Function And Mechanism Study Of Non-coding RNAs MiR-148a-3p And LincARDD1 In Decidualization Of Recurrent Implantation Failure

Chapter I Function and mechanism study of miR-148a-3p in decidualization of recurrent implantation failureBackground:Recurrent implantation failure(RIF),referred to failure to achieve a clinical pregnancy following repeated transfers with high quality embryos,is an intriguing clinical dilemma in reproductive medicine.Among multiple known causes of RIF,abnormal endometrial stromal cell decidualization is now considered as one of the primary causes responsible for implantation failure.Although numerous signaling molecules and pathways have been demonstrated to regulate decidual process,the local molecular mechanisms driving decidualization are still unclear.Furthermore,most of previous studies focused on protein-encoding genes in a functioning and receptive endometrium.It is still difficult to explain the complex and periodicity changes in endometrial stromal cell decidualization.MicroRNAs(miRNAs)are small single-stranded RNA molecules that function as important post-transcriptional regulators of gene expression and play significant roles in controlling a large variety of cellular processes,from cell proliferation,differetitation to apotosis.There is convincing evidence that miRNA dysregulation is associated with many pathological conditions and human diseases.Prior studies revealed miRNA signatures of pre-decidual versus post-decidual human endometrial stromal cells,or pre-receptive versus receptive endometrial tissues collected from healthy fertile females.However,the function and regulation mechanism of miRNAs in impaired decidualization of RIF remains poorly understood.Objective:To analyze decidualization-associated miRNA signatures in receptive endometrial tissues from RIF patients and female controls,and to investigate whether aberrant miRNA expression is involved in the defective decidualization of RIF.Methods:The miRCURY LNATM microRNA Array was utilized to screen different miRNA expressions in receptive endometrial tissues collected from 8 RIF patients and 10 female controls,and reverse transcription and quantitative real-time PCR(RT-qPCR)was used to quantify selected miRNAs and predicted miRNA target genes.Immortalized human endometrial stromal cells(HESCs)were cultured for proliferation and differentiation assays after enhancing miR-148a-3p expression or inhibiting putative target gene HOXC8 expression.RT-qPCR,western blot and luciferase reporter assays were performed to demonstrate the relationship between miR-148a-3p and HOXC8 gene.Results:MiR-148a-3p expression levels increased dramatically in receptive endometrial tissues of RIF patients compared with matched female controls.Both increased miR-148a-3p expression and decreased HOXC8 expression suppressed HESCs in vitro differentiation.Further analysis showed that overexpression of miR-148a-3p down-regulated HOXC8 mRNA and protein expressions through interacting with 3’ untranslated region of HOXC8 gene.Conclusions:Dysregulated miR-148a-3p may contribute to RIF by repressing endometrial stromal cell decidualization through HOXC8 gene.Chapter Ⅱ Function study of lincARDDl in decidualization of recurrent implantation failureBackground:Long noncoding RNAs(lncRNAs)are characterized by greater than 200 nucleotides in lengh and without evident protein coding function.LncRNAs can operate via various mechanisms,mainly including genomic imprinting,chromatin modification,transcriptional or post-transcriptional regulation.There is considerable evidence that lncRNAs are critical in multiple physiology and pathological events from cell proliferation and apoptosis to oncogenesis.Compared with the previor studies in miRNAs during human decidualization and embryo implantation processes,the function and regulation mechanism of lncRNAs in flawed decidualization of recurrent implantation failure(RIF)has not been explored.Objective:To profile the receptive endometrial lncRNA expression pattern in RIF,and to uncover the functional effects of dysregulated lncRNAs in the abnormal decidual process of RIF.Methods:The receptive endometrial tissues selected from RIF patients and female controls were used to delineate the lncRNA expression profile by Arraystar Human LncRNA Microarray v4.0 and the filtered lncRNAs were validated by reverse transcription and quantitative real-time PCR(RT-qPCR).The coding-potential analysis,nuclear/cytosol fractionation and fluorescence in situ hybridization assays were used for confirming the non-coding nature and subcellular localization of lncRNA-CTD-2659N19.9,named lincARDD1(lincRNA Activated in RIF with Decidualization Deficiency 1).The proliferation and differentiation assays were carried out in immortalized human endometrial stromal cells(HESCs)after enhancing lincARDD1 expression.Results:The lincARDDl expression levels were observably up-regulated in receptive endometrial tissues of RIF patients.LincARDDl was a long intergenic non-coding RNA without protein-coding potential and expressed in both cell nucleus and cytoplasm of stromal cells.Overexpression of lincARDD1 in HESCs decreased cell proliferation.Conclusions:Distorted lincARDDl may casuse RIF via suppressing endometrial stromal cell proliferation.Further mechanism investigations of lincARDDl in the pathogenesis of RIF could be worthwhile.Chapter III Predictive value of serum miRNA and lincARDD1 levels on HCG day for endometrial receptivityBackground:Impaired endometrial receptivity is employed in two-thirds of embryo implantation failures,and now considered to be an initial rate-limiting step for further improving the clinical pregnancy rate in reproductive practice.Therefore,accurate assessment of endometrial receptivity before embryo transfer will make for bettering pregnancy outcomes in assisted reproduction.At present,there are still no effective and convenient biomarkers for estimating endometrial receptivity.Benefited from the comprehensive investigations of non-conding RNAs(ncRNAs)in varied biological processes,the potential effects of circulating ncRNAs in disease prediction,diagnosis,treatment and prognosis are increasingly valued.Recently,more and more ncRNAs,especially microRNAs(miRNAs),have been demonstrated to associate with endometrial receptivity.The circulating receptivity-associated ncRNA expression lervels may be emerging predictive markers for evaluating e-ndometrial receptivity.Prior and our studies have discovered that miR-148a-3p,miR-181a,miR-222,miR-542-3p,miR-181b-5p,miR-30d and lincARDDl were referred to endometrial receptivity.No researches have been done to assess the predictive value of these ncRNA expression levels in serum for endometrial receptivity.Objective:To evaluate the predictive value of serum miRNA and lincARDD1 levels on human chorionic gonadotropin(HCG)administration day regarding endometrial receptivity before embryo transfer during in vitro fertilization/intracytoplasmic sperm injection(IVF/ICSI)treatment.Method:A total of 108 infertile females due to tubal or male factors were recruited in their first IVF/ICSI treatment cycle.The serum samples of all participants were collected on the day of HCG administration.According to whether transferred embryos implanted,these infertile females were divided into implantation and non-implantation groups.The serum receptivity-associated miRNAs(miR-148a-3p,miR-181 a,miR-222,miR-542-3p,miR-181 b-5p and miR-30d)and lincARDDl expression levels were quantified by transcription and quantitative real-time PCR(RT-qPCR).Result:The serum miR-542-3p expression level was significantly down-regulated in non-implantation group after adjusting endometrial thickness on HCG day.The receiver operating characteristic(ROC)curve analysis showed an area of 0.626 for serum miR-542-3p level in differentiating from implantation to non-implantaion(cut-off,0.0010;95%confidence interval,0.513-0.740;P<0.05).Further analysis revealed that serum miR-542-3p expression levels combined with endometrial thickness on HCG day could effectively recognizing implantation failure(area,0.669;95%confidence interval,0.562-0.776;P<0.05).Conclusion:Conjoint analysis of serum miR-542-3p level and endometrial thickness on HCG day could be of potential predictive value for assessing endometrial receptivity before embryo transfer in IVF/ICSI cycles,which might be helpful to predic and intervene endometrial receptivity in assisted reproductive clinical practice.