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The rheological properties of the extracellular fluid in the female reproductive tract vary spatiotemporally, however, the effect on the behaviour of epithelial cells that line the tract is unexplored. Here, we reveal that epithelial cells respond to the elevated viscosity of culture media by modulating their development and functionality to enhance cilia formation and coordination. Specifically, ciliation increases by 4-fold and cilia beating frequency decreases by 30% when cells are cultured at 100 mPa·s. Further, cilia manifest a coordinated beating pattern that can facilitate the formation of metachronal waves. At the cellular level, viscous loading activates the TRPV4 channel in the epithelial cells to increase intracellular Ca 2+ , subsequently decreasing the mitochondrial membrane potential level for ATP production to maintain cell viability and function. Our findings provide additional insights into the role of elevated tubal fluid viscosity in promoting ciliation and coordinating their beating-a potential mechanism to facilitate the transport of egg and embryo, suggesting possible therapeutic opportunities for infertility treatment., (© 2024. The Author(s).)

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 viscoelastic properties of the female reproductive tract influence sperm swimming behavior, but the exact role of these rheological changes in regulating sperm energetics remains unknown. Using high-speed dark-field microscopy, the flagellar dynamics of free-swimming sperm across a physiologically relevant range of viscosities is resolved. A transition from 3D to 2D slither swimming under an increased viscous loading is revealed, in the absence of any geometrical or chemical stimuli. This transition is species-specific, aligning with viscosity variations within each species' reproductive tract. Despite substantial drag increase, 2D slithering sperm maintain a steady swimming speed across a wide viscosity range (20-250 and 75-1000 mPa s for bull and human sperm) by dissipating over sixfold more energy into the fluid without elevating metabolic activity, potentially by altering the mechanisms of dynein motor activity. This energy-efficient motility mode is ideally suited for the viscous environment of the female reproductive tract., (© 2023 The Authors. Small Methods published by Wiley‐VCH GmbH.)

Alpha, beta, and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon, and zeta, however, remain poorly understood and their requirement in mammalian development untested. Herein we have used a spermatogenesis model to define epsilon tubulin (TUBE1) function in mice. We show that TUBE1 is essential for the function of multiple complex microtubule arrays, including the meiotic spindle, axoneme and manchette and in its absence, there is a dramatic loss of germ cells and male sterility. Moreover, we provide evidence for the interplay between TUBE1 and katanin-mediated microtubule severing, and for the sub-specialization of individual katanin paralogs in the regulation of specific microtubule arrays., (© 2024. The Author(s).)

Background: The widespread interest in male reproductive health (MRH), fueled by emerging evidence, such as the global decline in sperm counts, has intensified concerns about the status of MRH. Consequently, there is a pressing requirement for a strategic, systematic approach to identifying critical questions, collecting pertinent information, and utilizing these data to develop evidence-based strategies. The methods for addressing these questions and the pathways toward their answers will inevitably vary based on the variations in cultural, geopolitical, and health-related contexts. To address these issues, a conjoint ESHRE and Male Reproductive Health Initiative (MRHI) Campus workshop was convened., Objective and Rationale: The three objectives were: first, to assess the current state of MRH around the world; second, to identify some of the key gaps in knowledge; and, third, to examine how MRH stakeholders can collaboratively generate intelligent and effective paths forward., Search Methods: Each expert reviewed and summarized the current literature that was subsequently used to provide a comprehensive overview of challenges related to MRH., Outcomes: This narrative report is an overview of the data, opinions, and arguments presented during the workshop. A number of outcomes are presented and can be summarized by the following overarching themes: MRH is a serious global issue and there is a plethora of gaps in our understanding; there is a need for widespread international collaborative networks to undertake multidisciplinary research into fundamental issues, such as lifestyle/environmental exposure studies, and high-quality clinical trials; and there is an urgent requirement for effective strategies to educate young people and the general public to safeguard and improve MRH across diverse population demographics and resources., Limitations Reasons for Caution: This was a workshop where worldwide leading experts from a wide range of disciplines presented and discussed the evidence regarding challenges related to MRH. While each expert summarized the current literature and placed it in context, the data in a number of areas are limited and/or sparse. Equally, important areas for consideration may have been missed. Moreover, there are clear gaps in our knowledge base, which makes some conclusions necessarily speculative and warranting of further study., Wider Implications: Poor MRH is a global issue that suffers from low awareness among the public, patients, and heathcare professionals. Addressing this will require a coordinated multidisciplinary approach. Addressing the significant number of knowledge gaps will require policy makers prioritizing MRH and its funding., Study Funding/competing Interests: The authors would like to extend their gratitude to ESHRE for providing financial support for the Budapest Campus Workshop, as well as to Microptic S.L. (Barcelona) for kindly sponsoring the workshop. P.B. is the Director of the not-for-profit organization Global Action on Men's Health and receives fees and expenses for his work, (which includes the preparation of this manuscript). Conflicts of interest: C.J.D.J., C.L.R.B., R.A.A., P.B., M.P.C., M.L.E., N.G., N.J., C.K., AAP, M.K.O., S.R.-H., M.H.V.-L.: ESHRE Campus Workshop 2022 (Travel support-personal). C.J.D.J.: Cambridge University Press (book royalties-personal). ESHRE Annual Meeting 2022 and Yale University Panel Meeting 2023 (Travel support-personal). C.L.R.B.: Ferring and IBSA (Lecture), RBMO editor (Honorarium to support travel, etc.), ExSeed and ExScentia (University of Dundee), Bill & Melinda Gates Foundation (for research on contraception). M.P.C.: Previously received funding from pharmaceutical companies for health economic research. The funding was not in relation to this work and had no bearing on the contents of this work. No funding from other sources has been provided in relation to this work (funding was provided to his company Global Market Access Solutions). M.L.E.: Advisor to Ro, Doveras, Next, Hannah, Sandstone. C.K.: European Academy of Andrology (Past president UNPAID), S.K.: CEO of His Turn, a male fertility Diagnostic and Therapeutic company (No payments or profits to date). R.I.M.: www.healthymale.org.au (Australian Government funded not for profit in men's health sector (Employed as Medical Director 0.2 FET), Monash IVF Pty Ltd (Equity holder)). N.J.: Merck (consulting fees), Gedeon Richter (honoraria). S.R.-H.: ESHRE (Travel reimbursements). C.N.: LLC (Nursing educator); COMMIT (Core Outcomes Measures for Infertility Trials) Advisor, meeting attendee, and co-author; COMMA (Core Outcomes in Menopause) Meeting attendee, and co-author; International Federation of Gynecology and Obstetrics (FIGO) Delegate Letters and Sciences; ReproNovo, Advisory board; American Board of Urology Examiner; American Urological Association Journal subsection editor, committee member, guidelines co-author Ferring Scientific trial NexHand Chief Technology Officer, stock ownership Posterity Health Board member, stock ownership. A.P.: Economic and Social Research Council (A collaborator on research grant number ES/W001381/1). Member of an advisory committee for Merck Serono (November 2022), Member of an advisory board for Exceed Health, Speaker fees for educational events organized by Mealis Group; Chairman of the Cryos External Scientific Advisory Committee: All fees associated with this are paid to his former employer The University of Sheffield. Trustee of the Progress Educational Trust (Unpaid). M.K.O.: National Health and Medical Research Council and Australian Research Council (Funding for research of the topic of male fertility), Bill and Melinda Gates Foundation (Funding aimed at the development of male gamete-based contraception), Medical Research Future Fund (Funding aimed at defining the long-term consequences of male infertility). M.H.V.-L.: Department of Sexual and Reproductive Health and Research (SRH)/Human Reproduction Programme (HRP) Research Project Panel RP2/WHO Review Member; MRHI (Core Group Member), COMMIT (member), EGOI (Member); Human Reproduction (Associate Editor), Fertility and Sterility (Editor), AndroLATAM (Founder and Coordinator)., Competing Interests: C.J.D.J., C.L.R.B., R.A.A., P.B., M.P.C., M.L.E., N.G., N.J., C.K., A.P., M.K.O., S.R.-H., M.H.V.-L.: ESHRE Campus Workshop 2022 (Travel support—personal). C.J.D.J.: Cambridge University Press (book royalties—personal). ESHRE Annual Meeting 2022 and Yale University Panel Meeting 2023 (Travel support—personal). C.L.R.B.: Ferring and IBSA (Lecture), RBMO editor (Honorarium to support travel, etc.), ExSeed and ExScentia (University of Dundee), Bill & Melinda Gates Foundation (for research on contraception). M.P.C.: Previously received funding from pharmaceutical companies for health economic research. The funding was not in relation to this work and had no bearing on the contents of this work. No funding from other sources has been provided in relation to this work (funding was provided to his company Global Market Access Solutions). M.L.E.: Advisor to Ro, Doveras, Next, Hannah, Sandstone. C.K.: European Academy of Andrology (Past president UNPAID), S.K.: CEO of His Turn, a male fertility Diagnostic and Therapeutic company (No payments or profits to date). R.I.M.: www.healthymale.org.au (Australian Government funded not for profit in men’s health sector (Employed as Medical Director 0.2 FET), Monash IVF Pty Ltd (Equity holder)). N.J.: Merck (consulting fees), Gedeon Richter (honoraria). C.N.: LLC (Nursing educator); COMMIT (Core Outcomes Measures for Infertility Trials) Advisor, meeting attendee, and co‐author; COMMA (Core Outcomes in Menopause) Meeting attendee, and co‐author; International Federation of Gynecology and Obstetrics (FIGO) Delegate Letters and Sciences; ReproNovo, Advisory board; American Board of Urology Examiner; American Urological Association Journal subsection editor, committee member, guidelines co‐author Ferring Scientific trial NexHand Chief Technology Officer, stock ownership Posterity Health Board member, stock ownership. A.P.: Economic and Social Research Council (A collaborator on research grant number ES/W001381/1). Member of an advisory committee for Merck Serono (November 2022), Member of an advisory board for Exceed Health, Speaker fees for educational events organized by Mealis Group; Chairman of the Cryos External Scientific Advisory Committee: All fees associated with this are paid to his former employer The University of Sheffield. Trustee of the Progress Educational Trust (Unpaid). M.K.O.: National Health and Medical Research Council and Australian Research Council (Funding for research of the topic of male fertility), Bill and Melinda Gates Foundation (Funding aimed at the development of male gamete-based contraception), Medical Research Future Fund (Funding aimed at defining the long-term consequences of male infertility). M.H.V.-L.: Department of Sexual and Reproductive Health and Research (SRH)/Human Reproduction Programme (HRP) Research Project Panel RP2/WHO Review Member; MRHI (Core Group Member), COMMIT (member), EGOI (Member); Human Reproduction (Associate Editor), Fertility and Sterility (Editor), AndroLATAM (Founder and Coordinator)., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Juchnewitsch, Pomm, Dutta, Tamp, Valkna, Lillepea, Mahyari, Tjagur, Belova, Kübarsepp, Castillo-Madeen, Riera-Escamilla, Põlluaas, Nagirnaja, Poolamets, Vihljajev, Sütt, Versbraegen, Papadimitriou, McLachlan, Jarvi, Schlegel, Tennisberg, Korrovits, Vigh-Conrad, O’Bryan, Aston, Lenaerts, Conrad, Kasak, Punab and Laan.)

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.)

In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

An increasing number of genes are being described in the context of non-syndromic male infertility. Linking the underlying genetic causes of non-syndromic male infertility with clinical data from patients is important to establish new genotype-phenotype correlations. This process can be facilitated by using universal nomenclature, but no standardized vocabulary is available in the field of non-syndromic male infertility. The International Male Infertility Genomics Consortium aimed at filling this gap, providing a standardized vocabulary containing nomenclature based on the Human Phenotype Ontology (HPO). The "HPO tree" was substantially revised compared with the previous version and is based on the clinical work-up of infertile men, including physical examination and hormonal assessment. Some causes of male infertility can already be suspected based on the patient's clinical history, whereas in other instances, a testicular biopsy is needed for diagnosis. We assembled 49 HPO terms that are linked in a logical hierarchy and showed examples of morphological features of spermatozoa and testicular histology of infertile men with identified genetic diagnoses to describe the phenotypes. This work will help to record patients' phenotypes systematically and facilitate communication between geneticists and andrologists. Collaboration across institutions will improve the identification of patients with the same phenotypes, which will promote the discovery of novel genetic causes for non-syndromic male infertility., (© 2023. Springer Nature Limited.)

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.)

Meeting requirements for dietary proteins, especially of essential amino acids (EAAs), is critical for the life-long health of living organisms. However, defining EAA targets for preparing biologically-matched nutrition that satisfies metabolic requirements for protein remains challenging. Previous research has shown the advantages of 'exome matching' in representing the specific requirement of dietary AAs, where the target dietary AA profile was derived from in silico translation of the genome of an organism, specifically responsible for protein expression (the 'exome'). However, past studies have assessed these effects in only one sex, for few parameters (body mass and composition), and have used purified diets in which protein is supplied as a mixture of individual AAs. Here, for the first time, we utilise a computational method to guide the formulation of custom protein blends and test if exome matching can be achieved at the intact protein level, through blending standard protein ingredients, ultimately leading to optimal growth, longevity and reproductive function. Mice were provided ad libitum (ad lib) access to one of the four iso-energetic protein-limited diets, two matched and two mis-matched to the mouse exome target, and fed at a fixed protein energy level of 6.2%. During or following 13-weeks of feeding, the food intake, body growth, composition and reproductive functions were measured. Compared to the two mis-matched diets, male and female animals on the exome-matched diet with protein digestibility correction applied, exhibited significantly improved growth rates and final body mass. The feed conversion efficiency in the same diet was also increased by 62% and 40% over the worst diets for males and females, respectively. Male, not female, exhibited higher accretion of lean body mass with the matched, digestibility-corrected diet. All reproductive function measures in both sexes were comparable among diets, with the exception of testicular daily sperm production in males, which was higher in the two matched diets versus the mis-matched diets. The results collectively demonstrate the pronounced advantages of exome-matching in supporting body growth and improving feed conversion efficiency in both sexes. However, the potential impact of this approach in enhancing fertility needs further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Approximately 10%-15% of couples worldwide are infertile, and male factors account for approximately half of these cases. Teratozoospermia is a major cause of male infertility. Although various mutations have been identified in teratozoospermia, these can vary among ethnic groups. In this study, we performed whole-exome sequencing to identify genetic changes potentially causative of teratozoospermia. Out of seven genes identified, one, ATP/GTP Binding Protein 1 (AGTPBP1), was characterized, and three missense changes were identified in two patients (Affected A: p.Glu423Asp and p.Pro631Leu; Affected B: p.Arg811His). In those two cases, severe sperm head and tail defects were observed. Moreover, AGTPBP1 localization showed a fragmented pattern compared to control participants, with specific localization in the neck and annulus regions. Using murine models, we found that AGTPBP1 is localized in the manchette structure, which is essential for sperm structure formation. Additionally, in Agtpbp1-null mice, we observed sperm head and tail defects similar to those in sperm from AGTPBP1-mutated cases, along with abnormal polyglutamylation tubulin and decreasing △-2 tubulin levels. In this study, we established a link between genetic changes in AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

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.)

Thermal conditions in the developmental environment can substantially affect an individual's phenotype, particularly in egg-laying ectotherms. However, whether these effects persist into adulthood is rarely examined. To investigate this, we incubated delicate skink, Lampropholis delicata , eggs at either cool (22°C), mild (26°C) or hot (30°C) temperatures. After hatching, we measured growth, thermal performance curves of locomotor activity, and thermal sensitivity of resting metabolic rate of offspring as juveniles (4-6 weeks of age), sub-adults (approx. 200 days of age), and adults (approx. 2 years of age), and then measured developmental temperature impacts on male fertility. Incubation temperature had a lasting effect on growth and locomotor performance, with cool and hot incubation temperatures resulting in faster growth and larger maximum size, and hot incubation temperatures reducing locomotor performance at all timepoints. Effects on resting metabolic rate were only present in sub-adults, with a higher metabolic rate at high and average body mass and negative metabolic scaling exponent in cool-incubated lizards. Additionally, cool and hot incubation treatments resulted in shorter sperm midpieces and heads. Incubation temperature did not affect testis mass or sperm count. Overall, our results demonstrate that incubation temperature can have lasting effects on later life stages, highlighting the importance of maternal nest-site selection, but that some effects are age dependent. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm -deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

The development and function of male gametes is dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a crucial role in this process. Here, we sought to elucidate the roles of spastin, an as-yet-unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a crucial role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis and, commonly, a catastrophic loss of nuclear integrity. This work defines an essential role for spastin in regulating microtubule dynamics during spermatogenesis, and is of potential relevance to individuals carrying spastin variants and to the medically assisted reproductive technology industry., 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.

Background: Approximately 1 in 20 men are sub-fertile or infertile yet the aetiologies of male infertility remain largely unexplained. It is suggested that lifestyle choices and environmental factors contribute but research is limited. In particular, no study has evaluated early life exposures and subsequent male infertility. To address this knowledge gap, this study aims to characterise a cohort of men with idiopathic infertility and compare their general health, lifestyle choices and environmental exposures from teenage years onwards to men without reproductive abnormalities., Methods: Two groups of men (N = 500 cases; N = 500 controls), matched for age and socio-economic status, will be recruited from fertility clinics around Australia between June 2021 and June 2024. Men will be eligible if they are between 18 and 50 years, with a female partner less than 42 years, and have identified idiopathic male infertility (case) or are part of a couple with diagnosed female factor infertility but with no indication of compromised male fertility (control). Participants will complete an in-depth survey on general health, lifestyle and environmental exposures, reporting from teenage years onwards. An online medical data capture form will be used to gather fertility assessment information from participant medical records. Biological specimens of saliva (all study participants), blood and urine (optional) will be collected and stored for future genetic and epigenetic analysis. Differences in outcome measures between cases and controls will be determined using appropriate between groups comparisons. The relationship between explanatory variables and infertility will be analysed using multilevel modelling to account for clustering within fertility clinics., Discussion: This study addresses an important gap in research on the aetiology of male infertility and will provide a comprehensive profile of the lifestyle and environmental risk factors for male infertility, leading to provision of up-to-date health advice for male teenagers and adults about optimising their fertility., (© 2023. The Author(s).)

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification., (© 2022. The Author(s).)

Study Question: Is the metabolic health of men conceived using ICSI different to that of IVF and spontaneously conceived (SC) men?, Summary Answer: ICSI-conceived men aged 18-24 years, compared with SC controls, showed differences in some metabolic parameters including higher resting diastolic blood pressure (BP) and homeostasis model assessment for insulin resistance (HOMA-IR) scores, although the metabolic parameters of ICSI- and IVF-conceived singleton men were more comparable., What Is Known Already: Some studies suggest that IVF-conceived offspring may have poorer cardiovascular and metabolic profiles than SC children. Few studies have examined the metabolic health of ICSI-conceived offspring., Study Design, Size, Duration: This cohort study compared the metabolic health of ICSI-conceived men to IVF-conceived and SC controls who were derived from prior cohorts. Participants included 121 ICSI-conceived men (including 100 singletons), 74 IVF-conceived controls (all singletons) and 688 SC controls (including 662 singletons)., Participants/materials, Setting, Methods: Resting systolic and diastolic BP (measured using an automated sphygmomanometer), height, weight, BMI, body surface area and fasting serum metabolic markers including fasting insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol, triglycerides, highly sensitive C-reactive protein (hsCRP) and HOMA-IR were compared between groups. Data were analysed using multivariable linear regression adjusted for various covariates including age and education level., Main Results and the Role of Chance: After adjusting for covariates, compared to 688 SC controls, 121 ICSI-conceived men had higher diastolic BP (β 4.9, 95% CI 1.1-8.7), lower fasting glucose (β -0.7, 95% CI -0.9 to -0.5), higher fasting insulin (ratio 2.2, 95% CI 1.6-3.0), higher HOMA-IR (ratio 1.9, 95% CI 1.4-2.6), higher HDLC (β 0.2, 95% CI 0.07-0.3) and lower hsCRP (ratio 0.4, 95% CI 0.2-0.7) levels. Compared to 74 IVF-conceived singletons, only glucose differed in the ICSI-conceived singleton men (β -0.4, 95% CI -0.7 to -0.1). No differences were seen in the paternal infertility subgroups., Limitations, Reasons for Caution: The recruitment rate of ICSI-conceived men in this study was low and potential for recruitment bias exists. The ICSI-conceived men, the IVF-conceived men and SC controls were from different cohorts with different birth years and different geographical locations. Assessment of study groups and controls was not contemporaneous, and the measurements differed for some outcomes (BP, insulin, glucose, lipids and hsCRP)., Wider Implications of the Findings: These observations require confirmation in a larger study with a focus on potential mechanisms. Further efforts to identify whether health differences are due to parental characteristics and/or factors related to the ICSI procedure are also necessary., Study Funding/competing Interest(s): This study was funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. S.R.C. was supported through an Australian Government Research Training Program Scholarship. R.J.H. is supported by an NHMRC project grant (634457), and J.H. and R.I.M. have been supported by the NHMRC as Senior and Principal Research Fellows respectively (J.H. fellowship number: 1021252; R.I.M. fellowship number: 1022327). L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF Group and Ferring Australia, honoraria from Ferring Australia and travel fees from Merck Serono and MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and nonfinancial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.)

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Study Question: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)?, Summary Answer: One in 28 NOA cases carried an identifiable, medically actionable SF., What Is Known Already: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited., Study Design, Size, Duration: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study., Participants/materials, Setting, Methods: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed., Main Results and the Role of Chance: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population., Limitations, Reasons for Caution: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants., Wider Implications of the Findings: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA., Study Funding/competing Interest(s): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice., Methods: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry., Results: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present., Conclusions: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy., (© 2022. The Author(s).)

Sperm motility is a significant predictor of male fertility potential and is directly linked to fertilization success in both natural and some forms of assisted reproduction. Sperm motility can be impaired by both genetic and environmental factors, with asthenozoospermia being a common clinical presentation. Moreover, in the setting of assisted reproductive technology clinics, there is a distinct absence of effective and noninvasive technology to increase sperm motility without detriment to the sperm cells. Here, a new method is presented to boost sperm motility by increasing the intracellular rate of metabolic activity using high frequency ultrasound. An increase of 34% in curvilinear velocity (VCL), 10% in linearity, and 32% in the number of motile sperm cells is shown by rendering immotile sperm motile, after just 20 s exposure. A similar effect with an increase of 15% in VCL treating human sperm with the same setting is also identified. This cell level mechanotherapy approach causes no significant change in cell viability or DNA fragmentation index, and, as such, has the potential to be applied to encourage natural fertilization or less invasive treatment choices such as in vitro fertilization rather than intracytoplasmic injection., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Objective: To determine the semen quality and reproductive hormones of men conceived by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) compared with men conceived without assisted reproductive technology (ART)., Design: Cohort study., Setting: IVF centers in Victoria and the Western Australian Raine Study., Patient(s): Men conceived with IVF/ICSI and men conceived without ART aged 18-25 years., Intervention(s): Clinical review., Main Outcome Measure(s): The primary outcome was the prevalence of severe oligozoospermia (sperm concentration, <5 million/mL). The secondary outcomes were total sperm count, total and progressive motility, total motile count, normal morphology, and serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH)., Results: There was no difference in the prevalence of severe oligozoospermia between 120 men conceived with IVF/ICSI and 356 men conceived without ART (9% vs. 5.3%). Men conceived with IVF/ICSI had similar sperm concentration, total sperm count, and total motile count but lower mean total (55.3% vs. 60.6%) and progressive (44.7% vs. 53.9%) sperm motility with higher mean normal morphology (8.5% vs. 5.4%). Differences in progressive motility (ß, -9.9; 95% confidence interval [CI], -16.7 - -3.0), normal morphology (ß, 4.3; 95% CI, 3.0-5.7), and proportion with abnormal morphology (adjusted odds ratios, 0.1; 95% CI, 0.04-0.5) remained significant after adjusting for confounders. Men conceived with IVF/ICSI had lower mean FSH (3.3 IU/L) and LH (3.9 IU/L) levels and higher mean testosterone levels (19.1 nmol/L) than controls (4.2 IU/L, 11.0 IU/L, and 16.8 nmol/L)., Conclusion: This study of men conceived with IVF/ICSI found similar sperm output to men conceived without ART. Overall, the results are reassuring., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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.)

Sperm swim through the female reproductive tract by propagating a 3D flagellar wave that is self-regulatory in nature and driven by dynein motors. Traditional microscopy methods fail to capture the full dynamics of sperm flagellar activity as they only image and analyze sperm motility in 2D. Here, an automated platform to analyze sperm swimming behavior in 3D by using thin-lens approximation and high-speed dark field microscopy to reconstruct the flagellar waveform in 3D is presented. It is found that head-tethered mouse sperm exhibit a rolling beating behavior in 3D with the beating frequency of 6.2 Hz using spectral analysis. The flagellar waveform bends in 3D, particularly in the distal regions, but is only weakly nonplanar and ambidextrous in nature, with the local helicity along the flagellum fluctuating between clockwise and counterclockwise handedness. These findings suggest a nonpersistent flagellar helicity. This method provides new opportunities for the accurate measurement of the full motion of eukaryotic flagella and cilia which is essential for a biophysical understanding of their activation by dynein motors., (© 2022 The Authors. Small Methods published by Wiley-VCH GmbH.)

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).)