Correlation Study Of Differential Expression Of Sperm MicroRNA And Unexplained Recurrent Miscarriage

Background and Objectives:Recurrent spontaneous abortion(RSA)is a common multifactorial disease,but its etiology is not very clear so far.Usually,patients with recurrent miscarriage need to undergo tests for uterine factors,endocrine factors,immune factors,and thrombotic tendencies,as well as chromosome tests for both spouses.Even after systematic testing,about 50% of patients still cannot find the cause of miscarriage(unexplained recurrent spontaneous abortion,URSA).Sperm contributes 50% of the genetic material of the embryo,and the mi RNA carried by sperm plays an important regulatory role in the expression of the embryonic genome.Based on the above viewpoint,we propose the following hypothesis: differential expression of mi RNA in sperm may be one of the reasons for recurrent miscarriage.This topic first compared the differential expression of hsa-mi R-34c-5p,hsa-mi R-25-3p,hsa-mi R-375-3p(the above three mi RNAs are considered paternal mi RNAs in embryos)in the sperm of the spouse of patients with unexplained recurrent abortion with that of the control group,and then constructed the expression profile of mi RNA in the sperm of the spouse of patients with unexplained recurrent spontaneous abortion through high-throughput sequencing,and analyzed the target genes of the differentially expressed mi RNA using bioinformatics,Exploring the relationship between sperm mi RNA and unexplained recurrent miscarriage,providing scientific basis for the diagnosis and treatment of patients with recurrent miscarriage.Methods:Part 1:1.Research subjects and grouping: 19 spouses of URSA patients aged 23-45 from July2021 to February 2023 were selected as the case group,and 16 males who underwent pregnancy examination and had normal semen routine indicators during the same period were selected as the control group.2.Pretreatment of sperm samples and RNA extraction: After the collection of semen samples,they are treated with density gradient centrifugation combined with cell lysate to remove seminal plasma,round cells and impurities from semen.Subsequently,sperm RNA was extracted according to the instructions of the RNA extraction kit and the extraction results were tested.3.q RT-PCR quantitative detection: polyadenylation and reverse transcriptional synthesis of c DNA were performed on the qualified RNA,and the paternal mi RNAs of the two groups of samples were quantitatively detected with HOMO U6 as the internal reference gene.Part 2:1.Research subjects and grouping,pre-processing of sperm samples,and RNA extraction: 19 cases in the case group and 16 cases in the control group were selected,and the rest were the same as in Part 1.2.Establish the mi RNA expression profile of URSA patient’s spouse sperm: use high-throughput sequencing to establish the mi RNA expression profile of URSA patient’s spouse sperm,and DESeq2.0 software to analyze the sequencing results.Use | log2 FC |>1 and p-value<0.05 as screening conditions to screen differentially expressed mi RNAs,and draw the heatmap of differentially expressed mi RNAs.3.Bioinformatics analysis: predict target genes for differentially expressed mi RNAs,and screen for target genes under the condition of S ≥ 150,Δ G≤-30 kcal/mol。 Select the three mi RNAs with the highest predicted target gene scores,and draw a mi RNA target gene relationship diagram and target protein PPI network diagram.GO enrichment analysis and KEGG enrichment analysis take p-value ≤ 0.05 as the screening condition for significant enrichment,and draw the horizontal distribution map of differential mi RNA target genes at GO Level 2,the top 10 bar chart of GO enrichment analysis,the level distribution map of KEGG Level 2,and the top 20 bubble map of KEGG enrichment analysis.Results:Part 1:1.Compared with the control group,the expression of hsa-mi R-34c-5p in the sperm of the case group was significantly downregulated,and there was no statistically significant difference in the expression of hsa-mi R-375-3p and hsa-mi R-25-3p.Part 2:1.A total of 83 differentially expressed mi RNAs were detected in the sperm of the case group and the control group,of which 39 were significantly upregulated and 44 were significantly downregulated.2.A total of 68 out of 83 differentially expressed mi RNAs were successfully predicted to have 14614 target genes and 17111 target gene binding sites.Among them,hsa-mi R-486-3p,hsa-mi R-1285-3p,and hsa-mi R-146b-3p scored the highest in the prediction analysis of target genes.3.GO enrichment analysis results show that target genes are mainly enriched in cellular process,biological regulation,organelle part,and molecular synthetic and other life activities.KEGG enrichment analysis results show that target genes are mainly enriched in signal transduction,endocrine system,immune system and other pathways,of which PI3K-Akt signaling pathway,MAPK signaling pathway,Ras signaling pathway,and p53 signaling pathway are the most enriched target genes in signal transduction.The target genes of hsa-mi R-486-3p,hsa-mi R-1285-3p,and hsa-mi R-146b-3p are also mainly enriched in the above four pathways.The target gene of hsa-mi R-34c-5p is mainly enriched in the Ras signaling pathway.Conclusion:1.Compared with the control group,the expression of hsa-mi R-34c-5p in the sperm of URSA patients’ spouses was significantly downregulated.2.The sperm mi RNA expression profile of URSA patients’ spouses was constructed by high-throughput sequencing.3.Bioinformatics analysis found that the sperm of URSA patients’ spouses hsa-mi R-1285-3p,hsa-mi R-486-3p,hsa-mi R-146b-3p,and hsa-mi R-34c-5p may regulate the occurrence of RSA through the PI3K-Akt signaling pathway,MAPK signaling pathway,Ras signaling pathway,and p53 signaling pathway.