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This chapter discusses the need for innovative health literacy solutions to combat extensive chronic disease prevalence and costs. The authors explore the intersection of chronic disease management and health literacy. They provide specific examples of successful health literacy interventions for managing several highly prevalent chronic diseases. This is followed by suggestions on pairing research and practice to support effective disease management programs. In addition, the authors discuss strategies for collection and dissemination of knowledge gained from collaborations between researchers and practitioners. They identify current challenges specific to disseminating information from the health literacy field and offer potential solutions. The chapter concludes with a brief look at future directions and organizational opportunities to integrate health literacy practices to address the need for effective chronic disease management.

It is difficult to identify reliable reference genes for transcriptomic analyses in biofluids such as saliva. This situation is particularly relevant for the newborn population, where rapid development is associated with dynamic changes in gene expression. Real-time gene expression monitoring holds great promise for elucidating disrupted pathways that result in morbidities unique to this population, such as retinopathy of prematurity, but its impact depends on identifying stable and consistently expressed genes across a wide range of gestational ages. We extracted total RNA from 400 neonatal saliva samples (postconceptional ages: 32 5/7 to 48 2/7 weeks), converted it to cDNA, and pre-amplified and analyzed it by qPCR for three commonly used reference genes, ACTB, GAPDH, and YWHAZ. Relative quantification was determined using the Δ Ct method. Data were analyzed as a whole and also stratified by age and sex. Descriptive statistics and homogeneity of variance were performed to identify optimal reference genes. Data analyzed from all ages and both sexes showed significant expression variation for ACTB, while GAPDH and YWHAZ showed greater stability. Male infants exhibited increased expression variation compared to females for ACTB, but neither GAPDH nor YWHAZ showed significant variance for either sex. We suggest that ACTB is an unreliable reference gene for the newborn population. Males showed significantly more variation in ACTB expression compared to females, which suggests a sex-specific developmental role for this biomarker. By contrast, GAPDH and YWHAZ were less variable and therefore preferable for use in neonates. Our findings may improve the use of reference genes for the RT-qPCR platform in the newborn over a wide range of gestational ages, thereby minimizing the likelihood of erroneous interpretation of gene expression during rapid growth, development, and differentiation.

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell-free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.

Trisomies 18 and 21 are the two most common live born autosomal aneuploidies in humans. While the anatomic abnormalities in affected fetuses are well documented, the dysregulated biological pathways associated with the development of the aneuploid phenotype are less clear. Amniotic fluid (AF) cell-free RNA is a valuable source of biological information obtainable from live fetuses. In this study, we mined gene expression data previously produced by our group from mid-trimester AF supernatant samples. We identified the euploid, trisomy 18 and trisomy 21 AF transcriptomes, and analyzed them with a particular focus on the nervous system. We used multiple bioinformatics resources, including DAVID, Ingenuity Pathway Analysis, and the BioGPS Gene Expression Atlas. Our analyses confirmed that AF supernatant from aneuploid fetuses is enriched for nervous system gene expression and neurological disease pathways. Tissue analysis showed that fetal brain cortex and Cajal-Retzius cells were significantly enriched for genes contained in the AF transcriptomes. We also examined AF transcripts known to be dysregulated in aneuploid fetuses compared with euploid controls and identified several brain-specific transcripts among them. Many of these genes play critical roles in nervous system development. NEUROD2, which was downregulated in trisomy 18, induces neurogenic differentiation. SOX11, downregulated in trisomy 21, is a transcription factor that is essential for pan-neuronal protein expression and axonal growth of sensory neurons. Our results show that whole transcriptome analysis of cell-free RNA in AF from live pregnancies permits discovery of biomarkers of abnormal human neurodevelopment and advances our understanding of the pathophysiology of aneuploidy.

Amniotic fluid is a complex biological material that provides a unique window into the developing human. Residual amniotic fluid supernatant contains cell-free fetal RNA. The objective of this study was to develop an understanding of the amniotic fluid core transcriptome by analyzing the transcripts ubiquitously present in the amniotic fluid supernatant of euploid midtrimester fetuses.This was an in silico (computational) investigation using publicly available gene expression data previously produced by our group from 12 euploid midtrimester amniotic fluid samples. Functional analyses were performed using a web-based software analysis tool. Organ specificity was examined for each transcript using a gene expression atlas. For fetal organs not represented in the atlas, manual literature searching and the web-based software analysis tool were used to generate fetal organ-associated gene lists.There were 476 well-annotated genes present in 12 of 12 amniotic fluid samples. Functional analysis identified six physiologic systems represented in the amniotic fluid core transcriptome, including musculoskeletal and nervous system development and function and embryonic and organismal development. Mammalian target of rapamycin signaling was identified as a key canonical pathway. Twenty-three highly organ-specific transcripts were identified; six of these are known to be highly expressed in the fetal brain.Amniotic fluid cell-free fetal RNA can provide biological information on multiple fetal organ systems. The presence of fetal-brain specific transcripts in amniotic fluid suggests novel approaches to the study of developmental disorders that involve the central nervous system. The finding that the mammalian target of rapamycin signaling is enriched in midtrimester fetuses may have future applications in the study of fetal growth disorders.

Background: Gene expression profiling of the salivary supernatant is emerging as a new and important source of real-time, systemic, biological information. However, existing technologies prevent RNA extraction of small quantities found in neonatal salivary supernatant. Objective: The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant. Methods: Two saliva samples (10–100 µl) were serially collected from newborns (36–41 weeks’ gestation) (n = 13) and stabilized. Total RNA was extracted from salivary supernatant with the use of two modified extraction techniques: Qiagen RNAprotect® Saliva Mini Kit (method 1) and the QIAamp Viral RNA Mini Kit (method 2). Quantitative RT-PCR amplification for GAPDH was performed on extracted salivary samples. Statistical analyses were performed on mean threshold cycle (Ct) levels to compare RNA yield from each protocol. Paired microarray analyses were made between neonatal whole saliva and supernatant (n = 3) to discern gene expression differences between these biolayers. Results: mRNA was successfully extracted and amplified from all salivary supernatant samples. Extraction with method 2 yielded more RNA than with method 1 (p = 0.008). There was a 7.5% discordance between paired gene expression analyses for whole saliva and supernatant. Genes that were statistically significantly upregulated in supernatant highlighted 16 distinct biological functions not seen in whole saliva. Conversely, only two biological functions were unique to whole saliva. Conclusion: Neonatal cell-free salivary supernatant mRNA may be readily extracted and utilized on downstream applications. These technical enhancements allow for further exploration of the diagnostic potential of the neonatal salivary transcriptome.

Trisomy 18 is a common human aneuploidy that is associated with significant perinatal mortality. Unlike the well-characterized "critical region" in trisomy 21 (21q22), there is no corresponding region on chromosome 18 associated with its pathogenesis. The high morbidity and mortality of affected individuals has limited extensive investigations. In order to better understand the molecular mechanisms underlying the congenital anomalies observed in this condition, we investigated the in utero gene expression profile of second trimester fetuses affected with trisomy 18. Total RNA was extracted from cell-free amniotic fluid supernatant from aneuploid fetuses and euploid controls matched for gestational age and hybridized to Affymetrix U133 Plus 2.0 arrays. Individual differentially expressed transcripts were obtained by two-tailed t tests. Over-represented functional pathways among these genes were identified with DAVID and Ingenuity(®) Pathways Analysis. Results show that three hundred and fifty-two probe sets representing 251 annotated genes were statistically significantly differentially expressed between trisomy 18 and controls. Only 7 genes (2.8% of the annotated total) were located on chromosome 18, including ROCK1, an up-regulated gene involved in valvuloseptal and endocardial cushion formation. Pathway analysis indicated disrupted function in ion transport, MHCII/T cell mediated immunity, DNA repair, G-protein mediated signaling, kinases, and glycosylation. Significant down-regulation of genes involved in adrenal development was identified, which may explain both the abnormal maternal serum estriols and the pre and postnatal growth restriction in trisomy 18. Comparison of this gene set to one previously generated for trisomy 21 fetuses revealed only six overlapping differentially regulated genes. This study contributes novel information regarding functional developmental gene expression differences in fetuses with trisomy 18.

Neonates are exposed to elevated levels of reactive oxygen species as they transition from a hypoxic intrauterine to a normoxic extrauterine environment at birth. This increased oxidative stress is associated with neonatal morbidity. Current antioxidant supplementation treatment strategies have yet to translate into improved neonatal outcomes. Our understanding of a newborn's intricate redox balance, particularly at the genomic level, remains limited. Here, we performed genomic microarray analyses (approximately 14,500 genes) on extracted mRNA from umbilical cord whole blood at term gestation (n=10). Bioinformatic analyses identified 282 genes (2.0%) that were consistently present within the highest quintile of expressed genes. These genes were highly associated with oxidant stress and included superoxide dismutase 1, catalase, peroxiredoxins, and uncoupling proteins. Pathway analyses identified statistically significantly overrepresented functional pathways including "oxidative stress," "oxidative stress response mediated by nuclear factor-erythroid 2-related factor," "hypoxia-inducible factor signaling," and "mitochondrial dysfunction" (p

Background: There is an important need to develop noninvasive biomarkers to detect disease in premature neonates. Our objective was to determine if salivary genomic analysis provides novel information about neonatal expression of developmental genes. Methods: Saliva (50–200 μL) was prospectively collected from 5 premature infants at 5 time points: before, starting, and advancing enteral nutrition; at the introduction of oral feeds; and at advanced oral feeds. Salivary RNA was extracted, amplified, and hybridized onto whole-genomic microarrays. Results: Bioinformatics analyses identified 9286 gene transcripts with statistically significant gene expression changes across individuals over time. Of these genes, 3522 (37.9%) were downregulated, and 5764 (62.1%) were upregulated. Gene expression changes were highly associated with developmental pathways. Significantly downregulated expression was seen in embryonic development, connective tissue development and function, hematologic system development and function, and survival of the organism (10−14 < P < 10−3). Conversely, genes associated with behavior, nervous system development, tissue development, organ development, and digestive system development were significantly upregulated (10−11 < P < 10−2). Conclusions: Comparative genomic salivary analyses provide robust, comprehensive, real-time information regarding nearly all organs and tissues in the developing preterm infant. This innovative and noninvasive technique represents a new approach for monitoring health, disease, and development in this vulnerable patient population. By comparing these data in healthy infants with data from infants who develop medical complications, we expect to identify new biomarkers that will ultimately improve newborn care.

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.

Objective. To identify a candidate neonatal housekeeping gene and to determine the effects of pH and PaO2 on the stability of newborn gene expression in physiologically hypoxic and acidotic newborn blood.Methods. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification was performed for four commonly used housekeeping genes (GAPDH, β-actin, cyclophilin, 28S rRNA) on extracted RNA. Blood gas analyses determined pH and PaO2 levels.Results and conclusions. β-Actin was the least variable and GAPDH the most variable housekeeping gene studied. pH negatively correlated with gene expression levels. PaO2 levels did not significantly affect gene expression. These results inform selection of housekeeping genes for neonatal mRNA research.

The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Student's t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications.

To determine the kinetics of the short-term clearance of cell-free nucleic acids in maternal plasma, we conducted a prospective cohort study of 36 women who underwent first-trimester elective surgical termination. By using real-time polymerase chain reaction (PCR) amplification and measurement of the sex-determining region of the Y chromosome (SRY) gene (as a marker of fetal DNA), beta-hCG (a placental messenger RNA transcript), and glyceraldehyde phosphate dehydrogenase (GAPDH; as a marker of both total DNA and mitochondrial RNA), we found that cell-free nucleic acids in maternal plasma are not cleared within 90 minutes after the procedure, in contrast to the case of term delivery, in which levels decrease rapidly after birth.

Background: Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are novel biomarkers with many clinical applications. Amniotic fluid (AF) is a rich source of cffDNA. We investigated the biophysical characteristics of cffDNA in AF, hypothesizing that they would differ from cffDNA in maternal plasma. Methods: We obtained 10 mL of fresh AF supernatant from women carrying euploid fetuses (n = 39) and aneuploid fetuses (n = 4). To test the effects of storage and karyotype, samples from euploid fetuses (n = 19) and aneuploid fetuses with trisomies 21 (n = 16), 18 (n = 9), or 13 (n = 3); triploidy (n = 4); or monosomy X (n = 2) were frozen at −80 °C. AF cffDNA was characterized by real-time quantitative PCR amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and analysis of the DNA fragmentation signature. Results: We observed a significant correlation of concentration with gestational age for fresh AF cffDNA from euploid fetuses (R2 = 0.77, P Conclusions: Gestational age, karyotype, and sample storage time affect concentrations and fragment size of AF cff DNA. These effects may be attributable to fundamental differences in tissue sources, excretion modes, or kinetic pathways. Characteristic signature patterns for each common aneuploidy offer the possibility of using DNA fragmentation analysis as a means of triaging AF samples.

Background Previously, we showed that analysis of amniotic fluid (AF) supernatant cell-free fetal (cff) DNA using DNA microarrays (array-CGH) allows for detection of whole chromosome differences between test and reference DNA. Subsequent technical advances have increased both the yield and quality of extracted cffDNA. Here we determined whether array-CGH using smaller volumes of both fresh and frozen AF cffDNA could identify fetal aneuploidy. Methods CffDNA was extracted from 10 mL of residual AF supernatant. The test AF samples (n = 10) included one with a normal karyotype, and nine with the following fetal aneuploidies: trisomies 13 (n = 1), 18 (n = 3), 21 (n = 2), trisomy 9 mosaicism (47,XX,+ 9[18]/46,XX[2]), triploidy (69,XXY) and Turner syndrome (45,X). Results Array-CGH using AF cffDNA from aneuploid fetuses, compared to euploid reference AF cffDNA, detected whole chromosome aneuploidy in 8 of 9 cases tested, including the case of trisomy 9 mosaicism. The case of triploidy was not detected. Conclusions CffDNA extracted from 10 mL AF supernatant can be analyzed using array-CGH to correctly identify human chromosome abnormalities. This technology allows for rapid screening of AF samples for whole chromosomal changes by using routinely discarded supernatant, and may augment standard prenatal karyotyping techniques by providing additional molecular information. Copyright © 2007 John Wiley & Sons, Ltd.

Analysis of saliva for clinical monitoring and biomarker detection holds great promise for improving health care. Commercially available assays are not intended for use with neonates, however, and collection and processing of saliva for subsequent transcriptomic analysis presents unique challenges in this population. We compared RNA yield, quality, stability and RT-qPCR performance for two commonly used commercial systems: the Qiagen RNeasy Protect Saliva Mini Kit(®) and the DNA Genotek Oragene•RNA(®) assay. Two 10 μl saliva samples were collected from ten newborns and stabilized for each assay. Total RNA was extracted following incubation for 3, 10, 15 or 20 days. Total RNA extracted from each assay was analyzed for integrity, quality and quantity using the Agilent BioAnalyzer 2100. RT-qPCR was performed for the reference gene, GAPDH, to assess subsequent performance of the extracted RNA. Although the DNA Genotek extraction protocol required nearly twice the time of the Qiagen protocol, RNA integrity did not differ between the kits. RNA concentration using the DNA Genotek assay, however, was 3,264 pg/μl (range: 262 - 10,336 pg/μl) compared to 822.4 pg/μl (range: 0 - 1,856 pg/μl) for the Qiagen protocol. Linear regression analysis showed a stronger correlation between the threshold cycle and RNA concentration using DNA Genotek (r(2) = 0.356) compared to Qiagen (r(2) = 0.0331). Our results suggest that although the Qiagen assay may reduce overall extraction time, RNA yield and performance in subsequent transcriptomic analysis is more robust using the DNA Genotek assay.

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.

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.

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.

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

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.

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)

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.

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.

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.

Maspin is a serine protease inhibitor involved in regulating human placental trophoblast cell migration. Maspin has not been studied in preeclampsia (PE) or relative to the maternal-fetal immunological relationship, both of which may involve altered trophoblast migration. We examined maspin expression in placentas from in vitro fertilization (IVF) and egg donor (ED) pregnancies with and without PE. Exclusive to the chorionic plate, the number of maspin-positive extravillous trophoblasts was significantly decreased in IVF-PE vs. IVF (p = 0.005) and ED vs. IVF (p = 0.013). These data suggest maspin expression may be influenced by PE and/or the immunological dynamics of pregnancy. (C) 2013 Elsevier Ltd. All rights reserved.

To perform discovery-driven research on the neonatal salivary and cord blood transcriptomes.Two separate cohorts of infants were enrolled in this study. In one, cord blood (n = 10) and in the other, saliva samples (n = 10) were collected at term gestation. Total RNA was extracted, amplified and hybridized onto Affymetrix HG U133a gene expression microarrays. Following normalization, genes expressed in the highest quintile (≥ 80%) across all subjects in each biofluid were analyzed with Ingenuity Pathway Analysis. Over-represented pathways relating to organ specific development and physiological functions in the newborn were explored.There were 303 genes in neonatal saliva and 282 genes in umbilical cord blood that met statistical criteria. Of these, 114 were common to both biofluids. Pathway analyses revealed the important roles of redox balance, cellular proliferation, and smooth muscle relaxation. In blood, hematopoiesis and immune response pathways predominated. In saliva, pathways associated with the gastrointestinal system were highlighted.Neonatal cord blood and saliva provide a wealth of transcriptomic information. These normally discarded biofluids should be considered an important source of real-time gene expression data that may elucidate key pathways in neonatal physiology and pathology.

Background The current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate. Methodology/Principal Findings Salivary samples (n = 116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care.

Novel therapies are needed for the treatment of acute and chronic lung diseases, many of which are incurable. The use of exogenous stem cells has shown promise in both animal models and clinical trials. However, to date, the stem cell literature has under-recognized naturally acquired pregnancy-associated progenitor cells (PAPCs). These cells are found at sites of injury or disease in female tissues. They persist for decades after parturition in maternal blood and organs, with the largest number being found in the maternal lungs. Their presence there may be one explanation for the sex differences observed in the prevalence and prognosis of some lung diseases. Although the clinical significance of these cells is as yet unknown, the literature suggests that some of the PAPCs are stem cells or have stem cell-like properties. PAPCs harvested from the blood or organs of parous women could potentially be used as an alternate source of cells with regenerative properties for the woman herself or her children. Because PAPCs preferentially traffic to the maternal lung they may play a significant role in recovery or protection from lung disease. In this review article, we discuss ongoing research investigating the administration of both adult and placenta-derived stem cells to treat lung disease, and how PAPCs may also play an important future therapeutic role.

Fetal cell microchimerism (FCM) is the persistence of fetal cells in the maternal circulation and organs following pregnancy. Proposed hypotheses about the function of fetal cells in the pathogenesis of maternal cancer include promotion of tumorigenesis, protection by providing immunosurveillance, and participation in tissue repair. To date, studies of FCM and cancer have been primarily descriptive and quantitative. More research is needed to understand the cellular phenotype of the microchimeric cells in maternal tumors and whether they have a functional role. This research will require further study using a multidisciplinary approach, incorporating knowledge of the fetomaternal relationship, tumor biology, immunology, and clinical oncology. Cancer Res; 71(1); 8–12. ©2011 AACR.

The use of cell-free nucleic acids in the circulation of pregnant women for noninvasive prenatal diagnosis is arguably one of the hottest current topics in prenatal medicine. Between 1997 and the present era this field has gone from basic research to clinical application for diagnosis of fetal gender and Rhesus D status. Over the next few years it is likely that noninvasive prenatal diagnosis for Down syndrome will also be possible. Here we summarize current and future clinical applications of analyzing cell-free fetal DNA and RNA in both maternal and neonatal body fluids, including maternal plasma, serum, whole blood, amniotic fluid, and neonatal saliva. We describe methods to evaluate normal and abnormal fetal and neonatal development using gene expression microarrays. We also discuss the ways in which differentially-regulated gene lists can advance knowledge of both fetal and neonatal biology, as well as suggest novel possibilities for fetal and neonatal treatment.

To assess the relationship between first- and second-trimester cell-free DNA levels and maternal serum screening markers.First- and second-trimester residual maternal serum samples from 50 women were obtained. First-trimester (pregnancy-associated plasma protein A and beta-hCG) and second-trimester serum analytes (beta-hCG, alpha-fetoprotein, unconjugated estriol, and inhibin A) had been measured at the time of sample receipt. All fetuses were male as confirmed by birth records. Cell-free DNA was extracted and measured by real-time quantitative polymerase chain reaction amplification using glyceraldehyde phosphate dehydrogenase and DYS1 as markers of total DNA and fetal DNA, respectively. Determination of linear associations between first- and second-trimester serum markers and cell-free DNA levels using Pearson correlations was performed.Statistically significant correlations between first-trimester pregnancy-associated plasma protein A multiples of the median and both total (r=0.36, P=.016) and fetal (r=0.41, P=.006) DNA in the first trimester were observed. There were no significant correlations between first-trimester serum human chorionic gonadotropin or any second-trimester serum marker with DNA levels.Correlation between serum pregnancy-associated plasma protein A and first-trimester circulating cell-free fetal and total DNA levels is a novel finding. Pregnancy-associated plasma protein A is a glycoprotein of placental origin, and its correlation to cell-free fetal DNA in maternal serum suggests a common tissue origin through apoptosis of placental cells. However, because pregnancy-associated plasma protein A and cell-free DNA were only marginally correlated and cell-free DNA can be reliably detected in the first trimester, the addition of cell-free DNA to serum screening strategies may be helpful in predicting adverse pregnancy outcome.II.

Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N=36) and aneuploid (N=29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (-80 degrees C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification.

Circulating cell-free fetal deoxyribonucleic acids (cffDNAs) are promising biomarkers with various potential clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNAs. Further improvements to the original protocol for the extraction of cffDNAs from AF supernatant resulted in statistically significant higher yields of high-quality cffDNAs, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g., comparative genomic hybridization microarrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples compared with the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNAs from up to 10 samples in

Objective Cell-free DNA is a marker of cellular apoptosis and necrosis. We wished to determine if maternal smoking affects maternal and fetal serum cell-free DNA levels. Methods Case–control sets of stored second-trimester serum-screening samples from 27 smoking and 90 nonsmoking pregnant women were developed. Smoking status was confirmed by measuring serum cotinine levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and DYS1 levels were determined using real-time polymerase chain reaction (PCR) to measure total and fetal cell-free DNA, respectively. At delivery, medical records were reviewed to confirm gender and determine other factors that could affect DNA values. Results Smoking was associated with significantly elevated GAPDH levels compared with nonsmokers (median: 97 662 genome equivalents (GE)/mL vs 38 217 GE/mL; p = 0.018). DYS1 levels were not statistically significantly elevated in smokers (p = 0.29). Other factors that affected DYS1 levels included maternal age in nonsmokers only (r2 = 0.30, p = 0.013) and maternal Synthroid use (p = 0.0045) Conclusion Pregnant smokers have threefold higher levels of total cell-free DNA compared with pregnant nonsmokers. Maternal age and Synthroid exposure may also affect circulating cell-free fetal DNA levels. Copyright © 2008 John Wiley & Sons, Ltd.

Isolation, quantification, and genetic analysis of circulating plasma DNA have clinical applications in prenatal diagnosis, oncology, organ transplantation, posttrauma monitoring, and infectious disease. Recent technology has allowed the rapid isolation and purification of DNA from whole blood, plasma, serum, buffy coat, tissues, stool, and urine. With the advent of real-time polymerase chain reaction (PCR) amplification, extracted DNA not only can be easily identified to aid in clinical diagnoses, but also can be readily quantified to analyze ongoing clinical dynamics and aid in the medical prognoses of patients. Historically, identification of unique cell-free fetal DNA sequences has relied on the detection of paternally specific Y chromosome sequences owing to their relative ease in identification. However, any DNA sequence that is unique to the fetus has the potential to be amplified and quantified using real-time PCR. Our laboratory specializes in extraction of fetal DNA from maternal plasma with subsequent quantification with real-time PCR of paternally inherited sequences, such as the Y chromosome gene, SRY. The successful isolation and quantification of this DNA from plasma is dependent on three distinct protocols: plasma harvesting from whole blood, DNA extraction from cell-free plasma, and real-time PCR amplification and quantification of the SRY sequence.

Background: Fetal cell detection in maternal tissue requires an accurate, efficient, and reproducible microscopy method. Our objective was to compare manual scoring to a commercially available automated scanning system for the detection of chromosome signals by fluorescence in situ hybridization (FISH). Methods: X and Y chromosome FISH signals were detected on slides of calibrated mixtures of blood, paraffin-embedded liver sections, and post-termination blood. For manual scoring (400× magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200× magnification), duration of scanning, number of gallery images generated, duration of manual review of gallery images, and number of confirmed fetal cells were recorded. Results: From all slides the number of target fetal cells located by manual and automated microscopy was highly correlated (r = 0.90). However, automated scanning required on average 4-fold more time than manual scoring (P < 0.0001), with an average automated scanning time of 9.7 h per slide compared with 2.4 h per slide when scored manually. Conclusions: In general, the accuracy of automated and manual microscopy is comparable, although manual scoring is more efficient because of the level of magnification necessary for automated scanning of cells, and a large number of gallery images generated by automated scanning that must then be reviewed manually. This suggests that when rapid analysis is required (i.e., clinical situations), manual microscopy is preferable. In contrast, automated scanning may have advantages over manual microscopy when time constraints are less imposed (i.e., research situations).

Our laboratory continues to be actively involved in the development of new biomarkers for prenatal diagnosis using maternal blood and amniotic fluid. We have also developed a mouse model that demonstrates that cell-free fetal (cff) DNA is detectable in the pregnant maternal mouse. In human maternal plasma and serum we have analyzed factors that are important in the clinical interpretation of cff DNA levels. Maternal race, parity, and type of conception (natural or assisted) do not affect cff DNA levels, but maternal weight does. We have also analyzed the relationship between placental volume, using a three-dimensionsal ultrasound examination, and cff DNA levels. Surprisingly, there is no association between these values. Finally, we are using specific disease models (such as congenital diaphragmatic hernia and twin-to-twin transfusion) to understand the effects of gestational age and specific pathology on fetal gene expression by analyzing cell-free mRNA levels in maternal plasma. In the amniotic fluid we have focused on improvements in recovery of cff DNA and mRNA. By optimizing recovery we have made some interesting observations about differences in fetal DNA between blood and amniotic fluid. In addition, we have successfully hybridized cff DNA in amniotic fluid to DNA microarrays, permitting assessment of fetal molecular karyotype. We also have preliminary data on fetal gene expression in amniotic fluid. Finally, we remain actively involved in promoting noninvasive prenatal testing in the United States, such as encouraging the use of fetal DNA for fetal rhesus D assessment. On the other hand, we are cautious and concerned about the accuracy of "at-home" kits for fetal gender detection.

Objectives To evaluate whether cell-free fetal (cff) DNA in maternal plasma during the second trimester is a marker for developing pregnancy-associated complications. Two PCR techniques for the detection and quantitation of fetal DNA were compared. Methods Plasma samples were prospectively collected from 84 pregnant women carrying male fetuses before amniocentesis (14–29 weeks). We later recorded 26 pregnancies with complicated outcomes, including five cases of fetal chromosomal abnormalities. For statistical analysis, two overlapping subgroups A and B were made. Each group was separately compared for total and fetal DNA with a corresponding group considered normal using Wilcoxon rank sum test. Male fetal DNA concentration in maternal plasma was quantified using real-time quantitative polymerase chain reaction (PCR) of SRY sequences. The samples were also analyzed by quantitative fluorescent PCR (QF-PCR) using highly polymorphic short tandem repeat DNA sequences (STRs), and the percentage of relative fetal allele concentration in maternal alleles was calculated and compared to the fetal/total DNA ratio obtained by real-time PCR. Results Quantities of total and fetal circulating DNA were significantly correlated (r2 = 0.44, P < 0.0001) with a median total DNA concentration of 522 GE/mL (range 51–3047) and a median fetal DNA concentration of 8 GE/mL (range 0–879). Neither level was correlated with gestational age in pregnancies with normal (r2 = −0.05; P = 0.66, and r2 = 0.02; P = 0.88, respectively) and abnormal (r2 = 0.45; P = 0.17, and r2 = 0.11; P = 0.76, respectively) outcomes. Although both total and fetal DNA levels were always higher in women carrying pregnancies with chromosomal aberrations or having other pregnancy complications (P-values range from 0.028 to 0.267), these differences reached statistical significance only for total DNA levels between the group A and corresponding normal pregnancies (P = 0.028). The correlation between the fetal/total DNA ratio obtained by real-time PCR and the percentage of relative fetal allele concentration in maternal alleles obtained by QF-PCR was not found to be statistically significant (r2 = 0.04; P = 0.76). Conclusion Our results confirm the clinical value of fetal DNA measurement in maternal plasma during the second trimester as a supplement for the diagnosis of aneuploidies. Its use as a screening instrument for complications that develop later in pregnancy seems to be limited but needs further investigation. Although the QF-PCR assay has the advantage of being applicable to both female and male fetuses, this approach cannot be used for quantitation of cff DNA in maternal plasma samples. Copyright © 2006 John Wiley & Sons, Ltd.

Cell-free fetal nucleic acids (cffDNA) are present in maternal plasma and serum (1), but amniotic fluid (AF) also provides an attractive source of cffDNA. The concentration of cffDNA is 100- to 200-fold higher in AF than in maternal plasma/serum (2), but low yields of cffDNA compromise testing by techniques such as genomic microarrays, which require a minimum of 100 ng of DNA (3). For protocol optimization, we used 5 large-volume AF supernatant samples from patients who had undergone therapeutic amnioreduction for twin-twin transfusion syndrome. After optimization, we compared the DNA yield of the old and new protocols for freshly discarded AF supernatant samples from 29 euploid singleton pregnancies. Approval for this study was obtained from the Institutional Review Boards of Tufts-New England Medical Center and Women and Infant’s Hospital. The median gestational age at amniocentesis …

Much current research focuses on the properties and clinical applications of circulating nucleic acids (1). The recent discovery of cell-free RNA in the plasma and serum of cancer patients has generated considerable interest (2)(3)(4)(5)(6). Circulating RNA is surprisingly stable, and Ng et al. (7) recently showed that a considerable proportion of plasma mRNA species is particle associated and thus possibly protected from nuclease degradation (8). Fetal-derived mRNA has also been found in the plasma and serum of pregnant women (9)(10)(11) and in amniotic fluid (12), and has many potential clinical applications (13)(14). Amniotic fluid is routinely collected during amniocentesis for fetal chromosome analysis or fetal lung maturity studies. However, little is known regarding the biology of circulating fetal mRNA or fetal mRNA in amniotic fluid. In this report, we explore whether cell-free fetal nucleic acids in amniotic fluid have properties similar to circulating DNA and mRNA. Expanding on the work of Ng et al. (7), we hypothesized that cell-free fetal mRNA in amniotic fluid might be present in a particle-associated form and could thus be filterable, whereas the non-particle–associated form of DNA would be present in such high concentrations that there would be no significant reduction in its quantity by filtration. Additionally, we wished to compare the quantities of nucleic acids in amniotic fluid containing cells with the quantities in filtered and unfiltered cell-free supernatant. We hypothesized that whole amniotic fluid containing amniocytes would contain a significantly larger amount of DNA and RNA than cell-free amniotic fluid. This study was performed with Institutional Review Board approval from Tufts-New England Medical Center. Seven amniotic fluid samples with a minimum volume of 3 mL each were obtained from women undergoing scheduled amniocenteses. One sample originated from a woman …

Fetal cell microchimerism develops in all human pregnancies and has been associated with autoimmune diseases such as systemic sclerosis. It has been suggested that these disorders may be the consequence of an immune reaction between fetal and maternal cells in women after pregnancy. More recently, results from our laboratory suggest that microchimeric cells of fetal origin may differentiate into thyrocytes or hepatocytes in thyroid and liver specimens from women with non-autoimmune diseases. We therefore developed an alternative hypothesis suggesting that microchimeric stem cells may have the ability to participate in the maternal response to tissue injury.

Fetomaternal hemorrhage (FMH) during pregnancy or subsequent isoimmunization, caused by a breach of the placental barrier, could lead to an adverse perinatal outcome (1)(2). However, concerns have been raised regarding the accuracy of the standard Kleihauer–Betke test to quantify FMH (3). In a previously published study, we explored the possibility of using cell-free fetal DNA (fDNA) in maternal plasma as a novel marker of FMH after surgical and medical elective first-trimester termination of pregnancy (TOP) (4). In our earlier study, pre- and posttermination blood samples were drawn at various time points relative to TOP. To adjust for this confounder, we retrospectively applied a mathematical model to the analysis of the data. Projected pretermination fDNA values were calculated based on the anticipated linear increase of fDNA during pregnancy. Adjusted posttermination fDNA concentrations were calculated based on the reported 16-min clearance rate of circulating fDNA after delivery (5). Adjusted posttermination fDNA concentrations were then compared with the projected pretermination concentrations. In the present study, we specifically enrolled only pregnant women in the first trimester undergoing elective surgical TOP. In addition, the pre- and posttermination blood samples were drawn on the same day with a controlled time interval between TOP and the posttermination blood collection. The concentrations of circulating fDNA were then analyzed along with pertinent clinical information to better understand the dynamics of circulating fDNA after TOP. This study was approved by the Institutional Review Boards at Tufts-New England Medical Center and Boston University School of Medicine. Pregnant women in the first trimester of pregnancy undergoing elective surgical TOP at Boston Medical Center were enrolled. Gestational ages were ascertained by ultrasonography and were expressed as postmenstrual days. Paired blood samples were obtained before and immediately after the termination procedure. The blood samples were centrifuged at 800 g , after …

Objective Measurement of cell-free fetal (cff) DNA in maternal plasma may have clinical application in prenatal screening for fetal Down syndrome and preeclampsia. Little is known regarding the tissue of origin of these fetal-derived sequences. We tested the hypothesis that if the placenta is the major contributor of cff DNA, then an increased placental volume should be associated with higher maternal plasma cff DNA levels. Study design We enrolled 143 pregnant women who underwent first-trimester placental volume measurement using 3-dimensional ultrasonography. Cff DNA in maternal plasma on the day of the scan was quantified by real-time polymerase chain reaction (PCR) amplification of a Y-chromosome sequence. The association between measured placental volume and maternal plasma cff DNA levels was analyzed along with relevant clinical variables. Results The median (25th, 75th percentiles) maternal plasma cff DNA level was 16.9 genome equivalents (GE)/mL (10.8, 28.7). Raw values were adjusted for gestational age and maternal body mass index. The median (25th, 75th percentiles) placental volume was 53.2 mL (43.0, 64.7), and median placental quotient (ratio of placental volume to fetal crown-rump length) was 1 mm 2 (0.8, 1.1). Based on multivariate linear regression analyses, neither of the above placental measurements showed a significant association with maternal plasma cff DNA levels ( P =.43 and .43, respectively). A modest association was found between plasma cff DNA levels and gravidity ( P =.03). Conclusion Our data did not show a significant association between either the placental volume or placental quotient, and maternal plasma cff DNA levels. We speculate that it is the extent of placental apoptosis that primarily affects the amount of cff DNA released into the maternal circulation.

To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood samples (defined as the time between blood draw and the initial density gradient centrifugation step) compromises the ability to recover fetal cells, we performed a randomized comparison of immediate (within 4 hours of draw) versus delayed (between 18-24 hours of draw) processing. Four centers participated: two centers utilized flow cytometry (FLOW), and two centers utilized magnetic-activated cell sorting (MACS) techniques. Each center collected 34 samples. The outcome was the percentage of gamma positive (γ+) cells for FLOW or the number of nucleated red blood cells (NRBCs)for MACS, found in the final enriched cell population. Both outcomes reflect cell properties that are potentially fetal in origin, thus making them representative of the ability to recover fetal cells. Our results did not support our hypothesis that delay in processing compromises fetal cell recovery. Instead, in MACS processing, we observed an increase in recovered NRBCs when blood sample processing was delayed compared with immediate processing. There was no significant difference in 7 cells with FLOW. Time-related changes in the density of target cells, perhaps associated with their progress towards apoptosis during the delay period, may result in increased intact fetal cells with the study methods utilized. (J Soc Gynecol Investig 2004; 11:154-65)