Klara Dlouha

Absolute and Relative Quantification of Placenta-Specific MicroRNAs in Maternal Circulation with Placental Insufficiency–Related Complications

Placental insufficiency-related complications are one of the leading causes of maternal and perinatal morbidity and mortality. This study investigated the quantification of placenta-specific microRNAs (miRNAs) in the maternal circulation during gestation in a cohort of women with normally progressing pregnancies, the differentiation between placental insufficiency-related complications and normally progressing pregnancies, and the differentiation between placental insufficiency and normally progressing pregnancies during the early stages of gestation. Both absolute and relative quantification of placenta-specific miRNAs (ie, miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525, and miR-526a) was determined in 50 women with normally progressing pregnancies, 32 with complicated pregnancies [21 with preeclampsia with or without intrauterine growth retardation (IUGR) and 11 with IUGR], and 7 women with pregnancies at various gestational stages who later developed preeclampsia and/or IUGR using real-time PCR and a comparative C(T) method relative to normalization factor (ie, geometric mean of ubiquitous miR-16 and let-7d). Both quantification approaches revealed significant increases in extracellular placenta-specific miRNA levels over time in women with normally progressing pregnancies; however, they were not able to differentiate between normally progressing and complicated pregnancies at the time of preeclampsia and/or IUGR onset. Nevertheless, significant elevation of extracellular miRNA levels was observed during early gestation (ie, within the 12th to 16th weeks) in pregnancies with later onset of preeclampsia and/or IUGR. Early gestation extracellular miRNA screening can differentiate between women with normally progressing pregnancies and those who may later develop placental insufficiency-related complications.

Quantification of extracellular DNA using hypermethylated RASSF1A, SRY, and GLO sequences - evaluation of diagnostic possibilities for predicting placental insufficiency.

This study evaluated quantification of fetal extracellular DNA in maternal plasma for differentiation between cases at risk of onset of placental-insufficiency-related complications and normal pregnancies. Using real-time polymerase chain reaction, fetal (sex-determining region Y [SRY] and hypermethylated RASSF1A sequence) and total (beta-globin [GLO] gene) extracellular DNA was examined in 70 normal pregnancies, 18 at risk of placental-insufficiency-related pregnancy complications, 24 preeclampsia with or without (w or w/o) intrauterine growth retardation (IUGR) (median 34.0 week), and 11 IUGR (median 28.5 week). IUGR was diagnosed when estimated fetal weight was below the 10th percentile for evaluated gestational age. Although increased levels of extracellular DNA were detected in pregnancies with preeclampsia w or w/o IUGR relative to controls (RASSF1A, p < 0.001; SRY, p = 0.009; GLO, p < 0.001), quantities of fetal extracellular DNA in IUGR were not statistically significant (RASSF1A, p = 0.21; SRY, p = 0.2). RASSF1A, SRY, and GLO achieved 93.1%, 93.6%, and 92.1% accuracy for differentiation between normal pregnancy and preeclampsia w or w/o IUGR. Lower sensitivity was observed for pregnancies with onset of IUGR (RASSF1A, 60.0%; SRY, 80.0%; GLO, 72.7%), but did not influence final accuracy (RASSF1A, 91.6%; SRY, 92.5%; GLO, 89.5%). Among 18 patients at risk, 8 pregnancies involving 3 female and 5 male fetuses developed preeclampsia (n = 4), IUGR (n = 3), and chronic placentopathy causing hypoxia (n = 1). Elevation of extracellular DNA was demonstrated in 3/5 (SRY), 1/8 (hypermethylated RASSF1A), and 4/8 (GLO) patients at the earliest 26 weeks and at the latest 2 weeks before the onset of symptoms. These data indicate that fetal and total extracellular DNA concentrations can be significantly elevated in plasma of patients who later developed placental-insufficiency-related pregnancy complications. However, this is strongly individualized, and not a rule for all cases, and probably depends on the actual occurrence of excessive placental trophoblast apoptosis.

The Effect of DYS-14 Copy Number Variations on Extracellular Fetal DNA Quantification in Maternal Circulation

The aims of our research involved to investigate DYS-14 copy number variations in healthy males, to quantify extracellular DNA in maternal circulation in normal versus complicated pregnancies, and to study variations in the DYS-14 copy number in extracellular male fetal DNA. Fifty-five healthy males, 43 uncomplicated male singleton pregnancies (23 sampled at the 16th week and 20 sampled at the 36th week), and 15 pregnancies with placental insufficiency (PI)-related complications (mean 34.1 weeks) were analyzed using real-time PCR with DYS-14 sequence, sex determining region Y (SRY), and beta-globin (GLO) genes used as markers. Increased levels of extracellular DNA were detected in PI-related complications relative to gestational age-matched controls (SRY, p < 0.001; DYS-14, p = 0.007; GLO, p < 0.001). When the mean + 2SD (standard deviation) of controls was used as a cutoff, SRY, DYS-14, and GLO achieved 91.7%, 68.8%, and 94.4% accuracy, respectively, for differentiation between normal and complicated pregnancies. Considerable variations in the DYS-14 copy number in healthy males (mean 52.6) and extracellular DNA were found. A lower DYS-14 copy number was observed in PI-related complications (mean 83.5) compared to uncomplicated pregnancies (16th week: mean 114.2, p = 0.02; 36th week: mean 142.8, p = 0.04). The DYS-14 copy number was higher in extracellular DNA throughout gestation relative to healthy males. We concluded that, regarding interindividual copy number variations, the DYS-14 sequence is not an optimal marker for extracellular fetal DNA quantification for differentiation between normal and complicated pregnancies.

OP10.09: Heat shock protein gene expression in placental insufficiency related pregnancy complications—correlation with severity of the disease and Doppler‐determined umbilical artery PI

which in turn may cause less of a rise in heart rate (HR). Uterine artery Doppler (UtAD) provides a method of assessing risk for the development of pre-eclampsia. We examined the relationship between second trimester UtAD pulsatility index (PI), HR and birth weight. Methods: 99 women were recruited from the high-risk obstetric ultrasound clinic in the second trimester; median (± IQR) age and gestation were 33 (29–37) years and 23+6 (23+3–24+4) weeks respectively. Mean values of UtAD PI were recorded. Cardiovascular measurements including HR were performed at a later date (26+5 (25+6–28+0) weeks gestation). Gender specific z-scores were calculated from the unit’s previous 5 years’ births. Results: We found a significant positive correlation between HR and birth weight z-score, r = 0.22 (P = 0.03, 95% CI: 0.02–0.40). This correlation remained when 4 patients taking β-blockers were excluded, r = 0.21 (P = 0.04, 95% CI: 0.01–0.40). An inverse association was found between UtAD PI and HR r = −0.43 (P = 0.0001, 95% CI: 0.01–0.40). Conclusions: The novel finding is that lower second trimester HR is associated with lower birth weight; we hypothesize that this reflects inadequate cardiovascular adaptation to pregnancy. We confirm the previously described effect of HR on UtAD PI. However, it is not possible to determine whether this was a direct effect of HR on UtAD PI, or whether women with abnormal placentation are more likely to have a slower HR. This has obvious implications for predictive models using both UtAD and HR.

P12.02: 3D/4D ultrasound assessment of urogenital hiatus in women after elective and acute Caesaeran section

Objectives: We compared morphological parameters of urogenital hiatus in women after acute and elective Caesarean Section (CS). Methods: This is an open, prospective and non randomised study. All patients undergo 3D/4D ultrasound of the pelvic floor 6 weeks post partum, women are examined in supine position, after voiding. Patients are examined at rest, during pelvic muscle contraction and upon Valsava. Volumes are analyzed offline. Following parameters are measured: size of the urogenital hiatus (UGH, cm2) and parameter H (cm, measured as distance between the urethrovesical junction – UVJ and horizontal line going through the lower edge of the symphysis pubis). Results: We obtained data from 130 patients. 31 patients (23.8%) underwent elective CS, 99 patients (76.2%) delivered by acute CS. There was no statistically significant difference in measured parameters between patients after acute or elective surgery and there also wasn’t any difference when comparing acute CS in the first or second stage of labour. In patients after elective CS, mean UGH at rest was 11.92 (min. 7.67, ma. 17.68), H at rest was 3.2 (min. 2.4, max. 4.6), during contraction mean UGH was 10.56 (min. 6.92, max. 15.22), mean H was 3.56 (min. 2.72, max. 4.68) and upon Valsava mean UGH was 13.89 (min. 9.1, max. 18.74), mean H was 2.52 (min. 0.79, max. 3.51). In patients after acute CS mean UGH at rest was 12.3 (min. 7.79, max. 19.79), mean H was 3.26 (min. 2.1, max. 5), during contraction mean UGH was 10.5 (min. 6.98, max. 16.98), mean H was 3.58 (min. 2.28, max. 5.71) and upon Valsava mean UGH was 15.53 (min. 8.,22, max. 27.07) and mean H was 2.46 (min. 0, max. 4.18). Conclusions: There is no statistically significant difference in urogenital hiatus parameters measured by 4D ultrasound when comparing data from patients after elective and acute CS. We found no avulsion of the levator in women after CS.

OP34.01: Extracellular chromosome 21: derived microRNAs in maternal circulation: evaluation of their diagnostic potential for screening of Down syndrome

Objectives: Our aim was to determine the accuracy of a novel simple scoring system based on sonographic markers in differentiating between low and high risk forplacenta accreta (PA). Methods: All women who were referred to the Sheba Medical Center due to suspected PA were included, underwent a detailed ultrasound examination. A score was given based on the common sonographic findings of PA: loss of the hypoechoic retroplacental zone and placental lacunae. A score of 0–2 was defined as low risk and 3 was defined as high risk. Patients assigned to the high risk category underwent prophylactic pelvic artery catheterization before cesarean section and embolization if needed, whereas patients in the low risk category underwent simple cesarean section. Results: 71 women were included. PA was diagnosed clinically during surgery in 28 women, of whom 31 had a score of 3, and ruled out in 43 women, of whom only one had a score of 3. The sensitivity, specificity, positive predictive value and negative predictive value of our ultrasound-based scoring system in predicting PA were 90%, 97.5%93 and 95% respectively. Conclusions: A simple scoring system based on ultrasound alone can identify accurately a high risk population for PA who can benefit from prophylactic pelvic artery catheterization and embolization.

OP06.10: Absolute and relative quantification of placental specific microRNAs in maternal circulation with placental insufficiency related complications

Objectives: To investigate fetal and/or placental metabolism alterations in intrauterine growth restricted (IUGR) fetuses using a non-targeted metabolomic liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis of cord blood collected at birth. Methods: Cord blood samples were collected soon after birth from 22 IUGR and 21 appropriate for gestational age (AGA) fetuses. Serum samples were deproteinised by mixing with methanol at room temperature and centrifugation. Supernatants were lyophilized and reconstituted in water for analysis. LC-HRMS analyses were performed using an Orbitrap mass spectrometer hyphenated to a Surveyor Plus LC. Samples were injected on a 1.0 × 150 mm Luna C18 column. Spectra were collected in full scan mode at a resolution of approximately 30000. Data were acquired over the m/z range 50–1,000 with measurements performed in duplicate. To observe metabolic variation between the two sets of samples, LC-HRMS data were analyzed with principal component analysis (PCA) model. Results: Birth weight significantly differed from IUGR and AGA fetuses (P < 0.001). A large number of features (ionic species with specific retention times) were shown to differ between the two classes of samples. By comparison with available databases two main differentially expressed metabolites (phenylalanine and tryptophan) were detected. Logistic regression coupled to a receiver operating characteristic curve identified a cut-off value for phenylalanine and tryptophan, which gave an excellent discrimination between IUGR and AGA (sensitivity = 100%; specificity = 75%). Conclusions: The non-targeted metabolomic LC-HRMS analysis of cord blood collected at birth appears a promising tool for the identification of potential biomarkers of organ damage in IUGR.

P30.02: Placenta‐specific microRNAs in maternal circulation in normal pregnancies and those with placental insufficiency related complications

Results: In the first group intervillous lacuna were dilated in suprabasal area, the maternal placental surface was uneven; a significant myometrial thinness and an abnormal vascular architectonics were diagnosed in the placental zone. In the 2nd group no changes were reported. 7 children were delivered at 28–34 weeks and 3 – at 39. Vaginal bleeding during the pregnancy was in eight cases, five women were delivered prematurely because of the hemorrhage. Hysterectomy was needed in five patients of the 1st group (71%). The histology revealed a chorionic penetration into the myometrium, isthmus and the cervix. Invasion was normal in two cases (29%). In 2nd group no complications after delivery were registered. Morphological examination did not reveal any circulation abnormalities. Conclusions: Ultrasound examination and color Doppler help to suspect a placental penetration into myometrium. Sonographic criteria are dilatation of intervillous lacuna in suprabasal area, uneven maternal placental surface; a significant myometrial thinness and an abnormal vascular architectonics in the placental zone.

OC009: Evaluation of combined first‐trimester screening ‐ Czech multi‐center study

K. Dlouha1, I. Kucerova1, P. Calda2, D. Smetanova3, M. Brestak4, A. Hudec5, H. Viskova2, D. Stejskal3, J. Turek5, I. M. Malbohan6 1Institute for the Care of the Mother and Child, Prague, Czech Republic, 2Department of Obstetrics and Gynecology, First Medical Faculty, Charles University, Prague, Czech Republic, 3Gennet, Centre for Genetics and Assisted Reproduction, Prague, Czech Republic, 4Pronatal, Centre for Assisted Reproduction, Prague, Czech Republic, 5Department of Obstetrics and Gynecology, Medical Faculty in Pilsen, Charles University, Pilsen, Czech Republic, 6Department of Medical Biochemistry, First Medical Faculty, Charles University, Prague, Czech Republic