Gabriele Urban

Feto-maternal correlation of PTX3, sFlt-1 and PlGF in physiological and pre-eclamptic pregnancies

Objective: PTX3, sFlt-1 and PlGF levels in maternal blood are altered in some obstetric diseases, such as preeclampsia (PE). Nonetheless, only few data on their expression in the fetal compartment have been reported so far. Study Design: An observational study was performed by prospectively collecting maternal and fetal serum samples in 51 singleton pregnancies divided into two groups: 22 PE women and 29 healthy controls. The relationships between maternal and fetal marker serum levels were evaluated by Spearman correlation. Results: A feto-maternal correlation was neither identified for PTX3 in either PE or control groups (1.1 versus 3.8 ng/ml, p = 0.17 and 0.9 versus 1.3 ng/ml, p = 0.30, respectively), nor for sFlt-1 and PlGF in healthy pregnancies (158.2 versus 3326.0 pg/ml, p = 0.28 and 11.0 versus 230.9 pg/ml, p = 0.51). In contrast, PE patients showed a significant positive feto-maternal correlation for both sFlt-1 and PlGF (324.1 versus 10 825.0 pg/ml and 7.8 versus 31.6 pg/ml, respectively, p = 0.02 for both markers). Conclusion: According to our results, an independent fetal production of the analyzed soluble angiogenic markers can be hypothesized in pregnancies complicated by PE.

Elevated first trimester soluble fibrin polymer is associated with adverse pregnancy outcome in thrombophilic patients

Inherited and acquired thrombophilic conditions are associated with maternal thromboembolic events and a variety of adverse perinatal outcomes, including, preeclampsia, intrauterine growth restriction (<10th percentile), second and third trimester fetal loss, and abruptio placentae [1,2]. Currently, there is a paucity of functional or global tests that will provide insight as to the prediction of adverse pregnancy outcome [3,4]. Activation markers are of limited predictive value for adverse outcome [5]. Bombeli et al. [6] found that thrombin–antithrombin complexes (TAT) or D-dimers did not correlate with risk stratification in pregnant women based upon personal or family history of thrombosis and the presence of a thrombophilic condition. However, these authors did find that women with ongoing thrombosis during pregnancy had significantly elevated TAT and D-dimers with or without anticoagulant therapy. Coagulation activation markers such as prothrombin fragment 1.2 and TAT are increased with the progression of pregnancy to levels as seen in those patients with active thrombosis [4]. Bremme et al. [7] found that normal pregnancy (n=26 women) was associated with both increased thrombin activity, increasedsoluble fibrin levels (9.2–13.4 nmol/l), as well as fibrinolysis, as evidenced by increased levels of fibrin D-dimer (91–198 µg/l). Soluble fibrin polymer (SFP) has been shown to be a specific and sensitive marker of active clotting and thrombin generation in a group of nonpregnant individuals at high risk for thromboembolic event [8]. The purpose of this study was to determine if elevated SFP is an additional risk for adverse pregnancy outcome in thrombophilic patients. We conducted a retrospective cohort study among 39 patients with inherited and acquired thrombophilia (41 pregnancies) and 50 uncomplicated gestations. Thrombophilic women previously underwent a uniform evaluation for inherited and acquired thrombophilic conditions. A reference laboratory was used to evaluate the relevant thrombophilic conditions. Factor V Leiden, methylenetetrahydrofolate reductase gene mutation, and prothrombin gene mutation 20210 assay were performed by using multiplex allele-specific primers’ PCR amplification. We defined protein S as deficient if protein levels were below 40%, as determined by functional assay. Protein C and antithrombin were measured by chromogenic and antigenic assays, and respective levels below the laboratory’s normal reference range were considered abnormal. Homocysteine testing was evaluated by high performance liquid chromatography (HPLC), and values above the laboratory’s normal reference range were considered abnormal. Antiphospholipid antibody syndrome was diagnosed by established criteria [9]. Anticardiolipin and anti-beta 2 glycoprotein I antibodies were performed by enzyme-linked immunosorbent assay (ELISA), and values above the laboratory’s reference range were considered abnormal. The presence of the lupus anticoagulant was detected by screening with a sensitive activated partial thromboplastin time (APTT) and confirmed by the dilute Russel viper venom time with correction by adding the high concentration of phospholipids. This is felt to be a reliable confirmatory test for the lupus inhibitor. PAI-1 antigen and activity were measured by ELISA and chromogenic assay respectively, and levels three-fold above the upper limit of the reference range were considered abnormal. Thrombocytosis, as part of the myeloproliferative syndrome, was determined by the presence of an elevated platelet count, associated with abnormal platelet function studies, platelet flow cytometry, and compatible bone marrow findings, clinical presentation, such as splenomegaly, and other white cell or red cell abnormalities. Following informed consent and institutional review board approval, pregnant patients enrolled in the maternal fetal medicine faculty practice of New York University School of Medicine who underwent phlebotomy for other routine laboratory tests as part of their obstetrical care were asked to donate an additional aliquot of blood for SFP evaluation. Specimens were collected at routine blood draw and the plasma samples were stored at −80°C until analysis. Maternal plasma SFP concentrations were measured with a commercial ELISA [thrombus precursor protein (TpP); American Biogenetic Sciences, Inc., Copiague, New York, USA], as previously described [8]. The standard curve consistently had a R value of more than 0.98, the interassay and intraassay errors were less than 7%. Using a computerized obstetrical database, all thrombophilic patients, with an available blood sample obtained during their first prenatal visit and who met the criteria for thrombophilia, were identified and recruited. All charts were reviewed in detail to confirm the accuracy of the diagnosis. To establish a reference range for SFP in pregnancy, blood specimens from the first, second, and third trimesters were obtained from a group of healthy pregnant women with normal pregnancy outcome. Patients with thrombophilia were identified as having the following conditions: protein S deficiency (N = 8), factor V Leiden mutation R506Q (N=12), prothrombin gene mutation 20210A (N=6), antiphospholipid antibody syndrome (N=17), hyperhomocysteinemia (N=1), and thrombocythemia and myeloproliferative disorder with predominant manifestation of thrombocytosis (N=3). Five patients had two defects. Preeclampsia was defined by American College of Obstetricians and Gynecologist’s criteria [10]. Severe preeclampsia was defined when the following were present: systolic blood pressure (SBP) of at least 160mmHg or diastolic blood pressure (DBP) of at least 110mmHg and plus two or more protein on urinary dipstick. Severe preeclampsia was considered present if one of the blood pressure or proteinuria requirements was met along with either thrombocytopenia (platelet count<100 000/ml) or elevated liver enzymes (serum glutamic oxalo-acetic transaminase or serum glutamic pyruvic transaminase). The data were analyzed by Wilcoxon rank sum, t-test (DF, 1), and x2 (DF, 1) where appropriate. Level of significance was set at a P value less than 0.05. Receiver operator characteristic (ROC) curve analysis was performed to determine the optimum cutoff for SFP, to predict adverse pregnancy outcome. Thrombophilic patients had significantly lower birth weights and delivered earlier than controls, respectively: 2943 g ± 829 g vs. 3498 g ± 527 g and 37.0 weeks ± 4.5 weeks vs. 39.4 weeks ± 1.5 weeks (P<0.01). First trimester mean SFP maternal levels (mg/ml) were significantly higher among thrombophilic patients compared with controls (21.5 ± 3.7 vs. 3.7 ± 0.8mg/ml; P<0.02). The ROC curve of SFP to predict adverse pregnancy outcomes by using 3 mg/ml in the first trimester as cutoff (P = 0.03) produced a sensitivity of 83%, specificity 64%, positive predictive value of 57%, and negative predictive value of 97% for adverse pregnancy outcome. Five thrombophilic patients had at least two thrombophilic conditions, and their SFP values were higher than cases with one (26.4 ± 16.0; P<0.01). Even with heparin therapy, cases also had higher SFP values than controls in the second trimester (31.7 ± 8 vs. 10.9 ± 2; P<0.01) and third trimester (35.1 ± 16.1 vs. 12.9 ± 3; P<0.01). Table 1 shows the distribution of the adverse pregnancy outcomes in the thrombophilia group. Table 1 Distribution of adverse pregnancy outcome There were no significant differences between the thrombophilic patients (n=39) and controls (n=50) regarding mean maternal age, years_SD (32.6 ± 5.8 vs. 33.1 ± 5.6), median gravidity and range [3 (1–11) vs. 2 (0–9)], median parity and range [1 (0–4) vs. 0 (0–5)], nulliparity (17 vs. 16%), multiparity (83 vs. 84%), race expressed as percent Caucasian (80 vs. 83%), respectively. There was no significant difference in gestational age at blood draw [median (range) between the thrombophilic patients (n=41, 40, 39) and controls (n=50, 50, 50)] in the first trimester [10.2 (4.0–14.0) vs. 9.0 (4.6–13.0)], second trimester [21.3 (15.0–27.4) vs. 17.6 (15.3–26.9)], or the third trimester [33.5 (27.3– 40.4) vs. 29.7 (27.7–40.0)], respectively. Our study confirms the progressive increase of thrombin generation as pregnancy proceeds with a markedly accelerated thrombin generation in thrombophilic patients. Higher first-trimester SFP increases the risk for adverse pregnancy outcomes. Even some normal pregnant patients with uneventful pregnancies have elevated SFP values. The significance of this is not known at this time and may be reflective of normal biologic variability or indicative of indolent processes. SFP levels are inversely related to gestational age at delivery and birth weight confirming our previous data regarding multiple gestations [11]. Testing for SFP may be useful in predicting patients at risk for thrombophilia-induced poor pregnancy outcome and monitoring anticoagulation efficacy. After the completion of this study, the ELISA test for SFP became unavailable commercially. It employs a very specific antibody to SFP. Perhaps, other tests may be developed that will exhibit similar properties. Our data indicate that reintroduction of SFP measurement may have clinical utility, particularly if confirmed in a larger prospective study.

OP17.07: Doppler evaluation of wall shear rate (WSR), wall distension rate (WDR), turbulence index (TI) in preoperative ovarian cysts: A prospective study

Objectives: To evaluate prospectively an ultrasound-based scoring system as a method for triaging symptomatic women presenting with an adnexal mass for surgical treatment. Methods: 151 symptomatic women scheduled for surgical treatment at our institution between June 2003 and December 2007 were included in this prospective study. Patients were evaluated by transvaginal power Doppler ultrasound prior to surgery. Patients were classified as low-risk or high-risk for malignancy according an ultrasound-based scoring system. Patients with low risk for malignancy were scheduled for laparoscopy and patients for high risk for malignancy were scheduled for laparotomy. Some patients with high risk were scheduled for advanced oncologic laparoscopic surgery. Patients with low risk but tumor size > 10 cm were scheduled for laparotomy. Results: 82 women presented with pelvic pain, 8 had uterine bleeding and 61 referred symptoms suggestive for ovarian malignancy, such as abdominal swelling, bloating and abdominal discomfort. 75 (49.7%) masses were considered as ‘‘low risk’’ and treated by laparoscopy in 58 cases and by laparotomy in 7 cases, because emergency or associated pathology (All tumors were benign). 76 (50.3%) masses were considered as ‘‘high risk’’ and all treated by laparotomy (56 malignant and 4 benign tumors) or by advanced laparoscopy (16 malignant tumors). Ten (6.7%) tumors were considered as ‘‘low risk’’ but scheduled for primary laparotomy because of size > 10 cm (9 benign and 1 malignant). Sensitivity, specificity, PPV and NPV for this scoring system were 98.6%, 94.9%, 94.7% and 98.7%, respectively. The scoring system were more sensitive than patient’s complaints (98.6% vs 79.5%, P < 0.0001) and more specific than physical examination (94.9% vs 85.9%, P < 0.0001) Conclusions: Ultrasound based triage of symptomatic women diagnosed, as having an adnexal mass is effective for selecting surgical approach.

OC91: Multigate spectral Doppler analysis (MSDA) to study hemodynamic parameters during IVF treatment for egg donation cycles

Objectives: In this study multigate spectral Doppler analysis, a novel ultrasound technology that overcomes the limitations related to the use of a single sample volume, has been applied to the evaluation of hypogastric (Ha) and uterine (Ut) artery flow during IVF treatment, specifically egg donor and recipient cycles. Methods: MSDA using MyLab (Esaote, Florence) was carried out at baseline, during ovulation induction and at egg retrieval in the egg donors, and at baseline and embryo transfer time in the recipients. The analysis in real time included velocity profile (VP), relative wall distension rate (rWDR) and wall shear rate (WSR) of Ha and Ut in the donors and only in Ut for the recipients. Results: A total of 10 egg donors (mean age 24.3 ± 1.8 years) and six recipients (mean age 42.6 ± 3.4 years) were found eligible for the study and successfully evaluated. Compared to conventional spectral Doppler, MSDA revealed different shapes of velocity profiles. There were no significant differences in VP, rWDR and WSR between egg donors at the time of egg retrieval and recipients at the time of embryo transfer in Ut. The rWDR was inversely related to gravity, parity and hematocrit (Hct) (P < 0.05). The WSR of both Ha and Ut were correlated with Hct and the FSH at day 3 (rs = 0.9, P < 0.05). Recipients who conceived had a lower mean WSR (R = 338.3, L = 326.6) than those who did not (R = 512.9, L = 766.3; P < 0.05). WSR of recipients was inversely correlated with positive pregnancy test (rs = −0.878, P < 0.05) bilaterally, while WSR of egg donors was not. Conclusions: This is the first study to provide basic physiological values of VP, rWDR and WSR in patients undergoing ovulation induction for IVF. The measurements in egg donors are to be considered as reference values for patients undergoing IVF since they are young and their ovarian response optimal. Likewise, given the high pregnancy rate in the recipients, the results should be considered promising but not definitive.

OP16.10: Doppler evaluation of wall shear rate, wall distension rate and velocity profile in preoperative ovarian masses: preliminary results

Methods: Digitally stored B-mode sonographic images, from a random sample of 98 women with an adnexal mass submitted to surgery after B-mode transvaginal sonography, were evaluated by five different examiners with different degree of experience. Masses in which the echo features were highly characteristic of a given pathology, such as endometrioma, cystic teratoma, hemorrhagic cyst, hydrosalpinx, benign mucinous cystoadenoma, serous cyst, and peritoneal cysts were categorized as benign. Any cystic mass containing excrescences, thick septations, multiple irregular septations or solid mass (thick wall, thick septations, thick papillary projections, solid areas or mostly solid component) in which the echo architecture was not highly suggestive of benign histology was categorized as malignant. Intraobserver and interobserver agreement according to the level of experience was assessed by calculating the kappa index. Results: Of the 98 cases randomly selected, definitive histologic diagnoses were as follows: 70 (71%) benign and 28 (29%) malignant masses. Intraobserver agreement was good or very good for all examiners with different degrees of experience (kappa = 0.72–1). Interobserver agreement was good for all expert operators (kappa = 0.69–0.75). Interobserver agreement between experts and highly experienced operators was moderate or good (kappa = 0.51–0.63). Interobserver agreement between moderately experienced operators and experts was fair to moderate (kappa = 0.29–0.46). Interobserver agreement between moderately and highly experienced operators was fair (kappa = 0.33). Conclusions: Our results indicate that typical malignant patterns are very highly reproducible even in moderately experienced examiners. More experience was associated with better interobserver agreement.

OC142: Multigate spectral Doppler analysis, new application in maternal‐foetal science

intensities. During the 1st trimester the peak of higher intensities was increased. We did not find a difference between normal and high-risk pregnancies. In cases complicated by IUD we could not detect any signals. there was a significant decrease of all intensities during labour. Tocolytic drugs did not change the 3D-diagrams. Placental blood flow was significantly influenced by maternal breathing and in cases complicated by IUGR intensities decreased to 0 during expiration. Conclusions: Normal as well as reinforced maternal breathing influence the 3D-diagram and may help to detect IUGR in early pregnancy.