Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

Psychological responses of pregnant women to an infectious outbreak: a case-control study of the 2003 SARS outbreak in Hong Kong.

OBJECTIVE: The aim of the present study was to examine the behavioral and psychological responses of pregnant women during the 2003 severe acute respiratory syndrome (SARS) outbreak in Hong Kong. METHODS: Ethnographic interviews were first conducted to identify the common psychological and behavioral responses to the outbreak. This was followed by a case-control study of 235 consecutive pregnant women recruited during the SARS epidemic, and a historical cohort of 939 pregnant women recruited a year before the outbreak. Both cohorts completed standardized rating scales on depression, anxiety, and social support. RESULTS: Women in the SARS cohort adopted behavioral strategies to mitigate their risk of contracting infection. However, pregnant women tended to overestimate the risk of contracting SARS and nearly a third of the women were homebound. The anxiety level of the SARS cohort was slightly higher than that of the pre-SARS control. No statistical difference was found between the depression levels of the two cohorts. CONCLUSION: The improved social support experienced by pregnant women during SARS might have buffered the stress associated with an outbreak. However, clinicians should monitor for overestimation of infectious risk among pregnant women.

Detection of restriction enzyme-digested target DNA by PCR amplification using a stem-loop primer: application to the detection of hypomethylated fetal DNA in maternal plasma.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. METHODS: The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop-extended SERPINB5 molecules was confirmed by genotyping. RESULTS: From the real-time PCR results on maternal plasma, stem-loop-extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples. CONCLUSION: Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.

Hypermethylation of RASSF1A in human and rhesus placentas.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Maternal fear associated with pregnancy and childbirth in Hong Kong Chinese women.

BACKGROUND: Women's fear toward pregnancy and childbirth is a common and important health concern. This study examined the objects, causes, and manifestations of maternal fears and their associated demographic factors in a sample of Hong Kong Chinese pregnant women. METHODS: Three hundred Chinese pregnant women were recruited in an obstetric unit in Hong Kong in 2003. Data were collected using a 73-item questionnaire. Principal components factor analysis was applied to identify the objects, causes, and manifestations of fear toward pregnancy and childbirth. RESULTS: The mean maternal age was 30 (SD 5.6) years. All participants reported some degree of fear. The main objects of fear were "fear of childbirth" and "child's and mother's wellbeing." The first factor identified for causes of fear was "negative stories," followed by "negative attitude or mood." Regarding the various manifestations of fear, "stress symptoms" and "wish to avoid pregnancy and childbirth" ranked highest. Twenty-two percent of participants had considered requesting an elective cesarean section due to fear of childbirth. CONCLUSIONS: Even in a group of low-risk pregnant women, fear toward pregnancy and childbirth was frequently experienced. Better strategies to address women's psychological needs during pregnancy are warranted.

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.

Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. METHODS: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. RESULTS: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. CONCLUSION: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Thalassaemia screening in pregnancy.

PURPOSE OF REVIEW: This review provide an update on antenatal screening and diagnosis of thalassaemia disorders. RECENT FINDINGS: The topics covered are the effectiveness of antenatal screening programmes for thalassaemia, its prenatal diagnosis, molecular basis and laboratory findings, ultrasound screening for haemoglobin Bart's disease, and non-invasive prenatal diagnosis of thalassaemia. SUMMARY: Universal antenatal screening for thalassaemia carriers should be implemented in populations with a high prevalence of this condition. The appropriate measure to screen for alpha and beta thalassaemias remains mean cell haemoglobin (<27 pg) or mean corpuscular volume (<80 fl). A haemoglobin pattern and iron profile should follow if the red cell indices are low. In a population where alpha thalassaemia is prevalent, it is advisable to check the partner's mean cell haemoglobin or mean corpuscular volume as well. Further cascades of investigations will depend on these results and the prevalence of other haemoglobinopathies in that population. Invasive prenatal diagnosis remains the gold standard for diagnosis in high-risk couples. Provided expertise is available, ultrasound measurement of the cardiothoracic ratio appears a good screening tool for alpha thalassaemia major. Non-invasive prenatal diagnosis by identification of a paternal mutation in maternal plasma, although currently at the experimental stage, may be an option in the future.

Detection of the placental epigenetic signature of the maspin gene in maternal plasma.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

Circulating placental RNA in maternal plasma is associated with a preponderance of 5' mRNA fragments: implications for noninvasive prenatal diagnosis and monitoring.

BACKGROUND: The molecular characteristics of placental RNA circulating in maternal plasma are unknown. We investigated the integrity of circulating placental RNA in maternal plasma and tested the relevance of plasma RNA integrity for noninvasive prenatal diagnosis. METHODS: Six different placental transcripts and mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified for the 5' and 3' regions in maternal plasma by 1-step real-time reverse transcription-PCR (RT-PCR) assays. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. The rates of detection by the 5' and 3' assays for the beta-subunit of human chorionic gonadotropin (beta hCG) were assessed in maternal plasma samples collected from different gestational periods. RESULTS: For 5 of the 7 genes, the plasma mRNA concentrations measured by the 5' amplicons were significantly higher than those measured by the corresponding 3' amplicons. Every transcript under study demonstrated a higher rate of detection in the 5' assay than in the 3' assay in maternal plasma. In particular, the detection rate of beta hCG mRNA in maternal plasma was increased throughout gestation when the 5' assay was used. CONCLUSIONS: Circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma. Apart from its intrinsic biological interest, this information could have important implications for the development of new assays targeting fetal RNA markers for noninvasive prenatal diagnosis and monitoring.

Effect of hypoxia on urocortin production in human gestational trophoblasts in vitro.

PROBLEM: Urocortin is produced by the placenta throughout pregnancy but its regulation remains unknown. The effect of hypoxia on placental urocortin production is not known. The aim of this study was to determine the effect of in vitro hypoxia on human trophoblastic urocortin production. METHOD OF STUDY: Placental explants and primary cultures were incubated in anaerobe hypoxic bags for 24 h in a humidified incubator. Urocortin peptide secretion and mRNA (messenger RNA) production was determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Morphological and functional integrity was verified by immunohistochemical analysis of urocortin expression. Vascular endothelial growth factor expression was used to verify the generation of cellular hypoxia in our in vitro system. RESULTS: Hypoxia did not affect urocortin secretion or mRNA expression in explant and single-cell cultures. Production was greater from first trimester than term explants and from single-cell primary cultures more than from explant cultures. CONCLUSIONS: Hypoxia does not influence human placental urocortin secretion or mRNA expression in vitro.

Repeated courses of antenatal corticosteroids: is it justified?

The benefits of a single course of antenatal corticosteroids on neonatal outcomes are well established. There is, however, much controversy about how long this treatment should continue, and whether repeated courses should be administered if the women remain at risk for preterm delivery 7 days after the initial therapy. This review aims to discuss current evidence on the effectiveness and safety of repeated courses of antenatal corticosteroids.

Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma.

BACKGROUND: Fetal DNA has been detected in maternal plasma by the use of genetic differences between mother and fetus. We explore the possibility of using epigenetic markers for the specific detection of fetal DNA in maternal plasma. METHODS: A differentially methylated region in the human IGF2-H19 locus and a single-nucleotide polymorphism in this region were chosen for the study. The methylation status in this region is maintained in such a way that the paternal allele is methylated and the maternal allele is unmethylated. The single-nucleotide polymorphism was typed by direct sequencing of PCR products. The methylation status of this region was ascertained by bisulfite conversion and methylation-specific PCR. Differentially methylated fetal alleles were detected in maternal plasma by direct sequencing and a primer-extension assay. RESULTS: Women in the second (n = 21; 17-21 weeks) and third (n = 18; 37-42 weeks) trimesters of pregnancy were recruited. Among these 39 volunteers, the 16 who were heterozygous for the single-nucleotide polymorphism were chosen for further analysis. In 11 of these 16 cases, paternally inherited methylated fetal alleles were different from the methylated alleles of the respective mothers. Using direct sequencing, we detected paternally inherited methylated fetal DNA in 6 of 11 (55%) cases. In 8 of the 16 heterozygous cases, the fetuses possessed an unmethylated maternally inherited allele that was different from the unmethylated allele of the mother. Using a primer-extension assay, we detected fetal-derived maternally inherited alleles in maternal plasma of four of eight (50%) cases. CONCLUSIONS: These results represent the first use of fetal epigenetic markers in noninvasive prenatal analysis. These data may also have implications for the investigation of other types of chimerism.

The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia.

BACKGROUND: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia. METHODS: We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay. RESULTS: CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann-Whitney test, P <0.001). CONCLUSION: Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.

Prenatal exclusion of beta thalassaemia major by examination of maternal plasma.

The discovery of the presence of fetal DNA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of beta thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (-CTTT) mutation in the beta globin gene from maternal plasma by real-time PCR. The specificity and sensitivity of the allele-specific assay was confirmed by subjecting plasma, buffy coat, and amniotic fluid samples from 100 pregnancies to screening for the mutation. Subsequently, the assay was applied to the prenatal testing of eight fetuses at risk of beta thalassaemia major, the aim being to exclude fetal inheritance of paternally transmitted codon 41/42 mutation. The fetal genotype was completely concordant with conventional analysis and beta thalassaemia major was excluded in two of the pregnancies non-invasively.

Fetal DNA clearance from maternal plasma is impaired in preeclampsia.

BACKGROUND: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesized that impaired clearance of fetal DNA might contribute, at least in part, to the above-mentioned phenomenon. METHODS: We studied 7 preeclamptic and 10 control pregnant women. All had male fetuses. Serial blood samples were obtained from before delivery to 6 h postpartum. Male fetal DNA in maternal plasma was measured by real-time quantitative PCR for the SRY gene on the Y chromosome. RESULTS: The median fetal DNA concentrations before delivery were significantly higher in the preeclamptic women than in the controls (521 vs 227 genome-equivalents/mL for preeclamptic and control women, respectively; Mann-Whitney rank-sum test, P = 0.017). The median fetal DNA concentrations at 6 h after delivery were also significantly different between the two groups (208 vs 0 genome-equivalents/mL for preeclamptic and control women, respectively; Mann-Whitney rank-sum test, P = 0.002). A first-order clearance model was found to best describe the kinetics of maternal plasma fetal DNA clearance. Moreover, we observed a significant difference in the median apparent clearance half-lives of fetal DNA between the preeclamptic women (114 min) and controls (28 min; Mann-Whitney rank-sum test, P = 0.007). CONCLUSIONS: This study represents the first documentation of impaired fetal DNA clearance from maternal plasma in preeclampsia. Such an abnormality in circulating DNA clearance may also be present in other medical conditions associated with quantitative aberrations in circulating DNA concentrations.

mRNA of placental origin is readily detectable in maternal plasma.

The discovery of circulating fetal nucleic acid in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Thus far, a gender- and polymorphism-independent fetal-specific target that can be used for prenatal screening and monitoring in all pregnant women has not been reported. In addition, the origin of such circulating nucleic acid has remained unclear. Here we provide direct evidence that the placenta is an important source of fetal nucleic acid release into maternal plasma by demonstrating that mRNA transcripts from placenta-expressed genes are readily detectable in maternal plasma. The surprising stability of such placental mRNA species in maternal plasma and their rapid clearance after delivery demonstrate that such circulating mRNA molecules are practical markers for clinical use. The measurement of such plasma mRNA markers has provided a gender-independent approach for noninvasive prenatal gene expression profiling and has opened up numerous research and diagnostic possibilities.

Size distributions of maternal and fetal DNA in maternal plasma.

BACKGROUND: The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear. METHODS: We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene. RESULTS: The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann-Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma. CONCLUSION: Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.

Pregnancy and perinatal outcomes of women with severe acute respiratory syndrome.

OBJECTIVE: This study was undertaken to evaluate the pregnancy and perinatal outcomes of pregnant women with severe acute respiratory syndrome (SARS). STUDY DESIGN: All pregnant women (12) who presented with SARS in Hong Kong between February 1 and July 31, 2003, were included. The pregnancy and perinatal outcomes were collected. Evidence of perinatal transmission of virus was assessed with the SARS-associated coronavirus reverse-transcriptase polymerase chain reaction on cord blood, placenta tissue, and subsequent follow-up of the neonate on serology. RESULTS: Three deaths occurred among the 12 patients, giving a case fatality rate of 25%. Four of the 7 patients (57%) who presented in the first trimester had spontaneous miscarriage. Four of the 5 patients who presented after 24 weeks were delivered preterm. Two mothers recovered without delivery, but their ongoing pregnancies were complicated by intrauterine growth restriction. No newborn infant had clinical SARS and all investigations were negative for SARS. CONCLUSION: SARS during pregnancy is associated with high incidences of spontaneous miscarriage, preterm delivery, and intrauterine growth restriction. There is no evidence of perinatal SARS infection among infants born to these mothers.