Early prenatal diagnosis of Hb Lepore Boston-Washington and ß-thalassemia on fetal celomatic DNA.

INTRODUCTION: Analysis of fetal DNA in at risk couples for thalassemia is performed from fetal trophoblast or amniotic fluid cells. Although these procedures are in common use, the main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity develops around 4 weeks of pregnancy within the extraembryonic mesoderm and contains embryonic erythroid precursor cells as a source of fetal DNA that can be used to perform invasive prenatal diagnosis. METHODS: Celomatic fluids were obtained at 8 weeks of gestation in thirteen women with high-risk pregnancies. Twelve of these couples were at risk for Hb Lepore disease and ß-thalassemia and one couple represented a rare case in which both parents were carriers of Hb Lepore Boston-Washington. Fetal cells were isolated by micromanipulator and nested polymerase chain reactions were performed. RESULTS: The analysis was successfully performed in all examined cases. Two fetuses were found to have a compound heterozygosity for ß-thalassemia and Hb Lepore Boston-Washington, three fetuses were found to be carriers of ß-thalassemia, three fetuses of Hb Lepore, five were found without parental mutations. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. CONCLUSION: Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in celomatic fluid and demonstrate for the first time that prenatal diagnosis of ß-thalassemia and Hb Lepore may be feasible in an earlier time of pregnancy than other procedures.

Celomic Fluid: Laboratory Workflow for Prenatal Diagnosis of Monogenic Diseases.

BACKGROUND: Celomic fluid can be considered as an ultra-filtrate of maternal serum, containing a high protein concentration, urea, and many other molecules. It is an important transfer interface and a reservoir of nutrients for the embryo. Celomic fluid contains fetal cells that can be used for prenatal diagnosis of monogenic diseases in an earlier gestational period than villocentesis and amniocentesis. OBJECTIVE: The purpose of this study was to evaluate the characteristics of celomic fluid and to establish a workflow laboratory procedure for very early prenatal diagnosis of monogenic diseases. METHODS: Three hundred and eighty-five celomatic fluids were collected between the seventh and tenth week of gestation. We sampled 1 mL of celomic fluid in all cases. The embryo-fetal erythroid precursor cells were selected by the anti-CD71 microbead method or by a direct micromanipulator pick-up on the basis of their morphology. We amplified the extracted DNA using a nested polymerase chain reaction. Primers for short tandem repeat amplification were used to perform a quantitative fluorescent polymerase chain reaction evaluation to control maternal contamination. RESULTS: We observed maternal contamination in 95% of celomic fluids with a range between 5 and 100%. No fetal cells were observed in 0.78% of celomic fluids. The number of fetal cells ranged from a few units to several hundred. Isolation of embryo-fetal erythroblasts selected by the micromanipulator made diagnosis feasible in all cases. CONCLUSIONS: The selection of fetal cells by a micromanipulator and nested polymerase chain reaction analysis made celomatic fluid suitable for early prenatal diagnosis of monogenic disorders even in the presence of high maternal contamination and few fetal cells. The procedure reported in this study provides the opportunity for the use of celomic fluid sampled by celocentesis as an alternative to chorionic villi sampling and amniocentesis, to allow invasive prenatal diagnosis at a very early stage of pregnancy.

Very early prenatal diagnosis of Cockayne's syndrome by coelocentesis.

Cockayne's syndrome (CS) is a rare autosomal recessive multisystem disease characterised by early severe progression of symptoms. This study reports the feasibility of earlier prenatal diagnosis of CS by coelocentesis at 8 weeks of gestation respect to amniocentesis or villocentesis. Three couples at risk for CS asked to perform prenatal diagnosis by coelocentesis. Coelomic fluid was aspired from coelomic cavity in four singleton pregnancy at 8 weeks of gestation and 40 foetal cells were recovered by micromanipulator. Maternal DNA contamination was evaluated by quantitative fluorescent PCR (QF-PCR) and target regions of foetal DNA containing parental mutations of ERCC6 gene were amplified and sequenced. In all these cases, molecular analysis was possible. One foetus resulted affected of CS and the diagnosis was confirmed on placental tissue after voluntary abortion. In three cases, foetuses resulted carrier of a parental mutation and the results were confirmed after the birth. This study suggests that reliable prenatal diagnosis of CS could be performed using foetal cells present in coelomatic fluid in earlier pregnancy. Coelocentesis could be applied in prenatal diagnosis of CSs as well as for other monogenic diseases, at very early stage of pregnancy, if parental mutations are already known.Impact StatementWhat is already know on this subject? Previous studies utilising coelocentesis for prenatal determination of foetal sex reported variable success ranging from 58% to 95%, because of low total DNA content and presence of maternal cell contamination. This procedure has never been reported for early prenatal diagnosis at 8 weeks of gestation for rare genetically transmitted diseases such as Cockayne's syndrome.What do the results of this study add? This study demonstrates that coelomic fluid sampling combined with well-standardised laboratory procedures can be applied for prenatal diagnosis at eight weeks of gestation for any rare monogenic disease if molecular defects are known.What are the implications of these findings for clinical practice and/or further research? The findings of this study in at risk couples for monogenic diseases investigated by coelocentesis demonstrate that embryo-foetal cell selection from CF allows reliable and early prenatal diagnosis of diseases. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier than what can be achieved by the traditional procedures reducing anxiety of parents and provides the option for medical termination of affected cases at 8-10 weeks' gestation, which is less traumatic and safer than second-trimester surgical termination. Further research concerns the possibility to obtain foetal karyotype at eight weeks of gestation and the possibility of intrauterine corrective therapy.

Celocentesis for Early Prenatal Diagnosis in Couples at-Risk for ß-Thalassemia and Sicilian (δß)0-Thalassemia.

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δß)0-thalassemia [(δß)0-thal] deletion (NG_000007.3: g.64336_77738del13403) and ß-thalassemia (ß-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δß)0- and ß-thal, three fetuses were found to be carriers of ß-thal, four fetuses carriers of a Sicilian 뫧 deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δß)0-thal and ß-thal is feasible at an earlier time in pregnancy than other procedures.

Incidental Detection of a Chromosomal Aberration by Array-CGH in an Early Prenatal Diagnosis for Monogenic Disease on Coelomic Fluid.

BACKGROUND: Turner syndrome is a rare genetic condition in which a female is partly or completely missing an X chromosome. Signs and symptoms vary among those affected. In fetuses that survive at birth and without congenital malformations, the prognosis is usually positive, but it has high lethality in utero, especially in the first trimester of pregnancy. METHODS: We report a case of monosomy X detected during a prenatal diagnosis for beta thalassemia on coelomic fluid (CF) at the VIII week of gestation. Beta globin gene analysis, whole genome amplification (WGA), quantitative fluorescent PCR and array comparative genomic hybridization (array-CGH) were performed on DNA extracted from CF. RESULTS: A monoallelic pattern of all Short Tandem Repeats mapped on the X chromosome was found and array-CGH performed on WGA from a few fetal erythroblasts confirmed monosomy X. CONCLUSION: This report underlines the importance of an early prenatal diagnosis and the countless potentialities of array-CGH that could make definition of molecular karyotype possible from a few fetal cells, unlike conventional cytogenetic techniques that require a greater cellular content. This is the first report of a molecular karyotype obtained from two cells selected by micromanipulation of CF and defined at such an early gestational age.

Non-immune hydrops fetalis: Two case reports.

BACKGROUND: Fetal hydrops is a serious condition difficult to manage, often with a poor prognosis, and it is characterized by the collection of fluid in the extravascular compartments. Before 1968, the most frequent cause was the maternal-fetal Rh incompatibility. Today, 90% of the cases are non-immune hydrops fetalis. Multiple fetal anatomic and functional disorders can cause non-immune hydrops fetalis and the pathogenesis is incompletely understood. Etiology varies from viral infections to heart disease, chromosomal abnormalities, hematological and autoimmune causes. CASE SUMMARY: A 38-year-old pregnant woman has neck lymphoadenomegaly, fever, cough, tonsillar plaques at 14 wk of amenorrhea and a rash with widespread itching. At 27.5 wk a fetal ultrasound shows signs of severe anemia and hydrops. Cordocentesis is performed with confirmation of severe fetal anemia and subsequent fetal transfusion. The karyotype is 46, XX, array-comparative genome hybridization (CGH) negative, and infectious tests are not conclusive. In the following days there is a progressive improvement of the indirect signs of fetal anemia. At 33.6 wk, for relapse of severe fetal anemia, further fetal transfusions are necessary and an urgent cesarean section is performed. On the day 12 of life, for the detection of anemia, the newborn is subjected to transfusion of concentrated red blood cells and begins treatment with erythropoietin. Later there is a normalization of blood chemistry values and the baby does not need new transfusions. A 29-year-old pregnant woman, with Sjogren's syndrome and positive Anti-Ro/SSA antibodies, is subjected to serial fetal ecocardio for branch block. At 26.5 wk there is a finding of fetal ascites. Infectious disease tests on amniotic fluid are negative as well as quantitative fluorescent polymerase chain reaction, Array CGH. At cordocentesis Hb is 1.3 mmol/L, consequently fetal transfusion is performed. Also in this case, due to continuous episodes of relapse of fetal anemia with consequent transfusions, at 29.4 wk a cesarean section is performed. On day 9 of life, a treatment with erythropoietin is started in the newborn, but the baby needs three blood transfusions. The search for autoantibodies in the baby found SS-A Ro60 positive, SSA-Ro52 positive and SS-B negative. The hemoglobin values normalized after the disappearance of maternal autoantibodies. CONCLUSION: An attempt to determine the etiology of hydrops should be made at the time of diagnosis because the goal is to treat underlying cause, whenever possible. Even if the infectious examinations are not conclusive, but the pregnancy history is strongly suggestive of infection as in the first case, the infectious etiology must not be excluded. In the second case, instead, transplacental passage of maternal autoantibodies caused hydrops fetalis and severe anemia. Finally, obstetric management must be aimed at fetal support up to an optimal timing for delivery by evaluating risks and benefits to increase the chances of survival without sequelae.

Fetal aneuploidy diagnosed at celocentesis for early prenatal diagnosis of congenital hemoglobinopathies.

INTRODUCTION: Currently, prenatal diagnosis of genetic disorders requires chorionic villus sampling or amniocentesis carried out after 11 and 16 weeks of gestation, respectively. Celocentesis is a procedure for prenatal diagnosis that could be used from as early as 7 weeks. The present investigation evaluated the feasibility of performing diagnosis for monogenic diseases using celomic fluid containing cells of fetal origin. MATERIAL AND METHODS: Analysis consisted of 489 singleton pregnancies undergoing celocentesis for the prenatal diagnosis of hemoglobinopathies (n = 367) or before surgical termination of pregnancy for social indications (n = 122). Embryo-fetal cells were isolated from celomic fluid using CD71 antibodies or by micromanipulation. Quantitative fluorescent polymerase chain reaction of short tandem repeat sequences of chromosomes 13, 18, 21, X and Y were used to determine the presence of maternal DNA. RESULTS: 357/489 (73%) of celomic fluid samples were contaminated with maternal cells. In two cases, diagnosis was not possible due to the high contamination of celomic fluid. Eighty-seven (23.8%) fetuses were affected by hemoglobinopathies and, in five cases, chromosomal aneuploidies were found, including three cases of trisomy 21, one of trisomy 13 and one of triploidy. In all cases, the diagnosis of hemoglobinopathies and chromosomal abnormalities was confirmed by molecular and traditional cytogenetic analysis after amniocentesis, chorionic villus or placental tissue collection following pregnancy termination. CONCLUSIONS: The findings of this study demonstrate that embryo-fetal cell selection from celomic fluid allows reliable and early prenatal diagnosis of hemoglobinopathies and can give more information on any fetal aneuploidy following the control of maternal contamination by quantitative fluorescent-PCR.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

OBJECTIVE: The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. METHOD: Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin. RESULTS: Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. CONCLUSIONS: The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John Wiley & Sons, Ltd.

Prenatal diagnosis of a variant of the azygos venous system.

BACKGROUND: The azygos venous system consists of the azygos vein on the right side and the hemiazygos and accessory hemiazygos on the left side. The azygos vein runs through the abdominal cavity along the right side of the vertebral bodies, in a cranial direction, passes through the diaphragm and reaches the mediastinum, where it forms the arch of the azygos which flows into the superior vena cava. Along its course, the azygos vein communicates with the intercostal veins on the right, the hemiazygos vein that collects blood from the left lower intercostal veins, and accessory hemiazygos vein that drains into the left upper intercostal veins. The last two, at the level of the seventh thoracic vertebra, unite and end in the azygos vein. The accessory hemiazygos vein is normally included in the length between T4 and T8. The embryological origin of the accessory hemiazygos vein is the result of an expansion in the direction of the cranial hemiazygos vein, which comes from the left upper sovracardinale vein (Dudiak et al. in Semin Roentgenol 24(1):47-55, 1989; Radiographics 11(2):233-246, 1991; Webb et al. in Am J Roentgenol 139(1):157-161, 1982). FINDINGS: This case report describes a rare variant of azygos vein system identified in prenatal diagnosis and confirmed by postnatal ultrasonography. CONCLUSIONS: The observation of the patient has excluded hemodynamic alterations associated with vascular anomaly.

Embryo-fetal erythroid cell selection from celomic fluid allows earlier prenatal diagnosis of hemoglobinopathies.

OBJECTIVE: Celocentesis, which involves aspiration of celomic fluid at 7-9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid. METHOD: Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology. RESULTS: In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low (< 5%), and unambiguous results were obtained without the need for any preliminary procedures. In 160 (53%) cases, the contamination was between 5% and 60%, and selection of embryo-fetal erythroid precursors was successfully achieved by anti-CD71 MicroBeads. In 42 (13.9%) cases, the contamination was > 60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA. CONCLUSIONS: Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases.