The Study Of Genetic Diagnosis Analyses And Therapeutic Strategies Of Male-related Infertility

Objective:To explore the genetic causes of congenital obstructive azoospermia(OA),multiple morphological abnormalities of the sperm flagella(MMAF),and sperm-related fertilization failure,preliminarily explore the underlying mechanism,and systematically analyze the clinical outcomes.Methods:This single-center retrospective cohort study enrolled male infertility patients underwent whole-exome sequencing(WES)between January 2018 to December 2022.According to the etiology,the patients were divided into three categories:congenital OA,MMAF,and sperm-related fertilization failure.The genetic causes were identified by Sanger sequencing,and the pathogenicity of the gene mutations was analyzed by bioinformatics analysis tools and molecular biological techniques.Moreover,the effect of CFTR mutations and MMAF phenotype on the clinical outcome of congenital OA patients and MMAF patients were investigated,respectively.In addition,the efficacy and safety of the ionomycin and cycloheximide-based novel artificial oocyte activation(AOA)technology were evaluated in patients with repeated fertilization failure.Results:In Part Ⅰ,38 patients with congenital OA were enrolled.They carried 20 types of CFTR mutations,of which 6 were new mutations.Carrying two CFTR mutations did not significantly affect the embryo laboratory outcome,embryo clinical outcome,and neonatal outcome of congenital OA patients.In Part Ⅱ,38 patients with MMAF were enrolled and 23 of them underwent WES.Four causative genes,including DNAH1,CFAP43,FSIP2,and SPEF2,were identified in 10 of the 23 patients.DNAH1 mutations may affect the assembly of the axonemal central microtubules of sperm flagella,while MMAF phenotype did not affect acrosomal function.Although the embryo laboratory outcomes of MMAF patients were impaired,the subsequent clinical outcomes and neonatal outcomes were not affected.In Part III,3 patients with PLCZ1 mutations and 2 patients with ACTL7A mutations were enrolled.ACTL7A mutations may affect the expression of PLCζ1 in the sperms,meanwhile,the function of acrosome function was also impaired.In addition,six patients suffering from fertilization failure after ICSI and traditional AOA were enrolled.After novel AOA treatment,the fertilization rate was significantly improved and the blastocyst with normal morphology and karyotype could also be obtained.Conclusions:CFTR mutation is a common genetic cause of congenital OA,while clinical outcomes of congenital OA patients were not greatly affected by the presence of CFTR mutations.MMAF phenotype is caused by multiple pathogenic genes.ICSI treatment can help MMAF patients to achieve pregnancy,but the embryo laboratory outcomes were impaired.PLCZ1 and ACTL7A mutations were both the genetic causes of sperm-related fertilization failure.The ionomycin and cycloheximide-based novel AOA had a potential therapeutic effect on those couples experiencing fertilization failure.Our study provided support for the genetic diagnosis of male infertility and the foundation of therapeutic strategies.Part Ⅰ:The Analyses of Genetic Diagnosis and Pregnancy Outcome of Patients with Congenital Obstructive AzoospermiaObjective:To conduct an epidemiological survey of patients with congenital obstructive azoospermia(OA),screen for the causative genes,and analyze the pregnancy outcomes.Methods:In this retrospective study,we included patients with azoospermia who underwent whole-exome sequencing between January 2018 and December 2022.The semen parameters and baseline characteristics of congenital OA patients were investigated,and the carrying situation of cystic fibrosis transmembrane conductance regulator(CFTR)mutations was also analyzed.Then,baseline characteristics,embryonic laboratory outcomes,embryonic clinical outcomes,and neonatal outcomes were compared between the two groups of patients with congenital OA patients carrying two CFTR mutations and other congenital OA patients.Results:A total of 59 patients with azoospermia were included,and 30 of them were diagnosed with congenital OA.The semen of the majority of congenital OA patients was acidic with smaller semen volume and lower neutral alpha-glucosidase and fructose.Twenty-four congenital OA patients(63.2%)carried a total of 20 kinds of CFTR mutations,of which 6 were novel mutations.The mean carrier frequency of CFTR mutations was 1.71 per person,and the most frequent mutation(29.3%,12/41)was c.1210-12T[5]-c.121034TG[13].There were no significant differences regarding baseline characteristics,embryonic laboratory outcomes,embryonic clinical outcomes,and neonatal outcomes between congenital OA patients carrying two CFTR mutations(n=17)and other congenital OA patients(n=21).Conclusions:The genetic mutation pattern of the CFTR gene in the Chinese population is different from that of the Caucasian population.Although the clinical pregnancy outcomes of congenital OA patients with CFTR mutations were similar to other congenital OA patients,genetic counseling is still needed for congenital OA patients with CFTR mutations.This study provides a theoretical basis for the development of CFTR screening methods and genetic counseling guidelines for this population,as well as data to support the treatment of patients with congenital OA associated with CFTR mutations.MMAF patients.Part Ⅱ:The Analyses of Genetic Diagnosis and Pregnancy Outcome of Patients with Multiple Morphological Abnormalities of the Sperm FlagellaObjective:To screen the genetic causes of multiple morphological abnormalities of the sperm flagella(MMAF),explore the underlying mechanism,and analyze the pregnancy outcome and neonatal outcome of MMAF patients.Methods:This retrospective cohort study included MMAF patients who underwent assisted reproductive technology(ART)treatment between January 2018 and December 2022.The causative genes of MMAF patients were identified by whole-exome sequencing(WES)and Sanger sequencing,and the morphology and ultrastructure of sperm flagella were investigated.Then the effects of gene mutations on protein function were analyzed by bioinformatics analysis tools and immunofluorescence staining.After propensity score matching,the clinical baseline,embryo laboratory outcome,pregnancy outcome,neonatal outcome,cumulative clinical pregnancy rate,and cumulative live birth rate between MMAF patients and oligozoospermia controls were compared.Results:A total of 38 patients with MMAF were included,of which 23 patients underwent WES,and 4 causative genes were identified among ten MMAF patients.Seventy percent(7/10)of MMAF patients carried DNAH1 mutations.The expression of DNAH1,as well as SPAG6,a biomarker of the axonemal central microtubule,were significantly reduced in the sperms from patients with DNAH1 mutations.The results of the electron microscope showed that the axonemal central microtubules in the sperms from patients with DNAH1 mutations were missing.Moreover,the other 3 MMAF patients carried gene mutations in CFAP43,FSIP2,and SPEF2 genes,respectively.The acrosome functions of the sperms from these three patients were normal,and successful pregnancy and live birth can be achieved after ICSI treatment.After propensity score matching,the baseline characteristics of MMAF patients(n=38)and control group(n=131)were similar,while the normal fertilization rate(58.3%vs.71.3%,P<0.001),embryo cleavage rate(94.8%97.1%,P=0.030),blastocyst formation rate(50.9%vs.68.7%,P<0.001)and available blastocyst rate(33.8%vs.45.1%,P=0.001)of MMAF patients were significantly lower,compared with oligozoospermia controls.In addition,there were no statistical differences between the two groups regarding pregnancy outcomes and neonatal outcomes.Furthermore,Moreover,the CLPR in the MMAF group was lower than that in the control group according to the embryo transfer times(68.6%86.5%,P=0.033)and complete cycles(66.7%84.2%,P=0.020),while no significant differences were found in the neonatal outcomes.Conclusions:Causative genetic mutations were partly obligated to the pathogenesis of MMAF.DNAH1 mutations might cause the deletion of the central microtubule of the axoneme,resulting in the MMAF phenotype.The mutations of CFAP43,FSIP2,and SPEF2 genes are also the causative genes of MMAF.The prognosis of ICSI treatment may vary with the results of genetic diagnosis,and MMAF patients might have compromised ART outcomes.Therefore,for MMAF patients,systematic gene screening and genetic counseling can not only help clinicians to make the diagnosis but also provide clinical instruction according to the type of gene mutations.Part Ⅲ:Pathogenic Mechanism and Therapeutic Strategies of Patients with Sperm-related Fertilization FailureObjective:To clarify the causative genes of patients with sperm-related fertilization failure,explore the underlying mechanism,and investigate the efficacy and safety of novel artificial oocyte activation(AOA)in patients with repeated fertilization failure.Methods:This retrospective cohort study enrolled patients with sperm-related fertilization failure who underwent whole-exome sequencing(WES)between January 2018 and December 2022.The causative genes of patients were identified,and the effects of gene mutations on protein function were analyzed by bioinformatics analysis tools and immunofluorescence staining.Meanwhile,six couples experiencing repeated fertilization failure or low fertilization(<10%)after intracytoplasmic sperm injection(ICSI)and conventional calcium(Ca2+)ionophores-based ICSI-AOA were enrolled.The novel ICSIAOA,a combination of ionomycin and cycloheximide,was applied in these patients.The efficacy was evaluated by monitoring the development of embryos through Timelapse,and the safety was evaluated by whole-embryo aneuploidies analysis.Results:Three patients with PLCZ1 mutations and two patients with ACTL7A mutations were enrolled in this study.The patients with PLCZ1 mutations carried four kinds of pathogenic mutations,and the expression of PLCζ1 was absent in the sperms,resulting in fertilization failure.The patients with ACTL7A mutations carried 3 missense mutations,and the expression of ACTL7A and PLCζ1 was also absent,with the abnormality of acrosomal fluorescence signal.Compared with the regular ICSI group and the conventional ICSI-AOA group,the novel AOA method can significantly increase the fertilization rate from less than 10%up to approximately 50%in most cases.The normal distribution of PLCζ1 in the sperms of the cases indicated the absence of aberrant Ca2+ signaling activation.The results of the whole-embryo aneuploidies analysis indicated that oocytes receiving the novel AOA treatment had the potential to develop into blastocysts with normal karyotypes.Conclusions:PLCZ1 and ACTL7A mutations are the causative genes of s sperm-related fertilization failure.ACTL7A mutations may impair the expression of PLCζ1 in sperms,resulting in fertilization failure.The combination of ionomycin and cycloheximide could effectively improve the fertilization rate in the majority of patients suffering from repeated fertilization failure.This novel AOA method had a potential therapeutic effect on those couples experiencing fertilization failure,even after conventional AOA,which may surmount the severe fertilization deficiencies in patients with repeated low fertilization or total fertilization failure.