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Study Question: Do both ulipristal acetate (UPA) and mifepristone inhibit embryo-endometrial attachment at concentrations corresponding to the emergency contraception (EC) dose?Summary Answer: Both UPA and mifepristone at concentrations corresponding to the EC dose do not have an inhibitory effect on embryo implantation, although mifepristone at a higher concentration appeared to have such an effect.What Is Known Already: Levonorgestrel is commonly used for EC, but it only acts through inhibition of ovulation. UPA and mifepristone have higher efficacy as EC compared to levonorgestrel; while there is some suggestion that mifepristone may interfere with implantation, whether UPA has post-ovulatory action in inhibiting implantation is yet to be confirmed.Study Design, Size, Duration: An in vitro experimental study using trophoblastic spheroids made from JAr cell line as the embryo surrogate, and the Ishikawa cell line and primary human endometrial cells cultured to monolayer as the endometrial surrogate. The primary endometrial cells were collected from nine volunteer women in the mid-luteal phase with consent.Participants/materials, Setting, Methods: The study was conducted in a university gynaecology unit. The JAr and Ishikawa cell lines (or primary endometrial cells) were treated with graded concentrations of UPA (0, 0.04, 0.4 and 4 μM) or mifepristone (0, 0.1, 1 and 10 μM) for 24 h. Embryo-endometrial attachment was studied using an in vitro JAr spheroid-endometrial co-culture model. Expressions of progesterone receptor, β-catenin and glycogen synthase kinase 3 β (GSK-3β) were studied with real-time RT-PCR and Western blotting, respectively.Main Results and the Role Of Chance: In the Ishikawa experiments, there was no significant difference in the JAr spheroid attachment rate after treatment with UPA at 0 (93.0%), 0.04 (93.6%), 0.4 (93.4%) and 4 (91.4%) μM concentrations (P > 0.05); the attachment rate was reduced after treatment with mifepristone only at 10 μM (79.8%, P < 0.0001) but not at 0.1 (92.1%) or 1.0 (95.2%) μM concentrations. In the primary endometrial cell experiments, again no significant difference was observed in the JAr spheroid attachment rate after treatment with UPA 4 μM (42.6%) compared to control (46.5%, P > 0.05). Both UPA and mifepristone could significantly up-regulate progesterone receptor expression. There was no significant alteration in expression of β-catenin and GSK-3β after treatment with UPA 4 μM or mifepristone 10 μM (P > 0.05).Limitations, Reasons For Caution: The co-culture model is only a surrogate which may not fully represent the complicated process of embryo implantation in vivo, although there is no existing perfect model for studying implantation in vitro which fully resembles the latter.Wider Implications Of the Findings: The lack of inhibitory effect on embryo implantation by UPA and possibly mifepristone at concentrations corresponding to the EC dose is an important information for contraceptive counseling.Study Funding/competing Interest(s): We had free supply of the UPA compound used in this study from Laboratoire HRA Pharma. This work was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, The University of Hong Kong, Hong Kong. [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]

Study Question: What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation?Summary Answer: sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development.What Is Known Already: sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development.Study Design, Size, Duration: Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared.Participants/materials, Setting, Methods: Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively.Main Results and the Role Of Chance: sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized macrophages showed enhanced phagocytic activity. They also had higher expression and activity of indoleamine 2,3-dioxygenase 1, a phenotypic marker of decidual macrophages, which inhibited proliferation of autologous T-cells via induction of G0/G1 cell cycle arrest. In addition, sHLAG5-polarized macrophages had an increased secretion of interleukin-6 and C-X-C motif ligand 1, which inhibited interferon-γ production in T-cells and induction of trophoblast invasion, respectively.Limitations, Reasons For Caution: Most information on the phenotypes and biological activities of human decidual macrophages are based on past literatures. A direct comparison between sHLAG5-polarized macrophages and primary decidual macrophages is required to verify the present observations.Wider Implications Of the Findings: This is the first study on the role of sHLAG5 in macrophage differentiation. Further study on the mechanism that regulates the differentiation process of macrophages would enhance our understanding on the physiology of early pregnancy.Study Funding/competing Interests: This work was supported in part by the Hong Kong Research Grant Council Grant HKU774212 and the University of Hong Kong Grant 201309176126. The authors have no competing interests to declare.Trial Registration Number: Nil. [ABSTRACT FROM AUTHOR]

Background: Glycodelin-C is a glycodelin isoform isolated from the cumulus matrix. It stimulates spermatozoa-zona pellucida binding. Here, we report the isolation and characterization of a novel glycodelin interacting protein (GIP) from human cumulus matrix. Methods: GIP was purified by liquid chromatograph and identified by mass spectrometry. The interaction of GIP with glycodelin, matrix molecule and spermatozoa were investigated. Results: Mass spectrometry analysis suggested that GIP contained the N-terminal region of alpha2-macroglobulin, confirmed by western blot with anti-alpha2-macroglobulin antibody. GIP bound to native but not deglycosylated glycodelin-C in native gel electrophoresis, suggesting that the binding was glycosylation-dependent. GIP did not bind to capacitated and uncapacitated human spermatozoa. The cumulus cells could convert exogenous labeled alpha2-macroglobulin into GIP in vitro. GIP interacted with hyaluronic acid, a major component of the cumulus matrix. Glycodelin-C bound to hyaluronic acid-coated agarose beads in the presence of GIP. Human spermatozoa acquired the hyaluronic acid-GIP-bound glycodelin-C during incubation in vitro. Conclusion: The hyaluronic acid-GIP complex formed in the cumulus matrix retains and concentrates glycodelin-C in the cumulus matrix for displacing sperm-bound glycodelin-A and -F and stimulating the zona binding activity of the spermatozoa traversing through the cumulus mass. [ABSTRACT FROM AUTHOR]