Background:Low-intensity extracorporeal shockwave therapy (Li-ESWT) is thought to treat erectile dysfunction (ED) by stimulating neovascularization and nerve regeneration as demonstrated in animal models by histologically increased angiogenesis and neuronal-related growth factors, though corresponding human studies are limited. Aim:We hypothesized that Li-ESWT results in appreciable increases in growth factors in human tissues, and in this proof-of-concept study we aimed to determine whether markers for neovascularization and nerve regeneration can be detected in the corporal blood of men following Li-ESWT treatment. Methods:Patients were prospectively enrolled in a clinical trial of Li-ESWT for ED. Patients received 12 bi-weekly Li-ESWT treatments of 0.2 mJ/mm2 at 5 Hz, 1500 shocks delivered per treatment, with follow up at 1-2 weeks, 4-6 weeks, 3 months, and 6 months post-treatment. Cavernosal penile blood samples were obtained prior to treatment and at each visit post-treatment. The concentrations of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and brain-derived neurotropic factor (BDNF) in penile plasma samples were measured using enzyme-linked immunosorbent assay with specific commercial kits, following the protocols provided by the manufacturer. Outcomes:eNOS, nNOS, VEGF, and BDNF were detectable and demonstrated changes in cavernosal plasma samples following Li-ESWT treatment. Results:Twenty-five patients completed all five study visits. Mean patient age was 63. Mean baseline International Index of Erectile Function-Erectile Function score prior to treatment was 14.24 (±1.21). Corporal plasma samples were analyzed for eNOS, nNOS, VEGF, and BDNF using the enzyme-linked immunosorbent assay. Levels of eNOS, nNOS, and VEGF showed an upward trend following treatment but did not reach significance. BDNF levels were noted to decrease. Clinical Implication:Corporal blood aspirates may function as surrogates for histological studies to understand effects of Li-ESWT at the tissue level in humans. Strengths and Limitations:To our knowledge, this is first the molecular study in human tissues to attempt to quantify neurogenesis and neovascularization in penile tissue following Li-ESWT for ED. Although our sample size is small, we believe this represents a promising first step in understanding the effect of Li-ESWT at a tissue level in men. Conclusion:The clinical significance of our findings is currently unknown, but markers of neovascularization and neurogenesis are detectable in corporal plasma and may change following Li-ESWT. ClinicalTrials.gov  ID NCT04720755.

Male infertility affects one in 10 men, yet we have seen few changes in the diagnostic tests recommended in the guidelines in the recent decade. Here we review the literature to identify newly available or emerging diagnostic tools in male infertility. From “-omics” (such as genomic, transcriptomics, or proteomics), tests of sperm function, advanced and innovated imaging technology, and integrating artificial intelligence, there are many potential areas where these diagnostic tests can improve patient care. These tools will hopefully allow to continue to unlock the underlying pathophysiology and understand the heterogeneous nature of male infertility.

Major depressive disorder (MDD) and posttraumatic stress disorder (PTSD) are debilitating psychiatric conditions associated with poor health outcomes similarly observed in non-pathological aging. Ketamine is a dissociative anesthetic and NMDA receptor antagonist with demonstrated rapid reduction in symptoms associated with Treatment Resistant Depression (TRD) and PTSD. Ketamine’s effects on biological aging have not been extensively studied among patients with moderate to severe symptoms of depression and/or trauma. To address this gap, this study looked at the changes in non-epigenetic measures, DNA methylation levels, immune cell composition, and biological age based on various epigenetic biomarkers of aging, of 20 participants at baseline and after completion of a course of six ketamine 0.5 mg/kg infusions in individuals with MDD or PTSD. As expected, depression and PTSD scores decreased in participants following ketamine infusion treatments as measured by the PHQ-9 and PCL-5. We observed a reduction in epigenetic age in the OMICmAge, GrimAge V2, and PhenoAge biomarkers. In order to better understand the changes in epigenetic age, we also looked at the underlying levels of various Epigenetic Biomarker Proxies (EBPs) and surrogate protein markers and found significant changes following ketamine treatment. The results are consistent with existing literature on ketamine’s effects on different biomarkers. These results underline the ability of GrimAge V2, PhenoAge, and OMICmAge in particular, to capture signals associated with key clinical biomarkers, and add to the growing body of literature on ketamine’s epigenetic mechanisms and their effect on biological aging. Clinical Trial Code. NCT05294835.

Blood feeding provides essential nutrients for development and reproduction in hematophagous arthropods yet also initiates significant other physiological alterations in immune function. Immune cells, or hemocytes, are integral components of the arthropod innate immune system with notable roles in defining vector competence. Evidence suggests that both blood feeding and infection drive substantial changes in hemocyte phenotypes, including proliferation, immune activation, and differentiation, which directly and indirectly influence pathogen infection outcomes. These dynamics have fueled extensive research into hemocyte biology in recent years, which aided by emerging single-cell technologies and methods of phagocyte depletion, have provided novel molecular insights into hemocyte populations and additional support for their important contributions to parasite, virus, and bacterial infections. Despite this progress, many aspects of arthropod immune cell biology remain unclear. Focusing on mosquitoes and ticks as two of the most prominent and well-studied arthropod vectors, this review summarizes the effects of blood feeding and infection on mosquito and tick hemocytes, highlighting hemocyte classifications, and the known mechanisms by which hemocytes can have positive or negative impacts on vector-borne pathogen infection.

Tumor Necrosis Factor-α (TNF-α) is a proinflammatory cytokine and a master regulator of immune cell function in vertebrates. While previous studies have implicated TNF signaling in invertebrate immunity, the roles of TNF in mosquito innate immunity and vector competence have yet to be functionally explored. Herein, we confirm the identification of a conserved TNF-α pathway in Anopheles gambiae consisting of the TNF-α ligand, Eiger, and its cognate receptors Wengen and Grindelwald. Through gene expression analysis, RNAi, and in vivo injection of recombinant TNF-α, we provide direct evidence for the requirement of TNF signaling in regulating mosquito immune cell function by promoting granulocyte midgut attachment, increased granulocyte abundance, and oenocytoid rupture. Moreover, our data demonstrate that TNF signaling is an integral component of anti-Plasmodium immunity that limits malaria parasite survival. Together, our data support the existence of a highly conserved TNF signaling pathway in mosquitoes that mediates cellular immunity and influences Plasmodium infection outcomes, offering potential new approaches to interfere with malaria transmission by targeting the mosquito host.

PURPOSE:Non-obstructive azoospermia (NOA) is a common, but complex problem, with multiple therapeutic options and a lack of clear guidelines. Hence, there is considerable controversy and marked variation in the management of NOA. This survey evaluates contemporary global practices related to medical and surgical management for patients with NOA. MATERIALS AND METHODS:A 56-question online survey covering various aspects of the evaluation and management of NOA was sent to specialists around the globe. This paper analyzes the results of the second half of the survey dealing with the management of NOA. Results have been compared to current guidelines, and expert recommendations have been provided using a Delphi process. RESULTS:Participants from 49 countries submitted 336 valid responses. Hormonal therapy for 3 to 6 months was suggested before surgical sperm retrieval (SSR) by 29.6% and 23.6% of participants for normogonadotropic hypogonadism and hypergonadotropic hypogonadism respectively. The SSR rate was reported as 50.0% by 26.0% to 50.0% of participants. Interestingly, 46.0% reported successful SSR in <10% of men with Klinefelter syndrome and 41.3% routinely recommended preimplantation genetic testing. Varicocele repair prior to SSR is recommended by 57.7%. Half of the respondents (57.4%) reported using ultrasound to identify the most vascularized areas in the testis for SSR. One-third proceed directly to microdissection testicular sperm extraction (mTESE) in every case of NOA while others use a staged approach. After a failed conventional TESE, 23.8% wait for 3 months, while 33.1% wait for 6 months before proceeding to mTESE. The cut-off of follicle-stimulating hormone for positive SSR was reported to be 12-19 IU/mL by 22.5% of participants and 20-40 IU/mL by 27.8%, while 31.8% reported no upper limit. CONCLUSIONS:This is the largest survey to date on the real-world medical and surgical management of NOA by reproductive experts. It demonstrates a diverse practice pattern and highlights the need for evidence-based international consensus guidelines.

X-chromosome inactivation (XCI) is a fundamental mechanism in placental mammals that compensates for gene dosage differences between sexes. Using methylation levels of genes under XCI, we establish defective levels of XCI as a new source of interindividual variation among cancer types in females, characterized by a significant and consistent lowering of XIST expression and enrichment of differentially expressed genes under XCI. We show that defective XCI is an additive factor to the cancer risk of XCI escape deregulation in women. Defective XCI of more than 10% has an attributable risk of 40% among 12 different cancers from The Cancer Genome Atlas. Validations between independent studies of breast cancer samples show that defective XCI increases triple-negative subtype frequency, decreases survival rates, and is reduced by chemotherapy treatment. Mechanistically, it is associated with somatic mutations at TP53 and top MYC gains. In independent studies, defective XCI is detectable in blood and increases with aging, menopause, and cancer diagnosis.

Introduction: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. Methods: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. Results: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. Conclusion: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Age-related alterations in immune function are believed to increase risk for a host of age-related diseases leading to premature death and disability. Programming of the immune system by diet, lifestyle, and environmental factors occurs across the lifespan and influences both makeup and function of the immune system, including immunometabolism. This programming is believed to act in large part through epigenetic modification. Among dietary components that affect this process, polyphenols may play an outsized role. Polyphenols are a widely distributed group of plant nutrients consumed by humans. Certain foods possess distinctive and relatively higher levels of these compounds. One such food is Tartary buckwheat (fagopyrum tataricum), an ancient seed historically prized for its health benefits. It is suggested that the specific composition of polyphenols found in foods like Tartary buckwheat may lead to a unique impact on immunometabolic physiological pathways that could be interrogated through epigenetic analyses. The objective of this study was to investigate the epigenetic effects on peripheral immune cells in healthy individuals of a standardized polyphenol concentrate based on naturally occurring nutrients in Tartary buckwheat. This pilot clinical trial tested the effects of consuming 90 days of this concentrate in 50 healthy male (40%) and female (60%) participants aged 18–85 years using epigenetic age clocks and deconvolution methods. Analysis revealed significant intervention-related changes in multiple epigenetic age clocks and immune markers as well as population-wide alterations in gene ontology (GO) pathways related to longevity and immunity. This study provides previously unidentified insights into the immune, longevity and epigenetic effects of consumption of polyphenol-rich plants and generates additional support for health interventions built around historically consumed plants like Tartary buckwheat while offering compelling opportunities for additional research.Clinical trial registrationClinicalTrials.gov, Identifier: NCT05234203.

Background and Objective:In-vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) has become increasingly prevalent even in cases without significant male factor infertility; however, stagnant live-birth rates, both nationally and internationally, have driven more research into sperm selection. To date, nothing has replaced swim-up and density-gradient preparation methods and therefore we sought to review the state of the science. Methods:A PubMed search was performed between years of 1989 and 2024 for English research articles reporting data on sperm selection technology in assisted reproductive technology. Key Content and Findings:IVF with ICSI is increasingly prevalent even in men with normal semen parameters. Despite technologic advances and widespread use, reproductive outcomes with ICSI have been stagnant. This market for opportunity growth has allowed for sperm selection techniques to grow exponentially with heterogeneity in utilization and a paucity of positive reproductive outcomes. Swim-up and density-gradient centrifugation remain the most utilized sperm selection techniques. Various future technologies show promise including epigenetics, sperm biomarkers and a potential role of artificial intelligence; however, more research is needed. Conclusions:Given unchanged IVF success rates, sperm selection technologies hold promise to improve reproductive outcomes beyond traditional ICSI. At present, no technique has shown superiority to swim up and density centrifugation.

Background:Differentially methylated imprint control regions (ICRs) regulate the monoallelic expression of imprinted genes. Their epigenetic dysregulation by environmental exposures throughout life results in the formation of common chronic diseases. Unfortunately, existing Infinium methylation arrays lack the ability to profile these regions adequately. Whole genome bisulfite sequencing (WGBS) is the unique method able to profile these regions, but it is very expensive and it requires not only a high coverage but it is also computationally intensive to assess those regions. Findings:To address this deficiency, we developed a custom methylation array containing 22,819 probes. Among them, 9,757 probes map to 1,088 out of the 1,488 candidate ICRs recently described. To assess the performance of the array, we created matched samples processed with the Human Imprintome array and WGBS, which is the current standard method for assessing the methylation of the Human Imprintome. We compared the methylation levels from the shared CpG sites and obtained a mean R 2 = 0.569. We also created matched samples processed with the Human Imprintome array and the Infinium Methylation EPIC v2 array and obtained a mean R 2 = 0.796. Furthermore, replication experiments demonstrated high reliability (ICC: 0.799-0.945). Conclusions:Our custom array will be useful for replicable and accurate assessment, mechanistic insight, and targeted investigation of ICRs. This tool should accelerate the discovery of ICRs associated with a wide range of diseases and exposures, and advance our understanding of genomic imprinting and its relevance in development and disease formation throughout the life course.

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

Reactivation of retroelements in the human genome has been linked to aging. However, whether the epigenetic state of specific retroelements can predict chronological age remains unknown. We provide evidence that locus-specific retroelement DNA methylation can be used to create retroelement-based epigenetic clocks that accurately measure chronological age in the immune system, across human tissues, and pan-mammalian species. We also developed a highly accurate retroelement epigenetic clock compatible with EPICv.2.0 data that was constructed from CpGs that did not overlap with existing first- and second-generation epigenetic clocks, suggesting a unique signal for epigenetic clocks not previously captured. We found retroelement-based epigenetic clocks were reversed during transient epigenetic reprogramming, accelerated in people living with HIV-1, and responsive to antiretroviral therapy. Our findings highlight the utility of retroelement-based biomarkers of aging and support a renewed emphasis on the role of retroelements in geroscience.

INTRODUCTION:Iatrogenic injury during urethral catheterization is a common reason for inpatient urologic consultation and is associated with increased morbidity and resource utilization. Literature defining the patient population, interventions, or outcomes associated with traumatic catheterization is scarce.METHODS:We performed a retrospective review of consults for adult urethral catheterization at a single tertiary care center (July 2017-December 2019), with focus on patient characteristics and complications. Traumatic urethral catheterization was defined as catheterization by the primary team with at least 1 of these conditions: gross hematuria, meatal blood, or cystoscopic evidence of urethral trauma. Characteristics collected included urologic history, catheterization circumstances, procedural intervention, and subsequent visits.RESULTS:Three hundred urology consults for urethral catheterization were identified, including 98 (33%) traumatic events (5.3 incidents/1000 catheters placed). All traumatic catheterization consults were in men (median age 69 years). Most (71%) patients sustaining injury had significant urologic history (eg, benign prostatic hyperplasia, urethral stricture). Sixty-three (64%) consults were determined to be uncomplicated (not requiring any procedural intervention for catheter placement). Gross hematuria was the most common sequela (50% of patients). The 30-day catheter-associated urinary tract infection rate was 13%, and 2 patients developed sepsis. Complications required a total of 52 additional hospital admission days, 19 of which were intensive-care level, as well 113 outpatient urology visits.CONCLUSIONS:Traumatic urethral catheterization is associated with increased need for procedural intervention, risk of catheter-associated urinary tract infection, and additional resource utilization. Further studies on traumatic catheterization are needed to guide systemic efforts for minimizing injury and cost.

Abstract Introduction Low-intensity extracorporeal shockwave therapy (LiESWT) is thought to treat erectile dysfunction (ED) but the mechanism remains elusive. It is believed that LiESWT may work by stimulating neovascularization and nerve regeneration as demonstrated in animal models by histologically increased angiogenesis-related growth factors and neuronal factors including vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS) and brain derived-neurotropic factor (BDNF). Given its prolific use, a thorough understanding of the mechanism of action of this therapy at a cellular level is critical prior to recommendation of this therapy on a larger scale. Objective Objective data post LiESWT is sparse. In this study, we aimed to determine whether markers for neovascularization and nerve regeneration can be detected in the serum of penile tissue in men following LiESWT treatment. We hypothesized that LiESWT results in appreciable increases in vascular and neuronal factors post therapy. Methods Patients were prospectively enrolled in a clinical trial of LiESWT for ED. Patients received 12 bi-weekly LiESWT treatments of 0.2mJ/mm2 at 5Hz, 1500 shocks delivered per treatment, with follow up at 1-2 weeks, 4-6 weeks, 3 months and 6 months post-treatment. Cavernosal penile blood samples were obtained prior to treatment and at each visit post-treatment. The concentrations of eNOS, nNOS, VEGF, and BDNF in penile plasma samples were measured using Enzyme-Linked Immunosorbent Assay (ELISA) with specific commercial kits, following the protocols provided by the manufacturer. Results Twenty-five patients completed all five study visits. Mean patient age was 63. Mean baseline IIEF-EF score prior to treatment was 14.24 (±1.21). At the time of data analysis, plasma samples from 9 men were analyzed for BDNF, eNOS, and nNOS levels using the ELISA assay, and samples from all 25 men were analyzed for VEGF. BDNF levels demonstrated a sustained decrease after the first treatment (P = 0.0001, Figure 1A). Levels of eNOS were noted to trend upward, but no significant changes in eNOS, nNOS or VEGF levels were noted. Conclusions To our knowledge, this is the first study in human tissues to attempt to quantify objective measures of neurogenesis and neovascularization in penile tissue following LiESWT for ED. Though our N is small, our results suggest that LiESWT may decrease BDNF levels after initial LiESWT treatment while VEGF, eNOS and nNOS levels remain unchanged. The clinical significance of these findings is currently unknown, as our findings are not consistent with histological studies in animals which demonstrate upregulation of these markers in penile tissues. However, we believe this represents a promising first step in attempting to understand the effect of LiESWT at a tissue level in men undergoing LiESWT. Disclosure No.

IntroductionTicks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission.MethodsThis study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach.Results and discussionOur data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination.ConclusionOur results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tick-microbial interactions.