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.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males.

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.

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.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

How much, if anything, do we know about sperm chromosomes of Robertsonian translocation carriers?

In men with oligozoospermia, Robertsonian translocations (RobTs) are the most common type of autosomal aberrations. The most commonly occurring types are rob(13;14) and rob(14;21), and other types of RobTs are described as 'rare' cases. Based on molecular research, all RobTs can be broadly classified into Class 1 and Class 2. Class 1 translocations produce the same breakpoints within their RobT type, but Class 2 translocations are predicted to form during meiosis or mitosis through a variety of mechanisms, resulting in variation in the breakpoint locations. This review seeks to analyse the available data addressing the question of whether the molecular classification of RobTs into Classes 1 and 2 and/or the type of DD/GG/DG symmetry of the involved chromosomes is reflected in the efficiency of spermatogenesis. The lowest frequency value calculated for the rate of alternate segregants was found for rob(13;15) carriers (Class 2, symmetry DD) and the highest for rob(13;21) carriers (Class 2, DG symmetry). The aneuploidy values for the rare RobT (Class 2) and common rob(14;21) (Class 1) groups together exhibited similarities while differing from those for the common rob(13;14) (Class 1) group. Considering the division of RobT carriers into those with normozoospermia and those with oligoasthenozoospermia, it was found that the number of carriers with elevated levels of aneuploidy was unexpectedly quite similar and high (approx. 70%) in the two subgroups. The reason(s) that the same RobT does not always show a similar destructive effect on fertility was also pointed out.

Multiparametric Evaluation of Post-MI Small Animal Models Using Metabolic ([18F]FDG) and Perfusion-Based (SYN1) Heart Viability Tracers.

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [18F] fluorodeoxyglucose ([18F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography-computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [18F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes' expression, with Serpinb1c, Tnc and Nupr1, with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [18F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials.

Molecular Imaging of Human Skeletal Myoblasts (huSKM) in Mouse Post-Infarction Myocardium.

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.

Effect of acute isooxic hypercapnia on oxidative activity of systemic neutrophils in endotoxemic rabbits.

INTRODUCTION: Whether carbon dioxide (CO2) affects systemic oxidative phenomena under conditions of endotoxemia is not sufficiently clarified. The study aimed to assess the impact of moderate acute hypercapnia on the respiratory burst of circulating neutrophils in mechanically ventilated endotoxemic rabbits. MATERIAL AND METHODS: Twenty-four endotoxemic rabbits were mechanically ventilated with standard or CO2-enriched gas mixture in order to obtain isooxic hypercapnia. At a baseline point and following 180 min of hypercapnic ventilation, luminol-dependent chemiluminescence (CL) of circulating neutrophils and serum 2-thiobarbituric acid reactive substance (TBARS) concentrations were measured. Throughout the study, leukocyte and neutrophil counts, pH status, circulatory parameters and body temperature were also assessed. RESULTS: Following 180 min of hypercapnic ventilation, opsonized zymosan (OZ)-stimulated neutrophils showed lower CL vs. the control group (p = 0.004). Other parameters studied were not affected. CONCLUSIONS: Short-term isooxic hypercapnia in endotoxemic rabbits preserves circulating neutrophil count pattern and reactive oxygen species (ROS) generation, but it may reduce phagocytosis.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers-Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis.

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

Seminal Plasma Analysis of Oxidative Stress in Different Genitourinary Topographical Regions Involved in Reproductive Tract Disorders Associated with Genital Heat Stress.

The pathophysiological mechanisms responsible for male subfertility/infertility caused by or complicated by genital heat stress remains unclear in many respects. Because seminal plasma creates the environment for the proper functioning of spermatozoa, in this study, we verified the associations among standard spermiograms, seminal biochemical parameters (neutral alpha-glucosidase, fructose, and citric acid) and oxidative stress markers (total antioxidant capacity, catalase activity, superoxide dismutase activity, and malondialdehyde concentration) in distinct entities associated with male infertility with and without long-time exposure to local hyperthermia. We demonstrated that men exposed to prolonged environmental or clinically recognized local heat stress in adulthood may suffer from dysregulation of seminal antioxidant components, which can be directly associated with epididymal and prostate function. The comparative analysis of the studied parameters showed numerous correlations among all biochemical parameters (particularly neutral alpha-glucosidase) with low standard semen quality in almost all the investigated infertile groups. In light of the data obtained in this originally designed study, we conclude that more attention should be paid to the epididymis and accessory gland function in subfertile and infertile men exposed to genital heat stress, especially in the context of novel treatment algorithms (targeted therapies).

Viability and intracellular nitric oxide generation in the umbilical cord blood CD34+CD133- and CD34+CD133+ cell populations exposed to local anaesthetics.

Local anesthetics (LAs) are capable of influencing cell viability in systemic immunity and may also modify metabolism of those present in umbilical cord blood (UCB) following obstetric neuraxial analgesia and anaesthesia. Data regarding UCB immature cells, important for the neonate and critical for putative UCB transplantations, are lacking. LAs are capable of stimulating intracellular nitric oxide (NO) in human neutrophils; no information is available concerning newly perpetuated cells and its potential association with viability. The study aimed at assessing the LAs influence on the cell viability and intracellular NO production by UCB CD34+CD133- and CD34+ CD133+ cell populations. Mononuclear cells separated from UCB samples (n = 19) were incubated with bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM), and ropivacaine (0.0007, 0.007, 1.4 mM) for 4 h. Flow cytometry was applied for the assessment of cell viability and intracellular NO generation in CD34+CD133- and CD34+CD133+ cell populations using annexinV/7-AAD and DAF-2DA stainings, respectively. CD34+CD133+ cells showed less pronounced late apoptosis and necrosis as compared to CD34+CD133-population. Intracellular NO generation was comparable between both cell populations studied. LAs neither influenced cell viability nor changed NO production in either population. LAs do not interfere with viability and intracellular NO generation in the UCB CD34+CD133- and CD34+CD133+ cell populations.

New mutation causing androgen insensitivity syndrome - a case report and review of literature.

Androgen insensitivity syndrome (AIS) is a congenital disorder in which a defect in the androgen receptor (AR) gene leads to cellular resistance to androgens. Defects in the AR gene, located on the X chromosome, result in the development of a feminine phenotype in chromosomally male (46, XY) individuals. In this case report, we present a 44 years old patient with complete androgen insensitivity syndrome (CAIS) initially presenting with primary amenorrhea. The patient underwent a full clinical evaluation, revealing hypoplastic vagina and a lack of uterus and ovaries. Hormonal evaluation revealed markedly elevated testosterone, FSH, and LH serum concentrations. Diagnostic imaging, including pelvic MRI, confirmed the presence of two solid masses in the inguinal canals (right 26 × 13 mm, left 25 × 15 mm). The patient underwent genetic testing, revealing a 46 XY karyotype and an as of yet unprecedented androgen receptor mutation. The type of the mutation was a single-base exchange - the substitution from cytosine to thymine in chromosome X:66942710 position (referred to human reference genome GRCh37), which has resulted in an amino acid changes from leucine (CTT) to phenyloalanine (TTT) in ligand-binding domain.

Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics.

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

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.

Perspective in optimization of stem cell therapies for heart regeneration.

There is a variety of mechanisms(s) factor(s) that may influence stem cell therapies for heart regeneration. Among the best candidates for stem cell source are: mesenchymal stem cells (also those isolated from adipose tissue), cardiac cell progenitors (CPC) and descendants of iPSC cells. iPSC/s can be potentially beneficial although their pluripotent induction has been still in question due to: low propagation efficacy, danger of genomic integration/instability, biological risk of current vector system teratoma formation etc. which have been discussed in this review. Optimization protocols are required in order to enhance stem cells resistance to pathological conditions that they may encounter in pathological organ and to increase their retention. Combination between gene transfer and stem cell therapy is now more often used in pre-clinical studies with the prospect of subsequent clinical trials. Complementary substances have been contemplated to support stem cell viability (mainly anti-inflammatory and anti- apoptotic agents), which have been tested in animal models with promising results. Integration of nanotechnology both for efficient stem cell imaging as well as with the aim to provide cell supporting scaffolds seem to be inevitable for further development of cellular therapies. The whole organ (heart) reconstruction as well as biodegradable scaffolds and scaffold-free cell sheets have been also outlined.

The oral cavity - potential source of stem cells.

The purpose of this review is to present the current knowledge regarding the hierarchy of stem cells originating from the oral cavity, which could have a potential value when applied to regenerative stomatology. It must be particularly emphasized that the heterogenous nature of its biology and function within oral compartment may predispose them to different types of applications. Stem cells can be perceived as immature, primitive and unspecialized types of cells with the ability to proliferate, self-renew and differentiate into specialized progeny according to the compartmental signaling. Their presence in tissue reservoirs was already discovered in many organs and tissues as well as in the stomatognathic system. The oral cavity appears to be an exceptionally attractive site to acquire stem cells. The common presence and easy access to these cells in dental and peridental tissues provides a real chance to apply them for therapeutic purposes. Such an opportunity would also be neutral to bioethical and moral issues, assuming autologous stem cells employment. Many authors suspect that stem cells have epigenetic memory, so some of their features can be inherited through generations. They are not connected, however, with DNA sequence modifications. It is, therefore, justified to apply the cells, which have the oral cavity as their natural reservoir, in interventions associated with tissue engineering within the stomatognathic system. An increasing number of clinical trials, among which the number of randomized studies with large group of patients is progressively carried out, allows for a prediction that shortly therapeutic methods based on stem cells of dental origin may be implemented to the routine repertoire of clinical practice.

Meiotic and pedigree segregation analyses in carriers of t(4;8)(p16;p23.1) differing in localization of breakpoint positions at 4p subband 4p16.3 and 4p16.1.

PURPOSE: The purpose of this study was to compare meiotic segregation in sperm cells from two carriers with t(4;8)(p16;p23.1) reciprocal chromosome translocations (RCTs), differing in localization of the breakpoint positions at the 4p subband-namely, 4p16.3 (carrier 1) and 4p16.1 (carrier 2)-and to compare data of the pedigree analyses performed by direct method. METHODS: Three-color fluorescent in situ hybridization (FISH) on sperm cells and FISH mapping for the evaluation of the breakpoint positions, data from pedigrees, and direct segregation analysis of the pedigrees were performed. RESULTS: Similar proportions of normal/balanced and unbalanced sperm cells were found in both carriers. The most common was an alternate type of segregation (about 52 % and about 48 %, respectively). Unbalanced adjacent I and adjacent II karyotypes were found in similar proportions about 15 %. The direct segregation analysis (following Stengel-Rutkowski) of the pedigree of carriers of t(4;8)(p16.1;p23.1) was performed and results were compared with the data of the pedigree segregation analysis obtained earlier through the indirect method. The probability of live-born progeny with unbalanced karyotype for carriers of t(4;8)(p16.1;p23.1) was moderately high at 18.8 %-comparable to the value obtained using the indirect method for the same carriership, which was 12 %. This was, however, markedly lower than the value of 41.2 % obtained through the pedigree segregation indirect analysis estimated for carriers of t(4;8)(p16.3;p23.1), perhaps due to the unique composition of genes present within the 4p16.1-4p 16.3 region. CONCLUSIONS: Revealed differences in pedigree segregation analysis did not correspond to the very similar profile of meiotic segregation patterns presented by carrier 1 and carrier 2. Most probably, such discordances may be due to differences in embryo survival rates arising from different genetic backgrounds.

FISH and array CGH characterization of de novo derivative Y chromosome (Yq duplication and partial Yp deletion) in an azoospermic male.

This study presents a 28-year-old infertile male who was referred to the cytogenetic laboratory for chromosomal analysis after 4 years of regular unprotected intercourse in whom non-obstructive azoospermia was revealed. Standard cytogenetic G-banding was performed on metaphase spreads and a de-novo karyotype 46,X,der(Y)(q11.22;p11.3) was identified. This analysis was followed by flourescence in-situ hybridization(FISH) and array comparative genomic hybridization (aCGH). Finally, the patient's karyotype was identified as 46,X,der(Y)(qter→q11.221::p11.31→qter).ish der(Y) (qter+,pter-,SHOX+,SRY+,Ycen+,DYZ3+;DYZ1+,qter+).arrYq11.221q12(14,448,863-59,288,511) x2, Yp11.32p11.31(104,062-266,388) x0. It is proposed that de-novo derivative monocentric Y chromosome with duplicated region Y qter→q11.221::p11.31→qter with partial deletion of Yp PAR1 region most probably can perturb the conjugation of sex chromosomes during first meiotic division of spermatogenic arrested differentiation (development).