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The editorial "Antioxidants in Male and Animal Reproduction: Applications and Critical Issues" discusses the impact of oxidative stress and antioxidants on male infertility, emphasizing the role of antioxidants in improving sperm quality and fertility. The article highlights the importance of antioxidants in mitigating oxidative damage during semen manipulation and cryopreservation, with a focus on both human and animal models. Additionally, the editorial underscores the need for further research on the efficacy of antioxidants and their application in male reproductive health. [Extracted from the article]

Infertility represents a significant global health challenge, affecting more than 12% of couples worldwide, and most cases of infertility are caused by male factors. Several pathological pathways are implicated in male infertility. The main mechanisms involved are driven by the loss of reduction–oxidation (redox) homeostasis and the resulting oxidative damage as well as the chronic inflammatory process. Increased or severe oxidative stress leads to sperm plasma membrane and DNA oxidative damage, dysregulated RNA processing, and telomere destruction. The signaling pathways of these molecular events are also regulated by Nuclear factor-E2-related factor 2 (Nrf2). The causes of male infertility, the role of oxidative stress in male infertility and the Keap1-Nrf2 antioxidant pathway are reviewed. This review highlights the regulatory role of Nrf2 in the balance between oxidants and antioxidants as relevant mechanisms to male fertility. Nrf2 is involved in the regulation of spermatogenesis and sperm quality. Establishing a link between Nrf2 signaling pathways and the regulation of male fertility provides the basis for molecular modulation of inflammatory processes, reactive oxygen species generation, and the antioxidant molecular network, including the Nrf2-regulated antioxidant response, to improve male reproductive outcomes. [ABSTRACT FROM AUTHOR]

This review provides details on the role of centrin in human spermatozoa and in various forms of male infertility. Centrin is a calcium (Ca2+)-binding phosphoprotein that is located in the centrioles – which are typical structures of the sperm connecting piece and play a key role in centrosome dynamics during sperm morphogenesis – as well as in zygotes and early embryos during spindle assembly. In humans, three different centrin genes encoding three isoforms have been discovered. Centrin 1, the only one expressed in spermatozoa, seems to be lost inside the oocyte after fertilization. The sperm connecting piece is characterized by the presence of numerous proteins including centrin, that deserves particular attention because, in humans, it is enriched during maturation of the centrioles. In normal sperm, centrin 1 is visible as two distinct spots in the head–tail junction; however, in some defective spermatozoa, centrin 1 distribution is altered. Centrin has been studied in humans and animal models. Its mutations may lead to several structural alterations, such as serious defects in the connective piece and, subsequently, fertilization failure or incomplete embryonic development. However, the effects of these abnormalities on male fertility have not been fully studied. Because the presence and the function of centrin in the sperm connecting piece appears important for reproductive success, additional studies are needed to bring medical benefits in resolving some cases of idiopathic infertility. [ABSTRACT FROM AUTHOR]

Liposomes have been successfully used as drug-delivery vehicles, but there are no clinical studies on improved fertility and the few reported experimental studies have been performed in animal models far from humans. The aim of this paper was to study the effects of treatment with cationic, anionic and zwitterionic liposomes on our superior mammalian model of porcine prepubertal Sertoli cells (SCs) to find a carrier of in vitro test drugs for SCs. Porcine pre-pubertal SCs cultures were incubated with different liposomes. Viability, apoptosis/necrosis status (Annexin-V/Propidium iodide assay), immunolocalisation of β-actin, vimentin, the phosphorylated form of AMP-activated protein Kinase (AMPK)α and cell ultrastructure (Transmission Electron Microscopy, TEM) were analysed. Zwitterionic liposomes did not determine changes in the cell cytoplasm. The incubation with anionic and cationic liposomes modified the distribution of actin and vimentin filaments and increased the levels of the phosphorylated form of AMPKα. The Annexin/Propidium Iodide assay suggested an increase in apoptosis. TEM analysis highlighted a cytoplasmic vacuolisation. In conclusion, these preliminary data indicated that zwitterionic liposomes were the best carrier to use in an in vitro study of SCs to understand the effects of molecules or drugs that could have a clinical application in the treatment of certain forms of male infertility. [ABSTRACT FROM AUTHOR]

This brief report concerns the role of human sperm morphology assessment in different fields of male infertility: basic research, genetics, assisted reproduction technologies, oxidative stress. One of the best methods in studying sperm morphology is transmission electron microscopy (TEM) that enables defining the concept of sperm pathology and classifying alterations in non-systematic and systematic. Non-systematic sperm defects affect head and tail in variable ratio, whereas the rare systematic defects are characterized by a particular anomaly that marks most sperm of an ejaculate. TEM analysis and fluorescence in situ hybridization represent outstanding methods in the study of spermmorphology and cytogenetic in patients with altered karyotype characterizing their semen quality before intracytoplasmic sperm injection. In recent years, the genetic investigations on systematic sperm defects, made extraordinary progress identifying candidate genes whose mutations induce morphological sperm anomalies. The question if sperm morphology has an impact on assisted fertilization outcome is debated. Nowadays, oxidative stress represents one of themost important causes of altered spermmorphology and function and can be analyzed from two points of view: 1) spermatozoa with cytoplasmic residue produce reactive oxygen species, 2) the pathologies with inflammatory/oxidative stress background cause morphological alterations. Finally, sperm morphology is also considered an important endpoint in in vitro experiments where toxic substances, drugs, antioxidants are tested. We think that the field of spermmorphology is far frombeing exhausted and needs other research. This parameter can be still considered a valuable indicator of sperm dysfunction both in basic and clinical research. [ABSTRACT FROM AUTHOR]

In this review, the role of fatty acids (FA) in human pathological conditions, infertility in particular, was considered. FA and FA-derived metabolites modulate cell membrane composition, membrane lipid microdomains and cell signaling. Moreover, such molecules are involved in cell death, immunological responses and inflammatory processes. Human health and several pathological conditions are specifically associated with both dietary and cell membrane lipid profiles. The role of FA metabolism in human sperm and spermatogenesis has recently been investigated. Cumulative findings indicate F2 isoprostanes (oxygenated products from arachidonic acid metabolism) and resolvins (lipid mediators of resolution of inflammation) as promising biomarkers for the evaluation of semen and follicular fluid quality. Advanced knowledge in this field could lead to new scenarios in the treatment of infertility. [ABSTRACT FROM AUTHOR]

Purpose: Omentin-1/intelectin (ITLN)1 is an adipocytokine with both anti-inflammatory and anti-oxidative stress properties, and little is known about its role in male reproduction. This study was aimed at exploring the relationships among omentin-1/ITLN1, semen parameters and F2-isoprostanes (F2-IsoPs), a maker of oxidative stress, in groups of patients affected by different pathologies. In addition, omentin-1/ITLN1 immunolocalization was assessed in ejaculated spermatozoa and in tissues of male reproductive system. Patients and Methods: Semen samples of infertile patients with varicocele (n = 27), genitourinary infections (n = 17), idiopathic infertility (n = 15) and fertile men (n = 21) were analyzed following WHO guidelines, and seminal plasma were used to determine omentin-1/ITLN1 by ELISA and F2-IsoP levels by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Omentin-1/ITLN1 was localized in human sperm and in the tissue of male reproductive system. Results: Considering all participants, F2-IsoP and omentin-1/ITLN1 levels were positively correlated (p = 0.000), and both these indices were negatively correlated with sperm parameters. Infertile patients showed lower sperm parameters than fertile ones; varicocele and infection groups had significantly increased levels of F2-IsoPs (both p = 0.000) and omentin-1/ITLN1 (p = 0.000 and p = 0.001, respectively). Omentin-1/ITLN1 signal was located as a spot in the connecting piece (in 43.5% of cases midpiece was also labeled) of sperm from fertile men and in cytoplasmic residue and in the entire tail in sperm of patients with varicocele and genitourinary infections. A focal omentin-1/ITLN1 immunolabelling was evident in the basal area of epididymal tubule, and a diffuse signal was present in the seminal vesicle epithelium. Conclusion: Semen omentin-1/ITLN1 originates from seminal vesicles, its levels increase in inflammatory conditions and are negatively correlated with sperm parameters. For this reason, a sort of protective role of omentin-1/ITLN1 can be postulated, as this adipokine shows anti-inflammatory properties also in many other biological systems. Graphical [ABSTRACT FROM AUTHOR]

Abstract Background SPAG16 is a critical structural component of motile cilia and flagella. In the eukaryotic unicellular algae Chlamydomonas, loss of gene function causes flagellar paralysis and prevents assembly of the “9 + 2” axoneme central pair. In mice, we have previously shown that loss of Spag16 gene function causes male infertility and severe sperm motility defects. We have also reported that a heterozygous mutation of the human SPAG16 gene reduces stability of the sperm axonemal central apparatus. Methods In the present study, we analyzed DNA samples from 60 infertile male volunteers of Western European (Italian) origin, to search for novel SPAG16 gene mutations, and to determine whether increased prevalence of SPAG16 single nucleotide polymorphisms (SNPs) was associated with infertility phenotypes. Semen parameters were evaluated by light microscopy and sperm morphology was comprehensively analyzed by transmission electron microscopy (TEM). Results For gene analysis, sequences were generated covering exons encoding the conserved WD40 repeat region of the SPAG16 protein and the flanking splice junctions. No novel mutations were found, and the four SNPs in the assessed gene region were present at expected frequencies. The minor alleles were not associated with any assessed sperm parameter in the sample population. Conclusions Analysis of the SPAG16 regions encoding the conserved WD repeats revealed no evidence for association of mutations or genetic variation with sperm motility and ultrastructural sperm characteristics in a cohort of Italian infertile males.

One of the major causes of defective sperm function is oxidative stress, which limits the fertilizing potential of these cells as the result of collateral damage to proteins and lipids in the sperm plasma membrane. On this point, a derangement of both generation and neutralization of reactive oxygen species (ROS) is a recognized cause of male infertility. Antioxidant protection in sperm has been widely investigated, as well as the sperm composition of fatty acids (FA), which represents the preferred substrate for ROS, most frequently linked to the disease-related infertility. Isoprostanes are compounds derived from free radical-mediated oxidation of FAs. As such, they are considered an index of lipid oxidative damage and lipid mediators. This article discusses the role of isoprostanes as relevant factors both to sperm FA composition and sperm membrane integrity. Additionally, isoprostane's influence on sperm quality is reviewed. With reference to male reproductive dysfunction, increasing evidence indicates isoprostanes, detectable in biological fluids or sperm membrane, as the specific index of 1) exposure to chemical etiological agents, 2) oxidative damage, 3) reduced antioxidant response, and 4) sperm immaturity. OS: oxidative stress; ROS: reactive oxygen species; PUFAs: polyunsaturated fatty acids; ARA: arachidonic acid, F2-IsoPs; F2-isoprostanes, PLA2: phospholipase A2; NADPH: nicotinamide adenine dinucleotide phosphate; IVF: in vitro fertilization [ABSTRACT FROM AUTHOR]

[Display omitted] • Apelin was found in human spermatozoa. • Apelin was not detected in seminal fluid. • Apelin levels were increased in spermatozoa from men with varicocele and infections. • Apelin and APJ-R were immune-detected in sperm tail, cytoplasmic residues and post-acrosomal sheath. • Apelin and APJ-R were evident in Leydig cells and in a less extent inside the seminiferous tubule. Apelin/APJ receptor (R) is involved in many oxidative stress-induced pathological conditions. Since this system is not yet explored in male reproduction, we studied apelin/APJ-R in human semen and testis. Semen of 41 infertile patients with varicocele, genitourinary infections, unexplained infertility and 12 fertile men was analysed (WHO guidelines, 2021). Apelin was quantified by ELISA in seminal fluid and spermatozoa, interleukin (IL)-1β in seminal fluid. Apelin/APJ-R were immunolocalized in spermatozoa and testis. Apelin was present in spermatozoa and its levels were negatively correlated with normal sperm morphology% (r = -0.857; p < 0.001), and positively with IL-1β levels (r = 0.455; p < 0.001). Apelin and IL-1β concentrations were increased in patients' samples with varicocele (apelin p < 0.01; IL-1β p < 0.05) and infections (apelin p < 0.01; IL-1β p < 0.001). By logistic regression analysis, apelin (OR 1.310; p = 0.011) and IL-1β (OR 1.572; p = 0.005) were predictors of inflammatory diseases (varicocele, infections). Apelin and APJ-R immunofluorescence labels were weak in sperm tail of fertile men and intense along tail, cytoplasmic residues and post-acrosomal sheath of sperm from infertile men. In testis, apelin and APJ-R labels were evident in Leydig cells and weak inside the seminiferous tubule. Apelin/APJ-R system is present in human spermatozoa and testicular tissue and probably involved in human fertility. [ABSTRACT FROM AUTHOR]

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility’s therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore thein vitroeffects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology.Abbreviations:transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP [ABSTRACT FROM AUTHOR]

This study was aimed to address the possibility that Helicobacter pylori infection may play a detrimental role in semen quality of men with idiopathic infertility. Infection by H. pylori and by strains expressing CagA was determined in 80 male infertile patients by Western blotting and ELISA. Semen analysis was performed by light microscopy and transmission electron microscopy quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). Systemic levels of IL-6 and TNF-alpha were evaluated. Infertile patients infected with H. pylori showed a low sperm quality respective to uninfected patients. Particularly, in CagA-positive patients we observed a significant reduction in sperm motility and in the fertility index, while apoptosis and necrosis were increased. In these patients, the means of systemic TNF-alpha levels were higher than those of uninfected patients. The negative influence of CagA-positive H. pylori infection on sperm quality may help to understand the role of chronic infections in reproductive disorders. [ABSTRACT FROM AUTHOR]

Purpose To verify the prevalence of semen bacterial contamination and whether the contamination could decrease sperm quality. Methods Spermiogram, semen culture, and sperm transmission electron microscopy (TEM) analysis were performed. TEM data were elaborated using a mathematical formula that calculates a fertility index (FI)-able to define patients as fertile or infertile-and the percentage of sperm apoptosis, immaturity and necrosis. We aligned the amino acid sequence of beta-tubulin with protein of the most frequent species isolated from semen. Results Patients were divided according to the contaminating species; in each group, we observed fertile individuals, in whom the semen quality was similar to that of controls and infertile men whose sperm quality was significantly decreased, in terms of motility, FI, apoptosis and necrosis. Partial homology between β-tubulin and bacterial proteins was observed. Conclusion Sperm bacterial contamination is quite frequent and could contribute to the deterioration of the sperm quality of infertile men. [ABSTRACT FROM AUTHOR]

Background & objectives: Emotional stress plays a detrimental role on fertility. In this study male patients with idiopathic infertility were selected after evaluation of psychological stress to evaluate a positive effect of a stress therapy on their semen quality. Methods: A total of 20 patients with infertility were enrolled in the study and randomly divided in two groups. Ejaculates were examined by light and transmission electron microscopy ITEM;. Meiotic segregation was also investigated by fluorescence in situ hybridization (FISH). Ten patients were treated with Conveyer of Modulating Radiance (CRM) therapy and sperm characteristics and meiotic segregation were evaluated again three months at the end of treatment. Results: TEM data showed that, among sperm pathologies, necrosis and apoptosis were higher and the number of "healthy" sperm was significantly reduced in both groups of stressed men compared to reference values. The number of "healthy" sperm was significantly higher in the treated group after therapy, indicating a recovery of sperm quality, although no significant decrease in sperm pathologies was observed. FISH analysis showed that the mean frequencies of sex chromosomes disomies and diploidies significantly decreased after stress therapy. Interpretation & conclusions: The effects induced by stress also seem to include meiotic and structural alterations in sperm cells. The spermatogenic process was improved after a cycle of CRM therapy indicating that stress is an additional risk factor for idiopathic infertility. [ABSTRACT FROM AUTHOR]

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI–MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p < 0.001) and negatively correlated with mature sperm chromatin (p < 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen. [ABSTRACT FROM AUTHOR]

• The relevance of isoprostanoids in human semen quality was investigated. • F 2 -dihomo-IsoPs was significantly higher in varicocele group respect to fertile men. • F 2 -IsoPs positively correlated with F 2 -dihomo-IsoPs in the whole population. • F 4 -NeuroPs positively correlated with sperm necrosis in idiopathic infertility group. • Isoprostanoid semen levels appear to be associated with male infertility. The aim of this paper was to investigate the relevance of isoprostanoids i.e., F 2 -isoprostanes (F 2 -IsoPs), F 4 -neuroprostanes (F 4 -NeuroPs) and F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs) in semen quality. Isoprostanoid levels were detected in semen of fertile and infertile men with varicocele or idiopathic infertility. Semen quality was assessed by light microscopy and transmission electron microscopy; the relationships between isoprostanes and semen parameters were also explored. F 2 -IsoPs levels were significantly different in the varicocele group compared to idiopathic infertile group and fertile men (P < 0.01 and P < 0.001 respectively). Moreover, F 2 -dihomo-IsoP values were significantly higher in varicocele group respect to fertile men (P < 0.05). No significant statistical differences were found regarding F 4 -NeuroP concentrations. In the whole population, F 2 -IsoPs positively correlated with F 2 -dihomo-IsoPs and both isoprostanoids showed a positive correlation with immaturity and a negative correlation with sperm motility. F 2 -IsoP levels were positively correlated with the percentage of immaturity in infertile varicocele groups (P < 0.01) whereas a significant relationship between F 4 -NeuroP values and the percentage of sperm necrosis was shown in idiopathic infertility group (P < 0.01). A significant negative correlation of F 4 -NeuroPs with sperm morphology was detected in infertile varicocele subjects (P < 0.05). This study suggests that isoprostanoid semen levels appear to be associated with male infertility being related to the sperm quality and confirming the important role of fatty acids profiling in human sperm maturation. [ABSTRACT FROM AUTHOR]

Objective: To characterize the association of two systematic sperm defects. Design: Case report. Setting: University, Interdepartmental Centre for Research and Therapy of Male Infertility. Patient(s): Patient 1, 42 years old, and patient 2, 38 years old, both with severe asthenozoospermia. Intervention(s): Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, immunocytochemistry for tubulin, and fluorescence in situ hybridization (FISH) for chromosomes 18, X, and Y. Main Outcome Measure(s): Admixture of dysplasia of the fibrous sheath (DFS) and head-tail misalignment up to acephalic sperm detected by microscopic methods. Result(s): In both patients, DFS was present in incomplete form and was associated with acephalic sperm and abnormal head-tail attachment. In patient 2, spermatozoa were also affected by necrosis that may cause fragmentation leading to short flagella; submicroscopic examination allowed defining only the origin of these “stumpy” tails. Immunofluorescence confirmed the sperm alterations. FISH revealed an altered frequency of diploidy and disomy in patient 2 and a slight increase in diploidy in patient 1. Conclusion(s): The importance of ultrastructural sperm evaluation for correct identification of sperm pathologies is evident, particularly regarding assisted reproduction technology and genetic risk assessment. [ABSTRACT FROM AUTHOR]

Objective: To characterize a novel flagellar defect involving 98% of sperm tails. Design: Case report. Setting: Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. Patient(s): A 45-year-old infertile man with severe asthenozoospermia. Intervention(s): Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization (FISH) for chromosomes 9, 16, 18, X, and Y. Main Outcome Measure(s): Flagellar abnormalities detected by microscopical methods. Result(s): An apparent heterogeneity was observed: extremely elongated tails prone to ruptures; coiled tails at different levels with a strongly rolled axoneme or with a curl in the final flagellar segment; and V-shaped, isolated, bent tails. Transmission electron microscopy revealed the presence of normal heads, disorganized flagellar structures, and dynein deficiency. The FISH analysis was normal. Conclusion(s): We report a new sperm defect, characterized by abnormal elongation of the tail, which was prone to ruptures at different levels, concomitant with coiled tails, which were impossible to measure in length. This defect remained constant in different examined ejaculates and applied to the entire sperm population of a sterile man, the son of first-degree cousins, indicating a potential genetic origin. [Copyright &y& Elsevier]