The Clinical Study Of Seminal Plasma Exosomal Mirna As Novel Biomarkers For Male Infinity

BACKGROUD : The incidence of male infertility is increasing annually and has become a global health problem.The pathogenesis of male infertility is complex,which can be caused by congenital or secondary genitourinary system abnormalities,gonad infection,endocrine disorders,varicocele,mitochondrial dysfunction or immunological factors.Idiopathic infertility accounts for about 40% of these patients,whose molecular mechanism is not completely clear.At present,the clinical diagnosis of male infertility is mainly based on semen routine and biochemical indicators,but these tests lack of high diagnostic specificity and sensitivity,and also ignore the internal mechanism of male infertility.Although testicular biopsy has a high accuracy in the diagnosis of male infertility,especially azoospermia,puncture biopsy is invasive and may cause some complications.Therefore,it is still necessary to explore a new non-invasive diagnostic marker for male infertility.Recent studies have shown that seminal plasma contains a large number of exosomes,which come from a variety of cells in the male reproductive system.Since the mi RNA content of seminal plasma exosomes will change with the changes in cells of exosome origin,it can more accurately reflect the pathophysiological changes of reproductive organs,and can be used as a potential and reliable biomarker for male infinity.It has been reported that mi RNA plays an important physiological function in spermatogenesis,but its changes in spermatoplasmic exosomes in infertility have not been reported so far.OBJECTIVE: To comprehensively and systematically investigate the expression profile of seminal plasma exosomal mi RNAs in patients with male infinity and to identify the significantly changed mi RNAs in patients with male infinity,so as to evaluate its potential as an auxiliary biomarker for the diagnosis of male infertility.METHODS: A total of 108 cases of azoospermia,108 cases of asthenospermia and 108 cases of age-matched normal male seminal plasma samples were collected as research objects.Use UMI small RNA sequencing technology to detect the seminal plasma exosomal mi RNAs in these groups,for screening out the significantly changed mi RNAs in male infertility patients.Then exosomes were extracted one by one from all sperm samples with the kit,and individual verification was carried out in the rescreening group and the validation group by using the real-time quantitative PCR technique.Finally,ROC curve analysis,Spearman correlation analysis and Logistic regression analysis were used to evaluate the diagnostic value of the screened mi RNA in male infertility.RESULTS: UMI small RNA sequencing results showed that,compared with normal controls,the plasma exosomal mi RNA expression profile of male infinity was significantly changed.By using RT-q PCR for rescreening and validation,it was found that 5 mi RNA(mi R-891a-3p,mi R-890,mi R-891 b,mi R-892 b,mi R-202-5p)were significantly and stably down-regulated in seminal plasma exosomes of patients with azoospermia,and 2 mi RNA(mi R-7-5p and mi R-378e)were significantly and stably up-regulated in seminal plasma exosomes of patients with asthenospermia(?2-fold,P < 0.05).ROC curve analysis showed that the area under the ROC curve(AUC)for diagnosis of male infertility of 7 mi RNA was 0.686 ~ 0.778,among which mi R-892 b had the highest diagnostic accuracy.Furthermore,risk score analysis was performed to assess the associations between the combination of several mi RNAs.Statistical results showed that the AUC value of the combination of 3-mi RNA(mi R-891 b,mi R-892 b,mi R-202-5p)was 0.785(95% CI: 0.721 ~ 0.850),which had high diagnostic accuracy for azoospermia.The AUC of combination of mi R-7-5p and mi R-378 e in asthenospermia was also higher than that of other single mi RNA.In addition,Spearman correlation analysis showed that the 5 seminal plasma exosomal mi RNA which significantly down-regulated in patients with azoospermia were positively correlated with sperm concentration and sperm motility,while the 2 seminal plasma exosomal mi RNA significantly up-regulated in asthenospermia were negatively correlated with sperm motility.Binary logistic regression analysis showed that these 7 mi RNAs were all associated with male infertility and were potential risk factors(B > 0,OR > 1,P < 0.05).CONCLUSIONS: The miRNA expression profile of seminal plasma exosomes in male infertility patients was significantly different from that of normal controls,and 7 mi RNA showed significant changes in the seminal exosomes of male infertility,which are expected to serve as potential auxiliary diagnostic indicators of male infertility.