Federica Tarquini

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

Abstract 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 χ2 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. Chinese abstract 妊娠期糖尿病（GDM）是糖耐量受损的一种状况，妊娠期发病率为1%∼14%。发病率变化范围大是涉及单核苷酸多态性病理与母体体重为危险因素的反映。本研究对遗传组份和母体因素进行评估，从而确定发展为GDM较高风险的妇女。实验中240名妇女口服葡萄糖耐量试验阴性（—OGTT），38名妇女OGTT阳性（+OGTT）。通过SNP基因分型方法对单核苷酸多态性基因组ENPP1、NRF1、VEGF、CEBPA和PIK3R1进行分析，并进行了相关性研究，同时通过χ2检验分析病例组和对照组间基因型和等位基因频率的差异。OGTT阳性与孕前高体重指数和年龄密切相关。SNP中ENPP1基因与OGTT阳性相关，而其他变异基因与GDM无关联。与OGTT阴性相比，OGTT阳性患者中ENPP1纯合子和杂合子A等位基因频率不同。因此孕前体重指数（BMI）、年龄与A等位基因纯合基因型表达可显著增加OCTT阳性的风险。我们的研究结果表明年龄、孕前BMI与A等位基因的纯合型相关，这种相关性可显著增加糖耐量受损和GDM的发病风险。

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.

Maternal smoking does not affect the amount of cell-free fetal DNA in maternal plasma during the 1st trimester of pregnancy

Abstract CffDNA, from 344 non-smoking, 38 smoking and 33 ex-smoking pregnant women at 11 + 0–13 + 6 gestational weeks, was extracted and quantified by the multicopy DYS14, as the fetal DNA marker and using the quantitative real-time PCR 7300 detection system. The smoking habit was based on maternal self-report, confirmed by cotinine levels and male fetuses were verified by phenotype at birth. The genders of newborns were compared with DYS14-cffDNA analysis, achieving a 100% diagnostic accuracy of the test. A total of 177 non-smokers, 18 smokers and 22 ex-smoker pregnancies with male fetuses were identified by the cffDNA concentration. Results showed that smoking status was not associated with different amounts of DYS14-cffDNA (p = 0.159), suggesting the possibility of offering cffDNA testing to all pregnant women, even if they are active smokers or ex-smokers, and the test can be unadjusted for smoking status.

Caspase 3 activation and PARP cleavage in lymphocytes from newborn babies of diabetic mothers with unbalanced glycaemic control.

Several epidemiological studies showed that gestational diabetes mellitus is the most frequent metabolic disorder of pregnancy, the pathogenesis of which has yet to be completely clarified.

Diagnostic accuracy of non‐invasive prenatal sex determination: a large‐scale study

Non-invasive prenatal determination of fetal sex offers a promising alternative to invasive diagnosis of X-linked diseases and fetal disorders of ambiguous genitalia (1–4). Using free-fetal DNA (ffDNA) from maternal plasma (5) and different detection systems for Y chromosome (6–9), researchers achieved high diagnostic accuracy in fetal sex prediction during the first trimester of pregnancy without excluding variable percentage of false positives (FP) and false negatives (FN) (10, 11). We previously showed higher performance of DYS14 than SRY detection system without reaching an optimal diagnostic accuracy because of the low percentage of FN and FP (12). The aim of this study was to enhance the performance of our DYS14 detection system by introducing new key elements, increasing the volume of maternal plasma for DNA extraction and employing innovative interpretation criteria of results. Finally, we audited the overall diagnostic accuracy on a large-scale study to verify whether the new protocol ensured the correct fetal sex determination. We analyzed, in two different phases, plasma samples from 513 women at 10–15 weeks of gestation. During the first non-blinded phase, we established the best threshold value (TV) to discriminate male and female fetuses and, in the second blinded phase, we applied it to assess its diagnostic performance. Fetal sex was verified with the analysis of karyotype or confirmed with phenotype at birth. The study was approved by our Regional Ethical Committee. Genomic DNA was extracted from maternal plasma (500 μl in the first and 1000 μl in the second phase) using QIAmp DSP Virus kit (Qiagen, Hilden, Germany) and analyzed by real-time polymerase chain reaction (PCR) 7300 detection system (Applied Biosystems, Foster City, CA) using DYS14 detection system to measure the quantity of ffDNA (three replicates for each maternal sample) and telomerase reverse transcriptase detection system as a quality control. Real-time PCR [quantitative PCR (qPCR)] reaction was set up as previously described (12). Results were expressed as median values with range for a descriptive statistics and analyzed by using receiver operating characteristic (ROC) curves, calculated by spss software 17.0 (SPSS Inc., Chicago, IL), to set the TV, in terms of ffDNA concentration and number of DYS14positive replicates. Karyotype or phenotype at birth revealed that among the 115 pregnant women analyzed in the first phase of study, 55 delivered one daughter and 60 one son. By evaluation of qPCR results (Table 1), we set our first TV by analysis of ROC curve calculated using the number of DYS14-positive replicates (data not shown). However, the TV was not satisfactory, except for samples with 0, 1 or 2 DYS14-positive replicates that were clearly identified as female fetus. Only one sample from a male pregnancy had 0 DYS14-positive replicates. The critical point was the interpretation of those results with three DYS14-positive replicates, as they occurred in either female or male pregnancies. Two samples from female pregnancy gave three positive replicates but with a very low ffDNA concentration, whereas all the samples from male pregnancy gave three DYS14-positive replicates with elevated ffDNA concentration (Table 1). Therefore, we built a new ROC curve combining ffDNA concentration and the number of DYS14positive replicates and selecting only those samples with three DYS14-positive replicates (2 from female and 59 from male pregnancies). We individuated the best ffDNA concentration value as 1.42 GE/ml as it allowed us to reach a 100.00% diagnostic sensitivity [95% (confidence interval) CI: 93.9–100.0] and 100.00% diagnostic specificity (95% CI: 19.3–100.0) (Fig. 1). In the second phase of study, karyotype or phenotype at birth revealed that 208 delivered a son and 190 a daughter.

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.

Intrapartum test for detection of Group B Streptococcus colonization during labor

Abstract 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.

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.

P2.027 GeneXpert GBS and CT/NG Real Time PCR Assays as Innovative Tools For Cervico-Vaginal Infections’screening

Background Group B Streptococcus (GBS), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cervico-vaginal infections can be involved in pregnancy complications such as preterm birth and premature rupture of membrane (PROM). These infections can also be transmitted to the newborn during delivery leading to serious consequences. Therefore, CDC guidelines suggest microbial prenatal screening for administration of target prophylaxis based on culture results. To accurately predict the colonisation of genital tract, the test should be better performed during labour, because microbial presence may be transient/intermittent and re-colonisation can occur. GeneXpert®GBS and GeneXpert®CT/NG tests (Cepheid), fully-automated, easy-to-use and rapid PCR-assays (about 45 and 90 min, respectively) can be the right alternative to culture tests (at least 72 hours). This study evaluates the advantages of GeneXpert®GBS in the management of women, with unknown cervico-vaginal microbial status, during labour. Moreover, it assesses whether the prevalence of CT, NG and GBS infections is higher in pregnancy complicated by preterm labour or PROM. Methods During a four months’ period, all women with singleton pregnancy at beginning of labour either-term or preterm or PROM were enrolled. Exclusion criteria were planned caesarean section or recent use of systemic or topical antibiotics. Cervico-vaginal (for CT/NG) and vaginal-rectal (for GBS) swabs were collected from each patient and analysed by GeneXpert®GBS and GeneXpert®CT/NG assays on GeneXpert®System. Results CT/NG screening showed positive results only among PROM pregnancies (2.5% CT positive) while no positive results were found among preterm/term pregnancies. Among pregnant women analysed for GBS, 24.4% resulted positive and 75.6% negative. Only positive patients received IAP, instead of current guidelines, for which all patients would have been treated due to unknown GBS infection status. Conclusion With GeneXpert®GBS test, we could correctly manage all women and reduce administration of IAP. We calculated that the savings for the hospital was 3,500 EUR every three months.