Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.

PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.

Apigenin improves testosterone synthesis by regulating endoplasmic reticulum stress.

Obesity is a growing epidemic among reproductive-age men, which can cause and exacerbate male infertility by means of associated comorbidities, endocrine abnormalities, and direct effects on the fidelity and throughput of spermatogenesis. A prominent consequence of male obesity is a reduction in testosterone levels. Natural products have shown tremendous potential anti-obesity effects in metabolic diseases. This study aimed to investigate the potential of apigenin (AP) to alleviate testicular dysfunction induced by a high-fat diet (HFD) and to investigate the underlying mechanisms, focusing on endoplasmic reticulum stress (ERS) and testosterone synthesis. A murine model of obesity was established using HFD-fed mice. The effects of AP on obesity, lipid metabolism, testicular dysfunction, and ERS were assessed through various physiological, histological, and molecular techniques. Administration of AP (10 mg/kg) ameliorated HFD-induced obesity and testicular dysfunction in a mouse model, as evidenced by decreased body weight, improved lipid profiles and testicular pathology, and restored protein levels related to testosterone. Furthermore, in vitro studies demonstrated that AP relieved ERS and recovered testosterone synthesis in murine Leydig cells (TM3) treated with free fatty acids (FFAs). It was also observed that AP rescued testosterone synthesis enzymes in TM3 cells, similar to that observed with the inhibitor of the PERK pathway (GSK2606414). In addition, ChIP, qPCR, and gene silencing showed that the C/EBP homologous protein (CHOP) bound directly to the promoter region of steroidogenic STAR and negatively modulated its expression. Collectively, AP has remarkable potential to alleviate HFD-induced obesity and testicular dysfunction. Its protective effects are attributable partly to mitigating ERS and restoring testosterone synthesis in Leydig cells.

[The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction]. / N6-ç”²åŸºè ºå˜Œå‘¤ä¿®é¥°åŠå ¶è°ƒæŽ§å› å­åœ¨ç”·æ€§ç”Ÿæ®–ä¸­çš„ä½œç”¨.

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/ß-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

Role of varicocele repair in the era of assisted reproductive technologies: Lessons from 2000 cases of microsurgical varicocele repair.

Backgrounds: In an era of advanced maternal age, there is less conclusive evidence regarding the treatment outcomes of varicocele repair for assisted reproductive technology (ART). Progress in basic research on varicocele is notable whereas there are many clinically relevant points to discuss. Methods: Based on our experience with more than 2000 cases of microsurgical varicocele repair, we focused on the effectiveness of varicocele repair, pathophysiology, surgical approaches, contributions to ART, sperm DNA fragmentation, and varicocele-associated azoospermia in this review with the aim of identifying clearer directions for basic and clinical research on varicocele. Results: Microsurgical low ligation for varicocele repair is expected to remain the gold standard for surgical therapy. Based on the findings from a number of systematic reviews and meta-analyses, negative opinions regarding the efficacy of microsurgical varicocele repair in male infertility treatment have become virtually nonexistent. However, the majority of evidence regarding surgical indications and effectiveness pertains to improvements in semen parameters or non-ART pregnancy rates. Conclusions: Further understandings regarding to pathophysiology of varicocele will likely be gained through comprehensive genetic, transcriptomic, and epigenetic analyses using blood and testicular samples from humans and we hope to develop new diagnostic methods and pharmacotherapy.

Whole genome sequencing identifies a homozygous splicing variant in TDRKH segregating with non-obstructive azoospermia in an Iranian family.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

HSF5 Deficiency Causes Male Infertility Involving Spermatogenic Arrest at Meiotic Prophase I in Humans and Mice.

Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno-associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility.

Biogenic amines in the testis: sources, receptors and actions.

Biogenic amines are signaling molecules with multiple roles in the central nervous system and in peripheral organs, including the gonads. A series of studies indicated that these molecules, their biosynthetic enzymes and their receptors are present in the testis and that they are involved in the regulation of male reproductive physiology and/or pathology. This mini-review aims to summarize the current knowledge in this field and to pinpoint existing research gaps. We suggest that the widespread clinical use of pharmacological agonists/antagonists of these signaling molecules, calls for new investigations in this area. They are necessary to evaluate the relevance of biogenic amines for human male fertility and infertility, as well as the potential value of at least one of them as an anti-aging compound in the testis.

Does pyrethroid exposure lower human semen quality? a systematic review and meta-analysis.

Background: Pyrethroids are natural organic compounds extracted from flowers of pyrethrums and commonly used as domestic and commercial insecticides. Although it is effective in insect and parasitic control, its associated toxicity, including spermotoxicity, remains a challenge globally. Currently, the available reports on the effect of pyrethroids on semen quality are conflicting, hence an evaluation of its detrimental effect is pertinent. This study conducts a detailed systematic review and meta-analysis of the effects of pyrethroids on sperm quality. Materials and methods: The present study was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Using a pre-defined strategic protocol, an internet search was done using combined text words. The criteria for eligibility were selected based on Population, Exposure, Comparator, Outcome, and Study Designs (PECO) framework, and relevant data were collected. Appraisal was done using The Office of Health Assessment and Translation (OHAT) tool for the evaluation of the Risk of Bias and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group guidelines for the certainty of evidence. A quantitative meta-analysis was conducted with the Review Manager (RevMan). Results: Only 12 out of the 4, 050 studies screened were eligible for inclusion in this study. The eligible studies were from China (4), Japan (3), Poland (3), and United States (2). All the eligible studies were cross-sectional. A total of 2, 050 male subjects were included in the meta-analysis. Pyrethroid exposure significantly reduced sperm motility. Region-stratified subgroup analyses revealed that pyrethroid significantly reduced sperm motility among men in Poland and United States, and decreased sperm count among men in Japan. Pyrethroid exposure also reduced sperm concentration among men in Poland but increased sperm concentration among men in the United States. Conclusion: Although the study revealed inconsistent evidence on the detrimental effect of pyrethroids on semen quality, the findings showed that pyrethroids have deleterious potentials on sperm motility, count, and concentration. Studies focusing on the assessment of semen quality in pyrethroid-exposed men, especially at specific varying levels of exposure, and employing prospective cohort studies or controlled cross-sectional designs are recommended.

Toxic impacts of arsenic bioaccumulation on urinary arsenic metabolites and semen quality: A systematic and meta-analysis.

This study aims to investigate the effect of arsenic exposure on urinary levels of arsenic metabolites, semen parameters, and testosterone concentrations. A systematic comprehensive literature search was conducted up till 31st January 2024 using Embase, MEDLINE/Pubmed, and Scopus. This study adopted the Population Exposure Comparator Outcome and Study Design (PECOS) framework. Four studies with a total of 380 control subjects and 347 exposed men were included. Arsenic exposure significantly increased urinary levels of total arsenic (Mean Difference (MD) - 53.35 [95 % Confidence Interval (CI): - 100.14, - 6.55] P= 0.03), and reduced primary arsenic methylation index (PMI) (MD 0.22 [95 % CI: 0.14, 0.31] P< 0.00001), semen volume (MD 0.30 [95 % CI: 0.05, 0.54] P= 0.02) and total testosterone (MD 0.48 [95 % CI: 0.23, 0.73] P= 0.0002). In addition, arsenic exposure marginally reduced sperm concentration (MD 25.04 [95 % CI: - 45.42, 95.50] P= 0.49) and total sperm motility (MD 22.89 [95 % CI: - 14.15, 59.94] P= 0.23). The present meta-analysis demonstrates that arsenic exposure lowers semen quality and testosterone levels. Since the general human population is exposed to arsenic occupationally or domestically, adequate strategic measures should be put in place to limit arsenic exposure in an attempt to preserve semen quality. In addition, studies investigating interventions that may inhibit the bioaccumulation of arsenic in men who are exposed are recommended.

Vitamin B12 is associated with higher serum testosterone concentrations and improved androgenic profiles among men with infertility.

BACKGROUND: Infertility impacts 16% of North American couples, with male factor infertility contributing to ∼30% of cases. Reproductive hormones, especially testosterone, are essential for spermatogenesis. Age-independent population-level decline in testosterone concentrations over the past few decades has been proposed to be a result of diet and lifestyle changes. Vitamin B12 is present in the testes and has been suggested as an adjuvant nutritional therapy for male infertility due to its potential to improve sperm parameters. However, evidence examining the relationship between vitamin B12 and reproductive hormones is limited. OBJECTIVE: The objective was to cross-sectionally examine the relationship between serum vitamin B12 and male reproductive hormones (luteinizing hormone, follicular stimulating hormone, total testosterone, estradiol and prolactin). METHODS: Men with infertility (n = 303) were recruited from Mount Sinai Hospital in Toronto, Canada. Serum was analyzed for vitamin B12 and reproductive hormones. Statistical analyses included non-parametric Spearman's rank correlation coefficient, linear regression, logistic regression and effect modification by age and BMI linear regressions. RESULTS: An independent monotonic relationship between serum vitamin B12 and total testosterone (rho = 0.19, P = 0.001) was observed. Serum vitamin B12 was linearly associated with total testosterone (unadjusted ß = 0.0007, P = 0.008 and adjusted ß = 0.0005, P = 0.03). Compared to individuals in the lowest tertile of serum vitamin B12, those in the middle tertile (adjusted OR = 0.48, 95% CI [0.25, 0.93], P = 0.03) and the highest tertile (unadjusted OR = 0.41, 95% CI [0.22, 0.77], P = 0.005 and adjusted OR = 0.44, 95% CI [0.22, 0.87], P = 0.02) had reduced odds of testosterone deficiency. CONCLUSIONS: These findings suggest that among men with infertility, low serum vitamin B12 is associated with higher risk of testosterone deficiency and impaired androgenic hormonal profiles that impact spermatogenesis and consequently, fertility.

Micronutrient Antioxidants for Men (Menevit®) Improve Sperm Function by Reducing Oxidative Stress, Resulting in Improved Assisted Reproductive Technology Outcomes.

Oxidative stress (OS) affects men's health and impairs spermatogenesis. Micronutrient antioxidants are available for male infertility as complemental support; however, their efficacy remains debatable. This study aimed to investigate whether antioxidants can help to reduce sperm OS and improve semen analysis and quality. We included 171 male partners of couples planning to undergo assisted reproductive technology (ART). Male partners, aged 29-41 years, of couples intending to conceive were self-selected to take daily antioxidants (n = 84) containing folic acid and zinc, or not to take antioxidants (n = 52) for 6 months. We analyzed the alterations in serum oxidant levels, sperm parameters, OS, and deoxyribonucleic acid fragmentation after 3 and 6 months. Additionally, implantation, clinical pregnancy, and miscarriage rates after vitrified-warmed embryo transfer were compared between those taking antioxidants and those not taking them after 6 months. In men with high static oxidation-reduction potential (sORP), we observed a significant improvement in sperm concentration and sORP. The high-quality blastocyst rate tended to increase, and implantation and clinical pregnancy rates also significantly increased after 6 months of intervention. The micronutrient antioxidants could improve sperm function by reducing OS and improving ART outcomes. Therefore, micronutrient antioxidants may be a viable treatment option for male infertility.

Oxidative Stress-Associated Male Infertility: Current Diagnostic and Therapeutic Approaches.

Infertility is a prevalent global issue affecting approximately 17.5% of adults, with sole male factor contributing to 20-30% of cases. Oxidative stress (OS) is a critical factor in male infertility, disrupting the balance between reactive oxygen species (ROS) and antioxidants. This imbalance detrimentally affects sperm function and viability, ultimately impairing fertility. OS also triggers molecular changes in sperm, including DNA damage, lipid peroxidation, and alterations in protein expression, further compromising sperm functionality and potential fertilization. Diagnostic tools discussed in this review offer insights into OS markers, antioxidant levels, and intracellular ROS concentrations. By accurately assessing these parameters, clinicians can diagnose male infertility more effectively and thus tailor treatment plans to individual patients. Additionally, this review explores various treatment options for males with OS-associated infertility, such as empirical drugs, antioxidants, nanoantioxidants, and lifestyle modifications. By addressing the root causes of male infertility and implementing targeted interventions, clinicians can optimize treatment outcomes and enhance the chances of conception for couples struggling with infertility.

The emerging and diverse roles of F-box proteins in spermatogenesis and male infertility.

F-box proteins play essential roles in various cellular processes of spermatogenesis by means of ubiquitylation and subsequent target protein degradation. They are the substrate-recognition subunits of SKP1-cullin 1-F-box protein (SCF) E3 ligase complexes. Dysregulation of F­box protein­mediated proteolysis could lead to male infertility in humans and mice. The emerging studies revealed the physiological function, pathological evidence, and biochemical substrates of F-box proteins in the development of male germ cells, which urging us to review the current understanding of how F­box proteins contribute to spermatogenesis. More functional and mechanistic study will be helpful to define the roles of F-box protein in spermatogenesis, which will pave the way for the logical design of F-box protein-targeted diagnosis and therapies for male infertility, as the spermatogenic role of many F-box proteins remains elusive.

The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice.

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left-right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3+/- knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3+/- males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations.

Effects of acetate-producing Blautia wexlerae on oxidative stress and NLRP3 inflammasome in obesity-associated male infertility.

BACKGROUND: Obesity-associated male infertility is a common complication of obesity and has been increasing in prevalence. Blautia wexlerae has modulation effects on obesity. However, the action of B. wexlerae on obesity-associated male infertility is unclear. The nod-like receptor protein 3 (NLRP3) inflammasome has become a major target for addressing many diseases, including obesity-associated male infertility. This study aims to investigate the action of B. wexlerae on obesity-associated male infertility and the influence of B. wexlerae on NLRP3 inflammasome. MATERIALS AND METHODS: The fecal samples were collected from 60 infertile men with or without obesity and 30 healthy men. The obesity mice model was established through high-fat diet (HFD) induction. The mating assays evaluated the male infertility of obese mice. A mouse-derived spermatogonia (GC-1 spg) cell viability was detected using the Cell Counting Kit-8 assay. The reactive oxygen species (ROS) were assessed using flow cytometry. Furthermore, immunofluorescence, enzyme-linked immunosorbent assay, and western blotting were applied to measure the gene expressions. RESULTS: Blautia wexlerae was decreased and negatively correlated with interleukin-1 beta (IL-1ß) or IL-18 levels in infertile men with obesity. On the other hand, B. wexlerae improved the mating capability of obese male mice and suppressed oxidative stress and NLRP3 inflammasome via the activation of the acetate receptor. Furthermore, sodium acetate regulated oxidative stress and NLRP3 inflammasome via the activation of the acetate receptor in GC-1 spg cells in vitro. CONCLUSION: The administration of Blautia wexlerae improved obesity-associated male infertility and regulated oxidative stress and NLRP3 inflammasome activities. In general, its administration may be an effective strategy for the treatment of obesity-associated male infertility.

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

FANCM Gene Variants in a Male Diagnosed with Sertoli Cell-Only Syndrome and Diffuse Astrocytoma.

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

A comprehensive insight into the contribution of epigenetics in male infertility; focusing on immunological modifications.

Numerous recent studies have examined the impact epigenetics-including DNA methylation-has on spermatogenesis and male infertility. Differential methylation of several genes has been linked to compromised spermatogenesis and/or reproductive failure. Specifically, male infertility has been frequently associated with DNA methylation abnormalities of MEST and H19 inside imprinted genes and MTHFR within non-imprinted genes. Microbial infections mainly result in male infertility because of the immune response triggered by the bacteria' accumulation of immune cells, proinflammatory cytokines, and chemokines. Thus, bacterially produced epigenetic dysregulations may impact host cell function, supporting host defense or enabling pathogen persistence. So, it is possible to think of pathogenic bacteria as potential epimutagens that can alter the epigenome. It has been demonstrated that dysregulated levels of LncRNA correlate with motility and sperm count in ejaculated spermatozoa from infertile males. Therefore, a thorough understanding of the relationship between decreased reproductive capacity and sperm DNA methylation status should aid in creating new diagnostic instruments for this condition. To fully understand the mechanisms influencing sperm methylation and how they relate to male infertility, more research is required.

Unraveling the Impact of the PROCA1 Mutation in Male Infertility: Incorporating Whole Exome Sequencing in Teratozoospermia Patients and Analyzing Proca1 Knockout Mice.

In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.

Melatonin as a natural anti-inflammatory and anti-oxidant therapy in the testis: a focus on infertility and aging†.

Melatonin is a pineal hormone that regulates testicular activity (i.e. steroidogenesis and spermatogenesis) through two complementary mechanisms, indirect effects exerted via the hypothalamic-adenohypophyseal-axis and direct actions that take place on the different cell populations of the male gonad. The effects of increased age on the testis and the general mechanisms involved in testicular pathology leading to infertility are still only poorly understood. However, there is growing evidence that link testicular aging and idiopathic male infertility to local inflammatory and oxidative stress events. Because literature data strongly indicate that melatonin exhibits anti-inflammatory and anti-oxidant properties, this review focuses on the potential benefits exerted by this indoleamine at testicular level in male reproductive fertility and aging. Taking into account that the effects of melatonin supplementation on testicular function are currently being investigated, the overview covers not only promising prospects but also many questions concerning the future therapeutic value of this indoleamine as an anti-aging drug as well as in the management of cases of male infertility for which there are no medical treatments currently available.