Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Liu C ，Tu C ，Wang L ，Wu H ，Houston BJ ，Mastrorosa FK ，Zhang W ，Shen Y ，Wang J ，Tian S ，Meng L ，Cong J ，Yang S ，Jiang Y ，Tang S ，Zeng Y ，Lv M ，Lin G ，Li J ，Saiyin H ，He X ，Jin L ，Touré A ，Ray PF ，Veltman JA ，Shi Q ，O'Bryan MK ，Cao Y ，Tan YQ ，Zhang F ... -  《-》

A novel mutation in CFAP47 causes male infertility due to multiple morphological abnormalities of the sperm flagella.

A previous study suggested that loss of CFAP47 function is involved in multiple morphological abnormalities of the sperm flagella (MMAF) in humans and mice. However, the comprehensive role of CFAP47 in spermatogenesis is largely unknown. Whole-exome sequencing (WES) was conducted to identify pathogenic variant in two patients with MMAF. The functional effect of the identified mutations was investigated by immunofluorescence staining and western blotting. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. In this study, we identified a novel missense mutation (c.1414G>A; p.V472M) in CFAP47 in two unrelated patients with oligoasthenoteratozoospermia. Intriguingly, in addition to the MMAF phenotype very analogous to the previous report, the two patients notably presented abnormal morphology of sperm heads, the sperm mitochondrial sheath was obviously disorganized, and the sperm annulus were almost defective. Further functional experiments confirmed that the expression of CFAP47 was markedly reduced in the spermatozoa of the patients. Mechanism analysis suggested that CFAP47 might regulate the expression of CFAP65, CFAP69 and SEPTIN4 through their physical interactions and thus modulating sperm morphogenesis. we revealed a novel mutation in CFAP47 and further expanded the phenotype and mutation spectrum of CFAP47, as well as the potential mechanism of CFAP47 manipulating spermatogenesis, finally providing important guidance for genetic counseling and targeted treatment for CFAP47 mutation-related male infertility.

Liu M ，Dai S ，Zhang J ，Yang Y ，Shen Y ，Liu H ，Yang Y ，Jiang C ，Tian E ... -  《-》

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

He X ，Liu C ，Yang X ，Lv M ，Ni X ，Li Q ，Cheng H ，Liu W ，Tian S ，Wu H ，Gao Y ，Yang C ，Tan Q ，Cong J ，Tang D ，Zhang J ，Song B ，Zhong Y ，Li H ，Zhi W ，Mao X ，Fu F ，Ge L ，Shen Q ，Zhang M ，Saiyin H ，Jin L ，Xu Y ，Zhou P ，Wei Z ，Zhang F ，Cao Y ... -  《-》

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

Liu C ，Miyata H ，Gao Y ，Sha Y ，Tang S ，Xu Z ，Whitfield M ，Patrat C ，Wu H ，Dulioust E ，Tian S ，Shimada K ，Cong J ，Noda T ，Li H ，Morohoshi A ，Cazin C ，Kherraf ZE ，Arnoult C ，Jin L ，He X ，Ray PF ，Cao Y ，Touré A ，Zhang F ，Ikawa M ... -  《-》

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described. We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied. Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal "9 + 0" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI. Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Xu C ，Tang D ，Shao Z ，Geng H ，Gao Y ，Li K ，Tan Q ，Wang G ，Wang C ，Wu H ，Li G ，Lv M ，He X ，Cao Y ... -  《Reproductive Biology and Endocrinology》