Eva-Christine Weiss

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Cytokines and the risk of preterm delivery in twin pregnancies.

OBJECTIVE: To estimate the association between cytokine levels in twin pregnancies and risk of spontaneous preterm delivery, including the effect of progesterone treatment. METHODS: This secondary analysis of a randomized placebo-controlled trial investigating the effect of progesterone treatment on preterm delivery in twin pregnancies included 523 women with available dried blood spot samples collected before treatment with progesterone (n=258) or placebo (n=265) and after 4–8 weeks of treatment. Samples were analyzed for cytokines using a sandwich immunoassay. Cytokine levels in spontaneous preterm delivery at 34–37 weeks of gestation and spontaneous preterm delivery before 34 weeks of gestation were compared with delivery at 37 weeks of gestation or more for placebo-treated women. The association between interleukin (IL)-8 and risk of spontaneous preterm delivery before 34 weeks of gestation was estimated further, including comparison according to treatment. Statistical analyses included Kruskal-Wallis test, Mann-Whitney U test, linear regression, and Cox regression analysis. RESULTS: We found a statistically significant association between IL-8 and spontaneous preterm delivery. At 23–33 weeks of gestation, the median IL-8 level was 52 pg/mL (interquartile range 39–71, range 19–1,061) for term deliveries compared with 65 pg/mL (interquartile range 43–88, range 14–584) for spontaneous preterm delivery at 34–37 weeks of gestation and 75 pg/mL (interquartile range 57–102, range 22–1,715) for spontaneous preterm delivery before 34 weeks of gestation (P<.001). Risk of spontaneous preterm delivery was associated with a large weekly increase in IL-8 (hazard ratio 2.0, 95% confidence interval [CI] 1.2–3.3). There was no effect of progesterone treatment on IL-8 levels. Levels of IL-8 at 18–24 weeks of gestation were associated with a cervix less than 30 mm (odds ratio 1.8, 95% CI 1.2–2.7). CONCLUSION: Risk of spontaneous preterm delivery before 34 weeks of gestation is increased in women with high IL-8 levels. Progesterone treatment does not affect IL-8 levels. CLINICAL TRIAL REGISTRATION: EudraCT, https://eudract.ema.europa.eu, 2006-000503-41, and ClinicalTrials.gov, www.clinicaltrials.gov, NCT00329914. LEVEL OF EVIDENCE: II

Activated Protein C Resistance Assay and Factor V Leiden

The authors suggest that functional testing for activated protein C resistance is cheaper and more clinically relevant than genetic testing to detect a factor V Leiden mutation in identifying persons who are at risk for thromboembolism.

Short- and long-term perinatal outcome in twin pregnancies affected by weight discordance

The objective was to investigate the association between chorionicity‐specific intertwin birthweight discordance and adverse outcomes including long‐term follow up at 6, 18, and 48–60 months after term via Ages and Stages Questionnaire.

A novel factor V mutation causes a normal activated protein C ratio despite the presence of a heterozygous F5 R506Q (factor V Leiden) mutation

Activated protein C (APC) resistance (Dahlback et al, 1993) is a common risk factor for venous thromboembolism (Bucciarelli et al, 1999). This phenotype is highly associated with the F5 R506Q (F5 rs6025; factor V Leiden, FVL) mutation (Bertina et al, 1994). Heterozygous and homozygous discrepancies between F5 R506Q genotype and phenotype (APC resistance), known as pseudo-homozygosity, have been described (Castaman et al, 1997; Brugge et al, 2005). This incidental phenomenon suggests that, based on APC-resistance testing, there is a homozygous F5 R506Q mutation, yet, genetic testing only shows a heterozygous F5 R506Q mutation (Simioni et al, 1996). In such cases, polymorphisms located on the second factor V (FV) protein coding allele (i.e. wild-type for F5 R506Q) cause intracellular decreased FV protein production, leading to reduced FV activity (Castoldi et al, 1998). In contrast, the phenomenon “pseudo-wild-type” FVL, suggesting a wild-type F5 R506Q according to APC resistance testing but heterozygous F5 R506Q mutation by genetic testing, is infrequently described (Dargaud et al, 2003; Asselta et al, 2004). In these two studies, the polymorphisms responsible for the reduced FV protein levels were found to be located on the same FV protein coding allele, together with the F5 R506Q mutation.

OC01.01: Prevention of preterm delivery in twin gestations (PREDICT): a multicentre randomised placebo-controlled trial on the effect of vaginal micronised progesterone: Oral communication abstracts

L. Rode1,2, K. Klein3, K. Nicolaides4, E. Krampl-Bettelheim3, I. Vogel5, H. Larsen6, A. Holmskov7, K. Riis Andreasen8, N. Uldbjerg9, J. Ramb10, B. Bødker11, L. Skibsted12,2, L. Sperling13, S. Hinterberger14, L. Krebs15, H. Zingenberg16, E. Weiss17, I. Strobl18, L. Laursen19, J. Tranberg Christensen20, B. M. Hansen21, A. Lando21, A. Tabor1,2 1Department of Fetal Medicine 4002, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 2Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 3Department of Obstetrics & Gynecology, Medical University of Vienna, Vienna, Austria; 4Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, United Kingdom; 5Department of Clinical Genetics, Aarhus University Hospital Skejby, Aarhus, Denmark; 6Department of Obstetrics & Gynecology, Aalborg Hospital, Aalborg, Denmark; 7Department of Obstetrics & Gynecology, Viborg Hospital, Viborg, Denmark; 8Department of Obstetrics & Gynecology, Hvidovre Hospital, Hvidovre, Denmark; 9Department of Obstetrics & Gynecology, Aarhus University Hospital Skejby, Aarhus, Denmark; 10Department of Obstetrics & Gynecology, Sønderborg Hospital, Sønderborg, Denmark; 11Department of Obstetrics & Gynecology, Hillerød Hospital, Hillerød, Denmark; 12Department of Obstetrics & Gynecology, Roskilde University Hospital, Roskilde, Denmark; 13Department of Obstetrics & Gynecology, Herlev Hospital, Herlev, Denmark; 14Department of Obstetrics & Gynecology, General Hospital of Klagenfurt, Klagenfurt, Austria; 15Department of Obstetrics & Gynecology, Holbæk Hospital, Holbæk, Denmark; 16Department of Obstetrics & Gynecology, Glostrup Hospital, Glostrup, Denmark; 17Department of Obstetrics & Gynecology, Medical University of Graz, Graz, Austria; 18Department of Obstetrics & Gynecology, Medical University of Innsbruck, Innsbruck, Austria; 19Department of Obstetrics & Gynecology, Odense University Hospital, Odense, Denmark; 20Department of Obstetrics & Gynecology, Gentofte Hospital, Gentofte, Denmark; 21Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

OC06.01: Vaginal progesterone and the risk of preterm delivery in high‐risk twin gestations ‐ secondary analysis of a placebo‐controlled randomized trial

K. Klein1, L. Rode2,3, K. Nicolaides4, E. Krampl-Bettelheim1, H. Larsen5, A. Holmskov6, K. Riis Andreasen7, N. Uldbjerg8, J. Ramb9, B. Bodker10, L. Skibsted11, L. Sperling12, S. Hinterberger13, L. Krebs14, H. Zingenberg15, E. Weiss16, I. Strobl17, L. Laursen18, J. Tranberg Christensen19, I. Vogel20, B. M. Hansen21, A. Lando21, A. Tabor2,3 1Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria; 2Department of Fetal Medicine 4002, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 3Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 4Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, United Kingdom; 5Department of Obstetrics and Gynecology, Aalborg Hospital, Aalborg, Denmark; 6Department of Obstetrics and Gynecology, Viborg Hospital, Viborg, Denmark; 7Department of Obstetrics and Gynecology, Hvidovre Hospital, Hvidovre, Denmark; 8Department of Obstetrics and Gynecology, Aarhus University Hospital Skejby, Aarhus, Denmark; 9Department of Obstetrics and Gynecology, Sonderborg Hospital, Sonderborg, Denmark; 10Department of Obstetrics and Gynecology, Hillerod Hospital, Hillerod, Denmark; 11Department of Obstetrics and Gynecology, Roskilde University Hospital, Roskilde, Denmark; 12Department of Obstetrics and Gynecology, Herlev Hospital, Herlev, Denmark; 13Department of Obstetrics and Gynecology, General Hospital of Klagenfurt, Klagenfurt, Austria; 14Department of Obstetrics and Gynecology, Holbaek Hospital, Holbaek, Denmark; 15Department of Obstetrics and Gynecology, Glostrup Hospital, Glostrup, Denmark; 16Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria; 17Department of Obstetrics and Gynecology, Medical University of Innsbruck, Innsbruck, Austria; 18Department of Obstetrics and Gynecology, Odense University Hospital, Odense, Denmark; 19Department of Obstetrics and Gynecology, Gentofte Hospital, Gentofte, Denmark; 20Department of Clinical Genetics, Aarhus University Hospital Skejby, Aarhus, Denmark; 21Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities. Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood. Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208–0.026 μg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography. Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2–71.8%] of vehicle; cord: 73.4% [IQR: 70.2–91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 μg/ml; cord: 0.814 μg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels. Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates.

Thrombin Generation in Pregnancy

A well functioning hemostasis depends on the well-balanced interaction between many procoagulatory and inhibiting factors of the coagulation and the fibrinolytic system. During normal pregnancy this balance is shifted towards a state of hypercoagulability [1], which is more marked around term and in the immediate post partum period [2].