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.

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.

Peripheral CD300a+CD8+ T lymphocytes with a distinct cytotoxic molecular signature increase in pregnant women with chronic chorioamnionitis.

PROBLEM: CD300a is an immunomodulatory molecule of the immunoglobulin receptor superfamily expressed in the leukocytes of myeloid and lymphoid lineages. However, its biological function on CD8+ T lymphocytes remains largely unknown. This study was conducted to assess the biological significance of CD300a expression in T lymphocytes and to determine whether its expression in peripheral T lymphocytes changes in pregnant women presenting with antifetal rejection. METHODS OF STUDY: Microarray analysis was performed using total RNA isolated from peripheral CD300a+ and CD300a- T lymphocytes. Flow cytometric analysis of the peripheral blood samples of pregnant women and pathologic examination of the placentas were conducted. RESULTS: A large number of genes (N = 1245) were differentially expressed between CD300a- and CD300a+ subsets of CD8+ T lymphocytes, which included CCR7, CD244, CX3CR1, GLNY, GZMB, GZMK, IL15, ITGB1, KLRG1, PRF1, and SLAMF7. Gene ontology analysis of differentially expressed genes demonstrated enrichment of biological processes such as immune response, cell death, and signal transduction. CD300a expression in CD8+ T lymphocytes was coupled to a more cytotoxic molecular signature. Of note, the proportion of CD300a+CD8+ T lymphocytes increased in pregnant women with chronic chorioamnionitis (antifetal rejection of the chorioamniotic membranes; P < 0.05). CONCLUSION: The findings of this study strongly suggest an increase in systemic T-lymphocyte-mediated cytotoxicity in pregnant women with chronic chorioamnionitis as a manifestation of maternal antifetal rejection.

PP13, maternal ABO blood groups and the risk assessment of pregnancy complications.

BACKGROUND: Placental Protein 13 (PP13), an early biomarker of preeclampsia, is a placenta-specific galectin that binds beta-galactosides, building-blocks of ABO blood-group antigens, possibly affecting its bioavailability in blood. METHODS AND FINDINGS: We studied PP13-binding to erythrocytes, maternal blood-group effect on serum PP13 and its performance as a predictor of preeclampsia and intrauterine growth restriction (IUGR). Datasets of maternal serum PP13 in Caucasian (n = 1078) and Hispanic (n = 242) women were analyzed according to blood groups. In vivo, in vitro and in silico PP13-binding to ABO blood-group antigens and erythrocytes were studied by PP13-immunostainings of placental tissue-microarrays, flow-cytometry of erythrocyte-bound PP13, and model-building of PP13--blood-group H antigen complex, respectively. Women with blood group AB had the lowest serum PP13 in the first trimester, while those with blood group B had the highest PP13 throughout pregnancy. In accordance, PP13-binding was the strongest to blood-group AB erythrocytes and weakest to blood-group B erythrocytes. PP13-staining of maternal and fetal erythrocytes was revealed, and a plausible molecular model of PP13 complexed with blood-group H antigen was built. Adjustment of PP13 MoMs to maternal ABO blood group improved the prediction accuracy of first trimester maternal serum PP13 MoMs for preeclampsia and IUGR. CONCLUSIONS: ABO blood group can alter PP13-bioavailability in blood, and it may also be a key determinant for other lectins' bioavailability in the circulation. The adjustment of PP13 MoMs to ABO blood group improves the predictive accuracy of this test.

A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death.

Galectins are proteins that regulate immune responses through the recognition of cell-surface glycans. We present evidence that 16 human galectin genes are expressed at the maternal-fetal interface and demonstrate that a cluster of 5 galectin genes on human chromosome 19 emerged during primate evolution as a result of duplication and rearrangement of genes and pseudogenes via a birth and death process primarily mediated by transposable long interspersed nuclear elements (LINEs). Genes in the cluster are found only in anthropoids, a group of primate species that differ from their strepsirrhine counterparts by having relatively large brains and long gestations. Three of the human cluster genes (LGALS13, -14, and -16) were found to be placenta-specific. Homology modeling revealed conserved three-dimensional structures of galectins in the human cluster; however, analyses of 24 newly derived and 69 publicly available sequences in 10 anthropoid species indicate functional diversification by evidence of positive selection and amino acid replacements in carbohydrate-recognition domains. Moreover, we demonstrate altered sugar-binding capacities of 6 recombinant galectins in the cluster. We show that human placenta-specific galectins are predominantly expressed by the syncytiotrophoblast, a primary site of metabolic exchange where, early during pregnancy, the fetus comes in contact with immune cells circulating in maternal blood. Because ex vivo functional assays demonstrate that placenta-specific galectins induce the apoptosis of T lymphocytes, we propose that these galectins reduce the danger of maternal immune attacks on the fetal semiallograft, presumably conferring additional immune tolerance mechanisms and in turn sustaining hemochorial placentation during the long gestation of anthropoid primates.