Normal Metaphase Research Articles

An Integral R-Banded Karyotype Analysis System of Bone Marrow Metaphases Based on Deep Learning.

Conventional karyotype analysis, which provides comprehensive cytogenetic information, plays a significant role in the diagnosis and risk stratification of hematologic neoplasms. The main limitations of this approach include long turnaround time and laboriousness. Therefore, we developed an integral R-banded karyotype analysis system for bone marrow metaphases, based on deep learning. To evaluate the performance of the internal models and the entire karyotype analysis system for R-banded bone marrow metaphase. A total of 4442 sets of R-banded normal bone marrow metaphases and karyograms were collected. Accordingly, 4 deep learning-based models for different analytic stages of karyotyping, including denoising, segmentation, classification, and polarity recognition, were developed and integrated as an R-banded bone marrow karyotype analysis system. Five-fold cross validation was performed on each model. The whole system was implemented by 2 strategies of automatic and semiautomatic workflows. A test set of 885 metaphases was used to assess the entire system. The denoising model achieved an intersection-over-union (IoU) of 99.20% and a Dice similarity coefficient (DSC) of 99.58% for metaphase acquisition. The segmentation model achieved an IoU of 91.95% and a DSC of 95.79% for chromosome segmentation. The accuracies of the segmentation, classification, and polarity recognition models were 96.77%, 98.77%, and 99.93%, respectively. The whole system achieved an accuracy of 93.33% with the automatic strategy and an accuracy of 99.06% with the semiautomatic strategy. The performance of both the internal models and the entire system is desirable. This deep learning-based karyotype analysis system has potential in a clinical application.

Oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

ABSTRACT Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.

Effect on Antioxidants and Chromosomal Aberrations in the Offspring of Methyl Isocyanate Exposed Population of Bhopal

Bhopal gas tragedy was one of the worst industrial disasters that produced short-term as well as long-term health effects in the exposed population. The present study is an attempt to know about the genomic damage as well as the level of antioxidant caused by Methyl Isocyanate in the offspring of the exposed population and to know about the frequency of chromosomal aberrations in them. Offspring’s of the Methyl Isocyanate Exposed individuals living in the vicinity of 1.5 km from the Union Carbide’s pesticide plant were selected randomly from the study. Equal number of age (20 years – 50 years), sex (male / female) and locality matched non-exposed individuals were also enrolled as controls. All the subjects were non smokers and not employed at any type of chemical industry or a radiation department. Cytogenetic analysis was performed by standard protocol of lymphocyte culture as per as Moorhead et al. Chromosomal aberrations were observed. The antioxidant level of Vitamin E and Blood Glutathione was also recorded. Student’s t-test was performed to test the significance difference between means of chromosomal aberrations. The means were considered to be statistically significant at 1% level of probability. It was observed that the level of antioxidants (both Vitamin E by Jargar et al. and Blood Glutathione by Beutler et al.) was highly significant in the MIC Exposed and MIC Exposed F2 Generation Population as compared to MIC Non-Exposed Population. But, significant difference between MIC Exposed and MIC Exposed F2 Generation Population was observed. The mean percentage of normal metaphase in the Exposed group was significantly higher (p < 0.0001) as compared to Non-Exposed. Besides this, chromosomal aberrations (dicentrics and double minutes) were recorded at mitotic metaphase and were statistically elevated in the MIC Exposed Population. The present study indicates that the genomic instability still persists at a higher intensity among the Bhopal Gas Survivors. The higher incidence of chromosomal aberrations may act as the intermediate processes in the pathway of the progression of any genetic disorders and cancers. Hence, our study suggests that the Exposed Populations and their Offspring’s is more vulnerable and gas affected.
 Keywords: Chromosomal aberrations, Methyl isocyanate, Bhopal gas tragedy, Long-term genotoxicity, Exposed Population

Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

To treat couples with total fertilization failure (TFF) based on a combined oocyte- and sperm-related oocyte activation deficiency by optimizing oocyte response to chemical activation with calcium ionophore. Case report. Tertiary Hospital. Two couples with a history of TFF after intracytoplasmic sperm injection intracytoplasmic sperm injection (ICSI). To overcome oocyte-related oocyte activation deficiency (OAD), extended invivo/invitro oocyte maturation was performed to enhance ooplasmic maturity; to address sperm-related OAD, assisted gamete treatment (AGT) was performed to trigger oocyte activation. Treatment cycle outcomes for the 2 couples undergoing ICSI with extended oocyte maturation (EOM) and AGT. We identified 2 couples with TFF after ICSI because of a combined factor of OAD confirmed by phospholipase C zeta expression and genomic assessment. Initial AGT treatment alone failed to enhance fertilization, suggesting superimposed oocyte dysmaturity prohibiting oocytes from responding to chemical stimuli. To address this complex form of OAD, in couple 1, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after EOM; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved initially. Two embryos were thawed and transferred, yielding a monochorionic diamniotic twin pregnancy. Couple 2 underwent 3 ICSI cycles with EOM and AGT; 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). A total of 12 blastocysts were cryopreserved. A single 46XY blastocyst was thawed and transferred, resulting in a singleton pregnancy. Our study has demonstrated the usefulness of EOM by targeting spindle presence to enhance chemical responses to AGT.

P-270 Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

Abstract Study question How can we treat couples with complete and persistent fertilization failure with ICSI linked to a combination of oocyte- and sperm-related oocyte activation deficiency (OAD)? Summary answer By targeting spindle presence, we optimized oocyte response to chemical activation and enhanced fertilization. Genomic assessment confirmed gamete contribution. What is known already Total fertilization failure occurs in 1-3% of all intracytoplasmic sperm injection (ICSI) cases. In sperm-factor OAD, the lack of phospholipase C zeta (PLCζ) prevents the spermatozoon from initiating downstream calcium oscillation in the oocyte. In these cases, assisted gamete treatment (AGT), which exposes gametes to calcium ionophore, has been adopted to artificially trigger the influx of calcium ions and has been shown to effectively improve fertilization. However, AGT is limited to triggering an intracytoplasmic calcium influx and still requires optimal ooplasmic maturity. Study design, size, duration Over the past 17 months, we identified couples with compromised PLCζ and reported persistent fertilization failure with ICSI despite AGT treatment. We then devised a treatment plan comprising an extended in vitro culture (IVC) to pinpoint meiotic oocyte maturity confirmed by the presence of a meiotic II spindle and followed by AGT post-ICSI. Genomic assessment was also carried out. Participants/materials, setting, methods Two couples with recurrent and total fertilization failure even after AGT were included. PLCζ expression was assessed using immunofluorescence on ≥ 200 cells/specimen with a 30% threshold. In the follow-up cycles, IVC was extended for at least 8 hours between retrieval and ICSI. Metaphase II spindles were visualized by Oosight®. AGT was performed by exposing both spermatozoa and oocytes to calcium ionophore. NGS was performed on spermatozoa to identify gene mutations involved in fertilization. Main results and the role of chance We identified 2 couples (couple A: 37-year-old female, 39-year-old male; couple B: 32-year-old female, 33-year-old male) with the following semen parameters: average volume of 2.6 ml, concentration of 82.0x106/ml, 44% motility, and normal morphology of 3%. The oocyte maturation rate was 76.3% (45/59) but resulted in zero fertilized out of a total of 45 MII oocytes injected. In-house PLCζ assessment revealed a deficiency of oocyte activation factor at 12.9%. AGT treatment alone failed to enhance fertilization on a subsequent cycle, resulting in 0% (0/8) and 5.6% (1/18) fertilization rates for couples A and B, respectively. Couple A then underwent 3 ICSI cycles with extended IVC and AGT; upon examination of nuclear maturity, 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). To date, 12 blastocysts were cryopreserved. In couple B, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after extended IVC; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved. Overall, our treatment improved fertilization to an overall rate of 52.5% (31/59, P <0.00001). Genomic assessment of spermatozoa identified gene mutations involved in fertilization (ADAM15, ADAM30) and calcium channel activity (CATSPER1). Limitations, reasons for caution Assisted gamete treatment can enhance fertilization in cases of deficiency in PLCζ. However, chemical activation requires a responsive ooplasm that has reached meiotic maturity. These rare cases require precise diagnoses and tailored treatment techniques to address each aspect of sperm- and/or oocyte-factor OAD. Wider implications of the findings Our study has demonstrated the usefulness of extended IVC by targeting spindle presence to enhance chemical responses to AGT. Our findings show that although calcium ionophore can trigger the release of intracellular calcium and allow fertilization, a fully mature ooplasm is required. Trial registration number N/A

Simultaneous Occurrence Of Multiple Myeloma and Acute Myeloid Leukemia: A Dual Malignancy Of The Hematopoietic System

The occurrence of acute myeloid leukemia (AML) after the treatment for multiple myeloma (MM) is well described in several cases. The simultaneous presentation of MM and AML is very rare, with only 17 cases reported in articles found at PubMed.A 69 years old woman was sent at our institution for consultation due to pancitopenia at check-up. Past medical history was irrelevant. The CBC reported Hb 11.3 g/dL, Htc 33.8 %, ESR 32 mm/hr, Rouleaux formation +++, WBC 24.5 K/µL; differential showing nuetrophils 0.748 K/μL, lymphocytes 2.2 K/μL and myeloid blasts 20.1 K/µL, platelets 23.4 K/µL. The bone marrow aspiration was hypercellular with infiltration by myeloid blasts counting 95 %, and plasma cells with neoplastic morphology counting the remaining 5 %, no normal hematopoietic cells were observed. Hematological malignancy work up, also for MM, was performed, including flow cytometry (FC) and G band karyotype (GBK). FC revealed three populations: 1) representing 82.9 % that expressed CD11b +, CD13 ++, CD33 +, CD34 +, CD 38 +, HLA-DR + and MPO +; 2) representing 4.6 % and expressed CD38 +++, CD56 +, CD138 +++, kappa ++; 3) mixed population of normal B and T cells, representing 12.5 %. GBK showed 18 normal metaphases and 2 metaphases with t(11:14) (q13:q32) and del(13q). Stratification tests reported creatinin 2.3 mg/dL, , Ca 10.15 mg/dL, us-CRP 23 mg/dL, β-2-microglobulin 7.751 mg/L, albumin 2.35 g/dL, globulin 5.75 g/dL, serum protein electrophoresis with M-spike in the gamma region with 4.12 g/dL, positive Bence Jones protein, X-ray from the skull showed multiple lytic lesions. High risk MM and AML M4 were diagnosed. The patient did not accept any treatment and died few days later due a pulmonary hemorrhage.There is no pathophysiologic mechanism that explains the simultaneous occurrence. MM is a slowly disease, it may lead to chromosomal instability and immunosuppression with failure to eliminate clones of leukemic cells. No effective treatment is currently available, allogeneic stem cell transplantation is the best option. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. N/A.

11q23/MLL rearrangements in adult acute leukemia.

To detect the frequency, diagnostic and prognostic significance of 11q23/MLL rearrangements and to determine the chromosomes that are most frequently involved in 11q23/MLL abnormalities in adult acute leukemia (AL). Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140patients with acute myeloid leukemia (AML) and 57patients with acute lymphoblastic leukemia (ALL) were performed. The methods of conventional cytogenetics (GTG-banding) and fluorescence in situ hybridization were used. Chromosomal abnormalities in leukemia cells were found by conventional cytogenetic methods in 80 (57%) and 37 (65%) adult patients with AML and ALL, respectively. 11q23/MLL rearrangements were found in 7 (5%) and 8 (14%) patients with AML and ALL, respectively. Among them, 8 (53.4%) patients had translocations, 2 (13.3%)- had deletions and 5 (33.3%) patients had trisomies or tetrasomies of chromosome 11. With respect to the distribution of partner chromosomes involved in 11q23/MLL translocations chromosome 4was found to participate in 3 (37.5%) cases of 11q23/MLL translocations, 9- in 2 (25%) cases and chromosomes 10, 14and non-identified chromosome were involved in 1 (12.5%) case each. Nine patients (60%), besides abnormal ones, had 9-86% normal metaphases in their karyotypes. Of 15patients with 11q23/MLL rearrangements, 5 (33%) patients had only 11q23/MLL rearrangements, whereas other 10 (67%)- had additional cytogenetic abnormalities, besides 11q23/MLL rearrangements. Chromosomal abnormalities of various kinds were found in 57% and 65% adult patients with AML and ALL, respectively. The frequency of 11q23/MLL rearrangements in patients with AML and ALL was 5% and 14%, respectively. Since AL patients with 11q23/MLL rearrangements are attributed to cytogenetic categories of AL with a poor or intermediate risk prognosis, cytogenetic methods should be included in the standard examination of AL patients for diagnosis, prognosis and selection of the optimal treatment strategy.

Fluorescence In Situ Hybridization: A Sensitive Method for Trisomy 8 Detection in Bone Marrow Specimens

Trisomy 8 is a common anomaly in bone marrow (BM) cells of patients with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), or acute nonlymphocytic leukemia (ANLL). We studied the efficacy of fluorescence in situ hybridization (FISH) detection of trisomy 8 in patients with MPD, MDS, or ANLL using directly labeled fluorescent alpha-satellite and whole chromosome paint (WCP) DNA probes specific for chromosome 8. Using FISH, we analyzed interphase nuclei and metaphase spreads from randomized series of BM specimens from normal individuals and patients with varying proportions of trisomy 8 as determined by conventional cytogenetic analysis. The BM of all normal donors contained less than or equal to 2.0% nuclei with 3 interphase FISH signals and less than or equal to 1 metaphase with 3 WCP FISH signals. Ninety-five percent and 98% of BM specimens with at least two metaphase cells with trisomy 8 by cytogenetic analysis contained greater than 2.0% nuclei with 3 interphase FISH and greater than 2 metaphases with 3 WCP FISH signals, respectively. Thirteen patients had 1 in 20 or 1 in 30 metaphase cells with trisomy 8 by conventional cytogenetic studies. Of these patients, four had greater than 2.0% nuclei with 3 interphase FISH signals. The BM of all four patients contained positive metaphase FISH results. We then studied the usefulness of FISH analysis to detect occult trisomy 8 by analyzing BM nuclei from 144 patients who had MPD, MDS, or ANLL and either 20 normal metaphase cells or an abnormal karyotype without trisomy 8. Seven patients had greater than 2.0% nuclei with 3 interphase FISH signals (range, 2.10% to 3.40%) and six patients had 2 or more cells with trisomy 8 upon metaphase FISH or extensive conventional cytogenetic analysis. Our results show that interphase and metaphase FISH analyses are useful methods to detect trisomy 8 cells in BM specimens, especially for specimens with normal or uncertain conventional cytogenetic results.

Hypodiploidy in patients with acute lymphoblastic leukemia

Background. Acute lymphoblastic leukemia (ALL) is characterized by different clinical course and different sensitivity to therapy. Taking into consideration their significant prevalence an intensive search for new prognostic criteria is conducted that may determine individual prognosis and choose the most appropriate treatment approach for patients with ALL, who often require transfusion therapy and replacement therapy with blood components.&#x0D; Objective. To detect the frequency, diagnostic and prognostic significance of hypodiploidy in patients with ALL.&#x0D; Materials and methods. Standard cytogenetic investigation of bone marrow and/or peripheral bloodcells was performed according to the standard techniques from 57 adult patients with ALL.&#x0D; Results and discussion. Chromosomal aberrations of various kinds were found in 37 (65 %) patients with ALL. Among them presence of one karyotype abnormality was established in 9 (24 %) patients, two abnormalities – in 10 (27 %) and multiple structural and/or numerical changes (≥3) – in 18 (49 %). Samples from 20 (35 %) patients showed a normal female or male karyotype without cytogenetically visible changes. The most common abnormalities in ALL were: trisomy 8, rearrangements of 7q, 17p and 11q23, translocations t(4;11)(q21;q23), t(9;22)(q34;q11), marker chromosomes, acentric structures, hypodiploidy, hyperdiploidy, complex karyotype (≥3 changes) etc. Hypodiploidy was found in 2 (4 %) patients with ALL. One patient, except for abnormal ones, had normal metaphases in him karyotype. Of two patients with hypodiploidy, one had only numerical abnormalities, whereas other one had also structural cytogenetic aberrations, except the numerical changes, namely t(1;6)(q32;q27), add(12)(q24), del(17)(p11), r(17)(p13q25). Hypodiploidy is an unfavorable marker in ALL and a near haploidy is an extremely unfavorable factor.&#x0D; Conclusions. Cytogenetic abnormalities of various kinds were found in 37 (65 %) patients with ALL. The frequency of hypodiploidy was 4 %. ALL patients with hypodiploidy were classified into cytogenetic categories of ALL with a poor risk prognosis. Thus, cytogenetic investigations should be included in the standard examination of patients with ALL for diagnosis, prognosis and selection the optimal treatment strategy.

Fluorescence in Situ Hybridization (FISH) Utility for Risk Score Assessment in Patients With MDS With Normal Metaphase Karyotype

BackgroundCytogenetic profile is an essential parameter in myelodysplastic syndromes (MDS) risk stratification by both International Prognostic Symptom Score (IPSS) and Revised (R)-IPSS. Almost one-half of patients with MDS have normal cytogenetics by metaphase karyotype. Here we report the yield of MDS fluorescence in situ hybridization (FISH) panel detecting cytogenetic abnormalities in these patients and its impact on risk stratification. Patients and MethodsAmong patients with normal metaphase karyotype, we assessed those patients who had cytogenetic abnormalities detected by an MDS FISH panel, which included probes for del (5), del (7), del (20), trisomy 8, and del (17p). Risk stratification was calculated by both IPSS and R-IPSS. ResultsOf 1600 patients with MDS with normal metaphase karyotype, 53 (3%) patients had cytogenetic abnormality detected by MDS FISH panel. Integrating the MDS FISH panel cytogenetics (IPSS + FISH restaging) resulted in upstaging the score, where 53% of low-risk IPSS were upstaged to intermediate (int)-1, 56% of int-1 were upstaged to int-2, and 78% of int-2 were upstaged to high risk. Based on the R-IPSS, 61% of very low-risk patients, all low-risk patients, 92% of intermediate-risk patients, and 50% of high-risk patients with FISH abnormalities were upstaged, respectively. ConclusionThe yield of MDS FISH panel detecting cytogenetic abnormalities in patients with normal karyotype by G-banding is low and may not warrant ordering the panel in all patients. Among the 3% of patients with normal karyotype who had cytogenetic abnormality detected by FISH, the risk score assignment by IPSS and R-IPSS was upstaged.

Establishment and Characterization of a Stromal Cell Line Derived From a Patient With Thoracic Endometriosis

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10–7 mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and a-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.

Effects of Ferrocenyl 4-(Imino)-1,4-Dihydro-quinolines on Xenopus laevis Prophase I - Arrested Oocytes: Survival and Hormonal-Induced M-Phase Entry.

Xenopus oocytes were used as cellular and molecular sentinels to assess the effects of a new class of organometallic compounds called ferrocenyl dihydroquinolines that have been developed as potential anti-cancer agents. One ferrocenyl dihydroquinoline compound exerted deleterious effects on oocyte survival after 48 h of incubation at 100 μM. Two ferrocenyl dihydroquinoline compounds had an inhibitory effect on the resumption of progesterone induced oocyte meiosis, compared to controls without ferrocenyl groups. In these inhibited oocytes, no MPF (Cdk1/cyclin B) activity was detected by western blot analysis as shown by the lack of phosphorylation of histone H3. The dephosphorylation of the inhibitory Y15 residue of Cdk1 occurred but cyclin B was degraded. Moreover, two apoptotic death markers, the active caspase 3 and the phosphorylated histone H2, were detected. Only 7-chloro-1-ferrocenylmethyl-4-(phenylylimino)-1,4-dihydroquinoline (8) did not show any toxicity and allowed the assembly of a histologically normal metaphase II meiotic spindle while inhibiting the proliferation of cancer cell lines with a low IC50, suggesting that this compound appears suitable as an antimitotic agent.

New cellular imaging of oocytes and preimplantation embryos using Lumitein™: Evaluation of oocyte quality and new information on protein dynamics within the perivitelline space during the one-cell oocyte stage in mice.

The extracellular matrix between the oocyte and zona pellucida (ZP) plays an important role in mammalian fertilization and preserves the specific environment of the perivitelline space (PVS) during the development of a preimplantation embryo after fertilization. In this study, we applied a highly sensitive luminescent protein dye, Lumitein™, to observe the hydrophobic status of proteins in oocytes and preimplantation embryos. Lumitein™ is widely used for detecting denatured proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lumitein™ fluorescence was detected primarily in the PVS and degenerated first polar body of fresh normal metaphase II (MII) oocytes but much less within the ZP and ooplasm, which suggested a hydrophobic PVS environment in the MII oocytes. Unexpectedly, abnormally-shaped fresh or aged oocytes showed stronger fluorescence in the PVS, which reflected oocyte quality. Interestingly, 10 h after fertilization, the fluorescent signal in the PVS temporarily increased in a patched pattern that appeared and then disappeared by the two-cell stage. After the two-cell stage, the decreased fluorescent signal was maintained throughout the development of the preimplantation embryo. These results suggest new protein dynamics in the PVS during the one-cell stage of the oocyte. Thus, cellular imaging of oocytes and preimplantation embryos using Lumitein™ provides new information on protein dynamics.

Metaphase Cytogenetics for Risk Stratification in Newly Diagnosed Multiple Myeloma

Introduction: Abnormal metaphase cytogenetics are associated with inferior survival in newly diagnosed multiple myeloma (MM). These abnormalities are only detected in one third of cases due to the low proliferative rate of plasma cells. It is unknown if metaphase cytogenetics improve risk stratification when using contemporary prognostic models such as the revised international staging system (R-ISS), which incorporates interphase fluorescence in situ hybridization (FISH). Aims: The aims of this study were to 1) characterize the association between abnormalities on metaphase cytogenetics and overall survival (OS) in newly diagnosed MM treated with novel agents and 2) evaluate whether the addition of metaphase cytogenetics to R-ISS, age, and plasma cell labeling index (PCLI) improves model discrimination with respect to OS. Methods: We analyzed a retrospective cohort of 483 newly diagnosed MM patients treated with proteasome inhibitors (PI) and/or immunomodulators (IMID) who had metaphase cytogenetics performed prior to initiation of therapy. Abnormal metaphase cytogenetics were defined as MM specific abnormalities, while normal metaphase cytogenetics included constitutional cytogenetic variants, age-related Y chromosome loss, and normal metaphase karyotypes. Multivariable adjusted proportional hazards regression models were fit for the association between known prognostic factors and OS. Covariates associated with inferior OS on multivariable analysis included R-ISS stage, age ≥ 70, PCLI ≥ 2, and abnormal metaphase cytogenetics. We devised a risk scoring system weighted by their respective hazard ratios (R-ISS II +1, R-ISS III + 2, age ≥ 70 +2, PCLI ≥ 2 +1, metaphase cytogenetic abnormalities + 1). Low (LR), intermediate (IR), and high risk (HR) groups were established based on risk scores of 0-1, 2-3, and 4-5 in modeling without metaphase cytogenetics, and scores of 0-1, 2-3, and 4-6 in modeling incorporating metaphase cytogenetics, respectively. Survival estimates were calculated using the Kaplan-Meier method. Survival analysis was stratified by LR, IR, and HR groups in models 1) excluding metaphase cytogenetics 2) including metaphase cytogenetics and 3) including metaphase cytogenetics, with IR stratified by presence and absence of metaphase cytogenetic abnormalities. Survival estimates were compared between groups using the log-rank test. Harrell's C was used to compare the predictive power of risk modeling with and without metaphase cytogenetics. Results: Median age at diagnosis was 66 (31-95), 281 patients (58%) were men, median follow up was 5.5 years (0.04-14.4), and median OS was 6.4 years (95% CI 5.7-6.8). Ninety-seven patients (20%) were R-ISS stage I, 318 (66%) stage II, and 68 (14%) stage III. One-hundred and fourteen patients (24%) had high-risk abnormalities by FISH, and 115 (24%) had abnormal metaphase cytogenetics. Three-hundred and thirteen patients (65%) received an IMID, 119 (25%) a PI, 51 (10%) received IMID and PI, and 137 (28%) underwent upfront autologous hematopoietic stem cell transplantation (ASCT). On multivariable analysis, R-ISS (HR 1.59, 95% CI 1.29-1.97, p &lt; 0.001), age ≥ 70 (HR 2.32, 95% CI 1.83-2.93, p &lt; 0.001), PCLI ≥ 2, (HR 1.52, 95% CI 1.16-2.00, p=0.002) and abnormalities on metaphase cytogenetics (HR 1.35, 95% CI 1.05-1.75, p=0.019) were associated with inferior OS. IR and HR groups experienced significantly worse survival compared to LR groups in models excluding (Figure 1A) and including (Figure 1B) the effect of metaphase cytogenetics (p &lt; 0.001 for all comparisons). However, the inclusion of metaphase cytogenetics did not improve discrimination. Likewise, subgroup analysis of IR patients by the presence or absence of metaphase cytogenetic abnormalities did not improve risk stratification (Figure 1C) (p &lt; 0.001). The addition of metaphase cytogenetics to risk modeling with R-ISS stage, age ≥ 70, and PCLI ≥ 2 did not improve prognostic performance when evaluated by Harrell's C (c=0.636 without cytogenetics, c=0.642 with cytogenetics, absolute difference 0.005, 95% CI 0.002-0.012, p=0.142). Conclusions: Abnormalities on metaphase cytogenetics at diagnosis are associated with inferior OS in MM when accounting for the effects of R-ISS, age, and PCLI. However, the addition of metaphase cytogenetics to prognostic modeling incorporating these covariates did not significantly improve risk stratification. Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Alnylam: Research Funding; Celgene: Research Funding; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding. Kapoor:Celgene: Honoraria; Sanofi: Consultancy, Research Funding; Janssen: Research Funding; Cellectar: Consultancy; Takeda: Honoraria, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding. Leung:Prothena: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Omeros: Research Funding; Aduro: Membership on an entity's Board of Directors or advisory committees. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Cytotoxicity study and bioremediation of petroleum contaminated soil.

s. An experimental study was carried out to investigate the toxic effect of petroleum-contaminated soil by determining chromosomal aberration on mitosis of the onion root tip and demonstrating the role of mixtures or consortia of microbes in the detoxification of crude oil hydrocarbons. Soil samples were treated with different concentrations of petroleum (0, 2, 4, 8% of soil pot). All treatments with the exception of control were added 0.2 g consortium and one group was treated with only distilled water. To allow the degradation of oil components, all treatments were incubated in laboratory conditions for three periods (3,30,60 days). The results showed that chromosomal aberrations in root tip cells of Allium cepa are an abnormal anaphase with breakage, multipolar anaphase with breakage, normal metaphase and sticky metaphase, Mitotic index (MI) chromosomes that were calculated during prophase, metaphase and anaphase. The results show that adding a microbial consortium to the soil (0.2g/pot) where it recorded 600 divided cells per 1000 cells (IM=60) after incubation for 60 days, High level of crude oil 8% was caused decreasing of MI to 1.5, 3, 3.5 without any adding, while recording 35,46,50 (only distill water) in the control group. On the other hand, the high percentage removal of hydrocarbons by microbial activity with a low concentration of crude oil is 55% with increasing of time duration.

Establishment and Characterization of a Stromal Cell Line Derived From a Patient With Thoracic Endometriosis.

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10-7 mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and α-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.

Molecular Profile and Cytomorphological Manifestation of Isolated Y Loss in Myelodysplastic Syndromes

Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY.Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones.Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and <75%. Likewise, the VAF of molecular mutations did not significantly differ between MDS and pts without dysplasia according to LOY≥75% and <75%. Notably, for pts with LOY≥75% the number of mutated core genes was significantly higher in MDS compared to pts without dysplasia (p=0.024, Fig.4).Conclusions: Cytopenia with isolated LOY seems to comprise a heterogeneous mixture of cytomorphologic and molecular subtypes. We identified pts where LOY definitely is part of the MDS clone that also harbors molecular mutations, indicating that LOY is not only age-associated in these cases. The low total number of mutated genes corresponds to the previously described favorable prognosis of isolated LOY (Schanz et al, JCO 2012; Greenberg et al, Blood 2012). Our data support the current guidelines that LOY does not prove a hematologic disease in the absence of diagnostic morphologic features. In these cases LOY might fulfill the requirements of a clonal mutation as described for clonal hematopoiesis of indeterminate potential (CHIP) or CCUS. In summary, our data suggest that MDS with LOY shows a characteristic molecular mutation profile. [Display omitted] DisclosuresGerming:Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Follicular dynamics of glycerophospholipid and sphingolipid metabolisms in polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) is a common heterogeneous disease, affecting up to 5–10% women at reproductive age. Although PCOS patients could produce morphologically normal metaphase II oocytes undergoing assisted reproductive techniques (ART), oocyte developmental competence and embryo development have been impaired in following in-vitro fertilization (IVF) steps. Follicular fluid (FF) provides a variety of information in oocyte environment when oocytes grow. In the present work, based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), the metabolic signatures of PCOS FF have been compared with healthy women using untargeted metabolomics approach. Significant abundance differences of a series of glycerolipid, glycerophospholipids, sphingolipids, and carboxylic acids have been discovered. Among them, reduced levels of phosphatidylglycerolphosphate (PGP) and a triglyceride (TG) were highly related to the lower fertilization rate in PCOS; increased abundance of lysoPE and decreased amount of PC were significantly correlated with LH/FSH (ratio of luteinizing hormone to follicle stimulating hormone). Some metabolites, including decreased sphingolipids, glycerophospholipids, and fluctuated fatty acyls, also performed close relationship with other ART and clinical results. We concluded that dysfunctions in the metabolism of glycerolipid, glycerophospholipid, sphingolipid, and glycosphingolipid biosynthesis in PCOS patients’ follicles play a non-ignorable role in declining the 2 pronuclei (PN) fertilization rate during IVF procedure.

Transcriptome analysis of rhesus monkey failed-to-mature oocytes: deficiencies in transcriptional regulation and cytoplasmic maturation of the oocyte mRNA population.

Which different pathways and functions are altered in rhesus monkey oocytes that fail to mature after an ovulatory stimulus? Failed to mature (FTM) oocytes complete a large portion of the transition in transcriptome composition associated with normal maturation, but also manifest numerous differences that indicate incomplete transcriptional repression and cytoplasmic maturation affecting multiple processes. Oocyte maturation defects contribute to unexplained female infertility. Failure of some oocytes to undergo germinal vesicle breakdown or progress to second meiotic metaphase in response to an ovulatory stimulus can limit the number of high quality oocytes available for ART. The transcriptome of rhesus monkey oocytes that failed to mature (FTM; n = 11, 5 donors) in response to an ovulatory stimulus in vivo was compared to those of normal germinal vesicle stage (GV, n = 7, 2 donors) and metaphase II stage (MII, n = 7, 5 donors) oocytes by RNA-sequencing (RNAseq). Female rhesus monkeys of normal breeding age (6-12 years old) and with regular menstrual cycles were used. Animals underwent a controlled ovarian stimulation protocol for the collection of oocytes by ultrasound-guided needle aspiration of follicles. We obtained a high quality RNAseq dataset consisting of n = 7, n = 7, and n = 11 libraries for normal GV, normal MII and FTM oocytes, respectively. Total reads acquired were an average of 34 million for each GV sample, 41 million for each FTM sample and 59 million for each MII oocyte sample. Approximately 44% of the total reads were exonic reads that successfully aligned to the rhesus monkey genome as unique non-rRNA gene transcript sequences, providing high depth of coverage. Approximately 44% of the mRNAs that undergo changes in abundance during normal maturation display partial modulations to intermediate abundances, and 9.2% fail to diverge significantly from GV stage oocytes. Additionally, a small group of mRNAs are grossly mis-regulated in the FTM oocyte. Differential expression was seen for mRNAs associated with mitochondrial functions, fatty acid beta oxidation, lipid accumulation, meiosis, zona pellucida formation, Hippo pathway signaling, and maternal mRNA regulation. A deficiency DNA methyltransferase one mRNA expression indicates a potential defect in transcriptional silencing. All RNAseq data are published in the Gene Expression Omnibus Database (GSE112536). These results do not establish cause of maturation failure but reveal novel correlates of incompetence to mature. Transcriptome studies likely do not capture all post-transcriptional or post-translational events that inhibit maturation, but do reveal mRNA expression changes that lie downstream of such events or that are related to effects on upstream regulators. The use of an animal model allows the study of oocyte maturation failure independent of covariates and confounders, such as pre-existing conditions of the female, which is a significant concern in human studies. Depending on the legislation, it may not be possible to collect and study oocytes from healthy women; and using surplus oocytes from patients undergoing ART may introduce confounders that vary from case to case. FTM oocytes were at various stages of meiotic progression, so correlates of specific times of arrest are not revealed. All the FTM oocytes failed to respond appropriately to an ovulatory stimulus in vivo. Therefore, this analysis informs us about common transcriptome features associated with meiotic incompetence. These results reveal that some diagnostic markers of oocyte quality may not reflect developmental competence because even meiotically incompetent oocytes display many normal gene expression features. The results also reveal potential mechanisms by which maternal and environmental factors may impact transcriptional repression and cytoplasmic maturation, and prevent oocyte maturation. This work was supported by grants from the National Institutes of Health Office of Research Infrastructure Programs Division of Comparative Medicine Grants R24 [OD012221 to K.E.L., OD011107/RR00169 (California National Primate Research Center), and OD010967/RR025880 to C.A.V.]; the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD087166; MSU AgBioResearch, Michigan State University. Authors have nothing to disclose.