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.

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.

Effects of Hormone Therapy and Flavonoids Capable on Reversal of Menopausal Immune Senescence.

Menopause, probably the most important natural change in a woman's life and a major component of female senescence, is characterized, inter alia, by cessation of ovarian estrogen and progesterone production, resulting in a gradual deterioration of the female immune system. Hormone replacement therapy (HRT) is used in postmenopausal women to relieve some of the peri- and postmenopausal symptoms, while there is also evidence that the therapy may additionally partially reverse menopausal immune senescence. Flavonoids, and especially isoflavones, are widely used for the treatment of menopausal symptoms, although it is not at present clear whether they can reverse or alleviate other menopausal changes. HRT reverses the menopausal CD4/CD8 ratio and also limits the general peri- and postmenopausal inflammatory state. Moreover, the increased levels of interleukins (IL)-1ß, IL-6, and IL-8, as well as of tumor necrosis factor-α (TNF-α) are decreased after the initiation of HRT. However, some reports show no effect of HRT on IL-4, IL-10, and IL-12. It is thus evident that the molecular pathways connecting HRT and female immune senescence need to be clarified. Interestingly, recent studies have suggested that the anti-inflammatory properties of isoflavones possibly interact with inflammatory cytokines when applied in menopause treatments, thereby potentially reversing immune senescence. This narrative review presents the latest data on the effect of menopausal therapies, including administration of flavonoid-rich products, on age-associated immune senescence reversal with the aim of revealing possible directions for future research and treatment development.

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.

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.

Two novel variants in the TCF12 gene identified in cases with craniosynostosis.

Craniosynostosis (CS) is a condition where one or more of the cranial sutures fuse prematurely. It affects almost 1/2,000 newborns, and includes both syndromic and non-syndromic cases. To date, variants in over 70 different genes have been associated with the expression of CS. In this report, we describe two unrelated cases that presented with coronal CS. TCF12 sequencing analysis revealed novel frameshift nucleotide variants, which were evaluated as pathogenic according to the current guidelines for interpreting sequence variants. These findings expand the spectrum of TCF12 gene variants related with CS and support the importance of screening for such variants in patients with coronal synostosis.

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.

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.

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.

In situ assessment of PI3K and PTEN alterations in mycosis fungoides: correlation with clinicopathological features.

Deregulated signalling through phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in tumour initiation and progression. We have already shown that AKT is activated in skin lesions in Mycosis Fungoides (MF) and we herein further investigate the frequency and clinical significance of PTEN and PI3K at the protein and at the DNA level as well as the presence of AKT1 mutations in skin lesions from 50 patients with MF clinical stages I-IV in relation to clinicopathological features. Increased p-AKT expression correlated with poor prognosis in plaques (P = 0.0198), whereas p-AKT was an independent predictor of poor survival in the entire cohort (P = 0.017, HR = 1.012). PTEN cytoplasmic expression was found low or absent in all 77.3% of cases and inversely correlated with advanced clinical stages (P = 0.0744). Molecular analysis showed no AKT1 mutation, no PI3KCA copy number gain, only 1 case with PI3KCA mutation in exon 9 and 3 cases with PTEN mutations (7%) in exons 7, 8 and 5. The latter correlated with disease (P = 0.0253) and progression (P < 0.0001) free survival in tumour stage. Although activation of PI3K/AKT signalling pathway due to PTEN alterations is rarely attributed to abnormalities in PTEN, PI3K, and AKT1 genes, PTEN mutations exert a negative effect on patients' prognosis with tumours.

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms.

BACKGROUND: Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy. METHODS: Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients' clinical characteristics and pathological measures. RESULTS: In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied. CONCLUSIONS: Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

Investigation of FANCA mutations in Greek patients.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disease characterized by considerable heterogeneity. Fifteen subtypes are currently recognised and deletions of the Fanconi anemia complementation group A (FANCA) gene account for more than 65% of FA cases. We report on the results from a cohort of 166 patients referred to the Department of Medical Genetics of Athens University for genetic investigation after the clinical suspicion of FA. MATERIALS AND METHODS: For clastogen-induced chromosome damage, cultures were set up with the addition of mitomycin C (MMC) and diepoxybutane (DEB), respectively. Following a positive cytogenetic result, molecular analysis was performed to allow identification of causative mutations in the FANCA gene. RESULTS: A total of 13/166 patients were diagnosed with FA and 8/13 belonged to the FA-A subtype. A novel point mutation was identified in exon 26 of FANCA gene. CONCLUSION: In our study 62% of FA patients were classified in the FA-A subtype and a point mutation in exon 26 was noted for the first time.

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.

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.