Small Non-Coding RNAs as New Biomarkers to Evaluate the Quality of the Embryo in the IVF Process.

The increased interest in assisted reproduction through in vitro fertilization (IVF) leads to an urgent need to identify biomarkers that reliably highly predict the success of pregnancy. Despite advances in diagnostics, treatment, and IVF approaches, the 30% success rate of IVF seems insurmountable. Idiopathic infertility does not have any explanation for IVF failure especially when a patient is treated with a healthy competitive embryo capable of implantation and development. Since appropriate intercellular communication is essential after embryo implantation, the emergence of the investigation of embryonic secretome including short non-coding RNA (sncRNA) molecules is crucial. That's why biomarker identification, sncRNAs secreted during the IVF process into the blastocyst's cultivation medium, by the implementation of artificial intelligence opens the door to a better understanding of the bidirectional communication between embryonic cells and the endometrium and so the success of the IVF. This study presents a set of promising new sncRNAs which are revealed to predictively distinguish a high-quality embryo, suitable for an embryo transfer in the IVF process, from a low-quality embryo with 86% accuracy. The identified exact combination of miRNAs/piRNAs as a non-invasively obtained biomarker for quality embryo determination, increasing the likelihood of implantation and the success of pregnancy after an embryo transfer.

Selected In Situ Hybridization Methods: Principles and Application.

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

Effect of hypoxia factors gene silencing on ROS production and metabolic status of A375 malignant melanoma cells.

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O2•- with MitoTracker Red CMXRos. 1H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.

Bradykinin preconditioning affects the number of degenerated neurons and the level of antioxidant enzymes in spinal cord ischemia in rabbits.

Bradykinin preconditioning has been used for acquisition of tolerance after spinal cord ischemia. Rabbits were preconditioned intraperitoneally with bradykinin 48 h prior to 20 min of abdominal aorta ligation followed by 24 and 48 h of reperfusion. The activities of SOD and catalase were measured and Fluoro Jade B (FJB)-positive degenerated neurons were evaluated. The outcomes of Tarlov scoring system used to assess neurological functions showed significant improvement in bradykinin groups compared to the ischemic group. The number of FJB-positive degenerated neurons was decreased in ventral horns of both bradykinin groups. Significantly decreased activities of total SOD and mitochondrial Mn-SOD were also detected in both bradykinin groups versus ischemic group while CuZn-SOD and catalase activities were significantly decreased only in the bradykinin group after 24h of reperfusion versus ischemic group. These findings suggest that one of the possibilities of the neuroprotective effect of delayed bradykinin preconditioning against spinal cord ischemic injury could be realized by mitochondrial protection and decreased synthesis of Mn-SOD as well as by promotion of neuronal survival.

Hemodynamic parameters and heart rate variability during a tilt test in relation to gene polymorphism of renin-angiotensin and serotonin system.

PURPOSE: The aim of the study was to evaluate the renin-angiotensin system and serotonin transporter gene polymorphisms in relation to hemodynamic parameters and heart rate variability during a head-up tilt test (HUT) in patients with vasovagal syncope. METHODS: DNA was collected from 191 patients (mean age 44+/-18 years, 61 men, 130 women). The following gene polymorphisms were determined in genomic DNA: angiotensin-converting enzyme insertion/deletion polymorphism (I/D ACE), angiotensinogen gene polymorphism (M 235), angiotensin II receptor type 1 (ATR1) polymorphism (A 11666C), and polymorphism of serotonin transporter gene (5HTTLPR).Heart rate variability during HUT was assessed in 5-minute intervals by low frequency, high frequency, standard deviation of the normal-to-normal (SDNN), and root mean square successive difference parameters. RESULTS: AA genotype of A 1166C polymorphism was associated with lower minimal systolic blood pressure (SBP) and diastolic blood pressure (DBP) during HUT compared with other genotypes (minimal SBP: AA 59.6+/-21,8, AC 79.9+/-22.7, CC 65.4+/-22.7 mmHg, P=0.007), (minimal DBP: AA 36.4+/-22.7, AC 52.3+/-22.9, CC 45.4+/-19.5 mmHg, P=0.007).AA genotype was also associated with higher SDNN compared to other genotypes in the early phase of HUT (SDNN in 5 minutes of tilt: AA 59.7+/-24.6, AC 50.6+/-20.6, CC 46.0+/-13.2, P=0.01) and at syncope occurrence (SDNN: AA 71.0+/-20.9, AC 58.2+/-17.9, CC 58+/-10, P=0.04) CONCLUSION: AA genotype of A 1166C polymorphism in the ATR1 gene may be associated with hypotension and decline in sympathetic tone during HUT. Its role in genetic predisposition to vasovagal syncope cannot be excluded.