Identification of Urine Biomarkers to Improve Eligibility for Prostate Biopsy and Detect High-Grade Prostate Cancer.

PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50-75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa.

Expression of Indoleamine 2,3-Dioxygenase Induced by IFN-γ and TNF-α as Potential Biomarker of Prostate Cancer Progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested "in vitro" by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

Implication of vascular endothelial growth factor A and C in revealing diagnostic lymphangiogenic markers in node-positive bladder cancer.

Several lymphangiogenic factors, such as vascular endothelial growth factors (VEGFs), have been found to drive the development of lymphatic metastasis in bladder cancer (BCa).Here, we have analyzed the gene expression of lymphangiogenic factors in tissue specimens from 12 non-muscle invasive bladder cancers (NMIBC) and 11 muscle invasive bladder cancers (MIBC), considering tumor and tumor-adjacent normal bladder areas obtained from the same organs. We then compared the results observed in patients with those obtained after treating human primary bladder microvascular endothelial cells (MEC) with either direct stimulation with VEGF-A or VEGF-C or by co-culturing (trans-well assay) MEC with bladder cancer cell lines varying in VEGF-A and VEGF-C production based on tumor grade.The genes of three markers of lymphatic endothelial commitment and development (PDPN, LYVE-1 and SLP-76) were significantly overexpressed in tissues of MIBC patients showing positive lymphovascular invasion (LVI+), lymph node metastasis (Ln+) and tumor progression. Their expression was also significantly enhanced either after direct stimulation of MEC by VEGF-A and VEGF-C or in the trans-well assay with each bladder cancer cell line.SLP-76 showed the highest gene expression. Both VEGF-A and VEGF-C also enhanced the expression of SLP-76 protein in MEC. However, a correlation between increase of SLP-76 gene expression and the ability of MEC to migrate could only be seen after induction by VEGF-C.The significant expression of SLP-76 in LVI+/Ln+ progressive MIBC and its overexpression in MEC after VEGF-A and VEGF-C stimulation suggest the need to develop this regulator of developmental lymphangiogenesis as a diagnostic tool in BCa.

High VEGF-D and Low MMP-2 Serum Levels Predict Nodal-Positive Disease in Invasive Bladder Cancer.

BACKGROUND: To investigate stromal variables including angiogenesis, lymphangiogenesis, and matrix metalloproteinase (MMP) in the serum of patients with urothelial carcinoma of the bladder (UCB) and to evaluate their association with histopathological characteristics and clinical outcome. MATERIAL AND METHODS: Protein levels of vascular endothelial growth factors-A, -C, -D (VEGF-A/-C/-D), their receptors- VEGF-R2 and -R3 (VEGF-R2/-R3), and matrix metalloproteinases 2, -3, and -7 (MMP-2, MMP-3, MMP-7) were quantified in the blood serum samples of 71 patients with UCB before radical cystectomy (RC). Samples of patients with non-invasive UCB or no history of UCB were investigated as controls (n=20). Protein levels in the serum were measured using a flow cytometric cytokine assay. RESULTS: A positive association for VEGF-D (p<0.001) and an inverse association for MMP-2 (p=0.017) were observed in patients with positive lymph node (LN) status at the time of RC. VEGF-A (p<0.001), VEGF-C (p<0.001), MMP-2 (p<0.001), and MMP-7 (p=0.005) serum levels were different in serum of patients with invasive UCB compared with non-invasive UCB or healthy individuals. None of the serum markers were associated with disease progression. CONCLUSIONS: High VEGF-D and low MMP-2 serum levels predict LN metastasis in patients with UCB at the time of RC. VEGF-A, VEGF-C, MMP-2, and MMP-7 serum levels varied significantly between invasive and non-invasive disease as well as in comparison with healthy individuals. Clinical implementation of these marker serum measurements may be valuable to select high-risk patients with more invasive or nodal-positive disease.

Antibody response to BK polyomavirus as a prognostic biomarker and potential therapeutic target in prostate cancer.

Infectious agents, including the BK polyomavirus (BKPyV), have been proposed as important inflammatory pathogens in prostate cancer. Here, we evaluated whether the preoperative antibody response to BKPyV large T antigen (LTag) and viral capsid protein 1 (VP1) was associated with the risk of biochemical recurrence in 226 patients undergoing radical prostatectomy for primary prostate cancer. Essentially, the multivariate Cox regression analysis revealed that preoperative seropositivity to BKPyV LTag significantly reduced the risk of biochemical recurrence, independently of established predictors of biochemical recurrence such as tumor stage, Gleason score and surgical margin status. The predictive accuracy of the regression model was denotatively increased by the inclusion of the BKPyV LTag serostatus. In contrast, the VP1 serostatus was of no prognostic value. Finally, the BKPyV LTag serostatus was associated with a peculiar cytokine gene expression profile upon assessment of the cellular immune response elicited by LTag. Taken together, our findings suggest that the BKPyV LTag serology may serve as a prognostic factor in prostate cancer. If validated in additional studies, this biomarker may allow for better treatment decisions after radical prostatectomy. Finally, the favorable outcome of LTag seropositive patients may provide a potential opportunity for novel therapeutic approaches targeting a viral antigen.

Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia.

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor ß1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry.

PURPOSE: To examine the potential high throughput capability and efficiency of an automated DNA extraction system in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status. METHODS: A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray mass spectrometric system. RESULTS: We determined that as little as 15 pg of RHD-positive genomic DNA could be detected in a background of 585 pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and specificity of 96.1 and 96.1%, respectively. CONCLUSION: Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using real-time PCR.

PLAC4 and beta-HCG mRNA levels are not altered in the maternal circulation of pregnancies with trisomy 21.

OBJECTIVE: Beta-human chorionic gonadotropin (HCG) and pregnancy-associated plasma protein (PAPP-A) are placentally produced proteins whose levels are altered in pregnancies with trisomy 21. PLAC4 is located on chromosome 21 and its expression is restricted to the placenta. Here we investigated whether the levels of beta-HCG-, PAPP-A- and PLAC4 mRNA could be able to discriminate pregnancies whose fetus is affected by trisomy 21. METHOD: Hundred and forty-three blood samples from normal pregnancies and eight samples from trisomic pregnancies were collected. Total RNA was extracted from whole maternal blood, reverse-transcribed and the three mRNAs were quantified by real-time quantitative PCR. Hundred and nine controls were also tested for the serum levels of PAPP-A and HCG proteins. RESULTS: Beta-HCG and PLAC4 mRNAs were detected in all samples, in higher amounts than in plasma, whereas the detection rate for PAPP-A mRNA was below 10%. The levels of beta-HCG mRNA significantly correlated with the circulatory concentrations of the HCG protein. However, neither beta-HCG- nor PLAC4 mRNAs show a significant difference between cases and controls. CONCLUSION: Maternal blood levels of beta-HCG-, PLAC4- and PAPP-A mRNAs are not useful markers for the screening of pregnancies with trisomy 21 as their concentrations are either not significantly altered (beta-HCG and PLAC4) or too low to be detected (PAPP-A).

Isolation of serum nucleic acids for fetal DNA analysis: comparison of manual and automated extraction methods.

OBJECTIVES: To investigate the performance of an automated system for the extraction of cell-free DNA of maternal and fetal origin from stored serum samples for subsequent quantitative real-time polymerase chain reaction (PCR) analysis. METHODS: Thirty-two maternal blood samples between the early second trimester and term were obtained. Cell-free DNA was extracted from replicate stored sera using a column-based manual isolation procedure and with an automated system, the MagNA Pure LC Instrument. Real-time quantitative PCR for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and male-specific DYS14 loci was performed. RESULTS: The extraction yields for both total and fetal DNA and the quality of the purified nucleic acids were similar for the automated system or the manual procedure. However, the number of false-negative results in samples collected early in pregnancy was reduced with the automated extraction. Furthermore, the extraction rate by the automated system was highly reproducible over time. CONCLUSIONS: We validated the use of an automated extraction system for the isolation of fetal DNA from stored serum. This procedure might be exploited in the future for high-throughput non-invasive fetal gene analysis of archived serum samples.

Performance of a panel of maternal serum markers in predicting preeclampsia at 11-15 weeks' gestation.

OBJECTIVE: We evaluated whether a discriminant model of prediction based on quantitative distribution of a panel of biomolecules in maternal serum can discriminate normal pregnancies from those who will develop preeclampsia (PE) prior to onset of clinical symptoms at 11-15 weeks' gestation. METHODS: Case control study encompassing 56 women destined to develop PE cases matched 1:3 for gestational age with 168 controls. After multiple of median (MoM) conversion of all available markers, comprising total Activin A (t-activin A), P-selectin, and vascular endothelial growth factor receptor (VEGFR) the combined likelihood ratios generated for each marker were used to calculate, for each patient enrolled in the study, the odds of being affected given a positive results (OAPR) of developing PE. For all the analyses performed, the type II error was < 20% with a type I error fixed at 5%. RESULTS: Data were expressed in MoM of controls. P-selectin was identified as the marker with the best discriminant ability between controls and PE, followed by (t-activin A). No significant differences in VEGFR were observed between cases and controls. By using a 3% prevalence of PE (or, about 1:33) we found that the median OAPR of developing PE for the 56 cases was 1:9 or 10% (1:1-1:417). The median OAPR of PE for controls was 1:40 or 2.5% (range, 1:6-1:4205). Detection rate of the statistical model, with a 5% false-positive rate was 59%. CONCLUSION: This analysis revealed that maternal serum markers assessed at the first and second trimester of pregnancy in asymptomatic patients can improve the early detection of cases at higher risk of developing PE.

PLAC1 mRNA levels in maternal blood at induction of labor correlate negatively with induction-delivery interval.

OBJECTIVE: This study was conducted to determine whether, in low risk women having labor induced using prostaglandin gel (dinoprostone gel), there is a relationship between the concentration of mRNA for the PLAC1 gene (a trophoblast-specific gene) in maternal blood and the time elapsed between the first gel administration and spontaneous delivery. STUDY DESIGN: Blood was collected from 49 selected women at 40.2-41.4 weeks' gestation. Total RNA was extracted by means of an ABI Prism 6100 nucleic acid Prep Station and quantitative real-time PCR analysis was performed by use of a PE Applied Biosystems 5700 Sequence Detection System. Sequence data were obtained from the Genebank Sequence Database. To determine the amount of cDNA, the PLAC1 locus was used. RESULTS: Thirty women (61.2%) had a spontaneous delivery. A caesarean section, either for fetal dystocia or fetal distress, was performed in 19 (38.8%). The crude delivery rates of the women who ended up with a spontaneous delivery were 30% at 24 h and 43% at 48 h. Women (n=19) with a blood concentration of logPLAC1 mRNA>or=2.00 displayed a median time to delivery of 23.50h, (95% CI: 13.13-33.87) while those with a logPLAC1 mRNA<2.00 (n=30) had a median time of 54 h. (95% CI: 37.86-70.14; p=0.0043, log-rank test). By means of multivariate analysis, quantitative Bishop score (from 2 to 7) at the time of the first gel administration and logPLAC1 mRNA>or=2.00 were associated with a higher rate of delivery per unit of time with an odds ratio of 1.35 (95% CI: 1.07-1.71) and 3.48 (95% CI: 1.55-7.80), respectively. CONCLUSIONS: In induced term pregnancies, PLAC1 mRNA in maternal blood at the beginning of the treatment correlates with the time elapsed before delivery. This evidence demonstrates that the fetomaternal trafficking of nucleic acids is more consistent when the labor is about to begin.

PLAC1 mRNA in maternal blood correlates with Doppler waveform in uterine arteries in normal pregnancies at the second and third trimester.

Doppler analysis of the uterine arteries is currently used for pre-eclampsia (PE) screening. PLAC1 is a trophoblast-specific gene, and it is known that in normal pregnancies, trophoblastic cells are released into the maternal circulation, where specific trophoblastic mRNA can be detected. In PE, as in women who eventually develop PE, an abnormal passage of fetal and placental cells is also present. In this study, we aimed to verify whether, in normal pregnancies, Doppler waveform of the uterine arteries correlates with PLAC1 mRNA concentrations. Thirteen cases of normal pregnancies at 37 weeks' gestation (23-41) were enrolled in the study. PLAC1 mRNA was extracted from 2 mL of blood by ABI PRISM 6100 nucleic acid Prep Station (Applied Biosystems, Foster City, CA) and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed by a PE 5700 Sequence detection system. Bulk RNA from normal placental tissue was used as the reference curve, and the amount of PLAC1 mRNA in the study samples was then expressed as the "relative amount" of weight of placental tissue (ng/mL). The uterine arterial mean resistance index (RI) and presence/absence of a dicrote waveform were calculated by using a 5 MHz transabdominal probe (Tecnos, ESAOTE) at the uterine cervico-corporal junction. Doppler measurement was performed on the same day as blood collection. The median of the means of uterine arterial RI was 0.52 (0.39-0.68). RI of uterine arteries and PLAC1 mRNA were significantly correlated in a log-linear regression (R(2) = 0.483, P = 0.024). Our data support that in normal pregnancy, the passage of trophoblast material into the maternal circulation is correlated with the quantitative measurement of uterine hemodynamics.

Quantitative distribution of a panel of circulating mRNA in preeclampsia versus controls.

OBJECTIVE: The aim of this study was to evaluate whether the quantitative distribution of a panel of circulating mRNAs from maternal whole blood of normal pregnancies is statistically different from those complicated with preeclampsia (PE) with or without intrauterine growth restriction (IUGR). METHODS: Maternal whole blood of six subjects with mild or severe PE with or without IUGR and 30 matched controls (1:5 match for gestational age) were retrospectively examined for circulating mRNA markers. Seven specific mRNA markers were identified and chosen based on previous microarray mRNA expressions performed on placental tissue from normal and PE patients. They were human placental lactogen (hPL), inhibin A, KISS-1, pregnancy-associated plasma protein-A (PAPP-A), plasminogen activator inhibitor type 1 (PAI-1), selectin-P and vascular endothelial growth factor receptor (VEGFR), which were therefore quantified for statistical purposes. RESULTS: Median gestational age was 229 (178-283) and 232 (194-262) days for controls and cases respectively. All mRNA markers but PAPP-A, showed statistically different median values. They were hPL, inhibin A, KISS-1, PAI-1, Selectin-P, and VEGFR. Inhibin A, Selectin-P and VEGFR showed higher values than expected for controls. Instead, hPL, KISS-1 and PAI-1 values of PE patients were lower than those of controls. Selectin-P was the marker with the most aberrant difference, followed by VEGFR and KISS-1. CONCLUSION: This preliminary analysis revealed that the median values of a panel of mRNAs from the maternal blood of PE patients were different from those of the same gestational age control group at the third trimester. If prospective studies at the second trimester could detect a related marker sufficiently able to discriminate between affected and unaffected patients and thus detect the disease before its clinical onset, then a screening project using a panel of mRNAs would be feasible.

Total activin A in maternal blood as a marker of preterm delivery in low-risk asymptomatic patients.

OBJECTIVES: To retrospectively evaluate whether increased serum levels of total activin A (t-activin A) are found in women who subsequently experience preterm delivery (PTD). METHODS: Data on maternal serum t-activin A concentrations were available from a total of 84 singleton pregnant women and included 14 PTD pregnancies, each matched for gestational age and length of freezer storage, with 5 control pregnancies having term delivery (TD). Analyte values were expressed as multiple(s) of the control median. RESULTS: The median t-activin A for controls and cases was 1.00 +/- 0.45 and 1.27 +/- 0.53 MoM, respectively. Univariate analysis of the MoM values was performed using the Kaplan-Meier algorithm. Differences in the rate of delivery using a t-activin A MoM cut-off of > or = 1 SD (equivalent to 1.26 MoM) were analysed using the log rank test. The cumulative rate of PTD (< 37 weeks) was significantly higher for women with t-activin A concentrations > or = 1.26 MoM than those with t-activin A concentrations below this cut-off (40% vs.. 10%, p-value = 0.0218 log rank test). CONCLUSIONS: T-activin A concentration is higher in women who will develop PTD in a low-risk population. T-activin A values are inversely proportional to the time elapsed from blood test to delivery. Prospective studies would determine the precise discriminability of this marker for PTD and the best week for performing the blood test, allowing for a proper calculation of the detection rate and a positive predictive value.

Time profile of appearance and disappearance of circulating placenta-derived mRNA in maternal plasma.

BACKGROUND: Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied. METHODS: To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR. RESULTS: The earliest gestational age at which human placental lactogen and human chorionic gonadotropin beta-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min. CONCLUSIONS: Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.

Rapid clearance of mRNA for PLAC1 gene in maternal blood after delivery.

OBJECTIVE: To evaluate (1) whether the presence of mRNA for the specific trophoblast gene PLAC1 in maternal whole blood is pregnancy-specific, and (2) whether delivery would result in the clearance of mRNA from maternal blood. METHODS: Sixteen pregnant women at term (41 completed weeks' gestation) were enrolled in the study. Blood samples were obtained before the onset of labor and 24 h after delivery. Eight healthy donors (3 males and 5 non-pregnant women) were used as controls. Total RNA was extracted by means of ABI Prism 6100. A quantitative evaluation was obtained by means of real-time PCR. Wilcoxon test was used to evaluate differences between time intervals. RESULTS: Median concentrations of PLAC1 mRNA relative to the standardization curve (see below) were 44 (2.9-675) ng/ml and 0.48 (0.05-10.7) ng/ml respectively for pre- and post-delivery samples (p value <0.001). Male and non-pregnant female controls did not show any signal of cDNA amplification. CONCLUSION: mRNA transcripts from a placenta-expressed specific gene are detectable in maternal blood and rapidly disappear after delivery. Such an mRNA provides a gender-independent marker for non-invasive prenatal gene expression profiling, and can open new perspectives to monitor those conditions associated to trophoblast damage as well as preeclampsia.

Cell-free fetal DNA (SRY locus) concentration in maternal plasma is directly correlated to the time elapsed from the onset of preeclampsia to the collection of blood.

OBJECTIVE: To determine (1) if fetal DNA (fDNA) in the maternal circulation in women affected by preeclampsia correlates with the time elapsed from the onset of symptoms to the time of blood collection, and (2) if the inclusion of this variable improves the discrimination between affected and unaffected patients by using fDNA distributions. METHODS: Plasma were collected from 34 women at 33.7 +/- 3.9 weeks' gestation, affected by preeclampsia, and bearing a single male fetus. fDNA was extracted from 1.5-mL plasma samples, and the SRY and beta-globin gene were analyzed by real-time quantitative PCR. MoMs (multiple of the control median) were calculated by using a log equation of 102 normal cases. Log MoMs were then plotted against the time elapsed from onset of symptoms to blood collection (expressed in days) by means of a log-linear regression. Adjusted MoMs were then calculated. ROC curves were used to test the discrimination obtained by using adjusted MoMs. RESULTS: The median MoMs of controls and preeclamptic patients were 1.00 +/- 1.53 and 2.62 +/- 2.70 respectively. By plotting log MoM fDNA against the time elapsed from onset of symptoms to blood collection, we found a significant positive correlation, (p-value < 0.001, R2 = 0.55, F = 38.97, from 1 to 50 days). The adjusted median fDNA MoM was 2.66 +/- 2.50. Areas under the curves, as estimated by ROC curves, were 76.7 for unadjusted and 85.5 for adjusted MoMs respectively (p-value = 0.02). CONCLUSIONS: The effect of a further covariate showed that (1) fDNA passage from trophoblasts to maternal circulation for unit of time is proportional to the duration of the damage and that (2) increased discrimination can be obtained in comparison to normal subjects.