Advantages of current fetal neuroimaging and genomic technologies in prenatal diagnosis: A clinical case.

The diagnosis of prenatal microcephaly, as well as the possibility of underlining a genetic cause, is becoming more frequent thanks to advances in prenatal imaging and parallel massive sequencing. One case of primary microcephaly in three sibs demonstrates how complementary diagnostic exams can help to diagnose and establish the etiology.

Ultrasound and molecular prenatal diagnosis of Beckwith-Wiedemann syndrome: Two case reports.

Beckwith-Wiedemann syndrome (BWS) is a rare genetic disease, characterized by macrosomia, congenital malformations and tumor predisposition, associated with genetic and epigenetic alterations in the 11p15 region. Most cases are diagnosed after birth, with prenatal diagnosis being difficult and depending on the identification of specific ultrasound anomalies, namely macrosomia, macroglossia, omphalocele and renal dysplasia. Case 1: Ultrasound diagnosis at 13 weeks of isolated omphalocele with normal array. At 20 weeks, there were shortened fetal long bones, foot deformity, macroglossia, corpus callosum hypoplasia and bilateral nephromegaly. Due to the polymalformative syndrome, a termination of pregnancy (TOP) was performed. The anatomopathological study of the placenta identified mesenchymal dysplasia. The search for the methylation pattern of the 11p15 region by MS-MLPA was normal and the molecular study of the CDKN1C gene identified a likely pathogenic variant, inherited from the mother. Case 2: Morphological ultrasound at 21 weeks revealed macrosomia, macroglossia, omphalocele, bilateral renal dysplasia, and hydramnios. The cytogenetic study, after amniocentesis, was normal (46,XX karyotype). TOP was performed. The anatomopathological study of the fetus confirmed the described malformations and the one concerning the placenta identified placentomegaly. The search for the methylation pattern of the 11p15 region by MS-MLPA revealed abnormal methylation. These results confirmed the diagnosis of BWS in both cases. Prenatal ultrasound suspicion of this pathology is extremely important to guide the conduct in pregnancy and/or the prevention of perinatal complications. Shortened fetal long bones and foot deformity complement the broad spectrum of this syndrome. Positive molecular tests allow confirming the diagnosis, assessing the risk of recurrence and guiding the surveillance of future pregnancy.

Impact of fetal chromosomal disorders on maternal blood metabolome: toward new biomarkers?

OBJECTIVE: This study aimed at determining the relationship between fetal chromosomal disorders (CDs), including trisomy 21 (T21), and on first- and second-trimester maternal blood plasma, to identify the time-course metabolic adaptations to the conditions and the possible new plasma biomarkers. Furthermore, a definition of a joint circulatory (plasma) and excretory (urine) metabolic description of second-trimester CDs was sought. STUDY DESIGN: Plasma was obtained for 119 pregnant women: 74 controls and 45 CD cases, including 22 T21 cases. Plasma and lipid extracts (for T21 only) were analyzed by nuclear magnetic resonance spectroscopy, and data were handled by variable selection and multivariate analysis. Correlation analysis was used on a concatenated plasma/urine matrix descriptive of second-trimester CD, based on previously obtained urine data. RESULTS: CD cases were accompanied by enhanced lipid ß-oxidation (increased ketone bodies) and underutilization of glucose, pyruvate, and citrate. Lower circulating high-density lipoprotein levels were noted, along with changes in the proline and methanol in the first trimester, and also the urea, creatinine, acetate, and low-density lipoprotein plus very low-density lipoprotein in the second trimester and the different urea and creatinine levels, suggesting fetal renal dysfunction. In terms of plasma composition, T21 cases were indistinguishable from other CDs in the first trimester, whereas in the second trimester, increased methanol and albumin may be T21 specific. Furthermore, first-trimester lipid extracts of T21 showed decreased levels of 18:2 fatty acids, whereas in the second trimester, lower levels of 20:4 and 22:6 fatty acids were noted, possibly indicative of inflammation mechanisms. In both trimesters, high classification rates for CDs (88-89%) and T21 (85-92%) generally relied on variable selection of nuclear magnetic resonance data. Plasma/urine correlations confirmed most metabolic deviations and unveiled possible new ones regarding low-density lipoprotein plus very low-density lipoprotein, sugar, and gut-microflora metabolisms. CONCLUSION: This work partially confirmed previously reported data on first-trimester T21 and provided additional information on time-course metabolic changes accompanying CD and T21, in particular regarding plasma lipid composition. These results demonstrate the potential of plasma metabolomics in monitoring and characterizing CD cases; however, validation in larger cohorts is desirable.

Prediction of Gestational Diabetes through NMR Metabolomics of Maternal Blood.

Metabolic biomarkers of pre- and postdiagnosis gestational diabetes mellitus (GDM) were sought, using nuclear magnetic resonance (NMR) metabolomics of maternal plasma and corresponding lipid extracts. Metabolite differences between controls and disease were identified through multivariate analysis of variable selected (1)H NMR spectra. For postdiagnosis GDM, partial least squares regression identified metabolites with higher dependence on normal gestational age evolution. Variable selection of NMR spectra produced good classification models for both pre- and postdiagnostic GDM. Prediagnosis GDM was accompanied by cholesterol increase and minor increases in lipoproteins (plasma), fatty acids, and triglycerides (extracts). Small metabolite changes comprised variations in glucose (up regulated), amino acids, betaine, urea, creatine, and metabolites related to gut microflora. Most changes were enhanced upon GDM diagnosis, in addition to newly observed changes in low-Mw compounds. GDM prediction seems possible exploiting multivariate profile changes rather than a set of univariate changes. Postdiagnosis GDM is successfully classified using a 26-resonance plasma biomarker. Plasma and extracts display comparable classification performance, the former enabling direct and more rapid analysis. Results and putative biochemical hypotheses require further confirmation in larger cohorts of distinct ethnicities.

Following healthy pregnancy by NMR metabolomics of plasma and correlation to urine.

This work presents the first NMR metabolomics study of maternal plasma during pregnancy, including correlation between plasma and urine metabolites. The expected decrease in circulating amino acids early in pregnancy was confirmed with six amino acids being identified as required by the fetus in larger extents. Newly observed changes in citrate, lactate, and dimethyl sulfone suggested early adjustments in energy and gut microflora metabolisms. Alterations in creatine levels were also noted, in addition to creatinine variations reflecting alterations in glomerular filtration rate. Regarding plasma macromolecules, HDL and LDL+VLDL levels were confirmed to increase throughout pregnancy, although at different rates and accompanied by increases in fatty acid chain length and degree of unsaturation. Correlation studies suggested (a) an inverse relationship between lipoproteins (HDL and LDL+VLDL) and albumin, with a possible direct correlation to excreted (unassigned) pregnancy markers resonating at δ 0.55 and δ 0.63, (b) a direct link between LDL+VLDL and N-acetyl-glycoproteins, together with excreted marker at δ 0.55, and (c) correlation of plasma albumin with particular circulating and excreted metabolites. These results have unveiled specific lipoprotein/protein metabolic aspects of pregnancy with impact on the excreted metabolome and, therefore, provide an interesting lead for the further understanding of pregnancy metabolism.

Human plasma stability during handling and storage: impact on NMR metabolomics.

This work contributes to fill in some existing gaps in the knowledge of human plasma degradability during handling and storage, a paramount issue in Nuclear Magnetic Resonance (NMR) metabolomics. Regarding the comparison between heparin and EDTA anti-coagulant collection tubes, the former showed no interference of the polysaccharide, while conserving full spectral information. In relation to time/temperature conditions, room temperature was seen to have a large impact on lipoproteins and choline compounds from 2.5 hours. In addition, short-term storage at -20 °C was found suitable up to 7 days but, for periods up to 1 month, -80 °C was recommended. Furthermore, in the case of reusing plasma samples, no more than 3 consecutive freeze-thaw cycles were found advisable. Finally, the impact of long-term -80 °C storage (up to 2.5 years) was found almost negligible, as evaluated on a partially matched non-fasting cohort (n = 49), after having investigated the possible confounding nature of the particular non-fasting conditions employed.

Second trimester maternal urine for the diagnosis of trisomy 21 and prediction of poor pregnancy outcomes.

Given the recognized lack of prenatal clinical methods for the early diagnosis of preterm delivery, intrauterine growth restriction, preeclampsia and gestational diabetes mellitus, and the continuing need for optimized diagnosis methods for specific chromosomal disorders (e.g., trisomy 21) and fetal malformations, this work sought specific metabolic signatures of these conditions in second trimester maternal urine, using (1)H Nuclear Magnetic Resonance ((1)H NMR) metabolomics. Several variable importance to the projection (VIP)- and b-coefficient-based variable selection methods were tested, both individually and through their intersection, and the resulting data sets were analyzed by partial least-squares discriminant analysis (PLS-DA) and submitted to Monte Carlo cross validation (MCCV) and permutation tests to evaluate model predictive power. The NMR data subsets produced significantly improved PLS-DA models for all conditions except for pre-premature rupture of membranes. Specific urinary metabolic signatures were unveiled for central nervous system malformations, trisomy 21, preterm delivery, gestational diabetes, intrauterine growth restriction and preeclampsia, and biochemical interpretations were proposed. This work demonstrated, for the first time, the value of maternal urine profiling as a complementary means of prenatal diagnostics and early prediction of several poor pregnancy outcomes.

Mid-infrared (MIR) metabolic fingerprinting of amniotic fluid: a possible avenue for early diagnosis of prenatal disorders?

This work describes a mid-infrared (MIR) metabolic profiling study of 2nd trimester amniotic fluid in relation to selected prenatal disorders, with results focusing on fetal malformations (FM), preterm delivery (PTD) and premature rupture of membranes (PROM), the latter two conditions occurring later in pregnancy. Partial least squares-discriminant analysis (PLS-DA) models were obtained for FM and pre-PTD subject groups, supported by Monte Carlo Cross Validation (MCCV), and identified specific MIR profile changes. For pre-PROM subjects, minor changes were noted. MIR interpretation was assisted by intra- (MIR/MIR) and inter- (MIR/NMR) domain statistical correlation analysis, the results unveiling possible biomarker MIR signatures for FM and pre-PTD subjects. Biofluid MIR metabolic profiling holds enticing possibilities as a low cost, easy to use, rapid method and the results presented have shown its sensitivity to clinically diagnosed conditions such as FM, and to the pre-clinical stages of PTD. Specific improvement needs are discussed, namely regarding sample numbers and experimental reproducibility.

Following healthy pregnancy by nuclear magnetic resonance (NMR) metabolic profiling of human urine.

In this work, untargeted NMR metabonomics was employed to evaluate the effects of pregnancy on the metabolite composition of maternal urine, thus establishing a control excretory trajectory for healthy pregnancies. Urine was collected for independent groups of healthy nonpregnant and pregnant women (in first, second, third trimesters) and multivariate analysis performed on the corresponding NMR spectra. Models were validated through Monte Carlo Cross Validation and permutation tests and metabolite correlations measured through Statistical Total Correlation Spectroscopy. The levels of 21 metabolites were found to change significantly throughout pregnancy, with variations observed for the first time to our knowledge for choline, creatinine, 4-deoxyerythronic acid, 4-deoxythreonic acid, furoylglycine, guanidoacetate, 3-hydroxybutyrate, and lactate. Results confirmed increased aminoaciduria across pregnancy and suggested (a) a particular involvement of isoleucine and threonine in lipid oxidation/ketone body synthesis, (b) a relation of excreted choline, taurine, and guanidoacetate to methionine metabolism and urea cycle regulation, and (c) a possible relationship of furoylglycine and creatinine to pregnancy, based on a tandem study of nonfasting confounding effects. Results demonstrate the usefulness of untargeted metabonomics in finding biomarker metabolic signatures for healthy pregnancies, against which disease-related deviations may be confronted in future studies, as a base for improved diagnostics and prediction.

UPLC-MS metabolic profiling of second trimester amniotic fluid and maternal urine and comparison with NMR spectral profiling for the identification of pregnancy disorder biomarkers.

We report on the first untargeted UPLC-MS study of 2nd trimester maternal urine and amniotic fluid (AF), to investigate the possible metabolic effects of fetal malformations (FM), gestational diabetes mellitus (GDM) and preterm delivery (PTD). For fetal malformations, considerable metabolite variations were identified in AF and, to a lesser extent, in urine. Using validated PLS-DA models and statistical correlations between UPLC-MS data and previously acquired NMR data, a metabolic picture of fetal hypoxia, enhanced gluconeogenesis, TCA activity and hindered kidney development affecting FM pregnancies was reinforced. Moreover, changes in carnitine, pyroglutamate and polyols were newly noted, respectively, reflecting lipid oxidation, altered placental amino acid transfer and alterations in polyol pathways. Higher excretion of conjugated products in maternal urine was seen suggesting alterations in conjugation reactions. For the pre-diagnostic GDM group, no significant changes were observed, either considering amniotic fluid or maternal urine, whereas, for the pre-PTD group, some newly observed changes were noted, namely, the decrease of particular amino acids and the increase of an hexose (possibly glucose), suggesting alteration in placental amino acid fluxes and a possible tendency for hyperglycemia. This work shows the potential of UPLC-MS for the study of fetal and maternal biofluids, particularly when used in tandem with comparable NMR data. The important roles played by sampling characteristics (e.g. group dimensions) and the specific experimental conditions chosen for MS methods are discussed.

Lesão vascular da placenta condicionando RCIU e hidropisia fetal não imune em gestação gemelar / Placental vascular lesion as cause of IUGR and nonimmune fetal hydrops in twin pregnanc

As lesões vasculares da placenta constituem um grupo de entidades distintas, mas inter-relacionadas, em que se incluem os corioangiomas e a corangiomatose multifocal difusa. O corioangioma é uma lesão nodular expansiva com incidência de cerca de 1 por cento. A corangiomatose multifocal difusa é rara (0,2 por cento) e predominante em placentas em idade gestacional inferior a 32 semanas. Os autores apresentam um caso de gestação gemelar monocoriônica/biamniótica, no qual um dos fetos, à 26ª semana de gestação, apresentou quadro de restrição de crescimento intrauterino, hidropisia e anemia associado à formação tumoral da placenta com vascularização aumentada verificada pela doplervelocimetria. O estudo anatomopatológico da placenta permitiu o diagnóstico de corangiomatose multifocal difusa. Este raro caso de corioangiomatose multifocal difusa com forma de apresentação pré-natal mimetizando a de um corioangioma comprova que a detecção ultrassonográfica de um tumor da placenta com vascularização aumentada deve suscitar outras hipótese diagnóstica, além do corioangioma.

Placenta vascular lesions are a group of distinct yet related entities that include chorangiomas and diffuse multifocal chorangiomatosis. Chorangioma is an expansive nodular lesion with an incidence of about 1 percent. Diffuse multifocal chorangiomatosis is rare (0.2 percent) and mostly seen in placentas before the 32nd gestational week. The authors present a case of a monochorionic/biamniotic twin pregnancy, in which, at the 26th gestational week, one fetus developed intrauterine growth restriction (IUGR), hydrops, and anemia associated with a tumor of the placenta with increased vascularization in the Doppler study. Pathological examination of the placenta diagnosed diffuse multifocal chorangiomatosis. This rare case report of diffuse multifocal chorangiomatosis with prenatal manifestations resembling those of a chorangioma proves that prenatal ultrasound detection of a placenta tumor, with increased vascularization at Doppler study, must raise other diagnostic possibilities beside chorangioma.

Metabolic biomarkers of prenatal disorders: an exploratory NMR metabonomics study of second trimester maternal urine and blood plasma.

This work describes an exploratory NMR metabonomic study of second trimester maternal urine and plasma, in an attempt to characterize the metabolic changes underlying prenatal disorders and identify possible early biomarkers. Fetal malformations have the strongest metabolic impact in both biofluids, suggesting effects due to hypoxia (leading to hypoxanthine increased excretion) and a need for enhanced gluconeogenesis, with higher ketone bodies (acetone and 3-hydroxybutyric acid) production and TCA cycle demand (suggested by glucogenic amino acids and cis-aconitate overproduction). Choline and nucleotide metabolisms also seem affected and a distinct plasma lipids profile is observed for mothers with fetuses affected by central nervous system malformations. Urine from women who subsequently develop gestational diabetes mellitus exhibits higher 3-hydroxyisovalerate and 2-hydroxyisobutyrate levels, probably due to altered biotin status and amino acid and/or gut metabolisms (the latter possibly related to higher BMI values). Other urinary changes suggest choline and nucleotide metabolic alterations, whereas lower plasma betaine and TMAO levels are found. Chromosomal disorders and pre-preterm delivery groups show urinary changes in choline and, in the latter case, in 2-hydroxyisobutyrate. These results show that NMR metabonomics of maternal biofluids enables the noninvasive detection of metabolic changes associated to prenatal disorders, thus unveiling potential disorder biomarkers.

Accuracy of prenatal diagnosis in elective termination of pregnancy: 385 cases from 2000 to 2007.

Objective. To evaluate the quality of prenatal results in all cases of termination of pregnancy (TOP) due to fetal abnormalities in a tertiary prenatal diagnosis center. Material and Methods. Retrospective analysis of the 385 TOP performed on our department due to fetal abnormalities between January 1, 2000, and December 31, 2007. We compared all data for agreement between the ultrasound, genetic, and postmortem findings, regarding the abnormalities identified in the etiological diagnosis and its prognosis. Results. Chromosome abnormalities were the most common indication for TOP (39%), followed by abnormalities of CNS (20%), monogenic disorders (11%), sequences (9.6%), polimalformative syndromes (5.2%), and isolated congenital heart diseases (4%). Total agreement was 21%. Further abnormalities were identified in 79%. The data collected after TOP changed the etiologic diagnosis in 21% but the prognosis was changed in only one fetus. Discussion. This study corroborates the necessity of a multidisciplinary team in prenatal diagnosis centers. Their work remarkably improves the genetic counseling and represents an important aspect in quality control of the information given to a couple previously to a TOP.

[Placental vascular lesion as cause of IUGR and nonimmune fetal hydrops in twin pregnancy]. / Lesão vascular da placenta condicionando RCIU e hidropisia fetal não imune em gestação gemelar.

Placenta vascular lesions are a group of distinct yet related entities that include chorangiomas and diffuse multifocal chorangiomatosis. Chorangioma is an expansive nodular lesion with an incidence of about 1%. Diffuse multifocal chorangiomatosis is rare (0.2%) and mostly seen in placentas before the 32nd gestational week. The authors present a case of a monochorionic/biamniotic twin pregnancy, in which, at the 26th gestational week, one fetus developed intrauterine growth restriction (IUGR), hydrops, and anemia associated with a tumor of the placenta with increased vascularization in the Doppler study. Pathological examination of the placenta diagnosed diffuse multifocal chorangiomatosis. This rare case report of diffuse multifocal chorangiomatosis with prenatal manifestations resembling those of a chorangioma proves that prenatal ultrasound detection of a placenta tumor, with increased vascularization at Doppler study, must raise other diagnostic possibilities beside chorangioma.

Impact of prenatal disorders on the metabolic profile of second trimester amniotic fluid: a nuclear magnetic resonance metabonomic study.

This paper describes a metabonomic study of prenatal disorders using nuclear magnetic resonance (NMR) spectroscopy of amniotic fluid (AF) collected in the second trimester of pregnancy, to search for metabolite markers of fetal malformations, prediagnostic gestational diabetes (GD), preterm delivery (PTD), early rupture of membranes (PROM), and chromossomopathies. Fetal malformations were found to have the highest impact on AF metabolite composition, enabling statistical validation to be achieved by several multivariate analytical tools. Results confirmed previous indications that malformed fetuses seem to suffer altered energy metabolism and kidney underdevelopment. Newly found changes (namely in α-oxoisovalerate, ascorbate, creatinine, isoleucine, serine, threonine) suggest possible additional effects on protein and nucleotide sugar biosynthesis. Prediagnostic GD subjects showed an average increase in glucose and small decreases in several amino acids along with acetate, formate, creatinine, and glycerophosphocholine. Small metabolite changes were also observed in the AF of subjects eventually undergoing PTD and PROM, whereas no relevant changes were found for chromossomopathies (for which a low number of samples was considered). The potential value of these results for biochemical insight and prediction of prenatal disorders is discussed, as well as their limitations regarding number of samples and overlap of different disorders.

(1)H NMR based metabonomics of human amniotic fluid for the metabolic characterization of fetus malformations.

An NMR-metabonomic study of malformed fetuses was carried out through human amniotic fluid (HAF) analysis. Over 70 compounds were detected in control HAF by NMR. Possible confounding variables (fetus gender and gestational and maternal ages) were shown not to induce detectable compositional trends in the control group considered. Malformed fetuses showed variations in glucose, some amino acids and organic acids and proteins. In tandem with enzymatic assays, these NMR results suggest that changes in gycolysis and gluconeogenesis as well as kidney underdevelopment occur in the malformed fetuses studied here.