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ZusammenfassungDie Präeklampsie (PE) ist eine Systemerkrankung der Schwangerschaft und Teil des Spektrums der plazentaassoziierten Schwangerschaftserkrankungen. Sie ist durch einen neu aufgetretenen Bluthochdruck und eine weitere Organmanifestation, wie z. B. Proteinurie oder andere, bzw. pathologisch erhöhte PE-spezifische Markersysteme definiert. Entsprechend dem Manifestationszeitpunkt werden frühe und späte Formen unterschieden, welchen auch eine unterschiedliche Pathogenese zugrunde liegt. Insbesondere die frühen Formen können mit schweren Verläufen und Frühgeburtlichkeit einhergehen und sind, über die unmittelbaren peripartalen Komplikationen hinaus, auch mit einer erheblichen Langzeitmorbidität für Mutter und Kind assoziiert. Der PE-Screening-Test, der im ersten Trimenon durchgeführt wird, berechnet die Wahrscheinlichkeit für das Auftreten einer PE und wird aus dem A‑priori-Risiko aus mütterlichen Anamnesedaten sowie aus biophysikalischen (mittlerer arterieller Druck und Farbdoppler der Arteriae uterinae) und biochemischen Parametern („pregnancy-associated plasma protein A“, PAPP‑A, und „placental growth factor“, PLGF) errechnet. Diese Screeningmethode wurde für verschiedene Populationen validiert und von der International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) als effektivstes Instrument zur Identifikation von Risikopatientinnen anerkannt. Niedrig dosiertes Aspirin, d. h. 75–150 mg einmal täglich zur abendlichen Einnahme spätestens ab der 16. SSW, ist derzeit als einzig effektive Maßnahme zur Prävention der PE etabliert und bewirkt eine signifikante Risikoreduktion. Das PE-Screening ermöglicht nicht nur, Risikopatientinnen sehr früh in der Schwangerschaft (vor dem Auftreten klinischer Zeichen) zu identifizieren, sondern auch, durch die Gabe niedrig dosierten Aspirins eine nachweislich risikosenkende, prophylaktische Maßnahme einzuleiten.

Fetal neurosonography and the assessment of the posterior fossa have gained in importance during the last 2 decades primarily due to the development of high-resolution ultrasound probes and the introduction of 3 D sonography. The anatomical development of the posterior fossa can be visualized well with the newest ultrasound technologies. This allows better knowledge of the anatomical structures and helps with understanding of the development of malformations of the posterior fossa. In this article the longitudinal development of the posterior fossa structures will be reviewed. The embryologic description will be compared with ultrasound descriptions. These embryologic and anatomic illustrations form the basis for the screening and diagnosis of malformations of the posterior fossa. During the first trimester, screening for open spina bifida as well as cystic malformations of the posterior fossa is possible. In the second and third trimester, malformations of the posterior fossa can be subdivided into 3 groups: fluid accumulation in the posterior fossa (Dandy-Walker malformation, Blake’s pouch cyst, mega cisterna magna, arachnoid cyst, vermian hypoplasia), decreased cerebellar biometrics (volume) (cerebellar hypoplasia, pontocerebellar hypoplasia) and suspicious cerebellar anatomy (Arnold-Chiari malformation, rhombencephalosynapsis, Joubert syndrome). This algorithm, in combination with knowledge of normal development, facilitates the diagnostic workup of malformations of the posterior fossa. Die fetale Neurosonografie und damit auch die sonografische Beurteilung der Strukturen der Fossa posterior haben in den letzten beiden Jahrzehnten durch den Einsatz hochauflosender Ultraschallsonden und des 3D-Ultraschalls eine besondere Bedeutung erlangt. Durch die neuen Ultraschalltechnologien kann die embryologische Entwicklung der Fossa-posterior-Strukturen nachvollzogen werden. Dies erleichtert die Kenntnis der anatomischen Strukturen und tragt zum Verstandnis fur die Entstehung von Fehlbildungen der Fossa posterior bei. In der folgenden Ubersichtsarbeit wird die longitudinale Entwicklung der Fossa-posterior-Strukturen dargestellt. Dabei wird die embryologische Beschreibung mit der Darstellung im Ultraschall verglichen. Dieses embryologische und anatomische Wissen bildet die Grundlage fur das Screening und die Diagnose von Veranderungen und Fehlbildungen der Fossa posterior. Im Ersttrimester ist ein Screening fur Spina bifida aperta und zystische Fehlbildungen der Fossa posterior moglich. Im 2. und 3. Trimester konnen die Fehlbildungen der Fossa posterior in 3 Kategorien unterteilt werden: Fehlbildungen mit vermehrter Flussigkeit in der Fossa posterior (Dandy-Walker-Malformation, Blakes-pouch-Zyste, Megacisterna magna, Arachnoidalzyste, Vermishypoplasie), Fehlbildungen mit verminderter Biometrie bzw. vermindertem Volumen bei „uberwiegend“ normaler Anatomie des Zerebellums (zerebellare Hypoplasie, pontozerebellare Hypoplasie) und Fehlbildungen mit strukturell verandertem Zerebellum (Arnold-Chiari-Malformation, Rhombenzephalosynapsis, Joubert-Syndrom). Dieser Algorithmus und das Verstandnis der normalen Entwicklung ermoglichen ein systematisches Aufarbeiten der Fehlbildungen der Fossa posterior.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype. They are normally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is more suited to a high-throughput diagnostic service. This approach has been investigated in a small number of pilot studies and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype analysis, which subsequently confirms the rapid result and scans for other chromosome abnormalities not detected by the QF-PCR assay.

Wahrend einer gesunden Schwangerschaft andern sich die leberspezifischen Laborparameter (Transaminasen, γ-Glutamyltransferase, Bilirubin, Gallensauren, Cholinesterase, alkalische Phosphatase, Albumin, Prothrombinzeit) nicht. Lediglich die Albuminkonzentration im Serum fallt aufgrund des erhohten Blutvolumens (Normwert in der Schwangerschaft: 2,3–4,2 g/dl; Nadir im 3. Trimenon), und die Konzentration der alkalischen Phosphatase steigt, da mit der Plazenta ein zusatzlicher Produktionsort hinzukommt (Normwert in der Schwangerschaft: 38–229 U/l; Hochstwert im 3. Trimenon). Pathologische Veranderungen der Leberparameter finden sich in ca. 3–5 % aller Schwangerschaften. Zugrundeliegende Ursachen sind schwangerschaftsspezifische bzw. zufallig wahrend der Schwangerschaft erstmals aufgetretene oder vorbestehende Lebererkrankungen. Schwere Verlaufe sind selten, zeigen jedoch eine hohe Morbiditat und Mortalitat fur Mutter und Kind. Als schwangerschaftsspezifisch werden die Hyperemesis gravidarum (HG), die intrahepatische Cholestase (ICP), die akute Fettleber (AFLP) und eine Leberbeteiligung im Rahmen der Praeklampsie (P), Eklampsie (E) oder des HELLP-Syndroms definiert, mit z. T. trimesterspezifisch gehauftem Auftreten ( Tab. 1 ). Frauen mit einer Lebererkrankung in der Schwangerschaft bedurfen einer individuellen, spezialisierten und multidisziplinaren Betreuung in der Schwangerschaft und im Wochenbett.

Objective To identify obstetric risk factors and to elucidate the effect of prolonged rupture of the membranes on the development of cystic periventricular leukomalacia (PVL) in preterm infants. Methods A retrospective case–control study of 95 preterm infants with the diagnosis of PVL and 245 healthy controls matched for gestational age. A total of 52 antenatal, intrapartum and neonatal characteristics were studied by univariate methods and logistic regression. Results Preterm premature rupture of membranes (PPROM) (odds ratio 2.1 [95% CI 1.3–3.4], P =.003), gestational age at PPROM ( P =.025), prolonged rupture of membranes ( P P =.019) and antibiotics (1.9 [1.2–3.1], P =.008) were associated with PVL. The use of tocolytic agents >24 h ( P =.008), prolonged latency between the increase in maternal leukocyte count and birth ( P =.034), spontaneous onset of labor (1.8 [1.0–2.9], P =.026), vaginal delivery (1.7 [1.1–2.8], P =.029) and male gender (1.5 [1.0–2.0], P =.04) were found more frequently in PVL cases. Preeclampsia (0.4 [0.1–0.9], P =.034), hypertension at booking ( P =.009), sonographic IUGR ( P =.020), abnormal blood flow of the umbilical artery ( P =.032) and cesarean section without labor (0.5 [0.3–0.8], P =.006) were found less frequently. In logistic regression analysis, prolonged rupture of the membranes ( P =.748), preeclampsia ( P =.973), the use of antibiotics ( P =.617) and beta-sympathomimetic tocolytic agents ( P =.563) lost statistical significance, whereas birth weight ( P =.036) became significant. Conclusion PPROM and prolonged rupture of the membranes may provoke adverse effects on the neurodevelopmental outcome of the preterm fetus. These findings may have implications on the obstetric management of PPROM beyond 30 weeks of gestation. Cesarean section without labor was less likely associated with the diagnosis of PVL.

Objectives The aim of the study was the detection, quantification and correlation of cell-free fetal (cff) DNA in maternal urine and plasma in normal and complicated pregnancies during the third trimester. Methods One hundred and fifty-one urine and plasma samples obtained from 96 women carrying male fetuses, and 55 carrying female fetuses were collected and analyzed for cff-DNA using fluorescent PCR and quantitative real-time PCR. DNA was extracted from 1 mL maternal urine and analyzed with two different primer sets (SRY and DYS-14). The concentrations of cff and total DNA in maternal plasma were correlated with maternal and obstetric parameters using appropriate correlation analyses. Results Y-chromosome-specific sequences were detected in 31 of 96 (32.3%) urine samples collected from women pregnant with male fetuses using DYS-14 and in 6 of 96 (6.3%) urine samples using SRY as primers using real-time PCR. All 96 plasma samples obtained from women carrying male fetuses were positive for cff-DNA using real-time PCR. Cff-DNA exhibited a correlation with gestational age (R = 0.244; P = 0.018) and an inverse correlation with the latency between blood collection and birth (R = − 0.218; P = 0.036). Total DNA showed a correlation with placental weight (R = 0.182; P = 0.034) and pregnancy-associated complications (R = 0.280; P < 0.001). Conclusion Our data confirm that cff-DNA is cleared by the kidneys in detectable amounts, but due to its low concentration or problematic detection in maternal urine this source seems inappropriate for noninvasive prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.

Objectives The aim of this study was to investigate a possible relationship between fetal cell microchimerism and lichen sclerosus of the vulva. We searched for the presence of male cells and DNA in vulval tissue samples. Methods Paraffin-embedded skin biopsy samples from 15 women affected with vulval lichen sclerosus who gave birth to at least one son were analyzed for the presence of microchimeric male cells using fluorescence in situ hybridization (FISH) and fluorescent PCR. We included three lichen sclerosus samples originating from women without male offspring, six vulval specimens without pathological finding originating from autopsies and seven male gingival specimens as controls. Results Nucleated cells containing Y-chromosome specific sequences were neither detected at any site of the lesions nor in normal vulval specimens by using FISH. These results were confirmed by the use of PCR amplification demonstrating only DNA sequences specific for the X chromosome. No female microchimerism was detected in the male gingival samples. Conclusion Despite the limited number and size of the samples, we conclude that persistent male fetal cells are not involved in the pathogenesis of lichen sclerosus of the vulva, since we consistently could not detect Y-chromosome specific sequences by using two molecular techniques. Copyright © 2006 John Wiley & Sons, Ltd.

Molecular techniques have been developed for prenatal diagnosis of the most common chromosome disorders (trisomies 21, 13, 18 and sex chromosome aneuploidies) where results are available within a day or two. This involves fluorescence in situ hybridization (FISH) and microscopy analysis of fetal cells or quantitative fluorescence polymerase chain reaction (QF-PCR) on fetal DNA. Guidance is provided on the technological pitfalls in setting up and running these methods. Both methods are reliable, and the risk for misdiagnosis is low, although slightly higher for FISH. FISH is also more labour intensive than QF-PCR, the latter lending itself more easily to automation. These tests have been used as a preamble to full chromosome analysis by microscopy. However, there is a trend to apply the tests as 'stand-alone' tests for women who are at relatively low risk of having a baby with a chromosome disorder, in particular that associated with advanced age or results of maternal serum screening programmes. These women comprise the majority of those currently offered prenatal diagnosis with respect to fetal chromosome disorders and if introduced on a larger scale, the use of FISH and QF-PCR would lead to substantial economical savings. The implication, on the other hand, is that around one in 500 to one in 1000 cases with a mentally and/or physically disabling chromosome disorder would remain undiagnosed.

Objective The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. Methods CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbγ-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbγ and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. Results In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. Conclusion On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis. Copyright © 2003 John Wiley & Sons, Ltd.

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

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

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

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

Chromosome-specific DNA small tandem repeats (STRs), amplified by quantitative fluorescent polymerase chain reactions, have been successfully employed for the rapid prenatal detection of human major numeric aneuploidies. These assays – which can be performed in a few hours – can be used either as preliminary investigations aimed at relieving the anxiety of parents waiting for the results of conventional cytogenetic analysis or as prenatal diagnostic tests on samples retrieved from mothers at high risk of having an affected fetus according to previous biochemical or ultrasound tests. Here we discuss the possibility of performing prenatal diagnosis with this method as the only approach to detect numerical disorders affecting chromosomes 21,18,13,X and Y in populations where the use of conventional cytogenetic tests is hampered by technical and economical difficulties. The advantages of the quantitative fluorescent tests are that they are accurate, relatively economic and that a single operator can perform up to 40–50 prenatal diagnoses per day. Furthermore, chromosome-specific STRs, as well as primers for the detection of single gene defects, can be included in a multiplex assay. The tests can be carried out also on a very small number of amniotic or chorionic cells.

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.

Background: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC): class I, CD45 and cytokeratin expression. Methods: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. Results: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. Conclusions: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape. Cytometry 36:319–323, 1999. © 1999 Wiley-Liss, Inc.

Recently, maternal DNA was detected in umbilical cord blood using PCR amplification of minisatellite sequences. The presence of maternal DNA was demonstrated in 1% to 100% of umbilical cord blood samples. The objective of this study was to determine the frequency of cord blood contamination by maternal genetic material. We used fluorescent PCR amplification of highly polymorphic short tandem repeat (STR) markers to detect maternal DNA in umbilical cord plasma.

Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.

Fragestellung: Ziel unserer Studie was es, die Wertigkeit der sonographischen Messung der Endometriumdicke zur Fruherkennung des Endometriumkarzinoms zu uberprufen und unsere Ergebn

QF-PCR refers to the amplification of chromosome-specific polymorphic microsatellite markers using fluorescence-labelled primers, followed by semi-quantitative analysis of the products on a genetic analyser to determine copy number and/or imbalances of specific chromosomal material. This approach is now widely used for rapid prenatal diagnosis of the common trisomies. In addition, it can successfully detect maternal cell contamination and mosaicism in prenatal material.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype. They are normally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is more suited to a high-throughput diagnostic service. This approach has been investigated in a small number of pilot studies and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype analysis, which subsequently confirms the rapid result and scans for other chromosome abnormalities not detected by the QF-PCR assay.

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

The incidence of chromosomal abnormalities in liveborn infants has been established to be about 1 in 170 newborns (1). The most frequent chromosomal anomalies are aneuploidies involving chromosomes 21,18,13, and both sex chromosomes. Prenatal diagnosis of chromosomal disorders is performed by conventional cytogenetic analysis of fetal cells collected by amniocentesis, chorionic villus sampling, or fetal blood sampling. Cytogenetic techniques allow detection of chromosome aneuploidies with great accuracy. The major disadvantage of these procedures is that fetal cells must be cultured for up to two weeks before analysis. This interval of time places a significant emotional and/or clinical burden on the parents and the physician. Moreover, if the fetus is abnormal and there is a potential need for therapeutic measures, a rapid answer is of great importance. Attempts to perform rapid prenatal diagnostic tests by fluorescent in situ hybridization (FISH) on interphase nuclei of amniocytes are still hampered by technical difficulties 2), Consequently, this approach has not yet entered into the realm of routine diagnostic procedures.

Background Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. Methods CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-γ) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. Results In CB, 59% of events double positive for Hb-γ and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. Conclusions Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible. Cytometry 48:87–92, 2002. © 2002 Wiley-Liss, Inc.

Objective To develop a new method of RhD/d genotype determination using a quantitative fluorescent PCR (QF-PCR) assay. Methods Polymerase chain reaction amplification (PCR) of fragments of exon 7 of both the RHD and RHCE genes was performed from 32 amniotic fluid and 26 chorionic villus samples known to be heterozygous for the RHD gene, 74 peripheral blood samples of RhD-positive blood donors (homozy-gous or heterozygous) estimated by serologic typing and 24 RhD-negative fetal samples. The number of copies of the RHD gene in RhD-positive samples was determined by comparing the fluorescent intensities of the amplification products specific for the RHD and the RHCE genes. Results A ratio of fluorescent intensities of 1:1 clearly indicated D/D homozygous individuals whereas a ratio of 1:2 was demonstrated in samples from D/d heterozygous individuals. The mean fluorescent intensity ratio of the peak areas of homozygous samples was 1.12 (SD 0.128), the mean ratio of the peak areas of heterozygous samples was 0.51 (SD 0.060). Complete agreement was obtained between RhD/d typing by QF-PCR and RhD genotypes assessed by family studies and serological methods. Conclusions The fluorescent PCR-based DNA test allows easy, rapid and accurate determination of the zygosity for the RHD gene. This new technique provides useful information for the clinical management of pregnancies of sensitised RhD-negative mothers.

This is not the first editorial in Clinical Chemistry dedicated to the testing of urine for cell-free DNA. In 2000 Lo(1) highlighted the findings of Botezatu et al.(2), who were the first to describe a transfer of DNA across the kidney barrier into urine. The origins of detected transrenal DNA sequences were carcinomas, blood transfusions, and, in pregnant women, the fetus. Following this first report, some pioneering work was done in transplant medicine. Urinary microchimerism was found in women after receiving kidney transplants from males(3)(4). In a quantitative analysis it was demonstrated that transplant-derived DNA was increased during graft rejection, indicating a potential marker for transplant control(4). Later, attempts were made to detect cell-free fetal DNA in maternal urine. However, maternal urine analysis was faced with technical difficulties, and it was suggested that the concentration of transrenal DNA might be too low for standard PCR protocols. …

We report the results of a prospective study using quantitative fluorescent polymerase chain reaction (QF-PCR) and small tandem repeat markers (STR) for the rapid prenatal detection of aneuploidies in a group of pregnant women at increased risk of having fetuses with numerical chromosome disorders. Amniotic fluid samples (n = 52) were collected from mothers undergoing prenatal invasive testing for fetal abnormalities on ultrasonographic examination or abnormal maternal serum aneuploidy screening results. All samples were tested by cytogenetic analysis, but rapid diagnoses of aneuploidies were offered and performed using QF-PCR analysis with several STRs specific for chromosomes 21, 18, 13 and X. All cases with numerical chromosome aberrations involving chromosomes 21, 18 and 13 (n = 8) were correctly diagnosed. Three gonosomal aneuplodies (one 47,XXY and two 45,X) were not detected because they were uninformative for the X markers. Another sample with a deletion (46,XX,7q-), that the present assay was not designed to detect, was not identified. One sample was heavily contaminated with maternal blood and the results of the QF-PCR assays were uninformative. The remaining samples from normal fetuses provided QF-PCR patterns disomic for chromosomes 21, 18, 13 and X. Our study demonstrates that QF-PCR is a rapid method for the detection of common numerical chromosome disorders and it may play an important role in prenatal diagnosis for women at high risk for fetal aneuploidy.

The recovery of fetal cells from the maternal circulation represents a promising approach to noninvasive prenatal diagnosis. Advances in techniques of sensitive molecular genetic analysis have enabled the conclusive demonstration of the presence of fetal cells in maternal blood. In most pregnancies, there are few fetal cells detectable. In some abnormal pregnancies, there appears to be increased fetomaternal transfusion, which facilitates recognition of aneuploid fetal cells. This review article describes general strategies of fetal cell isolation, current technical challenges, and clinical applications that are envisioned for the future. The increased appreciation of fetal cell microchimerism, and its association with complications of pregnancy and the postpartum development of autoimmune disease, is also discussed.

Background: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility class I, CD45, and cytokeratin expression. Method: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. Experience: The sorting procedure was superior to the preferential lysis method within all tested conditions. In unfavorable dilutions, the male DNA could be identified only after cell sorting with flow cytometry. Conclusion: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there are only microtraces of sperm detectable after rape.

OBJECTIVE: We have developed a quantitative fluorescence multiplex polymerase chain reaction assay for the rapid detection of sex and aneuploidies involving chromosomes 21, 18, and 13. STUDY DESIGN: Samples of deoxyribonucleic acid ( n = 85) extracted from amniotic fluid, fetal tissues, and blood were investigated by multiplex polymerase chain reaction amplification of polymorphic small tandem repeat markers specific for chromosomes 21, 18, 13, and X. RESULTS: Quantitative analysis of the polymerase chain reaction products allowed us to distinguish between normal samples and samples with autosomal trisomies while sexing was performed simultaneously. From 85 samples only three produced unsatisfactory results with one of the two chromosome 13-specific markers. In these three cases the amplification of the other chromosome 13 marker always resulted in a correct normal pattern. CONCLUSION: Quantitative fluorescence multiplex polymerase chain reaction is a reliable and rapid method that allows prenatal diagnosis of the major numeric chromosomal abnormalities to be performed within 24 hours. (Am J Obstet Gynecol 1997;177:899-906.)

We have evaluated a rapid method that allows prenatal detection of Down's syndrome in less than 24 hours. DNA from uncultured amniotic fluid, fetal blood, and tissue samples was amplified with the small tandem repeat (STR) marker D21S11. Quantitative analysis of fluorescent STR products with evaluation of their sizes provided clear evidence for trisomy 21. Whilst most normal samples showed two amplification peaks of equal size, Down's syndrome samples were characterised by either three STR peaks or two peaks with a ratio of 2:1. Co-amplification with a non-polymorphic sequence allowed analysis of samples that were homozygous for the 21-derived STRs.