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Objective The objective of this study was to understand the biological pathways involved in twin–twin transfusion syndrome (TTTS) by performing global gene expression analysis of amniotic fluid (AF) cell-free RNA. Methods A prospective whole transcriptome microarray study analyzing cell-free RNA in AF from TTTS recipient twins and singleton controls was carried out. Significantly differentially regulated genes in TTTS cases (N = 8) versus matched controls (N = 8) were identified and pathways analyses performed. Significant gene expression differences between stage II TTTS recipients (N = 5) and stage III TTTS recipients with abnormal Doppler measurements (N = 5) were also analyzed. Results Analysis of paired data from TTTS cases and controls revealed differential expression of 801 genes, which were significantly enriched for neurological disease and cardiovascular system pathways. We also identified cardiovascular genes and pathways associated with the presence of critically abnormal Doppler measurements in stage III TTTS recipients. Conclusions This study provides the first transcriptome-wide data on the impact of TTTS on fetal development. Our results show that gene expression involving neurological and cardiovascular pathways are altered in recipient fetuses prior to surgical treatment. This has relevance for the origins of long-term complications seen in survivors and for the development of future fetal biomarkers. © 2013 John Wiley & Sons, Ltd.

It is now a well-established fact that fetal cells are present in the maternal circulation. The first evidence that fetal cells exist outside the fetoplacental unit was demonstrated in 1893 by Schmorl, who described the presence of trophoblast sprouts in the lungs of women who died of eclampsia (1). With the advent of sensitive molecular techniques, this finding has now been confirmed on numerous occasions. In 1969, Walknowska published a landmark study showing the detection of cells bearing a Y-chromosome in the blood of women carrying a male fetus (2). Since this report, the potential applications of using circulating fetal cells in prenatal screening have been researched extensively. Fetal cells represent a unique source of fetal DNA and RNA that can be used for the detection of fetal chromosomal aneuploidies and single gene disorders. In addition, new studies show that the fetoplacental unit is not as distinctly separate from the mother as was previously thought. In every pregnancy, there is trafficking of maternal and fetal cells and cell-free nucleic acids to and from the fetoplacental unit, respectively. Under normal circumstances, very few fetal cells enter the maternal bloodstream. However, in the case of many fetal or placental disorders, including aneuploidy or preeclampsia, an abnormally high number of fetal cells are present in the maternal circulation. Current data suggest that fetomaternal trafficking is a reflection not only of fetal health but also of placental development and pregnancy well-being. New evidence shows that the persistence of fetal cells in maternal tissues after delivery plays a role in subsequent maternal health. This chapter will discuss the role of circulating fetal cells in prenatal diagnosis and the relationship between abnormal fetomaternal trafficking of cells and the occurrence of disease in the woman both during gestation and postpartum.

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.

The objective of this study was to determine if fetal-maternal cell trafficking is affected by maternal immune competence and/or parental background strain using fluorescence-activated cell sorting (FACS). In our experience the sensitivity of FACS allows for the detection of 5 fetal in 10(7) maternal cells and assessment of cell surface phenotype. Wild-type C57BL/6J (n=18), FVB/NJ (n=15), and immunodeficient B6129S7-Rag1(tm1Mom)/J (n=16) female mice were mated to C57BL/6J males homozygous for the green fluorescent protein (GFP) transgene. Single cell suspensions of maternal lung, liver, spleen, bone marrow, and blood were analyzed between late gestation (day e16-18) and 1 day post-partum for the number of GFP-positive fetal cells in relation to 10(7) maternal cells and the percentage of GFP-positive cells that expressed the surface markers CD11b, CD29, CD34, CD44, or CD105. The highest relative proportions of GFP-positive fetal cells were observed in maternal lungs and livers from immunocompetent allogenic females. Among congenic matings, fetal cell microchimerism was higher in immunodeficient compared with immunocompetent females. Maternal strain and strain differences between the mother and father statistically significantly affected both the numbers of fetal cells and the relative distribution of cell types in maternal organs. The highest relative proportion of fetal cells was observed in allogenic matings with immunocompetent females. Since allogenic matings are more similar to those that occur in humans, future studies using animal models of microchimerism should consider incorporating this type of experimental design.

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.

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs.

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.

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.

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.

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 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: 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 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.

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 The realization that fetal cells pass into the maternal circulation and can survive for many years has raised the question of whether fetal microchimerism can cause subsequent disease in the mother. Available data suggest that fetal–maternal transfusion may be related to some autoimmune diseases, notably systemic sclerosis (SSc). The goal of the current work was to identify and quantify tissue-specific fetal microchimerism in women with SSc. Methods We analyzed multiple tissue specimens obtained at autopsy from women with SSc as well as women who had died of causes unrelated to autoimmunity, using fluorescence in situ hybridization to detect the presence of male cells in women with sons. Tissues analyzed included adrenal gland, heart, intestine, kidney, liver, lung, lymph node, pancreas, parathyroid, skin, and spleen. Results Male cells were observed in tissue from at least 1 site in each woman with SSc and were found most frequently in spleen sections. After spleen, male cells were observed most frequently in lymph node, lung, adrenal gland, and skin tissue. The only tissue type in which male cells were not seen in any patient was pancreatic tissue. Male cells were not observed in tissue from women who had died of causes unrelated to autoimmunity. Conclusion The results of this study suggest that fetal cells migrate from the peripheral circulation into multiple organs in women with SSc. All of the women studied had previously given birth to sons, so it is likely that these cells are of fetal origin. While the relevance of this finding to the pathogenesis of SSc remains to be elucidated, the presence of fetal cells in internal organs suggests that they could play a role in disease pathogenesis and that they may preferentially sequester in the spleen. The presence of these male cells may also be a result of disease, possibly through the migration of terminally differentiated and/or progenitor cells to areas of tissue damage.

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.

Fetal clonogenic erythroid cells may be present in maternal blood and serve as a source of fetal DNA for prenatal genetic diagnosis. Proliferating nucleated red cells in cultures from first and second trimester fetal blood contain only fetal haemoglobin (HbF; F+A− cells), whereas nucleated red cells from adult blood contain also adult haemoglobin (HbA; F+A+ or F−A+ cells). Thus, fetal red cells can be identified and flow sorted. However, a few adult cells are also F+A−, which reduces the purity of fetal cell isolation. Culture media optimized for erythropoiesis contain interleukin-3 (IL3). We show here that IL3 strongly stimulates the growth of F+ cells (both F+A− and F+A+) in cultures from adult blood but has only a comparatively small effect on F+A− cells in cultures from fetal blood. This difference is maintained in the unified conditions of co-cultures of adult and fetal cells, so that the purity of fetal cell sorts can be increased by omitting IL3 from the culture medium. We further show that IL3 accelerates the exhaustion of the long-term division potential of adult cells, allowing fetal secondary colonies to be identified by their size following a two-stage culture scheme. Thus, the choice to omit or include IL3 in the growth medium of maternal blood cultures should depend on whether fetal nucleated red cells are to be isolated by flow sorting after one week, or by picking secondary colonies after a later secondary culture stage. Copyright © 2000 John Wiley & Sons, Ltd.

During pregnancy, fetal cells cross into the maternal organs where they reside postpartum. Evidence from multiple laboratories suggests that these microchimeric fetal cells contribute to maternal tissue repair after injury. In mouse models, most injury experiments are performed during pregnancy; however, in a clinical setting most injuries or diseases occur postpartum. Therefore, experiments using animal models should be designed to address questions in the time period following delivery. In order to provide a baseline for such experiments, we analyzed the natural history of fetal cells in the postpartum maternal organs. Female C57BL/6J mice were mated to males homozygous for the enhanced green fluorescent protein gene. Fetal cells in the maternal lungs and bone marrow were identified by their green fluorescence using in a high-speed flow cytometer and their counts were compared between the lung and bone marrow. Spearman correlation analysis was used to identify relationships between the duration of time postpartum and the cell counts and ratio of live and dead cells. Our results show that fetal cells persist in these organs until at least three months postpartum in healthy female mice. We show a two-stage decline, with an initial two and a half-week rapid clearance followed by a trend of gradual decrease. Additionally, an increase in the ratio of live to dead cells within the lung over time suggests that these cells may replicate in vivo. The results presented here will inform the design of future experiments and may have implications for women's health.

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.

The transfer of fetal cells to maternal organs occurs in mouse and human pregnancy. Techniques such as polymerase chain reaction and flow cytometry do not permit study of fetal cell morphology or anatomic location. Using a green fluorescent protein (GFP) transgenic mouse model, our objective was to determine whether GFP+ signal emanates from intact or degraded fetal cells, and whether they have a characteristic appearance and location within maternal lung. Four wild-type female mice were mated to males homozygous for the Gfp transgene and studied at days e16–18. Controls were 2 females mated to wild-type males. Morphologic appearance and anatomic position of each GFP+ object within maternal lung was recorded. GFP signals were sufficiently bright to be visualized without anti-GFP antibody and were confirmed by confocal microscopy to be separate from fluorescent artifact. Of 438 GFP+ objects detected, 375 (85.6%) were from intact cells, and 63 (14.4%) were acellular. Four distinct categories of intact cells were observed. Of these, 23.2% had mononuclear morphology with a relatively large nucleus and GFP+ cytoplasm (Group A). An additional group of cells (10.1%) had mononuclear morphology and podocyte extensions (Group B). The remainder of cells had fragmented nuclei or cytoplasm. Both intact cells and acellular fragments were predominantly localized to the maternal alveolar septum (P

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.

Although the measurement of fetal proteins in maternal serum is part of standard prenatal screening for aneuploidy and neural tube defects, attempts to better understand the extent of feto-maternal protein trafficking and its clinical and biological significance have been hindered by the presence of abundant maternal proteins. The objective of this study was to circumvent maternal protein interference by using a computational predictive approach for the development of a noninvasive, comprehensive, protein network analysis of the developing fetus in maternal whole blood. From a set of 157 previously identified fetal gene transcripts, 46 were classified into known protein networks, and 222 downstream proteins were predicted. Statistically significantly over-represented pathways were diverse and included T-cell biology, neurodevelopment and cancer biology. Western blot analyses validated the computational predictive model and confirmed the presence of specific downstream fetal proteins in the whole blood of pregnant women and their newborns, with absence or reduced detection of the protein in the maternal postpartum samples. This work demonstrates that extensive feto-maternal protein trafficking occurs during pregnancy, and can be predicted and verified to develop novel noninvasive biomarkers. This study raises important questions regarding the biological effects of fetal proteins on the pregnant woman.

To determine whether chemically induced miscarriage affects fetomaternal trafficking in a mouse model, we measured the amount of fetal DNA present in various maternal organs by polymerase chain reaction amplification following exposure to lipopolysaccharide (LPS). As the frequency of fetal cells and the number of animals with detectable microchimerism following LPS injection were significantly increased, particularly in lung tissue compared to controls, with no signs of an inflammatory response, we conclude that LPS-induced miscarriage results in increased murine fetomaternal cell trafficking, supporting a relationship between fetal loss and the establishment of fetal cell microchimerism.

To better understand fetomaternal cell trafficking during pregnancy, we used a mouse model to determine the cell surface markers expressed on fetal cells, based on the hypothesis that fetal progenitor cells have the capacity to repair maternal organs, whereas more differentiated cells might initiate graft versus host disease. Wild-type females were mated to either homozygous or hemizygous transgenic males and euthanized in the peripartum period. Using dual color flow cytometry, we analyzed fetal transgene positive cells for the presence of nine markers (ITGAM, ITGB1, PECAM, CD34, CD44, PTPRC, ENG, SLAMF1, and CXCR4) to begin to identify the phenotype and degree of differentiation of fetal cells in nine maternal organs (lung, liver, spleen, blood, bone marrow, kidney, heart, thymus, and brain). Fetal cells were found in all maternal organs following either type of mating, albeit always at a higher frequency following mating with homozygous males. Some organs (e.g., lung and liver) had a wide variety of fetal cell markers present, while other organs (e.g., bone marrow and spleen) had a skewed distribution of fetal cell markers. Fetal cells in the murine pregnant female are diverse. Our results suggest that the fetal cells comprise a mixed population of progenitor and differentiated cells, with different relative proportions in different maternal organs. Future studies will address whether fetal cells cross the placental barrier in a differentiated state or as a homogenous population and subsequently differentiate in target maternal organs.

The purpose of the study was to understand in more detail the natural history of fetomaternal cell trafficking in healthy pregnant mice. Our goal was to identify the best target organs and days during pregnancy for further mechanistic studies of the role of fetal cells in maternal disease and injury. C57BL/6J wild-type virgin females (n = 54) were mated with congenic enhanced green fluorescent protein (EGFP) transgenic males. During pregnancy and after delivery, female mice were euthanized, and eight organs and blood were analyzed for the presence of fetal GFP+ cells with flow cytometry and real-time quantitative PCR. Maternal lungs, liver, and spleen were also analyzed by fluorescent stereomicroscopy. Fetal GFP+ cells were first found at low frequency at Embryonic Day 11, increased to a maximum at Embryonic Day 19, and decreased rapidly postpartum. These fetal cell dynamics were significantly reproducible among all mice studied. In addition, there was a consistent distribution of fetal cells within maternal organs, with lung, liver, blood, and spleen having the greatest concentrations; these were highly correlated at all time points (P < 0.0001). Maternal lung contained 10- to 100-fold more fetal cells than any other organ, and using all three techniques, the number of fetal cells detected was the most consistent and reproducible in this organ. Stereomicroscopy showed that within the lung, fetal cells were widely and apparently randomly distributed. Using a murine model, our data demonstrate that fetomaternal cellular trafficking occurs in reproducible patterns, is maximal near term delivery, and has predilection for the maternal lung..

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.

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.

In humans, maternal cells are present in the affected tissues of children with inflammatory myopathy, scleroderma, and neonatal lupus. It is unknown if maternal cell microchimerism (MCM) contributes to the pathology of disease. We sought to understand the factors that affect MCM to serve as a baseline for future mechanistic studies. Using a mouse model, we bred female mice transgenic for the luciferase (Luc) reporter gene to wild-type (WT) males. The WT offspring were sacrificed at various postnatal ages. DNA was extracted from multiple organs, and real-time PCR amplification was used to quantify Luc transgene as a marker for maternally derived cells. Sensitivity was one to two transgenic cells per 100,000 WT cells. MCM was noted in 85% of mice and 45% of tissues assayed. The average quantity of MCM was 158 maternal cells per 100,000 neonatal cells. The organs displaying the highest frequency and quantity of MCM were heart and lung (P < 0.001). Postnatal age up to 21 days did not appear to affect levels of MCM (P = 0.47), whereas increasing parity may increase levels of MCM. The data show that MCM is a common occurrence in healthy newborn mice, that it is present in their major organs, and that there are organ specific differences. This may represent differential migration of maternal cells or varying receptivity of specific fetal organs to microchimerism. Pregnancy history appears to play a role in maternal cell trafficking. The role of MCM in pregnancy and disease pathogenesis remains to be elucidated.

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.

BACKGROUND: In humans, fetal microchimeric cells transferred to maternal tissues during pregnancy can adopt a hepatocyte phenotype. Our objective was to determine whether fetal cells participate in the response to specific murine post-partum hepatic injuries. METHODS: Wild-type female mice were bred to males transgenic for the enhanced green fluorescent protein (GFP) (n = 42). Following delivery, we created models of chemical or surgical injury with carbon tetrachloride (CCl4) injection or by performing partial hepatectomy. Liver injury was assessed histologically. Fetal cells in maternal liver were detected and measured by real-time PCR amplification of the gfp transgene and by immunofluorescence using anti-GFP antibodies. RESULTS: PCR results showed that in chemical but not surgical injury, fetal GFP+ cells were detectable in maternal liver and spleen and that fetal cell presence was significantly increased over time following injury (4 versus 8 weeks, P = 0.006 for liver and P = 0.0006 for spleen). In some animals, following chemical injury, GFP+ cells were detected by immunofluorescence. CONCLUSIONS: The results of this preliminary study suggest that specific types of injury may elicit different fetal cell responses in maternal organs. There is a significant effect of time on fetal cell presence in liver and spleen. Furthermore, real-time PCR amplification is more sensitive than immunofluorescence for the detection of microchimeric fetal cells.

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.

BACKGROUND: Cell-free fetal DNA (fDNA) can be detected in maternal plasma throughout human pregnancy and is rapidly cleared after delivery. fDNA measurement has clinical application in many complications of pregnancy. Our aim was to determine if fDNA could be detected in maternal plasma during pregnancy in a mouse model system. We then compared the levels of fDNA during pregnancies in which the mother and fetus were either congenic or allogenic. METHODS: C57BL/6J (H-2b) or DBA/2J (H-2d) wild-type female mice were mated to C57BL/6J mice transgenic for the enhanced green fluorescent protein (gfp) and sacrificed while pregnant. C57BL/6J female mice that had previously given birth to three to six litters after mating with transgenic males were sacrificed after delivery. We used real-time quantitative PCR amplification to detect and measure gfp sequences in maternal plasma. RESULTS: fDNA was consistently detected in maternal plasma during pregnancy and was always absent after delivery [median 211 genome equivalents (GE)/ml vs 0 GE/ml, respectively, P=0.0001]. The level of fDNA was higher in allogenic matings compared to congenic matings (median 167 GE/ml/GFP + fetus vs 81 GE/ml/GFP + fetus, respectively). CONCLUSIONS: fDNA sequences can be reliably detected in maternal plasma during murine pregnancy. Our data lends further support to the use of nonhuman species to investigate the mechanisms involved in fetomaternal trafficking.

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)

Background: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for γ-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma. Methods: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma γ-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample. Results: γ-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P Conclusions: γ-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma γ-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination γ-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of γ-globin mRNA as a marker for fetomaternal hemorrhage.