Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity

STUDY QUESTION: Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions? SUMMARY ANSWER: We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples. WHAT IS KNOWN ALREADY: For >20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells. By using type-specific PDEIs, differential modulation of sperm motility may be achieved without adversely affecting other functions such as the acrosome reaction (AR). STUDY DESIGN, SIZE, DURATION: This was a basic medical research study examining sperm samples from normozoospermic donors and subfertile patients attending the Assisted Conception Unit (ACU), Ninewells Hospital Dundee for diagnostic semen analysis, IVF and ICSI. Phase 1 screened 43 commercially available compounds with reported PDEI activity to identify lead compounds that stimulate sperm motility. Samples were exposed (20 min) to three concentrations (1, 10 and 100 µM) of compound, and selected candidates (n = 6) progressed to Phase 2, which provided a more comprehensive assessment using a battery of in vitro sperm function tests.  PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy donors and subfertile patients were recruited at the Medical Research Institute, University of Dundee and ACU, Ninewells Hospital Dundee (ethical approval 08/S1402/6). In Phase 1, poor motility cells recovered from the 40% interface of the discontinuous density gradient were used as surrogates for patient samples. Pooled samples from three to four different donors were utilized in order to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds. During Phase 2 testing, semen samples from 23 patients attending for either routine diagnostic andrology assessment or IVF/ICSI were prepared and exposed to selected compounds. Additionally, 48 aliquots of prepared samples, surplus to clinical use, were examined from IVF (n = 32) and ICSI (n = 16) patients to further determine the effects of selected compounds under clinical conditions of treatment. Effects of compounds on sperm motility were assessed by computer-assisted sperm analysis. A modified Kremer test using methyl cellulose was used to assess sperm functional ability to penetrate into viscous media. Sperm acrosome integrity and induction of apoptosis were assessed using the acrosomal content marker PSA-FITC and annexin V kit, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In Phase 1, six compounds were found to have a strong effect on poor motility samples with a magnitude of response of ≥60% increase in percentage total motility. Under capacitating and non-capacitating conditions, these compounds significantly (P ≤ 0.05) increased the percentage of total and progressive motility. Furthermore, these compounds enhanced penetration into a cervical mucus substitute (P ≤ 0.05). Finally, the AR was not significantly induced and these compounds did not significantly increase the externalization of phosphatidylserine (P = 0.6, respectively). In general, the six compounds maintained the stimulation of motility over long periods of time (180 min) and their effects were still observed after their removal. In examinations of clinical samples, there was a general observation of a more significant stimulation of sperm motility in samples with lower baseline motility. In ICSI samples, compounds #26, #37 and #38 were the most effective at significantly increasing total motility (88, 81 and 79% of samples, respectively) and progressive motility (94, 93 and 81% of samples, respectively). In conclusion, using a two-phased drug discovery screening approach including the examination of clinical samples, 3/43 compounds were identified as promising candidates for further study. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating the results. Data for patients were from one assessment and thus the robustness of responses needs to be established. The n values for ICSI samples were relatively small. WIDER IMPLICATIONS OF THE FINDINGS: We have systematically screened and identified several compounds that have robust and effective stimulation (i.e. functional significance with longevity and no toxicity) of total and progressive motility under clinical conditions of treatment. These compounds could be clinical candidates with possibilities in terms of assisted reproductive technology options for current or future patients affected by asthenozoospermia or oligoasthenozoospermia

Can We Calculate Mean Arterial Pressure in Humans?

Mean arterial pressure (MAP) is either measured with an oscillometric cuff and then systolic (SBP) and diastolic (DBP) blood pressures are estimated from an unknown algorithm; or SBP and DBP are measured via auscultation and MAP calculated using measures of systolic pressure (SBP), diastolic pressure (DBP), and a form-factor (FF; equation: [(SBP-DBP)*FF]+DBP). The typical FF used is 0.33 though others (0.4) have been proposed. Recent work indicates that estimation of aortic MAP via a FF leads to inaccurate values and should therefore be interpreted with caution, whether this is the case for local MAP is unknown. While the implications for hypertension (HTN) diagnosis are minimal, the calculation of local MAP is essential to the study of blood pressure regulation and exercise hemodynamics in patient populations (e.g. heart failure). PURPOSE: To compare the calculation of local MAP using catheter waveforms and a FF, against MAP derived from the pressure-time integral (PTI; i.e. average pressure across the cardiac cycle) measured via radial arterial catheterization. METHODS: We analyzed radial arterial catheter waveforms from 39 patients (Age: 71±7 years; BMI: 38.4±6.7; Female: 66%; HTN prevalence: 97%) with heart failure with preserved ejection fraction (HFpEF) at rest and during cycling exercise at 20 Watts. We compared the PTI (from the catheter waveform) with the calculation of MAP from the peak and nadir of the same waveforms (5-beat averages) using the 0.33 and 0.4 FF’s in the FF equation. RESULTS: Compared to the PTI (91±13 mmHg), resting MAP was not significantly different when calculated using the 0.33 FF (91±11 mmHg, P\u3e0.999) but was higher when using the 0.4 FF (96±12 mmHg, PCONCLUSION:While the 0.33 FF provides an accurate assessment of MAP on average during rest and exercise in the radial artery in patients with HFpEF, the limits of agreement are large reflecting a lack of precision in measurement at an individual level. Indirect calculations of MAP via a FF may lead to inaccurate conclusions regarding the mechanisms of blood pressure regulation both at rest and during exercise testing in this population

Hospital admissions linked to SARS-CoV-2 infection in children and adolescents: cohort study of 3.2 million first ascertained infections in England

Objective To describe hospital admissions associated with SARS-CoV-2 infection in children and adolescents. Design Cohort study of 3.2 million first ascertained SARS-CoV-2 infections using electronic health care record data. Setting England, July 2020 to February 2022. Participants About 12 million children and adolescents (age <18 years) who were resident in England. Main outcome measures Ascertainment of a first SARS-CoV-2 associated hospital admissions: due to SARS-CoV-2, with SARS-CoV-2 as a contributory factor, incidental to SARS-CoV-2 infection, and hospital acquired SARS-CoV-2. Results 3 226 535 children and adolescents had a recorded first SARS-CoV-2 infection during the observation period, and 29 230 (0.9%) infections involved a SARS-CoV-2 associated hospital admission. The median length of stay was 2 (interquartile range 1-4) days) and 1710 of 29 230 (5.9%) SARS-CoV-2 associated admissions involved paediatric critical care. 70 deaths occurred in which covid-19 or paediatric inflammatory multisystem syndrome was listed as a cause, of which 55 (78.6%) were in participants with a SARS-CoV-2 associated hospital admission. SARS-CoV-2 was the cause or a contributory factor in 21 000 of 29 230 (71.8%) participants who were admitted to hospital and only 380 (1.3%) participants acquired infection as an inpatient and 7855 (26.9%) participants were admitted with incidental SARS-CoV-2 infection. Boys, younger children (<5 years), and those from ethnic minority groups or areas of high deprivation were more likely to be admitted to hospital (all P<0.001). The covid-19 vaccination programme in England has identified certain conditions as representing a higher risk of admission to hospital with SARS-CoV-2: 11 085 (37.9%) of participants admitted to hospital had evidence of such a condition, and a further 4765 (16.3%) of participants admitted to hospital had a medical or developmental health condition not included in the vaccination programme’s list. Conclusions Most SARS-CoV-2 associated hospital admissions in children and adolescents in England were due to SARS-CoV-2 or SARS-CoV-2 was a contributory factor. These results should inform future public health initiatives and research

Responsible Governance for Mental Health Research in Low Resource Countries

Blurb: Taghi Yasamy and colleagues identify challenges facing good research governance in low- and middle-income countries and provide suggestions for a way forward

Responsible Governance for Mental Health Research in Low Resource Countries

Blurb: Taghi Yasamy and colleagues identify challenges facing good research governance in low- and middle-income countries and provide suggestions for a way forward

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

30 pags., 10 figs., 1 tab.Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.Cancer Council NSW RG 11-07 Tracy M Bryan, Cancer Institute NSW Aaron Lavel Moye, Australian Research Council FL140100027 Antoine M van Oijen, Ernest and Piroska Major Foundation Scott B Cohen, Natural Sciences and Engineering Research Council of Canada, Masad J Damha Centre of Excellence for Innovation in Chemistry PERCH-CIC Siritron Samosorn Research Unit of Natural Products and Organic Synthesis for Drug Discovery NPOS 405/2560 Siritron Samosorn Cancer Council NSW RG 16-10 Tracy M Brya

Tissue Glucocorticoid Metabolism in Adrenal Insufficiency:A Prospective Study of Dual-release Hydrocortisone Therapy

Background: Patients with adrenal insufficiency (AI) require life-long glucocorticoid (GC) replacement therapy. Within tissues, cortisol (F) availability is under the control of the isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). We hypothesize that corticosteroid metabolism is altered in patients with AI because of the nonphysiological pattern of current immediate release hydrocortisone (IR-HC) replacement therapy. The use of a once-daily dual-release hydrocortisone (DR-HC) preparation, (Plenadren®), offers a more physiological cortisol profile and may alter corticosteroid metabolism in vivo.Study Design and Methods: Prospective crossover study assessing the impact of 12 weeks of DR-HC on systemic GC metabolism (urinary steroid metabolome profiling), cortisol activation in the liver (cortisone acetate challenge test), and subcutaneous adipose tissue (microdialysis, biopsy for gene expression analysis) in 51 patients with AI (primary and secondary) in comparison to IR-HC treatment and age- and BMI-matched controls.Results: Patients with AI receiving IR-HC had a higher median 24-hour urinary excretion of cortisol compared with healthy controls (72.1 µg/24 hours [IQR 43.6-124.2] vs 51.9 µg/24 hours [35.5-72.3], P = .02), with lower global activity of 11β-HSD2 and higher 5-alpha reductase activity. Following the switch from IR-HC to DR-HC therapy, there was a significant reduction in urinary cortisol and total GC metabolite excretion, which was most significant in the evening. There was an increase in 11β-HSD2 activity. Hepatic 11β-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11β-HSD1 in subcutaneous adipose tissue.Conclusion: Using comprehensive in vivo techniques, we have demonstrated abnormalities in corticosteroid metabolism in patients with primary and secondary AI receiving IR-HC. This dysregulation of pre-receptor glucocorticoid metabolism results in enhanced glucocorticoid activation in adipose tissue, which was ameliorated by treatment with DR-HC

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology

Differential glucocorticoid metabolism in patients with persistent versus resolving inflammatory arthritis

Introduction: Impairment in the ability of the inflamed synovium to generate cortisol has been proposed to be a factor in the persistence and severity of inflammatory arthritis. In the inflamed synovium, cortisol is generated from cortisone by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. The objective of this study was to determine the role of endogenous glucocorticoid metabolism in the development of persistent inflammatory arthritis. Methods: Urine samples were collected from patients with early arthritis (symptoms ≤12 weeks duration) whose final diagnostic outcomes were established after clinical follow-up and from patients with established rheumatoid arthritis (RA). All patients were free of disease-modifying anti-rheumatic drugs at the time of sample collection. Systemic measures of glucocorticoid metabolism were assessed in the urine samples by gas chromatography/mass spectrometry. Clinical data including CRP and ESR were also collected at baseline. Results: Systemic measures of 11β-HSD1 activity were significantly higher in patients with early arthritis whose disease went on to persist, and also in the subgroup of patients with persistent disease who developed RA, when compared with patients whose synovitis resolved over time. We observed a significant positive correlation between systemic 11β-HSD1 activity and ESR/CRP in patients with established RA but not in any of the early arthritis patients group. Conclusions: The present study demonstrates that patients with a new onset of synovitis whose disease subsequently resolved had significantly lower levels of systemic 11β-HSD1 activity when compared with patients whose synovitis developed into RA or other forms of persistent arthritis. Low absolute levels of 11β-HSD1 activity do not therefore appear to be a major contributor to the development of RA and it is possible that a high total body 11β-HSD1 activity during early arthritis may reduce the probability of disease resolution

Effect of pre-exposure use of hydroxychloroquine on COVID-19 mortality: a population-based cohort study in patients with rheumatoid arthritis or systemic lupus erythematosus using the OpenSAFELY platform.

BACKGROUND: Hydroxychloroquine has been shown to inhibit entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into epithelial cells in vitro, but clinical studies found no evidence of reduced mortality when treating patients with COVID-19. We aimed to evaluate the effectiveness of hydroxychloroquine for prevention of COVID-19 mortality, as opposed to treatment for the disease. METHODS: We did a prespecified observational, population-based cohort study using national primary care data and linked death registrations in the OpenSAFELY platform, which covers approximately 40% of the general population in England, UK. We included all adults aged 18 years and older registered with a general practice for 1 year or more on March 1, 2020. We used Cox regression to estimate the association between ongoing routine hydroxychloroquine use before the COVID-19 outbreak in England (considered as March 1, 2020) compared with non-users of hydroxychloroquine and risk of COVID-19 mortality among people with rheumatoid arthritis or systemic lupus erythematosus. Model adjustment was informed by a directed acyclic graph. FINDINGS: Between Sept 1, 2019, and March 1, 2020, of 194 637 people with rheumatoid arthritis or systemic lupus erythematosus, 30 569 (15·7%) received two or more prescriptions of hydroxychloroquine. Between March 1 and July 13, 2020, there were 547 COVID-19 deaths, 70 among hydroxychloroquine users. Estimated standardised cumulative COVID-19 mortality was 0·23% (95% CI 0·18 to 0·29) among users and 0·22% (0·20 to 0·25) among non-users; an absolute difference of 0·008% (-0·051 to 0·066). After accounting for age, sex, ethnicity, use of other immunosuppressive drugs, and geographical region, no association with COVID-19 mortality was observed (HR 1·03, 95% CI 0·80 to 1·33). We found no evidence of interactions with age or other immunosuppressive drugs. Quantitative bias analyses indicated that our observed associations were robust to missing information for additional biologic treatments for rheumatological disease. We observed similar associations with the negative control outcome of non-COVID-19 mortality. INTERPRETATION: We found no evidence of a difference in COVID-19 mortality among people who received hydroxychloroquine for treatment of rheumatological disease before the COVID-19 outbreak in England. Therefore, completion of randomised trials investigating pre-exposure prophylactic use of hydroxychloroquine for prevention of severe outcomes from COVID-19 are warranted. FUNDING: Medical Research Council