The microRNAome of pregnancy: deciphering miRNA networks at the maternal-fetal interface.

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy

miRNAs Assessed by Real-Time PCR.

<p>miRNAs Assessed by Real-Time PCR.</p

Assessment of Putative miRNA Target Genes by Real-Time PCR.

<p>Eleven mRNA targets of the miRNAs that were significantly elevated in arresting as compared to healthy endometrium in the microarray experiment were selected for quantification by Real-Time PCR. Each mRNA was relatively quantified against the control gene GAPDH. As miRNAs negatively regulate their mRNA targets, a decrease in mRNA transcripts in endometrium from arresting conceptus attachment sites was expected. Endometrium from healthy attachment sites (white bars, n = 6) was compared to endometrium from arresting sites (black bars, n = 6) by t-test. Bars on the graphs represent the mean plus the SEM. Data is presented on a logarithmic scale to display all genes on one graph. No statistically significant differences were observed, however each of the 11 mRNAs quantified was decreased in arresting endometrium. Statistical analyses revealed the power of the t-tests to be low, and this may have muted differences between the tissues.</p

mRNAs Assessed by Real-Time PCR.

<p>mRNAs Assessed by Real-Time PCR.</p

miRNA Expression during Porcine Pregnancy.

<p>Expression levels of miRNAs detected by microarray which were significantly different (<i>p</i><0.05) between non-pregnant endometrium (n = 4) as compared to endometrium associated with healthy conceptus attachment sites (A, n = 3), and arresting conceptus attachment sites (B, n = 3). Real-Time PCR validation was performed for several miRNAs, and significant fold changes are listed for comparison. A positive fold change indicates that the miRNA was elevated in non-pregnant endometrium. A negative fold change indicates a decrease in the miRNA in the non-pregnant endometrium. N/A: not assessed, N.S.: not significant.</p

miRNA Expression in Endometrium and Trophoblast.

<p>Expression levels of miRNAs detected by microarray which were significantly different (<i>p</i><0.05) between endometrium (n = 3) and trophoblast (n = 3) from isolated from healthy (A) and arresting conceptus attachment sites (B). Real-Time PCR validation was performed for several miRNAs, and significant fold changes are listed for comparison. A positive fold change indicates that the miRNA was elevated in endometrium. A negative fold change indicates a decrease in the miRNA in endometrium. N/A: not assessed, N.S.: not significant.</p