| Background and ObjectiveEpidemiological study demonstrated that human fertility was inclined to be deceased in these years.And now there were 10%-15%couples who were suffering from infertility.According to the reports of Beijing University,the infertility rate increased from 6%(1988)to 18%(2014)What’s more,the infertile couples in China account for 1/4-1/3 worldwidely and reached 20-30 million.Thus,WHO assumed that infertility,cardiology diseases and tumors were the most severe issues which were detrimental for human health.In these infertile couples,50%were male factors.And azoospermia was the most severe condition in male infertility.The azoospermia could be divided into three types:pre-testis,post-testis and testicular azoospermia Non-obstructive azoospermia due to testicular factors accounted for the highest proportion However,the etiology of NOA remained unclear.With the continuous development of genome sequencing technology and histopathological detection technology,whole exome sequencing and whole genome sequencing had been widely used in metabolic diseases such as tumors,whereas it was just initiated in reproductive diseases.This study aims to use NGS technology and testicular tissue staining technology to reveal new types of genetic variation associated with azoospermia and new mutation models;to construct a new method for fertility evaluation in patients with azoospermia;and to explore mechanism of proliferation and differentiation in spermatogonial stem cells(SSCs).And this study offered theoretical basis and research platform for the reconstruction of fertility in azoospermia patientsMaterials and MethodsI.Interpretation of novel variations associated with idiopathic NOA based on whole-exome sequencingClinical idiopathic NOA was selected to rule out known causes such as Chromosome disorders and infections.The study was enrolled according to its pathological type(SCOS,MA,and HS)with informed consent.Peripheral blood DNA was extracted and subjected to whole exome sequencing(WES).Bioinformatics analysis was performed to explore the pathogenic variations.Furthermore,the pathogenic variations were verified through Sanger sequencing Cyto-SCAN was used to detect CNV mutations in azoospermia patients.The dot mutation plasmid was constructed in vitro.Western Blot was used to evaluate the dot mutation effect in SYCE1 through transfection of the empty plasmid,overexpression plasmid and dot mutation plasmid2.Multi-site immunofluorescence whole-mount staining technique to evaluate seminiferous tubule inherent spermatogenesisClinically,patients with OA and NOA who underwent microsurgery were enrolled to obtain testicular tissue.The whole-mount staining technique was used to evaluate the fertility of azoospermia patients.HE staining and immunofluorescence staining were also performed on these azoospermia testicular tissues as control3.Identification and transcriptomics of human spermatogoniaHuman SSCs and differentiated spermatogonia were identified and isolated from OA patients And the transcriptome characteristics during the proliferation and differentiation of SSCs were compared through RNA-seq.Bioinformatics analysis was used to screen the key genes for SSCs proliferation regulation4.FGF5 regulates the proliferation of SSCs and related mechanismOur precious studies suggested that FGF5 was a key gene for the proliferation of SSCs.The effects and mechanisms of FGF5 on the proliferation of SSCs were evaluated by CCK-8,EDU,Phosphorylated Protein Chip and Western BlotResultsI.In this study,357 NOA patients were enrolled.Through WES technology,17 novel pathogenic variations in five genes were found and verified by Sanger sequencing.HE staining and immunohistochemistry confirmed that the clinical phenotype was consistent with the genotype.The cyto-SCAN results illustrated new mutation pattern of maturation arrest:CNV heterozygous deletion associated with heterozygous single base frameshift mutation.The SYCE1 and SYCE1 mutant plasmids were constructed in vitro and transfected into 293T,respectively.And the cells were subjected to protein extraction.The WB results showed that the SYCE1 mutant vector was reduced in size and decreased in expression compared with the SYCE1 overexpression vector,suggesting that the mutation is the cause of the disease in this patient.By comparing the probability of pathogenic variation diagnostic rate among SCO,MA and HS patients,it is found that pathogenic variation diagnostic rate of MA is much higher than that of SCOS and HS2.This study established a novel multi-site whole-mount seminiferous tubule staining method And fertility evaluation of testicular seminiferous tubules was performed in 57 patients with azoospermia,including 14 cases of OA and 43 cases of NOA.Comparing the results of HE staining and immunofluorescence staining,it was found that three kinds of staining results were consistent.But the novel fertility assessment approach was faster and accurate,allowing for a more accurate assessment of the presence of spermatogenic cells3.This study identified human SSCs through spermatogonial markers staining.And human SSCs and differentiated spermatogonia cells were isolated and sorted by MACS using membrane surface markers(GFRA1,FGFR3,and KIT).The RNA was extracted,and the purity was verified by RT-PCR.Distinct mRNA profiles were explored though RNA-seq.Also,two spermatogonial cell-specific expression genes were identified4.CCK-8 and EDU assay results revealed that FGF5 could promote proliferation of SSCs.And Phosphoprotein profiles Chip and Western Blot results showed that FGF5 promoted proliferation of SSCs via PI3K-AKT and MAPK pathwayConclusionThis study was closely integrated with male infertility clinic.Based on the whole exome sequencing technology and multi-site tissue whole-mount staining technology,this study established a novel method for reproductive genetic evaluation of male fertility and inherent spermatogenesis screening.Through screening 357 idiopathic non-obstructive azoospermia patients,17 novel pathogenic variations in 8 genes and new pathogenic genetic patterns associated with azoospermia were found.This study innovatively uses the whole-mount staining technology to evaluate inherent spermatogenesis of the seminiferous tubules Compared with the traditional methods,the overall staining technique is extensive,rapid and accurate,and the clinical application had broad prospectsBased on the spermatogonial cell markers,human SSCs were identified using broader markers And human spermatogonial transcriptome characteristics were illustrated using RNA-seq and bioinformatic analysis.Also this study revealed that FGF5 pathway is a new key pathway for SSCs proliferation and self-renewal,providing a novel target for long-term culture of human SSCs in vitro.Thus,this study provided a new experimental basis for spermatogenesis research and a novel platform for fertility reconstruction in azoospermia patients... |
PDF Full Text Request