Improving FISH diagnosis for preimplantation genetic aneuploidy screening.

BACKGROUND: In our routine programme of preimplantation genetic aneuploidy screening (PGS) by fluorescence in situ hybridization (FISH), nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) are analysed in two consecutive hybridization rounds. We also perform additional hybridization rounds for these chromosomes, using probes that bind to different loci, for non-conclusive results and for confirmation of certain aneuploidies. The aim of this study was to evaluate the impact of additional hybridization rounds on FISH accuracy. METHODS: This is a retrospective analysis of our FISH data from 1000 PGS cycles performed from December 2007 to December 2008 for various indications. In addition to the hybridization rounds described above, 132 of the embryos diagnosed as chromosomally abnormal were re-analysed on Day 5. RESULTS: A total of 2477 embryos were re-hybridized, 1496 due to non-conclusive results and 981 to confirm observed aneuploidies. After re-hybridization, 882 embryos (59%) were then diagnosed as normal, 600 embryos (40.1%) had a clear abnormality and only 14 embryos (0.9%) remained non-informative. From the 981 embryos in the latter group, 890 embryos had monosomies and, after re-hybridization 174 embryos (19.6%) were normal and 716 (80.5%) had confirmed monosomies. In contrast, re-hybridization confirmed 90 (98.9%) of the 91 observed trisomies. In addition, Day-5 re-analysis of abnormal embryos showed a higher rate of concordant diagnosis between Day 3 and Day 5 when re-hybridizations had been included on Day-3 (95 versus 82.7%; P= 0.0443), especially for the confirmation of monosomies (82.8 versus 61.0%; P = 0.0087). CONCLUSIONS: Our data indicate that additional hybridization rounds improve the accuracy of the diagnosis, increasing the number of chromosomally normal embryos available for transfer. Re-hybridization with additional probes as a standard approach to PGS could enhance the potential benefits of the technique.

Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome.

BACKGROUND: One of the described mechanisms leading to thrombosis in antiphospholipid syndrome (APS) is overexpression of tissue factor (TF) in the monocytes and endothelial cells of patients with antiphospholipid antibodies (aPL). Vascular endothelial growth factor (VEGF) may stimulate monocyte TF expression through its receptor, the tyrosine kinase Flt-1. OBJECTIVES: This study aimed to analyze the following in monocytes of 55 primary APS patients: VEGF and Flt-1 expression levels, their potential regulation by aPL, and the association of VEGF and Flt-1 expression with the increased TF expression found in APS patients. RESULTS: Purified monocytes from APS patients showed higher levels of VEGF and Flt-1 than healthy donors, which further correlated with immunoglobulin G (IgG) anticardiolipin titers and TF expression rank. Moreover, monocyte VEGF and Flt-1 levels were significantly higher in patients with than in patients without previous thrombosis. In vitro, IgG from APS patients increased monocyte VEGF and Flt-1 expression in a dose-dependent manner. VEGF and Flt-1 expression was significantly inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; this suggests the involvement of this kinase in the aPL-induced VEGF and Flt-1 upregulation. CONCLUSIONS: Our data show, for the first time in vivo, that monocytes from primary APS patients have an increased expression of VEGF and Flt-1. Furthermore, in vitro results indicated that this cytokine is produced by monocytes when treated with aPL, and that the p38 MAPK signaling pathway plays an important role. Thus, VEGF might act as a regulatory factor in aPL-mediated monocyte activation and TF expression, thereby contributing to the proinflammatory-prothrombotic phenotype of APS patients.

Klotho Prevents Translocation of NFκB.

Klotho protein is a ß-glucuronidase capable of hydrolyzing steroid ß-glucuronides. Two molecules are produced by the Klotho gene, a membrane bound form and a circulating form. This protein is recognized as an antiaging gene with pleiotropic functions. The activation of cellular systems is associated with the pathogenesis of several chronic and degenerative diseases associated with an inflammatory state. Inflammation is characterized by an activation of NFκB. Klotho suppresses nuclear factor NFκB activation and the subsequent transcription of proinflammatory genes. This review focuses on the current understanding of Klotho protein function and its relationship with NFκB regulation, emphasizing its potential involvement in the pathophysiologic process.

Search for DNA repair pathways in Drosophila melanogaster.

The knowledge about the existence of different pathways for the repairing of DNA lesions has made possible a better understanding of mutation processes. The double mutant method has been shown to be useful for grouping rad mutants in yeast. Through this method, three different groups of repair mechanisms were found: (a) RAD3 group corresponding to the excision repair of UV lesions, (b) RAD6 group corresponding to the translesion type of post-replication repair and (c) RAD52 group corresponding to the recombination type of post-replication repair. In this work, a search for a classification of Drosophila mus mutants in groups analogous to yeast RAD groups is done. Information obtained by double mutant studies was integrated with that obtained by biochemical, recombination, DNA damaging agent sensitivity and mutation studies. The following groups were found: (a) group of mei9 and mus201, analogous to RAD3, (b) group of mei41 and mus302 analogous to RAD52 and, (c) group of mus104 and mus101 analogous to RAD6. In addition, there are mutants that belong to a group corresponding to pre-replication repair of MMS lesions such as mus103, mus306 and mus207. As a peculiarity of Drosophila, it was found that interaction between pre- and post-replication repair mechanisms is indifferent and not synergistic as was found in yeast. A possible explanation could be a weaker control of post-replication repair mechanisms in Drosophila than in yeast. It is expected that this research could help for a better understanding of repair mechanisms in complex organisms.

Morphometric characterization and classification of alpaca sperm heads using the sperm-class analyzer computer-assisted system.

Sperm morphology has been identified as one characteristic which can be useful in the prediction of sperm fertility, therefore, we hope that this study aimed at establishing standardized morphological criteria might serve in future studies dealing with the search for sperm parameters which facilitate an estimation of sperm quality. For this purpose, ejaculates from fertile alpacas were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer (SCA) computer-aided image analysis system. We defined three morphological categories according to sperm head size (normal 50%, small 26%, large 24%) and five categories according to sperm head shape (normal 47%, pyriform 3%, short 20%, round 1%, long 29%). Sperm classification according to shape was performed by first morphometrically characterizing sperm heads clearly falling into each of the shape categories. Thereafter, discriminant analysis was performed on the data from these typical sperm heads and the resulting classification functions were used to categorize 2,200 spermatozoa from 11 alpacas. Classification of sperm heads by this method agreed in 88% of the cases with most of the misclassifications being due to pyriform heads classified as long heads. Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. At least 150 sperm heads should be evaluated to overcome sample size influence on sperm measurements. Significant differences in sperm morphometry were found between individuals (CV for morphometric parameters ranging from 1.3 to 13.0) and there were marked differences in the sperm morphological composition of the ejaculates. Within-animal CV ranged from 4.7 to 17.8 thus showing the high degree of sperm polymorphism present in the alpaca ejaculate.

Prosurvival effect of human wild-type alpha-synuclein on MPTP-induced toxicity to central but not peripheral catecholaminergic neurons isolated from transgenic mice.

In the present work we report the generation of a new line of alpha-synuclein (alpha-SYN) transgenic mice in which the human wild-type alpha-SYN cDNA is expressed under the control of a tyrosine hydroxylase (TH) promoter. We provide evidence that the ectopic protein is found in TH expressing neurons of both central and peripheral nervous systems. The transgene is expressed very early in development coinciding with the activity of the TH promoter and in the adult brain the human protein distributes normally to the nerve endings and cell bodies of dopaminergic nigral neurons without any evidence of abnormal aggregation. Our results indicate that expression of human wild-type alpha-SYN does not affect normal development or maintenance of TH immunoreactive nigral neurons, striatal dopamine content, or locomotor activity. Systemic administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a loss of TH immunoreactive nigral neurons and terminals and of dopamine levels to the same degree in both transgenic and non-transgenic adult mice. Intoxication also results in a similar loss of cardiac noradrenaline in both genotypes. Surprisingly, cultured transgenic ventral mesencephalic fetal dopaminergic neurons exhibit complete resistance to cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)) intoxication, without changes in dopamine transporter (DAT) surface levels. Interestingly, this protection is not observed in other populations of catecholaminergic neurons such as peripheral sympathetic neurons, despite their high sensitivity to MPP(+)in vitro.

Antiphospholipid syndrome and tissue factor: a thrombotic couple.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). Among the thrombogenic mechanisms proposed, it has been suggested that aPL can stimulate tissue factor (TF) expression by endothelial cells (ECs) and monocytes. Moreover, our in vivo studies have shown that APS patients (particularly those with thrombosis) have increased monocyte TF expression. Yet, the molecular mechanism(s) by which aPL induce TF expression has not been completely underscored. In a recent study, we have demonstrated that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-depenent nuclear translocation and activation of NFkappaB/Rel proteins. Understanding the intracellular mechanism(s) of aPL-mediated monocyte activation may help to establish new therapeutic approaches, such as selective inhibition of MAP kinases, to reverse the prothrombotic state in APS. Furthermore, the contribution of TF to a protrombotic state in the APS provides a renewed focus on antithrombotic therapies in current use, including the oral anticoagulation and, more recently, the use of statins, which have been proven to be effective in the inhibition of EC and monocyte TF-expression.

Síndrome de Guillain-Barré como forma de presentación de un linfoma no Hodgkin / Guillain-Barré syndrome as first presentation of non-Hodgkin lymphoma

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