Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoder

Children\u27s and caregivers\u27 perspectives about mandatory reporting of child maltreatment: A meta-synthesis of qualitative studies

Objective To systematically synthesise qualitative research that explores children\u27s and caregivers\u27 perceptions of mandatory reporting. Design We conducted a meta-synthesis of qualitative studies. Data sources Searches were conducted in Medline, Embase, PsycINFO, Cumulative Index to Nursing and Allied Health Literature, Criminal Justice Abstracts, Education Resources Information Center, Sociological Abstracts and Cochrane Libraries. Eligibility criteria English-language, primary, qualitative studies that investigated children\u27s or caregivers\u27 perceptions of reporting child maltreatment were included. All healthcare and social service settings implicated by mandatory reporting laws were included. Data extraction and synthesis Critical appraisal of included studies involved a modified checklist from the Critical Appraisal Skills Programme (CASP). Two independent reviewers extracted data, including direct quotations from children and caregivers (first-order constructs) and interpretations by study authors (second-order constructs). Third-order constructs (the findings of this meta-synthesis) involved synthesising second-order constructs that addressed strategies to improve the mandatory reporting processes for children or caregivers-especially when these themes addressed concerns raised by children or caregivers in relation to the reporting process. Results Over 7935 citations were retrieved and 35 articles were included in this meta-synthesis. The studies represent the views of 821 caregivers, 50 adults with histories of child maltreatment and 28 children. Findings suggest that children and caregivers fear being reported, as well as the responses to reports. Children and caregivers identified a need for improvement in communication from healthcare providers about mandatory reporting, offering preliminary insight into child-driven and caregiver-driven strategies to mitigate potential harms associated with reporting processes. Conclusion Research on strategies to mitigate potential harms linked to mandatory reporting is urgently needed, as is research that explores children\u27s experiences with this process

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety

Mandated reporters\u27 experiences with reporting child maltreatment: A meta-synthesis of qualitative studies

Objective To systematically search for research about the effectiveness of mandatory reporting of child maltreatment and to synthesise qualitative research that explores mandated reporters\u27 (MRS) experiences with reporting. Design As no studies assessing the effectiveness of mandatory reporting were retrieved from our systematic search, we conducted a meta-synthesis of retrieved qualitative research. Searches in Medline (Ovid), Embase, PsycINFO, Cumulative Index to Nursing and Allied Health Literature, Sociological Abstracts, Education Resources Information Center, Criminal Justice Abstracts and Cochrane Library yielded over 6000 citations, which were deduplicated and then screened by two independent reviewers. English-language, primary qualitative studies that investigated MRS\u27 experiences with reporting of child maltreatment were included. Critical appraisal involved a modified checklist from the Critical Appraisal Skills Programme and qualitative meta-synthesis was used to combine results from the primary studies. Setting All healthcare and social-service settings implicated by mandatory reporting laws were included. Included studies crossed nine high-income countries (USA, Australia, Sweden, Taiwan, Canada, Norway, Finland, Israel and Cyprus) and three middle-income countries (South Africa, Brazil and El Salvador). Participants: The studies represent the views of 1088 MRS. Outcomes Factors that influence MRS\u27 decision to report and MRS\u27 views towards and experiences with mandatory reporting of child maltreatment. Results Forty-four articles reporting 42 studies were included. Findings indicate that MRS struggle to identify and respond to less overt forms of child maltreatment. While some articles (14%) described positive experiences MRS had with the reporting process, negative experiences were reported in 73% of articles and included accounts of harm to therapeutic relationships and child death following removal from their family of origin. Conclusions The findings of this meta-synthesis suggest that there are many potentially harmful experiences associated with mandatory reporting and that research on the effectiveness of this process is urgently needed

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

Mandated reporters' experiences with reporting child maltreatment: a meta-synthesis of qualitative studies.

OBJECTIVE: To systematically search for research about the effectiveness of mandatory reporting of child maltreatment and to synthesise qualitative research that explores mandated reporters' (MRs) experiences with reporting. DESIGN: As no studies assessing the effectiveness of mandatory reporting were retrieved from our systematic search, we conducted a meta-synthesis of retrieved qualitative research. Searches in Medline (Ovid), Embase, PsycINFO, Cumulative Index to Nursing and Allied Health Literature, Sociological Abstracts, Education Resources Information Center, Criminal Justice Abstracts and Cochrane Library yielded over 6000 citations, which were deduplicated and then screened by two independent reviewers. English-language, primary qualitative studies that investigated MRs' experiences with reporting of child maltreatment were included. Critical appraisal involved a modified checklist from the Critical Appraisal Skills Programme and qualitative meta-synthesis was used to combine results from the primary studies. SETTING: All healthcare and social-service settings implicated by mandatory reporting laws were included. Included studies crossed nine high-income countries (USA, Australia, Sweden, Taiwan, Canada, Norway, Finland, Israel and Cyprus) and three middle-income countries (South Africa, Brazil and El Salvador). PARTICIPANTS: The studies represent the views of 1088 MRs. OUTCOMES: Factors that influence MRs' decision to report and MRs' views towards and experiences with mandatory reporting of child maltreatment. RESULTS: Forty-four articles reporting 42 studies were included. Findings indicate that MRs struggle to identify and respond to less overt forms of child maltreatment. While some articles (14%) described positive experiences MRs had with the reporting process, negative experiences were reported in 73% of articles and included accounts of harm to therapeutic relationships and child death following removal from their family of origin. CONCLUSIONS: The findings of this meta-synthesis suggest that there are many potentially harmful experiences associated with mandatory reporting and that research on the effectiveness of this process is urgently needed

Quality assurance and assessment frameworks of biosystems engineering studies

Regulatory instruments at the national level to ensure high quality are crucial to achieve and maintain a regional hub of higher education in Malta. While The Malta Qualifications Council and the National Commission for Higher Education are established and operational, the legal framework to set up a quality assurance agency and a quality assurance and licensing framework is in place, but the legislations is still awaiting approval. The University of Malta has set up internal quality assurance structures, The Programme Validation Committee monitors, reviews and recommends programmes for approval by Senate, The formation of the INSTITUTE OF EARTH SYSTEMS will facilitate the means through which a Bio Systems Engineering course could be offered, Furthermore the recent establishment of a Maltese Chamber of Agrologists could in theory eventually take up the role to grant professional accreditation,peer-reviewe

Engaging domestic abuse practitioners and survivors in a review of outcome tools – reflections on differing priorities

Researchers often develop and decide upon the measurement tools for assessing outcomes related to domestic abuse interventions. However, it is known that clients, service providers and researchers have different ideas about the outcomes that should be measured as markers of success. Evidence from non-domestic abuse sectors indicates that engagement of service providers, clients and researchers contributes to more robust research, policy and practice. We reflect on what we have learnt from the engagement of practitioners and domestic abuse survivors in a review of domestic abuse measurement tools where there were clear differences in priorities between survivors, practitioners and researchers about the ideal measurement tools. The purpose of this reflective article is to support the improvement of future outcome measurement from domestic abuse interventions, while ensuring that domestic abuse survivors do not relive trauma because of measurement

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycaemia on these processes remains poorly understood. Here we show that physiological levels of glucose (5mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in co-cultures at 17mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarised epithelium. 2D and 3D morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of RhoA-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signalling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes