Novel MEIOB pathogenic variants including a homozygous non-canonical splicing variant, cause meiotic arrest and human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is the most severe form of human male infertility, and the genetic causes of NOA with meiotic arrest remain largely unclear. In this study, we identified novel compound heterozygous MEIOB variants (c.814C > T: p.R272X and c.976G > A: p.A326T) and a previously undescribed homozygous non-canonical splicing variant of MEIOB (c.528 + 3A > C) in two NOA-affected individuals from two irrelevant Chinese families. MEIOB missense variant (p.A326T) significantly reduced protein abundance and nonsense variant (p.R272X) produced a truncated protein. Both of two variants impaired the MEIOB-SPATA22 interaction. The MEIOB non-canonical splicing variant resulted in whole Exon 6 skipping by minigene assay, which was predicted to produce a frameshift truncated protein (p.S111Rfs*32). Histological and immunostaining analysis indicated that both patients exhibited a similar phenotype as we previously reported in Meiob mutant mice, that is, absence of spermatids in seminiferous tubules and meiotic arrest. Our study identified three novel pathogenic variants of MEIOB in NOA patients, extending the mutation spectrum of the MEIOB and highlighting the contribution of meiotic recombination related genes in human fertility.

Performance evaluation of differential splicing analysis methods and splicing analytics platform construction.

A proportion of previously defined benign variants or variants of uncertain significance in humans, which are challenging to identify, may induce an abnormal splicing process. An increasing number of methods have been developed to predict splicing variants, but their performance has not been completely evaluated using independent benchmarks. Here, we manually sourced ∼50 000 positive/negative splicing variants from > 8000 studies and selected the independent splicing variants to evaluate the performance of prediction methods. These methods showed different performances in recognizing splicing variants in donor and acceptor regions, reminiscent of different weight coefficient applications to predict novel splicing variants. Of these methods, 66.67% exhibited higher specificities than sensitivities, suggesting that more moderate cut-off values are necessary to distinguish splicing variants. Moreover, the high correlation and consistent prediction ratio validated the feasibility of integration of the splicing prediction method in identifying splicing variants. We developed a splicing analytics platform called SPCards, which curates splicing variants from publications and predicts splicing scores of variants in genomes. SPCards also offers variant-level and gene-level annotation information, including allele frequency, non-synonymous prediction and comprehensive functional information. SPCards is suitable for high-throughput genetic identification of splicing variants, particularly those located in non-canonical splicing regions.

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.

ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

STUDY QUESTION: Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? SUMMARY ANSWER: A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. WHAT IS KNOWN ALREADY: ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. LIMITATIONS, REASONS FOR CAUTION: The absence of another independent pedigree to support our argument is a limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS: The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Pulmonary hypertension- a novel phenotypic hypothesis of Kabuki syndrome: a case report and literature review.

BACKGROUND: Pediatric pulmonary hypertension (PH) is a serious and rare disease that is often derived from genetic mutations. Kabuki syndrome (KS) is a chromosomal abnormality disease that has its origin in the mutation of lysine methyltransferase 2D(KMT2D). Recent evidence has shown that KMT2D mutations are associated with pediatric pulmonary disorders. However, the relationship between the clinical courses of PH and the KMT2D mutation is reported in extremely few cases. Therefore, in this paper, a case was presented and previous literature was reviewed for better understanding of the correlation between pediatric PH and KMT2D mutations. CASE PRESENTATION: A 3-year-old girl was transferred to our center for severe cough, shortness of breath, fatigue and fever. Physical examination revealed facial deformities and growth retardation. Echocardiography showed a small atrial septal defect (ASD), and right heart catheterization indicated a significant increase in pulmonary vascular pressure and resistance. The genetic test suggested that she had a KMT2D gene mutation. The patient was finally diagnosed with KS. She was given targeted drugs to reduce pulmonary vascular pressure, but the effect was unsatisfactory. CONCLUSIONS: KS can be complicated with multiple organ malformations and dysfunction. With the progress of next generation sequencing, an increasing number of new phenotypes related to KMT2D mutations have been reported. A bold hypothesis is proposed in this article, that is, PH may be a new phenotype associated with KMT2D mutations. It is suggested that KS and PH should be differentiated from each other to avoid delayed diagnosis and treatment in clinical practice. There is no specific drug for KS treatment. The prognosis of children with inherited PH is usually poor, and lung transplantation may increase their survival rates.

Novel mutations in DNAH17 cause sperm flagellum defects and their influence on ICSI outcome.

PURPOSE: To identify new mutations in DNAH17 that cause male infertility and analyze intracytoplasmic sperm injection (ICSI) outcomes in patients with DNAH17 mutations. METHODS: A total of five cases of new DNAH17 mutations exhibiting the multiple morphological abnormalities of the sperm flagella (MMAF) phenotype were identified through semen analysis and genetic testing. They were recruited at our reproductive medicine center from September 2018 to July 2022. Information on DNAH17 genetic mutations and ICSI outcomes was systematically explored following a literature review. RESULTS: Three novel compound mutations in DNAH17 were identified in patients with male infertility caused by MMAF. This study and previous publications included 21 patients with DNAH17 mutations. DNAH17 has been associated with asthenozoospermia and male infertility, but different types of DNAH17 variants appear to be involved in different sperm phenotypes. In 11 couples of infertile patients with DNAH17 mutations, there were 17 ICSI cycles and 13 embryo transplantation cycles. Only three men with DNAH17 variants ultimately achieved clinical pregnancy with their partners through ICSI combined with assisted oocyte activation (AOA). CONCLUSIONS: Loss-of-function mutations in DNAH17 can lead to severe sperm flagellum defects and male infertility. Patients with MMAF-harboring DNAH17 mutations generally have worse pregnancy outcomes following ICSI. ICSI combined with AOA may improve the outcome of assisted reproductive techniques (ARTs) for men with DNAH17 variants.

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.

Biallelic loss-of-function mutations in SEPTIN4 (C17ORF47), encoding a conserved annulus protein, cause thin midpiece spermatozoa and male infertility in humans.

Asthenoteratozoospermia is the primary cause of infertility in humans. However, the genetic etiology remains largely unknown for those suffering from severe asthenoteratozoospermia caused by thin midpiece defects. In this study, we identified two biallelic loss-of-function variants of SEPTIN4 (previously SEPT4) (Patient 1: c.A721T, p.R241* and Patient 2: c.C205T, p.R69*) in two unrelated individuals from two consanguineous Chinese families. SEPT4 is a conserved annulus protein that is critical for male fertility and the structural integrity of the sperm midpiece in mice. SEPT4 mutations disrupted the formation of SEPT-based annulus and localization of SEPTIN subunits in sperms from patients. The ultrastructural analysis demonstrated striking thin midpiece spermatozoa defects owing to annulus loss and disorganized mitochondrial sheath. Immunofluorescence and immunoblotting analyses of the mitochondrial sheath proteins TOMM20 and HSP60 further indicated that the distribution and abundance of mitochondria were impaired in men harboring biallelic SEPT4 variants. Additionally, we found that the precise localization of SLC26A8, a testis-specific anion transporter that colocalizes with SEPT4 at the sperm annulus, was missing without SEPT4. Moreover, the patient achieved a good pregnancy outcome following intracytoplasmic sperm injection. Overall, our study demonstrated for the first time that SEPT4 variants that induced thin midpiece spermatozoa defects were directly associated with human asthenoteratozoospermia.

Homozygous missense mutation in CCDC155 disrupts the transmembrane distribution of CCDC155 and SUN1, resulting in non-obstructive azoospermia and premature ovarian insufficiency in humans.

Non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) represent the most serious forms of human infertility caused by gametogenic failure. Although whole-exome sequencing (WES) has uncovered multiple monogenic causes of human infertility, our knowledge of the genetic basis of human gametogenesis defects remains at a rudimentary stage. Coiled-coil-domain-containing protein 155 (CCDC155) encodes a core component of the linker of the nucleoskeleton and cytoskeleton complex that is essential for modulating telomere-led chromosome movements during the meiotic prophase of mice. Additionally, Ccdc155 deficiency in mice causes infertility in both sexes with meiotic arrest. In this study, we applied WES to identify the pathogenic genes for 15 NOA and POI patients whose parents were consanguineous and identified a novel homozygous missense mutation in CCDC155 [c.590T>C (p.Leu197Pro)] in a pair of familial NOA and POI patients whose parents were first cousins. The affected spermatocytes were unable to complete meiotic division coupled with unresolved repair of the DNA double-strand break. This rare missense mutation with lesions in the conserved CC domain of CCDC155 blocked nuclear envelope (NE) distribution and subsequently prevented NE-specific enrichment of Sad1- and UNC84-domain-containing 1 either ex vivo or in vitro, eventually leading to disruptive NE anchoring of chromosome-induced meiotic arrest in both sexes. This study presents the first evidence of the necessity of the SUN1-CCDC155 complex during human meiosis and provides insight into the CCDC155 CC domain, thereby expanding the genetic spectrum of human NOA and POI and promoting adequate genetic counselling and appropriate fertility guidance for these patients.

A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

Loss of function mutation in DNAH7 induces male infertility associated with abnormalities of the sperm flagella and mitochondria in human.

Male infertility is an increasingly serious health problem affecting couples of reproductive age. Mutations in axoneme-associated genes cause male infertility. Dynein arm proteins are essential in sustaining normal axonemes and promote flagellar motility. However, the function of DNAH7 in male fertility in vivo remains unclear. Herein, we showed that DNAH7 disruption in humans results in male infertility, which was characterised by multiple morphological abnormalities of sperm flagella. The axoneme structure of the sperm from a DNAH7-deficient patient revealed the loss of inner dynein arms. Moreover, the mitochondria of the sperm flagella detached and dispersed outside the axoneme, leading to abnormalities in the mitochondrial sheath in the mid-piece region. Live birth was achieved via intracytoplasmic sperm injection. Thus, DNAH7 is critical for axoneme and mitochondrial development in human sperm. These findings further clarify the spectrum of DNAH7 biology and provide new insights for diagnosing infertility and treating patients harbouring DNAH7 mutations.

Identification of deleterious variants in patients with male infertility due to idiopathic non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe type of male infertility, affecting 1% of men worldwide. Most of its etiologies remain idiopathic. Although genetic studies have identified dozens of NOA genes, monogenic mutations can also account for a small proportion of idiopathic NOA cases. Hence, this genetic study was conducted to explore the causes of monogenic variants of NOA in a cohort of Chinese patients. METHODS: Following the screening using chromosomal karyotyping, Y chromosome microdeletion analyses, and sex hormone assessments, subsequent whole-exome sequencing analysis was performed in 55 unrelated idiopathic NOA patients with male infertility to explore potential deleterious variants associated with spermatogenesis. We also performed Sanger sequencing to demonstrate the variants. Testicular biopsy or microsurgical testicular sperm extraction was also performed to confirm the diagnosis of NOA and identify spermatozoa. Hematoxylin and eosin staining was performed to assess the histopathology of spermatogenesis. RESULTS: Abnormal testicular pathological phenotypes included Sertoli cell-only syndrome, maturation arrest, and hypospermatogenesis. Using bioinformatics analysis, we detected novel variants in two recessive genes, FANCA (NM_000135, c.3263C > T, c.1729C > G) and SYCE1 (NM_001143763, c.689_690del); one X-linked gene, TEX11 (NM_031276, c.466A > G, c.559_560del); and two dominant genes, DMRT1 (NM_021951, c.425C > T, c.340G > A) and PLK4 (NM_001190799, c.2785A > G), in eight patients, which corresponded to 14.55% (8/55) of the patients. CONCLUSION: This study presented some novel variants of known pathogenic genes for NOA. Further, it expanded the variant spectrum of NOA patients, which might advance clinical genetic counseling in the future.

Machine learning in a real-world PFO study: analysis of data from multi-centers in China.

PURPOSE: The association of patent foreman ovale (PFO) and cryptogenic stroke has been studied for years. Although device closure overall decreases the risk for recurrent stroke, treatment effects varied across different studies. In this study, we aimed to detect sub-clusters in post-closure PFO patients and identify potential predictors for adverse outcomes. METHODS: We analyzed patients with embolic stroke of undetermined sources and PFO from 7 centers in China. Machine learning and Cox regression analysis were used. RESULTS: Using unsupervised hierarchical clustering on principal components, two main clusters were identified and a total of 196 patients were included. The average age was 42.7 (12.37) years and 64.80% (127/196) were female. During a median follow-up of 739 days, 12 (6.9%) adverse events happened, including 6 (3.45%) recurrent stroke, 5 (2.87%) transient ischemic attack (TIA) and one death (0.6%). Compared to cluster 1 (n = 77, 39.20%), patients in cluster 2 (n = 119, 60.71%) were more likely to be male, had higher systolic and diastolic blood pressure, higher body mass index, lower high-density lipoprotein cholesterol and increased proportion of presence of atrial septal aneurysm. Using random forest survival (RFS) analysis, eight top ranking features were selected and used for prediction model construction. As a result, the RFS model outperformed the traditional Cox regression model (C-index: 0.87 vs. 0.54). CONCLUSIONS: There were 2 main clusters in post-closure PFO patients. Traditional cardiovascular profiles remain top ranking predictors for future recurrence of stroke or TIA. However, whether maximizing the management of these factors would provide extra benefits warrants further investigations.

Bi-allelic variants in DNAH10 cause asthenoteratozoospermia and male infertility.

PURPOSE: Multiple morphological abnormalities in the sperm flagella (MMAF) comprise a severe phenotype of asthenoteratozoospermia with reduced or absent spermatozoa motility. Whereas dozens of candidate pathogenic genes for MMAF have been identified, the genetic cause in a large proportion of patients is unknown. We attempted to identify novel genetic explanations for MMAF. METHODS: We performed whole-exome sequencing of patients with MMAF to identify pathogenic variants. The phenotypes of spermatozoa in patients carrying DNAH10 variants were investigated using haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy. The expression and location of DNAH10 and other spermatozoa structure-related proteins were analyzed using immunofluorescence assays. RESULTS: We found one homozygous frameshift DNAH10 variant (NM_207437: c.2514delG:p.L839*) and one compound heterozygous DNAH10 variant (NM_207437: c.10820 T > C:p.M3607T; c.12692C > T:p.T4231I) in two patients with MMAF. These variants were absent or rare in the general population. Haematoxylin and eosin staining and scanning electron microscopy revealed the significant disruption of sperm flagella in the patients. In addition, ultrastructural analysis by transmission electron microscopy showed significant inner dynein arm (IDA) deficiency in sperm flagella. Using immunofluorescence assays, we found a significant reduction in IDA-related proteins including DNAH10 and DNAH1. CONCLUSIONS: We identified putative novel pathogenic variants in DNAH10 for MMAF, which might advance the genetic diagnosis and clinical genetic counselling for male infertility.

Bi-allelic truncating variants in CFAP206 cause male infertility in human and mouse.

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.

Bi-allelic mutations in MCIDAS and CCNO cause human infertility associated with abnormal gamete transport.

Reduced generation of multiple motile cilia (RGMC) and the consequent primary ciliary dyskinesia (PCD) cause infertility due to a substantial reduction in the number of multiciliated cells (MCCs) in the efferent ducts (EDs)/oviducts. MCIDAS acts upstream of CCNO to regulate the biogenesis of basal bodies (BBs); therefore, both genes play a vital role in the multiciliogenesis of the reproductive tract epithelium. In this study, whole-exome sequencing was performed to identify the causative genes in 10 unrelated infertile patients with PCD: seven males and three females. Notably, homozygous frameshift mutations in MCIDAS (c.186dupT, p.Pro63Serfs*22) and CCNO (c.262_263insGGCCC, p.Gln88Argfs*8) were identified in one male and one female participant from two unrelated consanguineous families. Haematoxylin-eosin staining/scanning electron microscopy revealed abnormal MCCs in the mutated EDs/oviducts. Furthermore, transmission electron microscopy revealed significantly reduced BBs. Immunofluorescence staining showed the absence of MCIDAS and CCNO signals in the affected tissues and confirmed that MCIDAS acts upstream of CCNO in the context of multiciliogenesis in the reproductive tract epithelium. In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was successful, with a positive pregnancy outcome in both MCIDAS- and CCNO-mutated patients. Our results support the use of IVF/ICSI interventions to treat infertility due to RGMC in couples.

Novel variants in helicase for meiosis 1 lead to male infertility due to non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility; more than half of the NOA patients are idiopathic. Although many NOA risk genes have been detected, the genetic factors for NOA in majority of the patients are unknown. In addition, it is difficult to retrieve sperm from these patients despite using the microsurgical testicular sperm extraction (microTESE) method. Therefore, we conducted this genetic study to identify the potential genetic factors responsible for NOA and investigate the sperm retrieval rate of microTESE for genetically deficient NOA patients. METHODS: Semen analyses, sex hormone testing, and testicular biopsy were performed to categorize the patients with NOA. The chromosome karyotypes and Y chromosome microdeletion analyses were used to exclude general genetic factors. Whole exome sequencing and Sanger sequencing were performed to identify potential genetic variants in 51 patients with NOA. Hematoxylin and eosin staining (H&E) and anti-phosphorylated H2AX were used to assess the histopathology of spermatogenesis. Quantitative real time-polymerase chain reaction, western blotting, and immunofluorescence were performed to verify the effects of gene variation on expression. RESULTS: We performed whole exome sequencing in 51 NOA patients and identified homozygous helicase for meiosis 1(HFM1) variants (NM_001017975: c.3490C > T: p.Q1164X; c.3470G > A: p.C1157Y) in two patients (3.9%, 2/51). Histopathology of the testis showed that spermatogenesis was completely blocked at metaphase in these two patients carrying the HFM1 homozygous variants. In comparison with unaffected controls, we found a significant reduction in the levels of HFM1 mRNA and protein expression in the testicular tissues from these two patients. The patients were also subjected to microTESE treatment, but the sperms could not be retrieved. CONCLUSIONS: This study identified novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, and microTESE did not aid in retrieving sperms from these patients.

Differences in the Gut Microbiome of Women With and Without Hypoactive Sexual Desire Disorder: Case Control Study.

BACKGROUND: The gut microbiome is receiving considerable attention as a potentially modifiable risk factor and therapeutic target for numerous mental and neurological diseases. OBJECTIVE: This study aimed to explore and assess the difference in the composition of gut microbes and fecal metabolites between women with hypoactive sexual desire disorder (HSDD) and healthy controls. METHODS: We employed an online recruitment method to enroll "hard-to-reach" HSDD populations. After a stringent diagnostic and exclusion process based on DSM-IV criteria, fecal samples collected from 24 women with HSDD and 22 age-matched, healthy controls underwent microbiome analysis using 16S ribosomal RNA gene sequencing and metabolome analysis using untargeted liquid chromatography-mass spectrometry. RESULTS: We found a decreased abundance of Ruminococcaceae and increased abundance of Bifidobacterium and Lactobacillus among women with HSDD. Fecal samples from women with HSDD showed significantly altered metabolic signatures compared with healthy controls. The abundance of Bifidobacterium, Lactobacillus, and several fecal metabolites correlated negatively with the sexual desire score, while the number of Ruminococcaceae correlated positively with the sexual desire score in all subjects. CONCLUSIONS: Our analysis of fecal samples from women with HSDD and healthy controls identified significantly different gut microbes and metabolic signatures. These preliminary findings could be useful for developing strategies to adjust the level of human sexual desire by modifying gut microbiota. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1800020321; http://www.chictr.org.cn/showproj.aspx?proj=34267.

A novel hemizygous loss-of-function mutation in ADGRG2 causes male infertility with congenital bilateral absence of the vas deferens.

PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.