Sevgi Tercanli

Quantitative Proteomics Analysis of Maternal Plasma in Down Syndrome Pregnancies Using Isobaric Tagging Reagent (iTRAQ)

Currently no specific biomarkers exist for the screening of pregnancies at risk for down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in βHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are ‘digital PCR’ and ‘next-generation sequencing’ (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Three dimensional volume measurement of the cervix during pregnancy compared to conventional 2D-sonography

Objective: To compare the three dimensional (3D) volume assessment of the cervix with the conventional two‐dimensional cervical length measurement in a low and a high risk group for cervical incompetence. Methods: In an observational study, we investigated a group A of low risk pregnancies (no preterm contractions, no vaginal bleeding or vaginal infections and no history of preterm delivery) and a group B of high risk pregnancies (preterm contractions or PROM). All patients underwent a transvaginal ultrasound investigation with a 7.5 MHz probe using a three‐dimensional ultrasound system (Combison 530, Kretztechnik, Austria). After measuring the cervical length, the internal os and the funneling with the B‐mode, 3D‐volume was recorded twice by the same investigator using the same machine. Since 2D‐length measurement of the cervix has been established to be predictive for spontaneous preterm delivery, we wanted to test whether 3D‐volume assessment has a better discriminative power to differentiate a high‐risk from a low‐risk group. Therefore the 2D and 3D measurements (mm, resp. cm3) were compared between patient groups A and B using the two tailed Student t‐test and Fishers Exact test. Results: In 2D cervical length measurement the mean cervical length in group A was significantly longer than in group B: 41.1±8.61 mm and 27.77±10.42 mm, P=0.00000017. In 3D‐sonography the mean cervical volume was larger in group A, but the difference compared to group B was not significant: 47.71±18.38 mm and 39.90±12.57 mm, P=0.07. Conclusion: Contrary to our hypothesis cervical length measurement therefore was superior to cervical volume measurement assessed by 3D ultrasound for identifying women with increased risk of spontaneous preterm delivery. This may be due to the larger distribution of measurement values in the 3D group.

Spontaneous fetal loss rates in a non‐selected population

The objective of this work was to determine the rate of spontaneous fetal loss up to 28 weeks of gestation in uncomplicated pregnancies of a low-risk population after sonographically identified intact intrauterine pregnancy during the first trimester. Transvaginal ultrasounds were given to 2,534 women at between six and 12 weeks of gestation. Inclusion criteria were a positive fetal cardiac activity and no antecedent signs of vaginal bleeding. Gestational age was confirmed by measurement of the crown-rump length and/or biparietal diameter (BIP). Patients were followed until delivery or up to a fetal loss. The mean fetal loss rate between 12 and 28 weeks was 3.86% (n = 99). Fetal loss increased with maternal age: fetal loss rate under 20 yr: 2.94% (OR 0.75; CI 0.23-2. 46), 20-24 yr: 3.20% (OR 0.77; CI 0.48-1.23), 25-29 yr: 3.39% (OR 0.77; CI 0.50-1.19), 30-34 yr: 3.89% (OR 1.01; CI 0.59-1.71), 35-39 yr: 7.82% (OR 2.13; CI 1.04-4.32), 40-45 y: 50% (OR 13.84; CI 6.67-28.72) and > 45 yr: 50% (OR 13.05; CI 1.96-86.71) respectively. The frequency of spontaneous fetal loss before 28 weeks gestation was assessed systematically in a low-risk population. There was a very clear correlation with advancing maternal age. These data now can be used as background loss rate information for evaluating the safety of invasive prenatal diagnosis, and they will be more valid for this purpose than the available data taken from selected cohorts of women, such as those from hospital clinics or from infertility programs.

Charts for cervical length in singleton pregnancy

Objectives: To construct charts for cervical length in a low risk population measured by transvaginal ultrasonography. Methods: Pregnant women of an apparently normal population were seen in the ultrasound division of the University Womens Hospital Basel between 20 and 34 weeks of gestation and underwent once (one measurement per subject) a transvaginal ultrasound measurement of the cervix under standardized conditions. In order to establish normal values of the cervical length, finally only women who delivered spontaneously at term (>37 weeks of gestation) remained in the study. Exclusion criteria were preterm labor, multiple pregnancies, cerclage or surgical intervention prior to pregnancy. For statistical evaluation, regression analysis and calculation of 5th and 95th percentiles were performed. Results: A total of 669 cervical measurements were recorded. The number of measurements differed from 22 measurements at 23 weeks of gestation to 86 at 31 weeks of gestation. Cervical length gradually and significantly decreased as the gestational age progressed (between 20 and 34 weeks of gestation). New charts with the 5th, 50th and 95th percentile are presented and compared with previously published data. Conclusions: Our charts for cervical length in a limited risk population can be used for observing patients at high risk of preterm delivery and for clearly identifying a significant deviation or decline in the percentile for these subjects.

Cervical length assessment by ultrasound as a predictor of preterm labour—is there a role for routine screening?

Transvaginal ultrasonography has recently been shown to be an objective, reproducible and reliable method to assess the cervix and predict the risk of preterm delivery in high‐risk pregnancies. Assessment of the cervix includes cervical length measurement (CLM) and measurement of dilatation of the internal os in a dynamic functional examination. There is an inverse correlation between cervical length and the frequency of preterm delivery. The high negative predictive value avoids unnecessary interventions such as tocolysis or cerclage in high‐risk pregnancies. In contrast, a length of 25 mm or less at 28–30 weeks of gestation is associated with a significantly increased incidence of preterm delivery. Studies in women with high risk for preterm delivery, i.e. contractions, premature rupture of the membranes and history of preterm delivery, have shown a high sensitivity and a high positive predictive value, however in low‐risk groups they have failed to show a high sensitivity. From large observational studies in low‐risk populations we know that the 50th percentile of the cervical length is 35 mm at 24 weeks of gestation 3 . Advantages of CLM as a screening test include the fact that sonographical assessment of the cervix is a widely accepted and well‐standardised method, which requires only a relatively short period of training. Disadvantages of screening are two factors, the first being the low sensitivity of the test and the low prevalence of preterm deliveries in a low‐risk population, resulting in cut off values being set at a very low level (i.e. 5th percentile) in order to get acceptable specificity. Secondly, screening is only worthwhile if an effective preventive therapy is available. The debate about tocolysis and cerclage is not yet concluded. Therefore we would not currently recommend cervical length measurement as a screening tool—but as a routine method in high risk gravidas with or without symptoms. Further interest should be focused on scoring systems combining ultrasound with biochemical, endocrinological and maybe molecular cell methods such as the measurement of fetal DNA in maternal blood to prevent preterm deliveries in the general population.

Umbilical cord edema associated with patent urachus.

Umbilical cord anomalies can often be detected prenatally by ultrasound, but a definitive prenatal diagnosis is not always possible. We present a case with increasing edema of the Whartons jelly followed by the development of pseudocysts in the proximal umbilical cord due to a patent urachus. The first abnormal findings were detected by ultrasound in the 14th week of gestation. Differential diagnoses and their influence on surveillance and birth management are discussed. Copyright

Developments in laboratory techniques for prenatal diagnosis.

Ongoing trends in prenatal diagnosis aim at early, rapid, and ideally noninvasive diagnosis as well as at the improvement of risk-screening for aneuploidy. Interphase-fluorescence in situ hybridization and quantitative fluorescence polymerase chain reaction are efficient tools for the rapid exclusion of selected aneuploidies in addition to the established direct preparation of chromosomes from chorionic villi. Interphase fluorescence in situ hybridization has also made possible the diagnosis of selected chromosome abnormalities in single cells (e.g. in preimplantation genetic diagnosis) or noninvasive diagnosis. More complex multicolor fluorescence in situ hybridization approaches are currently being evaluated. Single cell polymerase chain reaction is the key technique for the molecular diagnosis of a growing number of monogenic conditions before implantation or, still more experimental, in fetal cells retrieved from the maternal circulation. New sources for noninvasive diagnosis came into play such as fetal DNA or cell nuclei in maternal plasma. The combination of biochemical parameters in the maternal serum, namely free beta-human chorionic gonadotropin with pregnancy associated plasma protein A and sonographic markers, has already dramatically increased the sensitivity of risk screening in the first trimester of pregnancy.

aCGH on chorionic villi mirrors the complexity of fetoplacental mosaicism in prenatal diagnosis

To describe the diagnostic performance of array comparative genomic hybridization (aCGH) in the presence of mosaicism in the fetoplacental unit using direct chorionic villi.

Influence of volume preloading on uteroplacental and fetal circulation during spinal anaesthesia for caesarean section in uncomplicated singleton pregnancies.

Objective: Effects of volume preloading during spinal anaesthesia for elective caesarean section on maternal blood pressure, feto-maternal circulation and fetal outcome. Patients and Methods: In a pilot study a randomised trial was performed in 22 healthy women with uncomplicated, singleton pregnancies at 36–40 weeks of gestation undergoing elective caesarean section under spinal anaesthesia. In the low volume group (group A) patients received 150 ml of crystalloid solution for preloading, in the high volume group (group B) they were given 15 ml/kg of crystalloid solution for preloading before the initiation of spinal anaesthesia. Maternal blood pressure was monitored intermittently. Hypotension was defined as a decrease in systolic pressure to less than 80% of the baseline value. The Doppler flow evaluation consisted of measurements from the uterine artery at the placental site, fetal umbilical artery and fetal middle cerebral artery. Pulsatility indices were derived before and after fluid preloading, and when spinal anaesthesia was established. The neonatal outcome was assessed by Apgar scores, arterial acid base status and neurologic and adaptive capacity scores (NACS). Results: The incidence of maternal hypotension in both groups was 45.5% (n = 10); 3 cases occurred in group A compared to 7 cases in group B (n.s.). There was no evidence that the high dose volume is useful in preventing maternal hypotension. The pulsatility indices of uterine arteries, umbilical arteries and middle cerebral arteries were not altered. Statistical analysis showed no changes in neonatal outcome concerning umbilical arterial pH, Apgar score and NACS (n.s.) between groups A and B. Conclusions: Our preliminary results suggest that high dose crystalloid volume preloading has no preventive function in the avoidance of maternal hypotension in healthy parturients undergoing elective caesarean section under spinal anaesthesia, and shows no harmful effects on neonatal outcome as long as maternal hypotension is corrected immediately. However, the statistical significance may reflect the small sample size, and larger series are needed before changing the current management.