| Part 1 Screening and identification of differentially expressed genes in cfs-mRNAs of meiosis arrest in NOA patients[Purpose] In order to establish a non-invasive method for the diagnosis of meiosis arrest in NOA patients,we screen differentially expressed cfs-mRNAs in NOA patients with testicular pathology diagnosed as meiosis arrest(MA)and non-meiosis arrest(non-MA).[Methods](1)We collected nine NOA patients’ semen whose testicular pathological results showed the arrest in meiosis(MA),and nine semen without testicular pathological results of arrest in meiosis(non-MA).We obtained written,informed consent from the participants.After seminal plasma separation,we extracted total RNA to detect the differentially expressed mRNAs through the microarray(Affymetrix HTA2.0 Array).(2)Eight genes were randomly selected from significantly different(p<0.05)mRNAs,and applied under RT-qPCR to verify the sequencing results in the same samples.(3)The expression of selected genes was screened;it was significantly decreased in patients with meiosis arrest(FC>1.5,p<0.05).(4)The source of candidate cfs-mRNAs was verified.The relative RTqPCR was used to verify further the source of cfs-mRNAs in normal males,complete obstructive azoospermia,and complete Sertoli cell syndrome.(5)Nested quantitative PCR was used to verify the differential expression of candidate genes in 53 MA patients and 27non-MA patients of similar samples.(6)ROC curve was established to find the cut-off point value of qPCR for the diagnosis of meiosis arrest.[Results](1)A total of 67529 mRNAs were detected by microarray in MA and non-MA groups.Compared with non-MA patients,175 genes were up-regulated,and 16258 mRNAs were down-regulated in MA patients(FC>2,p<0.05).Bioinformatics analysis revealed that these changes involve many important pathways related to biological processes,including Including cell proliferation and differentiation,spermatogenesis and apoptosis.(2)Eight randomly selected genes were verified by RT-qPCR in the same samples.The results showed the same expression trend as the results of the microarray.(3)According to the the selection standard(foldchange > 1.5,p < 0.05),we selected the candidate cfs-mRNAs.Based on the analysis of the GO genes related to spermatogenesis,meiosis,pick out 11 specificity expressed genes in the testicles,namely AKAP4,TNP1,ODF1,ESX1,KDM3 A,BOLL,TCP11,SOHLH2,SPEM1,and PRM2.Five genes were widely expressed in organs in the NCBI database,but the testis was highly expressed,and the prostate expression was less than 1/2 of the testis,namely ZMYND15,TDRKH,SETX,MCM8,and TUBB4 B.Also,the differentially expressed multiples of TNP1,ODF1,and PRM2 were all greater than 1.2,showing no statistical significance.These three genes are expressed in round sperm and sperm,respectively.Several studies had reported that the expression of these three genes in testicular samples could be used for testis pathological diagnosis of NOA or the prediction of the success rate of micro-TESE.(4)In order to know the source of candidate mRNAs,RT-qPCR was performed in the seminal plasma of healthy man(N),complete obstructive azoospermia(OA),and the Sertoli cell-only syndrome(SCOS).To compare the expression of cfs-mRNAs between three groups,and we selected nine genes compared to the healthy man,the genes were not expressed in OA and SCOS,or less than twice as much as of the healthy man.(5)To screen the diagnostic genes,the nested absolute quantitative RT-PCR was established to validate candidate genes in MA and non-MA.The mRNA BOLL(p<0.001)and TNP1(p<0.01)diagnosed the patient with MA.Compared with the non-MA patients,the seminal plasma expression level of MA patients significantly decreased and showed statistical significance.(6)ROC curve analysis revealed that the cut-off point value of BOLL≤367.0595(copies/μL)could help to diagnose the MA patients.The AUC was 0.819,p<0.001,the sensitivity was 84.2%,and the specificity was 78.3%.The cut-off value of TNP1 in the diagnosis of NOA was the absolute expression of TNP1≤62.3412(copies/μL),the AUC was 0.725,p<0.05,the sensitivity was 59.6%,and the specificity was 82.6%.[Conclusions] Our results showed a different expression profile of cfs-mRNAs in NOA patients with MA and non-MA.In this study.Some differentially expressed mRNAs in the microarrays play a significant role in meiosis of spermatogenesis,which provides a certain reference value for future research and experiments on gene function.The verification experiment of gene origin,which ensured the screened cell-free seminal mRNAs originated from the testis and expressed by germ cells.Through the nested RT-qPCR of large samples,BOLL/TNP1 was selected for the diagnosis of meiosis arrest.Part 2 Screening of cfs-mRNAs to predict the outcome of micro-TESE in NOA patients[Purpose] The establishment of nested RT-qPCR in the first part was used to analyze the role of candidate genes in predicting the success rate of micro-TESE sperm extraction.The correlation between testicular tissue and seminal plasma gene expression was analyzed.The success and failure of micro-TESE operation,micro-TESE success and failure predicted by BOLL,we analysis the clinical characteristics of the patients.[Methods](1)We collected 80 micro-TESE surgery patients’ semen samples,with which seminal plasma was separated,total RNA was extracted.According to the first part of the method of nested RT-qPCR,we selected eight candidate genes(AKAP4,BOLL,TCP11,TNP1,PRM2,ODF1,MCM8,and SETX)and verified in the seminal plasma of micro-TESE samples.(2)We collected patients with both testicular tissue and semen.After extracting RNA from tissues and seminal plasma,relatively quantitative RT-PCR was used to analyze the correlation of BOLL expression in testicular tissue and seminal plasma.(3)Age,semen volume,FSH,LH,T and testicular volume of the patients were collected,and non-paired T test was used to analyze the statistical between success and failure of micro-TESE,and BOLL predicted the success and failure of micro-TESE.[Results](1)After micro-TESE,Ninety-six seminal plasma from NOA patients were collected.The eight genes(AKAP4、 BOLL、 TCP11、 TNP1、PRM2、ODF1、MCM8、SETX)through the gene source verification were detected in the 80 samples.The seminal plasma expression of BOLL and MCM8 transcription in successful micro-TESE was significantly higher compared to failed micro-TESE(p<0.001).(2)The expression of BOLL mRNA in testicular and seminal plasma tissue of NOA patients was positively correlated(r=0.9389,p<0.01).(3)There were significant differences in semen volume between successful and unsuccessful patients with micro-tese sperm collection,but there were no significant differences in age,hormone levels or testicular volume.There were also no significant differences in age,semen volume,hormone levels and testicular volume between the patients who were successful and those who were not,as predicted by BOLL.[Conclusions](1)BOLL and MCM8 were differentially expressed in patients with successful and failed micro-TESE sperm extraction.Howeve,BOLL has a better clinical application value.(2)The expression level of BOLL mRNA in the testicular tissue correlated positively with the expression in seminal plasma.The expression of BOLL mRNA in the seminal plasma of testis could well reflect the expression of BOLL mRNA in testis.(3)The testicular volume of patients with MCM8 predicting the success of micro-TESE sperm retrieval was significantly greater than that of patients with micro-TESE sperm retrieval failure(p < 0.05).MCM8 predicting the outcome of micro-TESE may be more accurate.Hormone the level of semen volume and age of the patient may not have predictive value for the outcome of micro-TESE sperm retrieval. |
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