both P<0.05). The migration rate returned to normal levels when the glycodelin-forced-expressing HEC1-B cells were treated with glycodelin RNAi. Furthermore, forced expression of glycodelin in HEC1-B cells significantly increased the attachment of trophoblastic spheroids onto the endometrial epithelial cells (P<0.05). In summary, glycodelin suppressed endometrial cell migration and invasion but enhanced spheroid attachment., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects embryo development, implantation and fertility in humans. The underlying molecular mechanism by which TCDD suppresses implantation remains largely unknown. We used the trophoblastic spheroids (embryo surrogate)-endometrial cells co-culture assay to study the attachment of trophoblastic spheroids (BeWo and Jeg-3) onto the endometrial epithelial (RL95-2 and Ishikawa) cells. TCDD dose-dependently induced cytochrome P450 1A1 (Cyp1A1) expression in trophoblastic and endometrial epithelial cells. Moreover, TCDD at 1 and 10 nM suppressed β-catenin (a Wnt-signaling molecule) and E-cadherin expression, as well as spheroids attachment onto endometrial cells. Interestingly, activation of the canonical Wnt-signaling pathway via Wnt3a or lithium chloride reverted the suppressive effect of TCDD on β-catenin and E-cadherin expressions in the BeWo and RL95-2 cells, and restored the spheroids attachment rate to be comparable to the untreated controls. Taken together, TCDD induces Cyp1A1 expression, modulates the Wnt-signaling pathway and suppresses spheroids attachment onto endometrial cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

During placentation, the cytotrophoblast differentiates into the villous cytotrophoblast and the extravillous cytotrophoblast. The latter invades the decidualized endometrium. Glycodelin-A (GdA) is abundantly synthesized by the decidua but not the trophoblast. Previous data indicate that GdA suppresses the invasion of trophoblast cell lines by down-regulating proteinase expression and activities. This study addresses the signaling pathway involved in the above phenomenon. GdA was found to suppress phosphorylation of ERKs and expression of their downstream effector c-Jun, a component of the transcription factor activator protein-1 (AP-1). The involvement of ERKs and c-Jun in suppressing trophoblast invasion and biosynthesis of proteinases was confirmed by using siRNA knockdown and pharmacological inhibitors. Desialylation reduced binding affinity of GdA toward and invasion suppressive activities on the trophoblast. Co-immunoprecipitation showed that Siglec-6 on the trophoblast was the binding protein of GdA. The binding of GdA to Siglec-6 was sialic acid-dependent. Treatment with anti-Siglec-6 antibody abolished the invasion suppressive activities of GdA. These results show that GdA interacts with Siglec-6 to suppress trophoblast invasiveness by down-regulating the ERK/c-Jun signaling pathway.

Glycodelin-A (GdA) is a glycoprotein secreted from the endometrial glands and decidual glandular epithelium. Given its abundance and ubiquitous distribution in the first trimester uterus, GdA may be involved in early placental development via its modulatory effect on immune and trophoblast cells. GdA inhibits activation and proliferation, and induces apoptosis of T cells. By selectively inducing Th1 cell death, GdA may shift the Th1/Th2 ratio at the feto-maternal interface. This is also achieved indirectly through enhanced expression of Fas in the Th1 cells, thus making them vulnerable to cell death through Fas ligand expressed on trophoblast, endometrial, and activated T helper cells. GdA also promotes secretion of the Th2 cytokines IL-6 and IL-13 from NK cells, and induces immunological tolerance of dendritic cells and apoptosis of monocytes. Specific glycosylation is a prerequisite for the biological activities of GdA. Reduction in α2-6 sialylation of GdA, as in gestational diabetes, is associated with impairment of its T cell apoptosis-inducing activities. This review integrates recent studies on GdA and its role as a paracrine regulator in early pregnancy., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Glycodelin-A increased the secretion of interleukin-6, interleukin-13, and granulocyte-macrophage colony-stimulating factor from natural killer cells in the peripheral blood but does not affect their viability, cell death, and cytotoxicity. These data suggest that glycodelin-A contributes to the cytokine shift in early pregnancy., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Trophoblast invasion is crucial to placentation. The relationship between decidual glycodelin-A and trophoblast invasion is not known., Methods: Invasiveness of First trimester extravillous cytotrophoblast-1 (TEV-1) cell line, TEV-1, cells was determined by trans-well invasion assay. The gene expression, protein secretion and activities of the matrix metalloproteinase (MMP)-2 and -9, urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and plasminogen activator inhibitor (PAI-1) of glycodelin-A-treated cells were measured by quantitative PCR, ELISA and gel zymography, respectively., Results: Glycodelin-A bound to TEV-1 cells. At a concentration of 1 microg/ml, glycodelin-A, but not other glycodelin isoforms, suppressed the invasion of TEV-1 cells. The effect was glycosylation-dependent and was associated with reduction (P < 0.05) of MMP2, MMP9 and uPA activities in the conditioned medium from the treated culture. Glycodelin-A treatment suppressed the amount of MMP2 protein in the conditioned medium (P < 0.05) and MMP2 mRNA in the cells (P < 0.05), but did not affect that of MMP9. Glycodelin-A also significantly reduced the expression, secretion and activity of uPA (P < 0.05). The treatment did not affect the expression of TIMP-1, TIMP-2 or PAI-1, cell proliferation or survival of the cells., Conclusions: Glycodelin-A inhibits the invasion of extravillous cytotrophoblasts mainly by suppressing the activity of MMP2 and MMP9 in a glycosylation-dependent fashion.

Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings. [ABSTRACT FROM AUTHOR]

Study Question: Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation?Summary Answer: We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation.What Is Known Already: Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research.Study Design, Size, Duration: Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast.Participants/materials, Setting, Methods: Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-β inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells.Main Results and the Role Of Chance: After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture.Limitations, Reasons For Caution: The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles.Wider Implications Of the Findings: BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells. [ABSTRACT FROM AUTHOR]