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Borgbo T.,Laboratory of Reproductive Biology | Povlsen B.B.,Fertility Clinic | Andersen C.Y.,Laboratory of Reproductive Biology | Borup R.,Microarray Center | And 2 more authors.
Fertility and Sterility | Year: 2013

Objective To explore differences in follicle transcriptomes in patients having oocyte maturation with either a bolus of hCG or GnRHa. Design Cumulus cells (CC) and mural granulosa cells (MGC) were isolated from preovulatory follicles in patients undergoing controlled ovarian stimulation, prospectively randomized to GnRHa or hCG triggering. Setting University-based facilities for clinical services and research. Patient(s) Twenty women with indication for IVF or intracytoplasmic sperm injection treatment were randomly allocated to hCG or GnRH agonist (GnRHa) trigger. Intervention(s) MGC and CC were collected from individual follicles in connection with oocyte retrieval. Main Outcome Measure(s) RNA was extracted, labeled, amplified, and hybridized on HumanGene1.0ST GeneChip Affymetrix array. Expression data were robust multichip average normalized and compared using Partek and Ingenuity software. Array data were confirmed with reverse transcription-polymerase chain reaction analysis. Result(s) Comparing the transcriptomes between the groups, 391 and 252 genes were differentially expressed (fold change >1.5) in CC and MGC, respectively. The enriched bionetworks showed that CC genes highly represented "lipid metabolism and small molecule biochemistry" (network score, 41), while in MGC, the top network was "cardiovascular development and function and cellular movement" (network score, 50). For both CC and MGC, the regulator analysis suggested LH as the upstream regulator for the difference observed. In CC, the LH receptor was more highly expressed after GnRHa trigger, while in MGC, genes involved in angiogenesis such as angiopoietin 1 and semaphorin 3A were down- and up-regulated, respectively, in GnRHa- as compared with hCG-triggered patients. Conclusion(s) The comparisons between somatic cell transcriptomes from GnRHa- and hCG-triggered follicles showed significant functional differences in both CC (steroidogenesis) and MGC (angiogenesis) compartments. Copyright © 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.

Wissing M.L.,Fertility Clinic | Kristensen S.G.,Laboratory of Reproductive Biology | Andersen C.Y.,Laboratory of Reproductive Biology | Mikkelsen A.L.,Fertility Clinic | And 4 more authors.
Human Reproduction | Year: 2014

STUDY QUESTION: Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation? SUMMARY ANSWER: Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation. WHAT IS KNOWN ALREADY: Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows. STUDY DESIGN, SIZE, DURATION: Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbæk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay.MAIN RESULTS AND THE ROLE OF CHANCEE: leven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Müllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested-besides LH-TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response. LIMITATIONS, REASONS FOR CAUTION: The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle. WIDER IMPLICATIONS OF THE FINDINGS: These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers. © 2014 The Author.

Grondahl M.L.,Laboratory of Reproductive Biology | Grondahl M.L.,Copenhagen University | Andersen C.Y.,Laboratory of Reproductive Biology | Bogstad J.,Copenhagen University | And 3 more authors.
Molecular Human Reproduction | Year: 2012

During folliculogenesis, the granulosa cells differentiate into two cell types: cumulus cells (CCs) and mural granulosa cells (MGCs). The objective of the study was to generate and compare the transcriptomes of MGCs and CCs from the pre-ovulatory follicle to characterize the detailed profile of the two cell populations shortly before ovulation. Twenty-one IVF/ICSI patients undergoing controlled ovarian stimulation (COS) donated CCs and MGCs from individual follicles containing metaphase II oocytes. Cells were prepared immediately after recovery and mRNA was isolated for whole-genome gene expression analysis and reverse transcriptase-polymerase chain reactions. Paired (within the individual follicle) comparisons between the CC and MGC expression profiles were performed and corrected for multiple comparisons. A total of 1562 genes were differentially expressed by >2-fold (P < 0.01) in the two cell types. Of these, 156 genes were >8-fold changed and represented specialized cellular functional categories such as inflammatory response, extracellular matrix and cell-cell communication, whereas the 1406 genes were 2-8-fold changed and represented functional categories such as proliferation and lipid metabolism. Transcripts not previously linked to the follicle were found to be differentially expressed between CCs and MGCs, suggesting specialized function in these compartments, e.g. pepsinogen A was selectively expressed in MGCs, whereas ryanodine receptor-2 (RYR2) was selectively expressed in CCs. Positive correlations were present between expression levels of RYR2 and the amphiregulin and gap-junction proteins. In conclusion, the transcriptomes of corresponding CCs and MGCs from individual pre-ovulatory follicles clearly revealed two distinct cell types. New as well as known genes representing specific cell functions close to ovulation were highlighted. © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Devaux Y.,Laboratory of Cardiovascular Research | Vausort M.,Laboratory of Cardiovascular Research | Goretti E.,Laboratory of Cardiovascular Research | Nazarov P.V.,Microarray Center | And 7 more authors.
Clinical Chemistry | Year: 2012

BACKGROUND: Rapid and correct diagnosis of acute myocardial infarction (MI) has an important impact on patient treatment and prognosis. We compared the diagnostic performance of high-sensitivity cardiac troponin T (hs-cTnT) and cardiac enriched microRNAs (miRNAs) in patients with MI. METHODS: Circulating concentrations of cardiac-enriched miR-208b and miR-499 were measured by quantitative PCR in a case-control study of 510 MI patients referred for primary mechanical reperfusion and 87 healthy controls. RESULTS: miRNA-208b and miR-499 were highly increased in MI patients (>10 5-fold, P < 0.001) and nearly undetectable in healthy controls. Patients with ST-elevation MI (n=397) had higher miRNA concentrations than patients with non-ST-elevation MI (n =113) (P < 0.001). Both miRNAs correlated with peak concentrations of creatine kinase and cTnT (P <10 -9). miRNAs and hs-cTnT were already detectable in the plasma 1 h after onset of chest pain. In patients who presented <3 h after onset of pain, miR-499 was positive in 93% of patients and hs-cTnT in 88% of patients (P = 0.78). Overall, miR-499 and hs-cTnT provided comparable diagnostic value with areas under the ROC curves of 0.97. The reclassification index of miR-499 to a clinical model including several risk factors and hs-cTnT was not significant (P = 0.15). CONCLUSIONS: Circulating miRNAs are powerful markers of acute MI. Their usefulness in the establishment of a rapid and accurate diagnosis of acute MI remains to be determined in unselected populations of patients with acute chest pain. © 2011 American Association for Clinical Chemistry.

Vetter G.,University of Luxembourg | Saumet A.,French Institute of Health and Medical Research | Moes M.,University of Luxembourg | Vallar L.,Microarray Center | And 7 more authors.
Oncogene | Year: 2010

Epithelial to mesenchymal transition (EMT) is a key step toward metastasis. MCF7 breast cancer cells conditionally expressing the EMT master regulator SNAI1 were used to identify early expressed microRNAs (miRNAs) and their targets that may contribute to the EMT process. Potential targets of miRNAs were identified by matching lists of in silico predicted targets and of inversely expressed mRNAs. MiRNAs were ranked based on the number of predicted hits, highlighting miR-661, a miRNA with so far no reported role in EMT. MiR-661 was found required for efficient invasion of breast cancer cells by destabilizing two of its predicted mRNA targets, the cell-cell adhesion protein Nectin-1 and the lipid transferase StarD10, resulting, in turn, in the downregulation of epithelial markers. Reexpression of Nectin-1 or StarD10 lacking the 3′-untranslated region counteracted SNAI1-induced invasion. Importantly, analysis of public transcriptomic data from a cohort of 295 well-characterized breast tumor specimen revealed that expression of StarD10 is highly associated with markers of luminal subtypes whereas its loss negatively correlated with the EMT-related, basal-like subtype. Collectively, our non-a priori approach revealed a nonpredicted link between SNAI1-triggered EMT and the down-regulation of Nectin-1 and StarD10 through the up-regulation of miR-661, which may contribute to the invasion of breast cancer cells and poor disease outcome. © 2010 Macmillan Publishers Limited All rights reserved.

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