Abstract Study question What is the impact of escalating doses of common water-soluble antioxidant nutrients on semen parameters and fertility in healthy (CD1) and stressed (Gpx5-/-) mice? Summary answer High-dose supplementation of previously “thought-to-be-safe” common water-soluble antioxidant nutrients can cause significant DNA damage/impaired fertility in healthy mice. What is known already In assisted conception, men increasingly take preconceptual antioxidant supplements (even if they are generally healthy) to enhance the likelihood of a successful pregnancy. Recent published reviews suggest 79% of male fertility supplements include ingredients at dosages exceeding Food Safety Authorities’ Recommended Dietary Allowances (RDAs). While many fertility experts question the efficacy of such supplements, most still carry the notion that “dietary ingredients”, regardless of the doses given, pose little safety concerns. Moreover, without established RDAs for many natural substances such as carnitines, supplement makers often subscribe to the “more is better” philosophy, potentially compromising safety in their product formulations. Study design, size, duration This study evaluates escalating doses of common water-soluble antioxidant nutrients (Vitamin C, Zinc, Folate, and Carnitines) on redox status, semen parameters, genetic integrity, and fertility in healthy (CD1) and stressed (Gpx5 -/-) mice over 28 days. The treated groups (n = 6) were compared against pooled controls (n = 24). We analyzed the doses of these ingredients in commercial fertility supplements against our new “safety profiles” to highlight potential overdosing risks and asses the broader safety implications. Participants/materials, setting, methods Experimental ingredient exposure dosages were based on current US-FDA RDA/I declarations, commercial popularity, and/or use in clinical fertility settings. Animal dosage conversion calculations were based on standard pharmaceutical methodologies. Assessments of male mouse redox status and reproductive function included general sperm parameters (count, motility, viability, acrosome integrity), DNA parameters [oxidation (8-OHdG staining), fragmentation (TUNEL), decondensation (Toluidine Blue)], and blood analysis (total antioxidant capacity, glutathione, malondialdehyde). Pregnancy outcomes were assessed in Carnitine fed, CD1 mice. Main results and the role of chance In CD1 mice, escalating doses of individual micronutrients had minimal effect on semen parameters, except for a significant sperm count increase at the highest Carnitine dose. However, there were considerable adverse effects on sperm DNA integrity and blood plasma redox state. The high-dose Carnitine group notably exhibited reduced pregnancy rates, in terms of both impregnation and total pups born, compared to controls. Yet, there were no differences in the number of pups born or resorptions, per pregnant female, between the high-dose Carnitine group and controls. In GPX5-/- mice, general semen parameters were mostly unaffected. Intriguingly, Carnitine positively influenced sperm DNA integrity in this model, underscoring the established link between micronutrient deficiency, oxidative stress, and semen quality. In the analysis of 34 random male fertility supplements, almost every product contained at least one ingredient at a dose that was harmful according to our study. Alarmingly, 88% had at least two such ingredients, 68% had three or more, and roughly 33% contained four or more at doses considered unsafe. Limitations, reasons for caution Carnitines’ pungent odor may have affected the mating efficiency of carnitine exposed CD1 males. Due to synergy of action, antioxidants administered individually, even at the same dose, may exert a different biochemical profile than when in combination with other ingredients (as in most commercial formulations). Wider implications of the findings The majority of male fertility supplements often combine aggressive doses of antioxidant nutrients. Our results highlight the potential dangers that may arise from their use and provide a clear warning to fertility experts against routinely recommending random, unsubstantiated supplements, especially in the absence of prior oxidative stress assessment. Trial registration number not applicable

Abstract Study question Do common chemicals in plastic, BPAF and DBP, negatively impact sperm parameters and DNA integrity, and can oral antioxidant supplementation mitigate their impact? Summary answer Short-term, low-dose exposure to BPAF or DBP did not impact testicular/epididymal histology but caudal epididymal sperm parameters, particularly DNA integrity was significantly compromised. What is known already Plasticizers are among the most commonly produced chemicals worldwide with 90% of people showing detectable levels in their urine. As endocrine disruptors (EDCs), they act as estrogen mimetics but can also induce oxidative stress and form DNA adducts. Numerous studies have linked exposure to EDCs to impaired spermatogenesis, sperm DNA damage and male infertility. Limited studies suggest antioxidants may alleviate the reproductive toxicity of EDCs. This study measured the effects of short-term exposures to two common environmental EDCs (BPAF & DBP) on the post-testicular compartment epididymis and investigate the efficacy of an oral antioxidant supplement to safeguard sperm DNA integrity. Study design, size, duration Adult male CD1 mice were exposed to low doses (50mg/kg) of BPAF or DBP with or without co-administration of antioxidant supplementation (Fertilix®) for 14 days and evaluated after euthanasia. Participants/materials, setting, methods All experiments were performed at the University Clermont Auvergne, Clermont-Ferrand, France. Mice were supplemented with BPAF, DBP or/and Fertilix® in their drinking water. The results of exposure of males were evaluated against controls and included: testicular and epididymal histology, epididymal ROS status via 4-HNE levels in caput protein extracts, general semen parameters: (count, motility, viability, acrosome integrity), sperm nuclear/DNA parameters [decondensation (Toluidine blue stain), fragmentation (TUNEL test), oxidation (8-OHdG residue level)]. Main results and the role of chance The histology of the testis and epididymis was unaffected by the exposure conditions used. An oxidative stress situation was evident by the EDC exposures, demonstrated by increased level of 4-HNE adducts in the epididymal caput protein extracts. Viability, total and progressive motility, and acrosome integrity of caudal epididymal spermatozoa remained unaltered in exposed animals compared with controls. Exposure to BPAF but not DBP significantly decreased sperm count. Sperm nuclear/DNA integrity assessed by the percentages of sperm with decondensed and oxidized nuclei was significantly increased in EDC-exposed animals. Sperm DNA fragmentation did not significantly increase in any group of exposed animals. Co-administration with Fertilix® significantly corrected the defective sperm parameters (nuclear decondensation and DNA base oxidation) with the exception of sperm count which did not return to control levels in BPAF+ Fertilix® treated animals. These data confirm that even at low doses and short-term exposures, epididymal spermatozoa are susceptible to DNA damage induced by environmental ROS-generating EDCs, confirming its association with male infertility. Limitations, reasons for caution The translation of these findings to humans needs to be confirmed, although the nuclear compartment of human sperm is more susceptible to oxidative alterations than that of the mouse. Wider implications of the findings These data demonstrate the susceptibility of post-testicular sperm cells to common environmental chemicals. They also support the use of appropriate antioxidant supplementation as an effective therapeutic choice for individuals with multiple exposures to environmental stressors such as ROS-generating EDCs. Trial registration number not applicable

The maintenance of redox balance in the male reproductive tract is critical to sperm health and function. Physiological levels of reactive oxygen species (ROS) promote sperm capacitation, while excess ROS exposure, or depleted antioxidant defenses, yield a state of oxidative stress which disrupts their fertilizing capacity and DNA structural integrity. The guanine moiety is the most readily oxidized of the four DNA bases and gets converted to the mutagenic lesion 8-hydroxy-deoxyguanosine (8-OHdG). Numerous studies have also confirmed oxidative stress as a driving factor behind accelerated telomere shortening and dysfunction. Although a clear consensus has not been reached, clinical studies also appear to associate telomere integrity with fertility outcomes in the Assisted Reproductive Technology (ART) setting. Intriguingly, while sperm cellular and molecular characteristics make them more susceptible to oxidative insult than any other cell type, they are also the only cell type in which telomere lengthening accompanies aging. This article focuses on the oxidative stress response pathways to propose a mechanism in explanation of this apparent paradox.

Varicocele is characterized by testicular dysfunction that originates from hyperthermia and hypoxia, leading to defects in testicular tissue and altered spermatozoa structure and function. The varicocele testis is characterized by the presence of intracellular iron deposits that contribute to the associated oxidative stress. Therefore, we tested the hypothesis that administration of an iron-chelating agent, such as deferasirox (DFX), could potentially mitigate the consequences of varicocele on testicular tissue and spermatozoa. Using a well-established rat model of varicocele (VCL), we show that treatment with DFX partially improved the structure and function of the testis and spermatozoa. In particular, sperm motility was markedly restored whereas abnormal sperm morphology was only partially improved. No significant improvement in sperm count was observed that could be associated with the proapoptotic response observed following iron chelation treatment. No reduction in oxidative damage to spermatozoa was observed since lipid peroxidation and DNA integrity were not modified. This was suggested to be a result of increased oxidative stress. Finally, we also saw no indication of attenuation of the endoplasmic reticulum/unfolded protein (ER/UPR) stress response that we recently found associated with the VCL testis in rats.

Background In rodents and humans, vitamin D deficiency (VDD) is associated with altered sperm structure and function (primarily decreased motility and morphological abnormalities) that are primarily attributed to VDD-induced hypocalcemia. However, it is suspected that VDD has much more drastic effects on mammalian spermatozoa. Objectives The purpose of this study was to illustrate that VDD, depending on its severity and duration, can alter sperm nuclear integrity and can also lead to the loss of spermatozoa's ability to support embryonic development. Materials and methods A mouse model of induced VDD combining the action of a vitamin D-deficient diet, UV exposure limitation, and paricalcitol injections; a vitamin D2 analog that catabolizes endogenous vitamin D by increasing the expression of CYP24A, a member of the cytochrome P450 family, has been used to create different grades of VDD. Results We show that the most significant sperm defect recorded concerns the integrity of the paternal nucleus, which is both decondensed and fragmented in moderate-to-severe VDD situations. Consistent with the known consequences of fertilization with DNA-damaged spermatozoa, we show that paternal VDD decreases the ability of spermatozoa to optimally support fertilization and embryonic development. Discussion and Conclusion Given the worldwide high prevalence of VDD in humans, and although obtained in an animal model, the data presented here suggest that subfertile/infertile males may benefit from VDD testing and that attempts to correct serum vitamin D levels could be considered prior to conception, either naturally or through ART.

The Sperm Chromatin Structure Assay (SCSA®), in addition to identifying the DNA Fragmentation Index (DFI) also identifies High DNA satiability (HDS), supposed to reflect the nuclear compaction of spermatozoa. However, data on what exactly this parameter reveals, its relevance and usefulness are contradictory. In order to shed light on this situation, spermatozoa of a cohort (N = 397) of infertile men were subjected to the SCSA®, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling) and CMA3 (Chromomycin A3) tests. In a smaller subcohort (N = 100), aniline blue (AB) and toluidine blue (TB) staining were performed in addition. The objective of this study was thus to answer the question of whether HDS is a relevant and reliable parameter to be taken into account? HDS does not appear to be a reliable indicator of nuclear immaturity because it shows a weak correlation with the CMA3, AB and TB stains. The low correlation of HDS with sperm DNA fragmentation (TUNEL and SCSA®) and DNA condensation (CMA3, AB and TB) tests suggests that these two parameters could be decoupled. Unlike DFI and TUNEL, HDS has not been shown to correlate with classic clinical situations of male infertility (asthenozoospermia, teratozoospermia or astheno-teratozoospermia). HDS correlates poorly with most tests that focus specifically on the level of maturity of the sperm nucleus. To our knowledge, this study is the first to compare SCSA®, TUNEL, AB, TB and CMA3 assays on identical samples. It shows the potency, consistency and limitations of each test and the care that must be taken in their interpretation.

STUDY QUESTION Can a discriminant threshold be determined for human sperm DNA oxidation? SUMMARY ANSWER A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. WHAT IS KNOWN ALREADY Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa; optimized consensus protocols are lacking and no thresholds of normality have been established. STUDY DESIGN, SIZE, DURATION Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. PARTICIPANTS/MATERIALS, SETTING, METHODS This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). MAIN RESULTS AND THE ROLE OF CHANCE Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. LIMITATIONS, REASONS FOR CAUTION The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. WIDER IMPLICATIONS OF THE FINDINGS Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by institutional grants from the CNRS, INSERM and Université Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

STUDY QUESTION Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? SUMMARY ANSWER Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. WHAT IS KNOWN ALREADY Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. STUDY DESIGN, SIZE, DURATION The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. MAIN RESULTS AND ROLE OF CHANCE Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). LIMITATIONS, REASONS FOR CAUTION It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. WIDER IMPLICATIONS OF THE FINDINGS Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. STUDY FUNDING/COMPETING INTERESTS The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

ObjectiveCompare the cytotoxicity of several aldehyde byproducts of lipid peroxidation in human spermatozoa.DesignCytotoxicity and bioactivity of six aldehyde byproducts of lipid peroxidation were assessed in vitro on spermatozoa from healthy males. Acrolein (ACR), Heptadienal (HEPT), Hexanal (HEX), 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), 2-Octenal(OCT), and Valeraldehyde (VAL) were compared for their effects on cell health, as well as for their relative reactivity with glutathione (GSH). Adduction patterns of ACR, 4-HNE, and MDA were also studied. Untreated spermatozoa served as controls, and all measurements were repeated on at least three separate samples.Materials and MethodsSemen samples of healthy human volunteers were collected through the University of Newcastle's donor program. Spermatozoa were isolated and pooled samples were treated with increasing doses of aldehyde for 2 and 24 hours. Cytotoxicity at six concentrations (0, 12.5, 25, 50, 100, 200 μM) was assessed in vitro under physiological conditions using fluorocytometric assays for markers of cell health and viability, including mitochondrial production of Reactive Oxygen Species (ROS) and apoptosis (Annexin-V). Effects on motility and vitality were also assessed.ResultsAfter 2 hours of incubation, 4-HNE and ACR induced a dose-dependent reduction in motility, with a concurrent increase in mitochondrial ROS production (p<0.01). After 24 hours, 4-HNE, ACR, HEPT, and OCT increased mitochondrial ROS production and decreased motility, while incubation with ACR and 4-HNE increased Annexin-V levels in a dose-dependent manner (p<0.05). No observable cytotoxic effect was produced by HEX, MDA, or VAL at any dose, even after incubation for 24 hours. In a side-by-side comparison of motility, vitality, and ROS production, ACR was more cytotoxic than 4-HNE, while MDA produced no cytotoxic effect. Relative reactivity with GSH mirrored these effects, with ACR being the most reactive and HEX, MDA, and VAL being unreactive.ConclusionsThough aldehyde byproducts of lipid peroxidation are generally accepted to adversely impact cellular health, these data suggest considerable cytotoxic differences between species. These differences reflect the aldehydes' structures: the four cytotoxic species were unsaturated aldehydes, while MDA, VAL, and HEX exist as saturated aldehydes under the present testing conditions. ObjectiveCompare the cytotoxicity of several aldehyde byproducts of lipid peroxidation in human spermatozoa. Compare the cytotoxicity of several aldehyde byproducts of lipid peroxidation in human spermatozoa. DesignCytotoxicity and bioactivity of six aldehyde byproducts of lipid peroxidation were assessed in vitro on spermatozoa from healthy males. Acrolein (ACR), Heptadienal (HEPT), Hexanal (HEX), 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), 2-Octenal(OCT), and Valeraldehyde (VAL) were compared for their effects on cell health, as well as for their relative reactivity with glutathione (GSH). Adduction patterns of ACR, 4-HNE, and MDA were also studied. Untreated spermatozoa served as controls, and all measurements were repeated on at least three separate samples. Cytotoxicity and bioactivity of six aldehyde byproducts of lipid peroxidation were assessed in vitro on spermatozoa from healthy males. Acrolein (ACR), Heptadienal (HEPT), Hexanal (HEX), 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), 2-Octenal(OCT), and Valeraldehyde (VAL) were compared for their effects on cell health, as well as for their relative reactivity with glutathione (GSH). Adduction patterns of ACR, 4-HNE, and MDA were also studied. Untreated spermatozoa served as controls, and all measurements were repeated on at least three separate samples. Materials and MethodsSemen samples of healthy human volunteers were collected through the University of Newcastle's donor program. Spermatozoa were isolated and pooled samples were treated with increasing doses of aldehyde for 2 and 24 hours. Cytotoxicity at six concentrations (0, 12.5, 25, 50, 100, 200 μM) was assessed in vitro under physiological conditions using fluorocytometric assays for markers of cell health and viability, including mitochondrial production of Reactive Oxygen Species (ROS) and apoptosis (Annexin-V). Effects on motility and vitality were also assessed. Semen samples of healthy human volunteers were collected through the University of Newcastle's donor program. Spermatozoa were isolated and pooled samples were treated with increasing doses of aldehyde for 2 and 24 hours. Cytotoxicity at six concentrations (0, 12.5, 25, 50, 100, 200 μM) was assessed in vitro under physiological conditions using fluorocytometric assays for markers of cell health and viability, including mitochondrial production of Reactive Oxygen Species (ROS) and apoptosis (Annexin-V). Effects on motility and vitality were also assessed. ResultsAfter 2 hours of incubation, 4-HNE and ACR induced a dose-dependent reduction in motility, with a concurrent increase in mitochondrial ROS production (p<0.01). After 24 hours, 4-HNE, ACR, HEPT, and OCT increased mitochondrial ROS production and decreased motility, while incubation with ACR and 4-HNE increased Annexin-V levels in a dose-dependent manner (p<0.05). No observable cytotoxic effect was produced by HEX, MDA, or VAL at any dose, even after incubation for 24 hours. In a side-by-side comparison of motility, vitality, and ROS production, ACR was more cytotoxic than 4-HNE, while MDA produced no cytotoxic effect. Relative reactivity with GSH mirrored these effects, with ACR being the most reactive and HEX, MDA, and VAL being unreactive. After 2 hours of incubation, 4-HNE and ACR induced a dose-dependent reduction in motility, with a concurrent increase in mitochondrial ROS production (p<0.01). After 24 hours, 4-HNE, ACR, HEPT, and OCT increased mitochondrial ROS production and decreased motility, while incubation with ACR and 4-HNE increased Annexin-V levels in a dose-dependent manner (p<0.05). No observable cytotoxic effect was produced by HEX, MDA, or VAL at any dose, even after incubation for 24 hours. In a side-by-side comparison of motility, vitality, and ROS production, ACR was more cytotoxic than 4-HNE, while MDA produced no cytotoxic effect. Relative reactivity with GSH mirrored these effects, with ACR being the most reactive and HEX, MDA, and VAL being unreactive. ConclusionsThough aldehyde byproducts of lipid peroxidation are generally accepted to adversely impact cellular health, these data suggest considerable cytotoxic differences between species. These differences reflect the aldehydes' structures: the four cytotoxic species were unsaturated aldehydes, while MDA, VAL, and HEX exist as saturated aldehydes under the present testing conditions. Though aldehyde byproducts of lipid peroxidation are generally accepted to adversely impact cellular health, these data suggest considerable cytotoxic differences between species. These differences reflect the aldehydes' structures: the four cytotoxic species were unsaturated aldehydes, while MDA, VAL, and HEX exist as saturated aldehydes under the present testing conditions.

ObjectiveDoes Fertilix, a novel antioxidant formulation designed to alleviate oxidative stress in the male reproductive tract, reduce Sperm DNA Damage (SDD) and increase pregnancy rates in mouse models of Sperm Oxidative Stress (SOS)?DesignFertilix efficacy was evaluated in two, well-established mouse models of SOS; Glutathione Peroxidase 5 knockout (GPX-5 KO) mice and Scrotal Heat Shock (SHS) treatment, each with n=12, by independent laboratories. Mice were given Fertilix in their drinking water for 2 months (GPX-5 KO) and 2 weeks (SHS) and then compared with control groups for SDD and pregnancy rates.Materials and MethodsIn GPX-5 KO mice, SDD was evaluated by immunocytochemical detection of 8-Hydroxy-deoxy Guanosine (8-OHdG) residues, a biomarker of DNA oxidation. In the SHS model, each male's fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded.ResultsThe average background levels of 8-OHdG in WT mice is around 30%. This level doubles up to about 60% in transgenic male mice deficient in the antioxidant enzyme GPX-5. Our results indicate that a 2 month pretreatment of GPX-5 KO mice with Fertilix provides complete protection of sperm DNA against oxidation. In male mice exposed to the SHS model, only 35% (19/54) of partnered female mice got pregnant resulting in 169 fetuses. In contrast, male mice pretreated with Fertilix for 2 weeks yielded 74% (42/57) of partnered female mice becoming pregnant, resulting in 427 fetuses. The role of chance in obtaining supporting results for the efficacy of Fertilix in both models is minimal although it was not possible to ensure that every mouse took 100% of the product for the treatment period.ConclusionsOral administration of Fertilix significantly reduces SDD in GPX-5 knockout mice and restores pregnancy rates almost back to normal levels in mice subjected to SHS. These results, if confirmed in humans, will impact clinical fertility practice. Current clinical studies confirm moderate to severe SDD in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility, figures which are gravely concerning. Antioxidant supplementation will be an adjuvant therapy prior to undertaking ART procedures to improve fertilization rates, maintain a healthy pregnancy, and reduce de novo sporadic mutations being passed onto children. ObjectiveDoes Fertilix, a novel antioxidant formulation designed to alleviate oxidative stress in the male reproductive tract, reduce Sperm DNA Damage (SDD) and increase pregnancy rates in mouse models of Sperm Oxidative Stress (SOS)? Does Fertilix, a novel antioxidant formulation designed to alleviate oxidative stress in the male reproductive tract, reduce Sperm DNA Damage (SDD) and increase pregnancy rates in mouse models of Sperm Oxidative Stress (SOS)? DesignFertilix efficacy was evaluated in two, well-established mouse models of SOS; Glutathione Peroxidase 5 knockout (GPX-5 KO) mice and Scrotal Heat Shock (SHS) treatment, each with n=12, by independent laboratories. Mice were given Fertilix in their drinking water for 2 months (GPX-5 KO) and 2 weeks (SHS) and then compared with control groups for SDD and pregnancy rates. Fertilix efficacy was evaluated in two, well-established mouse models of SOS; Glutathione Peroxidase 5 knockout (GPX-5 KO) mice and Scrotal Heat Shock (SHS) treatment, each with n=12, by independent laboratories. Mice were given Fertilix in their drinking water for 2 months (GPX-5 KO) and 2 weeks (SHS) and then compared with control groups for SDD and pregnancy rates. Materials and MethodsIn GPX-5 KO mice, SDD was evaluated by immunocytochemical detection of 8-Hydroxy-deoxy Guanosine (8-OHdG) residues, a biomarker of DNA oxidation. In the SHS model, each male's fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. In GPX-5 KO mice, SDD was evaluated by immunocytochemical detection of 8-Hydroxy-deoxy Guanosine (8-OHdG) residues, a biomarker of DNA oxidation. In the SHS model, each male's fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. ResultsThe average background levels of 8-OHdG in WT mice is around 30%. This level doubles up to about 60% in transgenic male mice deficient in the antioxidant enzyme GPX-5. Our results indicate that a 2 month pretreatment of GPX-5 KO mice with Fertilix provides complete protection of sperm DNA against oxidation. In male mice exposed to the SHS model, only 35% (19/54) of partnered female mice got pregnant resulting in 169 fetuses. In contrast, male mice pretreated with Fertilix for 2 weeks yielded 74% (42/57) of partnered female mice becoming pregnant, resulting in 427 fetuses. The role of chance in obtaining supporting results for the efficacy of Fertilix in both models is minimal although it was not possible to ensure that every mouse took 100% of the product for the treatment period. The average background levels of 8-OHdG in WT mice is around 30%. This level doubles up to about 60% in transgenic male mice deficient in the antioxidant enzyme GPX-5. Our results indicate that a 2 month pretreatment of GPX-5 KO mice with Fertilix provides complete protection of sperm DNA against oxidation. In male mice exposed to the SHS model, only 35% (19/54) of partnered female mice got pregnant resulting in 169 fetuses. In contrast, male mice pretreated with Fertilix for 2 weeks yielded 74% (42/57) of partnered female mice becoming pregnant, resulting in 427 fetuses. The role of chance in obtaining supporting results for the efficacy of Fertilix in both models is minimal although it was not possible to ensure that every mouse took 100% of the product for the treatment period. ConclusionsOral administration of Fertilix significantly reduces SDD in GPX-5 knockout mice and restores pregnancy rates almost back to normal levels in mice subjected to SHS. These results, if confirmed in humans, will impact clinical fertility practice. Current clinical studies confirm moderate to severe SDD in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility, figures which are gravely concerning. Antioxidant supplementation will be an adjuvant therapy prior to undertaking ART procedures to improve fertilization rates, maintain a healthy pregnancy, and reduce de novo sporadic mutations being passed onto children. Oral administration of Fertilix significantly reduces SDD in GPX-5 knockout mice and restores pregnancy rates almost back to normal levels in mice subjected to SHS. These results, if confirmed in humans, will impact clinical fertility practice. Current clinical studies confirm moderate to severe SDD in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility, figures which are gravely concerning. Antioxidant supplementation will be an adjuvant therapy prior to undertaking ART procedures to improve fertilization rates, maintain a healthy pregnancy, and reduce de novo sporadic mutations being passed onto children.

3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine V11294 (1) has been identified as a lead structure, which selectively inhibits human lung PDE4 (436 nM) and is also active in a number of in vitro and in vivo models of inflammation. Here we describe the synthesis and pharmacology of a series of highly potent 8-[(benzyloxy)methyl]-substituted analogues, with potencies in the range 10-300 nM. In addition, several compounds showed interesting PDE4 subtype specificities, for example, the 3-thienyl derivative 5v, which showed 6-10 nM potency at PDE4B, D3, and D5 and a 20- to 200-fold selectivity over A and D2, respectively.

The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease.

We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes m1, M3 or M4. Here we describe allosteric effects at human M1–M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with 3H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at m1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at m1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPγS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.

In radioligand binding studies, it has been reported that brucine, N-chloromethyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M1, M3, and M4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acetylcholine (ACh) potency at M1 and M4 receptors respectively have been confirmed in guanosine-5'-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N-chloromethyl brucine on M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5'-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N-chloromethyl brucine with ACh on M4 receptor function was also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action of N-chloromethyl brucine on neurogenically released ACh binding at M3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes.

Substituted indoles have been condensed with N-benzyl-4-piperidone to give 3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles. Under basic conditions, 5-, 6-, and 7- (but not 4-) substituted indoles give reasonable yields of the product. For condensation with 4-substituted indoles, acidic conditions and the presence of at least a 3-fold excess of N-benzyl-4-piperidone are beneficial. Under basic conditions, the condensation of indoles with N-substituted-3-piperidones is highly regioselective with the regioselectivity depending on the nature of the N-substituent. 4-Substituted indoles do not react with N-substituted-3-piperidones under basic conditions but give a single product under acidic conditions.

We studied the interactions of strychnine, brucine, and three of the N-substituted analogues of brucine with [3H]N-methylscopolamine (NMS) and unlabeled acetylcholine at m1-m5 muscarinic receptors using equilibrium and nonequilibrium radioligand binding studies. The results were consistent with a ternary allosteric model in which both the primary and allosteric ligands bind simultaneously to the receptor and modify the affinities of each other. The compounds had Kd values in the submillimolar range, inhibited [3H]NMS dissociation, and showed various patterns of positive, neutral, and negative cooperativity with [3H]NMS and acetylcholine, but there was no predictive relationship between the effects. Acetylcholine affinity was increased approximately 2-fold by brucine at m1 receptors, approximately 3-fold by N-chloromethyl brucine at m3 receptors, and approximately 1.5-fold by brucine-N-oxide at m4 receptors. The existence of neutral cooperativity, in which the compound bound to the receptor but did not modify the affinity of acetylcholine, provides the opportunity for a novel form of drug selectivity that we refer to as absolute subtype selectivity: an agent showing positive or negative cooperativity with the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its affinities for the subtypes. Our results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetylcholine may yield compounds with various patterns of cooperativity across the receptor subtypes.

Two series of pentacyclic carbazolones, 22 and 23, have been synthesized utilizing a facile intramolecular Dielsminus signAlder reaction and are allosteric modulators at muscarinic acetylcholine receptors. Their affinities and cooperativities with acetylcholine and the antagonist N-methylscopolamine (NMS) at M(1)minus signM(4) receptors have been analyzed and compared. All of the synthesized compounds are negatively cooperative with acetylcholine. In contrast, the majority of the compounds exhibit positive cooperativity with NMS, particularly at M(2) and M(4) receptors. The subtype selectivity, in terms of affinity, was in general M(2) > M(1) > M(4) > M(3). The largest increases in affinity produced by a single substitution of the core structure were given by the 1-OMe (22b) and 1-Cl (22d) derivatives. The position of the N in the ring did not appear to be important for binding affinity or cooperativity. Two compounds 22y and 23i, both trisubstituted analogues, were the most potent compounds synthesized, with dissociation constants of 30minus sign100 nM for the M(2) NMS-liganded and unliganded receptor, respectively. The results indicate that the allosteric site, like the primary binding site, is capable of high-affinity interactions with molecules of relatively low molecular weight.