Search

Women are learning about their own health problems through fetal screening. Revise consent forms and raise awareness, urges Diana W. Bianchi.

Noninvasive prenatal testing (NIPT) using cell-free DNA is being offered to an increasing number of women. Comprehensive pre-test counseling is complicated by emerging information about the benefits and limitations of testing, as well as the potential to detect incidental findings. Genetic counselors are trained to facilitate informed decision-making; however, not all centers have access to these professionals. To aid in the informed consent process, we have summarized key points to be included in discussions with patients who are considering NIPT.

Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Summary and recommendations

'Omics analysis of large datasets has an increasingly important role in perinatal research, but understanding gene expression analyses in the fetal context remains a challenge. We compared the interpretation provided by a widely used systems biology resource (ingenuity pathway analysis [IPA]) with that from gene set enrichment analysis (GSEA) with functional annotation curated specifically for the fetus (Developmental FunctionaL Annotation at Tufts [DFLAT]).Using amniotic fluid supernatant transcriptome datasets previously produced by our group, we analyzed 3 different developmental perturbations: aneuploidy (Trisomy 21 [T21]), hemodynamic (twin-twin transfusion syndrome [TTTS]), and metabolic (maternal obesity) vs sex- and gestational age-matched control subjects. Differentially expressed probe sets were identified with the use of paired t-tests with the Benjamini-Hochberg correction for multiple testing (P.05). Functional analyses were performed with IPA and GSEA/DFLAT. Outputs were compared for biologic relevance to the fetus.Compared with control subjects, there were 414 significantly dysregulated probe sets in T21 fetuses, 2226 in TTTS recipient twins, and 470 in fetuses of obese women. Each analytic output was unique but complementary. For T21, both IPA and GSEA/DFLAT identified dysregulation of brain, cardiovascular, and integumentary system development. For TTTS, both analytic tools identified dysregulation of cell growth/proliferation, immune and inflammatory signaling, brain, and cardiovascular development. For maternal obesity, both tools identified dysregulation of immune and inflammatory signaling, brain and musculoskeletal development, and cell death. GSEA/DFLAT identified substantially more dysregulated biologic functions in fetuses of obese women (1203 vs 151). For all 3 datasets, GSEA/DFLAT provided more comprehensive information about brain development. IPA consistently provided more detailed annotation about cell death. IPA produced many dysregulated terms that pertained to cancer (14 in T21, 109 in TTTS, 26 in maternal obesity); GSEA/DFLAT did not.Interpretation of the fetal amniotic fluid supernatant transcriptome depends on the analytic program, which suggests that1 resource should be used. Within IPA, physiologic cellular proliferation in the fetus produced many "false positive" annotations that pertained to cancer, which reflects its bias toward adult diseases. This study supports the use of gene annotation resources with a developmental focus, such as DFLAT, for 'omics studies in perinatal medicine.

Numerous recent studies have shown the power of cell-free fetal RNA, obtained from amniotic fluid supernatant, to report on the development of the living fetus in real time. Examination of these transcripts on a genome-wide basis has led to new insights into the prenatal pathophysiology of multiple genetic, developmental, and environmental diseases. Each studied condition presents a unique, characteristic fetal transcriptome, which points to specific disrupted molecular pathways. These studies have also improved our knowledge of the normal development of the human fetus, revealing gestational age-related dynamic gene expression from a variety of organs. Analysis of the fetal transcriptome in normal and abnormal development has led to novel approaches for in utero prenatal treatment.

To describe the clinical experience with noninvasive prenatal testing for fetal sex chromosomes using sequencing of maternal plasma cell-free DNA in a commercial laboratory.A noninvasive prenatal testing laboratory data set was examined for samples in which fetal sex chromosomes were reported. Available clinical outcomes were reviewed.Of 18,161 samples with sex chromosome results, no sex chromosome aneuploidy was detected in 98.9% and the fetal sex was reported as XY (9,236) or XX (8,721). In 4 of 32 cases in which the fetal sex was reportedly discordant between noninvasive prenatal testing and karyotype or ultrasonogram, a potential biological reason for the discordance exists, including two cases of documented co-twin demise, one case of a maternal kidney transplant from a male donor, and one case of fetal ambiguous genitalia. In the remaining 204 samples (1.1%), one of four sex chromosome aneuploidies (monosomy X, XXX, XXY, or XYY) was detected. The frequency of false positive results for sex chromosome aneuploidies is a minimum of 0.26% and a maximum of 1.05%. All but one of the discordant sex chromosome aneuploidy results involved the X chromosome. In two putative false-positive XXX cases, maternal XXX was confirmed by karyotype. For the false-positive cases, mean maternal age was significantly higher in monosomy X (P.001) and lower in XXX (P=.008).Noninvasive prenatal testing results for sex chromosome aneuploidy can be confounded by maternal or fetal biological phenomena. When a discordant noninvasive prenatal testing result is encountered, resolution requires additional maternal history, detailed fetal ultrasonography, and determination of fetal and possibly maternal karyotypes.

Objectives: Non-invasive prenatal diagnosis using circulating fetal trophoblast cells has been challenging due to lack of a reproducible trophoblast-specific antibody. We investigated the use of three trophoblast cell-specific antibodies, HLA-G, placenta growth factor, and neuroD2, for the isolation of trophoblast cells from the maternal circulation. Methods: Trophoblast cells were isolated by density centrifugation from maternal blood samples (gestational age 10–20 weeks, n = 9). All women were carrying a male fetus. Following immunocytochemical staining with the trophoblast-specific antibodies, fluorescent in situ hybridization was performed, to verify whether any stained cells were indeed fetal. Results: The HLA-G antibody had a ubiquitous staining pattern, which was not specific for trophoblast cells. Neither the placenta growth factor nor the neuroD2 antibodies were able to identify any trophoblast cells. Following fluorescent in situ hybridization, no male cells were detected on any of the slides. Conclusion: The antibodies used in this study were unable to improve detection of trophoblast cells in the maternal circulation.

Objective The purpose of this study was to evaluate whether there is a nuchal translucency (NT) measurement, independent of gestational age, above which immediate diagnostic testing should be offered without waiting for first trimester serum markers. Study design Thirty-six thousand one hundred twenty patients had successful measurement of simple NT at 10 3/7 to 13 6/7 weeks and had first trimester serum screening. No risks were reported until second trimester serum screening was completed. Results Thirty-two patients (0.09%) had NT ≥4.0 mm; the lowest combined first trimester trisomy 21 risk assessment in euploid cases was 1 in 8 and among aneuploidy cases was 7 in 8. One hundred twenty-eight patients (0.3%) had simple NT ≥3.0 mm: the lowest combined first trimester trisomy 21 risk assessment of any patient in this group was 1 in 1479 and the lowest risk assessment among aneuploid cases was 1 in 2. Ten patients (8%) had first trimester trisomy 21 risk assessments lowered to less that 1:200 and none of these 10 cases had an abnormal outcome. Conclusion During first trimester Down syndrome screening, whenever an NT measurement of 3.0 mm or greater is obtained there is minimal benefit in waiting for serum screening results, and no benefit for NT of 4.0 mm or greater. Differentiation between cystic hygroma and enlarged simple NT (≥3.0 mm) is now a moot point as both are sufficiently high risk situations to warrant immediate CVS.

The aim of this study was to examine fetal gene expression in maternal plasma after fetoscopic intervention for twin-twin transfusion syndrome or congenital diaphragmatic hernia.Twelve women with pregnancies that were complicated by twin-twin transfusion syndrome and 10 women carrying fetuses with congenital diaphragmatic hernia were sampled before and sequentially after treatment. Levels of glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, and gamma globin messenger RNA were measured by real-time reverse transcriptase polymerase chain reaction amplification.At all time points, glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels were higher in the congenital diaphragmatic hernia cases than in the twin-twin transfusion syndrome cases (P.05), but during the immediate postoperative observation period, there were no significant changes in glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, or gamma globin messenger RNA levels in individual patients or patients who were grouped by procedure.Fetoscopic intervention of complicated pregnancies does not affect circulating fetal messenger RNA levels, which is in contrast to earlier observations that circulating fetal DNA levels increase after laser ablation for twin-twin transfusion syndrome. Plasma glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels could be a potential novel biomarker for fetal trauma.

Trisomy of the short arm of chromosome 12 is a rare chromosomal anomaly, with an estimated incidence of 1/50,000 births. It may present as a pure trisomy (complete or incomplete), as mosaic trisomy, or with other chromosomal abnormalities. Little is known from prior reports about the natural history and life expectancy of these individuals. In this study we describe the long-term outcome and the differences between patients with mosaic trisomy 12p compared to patients with complete trisomy. We present a series of 16 patients with trisomy 12p; 6 of them are older than 10 years. Most patients were born at term with normal or above normal birth weight. Seven were born with congenital anomalies, but no single anomaly was present in more than one individual. A clear and consistent dysmorphic facial pattern was apparent in all of the subjects. Most patients over 7 years old had a seizure disorder. All individuals exhibited developmental delay with speech affected more severely than motor skills. Six patients were described as "being social." Six had severe behavioral problems, and seven had significant sleep disturbances. Facial features of the three adult patients were different than the younger individuals. We show here that the outcome for patients with mosaic trisomy 12p is better than the outcome in complete trisomy 12p or in trisomy 12p with other chromosomal anomalies. We also provide recommendations for the long-term follow-up of patients with trisomy 12p.

In 2003 the National Institute of Child Health and Human Development (NICHD) sponsored a workshop entitled "Sharpening the Tools", which was designed to explore the then current state of prenatal diagnosis and screening using fetal cells in maternal blood. The goals of the workshop were to: review the then current state of the field and assess present capabilities, identify future research needs and challenges in this area, identify promising new and innovative approaches for future exploration, and provide a written summary of the conference for public distribution. The workshop featured brief presentations by experts from a wide range of scientific fields and by innovative bioengineering and technology leaders from academic centers and private industry. The workshop was divided into presentations on target cells, target approaches for separation, genetic and protein analysis, and "out of the box" (bioengineering) approaches. The passage of time since the workshop has allowed an objective assessment of where the research has progressed. A 2006 update on the field is included at the end of the summary.

BACKGROUND: Increased second-trimester levels of maternal serum HCG in IVF conceptions lead to an increased false-positive rate in Down syndrome screening. Increased levels of cell-free fetal DNA (cffDNA) in maternal plasma have been correlated with increased HCG levels. Our aim was to determine whether cffDNA levels are elevated in IVF pregnancies compared with natural pregnancies. METHODS: Sixteen archived second-trimester serum samples from IVF pregnancies were matched with five control samples from naturally conceived pregnancies per case, all carrying a singleton male fetus. cffDNA concentrations were me asured by real-time PCR amplification of a Y chromosome sequence and compared with four standard second trimester serum screening markers (-fetoprotein, estriol, HCG and inhibin A). RESULTS: Mean cffDNA levels for cases and controls were 57.9 and 57.1 genome equivalents/ml, respectively (P = 0.95). Mean observed rank (from 1 to 6) of cffDNA was 3.625 in the IVF conceived group, compared with an expected value of 3.5 (P = 0.53). No significant correlations w ere observed between cffDNA and serum markers. CONCLUSIONS: IVF does not affect levels of cffDNA, which appears to be independent of traditional screening markers (e.g. HCG). Therefore, cffDNA can be used as an additional serum marker (e.g. Down syndrome screening) without adjustment for IVF pregnancies.

In humans, fetal cells enter the maternal circulation during all pregnancies and can persist for decades. Human studies, however, are often limited by the number of subjects and the availability of healthy and diseased tissues for analysis. We sought to develop a murine model to establish the natural history of fetal cell microchimerism in various maternal tissues during and after healthy pregnancies resulting from congenic and allogenic matings. We bred C57BL/6J and DBA/2J virgin female mice to C57BL/6J males transgenic for the enhanced green fluorescent protein (GFP), which shows autosomal dominant inheritance with complete penetrance and is under the control of a ubiquitous chicken beta-actin promoter and a cytomegalovirus enhancer. During pregnancy and at different times after delivery, female mice were sacrificed. Tissues were collected and the presence of the gfp transgene and GFP+ cells was assessed by real-time quantitative PCR and by immunofluorescence. During pregnancy, microchimerism was detected in all tissues from mice carrying GFP+ fetuses. Fetal cells were often mononuclear. The frequency of fetal cells in the lungs was significantly higher compared to other tissues. The level of microchimerism was also significantly higher in congenic compared to allogenic matings. After delivery, the frequency of fetal cells decreased and fetal cells were undetectable at 2 and 3 weeks after the first delivery. However, some mice that had three gestations had detectable fetal cells 3 weeks after their last delivery. Using sensitive methods of detection, we demonstrate that fetal cell microchimerism occurs during all murine pregnancies. We describe a useful model for the study of the consequences of this phenomenon.

The objective was to estimate the effect of maternal age on obstetric outcomes. A prospective database from a multicenter investigation of singletons the FASTER trial was studied. Subjects were divided into 3 age groups: 1) less than 35 years 2) 35–39 years and 3) 40 years and older. Multivariable logistic regression analysis was used to assess the effect of age on outcomes after adjusting for race parity body mass index education marital status smoking medical history use of assisted conception and patient’s study site. A total of 36056 women with complete data were available: 28398 (79%) less than 35 years of age; 6294 (17%) 35–39 years; and 1364 (4%) 40 years and older. Increasing age was significantly associated with miscarriage (adjusted odds ratio {adjOR}2.0 and 2.4 for ages 35–39 years and age 40 years and older respectively) chromosomal abnormalities (adjOR 4.0 and 9.9) congenital anomalies (adjOR 1.4 and 1.7) gestational diabetes (adjOR 1.8 and 2.4) placenta previa (adjOR 1.8 and 2.8) and cesarean delivery (adjOR 1.6 and 2.0). Patients aged 35–39 years were at increased risk for macrosomia (adjOR 1.4). Increased risk for abruption (adjOR 2.3) preterm delivery (adjOR 1.4) low birth weight (adjOR 1.6) and perinatal mortality (adjOR 2.2) was noted in women aged 40 years and older. Increasing maternal age is independently associated with specific adverse pregnancy outcomes. Increasing age is a continuum rather than a threshold effect. (authors)

Rhesus (Rh) D blood group incompatibility between the pregnant woman and her fetus is a significant problem due to the possibility of maternal alloimmunization and consequent hemolytic disease of the newborn. The RhD-negative blood group is found in 15% of whites, 3-5% of black Africans, and is rare in Asians. Advances in both our understanding of the RHD locus and its variants, as well as technical improvements in the extraction and amplification of cell-free fetal DNA in maternal plasma, have led to incorporation of noninvasive diagnosis of RHD genotype into routine prenatal care in the United Kingdom, France, and the Netherlands. In this commentary we examine the experience to date with large-scale clinical trials performed in the European Union, describe approaches to reduce false-positive and false-negative results, and review ongoing research to standardize assays and reduce costs using automated assays. False-negative cases are mainly due to either a lack of fetal DNA in the maternal sample due to early gestation or insensitive methods. False-positive cases are due to genotypic variants observed in individuals of African ancestry. Noninvasive prenatal diagnosis of fetal Rhesus D genotype is sensitive and accurate and has been widely validated in Europe. The United States should begin to undertake clinical trials to bring this technology to patient care as soon as possible.

The presence and persistence of fetal cells in murine maternal tissue was first reported over 20 years ago, although it is only more recently that the occurrence and potential consequences of fetomaternal cell trafficking in humans have been fully appreciated. Fetal cell microchimerism is a growing field of investigation, although the data are contradictory relative to the health consequences of persistent fetal cells in maternal tissues. Understanding of the types of cells being transferred from fetus to mother, the location of these fetal cells within the various maternal tissue types, and the functionality of these cells may ultimately lead to measures to minimize or eliminate the deleterious effects of the cells, or to efforts to take advantage of the presence of these cells for therapeutic purposes. This review focuses on the origins of fetal cell microchimerism research and the different hypotheses regarding the consequences of persistent fetal cells in the mother, the various diseases that have been evaluated with respect to fetomaternal cell trafficking, the potential variables associated with the frequency, persistence and tissue distribution of fetal cells in maternal tissue, and an assessment of future direction in this innovative field of inquiry.

Analysis of cell-free fetal DNA (fDNA) and RNA in maternal plasma could be useful in the diagnosis and management of complications of pregnancy. In this review, we discuss our studies to investigate the potential of fetal nucleic acid measurement in maternal plasma as a marker of fetomaternal hemorrhage (FMH) after elective first-trimester termination of pregnancy (TOP). Using quantitative real-time PCR amplification of the DYS1 sequence, elevation of plasma fDNA levels after TOP was observed, especially in the late first trimester. This corresponds with the functional development of the placental vascular structure and fetal hematopoiesis. This Y sequence-based PCR amplification assay, however, limits the analysis to pregnant women carrying male fetuses. Therefore, we also developed a real-time quantitative reverse-transcriptase PCR assay of the γ-globin transcript as a marker of fetal erythroid cells. Although plasma γ-globin mRNA levels were decreased after TOP in many patients, an elevation was observed in some patients at greater than 9 weeks' gestation, which is consistent with the increase in plasma fDNA levels. Our data suggest that fetal hematopoietic cells contribute to the pool of fetal nucleic acids in the maternal circulation. Measurement of cell-free fetal nucleic acid levels in maternal plasma may have clinical application as a novel marker of FMH after 9 weeks of gestation.

Objective The purpose of this study was to determine whether cell-free fetal DNA is detectable in the cerebrospinal fluid of women during pregnancy and after delivery. Study design Cerebrospinal fluid was collected from 39 women who underwent an indicated spinal anesthesia procedure. Twenty-six samples were from women who carried at least 1 male fetus, and 13 samples were from women with only a female fetus. DNA was analyzed with the use of real-time polymerase chain reaction for DYS-1 (which represented male fetal DNA) and β-globin (which represented maternal and fetal DNA). Results β-Globin DNA was detected in all cerebrospinal samples. DYS-1 gene sequences were detected in 4 cerebrospinal fluid samples from women who had male fetuses (2 samples were from women who underwent cesarean delivery of singleton pregnancies, 1 sample was from a triplet pregnancy, and 1 sample was from a woman after delivery). No male DNA was detected in the cerebrospinal fluid of women who carried female fetuses. Conclusion Male fetal cells and/or cell-free fetal DNA is detectable in the cerebrospinal fluid of some pregnant women or some women after delivery.

Background: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. Methods: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. Results: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93–100%) with only four false-positive results obtained across the entire study. Conclusions: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

AB Purpose of review: Fetal cells enter the maternal circulation during most pregnancies and can persist in maternal blood and tissues after delivery. Concerns with regard to the histocompatibility of these fetal cells have raised the question of the long-term consequences of an immune response on maternal health. In the past few years, many investigators have demonstrated an association between the persistence of fetal cells in maternal tissues and blood and maternal autoimmune disease, especially systemic sclerosis. In this review we will summarize more recent data that provide a new insight into bi-directional feto-maternal cell trafficking. Recent findings: Persisting fetal cells have been found in the tissue of women affected with endocrine or infectious disease as well as healthy parous women. Summary: These data suggest the possibility that fetal microchimeric cells may also participate in the maternal physiological response to tissue injury. The medical consequences of pregnancy, therefore, appear to extend well beyond delivery. (C) 2003 Lippincott Williams & Wilkins, Inc.

Objectives On the basis of reports of maternal cells being detected in the umbilical cord blood of newborn infants, we tested the hypothesis that maternal cells can migrate out of the circulation into newborn tissues. Study design We studied autopsy material from 4 newborn infants who never received a blood transfusion and died during the first week of life. The study subjects' diagnoses were trisomy 21 with nonimmune hydrops, 46,XY, 4q+ with multiple congenital anomalies, Potter syndrome, and congenital ichthyosis. Fluorescence in situ hybridization analysis with X and Y chromosome–specific probes was performed on sections of paraffin-embedded tissue, including liver, spleen, thymus, thyroid, and skin. Results Female cells, as defined by the presence of intact nuclei with two X chromosome signals, were detected in multiple tissue types from all 4 male neonates. The number of female cells varied from 3 to 45 per slide. Conclusions Maternal cells enter the fetal circulation and are capable of migration to fetal and neonatal organs. This is of importance with regard to potential consequences of umbilical cord blood transplantation and postnatal development of autoimmune disease. (J Pediatr 2003;142:31-5)

The National Institute of Child Health and Human Development (NICHD) held a workshop on 27-28 July 2000 to bring together investigators working in the field of fetomaternal cellular and nucleic acid trafficking with the hope that this would stimulate further research into the biological implications of such phenomena.Invited speakers from all over the world presented their latest (unpublished) data. The conference proceedings were delayed until the present time to allow independent publication of the primary data.Bi-directional fetomaternal trafficking of cells and nucleic acids during pregnancy is now well established, through the use of molecular techniques including conventional and real-time polymerase chain reaction, as well as fluorescence in situ hybridization. In addition, human leukocyte antigen (HLA) is deposited in the skin of pregnant women. Fetomaternal trafficking is increased in some complications of pregnancy, such as pre-eclampsia, polyhydramnios, polymorphic eruption of pregnancy, preterm labor and specific fetal chromosome aneuploidies. Maternal cells and nucleic acids have been documented in umbilical cord blood and in autopsy tissue of non-transfused neonates. Fetal cells persist postpartum and may be associated with the development of disorders such as scleroderma, lichen planus, lupus and thyroid disease. The extent of fetomaternal trafficking may be affected by three generational HLA relationships. Thus, the consequences of pregnancy extend beyond gestation.

Maternal obesity affects 1 in 5 pregnant women in the United States1. Maternal obesity is associated with increased circulating total, but not fetal, cf DNA2. This may be a result of increased production or decreased clearance of cf DNA in obese women. It is more likely that this is due to increased production of total cfDNA, because decreased clearance would also likely lead to an increase in cell-free fetal cfDNA. In a prior study performed on obese pregnant women, we showed that active remodeling of adipose tissue via adipocyte necrosis and/or apoptosis of the stromal vascular fraction results in an increased release of cfDNA of maternal origin into the circulation3. The focus of the prior study was on the mechanisms underlying the release of the cfDNA. In this study we more closely examined the correlation between maternal weight and total DNA levels. This study was approved by the Institutional Review Boards at Tufts Medical Center and Metrohealth Medical Center. The samples are from the same cohort reported previously3, but the analysis is different. Briefly, sixteen obese (mean=39.2; pre-gravid BMI range 31–51) and 14 lean (mean 21.8; pre-gravid BMI range 17–24) women carrying male fetuses and 10 women carrying female fetuses (negative controls) were recruited at term (37–40 weeks) prior to an elective cesarean section. Written informed consent was obtained prior to obtaining the samples. Women with a multiple gestations, placenta previa or invasive placentation, labor, infection, fetal anomalies or aneuploidy, intrauterine growth restriction, or preeclampsia were excluded. Maternal peripheral venous blood was collected at MetroHealth Medical Center on admission to labor and delivery, prior to placement of an intravenous line for hydration. All subjects had the same instructions prior to admission and had nothing to eat or drink for 6–8 hours prior to the blood draw. The blood was collected in an EDTA tube and plasma was separated by centrifugation and kept frozen at −20°C prior to being shipped to Tufts Medical Center for further analysis. DNA was extracted from 400 uL of plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the blood and body fluid protocol. DNA was eluted in 50 μL of the elution buffer. Real time quantitative PCR amplification was performed as previously described4 to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS14 as markers of total and fetal DNA, respectively. All samples were analyzed in triplicate. Analysis was blinded, and a female processed and handled all samples so that there was no risk of contaminating samples with male DNA. Conversion of raw PCR data to genome equivalents per mL of plasma was performed using the methods of Lo et al.5 The levels of total and fetal cf DNA in plasma from lean and obese women were compared using the t test. In addition, the maternal plasma volume from both obese and lean subjects was adjusted for blood volume6 and compared using the t test, as prior experiments in our laboratory suggested an increased blood volume as a function of weight7. Finally, we performed a regression analysis between total and fetal cf DNA and maternal BMI. Analysis of the raw data from all subjects showed that there was a 1.7% increase in total cell-free DNA per BMI unit (kg/m2). Following adjustment for blood volume, there was a 3.2% increase per BMI unit (Table 1). When the subjects were categorized into lean and obese groups, however, there was a decrease in total cf DNA per BMI unit in lean women and an increase in obese women (Table 1 and Figure 1). Neither of these changes were statistically significant. However, this lack of significance may be a function of the small sample size. Further studies with more subjects are warranted to further characterize this relationship between cf DNA and BMI. Figure 1 Graph showing relationship between total cell-free DNA levels and body mass index [BMI]. The correlation coefficient between BMI and cfDNA for the lean population is -0.061 (p=0.41) and for the obese population is 0.23 (p=0.20). Table 1 Change in maternal cell-free DNA by body mass index In this study, we found a correlation between BMI and total DNA levels. In lean women, the decrease in total cf DNA likely reflects a dilutional effect seen in all pregnant women due to increasing plasma volume. Conversely, in obese women, the increased levels of total cf DNA may reflect the increased adipocyte necrosis3 and stromal vascular apoptosis that is significant enough to overcome the dilutional effect that occurs in pregnant women. Our results are similar to a previous study by Lapaire et al.2, in which a correlation was found between maternal BMI and total, but not fetal, second trimester cf DNA levels. There are several differences between our study and the Lapaire study, however. Firstly, the gestational ages of the study subjects were different. In the present study, maternal blood samples were drawn at term, in contrast to 20 to 21 weeks. Secondly, in the Lapaire et al. study there was no adjustment for maternal weight. We adjusted for the effect of maternal weight on blood volume using the method described by Lemmens et al.6 The present study and that of Wataganara et al.7 suggest that a correction factor for maternal weight is needed when cf DNA analyses are performed. It is standard practice to correct for maternal weight when performing serum screening for Down syndrome. Diagnostic laboratories typically derive their own regression curves for the relationship between serum analytes and maternal weight because of several factors, including differences in instrument sensitivity in the measurement of serum analytes, as well as differences in maternal weight distribution among centers8. Review of the literature, however, indicates that there is no consensus as to the best method to adjust serum markers, including cf nucleic acids, for maternal weight in cases of extreme obesity. We selected the Lemmens et al.6 formula because it correlated total blood volume with BMI, although that formula was used for quantification of blood volume in non-pregnant surgical patients. In summary, we show here that maternal BMI affects total cf DNA levels in both lean and obese pregnant women. In the future, raw DNA values may need to be adjusted for maternal BMI for adequate interpretation of clinical tests that involve assessment of the fetal fraction9. In addition, the presence of increased total cf DNA levels in obese pregnant women suggests that this analyte may be a biomarker for systemic problems during gestation that may impact both maternal and fetal health.

Increased levels of cell-free fetal DNA (f-DNA) in the maternal circulation are a potential noninvasive marker for fetal Down syndrome. Our objectives were to (1) determine whether f-DNA could be quantified by using archived serum and amniotic fluid, (2) examine whether serum f-DNA levels are elevated in Down syndrome pregnancies in a case-control series matched for gestational age and duration of sample storage, and (3) determine whether f-DNA levels are elevated in the amniotic fluid of Down syndrome fetuses.Eleven serum and six amniotic fluid samples previously collected and stored at -20 degrees C from gravid women carrying a 47,XY,+21 fetus were each paired with five matched control samples of identical specimen type from gravid women carrying a presumed euploid male fetus. f-DNA concentration was quantified blindly by real-time polymerase chain reaction amplification for a Y-chromosome sequence. Matched rank-sum analysis and analysis of variance were used for analysis.The mean observed rank of 5.0 in the Down syndrome group was significantly higher than expected (P/=.005). Adjusted mean serum f-DNA concentrations were 41.2 genomic equivalents (GE) per milliliter for the Down syndrome cases and 24.2 GE/mL for the euploid controls (P =.002). Differences among amniotic fluid samples were not statistically significant. There was a suggestion of a sample storage effect on f-DNA concentration on the order of -0.66 GE/mL per month (P =.071).Down syndrome pregnancies exhibit 1.7-fold higher levels of maternal serum cell-free f-DNA compared with matched controls. No such association is observed in amniotic fluid. Archived serum appears to be a useful source of clinical material for retrospective analyses but may require controlling for the duration of sample storage.

Objective: We conducted a trial to test if the blood of pregnant women contains fetal clonogenic erythroid cells the progeny of which can be identified and isolated by a newly developed flow-sorting procedure. Methods: We have previously demonstrated the identification of fetal nucleated red cells in cocultures of fetal and adult blood. The procedure is based on profiles of the correlated contents of fetal and adult hemoglobin (HbF and HbA, respectively), using antibodies specific for the different hemoglobin chains. In such profiles, fetal cells contain only HbF, while the vast majority of adult cells contain either only HbA or a combination of HbA and HbF. HbF+ HbA– cells are flow sorted and fetal cells identified by fluorescence in situ hybridization, using chromosome-specific probes. This technique provides a yield that approaches 100%, meaning that fetal cells will be found even if the culture contains only a single fetal erythroid colony among thousands of maternal colonies. Peripheral blood samples were obtained from 11 women carrying chromosomally normal male fetuses, from 5 women carrying trisomy 21 fetuses, and from 2 women carrying trisomy 18 fetuses. A further six samples came from women with an unknown fetal karyotype. As positive controls, we used blood samples drawn after termination procedures that tended to induce some fetomaternal hemorrhage. In parallel to the method being tested, we employed alternative techniques of fetal cell detection: one third of the mononuclear cell preparations from each maternal blood sample was not cultured but labeled with anti-HbF antibodies for flow sorting of F+ cells. Ten percent of the total harvested cell population of each culture was subjected to quantitative polymerase chain reaction analysis targeting a Y-chromosome-specific sequence. Results: Most posttermination blood samples yielded fetal cells with high purity which demonstrates the validity of the method. However, no fetal cells were found in any of the maternal blood samples with normal or abnormal pregnancies, neither before nor after culture. Conclusion: We conclude that a cell culture approach targeting clonogenic erythroid cells offers no advantage over established methods of direct isolation.

Objectives The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is a prospective, multicenter clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to diagnose or screen for fetal chromosome abnormalities. Methods Results of fluorescence in situ hybridization (FISH) analysis on interphase nuclei of fetal cells recovered from maternal blood were compared to metaphase karyotypes of fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). After the first 5 years of the study we performed a planned analysis of the data. We report here the data from 2744 fully processed pre-procedural blood samples; 1292 samples were from women carrying singleton male fetuses. Results Target cell recovery and fetal cell detection were better using magnetic-based separation systems (MACS) than with flow-sorting (FACS). Blinded FISH assessment of samples from women carrying singleton male fetuses found at least one cell with an X and Y signal in 41.4% of cases (95% CI: 37.4%, 45.5%). The false-positive rate of gender detection was 11.1% (95% CI: 6.1,16.1%). This was higher than expected due to the use of indirectly labeled FISH probes in one center. The detection rate of finding at least one aneuploid cell in cases of fetal aneuploidy was 74.4% (95% CI: 76.0%, 99.0%), with a false-positive rate estimated to be between 0.6% and 4.1%. Conclusions The sensitivity of aneuploidy detection using fetal cell analysis from maternal blood is comparable to single marker prenatal serum screening, but technological advances are needed before fetal cell analysis has clinical application as part of a multiple marker method for non-invasive prenatal screening. The limitations of the present study, i.e. multiple processing protocols, are being addressed in the ongoing study. Copyright © 2002 John Wiley & Sons, Ltd.

Objective: Umbilical cord blood is a source of hematopoietic stem cells for transplantation. Although the first clinical applications have been encouraging, concern has been raised about contamination of umbilical blood by maternal cells, which might constitute a theoretical risk of graft-versus-host disease. The aim of this study was to assess the frequency of maternal deoxyribonucleic acid (DNA) contamination in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA markers. Study Design: Fifty-seven mother/child pairs were tested for the presence of maternal DNA sequences in cord plasma. After delivery, cord blood samples were collected via gravity. Maternal specific alleles were detected by using polymerase chain reaction amplification of 9 highly polymorphic short tandem repeat markers (D21S11, D21S1411, D21S1412, D18S386, D18S535, MBP-A, MBP-B, D13S631, and D13S634). Results: All 57 mother-child pairs were informative for the identification of uniquely maternal alleles in at least 2 of 9 different short tandem repeat markers used per case. Uniquely maternal DNA sequences were found in 43 of 57 (75%) cord plasma samples. Conclusion: The results of our study demonstrate that maternal DNA is present in the majority of umbilical cord blood plasma samples. The technique described herein might have application in the screening of umbilical cord blood samples for the presence of contaminating maternal genetic material. (Am J Obstet Gynecol 2002;186:117-20.)

Objective Intact fetal cells isolated from maternal blood can be used for non-invasive gender determination and genetic diagnosis. Recent studies demonstrating a large amount of cell-free fetal DNA in maternal plasma suggest that the circulating fetal DNA may result from fetal cells undergoing apoptosis. In the present study we evaluated the potential role of Fas and Fas ligand (FasL) cell surface expression with respect to apoptosis induction in fetal cells isolated from maternal blood. Methods We flow sorted candidate fetal cells that were gamma chain-positive and Fas- or FasL-positive or -negative, and subsequently analysed them by fluorescence in situ hybridization (FISH) analysis using X and Y chromosome-specific probes. Results Among all gamma hemoglobin-positive cells, there was a significant difference in the percent of cells expressing Fas versus FasL (4.4 and 12.3, respectively). We found no significant correlation between the total number of fetal nucleated red blood cells (NRBCs) and gestational age or the presence of Fas- and FasL-positive cells. From approximately 7 ml of maternal peripheral blood, most of the confirmed fetal (XY) cells were found in the Fas- and FasL-negative sorted population; the average numbers were 12.8 and 15.7, respectively. Conclusion We conclude that fetal NRBCs express FasL more than Fas, although most fetal NRBCs in first trimester maternal blood samples do not express Fas or FasL. This suggests the absence of a functional Fas/FasL apoptotic system in fetal NRBCs, and that programmed cell death in these cells, which may lead to circulating fetal DNA in maternal plasma, probably occurs by another pathway. Copyright © 2002 John Wiley & Sons, Ltd.

Over the past thirty years there has been a significant increase in the use of prenatal diagnosis to improve perinatal outcome. Frequently, at least in developed countries, this involves prenatal sonography to image fetal anatomy and detect congenital anomalies. This has led to a primarily surgical approach to fetal and neonatal treatment. While effective, this strategy has revealed little about fetal functional development. Since 2005, our laboratory has explored the hypothesis that valuable fetal developmental gene expression information is present in amniotic fluid (AF). In pilot studies performed using large quantities of amniotic fluid obtained from pregnancies complicated by polyhydramnios, we showed that cell-free fetal messenger RNA (cff mRNA) could be successfully hybridized to gene expression microarrays. Comparative analyses of the arrays from different subjects demonstrated that fetal gene expression changes could be detected as a result of fetal sex, gestational age, and disease status [1]. Furthermore, we showed that cff mRNA in AF originated from the fetus and not the placenta, which suggested that AF could be used directly to analyze fetal development and well-being. AF is widely available but generally under-utilized [2]. It is obtained primarily for fetal chromosome analysis and alpha-fetoprotein measurement to diagnose open neural tube defects. AF is an excellent source of material for research, as large quantities of supernatant are obtained for clinical diagnosis and then discarded, samples generally have correlated medical record and karyotype information available, and there is no contamination by maternal nucleic acids [2]. AF provides a way to directly query fetal development from as early as 15 weeks of gestation. We have used cff mRNA in AF to identify new biomarkers of fetal disease, develop new insights into fetal pathophysiology, and to suggest new approaches to fetal treatment.

Under unseasonably hot and sunny Dutch skies, over 600 participants gathered in Amsterdam for the 15th International Conference on Prenatal Diagnosis and Fetal Therapy. What no amount of advance planning could have predicted, however, was that the Dutch national team would reach the final in the World Cup Football tournament to be played on the eve of the conference. Thus, meeting attendees arrived in the midst of a nation expressing its national pride by displaying its national color, orange, everywhere. The conference’s opening reception was canceled because of the game and its related celebrations; everyone was encouraged to watch the game. The International Society for Prenatal Diagnosis’ (ISPD) Special Interest Group (SIG) on Fetal Therapy hosted a party at a local tavern (Figure 1). Among the attendees were the scientific program co-chairs of the prior (2008) ISPD meeting, Professors Doug Wilson and Sylvie Langlois (Figure 2). Although the Dutch team lost to the Spaniards, the festive atmosphere continued throughout the meeting. In this March 2011 issue of Prenatal Diagnosis, we feature many of the presentations that were made at the ISPD 2010 meeting (others will appear in subsequent issues.) All the contributions, with the exception of the debates and reports from the SIGs, have undergone peer review. For additional perspectives on the conference, featuring the on-site impressions of Professors Gerard Visser (the Netherlands) and Irma Nippert (Germany), a free podcast is available at www.ispdhome.org. The best-attended parts of the meeting were the four debates, which are summarized in this issue. In the first one, ‘Is stem cell therapy ready for human fetuses?’ the basic scientist Christine Mummery argues that most stem cell therapies are not yet suitable for adults, let alone fetuses. Her opinion contrasts with that of the clinician, Magnus Westgren, who presents his group’s experience

In its successful annual cycle of controversies and debates, the International Society of Prenatal Diagnosis and Therapy once again addressed non-invasive prenatal testing (NIPT) by following up on the 2013 controversy, 'Should non-invasive DNA testing be the standard screening test for Down syndrome in all pregnant women'? with the proposition, 'NIPT for chromosomel abnormalities should be offered to women with low a priori risk'.

Objective One in three pregnant women in the United States is obese. Their offspring are at increased risk for neurodevelopmental and metabolic morbidity. Underlying molecular mechanisms are poorly understood. We performed a global gene expression analysis of mid-trimester amniotic fluid cell-free fetal RNA in obese versus lean pregnant women. Methods This prospective pilot study included eight obese (BMI≥30) and eight lean (BMI

Item does not contain fulltext

Methods for translating gene expression signatures into clinically relevant information have typically relied upon having many samples from patients with similar molecular phenotypes. Here, we address the question of what can be done when it is relatively easy to obtain healthy patient samples, but when abnormalities corresponding to disease states may be rare and one-of-a-kind. The associated computational challenge, anomaly detection, is a well-studied machine learning problem. However, due to the dimensionality and variability of expression data, existing methods based on feature space analysis or individual anomalously-expressed genes are insufficient. We present a novel approach, CSAX, that identifies pathways in an individual sample in which the normal expression relationships are disrupted. To evaluate our approach, we have compiled and released a compendium of public microarray data sets, reformulated to create a testbed for anomaly detection. We demonstrate the accuracy of CSAX on the data sets in our compendium, compare it to other leading anomaly-detection methods, and show that CSAX aids both in identifying anomalies and in explaining their underlying biology. We note the potential for the use of such methods in identifying subclasses of disease. We also describe an approach to characterizing the difficulty of specific expression anomaly detection tasks and discuss how one can estimate the feasibility of a specific task. Our approach provides an important step towards identification of individual disease patterns in the era of personalized medicine.

Background: The purpose of this study was to determine whether aneuploid fetal nucleated erythrocytes (NRBCs) could be detected in maternal blood through the use of fluorescent PCR amplification with polymorphic short tandem repeat (STR) markers as an alternative or complementary method to analysis by fluorescent in situ hybridization (FISH). Methods: Peripheral blood samples were obtained from women who had just undergone termination of pregnancy because of fetal trisomy 21 (three cases, 47,XY,+21; four cases, 47,XX,+21). Candidate fetal cells were isolated by flow-sorting by antibodies to the γ chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed by the use of chromosome-specific probes for X, Y, and 21. Fetal NRBCs, as defined by the presence of γ staining, characteristic morphology, and three chromosome 21 signals, along with maternal leukocytes, defined as γ negative and two chromosome 21 signals, were micromanipulated separately and subjected to fluorescent PCR amplification of chromosome 21 STR markers (D21S11, D21S1411, and/or D21S1412). Results: In five of seven cases analyzed, fetal NRBCs were aneuploid, as determined by the presence of triallelic or diallelic peaks of chromosome 21 sequences when compared with sequences from the maternal leukocytes. Conclusions: Fluorescent PCR amplification of STRs can detect fetal aneuploidy and may be useful in the setting of poor hybridization efficiency with FISH analysis. These results suggest that combined fetal aneuploidy and single-gene diagnoses by the use of DNA microarrays may be feasible in the near future.

Objective: To review the potential clinical diagnostic applications of fetal DNA analysis in maternal plasma or serum for noninvasive prenatal diagnosis and screening. Data Sources: We conducted a MEDLINE search of articles published between January 1970 and March 2000 using the key terms “fetal DNA,” “plasma,” and “serum.” Methods of Study Selection: All 369 articles describing the detection of fetal DNA in maternal plasma were reviewed. Results: The diagnostic use of circulating fetal DNA in maternal plasma is currently limited to genes that are present in the fetus but not in the mother. From a clinical perspective, the most advanced application is for noninvasive detection of fetal rhesus D (Rh[D]) genotype. The results of studies performed by four different groups showed that prenatal diagnosis of fetal Rh(D) status by molecular analysis of maternal plasma or serum is routinely possible beginning in the second trimester. Noninvasive fetal genotyping should be useful for the treatment of sensitized Rh(D)-negative women whose partners are heterozygous for the Rh(D) gene because no further diagnostic or therapeutic procedures are necessary if the fetus is Rh(D) negative. Future clinical applications of fetal DNA may be in its use as a screening test for Down syndrome, preeclampsia, or preterm labor. However, these applications currently rely on the detection of Y chromosomal sequences and consequently are limited presently to male fetuses. Conclusion: The recent discovery of high concentrations of fetal DNA in maternal plasma represents a promising noninvasive approach to prenatal diagnosis. Compared with the analysis of the cellular fraction of maternal blood, the analysis of fetal DNA extracted from maternal plasma has the advantage of being rapid, robust, and easy to perform. The fetal DNA detected is limited to the current pregnancy. However, universal fetal gene sequences must be identified that allow analysis of genetic material from both male and female fetuses. Study of fetal DNA in maternal plasma can improve our understanding of fetomaternal biology and physiology. The long-term effects of maternal exposure to relatively high amounts of foreign DNA are unknown but represent an exciting area for future inquiry.

Objective: To determine the benefits of antenatal diagnoses of fetal aneuploidy in women who continued their pregnancies. Methods: A questionnaire was mailed to 51 mothers of children with aneuploidy. Women whose fetuses were diagnosed prenatally comprised the study group and those whose infants were diagnosed at birth were controls. Outcomes measured included an assessment of pregnancy management, neonatal outcome, subjective measures of depression and anxiety, and evaluation of women’s emotional and physical experience of the pregnancy. For outcomes measured by nonparametric survey questions, 20 women were needed in each arm to achieve a power of 80% to detect a 2-point difference on a 6-point scale; for our neonatal outcomes, 100 women were needed in each arm to achieve 80% power to detect a difference in length of stay (less than 1 week versus greater than 1 week) or need for surgery. Results: Thirty-eight women (75%) responded. Most (86%) had children with trisomy 21. Seventeen women (45%) received their child’s diagnosis at birth; 21 (55%) had prenatal diagnoses. Demographic measures were similar except that women with prenatal diagnoses attended religious services more frequently (1–3 times per month versus once to several times per year, P = .04). Women with prenatal diagnosis had better perceptions of their physical experience of pregnancy (median score of 10 versus 6 on a 10-point visual analog scale, P = .005) and their emotional experience of the birth (median score of 7.5 versus 2, P = .001). Mental Health Inventory scores were similar between groups. Neonates without prenatal diagnoses were more likely to be transferred to tertiary centers after birth (70% versus 24%, P = .004); lengths of hospital stays and need for surgery were similar. Seventy-one percent (95% confidence interval [CI] 48, 89%) of women with prenatal diagnoses said they would have done nothing differently in the pregnancy compared with 29% (95% CI 10, 56%) of women with diagnoses at birth. Conclusion: Early knowledge of fetal aneuploidy is beneficial to women who continue their pregnancies. These results might be useful when counseling women who do not intend to terminate abnormal pregnancies, but are considering prenatal diagnosis.

Background The Ts1Cje model of Down syndrome is of particular interest for perinatal studies because affected males are fertile. This permits affected pups to be carried in wild-type females, which is similar to human pregnancies. Here we describe the early natural history and growth profiles of Ts1Cje embryos and neonates and determine if heart defects are present in this strain. Methods Pups were studied either on embryonic (E) day 15.5, or from postnatal (P) day 3 through weaning on P21. PCR amplification targeting the neomycin cassette (present in Ts1Cje) and Sry (present in males) was used to analyze pup genotypes and sex ratios. Body weights and lengths, as well as developmental milestones, were recorded in Ts1Cje mice and compared to their wild-type (WT) littermates. Histological evaluations were performed at E15.5 to investigate the presence or absence of heart defects. Pups were divided into two groups: Ts1Cje-I pups survived past weaning and Ts1Cje-II pups died at some point before P21. Results Ts1Cje mouse embryos showed expected Mendelian ratios (45.8%, n = 66 for Ts1Cje embryos; 54.2%, n = 78 for WT embryos). Histological analysis revealed the presence of ventricular septal defects (VSDs) in 21% of Ts1Cje E15.5 embryos. After weaning, only 28.2% of pups were Ts1Cje (185 Ts1Cje out of 656 total pups generated), with males predominating (male:female ratio of 1.4:1). Among the recovered dead pups (n = 207), Ts1Cje (63.3%, n = 131, p

Systemic lupus erythematosus (SLE) is a serious multisystem disease that has a striking propensity to affect women. The cause of SLE remains elusive. Fetomaternal cell trafficking, or the passage of fetal cells into the maternal circulation, is now a well-established phenomenon. In addition, fetal cells have been implicated in the development of preeclampsia and in the pathogenesis of scleroderma. We undertook this study to determine whether fetomaternal cell trafficking might also be involved in pathogenic processes in SLE. Fluorescence in situ hybridization analysis was performed using X and Y chromosome-specific probes on affected and unaffected tissue obtained at autopsy from a woman who had previously given birth to 2 males and who had died of complications of SLE. The goal of the analysis was to detect the presence of male cells of putative fetal origin. Male cells were found in every histologically abnormal tissue type that was examined, but were not found in histologically normal tissue. These data suggest that fetal cells may be associated with SLE. It is unclear whether their presence may be related to disease causation, an effect of disease progression, or unrelated to disease pathology. However, this case study is an important step toward understanding the potential relationship between fetomaternal cell trafficking and SLE pathology.

We investigated gene expression and functional differences between Ts1Cje mice and wild‐type (WT) littermates in adult cerebral cortex and hippocampus. These two brain regions are affected in people with Down syndrome, but have not been previously molecularly characterized in Ts1Cje mice. Total RNA was prepared from the brains of 8–10‐week‐old Ts1Cje mice (n = 6) and WT littermates (n = 5) and hybridized to Affymetrix 1.0 ST gene mouse arrays. Differentially regulated genes were identified and used to perform in silico functional analyses to better characterize dysregulated pathways in both brain regions. Hippocampus had more significantly differentially expressed genes compared with cortex (30 vs. 7 at a Benjamini‐Hochberg false discovery rate of 20%). We identified novel genes that were differentially regulated in adult brains, including Cyb5r1, Fsbp, Vmn2r110, Snd1 and Zhx2. Functional analyses in Ts1Cje mice highlighted the importance of NFAT signaling, oxidative stress, neuroinflammation and olfactory perception via G‐protein signaling. In a comparison of adult Ts1Cje and WT brains, we identified new genes and pathway differences in the cortex and hippocampus. Our analyses identified physiologically relevant pathways that can serve as targets for the development of future treatments to improve neurocognition in Down syndrome.

In human pregnancy, the constant turnover of villous trophoblast results in extrusion of apoptotic material into the maternal circulation. This material includes cell-free (cf) DNA, which is commonly referred to as "fetal", but is actually derived from the placenta. As the release of cf DNA is closely tied to placental morphogenesis, conditions associated with abnormal placentation, such as preeclampsia, are associated with high DNA levels in the blood of pregnant women. Over the past five years, the development and commercial availability of techniques of massively parallel DNA sequencing have facilitated noninvasive prenatal testing (NIPT) for fetal trisomies 13, 18, and 21. Clinical experience accrued over the past two years has highlighted the importance of the fetal fraction (ff) in cf DNA analysis. The ff is the amount of cell-free fetal DNA in a given sample divided by the total amount of cell-free DNA. At any gestational age, ff has a bell-shaped distribution that peaks between 10 and 20% at 10-21 weeks. ff is affected by maternal body mass index, gestational age, fetal aneuploidy, and whether the gestation is a singleton or multiple. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant; confined placental mosaicism has been increasingly reported as an underlying biologic explanation. Cell-free fetal DNA is a new biomarker that can provide information about the placenta and potentially be used to predict clinical problems. Knowledge gaps still exist with regard to what affects production, metabolism, and clearance of feto-placental DNA.

Fetal cells circulate in the blood of pregnant women. When the gestation is normal, fetal cells are low in number. Complications of pregnancy, such as pre-eclampsia, or fetal cytogenetic abnormalities, such as Down’s syndrome, increase fetomaternal transfusion. The isolation of fetal cells from maternal blood is currently under active investigation as a non-invasive method for prenatal diagnosis. The fetal cells that are most commonly used for non-invasive genetic diagnosis, the nucleated erythrocyte and the trophoblast, are highly differentiated and do not persist post-partum. In the context of studying fetal cells in maternal blood it was discovered that fetal progenitor cells originating from a prior pregnancy could also be detected. This led to the appreciation that unlike fetal DNA in plasma, which is cleared almost immediately following delivery, fetal cells persist for decades post-partum. Following pregnancy, labor, and delivery, a woman becomes a chimera. Transfused fetal stem and progenitor cells appear to be capable of further differentiation and migration to maternal organs. A further research agenda is needed to explore the newly appreciated phenomenon of bi-directional fetomaternal cell trafficking. Any consideration of the fetus as a patient must also consider the fetus as a potential source of therapeutic stem cells for the mother.

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.

Autosomal recessive polycystic kidney disease (ARPKD) is a hereditary condition with an estimated incidence of 1 in 20,000 live births. Various growth factors have been implicated in the causation of this disease. We describe a child with ARPKD whose levels of urinary basic fibroblast growth factor (bFGF) were markedly elevated. The concentrations of bFGF increased further following right nephrectomy, in response to the compensatory growth of the remaining kidney. We hypothesize that measurement of urinary bFGF may be useful as a noninvasive marker to assess progression of cystic renal development.

We have studied the effect of transforming growth factor beta (TGFbeta) on erythropoiesis in cultures from adult peripheral blood, using flow cytometric enumeration of fetal hemoglobin (HbF)-containing cells. TGFbeta caused a dramatic increase in the proportions of cells that accumulated HbF together with adult hemoglobin (HbA) (F+A+ cells). This highly significant (P