Identification Of Pathogenic Genes And Mechanism Of Male Infertility In Abnormal Flagella Of Sperm And Analysis Of Clinical Outcomes

Research background:Infertility is becoming an alarming social issue.It is estimated that 50 percent of infertile cases are attributable to the male partner,and15-30 percent of male infertility cases have an underlying genetic cause.Despite the considerable success achieved in identifying infertility genes in recent years,the genetic cause of a large number of male infertility cases remains undetermined.Asthenozoospermia(AZS)is the most common form of male infertility.AZS is responsible for up to 80% of male infertility cases,among which isolated AZS due to sperm defects is the most common cause of male infertility.AZS is often associated with oligospermia or teratozoospermia,accounting for about 19% of infertility.Multiple morphological abnormalities of sperm flagella(MMAF)is a special type of AZS,which is characterized by a heterogeneous combination of sperm flagella and severely impaired sperm motility.Presently,the pathogenesis of AZS is still very limited,and the mechanisms leading to reduced sperm motility and/or malformation are not fully understood.Therefore,underlying the genetic mechanism of male infertility caused by asthenozoospermia will better determine the pathogenesis of male infertility and provide new strategies for treatment.Aims: In this study,patients with primary male infertility were enrolled in the asthenozoospermia(abnormal flagella)cohort established by the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University and the MMAF cohort established jointly with other teams.Whole exon sequencing(WES)was used to explore the possible genetic etiology of asthenozoospermia,and the gene knockout mouse model was constructed to identify the new pathogenic genes of asthenozoospermia and explore its pathogenic mechanism.The clinical outcome of intracytoplasmic sperm injection(ICSI)was monitored to provide theoretical support and guidance for gene diagnosis and clinical genetic consultation of asthenozoospermia as well as the selection of clinical treatment plans.Methods: Blood samples were collected from 2 patients with severe asthenozoospermia(NK038 and NK062)from the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University.The two probands were from 2 unrelated families whose parents were not inbred.The parental blood samples of the two domestic probands were collected.Another blood sample was obtained from 1 MMAF patient(P1)from the Iran whose parents were consanguineous.The parents of the MMAF patient(P1)from the Middle East(Iran)were inbred(Cousins).Blood samples from the patient's parents were not collected.Two possible pathogenic genes were identified by WES from blood samples,and further verified by Sanger and a series of molecular experiments and ultrastructural observations(western blot(WB),real-time fluorescence quantitative PCR(RT-qPCR),immunofluorescence(IF)and electron microscopy(SEM ? TEM),etc.).The effects of gene mutations on the structure and function of sperm flagella were verified at transcription and translation levels,and the ICSI outcomes of 3 probands were monitored.Results: WES of 2 probands in the First Affiliated Hospital of Anhui Medical University revealed that they carried two novel potentially pathogenic missense mutations of the SLC26A8 gene(NK038 III?1: c.212G>T,p.Arg71Leu;c.290T>C,p.Leu97Pro/ NK 062 III?1: c.290T>C,p.Leu97Pro)and a functional deletion mutation(NK 062 III?1:C.1664 del T,P.le555Thrfs*11).Sanger sequencing confirmed that the SLC26A8 mutation affected two alleles of the proband and was inherited from their heterozygous parents.In addition,the paternal uncle of the proband in the NK062 family was also verified as a heterozygous carrier,who could reproduce normally.The variants identified above were all consistent with a recessive inheritance pattern of lineage co-disjunction with the sterile phenotype.These results indicated that the newly discovered SLC26A8 bi-allelic mutations were consistent with the recessive inheritance pattern.The progressive motility rate of the sperm from the 2 patients was less than 1%,and the thinning of the middle-piece of the sperm flagella(the mitochondrial position of the sperm)was obvious under light microscope and SEM.Statistics on the type and incidence of sperm flagella deformity showed that more than 70% of the middle part of the sperm flagella was thin.TEM results revealed that compared with normal sperm,the sperm flagella of SLC26A8 mutated patients displayed multiple defects,including the loss of mitochondrial sheath and annulus.To verify the molecular abnormalities of SLC26A8 bi-allelic mutations,sperm from 2patients were analyzed by IF and WB.IF results showed that the SLC26A8 signal was located in the annulus in the sperm of the control group,but no SLC26A8 signal was observed in the sperm of the patients.WB results of sperm protein showed that SLC26A8 protein expression was lost in sperm carrying SLC26A8 mutations.IF results showed that,SEPT4 was located in the annulus of flagella in the control group,but not in the flagella of the patient.HSP60 staining was mainly found in the middle-piece of the sperm flagella of the control group but significantly decreased in the sperm flagella from SLC26A8 mutanted patients.Meanwhile,the three-dimensional structure model of SLC26A8 protein was constructed.As a resut,the defect of the three-dimensional structure of the ? helix region of SLC26A8 mutant affected its stability.Follow-up of ICSI results of the 2 probands showed that all of them obtained pregnancy and delivered normally.A WES test was performed on the proband from the Iran and he was found to carry CFAP206 bi-allelic mutation(C.1430 dup A;Asp477lysfster15).RT-qPCR showed that CFAP206 was mainly expressed in testis,and the expression level in human airway cilia was low.The proband's semen parameters showed severe asthenoteratospermia.Morphological examination of sperm revealed that most of the sperm showed a short flagella,absent flagella,coiled flagella,and irregular flagella.In order to explore the effect of truncated variation of CFAP206 on the ultrastructure of human sperm flagella,we studied the sperm flagella by IF.RSPH1 staining was absent,dramatically reduced,or displayed an abnormal dotted and irregular pattern in the patient's sperm.In control sperm,WDR66 immunostaining decorated the full-length flagella,but in the CFAP206 patient,WDR66 staining was completely absent.The patient underwent ICSI for pregnancy but failed to get pregnant.To further confirm the association between CFAP206 gene mutation and asthenoteratospermia(MMAF),we constructed Cfap206 gene knockout mice(Cfap206-/-).Deleting Cfap206 gene did not affect the growth and basic life activities of mice.WB analyses were performed on the testicular tissue of Cfap206-/-mice to confirme that CFAP206 protein was completely absent.The fertility test of Cfap206-/-male mice showed that it could not produce offspring.Therefore,deletion of the Cfap206 gene leads to infertility in male mice.Compared with the control group,Cfap206-/-mice showed no significant difference in testicular/body weight ratio and testis by HE staining.Compared with wild-type mice sperm,Cfap206-/-mice sperm parameter analysis showed that the sperm count,sperm motility,and sperm forward motility were significantly decreased.Morphological examination and SEM observation showed that the abnormal sperm flagella were mainly characterized by coiled and bent flagella.The above results indicated that the sperm morphological results of Cfap206-/-mice were similar to those of the patient with CFAP206 truncated mutation.Further TEM observations showed that some longitudinal sections of the sperm of Cfap206-/-mice showed axonal distortion compared to the control group,which may be responsible for bending and coiling of the flagella.In addition,the observation of the flagella in cross section showed that the flagella were disorganized and had some defects,such as the absence of peripheral double microtubules and central microtubules;abnormal distribution of duplex microtubules or/and peripheral dense fibers.To observe the effect of Cfap206 gene knockout on early mouse embryo development,ICSI test was performed with sperm of Cfap206-/-mice.As a result,the2-cell cleavage rate and blastocyst rate of the fertilized oocytes injected with Cfap206-/-mice were significantly reduced,compared with wild-type mice.Conclusions: SLC26A8 bi-allelic variation can lead to severe asthenozoospermia characterized by loss of sperm flagella mitochondria and annulus.According to Sanger sequencing and pedigree investigation,SLC26A8 bi-allelic mutation was confirmed to be in line with recessive inheritance pattern,which was inconsistent with previous studies that considered SLC26A8 maybe a dominant inheritance.For patients with SLC26A8 bi-allelic variation,ICSI may be a feasible and effective treatment.The truncated mutation of CFAP206 bi-allelic can lead to severe asthenoteratospermia characterized by MMAF;The failure of ICSI in the patient with CFAP206 bi-allelic variation and the low blastocyst rate obtained after ICSI in Cfap206-/-mice suggest that CFAP206 deficiency may affect early embryo development.