Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Sperm chromatin structure assay (SCSA®) and flow cytometry-assisted TUNEL assay provide a concordant assessment of sperm DNA fragmentation as a function of age in a large cohort of approximately 10,000 patients.

BACKGROUND: Sperm DNA integrity is increasingly seen as a critical characteristic determining reproductive success, both in natural reproduction and in assisted reproductive technologies (ART). Despite this awareness, sperm DNA and nuclear integrity tests are still not part of routine examinations for either infertile men or fertile men wishing to assess their reproductive capacity. This is not due to the unavailability of DNA and sperm nuclear integrity tests. On the contrary, several relevant but distinct tests are available and have been used in many clinical trials, which has led to conflicting results and confusion. The reasons for this are mainly the lack of standardization between different clinics and between the tests themselves. In addition, the small number of samples analyzed in these trials has often weakened the value of the analyses performed. In the present work, we used a large cohort of semen samples, covering a wide age range, which were simultaneously evaluated for sperm DNA fragmentation (SDF) using two of the most frequently used SDF assays, namely the TUNEL assay and the sperm chromatin structure assay (SCSA®). At the same time, as standard seminal parameters (sperm motility, sperm morphology, sperm count) were available for these samples, correlations between age, SDF and conventional seminal parameters were analyzed. RESULTS: We show that the SCSA® and TUNEL assessments of SDF produce concordant data. However, the SDF assessed by TUNEL is systematically lower than that assessed by SCSA®. Regardless of the test used, the SDF increases steadily during aging, while the HDS parameter (High DNA stainability assessed via SCSA®) remains unchanged. In the cohort analyzed, conventional sperm parameters do not seem to discriminate with aging. Only sperm volume and motility were significantly lower in the oldest age group analyzed [50-59 years of age]. CONCLUSIONS: In the large cohort analyzed, SDF is an age-dependent parameter, increasing linearly with aging. The SCSA® assessment of SDF and the flow cytometry-assisted TUNEL assessment are well correlated, although TUNEL is less sensitive than SCSA®. This difference in sensitivity should be taken into account in the final assessment of the true level of fragmentation of the sperm nucleus of a given sample. The classical sperm parameters (motility, morphology, sperm count) do not change dramatically with age, making them inadequate to assess the fertility potential of an individual.

RéSUMé: CONTEXTE: l'intégrité de l'ADN des spermatozoïdes est de plus en plus considérée comme une caractéristique essentielle déterminant le succès de la reproduction, tant dans la reproduction naturelle que dans les techniques de reproduction assistée (AMP). Malgré cette prise de conscience, les tests d'intégrité nucléaire des spermatozoïdes ne font toujours pas partie des examens de routine pour les hommes infertiles ou fertiles souhaitant évaluer leur capacité de reproduction. Cette situation n'est pas due à l'indisponibilité des tests. Au contraire, plusieurs tests pertinents mais distincts sont disponibles et ont été utilisés dans de nombreux essais cliniques, ce qui a donné lieu à des résultats contradictoires et à une certaine confusion. Les raisons en sont principalement le manque de normalisation entre les différentes cliniques et entre les tests eux-mêmes. En outre, le petit nombre d'échantillons analysés dans ces essais a souvent affaibli la valeur des analyses effectuées. Dans le présent travail, nous avons utilisé une vaste cohorte d'échantillons, couvrant une large tranche d'âge, évalués simultanément pour la fragmentation de l'ADN des spermatozoïdes à l'aide de deux des tests les plus fréquemment utilisés, à savoir le test TUNEL et le test de la structure de la chromatine des spermatozoïdes (SCSA®). Parallèlement, comme les paramètres séminaux standard (motilité, morphologie, numération) étaient disponibles pour ces échantillons, les corrélations entre l'âge, le niveau de fragmentation et les paramètres séminaux conventionnels ont été analysées. RéSULTATS: Nous montrons que les évaluations SCSA® et TUNEL produisent des données concordantes. Cependant, le SDF évalué par TUNEL est systématiquement plus faible que celui évalué par SCSA®. Quel que soit le test utilisé, la fragmentation augmente régulièrement au cours du vieillissement, alors que le paramètre HDS (« High DNA stainability¼ évalué par le test SCSA®) reste inchangé. Dans la cohorte analysée, les paramètres spermatiques conventionnels ne semblent pas varier avec le vieillissement. Seuls le volume et la mobilité des spermatozoïdes étaient significativement plus faibles dans le groupe d'âge le plus élevé analysé [50­59 ans]. CONCLUSIONS: Dans la grande cohorte analysée, la fragmentation de l'ADN spermatique est un paramètre dépendant de l'âge, augmentant linéairement avec le vieillissement. L'évaluation du SDF par SCSA® et l'évaluation via le test TUNEL assistée par cytométrie de flux sont bien corrélées, bien que le TUNEL soit moins sensible que le SCSA®. Cette différence de sensibilité doit être prise en compte dans l'évaluation finale du niveau réel de fragmentation du noyau des spermatozoïdes d'un échantillon donné. Les paramètres classiques du sperme (motilité, morphologie, nombre de spermatozoïdes) ne changent pas de façon spectaculaire avec l'âge, ce qui les rend inadéquats pour évaluer le potentiel de fertilité d'un individu.

Enhanced Recovery After Surgery (ERAS®) Society Consensus Guidelines for Emergency Laparotomy Part 3: Organizational Aspects and General Considerations for Management of the Emergency Laparotomy Patient.

BACKGROUND: This is Part 3 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy using an enhanced recovery after surgery (ERAS) approach. This paper addresses organizational aspects of care. METHODS: Experts in management of the high-risk and emergency general surgical patient were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and MEDLINE database searches were performed for ERAS elements and relevant specific topics. Studies were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses and large cohort studies, and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. RESULTS: Components of organizational aspects of care were considered. Consensus was reached after three rounds of a modified Delphi process. CONCLUSIONS: These guidelines are based on best current available evidence for organizational aspects of an ERAS® approach to patients undergoing emergency laparotomy and include discussion of less common aspects of care for the surgical patient, including end-of-life issues. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Consensus Guidelines for Perioperative Care for Emergency Laparotomy Enhanced Recovery After Surgery (ERAS®) Society Recommendations Part 2-Emergency Laparotomy: Intra- and Postoperative Care.

BACKGROUND: This is Part 2 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy (EL) using an Enhanced Recovery After Surgery (ERAS) approach. This paper addresses intra- and postoperative aspects of care. METHODS: Experts in aspects of management of high-risk and emergency general surgical patients were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and Medline database searches were performed for ERAS elements and relevant specific topics. Studies on each item were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses, and large cohort studies and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. Some ERAS® components covered in other guideline papers are outlined only briefly, with the bulk of the text focusing on key areas pertaining specifically to EL. RESULTS: Twenty-three components of intraoperative and postoperative care were defined. Consensus was reached after three rounds of a modified Delphi Process. CONCLUSIONS: These guidelines are based on best available evidence for an ERAS® approach to patients undergoing EL. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa.

Abstract: Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation. Lay Summary: Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Sperm telomeres, oxidative stress, and infertility.

In brief: Oxidative stress is recognized as an underlying driving factor of both telomere dysfunction and human subfertility/infertility. This review briefly reassesses telomere integrity as a fertility biomarker before proposing a novel, mechanistic rationale for the role of oxidative stress in the seemingly paradoxical lengthening of sperm telomeres with aging. Abstract: 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, yields 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 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 for the explanation of this apparent paradox.

Science With Purpose: 50 Years of the Institute of Occupational Medicine.

The Institute of Occupational Medicine (IOM) was founded in 1969 by the then UK National Coal Board to complete its nation-wide epidemiological study of lung disease in coal miners, the Pneumoconiosis Field Research. The results quantified risks in the industry and were influential across the world in setting preventive standards. The research, based on epidemiology, was multidisciplinary from the start, and the IOM's broad scientific expertise was applied across many other industries with an increasing focus on environmental measurement and ergonomics. In 1990, as the coal industry declined, IOM became a self-funding research charity with a strong commercial arm. It has expanded its research, often with European collaborators and funding from governments, and has achieved wide recognition. This has most recently been applied during the pandemic in areas of hospital ventilation, personal protection, and viral exposure research, illustrating IOM's ability to respond to new environmental or occupational challenges.

Sperm oxidative stress in the context of male infertility: current evidence, links with genetic and epigenetic factors and future clinical needs.

Male infertility is a relatively common condition responsible for around 30% of all infertility cases and contributing to another 20%. Although the traditional semen profile is used to diagnose this condition, the criteria at the heart of this analysis are purely descriptive and shed no light on the underlying etiology. Recent research on the causes of male infertility have revealed the importance of three major factors including genetic and epigenetic mutations and a state of oxidative stress. The fundamental complexity of spermatogenesis, involving the coordinated action of over 2000 genes, plus the fact that any gene defect causing infertility will be heavily selected against, means that the genetic landscape for male infertility is characterized by a large number of different mutations each one of which is extremely rare. Such mutations can only remain within the population as a result of passage through the female germ line or transmission via heterozygous males. However, the most common genetic cause of male infertility, Y-chromosome deletions, cannot be propagated by either of these mechanisms and arises de novo in every instance. The presence of Y chromosome mutation in around 5% of severely oligozoospermic males suggests that the male germ line is under a great deal of stress, one form of which is oxidative stress. The latter impairs all aspects of sperm function and also disrupts the integrity of DNA in the sperm nucleus. An oxidative attack on sperm DNA not only generates strand breaks but, more importantly, oxidative base adducts that are extremely mutagenic. It is proposed that the persistence of these lesions into S-phase of the first mitotic division generates de novo mutations that are potentially capable of impacting the long-term health and wellbeing of the offspring including the latter's fertility. To address this issue, the field desperately needs simple validated tests of oxidative stress in the male germ line, that can then be used to direct the appropriate management of these patients.

Reactive Oxygen Species and Their Consequences on the Structure and Function of Mammalian Spermatozoa.

Significance: Among the 200 or so cell types that comprise mammals, spermatozoa have an ambiguous relationship with the reactive oxygen species (ROS) inherent in the consumption of oxygen that supports aerobic metabolism. Recent Advances: In this review, we shall see that spermatozoa need the action of ROS to reach their structural and functional maturity, but that due to intrinsic unique characteristics, they are, perhaps more than any other cell type, susceptible to oxidative damage. Recent studies have improved our knowledge of how oxidative damage affects sperm structures and functions. The focus of this review will be on how genetic and epigenetic oxidative alterations to spermatozoa can have dramatic unintended consequences in terms of both the support and the suppression of sperm function. Critical Issues: Oxidative stress can have dramatic consequences not only for the spermatozoon itself, but also, and above all, on its primary objective, which is to carry out fertilization and to ensure, in part, that the embryonic development program should lead to a healthy progeny. Future Directions: Sperm oxidative DNA damage largely affects the integrity of the paternal genetic material to such an extent that the oocyte may have difficulties in correcting it. Diagnostic and therapeutic actions should be considered more systematically, especially in men with difficulties to conceive. Research is underway to determine whether the epigenetic information carried by spermatozoa is also subject to changes mediated by pro-oxidative situations. Antioxid. Redox Signal. 37, 481-500.

Biocompatible Nanomaterials as an Emerging Technology in Reproductive Health; a Focus on the Male.

A growing body of research has confirmed that nanoparticle (NP) systems can enhance delivery of therapeutic and imaging agents as well as prevent potentially damaging systemic exposure to these agents by modifying the kinetics of their release. With a wide choice of NP materials possessing different properties and surface modification options with unique targeting agents, bespoke nanosystems have been developed for applications varying from cancer therapeutics and genetic modification to cell imaging. Although there remain many challenges for the clinical application of nanoparticles, including toxicity within the reproductive system, some of these may be overcome with the recent development of biodegradable nanoparticles that offer increased biocompatibility. In recognition of this potential, this review seeks to present recent NP research with a focus on the exciting possibilities posed by the application of biocompatible nanomaterials within the fields of male reproductive medicine, health, and research.

Apoptotic M540 bodies present in human semen interfere with flow cytometry-assisted assessment of sperm DNA fragmentation and oxidation.

BACKGROUND: The use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. It has been hypothesized that the observed discrepancies could be attributed to the presence of apoptotic bodies that can be labeled with merocyanine 540, the so-called M540 bodies. In order to verify this hypothesis and determine the accuracy of our in-house FC-assisted evaluation of spermatozoa parameters, we used FC to evaluate both the fragmentation of sperm DNA using the TUNEL assay and the oxidation of sperm DNA using the 8-OHdG assay on semen samples with or without M540 bodies. RESULTS: We show that the presence of M540 bodies lead to underestimation of both the level of sperm DNA fragmentation and sperm DNA oxidation when using FC assisted detection systems. We also observed that this situation is particularly pertinent in semen samples classified as abnormal with respect to the routine WHO semen evaluation as they appear to contain more M540 bodies than normal samples. CONCLUSIONS: We conclude that M540 bodies interfere with both FC-conducted assays designed to evaluate sperm nuclear/DNA integrity. Exclusion of these contaminants in unprepared semen samples should be performed in order to correctly appreciate the true level of sperm DNA/nuclear damage which is known to be a critical male factor for reproductive success.

RéSUMé: CONTEXTE: L'utilisation de la cytométrie en flux (CF) pour évaluer la fragmentation de l'ADN des spermatozoïdes via la technique TUNEL (Terminal transferase dUTP nick-end labelling) a montré des incohérences par rapport aux analyses conventionnelles par microscopie fluorescente. L'hypothèse a été émise que les divergences observées pourraient être attribuées à la présence de corps apoptotiques qui peuvent être marqués à la mérocyanine 540 (corps M540). Afin de vérifier cette hypothèse et de déterminer la précision de notre évaluation interne des paramètres des spermatozoïdes, nous avons mesuré par CF à la fois la fragmentation de l'ADN des spermatozoïdes en utilisant le test TUNEL et l'oxydation de l'ADN des spermatozoïdes en utilisant le test 8-OHdG sur des échantillons de sperme avec ou sans corps M540. RéSULTATS: Nous montrons que la présence des corps M540 entraîne une sous-estimation du niveau de fragmentation et d'oxydation de l'ADN des spermatozoïdes lors de l'utilisation de systèmes de détection assistée par CF. Nous avons également observé que cette situation est exacerbée dans les échantillons de sperme classés comme anormaux (selon les standards de l'OMS), car ces derniers semblent contenir plus de corps M540 que les échantillons normaux. CONCLUSIONS: Nous concluons que les corps M540 interfèrent avec les deux tests conduits par CF et conçus pour évaluer l'intégrité nucléaire des spermatozoïdes. L'exclusion de ces contaminants dans les échantillons de sperme non préparés devrait être considérée afin d'apprécier correctement le véritable niveau de dommages au noyau spermatique qui est connu pour être un facteur critique pour le succès reproductif.

Guidelines for Perioperative Care for Emergency Laparotomy Enhanced Recovery After Surgery (ERAS) Society Recommendations: Part 1-Preoperative: Diagnosis, Rapid Assessment and Optimization.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) protocols reduce length of stay, complications and costs for a large number of elective surgical procedures. A similar, structured approach appears to improve outcomes, including mortality, for patients undergoing high-risk emergency general surgery, and specifically emergency laparotomy. These are the first consensus guidelines for optimal care of these patients using an ERAS approach. METHODS: Experts in aspects of management of the high-risk and emergency general surgical patient were invited to contribute by the International ERAS® Society. Pubmed, Cochrane, Embase, and MEDLINE database searches on English language publications were performed for ERAS elements and relevant specific topics. Studies on each item were selected with particular attention to randomized controlled trials, systematic reviews, meta-analyses and large cohort studies, and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Recommendations were made on the best level of evidence, or extrapolation from studies on non-emergency patients when appropriate. The Delphi method was used to validate final recommendations. The guideline has been divided into two parts: Part 1-Preoperative Care and Part 2-Intraoperative and Postoperative management. This paper provides guidelines for Part 1. RESULTS: Twelve components of preoperative care were considered. Consensus was reached after three rounds. CONCLUSIONS: These guidelines are based on the best available evidence for an ERAS approach to patients undergoing emergency laparotomy. Initial management is particularly important for patients with sepsis and physiological derangement. These guidelines should be used to improve outcomes for these high-risk patients.

Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro.

Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro. LAY SUMMARY: Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules - called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.

Serum vitamin D content is associated with semen parameters and serum testosterone levels in men.

The present study aimed to evaluate the influence of serum vitamin D levels on semen quality and testosterone levels. This is a cross-sectional study conducted at Androscience, Science and Innovation Center in Andrology and High-Complex Clinical and Andrology Laboratory in Sao Paulo, Brazil, with 508 male patients, aged 18-60 years, from 2007 to 2017. Seminal parameters and serum sexual hormones were correlated with serum vitamin D concentrations in 260 men selected by strict selection criteria. Patients were divided into normozoospermic group (NZG, n = 124) and a group with seminal abnormalities (SAG, n = 136). Evaluation included complete physical examination, past medical history, habits and lifestyle factors, two complete seminal analysis with sperm functional tests, serum levels of 25-hydroxy-vitamin D3(25(OH)VD3), total and free testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone-binding globulin (SHBG), total cholesterol, homeostatic model assessment of insulin resistance (HOMA-IR) index, and karyotype. The mean concentration of 25(OH)VD3was significantly lower in the SAG (P < 0.001) and positively correlated with all baseline seminal parameters and total testosterone levels. In addition, serum vitamin D3concentration was found to be positively correlated with sperm concentration (ß= 2.103; P < 0.001), total number of spermatozoa with progressive motility (ß = 2.069; P = 0.003), total number of motile spermatozoa (ß = 2.571; P = 0.015), and strict morphology (ß = 0.056; P = 0.006), regardless of other variables. This is the first comparative study to address the issue of serum vitamin D3content between normozoospermic patients and those with sperm abnormalities. It clearly demonstrates a direct and positive relationship between serum vitamin D level and overall semen quality, male reproductive potential, and testosterone levels.

A novel approach to nonsurgical sterilization; application of menadione-modified gonocyte-targeting M13 bacteriophage for germ cell ablation in utero.

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox-cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide 'phage display library proved effective in the isolation of gonocyte-specific targeting clones. One such clone was modified via N-succinimidyl-S-acetylthioacetate (SATA) linkage to the N-terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof-of-concept experiments we applied the modified 'phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species.

The Importance of Oxidative Stress in Determining the Functionality of Mammalian Spermatozoa: A Two-Edged Sword.

This article addresses the importance of oxidative processes in both the generation of functional gametes and the aetiology of defective sperm function. Functionally, sperm capacitation is recognized as a redox-regulated process, wherein a low level of reactive oxygen species (ROS) generation is intimately involved in driving such events as the stimulation of tyrosine phosphorylation, the facilitation of cholesterol efflux and the promotion of cAMP generation. However, the continuous generation of ROS ultimately creates problems for spermatozoa because their unique physical architecture and unusual biochemical composition means that they are vulnerable to oxidative stress. As a consequence, they are heavily dependent on the antioxidant protection afforded by the fluids in the male and female reproductive tracts and, during the precarious process of insemination, seminal plasma. If this antioxidant protection should be compromised for any reason, then the spermatozoa experience pathological oxidative damage. In addition, situations may prevail that cause the spermatozoa to become exposed to high levels of ROS emanating either from other cells in the immediate vicinity (particularly neutrophils) or from the spermatozoa themselves. The environmental and lifestyle factors that promote ROS generation by the spermatozoa are reviewed in this article, as are the techniques that might be used in a diagnostic context to identify patients whose reproductive capacity is under oxidative threat. Understanding the strengths and weaknesses of ROS-monitoring methodologies is critical if we are to effectively identify those patients for whom treatment with antioxidants might be considered a rational management strategy.