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Vaiman D.,French Institute of Health and Medical Research | Vaiman D.,University of Paris Descartes | Gascoin-Lachambre G.,French Institute of Health and Medical Research | Gascoin-Lachambre G.,University of Paris Descartes | And 21 more authors.
PLoS ONE | Year: 2011

A low-protein diet applied during pregnancy in the rat results in intrauterine growth restricted (IUGR) fetuses. In humans, IUGR is associated with increased perinatal morbidity, higher incidence of neuro-developmental defects and increased risk of adult metabolic anomalies, such as diabetes and cardiovascular disease. Development and function of many organs are affected by environmental conditions such as those inducing fetal and early postnatal growth restriction. This phenomenon, termed "fetal programming" has been studied unconnectedly in some organs, but very few studies (if any) have investigated at the same time several organs, on a more comparative basis. However, it is quite probable that IUGR affects differentially most organ systems, with possible persistent changes in gene expression. In this study we address transcriptional alterations induced by IUGR in a multi-organ perspective, by systematic analysis of 20-days rat fetuses. We show that (1) expressional alterations are apparently stronger in organs functioning late in foetal or postnatal life than in organs that are functioning early (2) hierarchical classification of the deregulations put together kidney and placenta in one cluster, liver, lungs and heart in another; (3) the epigenetic machinery is set up especially in the placenta, while its alterations are rather mild in other organs; (4) the genes appear deregulated in chromosome clusters; (5) the altered expression cascades varies from organ to organ, with noticeably a very significant modification of the complement and coagulation cascades in the kidney; (6) we found a significant increase in TF binding site for HNF4 proteins specifically for liver genes that are down-regulated in IUGR, suggesting that this decrease is achieved through the action of HNF transcription factors, that are themselves transcriptionnally induced in the liver by IUGR (x 1.84 fold). Altogether, our study suggests that a combination of tissue-specific mechanisms contributes to bring about tissue-driven modifications of gene cascades. The question of these cascades being activated to adapt the organ to harsh environmental condition, or as an endpoint consequence is still raised. © 2011 Vaiman et al.

Lindgren I.,Lund University | Baath M.,Lund University | Uvebrant K.,Lund University | Dejmek A.,Skane University Hospital | And 9 more authors.
Human Reproduction | Year: 2016

STUDY QUESTION Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials? SUMMARY ANSWER The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF. WHAT IS KNOWN ALREADY The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context. STUDY DESIGN, SIZE, DURATION Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants. PARTICIPANTS/MATERIALS, SETTING, METHODS Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3′,5′-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035). LIMITATIONS, REASONS FOR CAUTION Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations. WIDER IMPLICATIONS OF THE FINDINGS Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed. STUDY FUNDING, COMPETING INTEREST(S) This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrol1ment of the subjects. The authors declare no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

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.

Borgbo T.,University of Aarhus | Borgbo T.,Laboratory of Reproductive Biology | Sommer Kristensen L.,Rigshospitalet | Lindgren I.,Lund University | And 2 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2014

Purpose: To provide an improved platform for simple, reliable, and cost-effective genotyping.Background: Modern fertility treatments are becoming increasingly individualized in an attempt to optimise the follicular response and reproductive outcome, following controlled ovarian stimulation. As the field of pharmacogenetics evolve, genetic biomarkers such as polymorphisms of the follicle stimulating hormone receptor (FSHR) may be included as a predictive tool for individualized fertility treatment. However, the currently available genotyping methods are expensive, time-consuming or have a limited analytical sensitivity. Here, we present a novel version of “competitive amplification of differentially melting amplicons” (CADMA), providing an improved platform for simple, reliable, and cost-effective genotyping.Methods: Two CADMA based assays were designed for the two common polymorphisms of the FSHR gene: rs6165 (c.919A > G, p. Thr307Ala, FSHR 307) and rs6166 (c.2039A > G, p. Asn680Ser, FSHR 680). To evaluate the reliability of the new CADMA-based assays, the genotyping results were compared with two conventional PCR based genotyping methods; allele-specific PCR (AS-PCR) and Sanger sequencing.Results: The genotype frequencies for both polymorphisms were 35 % (TT), 42 % (CT), and 23 % (CC), respectively. A 100 % accordance was observed between the CADMA-based genotyping results and sequencing results, whereas 5 discrepancies were observed between the AS-PCR results and the CADMA-based genotyping results. Comparing the CADMA-based assays to (AS-PCR) and Sanger sequencing, the CADMA based assays showed an improved analytical sensitivity and a wider applicability.Conclusions: The new assays provide a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practise. © 2014, Springer Science+Business Media New York.

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