Confined Placental Mosaicism in Chorionic Vilus Sampling - Case Report

Objectives: Chorionic Vilus Sampling (CVS) has several advantages over amniocentesis: it may be performed at an earlier gestational age, the results are quicker to obtain and there’s a lower miscarriage risk – 1%. However, the higher prevalence of discrepant fetal and vilus sampling material’s karyotype findings is a disadvantage of this technique – 0.5%. This is caused, amongst other causes, by placental mosaicism which consists of two genetically different cell lines. There are three types of placental mosaicism according to the abnormal cell line location: Type I – in the cytotrophoblast; Type II – in the vilus’ stroma; Type III – in both the above locations. Material and Methods: We present a case report about a 36-year-old pregnant woman going through our Department’s 1st trimester combined screening program; a CVS was performed, which showed Confined Placental Mosaicism (CPM). Results and Conclusion: Although the pregnant woman was in the low-risk group for aneuploidy, the patient wanted the cytogenetic study to be performed in order to reduce maternal anxiety. CVS was performed at the gestational age of 12 weeks + 5 days and the karyotype was 47XY+2/46XY. For the correct interpretation of this data an amniocentesis was performed at the gestational age of 15 weeks + 6 days, which showed a 46XY karyotype. We therefore conclude that the cytogenetic analysis of the CVS was the result of a CPM. A careful follow-up including fetal echocardiogram and seriated ultrasonographic monitoring was used to safely exclude malformations and fetal growth restriction. We verified no occurences throughout pregnancy, delivery and perinatal period. CVS practice was recently implemented in our country and has many advantages over amniocentesis. Besides the fact that an earlier gestational age usually means less affective bonding to the fetus and therefore makes medical termination of pregnancy somewhat less difficult, one should consider specific situations like the one reported in which CPM may be diagnosed. This condition is associated with increased risk of fetal growth restriction, so the clinician should be aware of the need for a more careful follow-up, since perinatal complications, which should be anticipated and treated, can be expected in 16-21% of these cases.

Análise dos resultados maternos e fetais dos procedimentos invasivos genéticos fetais: um estudo exploratório em Hospital Universitário

OBJETIVO: Caracterizar as indicações das gestantes que procuraram o serviço de Medicina Fetal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para realização de procedimentos invasivos diagnósticos e avaliar os resultados dos cariótipos fetais e de suas gestações. MÉTODOS: Estudo observacional retrospectivo das gestantes que realizaram biópsia de vilo corial (BVC), amniocentese e cordocentese no período de fevereiro de 2005 a dezembro de 2009. Não foram incluídos outros procedimentos diagnósticos ou procedimentos terapêuticos. O resultado da gestação foi obtido através de consulta de prontuário eletrônico e/ou físico e/ou contato telefônico. RESULTADOS: Foram realizados 713 procedimentos (113 BVC, 340 amniocenteses e 260 cordocenteses). A principal indicação para a realização dos procedimentos invasivos foi a presença de alterações estruturais nos fetos, seguido por valores aumentados da translucência nucal e pela idade materna avançada. O cariótipo fetal esteve alterado em 186 casos (26,1%). A trissomia do cromossomo 18 foi a aneuploidia mais comum, seguida pela trissomia do 21, a monossomia do X e a trissomia do cromossomo 13. Ocorreram 4,9% de abortamento, 25,7% de natimortos e 13% de neomortos. Oito gestantes optaram pela interrupção judicial, e 99% das gestantes cujos fetos não apresentavam malformação e que apresentavam cariótipo fetal normal tiveram nativivos.

ATTITUDES TOWARD AND UTILIZATION OF NON-INVASIVE PRENATAL TESTING FOR CHROMOSOME ANEUPLOIDY AMONG OB/GYNS

Prenatal diagnosis is traditionally made via invasive procedures such as amniocentesis and chorionic villus sampling (CVS). However, both procedures carry a risk of complications, including miscarriage. Many groups have spent years searching for a way to diagnose a chromosome aneuploidy without putting the fetus or the mother at risk for complications. Non-invasive prenatal testing (NIPT) for chromosome aneuploidy became commercially available in the fall of 2011, with detection rates similar to those of invasive procedures for the common autosomal aneuploidies (Palomaki et al., 2011; Ashoor et al. 2012; Bianchi et al. 2012). Eventually NIPT may become the diagnostic standard of care and reduce invasive procedure-related losses (Palomaki et al., 2011). The integration of NIPT into clinical practice has potential to revolutionize prenatal diagnosis; however, it also raises some crucial issues for practitioners. Now that the test is clinically available, no studies have looked at the physicians that will be ordering the testing or referring patients to practitioners who do. This study aimed to evaluate the attitudes of OB/GYN’s and how they are incorporating the test into clinical practice. Our study shows that most physicians are offering this new, non-invasive technology to their patients, and that their practices were congruent with the literature and available professional society opinions. Those physicians who do not offer NIPT to their patients would like more literature on the topic as well as instructive guidelines from their professional societies. Additionally, this study shows that the practices and attitudes of MFMs and OBs differ. Our population feels that the incorporation of NIPT will change their practices by lowering the amount of invasive procedures, possibly replacing maternal serum screening, and that it will simplify prenatal diagnosis. However, those physicians who do not offer NIPT to their patients are not quite sure how the test will affect their clinical practice. From this study we are able to glean how physicians are incorporating this new technology into their practice and how they feel about the addition to their repertoire of tests. This knowledge gives insight as to how to best move forward with the quickly changing field of prenatal diagnosis.

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.

Health and developmental outcome of children following prenatal diagnosis of confined placental mosaicism

Objective To determine the long-term health and development of a cohort of children in whom confined placental mosaicism (CPM) was diagnosed at prenatal diagnosis. Methods A retrospective cohort study was performed comparing 36 children in whom CPM had been diagnosed prenatally with 195 controls subjects in whom a normal karyotype had been detected prenatally. Data comprising birth information, health, health service utilisation, growth, development, behaviour, and the family were collected by a maternal questionnaire administered when the subjects were aged between 4 and 11 years. Results CPM cases did not differ from controls across a broad range of health measures and there were no major health problems or birth defects among the CPM group. No increase was detected in the incidence of intrauterine growth retardation (IUGR) among CPM cases; however, postnatal growth was reduced compared with controls (p = 0.047). Development and behaviour in CPM cases was similar to that of controls. Conclusions The prenatal diagnosis of CPM is not associated with an increased risk of birth defects or developmental problems, but may be associated with decreased growth. Copyright (C) 2006 John Wiley & Sons, Ltd.

Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration

OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.

New insights into the etiology of preeclampsia:identification of key elusive factors for the vascular complications

The incidence of preeclampsia is reduced by a third in smokers, but not in snuff users. Soluble Flt-1 (sFlt-1) and soluble endoglin (sEng) are increased prior to the clinical onset of preeclampsia. Animals exposed to high circulating levels of sFlt-1 and sEng elicit severe preeclampsia-like symptoms. Smokers have reduced circulating sFlt-1 and cigarette smoke extract decreases sFlt-1 release from placental villous explants. An anti-inflammatory enzyme, heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO), inhibit sFlt-1 and sEng release. Women with preeclampsia exhale less CO than women with normal pregnancies and HO expression decreases as the severity of preeclampsia increases. In contrast, sFlt-1 levels increase with increasing severity. More importantly, chorionic villous sampling from women at eleven weeks gestation shows that HO-1 mRNA expression is decreased in women who go on to develop preeclampsia. Collectively, these facts provide compelling evidence to support the proposition that the pathogenesis of preeclampsia is largely due to loss of HO activity. This results in an increase in inflammation and excessive elevation of the two key anti-angiogenic factors responsible for the clinical signs of preeclampsia. These findings provide strong evidence for a protective role of HO-1 in pregnancy and identify HO as a target for the treatment of preeclampsia. The cardiovascular drugs, statins, stimulate HO-1 expression and inhibit sFlt-1 release in vivo and in vitro, thus, they have the potential to ameliorate early onset preeclampsia. The StAmP trial is underway to address this and if positive, its outcome will lead to the very first therapeutic intervention to prolong affected pregnancies.

Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Postmortem measurement of human chorionic gonadotropin in vitreous humor and bile.

Postmortem human chorionic gonadotrophin (HCG) blood assay can confirm postmortem diagnosis of pregnancy or document situations in which HCG levels are elevated. In some cases, however, blood sampling is not possible at autopsy. In this study, HCG was quantified by enzyme-linked fluorescent assay (ELFA) in the bile (n = 5), vitreous humor (n = 4), and postmortem blood (n = 4) of five pregnant women. There were no false negatives in the pregnant subjects (n = 5) or false positives in controls (n = 34), enabling this test to be recommended for routine use in forensic contexts in which the detection of elevated HCG levels could be of interest.

Temporal variation of the phytoplankton community at short sampling intervals in the Mundaú reservoir, Northeastern Brazil

The aim of this study was to determine how abiotic factors drive the phytoplankton community in a water supply reservoir within short sampling intervals. Samples were collected at the subsurface (0.1 m) and bottom of limnetic (8 m) and littoral (2 m) zones in both the dry and rainy seasons. The following abiotic variables were analyzed: water temperature, dissolved oxygen, electrical conductivity, total dissolved solids, turbidity, pH, total nitrogen, nitrite, nitrate, total phosphorus, total dissolved phosphorus and orthophosphate. Phytoplankton biomass was determined from biovolume values. The role abiotic variables play in the dynamics of phytoplankton species was determined by means of Canonical Correspondence Analysis. Algae biomass ranged from 1.17×10(4) to 9.21×10(4) µg.L-1; cyanobacteria had biomass values ranging from 1.07×10(4) to 8.21×10(4) µg.L-1. High availability of phosphorous, nitrogen limitation, alkaline pH and thermal stability all favored cyanobacteria blooms, particularly during the dry season. Temperature, pH, total phosphorous and turbidity were key factors in characterizing the phytoplankton community between sampling times and stations. Of the species studied, Cylindrospermopsis raciborskii populations were dominant in the phytoplankton in both the dry and rainy seasons. We conclude that the phytoplankton was strongly influenced by abiotic variables, particularly in relation to seasonal distribution patterns.

Precision of distances and ordering of microsatellite markers in consensus linkage maps of chromosomes 1, 3 and 4 from two reciprocal chicken populations using bootstrap sampling

Some factors complicate comparisons between linkage maps from different studies. This problem can be resolved if measures of precision, such as confidence intervals and frequency distributions, are associated with markers. We examined the precision of distances and ordering of microsatellite markers in the consensus linkage maps of chromosomes 1, 3 and 4 from two F 2 reciprocal Brazilian chicken populations, using bootstrap sampling. Single and consensus maps were constructed. The consensus map was compared with the International Consensus Linkage Map and with the whole genome sequence. Some loci showed segregation distortion and missing data, but this did not affect the analyses negatively. Several inversions and position shifts were detected, based on 95% confidence intervals and frequency distributions of loci. Some discrepancies in distances between loci and in ordering were due to chance, whereas others could be attributed to other effects, including reciprocal crosses, sampling error of the founder animals from the two populations, F(2) population structure, number of and distance between microsatellite markers, number of informative meioses, loci segregation patterns, and sex. In the Brazilian consensus GGA1, locus LEI1038 was in a position closer to the true genome sequence than in the International Consensus Map, whereas for GGA3 and GGA4, no such differences were found. Extending these analyses to the remaining chromosomes should facilitate comparisons and the integration of several available genetic maps, allowing meta-analyses for map construction and quantitative trait loci (QTL) mapping. The precision of the estimates of QTL positions and their effects would be increased with such information.

Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling

Background: With nearly 1,100 species, the fish family Characidae represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems. The composition, phylogeny, and classification of Characidae is currently uncertain, despite significant efforts based on analysis of morphological and molecular data. No consensus about the monophyly of this group or its position within the order Characiformes has been reached, challenged by the fact that many key studies to date have non-overlapping taxonomic representation and focus only on subsets of this diversity. Results: In the present study we propose a new definition of the family Characidae and a hypothesis of relationships for the Characiformes based on phylogenetic analysis of DNA sequences of two mitochondrial and three nuclear genes (4,680 base pairs). The sequences were obtained from 211 samples representing 166 genera distributed among all 18 recognized families in the order Characiformes, all 14 recognized subfamilies in the Characidae, plus 56 of the genera so far considered incertae sedis in the Characidae. The phylogeny obtained is robust, with most lineages significantly supported by posterior probabilities in Bayesian analysis, and high bootstrap values from maximum likelihood and parsimony analyses. Conclusion: A monophyletic assemblage strongly supported in all our phylogenetic analysis is herein defined as the Characidae and includes the characiform species lacking a supraorbital bone and with a derived position of the emergence of the hyoid artery from the anterior ceratohyal. To recognize this and several other monophyletic groups within characiforms we propose changes in the limits of several families to facilitate future studies in the Characiformes and particularly the Characidae. This work presents a new phylogenetic framework for a speciose and morphologically diverse group of freshwater fishes of significant ecological and evolutionary importance across the Neotropics and portions of Africa.

Comparative analysis of two sampling techniques for pollen gathered by Nannotrigona testaceicornis Lepeletier (Apidae, Meliponini)

Pollen counts from samples taken from storage pots throughout one year (from October to September) were adjusted by Tasei's volumetric correction coefficient for the determination of pollen sources exploited by two colonies of Nannotrigona testaceicornis in Sao Paulo, Brazil. The results obtained by this sampling technique for seven months (December to June) were compared with those from corbicula load samples taken within the same period. This species visited a large variety of plant species, but few of them were frequently used. As a rule, pollen sources that appeared at frequencies greater than 1% were found with both sampling methods and significant positive correlations (Spearman correlation coefficient) were found between their values. The pollen load sample data showed that N. testaceicornis gathered pollen throughout the external activity period.