Seminal Microbiota of Idiopathic Infertile Patients and Its Relationship With Sperm DNA Integrity

Oxidative stress; Seminal microbiome; Sperm dna damageEstrés oxidativo; Microbioma seminal; Daño en el ADN de los espermatozoidesEstrès oxidatiu; Microbioma seminal; Dany a l'ADN dels espermatozoidesThe development of new biomarkers for human male infertility is crucial to improve the diagnosis and the prognosis of this disease. Recently, seminal microbiota was shown to be related to sperm quality parameters, suggesting an effect in human fertility and postulating it as a biomarker candidate. However, its relationship to sperm DNA integrity has not been studied yet. The aim of the present study is to characterize the seminal microbiota of a western Mediterranean population and to evaluate its relationship to sperm chromatin integrity parameters, and oxidative stress. For that purpose, 14 samples from sperm donors and 42 samples from infertile idiopathic patients were obtained and were analyzed to assess the composition of the microbiota through full-length 16S rRNA gene sequencing (Illumina MiSeq platform). Microbial diversity and relative abundances were compared to classic sperm quality parameters (macroscopic semen parameters, motility, morphology and concentration), chromatin integrity (global DNA damage, double-stranded DNA breaks and DNA protamination status) and oxidative stress levels (oxidation-reduction potential). The seminal microbiota observed of these samples belonged to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The most abundant genera were Finegoldia, Peptoniphilus, Anaerococcus, Campylobacter, Streptococcus, Staphylococcus, Moraxella, Prevotella, Ezakiella, Corynebacterium and Lactobacillus. To our knowledge, this is the first detection of Ezakiella genus in seminal samples. Two clusters of microbial profiles were built based on a clustering analysis, and specific genera were found with different frequencies in relation to seminal quality defects. The abundances of several bacteria negatively correlate with the sperm global DNA fragmentation, most notably Moraxella, Brevundimonas and Flavobacterium. The latter two were also associated with higher sperm motility and Brevundimonas additionally with lower oxidative-reduction potential. Actinomycetaceae, Ralstonia and Paenibacillus correlated with reduced chromatin protamination status and increased double-stranded DNA fragmentation. These effects on DNA integrity coincide in many cases with the metabolism or enzymatic activities of these genera. Significant differences between fertile and infertile men were found in the relative presence of the Propionibacteriaceae family and the Cutibacterium, Rhodopseudomonas and Oligotropha genera, which supports its possible involvement in male fertility. Our findings sustain the hypothesis that the seminal microbiome has an effect on male fertility.This research was funded by the European Regional Development Fund and Instituto de Salud Carlos III (Economy, Industry and Competitiveness Ministry, Madrid, Spain; Project PI14/00119) and Generalitat de Catalunya (2017SGR1796). Supported by the European Union’s Horizon 2020 Research and Innovation scheme under the Marie Skłodowska-Curie grant agreement No. 801342 (Tecniospring INDUSTRY; TECSPR-19-1-0003, to JR-M)

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

Generalitat de Catalunya 2019 FI_B2 00055The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (MeSO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of MeSO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both MeSO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for MeSO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification

Evidences of the Cerium Oxide-Catalysed DPF Regeneration in a Real Diesel Engine Exhaust

The active phase Ce0.5Pr0.5O2 has been loaded on commercial substrates (SiC DPF and cordierite honeycomb monolith) to perform DPF regeneration experiments in the exhaust of a diesel engine. Also, a powder sample has been prepared to carry out soot combustion experiments at laboratory. Experiments performed in the real diesel exhaust demonstrated the catalytic activity of the Ce–Pr mixed oxide for the combustion of soot, lowering the DPF regeneration temperature with regard to a counterpart catalyst-free DPF. The temperature for active regeneration of the Ce0.5Pr0.5O2-containing DPF when the soot content is low is in the range of 500–550 °C. When the Ce0.5Pr0.5O2-containing DPF is saturated with a high amount of soot, pressure drop and soot load at the filter reach equilibrium at around 360 °C under steady state engine operation due to passive regeneration. The uncoated DPF reached this equilibrium at around 440 °C. Comparing results at real exhaust with those at laboratory allow concluding that the Ce0.5Pr0.5O2-catalysed soot combustion in the real exhaust is not based on the NO2-assisted mechanism but is most likely occurring by the active oxygen-based mechanism.The authors thank the financial support of Generalitat Valenciana (Project Prometeo 2009/047), Spanish Ministry of Science and Innovation (project CIT-420000-2009-48) and EU (FEDER funding)

In vitro maturation in the presence of Leukemia Inhibitory Factor modulates gene and miRNA expression in bovine oocytes and embryos

Altres ajuts: OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems ; Natural Sciences and Engineering Research Council of CanadaMembers of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis

Key Genes of the Immune System and Predisposition to Acquired Hemophilia A: Evidence from a Spanish Cohort of 49 Patients Using Next-Generation Sequencing

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by the presence of autoantibodies against factor VIII (FVIII). As with other autoimmune diseases, its etiology is complex and its genetic basis is unknown. The aim of this study was to identify the immunogenetic background that predisposes individuals to AHA. HLA and KIR gene clusters, as well as KLRK1, were sequenced using next-generation sequencing in 49 AHA patients. Associations between candidate genes involved in innate and adaptive immune responses and AHA were addressed by comparing the alleles, genotypes, haplotypes, and gene frequencies in the AHA cohort with those in the donors' samples or Spanish population cohort. Two genes of the HLA cluster, as well as rs1049174 in KLRK1, which tags the natural killer (NK) cytotoxic activity haplotype, were found to be linked to AHA. Specifically, A*03:01 (p = 0.024; odds ratio (OR) = 0.26[0.06-0.85]) and DRB1*13:03 (p = 6.8 x 103, OR = 7.56[1.64-51.40]), as well as rs1049174 (p = 0.012), were significantly associated with AHA. In addition, two AHA patients were found to carry one copy each of the low-frequency allele DQB1*03:09 (nallele = 2, 2.04%), which was completely absent in the donors. To the best of our knowledge, this is the first time that the involvement of these specific alleles in the predisposition to AHA has been proposed. Further molecular and functional studies will be needed to unravel their specific contributions. We believe our findings expand the current knowledge on the genetic factors involved in susceptibility to AHA, which will contribute to improving the diagnosis and prognosis of AHA patients

MAB2.0 project: Integrating algae production into wastewater treatment

Different species of microalgae are highly efficient in removing nutrients from wastewater streams and are able to grow using flue gas as a CO2 source. These features indicate that application of microalgae has a promising outlook in wastewater treatment. However, practical aspects and process of integration of algae cultivation into an existing wastewater treatment line have not been investigated. The Climate-KIC co-funded Microalgae Biorefinery 2.0 project developed and demonstrated this integration process through a case study. The purpose of this paper is to introduce this process by phases and protocols, as well as report on the challenges and bottlenecks identified in the case study. These standardized technical protocols detailed in the paper help to assess different aspects of integration including biological aspects such as strain selection, as well as economic and environmental impacts. This process is necessary to guide wastewater treatment plants through the integration of algae cultivation, as unfavourable parameters of the different wastewater related feedstock streams need specific attention and management. In order to obtain compelling designs, more emphasis needs to be put on the engineering aspects of integration. Well-designed integration can lead to operational cost saving and proper feedstock treatment enabling algae growth

Photovoltaic tweezers an emergent tool for applications in nano and bio-technology

An overview of the work recently conducted by our group on the development and applications of photovoltaic tweezers is presented. It includes the analysis of the physical basis of the method and the main achievements in its experimental implementation. Particular attention will be paid to the main potential applications and first demonstrations of its use in nano- and bio-technology. Specifically: i) fabrication of metallic nanoestructures for plasmonic applications, ii) development of diffractive components, iii) manipulation and patterning (1D and 2D) of various types of bio-objects (spores or pollen...) and iv) effects of PV fields of LiNbO3 in tumour cells

Integrative genomic analysis of methylphenidate response in attention-deficit/hyperactivity disorder

Methylphenidate (MPH) is the most frequently used pharmacological treatment in children with attention-deficit/hyperactivity disorder (ADHD). However, a considerable interindividual variability exists in clinical outcome. Thus, we performed a genome-wide association study of MPH efficacy in 173 ADHD paediatric patients. Although no variant reached genome-wide significance, the set of genes containing single-nucleotide polymorphisms (SNPs) nominally associated with MPH response (P < 0.05) was significantly enriched for candidates previously studied in ADHD or treatment outcome. We prioritised the nominally significant SNPs by functional annotation and expression quantitative trait loci (eQTL) analysis in human brain, and we identified 33 SNPs tagging cis-eQTL in 32 different loci (referred to as eSNPs and eGenes, respectively). Pathway enrichment analyses revealed an over-representation of genes involved in nervous system development and function among the eGenes. Categories related to neurological diseases, psychological disorders and behaviour were also significantly enriched. We subsequently meta-analysed the association with clinical outcome for the 33 eSNPs across the discovery sample and an independent cohort of 189 ADHD adult patients (target sample) and we detected 15 suggestive signals. Following this comprehensive strategy, our results provide a better understanding of the molecular mechanisms implicated in MPH treatment effects and suggest promising candidates that may encourage future studies

Mortality and other adverse outcomes in patients with type 2 diabetes mellitus admitted for COVID-19 in association with glucose-lowering drugs: a nationwide cohort study

Background: Limited evidence exists on the role of glucose-lowering drugs in patients with COVID-19. Our main objective was to examine the association between in-hospital death and each routine at-home glucose-lowering drug both individually and in combination with metformin in patients with type 2 diabetes mellitus admitted for COVID-19. We also evaluated their association with the composite outcome of the need for ICU admission, invasive and non-invasive mechanical ventilation, or in-hospital death as well as on the development of in-hospital complications and a long-time hospital stay. Methods: We selected all patients with type 2 diabetes mellitus in the Spanish Society of Internal Medicine’s registry of COVID-19 patients (SEMI-COVID-19 Registry). It is an ongoing, observational, multicenter, nationwide cohort of patients admitted for COVID-19 in Spain from March 1, 2020. Each glucose-lowering drug user was matched with a user of other glucose-lowering drugs in a 1:1 manner by propensity scores. In order to assess the adequacy of propensity score matching, we used the standardized mean difference found in patient characteristics after matching. There was considered to be a significant imbalance in the group if a standardized mean difference > 10% was found. To evaluate the association between treatment and study outcomes, both conditional logit and mixed effect logistic regressions were used when the sample size was ≥ 100. Results: A total of 2666 patients were found in the SEMI-COVID-19 Registry, 1297 on glucose-lowering drugs in monotherapy and 465 in combination with metformin. After propensity matching, 249 patients on metformin, 105 on dipeptidyl peptidase-4 inhibitors, 129 on insulin, 127 on metformin/dipeptidyl peptidase-4 inhibitors, 34 on metformin/sodium-glucose cotransporter 2 inhibitor, and 67 on metformin/insulin were selected. No at-home glucose-lowering drugs showed a significant association with in-hospital death; the composite outcome of the need of intensive care unit admission, mechanical ventilation, or in-hospital death; in-hospital complications; or long-time hospital stays. Conclusions: In patients with type 2 diabetes mellitus admitted for COVID-19, at-home glucose-lowering drugs showed no significant association with mortality and adverse outcomes. Given the close relationship between diabetes and COVID-19 and the limited evidence on the role of glucose-lowering drugs, prospective studies are needed