In recent years, the application of LC/LAC in the treatment of oligoasthenoteratozoospermia of unknown origin has been proposed. Among them, the treatment of idiopathic male infertility is still controversial, and most of the clinical treatment still depends on empirical methods (Agarwal et al., 2020). Besides, NAC had a significantly greater increase in sperm concentration and ejaculate volume compared with the placebo group, and there was no significant difference in LC/LAC. The results indicated that LC/LAC and NAC had a considerable improvement in sperm motility and normal morphology compared with the placebo group. This meta-analysis aimed to confirm the efficacy of LC/LAC and NAC in men with idiopathic asthenozoospermia from the perspective of EBM. Two RCTs including 311 patients (155 patients in the NAC group and 156 patients in the placebo group) were used to estimate the level of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin. A series of z-stacks images were taken using confocal microscopy (Olympus fluoview FV3000, Tokyo, Japan) and the glial morphology was analyzed a 3D construction using Imaris software 7.0 (Bitplane, Oxford instrument company, AG, Zurich, Switzerland). Golgi staining was conducted in accordance with the instructions for the FD Rapid Golgistain™ Kits (FD NeuroTechnologies, Baltimore, MD) on unperfused brain tissue 62,63. To examine dendritic spine density, the brains were transferred to Golgi staining solution. Sections were treated with copper sulfate in ammonium acetate buffer to quench endogenous auto-fluorescence of the brain tissue . To determine cell apoptosis, the brain sections were processed following the commercial protocol for standard terminal deoxynucleotidyl transferase mediated biotinylated UTP nick end labelling (TUNEL) using the TACS 2TdT-Fluor in situ apoptosis detection kit (TACSÒ, Trevigen, R and D Systems, Minneapolis, MN, USA). Following this the brains were sliced by cryosection (Leica CM1950, Leica Biosystem Nussloch GmbH, Nussloch, Germany) to produce slices 20 μm thick for glial morphology and 10 μm thick for apoptosis. The whole brain was homogenized and mixed with 1.1 ml of 10% trichloroacetic acid (TCA) containing butylated hydroxytoluene (BHT) (50 ppm), heated at 90 °C for 30 minutes and cooled down to room temperature. Plasma glucose, cholesterol, triglyceride and HDL levels were measured using a colorimetric assay kit (Biotech, Bangkok, Thailand and Biovision, California, USA). The rats were fasted for five hours before they were anesthetized using isoflurane. Then rats were deeply anesthetized with isoflurane and terminated by decapitation. After acclimatization for a week, rats were fed on a normal diet (ND; 19.7% E fat) for 34 weeks. The dysbiosis of gut microbiota and cognitive decline were also reported in aging men , who developed testosterone deprivation. We found that ORX rats had significantly increased numbers of apoptotic cells as indicated by the increased number of TUNEL positive cells (Figure 5B and G). To determine hippocampal apoptosis, brain sections were further investigated using the TACS 2TdT-Fluor in situ apoptosis detection kit. All of these findings demonstrated that testosterone deprivation altered microglial and astrocyte morphology, and these changes were attenuated by all treatments. This increment was attenuated by treatment with NAC, inulin, or the combined therapy (Figure 4J). There are a few reports on the effects of dietary antioxidant supplementation on sperm DNA integrity. Significant reduction of serum LH, FSH and, significant increase of serum testosterone levels was observed after treatment with NAC compared to before treatment NAC oral supplementation may improve sperm parameters and oxidative/antioxidant status in infertile males. NAC is unlikely to significantly decrease testosterone levels and may even offer indirect support for hormonal balance through its antioxidant effects. Other studies haven’t found any significant changes in testosterone levels with NAC supplementation. Plasma was collected for biochemical analysis including plasma glucose, insulin, total cholesterol, HDL, triglyceride, and testosterone levels 28,29. These findings suggest that prebiotics, antioxidant and the combined prebiotics and antioxidant therapy could provide the beneficial effect on the brain similar to the use of testosterone replacement therapy in testosterone-deprived people. Relationship between sperm DNA fragmentation and sperm motility or abnormality. Comparison of FSH, LH, Testosterone, and prolactin before and after treatment with NAC. Red-staining sperm have intact DNA, while green-staining sperm have DNA fragmentation. Comparison of semen parameters before and after treatment with NAC. Comparison of age, duration of infertility, weight, height, and BMI before and after treatment with NAC. Seminal total antioxidant capacity (TAC) was measured using a commercially available kit (Zell Bio GmbH, Wurttemberg, Germany).
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