Isabela Nelly Machado

Fetal rhd genotyping from maternal plasma in a population with a highly diverse ethnic background

OBJECTIVE To establish the performance of conventional PCR as a noninvasive method for fetal genotyping, by free fetal DNA analysis of distinct RHD regions from maternal plasma, in a population of a diverse ethnic origin. METHODS We conducted a validity of the diagnostic test by analyzing 81 plasma samples from RhD-negative Brazilian pregnant women, from 4 to 41 gestational weeks. We tested for exon 10 and intron 4 gene regions by allele specific-PCR. Fetal RHD genotyping by PCR on maternal plasma was compared to serologic RhD typing in the neonatal period. RESULTS Samples were obtained as follows: 15 in the 1st, 37 in the 2nd and 29 in the 3rd trimester. General accuracy was 97.3%, sensitivity of 98.3% and specificity of 93.8%. CONCLUSIONS Conventional PCR is an accurate method for fetal RHD genotyping on maternal plasma, even in a population of mixed ethnic origin.

Congenital hydrocephalus: gestational and neonatal outcomes

PurposeTo evaluate gestational and neonatal outcomes in pregnancies complicated by fetal hydrocephalus.MethodsRetrospective analysis of 287 cases of fetal hydrocephalus followed at the Fetal Medicine Unit of the University of Campinas in the period of 1996 to 2006.ResultsMean maternal age was 25 years, mean gestational age at diagnosis was 27 weeks. There were 50 cases of isolated ventriculomegaly, 95 cases of Chiari II malformation and 142 cases of ventriculomegaly associated with other malformations. Preterm delivery and vaginal delivery were more frequent in the group of ventriculomegaly associated with other malformations. Cardiac, skeletal and renal malformations were the most common associated malformations. Cesarean section was common (95%) in the Chiari II group. Fetal and neonatal death occurred more frequently (29 and 68%, respectively) in the group of ventriculomegaly associated with other malformations. Chromosomal anomalies were present in 15% of 165 investigated cases.ConclusionsFetal and neonatal prognosis and outcome are associated with the presence of associated anomalies and aneuploidy.

Copy number imbalances detected with a BAC-based array comparative genomic hybridization platform in congenital diaphragmatic hernia fetuses.

Congenital diaphragmatic hernia (CDH) is a phenotypically and genetically heterogeneous disorder, with a complex inheritance pattern. Structural abnormalities of almost all chromosomes have been described in association with CDH. We made a molecular analysis through array comparative genomic hybridization (array CGH) of a group of fetuses with prenatal ultrasound diagnosis of CDH and normal G-banded karyotypes. A whole genome BAC-array CGH, composed of approximately 5000 BAC clones, was carried out on blood samples from fetuses with prenatal ultrasound diagnosis of CDH and a normal karyotype (500-band level). All potential cytogenetic alterations detected on the arrays were reported. The array CGH analysis showed copy number gains and losses in 10 of 12 cases. Eighty-five clones showed genomic imbalances, and 29 clones displayed described copy number variations. We identified a recurrent gain in 17q12 in two of 12 cases, which has not been previously described. Our results may contribute to determining the effectiveness and applicability of array CGH for prenatal diagnosis purposes, and also to elucidate the submicroscopic genomic instability of CDH fetuses.

Prenatal Genomic Profiling of Abdominal Wall Defects through Comparative Genomic Hybridization: Perspectives for a New Diagnostic Tool

Objectives: To describe the molecular analysis through comparative genomic hybridization (CGH) of fetuses with gastroschisis, and to observe if this technique could improve the resolution of the conventional cytogenetic techniques. Methods: Amniotic analysis of fetuses with gastroschisis, using both conventional (G-banding) and molecular (CGH) cytogenetics assays. Results: All of the seven fetuses studied displayed a normal G-band karyotype. Six fetuses displayed a normal disomic profile through CGH and one sample has displayed ish cgh enh 3q26→qter result (ICSN). The fetus with this imbalance of chromosome 3 was re-classified as a ruptured omphalocele, instead of gastroschisis, after birth. Conclusions: The molecular investigation through CGH technique can improve the resolution of the conventional karyotye analysis in cases of abdominal wall defects.

Evolução de 58 fetos com meningomielocele e o potencial de reparo intra-útero

RESUMO - Introducao:O diagnostico pre natal da meningomielocele (MM) permite melhor planejamento de sua abordagem e,mais recentemente ,um possivel reparo intra-utero. Objetivo:Descricao da evolucao perinatal de fetos com MM,acompanhadosem um centro de referencia em Medicina Fetal,identificando os possiveis fetos candidatos a cirurgia intra-uterina. Metodo:Analiseretrospectiva descritiva de 58 casos de MM fetal, atendidos no CAISM-UNICAMP, de janeiro de 1997 a dezembro de 2001,identificando-se os casos cuja indicacao de cirurgia fetal seria possivel. Resultados: Media da idade gestacional ao diagnosticode 29 semanas (17-39);nivel da lesao acima da regiao sacral em 85%;associacao com hidrocefalia em 86%;taxa de complicacoescirurgicas de 39%.Na evolucao,98% apresentaram bexiga neurogenica e 60% deficiencia neuro-mental.O potencial reparo intra-utero foi de 42%. Conclusao:MM esta associada a graves e frequentes sequelas.Quase um terco dos nossos casos poderiam tertido a cirurgia fetal como opcao terapeutica.PALAVRAS-CHAVE: diagnostico pre-natal, meningomielocele fetal, hidrocefalia, cirurgia fetal.Fetal myelomeningocele and the potential in-utero repair: follow-up of 58 fetusesABSTRACT - Introduction:Prenatal diagnosis of myelomeningocele (MM) allows planning its management and,recently,a possiblein utero repair. Objective: To describe the perinatal outcome of fetuses with MM, in a Fetal Medicine Unit, identifying possiblecandidates for the in utero surgical repair. Methodo:Retrospective and descriptive study of 58 cases of prenatally diagnosed MM,at CAISM-UNICAMP, from January 1997 to December 2001, identifying possible fetal candidates for in utero repair. Results: thediagnosis mean gestacional age was 29 weeks (17-39);level of lesions was above sacral region in 85%,association with hydrocepha lyin 86%. Surgical complications were present in 39% of the neonates. During follow-up, 98% presented neurogenic bladder and60% neurological/mental handicap. Twenty eight fetuses (42%) could have indication of in utero repair.Conclusion:MM isassociated with severe and frequent poor results.Almost one third of our cases could had fetal repair as a treatment choice.KEY WORDS: prenatal diagnosis, myelomeningocele, hydrocephaly, fetal surgery.

Anencephaly: do the pregnancy and maternal characteristics impact the pregnancy outcome?

Objective. To describe the characteristics of obstetric and perinatal outcome of a group of pregnancies complicated by an anencephalic fetus. Methods. Observational study including anencephalic fetuses, divided into groups according to the evolution of pregnancy: elective termination of pregnancy ETP; stillbirths (SBs); live births (LBs), and loss of follow-up. After a univariate description of the sample, some variables were compared using statistical tests. Results. 180 anencephalic fetuses were included. The mean maternal age was 25.3 years. In 71 fetuses (39%) were found additional anomalies. Comparing the groups, no statistical differences in maternal age (P = 0.5315), parity (P = 0.6070), number of previous abortion (P = 0.7464), fetal sex (P = 0.0502) and additional anomalies (P = 0.186) were found. Among those fetuses whose parents opted for continuation of pregnancy (n = 53), 20 spontaneous intrauterine deaths occurred (38%) and 33 were live births (62%). The average postnatal survival time was 51 minutes. There was no association between survival time and gestational age (P = 0.6125) or the presence of additional malformations (P = 0.1948). Conclusion. Results presented here could contribute to a better understanding of the natural history of this malformation, allowing obstetricians a more detailed discussion with the families.

Does the time between collecting and processing umbilical cord blood samples affect the quality of the sample

OBJECTIVE To assess the association between the time from umbilical cord blood collection until processing and the quality of the sample. METHODS Umbilical cord blood samples collected during the third stage of labor were placed in temperature-controlled boxes for the transport of biological material and sent to an umbilical cord blood bank, where the number of nucleated cells, viable cells and CD34+ cells were counted, and samples were additionally tested for contamination at the following time intervals: up to 24 hours, up to 48 hours and up to 72 hours following sampling. Data were analyzed using the multivariate analysis of variance (MANOVA) and compared using McNemars χ2 test. Significance was defined at p < 0.05. RESULTS Means and medians of the number of nucleated cells, viable cells and CD34+ cells decreased significantly (p < 0.0001) as a function of the increased time between sampling and analysis, the difference between 24 and 48 hours being less than the difference between 24 and 72 hours. A linear correlation was found between the mean number of viable cells and CD34+ cells at the three moments of analysis. Contamination testing was negative in all samples. CONCLUSION The increase in time interval from sampling until analysis negatively affected the number of nucleated cells, viable cells and CD34+ cells but was not associated with specimen contamination. A linear correlation was found between decrease in the number of viable cells and CD34+ cells.

Genomic imbalances detected through array CGH in fetuses with holoprosencephaly

OBJECTIVE Holoprosencephaly (HPE) is heterogeneous in pathogenesis, integrating genetic susceptibility with the influence of environmental factors. Submicroscopic aberrations may contribute to the etiology of HPE. Our aim was to report the molecular analysis of 4 fetuses with HPE and normal metaphase karyotype. METHOD A whole genome BAC-array based Comparative Genomic Hybridization (array CGH) was carried out in fetal blood samples. All potential cytogenetic alterations detected on the arrays were matched against the known copy number variations databases. RESULTS The array CGH analysis showed copy number gains and losses in all cases. We found a recurrent deletion in 15q14 (clone RP11-23J11) and in 15q22 (clone RP11-537k8) in 2 out 4 cases analyzed. We also observed submicroscopic gain in 6p21 in 3 out of 4 fetuses in nearby clones. All these regions were tested in known databases and no copy number variations have been described for them. CONCLUSION This is the first report of molecular characterization through a whole genome microarray CGH of fetuses with HPE. Our results may contribute to verify the effectiveness and applicability of the molecular technique of array CGH for prenatal diagnosis purposes, and contributing to the knowledge of the submicroscopic genomic instability characterization of HPE fetuses.

O intervalo de tempo entre coleta e processamento do sangue de cordão umbilical influencia na qualidade da amostra

Objective: To assess the association between the time from umbilical cord blood collection until processing and the quality of the sample. Methods: Umbilical cord blood samples collected during the third stage of labor were placed in temperature-controlled boxes for the transport of biological material and sent to an umbilical cord blood bank, where the number of nucleated cells, viable cells and CD34+ cells were counted, and samples were additionally tested for contamination at the following time intervals: up to 24 hours, up to 48 hours and up to 72 hours following sampling. Data were analyzed using the multivariate analysis of variance (MANOVA) and compared using McNemars X2 test. Significance was defined at p Objetivo: Avaliar a associacao do intervalo de tempo entre coleta e processamento do sangue de cordao umbilical e a qualidade da amostra. Metodos: As amostras de sangue de cordao umbilical, colhidas no terceiro periodo do parto, foram acondicionadas em caixas homologadas para transporte de material biologico, com monitoracao da temperatura, e enviadas a um banco de sangue de cordao umbilical, onde foram submetidas a contagem do numero de celulas nucleadas, do numero de celulas viaveis, do numero de celulas CD 34+ e pesquisa de contaminacao, nos intervalos de tempo de ate 24, ate 48 e ate 72 horas. Os dados foram analisados pelo teste de variância para medidas repetidas MANOVA e comparados por meio do teste do X2 de Mc Nemar, considerando-se nivel de significância de 5%. Resultados: As medias e as medianas do numero de celulas nucleadas, numero de celulas viaveis e numero de celulas CD34+ tiveram quedas significativas (p

Cytogenetic abnormalities in couples with a history of primary and secondary recurrent miscarriage: a Brazilian Multicentric Study

Abstract Objective: To evaluate the difference between chromosomal abnormalities between the gender of couples affected by Recurrent miscarriage (RM) and if there is an association between previous obstetric history and chromosomal abnormalities of the parents. Methods: Multicenter, retrospective, observational study from seven different RM clinics between 2006 and 2016. We enrolled 707 couples (1014 participants) with a history of RM. We compared the frequency of chromosomal abnormalities between groups of couples with primary and secondary RM and separated between women and their partners. Furthermore, we compared the prevalence of chromosomal abnormalities between groups based on the number of previous spontaneous abortions. Results: The overall prevalence of all cytogenetic abnormalities was 5.59% (n = 1414, women and their partners). Excluding cases of polymorphism and inversion of chromosome 9, which are considered variants of normality, the prevalence in all individuals was 2.26% (n = 32/1414). The comparative analysis of cases of chromosomal abnormalities among couples with primary and secondary RM based on the number of previous miscarriages (PM) revealed a similar frequency between groups. The statistical analysis of the total cases (primary PM + secondary PM) in these three groups were as follows: (a) couple, 2 pm versus 3 pm vs. ≥4 PM, p = .514; (b) women, 2 pm versus 3 pm vs. ≥4 PM, p = .347; and (3) partner, 2 pm versus 3 pm vs. ≥4 PM, p = .959. Chromosomal abnormalities were significantly more prevalent among women than among their partners (6.9 versus 4.2%; p = .027). Moreover, the distribution of leading chromosomal abnormalities among women was different compared with their partners. Among women, we observed these abnormalities in the following frequency order: mosaicism (38.8%), polymorphism (32.6%), translocation (16.3%), and inversion (12.3%). Among their partners, these abnormalities were polymorphism (73.3%), inversion (13.3%), mosaicism (6.7%), and translocation (6.7%). Conclusion: The number of PM and the history of full-term pregnancy does not correlate with an increase or decrease in the prevalence of cytogenetic abnormalities in couples with RM.