In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes.

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia.

BACKGROUND: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. OBJECTIVE: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). MATERIALS AND METHODS: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. RESULTS: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. DISCUSSION AND CONCLUSION: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.

Immunopathogenesis of endometriosis - a novel look at an old problem.

This review aims to cast a look at endometriosis as a chronic and progressive gynecological disease.Endometriosis-affected tissues show a variety of pathologic features: alterations in cell growth, apoptosis, activation, angiogenesis, cell adhesion, and cytokine production. Fresh endometriotic lesions are associated with induction of an inflammatory reaction represented by overproduction of prostaglandins (PGE2), metalloproteinases (MMP-2, -3, -9), cytokines (IL-1ß, IL-8, IFN-γ, TNF-α, MCP-1 and MIF) and adhesive molecules (ICAM-1, VCAM-1) and activation of synthesis of reactive oxygen and nitrogen species. The inflammatory process may lead to defective folliculogenesis by an altered follicular milieu. An increase in the number and change in function of macrophages, T- and B-lymphocytes and reduction of NK cells have been reported. Treg lymphocytes are known to play an extremely important role in controlling and modulating changes in the aberrant immune response in endometriosis. Dysregulation of the immune system results in both increased progression of endometriosis and its severity. In inflammatory conditions the immune cells provide immune defense at the local level - in peritoneal fluid - and could further cause: 1) a decrease of the number of NK CD16+ cells with expression of KIRs and an increase of NK CD57+; 2) increased numbers of CD8+ cells and CD11b- immature dendritic cells; 3) an increase of FoxP3 expression in the regulatory T cell (Treg) population; 4) an increase of macrophages activating T- and B-lymphocytes leading to elevated synthesis of cytokines and/or autoantibodies. We may conclude that endometriosis resembles an immunodependent disease with the autoimmune background and breakdown of immunosuppressive mechanisms. Further immunological investigations may open a new avenue to discover innovative immunomodulatory treatments of endometriosis.

Human live spermatozoa morphology assessment using digital holographic microscopy.

Digital holographic microscopy (DHM) was applied for the morphological assessment of live intact spermatozoa from fertile and infertile men directly after semen liquefaction. This method allowed us to study the sperm population directly from the sample droplet and not only from the focal plane of the microscope as in classical optical microscopy. The newly implemented 3-dimensional sperm morphological parameters (head height, acrosome/nucleus height, head/midpiece height) were included in morphological assessment of semen samples from fertile and infertile individuals. The values of the 3D parameters were less variable in fertile men than for infertile ones. DHM was also used to compare the morphological profiles of spermatozoa after applying the "swim-up" and gradient centrifugation techniques. During selection, the most statistically significant differences were observed after separation with a Percoll gradient of 90% and a 60-min "swim-up" procedure versus 'native' unfractionated samples. This shows that the developed methodology can be efficiently used for the selection of morphologically sound spermatozoa. The motility type for each spermatozoon was also assessed. The results indicate that the extension of the number of morphological parameters with new 3D parameters and the simultaneous assessment of sperm motility may be valuable addition to sperm examination.

Assessment of Immunological Potential of Glial Restricted Progenitor Graft In Vivo-Is Immunosuppression Mandatory?

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron and skeletal muscle loss and death. One of the promising therapeutic approaches is stem cell graft application into the brain; however, an immune reaction against it creates serious limitations. This study aimed to research the efficiency of glial restricted progenitors (GRPs) grafted into murine CNS (central nervous system) in healthy models and the SOD1G93A ALS disease model. The cellular grafts were administered in semiallogenic and allogeneic settings. To investigate the models of immune reaction against grafted GRPs, we applied three immunosuppressive/immunomodulatory regimens: preimplantation factor (PiF); Tacrolimus; and CTLA-4, MR1 co-stimulatory blockade. We tracked the cells with bioluminescence imaging (BLI) in vivo to study their survival. The immune response character was evaluated with brain tissue assays and multiplex ELISA in serum and cerebrospinal fluid (CSF). The application of immunosuppressive drugs is disputable when considering cellular transplants into the immune-privileged site/brain. However, our data revealed that semiallogenic GRP graft might survive inside murine CNS without the necessity to apply any immunomodulation or immunosuppression, whereas, in the situation of allogeneic mouse setting, the combination of CTLA-4, MR1 blockade can be considered as the best immunosuppressive option.

Transcription regulatory factor expression in T-helper cell differentiation pathway in eutopic endometrial tissue samples of women with endometriosis associated with infertility.

Endometriosis is a disease of epidemiological gravity of unknown primary reason. A complex of constitutional factors including the immune system has been considered as its background. The aim of the study was to identify Th1 and Th2 cells as well as the T-regulatory subset in the endometrium of women with endometriosis associated with infertility upon transcription factors expression. Expression of T-bet, GATA3, and Foxp3 genes was examined using a method of polymerase chain reaction (PCR) in the eutopic endometrial samples of 20 women with endometriosis associated with infertility and 20 women with infertility of tubal origin. An increase in mRNA expression for T-bet and GATA3 with prevailing mRNA level for T-bet and a decrease in Foxp3 expression were observed. In conclusion, the revealed changes in expression of transcription factors may indicate the imbalance between T-helper cells of the Th1 and Th2 type and elimination of regulatory function of T-cells, which can be one of the causes of endometriosis predisposing to the development of infertility associated with this disease.

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts.

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs' presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs' influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs' impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs' influence on treated skeletal myoblasts, not interfering with basic cell functions.