Analysis Of Amniotic Fluid Samples Research Articles

Optical genome mapping for detection of chromosomal aberrations in prenatal diagnosis.

Chromosomal aberrations are the most important etiological factors for birth defects. Optical genome mapping is a novel cytogenetic tool for detecting a broad range of chromosomal aberrations in a single assay, but relevant clinical feasibility studies of optical genome mapping in prenatal diagnosis are limited. We retrospectively performed optical genome mapping analysis of amniotic fluid samples from 34 fetuses with various clinical indications and chromosomal aberrations detected through standard-of-care technologies, including karyotyping, fluorescence in situ hybridization, and/or chromosomal microarray analysis. In total, we analyzed 46 chromosomal aberrations from 34 amniotic fluid samples, including 5 aneuploidies, 10 large copy number variations, 27 microdeletions/microduplications, 2 translocations, 1 isochromosome, and 1 region of homozygosity. Overall, 45 chromosomal aberrations could be confirmed by our customized analysis strategy. Optical genome mapping reached 97.8% concordant clinical diagnosis with standard-of-care methods for all chromosomal aberrations in a blinded fashion. Compared with the widely used chromosomal microarray analysis, optical genome mapping additionally determined the relative orientation and position of repetitive segments for seven cases with duplications or triplications. The additional information provided by optical genome mapping will be conducive to characterizing complex chromosomal rearrangements and allowing us to propose mechanisms to explain rearrangements and predict the genetic recurrence risk. Our study highlights that optical genome mapping can provide comprehensive and accurate information on chromosomal aberrations in a single test, suggesting that optical genome mapping has the potential to become a promising cytogenetic tool for prenatal diagnosis.

Development of a Rat Model of Intra-Amniotic Inflammation via Ultrasound-Guided Administration of a Triggering Agent in the Gestational Sac to Enable Analysis of Individual Amniotic Fluid Samples.

Objectives: To develop a rat model of intra-amniotic inflammation, characterized by the concentration of interleukin-6 in the amniotic fluid, induced by an ultrasound-guided transabdominal administration of lipopolysaccharide into individual gestational sacs. Methods: An ultrasound-guided transabdominal intra-amniotic administration of lipopolysaccharide or phosphate-buffered saline (PBS) as control was performed in rats on embryonic day 18. Only accessible gestational sacs with precise recording of their positions were injected. Twenty-four hours later, individual amniotic fluid samples were collected from the gestational sacs of laparotomized animals. The gestational sacs were divided into four subgroups: (i) with lipopolysaccharide: injected gestational sacs from rats undergoing lipopolysaccharide administration; (ii) without lipopolysaccharide: non-injected gestational sacs from rats undergoing lipopolysaccharide administration; (iii) with PBS: injected gestational sacs from rats undergoing PBS administration; and (iv) without PBS: non-injected gestational sacs from rats undergoing PBS administration. The concentration of interleukin-6 in individual amniotic fluid samples was assessed using ELISA. Results: In the group of five animals receiving lipopolysaccharide, 24 (33%) and 48 (77%) gestational sacs were and were not injected, respectively. The amniotic fluid was obtained from 21 (88%) injected and 46 (95%) non-injected sacs. In the control group of five animals receiving phosphate-buffered saline, 28 (35%) and 52 (75%) gestational sacs were and were not injected, respectively. The amniotic fluid was obtained from 18 (64%) injected and 50 (96%) non-injected sacs. No labor occurred, and only one fetal death was observed in a gestational sac injected with lipopolysaccharide. Differences in concentrations of interleukin-6 in the amniotic fluid were found among the subgroups of the gestational sacs (with lipopolysaccharide: median 762 pg/ml; without lipopolysaccharide: median 35.6 pg/ml; with PBS: median 35.6 pg/ml; and without PBS: median 35.6 pg/ml; p < 0.0001). Concentrations of interleukin-6 in the amniotic fluid from the gestational sacs with lipopolysaccharide were significantly higher than those in the three remaining subgroups (p < 0.0001). No differences in concentrations of interleukin-6 in the amniotic fluid were identified between the three remaining subgroups. Conclusion: The ultrasound-guided transabdominal intra-amniotic administration of lipopolysaccharide with a subsequent collection and analysis of amniotic fluid samples is feasible in rats. The intra-amniotic administration of lipopolysaccharide led to the development of intra-amniotic inflammation without leading to fetal mortality or induction of labor.

Prenatal diagnosis and genetic counseling in 19 cases with 22q11.2 microduplications

Patients with 22q11.2 microduplications have highly variable clinical phenotypes. The clinical manifestations and prognosis of 19 fetuses carrying 22q11.2 microduplications were analyzed. The fetuses were analyzed by single nucleotide polymorphism array (SNP array), which was followed by parental validation. Pregnancy outcome and clinical features of the newborns were analyzed in order to delineate genotype-phenotype correlation. Two fetuses were identified by karyotyping analysis of amniotic fluid samples. SNP array revealed that all have carried a 468.8 kb~3.4 Mb duplication in 22q11.2 region. Two couples have refused parental verification. Seven cases were inherited from the mother, 6 were from the father, and 4 cases were de novo in origin. Three couples opted termination of the pregnancy. One fetus perished at birth. Five newborns showed delayed growth, the remaining 10 were normal. The prenatal phenotype of fetuses carrying 22q11.2 microduplications are nonspecific, and the phenotypes of survivors may become more diverse along with increased age. Professional evaluation and long-term follow-up should be recommended.

Two novel ectodysplasin A gene mutations and prenatal diagnosis of X-linked hypohidrotic ectodermal dysplasia.

BackgroundHypohidrotic ectodermal dysplasia (HED) is mainly caused by ectodysplasin A (EDA) gene mutation. Fetus with genetic deficiency of EDA can be prenatally corrected. This study aimed at revealing the pathogenesis of two HED families and making a prenatal diagnosis for one pregnant female carrier.DesignsGenomic DNA was extracted from two HED patients and sequenced using whole exome sequencing (WES). The detected mutations were confirmed in patients and family members using Sanger sequencing. The expression of soluble ectodysplasin A1 (EDA1) protein was studied by western blot. The transcriptional activity of NF‐κB pathway was tested by dual luciferase assay. The genomic DNA of fetus was extracted from shed chorion cells and EDA gene was screened through Sanger sequencing.ResultsWe identified two novel EDA mutations: c.1136T>C (p.Phe379Ser) and c.[866G>C;868A>T] (p.[Arg289Pro;Ser290Cys]). Further examinations revealed that these two mutated EDA1 proteins showed completely impaired solubility, and the transcriptional NF‐κB activation induced by these missense mutant‐type EDA1 proteins was significantly reduced compared with wild‐type EDA1. Furthermore, the analysis of amniotic fluid samples from a pregnant heterozygote indicated that the fetus was a c.1136T>C mutation female carrier.ConclusionsThis study extended the mutation spectrum of X‐linked hypohidrotic ectodermal dysplasia (XLHED) and applied prenatal diagnosis for the pregnant carrier, which can be helpful in genetic counseling, prenatal diagnosis, and intervention for the XLHED family.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration≥2.6ng/mL. (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

Study on amniotic fluid metabolism in the second trimester of Trisomy 21.

BackgroundTrisomy 21 is a common aneuploid condition in humans and accounts for approximately one quarter of all aneuploid live births. To date, early diagnosis of Trisomy 21 remains a challenging task. Metabolomics may prove an innovative tool to study the early pathophysiology of Trisomy 21 at a functional level.MethodsUltra‐performance liquid chromatography coupled with mass spectrometer (UPLC‐MS) was used for untargeted metabolomic analysis of amniotic fluid samples from women having normal and trisomy 21 fetuses.ResultsMany significantly changed metabolites were identified between amniotic fluid samples from Trisomy 21 pregnancies and normal euploid pregnancies, such as generally lower levels of several steroid hormones and their derivatives, higher levels of glutathione catabolites coupled with lower levels of gamma‐glutamyl amino acids, and increased levels of phospholipid catabolites, sugars, and dicarboxylic acids. The identification of a human milk oligosaccharide in amniotic fluid may worth further investigation, since confirmation of this observation may have significant implications for regulation of fetal development.ConclusionsThe metabolisms in amniotic fluid from Trisomy 21 and normal pregnancies are quite different, and some of the significantly changed metabolites may be considered as candidates of early diagnostic biomarkers for Trisomy 21.

Preparation, Culture, and Analysis of Amniotic Fluid Samples.

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid may be used directly for biochemical analyses, fluorescence in situ hybridization (FISH), and isolation of DNA for molecular studies, including chromosomal microarray analysis (CMA). The fluid is typically cultured as a source of metaphase cells for chromosome analysis and to provide additional material for biochemical and DNA-based testing. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for metaphase chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies. Lastly, this unit describes a method for isolating DNA for CMA from uncultured amniotic fluid and cultured cells. © 2018 by John Wiley & Sons, Inc.

Genetic analysis and prenatal diagnosis of two Chinese families with split hand foot malformation

To identify genomic aberrations underlying pathogenesis of split hand foot malformation (SHFM) in two Chinese families, and to provide genetic counseling and prenatal diagnosis for them. Two sets of peripheral blood and amniotic fluid samples were collected from the patients. One was processed with routine culture and karyotype analysis. For another set, DNA was extracted and analyzed with array-based comparative genomic hybridization (array-CGH). Karyotype analysis of peripheral blood samples for both probands was normal. Karyotype analysis of the amniotic fluid from family 1 has found no abnormality. However, analysis of amniotic fluid samples from the second family showed del(7)(q21q22.1). By array-CGH analysis, both blood and amniotic fluid samples from the first family showed a 662.3 kb dup(10q24.31q24.32). Array-CGH analysis of the blood sample from the second family was normal, whilst analysis of amniotic fluid sample revealed a 19.97 Mb del(7q11.23q21.3). Array-CGH features high resolution, high accuracy and rapid diagnosis for unbalanced chromosomal aberration. The dup(10q24.31q24.32) and 19.97 Mb del(7q11.23q21.3) have been the cause of SHFM in the two families. Genetic counseling and prenatal diagnosis have been provided for both families in order to prevent this birth defect.

Preparation, Culture, and Analysis of Amniotic Fluid Samples

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid is used directly for biochemical analyses and as a source of fetal cells for biochemical assays, DNA testing, and chromosome studies. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies.

Determination of L-arginine in amniotic fluid by capillary zone electrophoresis

Conditions for the determination of L-arginine in a biological fluid using capillary zone electrophoresis were optimized. The effects of the pH of a running buffer, the time of sample injection into a capillary, the operating voltage, the temperature, and the wavelength on the results of the determination were studied. The procedure made it possible to evaluate the concentration of L-arginine over a range of 6–1000 μg/mL (c = 3 μg/mL; RSD = 2%). The duration of analysis including sample preparation was no longer than 30 min. The analysis of amniotic fluid samples in the cases of physiological pregnancy and pregnancy complicated by placental insufficiency demonstrated that the arginine content of amniotic fluid increased in the case of placental insufficiency, as compared to normal values.

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-β1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.

Preparation, Culture, and Analysis of Amniotic Fluid Samples

AbstractThe describes an in situ method for the preparation, culture, harvest, and analysis of amniotic fluid samples. In this procedure, cells are grown and harvested for metaphase spreads on glass coverslips. The metaphase spreads are analyzed without being removed from the coverslips. describes a harvesting technique that yields higher‐resolution banding. describes a method for growing more cells by passaging cells from a backup monolayer culture. An describes a flask method for the preparation, culture, harvest, and analysis of amniotic fluid samples that can be used instead of the in situ method. Accompanying is that describes subculturing cells from flasks.

Annex 6: Chromosome mosaicism in CVS and amniocentesis samples

Data on 1040 chorionic villus and 969 amniotic fluid samples were collected from women studied in the Canadian Multicentre Randomized Clinical Trial of Chorion Villus Sampling and Amniocentesis. Cytogenetic results were obtained from 98.0 per cent of chorionic villus samples and from 99.9 per cent of amniotic fluid samples. Level I mosaicism (a single cell with an abnormal karyotype) occurred frequently in both chorionic villus and amniotic fluid samples and appeared to have no clinical significance. Level II mosaicism occurred in 0.9 per cent of CVS mesenchyme and 1.5 per cent of amniotic fluid cultures and in general was not perceived to be of sufficient concern to warrant cytogenetic follow-up studies. Level III mosaicism was reported in 18 CVS cases (15 cytotrophoblast, 1 mesenchyme, and 2 with both cell methods) and in one amniotic fluid case. In all cases but one (fetus with trisomy 18), level III mosaicism was confined to the placenta. Maternal cell contamination occurring with a frequency of 6.4 per cent in the mesenchyme analyses was a concern. This study supports the final report of the Canadian Multicentre Randomized Clinical Trial of Chorion Villus Sampling and Amniocentesis. Cytogenetic analysis of chorionic villus samples appears to be an acceptable alternative to the analysis of amniotic fluid samples. However, because of mosaicism and maternal cell contamination concerns, the examination of both cytotrophoblast preparations and mesenchyme cultures from chorionic villus samples is recommended.

Spontaneous and induced chromosome breakage in chorionic villus samples: a cytogenetic approach to first trimester prenatal diagnosis of ataxia telangiectasia syndrome.

Patients with ataxia telangiectasia (AT) syndrome exhibit a high level of spontaneous chromosome aberrations, with hypersensitivity to gamma radiation and radiomimetic chemicals at the chromosomal and cellular level. Previously pregnancies at risk for AT have been screened solely by analysis of amniotic fluid samples. In this report we describe a cytogenetic approach to the prenatal diagnosis of AT using chorionic villus sampling (CVS). Levels of spontaneous and induced (gamma radiation and bleomycin) chromosome breakage were established in direct, semidirect, and culture preparations of CVS samples from normal pregnancies. The methods developed were then successfully applied to the screening of a pregnancy at risk for AT. Semidirect preparations showed normal levels of chromosome breakage, and this result was further confirmed in chorion, amniotic fluid, and lymphocyte cultures. In chorion villus samples, gamma radiation is probably the easiest and most reliable way of discriminating between unaffected fetuses and those with AT.

Evaluation and quality control of a monoclonal antibody based enzyme antigen immunoassay of acetylcholinesterase in amniotic fluid.

We have evaluated a new monoclonal antibody based enzyme antigen immunoassay (EAIA) for acetylcholinesterase in amniotic fluid, intended for the detection of open fetal abnormalities, especially open neural tube defects. With a sample volume of 50 microliters, the detection limit is 0.7 nkat/l and linearity is found up to an amount 200-times the detection limit. CV's are less than or equal to 6.6% within assays and less than or equal to 10.3% between assays; recoveries averaged 103%. The upper limit of the reference range for amniotic fluids from non-affected pregnancies is 8.5 nkat/l for clear specimens and 25.0 nkat/l for haemolysed specimens. Amniotic fluid specimens from 1473 patients (1388 normal and 85 pathological pregnancies) were examined with both the new EAIA and the original procedure using a polyclonal rabbit antiserum. The two procedures showed identical sensitivities and specificities of about 100%, except for amniotic fluid samples containing haemolysed blood. For the latter the new EAIA showed a specificity of 100% compared to 55.6% for the original procedure. We conclude that the new EAIA is accurate and reproducible and shows, compared with the original procedure, an increased specificity in the analysis of amniotic fluid samples containing haemolysed blood.

Capillary gas chromatographic profiling of total long-chain fatty acids cholesterol in biological materials

The profiling of total long-chain fatty acids and cholesterol in a variety of biological materials, using capillary gas chromatography with flame ionization detection, is described. The within-run precision and day-to-dy precision for fifteen fatty acids and cholesterol in erythrocyte samples were investigated. Quantitative data on the analysis of amniotic fluid samples collected from the women in the 30th week are given together with a correlation study on their lecithin/sphingomyelin and their palmitic acid/stearic ratios. In addition, the method was applied to lumbar cerebrospinal fluid, plasma, isolated leukemia blood cells and neuroblastoma tissue.