Pathogenesis Of Asthenozoospermia Research Articles

Shenqi Qiangjing Granules Ameliorate Asthenozoospermia in Mice by Regulating Ferroptosis through the METTL3/GPX4 Signaling Axis.

Asthenozoospermia is a leading cause of male infertility, yet current pharmacotherapies yield suboptimal outcomes, underscoring the urgent need for novel treatment modalities. Herein, we induced asthenozoospermic mouse models using busulfan and investigated the therapeutic effects of Shenqi Qiangjing Granules (SQ) on testicular pathology, serum sex hormone and steroidogenic enzyme levels, and ferroptosis. Furthermore, utilizing GC-1 spg cell lines, we elucidated the role of the METTL3-mediated m6A modification in GPX4 mRNA stability. Treatment with SQ or Fer-1 (an inhibitor of ferroptosis) significantly ameliorated testicular pathological injury, restored abnormal serum sex hormone levels, and enhanced testicular steroidogenic enzyme expression, highlighting the therapeutic potential of targeting ferroptosis in asthenozoospermia. In elucidating the molecular mechanism of METTL3 in ferroptosis, we found that METTL3 regulates GPX4 mRNA stability, subsequently impacting ferroptosis and sperm quality. Knockdown of METTL3 mimicked the effects of SQ treatment, while overexpression of METTL3 partially reversed SQ-mediated effects on ferroptosis and asthenozoospermia, underscoring the pivotal role of METTL3 in SQ therapy. In conclusion, the METTL3-GPX4-ferroptosis axis emerges as a novel regulatory pathway in the pathogenesis of asthenozoospermia. Targeting this axis, particularly through interventions such as SQ treatment, holds promise for the management of male infertility.

Relationship between seminal plasma cytokine levels and asthenozoospermia in infertile men

Background. Violation of male fertility can have various causes, one of which is a decrease in sperm motility. In the pathogenesis of asthenozoospermia, some bioactive substances, in particular cytokines, may be involved.Aim. To estimate the number of the most studied cytokines to date in the ejaculate of infertile men with normozoospermia and patients with asthenozoospermia.Materials and methods. We examined 38 infertile patients with normozoospermia and 10 men with asthenozoospermia, comparable in age. All participants in the study were performed a spermogram and an analysis to determine the amount of the cytokines in seminal plasma, such as interleukins 1β, 2, 4–8, 10, 12, 13, 17, G-CSF, GM-CSF, interferon γ, MCP-1, MIP-1β and tumor necrosis factor α. Then, using nonparametric statistics, the relationship between the level of the above cytokines and the presence of asthenozoospermia was analyzed.Results. As a result, we were able to find out that there is a relationship between the concentration of interleukin 6, interleukin 8 and MIP-1β and sperm motility.Conclusion. Further research is needed to assess whether these bioactive inflammatory mediators have potential relevance for use as targets in the treatment of male infertility.

Roles of CatSper channels in the pathogenesis of asthenozoospermia and the therapeutic effects of acupuncture-like treatment on asthenozoospermia.

Rationale: Idiopathic asthenozoospermia (iAZS) is one of the major causes of male infertility and has no effective therapeutic treatment. Understanding the potential mechanisms that cause it may be helpful in seeking novel targets and treatment strategies for overcoming the problem of low sperm motility in iAZS individuals.Methods: Computer-assisted semen analysis (CASA) was utilized to assess the sperm motility. RT-qPCR, Western blot, immunofluorescence staining, and calcium imaging analysis were performed to examine the expression and function of CatSper channels. Hyperactivation and acrosome reaction were used to evaluate the functional characteristics of epididymal sperm. In vivo fertility assay was applied to determine the fertility of rats. CatSper1 knockdown and overexpression experiments were performed to confirm the roles of CatSper channels in the pathogenesis of iAZS and the therapeutic effects of electroacupuncture (EA) treatment on AZS model rats.Results: Here, we reported a functional down-regulation of CatSper channel from CatSper1 to CatSper 4 in the sperm of both iAZS patients and ornidazole (ORN)-induced AZS model rats, and an impaired sperm function characterized by a reduction of protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in the epididymal sperm of AZS rats. Knockdown of CatSper1 in the testis tissues is sufficient to induce AZS in normal rats, and this action was validated by the reversal effects of CatSper1 overexpression. Transcutaneous electrical acupoint stimulation (TEAS) and electroacupuncture (EA) at 2 Hz frequency improve the sperm motility via enhancing the functional expression of CatSper channels in the sperm. Gene silencing CatSper1 in the sperm abolishes the therapeutic effects of 2 Hz-EA treatment on AZS rats.Conclusions: We conclude that a functional down-regulation of CatSper channel in the sperm may be a contributor or a downstream indicator for a portion of AZS, especially iAZS, while 2 Hz-TEAS or EA treatment has a therapeutic effect on iAZS through inducing the functional up-regulation of CatSper channels in the sperm. This study provides a novel mechanism for the pathogenesis of some AZS especially iAZS, and presents a potential therapeutic target of CatSper for iAZS treatment. Acupuncture treatment like TEAS may be used as a promising complementary and alternative medicine (CAM) therapy for male infertility caused by iAZS in clinical practice.

Ultrastructural Sperm Flagellum Defects in a Patient With CCDC39 Compound Heterozygous Mutations and Primary Ciliary Dyskinesia/Situs Viscerum Inversus.

Introduction: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease characterized by structural or functional motile cilia abnormalities. Up to 40 different genes seem, at the moment, to be involved in the pathogenesis of PCD. A number of ultrastructural defects have also been reported in sperm flagella, but the sperm mitochondrial membrane potential (MMP) has never been described in these cases.Aim: The aim of this study was to report the sperm MMP and ultrastructural abnormalities of the sperm flagella found in a patient with PCD and situs inversus (Kartagener syndrome) and its characterization from the genetic point of view.Methods: Transmission electronic microscopy (TEM) analysis was used to evaluate flagella ultrastructure. The genetic testing was performed by next-generation sequencing. Sperm DNA fragmentation and MMP were also evaluated by flow cytometry.Results: We report here the case of an 18-year-old male patient with PCD and situs inversus and severe oligo-astheno-teratozoospermia. TEM analysis of his spermatozoa showed an abnormal connecting piece. The mid piece appeared abnormally thickened, with cytoplasmic residue, dysplasia of fibrous sheath, loss of the outer dynein arms (ODAs), truncated inner dynein arms, and supernumerary outer fibers. The percentage of spermatozoa with fragmented DNA was normal, whereas a high percentage of spermatozoa had low MMP, suggesting an altered mitochondrial function. The genetic analysis showed the presence of c.610-2A > G, p.Arg811Cys compound heterozygous mutations in the CCDC39 gene.Conclusion: The case herein reported suggests that the high percentage of sperm with low MMP may play a role in the pathogenesis of asthenozoospermia in patients with Kartagener syndrome. In addition, we report, for the first time, the missense variant p.Arg811Cys in the CCDC39 gene in a patient with Kartagener syndrome. Although in silico analysis predicts its damaging potential, its clinical meaning remains unclear.

Outer dense fibers stabilize the axoneme to maintain sperm motility.

Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non‐motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down‐regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α‐tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α‐tubulin and protect the acetylation level of α‐tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α‐tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo‐expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.

Expression of NDUFA13 in asthenozoospermia and possible pathogenesis

Asthenozoospermia is a common cause of male infertility, which is characterized by reduced forward motility of spermatozoa. The cause and pathogenesis of asthenozoospermia are not fully understood. The purpose of this study was to investigate the expression of nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 (NDUFA13) in the spermatozoa of men with asthenozoospermia and its possible pathogenesis. Protein content of NDUFA13 in spermatozoa was measured by Western blot analysis. The results showed that NDUFA13 expression in spermatozoa was significantly lower in men with asthenozoospermic than in men with normozoospermia (P < 0.01). Immunofluorescence experiments showed that NDUFA13 was expressed predominantly in the sperm mid-piece. A lower mitochondrial membrane potential, a higher intracellular reactive oxygen species (ROS) level and more apoptotic cells were also detected in men with asthenozoospermia. NDUFA13-specific small interfering RNA was used in the mouse spermatocyte GC2-spd cell line to down-regulate the expression of NDUFA13. The knockdown of NDUFA13 in the GC2-spd cells caused a collapse of mitochondrial membrane potential, an increase in ROS level and more apoptotic cells. Our study showed that NDUFA13 deficiency may be associated with asthenozoospermia through the disturbance of spermatozoa mitochondrial membrane potential and by increasing apoptosis and intracellular ROS.

Quantification of CatSper1 expression in human spermatozoa and relation to functional parameters.

Is CatSper1 expression in human spermatozoa related to semen parameter values and sperm functions? CatSper1 expression is positively related to progressive and hyperactivated (HA) motility, [Ca(2+)]i responsiveness to progesterone but not the acrosome reaction (AR). The role of cationic channel of sperm (CatSper) in sperm functions is clear in animal models but less defined in human sperm cells. Current knowledge is mostly based on low specificity CatSper inhibitors showing agonistic and toxic effects on human spermatozoa and is thus of little help in clarifying the role of the CatSper channel in human sperm functions. CatSper1 protein expression was evaluated in 115 men undergoing semen analysis for couple infertility. CatSper1 expression was related to routine semen parameters, motility kinematic parameters and basal and progesterone-stimulated [Ca(2+)]i and the AR. CatSper1 expression was evaluated (n = 85 normozoospermic, n = 30 asthenozoospermic patients) by immunofluorescence coupled to flow cytometry leading to quantitative measurement of the percentage of ejaculated sperm cells expressing the protein. Semen analysis was evaluated according to World Health Organization guidelines. Kinematic parameters were evaluated by a computer-aided sperm analysis system. [Ca(2+)]i was measured by a spectrofluorimetric method in fura-2-loaded spermatozoa. The AR was evaluated in live sperm cells by fluorescent-labeled lectin. CatSper1 protein expression in spermatozoa was reduced in asthenozoospermic men (mean ± SD: 53.0 ± 15.5%, n = 30 versus 67.9 ± 17.1% in normozoospermic, n = 85, P < 0.01) and was significantly correlated with progressive (r = 0.36, P < 0.001), total (r = 0.35, P < 0.001) and HA (r = 0.41, P < 0.005) motility. In addition to a higher percentage of spermatozoa not expressing CatSper1, asthenozoospermic men showed a large number of spermatozoa with immunofluorescent signal localized outside the principal piece compared with those in normozoospermia. A significant positive correlation was found between CatSper1 protein expression and the increase of [Ca(2+)]i in response to progesterone (r = 0.36, P < 0.05, n = 40) but not with basal [Ca(2+)]i. No correlation was found with the AR, either basal or in response to progesterone. The study is partly descriptive. Furthermore, we cannot rule out the possibility that some round cells remain after a single round of 40% density gradient centrifugation or that this step may have removed some defective or slow swimming sperm, and therefore this preparation may not be representative of the entire sperm sample. Although our data suggest that CatSper1 may be a useful marker for infertility, and a possible contraceptive target, any clinical application is limited without further research. Our results demonstrate an association of CatSper1 expression with human sperm progressive and HA motility and provide preliminary evidence that lack of expression or mislocalization of CatSper1 in spermatozoa may be involved in the pathogenesis of asthenozoospermia. However, mechanistic studies are needed to confirm that the correlations between CatSper1 expression and sperm functions are causative. Supported by grants from Ministry of University and Scientific Research (PRIN project to E.B. and FIRB project to S.M.) and by Regione Toscana (to G.F.). L.T. was recipient of a grant from Accademia dei Lincei (Rome, Italy). The authors have no conflicts of interest to declare.

Seminal mast cells in infertile asthenozoospermic males

This work aimed to assess the possible association between the presence of seminal mast cells and asthenozoospermia. One hundred and seventy-six male subjects were investigated: group (Gr)1 (n=46) normozoospermic fertile controls, Gr2 (n=62) idiopathic asthenozoospermia, Gr3 (n=32) asthenozoospermia with scrotal varicocele and Gr4 (n=36) asthenozoospermia with leucocytospermia. Four smear slides were prepared for each semen sample to be stained with toluidine blue-pyronin to detect mast cells. A significant increase was shown in mast cell-positive samples among varicocele-associated and idiopathic asthenozoospermic patients in comparison with fertile controls. Seminal mast cells were also detected at higher frequency among smokers and in age group over 40 years. It is concluded that mast cells and their products may play a pivotal role in the pathogenesis of asthenozoospermia, possibly proposing a new goal for medical treatment of infertile males to pursue. In addition, this concept may in a way detain smoking as a cause of male infertility considering the clear abundance of mast cells in semen samples of smokers.

Incidence of sperm-tail tyrosine phosphorylation and hyperactivated motility in normozoospermic and asthenozoospermic human sperm samples

Our objective was to study the incidence of sperm-tail phosphotyrosine immunoreactivity in normozoospermic and asthenozoospermic human sperm samples, its association with sperm motion parameters, particularly hyperactivated motility, and its potential involvement in the pathogenesis of asthenozoospermia. The work was conducted as a prospective experimental study in the Sperm Biology and Andrology laboratories of the Jones Institute, a medical school-based fertility center. The study subjects were healthy fertile male donors (normozoospermic samples) and infertile patients (asthenozoospermic samples) attending the center. Recently ejaculated semen samples were washed twice to eliminate seminal plasma and a swim-up was performed to select the motile population which, in turn, was incubated up to 18 h at 37 degrees C in 3.5% human serum albumin-supplemented Ham's F10 to allow for capacitation. For evaluation, sperm aliquots were taken pre-swim-up (T0), immediately post swim-up (T1), at 6 h (T6), and 18 h (T18) of incubation. The main outcome measures were computer-analyzed sperm motion parameters and hyperactivated motility, and immunodetection of phosphotyrosine (PY)-containing proteins. During the capacitating incubation, normozoospermic samples displayed maximum motility, velocity, and hyperactivation at T6, significantly decreasing their values at T18. PY-proteins were located both at the tail and head of spermatozoa. Their expression increased progressively during the incubation, being present in about 70% of the sperm tails at T18. Asthenozoospermic samples showed an inability to respond to capacitation with an increase in motion parameters and PY-phosphorylation. At T6, both hyperactivation and PY-phosphorylation were significantly lower than in normal samples. Our results suggest that PY-phosphorylation of tail proteins is highly conspicuous in human spermatozoa, and increases its incidence in a time-dependent manner, as more sperm become capacitated. Asthenozoospermic samples displaying low percentages of motile sperm and altered motion characteristics showed a decreased incidence of PY-phosphorelated sperm. Tail protein PY-phosphorylation may be related to sperm movement, especially to hyperactivated motility and its deficiency may be associated to asthenozoospermia.

A repertoire of cytokines in human seminal plasma

The pathophysiological significance of seminal cytokines in sperm function is still controversial. We determined the repertoire of cytokines in seminal plasma obtained from men with or without abnormalities in semen and assessed the pathophysiological significance of seminal cytokines. After conventional analysis of semen samples obtained from 86 men, levels of seminal cytokines (interleukin [IL]-1α, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ, granulocyte colony-stimulating factor [G-CSF], macrophage CFS [M-CSF]) and granulocyte elastase were measured by an enzyme-linked immunosorbent assay. Leukocytospermia was defined as seminal plasma, which has ≥1000 ng/ml granulocyte elastase. Leukocytospermia was found in nine of 62 of the subjects in the normozoospermic group but in none of the 24 subjects showing abnormal sperm parameters (azoospermia, n=5; oligozoospermia, n=4; asthenozoospermia, n=15). The IL-8 level in the leukocytospermic group was significantly higher than those in the normal and oligozoospermic groups. IL-1α and TNF-α levels in the leukocytospermic group were significantly higher than those in the normal and asthenozoospermic groups. Although the G-CSF level in the leukocytospermic group was significantly higher than that in the normal group, high levels of M-CSF were detected in all groups. The IL-8 level was strongly correlated with IL-1α ( r=0.935, P<0.0001) and G-CSF ( r=0.916, P<0.0001) levels. Cytokines detected in seminal plasma are associated with the pathogenesis of leukocytospermia but not with the pathogenesis of asthenozoospermia and oligozoospermia.