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Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1 -/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility., (© 2024. The Author(s).)

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure., (© 2024 Houston et al.)

Currently, most men with infertility cannot be given an aetiology, which reflects a lack of knowledge around gamete production and how it is affected by genetics and the environment. A failure to recognize the burden of male infertility and its potential as a biomarker for systemic illness exists. The absence of such knowledge results in patients generally being treated as a uniform group, for whom the strategy is to bypass the causality using medically assisted reproduction (MAR) techniques. In doing so, opportunities to prevent co-morbidity are missed and the burden of MAR is shifted to the woman. To advance understanding of men's reproductive health, longitudinal and multi-national centres for data and sample collection are essential. Such programmes must enable an integrated view of the consequences of genetics, epigenetics and environmental factors on fertility and offspring health. Definition and possible amelioration of the consequences of MAR for conceived children are needed. Inherent in this statement is the necessity to promote fertility restoration and/or use the least invasive MAR strategy available. To achieve this aim, protocols must be rigorously tested and the move towards personalized medicine encouraged. Equally, education of the public, governments and clinicians on the frequency and consequences of infertility is needed. Health options, including male contraceptives, must be expanded, and the opportunities encompassed in such investment understood. The pressing questions related to male reproductive health, spanning the spectrum of andrology are identified in the Expert Recommendation., (© 2023. Springer Nature Limited.)

Katanins, a class of microtubule-severing enzymes, are potent M-phase regulators in oocytes and somatic cells. How the complex and evolutionarily crucial, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit KATNA1 and its close paralogue KATNAL1 together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagellum development, manchette regulation and sperm-epithelial disengagement. Finally, using proteomic approaches, we define the KATNA1, KATNAL1 and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal that the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for 'customised cutting' via neofunctionalisation and protective buffering via gene redundancy., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

A common herbicide, atrazine, is associated with poor health. Atrazine acts as an endocrine disruptor at supra-environmental levels. Little research, however, has been conducted regarding chronic exposure to environmental atrazine concentrations across generations. This study utilized comprehensive endpoint measures to investigate the effects of chronic exposure to a conservative atrazine concentration (0.02 ng/mL), measured in Australian waterways, on male mice fertility across two generations. Mice were exposed through the maternal line, from the pre-conception period and through the F1 and F2 generations until three or six months of age. Atrazine did not impact sperm function, testicular morphology nor germ cell parameters but did alter the expression of steroidogenic genes in the F1, down-regulating the expression of Cyp17a1 (Cytochrome P450 family 17, subfamily A member 1; p = 0.0008) and Ddx4 (DEAD-box helicase 4; p = 0.007), and up-regulating the expression of Star (Steroidogenic acute regulatory protein; p = 0.017). In the F2, atrazine induced up-regulation in the expression of Star ( p = 0.016). The current study demonstrates that chronic exposure to an environmentally relevant atrazine concentration perturbs testicular steroid-associated gene expression that varies across generations. Future studies through the paternal and combined parental lineages should be undertaken to further elucidate the multigenerational effects of atrazine on male fertility.

Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Thousands of genes are expressed during spermatogenesis and male infertility has a strong genetic component. Within this study, we focus on the role of Zfr2 in male fertility, a gene previously implicated in human male fertility. To date, very little is known about the role of ZFR2 in either humans or mice. To this end, the requirement for ZFR2 in male fertility was assessed using a knockout mouse model., Results: Zfr2 was found to be expressed in the testes of both humans and mice. Deletion of Zfr2 was achieved via removal of exon 2 using CRISPR-Cas9 methods. The absence of Zfr2 did not result in a reduction in any fertility parameters assessed. Knockout males were capable of fostering litter sizes equal to wild type males, and there were no effects of Zfr2 knockout on sperm number or motility. We note Zfr2 knockout females were also fertile., Conclusions: The absence of Zfr2 alone is not sufficient to cause a reduction in male fertility in mice., (Copyright © 2022. Published by Elsevier Inc.)

Competing Interests: None

Testicular-derived inhibin B (α/β B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (β A/B/β A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (β B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10 -5 ) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10 -4 ) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility., (© 2022. The Author(s).)

Background: Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syndrome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60-70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant increase in the number of male infertility NGS studies. These have revealed a considerable number of novel gene-disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype-phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a systematic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs., Objective and Rationale: In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systematically evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes., Search Methods: We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to 'male infertility' in combination with the word 'genetics' in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experimental quality, patient phenotype assessment and functional evidence based on gene expression, mutant in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are available at imigc.org., Outcomes: The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes., Wider Implications: Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability 'human male infertility genes' is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic testing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guidelines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our findings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expanding the patient groups for which genetic testing is recommended., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Study Question: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders?, Summary Answer: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 10 out of 21 patients (48%) and variants in novel candidate genes in seven additional patients (33%)., What Is Known Already: Severe sperm motility disorders are a form of male infertility characterised by immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum that do not affect viability. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 22 genes., Study Design, Size, Duration: We performed exome sequencing in 21 patients with severe sperm motility disorders from two different clinics., Participants/materials, Setting, Method: Two groups of infertile men, one from Argentina (n = 9) and one from Australia (n = 12), with clinically defined severe sperm motility disorders (motility <5%) and normal morphology values of 0-4%, were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on light and transmission electron microscopy. Sperm ultrastructural information was not available for the Australian cohort. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted., Main Results and Role of Chance: In 10 of 21 patients (48%), we identified pathogenic variants in known sperm assembly genes: CFAP43 (3 patients); CFAP44 (2 patients), CFAP58 (1 patient), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (55% and 42%, respectively). Furthermore, we identified patients with variants in the novel human candidate sperm motility genes: DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient., Large Scale Data: N/A., Limitations, Reasons for Caution: In this study, we described patients with either a homozygous or two heterozygous candidate pathogenic variants in genes linked to sperm motility disorders. Due to unavailability of parental DNA, we have not assessed the frequency of de novo or maternally inherited dominant variants and could not determine the parental origin of the mutations to establish in all cases that the mutations are present on both alleles., Wider Implications of the Findings: Our results confirm the likely causal role of variants in six known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility disorders (10/21 diagnosed; 48%). Furthermore, our analysis revealed six novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes., Study Funding/competing Interest(s): This project was supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., J.A.V. and R.I.M.L., The Netherlands Organisation for Scientific Research (918-15-667) to J.A.V., the Royal Society and Wolfson Foundation (WM160091) to J.A.V., as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. and Grants from the National Research Council of Argentina (PIP 0900 and 4584) and ANPCyT (PICT 9591) to H.E.C. and a UUKi Rutherford Fund Fellowship awarded to B.J.H., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

Background: Male infertility is a prevalent clinical presentation for which there is likely a strong genetic component due to the thousands of genes required for spermatogenesis. Within this study we investigated the role of the gene Scrn1 in male fertility. Scrn1 is preferentially expressed in XY gonads during the period of sex determination and in adult Sertoli cells based on single cell RNA sequencing. We investigated the expression of Scrn1 in juvenile and adult tissues and generated a knockout mouse model to test its role in male fertility., Results: Scrn1 was expressed at all ages examined in the post-natal testis; however, its expression peaked at postnatal days 7-14 and SCRN1 protein was clearly localized to Sertoli cells. Scrn1 deletion was achieved via removal of exon 3, and its loss had no effect on male fertility or sex determination. Knockout mice were capable of siring litters of equal size to wild type counterparts and generated equal numbers of sperm with comparable motility and morphology characteristics., Conclusions: Scrn1 was found to be dispensable for male fertility, but this study identifies SCRN1 as a novel marker of the Sertoli cell cytoplasm., (© 2021 American Association of Anatomists.)

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Male infertility is a heterogeneous condition of largely unknown etiology that affects at least 7% of men worldwide. Classical genetic approaches and emerging next-generation sequencing studies support genetic variants as a frequent cause of male infertility. Meanwhile, the barriers to transmission of this disease mean that most individual genetic cases will be rare, but because of the large percentage of the genome required for spermatogenesis, the number of distinct causal mutations is potentially large. Identifying bona fide causes of male infertility thus requires advanced filtering techniques to select for high-probability candidates, including the ability to test causality in animal models. The mouse remains the gold standard for defining the genotype-phenotype connection in male fertility. Here, we present a best practice guide consisting of (a) major points to consider when interpreting next-generation sequencing data performed on infertile men, and, (b) a systematic strategy to categorize infertility types and how they relate to human male infertility. Phenotyping infertility in mice can involve investigating the function of multiple cell types across the testis and epididymis, as well as sperm function. These findings will feed into the diagnosis and treatment of male infertility as well as male health broadly.

Globozoospermia is a rare form of male infertility where men produce round-headed sperm that are incapable of fertilizing an oocyte naturally. In a previous study where we undertook a whole exome screen to define novel genetic causes of globozoospermia, we identified homozygous mutations in the gene PDCD2L Two brothers carried a p.(Leu225Val) variant predicted to introduce a novel splice donor site, thus presenting PDCD2L as a potential regulator of male fertility. In this study, we generated a Pdcd2l knockout mouse to test its role in male fertility. Contrary to the phenotype predicted from its testis-enriched expression pattern, Pdcd2l null mice died during embryogenesis. Specifically, we identified that Pdcd2l is essential for post-implantation embryonic development. Pdcd2l -/- embryos were resorbed at embryonic days 12.5-17.5 and no knockout pups were born, while adult heterozygous Pdcd2l males had comparable fertility to wildtype males. To specifically investigate the role of PDCD2L in germ cells, we employed Drosophila melanogaster as a model system. Consistent with the mouse data, global knockdown of trus , the fly ortholog of PDCD2L , resulted in lethality in flies at the third instar larval stage. However, germ cell-specific knockdown with two germ cell drivers did not affect male fertility. Collectively, these data suggest that PDCD2L is not essential for male fertility. By contrast, our results demonstrate an evolutionarily conserved role of PDCD2L in development., (Copyright © 2020 Houston et al.)

Study Question: Can exome sequencing identify new genetic causes of globozoospermia?, Summary Answer: Exome sequencing in 15 cases of unexplained globozoospermia revealed deleterious mutations in seven new genes, of which two have been validated as causing globozoospermia when knocked out in mouse models., What Is Known Already: Globozoospermia is a rare form of male infertility characterised by round-headed sperm and malformation of the acrosome. Although pathogenic variants in DPY19L2 and SPATA16 are known causes of globozoospermia and explain up to 70% of all cases, genetic causality remains unexplained in the remaining patients., Study Design, Size, Duration: After pre-screening 16 men for mutations in known globozoospermia genes DPY19L2 and SPATA16, exome sequencing was performed in 15 males with globozoospermia or acrosomal hypoplasia of unknown aetiology., Participants/materials, Setting, Method: Targeted next-generation sequencing and Sanger sequencing was performed for all 16 patients to screen for single-nucleotide variants and copy number variations in DPY19L2 and SPATA16. After exclusion of one patient with DPY19L2 mutations, we performed exome sequencing for the 15 remaining subjects. We prioritised recessive and X-linked protein-altering variants with an allele frequency of <0.5% in the population database GnomAD in genes with an enhanced expression in the testis. All identified candidate variants were confirmed in patients and, where possible, in family members using Sanger sequencing. Ultrastructural examination of semen from one of the patients allowed for a precise phenotypic characterisation of abnormal spermatozoa., Main Results and Role of Chance: After prioritisation and validation, we identified possibly causative variants in eight of 15 patients investigated by exome sequencing. The analysis revealed homozygous nonsense mutations in ZPBP and CCDC62 in two unrelated patients, as well as rare missense mutations in C2CD6 (also known as ALS2CR11), CCIN, C7orf61 and DHNA17 and a frameshift mutation in GGN in six other patients. All variants identified through exome sequencing, except for the variants in DNAH17, were located in a region of homozygosity. Familial segregation of the nonsense variant in ZPBP revealed two fertile brothers and the patient's mother to be heterozygous carriers. Paternal DNA was unavailable. Immunohistochemistry confirmed that ZPBP localises to the acrosome in human spermatozoa. Ultrastructural analysis of spermatozoa in the patient with the C7orf61 mutation revealed a mixture of round heads with no acrosomes (globozoospermia) and ovoid or irregular heads with small acrosomes frequently detached from the sperm head (acrosomal hypoplasia)., Limitations, Reasons for Caution: Stringent filtering criteria were used in the exome data analysis which could result in possible pathogenic variants remaining undetected. Additionally, functional follow-up is needed for several candidate genes to confirm the impact of these mutations on normal spermatogenesis., Wider Implications of the Findings: Our study revealed an important role for mutations in ZPBP and CCDC62 in human globozoospermia as well as five new candidate genes. These findings provide a more comprehensive understanding of the genetics of male infertility and bring us closer to a complete molecular diagnosis for globozoospermia patients which would help to predict the success of reproductive treatments., Study Funding/competing Interest(s): This study was funded by The Netherlands Organisation for Scientific Research (918-15-667); National Health and Medical Research Council of Australia (APP1120356) and the National Council for Scientific Research (CONICET), Argentina, PIP grant 11220120100279CO. The authors have nothing to disclose., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Artificially generated radiofrequency-electromagnetic energy (RF-EME) is now ubiquitous in our environment owing to the utilization of mobile phone and Wi-Fi based communication devices. While several studies have revealed that RF-EME is capable of eliciting biological stress, particularly in the context of the male reproductive system, the mechanistic basis of this biophysical interaction remains largely unresolved. To extend these studies, here we exposed unrestrained male mice to RF-EME generated via a dedicated waveguide (905 MHz, 2.2 W/kg) for 12 h per day for a period of 1, 3 or 5 weeks. The testes of exposed mice exhibited no evidence of gross histological change or elevated stress, irrespective of the RF-EME exposure regimen. By contrast, 5 weeks of RF-EME exposure adversely impacted the vitality and motility profiles of mature epididymal spermatozoa. These spermatozoa also experienced increased mitochondrial generation of reactive oxygen species after 1 week of exposure, with elevated DNA oxidation and fragmentation across all exposure periods. Notwithstanding these lesions, RF-EME exposure did not impair the fertilization competence of spermatozoa nor their ability to support early embryonic development. This study supports the utility of male germ cells as sensitive tools with which to assess the biological impacts of whole-body RF-EME exposure.

Background: The sperm protein IZUMO1 (Izumo sperm-egg fusion 1) and its recently identified binding partner on the oolemma, IZUMO1R, are among the first ligand-receptor pairs shown to be essential for gamete recognition and adhesion. However, the IZUMO1-IZUMO1R interaction does not appear to be directly responsible for promoting the fusion of the gamete membranes, suggesting that this critical phase of the fertilization cascade requires the concerted action of alternative fusogenic machinery. It has therefore been proposed that IZUMO1 may play a secondary role in the organization and/or stabilization of higher-order heteromeric complexes in spermatozoa that are required for membrane fusion., Results: Here, we show that fertilization-competent (acrosome reacted) mouse spermatozoa harbor several high molecular weight protein complexes, a subset of which are readily able to adhere to solubilized oolemmal proteins. At least two of these complexes contain IZUMO1 in partnership with GLI pathogenesis-related 1 like 1 (GLIPR1L1). This interaction is associated with lipid rafts and is dynamically remodeled upon the induction of acrosomal exocytosis in preparation for sperm adhesion to the oolemma. Accordingly, the selective ablation of GLIPR1L1 leads to compromised sperm function characterized by a reduced ability to undergo the acrosome reaction and a failure of IZUMO1 redistribution., Conclusions: Collectively, this study characterizes multimeric protein complexes on the sperm surface and identifies GLIPRL1L1 as a physiologically relevant regulator of IZUMO1 function and the fertilization process.

The 2018 edition of the Society for Reproductive Biology's (SRB) Annual Meeting was a celebration of 50 years of Australian research into reproductive biology. The past 50 years has seen many important contributions to this field, and these advances have led to changes in practice and policy, improvements in the efficiency of animal reproduction and improved health outcomes. This conference review delivers a dedicated summary of the symposia, discussing emerging concepts, raising new questions and proposing directions forward. Notably, the symposia discussed in this review emphasised the impact that reproductive research can have on quality of life and the health trajectories of individuals. The breadth of the research discussed encompasses the central regulation of fertility and cyclicity, life course health and how the environment of gametes and embryos can affect subsequent generations, significant advances in our understanding of placental biology and pregnancy disorders and the implications of assisted reproductive technologies on population health. The importance of a reliable food supply and protection of endangered species is also discussed. The research covered at SRB's 2018 meeting not only recognised the important contributions of its members over the past 50 years, but also highlighted key findings and avenues for innovation moving forward that will enable the SRB to continue making significant contributions for the next 50 years.

As the use of mobile phone devices is now highly prevalent, many studies have sought to evaluate the effects of the radiofrequency-electromagnetic radiation (RF-EMR) on both human health and biology. While several such studies have shown RF-EMR is capable of inducing cellular stress, the physicobiological origin of this stress remains largely unresolved. To explore the effect of RF-EMR on the male reproductive system, we exposed cultured mouse spermatogonial GC1 and spermatocyte GC2 cell lines, as well as cauda epididymal spermatozoa to a waveguide generating continuous wave RF-EMR (1.8 GHz, 0.15 and 1.5 W/kg). This study demonstrated that a 4 h exposure is capable of inducing the generation of mitochondrial reactive oxygen species (ROS) in populations of GC1 (7 vs. 18%; p < 0.001) and GC2 cells (11.5 vs. 16 %; p < 0.01), identifying Complex III of the electron transport chain (ETC) as the potential source of electrons producing ROS. Assessing the generation of ROS in the presence of an antioxidant, penicillamine, as well as measuring lipid peroxidation via 4-hydroxynonenal levels, indicated that the elevated incidence of ROS generation observed under our exposure conditions did not necessarily induce an overt cellular oxidative stress response. However, exposure to RF-EMR at 0.15 W/kg for 3 h did induce significant DNA fragmentation in spermatozoa (that was no longer significant after 4 h), assessed by the alkaline comet assay ( p < 0.05). Furthermore, this fragmentation was accompanied by an induction of oxidative DNA damage in the form of 8-hydroxy-2'-deoxyguanosine, which was significant ( p < 0.05) after spermatozoa were exposed to RF-EMR for 4 h. At this exposure time point, a decline in sperm motility ( p < 0.05) was also observed. This study contributes new evidence toward elucidating a mechanism to account for the effects of RF-EMR on biological systems, proposing Complex III of the mitochondrial ETC as the key target of this radiation.

The reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposing male mice to a mild heat stress. For this purpose, adult male mice were exposed to an elevated ambient temperature of 35°C under two exposure models. The first involved acute exposure for 24 h, followed by recovery periods between 1 day and 6 weeks. The alternative heating regimen involved a daily exposure of 8 h for periods of 1 or 2 weeks. In our acute model, we identified elevated sperm mitochondrial ROS generation (P < 0.05), increased sperm membrane fluidity (P < 0.05), DNA damage in the form of single-strand breaks (P < 0.001), and oxidative DNA damage (P < 0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P < 0.001) and round spermatids (P < 0.001) isolated from testes after 1 day heat recovery. Despite these lesions, the spermatozoa of heat-treated mice exhibited no differences in their ability to achieve hallmarks of capacitation or to fertilize the oocyte and support development of embryos to the blastocyst stage (all P > 0.05). Collectively, our acute heat stress model supports the existence of heat susceptible stages of germ cell development, with the round spermatids being most perturbed and spermatogonial stem cells exhibiting resistance to this insult. Such findings were complemented by our chronic heat stress model, which further supported the vulnerability of the round spermatid population.

Study Question: What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility?, Summary Answer: While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function., What Is Known Already: CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4., Study Design, Size, Duration: In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization., Participants/materials, Setting, Methods: Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype., Main Results and the Role of Chance: While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis., Limitations Reasons for Caution: Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated., Wider Implications of the Findings: The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility., Large Scale Data: Not applicable., Study Funding and Competing Interest(s): This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

Mobile phone usage has become an integral part of our lives. However, the effects of the radiofrequency electromagnetic radiation (RF-EMR) emitted by these devices on biological systems and specifically the reproductive systems are currently under active debate. A fundamental hindrance to the current debate is that there is no clear mechanism of how such non-ionising radiation influences biological systems. Therefore, we explored the documented impacts of RF-EMR on the male reproductive system and considered any common observations that could provide insights on a potential mechanism. Among a total of 27 studies investigating the effects of RF-EMR on the male reproductive system, negative consequences of exposure were reported in 21. Within these 21 studies, 11 of the 15 that investigated sperm motility reported significant declines, 7 of 7 that measured the production of reactive oxygen species (ROS) documented elevated levels and 4 of 5 studies that probed for DNA damage highlighted increased damage due to RF-EMR exposure. Associated with this, RF-EMR treatment reduced the antioxidant levels in 6 of 6 studies that discussed this phenomenon, whereas consequences of RF-EMR were successfully ameliorated with the supplementation of antioxidants in all 3 studies that carried out these experiments. In light of this, we envisage a two-step mechanism whereby RF-EMR is able to induce mitochondrial dysfunction leading to elevated ROS production. A continued focus on research, which aims to shed light on the biological effects of RF-EMR will allow us to test and assess this proposed mechanism in a variety of cell types., (© 2016 Society for Reproduction and Fertility.)

Ambulatory ECG, approximately 18 to 24 hours in duration, were obtained from 113 male and 115 female clinically normal, purpose-bred Beagles. The ECG analyzed semi-automatically (44 males, 46 females) were evaluated for heart rate, ventricular ectopic complexes (VEC), bradycardia, and sinus pause; those analyzed by visual inspection (69 males, 69 females) were evaluated for VEC, second-degree atrioventricular block, and supraventricular escape complexes. Mean heart rate was highest at times of maximal human contact (eg, feeding, cleaning) and lowest at periods of no human contact. Bradycardia was observed in 27 of 44 males (61.4%) and 18 of 46 females (39.1%). Sinus pause was identified in 33 of 44 males (75%) and 30 of 46 females (65.2%). Frequency of bradycardia and sinus pause tended to vary inversely with mean heart rate. Ventricular ectopic complexes were detected in 18.8 to 26.1% of the ECG analyzed by use of either method. Although VEC runs and bigeminy were observed, most VEC were single and occurred sporadically. Second-degree atrioventricular block was observed in 6 of 69 males (8.7%) and 14 of 69 females (20.3%); episodes often were single and occurred sporadically. Supraventricular escape complex occurred in 2 of 69 females (2.9%). Multiple types of abnormal complexes were observed in 2 of 69 males (2.9%) and 6 of 69 females (8.7%). Among clinically normal Beagles, ambulatory electrocardiography detects a higher percentage of dogs with VEC, second-degree atrioventricular block, and supraventricular ectopic complexes than does resting electrocardiography.