Biochemical and Anthropometric Parameters for the Early Recognition of the Intrauterine Growth Restriction and Preterm Neonates at Risk of Impaired Neurodevelopment.

BACKGROUND: S100B and Tau are implicated with both brain growth and injury. Their urinary levels in 30-to-40-day-old full-term, preterm, IUGR, and preterm-IUGR subjects were measured to investigate their possible relationship with future delayed neurodevelopment. METHODS: Values were related to the neuro-behavioral outcome at two years of age, as well as to brain volumes and urinary NGF assessed at the same postnatal time point. RESULTS: Using the Griffiths III test, cognitive and motor performances were determined to establish subgroups characterized by either normal or impaired neuro-behavior. The latter included preterm, IUGR, and preterm-IUGR individuals who exhibited significantly higher and lower S100B and Tau levels, respectively, along with markedly reduced cerebral volumes and urinary NGF, as previously demonstrated. Contrary to NGF, however, Tau and S100B displayed a weak correlation with brain volumes. CONCLUSIONS: Delayed cognitive and motor performances observed in two-year-old preterm and IUGR-born individuals were also found to be associated with anomalous urinary levels of S100B and Tau, assessed at 30-40 days of the postnatal period, and their changes did not correlate with brain growth. Thus, our data suggests that, in addition to cerebral volumes and NGF, urinary S100B and Tau can also be considered as valuable parameters for the early detection of future neurodevelopmental abnormalities.

Microbiota-Associated HAF-EVs Regulate Monocytes by Triggering or Inhibiting Inflammasome Activation.

In pregnancy, human amniotic fluid extracellular vesicles (HAF-EVs) exert anti-inflammatory effects on T cells and on monocytes, supporting their immunoregulatory roles. The specific mechanisms are still not completely defined. The aim of this study was to investigate the ability of HAF-EVs, isolated from pregnant women who underwent amniocentesis and purified by gradient ultracentrifugation, to affect inflammasome activation in the human monocytes. Proteomic studies revealed that HAF-EV samples expressed several immunoregulatory molecules as well as small amounts of endotoxin. Surprisingly, metagenomic analysis shows the presence of specific bacterial strain variants associated with HAF-EVs as potential sources of the endotoxin. Remarkably, we showed that a single treatment of THP-1 cells with HAF-EVs triggered inflammasome activation, whereas the same treatment followed by LPS and ATP sensitization prevented inflammasome activation, a pathway resembling monocyte refractories. A bioinformatics analysis of microbiota-HAF-EVs functional pathways confirmed the presence of enzymes for endotoxin biosynthesis as well as others associated with immunoregulatory functions. Overall, these data suggest that HAF-EVs could serve as a source of the isolation of a specific microbiota during early pregnancy. Moreover, HAF-EVs could act as a novel system to balance immune training and tolerance by modulating the inflammasome in monocytes or other cells.

First trimester HtrA1 maternal plasma level and spontaneous preterm birth.

OBJECTIVES: High temperature requirement A1 (HtrA1) is a serine protease detected in maternal plasma and in placental tissues during normal gestation and in various pathological conditions. The purpose of this study was to determine whether the maternal plasma concentration of HtrA1 in first trimester, alone or combined with other maternal factors, can be used to identify women at risk for spontaneous preterm birth (SPTB). STUDY DESIGN: This is a cohort study on pregnant women at 12 weeks of gestation recruited between 2014 and 2016 and prospectively followed until delivery. One hundred and fifty-nine women were included in the study: 140 women delivered at term and 19 (11.9%) delivered spontaneously preterm. Plasma samples were assessed for HtrA1 by ELISA and data were compared between women which delivered at term with women which delivered preterm. A multiple logistic regression analysis was used to estimate the independent effect of women's characteristics on the probability of a SPTB. RESULTS: SPTB was significantly associated with log HtrA1 values at 12 weeks of gestation, BMI before pregnancy and physical activity. In particular, the probability of a SPTB increases of 79% for every added unit of log HtrA1, while decreases of 18% for every added unit of BMI. In addition, physical activity was found as an important protective factor. The ROC curve showed that the model had a good accuracy in predicting SPTB, with an AUC equal to 0.83 (95%CI: 0.73-0.91). CONCLUSIONS: Maternal plasma HtrA1 may be considered a marker of SPTB. In addition, our model indicates two factors that could be modified to reduce the risk of SPTB, i.e. BMI before pregnancy and maternal physical activity.

Pre-eclampsia predictive ability of maternal miR-125b: a clinical and experimental study.

Pre-eclampsia (PE) is a systemic maternal syndrome affecting 2-8% of pregnancies worldwide and involving poor placental perfusion and impaired blood supply to the foetus. It manifests after the 20th week of pregnancy as new-onset hypertension and substantial proteinuria and is responsible for severe maternal and newborn morbidity and mortality. Identifying biomarkers that predict PE onset prior to its establishment would critically help treatment and attenuate outcome severity. MicroRNAs are ubiquitous gene expression modulators found in blood and tissues. Trophoblast cell surface antigen (Trop)-2 promotes cell growth and is involved in several cancers. We assessed the PE predictive ability of maternal miR-125b in the first trimester of pregnancy by measuring its plasma levels in women with normal pregnancies and with pregnancies complicated by PE on the 12th week of gestation. To gain insight into PE pathogenesis we investigated whether Trop-2 is targeted by miR-125b in placental tissue. Data analysis demonstrated a significant association between plasma miR-125b levels and PE, which together with maternal body mass index before pregnancy provided a predictive model with an area under the curve of 0.85 (95% confidence interval, 0.70-1.00). We also found that Trop-2 is a target of miR-125b in placental cells; its localization in the basal part of the syncytiotrophoblast plasma membrane suggests a role for it in the early onset of PE. Altogether, maternal miR-125b proved a promising early biomarker of PE, suggesting that it may be involved in placental development through its action on Trop-2 well before the clinical manifestations of PE.

Association between first trimester plasma htra1 level and subsequent preeclampsia: A possible early marker?

INTRODUCTION: Preeclampsia (PE) is associated with risk of maternal and fetal mortality and morbidity. Several promising predictors of PE have been identified, but early pregnancy screening for PE remains insufficient, and randomized controlled trials that used biomarkers to identify high-risk women have been disappointed. Our aim is to identify a possible early marker of PE. METHODS: 158 women attending a routine antenatal care visit were recruited from 2014 to 2016 and prospectively followed until delivery (14 of whom had a diagnosis of PE). We have tested the plasma concentration of High temperature requirement factor A1 (HtrA1) at 12 weeks of gestation by ELISA technique in order to identify women at risk for developing PE. A multiple logistic regression analysis was used to estimate the independent effect of women' characteristics on the probability of developing PE. Likelihood ratio test and Hosmer-Lemeshow test were used to select the most parsimonious model and to evaluate the model's goodness of fit. Predictiveness of preeclampsia was estimated by ROC curve. RESULTS: PE cases had significantly higher BMI, before and after pregnancy, shorter gestational age at delivery and higher HtrA1values than healthy women. In addition, higher HtrA1 values in the first trimester maternal plasma, BMI before pregnancy and gestational age at delivery are significantly associated with subsequent development of PE. ROC curve showed a good accuracy in predicting preeclampsia, with an AUC of 0.83. CONCLUSIONS: These results suggest the HtrA1 as early predictive marker of PE having a strong clinical relevance for disease prevention.

Induction of the apoptotic pathway by oxidative stress in spontaneous preterm birth: Single nucleotide polymorphisms, maternal lifestyle factors and health status.

The purpose of the present study was to search for associations between spontaneous preterm birth (sPTB), single nucleotide polymorphisms (SNPs) associated with the apoptotic pathway as triggered by oxidative stress, maternal lifestyle and health status. SNP genotyping [rs7560 for c-Jun N-terminal kinase (JNK), rs9517320 for mammalian STE20-like protein kinase 3 (MST3), rs1049216 for caspase 3 (CASP3)] in the placenta and maternal blood of 300 controls with at-term birth and 43 cases of sPTB was performed. No association was identified in genotype frequencies or combinations of foetal/maternal genotypes between single SNPs and sPTB. The risk of sPTB was significantly reduced by physical activity and significantly increased by current hypertensive diseases, premature rupture of membranes (PROM) or preterm PROM (P-PROM) and previous sPTB. The TT/GA genotype of JNK/CASP3 in maternal blood and maternal health status (current hypertensive diseases, current PROM/P-PROM, previous sPTB) were independently associated with sPTB. The present findings suggested that, independently of other maternal factors, pregnant women carrying the TT/GA genotype of JNK/CASP3 were more susceptible to sPTB than women bearing the GT/GA (our reference) genotype; that the apoptotic pathway triggered by oxidative stress was involved; and that genetic and non-genetic factors contributed to sPTB. Knowledge of these aspects may aid to improve the management of pregnancies by indicating the lifestyle to be adopted on the basis of sPTB susceptibility.

Intrapartum test for detection of Group B Streptococcus colonization during labor.

PURPOSE: The purpose of this study was to evaluate the potential improvement of introducing an intrapartum test for the detection of Group B Streptococcus (GBS) during labor and to estimate its cost-effectiveness versus antepartum GBS screening culture. MATERIALS AND METHODS: Three hundred and thirteen women at beginning of labor, with unknown GBS status or with antepartum GBS screening culture were enrolled. A vaginal-rectal specimen was collected from each woman for GBS detection by real-time PCR. Results of intrapartum test and antepartum GBS screening culture were compared. RESULTS: Antepartum culture results did not always reflect the intrapartum maternal GBS colonization status since in 15.1% of the cases it was not concordant with intrapartum test. However, selecting only women, who underwent antepartum culture and intrapartum test at the same time, the percentage of concordance was 96.6%. Based on intrapartum test results, 74.9% of the total number of intrapartum antibiotic prophylaxis (IAP) was administered uselessly, while 1.9% of women did not receive IAP although they were positive to intrapartum test. Intrapartum test resulted less cost-effective than antepartum culture but it became more cost-effective at a cost threshold of about 16.00 €. CONCLUSIONS: The clinical introduction of intrapartum test could be a valuable mean for identification of GBS colonization during labor, allowing an appropriate management of mothers and neonates with consequent benefit for their health and with limited costs for Healthcare System.

Evaluation of quantitative fFn test in predicting the risk of preterm birth.

OBJECTIVE: To evaluate diagnostic accuracy of quantitative fetal fibronectin (qfFN) test in predicting preterm birth (PTB) risk <34 weeks' gestation or within 14 days from testing. We explored the predictive potential of the test in five-predefined PTB risk categories based on predefined qfFN thresholds (<10, 10-49, 50-199, 200-499 and ≥500 ng/mL). METHODS: Measurement of cervicovaginal qfFN with Rapid fFN 10Q System (Hologic) in 126 women with singleton pregnancy (23-33 weeks' gestation) reporting signs and symptoms indicative of preterm labour (PTL). RESULTS: For PTB prediction risk <34 weeks' gestation, sensitivity decreased from 100% to 41.7% and specificity increased from 0% to 99.1% with increasing fFN thresholds. Positive predictive value (PPV) increased from 9.5% to 83.3% with increasing qfFN thresholds, while negative predictive value (NPV) was higher than 90% among the fFN-predefined categories. Diagnostic accuracy results showed an area under a receiving operator characteristic (ROC) curve of 84.5% (95% CI, 0.770-0.903). For delivery prediction within 14 days from the testing, sensitivity decreased from 100% to 42.8% and specificity increased from 0% to 100% with increasing fFN thresholds. Diagnostic accuracy determined by the ROC curve was 66.1% (95% CI, 0.330-0.902). CONCLUSIONS: The QfFN thresholds of tests are a useful tool to distinguish pregnant women for PTB prediction risk <34 weeks' gestation.

Impact of Probiotic SYNBIO(®) Administered by Vaginal Suppositories in Promoting Vaginal Health of Apparently Healthy Women.

The purpose of this study was to investigate whether vaginal administration of probiotic Lactobacillus results in their colonization and persistence in the vagina and whether it promotes normalization and maintenance of pH and Nugent score. A single-arm, open-label controlled towards the baseline (pre-post) study including 35 apparently healthy women was conducted. Each woman was examined three times during the study. Women were instructed to receive daily for 7 days, the probiotic suppositories SYNBIO(®) gin (Lactobacillus rhamnosus IMC 501(®) and Lactobacillus paracasei IMC 502(®)). Vaginal swabs were collected during visit 1, 2, and 3 to determine the total lactobacilli count, the presence of the two administered bacteria, the measure of the pH, and the estimation of Nugent score. Evaluation of treatment tolerability was based on analysis of the type and occurrence of adverse events. The probiotic vaginal suppository was well tolerated and no side effects were reported. Intermediate Nugent score was registered in 40 % of women at visit 1 and these intermediate scores reverted to normal at day 7 (end of treatment) in 20 % of subjects. Administration of SYNBIO(®) gin contributed to a significant increase in the lactobacilli level at visit 2. Molecular typing revealed the presence of the two strains originating from SYNBIO(®) gin in 100 % of women at visit 2 and 34 % at visit 3. No significant changes were registered for pH between visits. The SYNBIO(®) gin product is safe for daily use in healthy women and it could be useful to restore and maintain a normal vaginal microbiota.

Non-Invasive Prenatal RHD Genotyping Using Cell-Free Fetal DNA from Maternal Plasma: An Italian Experience.

BACKGROUND: This study assessed the diagnostic accuracy of a non-invasive approach to fetal RHD genotyping using cell-free fetal DNA in maternal plasma and a combination of methodological strategies. METHODS: Real-time PCR (qPCR) was performed on 216 RhD-negative women between weeks 10+0 and 14+6 of gestation (1st qPCR). qPCR was repeated (2nd qPCR) to increase the amount of each sample for analysis, on 95 plasma aliquots that were available from first trimester blood collection (group 1) and on 13 samples that were collected between weeks 18+0 and 25+6 of gestation (group 2). qPCR was specific for exons 5 and 7 of the RHD gene (RHD5 and RHD7). The results were interpreted according to the number of positive replicates of both exons. RESULTS: 1st qPCR: diagnostic accuracy was of 93.3%. Diagnostic accuracy increased from 90.5% (1st qPCR) to 93.7% (2nd qPCR) in group 1 and from 84.6% (1st qPCR) to 92.3% (2nd qPCR) in group 2. These increments were not statistically significant. CONCLUSION: Our approach to RHD genotyping in early pregnancy yielded high diagnostic accuracy. Increasing the amount of DNA analyzed in each sample did not improve significantly the diagnostic accuracy of the test.

Body mass index associated to rs2021966 ENPP1 polymorphism increases the risk for gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a condition of impaired glucose tolerance occurring in 1-14% of all pregnancies. This wide range reflects pathological involvement of single nucleotide polymorphisms (SNPs) and maternal weight as risk factors. This study evaluated the association of genetic component and maternal factors to identify women with higher risk of developing GDM. About 240 pregnant women characterized by negative Oral Glucose Tolerance Test (-OGTT) and 38 with positive OGGT (+OGTT) were enrolled. SNPs for ENPP1, NRF1, VEGFA, CEBPA, and PIK3R1 were analyzed by SNP genotyping. An association study was performed and differences in genotype and allele frequencies between cases and controls were analyzed by χ(2) test. +OGTT was associated to high values of pre-gestational body mass index (BMI) and age. SNP for ENPP1 gene was associated to +OGTT, while genetic variants for other genes did not correlate to GDM. ENPP1 homozygous for A allele and heterozygous showed altered frequencies in +OGTT when compared with -OGTT. Association of both pre-gestational BMI and age with AA homozygous genotype increased significantly the risk to +OGTT. Our results demonstrate that correlation of age and pre-gestational BMI with homozygous for A allele increased significantly the risk of impaired glucose tolerance and GDM.

The potential usefulness of free fetal DNA in maternal blood for prenatal fetal gender determination in multiple pregnancies.

We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11-14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.

Identification of universal mRNA markers for noninvasive prenatal screening of trisomies.

OBJECTIVE: The discovery of placental transcripts in peripheral blood of pregnant women prompted us to investigate which was the most appropriate biological specimen, between plasma and serum, to easily detect them and to exploit hPL (human placental lactogen), betahCG (human chorionic gonadotrophin beta-subunit), LOC90625, and TFPI2 (tissue factor pathway inhibitor 2) levels in order to establish whether an abnormal variation degree of presence of these placental transcripts are likely to be associated to specific fetal trisomies. METHOD: RNA was extracted from plasma and serum samples of 255 pregnant women bearing euploid fetuses, 17 bearing fetuses affected by trisomy 21 and 10 with fetuses affected by trisomy 18. Placental transcript analysis was performed by real time RT-PCR using relative quantification. RESULTS: Results obtained from euploid samples showed that fetal transcripts were more abundant in plasma than in serum samples. Euploid samples had a placental transcript abundance distinguishable from those with trisomy 21 but not from those with trisomy 18. In particular, high betahCG abundance and advanced maternal age were significantly associated with trisomy 21 pregnancy. CONCLUSION: Plasma was the most suitable tool to be employed in the detection and dosage of placental transcripts. betahCG transcript together with maternal age could be a potential marker for noninvasive prenatal screening of fetal trisomy 21.

Persistence of male hematopoietic CD34+ cells in the circulation of women does not affect prenatal diagnostic techniques.

OBJECTIVE: We aimed to verify whether fetal microchimerism, because of persisting fetal hematopoietic CD34(+) cells from previous pregnancies, could interfere with the development of genetic tests based on using these cells, isolated from maternal blood for the diagnosis of fetal aneuploidies. STUDY DESIGN: CD34(+) cells, isolated from blood of parous women with at least 1 son and nulliparous women, were analyzed by using qualitative polymerase chain reaction (PCR), quantitative PCR, and fluorescence in situ hybridization (FISH) to establish whether these molecular techniques are concurrently capable of detecting circulating male DNA. RESULTS: By qualitative PCR, male DNA was found both in parous and nulliparous women, whereas by quantitative PCR and FISH analyses, no male DNA or male nuclei were revealed except in 1 cultured CD34(+) sample from a nulliparous woman. CONCLUSION: Fetal hematopoietic CD34(+) cells can be used in the noninvasive prenatal testing of fetal aneuploidies because the presence of fetal microchimerism does not affect fetal diagnosis in current pregnancies.

The best approach for early prediction of fetal gender by using free fetal DNA from maternal plasma.

OBJECTIVES: Detection of free fetal DNA (ffDNA) in maternal blood during pregnancy has given rise to the possibility of developing new noninvasive approaches for early prenatal diagnosis. On a large-scale study, two protocols of real-time polymerase chain reaction (PCR) were compared in order to establish which Y-specific locus, either multicopy DYS14 or single copy SRY sequence, was the most suitable for developing a test with high diagnostic efficiency for early fetal gender assessment. The second aim was to assess whether the combination of the two detection systems could increase the performance of the prenatal test. METHODS: We analyzed 145 plasma samples from healthy pregnant women between 11 and 12 weeks of singleton gestation. For each sample, fetal gender was determined by using both protocols (DYS14 and SRY) during the same real-time PCR run. RESULTS: The data obtained by the DYS14 and SRY assays showed an efficiency in fetal gender prediction of 97.9 and 80%, respectively. It is not advisable to combine the two protocols because this association does not help in further improvements in fetal gender prediction. CONCLUSIONS: DYS14 assay is the best approach for early fetal gender assessment because it is more sensitive, accurate, and efficient than the SRY assay.