Maddalena Smid

No evidence of fetal DNA persistence in maternal plasma after pregnancy

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.

Quantitative Analysis of Fetal DNA in Maternal Plasma in Pathological Conditions Associated with Placental Abnormalities

Abstract: An increased fetal DNA concentration in maternal plasma has been observed in placental pathological conditions associated with hypertension and preeclampsia. To confirm these data, we performed real‐time quantitative PCR on the SRY gene in a group of physiological and pathological male‐bearing pregnancies. In 78 physiological pregnancies, fetal DNA concentration in maternal plasma was 20.7, 13.4, 23.6, and 74.8 genome‐equivalents (g.e.)/mL during the first, second, and third trimesters and at term, respectively. In 10 preeclamptic women, fetal DNA concentration ranged from 59.3 to 615.2 g.e./mL (median: 332.9). In 7 women with preeclampsia and IUGR (intrauterine growth retardation), fetal DNA ranged from 96.5 to 859 g.e./mL (median: 146.8). In 4 women with IUGR and hypertension, fetal DNA ranged from 34 to 473.5 g.e./mL (median: 142.4). In 3 patients with IUGR, fetal DNA ranged from 168.6 to 519.7 g.e./mL (median: 308.1). In 2 patients with IUGR and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, fetal DNA concentration ranged from 105 to 394.1 g.e./mL (median: 249.7). Four women who developed preeclampsia some weeks later showed fetal DNA levels within the physiological range. These data suggest that increased fetal DNA concentrations might represent a valuable marker of placental abnormalities and suggest that this rise may precede clinical manifestation of preeclampsia by only a few weeks.

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of β-thalassemia

This study describes a novel approach to non-invasive pre-natal diagnosis of β-thalassemia based on microchip analysis of fetal DNA extracted from maternal plasma. The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent β-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases

Abstract: Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real‐time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male‐bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long‐term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild‐type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild‐type allele signals.

Fetal DNA in maternal plasma in twin pregnancies

Twin pregnancies have increased in recent years as assisted reproductive technologies have been adapted. The maternal age of women undergoing assisted reproduction is generally higher than that of women with spontaneous pregnancies. This also implies a higher risk of fetal aneuploidies. Furthermore, twin pregnancies are known to have a higher risk of developing complications such as preeclampsia, intrauterine growth retardation, and preterm labor [for a review, see Ref. (1)]. Of note is that women who have been trying for years to become pregnant and have finally undergone cumbersome therapeutic interventions could be particularly reluctant to expose their pregnancy to any invasive prenatal diagnostic procedure. Thus, noninvasive screening or diagnostic tests could be particularly welcome in this subset of pregnant women. Noncellular fetal DNA circulating in maternal plasma may represent a suitable source of fetal genetic material that can be obtained noninvasively. Several studies have shown a significantly higher concentration of fetal DNA in maternal plasma in some fetal aneuploidies and placental pathologies (2)(3)(4)(5)(6). Nevertheless, existing data about fetal DNA concentrations in maternal blood under physiologic and pathologic conditions refer only to singleton pregnancies, and reference values for twins are still missing. The origin of the fetal DNA released into maternal plasma is still unclear. Although there is evidence that some of the cell-free fetal DNA in the maternal plasma is derived from blood elements, several authors favor the hypothesis that the majority is derived from the placenta (7)(8). Interestingly, twin pregnancies may present with one or two placentas, and although dizygotic twins must have two placentas, monozygotic twins can be either mono- or bichorionic. It has been reported that monozygotic twins exhibit smaller uteroplacental junctional areas than do …

Evaluation of a panel of circulating DNA, RNA and protein potential markers for pathologies of pregnancy.

Abstract Background: Among markers of pregnancy complications, corticotropin-releasing hormone (CRH) mRNA, long pentraxin 3 (PTX3) protein and fetal and total DNA had been reported to be increased in the plasma of women with overt preeclampsia (PE). We developed an optimized protocol to evaluate whether concentrations of CRH mRNA, PTX3 mRNA and protein, fetal and/or total DNA are increased in fetal growth restriction (FGR), and whether they predict complications of pregnancy. Methods: The protocol included a preamplification step to enrich rare mRNA species. CRH and PTX3 mRNA, DNA and PTX3 protein were measured in the plasma of women with PE or FGR, in women at risk of developing these pathologies and in healthy women matched for gestational age. Results: CRH mRNA, fetal and/or total DNA and PTX3 protein were significantly increased in women with overt PE when compared to controls. Pregnant women who later developed PE or FGR during pregnancy showed total DNA levels that were significantly increased before the onset of both pathologies, while RNA markers were increased only in women who later developed PE. Conclusions: Our protocol for plasma RNA quantification may allow for the extension of a panel of predictive markers to be investigated in larger patient cohorts. Clin Chem Lab Med 2010;48:791–4.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR

Abstract:  The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer‐directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority‐mutated allele in the presence of an excess of wild‐type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild‐type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild‐type DNA in the beta‐globin gene region, where four beta‐thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild‐type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

HIF1A and MIF as potential predictive mRNA biomarkers of pre-eclampsia: a longitudinal prospective study in high risk population.

Abstract Background: Pre-eclampsia (PE) is a hypertensive multisystem disorder, causing significant fetal-maternal mortality and morbidity worldwide. This study aims to define possible longitudinal predictive mRNA markers involved in the main pathogenic pathways of PE: inflammation [macrophage migration inhibitory factor (MIF)], hypoxia and oxidative stress [hypoxia inducible factor 1-α subunit (HIF1A) and β-site APP-cleaving enzyme-2 (BACE2)] and endothelial dysfunction [endoglin (ENG), fms-related tyrosine kinase-1 (FLT1) and vascular endothelial growth factor (VEGF)]. Methods: Peripheral blood was collected from 33 singleton pregnancies characterized by a high cardiovascular profile risk sampled consecutively at 6–16; 17–23; 24–30; 31–34; ≥35 weeks followed by the Obstetrics and Gynecology Unit of the San Raffaele Hospital in Milan. A real-time quantitative PCR reaction was performed on plasma RNA. Results: Of the 33 women enrolled, nine developed PE. Until 23 weeks HIF1A was significantly higher in women who later developed PE compared to women who did not (p=0.049 and p=0.012 in the first and second blood collection). In the third time interval MIF (p=0.0005), FLT1 (p=0.024), ENG (p=0.0034) and BACE2 (p=0.044) appeared to be significantly increased while HIF1A was elevated even from 24 week onwards but not reaching the statistical significance. In the fourth time interval ENG mRNA still remained increased (p=0.037). Conclusions: HIF1A, marker of hypoxia and oxidative stress, and MIF, marker of inflammation, seemed to be the most promising RNA markers, suggesting that hypoxia, principally, and inflammation may play an important role in PE pathogenesis.

Predictive biomarkers of pre-eclampsia and effectiveness of preventative interventions for the disease.

Introduction: Pre-eclampsia (PE) is one of the most common pregnancy complication characterized by placental and maternal vascular dysfunction. It affects about 3 – 8% of women during the second half of pregnancy and represents one of the major causes of neonatal morbidity and mortality. The etiology of PE largely remains unknown. Areas covered: PE is considered a syndrome with multisystem involvement, so the ideal predictive test for it should utilize a combination of many predictors. Measurement in early pregnancy of a variety of biophysical and biochemical markers implicated in the pathophysiology of PE associated with clinical risk factors has been proposed to predict the development of the syndrome, thereby mitigating an adverse outcome. Expert opinion: The identification of reliable indicators is a clinically relevant issue that could result in early therapeutic intervention and leading to the prevention of maternal and fetal injuries before the manifestation of clinical signs. Many factors complicate the prevention of PE cases. Most are attributed to unknown etiology, the low predictive value of current screening tests and the several presentations of the disease. Although preventative treatments have been studied extensively, an effective intervention to avoid the development of PE has not yet been discovered.