A novel loss-of-function variant in PNLDC1 inducing oligo-astheno-teratozoospermia and male infertility.

Male infertility is a major reproductive disorder, which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality. To date, five male patients with biallelic loss-of-function (LOF) variants of PARN-like ribonuclease domain-containing exonuclease 1 ( PNLDC1 ) have been reported to experience infertility with nonobstructive azoospermia. The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia (OAT) in a patient from a Chinese Han family. Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant (NM_173516.2, c.142C>T, p.Gln48Ter) in PNLDC1 . Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype, including microcephaly, head tapering, and globozoospermia. Consistently, peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome. Furthermore, the disomy rate of chromosomes in the patient's spermatozoa was significantly increased compared with that of a fertile control sample. We reported an LOF variant of the PNLDC1 gene responsible for OAT.

Zhao SY ，Meng LL ，Du ZL ，Tan YQ ，He WB ，Wang X ... -  《-》

A novel homozygous LRRC6 mutation causes male infertility with asthenozoospermia and primary ciliary dyskinesia in humans.

Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes. To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved. The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations. In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband. This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.

Shi S ，Tang X ，Long S ，Yang J ，Wang T ，Wang H ，Hu T ，Shi J ，Huang G ，Qiao S ，Lin T ... -  《-》

A loss-of-function variant in ZCWPW1 causes human male infertility with sperm head defect and high DNA fragmentation.

Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify. An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay. The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband's sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband's spermatozoa have high DNA fragmentation. It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.

Song Y ，Guo J ，Zhou Y ，Wei X ，Li J ，Zhang G ，Wang H ... -  《-》

Broadening the ARMC2 mutational phenotype: linking multiple morphological abnormalities of the Flagella to Pulmonary Manifestations in Primary Ciliary Dyskinesia.

Severe asthenoteratozoospermia, a prevalent cause of male infertility, has increasingly been associated with ARMC2 variants that cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF). Although ARMC2 is also expressed in other ciliary structures, no studies have yet reported a link between ARMC2 gene variants and other symptoms of Primary Ciliary Dyskinesia (PCD). Here, we performed whole-exome sequencing (WES) on Chinese subjects with MMAF to identify potential genetic variants. Sanger sequencing was used to validate the candidate variants. Sperm morphology was assessed using modified hematoxylin and eosin (H&E) staining, and transmission electron microscopy (TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to evaluate variations in structural protein. Additionally, intracytoplasmic sperm injection (ICSI) was applied for assisted fertilization. We identified two compound heterozygous ARMC2 variants and one homozygous variant (P1: c.1030_1042del, p.T344fs/c.1331G > A, p.R444H; P2:c.1264C > T, p.R422X) in two unrelated individuals. Notably, in addition to MMAF, individual P2 exhibited classic symptoms of PCD in the lungs, including recurrent airway infections, bronchitis, and rhinosinusitis. Morphological and ultrastructural analyses of the spermatozoa obtained from the two individuals revealed dramatic disorganization in axonemal and peri-axonemal structures, as well as the absence of the axonemal central pair complex (CPC). Immunoblotting and immunofluorescence assays revealed the reduced expression of ARMC2 and the abnormality of various axonal structural proteins. Further assisted reproduction outcomes showed that one of the individuals conceived successfully after Intracytoplasmic Sperm Injection (ICSI). Overall, this study significantly expanded the mutational phenotype of ARMC2, marking the first discovery of PCD-related pulmonary phenotypes outside of the reproductive system. This work establishes the association between ARMC2 and typical PCD and lays the groundwork for further investigation into the molecular mechanisms of ARMC2 in both flagellogenesis and ciliogenesis.

Wu B ，Zhang W ，Yu H ，Ruan L ，Wang K ，Gu M ，Geng H ，Fang J ，Xu C ，Sheng Y ，Tan Q ，Shen Q ，Duan Z ，Wu H ，Hua R ，Guo R ，Wei Z ，Zhou P ，Xu Y ，Cao Y ，He X ，Li K ，Lv M ，Tang D ... -  《-》

Novel bi-allelic DNAH3 variants cause oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a widespread cause of male infertility. One of the usual clinical manifestations of OAT is multiple morphological abnormalities of the sperm flagella (MMAF), which are frequently associated with mutations and defects in the dynein family. However, the relationship between the newly identified Dynein Axonemal Heavy Chain 3 (DNAH3) mutation and oligonasthenospermia in humans has not yet been established. Whole exome sequencing, pathogenicity analysis, and species conservation analysis of mutation sites were conducted on two patients from different unrelated families with DNAH3 mutations. We identified representative mutation sites and predicted the protein structure following these mutations. The sperm characteristics of the two patients with DNAH3 mutations were verified using Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy. Additionally, mRNA and protein levels were assessed through RT-qPCR and Western blotting. The biallelic mutations in the first progenitor included a heterozygous deletion and insertion, c.6535_6536 delinsAC (to infect mutation (p.Asp2179Thr), and stop codon premutation, c.3249G > A (p.Trp1083Ter). In Family II, the patient (P2) harbored a DNAH3 heterozygous missense mutation, c. 10439G> A(p.Arg3480Gln), along with a stop codon premutation, (c.10260G > A; p.Trp3420Ter). Patients with premature termination of transcription or translation due to DNAH3 mutations exhibit OAT phenotypes, including fibrous sheath dysplasia and multiple tail malformations. We identified the representative sites after mutation, predicted the protein structure, and assessed changes in the protein levels of DNAH3 and related genes following mutations. Notably,a significant reduction in DNAH3 protein expression was validated in these patients. We may explore in the future how DNAH3 affects sperm motility and quality through regulatory mechanisms involving protein structural changes. Novel biallelic mutations in DNAH3, especially those resulting in a premature stop codon, may alter protein expression, structure, and active site, leading to spermatogenic failure and potentially inducing OAT. The discovery of new mutations in DNAH3 may be the key to the diagnosis and treatment of OAT.

Li S ，Zhang Z ，Xie L ，Zhao Y ，Chen H ，Zhang S ，Cai Y ，Ren B ，Liu W ，Tang S ，Sha Y ... -  《Frontiers in Endocrinology》