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BACKGROUND: mRNA vaccination is an effective, safe, and widespread strategy for protecting pregnant women against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, information on factors such as perinatal outcomes, safety, and coverage of mRNA vaccinations among pregnant women is limited in Japan. Therefore, this study aimed to investigate the perinatal outcomes, coverage, adverse effects, and short-term safety of mRNA vaccination as well as vaccine hesitancy among pregnant women. METHODS: We conducted a multicenter online survey of postpartum women who delivered their offspring at 15 institutions around Tokyo from October 2021 to March 2022. Postpartum women were divided into vaccinated and unvaccinated groups. Perinatal outcomes, COVID-19 prevalence, and disease severity were compared between the two groups. Adverse reactions in the vaccinated group and the reasons for being unvaccinated were also investigated retrospectively. RESULTS: A total of 1,051 eligible postpartum women were included. Of these, 834 (79.4%) had received an mRNA vaccine, while 217 (20.6%) had not, mainly due to concerns about the effect of vaccination on the fetus. Vaccination did not increase the incidence of adverse perinatal outcomes, including fetal morphological abnormalities. The vaccinated group demonstrated low COVID-19 morbidity and severity. In the vaccinated group, the preterm birth rate, cesarean section rate, and COVID-19 incidence were 7.2%, 33.2%, and 3.3%, respectively, compared with the 13.7%, 42.2%, and 7.8% in the unvaccinated group, respectively. Almost no serious adverse reactions were associated with vaccination. CONCLUSIONS: mRNA vaccines did not demonstrate any adverse effects pertaining to short-term perinatal outcomes and might have prevented SARS-CoV-2 infection or reduced COVID-19 severity. Concerns regarding the safety of the vaccine in relation to the fetus and the mother were the main reasons that prevented pregnant women from being vaccinated. To resolve concerns, it is necessary to conduct further research to confirm not only the short-term safety but also the long-term safety of mRNA vaccines.
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COVID-19 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Nascimento Prematuro , Recém-Nascido , Gravidez , Feminino , Humanos , Japão/epidemiologia , Gestantes , Cesárea , Estudos Retrospectivos , COVID-19/epidemiologia , COVID-19/prevenção & controle , SARS-CoV-2 , Nascimento Prematuro/epidemiologia , Vacinação/efeitos adversos , Inquéritos e QuestionáriosRESUMO
The incidence of chromosomal abnormalities in twin pregnancies is not well-studied. In this retrospective study, we investigated the frequency of chromosomal abnormalities in twin pregnancies and compared the incidence of chromosomal abnormalities in dichorionic diamniotic (DD) and monochorionic diamniotic (MD) twins. We used data from 57 clinical facilities across Japan. Twin pregnancies of more than 12 weeks of gestation managed between January 2016 and December 2018 were included in the study. A total of 2899 and 1908 cases of DD and MD twins, respectively, were reported, and the incidence of chromosomal abnormalities in one or both fetuses was 0.9% (25/2899) and 0.2% (4/1908) in each group (p = 0.004). In this study, the most common chromosomal abnormality was trisomy 21 (51.7% [15/29]), followed by trisomy 18 (13.8% [4/29]) and trisomy 13 (6.9% [2/29]). The incidence of trisomy 21 in MD twins was lower than that in DD twins (0.05% vs. 0.5%, p = 0.007). Trisomy 21 was less common in MD twins, even when compared with the expected incidence in singletons (0.05% vs. 0.3%, RR 0.15 [95% CI 0.04-0.68]). The risk of chromosomal abnormality decreases in twin pregnancies, especially in MD twins.
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Transtornos Cromossômicos , Síndrome de Down , Aneuploidia , Aberrações Cromossômicas , Transtornos Cromossômicos/epidemiologia , Transtornos Cromossômicos/genética , Síndrome de Down/epidemiologia , Síndrome de Down/genética , Feminino , Humanos , Gravidez , Gravidez de Gêmeos , Prevalência , Estudos Retrospectivos , Trissomia/genéticaRESUMO
AIM: We aimed to evaluate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of noninvasive prenatal testing (NIPT) in high-risk pregnant women. METHODS: Pregnant women who underwent GeneTech NIPT, the most commonly used NIPT in Japan, between January 2015 and March 2019, at Japan NIPT Consortium medical sites were recruited for this study. The exclusion criteria were as follows: pregnant women with missing survey items, multiple pregnancy/vanishing twins, chromosomal abnormalities in the fetus other than the NIPT target disease, and nonreportable NIPT results. Sensitivity and specificity were calculated from the obtained data, and maternal age-specific PPV and NPV were estimated. RESULTS: Of the 45 504 cases, 44 263 cases fulfilling the study criteria were included. The mean maternal age and gestational weeks at the time of procedure were 38.5 years and 13.1 weeks, respectively. Sensitivities were 99.78% (95% confidence interval [95% CI]: 98.78-99.96), 99.12% (95% CI: 96.83-99.76), and 100% (95% CI: 88.30-100) for trisomies 21, 18, and 13, respectively. Specificities were more than 99.9% for trisomies 21, 18, and 13, respectively. Maternal age-specific PPVs were more than 93%, 77%, and 43% at the age of 35 years for trisomies 21, 18, and 13, respectively. CONCLUSION: The GeneTech NIPT data showed high sensitivity and specificity in the detection of fetal trisomies 21, 18, and 13 in high-risk pregnant women, and maternal age-specific PPVs were obtained. These results could provide more accurate and improved information regarding NIPT for genetic counseling in Japan.
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Síndrome de Down , Teste Pré-Natal não Invasivo , Adulto , Feminino , Humanos , Japão , Laboratórios , Gravidez , Diagnóstico Pré-Natal , TrissomiaRESUMO
PURPOSE: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. METHODS: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing. RESULTS: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. CONCLUSIONS: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.
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OBJECTIVE: To evaluate the reasons for nonreportable cell-free DNA (cfDNA) results in noninvasive prenatal testing (NIPT), we retrospectively studied maternal characteristics and other details associated with the results. METHODS: A multicenter retrospective cohort study in pregnant women undergoing NIPT by massively parallel sequencing (MPS) with failed cfDNA tests was performed between April 2013 and March 2017. The women's data and MPS results were analyzed in terms of maternal characteristics, test performance, fetal fraction (FF), z scores, anticoagulation therapy, and other details of the nonreportable cases. RESULTS: Overall, 110 (0.32%) of 34 626 pregnant women had nonreportable cfDNA test results after an initial blood sampling; 22 (20.0%) cases had a low FF (<4%), and 18 (16.4%) cases including those with a maternal malignancy, were found to have altered genomic profile. Approximately half of the cases with nonreportable results had borderline z score. Among the women with nonreportable results because of altered genomic profile, the success rate of retesting using a second blood sampling was relatively low (25.0%-33.3%). Thirteen (11.8%) of the women with nonreportable results had required hypodermic heparin injection. CONCLUSIONS: The classification of nonreportable results using cfDNA analysis is important to provide women with precise information and to reduce anxiety during pregnancy.
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Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Natal/métodos , Projetos de Pesquisa , Trissomia/diagnóstico , Adulto , Reações Falso-Negativas , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Valor Preditivo dos Testes , Gravidez , Primeiro Trimestre da Gravidez/sangue , Primeiro Trimestre da Gravidez/genética , Segundo Trimestre da Gravidez/sangue , Segundo Trimestre da Gravidez/genética , Reprodutibilidade dos Testes , Projetos de Pesquisa/normas , Projetos de Pesquisa/estatística & dados numéricos , Estudos Retrospectivos , Fatores de Risco , Trissomia/genéticaRESUMO
AIM: The purpose of this study was to report the 3-year experience of a nationwide demonstration project to introduce non-invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan. METHODS: Tests were conducted to detect aneuploidy in high-risk pregnant women, and adequate genetic counseling was provided. The clinical data, test results, and pregnancy outcomes were recorded. We discuss the problems of NIPT on the basis of published reports and meta-analyses. RESULTS: From April 2013 to March 2016, 30 613 tests were conducted at 55 medical sites participating in a multicenter clinical study. Among the 30 613 women tested, 554 were positive (1.81%) and 30 021 were negative (98.1%) for aneuploidy. Of the 289, 128, and 44 women who tested positive for trisomies 21, 18, and 13, respectively, and underwent definitive testing, 279 (96.5%), 106 (82.8%), and 28 (63.6%) were determined to have a true-positive result. For the 13 481 women with negative result and whose progress could be traced, two had a false-negative result (0.02%). The tests were performed on the condition that a standard level of genetic counseling be provided at hospitals. CONCLUSION: Here, we report on the 3-year nationwide experience with NIPT in Japan. It is important to establish a genetic counseling system to enable women to make informed decisions regarding prenatal testing. Moreover, a welfare system is warranted to support women who decide to give birth to and raise children with chromosomal diseases.
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Aneuploidia , Testes para Triagem do Soro Materno/tendências , Feminino , Aconselhamento Genético , Humanos , Japão , Testes para Triagem do Soro Materno/ética , Testes para Triagem do Soro Materno/métodos , GravidezRESUMO
The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.
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DNA/genética , Marcadores Genéticos , Diagnóstico Pré-Natal , Trissomia/genética , Adulto , Peso Corporal , DNA/sangue , Feminino , Testes Genéticos/métodos , Idade Gestacional , Humanos , Gravidez , Diagnóstico Pré-Natal/métodos , Reprodutibilidade dos TestesRESUMO
The purpose of this study is to summarize the results from a survey on awareness of genetic counseling for pregnant women who wish to receive non-invasive prenatal testing (NIPT) in Japan. As a component of a clinical study by the Japan NIPT Consortium, genetic counseling was conducted for women who wished to receive NIPT, and a questionnaire concerning both NIPT and genetic counseling was given twice: once after pre-test counseling and again when test results were reported. The responses of 7292 women were analyzed. They expressed high satisfaction with the genetic counseling system of the NIPT Consortium (94%). The number of respondents who indicated that genetic counseling is necessary for NIPT increased over time. Furthermore, they highly valued genetic counseling provided by skilled clinicians, such as clinical geneticists or genetic counselors. The vast majority (90%) responded that there was sufficient opportunity to consider the test ahead of time. Meanwhile, women who received positive test results had a poor opinion and expressed a low-degree satisfaction. We confirmed that the pre-test genetic counseling that we conducted creates an opportunity for pregnant women to sufficiently consider prenatal testing, promotes its understanding and has possibilities to effectively facilitate informed decision making after adequate consideration. A more careful and thorough approach is considered to be necessary for women who received positive test results.
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Aconselhamento Genético , Conhecimentos, Atitudes e Prática em Saúde , Diagnóstico Pré-Natal , Inquéritos e Questionários , Adulto , Conscientização , Compreensão , Feminino , Humanos , Japão , Pessoa de Meia-Idade , Satisfação do Paciente , Gravidez , Diagnóstico Pré-Natal/métodos , Adulto JovemRESUMO
AIM: The aim of this study was to investigate the practicability and efficiency of lectin-based isolation of fetal erythroblasts for clinical use in non-invasive prenatal testing. METHODS: Peripheral blood samples were collected from 39 pregnant women. Leukocytes were removed with an anti-CD45 antibody after density gradient centrifugation. After blood cells were attached to slides by binding to a galactose-specific lectin and galactose-bound vinyl polymer, the slides were stained with May-Grünwald-Giemsa stain and cells were classified by automated image analysis based on their size and the nuclear area/cytoplasmic area ratio. In 14 samples from the women with male fetuses, fetal origin of the isolated erythroblasts was confirmed by detecting the Y chromosome using fluorescence in situ hybridization. In eight samples, single erythroblasts were collected by the laser capture microdissection technique for amplification of the sex-determining region Y gene to confirm fetal origin. RESULTS: Panning with an anti-CD45 antibody achieved stable removal of leukocytes without aggregation. In all samples, erythroblasts were successfully identified by automated image analysis (18-6000/10 mL of blood). The number of slides required to examine 10 mL of blood ranged from one to six, which was reasonable for clinical use. The Y chromosome was detected in 7.5-43.6% of erythroblasts by fluorescence in situ hybridization, and the sex-determining region Y gene was amplified in seven of eight samples. CONCLUSION: The combination of lectin-based erythroblast isolation and automated image analysis is a practical and efficient method for isolating fetal erythroblasts as a source of fetal genomes.
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Separação Celular/métodos , Eritroblastos , Sangue Fetal , Testes Genéticos/métodos , Lectinas/química , Testes para Triagem do Soro Materno/métodos , Anticorpos , Eritroblastos/química , Eritroblastos/citologia , Eritroblastos/imunologia , Feminino , Sangue Fetal/química , Sangue Fetal/citologia , Sangue Fetal/imunologia , Galactose/química , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Antígenos Comuns de Leucócito/imunologia , Masculino , GravidezRESUMO
For a parturient with breech presentation, an external cephalic version is sometimes done to enable vaginal delivery. Usually external cephalic version has been done without anesthesiologist's management. However there have been several reports indicating a benefit of anesthesia for external cephalic version. We report successful case of external cephalic version under combined spinal-epidural anesthesia followed by vaginal delivery.
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Anestesia Epidural , Anestesia Obstétrica , Raquianestesia , Versão Fetal , Adulto , Feminino , Humanos , GravidezRESUMO
Anterior urethral valves are a rare congenital anomaly associated with distal urethral obstruction, which can result in a poor prognosis. We report on the endoscopic creation of a fetal urethrotomy for obstructive uropathy resulting from anterior urethral valves. A 33-year-old woman was evaluated at 17 weeks gestation due to fetal megacystis. The diagnosis of anterior urethral valves was confirmed by the characteristic sonographic feature of a dilated membranous penile urethra. Oligohydramnios with normal-appearing kidneys and favorable urinary electrolytes led to fetal intervention. Ablation on the ventral site of the fetal penis for a cutaneous urethrotomy was performed using a YAG laser under a 1-mm fetoscope at 19 weeks gestation. Urine was drained from the incision and the dilated penis and the distended bladder shrunk with an increase in amniotic fluid. However, the fetus died unexpectedly on postoperative day 3, and chorioamnionitis was suspected as the etiology. While the outcome was unfavorable, our preliminary experience shows that fetal urethrotomy for obstructive uropathy can be achieved in utero using an endoscopic laser approach. Further experience will be required to evaluate the therapeutic value of this new procedure in the management of fetal anterior urethral valves.
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Doenças Fetais/cirurgia , Fetoscopia , Terapia a Laser , Uretra/cirurgia , Obstrução Uretral/cirurgia , Procedimentos Cirúrgicos Urológicos/métodos , Adulto , Autopsia , Corioamnionite/etiologia , Dilatação Patológica , Feminino , Morte Fetal , Doenças Fetais/diagnóstico por imagem , Fetoscopia/efeitos adversos , Idade Gestacional , Humanos , Terapia a Laser/efeitos adversos , Masculino , Oligo-Hidrâmnio/etiologia , Oligo-Hidrâmnio/cirurgia , Pênis/embriologia , Pênis/cirurgia , Gravidez , Resultado do Tratamento , Ultrassonografia Pré-Natal , Uretra/anormalidades , Uretra/diagnóstico por imagem , Obstrução Uretral/diagnóstico por imagem , Obstrução Uretral/embriologia , Procedimentos Cirúrgicos Urológicos/efeitos adversosRESUMO
Hypertensive disorders of pregnancy are known to be a risk factor for future cardiovascular diseases. In contrast, there is a paucity of data on the not so distant future prognosis of hypertensive disorders of pregnancy. In the present study, we evaluated the incidence of the diseases causing cardiovascular problems (hypertension, diabetes mellitus, dyslipidemia and metabolic syndrome) 5 years after delivery in Japanese women with hypertensive disorders of pregnancy. We performed a double-cohort study and compared medical conditions between women with and without a history of hypertensive disorders of pregnancy. A total of 1513 women who participated in the cohort study were invited to undergo a medical checkup 5 years after the index delivery, of whom 829 responded. After excluding pregnant and lactating women at the time of examination, 25 women with hypertensive disorders of pregnancy and 746 control subjects were analyzed. The incidence of hypertension was significantly higher among women with hypertensive disorders of pregnancy than women who were normotensive during pregnancy (24.0 vs. 2.5%, P<0.001). They were also at an increased risk of subsequent hypertension 5 years after the index delivery, after adjusting for confounding factors such as age, body mass index, family history of hypertension and salt intake (odds ratio 7.1, 95% CI, 2.0-25.6, P<0.003). These is no significant difference in the incidence of diabetes mellitus, dyslipidemia and metabolic syndrome. In conclusion, hypertensive disorders of pregnancy are strong risk factors for subsequent hypertension only 5 years after delivery.
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Hipertensão Induzida pela Gravidez/epidemiologia , Hipertensão/etiologia , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Diabetes Mellitus Tipo 2/epidemiologia , Dieta , Dislipidemias/epidemiologia , Feminino , Humanos , Hipertensão/epidemiologia , Hipertensão Induzida pela Gravidez/terapia , Incidência , Japão/epidemiologia , Síndrome Metabólica/epidemiologia , Pré-Eclâmpsia/epidemiologia , Gravidez , Fatores de RiscoRESUMO
OBJECTIVE: The purpose of this study is to compare the fetal fractions during non-invasive prenatal testing (NIPT) in singleton pregnancies according to gestational age and maternal characteristics to evaluate the utility of this parameter for the prediction of pregnancy complications including gestational diabetes mellitus (GDM) and hypertensive disorders of pregnancy (HDP). STUDY DESIGN: This study was a multicenter prospective cohort study. The present data were collected from women whose NIPT results were negative. The relationships between the fetal fractions and the gestational age, maternal weight and height, and incidences of miscarriage, preterm delivery, and pregnancy complications including GDM, HDP and placental abruption were assessed. RESULTS: A total of 5582 pregnant women with verified NIPT negative results were registered in the study. The demographic characteristics of the study populations were statistically analyzed, and the women with HDP tended to have a low fetal fraction in samples taken during early gestation. The area under the curve (AUC) in a receiver operating characteristic curve (ROC) analysis was 0.608 for women with HDP. CONCLUSION: A low fetal fraction on NIPT might be correlated with future HDP. However, predicting HDP during early pregnancy in women with a low fetal fraction might be difficult.
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Ácidos Nucleicos Livres/sangue , Testes para Triagem do Soro Materno , Complicações na Gravidez/sangue , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Gravidez , Estudos ProspectivosRESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent infections with certain types of bacteria and fungi. Presented herein is the case of a 29 year old woman with CGD who suffered from bacteria-associated haemophagocytic syndrome and a septic pulmonary embolism following a uterine infection and sepsis, caused by Burkholderia cepacia complex.
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Infecções por Burkholderia/complicações , Complexo Burkholderia cepacia/isolamento & purificação , Doença Granulomatosa Crônica/complicações , Linfo-Histiocitose Hemofagocítica/microbiologia , Embolia Pulmonar/microbiologia , Sepse/microbiologia , Adulto , Antibacterianos/uso terapêutico , Antifúngicos/uso terapêutico , Infecções por Burkholderia/tratamento farmacológico , Infecções por Burkholderia/microbiologia , Equinocandinas , Feminino , Humanos , Lipopeptídeos , Lipoproteínas/uso terapêutico , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Micafungina , Minociclina/uso terapêutico , Peptídeos Cíclicos/uso terapêutico , Sepse/complicações , Sepse/tratamento farmacológico , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
We previously reported the separation and recovery of nucleated red blood cells (NRBCs) in maternal blood using the lectin method. In the present study, we verified the lectin method and investigated the appearance of NRBCs during pregnancy. For the concentration of lectin soy bean agglutinin, 7 mL of maternal peripheral blood was collected from 20 subjects, and the relative fluorescence intensity was measured using flowcytometry; 50 mg/mL, used in previous studies, was the optimal concentration. The number of cells recovered at each step of the lectin method was also investigated by FACS using fluorescence-labeled CD11a and CD33, and the results showed the usefulness of the method. Next, 7 mL of maternal peripheral blood was collected from 292 women with a normal single pregnancy (389 specimens), and NRBCs were separated and recovered using the lectin method. NRBCs slightly increased over the course of pregnancy (y = 4.29x + 5.03, r2 = 0.11). When blood was collected multiple times in the same subjects, NRBCs increased in 63 of 77 subjects (83.1%, percent change: 2.4 +/- 19.0). No NRBCs were recovered in 17 subjects (4.7%). Regarding the relationship between fetomaternal disorders and the frequency of NRBCs, 89.4 +/- 92.6 cells appeared per 10 mL of maternal blood in the normal group, but NRBCs increased in patients with 18 trisomy, placenta previa, pre-eclampsia, intrauterine fetal death, and 21 trisomy. NRBC examination may play an assisting role not only in fetal diagnosis but also in fetomaternal diagnosis.
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Separação Celular/métodos , Eritrócitos/patologia , Sangue Fetal/citologia , Troca Materno-Fetal , Complicações na Gravidez/sangue , Núcleo Celular/patologia , Contagem de Eritrócitos , Feminino , Citometria de Fluxo/métodos , Fluoresceína-5-Isotiocianato , Idade Gestacional , Humanos , Lectinas , Lectinas de Plantas , Gravidez , Complicações na Gravidez/diagnóstico , Proteínas de SojaRESUMO
A fetal diagnostic method that is without risk to the embryo has been long awaited in the field of gene diagnosis. Establishment of non-invasive fetal diagnosis using maternal peripheral blood will greatly contribute to perinatal medical care. The lectin method that we have studied and developed selectively recovers nucleated red blood cells (NRBC) among fetal cells mixed in maternal peripheral blood. Maternal blood, 7 mL, was collected with ethylene diamine tetraacetic acid (EDTA) treatment in each week of gestation and subjected to preliminary concentration by density centrifugation (Histopaque [Histopaque-1077; Sigma Diagnostics, MO, USA], specific gravity: 1.095), and NRBC were separated and collected on slide glasses by the lectin method (soybean agglutinin [SBA]: 50 microg/mL). To investigate selective adhesion of the erythrocyte fraction, the SBA concentration was set to 50 microg/mL, and the cells were labeled with CD11a and CD33 (anti-white blood cell antibodies) and investigated by flowcytometry. Erythrocytes adhered at a high rate (87.0 +/- 9.7%) while the adhesion rates of granulocytes, monocytes, and lymphocytes were low, confirming the usefulness of the method for separation and recovery of the erythrocyte fraction. When recovery of NRBC was investigated using this method, a mean of 6.57 +/- 7.12 cells were recovered from 1 mL of maternal blood (May-Gluüwald-Giemsa stain). The number of recovered NRBC increased slowly with pregnancy, but differences were not significant. To confirm that recovered NRBC were derived from the fetus, NRBC were recovered by the lectin method in four patients suspected of 18 trisomy by echography and analyzed by fluorescence in situ hybridization. Three hybridization signals were detected in NRBC at a high frequency, showing that most cells were derived from the fetus and thus, fetal diagnosis may be possible. Since the procedure of the lectin method we have developed is simple, and high concentration efficiency can be obtained at a low cost, it may be clinically applicable.
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Eritroblastos/citologia , Sangue Fetal/citologia , Diagnóstico Pré-Natal/métodos , Separação Celular , Cromossomos Humanos Par 18 , Feminino , Humanos , Hibridização in Situ Fluorescente , Indicadores e Reagentes , Lectinas de Plantas , Gravidez , Proteínas de Soja , TrissomiaRESUMO
Protocadherin (Pcad) is a group of molecules obtained by polymerase chain reaction (PCR) utilizing the sequence that is well preserved in the extracellular domain of cadherin. Sano et al. analyzed Pcad (PC42,43) that had been cloned from rats, and found that it basically had homology to cadherin, but contained more than six cadherin repeats with a completely different intracellular domains (Sano et al. 1993). In the present study, of the Pcad (Pcad-1,2) cloned from a human cDNA library, as-yet-unspecified Pcad-2 was analyzed for expression in the human fetal central nervous system (CNS). Northern blot analysis of adult human tissue showed that Pcad-2 was expressed in the brain and the placenta, and that Pcad-2 mRNA was expressed in actively dividing neural tumor cell lines. Monoclonal antibodies against Pcad-2 were then made, and the CNS of fetuses were immunohistochemically stained. The expression was hardly visible at the 6th week of pregnancy, and began to become visible along the nerve fiber in the brain stem at the 8th week, and spread over the entire brain at the 11th week. At the 18th week, however, expression in the nerve fascicles, which had been visible by that time, was no longer visible or had decreased. These results suggest that Pcad-2 appears relatively early in the critical stage of development of the fetal CNS, and is involved in the induction, fasciculation, and extension of axons.
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Caderinas/metabolismo , Sistema Nervoso Central/metabolismo , Feto/metabolismo , Northern Blotting , Western Blotting , Proteínas Relacionadas a Caderinas , Caderinas/genética , Feminino , Humanos , Imuno-Histoquímica , Especificidade de Órgãos , Gravidez , RNA Mensageiro/metabolismoRESUMO
Non-invasive prenatal diagnostic methods posing no danger to the embryo have been desired for many years in the field of prenatal medicine. We are in the process of improving the lectin method that we developed for recovering fetus-derived nucleated red blood cells (NRBC) in the maternal peripheral blood. We previously used Ficoll density-gradient centrifugation for preliminary concentration in the lectin method. In the present study, we developed a molecular filter method, compared it with the Ficoll method, and tested its applicability to prenatal diagnosis. We tested the usefulness of a high molecular filter method for preliminary concentration. First, in a basic study, we prepared three kinds of non-woven cloth (NWC1-3) and a multi-porous filter (MP) to determine the optimal filter. Next, we compared the recovery rates of the Ficoll and filter methods as preliminary concentration methods in 34 normal pregnant women. Then, to examine whether the recovered NRBC were derived from the fetus, we attempted prenatal diagnosis by the fluorescence in situ hybridization (FISH) technique in 12 women pregnant with a male fetus (determined later by ultrasound) at between 8 and 16 weeks of gestation. Among the four devices used in the basic study of the high molecular filter method, NWC-2 had the best recovery rate. Therefore, we compared the numbers of NRBC recovered by the lectin method after preliminary concentration with NWC-2 or by Ficoll centrifugation, and found that the mean recovery rate of NWC-2 was 4.2 +/- 5.0 times as high as that of the Ficoll method, indicating that the NWC-2 filter method is superior as a preliminary concentration method. Next, FISH analysis of the 12 pregnant women with a male fetus for the Y chromosome showed that 19.5 +/- 12.8 NRBC were recovered, in 12.7 +/- 8.1 (63.6%) of which a Y signal was confirmed, suggesting the NWC-2 filter method can be applied to prenatal diagnosis. We consider the filter-lectin method to be a superior method for isolation and recovery of NRBC in the maternal blood which can be applied to prenatal diagnosis.
Assuntos
Eritroblastos/citologia , Feto , Núcleo Celular , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Gravidez , Diagnóstico Pré-Natal/métodosRESUMO
Fetal nucleated cells in maternal peripheral blood are a non-invasive source of fetal DNA for prenatal genetic diagnosis. However, the number of fetal cells present in maternal peripheral blood is very small. Therefore, fetal cell enrichment is generally considered necessary to allow detection and subsequent genetic analysis of the rare fetal cells. In the study presented here, we performed fetal cell separation from maternal blood using galactose-specific lectin to concentrate fetal nucleated red blood cells (FNRBCs), and attempted paternal diagnosis using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Fetal cell separation was performed using galactose-specific lectin on a PV-MeA coated slide. Twenty cells consisting of an NRBC and its surrounding 19 maternal cells were collected using laser microdissection for stable DNA amplification. DNA analysis was performed using three sequence tagged site markers (D13S270, D17S5, and D18S474) by PCR-SSCP. All seven cases were informative because they showed heterozygosity at least one locus in D13S270, D17S5, or D18S474, and paternal-specific bands were detected in all cases. These results suggest that our proposed lectin-laser-micromanipulation-PCR-SSCP method may contribute to the development of prenatal diagnosis.
Assuntos
DNA/sangue , Feto/metabolismo , Lectinas , Polimorfismo Conformacional de Fita Simples , Feminino , Humanos , Reação em Cadeia da Polimerase , GravidezRESUMO
ß-thalassemia is one of the most common genetic disorders worldwide. Concerted efforts are being made to prevent the disease, as the medical and economic burden of thalassemia represents a major public health problem. The molecular diagnosis of the ß-globin mutations that cause the disease currently involves a combination of classic methodologies. A microarray-based assay for parallel one-shot detection of mutations has been developed, but the assay remains too expensive for routine application. We developed a cost-effective plastic fiber-based DNA chip for the fast and reliable detection of 25 types of ß-thalassemia mutations. Assay conditions were established and genotyping was successfully performed on a genomic sample from a ß-thalassemia patient. Our data show that this ß-thalassemia genotyping chip is an advantageous platform for mass genotyping because of its low cost, rapid results, and reliability.