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.

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.

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.

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.

Diagnosis of trisomy 21 in fetal nucleated erythrocytes from maternal blood by use of short tandem repeat sequences.

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 gamma 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 gamma staining, characteristic morphology, and three chromosome 21 signals, along with maternal leukocytes, defined as gamma 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.

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats.

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.

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin.

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.

[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.

RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach.

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 (homozygous 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.

Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders.

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.

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.

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.

Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of 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 (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.

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples.

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.

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.