TSA reduces hESC motility and limits trophoblast outgrowth and invasion.

<p><b>A</b>, Wound healing assay was performed in 48 hour-treated hESCs at two different TSA concentrations (0.1 µM and 1 µM) or a vehicle (control). The % of migrating cells was calculated as the area covered by cells and is represented as an average of three independent experiments. Statistical analysis ** p<0.01. <b>B,</b> Invasion assays of the TSA-treated hESCs were conducted in collagen transwells. hESCs were allowed to invade for 48 h after being previously treated with a vehicle (control), or with 0.1 µM and 1 µM TSA. The histograms show a percentage of invading cells, with the invasion of the control cells designated as 100%. Representative bright field images of the cells invading the underside of the filter are shown below the histograms. Data represent the mean of three independent experiments. Statistical analysis,* p<0.05 and ** p<0.01. <b>C</b>, TSA-treated hESCs limit mouse embryos invasion potential. hESCs and the mouse embryo were marked by Vimentin (red) and E-Cadherin (green), respectively. Nuclei were marked by DAPI (blue). The invasion area was identified by the absence of stromal cells (red) and outlined by a white line. Bar size is 50 µm. <b>D</b>, 1 µM TSA-treated hESCs significantly decreased the area of embryo spreading. Data represent the mean areas of at least 8 embryos in three independent experiments. Statistical analysis, ** p<0.01. <b>E,</b> Schematic representation of the collagen transwell invasion assay used to measure the effect of the TSA-treated hESCs conditioned medium on human trophoblast cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030508#s2" target="_blank">Material and Methods</a> for procedure description. <b>F,</b> Histograms show the percentage of the invading cells, with invasion of the control cells designated as 100%. Representative bright field images are shown below the histograms. Representative bright field images of the cells invading the underside of the filter are shown below the histograms. Data represent the mean of three independent experiments.</p

Models of the TSA-treated hESC and decidual hESCs.

<p><b>A,</b> In control endometrial stromal cells, trophoblast invasion is massive due to low levels of TIMPs and high levels of MMPs and uPA proteases. <b>B,</b> TSA-treated hESCs inhibits HDAC and promotes histone acetylation at the promoters of TIMP-1 and TIMP-3 by increasing the transcription of these genes. In contrast, the levels and activities of MMPs and uPA decrease. These ECM modulators changes reduced the motility of hESCs and the invasion of trophoblast cells. <b>C</b>, In <i>in vitro</i> decidualized hESCs, the intracellular cAMP levels increase and promote histone acetylation at the TIMP-1 and TIMP-3 gene promoters. Moreover, the activity and level of MMPs and uPA decreases and, together, precisely control the invasion of trophoblast cells.</p

In vitro decidualized hESCs ECM modulators expression and Chip experiments at the TIMP-1 and TIMP-3 gene promoters.

<p><b>A</b>, Quantitative PCRs for MMP-2, MMP-9, TIMP-1, TIMP-3, uPA and RECK expression levels in 5 day-cAMP+MPA-treated hESCs.. <b>B</b>, Extracellular TIMP-1 and TIMP-3 Western blot analyses of the protein extracts of 24 hour-conditioned media from hESCs treated with cAMP and MPA for 5 days. Ponceau staining of the membranes was used as a protein loading control. Only the 60 kDa band of the ponceau gel is shown. Representative image of three different analyses is shown. <b>C</b> and <b>D</b>, Chromatin immunoprecipitation experiments using antibodies against global H3 and H4 acetylation were carried out on the TIMP-1 and TIMP-3 genes in 24 hour-cAMP+MPA-treated hESCs. Relative amounts of immunoprecipitated TIMP-1 and TIMP-3 promoter sequences compared to input chromatin were determined by real-time PCR. <b>E</b>, Quantitative PCR for the TIMP-1 and TIMP-3 expression levels in 48 hour-cAMP+MPA, 1 µM TSA and cAMP+MPA+1 µM TSA-treated hESCs. Note that there is no significant difference between these two conditions at the level of the TIMP-1 and TIMP-3 gene expressions. Data represent the mean of three independent experiments. Statistical analysis,* p<0.05, ** p<0.01, a p<0.05, b p>0.05.</p

List of the potential miRNA targets involved during differentiation and decidualization events.

<p>List of the potential miRNA targets involved during differentiation and decidualization events.</p

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization <em>In Vitro</em>

<div><p>Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during <em>in vitro</em> decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process <em>in vitro</em>. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.</p> </div

miRNAs differentially expressed identified in the <i>in vitro</i> decidualization model.

<p>List of the 26 more up-regulated miRNAs and the 17 more down-regulated miRNAs identified in the <i>in vitro</i> decidualization model. The p values and fold changes are indicated. The miRNAs previously identified by Qian et al, 2009, are shown in boldface.</p

Supplemental Material, supplemental_Figure_1 - NGS Analysis of Human Embryo Culture Media Reveals miRNAs of Extra Embryonic Origin

<p>Supplemental Material, supplemental_Figure_1 for NGS Analysis of Human Embryo Culture Media Reveals miRNAs of Extra Embryonic Origin by Immaculada Sánchez-Ribas, Patricia Diaz-Gimeno, Alicia Quiñonero, María Ojeda, Zaloa Larreategui, Agustín Ballesteros, and Francisco Domínguez in Reproductive Sciences</p

Dicer function during endometrial decidualization.

<p><b>A</b>, A representative Western blot of DICER protein levels in control siRNA decidualized hESCs (Control siRNA (cAMP+MPA)) and Dicer knockdown decidualized hESCs (Dicer siRNA (cAMP+MPA)) at 48 h and 72 h after decidual treatment. <b>B</b>, Prolactin, IGFBP-1 and MPIF-1 secretion levels in the non decidualized control hESCs, the control siRNA decidualized hESCs (Control siRNA (cAMP+MPA)) and the Dicer knockdown decidualized hESCs (Dicer siRNA (cAMP+MPA)) measured by ELISA at 48 h and 72 h after decidual treatment. Prolactin, IGFBP-1 and MPIF-1 secretion levels were normalized to the total amount of protein present in the media. Data represent the mean of four independent experiments. Error bars represent the SEM. Statistical analysis, * p<0.05. <b>C</b>, TIMP-3 secretion levels measured by Western blot in the control siRNA decidualized hESCs (Control siRNA (cAMP+MPA)) and the Dicer knockdown decidualized hESCs (Dicer siRNA (cAMP+MPA)) at 72 h after decidual treatment. Ponceau staining of membranes was used as a protein-loading control. Only the 60 kDa band of the ponceau gel is shown. Data represent the mean of four independent experiments. Error bars represent the SEM.</p

miR-96 and miR-135b role during endometrial decidualization.

<p><b>A,</b> Relative FOXO1 and HOXA10 mRNA levels quantified by RT-qPCR in control or miR-96 and miR-135b transfected mimics in decidualized hESCs 48 h and 72 h after decidual treatment. <b>B,</b> IGFBP-1 secretion levels in the non decidualized control hESCs, the control miRNA decidualized hESCs and miR-96, miR-135b or miR96 and miR-135b transfected mimics in decidualized hESCs measured by ELISA at 48 h after decidual treatment. IGFBP-1 secretion levels were normalized to the total amount of protein present in the media. Data represent the mean of three independent experiments. Error bars represent the SEM. Statistical analysis, * p<0.05.</p

miR-181, miR-183 and miR-200 miRNAs families members are similarly regulated during in vitro decidualization.

<p><b>A</b>, miR-95, miR-135b and miR-96 expression levels by quantitative PCR in hESCs treated with 17β-estradiol (E), progesterone (P), or both (E+P), for 9 days. <b>B</b>, miR-181 <b>C</b>, miR-200 and <b>D</b>, miR-183 family members’ expression in the E+P decidualized hESCs for 9 days if compared to the non decidualized control hESCs. <b>A</b>, <b>B</b>, <b>C</b>, and <b>D</b>, Data represent the mean of three independent experiments. Error bars represent the standard error of the mean (SEM). Statistical analysis,* p<0.05. The molecular pathways potentially regulated by the mir-181, miR-200 and miR-183 families with the potential target genes are listed below the histograms.</p