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Magnetic resonance imaging and angiography is the technique of choice in the diagnosis and follow-up of cerebral venous thrombosis: Thrombosis appears as an absence of flow void on spin echo images and lack of signal in angiographic techniques. The thrombus signal intensity is different on T1 and T2 spin echo weighted images and evolves according to hemoglobin degradation. Recognition of pitfalls and artefacts related to the different magnetic resonance imaging techniques employed is essential to interpret dural venous sinus thrombosis. In this paper the imaging of 27 patients with cerebral venous thromboses is reviewed. We describe the type of signal abnormalities, the different types of clot- and flow-related artefacts, and the indirect signs of cerebral venous thrombosis.

Summary Background Fetal cells can be found in the maternal circulation in most pregnancies. Fetal progenitor cells have been found to persist in the circulation of women many years after childbirth. We tested the hypothesis that microchimerism is involved in the pathogenesis of scleroderma. Scleroderma is of interest because of a strong female predilection, an increased incidence in the years after childbearing, and clinical similarities between scleroderma and chronic graft-versus-host disease after allogeneic bone-marrow transplantation. We also investigated whether HLA-compatibility of a child was associated with later development of scleroderma in the mother. Methods We enrolled 40 women who had previously given birth to at least one son—16 healthy controls, 17 scleroderma patients, and seven healthy sisters of patients. We used quantitative PCR to amplify a Y-chromosome-specific sequence in whole peripheral blood from these women. Also 32 controls with 58 children, and 21 scleroderma patients with 47 children were HLA genotyped. Findings The mean number of male cell DMA equivalents among controls was 0·38 cells per 16 ml whole blood (median 0 [range 0–2]) and 11·1 (6·0 [0–61]) among scleroderma patients (p=0-0007). Controls' youngest sons were born a mean of 15-4 years previously, and scleroderma patients' sons 18·5 years previously. Some scleroderma patients had concentrations of male DMA higher than those found in most pregnant women. HLA-class II compatibility of a child from the mother's perspective was more common among scleroderma patients than among controls, but was not essential for persistence of male DMA in maternal peripheral blood. Interpretation Low concentrations of male DMA can be detected in healthy women decades after the birth of a son. Microchimerism in scleroderma patients could be secondary to the underlying disease. However, the finding that HLA class II compatibility of a child was more common for scleroderma patients than for controls, supports the possibility that microchimerism may be involved in the pathogenesis of scleroderma.

In a proof of principle report elsewhere in this issue of Clinical Chemistry, Tong et al. (1) describe a novel approach to the noninvasive prenatal diagnosis of a human chromosomal abnormality by analyzing the allelic ratio of a polymorphism present within the methylated promoter of a DNA sequence on chromosome 18q21.3, maspin (SERPINB5) . This method differs from other approaches to noninvasive diagnosis of aneuploidy in that it is truly diagnostic, as opposed to being a screen. The technique described in this paper relies first on the isolation of cell-free fetal DNA from maternal plasma, and second, on the fact that there is differential methylation of maspin . In a previous report from the same laboratory group (2) maspin was shown to be hypomethylated (i.e., actively expressed) in placenta and hypermethylated (i.e., silenced) in maternal leukocytes, which are the source of most circulating cell-free DNA in plasma (3). By the method of bisulfite modification, unmethylated cytosine residues in the DNA sequence are converted to uracil, but methylated cytosine residues remain unchanged. This sequence difference is then exploited via methylation-specific PCR amplification, followed by allele-specific primer extension. A single-base variation [or single nucleotide polymorphism (SNP)] within the promoter sequence is then used as a focus for mass spectrometric analysis, with subsequent analysis of the ratio of copy numbers of the particular DNA sequence variation. In the present study, the U-maspin -156 SNP was used. A normal euploid fetus might be genotyped as AC at this locus. Fetuses with trisomy 18, if polymorphic, would be genotyped as having AAC or ACC. By measuring the ratio of A to …

OBJECTIVE: To determine whether the use of assisted reproductive technology (ART) is associated with an increase in chromosomal abnormalities, fetal malformations, or adverse pregnancy outcomes. METHODS: A prospective database from a large multicenter investigation of singleton pregnancies, the First And Second Trimester Evaluation of Risk trial, was examined. Subjects were divided into 3 groups: no ART use, use of ovulation induction (with or without intrauterine insemination), and use of in vitro fertilization (IVF). Multivariate logistic regression analysis was used to assess association between ART and adverse pregnancy outcomes (significance of differences was accepted at P < .05). RESULTS: A total of 36,062 pregnancies were analyzed : 34,286 (95.1%) were spontaneously conceived, 1,222 (3.4%) used ovulation induction, and 554 (1.5%) used IVF. There was no association between ART and fetal growth restriction, aneuploidy, or fetal anomalies after adjustment for age, race, marital status, years of education, prior preterm delivery, prior fetal anomaly, body mass index, smoking history, and bleeding in the current pregnancy. Ovulation induction was associated with a statistically significant increase in placental abruption, fetal loss after 24 weeks, and gestational diabetes after adjustment. Use of IVF was associated with a statistically significant increase in preeclampsia, gestational hypertension, placental abruption, placenta previa, and risk of cesarean delivery. CONCLUSION: Patients who undergo IVF are at increased risk for several adverse pregnancy outcomes. Although many of these risks are not seen in patients undergoing ovulation induction, several adverse pregnancy outcomes are still increased in this group. There was no increased incidence of fetal chromosomal or structural abnormalities in the women who used any type of ART compared with the women who conceived spontaneously. (Obstet Gynecol 2005;106:1039–45)

OBJECTIVE: Although polymorphonuclear leukocytes are the inflammatory cells most frequently recovered from the amniotic cavity in cases of suspected intrauterine infection, the source of these cells has not been definitively determined. We took advantage of the gender difference between the mother and her male fetus, and we report four cases in which amniotic fluid polymorphonuclear leukocytes were identified as fetal by fluorescence in situ hybridization with probes specific for X and Y chromosomes. Fetal membranes were intact at the time amniotic fluid was obtained in all cases. STUDY DESIGN: Amniotic fluid was obtained from women with male fetuses in premature labor with clinical or laboratory evidence of infection. Cytospin preparations of amniotic fluid samples with polymorphonuclear leukocytes were prepared and sequentially stained with fluorescent reagents. To determine which cells were polymorphonuclear leukocytes, all replicate samples were stained with the fluorescent nuclear stain 4′-6-diamidino-2-phenyl-indole. This allowed definition of the characteristic multilobed polymorphonuclear leukocytes nuclear morphologic features. The sample was then probed with a rhodamine-labeled probe specific for the X chromosome and a fluorescein-labeled probe specific for the Y chromosome to assess whether the polymorphonuclear leukocytes were male or female. RESULTS: Ninety percent to 99% of polymorphonuclear leukocytes identified by normal multiple lobed nuclear morphologic study on 4′-6-diamidino-2-phenyl-indole staining had an X and Y chromosome and were therefore fetal cells. CONCLUSION: These data demonstrate a fetal response during intraamniotic infection. Further investigation of the roles for maternal and fetal polymorphonuclear leukocytes in chorioamnionitis may provide valuable information about the critical interaction of the two immune responses in this setting. (Am J Obstet Gynecol 1997;176:77-81.)

1PDH-E2 is aberrantly expressed in the luminal surface of epithelial cells in the small bile duct only in patients with PBC and is the target of activated T lymphocytes. 2 In patients with PBC, precursor frequencies of PDH-E2-specific T cells isolated from the liver and hilar lymph nodes increase 100‐150 fold compared with peripheral blood mononuclear cells. 3 The immunodominant epitope that is recognised by MHC class II restricted CD4+ T cells in patients with PBC is aminoacids 163‐176 of PDH-E2. 4,5 The immunodominant epitope recognised by MHC class I restricted CD8+ T cells is similar (aminoacids 159‐167). 6 This peptide induces proliferation of specific MHC class I restricted CD8+ cytotoxic T lymphocytes from most patients with PBC who are positive for HLA-A*0201. The frequency of cytotoxic T lymphocytes recognising this epitope is increased ten-fold in liver compared with blood in patients with PBC. 6,7

The data accumulated thus far indicate that fetal NRBCs are the target cell type of choice in maternal blood for most investigators, although some groups continue to work with the trophoblast. Reports of persistent circulation of hematopoietic stem cells, lymphoid/myeloid progenitors, and lymphocytes mandate that removal of these cell types must occur before clinical diagnosis of the current pregnancy can be made. In selected cases, accurate detection of fetal aneuploidy has been made from fetal cells in maternal blood; the clinical evaluation sponsored by the National Institute of Child Health and Human Development will determine the sensitivity and specificity of cytogenetic diagnosis in a larger group of pregnant women, but this information will not be available for several years. At present, detection of uniquely fetal, paternally inherited gene polymorphisms or mutations such as the Rh(D) antigen is possible only because the mother lacks these genes; hence, maternal cell contamination does not hinder diagnosis. Currently the presence of large numbers of maternal cells in enriched samples precludes single-gene diagnosis for conditions in which the mother carries a mutant gene, because her cells are preferentially amplified and difficult to distinguish from those of the fetus. It is likely, however, that as techniques of individual fetal cell isolation are perfected, maternal cell contamination will no longer be an issue, and the entire fetal genome will become available for diagnosis and therapy. Pediatricians need to be aware of the progress of research in this field, because fetal cell isolation from maternal blood not only could change prenatal diagnosis but would change the amount of genetic information that arrives with a newborn infant at birth. The ultimate goal of this work is to diagnose noninvasively, in the first trimester, the common fetal aneuploidies and single-gene disorders, to permit in utero treatment, or to allow low-risk pregnant women carrying an abnormal fetus an opportunity for reproductive choice.

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.

amniotic fluid supernatant reveals major alterations in nervous and cardiovascular system gene expression LISA HUI, Heather Wick, Kenneth Moise, Anthony Johnson, Michael Belfort, Kirby Johnson, Diana Bianchi Tufts Medical Center, Mother Infant Research Institute, Boston, MA, Tufts University, Computer Science, Medford, MA, Baylor College of Medicine, Obstetrics and Gynecology, Houston, TX OBJECTIVE: To understand the molecular mechanisms underlying the recipient’s response to twin-twin transfusion syndrome (TTTS) and develop fetal biomarkers by performing functional genomic analysis of amniotic fluid supernatant (AFS). STUDY DESIGN: We have previously shown that AFS contains RNA from multiple fetal organs, including brain. This was a prospective study analyzing cell-free RNA transcripts in recipient AFS from women undergoing clinically-indicated laser surgery. Normal AFS was obtained from singleton fetuses undergoing genetic amniocentesis. The independent t test with the Benjamini-Hochberg correction was used to evaluate up-regulated genes in TTTS vs normal controls, and Quintero Stage III vs stage II cases. Functional analyses of the significantly up-regulated genes were performed with Ingenuity software. RESULTS: AFS samples from 8 recipient twins (4 Stage II and 4 Stage III, EGA 17-21 w) and 4 euploid controls (EGA 17-22 w) were included. Compared with controls, 811 genes were significantly upregulated in TTTS cases. The most statistically significant biological process that differed between cases and controls was nervous system development and function. There were 19 up-regulated nervous system genes in TTTS cases including microtubule-associated protein tau, a biomarker of adult neurodegeneration. Other important upregulated transcripts in TTTS were vascular endothelial growth factor 1 and angiotensin II receptor type 2. The most statistically significant up-regulated biological process in Stage III vs Stage II fetuses was cardiovascular disease. Among the 58 cardiovascular disease genes up-regulated in Stage III recipients were some specifically associated with hypertension and left ventricular dysfunction (TRPC4) and congenital pulmonary stenosis (NTRK3). CONCLUSION: Our study provides novel molecular evidence of the impact of TTTS on the developing nervous system. We also identified the cardiovascular genes specifically associated with Stage III vs Stage II disease. These results further our understanding of the pathophysiology of TTTS and provide potential prognostic fetal biomarkers. 424 Gender equality in fetal reduction (FR) patient decision preferences: major cultural change in the USA Mara Rosner, Stephanie Andriole, Avishai Alkalay, Rachel Greenbaum, Juliana Gebb, Mark Evans Montefiore Medical Center/Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, NY, Comprehensive Genetics, Genetic Counseling, New York, NY, Albert Einstein College of Medicine/ Montefiore Medical Center, Obstetrics and Gynecology & Women’s Health, Bronx, NY, Comprehensive Genetics, Ultrasound, New York, NY, Comprehensive Genetics & Mt. Sinai School of Medicine, Obstetrics & Gynecology, New York, NY OBJECTIVE: 20 years ago, a disproportionate % of gender preference requests for prenatal diagnosis, terminations, and reductions were from patients of ethnic groups that valued boys over girls. We refused to participate. As patient interest expanded to all ethnic groups and gender preference seemed to equalize, our bioethicist had us re-evaluate and incorporate patient preference. STUDY DESIGN: We prioritize FR decisions by: 1. documented major (M) anomaly; 2. suspicious or minor (m) findings; 3. if neither, we consider gender preference. We retrospectively reviewed patient choices in our last 400 CVS/FR cases starting with triplets or twins. Patients were categorized based on type of reduction (3i2, 3i1, or 2i1), presence or absence of M or m findings, and whether all fetuses were the same gender (SG) or a gender choice was possible. Higher order and non-CVS pregnancies were excluded. RESULTS: 178/321 (56%) had either M/m or SG. Of 3i2 with option, 71/79 reduced to M/F of which about 1/3rd had no preference. 1 chose MM and 7 chose FF. Most 3i1 included MZ twins for whom we reduced MZ in 33/35 cases. Of 20 3i1 with option, 10 chose M and 10 chose F. Of 44 2i1 with option, 20 chose M & 24 chose F. CONCLUSION: There has been a major shift in culture from a generation ago. Male preponderance has ended in the USA. The vast majority of our patients who reduced to 2 preferred one of each or had no preference. When reducing to 1, a non-significant trend towards female preference was found. In addition to identifying abnormalities, CVS prior to FR expands patient autonomy. Poster Session III Doppler Assessment, Fetus, Prematurity www.AJOG.org

Objective: The detection of fetal aneuploidy and gene mutations by analysis of fetal cells in maternal blood has demonstrated the feasibility of noninvasive prenatal diagnosis. Fetal cells are rare in the maternal circulation; all current methods used for their isolation also yield maternal cells. We developed a method that permits a quantitative assessment of the relative numbers of fetal and maternal cells. Study Design: Samples from 40 pregnant women were flow sorted with different monoclonal antibodies. Deoxyribonucleic acid was subsequently purified from candidate fetal cells; polymerase chain reaction was performed with synthetic primers specific for sequences on chromosomes Y and 7. Results: The maximum number of fetal cells detected was 52 in 1080 maternal cells. Fetal cell purity ranged from 0.001 % to 4.8%. Fetal cells were detected with antibodies to CD71, CD36, and glycophorin A. Conclusion: Quantitative polymerase chain reaction enables the determination of the purity and yield of fetal cells remaining after isolation from maternal blood, facilitating rapid comparisons between different cell separation techniques.

Since the presence of large amounts of circulating fetal DNA in maternal plasma and serum samples was first reported by Lo et al. in 1997 (1), a multitude of clinical applications for the noninvasive diagnosis of complications of pregnancy have been reported [reviewed in Ref. (2)]. These applications have largely focused on the quantification of male fetal DNA in maternal plasma/serum samples and its associated increase in conditions such as fetal trisomy 21, maternal hyperemesis gravidarum, preeclampsia, invasive placenta, and preterm labor (3)(4)(5)(6)(7). In addition, qualitative detection of uniquely fetal DNA sequences in maternal plasma/serum has facilitated the noninvasive prenatal diagnosis of Rhesus D genotype, myotonic dystrophy, and achondroplasia (8)(9)(10). Both of these approaches are feasible because fetal and maternal DNA can be distinguished from each other. This same approach, however, will not work in the noninvasive prenatal diagnosis of autosomal recessive disorders, in which a fetus shares a mutant allele with its mother. In this issue of Clinical Chemistry , Chiu et al. (11) describe a novel strategy to exclude the diagnosis of a recessive condition by identifying polymorphic markers that distinguish the uniquely paternal mutant and wild-type alleles. They amplified cell-free fetal DNA in maternal plasma at 11 and 17 weeks of gestation, and by comparison with DNA from the couple’s first affected child, were able to determine that the current pregnancy was unaffected. As a model system, these investigators chose the disease congenital adrenal hyperplasia (CAH), but the approach …

s / Placenta 32 (2011) S326–S340 S327 FETAL CELLS IN THE ADULT FEMALE FOLLOWING PREGNANCY: AN UNDER-APPRECIATED SOURCE OF PROGENITOR CELLS D.W. Bianchi Tufts University School of Medicine, Boston, MA, USA As a result of pregnancy, all human females acquire progenitor cells from their fetuses. These cells and their progeny survive for decades postpartum in maternal blood and organs (Bianchi et al. PNAS 1996; 93: 7058). Significant knowledge gaps exist as to whether these fetal cells have characteristics of embryonic or adult stem cells, and whether they have positive, negative, or neutral effects on the health of the mother. This is important, because these fetal cells may have unique therapeutic properties in maternal organs. Furthermore, the long-term presence of fetal cells in women may have broader significance, in that they may be an important factor in diseases that manifest gender-related differences. The long-range goal of my laboratory is to understand the consequences of feto-maternal cell trafficking to determine if pregnancy results in longterm beneficial health effects for the mother. Our central hypothesis is that fetal cells have phenotypically and functionally unique properties that can be developed and applied for novel therapeutic purposes. We are specifically testing this hypothesis by examining whether fetal cells contribute to repair of or recovery from tissue injury in the maternal lung in a murine model system. This organ was selected on the basis of our significant and reproducible murine data that demonstrate that the lung is the maternal organ that contains the most fetal cells. We are currently in the process of determining key factors that distinguish fetal cells from adult cells in maternal organs using immunohistochemistry and gene expression studies. doi:10.1016/j.placenta.2011.07.033 TRANSLATING TRANSPLANTATION TOLERANCE IN THE CLINIC: WHERE ARE WE, WHERE DO WE GO? K. Wood Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK Strategies for the induction of specific unresponsiveness to donor alloantigens currently under investigation in the clinic take advantage of two of the major mechanisms for the induction of tolerance to self antigens – deletion and immunoregulation/suppression. Deletion of donor antigen reactive cells from the immune repertoire of the recipient will potentially prevent or reduce the risk of the transplanted organ or cells from being attacked and rejected. Whereas, active control of immune responsiveness to donor antigens after transplantation by regulatory/suppressor T cells will enable tight control of the response directed against the transplant throughout the post-transplant course. After exposure to alloantigen in vivo and in vitro, alloantigen reactive immunoregulatory activity is enriched in a population of CD4+ T cells that express high levels of CD25, the a chain of the interleukin-2 receptor, and the transcription factor Foxp3. In vivo, common mechanisms underpin the activity of CD25+CD4+ Treg in both naive and manipulated adult hosts. We have identified a unique role for IFNg in the functional activity of CD25+CD4+ alloantigen reactive Treg during the development of operational tolerance to donor alloantigens in vivo that is consistent with observations showing that tolerance to alloantigens cannot be induced in the absence of IFNg. The identification and characterisation of Treg that can control immune responsiveness to alloantigens has opened up exciting opportunities for new therapies in transplantation. These observations may have important implications for the design of clinical protocols to induce allograft tolerance in adult recipients. doi:10.1016/j.placenta.2011.07.034 IMMUNOMODULATION BY MESENCHYMAL STEM CELLS AND CLINICAL EXPERIENCE C. Gotherstrom Karolinska Institutet, CLINTEC, Karolinska University Hospital Huddinge, Stockholm, Sweden Finding a suitable cell source is one of the main challenges in tissue engineering. Besides fulfilling the function of the reconstructed tissue, low immunogenicity is advantageous. Promising candidates are multipotent mesenchymal stromal cells, also known as mesenchymal stem cell (MSC), because of their low immunogenicity, differentiation potential and their capacity to be extensively expanded in culture in combination with a low oncogeneic risk in vivo. They differentiate into several mesodermal cell types, such as bone, cartilage and fat, but also into nonconventional mesenchymal lineages such as myogenic, endothelic, hepatic and neurogenic. In immunocompetent allogeneic/xenogeneic animal models, MSC engraft widely and demonstrate site-specific differentiation. MSC also produce important cytokines, growth factors and extra cellular components. In vitro, MSC induce little, if any, proliferation of allogeneic lymphocytes. MSC also inhibit T-cell proliferation to alloantigens and mitogens and prevent the development of cytotoxic T-cells. In vivo, they prolong skin allograft survival and have several immunomodulatory effects. MSC preferentially seem to home to damaged tissue and therefore have therapeutic potential. Stem cells are present at various stages of development, from the inner cell mass through fetal and finally, adult sources. MSC from the adult bone marrow are widely used in therapy today, as for example in the treatment of therapy-resistant severe acute graft-versus-host disease, where they have much potential. The identification of MSC in the human fetus raises the possibility of using a more primitive MSC population for transplantation purposes. Fetal MSC, like adult MSC, escape recognition by the immune system in vitro suggesting that they are not inherently immunogenic. Fetal MSC differ from adult MSC in some aspects, which may be advantageous for transplantation. They demonstrate marked expansive capacity, and cycle faster than adult MSC, having a doubling time of 30 hours over 20 passages (50 population doublings) without expressing a differentiated phenotype. Like their adult counterparts, fetal MSC possess the ability to differentiate into at least 3 different mesenchymal tissues: bone, cartilage, fat. More recent work suggests that they are more primitive than adult MSC, having longer telomeres and expressing embryonic pluripotency markers such as nanog and Oct4, differentiating more readily into bone and outside conventional lineage boundaries into skeletal muscle, and oligodendrocytes. Finally, it has been shown that there is an engraftment advantage of fetal liver compared to adult bone marrow cells in fetal recipients. In utero transplantation of human fetal MSC has shown promising results in mouse models of osteogenesis imperfect and Duchenne muscular dystrophy. This characteristic of MSC is the basis for their extensive potential for tissue repair and in other indications like therapy-resistant severe acute graftversus-host disease, treatment of rejection of organ allografts and in autoimmune disorders. doi:10.1016/j.placenta.2011.07.035 REGULATORY T CELLS IN PREGNANCY A.G. Betz Medical Research Council, Cambridge, UK Regulatory T cells play a crucial role in maternal fetal tolerance. Immediately after conception, their number in the blood, spleen and uterusdraining lymph nodes substantially increases. Whilst this initial expansion is independent of exposure to paternal alloantigen, their accumulation in the gravid uterus appears to be constrained to alloantigen specific regulatory T cells. Clearly, the role of this shift in the composition

Confined chorionic mosaicism, a discordance in the karyotype between the fetus and placenta, occurs in 1% of chorionic villus sampling (CVS) cases. While the cytogenetic discrepancies occurring between different fetal tissues may pose clinical dilemmas, they can also be viewed as a natural experiment to determine early cell lineage relationship in the human. We reviewed extensive data in experimental animals to define the origin of the human extraembryonic mesoderm. The extraembryonic mesoderm in humans is important component of the CVS culture preparation. Previously, the extraembryonic mesoderm was thought to originate in the cytotrophoblast or primitive streak. More recent evidence supports its origin from the yolk sac, which does not always correlate with the fetal karyotype. We formulated a model of early human cell lineage and employed it to clarify clinical cases of chorionic mosaicism in two large published studies. © 1993 Wiley-Liss, Inc.

One of President Bill Clinton's early decisions was to lift the ban on federally supported research on human fetal tissue. Under what circumstances can research on this material be justified?

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.

Over the past decade, cff nucleic acid detection has been increasingly used to diagnose genetic diseases and identify pregnancy complications, mainly in the research setting. Such advances have already provided valuable insights into the developing fetus and placenta. To date, two applications, gender determination and fetal RhD status, have already translated to clinical care. However, with evolving molecular technologies, additional clinical applications will be forthcoming. This review summarizes the characteristics of cell-free DNA and mRNA in maternal plasma and serum and the clinical application of them.

To estimate whether maternal thyroid hypofunction is associated with complications.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.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).Maternal thyroid hypofunction is not associated with a consistent pattern of adverse outcomes.II.

Maternal venous blood samples, obtained from six pregnant women, were used as a source of fetal nucleated erythrocytes (NRBC). Fetal cell enrichment was potentiated by flow sorting with the monoclonal antibodies TfR, Leu-4, and Leu-M3. Single copy Y chromosomal DNA sequences were detected in samples obtained from two women at 11 and 12 weeks' gestation. Y DNA sequences were absent in a subsequent sample from one of these women at 19 weeks and in two other women at 16 and 20 weeks. All four women delivered males. Y DNA sequences were not detected in two women who delivered females. By combining these results with prior data on the detection of Y chromosomal DNA sequences in maternal blood from male-bearing pregnancies, a relationship between gestational age and feto-maternal transfer of NRBC is suggested.

Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. We used monoclonal antibody against the transferrin receptor (TfR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 12 1/2-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations; 0.1-1 ng of fetal DNA was obtained in the 20-ml maternal samples. In 7/19 cases, a 222-bp band of amplified DNA was detected, consistent with the presence of male DNA in the isolated cells; 6/7 of these were confirmed as male pregnancies by karyotyping amniocytes. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, we were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further refinement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.

In this issue of the Journal,Lo et al. (1998) describe their experiences with the novel application of a real-time quantitative PCR assay designed to measure the concentration of fetal DNA in maternal plasma and serum samples. Their system, which uses a 5′ nuclease and fluorescent primers, permits continuous optical monitoring of the progress of an amplification reaction, and it is sensitive enough to detect the DNA in the equivalent of 1 male fetal cell against a background of 12,800 female-genome equivalents.

ContextNo molecular biological tests are available to monitor the ongoing development of human fetuses in vivo.ObjectiveTo determine whether cell-free fetal messenger RNA (mRNA) in amniotic fluid can be detected using oligonucleotide microarrays to study large-scale gene expression in living human fetuses, with analysis of sex, gestational age, and fetal pathology as variables.Design, Setting, and PatientsFour samples of cell-free amniotic fluid were analyzed from pregnant women between 20 and 32 weeks’ gestation and undergoing amnioreduction for polyhydramnios associated with twin-twin transfusion syndrome or hydrops fetalis (cases). The control consisted of 6 pooled amniotic fluid samples from women at 17 weeks’ gestation and undergoing genetic amniocentesis. After extraction from the normally discarded fraction of amniotic fluid, RNA was amplified twice, labeled, and analyzed using gene expression microarrays.Main Outcome MeasureRelative mRNA expression in cell-free samples of amniotic fluid from fetuses with polyhydramnios at different gestational ages vs cell-free amniotic fluid from a pooled control.ResultsThirty-six percent of 22 283 probe sets represented on the arrays were present in the cell-free amniotic fluid, and a median of 20% of all probe sets differed between cases and the pooled control. Only male samples expressed 1 Y chromosome transcript. The expression of some developmental transcripts, such as surfactant proteins, mucins, and keratins, changed with gestational age by up to 64-fold. A water transporter gene transcript was increased up to 18-fold in both twin-twin transfusion samples. Placental gene transcripts were not present in any samples.ConclusionsThis pilot study demonstrates that cell-free fetal mRNA can be extracted from amniotic fluid and successfully hybridized to gene expression microarrays. Preliminary analysis suggests that gene expression changes can be detected in fetuses of different sexes, gestational age, and disease status. Cell-free mRNA in amniotic fluid appears to originate from the fetus and not the placenta.

SEQUENCE ANALYSIS FOR THE GENDER-INDEPENDENT ASSESSMENT OF FETAL CELLS IN MATERNAL BLOOD DONGHYUN CHA, KIARASH KHOSROTEHRANI, DIANA BIANCHI, KIRBY JOHNSON, Pochon University, Obstetrics and Gynecology, Seoul, South Korea, Tufts University, Pediatrics, Boston, Massachusetts, Tufts University, Pediatrics, Obstetrics & Gynecology, Boston, Massachusetts OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (fNRBCs) 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 fNRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. STUDY DESIGN: fNRBCs 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 (4 cases), 18 (1 case), 13 (2 cases), and other genetic abnormalities (2 cases). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR 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 based on morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSION: 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. Women and Infants’ Hospital, Maternal Fetal Medicine, Providence, Rhode Island, University of North Carolina, Department of Obstetrics and Gynecology, Chapel Hill, North Carolina OBJECTIVE: Vaginal bleeding in the first trimester may result in a disruption in the maternal-fetal interface with subsequent transfer of hormones into the maternal circulation. Elevated maternal serum concentrations of AFP have been observed in the first and second trimester in women with a history of first trimester vaginal bleeding. Our goal was to examine the effect of first trimester vaginal bleeding on first trimester serum levels of PAPP-A, free b-hCG, and nuchal translucency (NT) using a prospectively-collected large database. STUDY DESIGN: Women enrolled in the FASTER trial had a NT measurement and PAPP-A and free b-hCG levels drawn at 10 3/7-13 6/7 weeks. Patients with fetal anomalies, a diagnosis of diabetes prior to becoming pregnant or who conceived with invitro fertilization were excluded from this analysis. Patients were divided into three groups: (1) no bleeding, (2) light bleeding, or (3) heavy bleeding. The log transformed multiples of the medians (MoMs) of NT, PAPPA, and free b-hCG for the three groups were compared using analysis of variance for crude effects and analysis of covariance adjusting for possible confounding variables. For presentation, the results were back transformed to medians in MoMs. RESULTS: The study included 34,548 patients: 29,639 patients without bleeding, 4341 patients with light bleeding, and 547 patients with heavy bleeding. Adjusted medians of the MoMs of the markers are listed in the table. CONCLUSION: First trimester vaginal bleeding does not appear to affect PAPP-A or free b-hCG levels nor does bleeding impact NT measurement in this large study.

ContextDuring pregnancy, fetal CD34+ cells enter the maternal circulation, persist for decades, and create a state of physiologic microchimerism. Many studies have confirmed the residual presence of fetal cells in maternal blood and tissues following pregnancy. Fetal cells may respond to maternal injury by developing multilineage capacity in maternal organs.ObjectiveTo verify that fetal microchimeric cells express markers of epithelial, leukocyte, and hepatocyte differentiation within maternal organs.Design, Setting, and PatientsArchived paraffin-embedded tissue section specimens from 10 women who had male offspring and were previously found to have high numbers of microchimeric cells, and 11 control women who had no prior male pregnancies. Male cells were identified by fluorescence in situ hybridization, using X and Y chromosome–specific probes, followed by histologic and immunochemical studies using anticytokeratin (AE1/AE3) as a marker of epithelial cells, anti-CD45 as a leukocyte marker, and heppar-1 as a hepatocyte marker.Main Outcome MeasurePercentage of microchimeric cells expressing nonhematopoietic markers.ResultsA total of 701 male (XY+) microchimeric cells were identified (mean [SD], 227 [128] XY+ cells per million maternal cells). In maternal epithelial tissues (thyroid, cervix, intestine, and gallbladder), 14% to 60% of XY+ cells expressed cytokeratin. Conversely, in hematopoietic tissues, such as lymph nodes and spleen, 90% of XY+ cells expressed CD45. In 1 liver sample, 4% of XY+ cells expressed heppar-1. Histologic and immunochemical evidence of differentiation, as assessed by independent observers, was highly concordant (κ = 0.72).ConclusionThe detection of microchimeric male cells, bearing epithelial, leukocyte, or hepatocyte markers, in a variety of maternal tissue specimens suggests the presence of fetal cells that may have multilineage capacity.