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Annals of Medicine Dec 2022Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological...
Combining metaphase cytogenetics with single nucleotide polymorphism arrays can improve the diagnostic yield and identify prognosis more precisely in myelodysplastic syndromes.
BACKGROUND
Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological aberrations, and a propensity for progression to acute myeloid leukaemia. Diagnosis and disease prognostic stratification are much based on genomic abnormalities. The traditional metaphase cytogenetics analysis (MC) can detect about 40-60% aberrations. Single-nucleotide polymorphism arrays (SNP-A) karyotyping can detect copy number variations with a higher resolution and has a unique advantage in detection of copy number neutral loss of heterozygosity (CN-LOH). Combining these two methods may improve the diagnostic efficiency and accuracy for MDS.
METHODS
We retrospectively analysed the data of 110 MDS patients diagnosed from January 2012 to December 2019 to compare the detection yield of chromosomal abnormalities by MC with by SNP-A, and the relationship between chromosomal abnormalities and prognosis.
RESULTS
Our results showed that SNP-A improved the detection yield of chromosomal aberrations compared with MC (74.5 vs. 55.5%, < .001). In addition, the positive yield could be further improved by combining MC with SNP-A to 77.3%, compared with MC alone. Univariate analysis showed that age >65 years, bone marrow blasts ≥5%, with acquired CN-LOH, new aberrations detected by SNP-A, TGA value > the median (81.435 Mb), higher risk by IPSS-R-MC, higher risk by IPSS-R-SNP-A all had poorer prognosis. More critically, multivariable analysis showed that age >65 years and higher risk by IPSS-R-SNP-A were independent predictors of inferior OS in MDS patients.
CONCLUSION
The combination of MC and SNP-A based karyotyping can further improve the diagnostic yield and provide more precise prognostic stratification in MDS patients. However, SNP-A may not completely replace MC because of its inability to detect balanced translocation and to detect different clones. From a practical point of view, we recommend the concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients on diagnosis and prognosis stratification.KEY MESSAGESSNP-A based karyotyping can further improve the MDS diagnostic yield and provide more precise prognostic stratification in MDS patients.Acquired CN-LOH is a characteristic chromosomal aberration of MDS, which should be integrated to the diagnostic project of MDS.The concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients can be recommended.
Topics: Aged; Humans; Chromosome Aberrations; Cytogenetic Analysis; DNA Copy Number Variations; Leukemia, Myeloid, Acute; Metaphase; Myelodysplastic Syndromes; Polymorphism, Single Nucleotide; Prognosis; Retrospective Studies
PubMed: 36148999
DOI: 10.1080/07853890.2022.2125173 -
Free Radical Biology & Medicine May 2013Here we show that hydroxyl radical ((•)OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH)....
Here we show that hydroxyl radical ((•)OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH). Metaphase-II mouse oocytes, obtained commercially, were grouped as follows: control, hydrogen peroxide (H2O2), Fe(II), and combined (Fe(II) +H2O2) treatments. After 7-10 min of incubation at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson χ(2) test and Fisher exact test were used to compare outcomes between controls and treated groups and also among the treated groups. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with a combination of H2O2 and Fe(II) (p<0.001); oocytes treated with H2O2 alone or Fe(II) alone showed no or few changes compared to control. Comparison of oocyte groups that received increasing concentrations of H2O2 and a fixed amount of Fe(II) showed that 70-80% demonstrated poor scores in both MT and CH when pretreated with 5 μM H2O2, and this increased up to 90-100% when treated with 10-20 μM H2O2. Hydroxyl radical generated by H2O2-driven Fenton reaction deteriorates the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the (•)OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states.
Topics: Animals; Cytoskeleton; Hydrogen Peroxide; Iron; Metaphase; Mice; Microtubules; Oocytes; Spindle Apparatus
PubMed: 23261938
DOI: 10.1016/j.freeradbiomed.2012.12.007 -
American Journal of Medical Genetics Mar 1987To evaluate the suggested nonrandom folding of Xq13-q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or...
To evaluate the suggested nonrandom folding of Xq13-q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or without X chromosome abnormalities (eight females and one male) were investigated with RBG-staining. A significant increase (p less than .05) in the number of arm folds (Xq13-q21) of the late-replicating X chromosome, particularly in early to mid-metaphase, was observed in four of eight females. Therefore, the stage of chromosome contraction was an important factor with more folds observed at the centromeres and in longer chromosomes in early metaphase compared with mid- to late metaphase. X chromosome folds were present in cells of subjects treated with or without bromodeoxyuridine. While our study agrees with the relationship of Xq13-q21 fold with the X-inactivation center, the correlation of extended chromosomes and folding limits this method as a sole indicator of X-inactivation in routine mid-metaphase, but is useful in the analysis of early metaphase chromosomes.
Topics: Dosage Compensation, Genetic; Female; Humans; Karyotyping; Male; Metaphase; Sex Chromosome Aberrations; X Chromosome
PubMed: 3565471
DOI: 10.1002/ajmg.1320260309 -
Nature Communications Apr 2019During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome...
During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.
Topics: Aneuploidy; Cell Line, Tumor; Centromere; Chromosome Segregation; HeLa Cells; Humans; Metaphase; Mitosis; Models, Biological; Spindle Apparatus
PubMed: 30988289
DOI: 10.1038/s41467-019-09578-z -
Journal of Visualized Experiments : JoVE Jan 2014Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in...
Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15).
Topics: Cells, Cultured; Chromosome Banding; Chromosomes, Human; Cytogenetic Analysis; Humans; Lymphocytes; Metaphase
PubMed: 24513647
DOI: 10.3791/50203 -
Journal of Microscopy May 2023A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK...
A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to Ca stable isotope in the extracellular buffer for 10 min. The Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 ( Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of Ca in 10 min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.
Topics: Metaphase; Calcium; Spectrometry, Mass, Secondary Ion; Cell Division; Cytoplasm
PubMed: 36864642
DOI: 10.1111/jmi.13182 -
Fertility and Sterility Apr 2023To evaluate whether metaphase I (MI) oocytes completing maturation in vitro to metaphase II ("MI-MII oocytes") have similar developmental competence as the sibling...
OBJECTIVE
To evaluate whether metaphase I (MI) oocytes completing maturation in vitro to metaphase II ("MI-MII oocytes") have similar developmental competence as the sibling metaphase II (MII) oocytes that reached maturity in vivo.
DESIGN
Retrospective cohort study.
SETTING
Academic medical center.
PATIENT(S)
A total of 1,124 intracytoplasmic sperm injection (ICSI) cycles from 800 patients at a single academic center between April 2016 and December 2020 with at least 1 MII oocyte immediately after retrieval and at least 1 sibling "MI-MII oocyte" that was retrieved as MI and matured to MII in culture before ICSI were included in the study.
INTERVENTION(S)
None.
MAIN OUTCOME MEASURE(S)
A total of 7,865 MII and 2,369 sibling MI-MII oocytes retrieved from the same individuals were compared for the fertilization and blastocyst formation rates. For patients who underwent single euploid blastocyst transfers (n = 406), the clinical pregnancy, spontaneous pregnancy loss, and live birth rates were compared between the 2 groups.
RESULT(S)
The fertilization rate was significantly higher in MII oocytes than in delayed matured MI-MII oocytes (75.9% vs. 56.1%). Similarly, the blastocyst formation rate was higher in embryos derived from MII oocytes than in those from MI-MII oocytes (53.8% vs. 23.9%). The percentage of euploid embryos derived from MII oocytes was significantly higher than that of those from MI-MII oocytes (49.2% vs. 34.7%). Paired comparison of sibling oocytes within the same cycle showed higher developmental competence of the MII oocytes than that of MI-MII oocytes. However, the pregnancy, spontaneous pregnancy loss, and live birth rates after a single euploid blastocyst transfer showed no statistically significant difference between the 2 groups (MII vs. MI-MII group, 65.7% vs. 74.1%, 6.4% vs. 5.0%, and 61.5% vs. 70.0%, respectively).
CONCLUSION(S)
Compared with oocytes that matured in vivo and were retrieved as MII, the oocytes that were retrieved as MI and matured to MII in vitro before ICSI showed lower developmental competence, including lower fertilization, blastocyst formation, and euploidy rates. However, euploid blastocysts from either cohort resulted in similar live birth rates, indicating that the MI oocytes with delayed maturation can still be useful even though the overall developmental competence was lower than that of their in vivo matured counterparts.
Topics: Pregnancy; Female; Humans; Male; Abortion, Spontaneous; Retrospective Studies; Metaphase; Semen; Oocytes; Fertilization in Vitro
PubMed: 36567036
DOI: 10.1016/j.fertnstert.2022.12.033 -
EMBO Reports Mar 2008Mature vertebrate oocytes typically undergo programmed arrest at the second meiotic cell cycle until they are signalled to initiate embryonic development at... (Review)
Review
Mature vertebrate oocytes typically undergo programmed arrest at the second meiotic cell cycle until they are signalled to initiate embryonic development at fertilization. Here, we describe the underlying molecular mechanisms of this second meiotic arrest and release in Xenopus, and compare and contrast them with their counterparts in mice.
Topics: Animals; Anura; Calcium Signaling; Homeostasis; Male; Meiosis; Metaphase; Mice; Spermatozoa
PubMed: 18311174
DOI: 10.1038/embor.2008.22 -
Scientific Reports Mar 2017Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare...
Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n = 240), with and without cumulus cells (CCs), were exposed for 4 hours to D-galactose (2 μM), galactitol (11 μM) and galactose 1-phosphate (0.1 mM), (corresponding to plasma concentrations in patients on galactose-restricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients.
Topics: Animals; Embryonic Development; Galactitol; Galactose; Galactosephosphates; Metaphase; Mice; Oocytes; Spindle Apparatus
PubMed: 28331195
DOI: 10.1038/s41598-017-00159-y -
The Journal of Cell Biology Jan 2016The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that... (Comparative Study)
Comparative Study
The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration.
Topics: Animals; Cell Line, Transformed; Drosophila; Humans; Imaging, Three-Dimensional; Metaphase; Microscopy, Fluorescence; Microtubules; Spindle Apparatus
PubMed: 26783303
DOI: 10.1083/jcb.201506128