Detection of maternal DNA in umbilical cord plasma by fluorescent PCR amplification of short tandem repeat sequences.

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.

Fetal DNA in maternal plasma: emerging clinical applications.

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.

Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault.

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.

First trimester prenatal diagnosis: fetal cells in the maternal circulation.

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.

Preterm twin gestation and cystic periventricular leucomalacia.

OBJECTIVE: To identify risk factors for the development of cystic periventricular leucomalacia (PVL) in twin gestation. DESIGN: Retrospective case-control study. SETTING: Tertiary care university hospital, Department of Paediatrics, Division of Neonatology, Graz, Austria. PATIENTS: Preterm twin gestations with one sibling having developed cystic PVL, diagnosed by ultrasound scans, compared with their co-twins without PVL, in hospital between 1988 and 2000. MAIN OUTCOME MEASURES: Perinatal and postnatal risk factors for the development of PVL. RESULTS: Eighteen preterm twin gestations were included. Monochorionicity was evident in 47% of the pregnancies, and twin to twin transfusion syndrome occurred in two cases (11%). Fetal distress correlated inversely with PVL (15% v 53%, p = 0.019, relative risk (RR) = 2.057, 95% confidence interval (CI) = 1.067 to 3.968). Hypocarbia with Pco(2) levels below 30 mm Hg (4 kPa) was diagnosed in 29% of the cases compared with 6% of the controls (p = 0.038, RR = 1.944, 95% CI = 1.113 to 3.396). There were no significant differences between groups with regard to premature rupture of the membranes, early onset infection, respiratory distress syndrome, mechanical ventilation, arterial hypotension, persistent ductus arteriosus, and hyperbilirubinaemia. Asphyxia was only evident in three controls. Three infants died and another three were lost to follow up. None of the cases compared with 62% of the controls were diagnosed as having developed normally (p < 0.001), and 14 cases (82%) compared with two controls (15%) developed cerebral palsy (p < 0.001). CONCLUSION: Hypocarbia was the only risk factor strongly associated with cystic PVL. The general outcome of the infants was poor.

Diagnosis of chromosomal aneuploidies using quantitative fluorescent PCR.

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.

Characterisation of a 19-year-old "long-term survivor" with Edwards syndrome.

Trisomy 18 is the second most frequent autosomal aneuploidy affecting about 1 in 8,000 new-borns. Similar to trisomy 13 more than 90% of the patients die within the first year. Main causes of death are failure of vital organ function, in most cases of brain, heart, kidney, and gut, sometimes combined with severe infections. The degree to which essential organs are affected at birth and the clinical course differ considerably. Unknown genetic factors and various environmental effects are most likely involved. A less severe course of Edwards syndrome can be caused by a partial trisomy due to a deletion of the extra chromosome 18 or somatic mosaicism with a trisomic and a normal cell-line in the patient. In this report conventional chromosome analysis, FISH, and QF-PCR have been performed on a 19-year-old female patient with trisomy 18 to investigate a large number of cells including non-mitotic cells from various different tissues. This study supports evidence for an apparently pure form of trisomy 18 in this "long-living" patient with Edwards syndrome.

[Value of ultrasound in early detection of endometrial carcinoma]. / Stellenwert der Sonographie bei der Früherkennung des Endometriumkarzinoms.

OBJECTIVE: The purpose of our study was to examine the diagnostic value of the biometry of the endometrium for the early detection of endometrial carcinomas in postmenopausal women as well as to compare our results with those of other authors. METHODS: In 69 post menopausal women the endometrial thickness measured by vaginosonography before diagnostic curettage or hysterectomy was compared with the histological results. RESULTS: Using a cutoff value of 6 mm, a sensitivity of 96%, a specificity of 24%, a positive predictive value of 16%, and a negative predictive value of 97% were established. CONCLUSIONS: Endovaginal ultrasound evaluation of the endometrial thickness is not specific enough to be used screening method for the early detection of endometrial carcinoma.

[The triple test scenario for Styria. With data of the Styria Abnormalities Register]. / Triple-Test-Szenario für die Steiermark. Mit Daten des Steirischen Fehlbildungsregisters.

OBJECTIVE: The aim of the study was to clarify by a cost-effectiveness analysis, if a triple-marker screening for trisomy 21 (triple test) should be established in Austria. METHODS: The published triple-test results of the last years were combined with the data of the Styrian Malformation Register covering the years 1985-1992. The cost-effectiveness analysis was based on total costs of prenatal diagnosis, costs per fetus diagnosed as affected, the number of affected fetuses detected, and the number of procedure-related losses. RESULTS: If low costs are given priority, the triple test should be offered to women 35 years of age or older. If a high detection rate is given top priority, the test should be offered to all pregnant women. CONCLUSION: The results suggest that the present policy of maternal age screening in Austria should be replaced by maternal serum screening.

[The pregnant patient in dental care. Survey results and therapeutic guidelines]. / Die schwangere Patientin in zahnärztlicher Behandlung. Umfrageergebnisse und therapeutische Richtlinien.

In a telephone survey using a standardized questionnaire, 78 resident dentists in Germany, Switzerland and Austria were interviewed with respect to several aspects of the dental treatment of pregnant women. Only 58% of the interviewees decided clearly in favour of local anaesthetics, 59% supported the use of analgesics, 70% a possible antibiotic therapy and 33% a radiological examination during pregnancy. In addition, according to references in the specialist literature guidelines for the dental treatment, drug therapy and radiological diagnosis of pregnant women are presented. The local anaesthetics should have a high plasma protein bonding (articain, bupivacain, etidocain) and a minimum adrenaline concentration. Paracetamol is the analgesic of choice. If an antibiotic treatment is required, penicillin, cephalosporin and erythromycin are recommended. In particular during the first three-month period, radiological examinations should be restricted to the absolute minimum and performed only if no reasonable alternative is available, even though the radiological burden on the foetus falls 500,000 times short of the limit value of 50 mgray (5 rad) in the case of a microradiogram, and 50,000 times short of the limit value in the case of an orthopantomogram.

Genetic variations in fetal and maternal tumor necrosis factor-α and interleukin 10: is there an association with preterm birth or periventricular leucomalacia?

OBJECTIVE: The aim of the study was to identify whether tumor necrosis factor-α (TNF-α) (-308) and interleukin (IL)-10 (-1082; -819) genotypes were associated with preterm delivery and cystic periventricular leucomalacia (PVL). STUDY DESIGN: Venous blood, buccal swabs or cord blood were collected from mother/child pairs with infants born at term (200) or preterm (106) in the presence and absence of neonatal PVL and of premature infants with PVL (7). Extracted genomic DNA served as template for determination of IL-10 (-1082), IL-10 (-819) and TNF-α (-308) genotypes by allele-specific PCR. RESULT: No significant difference was observed in the frequencies of IL-10 (-1082), IL-10 (-819) and TNF-α (-308) genotypes in mothers or in children of term versus preterm deliveries with or without PVL. CONCLUSION: Maternal and infant IL-10 (-1082, -819) and TNF-α (-308) genotypes are not indicative for an increased risk of preterm birth or the development of PVL in premature newborns.

SRY-specific cell free fetal DNA in maternal plasma in twin pregnancies throughout gestation.

OBJECTIVES: Levels of SRY-specific cell free fetal DNA (SRY-cffDNA) in maternal plasma were investigated in twin pregnancies with two male fetuses versus one male and one female fetus and singleton male pregnancies during second and third trimester. The aim was to evaluate at which gestational age the amount of SRY-cffDNA reflects the number of fetuses and placentas respectively. METHODS: 251 venous blood samples were analyzed from a total of 178 women with male or mixed-gender twin pregnancies and male singleton pregnancies in the second and the third trimester. The concentration of SRY-cffDNA was determined by quantitative real time PCR using the Y-chromosome specific SRY assay. For statistical analysis these three groups were divided into four subgroups according to their gestational age. RESULTS: During second trimester levels of SRY-cffDNA showed no differences between twin and singleton pregnancies. After 28 weeks SRY-cffDNA of male twin pregnancies was significantly increased compared to singleton male pregnancies and mixed-gender twin pregnancies with no differences between the latter two. CONCLUSION: The level of SRY-cffDNA in maternal serum of twin pregnancies reflects the number of fetuses only during the third trimester. Hence its use as a diagnostic tool for complications related to altered SRY-cffDNA levels in twin pregnancies should be evaluated at different weeks of gestation, especially during the second trimester.

Maternal interleukin-6 (-174) C/C polymorphism is associated with chorioamnionitis and cystic periventricular leucomalacia of the preterm infant.

OBJECTIVE: To evaluate the association between maternal interleukin (IL)-6 G(-174)C polymorphism and cystic periventricular leukomalacia (cPVL) of the preterm newborn. STUDY DESIGN: After searching a local database, we recruited 132 preterm infants with diagnosis of cPVL, 44 Caucasian mothers were also recruited to participate in this candidate gene-association study at a single teritary care center. Data related to maternal IL-6 G(-174)C polymorphisms were compared with 41 controls, and furthermore compared with data from umbilical cord blood samples from a consecutive birth cohort of 395 healthy newborns, and published data from Caucasian populations including 1104 adults, respectively. In addition, subgroup analysis was performed in cases with either history of preterm premature rupture of the membranes (PPROM) or clinical chorioamnionitis (CCA). IL-6 genotyping was performed using an allele-specific polymerase chain reaction technique. RESULT: Frequencies of the IL-6 G(-174)C polymorphisms did not differ between cases (GG, 29.5%; GC, 54.5% and CC, 15.9%) and controls (GG, 34.2; GC, 51.2 and CC, 14.6%). Subgroup analysis of 31 cases with history of PPROM (GG, 25.8; GC, 54.8 and CC 19.4%) and controls did not reveal significant differences, but a significantly higher frequency of the CC genotype was found in 23 cases with a history of CCA (34.8%) compared with controls by either univariate (P=0.032; odds ratio 3.11, 95% confidence interval (CI) 1.11 to 8.68) or multivariate analysis (P=0.049, odds ratio 2.54, 95% CI 1.01 to 6.45). These data were confirmed by a comparing the CC genotype frequency to 395 term controls (CC 14.7%, P=0.005) and to the mean CC genotype frequency of 1104 Caucasian adults (CC 15.6%, P<0.0001). CONCLUSION: Frequencies of the IL-6 G(-174)C polymorphisms did not differ between groups. Subgroup analysis revealed an association of the CC genotype with CCA and cPVL in the preterm newborn.

Role of microchimerism in the pathogenesis of oral lichen planus.

OBJECTIVE: Microchimerism of persistent fetal cells has been implicated in some cell-mediated autoimmune diseases. This study examines the hypothesis that fetal microchimerism plays a role in the pathogenesis of lichen planus (LP) affecting the oral cavity. STUDY DESIGN: Mucosal biopsies of 12 women with oral LP (OLP) were tested for the presence of both male cells and male DNA originating from prior pregnancies or prior blood transfusions. Six male patients with OLP served as a control group. Biopsies were analyzed for the presence of Y-chromosome-positive cells by fluorescence in situ hybridization (FISH) with X- and Y-specific DNA probes. To confirm the FISH findings, we used fluorescent polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from mucosal lesions. RESULTS: Using FISH technology, all the 18 biopsy samples showed good hybridization results. In females, Y-chromosome-specific signals were not detected by FISH at any site of the lesions. PCR amplification demonstrated a single peak at the locus specific for the X-chromosome. CONCLUSION: Male DNA microchimerism was not present in any of the investigated lesions, suggesting that microchimerism because of persisting male fetal cells is unlikely to play a major role in the pathogenesis of OLP.

Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction.

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.

Rapid detection of selected aneuploidies by quantitative fluorescent PCR.

Selected aneuploidies can be rapidly diagnosed by the analysis of fluorescent polymerase chain reaction (PCR) products of chromosome-specific and highly polymorphic small tandem repeats (STRs). The quantitative STR patterns obtained from samples of normal individuals are markedly different from those seen when patients with aneuploidies involving chromosome X, or trisomies of chromosomes 21 and 18, are tested. For example, while samples from normal subjects--tested with a chromosome 21-derived STR (D21S11)--show two fluorescent PCR peaks with similar activities in a 1:1 ratio, the analysis of samples from patients with trisomy 21 reveals the presence of either three peaks (ratio 1:1:1), or two peaks with a ratio of 2:1. The use of an internal non-polymorphic marker allows identification of trisomic samples with three copies of the same allele. This rapid approach (24 hours) is particularly valuable when applied to prenatal diagnosis of chromosomal abnormalities since it reduces the time of anxiety of the parents waiting for the results of the conventional cytogenetic tests, which require several weeks.

[Diagnosis and therapy of fetal thyroid gland dysfunction in primary maternal disease]. / Die Diagnostik und Therapie fetaler Schilddrüsendysfunktion bei maternaler Grunderkrankung.

Fetal and maternal thyroid function are working independently under physiologic conditions. In case of maternal autoimmune hyperthyroidism during pregnancy there is an up to 12% chance for the fetus to develop thyroid dysfunction, mediated by the transplacental passage of maternal immunoglobulins directed against TSH receptors in the fetal thyroid gland. This may lead to intrauterine growth retardation, craniosynostosis, preterm delivery, perinatal death, etc. Sonography and fetal blood sampling provide important information to detect fetuses at risk and allow intrauterine therapy. Furthermore prenatal diagnosis is important in case of maternal antithyroid drug ingestion possibly leading to fetal hypothyroidism and goitre. The cooperation of specialists for internal and fetal medicine is required for the management of maternal thyroid disease in pregnancy.

[Effect of ultrasound examination on fetal version of breech presentation]. / Der Einfluss der Ultraschalluntersuchung bei der äusseren Wendung der Beckenendlage.

External version was attempted in 70 pregnancies with foetuses in breech presentation near term. Version was successful in 50 patients (71%), 40 of whom delivered vaginally (80%). Of the 20 patients in whom version was not successful, only 7 patients (35%) delivered vaginally. 2 patients required immediate Caesarean section after attempted version because of ominous foetal heart rate patterns. We analysed the sonographic parameters associated with successful version. The location of the placenta, amount of amniotic fluid, foetal biometry (estimated foetal weight, cephalic circumference, abdominal diameter, femur length) and extension or flexion of the foetal head were analysed. Only extended legs correlated with the success of the procedure. The results suggest, that ultrasonography is an important prerequisite for successful version of a foetus in breech presentation near term.