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This study aims to investigate the role of ferroptosis, an iron-dependent form of regulated cell death, in male infertility. The motivation behind this research stems from the increasing recognition of oxidative stress and iron metabolism dysregulation as critical factors in male reproductive health. In this study, 28 infertile patients (grouped by the presence of urogenital infections or varicocele) and 19 fertile men were selected. Spermiograms were performed by light microscopy (WHO, 2021). Testosterone, ferritin, transferrin-bound iron, transferrin, and F 2 -isoprostanes (F 2 -IsoPs) were detected in seminal plasma. Glutathione peroxidase 4 (GPX4) and acyl coenzyme A synthetase long chain family member 4 (ACSL4) were also assessed in sperm cells using enzyme-linked immunosorbent assays (ELISA). All the variables were correlated (statistically significant Spearman's rank correlations) in the whole population, and then the comparison between variables of the different groups of men were carried out. Seminal ferritin and transferrin positively correlated with seminal F 2 -IsoPs, which had positive correlations with ACSL4 detected in sperm cells. Ferritin and ACSL4 negatively correlated with the seminal parameters. No correlation was detected for GPX4. Comparing the variables in the three examined groups, elevated levels of ACSL4 were observed in infertile patients with urogenital infections and varicocele; GPX4 levels were similar in the three groups. These results suggested a mechanism of ferroptosis, identified by increased ACSL4 levels and the occurrence of lipid peroxidation. Such events appear to be GPX4-independent in reproductive pathologies such as varicocele and urogenital infections.

F 2 -isoprostanes (F 2 -IsoPs), byproducts of arachidonic acid oxidation, are one of the most reliable indices for assessing lipid peroxidation in vivo. This study aimed at evaluating the seminal F 2 -IsoP level in 147 patients with different reproductive conditions (varicocele, urogenital infection, idiopathic infertility) and 45 fertile controls to establish a cut-off value discriminating physiological and pathological ranges. Semen analyses were performed following WHO guidelines; F 2 -IsoP levels were measured by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Considering the whole group of patients, F 2 -IsoPs correlated negatively with normal morphology (r = -0.283, p < 0.01), viability (r = -0.245, p < 0.01), total progressive motility (r = -0.309, p < 0.01) and rapid motility (r = -0.535, p < 0.01). The area under the ROC curve for F 2 -IsoP levels was 0.839, indicating a good performance of the test; the Youden index showed a cut-off value of 29.96 ng/mL. Fertile men (except one) were distributed in the group of patients with F 2 -IsoP level < 29.96 ng/mL. Varicocele and urogenital infection groups showed the highest levels of F 2 -IsoPs in semen. For the first time, a cut-off for F 2 -IsoPs is identified in human semen. It allows discriminating different male infertility conditions by the semen F 2 -IsoP amounts, as an additional parameter for clinical evaluation.

Cytokines are physiological seminal components and their abnormal levels, reported in different pathological conditions, negatively influence the sperm function. We analysed the relationship between interleukin (IL)-6 and IL-33 levels and lipid peroxidation (LPO), measured both in semen and sperm lysate, in 44 human semen samples. The semen analysis was performed following the WHO guidelines. Seminal IL-6 and IL-33 concentrations were assessed by ELISA and LPO was evaluated measuring malondialdehyde (MDA) both in seminal plasma and viable spermatozoa. Two small groups of patients with varicocele and infection were extrapolated from the cases analysed and the variables compared with those of a group of control. IL-33 levels were undetectable in all samples and IL-6 levels were positively correlated with both seminal and sperm MDA concentrations (p < 0.01) and negatively with sperm parameters (p < 0.01). Seminal and sperm MDA levels were both negatively correlated with sperm parameters (p < 0.01). IL-6 and semen MDA showed an exponential positive relationship, whereas MDA values measured in viable spermatozoa were low until IL-6 amount reached a concentration of >30 pg/mL, rising consistently. By comparing the variables in the groups, we confirmed that a high IL-6 concentration in the varicocele and infection groups was concomitant with an increase of seminal MDA levels, but also with MDA measured in viable spermatozoa, which represents the novelty of this study. We identified the IL-6 threshold, beyond which sperm MDA concentration rises concomitantly with the increase of IL-6 concentration. Other studies are needed, considering the increasing number of patients with different pathologies affecting male infertility., (© 2021. The Author(s).)

The evaluation of the seminal plasma plays a relevant role in the definition of male infertility and in assisted reproduction outcomes; for this reason, it would be recommended to find biochemical markers able to characterize sperm pathology. In this study, 53 infertile patients (grouped by the presence leukocytospermia, idiopathic infertility, or varicocele) and 10 fertile men were selected. Spermiogram was performed by light microscopy, and sperm ultrastructure was evaluated by transmission electron microscopy (TEM) mathematically elaborated. Testosterone (TESTO), estradiol (E2), ferritin (FERR), iron (Fe), transferrin (TRSF), triglycerides (TRG), cholesterol (CHOL), and isoprostanes (F 2 -IsoPs) were detected in seminal plasma. Sperm characteristics and biochemical components were correlated by Spearman's rank correlation coefficient in the whole population and in each group. The levels of TESTO and E2 were positively correlated with sperm quality in particular, and E2 was correlated with fertility index expressing the number of sperm free of ultrastructural defects evaluated by TEM. On the contrary, the indices of iron metabolism (FERR, Fe, and TRSF) were positively associated with low sperm quality and sperm necrosis, particularly in leukocytospermia and varicocele groups, pathologies in which an inflammatory status and oxidative stress condition are present. The study of the seminal plasma composition deserves attention because the levels of the various components seem to be associated with specific reproductive pathologies.

In the last two decades, the human sperm count linearly decreased in Western countries. Health problems, lifestyle, pollutants, and dietary behaviours are considered as the main risk factors, and the unbalance of dietary n -6/ n -3 fatty acids is one of the most relevant. The aim of the present research is to study the effect of different dietary sources of n -3 polyunsaturated fatty acids (PUFA) on reproductive traits using rabbit buck as the animal model. Fifteen rabbit bucks were assigned to three experimental groups: the control group, the FLAX group fed 10% extruded flaxseed, and the FISH group fed 3.5% fish oil for 110 days (50-day adaptation and 60-day experimental periods). Semen samples were collected weekly, whereas blood was collected every two weeks for the analytical determination of semen traits, oxidative status, fatty acid profiles, isoprostanes, neuroprostanes, and the immunocytochemistry of docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) acid. At the end of the trial, the rabbits were killed and the testes were removed and stored for the analysis of fatty acid profile and immunocytochemistry. Results showed that dietary administration of n -3 PUFA improved the track speed of the sperm and increased the n -3 long-chain PUFA mainly confined in the sperm tail. Seminal plasma increased the thiobarbituric reactive substances (TBARs) by three times in the groups fed supplemental n -3, whereas the F 2 -isoprotanes (F 2 -IsoPs) and F 4 -neuroprostanes (F 4 -NeuroPs) were lower and higher, respectively, in both supplemented groups than in the control. The testes and sperm showed a higher DHA and EPA distribution in rabbits from the n -3 supplemented groups compared with the control. In conclusion, supplemental dietary n -3 PUFA improved sperm motion traits and resulted in an enrichment of membrane fatty acid in the sperm and testes of the rabbits. However, such an increased amount of PUFA negatively affected the sperm oxidative status, which was mainly correlated with the generation of F 4 -NeuroPs with respect to F 2 -IsoPs. Accordingly, the latter cannot be considered a good marker of oxidation when diets rich in n -3 PUFA are provided., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Cesare Castellini et al.)

Resistin is an adipokine involved in inflammation and able to induce the expression of other proinflammatory cytokines. It is known that, in human semen, resistin is correlated with inflammatory cytokines and sperm quality. The aim of this prospective study was to explore the potential relationship between resistin, lipid peroxidation (LPO), catalase (CAT) activity, and reduced and oxidized glutathione (GSH/GSSG) ratio in semen samples of infertile patients with leukocytospermia (no. 19), infertile patients with varicocele (no. 17), and fertile men (no. 17). Semen analysis was performed following the WHO guidelines, and sperm apoptosis and necrosis were evaluated with annexin V/propidium iodide assay. Seminal plasma samples were used to determine resistin levels by an immunological method, MDA concentration by a HPLC analysis with UV detection, GSH/GSSG ratio by an enzymatic method, CAT activity by a spectrophotometric method. The results showed that, in both groups of infertile patients, semen parameters were significantly reduced ( P < 0.001) and sperm apoptosis and necrosis percentages were increased. Resistin levels were significantly higher in leukocytospermia and varicocele groups ( P < 0.001 and P < 0.01, respectively) as well as MDA concentration ( P < 0.001) compared to controls. The MDA level was also significantly increased in the leukocytospermia group versus the varicocele group ( P < 0.05). The GSH/GSSG ratio was higher in fertile controls than the leukocytospermia group ( P < 0.05) and the varicocele group ( P < 0.001) and in the leukocytospermia group versus the varicocele group ( P < 0.05). Both the leukocytospermia and varicocele groups showed increased values of CAT activities ( P < 0.001) than controls. Briefly, the correlation between variables, calculated in the whole patient population, showed that resistin levels positively correlated with MDA levels, CAT activity, sperm apoptosis, and necrosis and negatively with sperm parameters and GSH/GSSG ratio. These results support an active role of resistin in an inflammatory process causing LPO, increase of CAT activity, and decrease of GSH/GSSG ratio in seminal plasma of infertile men vs. fertile controls., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the reported research., (Copyright © 2019 Elena Moretti et al.)

Ghrelin and obestatin are involved in many biological functions including reproduction. Growing evidences suggest that both peptides could exert protective and antioxidant activities. In this study, the relationships between ghrelin/obestatin, interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α ), malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), expressed as the GSH/GSSG ratio, catalase (CAT), and semen parameters in infertile patients with varicocele or leukocytospermia and controls were investigated. Fifty-six infertile patients (32 with leukocytospermia and 24 with varicocele) and 14 controls participated in this study. Semen analysis was performed following the WHO guidelines. Apoptotic and necrotic sperm were scored by annexin V/propidium iodide assay. Seminal plasma samples were used for the following determinations: ghrelin, obestatin, IL-6, and TNF- α were measured by an immunological method, GSH/GSSG by an enzymatic method, and CAT by spectrophotometric determination. With respect to controls, both the leukocytospermia and varicocele groups showed altered sperm parameters, significantly increased sperm apoptosis ( P = 0.009 and P = 0.011, respectively), IL-6 ( P = 0.0001 and P = 0.004, respectively), and TNF- α levels ( P = 0.0001 and P = 0.002, respectively); both groups had significantly decreased levels of ghrelin ( P = 0.0001), obestatin ( P = 0.0001 and P = 0.006, respectively), and GSH/GSSG ratio ( P = 0.003 and P = 0.0001, respectively). The MDA concentration was significantly increased in the leukocytospermia group vs. controls ( P = 0.0001), in the varicocele group vs. controls ( P = 0.011), and in the leukocytospermia group vs. the varicocele group ( P = 0.008). CAT activity was augmented in both the leukocytospermia and varicocele groups ( P = 0.0001) vs. controls. The results indicate that both ghrelin and obestatin may play a protective role in human semen and this effect is probably due to their antioxidant properties., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2019 Lucia Micheli et al.)

The aim of the study was to assess whether abnormal levels of seminal biochemical components could be associated with semen alterations and infertility. In this study, 92 human ejaculates from selected men were analyzed. Albumin, estradiol, ferritin, total proteins (TP), folic acid (FA), vitamin B12, alkaline phosphatase (ALP), creatine kinase (CK), gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase were evaluated. Semen parameters and biochemical components of the 92 samples were correlated bySpearman's rho coefficient. Albumin showed a negative correlation with sperm progressive motility and vitality (P < 0.05), CK with sperm concentration and vitality (P < 0.05), ferritin with sperm morphology (P < 0.05). FA negatively correlated with sperm concentration (P < 0.05) and GGT with sperm motility (P < 0.05). The values of biochemical components were compared for each semen parameters (concentration, motility, morphology, vitality) in samples ≤5th percentile with those >5th percentile and in patients with/without leukocytospermia, presence/absence of germ cells, increased/normal viscosity by Mann Whitney U test. The albumin (P < 0.001) and TP (P < 0.05) levels and the GGT activity (P < 0.001) were significantly higher in patients with sperm motility ≤5th percentile. Patients with sperm vitality ≤5th percentile showed increased albumin concentration (P < 0.01) and the CK activity (P < 0.001). The presence of germ cells in semen was concomitant with high values of ferritin (P < 0.01); the ALP activity (P < 0.01) and FA level (P < 0.001) were decreased in hyperviscous semen. The FA and estradiol levels were significantly decreased in the smoker group compared to those measured in the non-smoker group. Subjects were grouped in infertile patients and men with unknown reproductive potential. Infertile patients albumin and ferritin were significantly increased (P < 0.05). This study suggests that some biochemical components may be associated with human seminal pathological conditions. Abbreviations: ALP: alkaline phosphatase; LDH: lactate dehydrogenase; GGT: γ-glutamyl transferase; CK: creatine kinase; ACP: acid phosphatase; ALB: albumin; TP: total proteins; FERR: ferritin, E: estradiol; FOL: folic acid; B12: vitamin B12; FSH: follicle stimulating hormone; LH: luteinizing hormone; T: testosterone; BMI: body mass index; WHO: World Health Organization.

Polyunsaturated fatty acid damages lead to alterations in sperm function. This study aimed to investigate the involvement of F 2 -isoprostanes (F 2 -IsoPs), oxidized lipid products from arachidonic acid, in sperm quality impairment. For this purpose, F 2 -IsoP levels in semen and F 2 -IsoP localization in spermatozoa were explored in infertile subjects affected by idiopathic infertility or varicocele, as well as in fertile men. As compared to fertile men, in the idiopathic infertility and varicocele groups, sperm concentration, motility, morphology, viability, and fertility index were significantly lower and the mean scores concerning sperm apoptosis, necrosis, and immaturity were significantly higher. The idiopathic infertile group showed a reduction in sperm motility and fertility index, as well as an increase of apoptosis and necrosis percentages, in comparison to the varicocele group. The varicocele group showed the highest levels of F 2 -IsoPs, a significant increase of sperm immaturity, and a significant correlation between F 2 -IsoP levels and sperm immaturity. 8-Iso Prostaglandin F 2 α , biomarker of in vivo F 2 -IsoP, was clearly localized in sperm midpiece and cytoplasmic residues. Data show that F 2 -IsoP formation is relevant in semen and sperm from infertile patients with varicocele and high percentage of immaturity, suggesting that a correct fatty acid integrity is needed for sperm maturation.

Objective: To explore the relationships between seminal interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) with semen parameters, sperm apoptosis, and necrosis in subjects infected by Helicobacter pylori (HP) expressing and not expressing CagA., Methods: In 109 selected patients, HP infection and seminal levels of IL-6 and TNF-α were determined using enzyme-linked immunosorbent assays. Western blotting was used to detect antibodies to CagA. Semen parameters were determined following World Health Organization guidelines and sperm apoptosis and necrosis by annexin V and propidium iodide assay., Results: Twenty-eight subjects were infected by HP (HP+); among them, 12 were CagA seropositive (CagA+) and 16 were negative (CagA-). Eighty-one men were HP seronegative (HP-). Semen TNF-α and IL-6 concentrations were increased in HP+ vs HP- groups (TNF-α: 41 pg/mL vs 27 pg/mL; IL-6: 11 pg/mL vs 5 pg/mL; P <.01). In comparison to the HP- group, CagA+ group showed reduced sperm motility (24% vs 32% motile sperm; P <.05), enhanced necrosis (33.5% vs 21% necrotic sperm; P <.05), and increased cytokines levels (TNF-α: 46 pg/mL vs 27 pg/mL; P <.01; IL-6: 17.5 pg/mL vs 5 pg/mL; P <.01). Sperm motility of CagA+ group was lower vs CagA- group (24% vs 36.5% motile sperm; P <.05). Both IL-6 and TNF-α levels positively correlated with the percentage of necrotic sperm (P <.001)., Conclusion: CagA+ HP infection increases semen levels of inflammatory cytokines, which may reduce sperm motility and determine sperm damage and contribute to reduce the reproductive potential in men., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Helicobacter pylori (HP) infection might have negative effects on the semen parameters of infertile men. We explored the possibility that this infection can influence systemic and seminal levels of ghrelin and obestatin, hormones mainly produced by the stomach. Ghrelin and obestatin exert many activities, including the regulation of reproductive biology, and are present in many organs and fluids, including human semen. In 78 men, we determined HP infection and cytotoxin-associated gene A protein (CagA) status by enzyme-linked immunosorbent assay and Western blotting, semen quality following World Health Organization guidelines, and ghrelin and obestatin levels in the blood stream (47 subjects) and semen by radioimmunoassay. Twenty-seven men (34.6%) were infected (HP+) and 11 out of 27 infected men (40.7%) were seropositive for CagA (CagA+). Sperm motility was significantly reduced in HP+/CagA+ men compared with HP+/CagA- men (P < .01). Ghrelin semen levels were decreased in HP+ men compared with uninfected individuals (P < .05), whereas they were increased in HP+/CagA+ men compared with HP+/CagA- subjects (P < .01). Ghrelin semen concentrations in HP+/CagA- men were lower than those measured in uninfected subjects (P < .001). Semen obestatin concentration was increased, in a nonsignificant manner, in HP+/CagA+ men. The obestatin levels were approximately 4 times higher than those of ghrelin in semen and approximately half the levels of ghrelin in serum specimens of all the analyzed groups. No significant differences were found in systemic levels of ghrelin and obestatin in HP+ to uninfected individuals. HP infection may influence the ghrelin seminal concentrations, probably as a response to a negative effect of infection on the semen quality.

Aquaporins (AQPs) are a family of 13 small hydrophobic trans-membrane proteins expressed in numerous tissues and cells. Some AQPs work as strict water channels, others are permeable to a range of substances, including glycerol. In the male reproductive system their localization in testis, efferent ducts, epididymis, and spermatozoa has been described. We studied the distribution of AQP7 in ejaculated human sperm and the relationship between AQP7 labeling and sperm characteristics. Semen samples from 33 men were examined by light and transmission electron microscopy (TEM). TEM data were quantified using a mathematical formula that calculates a fertility index (FI) and the percentages of sperm apoptosis, immaturity, and necrosis. Immunocytochemistry with a polyclonal antibody anti-AQP7 was performed on the sperm samples. Normal sperm were labeled in the pericentriolar area, midpiece, equatorial segment, and weakly in the tail (grade 1). Abnormal sperm showed a diffuse low intensity of fluorescence evident in the cytoplasmic residues, coiled tails, in the entire head, and acrosome (grade 2). A high number of motile sperm obtained by swim up were labeled in a dotted manner in the mitochondria. A significant positive correlation was found between the spermatozoa with AQP7 grade 1 labeling and the percentage of normal form (P<0.008), progressive motility and FI (P<0.005); a negative correlation was noted with the percentages of cytoplasmic residues (P<0.010) and immaturity (P<0.006) and coiled tails (P<0.012). The link between AQP7 distribution and sperm morphology and the particular dotted labeling in swim up selected motile sperm are novel and deserve additional studies.

Objective: To investigate the occurrence of ghrelin and obestatin in human semen., Design: Prospective study., Setting: University, center for research and therapy of male infertility., Patient(s): 112 consecutively selected men., Intervention(s): Family history, clinical and physical examination, radioimmunoassay for ghrelin and obestatin determinations, semen analysis, annexinV/propidium iodide assay., Main Outcome Measure(s): Ghrelin and obestatin detected in the semen and relationships with semen parameters and conditions influencing semen quality (smoking, varicocele, ex varicocele, leukocytospermia)., Result(s): The levels of both peptides in semen were higher versus serum. Linear correlations between ghrelin and obestatin levels in serum and in semen were observed. Serum ghrelin levels were negatively correlated with the men's ages. Semen obestatin levels were positively correlated with sperm concentration and motility. Obestatin levels were decreased in the semen of smokers and in the presence of leukocytospermia., Conclusion(s): This is the first study on the presence of obestatin in human semen and its relationship with sperm concentration and motility, suggesting a possible role of the peptide in controlling cell proliferation and survival. Further investigations are required to explore the exact role of obestatin and ghrelin in human semen., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the presence of the different isoforms of tocopherol (T) in seminal plasma (P) and in the sperm fractions of individuals with abnormal (group 1) and normal (group 2) sperm parameters; the relationships between these isoforms and conventional sperm parameters were also explored. Two vitamin E homologues, α-T and δ-T, were identified in the semen of all participants. Although α-T and δ-T concentrations were similar in the semen of the 2 groups, group 1 showed a lower α-T ratio (S/P) (0.90 vs. 1.20, P < .001) and δ-T ratio (0.86 vs 1.13, P = .007) than group 2. In addition, both T ratios were correlated with the percentage of viable cells, detected by eosin staining. These results suggested that α-T and δ-T are not homogeneously distributed in the semen fractions; in normal semen they are more concentrated in the sperm membrane, whereas in abnormal semen the damaged sperm cells may release both Ts in the plasma. To verify whether sperm membrane breakage could alter α-T and δ-T distribution between the seminal plasma and the spermatozoa, normal sperm samples were sonicated; after sonication a consistent sperm plasma membrane fragmentation, highlighted by transmission electron microscopy, and a concomitant release of α-T and δ-T were observed. In conclusion, the Ts coupled directly with the sperm membrane seem to play the main protective role in the semen, and the release of α-T and δ-T in the P fraction is probably an index of lower antioxidant power and sperm quality.

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 beta-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.

Cryptorchidism is a pathological condition defined as the failure of the testis to descend into the scrotum, the location of the cryptorchid testis can be in the inguinal canal or in the prescrotal and abdominal area, sometimes resulting in atrophic seminiferous tubules. The aim of this study was to analyze semen quality of men who underwent orchidopexy for unilateral or bilateral cryptorchidism during childhood. Semen quality was investigated by light microscopy to evaluate sperm concentration and motility. Sperm morphology was performed by transmission electron microscope (TEM), and the data were mathematically elaborated. The presence of Y microdeletions was investigated by polymerase chain reaction. The effect of cryptorchidism on meiosis was explored by fluorescence in situ hybridization (FISH). The incidence of azoospermia was higher in the group with bilateral compared with unilateral cryptorchidism, and semen parameters were better in the unilateral group. Sperm pathologies detected by TEM indicated a severe deterioration of sperm quality in both groups. Necrosis and apoptosis appeared to be the most frequent pathologies, and their values reached statistical significance compared with those from fertile controls. The presence of chromosome Y microdeletions in patients with cryptorchidism and severe spermatogenetic defects is controversial. No microdeletions were found in this study. FISH values indicated that the mean percentage of gonosome disomies and diploidies were generally out of normal range, indicating a severe disturbance of meiotic segregation. The effects induced by cryptorchidism resolved in childhood seem to include a spermatogenetic impairment, leading to recommendation of detailed ultrastructural and chromosomal sperm analyses before undertaking assisted reproductive techniques.

Nerve growth factor (NGF) signalling affects spermatogenesis and mature sperm traits. In this paper, we aimed to evaluate the distribution and the role of NGF and its receptors (p75NTR and TrKA) on the reproductive apparatus (testis and epididymis) and sperm of fertile men (F) and men with different pathologies, namely varicocele (V) and urogenital infections (UGIs). We collected semen samples from 21 individuals (31–40 years old) subdivided as follows: V (n = 7), UGIs (n = 7), and F (n = 7). We submitted the semen samples to bacteriological analysis, leucocyte identification, and analysis of sperm parameters (concentration, motility, morphology, and viability). We determined the seminal plasma levels of NGF, interleukin 1β (IL-1β), and F2-isoprostanes (F2-IsoPs), and the gene and protein expression of NGF receptors on sperm. We also used immunofluorescence to examine NGF receptors on ejaculated sperm, testis, and epididymis. As expected, fertile men showed better sperm parameters as well as lower levels of NGF, F2-IsoPs, and IL-1β compared with men with infertility. Notably, in normal sperm, p75NTR and TrKA were localised throughout the entire tail. TrKA was also found in the post-acrosomal sheath. This localisation appeared different in patients with infertility: in particular, there was a strong p75NTR signal in the midpiece and the cytoplasmic residue or coiled tails of altered ejaculated sperm. In line with these findings, NGF receptors were intensely expressed in the epididymis and interstitial tissue of the testis. These data suggest the distinctive involvement of NGF and its receptors in the physiology of sperm from fertile men and men with infertility, indicating a possible role for new targeted treatment strategies. [ABSTRACT FROM AUTHOR]

Apelin and other novel adipokines have been associated with normal and pathological reproductive conditions in humans and animals. In this paper, we used a rabbit model to investigate if apelin and resolvin (RvD1) in testis and sperm are associated with the oxidative status of semen and serum testosterone of rabbits fed different diets enriched with flaxseed (alpha-linolenic acid, ALA) or with fish oil (eicosapentaenoic acid, EPA, docosapentaenoic acid, DPAn-3, and docosahexaenoic acid, DHA). Apelin and RvD1 were detected by ELISA and apelin and the apelin receptor by immunofluorescence. Increased levels of apelin in testes from both enriched diets were shown, particularly in the interstitial tissue of the FLAX group. The FLAX diet enhanced serum testosterone, and both enriched diets showed higher levels of malondialdehyde and RvD1 in the testis. In ejaculated sperm, apelin and its receptor were localized in the entire tail of the control and both treated groups. The ryanodine receptor was investigated in rabbit testis; the fluorescent signal was increased in mature elongated spermatids of the FLAX group. In conclusion, this data seems to indicate that FLAX increases the amount of apelin in testis, suggesting an involvement of this adipokine in male reproduction and probably a role in the resolution of the inflammatory status. [ABSTRACT FROM AUTHOR]

Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted 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]

Human seminal fluid is a complex mixture of secretions originated from epididymis and the male accessory sex glands. It contains a variety of both inorganic and organic components, among which proteins are a major part of the high molecular-mass substances. In this study, 83 human seminal plasma samples were analyzed using a combined Nuclear Magnetic Resonance (NMR) Spectroscopy and Principal Component Analysis (PCA) approach to discriminate patients in relation to semen characteristics and/or conditions affecting the fertility status. Results showed a discrimination between patients with leukocytospermia and with the concomitant presence of varicocele/ex varicocele and leukocytospermia. Patients with testicular cancer, necrozoospermia, and azoospermia were separated from the other patient clusters. In addition, a differentiation of semen quality was also possible. This study represents to first use of sperm parameters together with NMR data as variables in the PCA analysis. Furthermore, this methodology allows the identification of the metabolites which play the most important role in identifying differences among human seminal plasma samples. [ABSTRACT FROM AUTHOR]

Semen from 33 patients were evaluated by light microscopy (LM) obtaining sperm concentration, percent motility, percentage of sperm with normal morphology (PAP staining), and percentage of dead sperm (Eosin Y stained). The samples were observed by polarizing microscopy (PM), that evaluates sperm morphology and the viability by birefringence of organelles, and it provides a PM index (percentage of birefringent, viable, motile sperm) and a percentage of dead, non-birefringent sperm. Sperm were processed for transmission electron microscopy (TEM) and TEM data were elaborated with a mathematical formula able to provide a fertility index (FI, number of sperm free of structural defects) and percentages of sperm immaturity and necrosis (dead sperm). To test the reliability of these techniques, the values of normal acrosome, nucleus, midpiece, and tail and the presence of cytoplasmic residues obtained with the three methods were compared. With the exception of cytoplasmic residues ( P = 0.40), significant differences in the evaluation of each organelle were observed and TEM analysis resulted as the most stringent screening. In addition, relationships among relevant sperm variables were investigated. Motility showed positive correlations with the percentage of normal tail, midpiece, and PM index ( P < 0.01), but it exhibited negative correlations with indices of sperm death (non-birefringent sperm: P < 0.05; percentage of eosin Y stained sperm: P < 0.05; necrosis: P < 0.01), which were positively correlated with each other ( P < 0.01). Positive correlations were found between indices expressing normal sperm morphology: FI with PM index ( P < 0.01) and with the percentage of normal sperm (PAP staining) (P < 0.01), which in turn were correlated with the PM index ( P < 0.001). Sperm immaturity showed positive correlations ( P < 0.01) with the presence of cytoplasmic residues detected with the three methods. In conclusion, LM, PM, and TEM are reliable techniques in evaluating sperm quality. PM appears to offer several advantages 'midway' between LM and TEM and it should be considered in sperm analysis. [ABSTRACT FROM AUTHOR]

Semen parameters were evaluated by a variety of tests. A polarizing microscope (PM) which allows, in a single step, for the evaluation of the number, motility, viability of sperm using the phenomenon of birefringence was used. This approach avoids a Papanicolaou staining procedure modified for sperm (PAP) performed in fixed material. The aim of this study was to examine the birefringence of sperm structures, in live cells, for the direct analysis of morphology on fresh samples. Semen samples from 15 men of recently proven fertility and from 66 male patients who attended our center for semen analysis were examined by polarization microscope (PM) analysis in order to find an index representing a pool of sperm with normal morphology and progressive motility. The receiver operating characteristics (ROC) curves were used to determine the performance of the proposed diagnostic method. The difference between the two areas under the ROC curves (PAP=0.76 and PM=0.82) was quantitatively not significant ( P=0.308); however, the curve of the PM method was always higher than the curve of PAP, revealing that, qualitatively, PM was more sensitive than PAP. The PM index can represent the percentage of motile sperm with normal morphology, which is the actual pool of sperm that can reach and fertilize the oocyte. We suggest a PM diagnostic cut-off value of 20%, since this value was the lowest found for individuals of proven fertility. [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]

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]

F4-neuroprostanes (F4-NeuroPs), derived from the oxidative metabolization of docosahexaenoic acid (DHA), are considered biomarkers of oxidative stress in neurodegenerative diseases. Neurons and spermatozoa display a high DHA content. NeuroPs might possess biological activities. The aim of this in vitro study was to investigate the biological effects of chemically synthetized 4-F4t-NeuroP and 10-F4t-NeuroP in human sperm. Total progressive sperm motility (p < 0.05) and linearity (p = 0.016), evaluated by a computer-assisted sperm analyzer, were significantly increased in samples incubated with 7 ng F4-NeuroPs compared to non-supplemented controls. Sperm capacitation was tested in rabbit and swim-up-selected human sperm by chlortetracycline fluorescence assay. A higher percentage of capacitated sperm (p < 0.01) was observed in samples incubated in F4-NeuroPs than in the controls. However, the percentage of capacitated sperm was not different in F4-NeuroPs and calcium ionophore treatments at 2 h incubation. The phosphorylated form of AMPKα was detected by immunofluorescence analysis; after 2 h F4-NeuroP incubation, a dotted signal appeared in the entire sperm tail, and in controls, sperm were labeled in the mid-piece. A defined level of seminal F4-NeuroPs (7 ng) showed a biological activity in sperm function; its addition in sperm suspensions stimulated capacitation, increasing the number of sperm able to fertilize. [ABSTRACT FROM AUTHOR]

Objective: To characterize and describe the ontogenesis of a rare flagellar defect affecting the whole sperm population of a sterile man.Design: Case report.Setting: Regional referral center for male infertility in Siena, Italy.Patient(s): A 28-year-old man with severe asthenozoospermia.Intervention(s): Physical and hormonal assays, semen analysis, and testicular biopsy.Main outcome measure(s): Semen samples and testicular biopsies were analyzed by light and transmission electron microscopy; immunocytochemical study with anti-β-tubulin and anti-AKAP 82 antibodies was performed to detect the presence and distribution of proteins.Result(s): Ultrastructural analysis of ejaculated spermatozoa and testicular biopsy revealed absence of the fibrous sheath in the principal-piece region of the tail. Fibrous sheath-like structures were observed in cytoplasmic residues and residual bodies released by spermatids in the seminiferous epithelium. Other anomalies observed were supplementary axonemes and mitochondrial helix elongation. These features were confirmed by immunocytochemical staining.Conclusion(s): This rare sperm tail defect, characterized by absence of the fibrous sheath, presence of supplementary axonemes, and an abnormally elongated midpiece, originates in the seminiferous tubules during spermiogenesis, as detected in testicular biopsy sections. These defects occur in the whole sperm population, and therefore a genetic origin could be suggested. [Copyright &y& Elsevier]