Determination of source and quantity of DNA in spent embryo culture medium

Tiivistelmä: Tutkimuksen tausta ja tavoitteet: Alkiodiagnostiikka hedelmöityshoidoissa on enenemässä määrin tarpeellinen erityisesti ensisynnyttäjien keski-iän noustessa, jolloin alkioiden kromosomipoikkeavuudet lisääntyvät ja raskaaksi tuleminen hankaloituu. Tällä hetkellä kromosomien seulontatutkimukset perustuvat alkiosta otettuun biopsiaan. Tuoreissa tutkimuksissa alkioiden viljelyliuoksesta on löydetty alkioiden DNA:ta ja näin ollen viljelyliuoksen käyttöä alkiodiagnostiikassa on tarpeen tutkia. Tutkimushypoteesina oli oletus, että alkioperäistä DNA:ta löydetään viljelyliuoksesta ja siitä voidaan tehdä päätelmiä alkion laadusta. Tämän tutkimuksen tarkoituksena oli optimoida DNA:n eristysmenetelmä viljelyliuoksesta, ja osoittaa, että DNA on peräisin alkiosta käyttäen Y-kromosomaalisen TSPY-geenin määritystä liuoksesta sekä kromosomiseulontaa verraten tuloksia jo analysoituihin alkiodiagnostiikan tuloksiin. Tutkimuksessa analysoitiin lisäksi alkioista saatavaa kuvamateriaalia kontaminaatiolähteistä ja alkion kehitysominaisuuksista EmbryoScope®-inkubaattorista, verraten sitä viljelyliuoksesta eristetyn DNA:n määrään. Menetelmät: Suolasaostusta ja NucleoSpin plasma XS -kittiä verrattiin DNA:n eristyksessä käyttäen qPCR-menetelmää, elektroforeesia sekä absorbanssi- ja pitoisuusmittauksia NanoDropilla. Viljelyliuokset kerättiin alkion viljelyn päättyessä, DNA eristettiin ja sen kokonaismäärää arvioitiin käyttäen Alu4-aluketta sekä TSPY-alukkeita osoittamaan Y-kromosomaalinen DNA liuoksista qPCR:llä. Kuva-analyysissä käytettiin lisäksi IBM SPSS -tilasto-ohjelmaa. Kromosomiseulontaan käytettiin aCGH-menetelmää ja sen tuloksia verrattiin aiemmin analysoituihin NGS-tuloksiin. Tutkimustulokset: NucleoSpin plasma XS -kitti oli herkempi menetelmä sekä DNA:n saannin, puhtauden, että toistettavuuden osalta. Kumulussolujen (p<0,001), kuten myös kuolleiden solujen määrä (p<0,019) nosti DNA:n määrää viljelyliuoksessa. Havaittiin myös, että mitä pidempään alkiota viljeltiin (4, 5 tai 6 päivää), sitä suurempi oli DNA:n määrä liuoksessa (p<0,001). Muiden tekijöiden, kuten siittiöiden, ei havaittu merkitsevästi vaikuttavan DNA:n määrään. Kromosomiseulonnan tulokset viljelyliuoksesta aCGH-tekniikalla eivät vastanneet NGS-tekniikan tuloksia biopsioista. Johtopäätökset: NucleoSpin plasma XS -kitti toimi suolasaostusta paremmin DNA:n eristyksessä saannin ja puhtauden sekä toistettavuuden osalta. Kumulussolujen, alkion viimeisen viljelypäivän sekä viljelyssä havaittujen kuolleiden solujen määrän havaittiin lisäävän DNA:n määrää liuoksessa. Muiden tekijöiden ei havaittu merkitsevästi vaikuttavan DNA:n määrään liuoksessa. Lisää tutkimuksia on tarpeen tehdä suuremmilla näytteiden määrillä. Kromosomiseulonnan tulokset eivät vastanneet NGS-tuloksia, joten menetelmän optimointi on edelleen tarpeen. Abstract: Background and aims: Infertility is more common phenomenon especially due to increase in maternal age. Embryo diagnostics is necessary within the couple with repetitive miscarriages and disruption of fertilization. Current procedures for embryo chromosomal screening require still an invasive biopsy of the embryo. There have been promising results of embryo´s spent medium use in analyzing embryo´s DNA and that field is necessary to be investigated more. The hypothesis of this study was to expect embryonic DNA to be found in spent medium and be demonstrated to correlate with the quality of the embryo. The purpose of this study was to optimize DNA extraction method from embryo culture medium and indicate that DNA in spent medium originated from embryo determining Y-chromosomal TSPY gene as marker and comparing aCGH results of spent media to NGS results of chromosomal screening. The aim was also to investigate if the evaluated events from time-lapse EmbryoScope® device correlate with amount of DNA. Especially, feasible DNA contamination sources and factors as well as embryo development properties were analyzed to explore their impact to released DNA amount in spent media. Methods: Salt precipitation method and NucleoSpin plasma XS kit (Macherey-Nagel) for DNA extraction were compared using electrophoresis, qPCR and NanoDrop measurements. Embryo´s spent media from D4 to D6 embryo cultures were collected. DNA from spent media was determined using Alu4 as target to evaluate DNA amount and TSPY to determine Y-chromosomal DNA. Statistical analyses of time-lapse incubator images were used to investigate relation between contamination sources and embryo properties with DNA amount in spent media. IBM SPSS was used for statistical analyses. To compare chromosomal screening of spent medium with NGS results, aCGH (Agilent) was used. Results: Yield and purity of the extracted DNA as well as repeatability of the method were better using NucleoSpin plasma XS kit than using salt precipitation method. Cumulus cells as contaminant DNA source (p<0,001), as well as lysed cells (p<0,019) were observed to increase the DNA amount in spent medium. Culture time was demonstrated to increase the DNA amount among D4, D5 and D6 embryo culture medium samples. Other evaluated factors had no impact on DNA amount in spent medium. Results of spent medium chromosomal screening using aCGH were not consistent with chromosomal screening results of biopsy by NGS. Conclusions: DNA extraction using NucleoSpin plasma XS kit was more accurate. Cumulus cells and lysed cells, were demonstrated to increase DNA amount in spent medium. The culture time had also impact on DNA amount in culture medium. Other factors, such as sperm cells, had no impact on DNA amount, but more studies must be done because the number of samples was low. Also, the procedure of aCGH must be optimized because or re-evaluated the results were confusing

Matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 predict incident cardiovascular disease events and all-cause mortality in a population-based cohort

Background Extracellular matrix degrading proteases and their regulators play an important role in atherogenesis and subsequent plaque rupture leading to acute cardiovascular manifestations. Design and methods In this prospective cohort study, we investigated the prognostic value of circulating matrix metalloproteinase-8, tissue inhibitor of matrix metalloproteinase-1 concentrations, the ratio of matrix metalloproteinase-8/ tissue inhibitor of matrix metalloproteinase-1 and, for comparison, myeloperoxidase and C-reactive protein concentrations for incident cardiovascular disease endpoints. The population-based FINRISK97 cohort comprised 7928 persons without cardiovascular disease at baseline. The baseline survey included a clinical examination and blood sampling. During a 13-year follow-up the endpoints were ascertained through national healthcare registers. The associations of measured biomarkers with the endpoints, including cardiovascular disease event, coronary artery disease, acute myocardial infarction, stroke and all-cause death, were analysed using Cox regression models. Discrimination and reclassification models were used to evaluate the clinical implications of the biomarkers. Results Serum tissue inhibitor of matrix metalloproteinase-1 and C-reactive protein concentrations were associated significantly with increased risk for all studied endpoints. Additionally, matrix metalloproteinase-8 concentration was associated with the risk for a coronary artery disease event, myocardial infarction and death, and myeloperoxidase concentration with the risk for cardiovascular disease events, stroke and death. The only significant association for the matrix metalloproteinase-8/ tissue inhibitor of matrix metalloproteinase-1 ratio was observed with the risk for myocardial infarction. Adding tissue inhibitor of matrix metalloproteinase-1 to the established risk profile improved risk discrimination of myocardial infarction (p=0.039) and death (0.001). Both matrix metalloproteinase-8 (5.2%, p <0.001) and tissue inhibitor of matrix metalloproteinase-1 (12.9%, p <0.001) provided significant clinical net reclassification improvement for death. Conclusions Serum matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 can be considered as biomarkers of incident cardiovascular disease events and death.Peer reviewe

IL-4, IL-13 and IFN-γ -induced genes in highly purified human neutrophils

Interleukin (IL)-4 and IL-13 are related cytokines with well-known specific roles in type 2 immune response. However, their effects on neutrophils are not completely understood. For this, we studied human primary neutrophil responses to IL-4 and IL-13. Neutrophils are dose-dependently responsive to both IL-4 and IL-13 as indicated by signal transducer and activator of transcription 6 (STAT6) phosphorylation upon stimulation, with IL-4 being more potent inducer of STAT6. IL-4-, IL-13- and Interferon (IFN)-γ-stimulated gene expression in highly purified human neutrophils induced both overlapping and unique gene expression in highly purified human neutrophils. IL-4 and IL-13 specifically regulate several immune-related genes, including IL-10, tumor necrosis factor (TNF) and leukemia inhibitory factor (LIF), while type1 immune response-related IFN-γ induced gene expression related for example, to intracellular infections. In analysis of neutrophil metabolic responses, oxygen independent glycolysis was specifically regulated by IL-4, but not by IL-13 or IFN-γ, suggesting specific role for type I IL-4 receptor in this process. Our results provide a comprehensive analysis of IL-4, IL-13 and IFN-γ -induced gene expression in neutrophils while also addressing cytokine-mediated metabolic changes in neutrophils.publishedVersionPeer reviewe

Thirty-One Novel Biomarkers as Predictors for Clinically Incident Diabetes

The prevalence of diabetes is increasing in all industrialized countries and its prevention has become a public health priority. However, the predictors of diabetes risk are insufficiently understood. We evaluated, whether 31 novel biomarkers could help to predict the risk of incident diabetes.The biomarkers were evaluated primarily in the FINRISK97 cohort (n = 7,827; 417 cases of clinically incident diabetes during the follow-up). The findings were replicated in the Health 2000 cohort (n = 4,977; 179 cases of clinically incident diabetes during the follow-up). We used Cox proportional hazards models to calculate the relative risk of diabetes, after adjusting for the classic risk factors, separately for each biomarker. Next, we assessed the discriminatory ability of single biomarkers using receiver operating characteristic curves and C-statistics, integrated discrimination improvement (IDI) and net reclassification improvement (NRI). Finally, we derived a biomarker score in the FINRISK97 cohort and validated it in the Health 2000 cohort. A score consisting of adiponectin, apolipoprotein B, C-reactive protein and ferritin almost doubled the relative risk of diabetes in the validation cohort (HR per one standard deviation increase 1.88, p = 2.8 e-5). It also improved discrimination of the model (IDI = 0.0149, p<0.0001) and reclassification of diabetes risk (NRI = 11.8%, p = 0.006). Gender-specific analyses suggested that the best score differed between men and women. Among men, the best results were obtained with the score of four biomarkers: adiponectin, apolipoprotein B, ferritin and interleukin-1 receptor antagonist, which gave an NRI of 25.4% (p<0.0001). Among women, the best score included adiponectin, apolipoprotein B, C-reactive protein and insulin. It gave an NRI of 13.6% (p = 0.041).We identified novel biomarkers that were associated with the risk of clinically incident diabetes over and above the classic risk factors. This gives new insights into the pathogenesis of diabetes and may help with targeting prevention and treatment

Low IL-13Rα1 expression on mast cells tunes them unresponsive to IL-13

Cytokine-mediated mast cell regulation enables precise optimization of their own proinflammatory cytokine production. During allergic inflammation, interleukin (IL)-4 regulates mast cell functions, tissue homing, and proliferation, but the direct role of closely related IL-13 for mast cell activation remains unclear. Previous work has shown that mast cells are potent IL-13 producers, but here we show that mouse mast cells do not directly respond to IL-13 by Stat6 activation, as they do not express measurable amount of IL-13 receptor α1 (IL-4Rα1) messenger RNA. Consequently, IL-4 responses are mediated via type I IL-4R (IL-4/IL4Rα/γC), and IL-4-induced Stat6 activation is abolished in γC-deficient mast cells. Type II IL-4R deficiency (IL-13Rα1 knockout) has no effect on IL-4-induced Stat6 activation. In basophils, both IL-4 and IL-13 induce Stat6 activation in wild-type and γC-deficient cells, while in type II IL-4R-deficient basophils, IL-4 signaling is impaired at low ligand concentration. Thus, mast cell and basophil sensitivity to IL-4/IL-13 is different, and in mast cells, lack of IL-13Rα1 expression likely explains their unresponsiveness to IL-13.publishedVersionPeer reviewe

RadoNorm – towards effective radiation protection based on improved scientific evidence and social considerations – focus on RADON and NORM

RadoNorm aims to manage risks from exposures to radon and naturally occurring radioactive material (NORM) to promote effective radiation protection based on improved scientific evidence and social considerations. It supports the European Member States and the EU Commission (EC) in implementing the Basic Safety Standards for protection against ionising radiation hazards at the legislative, executive, and operational levels (Directive 2013/59/EURATOM). The project is grounded on (1) implementation of multidisciplinary and innovative research and technologies, (2) integration of education and training, and (3) dissemination of project results targeting a broad stakeholder community including the public, regulators, and policymakers. The objectives are achieved through scientific research-related topics (exposure, dosimetry, biology, epidemiology, societal aspects), cross-cutting topics (education and training, dissemination, ethics) and project management. The project will yield guidelines at legal, executive and operational levels. It will enable consolidated and harmonised decision-making in the field of radiation protection, considering societal aspects and sustainable knowledge transfer. The project contributes to EC activities to strengthen radiation protection in a consistent and joint manner, as has already been done through the establishment of radiation protection platforms, the promotion of projects (e.g., DoReMi, OPERRA) and the partnership CONCERT-EJP. The outcomes may also impact future recommendations

Assessment of causality of natriuretic peptides and atrial fibrillation and heart failure : a Mendelian randomization study in the FINRISK cohort

Aims Natriuretic peptides are extensively studied biomarkers for atrial fibrillation (AF) and heart failure (HF). Their role in the pathogenesis of both diseases is not entirely understood and previous studies several single-nucleotide poly-morphisms (SNPs) at the NPPA-NPPB locus associated with natriuretic peptides have been identified. We investigated the causal relationship between natriuretic peptides and AF as well as HF using a Mendelian randomization approach. Methods and results N-terminal pro B-type natriuretic peptide (NT-proBNP) (N= 6669), B-type natriuretic peptide (BNP) (N= 6674), and mid-regional pro atrial natriuretic peptide (MR-proANP) (N= 6813) were measured in the FINRISK 1997 cohort. N=30 common SNPs related to NT-proBNP, BNP, and MR-proANP were selected from studies. We performed six Mendelian randomizations for all three natriuretic peptide biomarkers and for both outcomes, AF and HF, separately. Polygenic risk scores (PRSs) based on multiple SNPs were used as genetic instrumental variable in Mendelian randomizations. Polygenic risk scores were significantly associated with the three natriuretic peptides. Polygenic risk scores were not significantly associated with incident AF nor HF. Most cardiovascular risk factors showed significant confounding percentages, but no association with PRS. A causal relation except for small causal betas is unlikely. Conclusion In our Mendelian randomization approach, we confirmed an association between common genetic variation at the NPPA-NPPB locus and natriuretic peptides. A strong causal relationship between natriuretic peptides and incidence of AF as well as HF at the community-level was ruled out. Therapeutic approaches targeting natriuretic peptides will therefore very likely work through indirect mechanisms.Peer reviewe

Effect of inactivated nature-derived microbial composition on mouse immune system

Introduction: The hygiene hypothesis suggests that decrease in early life infections due to increased societal-level hygiene standards subjects one to allergic and autoimmune diseases. In this report, we have studied the effect of sterilized forest soil and plant-based material on mouse immune system and gut microbiome. Methods: Inbred C57Bl/6 mice maintained in normal sterile environment were subjected to autoclaved forest soil-derived powder in their bedding for 1 h a day for 3 weeks. Immune response was measured by immune cell flow cytometry, serum cytokine enzyme-linked immunoassay (ELISA) and quantitative polymerase chain reaction (qPCR) analysis. Furthermore, the mouse gut microbiome was analyzed by sequencing. Results: When compared to control mice, mice treated with soil-derived powder had decreased level of pro-inflammatory cytokines namely interleukin (IL)-17F and IL-21 in the serum. Furthermore, splenocytes from mice treated with soil-derived powder expressed less IL-1b, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF) upon cell activation. Gut microbiome appeared to be stabilized by the treatment. Conclusions: These results provide insights on the effect of biodiversity on murine immune system in sterile environment. Subjecting mice to soil-based plant and microbe structures appears to elicit immune response that could be beneficial, for example, in type 2 inflammation-related diseases, that is, allergic diseases.Peer reviewe

Genome-Wide Association Study Implicates Atrial Natriuretic Peptide Rather Than B-Type Natriuretic Peptide in the Regulation of Blood Pressure in the General Population

Background Cardiomyocytes secrete atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in response to mechanical stretching, making them useful clinical biomarkers of cardiac stress. Both human and animal studies indicate a role for ANP as a regulator of blood pressure with conflicting results for BNP. Methods and Results We used genome-wide association analysis (n=6296) to study the effects of genetic variants on circulating natriuretic peptide concentrations and compared the impact of natriuretic peptide-associated genetic variants on blood pressure (n=27059). Eight independent genetic variants in 2 known (NPPA-NPPB and POC1B-GALNT4) and 1 novel locus (PPP3CC) associated with midregional proANP (MR-proANP), BNP, aminoterminal proBNP (NT-proBNP), or BNP:NT-proBNP ratio. The NPPA-NPPB locus containing the adjacent genes encoding ANP and BNP harbored 4 independent cis variants with effects specific to either midregional proANP or BNP and a rare missense single nucleotide polymorphism in NT-proBNP seriously altering its measurement. Variants near the calcineurin catalytic subunit gamma gene PPP3CC and the polypeptide N-acetylgalactosaminyltransferase 4 gene GALNT4 associated with BNP:NT-proBNP ratio but not with BNP or midregional proANP, suggesting effects on the post-translational regulation of proBNP. Out of the 8 individual variants, only those correlated with midregional proANP had a statistically significant albeit weak impact on blood pressure. The combined effect of these 3 single nucleotide polymorphisms also associated with hypertension risk (P=8.2x10(-4)). Conclusions Common genetic differences affecting the circulating concentration of ANP associated with blood pressure, whereas those affecting BNP did not, highlighting the blood pressure-lowering effect of ANP in the general population.Peer reviewe

Genome-Wide Association Study Implicates Atrial Natriuretic Peptide Rather Than B-Type Natriuretic Peptide in the Regulation of Blood Pressure in the General Population

Background Cardiomyocytes secrete atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in response to mechanical stretching, making them useful clinical biomarkers of cardiac stress. Both human and animal studies indicate a role for ANP as a regulator of blood pressure with conflicting results for BNP. Methods and Results We used genome-wide association analysis (n=6296) to study the effects of genetic variants on circulating natriuretic peptide concentrations and compared the impact of natriuretic peptide-associated genetic variants on blood pressure (n=27059). Eight independent genetic variants in 2 known (NPPA-NPPB and POC1B-GALNT4) and 1 novel locus (PPP3CC) associated with midregional proANP (MR-proANP), BNP, aminoterminal proBNP (NT-proBNP), or BNP:NT-proBNP ratio. The NPPA-NPPB locus containing the adjacent genes encoding ANP and BNP harbored 4 independent cis variants with effects specific to either midregional proANP or BNP and a rare missense single nucleotide polymorphism in NT-proBNP seriously altering its measurement. Variants near the calcineurin catalytic subunit gamma gene PPP3CC and the polypeptide N-acetylgalactosaminyltransferase 4 gene GALNT4 associated with BNP:NT-proBNP ratio but not with BNP or midregional proANP, suggesting effects on the post-translational regulation of proBNP. Out of the 8 individual variants, only those correlated with midregional proANP had a statistically significant albeit weak impact on blood pressure. The combined effect of these 3 single nucleotide polymorphisms also associated with hypertension risk (P=8.2x10(-4)). Conclusions Common genetic differences affecting the circulating concentration of ANP associated with blood pressure, whereas those affecting BNP did not, highlighting the blood pressure-lowering effect of ANP in the general population.Peer reviewe