Non-invasive prenatal rhesus D genotyping using cell-free foetal DNA

Background & objectives: Non-invasive prenatal diagnosis (NIPD) of rhesus D (RHD) genotype using cell-free foetal DNA is extensively used in many developed countries. Studies on NIPD from India are scarce. The aim of the present study was to evaluate the performance of non-invasive foetal RHD genotyping by targeting exon 10 of the RHD gene using cell-free DNA. Methods: DNA was extracted from the maternal plasma of alloimmunized and non-alloimmunized women between 7 and 34 wk of gestation. RHD sequence was determined by quantitative real time polymerase chain reaction (PCR). Results were compared with RhD phenotype obtained from cord blood samples of neonates. Results: A total of 135 samples from RhD-negative pregnant women were collected. The foetal RHD status was conclusive in all 135 (100%) cases. The highest number of cases reported for RHD genotyping were from Punjab (38.5%) followed by Haryana (24.4%), Himachal Pradesh (17.0%) and Chandigarh Union Territory (13.3%). The non-invasive test correctly predicted the foetal RhD phenotype in 133 of 135 cases, making the accuracy of the test as 98.51 per cent [95% confidence interval (CI): 97.90-99.50%]. The overall sensitivity and specificity of the test were 99.18 per cent (95% CI: 95.52-99.98%) and 92.31 per cent (95% CI: 63.97-99.81%), respectively, with negative and positive predictive values of 99.80 per cent (95% CI: 94.85-99.87%) and 96.31 per cent (95% CI: 62.87-98.84%), respectively. Interpretation & conclusions: Non-invasive foetal RHD determination by single-exon quantitative PCR exhibited high accuracy and could be used in routine clinical practice after confirmatory studies are done.

Midtrimester maternal plasma concentrations of angiopoietin 1, angiopoietin 2, and placental growth factor in pregnant women who subsequently develop preeclampsia

OBJECTIVE: The aim of this study was to compare midtrimester maternal plasma concentrations of angiopoietin 1, angiopoietin 2, and placental growth factor between pregnant women who subsequently developed preeclampsia and those who did not. METHODS: Midtrimester maternal plasma was collected and stored at -70degrees C when genetic amniocentesis was performed. Cases included 37 samples of individual who subsequently developed preeclampsia, and matched controls were from individuals who did not develop preeclampsia. Angiopoietin 1, angiopoietin 2, and placental growth factor concentrations were measured by the enzyme-linked immunosorbent assay method and were compared using the Mann-Whitney U-test. A P-value <0.05 was considered significant. RESULTS: In pregnant women who subsequently developed preeclampsia, midtrimester maternal plasma concentrations of angiopoietin 1 and angiopoietin 2 were significantly higher and placental growth factor concentrations were significantly lower than in women who did not develop preeclampsia (angiopoietin 1: 10.6 [3.1-19.7] vs. 7.8 [0.9-24.4] ng/mL, P=0.031; angiopoietin 2: 31.0 [4.7-81.2] vs. 18.4 [4.2-49.7] ng/mL, P<0.001; placental growth factor: 87.1 [14.2-774.3] vs. 148.8 [57.2-425.6] pg/mL, P<0.001). Within the case group who subsequently developed preeclampsia, the placental growth factor was significantly lower in those who had fetal growth restrictions than in those who did not (placental growth factor: 72.5 [14.2-774.3] vs. 140.9 [44.2-257.5] pg/mL, P=0.003). CONCLUSION: Midtrimester maternal plasma concentrations of angiopoietin 1, angiopoietin 2, and placental growth factor may be associated with the subsequent development of preeclampsia.

Extracellular chromosome 21-derived microRNAs in euploid & aneuploid pregnancies.

Background & objectives: Trisomy 21 is the most common chromosomal aneuploidy in live born infants. Recently, the over expression of chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802) in human fetal hippocampus and heart samples from individuals with Down syndrome was observed. Therefore, concentrations and expression profile of extracellular chromosome 21-derived microRNAs were studied to verify their ability to distinguish noninvasively between pregnancies bearing euploid fetuses and those affected with Down syndrome. Methods: RNA enriched for small RNAs was isolated from plasma samples of 12 pregnant women with high risk of bearing Down syndrome foetuses (median gestation 18.5 wk), 12 women with normal course of gestation and 10 non-pregnant women. MicroRNA transcribed into cDNA using specific stem-loop primer was detected using real-time PCR assay. Simulation experiments using RNA pools of healthy non-pregnant individuals and aneuploid amniotic fluid samples in descending dilution ratio ranging from 1:1 to 1000:1 were used to test the detection limit of the technique for overexpressed chromosome 21-derived microRNAs specific for Down syndrome. The expression profile of the gene encoding microRNA was studied through the relative gene expression using the comparative Ct (threshold cycle) method. Concentrations of individual microRNAs were subtracted from the calibration curves in the course of analyses and expressed as pg of total RNA per milliliter of plasma. Results: Four of the five extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) were reliably detected in plasma samples. Simulation experiments revealed the detection limit of aneuploidy at a ratio 100:1 for let-7c, miR-125b-2 and miR-155, and a ratio of 1000:1 for miR-99a. Overexpression of extracellular miR-99a, miR-125b-2 and miR-155 was observed in pregnant women compared to non-pregnant women. Similarly, increased concentrations of extracellular miR-99a and miR-125b-2 were detected in pregnant women than in non-pregnant women. The concentrations and relative gene expression of extracellular chromosome 21-derived microRNAs did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. Interpretation & conclusions: Analysis of extracellular chromosome 21-derived microRNAs has no benefit for screening programmes and non-invasive diagnosis of Down syndrome.

Utilização de ácidos nucleicos fetais livres no plasma materno para o diagnóstico pré-natal: realidade do Brasil neste cenário / Use of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis: the reality of this scenario in Brazil

A descoberta de ácidos nucleicos fetais livres no plasma de gestantes possibilitou o desenvolvimento de novos testes de diagnóstico pré-natal não invasivo para a determinação do sexo e do Rh fetal. Esses testes foram implantados no sistema de saúde pública de diversos países da Europa há mais de cinco anos. As novas possibilidades de aplicação diagnóstica dessas tecnologias são a detecção de aneuploidias cromossômicas fetais, de doenças monogênicas fetais e de distúrbios relacionados com a placenta, temas pesquisados intensivamente por diversos grupos ao redor do mundo. O objetivo deste estudo é expor a situação brasileira no âmbito de pesquisa e utilização clínica dos testes disponíveis comercialmente que utilizam esses marcadores moleculares plasmáticos, ressaltando as vantagens, tanto econômicas quanto de segurança, que os testes não invasivos têm em relação aos atualmente utilizados em nosso sistema de saúde pública.

The discovery of cell-free fetal nucleic acids in the plasma of pregnant women has allowed the development of new, noninvasive prenatal diagnostic tests for the determination of fetal gender and Rh. These tests have been implemented in the public health system in several countries of Europe for over five years. The new possibilities for diagnostic use of these technologies are the detection of fetal chromosomal aneuploidies, monogenic fetal disorders, and placental-related disorders, subjects that have been intensively studied by several groups around the world. The aim of this review was to assess the Brazilian research and clinical scenarios regarding the utilization of commercially available tests that use these plasma markers, stressing the advantages, both economic and safety-related, that non-invasive tests have when compared to those currently used in the Brazilian public health system.

Noninvasive Prenatal Diagnosis using Cell-Free Fetal DNA in Maternal Plasma: Clinical Applications

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

Application of MiniSTR in the detection of fetal DNA in maternal plasma / 中国输血杂志

Objective To develop an efficient method for detecting the short tandem repeat(STR) of fetal DNA in maternal plasma by miniSTR technique.Methods A total of 9 blood samples form pregnant women from 11 to 27 weeks of gestation were collected.Each isolated total plasma DNA was amplified in single multiplex using the ABI MiniFilerTM kit,which could simultaneously genotype the 9 miniSTR loci,including D13S317,D7S820,D2S1338,D21S11,D16S539,D18S51,CSFIPO,FGA and Amelogenin,and the PCR products were detected by using ABI PrismTM 3100 DNA Sequencer.The allelic designation of each STR locus was accomplished using the GeneMapper ID 3.2 software.Results Father-origin fetal STR allele was detected in all the 9 plasma DNA samples.An average of 3.1 fetal STR alleles of the 8 autosomal STR loci was observed in each of the 9 plasma DNA samples.As for the Amelogenin locus,Amelogenin Y allele was detected in 5 plasma DNA samples from pregnancies with male fetus,and allelic peak height values were all over 50 RFU,according to ABI Mini FilerTM PCR conditions,and the ratio of Amelogenin Y allele peak height value to Amelogenin X allele peak height value was 8.45%.However,no Amelogenin Y allele was detected in other 4 plasma DNA samples from pregnant women with female fetus.Conclusion The miniSTR technique is suitable for STR genotyping using fetal DNA in maternal plasma,and it suggests a broad application in noninvasive molecular prenatal diagnosis.

Hormona del crecimiento y prolactina en líquido amniótico y plasma materno durante el embarazo normal / Growth hormone and prolactin in maternal plasma and amniotic fluid during normal gestation

Objective. To measure the concentration of human growth hormone (GH) and prolactin (PRL) In maternal plasma (MP) and amniotic fluid (AF) during gestation and to evaluate their correlation. Methods. HC and PRL were measured by radioimmunoassay in 60 healthy women with uncomplicated singleton pregnancies at 16-36 weeks of gestation (WG). Trends of both hormones were estimated throughout pregnancy. The correlation of either hormone measured in different fluids was also estimated. Results. GH in AF (AF-GH) decreased significantly throughout pregnancy; (AF-GH = 21.32 - 0.54 X WG, r = -0.72 [95% confidence intervals (95% CI) -0.57-0.82], p = 0.001), and increased in MP (MP-GH); (MP-GH = 2.73 +0.11 X WG, r = 0.45 [95% CI, 0.21-0.63]p = 0.05). The correlation between MP-GH and AF-GH was, (AF-GH = 16.28 - 1.54 x - M-PGH, r = -0.47 [95% CI, -0.64 --0.21]; p = 0.01). PRL values did not show significant differences neither in AF (AF-PRL / WG, r = 0.06, p = 0.6) nor in MP (MP-PRL / WG, r = 0.25, p = 0.14) during pregnancy, being AF-PRL (mean 151.3 ng/mL, SD 34.2 ng/mL) significantly higher than MP-PRL (mean 119.3 ng/mL, SD 55.4 ng/mL) (p = 0.006) in all the studied period. Conclusion. AF-GH and MP-GH showed a significant negative correlation during pregnancy. PRL measured in AF and PM did not show changes throughout gestation being AF-PRL significantly higher than MP-PRL.

Objetivo. Medir los valores de hormona del crecimiento (HC) y prolactina (PRL) en el líquido amniótico (LA) y en el plasma materno (PM) durante el embarazo normal y analizar sus asociaciones. Métodos. HC y PRL fueron medidas en el LA y en el PM por medio de radioinmunoanálisis en 60 mujeres con embarazo único y sin complicaciones entre 16 y 36 semanas de gestación (SG). Se evaluaron las tendencias de ambas hormonas a lo largo del embarazo y la correlación entre los valores obtenidos en ambos compartimentos. Resultados. La HC en LA (HCLA) disminuyó en forma significativa durante el embarazo (HCLA = 21.32 - 0.54 X SG, r = -0.72 [intervalos de confianza al 95% (IC 95%) -0.57-0.82], p = 0.001) y en PM (HCPM) aumentó (HCPM = 2.73 + 0.11 X SG, r = 0.45 [IC 95%, 0.21-0.63] p = 0.05). La correlación entre los valores de HC en LA y en PM fue: (HCLA = 16.28-1.54 x -HCPM, r = -0.47 [IC 95% -0.64 -0 -0.21]; p = 0.01). La concentración de PRL a lo largo del embarazo tanto en LA (PRL-LA/SG, r = 0.06, p = 0.6) como en PM (PRL-PM/SG r = 0.25, p = 0.14) no cambió y fue significativamente más alta en LA que en PM (LA; media 151.3 ng/mL, DE 34.2 ng/ inL, PM; media 119.3 ng/mL, DE 55.4 ng/mL, respectivamente, p = 0.006). Conclusiones. HC en PM y HC en LA presentan una correlación inversa a lo largo del embarazo, en tanto que los valores de PRL en ambos compartimentos no cambian, siendo significativamente más elevados en el LA.

The diagnostic potential of maternal plasma in detecting fetal diseases by DNA test.

Conventionally, DNA based investigations for fetal diseases are done by chorionic villous sampling and amniocentesis. Both are invasive techniques. Recently, molecular diagnosis has also been made possible in early pregnancy from maternal blood which is noninvasive and advantageous. Most of the researches have tried to identify the Y chromosome marker(s) to detect a male fetus and paternally inherited allele. This is currently helpful to detect a very few genetic disorders including Rh D status in Rh negative women in early pregnancy and preeclampsia a few weeks preceding the clinical onset. This is a potential area for prenatal diagnosis in future.

Maternal Plasma Homocysteine Levels and Pregnancy Outcomes / 대한지역사회영양학회지

Elevated maternal plasma homocysteine concentrations have been associated with adverse pregnancy outcomes, including birth defects, low birth weight, preeclampsia, spontaneous abortion, placental abruption, and other maternal or fetal complications. The purpose of this study was to assess the maternal plasma homocysteine level during pregnancy and to investigate the relationship between the plasma homocysteine concentrations and pregnancy outcomes. Venous blood samples were drawn from 82 pregnant women who were grouped with gestational age, 1st trimester (n = 26), 2nd trimester (n = 27) and 3rd trimester (n = 29). The concentration of plasma homocysteine was analyzed by HPLC, and pregnancy outcomes including gestational length, maternal weight gain, infant birth weight, and Apgar score were collected with the medical records of the pregnant women. The levels of plasma homocysteine of the pregnant women at the 1st, 2nd, and 3rd trimester were 5.7 +/- 3.7 micronmol/L, 5.6 +/- 4.1 micronmol/L and 7.0 +/- 4.5 micronmol/L, respectively, which had not showed any significant difference. The result of this study showed that in case of the pregnant women at the 1st trimester, the maternal plasma homocysteine level of the pregnant women whose gestational length was less than 38 weeks was significantly high (p < 0.01) compared to that of the pregnants whose gestational length was more than 38 weeks. And also, the level of homocysteine of the pregnant women at the 2nd trimester was significantly low when the maternal weight gain was high (p < 0.05). These findings suggest that maternal plasma homocysteine level at early stage of gestation will be a predicter of gestational length and maternal weight gain.

When Can We Identify Fetal Male Gene By Using Maternal Plasma DNA? / 대한산부인과학회잡지

OBJECTIVE: To know when we can identify fetal Y chromosome SRY gene by using fetal DNA in maternal plasma. METHODS: DNA from maternal plasma of 62 pregnant women (48: inpatients, 14: outpatients) underwent a sensitive Y-PCR assay to identify SRY gene of Y chromosome. RESULTS: Of the patients, fetus-derived Y sequences were detected in 37 (88.1%) of the 42 maternal plasma samples from women bearing male fetuses. One of the 20 women bearing female fetuses had positive result from plasma DNA. Seventh gestational week was the earliest gestation of gender identification. CONCLUSION: We could identify fetal gender using fetal DNA in maternal plasma (sensitivity 88.1%). The earlist to detect was 7th gestational week.

Identification of Fetal Gender Using Maternal Plasma DNA / 대한산부인과학회잡지

OBJECTIVE: To identify fetal gender using fetal DNA in maternal plasma. METHODS: DNA from maternal plasma of 55 pregnant women(47: inpatients, 8: outpatients) underwent a sensitive Y-PCR assay to identify gender. RESULTS: Of the inpatients, fetus-derived Y sequences were detected in 26(80.6%) of the 31 maternal plasma samples from women bearing male fetuses. None of the 16 women bearing female fetuses had positive results from plasma DNA. Eighteen weeks is earliest gestation of gender identification. Of the outpatients(GA 8-11 weeks), fetus-derived Y sequences were detected in 7 of the 8 maternal plasma. Only one patient's fetal gender(GA 9 weeks) was identified. The others were not identified at this moment. CONCLUSION: We identified fetal DNA in maternal plasma. The sensitivity of Y-PCR was 80.6% in women bearing male fetus and the specificity was 100% in women bearing female fetus.

The Obligatory Role of the Chorionic Membranes in the Synthesis of Myometrial cGMP during Pregnancy and Its Inhibition by Maternal and Fetal Plasma / 대한산부인과학회잡지

The Obligatory Role of the Chorionic Membranes in the Synthesis of Myometrial cGMP during Pregnancyand Its Inhibition by Maternal and Fetal Plasma The mechanism of uterine quiescence during pregnancy and initiation of labor is unknown. In previousreport, we demonstrated myometrial cGMP rises dramatically during pregnancy and then declines just priorto the onset of labor. Further, pregnancy decrease myometrial soluble guanylate cyclase activity whileenhancing particulate activity. Theses findings suggest a natriureitc peptide(NP) is responsible for theincrease in cGMP. This study was undertaken to evaluate the source of that NP. We incubatedmyometrium from term guinea pigs in oxygenated buffer in the absence/presence of either amnionicmembranes(AM, 400mg), chorionic membrane(CM, 400mg), fetal guinea pig plasma(FP, 1ml), maternalguinea pig plasama(MP, 1ml), or a combination. After incubation, cGMP(mol/mg portein) was measuredby radioimmuno assay. Both AM and CM(CM>AM, significantly) increased myometrial cGMP. Thestimulation of myometrial cGMP by the CM has a significant linear relationship between fetal weight andthe greatest cGMP increase occurred between 51~60 days after which it showed a trend to decrease. BothFP and MP decrease myometrial cGMP and inhibition of cGMP by MP increase significantly withadvancing gestational age.Our date indicate that myometrial cGMP is stimulated by a compound produced by the CM and the actionof this compound is inhibited by a substance produced by the mother and fetus at the end of a normalpregnancy.