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1.
PLoS One ; 8(3): e58502, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23516492

RESUMO

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100-300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50-150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.


Assuntos
Microambiente Celular , Implantação do Embrião , Endométrio/citologia , Exossomos/metabolismo , Biomarcadores/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Membranas Intracelulares/metabolismo , Ciclo Menstrual/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Muco/metabolismo
2.
PLoS One ; 7(12): e53098, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23300868

RESUMO

BACKGROUND: Synchronous development of the endometrium (to achieve a receptive state) and of the embryo is essential for successful implantation and ongoing pregnancy. Endometrial receptivity exists only for a finite time in a menstrual cycle and the endometrium is refractory to embryo implantation outside of this window. Administration of hormones to stimulate multifollicular development within the ovary, integral to the majority of assisted reproduction (ART) protocols, dramatically alters the hormonal milieu to which the endometrium is exposed versus normal menstrual cycles. Endometrial maturation may be profoundly affected by this altered endocrine environment. AIM: Compare endometrial histology in fertile women, fertile women undergoing hormonal stimulation for oocyte donation and infertile women undergoing fresh embryo transfers in an ART cycle with further comparisons between women who did or did not become pregnant. Examine the presence of leukocytes and markers of endometrial maturation. METHODS: Endometrial histology was examined by hematoxylin and eosin staining with a semi quantitative scoring method developed to compare histological appearance of tissues. The presence of leukocytes and developmental markers was examined by immunohistochemistry and scored. RESULTS: Endometrial histology was dramatically altered upon stimulation for ART. However, those women who became pregnant presented with significantly less alterations in histological endometrial maturation. Numbers and activation status of leukocyte populations were also altered within the endometria stimulated for ART, with neutrophils undergoing degranulation, usually observed only pre-menstrually. CONCLUSION: We propose that such developmental changes render the endometrium hostile to the embryo and that modifications to ART protocols should be considered to take account of the requirement for endometrial receptivity and hence increase pregnancy rates.


Assuntos
Implantação do Embrião/fisiologia , Endométrio/patologia , Infertilidade Feminina/patologia , Adulto , Endométrio/metabolismo , Feminino , Fertilização in vitro , Humanos , Infertilidade Feminina/metabolismo , Leucócitos/metabolismo , Leucócitos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Gravidez , Taxa de Gravidez , Prolactina/metabolismo , Receptores de Progesterona/metabolismo , Estudos Retrospectivos
3.
J Proteome Res ; 9(12): 6256-64, 2010 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-20925431

RESUMO

Endometrial secretions in the uterine cavity contain mediators important for endometrial receptivity and embryo implantation. Unbiased analysis of uterine fluid from a receptive versus nonreceptive time of the menstrual cycle and in fertile and infertile women will provide new insights into uterine receptivity. We hypothesized that proteomic analysis of human uterine lavages would identify proteins important for the establishment of pregnancy in humans. Lavages collected from fertile (n = 7) and infertile (n = 8) women during the midsecretory (MS) phase, and from fertile women during the midproliferative (MP) (n = 7) phase, were assessed using 2D-differential in gel electrophoresis (2D-DiGE) over a pI 4-7 range. Statistical analysis revealed 7 spots that were significantly decreased in the MP compared to the MS phase, while 18 spots showed differential expression between fertile and infertile women. A number of proteins were identified by mass spectrometry, including antithrombin III and alpha-2-macroglobulin, whose production was confirmed in endometrial epithelium. Their staining pattern suggests roles during embryo implantation. Assessment of the human endometrial secretome has identified differences in the protein content of uterine fluid with respect to receptivity and fertility.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Endométrio/metabolismo , Proteínas/análise , Proteômica/métodos , Adulto , Sequência de Aminoácidos , Antitrombina III/análise , Antitrombina III/metabolismo , Feminino , Fertilidade , Humanos , Infertilidade Feminina , Dados de Sequência Molecular , Gravidez , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Útero/metabolismo , alfa-Macroglobulinas/análise , alfa-Macroglobulinas/metabolismo
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