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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(11): 1041-1044, 2021 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-34729739

RESUMO

OBJECTIVE: To explore the clinical effect of expanded non-invasive prenatal testing (NIPT-plus) for prenatal screening. METHODS: The screening result, prenatal diagnosis and pregnancy outcome of 3700 pregnant women who volunteered NIPT-plus screening at our center from September 2018 to December 2019 were reviewed. RESULTS: Among the 3700 pregnant women, 74(2.0%) were scored positive for clinically significant fetal chromosomal abnormalities and underwent NIPT-plus screening. Sixty three women with a high risk underwent invasive prenatal diagnosis, among whom 19 were diagnosed, which yielded a positive predictive value (PPVs) of 30.2% (19/63). Statistical analysis showed that NIPT-plus has higher PPVs for common aneuploidies and low-to-medium PPVs for sex chromosome aneuploidies and microdeletion/microduplication syndromes. CONCLUSION: As a screening technique, NIPT-plus has broader applications compared with conventional techniques, and has reference value for clinicians and pregnant women during pregnancy.


Assuntos
Aneuploidia , Diagnóstico Pré-Natal , Aberrações Cromossômicas , Feminino , Humanos , Gravidez , Resultado da Gravidez , Aberrações dos Cromossomos Sexuais
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(11): 1045-1050, 2021 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-34729740

RESUMO

OBJECTIVE: To assess the clinical value of non-invasive prenatal testing (NIPT) for the screening of trisomy and copy number variations (CNVs) of chromosomes 21, 18 and 13. METHODS: From January 2015 to December 2019, 40 628 pregnant women underwent NIPT testing using high-throughput sequencing and bioinformatics analysis to test the cell-free fetal DNA in maternal plasma. High-risk pregnant women underwent invasive prenatal diagnosis, while low-risk ones were followed up by telephone. RESULTS: The three most common indications included intermediate risk of serological screening, high risk of serological screening and advanced maternal age. Among all pregnant women, 257 cases were detected as trisomy 21, 18 and 13 (170, 49 and 38 cases, respectively). 227 cases chose invasive prenatal diagnosis, with respectively 122, 28 and 10 cases confirmed. The positive predictive value (PPV) was 81.33% (122/150), 65.12% (28/43), 29.41% (10/34), respectively. Two false negative cases of trisomy 18 were found during follow-up. Meanwhile, NIPT has detected 46 cases (15, 16 and 15 cases, respectively) CNVs on chromosomes 21, 18 and 13, among which 37 cases underwent invasive prenatal diagnosis. There were 5, 3 and 5 positive cases, which yielded a PPV of 41.67% (5/12), 25%(3/12) and 33.33%(5/15), respectively. Two other chromosome CNVs were accidentally discovered among the false positive samples. CONCLUSION: The incidence of chromosomal abnormalities in the serological screening high-risk group was 52.02%, which was significantly higher than other groups. NIPT has a high sensitivity and specificity for the screening of trisomies 21, 18 and 13, while its accuracy for detecting CNVs of chromosomes 21, 18 and 13 needs to be improved. As a screening method, NIPT has a great clinical value, though there are still limitations of false positive and false negative results.Comprehensive pre- and post-test genetic counseling should be provided to the patients.


Assuntos
Transtornos Cromossômicos , Síndrome de Down , Aneuploidia , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos , Variações do Número de Cópias de DNA , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal , Trissomia/diagnóstico , Trissomia/genética , Síndrome da Trissomía do Cromossomo 18/genética
3.
Fertil Steril ; 116(5): 1203-1204, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34756325

RESUMO

This month's Views and Reviews focuses on different aspects of the diagnosis of mosaicism. The first piece was written by Treff and Marin, who discuss the methodologic challenges of preimplantation genetic testing for aneuploidies and how the diagnosis of "mosaic" has led to a significant reduction in the accuracy of such testing. The second article by Viotti et al. provides an excellent overview of outcome data from >1,000 mosaic embryo transfers. The investigators make a strong case for three categorizations of embryos: euploid, aneuploid, and mosaic, and that use of mosaic embryos will prevent the discard of embryos that may lead to live birth. The final piece in this series was written by Besser et al., who discussed the various society recommendations for genetic counseling and prenatal diagnostic testing after mosaic embryo transfer and question whether these are truly data-driven. This collection of pieces will give the reader a deeper understanding of the controversy and management of mosaic embryo transfer.


Assuntos
Blastocisto/patologia , Testes Genéticos , Infertilidade/terapia , Mosaicismo , Diagnóstico Pré-Natal , Técnicas de Reprodução Assistida/efeitos adversos , Aneuploidia , Transferência Embrionária , Feminino , Aconselhamento Genético , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Masculino , Valor Preditivo dos Testes , Gravidez , Reprodutibilidade dos Testes , Resultado do Tratamento
4.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(12): 1171-1175, 2021 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-34839500

RESUMO

OBJECTIVE: To analyze the cause and pregnancy outcome for non-reportable cell-free DNA (cfDNA) results during non-invasive prenatal testing (NIPT). METHODS: cfDNA was extracted from maternal plasma from 5898 singleton pregnancies at 12 to 22 gestational weeks and underwent NIPT with strict quality control standards. For those with sub-standard results, redraw or invasive prenatal procedures were recommended. RESULTS: Among the 5898 cases, 32 have failed for the initial NIPT, including 17 cases with substandard cffDNA%, 10 cases with data fluctuation after twice library constructing and sequencing, and 5 cases with unidentifiable sex chromosome abnormalities. For these 32 cases, 2 directly underwent amniocentesis, and karyotyping analysis showed both were normal. Six of the 30 redrawn cases finally turned out to be nonreportable. The final nonreportable rate was therefore 0.1% (8/5898). Of the redrawn cases, 1 trisomy 21, 1 trisomy 18 and 1 trisomy 13 high risk-cases were identified, which were all confirmed to be false positive. Among the 6 nonreportable cases, 2 women underwent invasive prenatal testing, and 1 was found to have a normal fetal karyotype, while another was found to have an abnormal karyotype of mos45,X[32]/46,XY[18]. The other 4 nonreportable cases who did not accept invasive prenatal testing have all reported normal child development at follow-up. CONCLUSION: The main reason for nonreportable NIPT results was low cffDNA%. The high success rate of the redrawn cases has effectively increased the overall NIPT success rate and reduced the number of the cases necessitating invasive prenatal diagnosis. The initially nonreportable women may consider retesting after careful counseling with informed consent.


Assuntos
Teste Pré-Natal não Invasivo , Aneuploidia , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Gravidez , Diagnóstico Pré-Natal , Trissomia , Síndrome da Trissomía do Cromossomo 18/genética
5.
J Equine Vet Sci ; 105: 103720, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34607685

RESUMO

A 4-year-old Colombian Creole mare was presented for diagnosis because the external orifice of her cervix was not detectable when a uterine lavage as therapy for uterine fluid accumulation was attempted. Clinical and ultrasonographic evaluation of the genital tract revealed that ovaries were of normal size and showed structures suggestive of regular ovarian activity. However, granular free-floating fluid material distending the uterus was detected by ultrasound. Upon vaginal examination, the normal external cervical morphology was not evident. The vagina ended in a blind bag with a small papilla with no evident external cervical os. Cytology of uterine fluid obtained by transvaginal aspiration showed findings compatible with mucometra. Cytogenetic analysis revealed an abnormal karyotype (63,X and 64,XX both 45% and 65,XXX 10%). A diagnosis of congenital segmental cervical aplasia was proposed possibly related to the mosaicism detected. To our knowledge, this is the first case of this reproductive pathology in a mare with regular ovarian activity and confirmed aneuploidy in mosaic form of the X sex chromosome.


Assuntos
Doenças dos Cavalos , Útero , Aneuploidia , Animais , Colômbia , Feminino , Cavalos , Mosaicismo , Cromossomo X
6.
Int J Mol Sci ; 22(19)2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34638739

RESUMO

Numerical chromosomal aberrations in sperm are considered to be a major factor in infertility, early pregnancy loss and syndromes with developmental and cognitive disabilities in mammals, including primates. Despite numerous studies in human and farm animals, the incidence and importance of sperm aneuploidies in non-human primate remains mostly undetermined. Here we investigated the incidence and distribution of sperm aneuploidy in chimpanzees (Pan troglodytes), the species closest to human. We identify evolutionary conserved DNA sequences in human and chimpanzee and selected homologous sub-telomeric regions for all chromosomes to build custom probes and perform sperm-FISH analysis on more than 10,000 sperm nuclei per chromosome. Chimpanzee mean autosomal disomy rate was 0.057 ± 0.02%, gonosomes disomy rate was 0.198% and the total disomy rate was 1.497%. The proportion of X or Y gametes was respectively 49.94% and 50.06% for a ratio of 1.002 and diploidy rate was 0.053%. Our data provide for the first time an overview of aneuploidy in non-human primate sperm and shed new insights into the issues of aneuploidy origins and mechanisms.


Assuntos
Aneuploidia , Cromossomos de Mamíferos/genética , Hibridização in Situ Fluorescente , Espermatozoides , Animais , Humanos , Masculino , Pan troglodytes
7.
In Vivo ; 35(6): 3449-3457, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34697181

RESUMO

BACKGROUND/AIM: Preimplantation genetic testing (PGT) for chromosomal screening, based on embryo biopsy, has significant limitations. Cell-free DNA (cf-DNA) has been detected in spent culture medium (SCM), opening new horizons for the development of non-invasive PGT (ni-PGT). In this study, we evaluated the diagnostic performance of ni-PGT for aneuploidy (niPGT-A), comparing the results of trophectoderm biopsies (TE) and respective SCM from individually cultured embryos via Next Generation Sequencing (NGS). MATERIALS AND METHODS: Forty fresh embryos were analyzed. TE and SCM from blastocysts were collected and analyzed. RESULTS: We detected cfDNA in 100% of samples tested. The overall concordance rate between the ni-PGT-A and PGT-A was 27/33 (81.8%). The full concordance rate was 21/33 (63.6%). The aneuploidy agreement was 91.66%, and the euploidy agreement was 76.19%. CONCLUSION: We found a good accordance between TE and SCM analysis, suggesting that niPGT-A could be a reliable alternative for chromosomal abnormalities assessment of in vitro cultured embryos.


Assuntos
Ácidos Nucleicos Livres , Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto , Ácidos Nucleicos Livres/genética , Feminino , Testes Genéticos , Humanos , Gravidez
8.
Rom J Morphol Embryol ; 62(1): 191-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34609421

RESUMO

INTRODUCTION: An estimated 12.5% of couples experiencing fertility problems and almost 12% of reproductive age women have turned to health services at least once due to infertility. First trimester miscarriage is the most common clinical manifestation of infertility associated with a genetic cause. PATIENTS, MATERIALS AND METHODS: The scientific research was conducted at A.S. Medical Center in Bucharest, Romania, between January 2016 and December 2018, on a representative group of 1264 Caucasian patients diagnosed with infertility, from which the study group was selected, consisting of 273 patients who were further genetically investigated. RESULTS: Chromosomal instability, identified in 14% of patients, has been encountered most frequently in women (7%), and least often in fetuses (2%), unlike other chromosomal anomalies, identified in 55% of patients, which were more common in fetuses (27%) and least frequently in men (9%). Recurrent pregnancy loss due to genetic causes was identified in 53% of cases, being determined by chromosomal instability in 16% of cases and by other chromosomal anomalies in 37% of cases. Infertility due to a genetic cause was identified in 83% of cases, being determined by chromosomal instability in 17% of cases and by other chromosomal anomalies encountered in 66% of cases. In genetic risk pregnancies in evolution, fetal chromosomal anomalies were detected in 94% of cases, the most frequent being aneuploidy and polyploidy. Cytogenetic studies carried out on tissue fragments taken from aborted products of conception revealed the presence of a genetic cause in 57% of cases, an abnormal chromosome number being the most common (36%). The analysis of microdeletions of the long arm of the Y chromosome indicated that 5.5% of men with infertility are affected by this condition. CONCLUSIONS: Although genetic tests are considered complex and expensive laboratory investigations, they are crucial in identifying the etiology of over 40% of infertility cases associated with genetic factors, as well as in the correct and effective management of infertility.


Assuntos
Infertilidade , Aneuploidia , Aberrações Cromossômicas , Feminino , Feto , Testes Genéticos , Humanos , Masculino , Gravidez
9.
Curr Oncol ; 28(5): 3717-3728, 2021 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-34677235

RESUMO

Amplification (amp) of MET can be observed in cases of focal gene copy number gain, such as MET-driven amp, or with a gain of chromosome 7, such as aneuploidy. Several studies have shown that only high-level focal MET amp (MET/CEP7 ratio ≥5) is oncogenic, with such tumors responding to targeted therapy. However, there are few reports on how to distinguish between focal amplification and aneuploidy using next-generation sequencing (NGS). A total of 1025 patients with advanced solid tumors (typically pre-treated) were tested with a non-invasive comprehensive cfDNA NGS panel (Guardant360) from July 2014 to June 2019. Since bioinformatics upgrades of Guardant360 were undergoing in September 2018, focal MET amp was determined by our independent algorithm using the cohorts tested before September 2018 (291 patients), and validation was performed in the remaining cohort (734 patients). MET alterations (alts) associated with aberrant signaling were found in 110 patients (10.7%) among nine different cancer types, most commonly in non-small cell (12.2%, 62/510) and small cell (33.3%, 3/9) lung cancers, gastroesophageal cancer (19.4%, 7/36), and prostate adenocarcinoma (15.6%; 5/32). Among 291 patients tested before September 2018, 37 (12.7%) had MET alts. Among these, 24 (64.9%) had amps, 5 (13.5%) had exon 14 skipping, and 13 (35.1%) had single nucleotide variants (SNVs). Co-alterations, such as amp + SNVs, were found in four samples (10.8%). Among 24 MET amps, 29.2% (7/24) were focal according to our algorithm. MET copy number was significantly higher with focal amp compared to non-focal amp (mean copy number 3.26 vs. 2.44, respectively, p = 0.00304). In 734 patients tested after September 2018, our definition of focal MET amp was detected in 4.2% (31/734). Overall, focal amplification based on our algorithm was 3.7% (=38/1025). This study describes an approach to distinguish focal and non-focal MET amplification using comprehensive genomic profiling of cfDNA in advanced cancer patients. Focal MET amp accounted for ~30% of all MET amp, which was found in 3.7% of patients with diverse cancers and was associated with a higher plasma copy number. Clinical studies are warranted to assess the clinical utility of targeted therapies for tumors with focal MET amplification detected by NGS of cfDNA.


Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Aneuploidia , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Genômica , Humanos , Neoplasias/diagnóstico , Neoplasias/genética
11.
Fertil Steril ; 116(5): 1212-1219, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34627598

RESUMO

Chromosomal mosaicism, the coexistence of cells with different chromosomal content, has been documented in human embryos for 3 decades. Early versions of preimplantation genetic testing for aneuploidy (PGT-A) did not measure mosaicism, either because typically only a single cell was assessed or because the technique could not accurately identify it. Although this led to a straightforward diagnosis (an embryo was considered either normal or abnormal), it simply avoided the issue and, in hindsight, may have led to numerous misdiagnoses with negative clinical consequences. Modern PGT-A evaluates a multicellular biopsy specimen with techniques capable of recognizing intermediate copy number signals for chromosomes or subchromosomal regions. We are, therefore, inevitably confronted with the issue of mosaicism and the challenge of managing embryos producing such results in the clinic. Here we discuss recent data showing that not only mosaicism in general, but specific features of mosaicism detected with PGT-A, are associated with variable clinical outcomes. The conclusion is evident: mosaicism should be considered for more informed and improved embryo selection in the clinic.


Assuntos
Blastocisto/patologia , Testes Genéticos , Infertilidade/terapia , Mosaicismo , Diagnóstico Pré-Natal , Técnicas de Reprodução Assistida/efeitos adversos , Aneuploidia , Transferência Embrionária , Feminino , Aconselhamento Genético , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Masculino , Valor Preditivo dos Testes , Gravidez , Reprodutibilidade dos Testes , Resultado do Tratamento
12.
Klin Lab Diagn ; 66(10): 603-609, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34665946

RESUMO

One of the causes of spontaneous pregnancy termination, infertility, and birth of children with development delay and malformations are chromosomal abnormalities (CA) as well as spontaneous aneuploidies in gametes of phenotypically normal parents. Often couples with reproductive problems, as well as spouses one of whom is a carrier of CA, turn to the programs of assisted reproductive technologies (ART) for preimplantation evaluation of the zygote chromosomal status. As part of ART programs, parental gametes are examined to assess the level of spontaneous aneuploidy. As a rule, the most accessible material for analysis is the ejaculate. Fluorescent in situ hybridization (FISH) is used to examine male gametes obtained from the ejaculate. However, this FISH-analysis has a number of limitations and difficulties because of the peculiarities of the sperm head structure, namely the supercondensed state of chromosome chromatin. In order to optimize the FISH protocol, five different protocols were used for pre-hybridization processing of ejaculate samples obtained from nine phenotypically normal men. A comparative analysis of hybridization efficiency showed that the protocol using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a decondensation agent was the most effective for subsequent molecular cytogenetic studies. The developed hybrid protocol combining proteolytic pretreatment, TCEP and thermal decondensation can be used when other protocols for pre-hybridization treatment of ejaculate preparations are not effective.


Assuntos
Infertilidade Masculina , Aneuploidia , Criança , Aberrações Cromossômicas , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Gravidez , Espermatozoides
13.
J Transl Med ; 19(1): 416, 2021 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-34625066

RESUMO

BACKGROUND: In vitro oocyte maturation (IVM) is being increasingly approached in assisted reproductive technology (ART). This study aimed to evaluate the quality of embryos generated by in-vitro matured immature follicles, as a guideline for further clinical decision-making. METHODS: A total of 52 couples with normal karyotypes underwent in vitro fertilization, and 162 embryos were donated for genetic screening. Embryos in IVF group were generated by mature follicles retrieved during gonadotrophin-stimulated in vitro fertilization (IVF) cycles. And embryos in IVM group were fertilized from IVM immature oocytes. RESULTS: The average age of the women was 30.50 ± 4.55 years (range 21-42 years) with 87 embryos from IVF group and 75 embryos from IVM group. The rate of aneuploid with 28 of the 87 (32.2%) embryos from IVF group and 21 of the 75 (28%) embryos from IVM group, with no significant difference. The frequency of aneuploid embryos was lowest in the youngest age and increased gradually with women's age, whether in IVF group or IVM group and risen significantly over 35 years old. The embryos with morphological grade 1 have the lowest aneuploidy frequency (16.6%), and increase by the grade, especially in IVF group. In grade 3, embryos in IVM group were more likely to be euploid than IVF group (60% vs 40%, respectively). CONCLUSIONS: IVM does not affect the quality of embryos and does not increase the aneuploidy rate of embryos. It is clinically recommended that women more than 35 years have a high aneuploidy rate and recommended to test by PGS (strongly recommended to screened by PGS for women more than 40 years). Women aged less than 35 years old for PGS according to their physical and economic conditions. Embryo with poor quality is also recommended to test by PGS, especially for grade III embryos.


Assuntos
Aneuploidia , Técnicas de Maturação in Vitro de Oócitos , Adulto , Cromossomos , Feminino , Fertilização In Vitro , Humanos , Oócitos , Adulto Jovem
14.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(9): 845-848, 2021 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-34487527

RESUMO

OBJECTIVE: To assess the application value of mapping allele with resolved carrier status (MaReCs) technique for preimplantation genetic testing (PGT). METHODS: The characteristics of MaReCs for PGT and outcome of patients were retrospectively analyzed. RESULTS: Compared with those who could not use the technique, carriers who have used the MaReCs technique were younger, had significantly higher level of anti-Mullerian hormone, more antral follicles, occytes, mature occytes, biopsied embryos and euploid embryos, and lower risks for de novo chromosomal abnormality (P<0.05). It was necessary for couples with fewer oocytes, mature oocytes and balstocyst to preserve discarded embryos to facilitate the test. Carriers who have used the MaReCs technique had higher clinical pregnancy rate and abortion rate compared with those undergoing routine PGT, albeit no significant difference was found between the two groups (P> 0.05). Carriers undergoing MaReCs test could preferentially select embryos with normal chromosome structures for the transfer. CONCLUSION: Application of MaReCs has a prerequisite for having a minimum number of occytes and biopsied embryos and using discarded embryos sometimes. MaReCs is efficient for the detection of carrier status of embryos and attaining higher rate of pregnancy and live birth, which can significantly improve the outcome for couples carrying chromosomal translocations.


Assuntos
Diagnóstico Pré-Implantação , Translocação Genética , Alelos , Aneuploidia , Blastocisto , Feminino , Fertilização In Vitro , Testes Genéticos , Humanos , Gravidez , Estudos Retrospectivos
15.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(9): 895-899, 2021 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-34487540

RESUMO

OBJECTIVE: To explore whether it is necessary to choose NIPT-plus for the prenatal screening of pregnant women. METHODS: The results of NIPT and NIPT-plus sequencing data, fetal DNA concentration, prenatal diagnosis and pregnancy outcome of 50 pregnant women were compared. RESULTS: Compared with NIPT, NIPT-plus attained similar fetal DNA concentration and a 4.4-fold increase in sequencing data. NIPT was able to detect 4 cases of 21-trisomy, 2 cases of 18-trisomy, and 9 cases of sex chromosome aneuploidies (SCAs) signaled by NIPT-plus, but missed one 18-trisomy, and failed to detect rare chromosome aneuploidies (RCAs) and microdeletion/microduplication syndromes (MMS). The PPVs of NIPT-plus for 21-trisomy, 18-trisomy, SCAs, MMS and RCAs were 100%, 100%, 44.4%, 30.4% and 0%, respectively. And those of NIPT for 21-trisomy, 18-trisomy, and SCAs were 100%, 100%, and 44.4%, respectively. CONCLUSION: It is necessary for pregnant women to select NIPT-plus to improve the detection rate of common trisomies, SCAs and disease-specific MMS, therefore reduce the occurrene of birth defect.


Assuntos
Aneuploidia , Gestantes , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal , Trissomia , Síndrome da Trissomia do Cromossomo 13 , Síndrome da Trissomía do Cromossomo 18
17.
Molecules ; 26(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34500570

RESUMO

Thymoquinone (TQ) is the main biologically active constituent of Nigella sativa. Many studies have confirmed its anticancer actions. Herein, we investigated the different anticancer activities of, and considered resistance mechanisms to, TQ. MTT and clonogenic data showed TQ's ability to suppress breast MDA-MB-468 and T-47D proliferation at lower concentrations compared to other cancer and non-transformed cell lines tested (GI50 values ≤ 1.5 µM). Flow-cytometric analyses revealed that TQ consistently induced MDA-MB-468 and T-47D cell-cycle perturbation, specifically inducing pre-G1 populations. In comparison, less sensitive breast MCF-7 and colon HCT-116 cells exhibited only transient increases in pre-G1 events. Annexin V/PI staining confirmed apoptosis induction in MDA-MB-468 and HCT-116 cells, which was continuous in the former and transient in the latter. Experiments revealed the role of reactive oxygen species (ROS) generation and aneuploidy induction in MDA-MB-468 cells within the first 24 h of treatment. The ROS-scavenger NAD(P)H dehydrogenase (quinone 1) (NQO1; DT-diaphorase) and glutathione (GSH) were implicated in resistance to TQ. Indeed, western blot analyses showed that NQO1 is expressed in all cell lines in this study, except those most sensitive to TQ-MDA-MB-468 and T-47D. Moreover, TQ treatment increased NQO1 expression in HCT-116 in a concentration-dependent fashion. Measurement of GSH activity in MDA-MB-468 and HCT-116 cells found that GSH is similarly active in both cell lines. Furthermore, GSH depletion rendered these cells more sensitive to TQ's antiproliferative actions. Therefore, to bypass putative inactivation of the TQ semiquinone metabolite, the benzylamine analogue was designed and synthesised following modification of TQ's carbon-3 atom. However, the structural modification negatively impacted potency against MDA-MB-468 cells. In conclusion, we disclose the following: (i) The anticancer activity of TQ may be a consequence of ROS generation and aneuploidy; (ii) Early GSH depletion could substantially enhance TQ's anticancer activity; (iii) Benzylamine substitution at TQ's carbon-3 failed to enhance anticancer activity.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Aneuploidia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HCT116 , Humanos , Células MCF-7 , NAD(P)H Desidrogenase (Quinona)/metabolismo , Nigella sativa/química
18.
Nat Commun ; 12(1): 5184, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465782

RESUMO

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.


Assuntos
Aneuploidia , Mutação com Ganho de Função , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação com Perda de Função , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo
19.
J Vis Exp ; (175)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34570097

RESUMO

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.


Assuntos
Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto , Cromossomos , Feminino , Fertilização In Vitro , Testes Genéticos , Humanos , Ploidias , Gravidez
20.
J Cell Biol ; 220(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34505874

RESUMO

Fertilization often triggers the final step of haploidization of the female gamete genome. In this issue, Mori et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202012001) identify two successive actin-dependent mechanisms that delay fusion of maternal and paternal chromosomes, preventing inadvertent elimination of paternal chromosomes together with maternal ones.


Assuntos
Actinas , Cromossomos , Aneuploidia , Feminino , Humanos
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