Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

Predictors of successful salvage microdissection testicular sperm extraction (mTESE) after failed initial TESE in patients with non-obstructive azoospermia: A systematic review and meta-analysis.

BACKGROUND: There has been no systematic review and meta-analysis to analyze and summarize the predictive factors of successful sperm extraction in salvage microdissection testicular sperm extraction. OBJECTIVES: We aimed to investigate the factors predicting the result of salvage microdissection testicular sperm extraction in patients with non-obstructive azoospermia who failed the initial microdissection testicular sperm extraction or conventional testicular sperm extraction. MATERIALS AND METHODS: We conducted a systematic literature search in PubMed, Web of Science, EMBASE, and the Cochrane Library for literature that described the characteristics of patients with non-obstructive azoospermia who underwent salvage microdissection testicular sperm extraction after failing the initial microdissection testicular sperm extraction or conventional testicular sperm extraction published prior to June 2022. RESULTS: This meta-analysis included four retrospective studies with 332 patients with non-obstructive azoospermia who underwent a failed initial microdissection testicular sperm extraction and three retrospective studies with 177 non-obstructive azoospermia patients who underwent a failed conventional testicular sperm extraction. The results were as follows: among non-obstructive azoospermia patients whose first surgery was microdissection testicular sperm extraction, younger patients (standard mean difference: -0.28, 95% confidence interval [CI]: -0.55 to -0.01) and those with smaller bilateral testicular volume (standard mean difference: -0.55, 95% CI: -0.95 to -0.15), lower levels of follicle-stimulating hormone (standard mean difference: -0.86, 95% CI: -1.18 to -0.54) and luteinizing hormone (standard mean difference: -0.68, 95% CI: -1.16 to -0.19), and whose testicular histological type was hypospermatogenesis (odds ratio: 3.52, 95% CI: 1.30-9.53) were more likely to retrieve spermatozoa successfully, while patients with Sertoli-cell-only syndrome (odds ratio: 0.41, 95% CI: 0.24-0.73) were more likely to fail again in salvage microdissection testicular sperm extraction. Additionally, in patients who underwent salvage microdissection testicular sperm extraction after a failed initial conventional testicular sperm extraction, those with testicular histological type of hypospermatogenesis (odds ratio: 30.35, 95% CI: 8.27-111.34) were more likely to be successful, while those with maturation arrest (odds ratio: 0.39, 95% CI: 0.18-0.83) rarely benefited. CONCLUSION: We found that age, testicular volume, follicle-stimulating hormone, luteinizing hormone, hypospermatogenesis, Sertoli-cell-only syndrome, and maturation arrest were valuable predictors of salvage microdissection testicular sperm extraction, which will assist andrologists in clinical decision-making and minimize unnecessary injury to patients.

Genetic mechanisms of fertilization failure and early embryonic arrest: a comprehensive review.

BACKGROUND: Infertility and pregnancy loss are longstanding problems. Successful fertilization and high-quality embryos are prerequisites for an ongoing pregnancy. Studies have proven that every stage in the human reproductive process is regulated by multiple genes and any problem, at any step, may lead to fertilization failure (FF) or early embryonic arrest (EEA). Doctors can diagnose the pathogenic factors involved in FF and EEA by using genetic methods. With the progress in the development of new genetic technologies, such as single-cell RNA analysis and whole-exome sequencing, a new approach has opened up for us to directly study human germ cells and reproductive development. These findings will help us to identify the unique mechanism(s) that leads to FF and EEA in order to find potential treatments. OBJECTIVE AND RATIONALE: The goal of this review is to compile current genetic knowledge related to FF and EEA, clarifying the mechanisms involved and providing clues for clinical diagnosis and treatment. SEARCH METHODS: PubMed was used to search for relevant research articles and reviews, primarily focusing on English-language publications from January 1978 to June 2023. The search terms included fertilization failure, early embryonic arrest, genetic, epigenetic, whole-exome sequencing, DNA methylation, chromosome, non-coding RNA, and other related keywords. Additional studies were identified by searching reference lists. This review primarily focuses on research conducted in humans. However, it also incorporates relevant data from animal models when applicable. The results were presented descriptively, and individual study quality was not assessed. OUTCOMES: A total of 233 relevant articles were included in the final review, from 3925 records identified initially. The review provides an overview of genetic factors and mechanisms involved in the human reproductive process. The genetic mutations and other genetic mechanisms of FF and EEA were systematically reviewed, for example, globozoospermia, oocyte activation failure, maternal effect gene mutations, zygotic genome activation abnormalities, chromosome abnormalities, and epigenetic abnormalities. Additionally, the review summarizes progress in treatments for different gene defects, offering new insights for clinical diagnosis and treatment. WIDER IMPLICATIONS: The information provided in this review will facilitate the development of more accurate molecular screening tools for diagnosing infertility using genetic markers and networks in human reproductive development. The findings will also help guide clinical practice by identifying appropriate interventions based on specific gene mutations. For example, when an individual has obvious gene mutations related to FF, ICSI is recommended instead of IVF. However, in the case of genetic defects such as phospholipase C zeta1 (PLCZ1), actin-like7A (ACTL7A), actin-like 9 (ACTL9), and IQ motif-containing N (IQCN), ICSI may also fail to fertilize. We can consider artificial oocyte activation technology with ICSI to improve fertilization rate and reduce monetary and time costs. In the future, fertility is expected to be improved or restored by interfering with or supplementing the relevant genes.

Eif4enif1 haploinsufficiency disrupts oocyte mitochondrial dynamics and leads to subfertility.

Infertility affects couples worldwide. Premature ovarian insufficiency (POI) refers to loss of ovarian function before 40 years of age and is a contributing factor to infertility. Several case studies have reported dominant-inherited POI symptoms in families with heterozygous EIF4ENIF1 (4E-T) mutations. However, the effects of EIF4ENIF1 haploinsufficiency have rarely been studied in animal models to reveal the underlying molecular changes related to infertility. Here, we demonstrate that Eif4enif1 haploinsufficiency causes mouse subfertility, impairs oocyte maturation and partially arrests early embryonic development. Using dual-omic sequencing, we observed that Eif4enif1 haploinsufficiency significantly altered both transcriptome and translatome in mouse oocytes, by which we further revealed oocyte mitochondrial hyperfusion and mitochondria-associated ribonucleoprotein domain distribution alteration in Eif4enif1-deficient oocytes. This study provides new insights into the molecular mechanisms underlying clinical fertility failure and new avenues to pursue new therapeutic targets to address infertility.

Deficiency in AK9 causes asthenozoospermia and male infertility by destabilising sperm nucleotide homeostasis.

BACKGROUND: Asthenozoospermia is the primary cause of male infertility; however, its genetic aetiology remains poorly understood. Adenylate kinase 9 (AK9) is highly expressed in the testes of humans and mice and encodes a type of adenosine kinase that is functionally involved in cellular nucleotide homeostasis and energy metabolism. We aimed to assess whether AK9 is involved in asthenozoospermia. METHODS: One-hundred-and-sixty-five Chinese men with idiopathic asthenozoospermia were recruited. Whole-exome sequencing (WES) and Sanger sequencing were performed for genetic analyses. Papanicolaou staining, Haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were used to observe the sperm morphology and structure. Ak9-knockout mice were generated using CRISPR-Cas9. Sperm adenosine was detected by liquid chromatography-mass spectrometry. Targeted sperm metabolomics was performed. Intracytoplasmic sperm injection (ICSI) was used to treat patients. FINDINGS: We identified five patients harbouring bi-allelic AK9 mutations. Spermatozoa from men harbouring bi-allelic AK9 mutations have a decreased ability to sustain nucleotide homeostasis. Moreover, bi-allelic AK9 mutations inhibit glycolysis in sperm. Ak9-knockout male mice also presented similar phenotypes of asthenozoospermia. Interestingly, ICSI was effective in bi-allelic AK9 mutant patients in achieving good pregnancy outcomes. INTERPRETATION: Defects in AK9 induce asthenozoospermia with defects in nucleotide homeostasis and energy metabolism. This sterile phenotype could be rescued by ICSI. FUNDING: The National Natural Science Foundation of China (82071697), Medical Innovation Project of Fujian Province (2020-CXB-051), open project of the NHC Key Laboratory of Male Reproduction and Genetics in Guangzhou (KF202004), Medical Research Foundation of Guangdong Province (A2021269), Guangdong Provincial Reproductive Science Institute Innovation Team grants (C-03), and Outstanding Young Talents Program of Capital Medical University (B2205).

Novel mutations of TEX11 are associated with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) affects 10% of infertile men worldwide, and genetic studies revealed that there are plenty of monogenic mutations that responsible for a part of idiopathic NOA cases. Testis-expressed gene 11 (TEX11) is an X-linked meiosis-specific gene, many pathogenic variants in TEX11 have been detected in NOA patients, and the deficiency of this gene can cause abnormal meiotic recombination and chromosomal synapsis. However, many NOA-affected cases caused by TEX11 mutation remain largely unknown. This study reported three novel TEX11 mutations (exon 5, c.313C>T: p.R105*), (exon 7, c.427A>C: p.K143Q) and (exon 29, c.2575G>A: p.G859R). Mutations were screened using whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon. Histological analysis of testicular biopsy specimens revealed a thicker basement membrane of the seminiferous tubules and poorly developed spermatocytes, and no post-meiotic round spermatids or mature spermatozoa were observed in the seminiferous tubules of patients with TEX11 mutation. Conclusion: This study presents three novel variants of TEX11 as potential infertility alleles that have not been previously reported. It expanded the variant spectrum of patients with NOA, which also emphasizes the necessity of this gene screening for the clinical auxiliary diagnosis of patients with azoospermia.

TENT5D disruption causes oligoasthenoteratozoospermia and male infertility.

BACKGROUND: Oligoasthenoteratozoospermia (OAT) is one of the most complex aggregators of male gametic problems. However, the genetic etiology of OAT is still largely unknown. OBJECTIVES: To reveal the new genetic factors responsible for male infertility owning to OAT and reveal the outcomes of the affected patients from intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: Two infertile men with typical OAT were recruited in 2018 and retrospected a cohort that included 47 patients with OAT from 2013 to 2021. Fifty healthy men with proven fertility served as control subjects. To identify the novel pathogenic variants, whole-exome sequencing and Sanger sequencing were used. In silico analysis revealed the affecting of the variants. Field emission scanning electron microscopy was employed to observe the morphological defects of the spermatozoa. Immunofluorescence was used to analyze the expression and localization of the related protein. CRISPR/Cas9 was used to generate the mouse model. ICSI was used as a treatment for the patients and to assess the effects of the pathogenic variant on fertilization and embryo development. RESULTS: We identified a loss-of-function mutation NM_001170574.2:c.823G > T (p.Glu275*) in X-linked TENT5D from two patients with OAT. This variant is highly deleterious and has not been found in the human population. The count of patients' spermatozoa is dramatically decreased and displays multiple morphologic abnormalities with poor motility. Tent5d knockout mice are infertile and exhibit parallel defects. ICSI could rescue the infertility of the Tent5d knockout male mice. Moreover, the proband was treated with ICSI and achieved a successful pregnancy outcome for the first time. Subsequent mutation screening identified no TENT5D mutations among 47 additional patients with OAT and 50 control subjects. CONCLUSION: Mutation in TENT5D results in OAT and male infertility, and this terrible situation could be rescued by ICSI.

Biallelic mutations in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans and mice.

BACKGROUND: Asthenozoospermia is a major factor contributing to male infertility. The mitochondrial sheath (MS), an important organelle in the midpiece of spermatozoa, is crucial to sperm motility. ARMC12 is a mitochondrial peripheral membrane protein. Deletion of Armc12 impairs the arrangement of MS and causes infertility in mice. However, the role of ARMC12 in human asthenozoospermia remains unknown. OBJECTIVE: To study the genetic defects in patients with asthenozoospermia. METHODS: A total of 125 patients with asthenozoospermia and 120 men with proven fertility were recruited. Whole-exome sequencing and Sanger sequencing were performed for genetic analysis. Papanicolaou staining, HE staining, immunofluorescent staining, transmission electron microscopy and field emission scanning electron microscopy were employed to observe the morphological and structural defects of the spermatozoa and testes. Armc12-knockout mice were generated using the CRISPR-Cas9 system. Intracytoplasmic sperm injection was used to treat the patients. RESULTS: Biallelic ARMC12 mutations were identified in three patients, including homozygous mutations in two siblings from a consanguineous family and compound heterozygous mutations in one sporadic patient. ARMC12 is mainly expressed in the midpiece of elongated and late spermatids in the human testis. The patients' spermatozoa displayed multiple midpiece defects, including absent MS and central pair, scattered or forked axoneme and incomplete plasma membrane. Spermatozoa from Armc12-/- mice showed parallel defects in the midpiece. Moreover, two patients were treated with intracytoplasmic sperm injection and achieved good outcomes. CONCLUSION: Our findings prove for the first time that defects in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans.

TBC1D21 is an essential factor for sperm mitochondrial sheath assembly and male fertility‡.

During spermiogenesis, the formation of the mitochondrial sheath is critical for male fertility. The molecular processes that govern the development of the mitochondrial sheath remain unknown. Whether TBC1D21 serves as a GTPase-activating protein (GAP) for GTP hydrolysis in the testis is unclear, despite recent findings indicating that it collaborates with numerous proteins to regulate the formation of the mitochondrial sheath. To thoroughly examine the property of TBC1D21 in spermiogenesis, we applied the CRISPR/Cas9 technology to generate the Tbc1d21-/- mice, Tbc1d21D125A R128K mice with mutation in the GAP catalytic residues (IxxDxxR), and Tbc1d21-3xFlag mice. Male Tbc1d21-/- mice were infertile due to the curved spermatozoa flagella. In vitro fertilization is ineffective for Tbc1d21-/- sperm, although healthy offspring were obtained by intracytoplasmic sperm injection. Electron microscopy revealed aberrant ultrastructural changes in the mitochondrial sheath. Thirty-four Rab vectors were constructed followed by co-immunoprecipitation, which identified RAB13 as a novel TBC1D21 binding protein. Interestingly, infertility was not observed in Tbc1d21D125A R128K mice harboring the catalytic residue, suggesting that TBC1D21 is not a typical GAP for Rab-GTP hydrolysis. Moreover, TBC1D21 was expressed in the sperm mitochondrial sheath in Tbc1d21-3xFlag mice. Immunoprecipitation-mass spectrometry demonstrated the interactions of TBC1D21 with ACTB, TPM3, SPATA19, and VDAC3 to regulate the architecture of the sperm midpiece. The collective findings suggest that TBC1D21 is a scaffold protein required for the organization and stabilization of the mitochondrial sheath morphology.

Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

Deleterious variants in TAF7L cause human oligoasthenoteratozoospermia and its impairing histone to protamine exchange inducing reduced in vitro fertilization.

Introduction: Oligoasthenoteratozoospermia (OAT) is a major cause of infertility in males. Only a few pathogenic genes of OAT have been clearly identified till now. A large number of OAT-affected cases remain largely unknown. Methods: Here, Whole-exome sequencing (WES) in 725 idiopathic OAT patients was performed. Ejaculated spermatozoa by OAT patients were microinjected into mouse oocytes to estimate fertilization potential. Diff-quick staining and transmission electron microscopy were performed to evaluate sperm morphology and ultrastructure. The protein expression level and localization In vitro were detected by Western Blotting and Immunocytochemistry. Results: We identified four X-linked hemizygous deleterious variants of TAF7L-namely, c.1301_1302del;(p.V434Afs*5), c.699G>T;(p.R233S), c.508delA; (p. T170fs), c.719dupA;(p.K240fs) -in five probands. Intracytoplasmic sperm injection (ICSI) were carried out in M1, M2-1and M3 patient's wife. However only M1 patient's wife became pregnant after embryo transfer. In vitro study demonstrated significantly reduced fertilization ability in patient with TAF7L mutation. The TAF7L mutation let to abnormal sperm head and impaired histone-to protamine exchange. Variant 719dupA (p. K240fs) resulted in producing a truncated TAF7L protein and localized massively within the nucleus. In addition, TAF7L expression were not able to be detected due to variants c.1301_1302del (p. V434Afs*5) and c.508delA (p. T170fs) In vitro. Conclusion: Our findings support that TAF7L is one of pathogenic genes of OAT and deleterious mutations in TAF7L may cause impaired histone-to-protamine affected the chromatin compaction of sperm head.

Homozygous mutation in DNALI1 leads to asthenoteratozoospermia by affecting the inner dynein arms.

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of sperm flagella and defects in these proteins generally impair the axoneme structure and affect sperm flagella function. In this study, we performed whole exome sequencing for a cohort of 126 infertile patients with asthenozoospermia and identified homozygous DNALI1 mutation in one patient from a consanguineous family. This identified homozygous mutation was verified by Sanger sequencing. In silico analysis showed that this homozygous mutation is very rare, highly pathogenic, and very conserved. Sperm routine analysis confirmed that the motility of the spermatozoa from the patient significantly decreased. Further sperm morphology analysis showed that the spermatozoa from the patient exhibited multiple flagella morphological defects and a specific loss in the inner dynein arms. Fortunately, the patient was able to have his child via intracytoplasmic sperm injection treatment. Our study is the first to demonstrate that homozygous DNALI1 mutation may impair the integration of axoneme structure, affect sperm motility and cause asthenoteratozoospermia in human beings.

Bi-allelic mutations in DNAH7 cause asthenozoospermia by impairing the integrality of axoneme structure.

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of both the cilia and flagella, and defects in these proteins generally impair the axoneme structure and cause primary ciliary dyskinesia. But relatively little is known about the influence of dynein protein arm defects on sperm flagella function. Here, we recruited 85 infertile patients with idiopathic asthenozoospermia and identified bi-allelic mutations in DNAH7 (NM_018897.3) from three patients using whole-exome sequencing. These variants are rare, highly pathogenic, and very conserved. The spermatozoa from the patients with DNAH7 bi-allelic mutations showed specific losses in the inner dynein arms. The expression of DNAH7 in the spermatozoa from the DNAH7-defective patients was significantly decreased, but these patients were able to have their children via intra-cytoplasmic sperm injection treatment. Our study is the first to demonstrate that bi-allelic mutations in DNAH7 may impair the integrality of axoneme structure, affect sperm motility, and cause asthenozoospermia in humans. These findings may extend the spectrum of etiological genes and provide new clues for the diagnosis and treatment of patients with asthenozoospermia.

Pathogenic Variants in ACTRT1 Cause Acephalic Spermatozoa Syndrome.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia, but its pathogenesis is largely unknown. Here, we performed whole-exome sequencing for 34 patients with acephalic spermatozoa syndrome and identified pathogenic variants in the X-linked gene, ACTRT1, in two patients. Sanger sequencing confirmed the pathogenic variants of ACTRT1 in the patients. Both pathogenic variants of ACTRT1 were highly conserved, and in silico analysis revealed that they were deleterious and rare. Actrt1-knockout mice exhibited a similar acephalic spermatozoa phenotype. Therefore, we speculated that mutations in ACTRT1 account for acephalic spermatozoa syndrome. Moreover, the patients in this study conceived their children through artificial insemination. This study provides further insights for clinicians and researchers regarding the genetic etiology and therapeutic strategies for acephalic spermatozoa patients with pathogenic variants in ACTRT1.

Biallelic mutations in KATNAL2 cause male infertility due to oligo-astheno-teratozoospermia.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, and most of idiopathic OAT patients are thought to be caused by genetic defects. Here, we recruited 38 primary infertile patients with the OAT phenotype and 40 adult men with proven fertility for genetic analysis and identified biallelic mutations of KATNAL2 by whole-exome sequencing in two cases. F013/II:1, from a consanguineous family, carried the KATNAL2 c.328C > T:p.Arg110X homozygous mutations. The other carried c.55A > G: p.Lys19Glu and c.169C > T: p Arg57Trp biallelic mutations. None of the KATNAL2 variants were found in the 40 adult men with proven fertility. The spermatozoa from patients with KATNAL2 biallelic mutations exhibited conspicuous defects in maturation, head morphology, and the structure of mitochondrial sheaths and flagella. KATNAL2 was mainly expressed in the pericentriolar material and mitochondrial sheath of the spermatozoa from control subjects, but it was undetectable in the spermatozoa from the patients. Furthermore, Katnal2 null male mice were infertile and displayed an OAT phenotype. Our results proved that the biallelic mutations in KATNAL2 cause male infertility and OAT in humans for the first time, to our knowledge, which could enrich the genetic defect spectrum of OAT and be beneficial for its accurate genetic screening and clinical diagnosis.

[Analysis of a patient with primary ciliary dyskinesia caused by DNAH5 variants].

OBJECTIVE: To explore the genetic basis for a patient with primary ciliary dyskinesia (PCD). METHODS: High-throughput sequencing and bioinformatic analysis were carried out to identify pathogenic variant in the patient. Suspected variant was verified by Sanger sequencing among the family members, and intracytoplasmic sperm injection (ICSI) was used to achieve the pregnancy. RESULTS: The patient had obstructive azoospermia, measurement of nasal NO exhalation at 84 ppb, and typical symptoms of PCD in nasal sinuses and lungs. DNA sequencing showed that he had carried biallelic variants of the DNAH5 gene, namely c.1489C>T (p.Q497X) in exon 11 and c.6304C>T (p.R2102C) in exon 38. His wife achieved clinical pregnancy with the assistance of ICSI. CONCLUSION: Above finding has enriched the spectrum of DNAH5 gene variants, though the latters did not affect the outcome of pregnancy by ICSI.