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Title: The role of microRNAs in parturition
Author: Cook, Joanna Rachel
ISNI:       0000 0004 6423 5554
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Introduction Preterm birth is a common obstetric syndrome with multiple potential causes. The consequences of preterm delivery are significant for the baby, their family and wider society. Our understanding of the biological mechanisms underlying preterm birth is incomplete and there is no available strategy to predict preterm labour in asymptomatic primiparous women. MicroRNAs (miRNAs) are small, non-coding RNA molecules, which regulate gene expression at the post-transcriptional level. MiRNAs play a modulatory role in pathways leading to labour onset, and are shed from their tissue of origin in to the peripheral circulation, where they are stable and easily measurable. Methods Myometrial biopsies were collected from pregnant women undergoing either prelabour or intrapartum oxytocin augmented caesarean section. Tissue samples were subjected to miRNA profiling using microarray. Differentially expressed miRNAs were identified and then validated using RT-PCR and expression was then compared with samples from women in spontaneous labour. Human uterine myocytes were cultured and subjected to electroporation transfection experiments which examined the functional effects of earlier identified labour/oxytocin associated miRNAs. Plasma samples and cervical length measurements were prospectively collected from women in the second trimester of pregnancy. Plasma miRNA profiling was undertaken using the nCounterTM assay to describe expression across gestation in uncomplicated pregnancies and then compared with samples from women who went on to display cervical shortening or deliver preterm. MiRNAs predictive of later cervical shortening were identified, and validated with RT-PCR. ROC curves were constructed. Results 494 miRNAs were detected in human myometrium and six were differentially expressed in samples from women receiving oxytocin. Four of the validated miRNAs (hsa-miR-146a-5p, hsa-miR-146b-3p, hsa-miR-196b-3p and hsa-miR-876-5p) were expressed in primary human myocytes and oxytocin treatment of these cells replicated the directional changes observed in vivo. Additionally, hsa-miR-146b-3p was increased in both oxytocin receiving and spontaneous labouring samples. Myocytes treated with IL1β demonstrated increased expression of hsa-miR-146a-5p and hsa-miR-146b-3p. Knockdown of the p65 NFκB subunit resulted in reduced expression of hsa-miR-146a-5p whilst overexpression of the p65 NFκB subunit resulted in increased expression of both hsa-miR-146a-5p and hsa-miR-146b-3p. Myocytes transfected with hsa-miR-146a-5p and hsa-miR-146b-3p mimics exhibited a tendancy in suppression at the mRNA level of IL1β mediated NFκB dependent genes: CCR1, MMP10, IL8R, CCL4 and OXTR. Overexpression of hsa-miR-146a-5p resulted in a tendency of reduction of the IL1β induced inflammatory cytokines. Fifty-six miRNAs were expressed in maternal plasma samples and the profile was stable across gestation in uncomplicated pregnancies. Levels of nine miRNAs were significantly higher in plasma from women who went on to exhibit cervical shortening. Of the nine, hsa-miR-150-5p had both the highest AUC (0.86) and the greatest detection rate at a given false positive rate (FPR). The minimum FPR required for hsa-miR-150-5p to achieve 100% sensitivity is 39%. Hsa-miR-150-5p appears to be the strongest candidate biomarker to predict cervical shortening and therefore cervical weakness associated preterm labour. Conclusions This thesis describes how oxytocin alters the expression of a set of myometrial miRNAs and identifies a further role for oxytocin as a signalling molecule involved in the regulation of gene expression during parturition. This thesis also demonstrates that hsa-miR-146a-5p and hsa-miR-146b-3p are regulated by NFκB in human myocytes and that these miRNAs may, in turn, regulate NFκB dependent genes which are key to the inflammatory process of parturition. Furthermore, this work identifies a panel of nine plasma miRNAs which predict cervical shortening from 12 weeks gestation, prior to ultrasonographic evidence of cervical change. The second trimester is a key period in pregnancy when pathological changes can be identified, but time remains to deliver outcome-modifying interventions.
Supervisor: Terzidou, Vasso ; MacIntyre, David ; Bennett, Phillip Sponsor: Action Medical Research ; Genesis Research Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral