Overview of Chromosome Abnormalities in First Trimester Miscarriages: A Series of 1,011 Consecutive Chorionic Villi Sample Karyotypes

In order to contribute to the knowledge of type and frequency of chromosome abnormalities in early pregnancy losses, we analyzed the cytogenetic results from a large series of first trimester miscarriages, using a diagnostic approach with a high success rate and no maternal contamination. A total of 1,119 consecutive chorionic villi samples were obtained before evacuation, and karyotypes were prepared after short-term culture (STC). In 603 samples, a long-term culture (LTC) was also performed. The overall and individual frequencies of the different types of chromosome abnormalities were established, including placental mosaicisms, and their relationship with maternal age and gestational weeks was assessed. An abnormal karyotype was detected in 70.3% of the samples. Single autosomal trisomy was the most frequent abnormality (64.6% of the abnormal cases), followed by triploidy (13.1%) and monosomy X (10.4%). Chromosome rearrangements were found in 5.2%, combined abnormalities in 8.9%, and placental mosaicism in 3.5% of the cases with STC and LTC performed. Individual trisomies behaved differently with respect to maternal age and intrauterine survival. Due to the combination of STC and LTC, our study offers reliable information on the incidence and type of chromosome abnormalities and placental mosaicism in miscarriages and contributes to define the cytogenetic implication in their etiology.

Earlier Antenatal Diagnosis of Hemoglobinopathies By Coelocentesis

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 (100 samples) were successfully analyzed without preliminary treatment. In samples with >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.

Microsatellite Markers within —SEA Breakpoints for Prenatal Diagnosis of HbBarts Hydrops Fetalis

Background: We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrops fetalis and exclusion of maternal contamination. Methods: We developed a multiplex quantitative fluorescent PCR (QF-PCR) test that detects the presence/ absence of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia (—SEA) deletion. HbBarts hydrops fetalis (—SEA/—SEA) is diagnosed by absence of both markers, and maternal contamination of fetal DNA is excluded by absence of noninherited maternal alleles. Fetal and parental DNA samples from 50 families were analyzed in a blinded clinical validation study, and QF-PCR results were compared with their respective molecular genotypes. Results: The multiplex QF-PCR results included correct diagnoses of HbBarts hydrops fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (αα/—SEA) or unaffected homozygotes in 18. Misidentification as unaffected occurred for 1 carrier. Sensitivity for diagnosis of HbBarts hydrops fetalis was 100% [lower 95% confidence interval, 76.2%], and specificity was 100% (lower 95% confidence interval, 92.6%). None of the samples tested showed any traces of noninherited maternal alleles; thus false-positives because of maternal contamination were eliminated. Conclusions: In this QF-PCR method, detection of maternally and paternally inherited fetal alleles allowed diagnosis of the double-deletion syndrome, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination of the fetal genetic material. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrops fetalis.