Villous Sampling Research Articles

Emerging role for dysregulated decidualization in the genesis of preeclampsia

In normal human placentation, uterine invasion by trophoblast cells and subsequent spiral artery remodeling depend on cooperation among fetal trophoblasts and maternal decidual, myometrial, immune and vascular cells in the uterine wall. Therefore, aberrant function of anyone or several of these cell-types could theoretically impair placentation leading to the development of preeclampsia. Because trophoblast invasion and spiral artery remodeling occur during the first half of pregnancy, the molecular pathology of fetal placental and maternal decidual tissues following delivery may not be informative about the genesis of impaired placentation, which transpired months earlier. Therefore, in this review, we focus on the emerging prospective evidence supporting the concept that deficient or defective endometrial maturation in the late secretory phase and during early pregnancy, i.e., pre-decidualization and decidualization, respectively, may contribute to the genesis of preeclampsia. The first prospectively-acquired data directly supporting this concept were unexpectedly revealed in transcriptomic analyses of chorionic villous samples (CVS) obtained during the first trimester of women who developed preeclampsia 5 months later. Additional supportive evidence arose from investigations of Natural Killer cells in first trimester decidua from elective terminations of women with high resistance uterine artery indices, a surrogate for deficient trophoblast invasion. Last, circulating insulin growth factor binding protein-1, which is secreted by decidual stromal cells was decreased during early pregnancy in women who developed preeclampsia. We conclude this review by making recommendations for further prospectively-designed studies to corroborate the concept of endometrial antecedents of preeclampsia. These studies could also enable identification of women at increased risk for developing preeclampsia, unveil the molecular mechanisms of deficient or defective (pre)decidualization, and lead to preventative strategies designed to improve (pre)decidualization, thereby reducing risk for preeclampsia development.

Pharmacokinetics and Placental Transfer of Elvitegravir, Dolutegravir, and Other Antiretrovirals during Pregnancy.

The integrase inhibitors elvitegravir (EVG) and dolutegravir (DTG) rapidly decrease the plasma HIV-1 viral load, a key factor in the prevention of maternal-to-fetal transmission of HIV-1. No data have been reported on the concentrations of these drugs in cord blood, maternal peripheral blood mononuclear cells (PBMCs), or placental tissue in pregnant women. We present in vivo pharmacokinetic data on antiretrovirals (ARV) within maternal and cord blood and within placentae from HIV-1-infected pregnant women. Maternal blood and cord blood were obtained from women receiving EVG, cobicistat, tenofovir disoproxil fumarate, and emtricitabine as a single fixed-dose combination formulation or DTG as part of a combination regimen. Plasma and PBMCs from maternal and cord blood were obtained along with villous placental samples. Drug concentrations were simultaneously determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Utilizing medians and ranges to interpret our data, we compared the drug concentration ratios between different matrices (maternal and cord blood plasma, PBMCs, and placenta). All five agents transferred from maternal into fetal circulation via the placenta. Concentration ratios for EVG, cobicistat, tenofovir, and emtricitabine (n = 10) and DTG (n = 3) were determined between cord plasma and placenta, cord and maternal plasma, and cord PBMCs and maternal PBMCs. TFV moves from maternal plasma through the placenta to the cord blood and then into cord PBMCs, where it is phosphorylated into its active forms (TFV diphosphate). These five ARVs were detected in each of the compartments, highlighting transfer of these agents from the maternal into the fetal circulation.

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells.

Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. APPROACH AND RESULTS: BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks' gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 (ANGPT4), platelet-derived growth factor receptor α (PDGFRA), tumor necrosis factor superfamily member 15 (TNFSF15), angiogenin (ANG), serpin family C member 1 (SERPIN1), angiopoietin 2 (ANGPT2), and CXC motif chemokine 12 (CXCL12) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies.

PREVENTION OF THALASSEMIA;

Objectives: The study was conducted to determine role of CVS in preventionof thalassemia and its sensitivity and specificity in those families with thalassemia positivehistory. Period: Three months. Study Design: Cross sectional study. Material and Methods:130 women who had thalassemic children and had come to transfusion centers for bloodtransfusion. Questionnaire was used for interview. Results: out of the total 130 women, 101were aware of CVS technique in which 50 had CVS. In 50 CVS, 15 had positive result while 35had negative result. In 15 positive cases 11 had abortion while four positive women refusedto abort. Out of the four women who completed their term, three were true positive while onecase was false positive. In 35 negative case results, 32 were true negative and three werefalse negative. Conclusion: Thalassemia is the commonest genetic disorder worldwide. It istransmitted from those parents who are carrier of thalassemia. Chorionic villous sampling playsimportant role in prevention of thalassemia during 1st trimester of pregnancy.

English

Objectives: The study was conducted to determine role of CVS in preventionof thalassemia and its sensitivity and specificity in those families with thalassemia positivehistory. Period: Three months. Study Design: Cross sectional study. Material and Methods:130 women who had thalassemic children and had come to transfusion centers for bloodtransfusion. Questionnaire was used for interview. Results: out of the total 130 women, 101were aware of CVS technique in which 50 had CVS. In 50 CVS, 15 had positive result while 35had negative result. In 15 positive cases 11 had abortion while four positive women refusedto abort. Out of the four women who completed their term, three were true positive while onecase was false positive. In 35 negative case results, 32 were true negative and three werefalse negative. Conclusion: Thalassemia is the commonest genetic disorder worldwide. It istransmitted from those parents who are carrier of thalassemia. Chorionic villous sampling playsimportant role in prevention of thalassemia during 1st trimester of pregnancy.

Diagnóstico prenatal de anomalías cromosómicas Biopsia de vellosidades coriales y amniocentesis para cariotipo fetal

Introducción: El diagnóstico prenatal temprano de anomalías cromosómicas requiere de técnicas invasivas, como la biopsia de vellosidades coriales (BVC) y la amniocentesis (AMC), con el fin de obtener células fetales, cultivarlas y obtener el cariotipo en los fetos con riesgo alto para estas anomalías, identificadas mediante marcadores ecográficos y bioquímicos desde las 11 semanas. Reportamos nuestra experiencia hasta junio del año 2016. Diseño: Estudio descriptivo longitudinal. Institución: Instituto Latinoamericano de Salud Reproductiva (ILSAR), Lima, Perú. Participantes: Fetos de primer y segundo trimestres del embarazo. Intervenciones: Se analizó los resultados del estudio de 400 fetos que cursaban el primer y segundo trimestre y que tenían riesgo alto para anomalías cromosómicas (mayor de 1/270 inicialmente y 1/100 desde el año 2012), resultado cuantificado utilizando la base de datos del Fetal Test de España más el resultado de los marcadores bioquímicos (riesgo combinado). El análisis del cariotipo fetal se realizó en muestras obtenidas por medio de 338 amniocentesis genéticas y 62 biopsias de vellosidades coriales realizadas durante el período comprendido de enero 2003 a junio 2016 en nuestro centro ILSAR. Principales medidas de resultados: Presencia de arcadores ecográficos y normalidad de los cariotipos. Resultados: Los marcadores ecográficos encontrados con mayor frecuencia fueron: higroma quístico (35,8%), translucencia nucal aumentada (13%), ductus venoso con onda de velocidad de flujo anormal (8,5%), dos o más marcadores asociados a anomalías fetales (13,7%). De 400 muestras estudiadas, 141 (35%) fueron cariotipos anormales: 64 (45%) T21, 35 (25%) T18, 21 (15%) 45X, 7 (5%) T13, 14 (10%) otras anomalías. No hubo complicación importante alguna atribuida al procedimiento invasivo. Conclusiones: En los fetos con riesgo alto para anomalías cromosómicas estudiados, el 35% tuvo cariotipo anormal, siendo las más frecuentes las trisomías de los cromosomas 21 y 18, seguidas de la monosomía del cromosoma X. El higroma quístico, la translucencia nucal aumentada y la presencia de 2 o más marcadores asociados a anomalías fetales fueron los hallazgos más frecuentes en la determinación del riesgo ecográfico. El higroma quístico mostró el mayor valor predictivo para anomalías cromosómicas.

НАРУШЕНИЕ ПРЕОБРАЗОВАНИЯ АНДРОСТЕНДИОЛА В ПЛАЦЕНТЕ ПРИ БЕРЕМЕННОСТИ, ОСЛОЖНЕННОЙ РЕАКТИВАЦИЕЙ ЦИТОМЕГАЛОВИРУСНОЙ ИНФЕКЦИИ В ПЕРВОМ ТРИМЕСТРЕ

The aim is to analyze androstenediol converting in villous chorions at gestation against cytomegalovirus infection activation during the first trimester of pregnancy. 48 villous chorions samples taken during medical abortion at 8-10th weeks of gestation of women with chronic cytomegalovirus reactivation during pregnancy became the material for study, and 35 villous chorions samples were taken as the control. The results of pregnant women examination were analyzed from the point of cytomegalovirus infection activity with immune-enzyme method by the presence of IgM antibodies or by the value of 4-times and more growth of IgG antibodies titer in paired sera in dynamics in 10 days. IgG avidity index to cytomegalovirus was more than 75% in all women. Androstenediol converting was assessed with histochemical methods. Quantitative evaluation of the reaction products was carried out on sections under the microscope MT (Japan) associated with software-hardware complex «SCION Corporation» (USA). Histochemical reaction product of the androstenediol dehydrogenase was well detected in cyto- and syncytiotrophoblast of chorious villi. Reaction activity in the material from women with reactivation of cytomegalovirus infection during pregnancy was low, which indicated a reduction in the reserve for the synthesis of estrogen. The concentration of estriol and estradiol in villous chorions during reactivation of cytomegalovirus infection decreased till 24.9±2.01 nmol/l and 15765.1±107.77 pmol/l, respectively.

Maternal prenatal depression is associated with decreased placental expression of the imprinted gene PEG3.

Maternal prenatal stress during pregnancy is associated with fetal growth restriction and adverse neurodevelopmental outcomes, which may be mediated by impaired placental function. Imprinted genes control fetal growth, placental development, adult behaviour (including maternal behaviour) and placental lactogen production. This study examined whether maternal prenatal depression was associated with aberrant placental expression of the imprinted genes paternally expressed gene 3 (PEG3), paternally expressed gene 10 (PEG10), pleckstrin homology-like domain family a member 2 (PHLDA2) and cyclin-dependent kinase inhibitor 1C (CDKN1C), and resulting impaired placental human placental lactogen (hPL) expression. A diagnosis of depression during pregnancy was recorded from Manchester cohort participants' medical notes (n = 75). Queen Charlotte's (n = 40) and My Baby and Me study (MBAM) (n = 81) cohort participants completed the Edinburgh Postnatal Depression Scale self-rating psychometric questionnaire. Villous trophoblast tissue samples were analysed for gene expression. In a pilot study, diagnosed depression during pregnancy was associated with a significant reduction in placental PEG3 expression (41%, p = 0.02). In two further independent cohorts, the Queen Charlotte's and MBAM cohorts, placental PEG3 expression was also inversely associated with maternal depression scores, an association that was significant in male but not female placentas. Finally, hPL expression was significantly decreased in women with clinically diagnosed depression (44%, p < 0.05) and in those with high depression scores (31% and 21%, respectively). This study provides the first evidence that maternal prenatal depression is associated with changes in the placental expression of PEG3, co-incident with decreased expression of hPL. This aberrant placental gene expression could provide a possible mechanistic explanation for the co-occurrence of maternal depression, fetal growth restriction, impaired maternal behaviour and poorer offspring outcomes.

Chorionic villous sampling through transvaginal ultrasound approach: A retrospective analysis of 1138 cases.

The aim of this study was to evaluate the effectiveness and safety of a transvaginal approach for chorionic villous sampling (CVS). We carried out a retrospective data analysis of all the transvaginal CVS procedures performed for the purpose of prenatal diagnosis in a university-level referral center between January 2000 and December 2014. Women underwent the prenatal testing between 10 and 17 weeks of gestation mainly for hematological disorders involving single gene defects. The main outcomes were successful sampling rate, maternal contamination rate, post-procedure complications rates, and immediate fetal loss rate (<14 days post-procedure). A total of 1138 transvaginal CVS were performed during the study period and were available for analysis. The sampling success rate after the first attempt was 98.5% (1121/1138) and the overall success rate was 99.6% (1133/1138). The maternal contamination rate was 0.4% (5/1138). While two patients had vaginal bleeding (0.2%), fresh retroplacental collection was noted in four patients (0.4%) post-procedure. None of the patients developed ascending uterine infection following CVS. The immediate fetal loss rate was 0.2% (2/1138). Transvaginal approach is associated with high sampling success, along with low rates of maternal contamination and post-procedure complications; hence, it can be offered as an effective alternative method of CVS.

The Iranian Integrated Maternal Health Care Guideline Based on Evidence-Based Medicine and American Guidelines: A Comparative Study

Background: There is a little consistency among prenatal care guidelines. An evidence-based guideline can be used to improve the quality of the mother and child health care. This study was designed to compare the consistency of the Iranian integrated maternal health care guideline with evidence-based prenatal care manuals and American guidelines. Methods: In this comparative study, the Iranian guideline recommendations were compared with the content of American prenatal care according to obstetrics and gynecology recommendations of the U.S. preventive services task force and evidence-based prenatal care. The strength of the recommendations was assessed based on evidence-based medicine. Results: In this study, 71 recommendations were compared in four parts. About 18.3 percent of the recommendations were consistent with all guidelines. Screening for group B streptococcus, performing chorionic villous sampling and amniocentesis for at risk women, and anomaly screening were not mentioned in the Iranian guideline. The Iranian guideline was consistent with the content of U.S. preventive services task force in 38% of the recommendations, American Obstetricians and Gynecologists expert panel guidelines in 75.6% of the recommendations, and with evidence-based care in 66.2% of the recommendations. Conclusions: Although there was a little consistency among guidelines, the Iranian guideline had a good consistency with the American guidelines and evidence-based care recommendations. Updating clinical tests and the prenatal screening part of the Iranian guideline based on the results of this study may increase the effectiveness of the prenatal care and ultimately improve the mother and child’s health.

Cytogenetic highlights and transitions.

Medical cytogenetics, genetic diagnostics, and medical genetics had their origins in the late 1950's, as evaluation of human chromosomes became possible, and it was recognized that chromosomal abnormalities could cause a variety of clinical phenotypes. Dr. Laird Jackson began his medical and scientific career just as this field was emerging and he was an early adopter and driver of several key trends in the development of these fields, notably in the area of prenatal diagnostics. Laird's greatest impact was in his work to demonstrate the clinical utility of amniocentesis, chorionic villous sampling, and chromosomal microarray analysis for prenatal diagnosis. © 2016 Wiley Periodicals, Inc.

Placental PHLDA2 expression is increased in cases of fetal growth restriction following reduced fetal movements.

BackgroundMaternal perception of reduced fetal movements (RFM) is associated with increased risk of fetal growth restriction (FGR) and stillbirth, mediated by placental insufficiency. The maternally expressed imprinted gene PHLDA2 controls fetal growth, placental development and placental lactogen production in a mouse model. A number of studies have also demonstrated abnormally elevated placental PHLDA2 expression in human growth restricted pregnancies. This study examined whether PHLDA2 was aberrantly expressed in placentas of RFM pregnancies resulting in delivery of an FGR infant and explored a possible relationship between PHLDA2 expression and placental lactogen release from the human placenta.MethodsVillous trophoblast samples were obtained from a cohort of women reporting RFM (N = 109) and PHLDA2 gene expression analysed. hPL levels were assayed in the maternal serum (N = 74).ResultsPlacental PHLDA2 expression was significantly 2.3 fold higher in RFM pregnancies resulting in delivery of an infant with FGR (p < 0.01), with highest levels of PHLDA2 expression in the most severe cases. Placental PHLDA2 expression was associated with maternal serum hPL levels (r = −0.30, p = 0.008, n = 74) although this failed to reach statistical significance in multiple linear regression analysis controlling for birth weight (p = 0.07).ConclusionsThese results further highlight a role for placental PHLDA2 in poor perinatal outcomes, specifically FGR associated with RFM. Furthermore, this study suggests a potential relationship between placental PHLDA2 expression and hPL production by the placenta, an association that requires further investigation in a larger cohort.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0279-1) contains supplementary material, which is available to authorized users.

Unique trophoblast stem cell- and pluripotency marker staining patterns depending on gestational age and placenta-associated pregnancy complications

ABSTRACTPreeclampsia (PE) and intrauterine growth retardation (IUGR) are rare but severe pregnancy complications that are associated with placental insufficiency often resulting in premature birth. The clinical pathologies are related to gross placental pathologies and trophoblastic deficiencies that might derive from inflammatory processes and oxidative stress injury. The mesenchymal core of placental villi has been identified as a possible niche for trophoblast progenitor cells that are called upon to replenish the injured syncytiotrophoblast layer. These progenitor cells are known to express trophoblast stem cell (CDX2) and pluripotency (SOX2, NANOG and OCT4A) markers, however only little data is available characterizing the expression of these transcription factors beyond the blastocyst stage. We aimed to describe the expression of these factors in healthy 1st and 3rd trimester placentae as well as PE, IUGR and combined PE+IUGR placentae. We analyzed 8 respective samples derived from 1st trimester (elective abortions), and 3rd trimester (healthy controls, PE, IUGR and combined PE+IUGR). We accomplished immunoperoxidase staining to detect the stem cell markers: CDX2 (trophectoderm), SOX2, NANOG and OCT4A (embryonal). Immunoreative scoring was used for objective analyses of staining patterns. All markers display clearly elevated signals in 1st trimester villous samples as compared to healthy 3rd trimester counterparts. Especially CDX2 and NANOG were specific to the cytotrophoblast layer and the mesenchymal core. Specific and differential expression patterns were visible in the villous/extravillous compartment of each placenta-associated pregnancy complication (PE: pan elevated expression; IUGR elevated SOX2 in basal plate; combined PE+IUGR pan loss of expression). Reduction of stem cell transcription factor expression in term placentae indicates temporal regulation, and probably a specific function which is yet to be elucidated. The differential expression patterns within placentae complicated with placenta-associated pregnancy complications indicate that PE, IUGR and combined PE+IUGR are separate entities. It is unclear whether the alterations are the cause or the effect of the clinical pathology.

Confined placental mosaicism in short term culture

Finding of fetal chromosomal mosaicism complicates genetic counseling, as well as pregnancy management. The aim of this study was to determine the risk of confined placental mosaicism in short term culture of chorionic villous samples. We conducted a retrospective review of karyotype analysis results obtained after chorionic villous sampling (CVS) in two years period. A 420 samples of chorionic villi were taken transabdominally and obtained by a semidirect method (overnight incubating culture). All fetuses with CVS mosaicism were under the intensive perinatal care. In all cases of chromosome mosaicism the additional karyotyping was performed from fetal blood samples after 22nd gestational week in order to exclude true fetal mosaicism. After delivery newborns were examined by experienced pediatrician. From 420 analyzed samples in 11 (2,6%) cases we found placental mosaicism. No anomalies were seen in genetic sonogram of this fetuses and mosaicism was confirmed only in one case. Confined placental mosaicism (CPM) was found in 2,1% (9/420) of all analyzed cases, and it made 90% of all placental mosaicism. In 60% (6/10) of placental mosaicism cases we found mosaicism with single aberrant cell. Trisomy 21 mosaicism was the most frequent aberration found in 30% of cases. Finding of mosaicism in chorionic villi sample is at special importance for genetic counseling, because every case has to be reveled individually regarding the type and level of mosaicism. Anyway, in every case of placental mosaicism intensive antenatal monitoring is necessary, with additional chromosome analysis from different tissue in consideration of previous findings.

Assessment of high resolution melt analysis feasibility for evaluation of beta-globin gene mutations as a reproducible, cost-efficient and fast alternative to the present conventional method.

Background:Beta-thalassemia is the most prevalent monogenic disease throughout the world. It was the first genetic disorder nominated for nation-wide prevention programs involving population screening for heterozygotes and prenatal diagnosis (PND) in Iran. Due to the high prevalence of beta-thalassemia, the shift from conventional mutation detection methods to more recently developed techniques based on novel innovative technologies are essential. We aimed to develop a real-time polymerase chain reaction (PCR) based protocol using high resolution melting (HRM) analysis for diagnosis of common beta-thalassemia mutations.Materials and Methods:Forty DNA samples extracted from peripheral blood of suspected beta-thalassemia carriers participated in this study were subjected to amplification refractory mutation system (ARMS). We then used 20 of these samples for HRM optimization. When 100% sensitivity and specificity was obtained with HRM procedure, we applied the technique for mutation detection on another remaining 20 samples as thalassemia cases with unknown mutations (detected mutations with ARMS-PCR kept confidential). Finally, the HRM procedure applied on 2 chorionic villous sample (CVS) biopsied from 12 weeks gestational age pregnant women for routine PND analysis.Results:In the first step of study, Fr 8/9 (+G), IVSI-1 (G > A), IVSI-5 (G > C), IVSI-110 (G > A), and CD44 (−C) mutations were diagnosed in samples under study using ARMS-PCR technique. Finally, the HRM procedure applied on 20 unknown samples and 2 CVS The results of HRM were in complete concordance with ARMS and confirmed by sequencing.Conclusions:The advantages of HRM analysis over conventional methods is high throughput, rapid, accurate, cost-effective, and reproducible.

Importance of Additional Mutation Analysis in Chorionic Villous Sample for Prenatal Diagnosis of Thalassemia

Introduction:β thalassaemia is a genetic disorder of β globiin gene synthesis. Its presentation ranges from transfusion dependent to non transfusion dependent thalassaemia. Non transfusion dependent thalassaemia can have normal life with proper care whereas only curative option for transfusion dependent thalassaemia is bone marrow transplantation which is costly as well as requiring technical expertise. Carrier screening before marriage/conception, antenatal screening and prenatal diagnosis are the only way to prevent birth of a child with thalassaemia. Objective of prenatal diagnosis is to identify homozygous or double heterozygous baby by mutation study. However clinical course after birth may get altered depending on associated other mutation. Till date these associated mutations are not considered in cases of prenatal diagnosis. However, this approach may avoid termination of the baby. This study was carried out retrospectively in the prenatal samples to detect presence of phenotype-modifying mutations.Method and Material:The study was conducted at the Institute of Haematology and Transfusion Medicine, Medical College, Kolkata. Study population consisted of 145 CVS samples and samples of carrier mother and father. Carrier status was detected by HPLC and confirmed by ARMS- PCR for β thalassaemia mutation. Co- inheritance of α- thalassaemia was assessed by GAP- PCR and polymorphism with haplotype assessment was done by RFLP-PCR. 36 parent samples were analyzed for mutation for the carrier status and also for presence of α and Xmn 1 polymorphism.Result:Out of 145 CVS, 8.96% (13 samples) were found to be homozygous, 12.41% (18 samples) double heterozygous, 51.72% (75 samples) heterozygous, 23.44% (34 samples) normal and 3.44% (5 samples) with uncharacterized for β mutation. Our results designated that IVS 1-5 was the most common beta mutation in the state of West Bengal population (Table 1). Co-inheritance of α 3.7 deletion was found in only three affected CVS. Xmn1 polymorphism was detected in 9 CVS samples. Out of these, 3 were homozygous and 6 were heterozygous. Both co-inheritance of α mutation and xmn1 polymorphism were detected in 22 parents sample and 18 affected CVS sample ( Table: 2). Out of 31 affected sample, 18 samples showed presence of Xmn 1 and 4 for α deletion. Additional disease modifying mutations were detected in 6 samples (19.35%). In cases where the 18 parents' sample were positive for Xmn1 polymorphism and 3 for α deletion in CVS sample, either homozygous or double heterozygous for parents' mutation.Table 1Common β globin gene mutation of CVS samplesBeta genotypeHomozygoteHeterozygoteCompound HeterozygoteNormalUncommonIVS 1-5 (G→ C)748-----CD26(G→A) HbE1135----CD15(G→A)89-----CD30 (G→ C)5--------CD41/42(-CTTT)23----CD8/9(+G)3--------CD6 (A → T)22----Total137553418Table 2Affected CVS with additional mutations:Additional mutationsHomozygous CVSDouble heterozygous CVSParentsα deletion1--7Homozygous Xmn1 +/+113Heterozygous Xmn 1 -/+9312α deletion + Homozygous Xmn1 +/+--1--α deletion + Homozygous Xmn1 -/+--2--Discussion:Common thalassaemia subtypes prevalent in West Bengal are HbE beta and Beta thalassaemia . In cases where both the parents are carrier, beta mutation or mutation for HbE was first analyzed and subsequent decision for termination of baby was taken on the presence of both these mutations in the CVS sample. But phenotype is determined by presence of additional mutations like co-inheritance of α mutation and Xmn 1 polymorphism. From our study it became evident that around 19.35% cases the baby was expected to have intermedia or NTDT phenotype. Complete genotype analysis of CVS samples in prenatal diagnosis can save a life and that give a baby to the parents. However, further exploration with larger number of patients and post natal follow up is necessary to reach a definite conclusion.Conclusion:Alpha deletion and Polymorphism detection should be employed for prenatal diagnosis. While developing such program in the population, we save of multiple neonates. DisclosuresNo relevant conflicts of interest to declare.

Differential expression of human placental PAPP-A2 over gestation and in preeclampsia

IntroductionPregnancy Associated Plasma Protein A2 (PAPP-A2) is a pregnancy related insulin-like growth factor binding protein-5 (IGFBP-5) protease, known to be elevated in preeclampsia. As the insulin-like growth factors are important in human implantation and placentation, we sought to determine the expression pattern of PAPP-A2 over human gestation in normal and preeclamptic pregnancies to evaluate its role in placental development and the pathogenesis of preeclampsia. MethodsPlacental basal plate and chorionic villi samples, maternal and fetal cord blood sera were obtained from preeclamptic and control pregnancies. Formalin-fixed tissue sections from across gestation were stained for cytokeratin-7, HLA-G, and PAPP-A2. PAPP-A2 immunoblot analysis was also performed on protein lysates and sera. ResultsPAPP-A2 expression is predominately expressed by differentiated trophoblasts and fetal endothelium. Chorionic villi show strong expression in the first trimester, followed by a progressive decrease in the second trimester, which returns in the third trimester. PAPP-A2 expression is not impacted by labor. PAPP-A2 is increased in the basal plate, chorionic villi and maternal sera in preeclampsia compared to controls, but is not detectable in cord blood. DiscussionPAPP-A2 is differentially expressed in different trophoblast populations and shows strong down regulation in the mid second trimester in chorionic villous samples. Both maternal sera and placental tissue from pregnancies complicated by preeclampsia show increased levels of PAPP-A2. PAPP-A2 levels are not altered by labor. Additionally, PAPP-A2 cannot be detected in cord blood demonstrating that the alterations in maternal and placental PAPP-A2 are not recapitulated in the fetal circulation.

Detection of paternally inherited fetal point mutations for β-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment

Objective: To design and evaluate a noninvasive protocol for prenatal diagnosis (PND) of β-thalassemia, using cell free fetal DNA (cff-DNA) in maternal circulation. Traditional current PND which is mainly based on chorionic villous sampling (CVS), amplification refractory mutation system and sequencing holds as gold standard.Methods: Ten thalassemia trait couples with distinct mutations for the husband and wife were included in this study. The mutations in carrier fathers were IVSI-1, IVSI-5, FR8/9 and CD44. After maternal plasma isolation and free DNA extraction, all samples subjected to designed protocol including DNA size separation on agarose gel, elution of DNA from the gel slices using a simple and efficient manual purification method, with or without whole genome amplification and the detection method was allele-specific real-time PCR.Results: Presence or absence of the paternal mutant allele was correctly determined in all of cases and the accuracy of designed protocol was determined 100%.Conclusions: The protocol described here is very simple, inexpensive and easy to perform, but with satisfactory accuracy in detection of paternal mutations in cff-DNA. Due to the risk of fetal loss with current invasive sampling for PND, a noninvasive alternative is highly demanded in clinical setting.

Use of High-Performance Liquid Chromatography for Prenatal Diagnosis of a-Thalassemia (Hemoglobin Bart's) Hydrops Fetalis in Fetal Cord Blood Sample

The gold standard for prenatal diagnosis of α-thalassemia is mutational analysis of fetal cells by chorionic villous sampling or amniocentesis. However, these are time-consuming tests and thus, in the mid-trimester the diagnosis can be made expeditiously by percutaneous umbilical cord blood sampling with high-performance liquid chromatography (HPLC) analysis of the fetal hemoglobins. We present a case of Bart’s …

First trimester ultrasound screening for Down syndrome based on maternal age, fetal nuchal translucency and different combinations of the additional markers nasal bone, tricuspid and ductus venosus flow.

To examine the performance of screening for Down syndrome based on maternal age, fetal nuchal translucency (NT) and different combinations of the additional ultrasound parameters: nasal bone (NB), tricuspid flow (TF) and ductus venosus (DV). Retrospective study at the University of Tuebingen, Germany including women who underwent chorionic villous sampling between 2008 and 2014. Prior to invasive testing, the crown rump length, NT, NB, TF and DV were measured. In each case, the added value of the additional markers NB, TF and DV were compared with screening for trisomy 21 based on maternal age (MA) and NT thickness alone. A total of 1916 pregnancies met the inclusion criteria, including 1823 fetuses with a normal karyotype and 93 with trisomy 21. Screening based on MA, fetal NT and one, two and three of the additional ultrasound markers resulted in a detection rate of about 80%, 87% and 94%, respectively for a false positive rate of 3%. Detection rates for trisomy 21 in first trimester ultrasound screening are substantially higher if all three additional markers rather than just one are assessed.