Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility.

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.

Undiagnosed RASopathies in infertile men.

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.

Metabolic profiling identifies Qrich2 as a novel glutamine sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

In our prior investigation, we discerned loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to various morphological abnormalities in sperm flagella and male infertility. The Qrich2 knockout (KO) in mice also exhibits multiple morphological abnormalities of the flagella (MMAF) phenotype with a significantly decreased sperm motility. However, how ORICH2 regulates the formation of sperm flagella remains unclear. Abnormal glutamylation levels of tubulin cause dysplastic microtubules and flagella, eventually resulting in the decline of sperm motility and male infertility. In the current study, by further analyzing the Qrich2 KO mouse sperm, we found a reduced glutamylation level and instability of tubulin in Qrich2 KO mouse sperm flagella. In addition, we found that the amino acid metabolism was dysregulated in both testes and sperm, leading to the accumulated glutamine (Gln) and reduced glutamate (Glu) concentrations, and disorderly expressed genes responsible for Gln/Glu metabolism. Interestingly, mice fed with diets devoid of Gln/Glu phenocopied the Qrich2 KO mice. Furthermore, we identified several mitochondrial marker proteins that could not be correctly localized in sperm flagella, which might be responsible for the reduced mitochondrial function contributing to the reduced sperm motility in Qrich2 KO mice. Our study reveals a crucial role of a normal Gln/Glu metabolism in maintaining the structural stability of the microtubules in sperm flagella by regulating the glutamylation levels of the tubulin and identifies Qrich2 as a possible novel Gln sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Investigation of sperm hsa-mir-145-5p and MLH1 expressions, seminal oxidative stress and sperm DNA fragmentation in varicocele.

BACKGROUND: Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. METHODS: Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. RESULTS: Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. CONCLUSION: This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.

Cancer risk and male Infertility: Unravelling predictive biomarkers and prognostic indicators.

In recent years, there has been a global increase in cases of male infertility. There are about 30 million cases of male infertility worldwide and male reproductive health is showing rapid decline in last few decades. It is now recognized as a potential risk factor for developing certain types of cancer, particularly genitourinary malignancies like testicular and prostate cancer. Male infertility is considered a potential indicator of overall health and an early biomarker for cancer. Cases of unexplained male factor infertility have high levels of oxidative stress and oxidative DNA damage and this induces both denovo germ line mutations and epimutations due to build up of 8-hydroxy 2 deoxygunaosine abase which is highly mutagenic and also induces hypomethylation and genomic instability. Consequently, there is growing evidence to explore the various factors contributing to an increased cancer risk. Currently, the available prognostic and predictive biomarkers associated with semen characteristics and cancer risk are limited but gaining significant attention in clinical research for the diagnosis and treatment of elevated cancer risk in the individual and in offspring. The male germ cell being transcriptionally and translationally inert has a highly truncated repair mechanism and has minimal antioxidants and thus most vulnerable to oxidative injury due to environmental factors and unhealthy lifestyle and social habits. Therefore, advancing our understanding requires a thorough evaluation of the pathophysiologic mechanisms at the DNA, RNA, protein, and metabolite levels to identify key biomarkers that may underlie the pathogenesis of male infertility and associated cancer. Advanced methodologies such as genomics, epigenetics, proteomics, transcriptomics, and metabolomics stand at the forefront of cutting-edge approaches for discovering novel biomarkers, spanning from infertility to associated cancer types. Henceforth, in this review, we aim to assess the role and potential of recently identified predictive and prognostic biomarkers, offering insights into the success of assisted reproductive technologies, causes of azoospermia and idiopathic infertility, the impact of integrated holistic approach and lifestyle modifications, and the monitoring of cancer susceptibility, initiation and progression. Comprehending these biomarkers is crucial for providing comprehensive counselling to infertile men and cancer patients, along with their families.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

A loss-of-function variant in ZCWPW1 causes human male infertility with sperm head defect and high DNA fragmentation.

BACKGROUND: Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify. METHODS: An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay. RESULTS: The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband's sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband's spermatozoa have high DNA fragmentation. CONCLUSIONS: It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.

Effect of smoking on methylation and semen parameters.

One type of epigenetic modification is genomic DNA methylation, which is induced by smoking, and both are associated with male infertility. In this study, the relationship between smoking and CHD5 gene methylation and semen parameters in infertile men was determined. After the MS-PCR of blood in 224 samples, 103 infertile patients (62 smokers and 41 non-smokers) and 121 fertile men, methylation level changes between groups and the effect of methylation and smoking on infertility and semen parameters in infertile men were determined. The results showed that there is a significant difference in the methylation status (MM, MU, UU) of the CHD5 gene between the patient and the control group, and this correlation also exists for the semen parameters (p < .001). The average semen parameters in smokers decreased significantly compared to non-smokers and sperm concentration was (32.21 ± 5.27 vs. 55.27 ± 3.38), respectively. MM methylation status was higher in smokers (22.5%) compared to non-smokers (14.6%). Smoking components affect the methylation pattern of CHD5 gene, and smokers had higher methylation levels and lower semen parameters than non-smokers, which can be biomarkers for evaluating semen quality and infertility risk factors. Understanding the epigenetic effects of smoking on male infertility can be very useful for predicting negative consequences of smoking and providing therapeutic solutions.

Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification.

There is strong evidence that obesity is a risk factor for poor semen quality. However, the effects of multigenerational paternal obesity on the susceptibility to cadmium (a reproductive toxicant)-induced spermatogenesis disorders in offspring remain unknown. Here, we show that, in mice, spermatogenesis and retinoic acid levels become progressively lower as the number of generations exposed to a high-fat diet increase. Furthermore, exposing several generations of mice to a high fat diet results in a decrease in the expression of Wt1, a transcription factor upstream of the enzymes that synthesize retinoic acid. These effects can be rescued by injecting adeno-associated virus 9-Wt1 into the mouse testes of the offspring. Additionally, multigenerational paternal high-fat diet progressively increases METTL3 and Wt1 N6-methyladenosine levels in the testes of offspring mice. Mechanistically, treating the fathers with STM2457, a METTL3 inhibitor, restores obesity-reduced sperm count, and decreases Wt1 N6-methyladenosine level in the mouse testes of the offspring. A case-controlled study shows that human donors who are overweight or obese exhibit elevated N6-methyladenosine levels in sperm and decreased sperm concentration. Collectively, these results indicate that multigenerational paternal obesity enhances the susceptibility of the offspring to spermatogenesis disorders by increasing METTL3-mediated Wt1 N6-methyladenosine modification.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential.

PURPOSE: To identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates. MATERIALS AND METHODS: Sperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule. RESULTS: Overall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci. CONCLUSIONS: Sperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Advocating hormonal treatment to prevent adult in-fertility in patients diagnosed with congenital un-descended testes.

In 2007 the Nordic group came to the following unanimous conclusions: In general, hormonal treatment is not recommended, considering the poor immediate results and the possible long-term adverse effects on spermatogenesis. Thus, surgery is to be preferred. However, defective mini puberty inducing insufficient gonadotropin secretion is one of the most common causes of nonobstructive azoospermia in men suffering from congenital isolated unilateral or bilateral cryptorchidism. The extent of alteration in the unilateral undescended testis correlate with the contralateral descended testis, indicating that unilateral cryptorchidism is a bilateral disease. Idiopathic central hypogonadism explains the phenomenon of defective mini puberty in otherwise healthy cryptorchid boys. We therefore recommend hormonal treatment for cryptorchid boys with defective mini puberty. Gonadotropin releasing hormone agonist (GnRHa) treatment following surgery to correct cryptorchidism restores mini puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. Several genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed in both the brain and testis. At the molecular level, there is no convincing evidence that heat shock is responsible for the observed pathological testicular changes. Thus, impaired transformation of gonocytes is not the result of temperature stress but rather a hormonal imbalance. Cryptorchidism should therefore be considered a serious andrological problem that cannot be successfully treated by early orchidopexy alone.

A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Compound heterozygous mutations in CFTR causing congenital bilateral absence of the vas deferens in a Chinese pedigree.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disorder rarely found in Asian populations. Most males with CF are infertile because of obstructive azoospermia (OA) caused by congenital bilateral absence of the vas deferens (CBAVD). Compound heterozygous mutations of cystic fibrosis transmembrane conductance regulator (CFTR) are among the most common pathogenic factors in CBAVD. However, few genealogical analyses have been performed. METHODS: In this study, whole-exome sequencing and cosegregation analysis were performed in a Chinese pedigree involving two siblings with CBAVD. Moreover, in vitro gene expressions were used to analyze the pathogenicity of a novel CFTR mutation. RESULTS: We identified compound heterozygous mutations of CFTR comprising the known disease-causing variant c.1210-11T>G (also known as IVS9-5 T) and c.2144delA;p.q715fs in two siblings with CBAVD. To verify the effects in vitro, we transfected vectors expressing wild-type and mutated CFTR into 293T cells. The results showed that the CFTR protein containing the frameshift mutation (c.2144delA) was 60 kD smaller. With testicular sperm aspiration/intracytoplasmic sperm injection-embryo transfer (TESA/ICSI-ET), both CBAVD patients fathered healthy offspring. CONCLUSION: Our study revealed that compound heterozygous mutations of CFTR are involved in CBAVD, expanding the known CFTR gene mutation spectrum of CBAVD patients and providing more evidence that compound heterozygous mutations can cause familial CBAVD.

Advancements in the ERK1/2 Signaling Pathway Affecting Male Reproduction.

Male infertility, age-related changes, and tumors have been increasingly studied in the field of male reproductive health due to the emergence of environmental stressors, declining fertility rates, and aging populations. Numerous studies have demonstrated that the ERK1/2 signaling pathway plays a significant role in male reproduction. The ERK1/2 pathway is associated with several signaling pathways and has a complex interplay that influences the spermatogenic microenvironment, sperm viability, gonadal axis regulation, as well as resistance to testicular aging and tumors. Moreover, the ERK1/2 pathway directly or indirectly regulates testicular somatic cells, which are crucial for maintaining spermatogenesis and microenvironment regulation. Given the critical role of the ERK1/2 signaling pathway in male reproductive health, comprehensive exploration of its multifaceted effects on male reproduction and underlying mechanisms is necessary. This study aims to provide a solid foundation for in-depth research in the field of male reproduction and further enhance the reproductive health of males.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.