Identification Of Pathogenic Genes And Strategy Of Clinical Treatment In Male Infertility Patients With Primary Ciliary Dyskinesia

Objective（1）Using whole exome sequencing technology to identify the pathogenic genes of male infertility patients with primary ciliary dyskinesia（PCD）.（2）To observe the clinical pregnancy outcomes of male infertility PCD patients with intracytoplasmic sperm injection（ICSI）.Methods（1）PCD patients with primary infertility treated in the reproductive medicine center of the First Affiliated Hospital of Anhui Medical University from February 2018 to October 2019 were recruited in this study.The general clinical data of all subjects were collected by questionnaires.Peripheral blood of patients and their parents was taken,and semen samples from patients were also taken.DNA was extracted from peripheral blood for whole exome sequencing and Sanger sequencing.The sequencing data were analyzed to identify the pathogenic genes,then HE staining,scanning electron microscopy,transmission electron microscopy,q PCR and sperm immunofluorescence experiments were used to verify the pathogenicity of candidate genes.（2）ICSI fertility treatments were applied in six patients of PCD,motile sperms in the semen were the first choice for ICSI.If motile sperms were unsearchable,sperm viability was assessed by using the ICSI pipette to test the elasticity of the sperm tail.Then the fertilization rate,cleavage rate and embryo development rate were observed the pregnancy outcomes were followed up.The ICSI outcomes of those patients were compared with eight DNAH1-mutated patients and 215 oligo-asthenospermia（OAT）patients.Results（1）Gene mutations were found in three PCD patients by using whole exome sequencing,including a novel homozygous nonsynonymous DNAAF1 mutation c.T440G（p.L147R）,a novel homozygous nonsynonymous RSPH3 mutation（c.C799T: p.Arg267Cys）and the compound heterozygous SPAG6 mutations（c.143₁45del: p.48₄9del,c.585 del A: p.Lys196Serfs*6）,Sanger sequencing confirmed that these mutations were Co-isolated in each patient’s familie.HE staining and SEM showed that the sperms of the DNAAF1-mutated patient were a mixed phenotype of malformations and normal flagella,and the sperm flagellum of patients with SPAG6 and RSPH3 mutations were multiple malformations.TEM showed that the ultrastructure of sperm flagella in the DNAAF1-mutated patient was the absence of inner and outer dynein arms or the absence of dynein arms with disordered arrangement of microtubules,the ultrastructure in the RSPH3-mutated patient was a complete or partial loss of radial spokes,and the ultrastructure in the SPAG6-mutated patient was "9+0" or "9+1" structure.The results of sperm q PCR and immunofluorescence experiments showed that sperm m RNA expression levels and protein degradation were significantly reduced in patients with DNAAF1,RSPH3,and SPAG6 mutations.（2）All six patients underwent seven ICSI cycles.The fertilization rate,blastocyst development rate,and clinical pregnancy rate were 69.5%,49.1%,and 71.4%,five of the six couples got healthy children born after ICSI.The statistical result showed that there were no significant difference between the PCD group and other two groups in the fertilization rate,cleavage rate,embryo development rate（8 cells and blastocysts）,implantation rate and clinical pregnancy rate.Conclusion The novel mutations in ciliopathy-related DNAAF1,RSPH3,and SPAG6 cause PCD and sterility.ICSI is an effective method to obtain biological progeny for male infertility patients with PCD.