Asthenoteratospermia is a significant cause of male infertility. FAM71D (Family with sequence similarity 71, member D), as a novel protein exclusively expressed in the testis, has been found to be associated with sperm motility. However, the association of FAM71D mutation with male infertility has yet to be examined. Here, we conducted whole-exome sequencing and identified a homozygous missense mutation c.440G > A (p. Arg147Gln) of FAM71D in an asthenoteratospermia-affected man from a consanguineous family. The FAM71D variant is extremely rare in human population genome databases and predicted to be deleterious by multiple bioinformatics tools. Semen analysis indicated decreased sperm motility and obvious morphological abnormalities in sperm cells from the FAM71D-deficient man. Immunofluorescence assays revealed that the identified FAM71D mutation had an important influence on the assembly of sperm structure-related proteins. Furthermore, intra-cytoplasmic sperm injection (ICSI) treatment performed on the infertile man with FAM71D variant achieved a satisfactory outcome. Overall, our study identified FAM71D as a novel causative gene for male infertility with asthenoteratospermia, for which ICSI treatment may be suggested to acquire good prognosis. All these findings will provide effective guidance for genetic counselling and assisted reproduction treatments of asthenoteratospermia-affected subjects.

OBJECTIVE: Asthenoteratospermia is characterized by malformed spermatozoa with motility defects, which results in male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a hallmark of asthenoteratospermia. The genetic causes of MMAF, however, are unknown in about one-third of cases. Which other MMAF-associated genes are waiting to be discovered?
METHODS: Whole-exome sequencing was conducted to identify causative genes in a man with MMAF. Immunofluorescence staining and western blot were applied to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF.
RESULTS: Sanger sequencing of the family demonstrated that the infertile man carried a homozygous DNAH17 variant (c. 4810C>T [p.R1604C]). The obviously decreased DNAH17 expression was observed in HEK293T cells transfected with MUT-DNAH17 plasmid compared with cells with WT-DNAH17 plasmid. Immunofluorescence analysis showed that this mutation induced significant decrease in DNAH17 expression, which negatively affected the DNAH8 expression in the patient\'s spermatozoa. Moreover, the outcome of ICSI in the patient was unsuccessful.
CONCLUSIONS: Our study revealed a novel homozygous missense mutation in DNAH17 involved in MMAF phenotype. The finding of the novel mutation in DNAH17 enriches the gene variant spectrum of MMAF, further contributing to diagnosis, genetic counselling and prognosis for male infertility.

BACKGROUND: Asthenoteratospermia with multiple morphological abnormalities in the sperm flagella (MMAF) is a significant cause of male infertility. WDR19 is a core component in the IFT-A complex and has a critical role in intraflagellar transport. However, the role of WDR19 mutations in male infertility has yet to be examined.
RESULTS: We performed whole exome sequencing (WES) for 65 asthenoteratospermia individuals and identified a proband who carried a homozygous WDR19 (c.A3811G, p.K1271E) mutation from a consanguineous family. Systematic examinations, including CT scanning and retinal imaging, excluded previous ciliopathic syndromes in the proband. Moreover, semen analysis of this patient showed that the progressive rate decreased to zero, and the sperm flagella showed multiple morphological abnormalities. Scanning and transmission electron microscopy assays indicated that the ultrastructure of sperm flagella in the patient was completely destroyed, while immunofluorescence revealed that WDR19 was absent from the sperm neck and flagella. Moreover, IFT140 and IFT88, predicted to interact with WDR19 directly, were mis-allocated in the WDR19-mutated sperm. Notably, the MMAF subject harboring WDR19 variant and his partner successfully achieved clinical pregnancy through intracytoplasmic sperm injection (ICSI).
CONCLUSIONS: We identified WDR19 as a novel pathogenic gene for male infertility caused by asthenoteratospermia in the absence of other ciliopathic phenotypes, and that patients carrying WDR19 variant can have favorable pregnancy outcomes following ICSI.

OBJECTIVE: To investigate the relation between mutations in ciliopathy-related SPAG6 and RSPH3 and male infertility with severe asthenoteratospermia characterized by multiple flagellar malformations and reveal the intracytoplasmic sperm injection (ICSI) outcomes of those primary ciliary dyskinesia (PCD) patients.
METHODS: Whole-exome sequencing was applied to identify the pathogenic genes for the five PCD patients. The ICSI outcomes of those patients were compared with eight DNAH1-mutated patients and 215 oligo-asthenospermia (OAT) patients.
RESULTS: We identified, for the first time, the compound heterozygous SPAG6 mutations (c.143_145del: p.48_49del, c.585delA: p.Lys196Serfs*6) in a sporadic PCD patient. Further, a novel homozygous nonsynonymous RSPH3 mutation (c.C799T: p.Arg267Cys) was identified in another PCD patient with consanguineous parents. The pathogenicity of these mutations in the assembly of sperm flagella was confirmed by flagellar ultrastructure analysis, immunofluorescence, and quantitative real-time PCR. All five patients underwent six ICSI cycles. The fertilization rate, blastocyst development rate, and clinical pregnancy rate were 69.3%, 50.0%, and 66.7%, respectively. Four of the five couples, including the subjects carrying mutations in SPAG6 or RSPH3, got healthy children born after ICSI. Additionally, the ICSI outcomes of the five PCD couples were statistically comparable with those of the eight DNAH1-mutated couples and the 215 OAT couples.
CONCLUSIONS: Mutations in ciliopathy-related SPAG6 and RSPH3 cause severe asthenoteratospermia characterized by multiple flagellar malformations, resulting in sterility. ICSI is an optimal management with a positive pregnancy outcome.

Asthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes.
Two asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF.
Our study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.

As a type of severe asthenoteratospermia, multiple morphological abnormalities of the flagella (MMAF) are characterized by the presence of immotile spermatozoa with severe flagellar malformations. MMAF is a genetically heterogeneous disorder, and the known MMAF-associated genes can only account for approximately 60% of human MMAF cases. Here we conducted whole-exome sequencing and identified bi-allelic truncating mutations of the TTC29 (tetratricopeptide repeat domain 29) gene in three (3.8%) unrelated cases from a cohort of 80 MMAF-affected Han Chinese men. TTC29 is preferentially expressed in the testis, and TTC29 protein contains the tetratricopeptide repeat domains that play an important role in cilia- and flagella-associated functions. All of the men harboring TTC29 mutations presented a typical MMAF phenotype and dramatic disorganization in axonemal and/or other peri-axonemal structures. Immunofluorescence assays of spermatozoa from men harboring TTC29 mutations showed deficiency of TTC29 and remarkably reduced staining of intraflagellar-transport-complex-B-associated proteins (TTC30A and IFT52). We also generated a Ttc29-mutated mouse model through the use of CRISPR-Cas9 technology. Remarkably, Ttc29-mutated male mice also presented reduced sperm motility, abnormal flagellar ultrastructure, and male subfertility. Furthermore, intracytoplasmic sperm injections performed for Ttc29-mutated mice and men harboring TTC29 mutations consistently acquired satisfactory outcomes. Collectively, our experimental observations in humans and mice suggest that bi-allelic mutations in TTC29, as an important genetic pathogeny, can induce MMAF-related asthenoteratospermia. Our study also provided effective guidance for clinical diagnosis and assisted reproduction treatments.

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes.