RESUMO
STUDY QUESTION: Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child? SUMMARY ANSWER: A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed. WHAT IS KNOWN ALREADY: The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting. STUDY DESIGN, SIZE, DURATION: Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833). MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)). LIMITATIONS, REASONS FOR CAUTION: It is possible that some residual confounding remains. WIDER IMPLICATIONS OF THE FINDINGS: Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth. STUDY FUNDING/COMPETING INTEREST(S): Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Nascimento Prematuro , Blastocisto , Estudos de Coortes , Transferência Embrionária/métodos , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Estudos RetrospectivosRESUMO
STUDY QUESTION: To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER: Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence. WHAT IS KNOWN ALREADY: Several studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations. STUDY DESIGN, SIZE, DURATION: Infertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Moderate intra-class correlation coefficients (0.16-0.31) were observed for all parameters except duration of the 3-cell stage, which demonstrates that embryos from one patient elicit clustering at a patient level. No single patient- and treatment-related factor was found to systematically influence the timing from cleavage to blastocyst stage, which indicates that no individual patient-related factor can be identified that separately explains the clustering throughout the entire developmental stages. The blastocyst parameters were more affected by patient-related factors than cleavage stage parameters, as tEB occurred significantly later with older age (0.29 h/year (95% confidence interval: CI 0.03; 0.56)), while both tEB and tFB occurred significantly later with increasing dose of FSH (tEB: 0.12 h/100 IU FSH (95% CI 0.01;0.24); tFB 0.14 h/100 IU FSH (95% CI 0.03;0.27)) and with more previous attempts (tEB: 1.2 h/attempt (95% CI 0.01;2.5); tFB 1.4 h/attempt (0.10;2.7)). Fertilization method affected timing of the first division, with ICSI embryos cleaving significantly faster than IVF embryos (-3.6% (95% CI -6.4; -0.77)), whereas no difference was found in the subsequent divisions. The univariable regression analysis identified female age, cumulative FSH dose, degree of blastocyst expansion, score of the inner cell mass and timing of full blastocyst formation as predictors of live birth. The timing of full blastocyst formation (tFB) did not remain significant when adjusting for age, number of previous cycles and cumulative FSH dose, which were the parameters shown to influence tFB in the mixed regression model. LIMITATIONS, REASONS FOR CAUTION: Only good prognosis patients were enrolled, so these results may not be generalized to all infertile women. Not all patient-related factors were investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline the importance of treating embryos as dependent observations and suggest a high risk of patient-based confounding in retrospective studies. The impact of confounders and the embryo origin needs to be addressed in order to apply appropriate statistical models in observational studies. Furthermore, this observation emphasizes the need for RCTs for evaluating use of time-lapse parameters for embryo selection. STUDY FUNDING/COMPETING INTERESTS: Funding for the cohort study was provided by the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and NordicInfu Care research grant. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from MSD and Ferring. K.K. is funded by a grant from the Danish Council for Independent Research Medical Sciences. The authors declare no competing interest.
Assuntos
Blastocisto/citologia , Desenvolvimento Embrionário , Técnicas de Reprodução Assistida , Adulto , Estudos de Coortes , Feminino , Fertilização , Fertilização in vitro , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/uso terapêutico , Humanos , Modelos Lineares , Nascido Vivo , Idade Materna , Análise Multivariada , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
STUDY QUESTION: Does the metabolomic profile, obtained with nuclear magnetic resonance (NMR), of spent culture media from human embryos correlate with reproductive potential in a cohort of good prognosis patients? SUMMARY ANSWER: In a large cohort of single transferred blastocysts from a homogeneous group of good prognosis patients, we find a high degree of individual variation in the metabolome that, however, has no relation to pregnancy outcome. WHAT IS KNOWN ALREADY: Differences among various specific metabolites have been linked to reproductive potential. Although results from retrospective near infrared (NIR) spectroscopy analyses of spent culture medias from transferred embryos were promising, randomized controlled trials were unable to demonstrate that NIR analysis improved pregnancy rates. Therefore, a more detailed investigation of the relation between embryo metabolism and reproductive potential is required. NMR is a powerful technique that provides detailed structural and dynamic information. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted at the Fertility Clinic, Aarhus University Hospital between February 2011 and July 2012. Infertile patients aged <38 years without endometriosis were offered participation and their embryos were included if greater than or equal to eight oocytes were retrieved. In total, 161 infertile patients were included in the cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spent culture media was collected on Days 3 and 5 after oocyte retrieval from 148 single transferred embryos. NMR spectra were obtained from 12 µl of spent media. Data were quantitatively analysed using multivariate analysis with respect to pregnancy outcome, defined as a live fetus by ultrasound in gestational Week 8, along with patient and treatment related variables such as embryo score, age, BMI, fertilization method and cause of infertility. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 148 cycles were included in the analysis [embryo transfer cancelled (n = 12), no media collected (n = 1)]. Clinical pregnancy was confirmed in 47 patients (32%). We obtained high quality NMR spectra for 141 Day 3 and 137 Day 5 samples. Our spectra show a high degree of individual variation. Multivariate data analysis was performed on spectral data with several different pre-processing combinations, i.e. binning, alignment, normalization and scaling in the attempt to develop a valid prediction model. Different strategies of multivariate analysis showed, however, no correlation between the NMR profiles and pregnancy outcome, patient or treatment characteristics. No model could therefore be developed for prediction of pregnancy outcome. We conclude that within this group of good prognosis patients, large-scale metabolic variations between embryos detected with NMR have no apparent association with pregnancy outcome. LIMITATIONS, REASONS FOR CAUTION: Although this study is the largest we know of using NMR to investigate metabolomic profiles of single-transferred embryos, there may be differences that would be detected with a larger study. When analysing such a small sample volume, even small variations in the amount of media and dilution may introduce a large uncertainty in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our study questions the usefulness of the entire metabolome for embryo selection, which should direct the search for viability markers in the culture media towards individual components. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation. Research at the Fertility Clinic, Aarhus Universtity Hospital is supported by an unrestricted grant from MSD and Ferring. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: NCT01139268.
Assuntos
Meios de Cultura/metabolismo , Infertilidade Feminina/metabolismo , Transferência de Embrião Único , Adulto , Técnicas de Cultura Embrionária , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Metabolômica , Gravidez , Resultado da Gravidez , PrognósticoRESUMO
STUDY QUESTION: Do early time-lapse parameters predict which embryos will develop to high-quality blastocysts and does timing of development differ between embryos that implant and those that do not. SUMMARY ANSWER: Development to high-quality blastocysts could be predicted within the first 48 h of culture, whereas time-lapse parameters could not predict pregnancy. WHAT IS KNOWN ALREADY: Historical cohort studies on embryos from unselected groups of patients have suggested several putative kinetic markers of viability. Before well-designed randomized studies can be conducted, relevant selection models based on solid data must be developed. So far conclusions from the previous studies are ambiguous. STUDY DESIGN, SIZE, DURATION: A prospective cohort study conducted from February 2011 to June 2012. A total of 571 ICSI embryos from 92 patients were included in the blastocyst development analysis and 84 single embryo transfers were included in the pregnancy outcome analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos from women aged <38 years, with no endometriosis and ≥ 8 oocytes retrieved. University affiliated clinic. Embryos were cultured in a time-lapse incubator till Day 6. Logistic regression analysis was performed with variables selected based on indication. MAIN RESULTS AND THE ROLE OF CHANCE: Duration of the first cytokinesis, duration of the 3-cell stage and direct cleavage to 3-cells predicted development to high-quality blastocyst. We found no difference in timing between implanted and non-implanted embryos. LIMITATIONS, REASONS FOR CAUTION: A larger study might detect differences in timing between implanted and non-implanted embryos. The cohort consisted of good prognosis patients only and may not be representative of the entire IVF population. WIDER IMPLICATIONS OF THE FINDINGS: Our results in context with the lack of consistency in previous studies and the presumed influences of different external factors indicate that a universal algorithm for optimal timing of development might not be feasible. The apparent negative significance of division patterns that differ from the expected may imply that time-lapse will facilitate de-selection of embryos. STUDY FUNDING/COMPETING INTEREST(S): Funding for the present study was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and by an unrestricted grant from MSD and Ferring. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: The study was registered at ClinicalTrial.gov with accession number NCT01139268.
Assuntos
Blastocisto/citologia , Desenvolvimento Embrionário , Adulto , Estudos de Coortes , Transferência Embrionária , Feminino , Humanos , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Prognóstico , Imagem com Lapso de TempoRESUMO
OBJECTIVE: When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples. METHODS: Using methylation-specific multiplex ligation-dependent probe amplification, we measured methylation levels at different imprinted sites. RESULTS: We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed. CONCLUSIONS: We present methylation-specific multiplex ligation-dependent probe amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 h; it is inexpensive, simple and easy to use, and parental samples are not needed.
Assuntos
Metilação de DNA/genética , Mola Hidatiforme/genética , Reação em Cadeia da Polimerase Multiplex , Pais , Diagnóstico Pré-Natal/métodos , Triploidia , Feminino , Humanos , Mola Hidatiforme/diagnóstico , Cariotipagem , Masculino , Gravidez , Complicações na Gravidez/genética , Reprodutibilidade dos Testes , Fatores de Risco , Neoplasias Uterinas/genéticaRESUMO
BACKGROUND: This report presents a case where electroejaculation (EEJ) was used for semen cryopreservation (SCP), prior to gonadotoxic anti-cancer treatment in a 14-year old boy diagnosed with Hodgkins disease. METHOD: Two sessions of EEJ were performed with an interval of 48 hours. RESULTS: No complications were seen and the procedures resulted in nine frozen straws of motile spermatozoa. CONCLUSION: EEJ is a safe and feasible procedure for SCP in an adolescent cancer patient who is unable to masturbate or use penile vibratory stimulation (PVS).
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Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Criopreservação , Estimulação Elétrica/métodos , Preservação da Fertilidade/métodos , Doença de Hodgkin/tratamento farmacológico , Infertilidade Masculina/induzido quimicamente , Preservação do Sêmen/métodos , Manejo de Espécimes/métodos , Adolescente , Anestesia Geral , Ciclofosfamida/administração & dosagem , Dacarbazina/administração & dosagem , Doxorrubicina/administração & dosagem , Etoposídeo/administração & dosagem , Humanos , Masculino , Vincristina/administração & dosagemRESUMO
In this report we describe a partial triplication (7q) and other structural aberrations found in a child with acute lymphoblastic leukemia (ALL), and we demonstrate the importance of PRimed IN Situ labeling (PRINS) and chromosome painting as a support to banding analysis for the clarification of complex structural chromosome rearrangements. Initially, the der(7) was interpreted as der(7)t(1;7;7). However, PRINS and chromosomes painting showed that der(7) consisted entirely of chromosome 7 material. Further, a derivative chromosome interpreted by banding analysis as a der(17)t(?1;17) was shown to be der(17)t(13;17) by the newly developed PRINS painting technique.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 7 , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Criança , Bandeamento Cromossômico , Humanos , Hibridização In Situ , MasculinoRESUMO
We used the molecular cytogenetic in situ techniques chromosome painting and PRimed IN Situ labeling (PRINS) to elaborate the cytogenetic observations in two cases of the rare aberration der(16)t(1;16), which occurs in a wide variation of hematologic and nonhematologic malignancies [1-3]. Review of the literature showed that, in contrast to the chromosome 1 breakpoint, the breakpoint on chromosome 16 is associated with diagnosis as well as patient age.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 1 , Técnicas Genéticas , Hibridização in Situ Fluorescente , Translocação Genética , Adulto , Aberrações Cromossômicas , Feminino , Humanos , CariotipagemRESUMO
PRimed IN Situ labeling (PRINS) is a fast and sensitive alternative to fluorescence in situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.
Assuntos
Ácidos Nucleicos/química , Cromossomos Humanos , Primers do DNA , Sondas de DNA , DNA Satélite , Feminino , Técnicas Genéticas , Humanos , Hibridização in Situ FluorescenteRESUMO
BACKGROUND AND DESIGN: Enumeration of malignant cells in Sézary syndrome often relies on the identification of the Sézary cell nucleus. This morphologic method is, however, nonspecific and unreliable in the enumeration of the proportion of malignant lymphocytes in peripheral blood of patients with Sézary syndrome. Malignant lymphocytes of patients with mycosis fungoides and Sézary syndrome are often characterized by multiple chromosome aberrations. Herein, we demonstrate that fluorescent in situ hybridization can visualize and accurately enumerate malignant aneuploid mononuclear cells in a patient with Sézary syndrome. RESULTS: Fluorescent in situ hybridization demonstrated that 90% of the mononuclear cells in the patient with Sézary syndrome showed numerical aberrations for both chromosome 7 and X, a figure confirmed by flow cytometry. CONCLUSION: Fluorescent in situ hybridization may be a valuable tool to visualize and enumerate aneuploid tumor cells in patients with cutaneous T-cell lymphoma.
Assuntos
Hibridização in Situ Fluorescente , Síndrome de Sézary/genética , Neoplasias Cutâneas/genética , Linfócitos T/patologia , Antígenos CD8 , Aberrações Cromossômicas , Cromossomos Humanos Par 17 , DNA de Neoplasias/análise , Citometria de Fluxo , Humanos , Cariotipagem , Metáfase , Ploidias , Síndrome de Sézary/patologia , Neoplasias Cutâneas/patologia , Linfócitos T/ultraestrutura , Células Tumorais CultivadasRESUMO
To illustrate the advantage of fluorescence in situ hybridization (FISH) in clinical genetics, we have chosen six clinical cases from our routine laboratory where we have used molecular cytogenic techniques to optimise the cytogenetic analysis. Using centromere-specific DNA probes and whole chromosome DNA libraries either obtained from somatic cell hybrids or generated from flow-sorted chromosomes, we have been able to identify small marker chromosomes, chromosomal duplications and inversions, to determine the ploidy in interphase nuclei, and to characterize subtle chromosomal translocations. We conclude that molecular cytogenetics is a valuable technique that should be used as a supplement to conventional cytogenetics to reduce the number of unresolved karyotypes in clinical genetics.
Assuntos
Aberrações Cromossômicas/fisiologia , Hibridização in Situ Fluorescente , Adolescente , Adulto , Aneuploidia , Núcleo Celular/fisiologia , Pré-Escolar , Inversão Cromossômica , Feminino , Marcadores Genéticos , Humanos , Interfase/genética , Cariotipagem , Masculino , Translocação GenéticaRESUMO
This article describes a case of 46,XX male, the most frequent form of sex reversal syndromes in humans. A method of identifying Y chromosome material in these and other patients with structural chromosomal abnormalities involving chromosome Y is given. Chromosomes from a phenotypically normal male child without any congenital malformation, where prenatal diagnosis revealed the female karyotype 46,XX, were analysed using fluorescence in situ hybridisation (FISH). The analysis revealed an X chromosome, containing Y chromosome sequences on the tip of the short arm. The sequences are not normally visible in conventional cytogenetic analyses of XX males. The breakpoint on Y was determined to be in the region of Yp11.2, which is proximal for the putative sex determining gene on Y. The results are consistent with theories of abnormal crossing-over during the paternal meiotic cell division where meiotic recombination can give rise to structural abnormalities, which can then cause sex reversal syndromes. Prenatal diagnosis of structural sex chromosome abnormalities has become available using the FISH method.
Assuntos
Hibridização in Situ Fluorescente , Aberrações dos Cromossomos Sexuais/genética , Cromossomo X , Cromossomo Y , Adulto , Sondas de DNA , Feminino , Humanos , Masculino , Fenótipo , Diagnóstico Pré-Natal/métodos , Aberrações dos Cromossomos Sexuais/diagnóstico , Troca de Cromátide IrmãRESUMO
INTRODUCTION: Preimplantation genetic diagnosis (PGD) is a possible alternative to prenatal diagnosis, whereby families with serious inherited diseases can avoid having children with the disease. The genetic diagnosis is performed on embryos before implantation and therefore implies IVF. Hence, PGD offers the possibility of transferring embryos without disease, thereby avoiding termination of pregnancy owing to an affected fetus. MATERIAL AND METHODS: Activities at the Centre for Preimplantation Genetic Diagnosis at Aarhus University Hospital since its opening in February 1999 are described. The fluorescent in situ hybridisation (FISH) technique was used for sex selection (hemophilia A and Duchenne's muscular dystrophy) and translocations. The polymerase chain reaction (PCR) was used for cystic fibrosis. RESULTS: Of 20 PGD cycles started, 15 were successful in terms of transference of healthy or carrier embryos. A positive pregnancy test was found after six of 15 embryo transfers (40%) with two subsequent clinical pregnancies. CONCLUSIONS: The present pregnancy rates with PGD are comparable to those following IVF; the clinical pregnancy rate may seem low, but the cycle numbers are small. Preimplantation genetic diagnosis seems to be a realistic alternative for selected genetic diseases, in cases where the couple find abortion unacceptable.
Assuntos
Predisposição Genética para Doença , Diagnóstico Pré-Implantação , Adulto , Blastômeros/ultraestrutura , Aberrações Cromossômicas/diagnóstico , Aberrações Cromossômicas/genética , Transtornos Cromossômicos , Dinamarca , Feminino , Fertilização in vitro , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Implantação/métodosAssuntos
Sondas de DNA , Hibridização in Situ Fluorescente/métodos , Sequência de Bases , Cromossomos Humanos , Primers do DNA/genética , Sondas de DNA/genética , Citometria de Fluxo , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Coloração e Rotulagem/métodosAssuntos
Primers do DNA , Hibridização In Situ/métodos , RNA/genética , Animais , Sequência de Bases , Biotina , Linhagem Celular , Primers do DNA/genética , Fixadores , Citometria de Fluxo , Cadeias kappa de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , RNA/isolamento & purificação , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA , Coloração e Rotulagem/métodosRESUMO
Digestion of porcine DNA with the restriction enzymes SnoI and BamHI and subsequent gel electrophoresis revealed the presence of a repeat fragment of approximately 340 bp in length. When this fragment was cloned and used to probe a Southern blot containing pig DNA that had been digested with various enzymes, a ladder pattern of bands based on a unit of 340 bp was observed. Three individual BamHI repeat units were sequenced; each monomer unit was approximately 90% identical in sequence to the other two monomers. Double digestion did not alter the ladder pattern, indicating a differential distribution of restriction enzyme sites within the repeat family. We demonstrate by Southern blotting of DNAs from a panel of pig x rodent hybrid cell lines that the differential restriction site distribution has a chromosomal basis. We show that this repeat family hybridizes to the centromeres of all pig chromosomes other than the Y chromosome. However, when stringency was raised, hybridization persisted only to certain metacentric chromosomes.
Assuntos
Centrômero , Cromossomos , Sequências Repetitivas de Ácido Nucleico , Animais , Southern Blotting , DNA/genética , DNA/isolamento & purificação , Enzimas de Restrição do DNA , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , SuínosRESUMO
PURPOSE: The aim of this study was to develop a multiplex PCR protocol, which could be suitable for screening of microdeletions in the three azoospermia factor (AZF) regions on the Y chromosome. METHODS: In the screening protocol, 36 known sequence tagged site (STS) primer pairs were first tested in single PCR reactions and thereafter combined in multiplex PCR to test for specificity and sensitivity in order to develop a stable and reliable multiplex PCR assay to detect Y microdeletions. RESULTS: Of the 36 primers tested, 11 turned out not to be specific or produced PCR products that were too weak, and they were therefore not used in the multiplex PCR. The remaining 25 STSs were selected on the basis of their ability to be reproducibly amplified with each other using identical amplification conditions. Five multiplex sets, each consisting of five primer pairs, were established in the multiplex PCR setup. CONCLUSION: The multiplex PCR protocol presented in this study is an easy and reliable method for detection of Y chromosome microdeletions and could be used for screening of infertile men to allow genetic counseling about the risk of transmitting infertility from father to son.