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BACKGROUND: Nectins are cell adhesion molecules that play a pivotal role in adherens junctions and tight junctions. Our previous study using whole-genome oligonucleotide microarrays revealed that nectin-4 was upregulated in pre-eclamptic placentas. We investigated the role of nectin-4 in the etiology of pre-eclampsia. METHODS: We investigated the expression of nectin-4 using real-time RT-PCR, western blot and immunostaining. Additionally, we performed matrigel invasion assay and cytotoxicity assay using cells overexpressing the nectin-4. RESULTS: NECTIN4 transcripts were elevated in pre-eclamptic placentas relative to uncomplicated pregnancies. Nectin-4 protein levels in pre-eclamptic placentas were higher on a semi-quantitative western blot. Nectin-4 was localized at the apical cell membrane in syncytiotrophoblast cells and not at the adherens junctions. Nectin-4 was also detected in cytotrophoblasts and a subset of cells in the decidua. Nectin-4 overexpressing trophoblast cells migrated normally in the matrix. However, Natural killer (NK) cells showed a strong cytotoxic effect against nectin-4 overexpressing trophoblast cells. No causative genetic variation was evident in the NECTIN4 gene from a pre-eclamptic placenta. CONCLUSIONS: There are as yet unknown factors that induce nectin-4 overexpression in trophoblast cells that may contribute to abnormal placentation via an aberrant immune response and the onset of a pre-eclamptic pregnancy.
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Moléculas de Adesão Celular/genética , Decídua/imunologia , Pré-Eclâmpsia/genética , RNA Mensageiro/genética , Trofoblastos/imunologia , Adulto , Estudos de Casos e Controles , Moléculas de Adesão Celular/imunologia , Cesárea , Citotoxicidade Imunológica , Decídua/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/patologia , Pré-Eclâmpsia/cirurgia , Gravidez , RNA Mensageiro/imunologia , Trofoblastos/patologiaRESUMO
AIM: Patients with an ultra-short uterine cervix as a result of large conization, repeated conization or radical trachelectomy (RT), are at high risk of preterm premature rupture of the membrane, which leads to preterm birth. We have commenced performing transabdominal cerclage (TAC) of the uterine cervix for these patients. In this study, we examined the safety of TAC and its impact on pregnancy. METHODS: We have performed TAC in 11 patients before pregnancy: in six after large cervical operations, such as repeated conization; and in five for difficulties with cervical cerclage after RT. After laparotomy, a Teflon thread was placed in the avascular space between the uterine vessels and the uterine muscle, and tied. The clinical course of the patients after TAC and their pregnancy course were retrospectively reviewed. RESULTS: TAC was performed safely without any complications. The mean operative duration was 53 ± 10 min, and the mean blood loss during the operation was 49 ± 64 mL. Seven women conceived within 2 years after TAC. Their pregnancy courses were favorable. Five of the women underwent scheduled cesarean sections, while two pregnancies are ongoing. CONCLUSIONS: Although there are risks of various complications as a result of the use of non-absorbable thread and the need for two extra laparotomies, TAC can be a safe and useful option for patients who show cervical incompetence after large uterine cervical operations, such as RT or large conization.
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Parede Abdominal/cirurgia , Cerclagem Cervical/métodos , Colo do Útero/patologia , Colo do Útero/cirurgia , Conização/métodos , Avaliação de Processos e Resultados em Cuidados de Saúde , Resultado da Gravidez , Adulto , Cerclagem Cervical/efeitos adversos , Feminino , Humanos , Laparotomia , Gravidez , Estudos RetrospectivosRESUMO
BACKGROUND: In the present study, we report on a couple who underwent prenatal genetic diagnosis for autosomal recessive polycystic kidney disease (ARPKD). CASE PRESENTATION: This healthy couple had previously had a healthy boy but had experienced two consecutive neonatal deaths due to respiratory distress resulting from pulmonary hypoplasia caused by oligohydramnios. The woman consulted our facility after she realized she was pregnant again. We promptly performed a carrier test for the PKHD1 gene by target exome sequencing of samples from the couple. A pathogenic mutation was identified only in the paternal allele (c.9008C>T, p.S3003F). The mutation was confirmed by Sanger sequencing of the DNA from formalin-fixed, paraffin-embedded, kidney tissue of the second neonate patient and was not found in the healthy sibling. We then performed haplotype analyses using microsatellite markers scattered throughout the PKHD1 gene. DNA from the amniocentesis was determined to belong to a carrier, and the couple decided to continue with the pregnancy, obtaining a healthy newborn. Subsequent detailed examination of the exome data suggested higher read depth at exons 45 and 46. Multiplex ligation-dependent probe amplification allowed identification of duplication of these two exons. This case suggests the potential usefulness of target exome sequencing in the prenatal diagnosis of the PKHD1 gene in ARPKD. CONCLUSIONS: This is the first report of intragenic duplication in the PKHD1 gene in ARPKD.
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Mutação , Rim Policístico Autossômico Recessivo/diagnóstico , Rim Policístico Autossômico Recessivo/genética , Receptores de Superfície Celular/genética , Amniocentese/métodos , Exoma , Feminino , Humanos , Masculino , Gravidez , Análise de Sequência de DNA , Adulto JovemRESUMO
Objectives: Nectin-4 is a cell adhesion molecule with vital functions at adherens and tight junctions. Cumulative evidence now indicates that the NECTIN4 gene is overexpressed in a variety of cancers, and that the nectin-4 protein is both a disease marker and therapeutic target in a subset of these cancers. We previously demonstrated that NECTIN4 is overexpressed in placenta during pre-eclamptic pregnancy, which is one of the most serious obstetric disorders. Methods: Nectin-4 protein levels were measured in maternal sera from pregnant women with pre-eclampsia and its related disorder, unexplained fetal growth retardation. Results: Maternal serum concentrations of nectin-4 were significantly elevated in pre-eclamptic women compared with those with an uncomplicated normotensive pregnancy. However, no increase was observed in pregnancies with unexplained fetal growth retardation. Serum nectin-4 levels were higher in cases with early-onset pre-eclampsia that generally showed more severe clinical symptoms, but levels were not correlated to other clinical indicators of disease severity. Conclusions: Nectin-4 is a potential new diagnostic and predictive biomarker for severe pre-eclampsia.
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Objectives: Fetal human cytomegalovirus (HCMV) infection might be involved in fetal growth restriction (FGR). Maternal serostatus and the prevalence of congenital HCMV infection are affected by various factors, such as socioeconomic status and ethnicity. Therefore, the prevalence of congenital HCMV-related FGR should be examined in each region. Methods: Seventy-eight cases of FGR with delivery between January 2012 and January 2017 at Fujita Health University Hospital were studied. Twenty-one non-FGR cases were also included as a control group. Placental sections obtained from the FGR and control cases were immunostained with two primary antibodies for detecting immediate early antigens. Results: Nineteen placental samples from FGR cases with another etiology were excluded. Finally, 59 placental samples from FGR cases of unknown etiology were included in the pathological analysis. Four of 59 (6.8%) placental samples were positive for HCMV antigen. All four positive cases were stained with the M0854 antibody, and there were no positive case with the MAB810R antibody. Neither maternal nor infantile clinical features were different between the HCMV-positive and -negative FGR cases. A pathological examination showed a hematoma in three of four cases and infarction in two of four cases. Conclusions: HCMV antigen was detected in 6.8% of placental samples obtained from FGR cases without an obvious etiology. No remarkable maternal or neonatal clinical features discriminated HCMV-related FGR from FGR due to other causes. Vasculitis and inflammation might play important roles in the pathogenesis of HCMV-related FGR.
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BACKGROUND: FLT1 is one of the significantly overexpressed genes found in a pre-eclamptic placenta and is involved with the etiology of this disease. METHODS: We conducted genome-wide expression profiling by RNA-seq of placentas from women with pre-eclampsia and those with normotensive pregnancy. RESULTS: We identified a lncRNA gene, MG828507, located ~80 kb upstream of the FLT1 gene in a head-to-head orientation, which was overexpressed in the pre-eclamptic placenta. MG828507 and FLT1 are located within the same topologically associated domain in the genome. The MG828507 mRNA level correlated with that of the FLT1 in placentas from pre-eclamptic women as well as in samples from uncomplicated pregnancies. However, neither the overexpression nor knockdown of MG828507 affected the expression of FLT1. Analysis of pre-eclampsia-linking genetic variants at this locus suggested that the placental genotype of one variant was associated with the expression of MG828507. The MG828507 transcript level was not found to be associated with maternal blood pressure, but showed a relationship with birth and placental weights, suggesting that this lncRNA might be one of the pivotal placental factors in pre-eclampsia. CONCLUSION: Further characterization of the MG828507 gene may elucidate the etiological roles of the MG828507 and FLT1 genes in pre-eclampsia in a genomic context.
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Objectives: Alterations in the vaginal bacterial flora reflect the status of various obstetric conditions and are associated with mechanisms that underlie certain pregnancy-associated complications. These changes are also a predictive biomarker for clinical outcomes of these adverse events. Methods: We examined the vaginal microbiome in samples from pregnant Japanese women with preterm labor. Results: The microbiota composition in preterm delivery (PD) samples differed from those of control or threatened preterm delivery (TPD) samples in principal component analysis. An increase in Firmicutes and a decrease in Actinobacteria were significantly associated with PD only (both P<0.01). In the Firmicutes phylum, Lactobacillus tended to be abundant, and the abundance of L. iners and L. crispatus was especially high, whereas the L. gasseri population was low in PD samples. Longitudinal analysis showed that the abundance of L. iners decreased after commencing tocolytic treatment in TPD samples compared with before treatment, but it remained high in PD samples. Conclusions: The vaginal microbiome may be a useful prognostic indicator of preterm labor and a monitoring tool for tocolytic treatment to prevent preterm birth.
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BACKGROUND: It has been well documented that pre-eclampsia and unexplained fetal growth restriction (FGR) have a common etiological background, but little is known about their linkage at the molecular level. The aim of this study was to further investigate the mechanisms underlying pre-eclampsia and unexplained FGR. METHODS: We analyzed differentially expressed genes in placental tissue from severe pre-eclamptic pregnancies (n = 8) and normotensive pregnancies with or (n = 8) without FGR (n = 8) using a microarray method. RESULTS: A subset of the FGR samples showed a high correlation coefficient overall in the microarray data from the pre-eclampsia samples. Many genes that are known to be up-regulated in pre-eclampsia are also up-regulated in FGR, including the anti-angiogenic factors, FLT1 and ENG, believed to be associated with the onset of maternal symptoms of pre-eclampsia. A total of 62 genes were found to be differentially expressed in both disorders. However, gene set enrichment analysis for these differentially expressed genes further revealed higher expression of TP53-downstream genes in pre-eclampsia compared with FGR. TP53-downstream apoptosis-related genes, such as BCL6 and BAX, were found to be significantly more up-regulated in pre-eclampsia than in FGR, although the caspases are expressed at equivalent levels. CONCLUSIONS: Our current data indicate a common pathophysiology for FGR and pre-eclampsia, leading to an up-regulation of placental anti-angiogenic factors. However, our findings also suggest that it may possibly be the excretion of these factors into the maternal circulation through the TP53-mediated early-stage apoptosis of trophoblasts that leads to the maternal symptoms of pre-eclampsia.
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Retardo do Crescimento Fetal/genética , Perfilação da Expressão Gênica , Placenta/metabolismo , Pré-Eclâmpsia/genética , Feminino , Humanos , Análise em Microsséries , Gravidez , Proteína Supressora de Tumor p53/genéticaRESUMO
Serum 25-hydroxyvitamin D (25-OHD) concentrations are thought to accurately reflect vitamin D stores, and vitamin D deficiency causes secondary hyperparathyroidism, irreversible bone loss, and increased risk of fracture. Recent studies suggest that decrease of serum 25-OHD level in mothers could increase the risk of preeclampsia, cesarean section, and craniotabes. Furthermore, this deficiency may affect bone mass and the incidence of neuromuscular diseases of their children in the future. In the present study, the serum concentration of 25-OHD in 93 pregnant women after the 30th week of their gestation was determined by direct radioimmunoassay. Mean 25-OHD levels in spring, summer, fall, and winter were 14.3 ± 5.1, 15.7 ± 6.4, 13.7 ± 5.1, and 13.9 ± 4.2 ng/ml, respectively. Severe vitamin D deficiency (25-OHD < 10 ng/ml) was found in 10 of these 93 women. Overall, hypovitaminosis D, which was defined as serum 25-OHD concentration equal to or less than 20 ng/ml, was revealed in 85 mothers (89.5%). Serum 25-OHD levels were not associated with either intact parathyroid hormone or corrected calcium concentrations, but were negatively associated with serum type I collagen N-terminal telopeptide and bone-specific alkaline phosphatase in these subjects. Mothers with threatened premature delivery had significantly lower 25-OHD levels (11.2 ± 3.2 ng/ml) than those in mothers with normal delivery (15.6 ± 5.1 ng/ml). In conclusion, the present data suggest a high prevalence of hypovitaminosis D in perinatal pregnant Japanese women throughout the year, which seems to affect bone metabolism and to be associated with threatened premature delivery.
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Trabalho de Parto Prematuro/epidemiologia , Deficiência de Vitamina D/epidemiologia , Adolescente , Adulto , Povo Asiático , Feminino , Humanos , Pessoa de Meia-Idade , Trabalho de Parto Prematuro/sangue , Hormônio Paratireóideo/sangue , Gravidez , Radioimunoensaio , Vitamina D/análogos & derivados , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Adulto JovemRESUMO
We present three extremely rare cases of spontaneous spinal epidural hematoma occurring in pregnancy. The patients developed progressive paralysis of the upper and lower limbs and the diagnoses were confirmed by magnetic resonance imaging. Urgent decompression is required to prevent neurological sequelae. The pregnancy may either be continued or delivered depending on the gestational age and severity of the disorder. Pregnancy-induced structural changes of the vascular walls and hemodynamic changes may play a role in the pathogenesis of spontaneous spinal epidural hematoma.
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Hematoma Epidural Espinal/diagnóstico , Paresia/etiologia , Complicações na Gravidez/diagnóstico , Adulto , Descompressão Cirúrgica , Feminino , Hematoma Epidural Espinal/complicações , Hematoma Epidural Espinal/cirurgia , Humanos , Gravidez , Complicações na Gravidez/cirurgia , Resultado do TratamentoRESUMO
OBJECTIVE: The proprotein convertase furin is known to be involved in the processing of pro-B-type natriuretic peptide (proBNP) and prorenin receptor (PRR), suggesting that it has a potential function in blood pressure regulation. We investigated the role of furin in the etiology of pre-eclampsia and its related disorder, unexplained fetal growth restriction (FGR) without hypertension. METHODS: We evaluated serum and placental furin levels in pre-eclampsia, FGR and uncomplicated pregnancy. Additionally, we investigated the correlation between the serum furin levels and products of furin enzymatic activity or clinical parameters. RESULTS: We demonstrated that the maternal circulation in cases of pre-eclampsia and FGR had lower levels of soluble furin than uncomplicated pregnancies. Both NT-proBNP and soluble PRR were elevated in pre-eclampsia, whereas only soluble PRR was at higher levels in unexplained FGR. Linear regression analysis revealed a negative correlation between the serum furin level and that of NT-proBNP or soluble PRR. While we observed that the serum furin or soluble PRR level correlated with blood pressure, a stronger correlation was observed with birth and placental weights. Further to this, the FURIN mRNA levels were significantly reduced in placental pre-eclamptic placentas as well as in FGR cases. CONCLUSION: These data suggest the possibility that reduced levels of furin may be the result of a negative feedback from the activation of the renin-angiotensin pathway that leads to feto-placental dysfunction with or without maternal hypertension. This may represent an etiologic pathway of pre-eclampsia and unexplained FGR.
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Retardo do Crescimento Fetal/sangue , Furina/análise , Pré-Eclâmpsia/sangue , Receptores de Superfície Celular/análise , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Feminino , Retardo do Crescimento Fetal/diagnóstico , Retardo do Crescimento Fetal/epidemiologia , Furina/sangue , Humanos , Japão/epidemiologia , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/epidemiologia , Gravidez , Receptores de Superfície Celular/sangue , Receptor de Pró-ReninaRESUMO
BACKGROUND: Soluble fms-like tyrosine kinase 1 (sFlt-1) is believed to be a prominent component in the pathogenesis of pre-eclampsia, although the precise etiology has remained elusive. In this study, the etiological role of FLT1 variant was further validated in pre-eclampsia by examining this association in a Japanese sample population. METHODS: The genotypes of three variants (rs4769613, rs12050029 and rs149427560) were examined in the upstream region of the FLT1 gene in placentas from pre-eclamptic (n=47) or normotensive control (n=49) pregnancy samples. Additionally, FLT1 mRNA levels in placenta were determined by qRT-PCR. ELISA was further used to detect circulating sFlt-1 levels in maternal sera. The intergroup comparisons were made using the Mann-Whitney U test or one way analysis of variance and P values of less than 0.05 were considered statistically significant. RESULTS: First, the rs4769613 (C>T) and rs12050029 (G>A) genotypes were examined in placentas but no significant differences were found in the genotype or allele-type frequencies. Next, nearby short tandem repeat, rs149427560, was examined which manifested four size variants. In the genotypewise analysis, the frequency of the 474/476 heterozygote was significantly lower in pre-eclampsia (p<0.05). As expected, the FLT1 mRNA levels were significantly elevated in the pre-eclamptic placentas and sFlt-1 was higher in pre-eclamptic maternal sera. However, the genotype of these variants did not affect the FLT1 mRNA or serum sFlt-1 levels. CONCLUSION: Our findings did not support the hypothesis that genetic variations around the FLT1 gene affect the subtle expression changes underlying the etiologic pathway of pre-eclampsia. The hypothesis deserves further investigation through a larger sample size.
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BACKGROUND: Defective nitric oxide (NO)-mediated vasodilation is widely regarded as an underlying cause of hypertension in pre-eclampsia, although there are also arguments against this hypothesis. METHODS: We examined both the mRNA levels and the presence of a Glu298Asp substitution in the NO synthase (NOS) gene, as well as the NO metabolite concentration, in placentas and maternal sera from women with pre-eclampsia and in normotensive pregnant controls (25-40 vs. 24-41 weeks of gestation). RESULTS: Pre-eclamptic and control placentas did not show any significant differences in their NO metabolite levels or their NOS expression levels as measured by quantitative RT-PCR. In addition, we did not find any association between pre-eclampsia and the occurrence of the Glu298Asp amino acid substitution in the NOS gene. In contrast, high maternal circulating NO metabolites were evident in severe pre-eclampsia (p < 0.0001). Although a positive correlation between circulating NO metabolites and blood pressure was not observed, uterine artery resistance measured by ultrasound was found to positively correlate with the maternal NO levels. CONCLUSIONS: Our current data suggest that an altered placental NOS pathway is unlikely to be the primary cause of pre-eclampsia and that the activation of this pathway is possibly in response to maternal symptoms.
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Óxido Nítrico/metabolismo , Placenta/química , Pré-Eclâmpsia/etiologia , Adulto , Ácido Aspártico/genética , Estudos de Casos e Controles , GMP Cíclico/sangue , Feminino , Idade Gestacional , Ácido Glutâmico/genética , Humanos , Nitratos/sangue , Óxido Nítrico/sangue , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III/genética , Placenta/enzimologia , Polimorfismo de Nucleotídeo Único , Pré-Eclâmpsia/enzimologia , Gravidez , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: The purpose of this study was to assess the clinical features and characteristics of the blood flow in uterine vascular abnormalities using ultrasound and magnetic resonance imaging (MRI). METHODS: A total of 17 women were diagnosed with uterine vascular abnormalities by ultrasound. The clinical characteristics of the patients and the distribution and waveform of the intrauterine vessels were examined using transvaginal gray-scale and Doppler ultrasonography, spin-echo MRI, and MR angiography. RESULTS: The average age of the 17 subjects was 44.3 years, and 5 were postmenopausal women. The number of pregnancies and deliveries was 2.0 and 1.7, respectively. Of the 17 subjects, 7 had a moderate or severe grade of dysmenorrhea and 7 had a history of vascular disease. In all subjects, vaginal ultrasound demonstrated tubular or numerous tortuous anechoic areas in the uterine wall, and Doppler ultrasound showed that the tubular or numerous dilated tortuous vessels had an atypical wave flow, unlike that of an artery or a vein. The distribution of displayed flow varied, and the waveforms of the Doppler ultrasound displayed three patterns. The averages of the pulse Doppler flow indices showed low impedance in the abnormal uterine vessel and the uterine artery, especially in cases of true arteriovenous malformations. MR angiography demonstrated distinct, tortuous, and coiled vascular channels in the pelvis during and just after the arterial phase. CONCLUSION: Characterization of the clinical features of uterine vascular abnormalities is considered to be valuable for obstetricians and gynecologists.
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Examination of maternal plasma cell-free DNA (cfDNA) for noninvasive prenatal testing for fetal trisomy is a highly effective method for pregnant women at high risk. This can be also applied to fetal gender determination in female carriers of severe X-linked disease. Polymerase chain reaction (PCR) analysis is a relatively simpler and less expensive method of detecting Y chromosome-specific repeats (Y-specific PCR; YSP), but is limited by the risk of false-negative results. To address this, we have developed a combined strategy incorporating YSP and an estimation of the fetal DNA fraction. Multiplex PCR for 30 single nucleotide polymorphism (SNP) loci selected by high heterozygosity enables the robust detection of the fetal DNA fraction in cfDNA. The cfDNA sample is first subjected to YSP. When the YSP result is positive, the fetus is male and invasive testing for an X-linked mutation is then required. When the YSP result is negative, the cfDNA sample is analyzed using multiplex PCR. If fetal DNA is then found in the cfDNA, invasive testing is not then required. If the multiplex PCR analysis of cfDNA is negative for fetal DNA, the fetal gender cannot be determined and invasive testing is still required. Our technique provides a potentially effective procedure that can help to avoid unnecessary invasive prenatal testing in some female carriers of severe X-linked disease.
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Ácidos Nucleicos Livres/genética , Cromossomos Humanos X/química , Cromossomos Humanos Y/química , Síndrome de Down/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Diagnóstico Pré-Natal/métodos , Adulto , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Síndrome de Down/sangue , Síndrome de Down/genética , Feminino , Feto , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex/métodos , Polimorfismo de Nucleotídeo Único , Gravidez , Primeiro Trimestre da Gravidez , TrissomiaRESUMO
Thanatophoric dysplasia and achondroplasia are allelic disorders caused by a constitutively active mutation in the FGFR3 gene. Because thanatophoric dysplasia is a lethal disorder and achondroplasia is non-lethal, they need to be distinguished after ultrasound identification of fetal growth retardation with short limbs. Accordingly, we have developed a noninvasive prenatal test using cell-free fetal DNA in the maternal circulation to distinguish thanatophoric dysplasia and achondroplasia. A multiplex PCR system encompassing five mutation hotspots in the FGFR3 gene allowed us to efficiently identify the responsible mutation in cell-free DNA in all examined pregnancies with a suspected thanatophoric dysplasia or achondroplasia fetus. This system will be helpful in the differential diagnosis of thanatophoric dysplasia and achondroplasia in early gestation and in couples concerned about the recurrence of thanatophoric dysplasia due to germinal mosaicism.
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Acondroplasia/genética , Ácidos Nucleicos Livres/genética , Retardo do Crescimento Fetal/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Pré-Natal/métodos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Displasia Tanatofórica/genética , Acondroplasia/sangue , Acondroplasia/diagnóstico por imagem , Acondroplasia/patologia , Adulto , Sequência de Bases , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Diagnóstico Diferencial , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/diagnóstico por imagem , Retardo do Crescimento Fetal/patologia , Feto , Expressão Gênica , Humanos , Mosaicismo , Mutação , Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/sangue , Displasia Tanatofórica/sangue , Displasia Tanatofórica/diagnóstico por imagem , Displasia Tanatofórica/patologia , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
Pregnancy-associated plasma protein-A and -A2 (PAPP-A and -A2) are proteases that cleave insulin-like growth factor-binding proteins (IGFBPs), resulting in local activation of IGF signaling pathways. Here, we examined PAPP-A and -A2 mRNA and protein levels in placenta and maternal sera from women with pre-eclampsia and compared them with samples from uncomplicated pregnancy. PAPP-A2 but not PAPP-A mRNA and protein were elevated in pre-eclamptic placenta (P < 0.01). PAPP-A2 is normally produced in placental syncytiotrophoblast cells and maternal decidua. PAPP-A2 in syncytiotrophoblast cells was dramatically increased in pre-eclampsia. Maternal serum concentrations of PAPP-A2 but not PAPP-A were also significantly elevated in pre-eclampsia as compared with uncomplicated pregnancy. mRNA levels of IGFBP5, a specific substrate for PAPP-A2 protease activity, were also significantly increased, suggesting a potential role for IGFBP5 in fetal and placental growth suppression during pre-eclampsia. However, IGFBP5 protein levels were not increased in placenta from pre-eclampsia, possibly due to cleavage by up-regulated PAPP-A2. These data might imply that PAPP-A2 may be up-regulated in pre-eclamptic pregnancy to compensate for IGFBP5-mediated suppression of the IGF pathway, although final birthweights are still low in pre-eclamptic pregnancy.
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Pré-Eclâmpsia/sangue , Proteína Plasmática A Associada à Gravidez/genética , Proteína Plasmática A Associada à Gravidez/metabolismo , Adulto , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Idade Gestacional , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/genética , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/ß-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.
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Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.