In Vitro Effects of Charged and Zwitterionic Liposomes on Human Spermatozoa and Supplementation with Liposomes and Chlorogenic Acid during Sperm Freezing.

Semen handling and cryopreservation induce oxidative stress that should be minimized. In this study, human semen was supplemented during cryopreservation with formulations of handmade liposomes and chlorogenic acid (CGA), an antioxidant compound. Zwitterionic (ZL), anionic (AL), and cationic (CL) liposomes were synthesized and characterized. Three aliquots of swim-up-selected sperm were incubated with ZL, AL, and CL (1:10,000), respectively. The percentages of sperm with progressive motility, high mitochondrial membrane potential (MMP; JC-1), double-stranded DNA (dsDNA acridine orange), and acrosome integrity (Pisum sativum agglutinin) were assessed. Then, human semen was frozen using both 1:10,000 ZL and CGA as follows: freezing medium/empty ZL (EL), freezing medium/empty ZL/CGA in the medium (CGA + EL), freezing medium/CGA loaded ZL (CGA), freezing medium (CTR). The same sperm endpoints were evaluated. ZL were the most tolerated and used for semen cryopreservation protocols. All the supplemented samples showed better endpoints versus CTR (p < 0.001). In particular, spermatozoa from the CGA and CGA + EL A samples showed increased motility, dsDNA, and acrosome integrity versus CTR and EL (p < 0.001; motility EL vs. CGA + EL p < 0.05). ZL and CGA can improve post-thaw sperm quality, acting on both cold shock effect management and oxidative stress. These findings open new perspectives on human and animal reproduction.

Redox Homeostasis and Nrf2-Regulated Mechanisms Are Relevant to Male Infertility.

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.

A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology.

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 1ß (IL-1ß), 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-1ß 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.

Lung ultrasound supports clinical evaluation of feeding competence development in preterm neonates.

Introduction: The achievement of alimentary competencies is a milestone in the development of preterm neonates. Ten percent of neonates <37 weeks of gestational age and 25% of those VLBW experience swallowing disorders, with an increased risk of problems in the early phase of life (failure to thrive, growth retardation, inhalation, and consequent risk of pulmonary infection) and later in life due to delayed development of oromotor skills.The main diagnostic tools for swallowing disorders are endoscopic (fiber-optic endoscopic examination of swallowing, FEES) or radiographic (videofluoroscopic swallowing study, VFSS) exams. Given the invasiveness of these methods and the bias due to rheologic differences between bolus and contrast medium, FEES and VFSS are poorly reproducible. Moreover, neither of the technique is capable of detecting post-meal inhalations, especially microinhalations or those consequent to a whole meal rather than to a single swallowing.Lung ultrasound (LUS) is a widespread, repeatable, safe, fast point-of-care tool and we reported previous encouraging results in detecting silent and overt inhalation related to the meal in children with dysphagia/gastroesophageal reflux disease (GERD) risk factors. Methods: We report a pilot study, that investigated LUS approach (performing imaging before and after meals) to assess feeding competence development in a cohort of n. 19 newborns <32 weeks of age. Results: Meal monitoring by LUS did not show any significant difference in scoring before/after eating. The achievement of full enteral feeding correlates with GA at birth (p < 0.001) but not with LUS scoring. The introduction of the first meal by bottle correlates both with gestational age (p < 0.001) and ultrasound scores (p = 0.004). LUS score at 7 days of life resulted predictive for length of invasive/non-invasive respiratory support (p = 0.002) and length of oxygen supply (p = 0.001), while LUS score at 48 h of life did not (p n.s.). Discussion: Our study suggests that the development of oral feeding skills is not strictly dependent on gestational age. Moreover, our research suggests the predominant role of LUS in predicting the time of readiness to oral feeding, as the LUS score can be a marker of respiratory and lung wellness, and consequently a predictor of neonate stability during deglutitory apnea.

MicroRNA as Possible Mediators of the Synergistic Effect of Celecoxib and Glucosamine Sulfate in Human Osteoarthritic Chondrocyte Exposed to IL-1ß.

This study investigated the role of a pattern of microRNA (miRNA) as possible mediators of celecoxib and prescription-grade glucosamine sulfate (GS) effects in human osteoarthritis (OA) chondrocytes. Chondrocytes were treated with celecoxib (1.85 µM) and GS (9 µM), alone or in combination, for 24 h, with or without interleukin (IL)-1ß (10 ng/mL). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and reactive oxygen species (ROS) by cytometry, nitric oxide (NO) by Griess method. Gene levels of miRNA, antioxidant enzymes, nuclear factor erythroid (NRF)2, and B-cell lymphoma (BCL)2 expressions were analyzed by quantitative real time polymerase chain reaction (real time PCR). Protein expression of NRF2 and BCL2 was also detected at immunofluorescence and western blot. Celecoxib and GS, alone or in combination, significantly increased viability, reduced apoptosis, ROS and NO production and the gene expression of miR-34a, -146a, -181a, -210, in comparison to baseline and to IL-1ß. The transfection with miRNA specific inhibitors significantly counteracted the IL-1ß activity and potentiated the properties of celecoxib and GS on viability, apoptosis and oxidant system, through nuclear factor (NF)-κB regulation. The observed effects were enhanced when the drugs were tested in combination. Our data confirmed the synergistic anti-inflammatory and chondroprotective properties of celecoxib and GS, suggesting microRNA as possible mediators.

Can Dietary n-3 Polyunsaturated Fatty Acids Affect Apelin and Resolvin in Testis and Sperm of Male Rabbits?

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.

Apelin is found in human sperm and testis and is raised in inflammatory pathological conditions.

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)-1ß 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-1ß levels (r = 0.455; p < 0.001). Apelin and IL-1ß concentrations were increased in patients' samples with varicocele (apelin p < 0.01; IL-1ß p < 0.05) and infections (apelin p < 0.01; IL-1ß p < 0.001). By logistic regression analysis, apelin (OR 1.310; p = 0.011) and IL-1ß (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.

Human Sperm as an In Vitro Model to Assess the Efficacy of Antioxidant Supplements during Sperm Handling: A Narrative Review.

Spermatozoa are highly differentiated cells that produce reactive oxygen species (ROS) due to aerobic metabolism. Below a certain threshold, ROS are important in signal transduction pathways and cellular physiological processes, whereas ROS overproduction damages spermatozoa. Sperm manipulation and preparation protocols during assisted reproductive procedures-for example, cryopreservation-can result in excessive ROS production, exposing these cells to oxidative damage. Thus, antioxidants are a relevant topic in sperm quality. This narrative review focuses on human spermatozoa as an in vitro model to study which antioxidants can be used to supplement media. The review comprises a brief presentation of the human sperm structure, a general overview of the main items of reduction-oxidation homeostasis and the ambivalent relationship between spermatozoa and ROS. The main body of the paper deals with studies in which human sperm have been used as an in vitro model to test antioxidant compounds, including natural extracts. The presence and the synergic effects of different antioxidant molecules could potentially lead to more effective products in vitro and, in the future, in vivo.

Focus on centrin in normal and altered human spermatozoa.

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.

Follicular Fluid Components in Reduced Ovarian Reserve, Endometriosis, and Idiopathic Infertility.

Follicular fluid (FF) molecules, and their increase or decrease, can contribute to appropriate follicular growth and oocyte maturation, thus being related to female infertility conditions. In this paper, we studied the changes and the relationships of some biochemical components, hormones, antioxidant enzymes, F2-Isoprostanes (F2-IsoPs), and resolvin (Rv) D1 in the FF of infertile women with different reproductive conditions such as endometriosis, reduced ovarian reserve, and idiopathic infertility during assisted reproductive techniques (ART). In the whole population, positive correlations between albumin (ALB)/iron (Fe), ALB/beta-2-microglobulin (B2MG), and F2-IsoPs/RvD1 were detected in the FF. In FF from aged women, increased levels of follicle stimulating hormone (FSH) and reduced anti-Müllerian hormone (AMH) levels were associated with a worse oocyte quality. The negative ART outcome was influenced by patient age and AMH, B2MG, and FSH levels. Moreover, the reduced ovarian reserve condition was characterised by a significant decrease in oocyte number and quality, AMH amount, and lactate dehydrogenase (LDH) activity, as well as by an increase in age and FSH levels. In the presence of endometriosis, high levels of MDA and RvD1 were detected in FF, with a decrease in luteinising hormone (LH). Finally, among the molecules examined, none characterised the condition of idiopathic infertility. These data could support the identification of new FF markers in different reproductive disorders, suggesting the need for personalised therapeutic approaches and optimised ART outcomes. In particular, the evaluation of resolvins and lipid mediators in FF could be a promising field of investigation with which to understand the entity of oxidative stress and inflammation in some female infertility conditions.

F4-Neuroprostane Effects on Human Sperm.

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.

Effects and Mechanisms Activated by Treatment with Cationic, Anionic and Zwitterionic Liposomes on an In Vitro Model of Porcine Pre-Pubertal Sertoli Cells.

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 ß-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.

The relevance of sperm morphology in male infertility.

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 sperm morphology 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 the most important causes of altered sperm morphology 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 sperm morphology is far from being exhausted and needs other research. This parameter can be still considered a valuable indicator of sperm dysfunction both in basic and clinical research.

Lung ultrasound in young children with neurological impairment: A proposed integrative clinical tool for deaeration-detection related to feeding.

Infants and children with neurological impairment, such as cerebral palsy (CP), often experience abnormal ingestion functions, including oropharyngeal dysphagia and gastroesophageal reflux disease, which led to aspiration-related respiratory complications, morbidity, hospitalization, or death. There is a lack of evidence-based, repeatable, infant-friendly instrumental procedures to assess aspiration-risk in infants with CP or other neurological disorders, with also a lack of clinical assessment measures to support the use of more invasive diagnostic techniques. To this purpose, in the current study we explore the feasibility of lung ultrasound (LUS), to assess lung deaeration possibly related to aspiration during meal, in a cohort of 35 subjects affected by CP or other encephalopathies, and 10 controls in the same age-range. We coupled LUS procedure with meal caregiver administration for each child. Our results support the feasibility of this innovative approach in the clinical setting. Exploratory findings revealed a number of lung abnormalities likely related to abnormal ingestion function in subjects. Subgroup analyses revealed possible differences in LUS abnormalities between CP and other encephalopathies, possibly related to different mechanism of disease or dysfunction. Also, some evidences arose about the possible relationship between such LUS abnormalities and feeding and swallowing abilities in CP or other encephalopathies. LUS showed preliminarily feasibility and effectiveness in detecting meal-related LUS abnormalities in a dynamic manner in the clinical setting. This approach demonstrated usefulness as a potential tool for improving assessment and management in complex care of infants and young children with severe neurological disorders.

Oxidation of Polyunsaturated Fatty Acids as a Promising Area of Research in Infertility.

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.

Seminal Levels of Omentin-1/ITLN1 in Inflammatory Conditions Related to Male Infertility and Localization in Spermatozoa and Tissues of Male Reproductive System.

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.

Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis.

The metabolism of polyunsaturated fatty acids (PUFAs) plays an important role in male reproduction. Linoleic and alpha-linolenic acids need to be provided in the diet and they are converted into long chain polyunsaturated fatty acids by steps of elongation and desaturation, exerted by elongases 2 (ELOVL2) and 5 (ELOVL5) and Δ5- (FADS1) and Δ6-desaturase (FADS2). This study aims to assess the gene expression and localization of enzymes involved in the synthesis of n-3 and n-6 long-chain PUFAs in control rabbits and those fed diets containing 10% extruded flaxseed. Enzyme and PUFA localization were assessed in the testes and epididymis by immunofluorescence. Testes showed high gene expression of FADS2, ELOVL2 and ELOVL5 and low expression of FADS1. Intermediate metabolites, enzymes and final products were differently found in Leydig, Sertoli and germinal cells. FADS2 was localized in interstitial cells and elongated spermatids; ELOVL5 in meiotic cells; FADS1 was evident in interstitial tissue, Sertoli cells and elongated spermatids; ELOVL2 in interstitial cells. Epididymal vesicles were positive for FADS1, ELOVL2 and ELOVL5 as well as docosahexaenoic, eicosapentaenoic, and arachidonic acids. This knowledge of fatty acids (FA) metabolism in spermatogenesis and the influence of diet on FA profile could help identify causes of male infertility, suggesting new personalized therapy.

A Study to Validate the Relevance of Semen F2-Isoprostanes on Human Male Infertility.

F2-isoprostanes (F2-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 F2-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; F2-IsoP levels were measured by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Considering the whole group of patients, F2-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 F2-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 F2-IsoP level < 29.96 ng/mL. Varicocele and urogenital infection groups showed the highest levels of F2-IsoPs in semen. For the first time, a cut-off for F2-IsoPs is identified in human semen. It allows discriminating different male infertility conditions by the semen F2-IsoP amounts, as an additional parameter for clinical evaluation.

Fatty Acid Oxidation and Pro-Resolving Lipid Mediators Are Related to Male Infertility.

Specialized pro-resolving lipid mediators regulate the resolution of acute inflammation. They are formed by enzymatic oxygenation of polyunsaturated fatty acids and are divided into families including lipoxins, resolvins, protectins, and maresins. Resolvin D1 (RvD1), produced by docosahexaenoic acid, exerts anti-inflammatory and pro-resolving activities. This research aimed to investigate the implication of seminal RvD1 in human infertility. Infertile patients (n° 67) were grouped based on pathological reproductive conditions as idiopathic infertility, varicocele, and leukocytospermia; the fourth group was composed of fertile men (n° 18). Sperm characteristics were evaluated by light microscopy (WHO guidelines) and by transmission electron microscopy (TEM). The seminal levels of RvD1 and F2-isoprostane (F2-IsoPs) were dosed. In twenty men (6 fertile men, 8 with varicocele, 6 with leukocytospermia) seminal phospholipase A2, iron, cholesterol, transferrin, estradiol, ferritin, testosterone, and sperm membrane fatty acids were detected. The results indicated that: (i) RvD1 amount was positively correlated with F2-IsoPs and reduced sperm quality; (ii) RvD1 levels were significantly higher in patients with leukocytospermia, varicocele, and idiopathic infertility compared to fertile men; (iii) RvD1 increased along with other markers of oxidative stress and inflammation as fatty acids content and clinical biomarkers. This study suggests a panel of inflammatory markers and lipid mediators for a diagnosis of inflammatory status and a subsequent appropriate therapeutic approach.

F4-Neuroprostanes: A Role in Sperm Capacitation.

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.