Embryonic miRNA Profiles of Normal and Ectopic Pregnancies

<div><p>Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs) and controlled abortions (voluntary termination of pregnancy; VTOP). Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p) and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223) in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.</p></div

Embryonic miRNA Profiles of Normal and Ectopic Pregnancies - Table 1

<p>Patient characteristics for the samples used for the array studies. Data is presented as the mean plus or minus (±) standard error mean (SEM), or as the number and its corresponding percentage; ns  =  not significant. Gestational age (in days), gravidity, the number of births, history of previous spontaneous or induced abortions, and the number of induced abortions were evaluated with the Mann–Whitney U test, while the history of previous ectopic pregnancies, abdominal or pelvic surgeries, infertility, or use of an IUD or progestogen contraception was evaluated using Fisher's exact test.</p

Trophoblast cell lines transfection with miR-196 and miR223.

<p>JEG3 cell line was used to check the inhibition effects of miR-196b and miR-223 mimics over predicted gene targets. miRNA expression levels were checked after mimic transfection by Real Time PCR and compared to scramble expression (A). JEG3 cells transfected with miR-196b mimic showed a sharp decrease of ITGA2 after 48 and 72 hours of transfection, while GALNT13 showed a slighter decrease (B). A significant ITGA2 expression was observed GALNT1 in JEG3 cells after 48 and 72 hours of miR-223 transfection (C). All data are presented as relative miRNA expression levels. *<i>p</i>-value <0.05.</p

Validation of miRNAs in ectopic pregnancies and controls by real time PCR.

<p>21 VTOP and 15 EP embryonic samples were used for the validation study. We observed a very significant increase (p<0.001) in the EP tissue samples compared to VTOP samples for hsa-miR-451(A) and hsa-miR-223 (B) expression, while hsa-miR-196b (D) showed a significant (p<0.05) downregulation compared to VTOP controls. No statistical differences in expression were found for hsa-miR-30a (C). All data are presented as relative miRNA expression levels. *<i>p</i>-value <0.05; **<i>p</i>-value <0.01; ***<i>p</i>-value <0.001. Box plots represent the first quartile, median and third quartile; error bars show maximum and minimum relative expression levels.</p

Predicted genes and gene validation.

<p>(A) The most significant KEGG pathways for mir-196b, 223, and 451 miRNAs, together with the targeted genes for each miRNA and their mirV score are shown. (B) Real time PCRs were performed on EP and VTOP embryonic tissues for each of the predicted target genes: GALNT7, GALNT13, GALNT1, ITGA2, and COL1A2. GALNT13 and ITGA2 were found to be significantly upregulated in EP samples compared to VTOP samples. (C) hsa-miR-196b is encoded in an intron of the HOXA9 gene and inhibits the expression of this gene.</p

A list of differentially expressed miRNAs in embryonic tissues. Principal component analysis (PCA) and clustering analysis.

<p>(A) List of significantly altered miRNAs in pregnancy-derived tissues from ectopic pregnancies (EPs) compared to voluntary termination of pregnancy (VTOP) tissues using microarray analysis. Negative fold-change values represent downregulation and positive values represent upregulation. (B) Clustering and heat map analysis was performed using MeV software to cluster samples into hierarchies using Pearson correlation. All the samples classify well into two different populations (EP and VTOP) according to the expression of these seven miRNAs. The expression levels of these miRNAs were standardized and are represented as a heat map, showing variation ranging from high expression (red) to low expression (green) in each of the samples analyzed. (C) Three dimensional supervised PCA (which determines the key variables within the data set that explain the differences between the samples) for all the 16 tissue samples included in the microarray study, based on the expression profiles of the seven differentially expressed miRNAs. EP samples are clearly distinguished from VTOP sample sets at a separate position in the PCA analysis, and moreover, the EP samples and the VTOP samples clearly cluster together.</p

Embryonic miRNA Profiles of Normal and Ectopic Pregnancies - Table 2

<p>The patient characteristics for the samples used for the real time PCR analysis. Data is presented as the mean plus or minus (±) the standard error mean (SEM) or as the number and its corresponding percentage; ns  =  not significant. Gestational age (in days), gravidity, the number of births, history of previous spontaneous or induced abortions, and the number of induced abortions were evaluated with the Mann–Whitney U test while any history of previous ectopic pregnancies, abdominal or pelvic surgeries, infertility, or use of IUD or progestogen contraception was evaluated using Fisher's exact test.</p

Altered expression of LIN28B and Let-7a in embryonic tissue from early ectopic pregnancies.

<p>Relative levels of <i>LIN28B</i> mRNA (<b>A</b>) and <i>Let-7</i> miRNA (<b>B</b>) were assayed in samples from VTOP controls and ectopic pregnancy patients. For presentation, the data were grouped in two gestation age ranges: ≤6-week vs. 7–9 week gestational samples. Data are presented as mean ± SEM. Expression levels were quantitatively analyzed using the 2ΔΔCt method and were normalized to values from ≤6-week VTOP (control) samples. Data were analyzed by two-way ANOVA, followed by individual Newman-Keuls tests; **, P<0.01 vs. corresponding values in control samples (effect of EP); a, P<0.01 vs. corresponding values in <6-week samples (effect of gestational age).</p

Expression profiles of LIN28B and several miRNAs in embryonic tissue during normal placentation at early gestational periods.

<p>Relative mRNA and miRNA levels were assayed in placental samples from normal pregnancies, obtained from women undergoing voluntary termination of pregnancy (VTOP) at gestational ages ranging from week-5 to week-9 after amenorrhea. Samples from week-5 and −6 of amenorrhea were grouped as ≤6-week gestational samples. Targets analyzed were: <i>LIN28B</i> (<b>A</b>), <i>Let-7a</i> (<b>B</b>), <i>mir-132</i> (<b>C</b>), <i>mir-145</i> (<b>D</b>) and <i>mir-323-3p</i> (<b>E</b>). Data are presented as mean ± SEM. Expression levels were quantitatively analyzed using the 2ΔΔCt method and were normalized to values from ≤6-week gestational samples. *, P<0.05; **, P<0.01; and ***, P<0.001 vs. values in ≤6-week gestational samples (One-way ANOVA followed by Newman-Keuls test).</p