ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice.

Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.

A novel homozygous missense TTC12 variant identified in an infertile Pakistani man with severe oligoasthenoteratozoospermia and primary ciliary dyskinesia.

TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.

Homozygous variant in DRC3 (LRRC48) gene causes asthenozoospermia and male infertility.

Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility.

Decreased embryo developmental potential and lower cumulative pregnancy rate in men with multiple morphological abnormalities of the sperm flagella.

Objective: Multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by abnormal flagellar phenotypes, which is a particular kind of asthenoteratozoospermia. Previous studies have reported a comparable intracytoplasmic sperm injection (ICSI) outcome in terms of fertilization rate and clinical pregnancy rate in patients with MMAF compared with those with no MMAF; however, others have conflicting opinions. Assisted reproductive technology (ART) outcomes in individuals with MMAF are still controversial and open to debate. Methods: A total of 38 patients with MMAF treated at an academic reproductive center between January 2014 and July 2022 were evaluated in the current retrospective cohort study and followed up until January 2023. Propensity score matching was used to adjust for the baseline clinical characteristics of the patients and to create a comparable control group. The genetic pathogenesis of MMAF was confirmed by whole exome sequencing. The main outcomes were the embryo developmental potential, the cumulative pregnancy rate (CLPR), and the cumulative live birth rate (CLBR). Results: Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in patients with MMAF. Laboratory outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyst rate, followed a trend of decline in the MMAF group (p < 0.05). Moreover, according to the embryo transfer times and complete cycles, the CLPR in the cohort of MMAF was lower compared with the oligoasthenospermia pool (p = 0.033 and p = 0.020, respectively), while no statistical differences were observed in the neonatal outcomes. Conclusion: The current study presented decreased embryo developmental potential and compromised clinical outcomes in the MMAF cohort. These findings may provide clinicians with evidence to support genetic counseling and clinical guidance in specific patients with MMAF.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

A novel homozygous nonsense variant of AK7 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.

Identification of novel homozygous asthenoteratospermia-causing ARMC2 mutations associated with multiple morphological abnormalities of the sperm flagella.

PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.

Bi-allelic variants in chromatoid body protein TDRD6 cause spermiogenesis defects and severe oligoasthenoteratozoospermia in humans.

BACKGROUND: The association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing and Sanger sequencing were employed to identify potential pathogenic variants of TDRD6 in infertile men. Histology, immunofluorescence, immunoblotting and ultrastructural analyses were conducted to clarify the structural and functional abnormalities of sperm in mutated patients. Tdrd6-knockout mice were generated using the CRISPR-Cas9 system. Total RNA-seq and single-cell RNA-seq (scRNA-seq) analyses were used to elucidate the underlying molecular mechanisms, followed by validation through quantitative RT-PCR and immunostaining. Intracytoplasmic sperm injection (ICSI) was also used to assess the efficacy of clinical treatment. RESULTS: Bi-allelic TDRD6 variants were identified in five unrelated Chinese individuals with OAT, including homozygous loss-of-function variants in two consanguineous families. Notably, besides reduced concentrations and impaired motility, a significant occurrence of acrosomal hypoplasia was detected in multiple spermatozoa among five patients. Using the Tdrd6-deficient mice, we further elucidate the pivotal role of TDRD6 in spermiogenesis and acrosome identified. In addition, the mislocalisation of crucial chromatoid body components DDX4 (MVH) and UPF1 was also observed in round spermatids from patients harbouring TDRD6 variants. ScRNA-seq analysis of germ cells from a patient with TDRD6 variants revealed that TDRD6 regulates mRNA metabolism processes involved in spermatid differentiation and cytoplasmic translation. CONCLUSION: Our findings strongly suggest that TDRD6 plays a conserved role in spermiogenesis and confirms the causal relationship between TDRD6 variants and human OAT. Additionally, this study highlights the unfavourable ICSI outcomes in individuals with bi-allelic TDRD6 variants, providing insights for potential clinical treatment strategies.

Effect of moxa smoke on sperm parameters and oxidative stress in rats with asthenozoospermia.

Asthenozoospermia is a leading cause of male infertility, characterized by reduced sperm motility. In this study, we determined sperm motility and the activities of antioxidant enzymes and oxidation products in the testis of rats with ornidazole (ORN)-induced asthenozoospermia and further examined and compared the differential effects of moxa smoke (MS) and cigarette smoke (CS) on sperm motility and oxidative stress (OS) of asthenozoospermic rats. The smoke intervention was initiated 11 days after intragastric administration of ORN, followed by the examination of testis index, sperm parameters, OS-related gene levels, and testicular histopathology. Sperm motility and antioxidant enzyme activities, as well as oxidation products significantly decreased in ORN-induced rats compared with MS-treated rats (p < .05-.001). MS treatment restored the reduced sperm motility and activities of glutathione peroxidase, superoxide dismutase, and catalase, but increased the malondialdehyde and nitric oxide synthetase levels in ORN-induced rats (p < .05-.001). Also, the histopathological changes in the testis of ORN-induced rats were improved by MS treatment. The study highlighted that MS was an effective factor in moxibustion therapy, which notably improved the sperm motility of asthenozoospermic rats by inhibiting OS in the reproductive system.

The Activated AMPK/mTORC2 Signaling Pathway Associated with Oxidative Stress in Seminal Plasma Contributes to Idiopathic Asthenozoospermia.

Asthenozoospermia is a common form of abnormal sperm quality in idiopathic male infertility. While most sperm-mediated causes have been investigated in detail, the significance of seminal plasma has been neglected. Herein, we aimed to investigate the possible pathogenic factors leading to decreased sperm motility based on seminal plasma. Semen was collected from normo- (NOR, n = 70), idiopathic oligo- (OLI, n = 57), and idiopathic asthenozoospermic (AST, n = 53) patients. Using attenuated total reflection-Fourier transform infrared coupled with chemometrics, distinct differences in the biochemical compositions of nucleic acids, protein structure (amides I, II, and III), lipids, and carbohydrates in seminal plasma of AST were observed when compared to NOR and OLI. Compared with NOR and OLI, the levels of peptide aggregation, protein phosphorylation, unsaturated fatty acid, and lipid to protein ratio were significantly increased in AST; however, the level of lipid saturation was significantly decreased in seminal plasma of AST. Compared with NOR, the levels of ROS, MDA, 8-iso-prostaglandin F2α (8-isoPGF2α), and the ratio of phospho-AMPKα/AMPKα1 were significantly increased in AST; however, the levels of SOD, glutathione S-transferase (GSTs), protein carbonyl derivative (PC), and the ratio of phospho-Rictor/Rictor were significantly decreased in seminal plasma of AST. Changes of the AMPK/mTORC2 signaling in the seminal microenvironment possibly induce abnormal glucose and lipid metabolism, which impairs energy production. Oxidative stress potentially damages seminal plasma lipids and proteins, which in turn leads to impaired sperm structure and function. These findings provide evidence that the changes in seminal plasma compositions, oxidative stress, and activation of the AMPK/mTORC2 signaling contribute to the development of asthenozoospermia.

Sperm mRNAs as potential markers of male fertility.

Advances in transcriptomic technologies are contributing to an increased understanding of the role of spermatozoal RNA in sperm physiology. Although sperm transcriptomic studies have delivered large amounts of valuable information, no new male fertility biomarkers have emerged from such studies to date. This review summarizes current knowledge about the potential relevance of certain mRNA as biomarkers, focusing on comparative studies of human spermatozoa transcriptomic profiles from fertile and pathological semen samples. Asthenozoospermia is the semen aberrant condition that has been most exhaustively investigated to date. We cross-analyzed findings from three different studies on the transcriptome of asthenozoospermic semen samples and identified 100 transcripts that were consistently differentially expressed and that consequently are candidates for characterizing the molecular source of this sperm anomaly. The potential use of sperm mRNAs as predictors of outcomes of assisted reproductive technologies (ART) is also reviewed. Improving the understanding of the human spermatozoa mRNA content is expected to improve the evaluation and diagnosis of infertile men, and ultimately facilitate the selection of the best treatment to overcome infertility.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms.

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Homozygous mutations in CCDC34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

BACKGROUND: Oligoasthenoteratozoospermia is a typical feature of sperm malformations leading to male infertility. Only a few genes have been clearly identified as pathogenic genes of oligoasthenoteratozoospermia. METHODS AND RESULTS: Here, we identified a homozygous frameshift variant (c.731dup, p.Asn244Lysfs*3) in CCDC34, which is preferentially expressed in the human testis, using whole-exome sequencing in a cohort of 100 Chinese men with multiple morphological abnormalities of the sperm flagella (MMAF). In an additional cohort of 167 MMAF-affected men from North Africa, Iran and France, we identified a second subject harbouring a homozygous CCDC34 frameshift variant (c.799_817del, p.Glu267Lysfs*72). Both affected men presented a typical MMAF phenotype with an abnormally low sperm concentration (ie, oligoasthenoteratozoospermia). Transmission electron microscopy analysis of the sperm flagella affected by CCDC34 deficiency further revealed dramatic disorganisation of the axoneme. Immunofluorescence assays of the spermatozoa showed that CCDC34 deficiency resulted in almost absent staining of CCDC34 and intraflagellar transport-B complex-associated proteins (such as IFT20 and IFT52). Furthermore, we generated a mouse Ccdc34 frameshift mutant using CRISPR-Cas9 technology. Ccdc34-mutated (Ccdc34mut/mut ) male mice were sterile and presented oligoasthenoteratozoospermia with typical MMAF anomalies. Intracytoplasmic sperm injection has good pregnancy outcomes in both humans and mice. CONCLUSIONS: Our findings support that CCDC34 is crucial to the formation of sperm flagella and that biallelic deleterious mutations in CCDC34/Ccdc34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

Novel biallelic mutations in SLC26A8 cause severe asthenozoospermia in humans owing to midpiece defects: Insights into a putative dominant genetic disease.

To investigate the genetic cause of male infertility characterized by severe asthenozoospermia, two unrelated infertile men with severe asthenozoospermia from nonconsanguineous Chinese families were enrolled, and whole exome sequencing were performed to identify the potential pathogenic mutations. Novel compound heterozygous mutations (NK062 III-1: c.290T>C, p.Leu97Pro; c.1664delT, p.Ile555Thrfs*11/NK038 III-1: c.212G>T, p.Arg71Leu; c.290T>C, p.Leu97Pro) in SLC26A8 were identified. All mutations were inherited from their heterozygous parents and are predicted to be disease-causing by sorts intolerant from tolerant, PolyPhen-2, Mutation Taster, and Combined Annotation Dependent Depletion. In silico mutant SLC26A8 models predict that mutations p.Leu97Pro and p.Arg71Leu cause changes in the α-helix, which may result in functional defects in the protein. Notably, heterozygous male carriers of each mutation in both families were able to reproduce naturally, which is inconsistent with previous reports. Ultrastructural analysis revealed severe asthenozoospermia associated with absence of the mitochondrial sheath and annulus in spermatozoa from both the probands, and both structural defects were verified by HSP60 and SEPT4 immunofluorescence analysis. SLC26A8 levels were significantly reduced in spermatozoa from patients harboring biallelic SLC26A8 mutations, and both patients achieved good prognosis following intracytoplasmic sperm injection. Our findings indicate that mutations in SLC26A8 could manifest as a recessive genetic cause of severe asthenozoospermia and male infertility.

Evaluating the therapeutic effect and toxicity of theophylline in infertile men with asthenoteratozoospermia: a double-blind, randomized clinical trial study.

Theophylline as a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor (cAMP-PDEI) elevates cAMP levels. We aimed to evaluate the therapeutic effect and toxicity of theophylline on the sperm parameters, oxidative stress (OS), and inflammation in asthenoteratozoospermic men. Sixty asthenoteratozoospermic patients were divided into groups of placebo and theophylline (200 mg/day). After 3 months of oral treatment, sperm parameters, viability, and DNA fragmentation were analyzed by the CASA system, eosin nigrosin staining, sperm DNA fragmentation kit, respectively. The seminal plasma level of reactive oxygen species (ROS) of neat semen samples, malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) was assessed. Data were analyzed statistically using the independent samples t-test and the paired t-test and the means were considered significantly different at p < 0.05. Sperm motility, viability, and the number of sperms with normal morphology and the seminal plasma level of TAC and IL-10 and also sperm DNA fragmentation increased significantly in the theophylline group compared to the placebo. The MDA, TNF-α, and ROS levels decreased significantly in the theophylline group compared to the placebo. Theophylline improved sperm parameters, reduced OS and inflammation, but also created genotoxicity and increased sperm DNA fragmentation. Therefore, to benefit from the desired effects of theophylline and inhibit the toxicity of it in the treatment of men with asthenoteratozoospermia, it is suggested to be used simultaneously with another antioxidant to protect sperm DNA from fragmentation.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms / 亚洲男科学杂志(英文版)

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

A novel mutation in DNAH17 is present in a patient with multiple morphological abnormalities of the flagella.

RESEARCH QUESTION: 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? DESIGN: 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. RESULT: 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. CONCLUSION: 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.

A network pharmacology approach to determine the underlying mechanisms of action of Yishen Tongluo formula for the treatment of oligoasthenozoospermia.

Oligoasthenozoospermia is a complex disease caused by a variety of factors, and its incidence is increasing yearly worldwide. Yishen Tongluo formula (YSTLF), created by Professor Sun Zixue, has been used to treat oligoasthenozoospermia in clinical practice for several decades with a good therapeutic effect. However, the chemical and pharmacological profiles of YSTLF remain unclear and need to be elucidated. In this study, a network pharmacology approach was applied to explore the potential mechanisms of YSTLF in oligoasthenozoospermia treatment. All of the compounds in YSTLF were retrieved from the corresponding databases, and the bioactive ingredients were screened according to their oral bioavailability (OB) and drug-likeness (DL). The potential proteins of YSTLF were obtained from the traditional Chinese medicine systems pharmacology (TCMSP) database and the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) database, while the potential genes of oligoasthenozoospermia were obtained from the GeneCards database and the DisGeNET database. The STRING database was used to construct an interaction network according to the common targets identified by the online tool Venny for YSTLF and oligoasthenozoospermia. The topological characteristics of nodes were visualized and analyzed through Cytoscape. Biological functions and significant pathways were determined and analyzed using the Gene Ontology (GO) knowledgebase, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Metascape. Finally, the disease-formula-compound-target-pathway network was constructed by Cytoscape. A total of 106 bioactive ingredients and 134 potential targets from YSTLF were associated with oligoasthenozoospermia or considered to be therapeutically relevant. Pathway analysis indicated that the PI3K/Akt, MAPK and apoptosis signaling pathways were significant pathways involved in oligoasthenozoospermia. In conclusion, the current study expounded the pharmacological actions and molecular mechanisms of YSTLF in treating oligoasthenozoospermia from a holistic viewpoint. The potential molecular mechanisms were closely related to antioxidative stress, antiapoptosis and anti-inflammation, with TNF, CCND1, ESR1, NFKBIA, NR3C1, MAPK8, and IL6 being possible targets. This network pharmacology prediction may offer a helpful tool to illustrate the molecular mechanisms of the Chinese herbal compound YSTLF in oligoasthenozoospermia treatment.