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Taiwanese Journal of Obstetrics &... Jul 2022To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte...
OBJECTIVE
To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte dysmorphisms.
MATERIALS AND METHODS
Retrospective analysis of the morphologies of metaphase II (MII) human oocytes, which had developed into 590 biopsied blastocysts derived from 109 patients that received preimplantation genetic testing for aneuploidies (PGT-A) cycles between March 2013 and December 2017. The euploid rate of blastocysts that originated from morphologically abnormal or normal oocytes were analyzed. The chromosome status of the blastocysts was determined and analyzed by array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) following trophectoderm biopsy.
RESULTS
According to the odds ratios obtained for each oocyte morphotype, no statistically significant relationship was found between oocyte dysmorphisms and euploid rate. Specifically, although SERc-positive oocytes had a higher rate of arrest at two pronuclei, or 2 PN (26.7% vs. 19.4%, p > 0.05), the blastocyst formation rate was not affected as compared with SERc-negative oocytes (40.0% vs. 38.6%, p > 0.05). Among nine euploid embryos derived from oocytes with SERc, three single euploid embryo transfers were performed, of which one resulted in blighted ovum, and two resulted in the births of two healthy, singleton term babies.
CONCLUSION
The results presented here suggest that oocyte dysmorphisms do not affect the euploidy rate of the blastocyst. The occurrence of SERc in the oocyte does not seem to impair the developing blastocyst nor does it interfere with good embryo formation rate and euploid rate. Thus, the embryos derived from SERc-positive oocytes could still be considered for embryo transfer if there are no other embryos available.
Topics: Betahistine; Blastocyst; Comparative Genomic Hybridization; Endoplasmic Reticulum, Smooth; Female; Humans; Metaphase; Oocytes; Pregnancy; Retrospective Studies
PubMed: 35779904
DOI: 10.1016/j.tjog.2021.03.044 -
The Journal of Cell Biology Jun 2023Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes...
Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant CENP-A. CENP-A nucleosome assembly is uncoupled from replication and occurs in G1, but how cells control this timing is incompletely understood. The formation of CENP-A nucleosomes in vertebrates requires CENP-C and the Mis18 complex which recruit the CENP-A chaperone HJURP to centromeres. Using a cell-free system for centromere assembly in X. laevis egg extracts, we discover two activities that inhibit CENP-A assembly in metaphase. HJURP phosphorylation prevents the interaction between HJURP and CENP-C in metaphase, blocking the delivery of soluble CENP-A to centromeres. Non-phosphorylatable mutants of HJURP constitutively bind CENP-C in metaphase but are not sufficient for new CENP-A assembly. We find that the M18BP1.S subunit of the Mis18 complex also binds to CENP-C to competitively inhibit HJURP's access to centromeres. Removal of these two inhibitory activities causes CENP-A assembly in metaphase.
Topics: Animals; Autoantigens; Carrier Proteins; Centromere; Centromere Protein A; Metaphase; Nucleosomes; Phosphorylation; Xenopus laevis; Xenopus Proteins; DNA-Binding Proteins
PubMed: 37141119
DOI: 10.1083/jcb.202110124 -
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 -
Reproductive Biology and Endocrinology... Dec 2022High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone that is highly conserved from bacteria to humans. It plays an important role in...
BACKGROUND
High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone that is highly conserved from bacteria to humans. It plays an important role in mitochondrial homeostasis and apoptosis. In this study, we investigated the role of HtrA2 in mouse oocyte maturation.
METHODS
The role of HtrA2 in mouse oocyte maturation was investigated by employing knockdown (KD) or overexpression (OE) of HtrA2 in young or old germinal vesicle (GV) oocytes. We employed immunoblotting, immunostaining, fluorescent intensity quantification to test the HtrA2 knockdown on the GV oocyte maturation progression, spindle assembly checkpoint, mitochondrial distribution, spindle organization, chromosome alignment, actin polymerization, DNA damage and chromosome numbers and acetylated tubulin levels.
RESULTS
We observed a significant reduction in HtrA2 protein levels in aging germinal vesicle (GV) oocytes. Young oocytes with low levels of HtrA2 due to siRNA knockdown were unable to complete meiosis and were partially blocked at metaphase I (MI). They also displayed significantly more BubR1 on kinetochores, indicating that the spindle assembly checkpoint was triggered at MI. Extrusion of the first polar body (Pb1) was significantly less frequent and oocytes with large polar bodies were observed when HtrA2 was depleted. In addition, HtrA2 knockdown induced meiotic spindle/chromosome disorganization, leading to aneuploidy at metaphase II (MII), possibly due to the elevated level of acetylated tubulin. Importantly, overexpression of HtrA2 partially rescued spindle/chromosome disorganization and reduced the rate of aneuploidy in aging GV oocytes.
CONCLUSIONS
Collectively, our data suggest that HtrA2 is a key regulator of oocyte maturation, and its deficiency with age appears to contribute to reproduction failure in females.
Topics: Female; Mice; Humans; Animals; Tubulin; Oocytes; Meiosis; Metaphase; Aging
PubMed: 36539842
DOI: 10.1186/s12958-022-01048-4 -
Scientific Data Feb 2023Chromosomes are a principal target of clinical cytogenetic studies. While chromosomal analysis is an integral part of prenatal care, the conventional manual...
Chromosomes are a principal target of clinical cytogenetic studies. While chromosomal analysis is an integral part of prenatal care, the conventional manual identification of chromosomes in images is time-consuming and costly. This study developed a chromosome detector that uses deep learning and that achieved an accuracy of 98.88% in chromosomal identification. Specifically, we compiled and made available a large and publicly accessible database containing chromosome images and annotations for training chromosome detectors. The database contains five thousand 24 chromosome class annotations and 2,000 single chromosome annotations. This database also contains examples of chromosome variations. Our database provides a reference for researchers in this field and may help expedite the development of clinical applications.
Topics: Female; Humans; Pregnancy; Chromosomes; Metaphase
PubMed: 36823215
DOI: 10.1038/s41597-023-02003-7 -
Proceedings of the National Academy of... Nov 2018Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their...
Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.
Topics: Anaphase; Animals; Cartilage; Cell Cycle; Cell Polarity; Chick Embryo; Chondrocytes; Cytokinesis; Discs Large Homolog 1 Protein; Embryonic Development; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humans; Metaphase; Mice; Mice, Knockout; Microscopy, Fluorescence; Mitosis; Morphogenesis; Vertebrates
PubMed: 30377270
DOI: 10.1073/pnas.1713959115 -
F&S Science Feb 2022To determine if a cytoplasmic diameter of ≥130 μm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities...
OBJECTIVE
To determine if a cytoplasmic diameter of ≥130 μm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers.
DESIGN
Case-control study.
SETTING
Private in vitro fertilization clinic.
PATIENT(S)
The subjects were women aged 20-49 years who underwent oocyte retrieval after ovarian stimulation from January 2014 to December 2020.
INTERVENTION(S)
None.
MAIN OUTCOME MEASURE(S)
The oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers.
RESULT(S)
Among the 254,337 oocytes examined, 561 (0.22%) had a diameter of ≥130 μm. The mean diameter ranges in the normal-sized metaphase II (MII) oocytes (MII group) and GO group were 103.0-119.0 and 132.3-175.9 μm. Spindle size could be measured in 6 GOs with 1 spindle (GO1), 10 GOs with 2 spindles (GO2), and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than in the GO2 and MII groups. The median numbers of centromeres were 86 in GOs with 1 spindle and 42 in each spindle for GOs with 2 spindles among 11 GO1s and 5 GO2s.
CONCLUSION(S)
This study is the first to define GOs as oocytes with a diameter of ≥130 μm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.
Topics: Case-Control Studies; Cytoplasm; Female; Humans; Male; Metaphase; Oocytes; Spindle Apparatus
PubMed: 35559990
DOI: 10.1016/j.xfss.2021.11.004 -
Proceedings of the National Academy of... Feb 2016The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture...
The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.
Topics: Animals; Chickens; Chromatin; Chromosomes, Human; Erythrocytes; HeLa Cells; Humans; Metaphase; Microscopy, Electron; Nucleosomes
PubMed: 26787893
DOI: 10.1073/pnas.1518280113 -
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 -
Scientific Reports Oct 2018Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified...
Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.
Topics: Adult; Cumulus Cells; Female; Forkhead Box Protein M1; Genes, cdc; Humans; Metaphase; MicroRNAs; Ovarian Follicle; Ovulation; RNA, Messenger; Transcriptome
PubMed: 30353018
DOI: 10.1038/s41598-018-33807-y