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OBJECTIVE Celocentesis is an invasive technique that can provide prenatal diagnosis of single-gene disorders, from as early as 7 weeks' gestation. The objective of this study was to examine the safety of celocentesis. METHODS In this prospective study, celocentesis was performed for prenatal diagnosis of hemoglobinopathy in 402 singleton pregnancies in which both parents were carriers of β-thalassemia or sickle cell disease trait. We assessed procedure-related maternal discomfort or pain, success of sampling and obtaining results, pregnancy outcome and postnatal follow-up. RESULTS First, celocentesis was carried out at a median gestational age of 8.6 (range, 6.9-9.9) weeks and celomic fluid was successfully aspirated in 99.8% of cases. Second, 67% of women had no or only mild discomfort, 18% had moderate discomfort, 12% had mild-to-moderate pain and 3% had severe pain. Third, prenatal diagnosis from analysis of the celomic fluid was successful in 93.8% cases, and in the last 121 cases, it was always successful. Fourth, in all cases of successful sampling and analysis of celomic fluid, the diagnosis was concordant with results obtained from additional prenatal or postnatal testing. Fifth, in addition to diagnosis of hemoglobinopathy, quantitative fluorescence polymerase chain reaction analysis, which was performed to evaluate maternal contamination using several markers for chromosomes X, Y, 21, 18 and 13, led to the accurate diagnosis of chromosomal aneuploidy. Sixth, in all cases of an affected fetus diagnosed by celocentesis in which the parents chose termination of pregnancy, this was carried out

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 was extracted from celomic fluid cells showed the same morphology and quantitative fluorescentPCR 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 make 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-CGH. This article is protected by copyright. All rights reserved.

Coelomic fluid (CF) is the earliest dynamic and complex fluid of the gestational sac. CF contains maternal cells and proteins produced by embryonic cells, tissues and excretions. The biochemical composition of CF is modified throughout the first trimester of pregnancy and its protein profile reflects both physiological/pathological changes affecting the embryo and mother. Identification of variations in the balance of proteins might indicate particular types of pathologies, or ascertain specific genetic disorders. A platform utilizing protein enrichment procedures coupled with shotgun identification and iTRAQ differentiation provided the identification and quantitation of 88 unique embryonic proteins. It is relevant to note that chromosome X protein CXorf23 was found suggesting the embryo sex. Foetal sex was determined by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) on coelomic cells, foetal tissues and maternal white blood cells, with a 100% concordance rate between iTRAQ-MS/MS and QF-PCR data. The functional associations among the identified proteins were investigated using STRING database. Open Targets Platform showed as significant the following therapeutic areas: nervous, respiratory, eye and head system disease.

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 ( 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. © 2016 John Wiley & Sons, Ltd.

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.

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.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.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.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 WileySons, Ltd.

At 5-12 weeks of gestation the amniotic sac is surrounded by celomic fluid which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound-guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of hemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7-9 weeks' gestation in 26 singleton pregnancies at risk for hemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for hemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks' gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis, although further work is necessary to improve widespread use and safety of the procedure..

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.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.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 was60%, 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.Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases.

Summary Background Haemoglobinopathies are a major public health problem in Sicily: it was estimated a frequency of 1/245 couples are at risk of haemoglobinopathies. This paper reviews legislative actions, prevention activities, carrier screening, genetic counselling, foetal sampling and laboratory methodology analysis evolution reporting the results of 30 years of prevention actions to assess the efficiency of our preventative programme in the control of haemoglobinopathies in Sicily. Methods This programme consisted principally of five phases: legislative actions, public awareness campaign, carrier screening, genetic counselling and prenatal diagnosis. Results These programmes have been very effective, which we can see from a greater public awareness of thalassaemia and its prevention in the target population furthermore by a marked decline in the incidence of thalassaemia major and sickle cell anaemia from 1 in 245 live births in the absence of prevention to 1 in 2000, with a reduction in about 85%. The residual cases were because of a conscious choice by expecting parents in relation to improved life expectancy as well as improved quality of life of the affected patients. Conclusion The study suggests that public health authorities should act and invest in a similar programme for prevention of thalassaemia, as well as in relation to the increased survival of patients and the consequent organ complications.

Prenatal diagnosis of hemoglobinopathies involves the study of fetal material from blood, amniocytes, trophoblast coelomatic cells and fetal DNA in maternal circulation. Its first application dates back to the 70s and it involves globin chain synthesis analysis on fetal blood. In the 1980s molecular analysis was introduced as well as amniocentesis and chorionic villi sampling under high-resolution ultrasound imaging. The application of direct sequencing and polymerase chain reactionbased methodologies improved the DNA analysis procedures and reduced the sampling age for invasive prenatal diagnosis from 18 to 16–11 weeks allowing fetal genotyping within the first trimester of pregnancy. In the last years, fetal material obtained at 7–8 weeks of gestation by coelocentesis and isolation of fetal cells has provided new platforms on which to develop diagnostic capabilities while non-invasive technologies using fetal DNA in maternal circulation are starting to develop.

The use of hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy, although this approach has provided heterogeneous results. In this paper we have undertaken molecular, phenotypic and functional studies aimed at identifying the presence of fully competent T lymphocytes in samples of fetal livers and cord blood. We found mature VDJ TCR beta chain transcripts in fetal liver cells taken from 7 to 16 weeks of gestation and a similar pattern was detected in cord blood cells sampled from 13.5 to 20.5 weeks of gestation. A Vbeta8 gene sequence comparable to that detected in adult PBMC was found in fetal liver samples at 9 or 17 weeks gestation. PreTalpha message was detected in all samples and its expression decreased in fetal blood samples with increasing gestational age while Calpha message appeared at 9.4 weeks and its expression increased during gestational age. T cell clones obtained from fetal liver cells showed a mature TCR alphabeta+, CD8+ phenotype and displayed strong alloreactivity against allo-MHC class I molecules. The presence of alloreactive T lymphocytes may explain the failure to engraft in fetuses older than 13 to 16 weeks and may provide insights into fetal liver transplantation. Bone Marrow Transplantation (2000) 25, 135-141.

At 5-12 weeks of gestation the amniotic sac is surrounded by celomic fluid, which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound-guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of haemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7-9 weeks gestation in 26 singleton pregnancies at risk for haemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for haemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis before widespread use.

X-linked hydrocephalus is due to mutations in the L1 neuronal cell adhesion molecule (L1CAM) gene. L1 protein plays a key role in neurite outgrowth, axonal guidance, and pathfinding during the development of the nervous system. We report on a familial case diagnosed by prenatal ultrasonographic examination, with cerebellar hypoplasia, agenesis of the corpus callosum, and the bilateral overlapping of the second and third fingers of the hand. Sequencing of the L1CAM gene showed a novel missense mutation in exon 14: transition of a guanine to cytosine at position 1777 (c.1777G > C), which led to an amino acid change of alanine to proline at position 593 (Ala593Pro) in the sixth immunoglobulin domain of the L1 protein. The L1CAM mutation testing should be considered in fetuses with ultrasonographic signs of hydrocephalus and a positive family history compatible with X-linked inheritance. We agree with previous reports that suggest also considering limb abnormalities other than adducted thumbs in addition to classical neurological disgenesis, as characteristic for L1-disease

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound-guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post-fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound-guided puncture. J Cell Physiol

Coelocentesis offers a new opportunity for gaining access to the human embryos from 28 d postfertilization. However, while some studies about its biochemical composition have been reported, our knowledge about immunological pattern of this compartment is still limited. For this reason, we studied the human coelomic fluids sampled from 6.6 to 10 wk of gestation. The majority of cellular population consisted in mesenchymal/epithelial cells. In fluids sampled before 10 wk we found only a preT Cell Receptor expression and an absence or a very low frequency of B lymphocytes, T lymphocytes and NK (natural killer) antigens. These preliminary data suggest that the immunological system in human embryos could be in the ideal conditions to start a process of tolerance induction. Eur J Haematol

In countries with a high prevalence of hemoglobinopathy carriers, the only realistic approach to control the birth of new patients with thalassemia major or sickle cell disease is population screening in combination with invasive prenatal diagnosis[1]. In the early 1990's, molecular definition of the thalassemia defects, development of procedures for their detection by DNA analysis and introduction of amniotic fluid sampling (amniocentesis) or chorionic villus sampling (CVS) led to early prenatal diagnosis at 16 and 11 weeks, respectively [2]. An alternative technique for earlier diagnosis is celocentesis [3]. At the end of week 4 of gestation, the developing exocoelomic cavity (ECC) splits the extra-embryonic mesoderm into two layers, the somatic mesoderm lining the trophoblast and the splanchnic mesoderm covering the secondary yolk sac and the embryo. The ECC is the largest anatomical space inside the gestational sac between 5 and 9 weeks of gestation and is surrounded by celomic fluid (CF), which contains cells of fetal origin [4-5]. This fluid can be sampled by a technique that involves the ultrasound-guided insertion of a needle through the vagina from as early as 7 weeks of gestation. Previous studies utilizing coelocentesis for prenatal determination of single-gene defects reported variable success ranging from 58 to 95% because of presence of maternal cell contamination (MCC) [6]. In this work we demostred as this problem can be overcome through the identification of embryo-fetal erythroid precursors presented in the coelomatic fluid and their specific selection. 302 couples from different regions of Italy, at risk for ß thalassemia or sickle cell disease asked for prenatal diagnosis by coelocentesis that was carried out at between 6+6 and 9+2 weeks' gestation. Coelomic fluid samples with no or very low (5% maternal contamination, two different procedures were used to isolate embryo-fetal cells: the first technique involved positive selection of embryo-fetal erythroid precursors by anti-CD71 MicroBeads (160 samples), the second procedure was through the use of a micromanipulator (42 samples). In 68/300 (22.6%) cases the fetus was affected by ß-thalass/ß-thalassemia and 66 women chosen to terminate the pregnancy. Two families decided to continue the pregnancy for ethical reasons despite the documented presence of an affected fetus. The antenatal diagnosis was confirmed in all 66 cases by molecular analysis of placental tissue after termination (Table 1). In 232/300 (77.4%) cases the fetus was diagnosed as being normal or a carrier for ß-thalassemia or sicke cell disease. The results obtained after coelocentesis were confirmed by amniocentesis or postnatally. In two case (0.66%) was no obtained a reliable diagnosis because no fetal cells were found (Table 1). The findings of this study in a large number of pregnancies investigated by coelocentesis, demonstrate that embryo-fetal erytroid cell selection from coelomatic fluid allows reliable and earlier prenatal diagnosis of hemoglobinopathies. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier respect to traditional procedures reducing anxiety of parents and from a clinical point of view this procedure would allow women to undergo medical TOP at 8-10 weeks of gestation which is less traumatic and safer than second trimester surgical TOP. Table 1. Overall results of prenatal diagnosis of hemoglobinopathies by coelocentesis. Number of coelocentesis effectuated 302 Results of Prenatal diagnosis: Affected 68 Carrier of hemoglobinopathies 161 Non Affected 71 diagnosis not reiable 2 Control post coelocentesis: Placental tissue after termination 66 CVS or amniocentesis 153 Postnatal test 61 Awaiting 20 Coelocentesis diagnostic errors 0 Disclosures No relevant conflicts of interest to declare.

Five hundred cordocenteses were performed between 12 and 21 weeks. The indications were thalassaemia (386), rapid karyotyping (97), feto-maternal allo-immunization (10), rubella (6), and toxoplasmosis (1). One hundred and ten pregnancies underwent termination on the basis of the result, while 20 of the 370 pregnancies intended to continue were lost to follow-up. Amongst these were 16 fetal losses (4.3 per cent) and 22 premature deliveries (5.9 per cent); no other complications were reported. Four adverse prognostic factors were identified: (a) cord bleeding; (b) fetal bradycardia; (c) prolonged procedure time; and (d) anterior insertion of the placenta. There was no 'obvious' difference in fetal loss rate with advancing gestation until 19-21 weeks, when the risk of fetal loss decreased to 2.5 per cent.

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.