An Exploratory Study Of PiRNAs As Biomarkers For Diagnosing Residual Spermatozoa In The Testis Of NOA Patients

Part Ⅰ Pi RNA expression profile in testicular tissue from non-obstructive azoospermia patients was performed using the second-generation high-throughput sequencing technology after micro-dissection testicular sperm extraction finding sperm and those without sperm.[Purpose] Pi RNA is a kind of small non-coding RNA with a length of about 30 nucleotides,its major specificity interacts with piwi subfamily proteins to form the piRNA-induced silencing complex(pi RISC)that regulates gene silencing and are specifically expressed in mammalian germ cells.Although piRNA plays an important role in the regulation of spermatogenesis,such as regulating the expression of the corresponding m RNA through ubiquitination degradation and inhibiting transposons,as well as the methylation modification of the target gene,little is known about it in male infertility.Therefore in this study,in order to explore if the piRNA expression profile from testicular tissue can be as molecular markers of clinical diagnosis of male infertility,we systematically examined non obstructive no sperm(non-obstructive azoospermia,NOA)patients after micro-dissection testicular sperm extraction(micro-TESE)finding sperm and those without sperm of testicular tissue source piRNA expression,analysis show significant different expressed piRNA,as a biomarker to predict whether there are residual spermatogenic sites in testicular tissue of NOA patients,it can reduce the psychological and economic burden of patients,as well as unnecessary physical injuries.[Methods] Testicular tissue samples were collected from NOA patients after micro-TESE with and without spermatozoa.Total RNA was extracted from samples of each group,and small-RNA library was established after qualified quality inspection.Sequencing was conducted using Illumina X10,the second-generation high-throughput sequencing platform.Finally,the piRNA with significantly different expression level in the sequencing results was verified by q RT-PCR.[Results] A total of 18,324 piRNAs were detected by high-throughput sequencing of testicular tissue.Of these,959 piRNAs showed significant changes in the expression between the spermatozoa found and the spermatozoa not found.Of these,951 piRNAs were significantly down-regulated and 8 significantly up-regulated in the spermatozoa not found.In addition,553 piRNAs with specific testicular expression are found only in the spermatozoa found and not in the spermatozoa found,suggesting that these 553 piRNAs may be used as molecular markers for predicting whether sperm can be found during micro-TESE.Finally,a total of 20 piRNAs with significant changes in expression were selected to verify the sequencing results by q RT-PCR.Bioinformatics analysis revealed that these piRNAs involved many pathways involved in important biological processes,including apoptosis,cell proliferation,and differentiation.[Conclusions] Our results confirmed piRNA’s different expression profiles in testicular tissue of NOA patients with and without successfully obtained sperm.This study provides further evidence for piRNA’s role in the regulation of spermatogenesis in male and provides important clues for the determination of effective biomarkers for predicting residual spermatogenic sites in testicular tissue of NOA patients in assisted reproductive therapy.Part Ⅱ: Pi RNA expression in testicular tissue of normal and non-obstructive azospermia(NOA)patients by micro-TESE with and without spermatozoa was detected in seminal plasma[Purpose] Found in patients with NOA find expression level sperm group compared with the sperm was not found significant change piRNA,and put these piRNA as NOA patients micro-TESE with preoperative predicting success to sperm biological targets,so that to improve the success rate of micro-TESE and reduce patients’ physical and mental damage due to surgery.[Methods] According to foldchange > 2 times,reads number > 300 standard of screening,find expression in high-throughput sequencing results measuring piRNA have significant difference,and the sifting 20 piRNA,use q RT-PCR in patients with NOA USRG and SSRG validated,then from the literature reports from adult male normal testicular tissue piRNA compared with the 20 piRNA,four piRNA(hsa-pir-6254,hsa-pir-5026,hsa-pir-11482,and hsa-pir-17765)were selected from these four piRNA.The expression levels of these four piRNA in the spermary of NOA patients and normal adult males were detected by q RT-PCR.[Results] These four piRNA(hsa-pir-6254,hsa-pir-5026,hsa-pir-11482 and hsa-pir-17765)were found to be derived from the seminal plasma of the epididymis or testes of normal adult males,and there were significant differences in the expression levels of hsa-pir-6254 and hsa-pir-17765 between normal adult males and NOA patients(P < 0.05).[Conclusions] These four piRNA(hsa-pir-6254,hsa-pir-5026,hsa-pir-11482 and hsa-pir-17765)with spermatic plasma sources are expected to be used as biomarkers for the preoperative prediction of the success of sperm collection in NOA patients with micro-TESE.