Testis transcriptome analysis in male infertility: new insight on the pathogenesis of oligo-azoospermia in cases with and without AZFc microdeletion

<p>Abstract</p> <p>Background</p> <p>About 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the Y chromosome (Yq). Despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. In order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in 16 patients carrying an AZFc microdeletion or affected by idiopathic infertility. Hierarchical clustering was performed pooling the data set from 26 experiments (16 patients, 10 replicates).</p> <p>Results</p> <p>An intriguing and unexpected finding is that all the samples showing the AZFc deletion cluster together irrespectively of their testicular phenotypes. This cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mRNA storage. Interestingly, the four idiopathic patients present in the cluster showed no testicular expression of <it>DAZ </it>despite the absence of AZFc deletion in the peripheral blood.</p> <p>Conclusions</p> <p>Our expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mRNA storage. Our data suggest that a lack of testicular DAZ gene expression may be the trigger of such mechanism. Furthermore, the presence of AZFc deletions in mosaic or the loss of function of AZFc genes in absence of Yq deletion can perhaps explain these findings. Finally, based on our data, it is intriguing to hypothesize that <it>DAZ </it>gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing Yq deletions in peripheral blood.</p

Embryoid body in vitro formation from human amniotic fluid stem cells (AFSCs): an ultrastructural study

Amniotic fluid stem cells (AFSCs) harbour the potential to differentiate into cells of any of the three germ layers and to form embryoid bodies (EBs) without inducing teratoma formation (De Coppi et al., 2007; Antonucci et al., 2012). However, no previous findings have been reported regarding embryoid body in vitro development and ultrastructural organization. Thus, this was the aim of our study. Amniotic fluid samples were obtained from women undergoing amniocentesis for prenatal diagnosis at 16-19 weeks of pregnancy after written informed consent and the local ethical committee approval. Human AFSCs were cultured up to the 8th passage and analysed with RT-PCR for the expression of pluripotency markers. Some cellular pools were cultured in suspension in uncoated Petri dishes (hanging drop method) to obtain EB formation. After 5 days of culture, the appearance of EBs of different size was observed with phase contrast microscopy and monitored up to 10-15 days of culture. In parallel, EB standard embedding in paraffin for light microscopy or in epoxy resin for transmission electron microscopy was performed. RT-PCR analysis revealed the presence of classical markers of pluripotency (OCT4, NANOG, SOX2) in AFSCs at the 2th-8th passage, whereas specific markers of the three embryonic germ layers were detected in EB specimens. Morphological assays of three-dimensional aggregates demonstrated the presence of solid structures only at the beginning of the culture whereas signs of apoptotic cell death accompanied by the secretion of an amorphous substance were soon detected. These features were preliminary to the development, at later culture time intervals, of an inner hollow cavity surrounded by a crown of flat cells displaying a number of electron dense granules and highly resembling trophoblastic cells

Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules

Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

<p>Abstract</p> <p>Background</p> <p>Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.</p> <p>Results</p> <p>The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.</p> <p>Conclusion</p> <p>The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.</p

A morpho-functional analysis of embryoid body-like structures from human amniotic fluid-derived stem cells (AFSCs) unselected for c-kit

Human AFSCs, a novel class of stem cells sharing characteristics of both embryonic and adult stem cells, harbour high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells (ESCs). The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the main step in the differentiation of pluripotent embryonic stem cells. The purpose of this study was to investigate whether human AFSCs, unselected for c-kit, have features of pluripotency. With this aim, we evaluated both AFSC ability to form in vitro EB structures and transcriptional profiles of genes typically expressed in human ESCs. Total AFSCs were cultured in suspension in uncoated Petri dishes for EB formation, whose incidence was assessed in 5 independent experiments. EB-like structures were observed and morphometrically analysed under a LEICA phase contrast microscope equipped with a CoolSnap videocamera. A number of samples were processed for alkaline phosphatase (AP) or haematoxylin-eosin staining, immunofluorescence and transmission electron microscopy to follow-up morphology and markers of pluripotency. As to the expression studies, RNA was extracted from AFSCs at the 3th, 4th, 5th and 8th passage in culture and the presence of ESC and primordial germ cell (PGC) specific markers was assessed with RT-PCR. As early as after 5 days of culture we were able to observe the formation of EB-like solid structures of different size progressively increasing at later time intervals of incubation in cell culture medium (10-15 days). At these later time points EB aggregates showed the presence of an internal cavity and were surrounded by a wide cohort of bigger cells detaching from them. Both early and late time EBs were positive for alkaline phosphatase (AP) staining and specific markers of pluripotency (OCT4 and SOX2). The parallel analysis of AFSCs with RT-PCR demonstrated the presence of ESC and PGC specific gene transcripts and, moreover, the expression of alternatively spliced genes also detectable in EB cells. These findings demonstrate that AFSCs are a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development

Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases

In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases

Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.

Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%