RESUMO
Endocrine-disrupting chemicals (EDCs) might contribute to the increase in female-specific cancers in Western countries. 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) is considered the "prototypical toxicant" to study EDCs' effects on reproductive health. Epigenetic regulation by small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), is crucial for controlling cancer development. The aim of this study was to analyze transcriptional activity and sncRNA expression changes in the KGN cell line after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD in order to determine whether sncRNAs' deregulation may contribute to transmitting TCDD effects to the subsequent cell generations (day 9 and day 14 after chronic exposure). Using Affymetrix GeneChip miRNA 4.0 arrays, 109 sncRNAs were found to be differentially expressed (fold change < -2 or >2; p-value < 0.05) between cells exposed or not (control) to TCDD for 3 h and 72 h and on day 9 and day 14 after chronic exposure. Ingenuity Pathway Analysis predicted that following the acute and chronic exposure of KGN cells, sncRNAs linked to cellular development, growth and proliferation were downregulated, and those linked to cancer promotion were upregulated on day 9 and day 14. These results indicated that TCDD-induced sncRNA dysregulation may have transgenerational cancer-promoting effects.
Assuntos
Disruptores Endócrinos , MicroRNAs , Neoplasias , Dibenzodioxinas Policloradas , Pequeno RNA não Traduzido , Humanos , Feminino , MicroRNAs/genética , Dibenzodioxinas Policloradas/toxicidade , Epigênese Genética , Células da GranulosaRESUMO
BACKGROUND: Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. METHODS: Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. RESULTS: In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. CONCLUSIONS: The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.
Assuntos
Células do Cúmulo/citologia , Regulação da Expressão Gênica , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Adulto , Técnicas de Cultura Embrionária/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Técnicas de Maturação in Vitro de Oócitos , Análise de Sequência com Séries de Oligonucleotídeos , Oogênese/fisiologia , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/terapia , Reação em Cadeia da Polimerase/métodos , Fatores de Tempo , Transcrição GênicaRESUMO
The appreciation of endometrial receptivity is a crucial step in assisted reproductive technology as implantation failures are thought to result, in large part, from abnormal endometrial receptivity. Using emerging omics technologies, investigators have begun to define both molecular signatures and specific biomarkers of receptive endometrium. The aim of this review was to analyse the new perspectives brought to the appreciation of endometrial receptivity by transcriptomic and proteomic technologies, involving the analysis of gene- or protein-expression-profile shifts between the pre-receptive and receptive secretory stages and how they might lead to new strategies for endometrial receptivity assessments. The use of omics as molecular tools to determine the effects of stimulation protocols on endometrial gene expression and clinical outcomes has also been investigated.
Assuntos
Endométrio/metabolismo , Regulação da Expressão Gênica , Proteômica/métodos , Transcrição Gênica , Biomarcadores/metabolismo , Biópsia , Implantação do Embrião , Feminino , Fertilização in vitro/métodos , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Fenótipo , GravidezRESUMO
Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.
Assuntos
Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Leucemia de Células B/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Fosforilação Oxidativa , Trifosfato de Adenosina/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/imunologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo/imunologia , Glutamina/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Jurkat , Leucemia L1210 , Leucemia de Células B/enzimologia , Leucemia de Células B/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/genética , Regulação para Cima/imunologiaRESUMO
This is a retrospective study to evaluate if the miRNome profile of endometrium samples collected during the implantation window predicts Assisted Reproduction Technology (ART) outcomes. We first investigated the endometrial miRNome profile according to the receptivity status in 20 patients with repeated implantation failures (RIF) (discovery cohort). After customized embryo transfer, the miRNome profiles of receptive patients with a positive or negative ß-hCG, and with early miscarriage or live birth were analysed. Some differentially expressed miRNAs were selected for validation by RT-qPCR in endometrial samples from 103 RIF patients (validation cohort). Analysis of the different miRNome profiles identified endometrial receptivity, implantation failure, and early miscarriage-associated miRNA signatures that included 11, 261, and 76 miRNAs, respectively. However, only four miRNAs associated with the endometrial receptivity status (miR-455-3p and miR-4423-3p) and implantation failure (miR-152-3p and miR-155-5p) were significantly validated in endometrial samples. The miRNome profile of endometrial tissues during the implantation window can predict the pregnancy outcome. These data are crucial for opening new perspectives to predict implantation failure and consequently, to increase ART success.
Assuntos
Aborto Espontâneo , MicroRNAs , Implantação do Embrião/genética , Endométrio , Feminino , Humanos , MicroRNAs/genética , Gravidez , Estudos RetrospectivosRESUMO
In women, up to 99.9% of the oocyte stockpile formed during fetal life is decimated by apoptosis. Apoptotic features are also detected in human preimplantation embryos both in vivo and in vitro. Despite the important consequences of cell death processes to oocyte competence and early embryonic development, little is known about its genetic and molecular control. B cell lymphoma-2 (BCL2) family proteins are major regulators of cell death and survival. Here, we present a literature review on BCL2 family expression and protein distribution in human and animal oocytes and early embryos. Most of the studies focused on the expression of two antagonistic members: the founding and survival family member BCL2 and its proapoptotic homolog BAX. However, recent transcriptomic analyses have identified novel candidate genes related to oocyte and/or early embryonic viability (such as BCL2L10) or commitment to apoptosis (e.g. BIK). Interestingly, some BCL2 proteins appear to be differentially distributed at the subcellular level during oocyte maturation and early embryonic development, a process probably linked to the functional compartmentalization of the ooplasm and blastomere. Assessment of BCL2 family involvement in regulating the survival of human oocytes and embryos may be of particular value for diagnosis and assisted reproductive technology. We suggest that implications of not only aberrant gene expression but also abnormal subcellular protein redistribution should be established in pathological conditions resulting in infertility.
Assuntos
Blastocisto/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Animais , Blastocisto/patologia , Morte Celular , Sobrevivência Celular , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Infertilidade/genética , Infertilidade/metabolismo , Infertilidade/fisiopatologia , Oócitos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Understanding whether SARS-CoV-2 could infect cells and tissues handled during ART is crucial for risk mitigation, especially during the implantation window when either endometrial biopsies are often practiced for endometrial receptivity assessment or embryo transfer is performed. To address this question, this review analyzed current knowledge of the field and retrospectively examined the gene expression profiles of SARS-CoV-2-associated receptors and proteases in a cohort of ART candidates using our previous Affymetrix microarray data. Human endometrial tissue under natural and controlled ovarian stimulation cycles and preimplantation embryos were analyzed. A focus was particularly drawn on the renin-angiotensin system, which plays a prominent role in the virus infection, and we compared the gene expression levels of receptors and proteases related to SARS-CoV-2 infection in the samples. High prevalence of genes related to the ACE2 pathway during both cycle phases and mainly during the mid-secretory phase for ACE2 were reported. The impact of COS protocols on endometrial gene expression profile of SARS-CoV-2-associated receptors and proteases is minimal, suggesting no additional potential risks during stimulated ART procedure. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level. Specimens from female genital tract should be considered as potential targets for SARS-CoV-2, especially during the implantation window.
RESUMO
Oxygen (O2) concentration is approximately 5% in the fallopian tube and 2% in the uterus in humans. A "back to nature" approach could increase in vitro fertilization (IVF) outcomes. This hypothesis was tested in this monocentric observational retrospective study that included 120 couples who underwent two IVF cycles between 2014 and 2019. Embryos were cultured at 5% from day 0 (D0) to D5/6 (monophasic O2 concentration strategy) in the first IVF cycle, and at 5% O2 from D0 to D3 and 2% O2 from D3 to D5/6 (biphasic O2 concentration strategy) in the second IVF cycle. The total and usable blastocyst rates (44.4% vs. 54.8%, p = 0.049 and 21.8% vs. 32.8%, p = 0.002, respectively) and the cumulative live birth rate (17.9% vs. 44.1%, p = 0.027) were significantly higher with the biphasic (5%-2%) O2 concentration strategy. Whole transcriptome analysis of blastocysts donated for research identified 707 RNAs that were differentially expressed in function of the O2 strategy (fold-change > 2, p value < 0.05). These genes are mainly involved in embryo development, DNA repair, embryonic stem cell pluripotency, and implantation potential. The biphasic (5-2%) O2 concentration strategy for preimplantation embryo culture could increase the "take home baby rate", thus improving IVF cost-effectiveness and infertility management.
Assuntos
Coeficiente de Natalidade , Blastocisto/metabolismo , Técnicas de Cultura Embrionária/métodos , Fertilização in vitro/métodos , Infertilidade/terapia , Nascido Vivo , Oxigênio/metabolismo , Adulto , Análise Custo-Benefício , Implantação do Embrião/genética , Transferência Embrionária/métodos , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro/economia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Estudos Retrospectivos , Transcriptoma/genética , Resultado do TratamentoRESUMO
The aim of this prospective study was to evaluate outcome benefits expected in repeated implantation failure (RIF) patients (n = 217) after customized embryo transfer based upon identification of the receptivity window by transcriptomic approach using the Win-Test. In this test, the expression of 11 endometrial genes known to be predictive of endometrial receptivity is assessed by RT-PCR in biopsies collected during the implantation window (6-9 days after the spontaneous luteinizing hormone surge during natural cycles, 5-9 days after progesterone administration during hormone replacement therapy cycles). Then, patients underwent either customized embryo transfer (cET, n = 157 patients) according to the Win-Test results or embryo transfer according to the classical procedure (control group, n = 60). Pregnancy and live birth rates were compared in the two groups. The Win-Test showed that in 78.5% of women, the receptivity window lasted less than 48 h, although it could be shorter (< 24 h, 9.5%) or longer (> 48 h, 12%). This highlighted that only in 20% of patients with RIF the endometrium would have been receptive if the classical embryo transfer protocol was followed. In the other 80% of patients, the receptivity window was delayed by 1-3 days relative to the classical timing. This suggests that implantation failure could be linked to inadequate timing of embryo transfer. In agreement, both implantation (22.7% vs. 7.2%) and live birth rates per patient (31.8% vs. 8.3%) were significantly higher in the cET group than in the control group. cET on the basis of the Win-Test results could be proposed to improve pregnancy and live birth rates.ClinicalTrials.gov ID: NCT04192396; December 5, 2019, retrospectively registered.
Assuntos
Criopreservação , Implantação do Embrião/genética , Transferência Embrionária/efeitos adversos , Fertilização in vitro/efeitos adversos , Perfilação da Expressão Gênica , Infertilidade/terapia , Transcriptoma , Adulto , Feminino , França , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Nascido Vivo , Gravidez , Taxa de Gravidez , Estudos Prospectivos , Fatores de Tempo , Falha de TratamentoRESUMO
Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst-like structures suggesting cholangiocyte-type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte-specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor-beta (TGF-beta) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF-beta on hepatocytic lineage differentiation.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula , Forma Celular , Fígado/citologia , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Humanos , Laminina/metabolismo , Fígado/fisiologia , Camundongos , Organoides/citologia , Organoides/metabolismo , Proteoglicanas/metabolismo , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta/genéticaRESUMO
The impact of gonadotropin-releasing hormone (GnRH) agonist long compared with GnRH antagonist protocols, under in vitro fertilization conditions on endometrial receptivity, is still debated. Therefore, we compared the effect of both GnRH antagonist and agonist long protocols on the endometrial receptivity by analyzing, to our knowledge for the first time, the global gene expression profile shift during the prereceptive and receptive stages of stimulated cycles under the two GnRH analogue protocols compared with natural cycles in the same patients. For the same normal-responder patients, endometrial biopsies were collected on the day of oocyte retrieval and on the day of embryo transfer after human chorionic gonadotropin administration of a stimulated cycle with either GnRH agonist long or GnRH antagonist protocols and compared with the prereceptive and receptive stages of a natural cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved during the prereceptive stage to the receptive endometrial transition of stimulated and natural cycles were analyzed and compared for each patient. Both protocols affect endometrial receptivity in comparison with their natural cycle in the same patients. Major differences in endometrial chemokines and growth factors under stimulated cycles in comparison with natural cycles were observed. Such an effect has been associated with gene expression alterations of endometrial receptivity. However, the endometrial receptivity under the GnRH antagonist protocol was more similar to the natural cycle receptivity than that under the GnRH agonist protocol.
Assuntos
Endométrio/fisiopatologia , Fertilização in vitro , Indução da Ovulação/efeitos adversos , Biomarcadores/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Gonadotropina Coriônica/administração & dosagem , Protocolos Clínicos/normas , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Fertilização in vitro/efeitos adversos , Fertilização in vitro/métodos , Perfilação da Expressão Gênica , Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/administração & dosagem , Humanos , Infertilidade/fisiopatologia , Infertilidade/terapia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Indução da Ovulação/métodosRESUMO
Single-embryo transfer is becoming increasingly common in in vitro fertilization (IVF) treatment as a means of reducing multiple pregnancy rates leading to a higher incidence of medical, perinatal and neonatal complications. Consequently, selecting embryos with the highest implantation potential is of great importance in assisted reproductive technology. To date, the choice of the best embryos to transfer is based on subjective morphological parameters. However, as judged by their subjective aspect, movement towards more sophisticated technologies to select the most competent oocytes and/or embryos with the greatest implantation potential have become available, including emerging 'omics' sciences, such as genomics, transcriptomics, proteomics and metabolomics. In this way, the study of the cumulus cells (CCs) transcriptomic profile offers the opportunity, by a non-invasive method, to predict oocyte and embryo competence because bidirectional traffic between CCs and the oocyte is very important for the acquisition of this competence. Using either RT-PCR or DNA microarrays, some studies have provided evidence for the genes expressed in CCs presenting potential biomarkers to predict embryo quality and pregnancy outcomes. This review provides an overview of the current knowledge about CCs as biomarkers for oocyte and embryo selection under an IVF program.
Assuntos
Biomarcadores/análise , Células do Cúmulo/metabolismo , Resultado da Gravidez , Técnicas de Reprodução Assistida , Feminino , Humanos , Análise em Microsséries , GravidezRESUMO
Repression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N-methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins, which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrometry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs-/- mice suggest that Coprs is implicated in genome surveillance mechanisms.
Assuntos
Chaperonas de Histonas/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Espermatócitos/crescimento & desenvolvimento , Espermatogênese/genética , Animais , Masculino , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/metabolismo , Espermatócitos/metabolismoRESUMO
In assisted reproduction, about 30% of embryo implantation failures are related to inadequate endometrial receptivity. To identify molecules involved in endometrial receptivity acquisition, we investigated, using a SELDI-TOF approach, the protein expression profile of early-secretory and mid-secretory endometrium samples. Among the proteins upregulated in mid-secretory endometrium, we investigated the function of S100A10 in endometrial receptivity and implantation process. S100A10 was expressed in epithelial and stromal cells of the endometrium of fertile patients during the implantation windows. Conversely, it was downregulated in the mid-secretory endometrium of infertile patients diagnosed as non-receptive. Transcriptome analysis of human endometrial epithelial and stromal cells where S100A10 was silenced by shRNA revealed the deregulation of 37 and 256 genes, respectively, related to components of the extracellular matrix and intercellular connections. Functional annotations of these deregulated genes highlighted alterations of the leukocyte extravasation signaling and angiogenesis pathways that play a crucial role during implantation. S100A10 silencing also affected the migration of primary endometrial epithelial and stromal cells, decidualization and secretory transformation of primary endometrial stromal cells and epithelial cells respectively, and promoted apoptosis in serum-starved endometrial epithelial cells. Our findings identify S100A10 as a player in endometrial receptivity acquisition.
Assuntos
Anexina A2/metabolismo , Endométrio/metabolismo , Proteínas S100/metabolismo , Adulto , Apoptose , Adesão Celular , Movimento Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Regulação para Baixo , Implantação do Embrião , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Espectrometria de Massas , Ciclo Menstrual , Fenótipo , Gravidez , Proteômica , Células Estromais/metabolismo , Transcriptoma/genética , Trofoblastos/citologiaRESUMO
Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-X(L), yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-X(L) failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-X(L).
Assuntos
Apoptose/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Animais , Eletrofisiologia , Citometria de Fluxo , Células HeLa , Hepatócitos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Canais Iônicos/metabolismo , Células Jurkat , Lipossomos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/síntese química , Permeabilidade/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The impact of a premature elevation of serum progesterone level, the day of hCG administration in patients under controlled ovarian stimulation during IVF procedure, on human endometrial receptivity is still debated. In the present study, we investigated the endometrial gene expression profile shifts during the prereceptive and receptive secretory stage in patients with normal and elevated serum progesterone level on the day of hCG administration in fifteen patients under stimulated cycles. Then, specific biomarkers of endometrial receptivity in these two groups of patients were tested. Endometrial biopsies were performed on oocyte retrieval day and on day 3 of embryo transfer, respectively, for each patient. Samples were analysed using DNA microarrays and qRT-PCR. The endometrial gene expression shift from the prereceptive to the receptive stage was altered in patients with high serum progesterone level (>1.5 ng/mL) on hCG day, suggesting accelerated endometrial maturation during the periovulation period. This was confirmed by the functional annotation of the differentially expressed genes as it showed downregulation of cell cycle-related genes. Conversely, the profile of endometrial receptivity was comparable in both groups. Premature progesterone rise alters the endometrial gene expression shift between the prereceptive and the receptive stage but does not affect endometrial receptivity.
Assuntos
Gonadotropina Coriônica/administração & dosagem , Transferência Embrionária , Endométrio/metabolismo , Progesterona/sangue , Substâncias para o Controle da Reprodução/administração & dosagem , Adulto , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Endométrio/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , HumanosRESUMO
In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality.
Assuntos
Células do Cúmulo/metabolismo , Hormônio Foliculoestimulante/administração & dosagem , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Gonadotropinas/administração & dosagem , Ensaios Clínicos como Assunto , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismoRESUMO
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Diferenciação Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Ectoderma/citologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Trofoblastos/metabolismo , Trofoblastos/fisiologiaRESUMO
In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.