Search

Objective: Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA., Methods: Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender., Results: Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels., Conclusions: While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

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., (Copyright 2010. Published by Elsevier Inc.)

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 microL) 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.

Objective: To determine placental pathology and immune response at the maternal-fetal interface in pregnancies conceived by IVF via egg donation compared with nondonor IVF pregnancies., Design: Retrospective case-control study., Setting: Academic medical center., Patient(s): The study population included 20 egg donor and 33 nondonor IVF pregnancies of >24 weeks' gestation., Intervention(s): None., Main Outcome Measure(s): Perinatal complications (gestational hypertension, abruption, preterm delivery, cesarean section), microscopic features indicating an immune response and trophoblast damage, and characterization of inflammatory cells using immunohistochemistry., Result(s): There was an increase in gestational hypertension and preterm delivery in egg donor pregnancies. Dense fibrinoid deposition in the basal plate with severe chronic deciduitis containing significantly increased numbers of T helper and natural killer cells were demonstrated in egg donor placentas. Trophoblast damage was also increased in the preterm egg donor group., Conclusion(s): There are significant histological and immunohistochemical differences between the placentas of egg donor and nondonor IVF pregnancies. The increased immune activity and fibrinoid deposition at the maternal-fetal interface of egg donor pregnancies could represent a host versus graft rejection-like phenomenon., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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.

Objective: To prospectively study the addition of array comparative genomic hybridization (CGH) to the prenatal evaluation of fetal structural anomalies., Methods: Pregnant women carrying fetuses with a major structural abnormality were recruited at the time of invasive procedure for chromosome analysis. Only women whose fetuses had a normal karyotype (n = 50) were subsequently evaluated by array CGH using one of two arrays (1887 clones covering 622 loci or subsequently 4685 clones covering 1500 loci)., Results: The mean gestational age of the fetuses was 24.5 weeks (range 11-38 weeks). The most prevalent anomalies were cardiac, central nervous system, skeletal, and urogenital. The median turnaround time for culturing and array CGH diagnosis was 18 days (range 2-72). Four of 50 fetuses had abnormal array results. One (2%) was clinically significant and three (6%) were inherited or benign variants., Conclusions: Array CGH studies in fetuses with sonographic anomalies and normal metaphase karyotype detected clinically significant copy number alterations in 1 of 50 cases. This percentage (2%) is consistent with prior prenatal reports. Further studies are warranted to more precisely identify which fetal anomalies are associated with copy number alterations of clinical significance., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Objective: To estimate the effectiveness of second-trimester genetic sonography in modifying Down syndrome screening test results., Methods: The First and Second Trimester Evaluation of Risk (FASTER) aneuploidy screening trial participants were studied from 13 centers where a 15- to 23-week genetic sonogram was performed in the same center. Midtrimester Down syndrome risks were estimated for five screening test policies: first-trimester combined, second-trimester quadruple, and testing sequentially by integrated, stepwise, or contingent protocols. The maternal age-specific risk and the screening test risk were modified using likelihood ratios derived from the ultrasound findings. Separate likelihood ratios were obtained for the presence or absence of at least one major fetal structural malformation and for each "soft" sonographic marker statistically significant at the P<.005 level. Detection and false-positive rate were calculated for the genetic sonogram alone and for each test before and after risk modification., Results: A total of 7,842 pregnancies were studied, including 59 with Down syndrome. Major malformations and 8 of the 18 soft markers evaluated were highly significant. The detection rate for a 5% false-positive rate for the genetic sonogram alone was 69%; the detection rate increased from 81% to 90% with the combined test, from 81% to 90% with the quadruple test, from 93% to 98% with the integrated test, from 97% to 98% with the stepwise test, and from 95% to 97% with the contingent test. The stepwise and contingent use of the genetic sonogram after first-trimester screening both yielded a 90% detection rate., Conclusion: Genetic sonography can increase detection rates substantially for combined and quadruple tests and more modestly for sequential protocols. Substituting sonography for quadruple markers in sequential screening was not useful., Level of Evidence: II.

Large (>90%) for gestational age (LGA) fetuses are usually identified incidentally. Detection of the LGA fetus should first prompt the provider to rule out incorrect dates and maternal diabetes. Once this is done, consideration should be given to certain overgrowth syndromes, especially if anomalies are present. The overgrowth syndromes have significant clinical and molecular overlap, and are associated with developmental delay, tumors, and other anomalies. Although genetic causes of overgrowth are considered postnatally, they are infrequently diagnosed prenatally. Here, we review prenatal sonographic findings in fetal overgrowth syndromes, including Pallister-Killian, Beckwith-Wiedemann, Sotos, Perlman, and Simpson-Golabi-Behmel. We also discuss prenatal diagnosis options and recurrence risks.

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.

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.

Background: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates., Objective: To characterise genetic and clinical findings in patients with SIX3 mutations., Methods: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish., Results: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%., Conclusions: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype-phenotype correlation, as shown by functional studies using animal models.

Chimerism in an individual refers to the coexistence of cells arising from two distinct organisms. It can arise iatrogenically via transplant or blood transfusion, and physiologically via twin to twin transfer, or from trafficking between mother and fetus during pregnancy. Many of the diseases associated with microchimerism affect the endocrine system (e.g., autoimmune thyroid disease and diabetes mellitus type 1). Microchimerism is relevant to endocrine pathology because (a) it is associated with pregnancy, a condition of complex endocrine physiology; (b) materno-fetal and feto-maternal cellular migration must involve the placenta, itself an endocrine organ; and (c) in some species, chimerism results in states of intersexuality, a condition intimately involved with endocrine physiology. Studies of feto-maternal microchimerism in the thyroid have documented the presence of fetal cells in association with Hashimoto thyroiditis, Graves' disease, thyroid adenoma, and papillary thyroid carcinoma. Studies of materno-fetal microchimerism have documented the presence of maternal cells in juvenile diabetes and other pediatric conditions. Microchimerism plays a potential role in the repair of diseased thyroid and pancreatic tissues.

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.

Objective: Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high-throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood., Material and Methods: NRBCs were isolated from the peripheral blood of 58 pregnant women using a microfluidic process that consists of a microfluidic chip for size-based cell separation and a magnetic device for hemoglobin-based cell isolation., Results: The microfluidic-magnetic combination removes nontarget red blood cells and white blood cells at a very high efficiency (approximately 99.99%). The device successfully identified NRBCs from the peripheral blood of 58/58 pre-termination samples with a mean of 37.44 NRBC/mL (range 0.37-274.36 NRBC/mL). These results were compared with those from previous studies., Conclusion: The microfluidic device results in an approximate 10- to 20-fold enrichment of NRBCs over methods described previously. The reliability of isolation and the purity of the NRBC product have the potential to enable the subsequent application of molecular diagnostic assays., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

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: To identify a candidate neonatal housekeeping gene and to determine the effects of pH and PaO(2) 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, beta-actin, cyclophilin, 28S rRNA) on extracted RNA. Blood gas analyses determined pH and PaO(2) levels., Results and Conclusions: Beta-Actin was the least variable and GAPDH the most variable housekeeping gene studied. pH negatively correlated with gene expression levels. PaO(2) levels did not significantly affect gene expression. These results inform selection of housekeeping genes for neonatal mRNA research.

Objective: To estimate whether maternal thyroid hypofunction is associated with complications., Methods: A total of 10,990 patients had first- and second-trimester serum assayed for thyroid-stimulating hormone (TSH), free thyroxine (freeT4), and antithyroglobulin and antithyroid peroxidase antibodies. Thyroid hypofunction was defined as 1) subclinical hypothyroidism: TSH levels above the 97.5th percentile and free T4 between the 2.5th and 97.5th percentiles or 2) hypothyroxinemia: TSH between the 2.5th and 97.5th percentiles and free T4 below the 2.5th percentile. Adverse outcomes were evaluated. Patients with thyroid hypofunction were compared with euthyroid patients (TSH and free T4 between the 2.5th and 97.5th percentiles). Patients with and without antibodies were compared. Multivariable logistic regression analysis adjusted for confounders was used., Results: Subclinical hypothyroidism was documented in 2.2% (240 of 10,990) in the first and 2.2% (243 of 10,990) in the second trimester. Hypothyroxinemia was documented in 2.1% (232 of 10,990) in the first and 2.3% (247 of 10,990) in the second trimester. Subclinical hypothyroidism was not associated with adverse outcomes. In the first trimester, hypothyroxinemia was associated with preterm labor (adjusted odds ratio [aOR] 1.62; 95% confidence interval [CI] 1.00-2.62) and macrosomia (aOR 1.97; 95% CI 1.37-2.83). In the second trimester, it was associated with gestational diabetes (aOR 1.7; 95% CI 1.02-2.84). Fifteen percent (1,585 of 10,990) in the first and 14% (1,491 of 10,990) in the second trimester had antithyroid antibodies. When both antibodies were positive in either trimester, there was an increased risk for preterm premature rupture of membranes (P=.002 and P<.001, respectively)., Conclusion: Maternal thyroid hypofunction is not associated with a consistent pattern of adverse outcomes., Level of Evidence: II.

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.

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 (r(2) = 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 (c) 2008 John Wiley & Sons, Ltd.)

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., ((c) 2008 International Society for Analytical Cytology)

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 <3 h. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g., to reduce potential contamination). The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material. Furthermore, the improvements allow for exploration of widely unknown genetic, pathophysiological, and kinetic issues of cell-free fetal DNA in AF.

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: 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 (400x magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200x 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).

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.

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 degrees 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 (R(2) = 0.77, P <0.0001) but not for frozen cffDNA (P = 0.63). The median amount of cffDNA in frozen euploid samples was significantly lower than in fresh samples (P <0.0001). After adjustment for gestational age, there was a statistically significant decrease in the median amount of cffDNA in frozen aneuploidy samples compared with frozen euploid samples (P = 0.0005). Analysis of the cffDNA size distribution showed different and qualitatively unique patterns for each karyotype., 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: 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 (CCl(4)) 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.

The ability to detect cell-free fetal nucleic acids in pregnant women has greatly evolved over the past decade. Dozens of papers have explored the biology, kinetics, and clinical significance of both cell-free fetal DNA and mRNA in the maternal circulation. As a result, our overall understanding of fetal nucleic acid trafficking has expanded. To date, two applications, gender determination and fetal RhD status, have translated into clinical medicine. However, with advanced molecular techniques such as mass spectrometry, real-time quantitative polymerase chain reaction, and gene expression arrays, the ease with which fetal genes can be detected within the mother has greatly improved. Newly identified placental and fetal mRNA transcripts as well as an epigenetically modified placental DNA marker, maspin, have universal applicability. Global expression analyses of fetal mRNA in both amniotic fluid and blood provide new insights into fetal development and pathology. Prenatal diagnosis is poised to evolve from detection of aneuploidy to detection of deviation from normal development, which should provide novel opportunities for fetal treatment., ((c) 2007 Wiley-Liss, Inc.)

Trafficking of cells between the fetus and its mother provides indirect clues to the underlying pathophysiology of pregnancy. Georg Schmorl first documented the presence of fetal cells in the maternal body and emphasized the importance of the placenta in eclampsia. Although his classic paper, written in 1893, is widely cited today, few investigators have actually read the paper, as it was published in German [Schmorl G., Pathologisch-anatomische Untersuchungen über Puerperal-Eklampsie. Verlag FCW Vogel, Leipzig; 1893]. Our goal was to translate the paper into English and critically re-evaluate its conclusions from a 21st century perspective. Schmorl was remarkably astute in his assessment of the pathologic changes that were seen in the 17 women on whom he performed complete autopsies. He found similar severe changes in all of the women, implying a common pathogenesis. This was in direct contrast to the then current doctrine. He was the first to observe the presence of thrombi containing multinucleated syncytial giant cells in the lungs of the women and speculated that they were of placental origin. To support his hypothesis he performed animal experiments. He also recognized that feto-maternal trafficking occurred in normal gestations but was increased in pregnancies affected by eclampsia. Using sophisticated molecular techniques we can now precisely confirm what Schmorl so elegantly described.

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.

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.

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.

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

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 (r(2) = 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 (r(2) = -0.05; P = 0.66, and r(2) = 0.02; P = 0.88, respectively) and abnormal (r(2) = 0.45; P = 0.17, and r(2) = 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 (r(2) = 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.

Considerable quantities of cell-free fetal DNA circulate in the maternal blood during human pregnancy, but the origin of the DNA remains uncertain. Circumstantial evidence suggests the placenta is the principal source, so we tested the hypothesis that release occurs from the syncytiotrophoblast after the induction of apoptotic changes. Villous explants from normal placentas delivered by elective caesarean section were cultured under normoxic conditions (10% oxygen) for up to 20 hours or exposed to hypoxia (0.5% oxygen) for 1 hour followed by reoxygenation. The concentration of beta-globin cell-free DNA in the supernatant, measured using real-time polymerase chain reaction methodology, was significantly increased at 20 hours after hypoxia-reoxygenation. Release was associated with increased apoptosis, confirmed by increased activation of caspase-3 on Western blotting, and immunolocalized to the syncytiotrophoblast; necrosis was also evidenced by release of lactate dehydrogenase. Both release of cell-free DNA and apoptosis could be significantly reduced by the addition of antioxidant vitamins C and E to the culture medium. This study provides the first evidence of a mechanistic and quantitative link between placental apoptosis/necrosis and release of cell-free DNA, hence confirming that maternal serum/plasma concen-trations of cell-free DNA may act as a biomarker of trophoblast well-being during pregnancy.

Objective: 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., Study Design: 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., Results: 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., Conclusion: Fetoscopic intervention of complicated pregnancies does not affect circulating fetal messenger RNA levels, which is in contrast to earlier observations that circulating fetal DNA levels increase after laser ablation for twin-twin transfusion syndrome. Plasma glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels could be a potential novel biomarker for fetal trauma.

Trisomy of the short arm of chromosome 12 is a rare chromosomal anomaly, with an estimated incidence of 1/50,000 births. It may present as a pure trisomy (complete or incomplete), as mosaic trisomy, or with other chromosomal abnormalities. Little is known from prior reports about the natural history and life expectancy of these individuals. In this study we describe the long-term outcome and the differences between patients with mosaic trisomy 12p compared to patients with complete trisomy. We present a series of 16 patients with trisomy 12p; 6 of them are older than 10 years. Most patients were born at term with normal or above normal birth weight. Seven were born with congenital anomalies, but no single anomaly was present in more than one individual. A clear and consistent dysmorphic facial pattern was apparent in all of the subjects. Most patients over 7 years old had a seizure disorder. All individuals exhibited developmental delay with speech affected more severely than motor skills. Six patients were described as "being social." Six had severe behavioral problems, and seven had significant sleep disturbances. Facial features of the three adult patients were different than the younger individuals. We show here that the outcome for patients with mosaic trisomy 12p is better than the outcome in complete trisomy 12p or in trisomy 12p with other chromosomal anomalies. We also provide recommendations for the long-term follow-up of patients with trisomy 12p., (Copyright 2006 Wiley-Liss, Inc.)

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 measured by real-time PCR amplification of a Y chromosome sequence and compared with four standard second trimester serum screening markers (alpha-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 were 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.

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

Objective: This study was conducted to determine whether there is a relationship between the concentration of fetal cell-free DNA in maternal serum and the duration of pregnancy in women who are at high risk for preterm delivery because of either preterm labor or preterm premature rupture of the membranes., Study Design: Sera were collected and frozen from 71 women with a male fetus. Maternal serum fetal cell-free DNA concentration was measured with the use of real-time polymerase chain reaction amplification of DYS1. Fetal cell-free DNA concentrations were converted to multiples of the median. The following groups were studied: group 1: women with preterm labor and intact membranes who were delivered at > or = 36 weeks of gestation (n = 21); group 2: women with preterm labor who were delivered at <36 weeks of gestation (n = 29); and group 3: women with preterm premature rupture of the membranes in labor (n = 20) or not in labor (n = 1) who were delivered prematurely (<36 weeks of gestation). Kaplan-Meier and Cox regression analyses were used to analyze the relationship between fetal cell-free DNA concentrations and the likelihood of preterm delivery., Results: A cut-off value for fetal cell-free DNA of 1.82 multiples of the median was chosen for analysis. The cumulative rate of early preterm delivery (<30 weeks of gestation) was significantly higher for women with fetal cell-free DNA concentrations of > or = 1.82 multiples of the median than those with fetal cell-free DNA concentrations below this cut-off (45% [95% CI, 36%-74%] vs 18% [95% CI, 11%-25%]; P = .008]. The cumulative rate of preterm delivery (<36 weeks of gestation) was also significantly higher at > or = 1.82 multiples of the median (73% [95% CI, 52%-93%] vs 66% [95% CI, 54%-79%]; P = .02). After adjustment for covariates, Cox analysis showed that fetal cell-free DNA at > or = 1.82 multiples of the mechanisms of disease that are associated with a mean hazard rate of delivery of 1.57 (P = .005)., Conclusion: High concentrations of fetal cell-free DNA in maternal serum are associated with an increased risk of spontaneous preterm delivery. This observation may have implications for the understanding of the mechanisms of disease that is associated with preterm labor.

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 mm2 (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.

Objective: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fetal NRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin., Methods: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (n=4), 18 (n=1), 13 (n=2), and other genetic abnormalities (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR (polymerase chain reaction) amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms., Results: In all cases, candidate fetal NRBCs were accurately identified on the basis of morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and nonshared polymorphic DNA alleles when compared to DNA isolated from maternal cells., Conclusions: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

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.

Fetal cells circulate in pregnant women and persist in blood and tissue for decades post-partum. The mother thus becomes chimeric. Factors that may influence such fetal cell microchimerism include histocompatibility, fetal or placental abnormalities, or a reproductive history that includes miscarriage or elective termination. Fetal cell microchimerism is associated with some maternal autoimmune diseases, such as systemic sclerosis. Moreover, a novel population of fetal cells, the pregnancy-associated progenitor cells (PAPCs), appears to differentiate in diseased or injured maternal tissue. The cellular origin of these cells is at present unknown but could be a hematopoietic stem cell, a mesenchymal stem cell, or a novel cell type. Pregnancy therefore results in the acquisition of cells with stem-cell-like properties that may influence maternal health post-partum. Rather than triggering disease, these cells may instead combat it.