High percentage of midpiece defects in Brangus bull sperm with no reduction in sperm kinematics.

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.

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

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

Age-Related Changes in Sperm Morphology and Analysis of Multiple Sperm Defects.

BACKGROUND: Analysis of sperm morphology defects (amorphous heads, abnormal acrosome, etc.) is useful for estimating the efficiency of spermiogenesis and sperm maturation. An advanced paternal age (more than 40 years) is associated with decreasing sperm count and reduced motility; however, there is little information on the effect of aging relating to sperm morphological defects. Moreover, searching for stable combinations of certain morphological defects in the same sperm can be useful for better understanding spermiogenesis. The aim of the study was to investigate age-related changes in sperm morphology and the prevalence of certain combinations of sperm morphological defects in men from the general population. METHODS: Sperm morphology was assessed in 1266 volunteers from the Russian urban general population in different age groups (18-19, 20-24, 25-29, 30-34, 35-40, and over 40 years old). Two hundred sperm were evaluated from each semen sample (about 250 thousand spermatozoa in total). Sperm defects were classified according to the WHO laboratory manual (WHO, 2010). The total percentage of each sperm defect and the frequency of different combinations of sperm morphological anomalies for each age group were counted. Additionally, a similar analysis was performed for the groups of normospermia and pathozoospermia. RESULTS: The frequency of coiled and short sperm tails increased in men over 40 years old compared to younger subjects; however, aging did not affect the percentage of morphologically normal sperm. It was shown that the combination of a misshaped head (amorphous, pyriform, and elongated) with a postacrosomal vacuole, acrosome defect, excess residual cytoplasm, or any anomaly of the midpiece or tail in the same spermatozoon were not random combinations of independent solitary defects. The increased frequency of combinations of coiled tails with amorphous, elongated, or vacuolated heads was observed in men older than 40 years. Sperm morphological defects, such as severely deformed heads (pyriform, elongated, and round) were more common in men with pathozoospermia compared to normospermic subjects. CONCLUSIONS: An age-related impairment in sperm morphology was found. Stable combinations of head defects with anomalies in the acrosome, midpiece or tail suggest that these defects may be the result of a general violation in the morphogenetic mechanism.

A caffeine pre-treatment and sole effect of bone-marrow mesenchymal stem cells-derived conditioned media on hyperglycemia-suppressed fertilization.

As a common metabolic disorder, hyperglycemia (HG) affects and disrupts the physiology of various systems in the body. Transplantation of mesenchymal stem cells (MSCs) has been used to control the complications of disease. Most of the therapeutic properties of MSCs are attributed to their secretome. This study aimed to investigate the effects of conditioned media extracted from sole or caffeine pre-treated bone-marrow-derived MSCs on hyperglycemia-induced detrimental impact on some aspects of reproduction. The HG was induced by intraperitoneally injection of streptozotocin (65 mg/kg) and nicotinamide (110 mg/kg). Twenty-four male Wistar rats (190 ± 20 g) were divided into control, HG, and the hyperglycemic groups receiving conditioned media of proliferated MSCs solely (CM) or MSCs pre-treated with caffeine (CCM). During the 49-day treatment, body weight and blood glucose were measured weekly. Finally, HbA1c, spermatogenesis development, sperm count, morphology, viability, motility, chromatin condensation, and DNA integrity were examined. Also, testicular total antioxidant capacity (TAC), malondialdehyde, sperm fertilization potential, and pre-implantation embryo development were evaluated. A one-way ANOVA and Tukey's post-hoc tests were used to analyze the quantitative data. The p < 0.05 was considered statistically significant. The CM and with a higher efficiency, the CCM remarkably (p < 0.05) improved body weight and HG-suppressed spermatogenesis, enhanced sperm parameters, chromatin condensation, DNA integrity, and TAC, reduced HbA1c, sperm abnormalities, and malondialdehyde, and significantly improved pre-implantation embryo development versus HG group. The conditioned media of MSCs solely (CM) and more effectively after pre-treatment of MSCs with caffeine (CCM) could improve spermatogenesis development, sperm quality, pre-implantation embryo development, and testicular global antioxidant potential during hyperglycemia.

Mutations in CCIN cause teratozoospermia and male infertility.

Teratozoospermia is usually associated with defective spermiogenesis and is a disorder with considerable genetic heterogeneity. Although previous studies have identified several teratozoospermia-associated genes, the etiology remains unknown for a majority of affected men. Here, we identified a homozygous missense mutation and a compound heterozygous mutation of CCIN in patients suffering from teratozoospermia. CCIN encodes the cytoskeletal protein Calicin that is involved in the formation and maintenance of the highly regular organization of the calyx of mammalian spermatozoa, and has been proposed to play a role in sperm head structure remodeling during the process of spermiogenesis. Our morphological and ultrastructural analyses of the spermatozoa obtained from all three men harboring deleterious CCIN mutants reveal severe head malformation. Further immunofluorescence assays unveil markedly reduced levels of Calicin in spermatozoa. These patient phenotypes are successfully recapitulated in mouse models expressing the disease-associated variants, confirming the role of Calicin in male fertility. Notably, all mutant spermatozoa from mice and human patients fail to adhere to the zona mass, which likely is the major mechanistic reason for CCIN-mutant sperm-derived infertility. Finally, the use of intra-cytoplasmic sperm injections (ICSI) successfully makes mutated mice and two couples with CCIN variants have healthy offspring. Taken together, our findings identify the role of Calicin in sperm head shaping and male fertility, providing important guidance for genetic counseling and assisted reproduction treatments.

Chromosome 17 translocation affects sperm morphology: Two case studies and literature review.

We present two cases of infertile males with teratozoospermia stemming from chromosome 17 translocation. The patients present karyotypes that have not been previously reported. Genes located on breakpoints (17p11.2, 9q31, and 11p15) were analysed to find the probable mechanism affecting sperm morphology. Our results suggest that ALKBH5, TOP3A, and LLGL1 interactions may be an underlying cause of abnormal sperm head morphology. Translocation of chromosome 17 occurred in conjunction with chromosome 9 and chromosome 11 translocation in the two cases, resulting in oligozoospermia and asthenozoospermia, respectively. These abnormal phenotypes may involve meiosis- and motility-related genes such as LDHC, DNHD1, UBQLN3, and NUP98. Translocation is thus a risk factor for sperm morphological abnormalities and motility deficiency. The interaction network of 22 genes on breakpoints suggests that they contribute to spermatogenesis as a group. In conclusion, this study highlighted the importance of investigating genes linked to sperm morphology, together with chromosome 17 translocation and reproductive risks. For patients interested in screening before a future pregnancy, we recommend preimplantation genetic diagnosis to reduce the risk of karyotypically unbalanced foetuses and birth defects.

Molecular Alterations and Severe Abnormalities in Spermatozoa of Young Men Living in the "Valley of Sacco River" (Latium, Italy): A Preliminary Study.

The Valley of Sacco River (VSR) (Latium, Italy) is an area with large-scale industrial chemical production that has led over time to significant contamination of soil and groundwater with various industrial pollutants, such as organic pesticides, dioxins, organic solvents, heavy metals, and particularly, volatile organic compounds (VOCs). In the present study, we investigated the potential impact of VOCs on the spermatozoa of healthy young males living in the VSR, given the prevalent presence of several VOCs in the semen of these individuals. To accomplish this, spermiograms were conducted followed by molecular analyses to assess the content of sperm nuclear basic proteins (SNBPs) in addition to the protamine-histone ratio and DNA binding of these proteins. We found drastic alterations in the spermatozoa of these young males living in the VSR. Alterations were seen in sperm morphology, sperm motility, sperm count, and protamine/histone ratios, and included significant reductions in SNBP-DNA binding capacity. Our results provide preliminary indications of a possible correlation between the observed alterations and the presence of specific VOCs.

Novel Compound Heterozygous Mutation in FSIP2 Causes Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) and Male Infertility.

Multiple morphological abnormalities of the sperm flagella (MMAF), characteristic with bent, short, coiled, absent, and abnormal caliber flagella, is an important basis of male infertility. Genetic factors account for a large proportion of patients with MMAF. The fibrous sheath interacting protein 2 (FSIP2) has a significant function in the spermatogenesis and flagellar motility. In our study, a novel compound heterozygous mutation (c.1494C > A, p.C498* and c.11020_11024del, p.Tyr3675Cysfs*3) in FSIP2 gene was identified in an infertile male patient with MMAF. H&E staining presented typical MMAF phenotype and thick neck, midpiece in the patient's sperm cells. Transmission electron microscopy observation showed abnormal mitochondrial arrangement and disorganization and dysplastic of the fibrous sheath (FS), which were verified again under light microscopy. Immunofluorescence (IF) analysis of FISP2 expression showed that FSIP2 was absent in the flagellum of the patient's sperm cells. Our findings will be helpful to the precise diagnosis of MMAF and male infertility and enrich the mutational spectrum of FSIP2 gene.

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described. METHODS: We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied. RESULTS: Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal "9 + 0" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI. CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Paternal acrylamide exposure induces transgenerational effects on sperm parameters and learning capability in mice.

Acrylamide (AA) has been shown to have neurological and reproductive toxicities, but little is known about transgenerational effects of AA. In this study, male C57BL/6 mice were exposed to AA (0.01, 1, 10 µg/mL) and its metabolite glycidamide (GA, 10 µg/mL) in drinking water, which were then mated with unexposed female mice to produce F1 and F2 generations. We found that both AA and GA at high concentrations decreased sperm motility in F0 mice and increased sperm malformation rates in mice from all the three generations. In addition, AA and GA increased sperm reactive oxygen species as well as decreased serum testosterone levels, and increased the escape latency time in exposed mice and their offspring. We further found that AA-induced mRNA expression changes in the hippocampus of F0 mice persist to the F2 generation. In the sperm of F0 mice, AA induced significant DNA methylation changes in genes involved in neural and reproduction; the mRNA expression levels of Dnmt3b, a DNA methyltransferase, were dramatically decreased in the testes of F0 and F1 mice. In conclusion, our study indicates that paternal AA exposure leads to DNA methylation-mediated transgenerational adverse effects on sperm parameters and leaning capability in mice.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Abnormal sperm morphology is associated with sensitization to inhaled allergens.

BACKGROUND: Allergy is associated with the loss of tolerance of environmental antigens, combined with a pathological immune response. There were no studies up to date that would show whether the quality of semen decreases in people with allergic diseases. MATERIAL AND METHODS: The research included men who reported to the Gynecological Outpatient Clinic due to reproductive difficulties, defined as the lack of pregnancy after one year of regular intercourse. Semen quality was assessed according to the World Health Organization (WHO) standard. All patients underwent skin prick tests with the most important inhalation allergens (such as hazel, silver birch, mugwort, rye, dog, cat, Dermatophagoides farinae, Dermatophagoides pteronyssinus, alder, Alternaria alternata, Cladosporium herbarum, and grass mix). The data was statistically analyzed. RESULTS: Results of 52 patients aged 25-52 years (34.62 ± 4.96) were analyzed. The mean BMI (Body mass index) was 28.25 (+ -3.77). It was found that 38 men (73%) had increased body weight, and 14 men (26.9%) were obese (BMI > = 30). 13 patients were smokers (25%), and 24 patients (46%) had skin tests positive for at least one inhaled allergen. Sperm tail defects were statistically more significant in patients allergic to birch, rye, cat, alder, and grass. In patients allergic to Alternaria alternata, head defects were statistically more significant (p < .05). No association was found between allergy to house dust mites, mugwort, hazel, and dogs and the deterioration of semen. CONCLUSION: Allergy due to inhalation allergens had an influence on the quality of male semen. Further research is necessary to establish the immunological bases of this phenomenon.

Sperm quality and absence of SARS-CoV-2 RNA in semen after COVID-19 infection: a prospective, observational study and validation of the SpermCOVID test.

OBJECTIVE: To study the contagiousness of sperm and its influence on fertility after recovery from COVID-19 infection. DESIGN: Prospective cohort study. SETTING: University medical center. PATIENT(S): One hundred twenty Belgian men who had recovered from proven COVID-19 infection. INTERVENTION(S): No intervention was performed. MAIN OUTCOME MEASURE(S): Semen quality was assessed using the World Health Organisation criteria. DNA damage to sperm cells was assessed by quantifying the DNA fragmentation index and the high density stainability. Finally antibodies against SARS-CoV2 spike-1 antigen, nuclear and S1-receptor binding domain were measured by Elisa and chemilumenscent microparticle immunoassays, respectively. RESULT(S): SARS-CoV-2 RNA was not detected in semen during the period shortly after infection nor at a later time. Mean progressive motility was reduced in 60% of men tested shortly (<1 month) after COVID-19 infection, 37% of men tested 1 to 2 months after COVID-19 infection, and 28% of men tested >2 months after COVID-19 infection. Mean sperm count was reduced in 37% of men tested shortly (<1 month) after COVID-19 infection, 29% of men tested 1 to 2 months after COVID-19 infection, and 6% of men tested >2 months after COVID-19 infection. The severity of COVID-19 infection and the presence of fever were not correlated with sperm characteristics, but there were strong correlations between sperm abnormalities and the titers of SARS-CoV-2 IgG antibody against spike 1 and the receptor- binding domain of spike 1, but not against nucleotide, in serum. High levels of antisperm antibodies developed in three men (2.5%). CONCLUSION(S): Semen is not infectious with SARS-CoV-2 at 1 week or more after COVID-19 infection (mean, 53 days). However, couples with a desire for pregnancy should be warned that sperm quality after COVID-19 infection can be suboptimal. The estimated recovery time is 3 months, but further follow-up studies are under way to confirm this and to determine if permanent damage occurred in a minority of men.

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient.

RESEARCH QUESTION: Male infertility is a widespread symptom in patients with primary ciliary dyskinesia (PCD). PCD-related male infertility is often caused by asthenozoospermia, with barely normal sperm morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenozoospermia, characterized by various malformed morphologies of sperm flagella. To date, a limited number of genes have been suggested to be involved in the pathogenesis of both PCD and MMAF. What other genes associated with both PCD and MMAF are waiting to be discovered? DESIGN: Whole-exome sequencing (WES) was performed to identify the pathogenic mutation associated with MMAF in a PCD patient. Peripheral venous blood and semen samples were collected from the PCD patient. Transmission electron microscopy (TEM), immunofluorescence staining and western blotting were conducted to confirm the pathogenicity of the identified mutation. RESULTS: A novel homozygous mutation in CCDC39, c.983 T>C (p. Leu328Pro), was identified in two PCD-affected siblings of a consanguineous family showing a typical PCD phenotype, while the proband was infertile, which is associated with characterized MMAF. Furthermore, TEM revealed the abnormal ultrastructure of the patient's sperm flagella. Moreover, immunofluorescence staining revealed that CCDC39 was almost undetectable in the spermatozoa, which was further confirmed by western blotting. The outcome of intracytoplasmic sperm injection (ICSI) in the patient with the CCDC39 mutation was also favourable. CONCLUSION: This study demonstrates that a novel loss-of-function mutation of CCDC39 is involved in the pathogenesis of PCD and MMAF and initially reported that ICSI treatment has a good outcome. Therefore, the novel variant of CCDC39 contributes to the genetic diagnosis, counselling and treatment of male infertility in PCD patients with MMAF phenotype.

A novel stop-gain mutation in ARMC2 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Male infertility is a global issue worldwide and multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most severe forms of the qualitative sperm defects with a heterogeneous genetic cause that has not been completely understood. Can whole-exome sequencing (WES) reveal novel genetic causes contributing to MMAF in a consanguineous Pakistani family, comprising three infertile brothers? DESIGN: WES and bioinformatic analysis were conducted to screen potential pathogenic variants. The identified variant was validated by Sanger sequencing in all available family members Transmission electron microscopy analyses was carried out to examine the flagella ultrastructure of spermatozoa from patient. RESULTS: WES and Sanger sequencing identified a novel homozygous stop-gain mutation (ENST00000392644.4, c.182C>G, p.S61X) in ARMC2, which is expected to lead to loss of protein functions. Transmission electron microscopy analyses revealed that the flagellar ultrastructure of the patient's spermatozoa was disorganized along with a complete absence of central pair complex (CPC), suggesting that ARMC2 is involved in the assembly, stability of the axonemal complex, or both, particularly the CPC. CONCLUSION: We report that a familial stop-gain mutation in ARMC2 is associated with male infertility in humans caused by MMAF accompanied with loss of CPCs and axonemal disorganization. We provide genetic evidence that ARMC2 is essential for human spermatogenesis and its mutation may be pathogenic for MMAF. These findings will improve the knowledge about the genetic basis of MMAF and provide information for genetic counselling of this disease.

A novel mutation in DNAH17 is present in a patient with multiple morphological abnormalities of the flagella.

RESEARCH QUESTION: Asthenoteratospermia is characterized by malformed spermatozoa with motility defects, which results in male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a hallmark of asthenoteratospermia. The genetic causes of MMAF, however, are unknown in about one-third of cases. Which other MMAF-associated genes are waiting to be discovered? DESIGN: Whole-exome sequencing was conducted to identify causative genes in a man with MMAF. Immunofluorescence staining and western blot were applied to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. RESULT: Sanger sequencing of the family demonstrated that the infertile man carried a homozygous DNAH17 variant (c. 4810C>T [p.R1604C]). The obviously decreased DNAH17 expression was observed in HEK293T cells transfected with MUT-DNAH17 plasmid compared with cells with WT-DNAH17 plasmid. Immunofluorescence analysis showed that this mutation induced significant decrease in DNAH17 expression, which negatively affected the DNAH8 expression in the patient's spermatozoa. Moreover, the outcome of ICSI in the patient was unsuccessful. CONCLUSION: Our study revealed a novel homozygous missense mutation in DNAH17 involved in MMAF phenotype. The finding of the novel mutation in DNAH17 enriches the gene variant spectrum of MMAF, further contributing to diagnosis, genetic counselling and prognosis for male infertility.

Pathogenesis of acephalic spermatozoa syndrome caused by splicing mutation and de novo deletion in TSGA10.

PURPOSE: To identify the genetic causes for acephalic spermatozoa syndrome. METHODS: Whole-exome sequencing was performed on the proband from a non-consanguineous to identify pathogenic mutations for acephalic spermatozoa syndrome. Quantitative real-time polymerase chain reaction and whole genome sequencing were subjected to detect deletion. The functional effect of the identified splicing mutation was investigated by minigene assay. Western blot and immunofluorescence were performed to detect the expression level and localization of mutant TSGA10 protein. RESULTS: Here, we identified a novel heterozygous splicing mutation in TSGA10 (NM_025244: c.1108-1G > T), while we confirmed that there was a de novo large deletion in the proband. The splicing mutation led to the skipping of the exon15 of TSGA10, which resulted in a truncated protein (p. A370Efs*293). Therefore, we speculated that the splicing mutation might affect transcription and translation without the dosage compensation of a normal allele, which possesses a large deletion including intact TSGA10. Western blot and immunofluorescence demonstrated that the very low expression level of truncated TSGA10 protein led the proband to present the acephalic spermatozoa phenotype. CONCLUSION: Our finding expands the spectrum of pathogenic TSGA10 mutations that are responsible for ASS and male infertility. It is also important to remind us of paying attention to the compound heterozygous deletion in patients from non-consanguineous families, so that we can provide more precise genetic counseling for patients.

Testicular deficiency associated with exposure to cypermethrin, imidacloprid, and chlorpyrifos in adult rats.

The testicular deficiency associated with exposure to three widely used insecticides in Egyptian agriculture was evaluated. Animals were orally treated with sub-lethal dose (1/50 of the oral LD50) of cypermethrin (CYP), imidacloprid (IMC), and chlorpyrifos (CPF) at 5, 9 and 1.9 mg/kg/day, respectively, five times a week for one month. The CYP, IMC, and CPF exposure resulted in a significant decline in animal body weight, sperm count, motility, normality, and viability with increased head and tail deformities. Significant reduction in serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), testis superoxide dismutase (SOD), and reduced glutathione (GSH) levels. In contrast, catalase (CAT), lipid peroxidation (LPO), and protein carbonyl content (PCC) levels were significantly stimulated. Jointly, obtained results were confirmed by microscopic examination of testis sections. The present data concluded that the CYP, IMC, and CPF have a public health impact and violently interferes with male rat reproductive system.

IZUMO family member 3, IZUMO3, is involved in male fertility through the acrosome formation.

Many factors are involved in acrosome biogenesis in order for appropriate acrosome formation to occur. Here, we demonstrate that IZUMO family member 3, IZUMO3, plays an important role in acrosome biogenesis, as proven by gene disruption experiments. A loss of IZUMO3 in round spermatids affects acrosomal granule positioning due to lack of acrosomal granule contact with the inner acrosomal membrane, leading to the formation of grossly malformed spermatozoa associated with male subfertility. Thus, we suggest that mammalian spermiogenesis needs an elaborate acrosome biogenesis through IZUMO3 involvement.

Sperm parameters and mitochondrial DNA sequence variants among patients at a fertility clinic in Ghana.

PURPOSE: The present study sought to investigate the common abnormalities and mtDNA mutations in the sperm of Ghanaian men attending the fertility Clinic at the Korle-Bu Teaching Hospital (KBTH). The study therefore provides a baseline data mtDNA mutations in a cross-section of Ghanaian men on referral to the fertility clinic at the KBTH. MATERIALS AND METHODS: The semen of 55 men attending the fertility clinic were collected from the Urology and the Obstetrics and Gynaecology Departments of the KBTH. Demographic and clinical data were also collected using questionnaires. Semen analyses were performed and were followed by amplification and purification of mtDNA from total DNA extracted from the semen. Sequencing of the mtDNA amplicons was performed using the next generation sequencer (Illumina-MiSeq). RESULTS: Asthenozoospermia, oligospermia and oligoasthenoteratozoospermia were observed in 1.79%, 5.36% and 28.57%, respectively, of the study participants. There was no association between drinking and/or smoking and history of gonorrhea infection on sperm status/morphology. A total of 785 point mutations were detected in the non-coding control regions, rRNA genes, tRNA genes and the coding regions of the mtDNA samples from the participants. Amongst these mutations, 16 transition mutations were predominantly detected in the mtDNA samples. Missense mutations that were present in only specific sperm abnormalities were identified and they may contribute to infertility in the study population. CONCLUSION: The present study has identified various abnormal sperm phenotypes that are prevalent in the study population and provided a baseline data on mtDNA mutations in the spermatozoa of the patients. A wide range of sperm abnormalities were detected in the study population with no association with life style or history of gonorrhea infection. The mtDNA point mutations detected in the selected genes that were analysed were mostly transition mutations. These transition mutations might be critical for the development of abnormal sperm phenotypes underlying male infertility in the Ghanaian population.