First Trimester Maternal Plasma Aberrant miRNA Expression Associated with Spontaneous Preterm Birth.

Spontaneous Preterm Delivery (sPTD) is one of the leading causes of perinatal mortality and morbidity worldwide. The present case−control study aims to detect miRNAs differentially expressed in the first trimester maternal plasma with the view to identify predictive biomarkers for sPTD, between 320/7 and 366/7 weeks, that will allow for timely interventions for this serious pregnancy complication. Small RNA sequencing (small RNA-seq) of five samples from women with a subsequent sPTD and their matched controls revealed significant down-regulation of miR-23b-5p and miR-125a-3p in sPTD cases compared to controls, whereas miR-4732-5p was significantly overexpressed. Results were confirmed by qRT-PCR in an independent cohort of 29 sPTD cases and 29 controls. Statistical analysis demonstrated that miR-125a is a promising early predictor for sPTL (AUC: 0.895; 95% CI: 0.814-0.972; p < 0.001), independent of the confounding factors tested, providing a useful basis for the development of a novel non-invasive predictive test to assist clinicians in estimating patient-specific risk.

Plasma biomarkers for the identification of women at risk for early-onset preeclampsia.

BACKGROUND: To identify potential biomarkers in the 1st trimester of pregnancy for the identification of women destined to develop early onset preeclampsia (EOPE). METHODS: Blood samples were obtained from pregnant women at 11-13 weeks of gestation. Women were followed up until delivery. Five samples from EOPE complicated pregnancies and 5 from unaffected ones were analysed using 2-DE and MALDI-TOF-TOF MS/MS. The altered expression of selected proteins was verified by ELISA in an extended sample cohort. RESULTS: Twelve proteins were differentially expressed in the plasma of women who subsequently developed EOPE as compared to controls. Alpha-1-antitrypsin (A1AT), CD5 antigen-like molecule (CD5L) Keratin, type I cytoskeletal 9 (K1C9), Myeloid cell nuclear differentiation antigen (MNDA), Transferrin (TRFE) and Vitamin D-binding protein (VTDB) were up-regulated with fold changes 3.14, 2.18, 1.53, 1.53, 4.26 3.38 respectively, whereas Alpha-2-HS-glycoprotein (FETUA), Beta-2-glycoprotein 1 (APOH), Complement factor B (CFAB), Haptoglobin (HPT), Vitronectin (VTNC) and Zinc-alpha-2-glycoprotein (ZA2G) were down-regulated with fold changes -0.38, -0.76, -0.24, -0.47, -0.23, and -0.50 respectively. The down-regulation of APOH, VTNC and HPT was verified using ELISA. CONCLUSIONS: The differentially expressed proteins represent potential biomarkers for the early screening for EOPE. Follow-up experiments however are necessary for evaluation.

RASSF1A in maternal plasma as a molecular marker of preeclampsia.

OBJECTIVES: This study aimed to quantitate cell free (cf) and cell free fetal (cff) DNA in maternal plasma by determining RASSF1A levels before and after enzyme digestion in women who subsequently developed preeclampsia (PE) and compare them with uncomplicated pregnancies. METHODS: Twenty-four samples from pregnant women who developed PE and 48 samples from women with uncomplicated pregnancies were analysed. Blood samples were obtained at 11-13 weeks. cfDNA was determined by quantifying RASSF1A using qRT-PCR. A second qRT-PCR was performed following methylation-sensitive enzyme digestion by BstUI, to quantitate hypermethylated RASSF1A sequences of fetal origin. ACTB gene was used as control to confirm complete enzyme digestion. RESULTS: cfDNA and cffDNA levels were significantly increased in women who developed PE as compared with uncomplicated pregnancies (median cfDNA: 9402 vs 2698, median cffDNA: 934.5 vs 62, respectively). Following operating characteristic curve analysis, cut-off values of 7486 Εq/mL for cfDNA and 512 Εq/mL for cffDNA were chosen, which provided a sensitivity of 75% and 100% and specificity of 98% and 100%, respectively, to identify women at risk for PE. CONCLUSIONS: The study demonstrates potential use of cfDNA and cffDNA in maternal plasma as markers for the early prediction of women at risk for PE.

First trimester maternal plasma proteomic changes predictive of spontaneous moderate/late preterm delivery.

OBJECTIVE: Identification of differentially expressed proteins (DEPs) in first trimester maternal plasma between pregnant women with a subsequent spontaneous moderate/late Preterm Delivery (sPTD) and women who delivered at term. The sPTD group consisted of women who delivered between 32°/7 and 366/7 weeks of gestation. METHODS: Isobaric tags for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS was used for the analysis of five first trimester maternal plasma samples obtained from women with a subsequent moderate/late preterm sPTD and five women with term deliveries. Enzyme-linked immunosorbent assay (ELISA) was further applied in an independent cohort of 29 sPTD cases and 29 controls to verify the expression levels of selected proteins. RESULTS: 236 DEPs, mainly linked to coagulation and complement cascade, were identified in first trimester maternal plasma obtained from the sPTD group. Decreased levels of selected proteins, namely, VCAM-1, SAA, and Talin-1, were further confirmed using ELISA, highlighting their potential as candidate predictive biomarkers for sPTD at32°/7 and 366/7 weeks of gestation. CONCLUSION: First trimester maternal plasma proteomic analysis revealed protein changes associated with subsequent moderate/late preterm sPTD.

Quantitative Comparative Proteomics Reveals Candidate Biomarkers for the Early Prediction of Gestational Diabetes Mellitus: A Preliminary Study.

AIM: To identify differentially expressed proteins (DEPs) in 1st trimester maternal plasma between pregnant women at risk for gestational diabetes mellitus (GDM) and uncomplicated controls. MATERIALS AND METHODS: First-trimester plasma from five women who developed GDM and five from non-diabetic ones were analyzed using isobaric tag for relative and absolute quantitation - labeled proteomics. Enzyme-linked immunosorbent assay was further applied in an independent cohort of 25 GDM cases and 25 controls for verification. RESULTS: Prenylcysteine oxidase 1 (PCYOX1), beta-ala-his dipeptidase (CNDP1), extracellular matrix protein 1 (ECM1), basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2), thrombospondin-4 (TSP-4) demonstrated significant differences in expression between the two groups (p<0.05). DEPs are mainly associated with complement and coagulation cascades. CONCLUSION: The reported plasma proteomic changes represent potential biomarkers for the early identification of women at risk for GDM. Future studies using larger and more diverse cohorts are necessary to assess the clinical utility of these findings.

Deep Sequencing Identified Dysregulated Circulating MicroRNAs in Late Onset Preeclampsia.

BACKGROUND/AIM: To characterize global microRNA (miRNA) expression profile in the first trimester maternal plasma of women who subsequently develop late-onset preeclampsia (LOPE) compared to uncomplicated pregnancies. MATERIALS AND METHODS: Five first trimester plasma samples from women who developed LOPE and 5 controls were analyzed using next generation sequencing technology (NGS) followed by target prediction, Gene Ontology analysis and pathway identification. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for confirmation in an independent cohort of 12 LOPE cases and 12 controls. RESULTS: miR-23b-5p and miR-99b-5p were down-regulated by >1.5 fold in LOPE complicated pregnancies (p value <0.05) compared to controls. Target prediction showed that the major targets of these miRNAs are associated with glycometabolism and immune response. CONCLUSION: miR-23b-5p and miR-99b-5p are possibly implicated in the pathogenic mechanisms leading to the induction of LOPE and may serve as candidate non-invasive biomarkers for early prediction and prevention.

Dysregulated placental microRNAs in Early and Late onset Preeclampsia.

INTRODUCTION: To determine the miRNA expression profile in placentas complicated by Preeclampsia (PE) and compare it to uncomplicated pregnancies. METHODS: Sixteen placentas from women with PE, [11 with early onset PE (EOPE) and 5 with late onset PE (LOPE)], as well as 8 placentas from uncomplicated pregnancies were analyzed using miRNA microarrays. For statistical analyses the MATLAB® simulation environment was applied. The over-expression of miR-518a-5p was verified using Quantitative Real-Time Polymerase Chain Reaction. RESULTS: Forty four miRNAs were found dysregulated in PE complicated placentas. Statistical analysis revealed that miR-431, miR-518a-5p and miR-124* were over-expressed in EOPE complicated placentas as compared to controls, whereas miR-544 and miR-3942 were down-regulated in EOPE. When comparing the miRNA expression profile in cases with PE and PE-growth restricted fetuses (FGR), miR-431 and miR-518a-5p were found over-expressed in pregnancies complicated by FGR. DISCUSSION: Since specific miRNAs can differentiate EOPE and LOPE from uncomplicated placentas, they may be considered as putative PE-specific biomarkers. MiR-518a-5p emerged as a potential diagnostic indicator for EOPE cases as well as for PE-FGR complicated placentas, indicating a potential link to the severity of the disease.

miRNAs in pregnancy-related complications.

MicroRNAs (miRNAs) constitute a highly conserved class of small non-coding RNAs, involved in post-transcriptional regulation processes by modifying the expression of specific mRNAs. During placental development, cell differentiation, adhesion, migration, apoptosis and angiogenesis are regulated by specific miRNAs and aberrant expression has been associated with the pathogenesis of pregnancy-related complications. Recent studies focusing on placental and maternal peripheral blood miRNA profiling showed different expression between normal and complicated pregnancies, providing valuable information about the pathophysiological role of miRNAs and identifying potential biomarkers for monitoring pregnancy complications. This review summarizes the current knowledge in the field and presents the possible use of miRNAs as biomarkers for early detection and monitoring of these complications.

Urine proteomic studies in preeclampsia.

Preeclampsia (PE) is a multisystem disorder of pregnancy that develops after 20 wk of gestation in previously normotensive women and complicates 5-8% of pregnancies. This rapidly progressive syndrome is usually diagnosed when the mother develops hypertension and proteinuria. The only effective treatment is delivery of the baby although early low-dose aspirin has been shown to significantly reduce the risk for PE. Recent advances in proteomic methods of protein separation, identification, and quantitation may allow for the identification of proteins and peptides that could facilitate early detection of disease, improve assessment of prognosis, and allow closer monitoring of women at risk for PE. This review summarizes all currently available markers for prediction and diagnosis of PE and presents urine proteomic studies performed for the identification of novel biomarkers.

Validation of serum biomarkers derived from proteomic analysis for the early screening of preeclampsia.

AIM: To examine the potential value of previously identified biomarkers using proteomics in early screening for preeclampsia (PE). METHODS: 24 blood samples from women who subsequently developed PE and 48 from uncomplicated pregnancies were obtained at 11-13 weeks and analysed after delivery. Cystatin-C, sVCAM-1, and Pappalysin-1 were quantified by ELISA. Maternal characteristics and medical history were recorded. RESULTS: Median values of Cystatin-C, sVCAM-1, and Pappalysin-1 in the PE group as compared to controls were 909.1 gEq/mL versus 480.0 gEq/mL, P = .000, 832.0 gEq/mL versus 738.8 gEq/mL, P = .024, and 234.4 gEq/mL versus 74.9 gEq/mL, P = .064, respectively. Areas under the receiver-operating characteristic curves (AUC, standard error (SE)) for predicting PE were Cystatin-C: 0.90 (SE 0.04), VCAM-1: 0.66 (SE 0.074), and Pappalysin-1: 0.63 (SE 0.083). To discriminate between cases at risk for PE and normal controls, cut-off values of 546.8 gEq/mL for Cystatin-C, 1059.5 gEq/mL for sVCAM-1, and 220.8 gEq/mL for Pappalysin-1 were chosen, providing sensitivity of 91%, 41%, and 54% and specificity of 85%, 100%, and 95%, respectively. CONCLUSIONS: sVCAM-1 and Pappalysin-1 do not improve early screening for PE. Cystatin-C, however, seems to be associated with subsequent PE development, but larger studies are necessary to validate these findings.

Review: advances in non-invasive prenatal diagnosis.

The invasive procedures amniocentesis and chorionic villus sampling are routinely applied in pregnancies at risk for fetal genetic disorders and the results obtained are the gold standard for prenatal diagnosis. These procedures have an approximately 0.5-1% risk for fetal loss and are mainly used in cases at risk for fetal chromosomal abnormalities and single-gene disorders. Identification of cell-free fetal nucleic acids (DNA and RNA) in maternal plasma and the recognition that they represent a useful source of fetal genetic material for prenatal diagnosis has led to intensive efforts to develop non-invasive prenatal testing. This review summarizes recent developments in the field of non-invasive prenatal diagnosis through the use of cell-free fetal nucleic acids in maternal circulation during pregnancy and provides an overview of the possibilities for future clinical applications.

Non-invasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma: Progress overview beyond predictive and personalized diagnosis.

The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma allowed for the development of alternative methodologies that may facilitate safe non-invasive prenatal diagnosis (NIPD). The low concentration of cffDNA in maternal plasma, however, and the coexistence of maternal DNA limit its clinical application to the detection or exclusion of fetal targets that are not present in the mother, such as Y chromosome sequences, the RHD gene in a RhD-negative woman and genetic conditions inherited from the father. Strategies for NIPD of monogenic disorders and fetal chromosomal aneuploidies have also been achieved using next-generation sequencing and could be introduced to the clinics as soon as cost-effective and high throughput protocols are developed.

A multiplex PCR for non-invasive fetal RHD genotyping using cell-free fetal DNA.

AIM: To design a protocol for non-invasive prenatal diagnosis of fetal Rhesus D (RhD) status. MATERIALS AND METHODS: A total of 112 single lymphocytes were used to test the efficiency of the assay. The protocol was validated using blood samples from 84 RhD-negative pregnant women at 7-24 weeks of gestation. Cell-free DNA (cfDNA) was enzymatically digested using AciI and analyzed by a polymerase chain reaction (PCR) that allowed simultaneous amplification of RHD exons 7 and 10, SRY, RASFF1A and ACTB. RESULTS: On the one genome-equivalent level, the efficiency of the protocol was ≥ 94.6% for each locus amplified. Conclusive results from the first set of PCRs were obtained for 79 cases with one false-positive. In five cases the analysis was repeated and, subsequently, all cases were accurately diagnosed. CONCLUSION: The proposed protocol is rapid, applicable in most molecular diagnostic laboratories and provides the basis for non-invasive examination of fetal RhD with 96.7% specificity and 100% sensitivity.