Fetal Blood Cells Research Articles

Noninvasive prenatal diagnosis for single gene disorders.

Noninvasive prenatal diagnosis for single gene disorders is coming to fruition in its clinical utility. The presence of cell-free DNA in maternal plasma has been recognized for many years, and a number of applications have developed from this. Noninvasive prenatal diagnosis for single gene disorders has lagged behind due to complexities of technology development, lack of investment and the need for validation samples for rare disorders. Publications are emerging demonstrating a variety of technical approaches and feasibility of clinical application. Techniques for analysis of cell-free DNA including digital PCR, next-generation sequencing and relative haplotype dosage have been used most often for assay development. Analysis of circulating fetal cells in the maternal blood is still being investigated as a viable alternative and more recently transcervical trophoblast cells. Studies exploring ethical and social issues are generally positive but raise concerns around the routinization of prenatal testing. Further work is necessary to make testing available to all patients with a pregnancy at risk of a single gene disorder, and it remains to be seen if the development of more powerful technologies such as isolation and analysis of single cells will shift the emphasis of noninvasive prenatal diagnosis. As testing becomes possible for a wider range of conditions, more ethical questions will become relevant.

Evidence for Feasibility of Fetal Trophoblastic Cell-Based Noninvasive Prenatal Testing

(Abstracted from Prenat Diagn 2016;36:1–11) The potential use of fetal cells in maternal blood for prenatal diagnosis has been a promising technology for decades. Several strategies have been tried and tested for recovering fetal cells, although they are inconsistent and not reproducible.

Tissue specific expression of human fatty acid oxidation enzyme genes in late pregnancy.

BackgroundAbnormal fatty acid oxidation (FAO) is associated with maternal and fetal complications during pregnancy. The contribution of maternal and fetal tissues to FAO capacity during late pregnancy is important to understand the pathophysiology of pregnancy-associated complications. The aim of this study was to determine the expression levels of mitochondrial FAO enzymes in maternal and fetal tissues during late normal pregnancy.MethodsWe have measured by Real-time PCR the levels of long- and medium -chain acyl-CoA dehydrogenase (LCHAD and MCAD), two acyl-CoA dehydrogenases that catalyze the initial step in the mitochondrial FAO spiral.ResultsLCHAD and MCAD were expressed in maternal skeletal muscle, subcutaneous adipose tissue, placenta, and maternal and fetal blood cells. LCHAD gene expression was four- to 16-fold higher than MCAD gene expression in placenta, adipose tissue and skeletal muscle. In contrast, MCAD gene expression was ~5-fold higher in fetal blood than maternal blood (p = 0.02), whereas LCHAD gene expression was similar between fetal blood and maternal blood (p =0.91).ConclusionsLCHAD and MCAD are differentially expressed in maternal and fetal tissues during normal late pregnancy, which may represent a metabolic adaptation in response to physiological maternal dyslipidemia during late pregnancy.

Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women.

Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X). Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.

Aneuploidy screening using circulating fetal cells in maternal blood by dual-probe FISH protocol: a prospective feasibility study on a series of 172 pregnant women.

BackgroundA long sought goal in medical genetics has been the replacement of invasive procedures for the detection of chromosomal aneuploidies by isolating and analyzing fetal cells or free fetal DNA from maternal blood, avoiding risk to the fetus. However, a rapid, simple, consistent, and low‐cost procedure suitable for routine clinical practice has not yet been achieved. The purpose of this study was to assess the feasibility of predicting fetal aneuploidy by applying our recently established dual‐probe FISH protocol to fetal cells isolated and enriched from maternal blood.MethodsA total of 172 pregnant women underwent prospective testing for fetal aneuploidy by FISH analysis of fetal cells isolated from maternal blood. Results were compared with the karyotype determined through invasive procedures or at birth.ResultsSeven of the samples exhibited fetal aneuploidy, which was confirmed by invasive prenatal diagnosis procedures. After enrichment for fetal cells, the frequency of trisomic cells was at least double in samples from aneuploid pregnancies (range 0.38–0.90%) compared to samples from normal pregnancies (≤0.18%). One false negative result was also obtained.ConclusionsNoninvasive prenatal aneuploidy screening using fetal cells isolated from maternal blood is feasible and could substantially reduce the need for invasive procedures.

Endothelial progenitor cells improve the quality of transplanted hematopoietic stem cells and maintain longer term effects in mice.

Engraftment of hematopoietic stem cells (HSCs) is a pre-requisite for the success of hematopoietic stem cell transplantation (HSCT). Fetal blood cell (FBC)-derived endothelial progenitor cells (EPCs) are known to facilitate HSC reconstitution in the early phase. However, longer term effects on HSCs remain unclear. The purpose of this study was to evaluate the effect of EPCs on the quality of transplanted hematopoietic stem cells in mouse HSCT model. BALB/c mice were randomly divided into four groups, namely, control, total body irradiation only, HSCT, and HSCT + EPCs (with infusion of 5 × 105 EPCs). Mice was sacrificed on days 7, 14, 21, and 28 post-HSCT for the analysis of the bone marrow pathology by H&E staining, measurement of c-kit+sca-1+, c-kit+, apoptosis, and necrosis by flow cytometry as well as colony formation assay. Secondary transplantation involved the injection of transplanted BALB/c-derived HSCs into new TBI-treated BALB/c mice. Compared with HSCT, EPCs infusion promoted the differentiation and reduced apoptosis of transplanted HSCs, possibly through promotion of vascular repair of the bone marrow microenvironment via differentiation into the bone marrow endothelial cells. Significantly, EPCs' effect on HSCs was maintained for a long period as demonstrated using a secondary transplantation approach. These data revealed EPCs improved the quality and quantity of transplanted HSCs and maintained their effects over the longer term, suggesting a novel approach to improve HSCT efficiency and outcomes.

Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response.

Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for further studies to determine the limit of this response that definitely changes the fate of a fetus under these conditions of cellular stress.

Abnormal labyrinthine zone in the Hectd1-null placenta

IntroductionThe labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. MethodsThe structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. ResultsHectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). DiscussionTogether these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta.

Sex determination of ovine embryos by SRY and amelogenin (AMEL) genes using maternal circulating cell free DNA

Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. Some of the existing methods depend only on the detection of Y-chromosome specific sequences. However, the detection of Y and X-chromosome specific sequences is advantageous. In the present study the accuracy of sex determination by SRY (sex-determining region Y) and AMEL (Amelogenin) gene detection was assessed using a polymerase chain reaction (PCR) of DNA extracted from free fetal cells in maternal blood, which is noninvasive for fetus and easier to collect. The PCR amplification of SRY primers produced a single band of 171bp from ewes bearing a male fetus, whereas no band was amplified from the DNA extracted from ewes pregnant to a female fetus. Moreover, two bands of 182 and 242bp in male and a single band of 242 in female fetuses were produced by AMEL gene primers in the PCR reaction. Using this technique 100% of samples were successfully sexed, excluding twins. In conclusion, we demonstrated that sex determination using DNA of free fetal cells in maternal plasma is efficient using both SRY and AMEL gene sequences. It also is evident that this method is not suitable for sex determination of twin pregnancies.

The Number of Endovascular Trophoblasts in Maternal Blood Increases Overnight and after Physical Activity: An Experimental Study

Introduction: Fetal cells in maternal blood may be used for noninvasive prenatal diagnostics, although their low number is a challenge. This study's objectives were to evaluate whether physical activity, transabdominal and transvaginal ultrasound scans of the uterus, as well as overnight or day-to-day variation affect the number of isolated fetal cells, more specifically the presumed endovascular trophoblast (pEVT). Material and Methods: In each of 3 different experiments, 10 normal singleton pregnant women (gestational age 10⁺⁴-14⁺⁴ weeks) participated. The number of pEVTs was assessed in 30-36 ml blood using specific markers for enrichment and identification. Results: The number of pEVTs increased overnight (p = 0.001) from a median of 1.5 to 3.5 and even further to a median of 6.0 after 30 min of physical activity (p = 0.04) but was not affected by transabdominal and transvaginal ultrasound scans. Repeated sampling showed that the interindividual variation of pEVTs was higher than the intraindividual variation (p < 0.001). However, even in pregnant women with a consistently low number of pEVTs, isolation of the pEVTs for prenatal diagnoses was possible in all cases by doing 2 separate blood samplings a few days apart. Discussion: The number of pEVTs identified in maternal blood can be increased by presampling conditions or repeated sampling.

Outside-in? Acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood

The preterm birth syndrome (delivery before 37 weeks gestation) is a major contributor to the global burden of perinatal morbidity and death. The cause of preterm birth is complex, multifactorial, and likely dependent, at least in part, on the gestational age of the fetus. Intrauterine infection is frequent in preterm deliveries that occur at <32 weeks gestation; understanding how the fetus responds to proinflammatory insult will be an important step towards early preterm birth prevention. However, animal studies of infection and inflammation in prematurity commonly use older fetuses that possess comparatively mature immune systems. Aiming to characterize acute fetal responses to microbial agonist at a clinically relevant gestation, we used 92-day-old fetuses (62% of term) to develop a chronically catheterized sheep model of very preterm pregnancy. We hypothesized that any acute fetal systemic inflammatory responses would be driven by signaling from the tissues exposed to Escherichia coli lipopolysaccharide that is introduced into the amniotic fluid. Eighteen ewes that were carrying a single fetus at 92 days of gestation had recovery surgery to place fetal tracheal, jugular, and intraamniotic catheters. Animals were recovered for 24 hours before being administered either intraamniotic E coli lipopolysaccharide (n = 9) or sterile saline solution (n = 9). Samples were collected for 48 hours before euthanasia and necroscopy. Fetal inflammatory responses were characterized by microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Intraamniotic lipopolysaccharide reached the distal trachea within 2 hours. Lipopolysaccharide increased tracheal fluid interleukin-8 within 2 hours and generated a robust inflammatory response that was characterized by interleukin-6 signaling pathway activation and up-regulation of cell proliferation but no increases in inflammatory mediator expression in cord blood RNA. In very preterm sheep fetuses, lipopolysaccharide stimulates inflammation in the fetal lung and fetal skin and stimulates a systemic inflammatory response that is not generated by fetal blood cells. These data argue for amniotic fluid-exposed tissues that play a key role in driving acute fetal and intrauterine inflammatory responses.

Anti-D administration in pregnancy for preventing Rhesus alloimmunisation.

During pregnancy, a Rhesus negative (Rh-negative) woman may develop antibodies when her fetus is Rhesus positive (Rh-positive). These antibodies may harm Rh-positive babies. To assess the effects of antenatal anti-D immunoglobulin on the incidence of Rhesus D alloimmunisation when given to Rh-negative women without anti-D antibodies. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 May 2015) and reference lists of retrieved studies. Randomised trials in Rh-negative women without anti-D antibodies given anti-D after 28 weeks of pregnancy, compared with no treatment, placebo or a different regimen of anti-D. Two review authors independently assessed trials for inclusion and risk of bias, extracted data and checked them for accuracy. We included two trials involving over 4500 women, comparing anti-D prophylaxis with no anti-D during pregnancy in this review. Overall, the trials were judged to be at moderate to high risk of bias. The quality of the evidence for pre-specified outcomes was also assessed using the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) approach.In regards to primary review outcomes, there did not appear to be a clear difference in the risks of immunisation when women who received anti-D at 28 and 34 weeks' gestation were compared with women who were not given antenatal anti-D: risk ratio (RR) for incidence of Rhesus D alloimmunisation during pregnancy was 0.42 (95% confidence interval (CI) 0.15 to 1.17, two trials, 3902 women; GRADE: low quality evidence); at birth of a Rh-positive infant the RR was 0.42 (95% CI 0.15 to 1.17, two trials, 2297 women); and within 12 months after birth of a Rh-positive infant the average RR was 0.39 (95% CI 0.10 to 1.62, two trials, 2048 women; Tau²: 0.47; I²: 39%; GRADE: low quality evidence). Neither of the trials reported on incidence of Rhesus D alloimmunisation in subsequent pregnancies.Considering secondary outcomes, in one trial, women receiving anti-D during pregnancy were shown to be less likely to register a positive Kleihauer test (which detects fetal cells in maternal blood) in pregnancy (at 32 to 25 weeks) (RR 0.60, 95% CI 0.41 to 0.88; 1884 women; GRADE: low quality evidence) and at the birth of a Rh-positive infant (RR 0.60, 95% CI 0.46 to 0.79; 1189 women; GRADE: low quality evidence). No clear differences were seen for neonatal jaundice (RR 0.26, 95% CI 0.03 to 2.30; 1882 infants; GRADE: very low quality evidence). Neither of the trials reported on adverse effects associated with anti-D treatment. Existing studies do not provide conclusive evidence that the use of anti-D during pregnancy benefits either mother or baby in terms of incidence of Rhesus D alloimmunisation during the pregnancy or postpartum, or the incidence of neonatal morbidity (jaundice) (low to very low quality evidence). However women receiving anti-D may be less likely to register a positive Kleihauer test in pregnancy and at the birth of a Rh-positive infant (low quality evidence). Fewer women who receive anti-D during pregnancy may have Rhesus D antibodies in a subsequent pregnancy, with benefits for the baby, however this needs to be tested in studies of robust design.

Telomere homeostasis in trophoblasts and in cord blood cells from pregnancies complicated with preeclampsia

Telomeres are nucleoprotein structures, essential for chromosome stability and cell survival. Telomeres are progressively shortened with each cell division and by environmental factors. Telomere loss has been linked to age and stress-induced premature senescence. Dysfunctional telomeres tend to form aggregates, which consist of the end-to-end fusion of telomeres. Telomere elongation is carried out by telomerase, which is a specific reverse transcriptase capable of adding telomeric repeats to chromosome termini. The TERC gene encodes the RNA template of the telomerase. Another compensatory mechanism that is enhanced in response to telomere shortening and senescence is the telomere capture (TC). Telomere shortening and elevated aggregate formation have been observed in trophoblasts from pregnancies complicated with preeclampsia (PE). We opted to study mechanisms of telomere shortening in trophoblasts from pregnancies complicated with PE and to assess telomere length and homeostasis in fetal cord blood cells from PE pregnancies. Placental specimens and cord blood samples from uncomplicated pregnancies and from pregnancies complicated with PE were collected. Staining with 4',6-diamidino-2-phenylindole was used to assess nuclear fragmentation: senescence-associated heterochromatin foci (SAHF). Fluorescence in situ hybridization was used to evaluate TERC gene copy number and TC. Telomere length and aggregate formation were assessed in cord blood using quantitative fluorescence in situ hybridization. Nonparametric Kruskal-Wallis and Mann-Whitney U tests were applied to test the differences between the study groups. Nine samples from pregnant patients with PE without intrauterine growth restriction and 14 samples from uncomplicated pregnancies that served as controls were collected. In cord blood cells, no differences were observed in telomere length, aggregate formation, TERC copy number, TC, or SAHF between PE and controls. In PE trophoblasts the percentage of cells with SAHF was higher in PE trophoblasts compared to controls (56.8 SD = 10.5% vs 35.2 SD = 10.7%, P = .028). The percentage of cells with abnormal TERC copy number was increased in PE trophoblasts compared to controls (31 ± 3.6% vs 12.97 SD = 5%, P = .004) as well as the percentage of cells with TC (27.4 SD = 9.4% vs 16 SD = 4.67%, P = .028). We suggest that telomere shortening in PE trophoblasts is linked to cellular increased senescence. Alterations in telomere homeostasis mechanisms are present in such cases. These findings support the role of telomeres in the pathogenesis of trophoblastic dysfunction in PE. The lack of telomere shortening, modified telomere homeostasis mechanisms, and increased senescence in cord blood from pregnancies complicated with PE suggests that these processes are probably restricted primarily to the placenta.

OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo.

Rapid Aneuploidy Detection of Chromosomes 13, 18, 21, X and Y Using Quantitative Fluorescent Polymerase Chain Reaction with Few Microdissected Fetal Cells

Objectives: Analysis of DNA from small numbers of cells, such as fetal cells in maternal blood, is a major limiting factor for their use in clinical applications. Traditional methods of single-cells whole genome amplification (SCs-WGA) and accurate analysis have been challenging to date. Our purpose was to assess the feasibility of using a few fetal cells to determine fetal sex and major chromosomal abnormalities by quantitative fluorescent polymerase chain reaction (QF-PCR). Methods: Cultured cells from 26 amniotic fluid samples were used for standard DNA extraction and recovery of 5 fetal cells by laser-capture microdissection. SCs-WGA was performed using the DNA from the microdissected cells. PCR amplification of short tandem repeats specific for chromosomes 13, 18, 21, X and Y was performed on extracted and amplified DNA. Allele dosage and sexing were quantitatively analyzed following separation by capillary electrophoresis. Results: Microsatellite QF-PCR analysis showed high concordance in chromosomal copy number between extracted and amplified DNA when 5 or more cells were used. Results were in concordance with that of conventional cytogenetic analysis. Conclusion: Satisfactory genomic coverage can be obtained from SCs-WGA. Clinically, SCs-WGA coupled with QF-PCR can provide a reliable, accurate, rapid and cost-effective method for detection of major fetal chromosome abnormalities.

Fetal gender and several cytokines are associated with the number of fetal cells in maternal blood--an observational study.

ObjectiveTo identify factors influencing the number of fetal cells in maternal blood.MethodsA total of 57 pregnant women at a gestational age of weeks 11–14 were included. The number of fetal cells in maternal blood was assessed in 30 ml of blood using specific markers for both enrichment and subsequent identification.ResultsParticipants carrying male fetuses had a higher median number of fetal cells in maternal blood than those carrying female fetuses (5 vs. 3, p = 0.04). Certain cytokines (RANTES, IL-2 and IL-5) were significantly associated with the number of fetal cells in maternal blood.ConclusionThe number of fetal cells in maternal blood is associated with certain cytokines and fetal gender.

Maternal intravenous administration of azithromycin results in significant fetal uptake in a sheep model of second trimester pregnancy.

Treatment of intrauterine infection is likely key to preventing a significant proportion of preterm deliveries before 32 weeks of gestation. Azithromycin (AZ) may be an effective antimicrobial in pregnancy; however, few gestation age-approriate data are available to inform the design of AZ-based treatment regimens in early pregnancy. We aimed to determine whether a single intra-amniotic AZ dose or repeated maternal intravenous (i.v.) AZ doses would safely yield therapeutic levels of AZ in an 80-day-gestation (term is 150 days) ovine fetus. Fifty sheep carrying single pregnancies at 80 days gestation were randomized to receive either: (i) a single intra-amniotic AZ administration or (ii) maternal intravenous AZ administration every 12 h. Amniotic fluid, maternal plasma, and fetal AZ concentrations were determined over a 5-day treatment regimen. Markers of liver injury and amniotic fluid inflammation were measured to assess fetal injury in response to drug exposure. A single intra-amniotic administration yielded significant AZ accumulation in the amniotic fluid and fetal lung. In contrast, repeated maternal intravenous administrations achieved high levels of AZ accumulation in the fetal lung and liver and a statistically significant increase in the fetal plasma drug concentration at 120 h. There was no evidence of fetal injury in response to drug exposure. These data suggest that (i) repeated maternal i.v. AZ dosing yields substantial fetal tissue uptake, although fetal plasma drug levels remain low; (ii) transfer of AZ from the amniotic fluid is less than transplacental transfer; and (iii) exposure to high concentrations of AZ did not elicit overt changes in fetal white blood cell counts, amniotic fluid monocyte chemoattractant protein 1 concentrations, or hepatotoxicity, all consistent with an absence of fetal injury.

A new marker set that identifies fetal cells in maternal circulation with high specificity.

Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. Fetal cell candidates could be detected in 91% of the samples, and in 85% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100% if three or more fetal cells with FISH signals could be found in a sample. The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic.

Validation of automatic scanning of microscope slides in recovering rare cellular events: application for detection of fetal cells in maternal blood

Detection of rare fetal cells (FCs) in the maternal circulation could be used for non-invasive prenatal diagnosis. Considering that FCs in maternal blood are present in extremely low frequency, manual scanning is cumbersome, time-consuming, and unsuitable for clinical applications. As an alternative, we optimized a custom-made classifier for automatic detection of FCs. Using MetaSystems' automated platform, we developed a robust detection algorithm and validated its efficiency on retrieval of rare XY cells in a pure population of XX cells. Slides were scanned for presence of predefined XY cells after fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS). Retrieval of FCs was also performed on samples from maternal blood. The efficiency of detection of rare XY cells was 88% using FISH (117/133) in comparison with 78% (53/68) with PRINS. FC frequencies per 1 mL of maternal blood ranged from 3 to 6 FCs in normal pregnancies versus 13 to 21 FCs in Down syndrome pregnancies. Automatic scanning was more efficient and consistent than manual scanning for detection of rare FCs and required considerably less operator time. Automatic scanning using FISH is more sensitive than that using PRINS. The study validates automatic scanning retrieval of FCs from maternal blood.

Mosaic tetrasomy 9p at amniocentesis: Prenatal diagnosis, molecular cytogenetic characterization, and literature review

ObjectiveThis study was aimed at prenatal diagnosis of mosaic tetrasomy 9p and reviewing the literature. Materials and methodsA 37-year-old woman underwent amniocentesis at 20 weeks' gestation because of advanced maternal age and fetal ascites. Cytogenetic analysis of cultured amniocytes revealed 21.4% (6/28 colonies) mosaicism for a supernumerary i(9p). Repeat amniocentesis was performed at 23 weeks' gestation. Array comparative genomic hybridization, interphase fluorescence in situ hybridization, and quantitative fluorescent polymerase chain reaction were applied to uncultured amniocytes, and conventional cytogenetic analysis was applied to cultured amniocytes. ResultsArray comparative genomic hybridization analysis of uncultured amniocytes detected a genomic gain at 9p24.3–9q21.11. Interphase fluorescence in situ hybridization analysis of uncultured amniocytes using a 9p24.3-specific probe RP11-31F19 (spectrum red) showed four red signals in 47.1% (49/104 cells) in uncultured amniocytes. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XX, +idic(9)(pter→q21.11::q21.11→pter)[4]/46,XX[20] and 16.7% (4/24 colonies) mosaicism for tetrasomy 9p. Quantitative fluorescent polymerase chain reaction confirmed a maternal origin of tetrasomy 9p. The pregnancy was terminated, and a malformed fetus was delivered with hydrops fetalis and facial dysmorphism. The fetal blood cells had 32.5% (13/40 cells) mosaicism for tetrasomy 9p. ConclusionMosaic tetrasomy 9p at amniocentesis can be associated with fetal ascites and hydrops fetalis. The mosaic level of tetrasomy 9p may decrease after long-term tissue culture in amniocytes in case of mosaic tetrasomy 9p.