Sperm bauplan and function and underlying processes of sperm formation and selection.

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.

Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility.

Regulatory T cells (Tregs) modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, we show that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. We found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of antisperm antibodies (ASA). In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects. Deciphering these immunoregulatory mechanisms may help to design new strategies to treat male infertility, as well as to identify potential targets for immunocontraception.

Exploring novel function of Gpx5 antioxidant activity: Assisting epididymal cells secrete functional extracellular vesicles.

Glutathione peroxisomal-5 (Gpx5) promotes the elimination of H2O2 or organic hydrogen peroxide, and plays an important role in the physiological process of resistance to oxidative stress (OS). To directly and better understand the protection of Gpx5 against OS in epididymal cells and sperm, we studied its mechanism of antioxidant protection from multiple aspects. To more directly investigate the role of Gpx5 in combating oxidative damage, we started with epididymal tissue morphology and Gpx5 expression profiles in combination with the mouse epididymal epithelial cell line PC1 (proximal caput 1) expressing recombinant Gpx5. The Gpx5 is highly expressed in adult male epididymal caput, and its protein signal can be detected in the sperm of the whole epididymis. Gpx5 has been shown to alleviate OS damage induced by 3-Nitropropionic Acid (3-NPA), including enhancing antioxidant activity, reducing mitochondrial damage, and suppressing cell apoptosis. Gpx5 reduces OS damage in PC1 and maintains the well-functioning extracellular vesicles (EVs) secreted by PC1, and the additional epididymal EVs play a role in the response of sperm to OS damage, including reducing plasma membrane oxidation and death, and increasing sperm motility and sperm-egg binding ability. Our study suggests that GPX5 plays an important role as an antioxidant in the antioxidant processes of epididymal cells and sperm, including plasma membrane oxidation, mitochondrial oxidation, apoptosis, sperm motility, and sperm-egg binding ability.

The interactome of cystic fibrosis transmembrane conductance regulator and its role in male fertility: A critical review.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate (cAMP)-regulated chloride and bicarbonate ion channel found in many human cells. Its unique biochemical characteristics and role as a member of the adenosine triphosphate (ATP)-binding cassette transporters superfamily are pivotal for the transport of several substrates across cellular membranes. CFTR is known to interact, physically and functionally, with several other cellular proteins. Hence, its properties are essential for moving various substances across cell membranes and ensuring correct cell functioning. Genetic mutations or environmental factors may disrupt CFTR's function resulting in different possible phenotypes due to gene variations that affect not only CFTR's function, localization, and processing within cells, but also those of its interactors. This has been reported as an underlying cause of various diseases, including cystic fibrosis. The severe clinical implications of cystic fibrosis have driven intense research into the role of CFTR in lung function but its significance to fertility, particularly in men, has been comparatively understudied. However, ongoing and more recent research into CFTR and its interacting proteins in the testis or specific testicular cells is beginning to shed light on this field. Herein, we provide a comprehensive and up-to-date overview of the CFTR, its interactome, and its crucial role in male reproduction, highlighting recent discoveries and advancements in understanding the molecular mechanisms involved. The comprehension of these complex interactions may pave the way for potential therapeutic approaches to improve fertility of men suffering from alterations in the function of CFTR.

LRRC52 is likely a functional component of human KSper†.

Completion of fertilization is orchestrated by various ion channels in sperm membrane. Hyperpolarization of membrane potential, an indispensable event during the capacitation process, is dominated by sperm potassium channel (KSper). In addition to sperm-specific SLO3, which forms the channel pore, the auxiliary subunit leucine-rich-repeat-containing protein 52 (LRRC52) is required to form mKSper to function under physiological conditions. However, in human sperm, although most evidence supports that hSLO3 is the pore-forming subunit, whether hLRRC52 contributes to hKSper conductance and modulates sperm function remains to be understood. Here, using an extracellular segment that is homologous between mice and humans as an antigen, we developed a polyclonal antibody designed as LID1 that specifically detected mLRRC52 and performed co-immunoprecipitation with mSLO3. Additionally, patch-clamp recordings of mouse sperm showed that, physiological activation of mKSper and sperm functions were dramatically attenuated after treatment with LID1, indicating that LID1 functionally disrupted the regulation of mLRRC52 on mKSper. Next, LID1 was used to investigate the significance of hLRRC52 for hKSper activation. As a result, hLRRC52 was expressed in human sperm and might be assembled with hSLO3. More importantly, LID1 inhibited hKSper currents and depolarized sperm membrane potential, supporting essential modulation of hLRRC52 in hKSper. Ca2+ signaling of human sperm was also compromised in the presence of LID1, which impaired sperm motility and acrosome reaction. Because LID1 specifically inhibited both mKSper and hKSper but not mCatSper or hCatSper, our results suggest that hLRRC52 functions as an important component of hKSper and regulates sperm physiological functions.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Epigenetic transgenerational effects of PM2.5 collected from southern Taiwan on sperm functions and DNA methylation in mouse offspring.

During respiration, particulate matter with a diameter of 2.5 µm or less (PM2.5) suspended in the atmosphere enters the terminal alveoli and blood. PM2.5 particles can attach to toxic substances, resulting in health problems. Limited information is available regarding the effects of prenatal exposure to water-soluble PM2.5 (WS-PM2.5) and water-insoluble PM2.5 (WI-PM2.5) on male reproduction. In addition, whether exposure to these particles has transgenerational effects remains unknown. We investigated whether prenatal exposure to WS-PM2.5 and WI-PM2.5 disrupts sperm function in generations F1, F2, and F3 of male mice. Pregnant BALB/c mice were treated using intratracheal instillation on gestation days 7, 11, and 15 with 10 mg of a water extract or insoluble PM2.5. On postnatal day 105, epididymal sperm count, motility, morphology, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, the sperm chromatin DNA fragmentation index (DFI), and testicular DNA methyltransferase (Dnmt) levels were evaluated in all generations. Whole-genome bisulfite sequencing was used to analyze the DNA methylation status of generation F3. According to the results, exposure to WS-PM2.5 affected sperm morphology, ROS production, and mean DFI in generation F1; ROS production and mean DFI in generation F2; and sperm morphology and MMP in generation F3. Similarly, exposure to WI-PM2.5 affected sperm morphology, ROS production, mean DFI, %DFI, and Dnmt1 expression in generation F1; sperm morphology, MMP, and ROS production in generation F2; and sperm morphology, ROS, and %DFI in generation F3. Two hypermethylated genes, PRR16 and TJP2, were observed in the WS-PM2.5 and WI-PM2.5 groups, two hypomethylated genes, NFATC1 and APOA5, were observed in the WS-PM2.5 group, and two hypomethylated genes, ZFP945 and GSE1, were observed in the WI-PM2.5 group. Hence, prenatal exposure to PM2.5 resulted in transgenerational epigenetic effects, which may explain certain phenotypic changes in male reproduction.

Comparative analysis of calcium-sensing receptor (CaSR) expression and function in normal and abnormal human sperm and spermatogenic cells.

The calcium-sensing receptor (CaSR) is a critical mediator of calcium homeostasis in various tissues. Its role in human reproduction, especially in sperm function and male fertility, remains not fully elucidated. This study investigates the expression patterns of CaSR in normal and abnormal sperm and spermatogenic cells and evaluates its potential effect on sperm motility and morphology. Using immunohistochemistry (IHC), quantitative PCR (qPCR), we assessed the expression levels of CaSR in normal sperm, spermatogonia, and cases of asthenozoospermia, oligozoospermia, and teratozoospermia. In vitro functional assays were performed to analyze the effects of CaSR modulation on sperm motility under varying conditions, including the presence of specific CaSR agonists and antagonists. Our study revealed distinct patterns of CaSR expression in normal sperm and spermatogonia compared with those in abnormal sperm samples, particularly in cases of asthenozoospermia, oligozoospermia, and teratozoospermia. A marked decrease in CaSR expression was evident in these abnormal samples, highlighting its significance in normal sperm functionality. Functional assays further elucidated the role of CaSR in sperm motility. Activation of CaSR through specific agonists enhanced sperm motility, while inhibition by antagonists led to reduced motility. Our findings suggest that CaSR plays a significant role in maintaining sperm functionality and that changes in its expression may be associated with male infertility. These insights into the molecular underpinnings of sperm physiology highlight CaSR as a potential therapeutic target for treating certain forms of male infertility.

The Role of Oxidative Stress and DNA Hydroxymethylation in the Pathogenesis of Benzo[a]pyrene-Impaired Reproductive Function in Male Mice.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is known to cause teratogenesis. Environmental exposure of BaP has led to wide public concerns due to their potential risk of reproductive toxicity. However, the exact mechanism is still not clear. We aimed to explore the alterations of oxidative stress and DNA hydroxymethylation during BaP-impaired reproductive function. BALB/c mice were intragastrically administered with different doses of BaP (0.01, 0.1, and 1 mg/kg/day, once a day), while control mice were administered with corn coil. Then, the reproductive function, alterations of oxidative stress, DNA methylation, and DNA hydroxymethylation of testis tissues were evaluated. We found that BaP caused obvious histopathological damages of testis tissues. As for sperm parameters after BaP administration, testis weight and the rate of teratosperm were increased, as well as sperm count and motility were decreased. In mechanism, BaP upregulated HO-1 and MDA levels and downregulated SOD and CAT activity and GSH content in testis tissues, indicating that oxidative stress was induced by BaP. Furthermore, a significant induction of hydroxymethylation and inhibition of methylation were observed in testis tissues after BaP exposure. Collectively, BaP-induced oxidative stress and hydroxymethylation were involved in impairing reproductive function, which may be the mechanism of the male infertility.

Research progress on the effect of sperm chromatin integrity on function and its detection methods.

Sperm chromatin not only carries genetic information such as paternal DNA, but also carries structural proteins, epigenetic information, and higher-order chromatin structures (such as matrix attachment regions and telomeres), etc. These information play an important role in embryonic development. This article mainly reviews the effects of these different information carried by sperm chromatin on sperm function and embryonic development and the research progress of related detection methods, in order to provide a theoretical basis and scientific diagnosis and treatment strategies for the etiology screening of clinical infertility, embryo arrest and recurrent miscarriage, so as to improve the pregnancy outcomes of natural conception and assisted reproduction. Keywords: sperm chromatin; epigenetics; sperm DNA damage; sperm function; higher-order chromatin structures.

Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 µM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 µM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, µm) and curvilinear velocity (VCL, µm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 µM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Catalyzing momentum in male contraceptive development†.

Globally, nearly half of all pregnancies are unplanned. Male contraceptives offer the potential to decrease unintended pregnancy and introduce contraceptive equity, but decades of research have yet to bring a novel product to market. New funding avenues from the philanthropic sector seek to stimulate research in male contraceptives through investments, grants, and support for trainees alongside other programs that encourage product development and ultimately commercialization. This Forum outlines the purpose of and funding opportunities provided by Male Contraceptive Initiative, a funding agency and non-profit focused on the advancement of non-hormonal, reversible contraceptive technologies for those who produce sperm.

Autophagy is activated in human spermatozoa subjected to oxidative stress and its inhibition impairs sperm quality and promotes cell death.

STUDY QUESTION: Does oxidative stress (OS) activate autophagy in human sperm? SUMMARY ANSWER: Human spermatozoa subjected to OS activate an autophagic response. WHAT IS KNOWN ALREADY: Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. STUDY DESIGN, SIZE, DURATION: Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. PARTICIPANTS/MATERIALS, SETTING, METHODS: For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. WIDER IMPLICATIONS OF THE FINDINGS: Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

STUDY QUESTION: What effects did treatment using hyaluronic acid (HA) binding/selection prior to ICSI have on clinical outcomes in the Hyaluronic Acid Binding sperm Selection (HABSelect) clinical trial? SUMMARY ANSWER: Older women randomized to the trial's experimental arm (selection of sperm bound to immobilized (solid-state) HA) had the same live birth rates as younger women, most likely a result of better avoidance of sperm with damaged DNA. WHAT IS KNOWN ALREADY: Recent randomized controlled trials (RCTs) investigating the efficacy of HA-based sperm selection prior to ICSI, including HABSelect, have consistently reported reductions in the numbers of miscarriages among couples randomized to the intervention, suggesting a pathological sperm-mediated factor mitigated by prior HA-binding/selection. The mechanism of that protection is unknown. STUDY DESIGN, SIZE, DURATION: The original HABSelect Phase 3 RCT ran from 2014 to 2017 and included 2752 couples from whom sperm samples used in control (ICSI) and intervention (Physiological IntraCytoplasmic Sperm Injection; PICSI) arms of the trial were stored frozen for later assessment of DNA quality (DNAq). The trial overlapped with its mechanistic arm, running from 2016 to 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: As miscarriage reduction was a significant secondary outcome of the trial, samples (n = 1247) selected for the mechanistic analysis were deliberately enriched for miscarriage outcomes (n = 92 or 7.4%) from a total of 154 miscarriages (5.6%) among all (n = 2752) couples randomized by stratified random sampling. Values from fresh semen samples for sperm concentration (mml), percentage forward progressive motility and percentage HA-binding score (HBS) were obtained before being processed by differential density gradient centrifugation or (rarely) by swim-up on the day of treatment. Surplus sperm pellets were recovered, aliquoted and cryopreserved for later analysis of DNAq using slide-based Comet, TUNEL, acridine orange (AO) and the sperm chromatin dispersion (SCD) assays. Following their classification into normal and abnormal sample subcategories based on reference values for sperm concentration and motility, relationships with HBS and DNAq were examined by Spearman correlation, Student's t-tests, Mann Whitney U tests, and logistic regression (univariable and multivariable). Parsimonious selection enabled the development of models for exploring and explaining data trends. Potential differences in future cumulative pregnancy rates relating to embryo quality were also explored. MAIN RESULTS AND THE ROLE OF CHANCE: Results from the 1247 sperm samples assayed for HBS and/or DNAq, generated data that were considered in relation to standard physiological measures of (sperm) vitality and to treatment outcomes. All measures of HBS and DNAq discriminated normal from abnormal sperm samples (P < 0.001). SCD correlated negatively with the Comet (r = -0.165; P < 0.001) and TUNEL assays (r = -0.200; P < 0.001). HBS correlated negatively with AO (r = -0.211; P < 0.001), Comet (r = -0.127; P < 0.001) and TUNEL (r = -0.214; P < 0.001) and positively with SCD (r = 0.255; P < 0.001). A model for predicting live birth (and miscarriage) rates included treatment allocation (odds ratio: OR 2.167, 95% CI 1.084-4.464, P = 0.031), female age (OR 0.301, 95% CI 0.133-0.761, P = 0.013, per decade) and the AO assay (OR 0.79, 95% CI 0.60-1. 02.761, P = 0.073, per 10 points rise). A model predicting the expected rate of biochemical pregnancy included male age (OR 0.464, 95% CI 0.314-0.674, P < 0.001, per decade) and the SCD assay (OR 1.04, 95% CI 1.007-1.075, P = 0.018, per 10 point rise). A model for conversion from biochemical to clinical pregnancy did not retain any significant patient or assay variables. A model for post-injection fertilization rates included treatment allocation (OR 0.83, 95% CI 0.75-0.91, P < 0.001) and the Comet assay (OR 0.950, 95% CI 0.91-1.00, P = 0.041). LIMITATIONS, REASONS FOR CAUTION: HABSelect was a prospective RCT and the mechanistic study group was drawn from its recruitment cohort for retrospective analysis, without the full benefit of randomization. The clinical and mechanistic aspects of the study were mutually exclusive in that measures of DNAq were obtained from residual samples and not from HA-selected versus unselected sperm. Models for fitting mechanistic with baseline and other clinical data were developed to compensate for variable DNAq data quality. HABSelect used a solid-state version of PICSI and we did not assess the efficacy of any liquid-state alternatives. PICSI reduced fertilization rates and did not improve the outlook for cumulative pregnancy rates. WIDER IMPLICATIONS OF THE FINDINGS: Notwithstanding the interventional effect on fertilization rates and possibly blastocyst formation (neither of which influenced pregnancy rates), poor sperm DNAq, reflected by lower HBS, probably contributed to the depression of all gestational outcomes including live births, in the HABSelect trial. The interventional avoidance of defective sperm is the best explanation for the equalization in live birth rates among older couples randomized to the trial's PICSI arm. As patients going forward for assisted conception cycles globally in future are likely to be dominated by an older demographic, HA-based selection of sperm for ICSI could be considered as part of their treatment plan. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the National Institute for Health Research (NIHR) EME (Efficacy and Mechanism Evaluation)-11-14-34. National Research Ethics Service approval 11/06/2013: 13/YH/0162. S.L. is CEO of ExamenLab Ltd (company number NI605309). TRIAL REGISTRATION NUMBER: ISRCTN99214271.

Oxidative Stress-Induced Male Infertility: Role of Antioxidants in Cellular Defense Mechanisms.

Male infertility is linked to several environmental and mutagenic factors. Most of these factors, i.e., lifestyle, radiations, and chemical contaminations, work on the fundamental principles of physics, chemistry, and biology. Principally, it may induce oxidative stress (OS) and produce free radicals within the cells. The negative effect of OS may enhance the reactive oxygen species (ROS) levels in male reproductive organs and impair basic functions in a couple's fertility. Evidence suggests that infertile men have significantly increased ROS levels and a reduced antioxidant capacity compared with fertile men. Although, basic spermatic function and fertilizing capacity depend on a delicate balance between physiological activity of ROS and antioxidants to protect from cellular oxidative injury in sperm, that is essential to achieve pregnancy. The ideal oxidation-reduction (REDOX) equilibrium requires a maintenance of a range of ROS concentrations and modulation of antioxidants. For this reason, the chapter focuses on the effects of ROS in sperm functions and the current concepts regarding the benefits of medical management in men with diminished fertility and amelioration of the effect to improve sperm function. Also, this evidence-based study suggests an increasing rate of infertility that poses a global challenge for human health, urging the need of health care professionals to offer a correct diagnosis, comprehension of the process, and an individualized management of the patients.

Genipin improves lipid metabolism and sperm parametersin obese mice via regulation of miR-132 expression.

Obesity has now surpassed malnutrition and infectious diseases as the most significant contributor to health problems worldwide. In particular, obesity is associated with several metabolic disorders, including hyperlipidemia, hepatic steatosis, and subfertility. Genipin (GNP), the aglycone of geniposide, is isolated from the extract of the traditional Chinese medicine Gardenia jasminoides Ellis and has been used in traditional oriental medicine against several inflammation-driven diseases. However, the effect and molecular mechanism of GNP on obesity-associated dyslipidemia and sperm dysfunction still need to be explored. In this study, we detect the effects of GNP on hyperlipidemia, hepatic lipid accumulation and sperm function using a high-fat diet (HFD)-induced obese mouse model. We find that obese mice treated with GNP show an improvement in body weight, serum triglyceride levels, serum hormone levels, serum inflammatory cytokines, hepatic steatosis and sperm function. At the molecular level, HFD/GNP diversely regulates the expression of miR-132 in a tissue-specific manner. miR-132 further targets and regulates the expression of SREBP-1c in liver cells, as well as the expressions of SREBP-1c and StAR in Leydig cells in the testis, thus modifying lipogenesis and steroidogenesis, respectively. Collectively, our data demonstrate that GNP shows a broad effect on the improvement of HFD-induced metabolic disorder and sperm dysfunction in male mice by tissue-specific regulation of miR-132. Our findings reveal the function GNP in ameliorating hepatic lipid metabolism and sperm function and suggest that this compound is a versatile drug to treat metabolic disorders.

Involvement of Ca2+ and ROS signals in nickel-impaired human sperm function.

As one of the main environmental pollutants and occupational hazards, nickel has been reported to have mutagenic, carcinogenic, and teratogenic properties, as well as reproductive toxicity. However, how nickel affects human reproduction is still unclear. In this study, the toxicity of nickel on human sperm and the underlying mechanisms were evaluated in vitro. We found that NiCl2 (10, 50, and 250 µM) impaired sperm total motility and progressive motility in a dose- and time-dependent manner. In addition, sperm hyperactivation and the ability of human sperm to penetrate a viscous medium were found to be compromised after nickel exposure. Mechanically, NiCl2 significantly inhibited the basal intracellular Ca2+ signaling. Besides, reactive oxygen species (ROS), superoxide, and malondialdehyde levels were increased in human sperm after exposure to different concentrations of NiCl2. Consistently, eliminating excess ROS by N-acetyl-L-cysteine or tocopherol significantly alleviated nickel-impaired sperm motility. Taken together, these results revealed that nickel could compromise sperm functions by interfering with Ca2+ signaling and inducing excessive oxidative stress. These findings suggest that, in the high and occupational nickel exposure environments, the contribution of nickel toxicity to the males who wish to preserve their fertility is worthy of careful evaluation.

Microfluidic devices employing chemo- and thermotaxis for sperm selection can improve sperm parameters and function in patients with high DNA fragmentation.

Conventional sperm processing uses centrifugation has a negative effect on sperm parameters and DNA integrity. We designed and fabricated a novel microfluid device based on chemotaxis and thermotaxis, and compared it with the swim-up method. Twenty normal samples with high DNA fragmentation were included. Each sample was divided into four groups: Group 1, control, Group 2: sperm selection by thermotaxis, Group 3: sperm selection by chemotaxis, and Group 4: sperm selection with thermotaxis and chemotaxis. We used cumulus cells in a microfluid device to create chemotaxis, and, two warm stages to form a temperature gradient for thermotaxis. The spermatozoa were assessed based on the concentration, motility, and fine morphology using Motile Sperm Organelle Morphology Examination, mitochondrial membrane potential (MMP), acrosome reaction (AR), and sperm DNA fragmentation. Concentration (22.40 ± 5.39 vs. 66.50 ± 19.21; p < 0.001) and DNA fragmentation (12.30 ± 3.96% vs. 17.95 ± 2.89%; p < 0.001) after selection in the chemotaxis and thermotaxis microfluid device were significantly lower than control group. The progressive motility (93.75 ± 4.39% vs. 75.55 ± 5.86%, p < 0.001), normal morphology (15.45 ± 2.50% vs. 10.35 ± 3.36, p < 0.001), MMP (97.65 ± 1.81% vs. 94 ± 3.89%, p = 0.02), and AR status (79.20 ± 5.28% vs. 31.20 ± 5.24%, p < 0.001) in the chemotaxis and thermotaxis microfluid device were significantly increased compared to control group. According to these findings, spermatozoa that have penetrated the cumulus oophorus have better morphology and motility, as well as acrosome reactivity and DNA integrity.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 µM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 µM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.