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Search Results: 1 - 10 of 288637 matches for " Hilary O. D. Critchley "
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Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast
W. Colin Duncan, Julie L. V. Shaw, Stewart Burgess, Sarah E. McDonald, Hilary O. D. Critchley, Andrew W. Horne
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023595
Abstract: The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy.
TGFβ1 Attenuates Expression of Prolactin and IGFBP-1 in Decidualized Endometrial Stromal Cells by Both SMAD-Dependent and SMAD-Independent Pathways
Nicole M. Kane,Marius Jones,Jan J. Brosens,Rodney W. Kelly,Philippa T. K. Saunders,Hilary O. D. Critchley
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0012970
Abstract: Decidualization (differentiation) of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor β1 (TGFβ1) canonically propagates its actions via SMAD signalling. A role for TGFβ1 in decidualization remains to be established and published data concerning effects of TGFβ1 on markers of endometrial decidualization are inconsistent.
Evidence from a Mouse Model That Epithelial Cell Migration and Mesenchymal-Epithelial Transition Contribute to Rapid Restoration of Uterine Tissue Integrity during Menstruation
Fiona L. Cousins, Alison Murray, Arantza Esnal, Douglas A. Gibson, Hilary O. D. Critchley, Philippa T. K. Saunders
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086378
Abstract: Background In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. Methodology A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Principal Findings Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. Conclusions/Significance These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These insights may inform development of new therapies to induce rapid healing in the endometrium and other tissues and offer hope to women who suffer from heavy menstrual bleeding.
CB1 Expression Is Attenuated in Fallopian Tube and Decidua of Women with Ectopic Pregnancy
Andrew W. Horne, John A. Phillips, Nicole Kane, Paula C. Lourenco, Sarah E. McDonald, Alistair R. W. Williams, Carlos Simon, Sudhansu K. Dey, Hilary O. D. Critchley
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003969
Abstract: Background Embryo retention in the Fallopian tube (FT) is thought to lead to ectopic pregnancy (EP), a considerable cause of morbidity. In mice, genetic/pharmacological silencing of cannabinoid receptor Cnr1, encoding CB1, causes retention of embryos in the oviduct. The role of the endocannabinoids in tubal implantation in humans is not known. Methods and Findings Timed FT biopsies (n = 18) were collected from women undergoing gynecological procedures for benign conditions. Endometrial biopsies and whole blood were collected from women undergoing surgery for EP (n = 11); management of miscarriage (n = 6), and termination of pregnancy (n = 8). Using RT-PCR and immunohistochemistry, CB1 mRNA and protein expression levels/patterns were examined in FT and endometrial biopsies. The distribution of two polymorphisms of CNR1 was examined by TaqMan analysis of genomic DNA from the whole blood samples. In normal FT, CB1 mRNA was higher in luteal compared to follicular-phase (p<0.05). CB1 protein was located in smooth muscle of the wall and of endothelial vessels, and luminal epithelium of FT. In FT from women with EP, CB1 mRNA expression was low. CB1 mRNA expression was also significantly lower (p<0.05) in endometrium of women with EP compared to intrauterine pregnancies (IUP). Although of 1359G/A (rs1049353) polymorphisms of CNR1 gene suggests differential distribution of genotypes between the small, available cohorts of women with EP and those with IUP, results were not statistically significant. Conclusions CB1 mRNA shows temporal variation in expression in human FT, likely regulated by progesterone. CB1 mRNA is expressed in low levels in both the FT and endometrium of women with EP. We propose that aberrant endocannabinoid-signaling in human FT leads to EP. Furthermore, our finding of reduced mRNA expression along with a possible association between polymorphism genotypes of the CNR1 gene and EP, suggests a possible genetic predisposition to EP that warrants replication in a larger sample pool.
Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines
Irene Cervelló, Aymara Mas, Claudia Gil-Sanchis, Laura Peris, Amparo Faus, Philippa T. K. Saunders, Hilary O. D. Critchley, Carlos Simón
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0021221
Abstract: Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1–7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12–15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45?) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.
Shotgun Proteomics Identifies Serum Fibronectin as a Candidate Diagnostic Biomarker for Inclusion in Future Multiplex Tests for Ectopic Pregnancy
Jeremy K. Brown, Katarina B. Lauer, Emily L. Ironmonger, Neil F. Inglis, Tom H. Bourne, Hilary O. D. Critchley, Andrew W. Horne
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0066974
Abstract: Ectopic pregnancy (EP) is difficult to diagnose early and accurately. Women often present at emergency departments in early pregnancy with a ‘pregnancy of unknown location’ (PUL), and diagnosis and exclusion of EP is challenging due to a lack of reliable biomarkers. The objective of this study was to identify novel diagnostic biomarkers for EP. Shotgun proteomics, incorporating combinatorial-ligand library pre-fractionation, was used to interrogate pooled sera (n = 40) from women undergoing surgery for EP, termination of viable intrauterine pregnancy and management of non-viable intrauterine pregnancy. Western blot was used to validate results in individual sera. ELISAs were developed to interrogate sera from women with PUL (n = 120). Sera were collected at time of first symptomatic presentation and categorized according to pregnancy outcome. The main outcome measures were differences between groups and area under the receiver operating curve (ROC). Proteomics identified six biomarker candidates. Western blot detected significant differences in levels of two of these candidates. ELISA of sera from second cohort revealed that these differences were only significant for one of these candidates, fibronectin. ROC analysis of ability of fibronectin to discriminate EP from other pregnancy outcomes suggested that fibronectin has diagnostic potential (ROC 0.6439; 95% CI 0.5090 to 0.7788; P>0.05), becoming significant when ‘ambiguous’ medically managed PUL excluded from analysis (ROC 0.6538; 95% CI 0.5158 to 0.7918; P<0.05). Fibronectin may make a useful adjunct to future multiplex EP diagnostic tests.
Quantitative Serial MRI of the Treated Fibroid Uterus
Kirsty I. Munro, Michael J. Thrippleton, Alistair R. W. Williams, Graham McKillop, Jane Walker, Andrew W. Horne, David E. Newby, Richard A. Anderson, Scott I. Semple, Ian Marshall, Steff C. Lewis, Robert P. Millar, Mark E. Bastin, Hilary O. D. Critchley
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0089809
Abstract: Objective There are no long-term medical treatments for uterine fibroids, and non-invasive biomarkers are needed to evaluate novel therapeutic interventions. The aim of this study was to determine whether serial dynamic contrast-enhanced MRI (DCE-MRI) and magnetization transfer MRI (MT-MRI) are able to detect changes that accompany volume reduction in patients administered GnRH analogue drugs, a treatment which is known to reduce fibroid volume and perfusion. Our secondary aim was to determine whether rapid suppression of ovarian activity by combining GnRH agonist and antagonist therapies results in faster volume reduction. Methods Forty women were assessed for eligibility at gynaecology clinics in the region, of whom thirty premenopausal women scheduled for hysterectomy due to symptomatic fibroids were randomized to three groups, receiving (1) GnRH agonist (Goserelin), (2) GnRH agonist+GnRH antagonist (Goserelin and Cetrorelix) or (3) no treatment. Patients were monitored by serial structural, DCE-MRI and MT-MRI, as well as by ultrasound and serum oestradiol concentration measurements from enrolment to hysterectomy (approximately 3 months). Results A volumetric treatment effect assessed by structural MRI occurred by day 14 of treatment (9% median reduction versus 9% increase in untreated women; P = 0.022) and persisted throughout. Reduced fibroid perfusion and permeability assessed by DCE-MRI occurred later and was demonstrable by 2–3 months (43% median reduction versus 20% increase respectively; P = 0.0093). There was no apparent treatment effect by MT-MRI. Effective suppression of oestradiol was associated with early volume reduction at days 14 (P = 0.041) and 28 (P = 0.0061). Conclusion DCE-MRI is sensitive to the vascular changes thought to accompany successful GnRH analogue treatment of uterine fibroids and should be considered for use in future mechanism/efficacy studies of proposed fibroid drug therapies. GnRH antagonist administration does not appear to accelerate volume reduction, though our data do support the role of oestradiol suppression in GnRH analogue treatment of fibroids. Trial Registration ClinicalTrials.gov NCT00746031
Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification  [PDF]
David J. Bunyan, Hilary M. S. Bullman, Margaret Lever, Sasi D. Saminathan, Wee Teik Keng, Roziana Araffin, David O. Robinson
Open Journal of Genetics (OJGen) , 2011, DOI: 10.4236/ojgen.2011.12003
Abstract: We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
Innate immune defences in the human endometrium
Anne E King, Hilary OD Critchley, Rodney W Kelly
Reproductive Biology and Endocrinology , 2003, DOI: 10.1186/1477-7827-1-116
Abstract: The key role of the human endometrium is to orchestrate the events that lead to fertilization, implantation and pregnancy. The prevention of uterine infection is crucial to successful human reproduction and although the endometrium must function as an efficient mucosal barrier the passage of spermatozoa has to be accommodated. The innate immune system in endometrium, as elsewhere, must protect against infection while also signalling the presence of a pathogen to the acquired immune system in the event that infection does occur. Natural anti-microbials are gene-encoded peptides that are key mediators of the innate immune system and the primary focus of this review is to describe their expression in the human endometriumThe human endometrium undergoes characteristic cyclical changes in response to the steroid hormones, oestradiol and progesterone. These have been described in detail by Noyes et al [1]. In the first half of the menstrual cycle (proliferative phase, days 4–13), when oestradiol concentrations are increasing due to the development of a follicle, the endometrium undergoes proliferation. Ovulation occurs on day 14 of the cycle, the corpus luteum forms and progesterone concentrations increase thereafter. Under the influence of progesterone endometrial growth ceases and differentiation occurs in preparation for implantation and pregnancy. The secretory function of the endometrial glands increases with maximal secretion in the mid secretory phase (days 19–23) around day 20. In the late secretory phase (days 24–28) spiral arteriole differentiation occurs and the stromal cells in immediate proximity to these vessels differentiate (decidualize). The nuclei of these cells become enlarged and the cytoplasmic volume increases. These changes also occur in the stromal cells below the surface epithelium and, if pregnancy occurs, the entire endometrium decidualizes. In the absence of implantation menstruation occurs resulting in tissue breakdown and repair allowing rege
Pathogenesis of Endometriosis and Uterine Fibroids
Pasquapina Ciarmela,Hilary Critchley,Gregory M. Christman,Fernando M. Reis
Obstetrics and Gynecology International , 2013, DOI: 10.1155/2013/656571
Abstract:
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