Deregulation of miR-324/KISS1/kisspeptin in early ectopic pregnancy: mechanistic findings with clinical and diagnostic implications.

BACKGROUND: Ectopic pregnancy is a life-threatening condition for which novel screening tools that would enable early accurate diagnosis would improve clinical outcomes. Kisspeptins, encoded by KISS1, play an essential role in human reproduction, at least partially by regulating placental function and possibly embryo implantation. Kisspeptin levels are elevated massively in normal pregnancy and reportedly altered in various gestational pathologic diseases. Yet, the pathophysiologic role of KISS1/kisspeptin in ectopic pregnancy has not been investigated previously. OBJECTIVE: The purpose of this study was to evaluate changes of KISS1/kisspeptin levels in ectopic pregnancy and their underlaying molecular mechanisms and to ascertain the diagnostic implications of these changes. STUDY DESIGN: A total of 122 women with normal pregnancy who underwent voluntary termination of pregnancy and 84 patients who experienced tubal ectopic pregnancy were recruited. Measurements of plasma kisspeptins and KISS1 expression analyses in human embryonic/placental tissue were conducted in ectopic pregnancy and voluntary termination of pregnancy control subjects during the early gestational window (<12 weeks). Putative microRNA regulators of KISS1 were predicted in silico, followed by expression analyses of selected microRNAs and validation of repressive interactions in vitro. Circulating levels of these microRNAs were also assayed in ectopic pregnancy vs voluntary termination of pregnancy. RESULTS: Circulating kisspeptins gradually increased during the first trimester of normal pregnancy but were reduced markedly in ectopic pregnancy. This profile correlated with the expression levels of KISS1 in human embryonic/placental tissue, which increased in voluntary termination of pregnancy but remained suppressed in ectopic pregnancy. Bioinformatic predictions and expression analyses identified miR-27b-3p and miR-324-3p as putative repressors of KISS1 in human embryonic/placental tissue at <12 weeks gestation, when expression of microRNAs was low in voluntary termination of pregnancy control subjects but significantly increased in ectopic pregnancy. Yet, a significant repressive interaction was documented only for miR-324-3p, occurring at the predicted 3'-UTR of KISS1. Interestingly, circulating levels of miR-324-3p, but not of miR-27b-3p, were suppressed distinctly in ectopic pregnancy, despite elevated tissue expression of the pre-microRNA. A decision-tree model that used kisspeptin and miR-324-3p levels was successful in discriminating ectopic pregnancy vs voluntary termination of pregnancy, with a receiver-operating characteristic area under the curve of 0.95±0.02 (95% confidence interval). CONCLUSION: Our results document a significant down-regulation of KISS1/kisspeptins in early stages of ectopic pregnancy via, at least partially, a repressive interaction with miR-324-3p. Our data identify circulating kisspeptins and miR-324-3p as putative biomarkers for accurate screening of ectopic pregnancy at early gestational ages.

Developmental Changes in Genome Replication Progression in Pluripotent versus Differentiated Human Cells.

DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and differentiated cells. Using single-cell microscopic approaches, we map the spatio-temporal genome replication as a function of chromatin marks/compaction level. Furthermore, we mapped the replication timing of subchromosomal tandem repeat regions and interspersed repeat sequence elements. Albeit the majority of these genomic repeats did not change their replication timing from pluripotent to differentiated cells, we found developmental changes in the replication timing of rDNA repeats. Comparing single-cell super-resolution microscopic data with data from genome-wide sequencing approaches showed comparable numbers of replicons and large overlap in origins numbers and genomic location among developmental states with a generally higher origin variability in pluripotent cells. Using ratiometric analysis of incorporated nucleotides normalized per replisome in single cells, we uncovered differences in fork speed throughout the S phase in pluripotent cells but not in somatic cells. Altogether, our data define similarities and differences on the replication program and characteristics in human cells at different developmental states.

Embryonic miRNA profiles of normal and ectopic pregnancies.

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.

The Lin28/Let-7 system in early human embryonic tissue and ectopic pregnancy.

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7-9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤ 6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤ 6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.