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Resistance of aspirin during and after pregnancy: A longitudinal cohort study
Pregnancy Hypertension 19 (2020) 25–30
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Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Resistance of aspirin during and after pregnancy: A longitudinal cohort study☆
T
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Jeske M. bij de Wega, , Carolien N.H. Abheidena, Wessel W. Fuijkschotb, Ankie M. Harmszec, Marjon A. de Boera, Abel Thijsb, Johanna I.P. de Vriesa a
Amsterdam UMC, location VUmc, Department of Obstetrics and Gynaecology, Reproduction and Development, De Boelelaan 1117, Amsterdam, the Netherlands Amsterdam UMC, location VUmc, Department of Internal Medicine, Amsterdam Cardiovascular Sciences, De Boelelaan 1117, Amsterdam, the Netherlands c Amsterdam UMC, location VUmc, Department of Clinical Pharmacology and Pharmacy, De Boelelaan 1117, Amsterdam, the Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspirin Drug resistance HELLP Pre-eclampsia Pregnancy Preventive therapy
Objectives: The objective of this study is to investigate possible changes in aspirin resistance during and after pregnancy over time. Study design: A longitudinal cohort study in obstetric high risk women with an indication for aspirin usage during pregnancy to prevent placenta mediated pregnancy complications. Main outcome measures: Aspirin resistance measured in the first, second and third trimester of pregnancy and at least three months postpartum by four complementary test: PFA-200, VerifyNow®, Chronolog light transmission aggregometry (Chronolog LTA) and serum thromboxane B2 (TxB2) level measurements. Correlation between the devices was investigated. Results: In total, 23 pregnant women participated in the present study. Aspirin resistance according to the PFA200, VerifyNow®, Chronolog LTA and serum TxB2, was 30.4%, 17.4%, 26.1% and 23.8% respectively. Resistance by any device was 69.6%. Aspirin resistance measured by the VerifyNow®, Chronolog LTA, serum TxB2 and aspirin resistance by any device during pregnancy was demonstrated more frequently than aspirin resistance after pregnancy. Correlation between the different devices was weak. Conclusion: Aspirin resistance was found in a considerable part of the participants. Considerable variation between participants, within participants over time and between the different devices was found. Prevalence of aspirin resistance during pregnancy differs from after pregnancy. More research on aspirin resistance and clinical obstetric outcome is needed.
1. Introduction Hypertensive disorders of pregnancy (HDP) are an important cause of maternal and neonatal morbidity and mortality [1,2]. It is thought that poor spiral artery remodelling contributes to the onset of HDP [3]. Poor spiral artery remodelling is primary linked to early-onset HDP. Women at risk of early-onset HDP have an indication to use aspirin during pregnancy [4,5] which reduces the risk for recurrent early onset HDP [6,7]. Aspirin resistance is the phenomenon of a less effective response to aspirin in some patients. Prevalence of aspirin resistance in populations of men and non-pregnant women with cardiovascular diseases of about 5 up to 65% is reported, depending on which test and cut-off values are used [8–11]. Aspirin resistance during pregnancy is investigated in four
cohort studies and one review showing that around one third is resistant to aspirin, depending on the cut-off values and definition used [11–15]. In three studies the timing of measurement of aspirin resistance was not standardized which might be a possible confounder [12,14,15]. Aspirin resistance may change during pregnancy by the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [16–18]. All these factors can have an effect on pharmacokinetics and/or pharmacodynamics and therefore lead to possible differences in occurrence of aspirin resistance over time during pregnancy. Finally, non-adherence to aspirin can influence results of aspirin resistance measurements [19,20]. Aspirin resistance could be an explanation of (recurrent) HDP despite the use of aspirin. Bujold et al commented that the beneficial
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The work has been carried out in the Amsterdam UMC, location VUmc, Amsterdam, the Netherlands. Corresponding author at: Department of Obstetrics and Gynaecology, Amsterdam UMC, location VUmc, P.O. Box 7057, 1007 MB Amsterdam, the Netherlands. E-mail address: [email protected] (J.M. bij de Weg).
https://doi.org/10.1016/j.preghy.2019.11.008 Received 28 June 2019; Received in revised form 23 October 2019; Accepted 25 November 2019 2210-7789/ © 2019 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
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enzyme immunoassay kit (Assay Designs®, Ann Arbor, MI, USA). PFA-200 measures the process of primary hemostasis [24]. The system measures platelet plug formation in a capillary through which citrated whole blood is passed. The closure time (CT) is measured: the time needed for complete obstruction of the capillary. A CT of ≤150 s is used for the dichotomous definition of aspirin resistance [12,14]. PFA has a theoretical maximum of 300 s, therefore any CT > 300 s was reported as 301 s. The Collagen/Epinephrine cartridge is used. VerifyNow® utilizes arachidonic acid as an agonist to measure the antiplatelet effect of aspirin specifically along the pathway of inhibiting cyclooxygenase-1. Change in light transmittance in patient’s whole blood sample is measured in an aspirin cartridge. Results are displayed in Aspirin Reactions Units (ARU) [25]. An ARU of ≥550 is used for the dichotomous definition of aspirin resistance [26,27]. Chronolog LTA measures the light transmittance of platelet rich plasma which is influenced by platelet aggregation stimulated by arachidonic acid [28]. Platelet aggregation is reduced by lesser production of TxA2 through addition of arachidonic acid. In women resistant to aspirin, more aggregation occurs and the light transmittance is increased. The percentage of maximal aggregation is measured and a value > 22% is defined as aspirin resistant [29–31]. Serum TxB2 is a direct measure of the capacity of platelets to synthesize thromboxane A2 (TxA2) and a specific measure of the pharmacological effect of aspirin on platelets. Blood samples are placed in a stove for one hour at 37 degrees Celsius, directly after collection. Samples are spinned with 3000 rotations per minute for 10 min and serum samples were stored at −80 degrees Celsius. Thereafter, samples were analyzed by enzyme immunoassay in the laboratory for hematology, unit thrombosis and hemostasis of the Radboud University Medical Center in Nijmegen, the Netherlands. A TxB2 concentration in the highest quartile is defined as aspirin resistant [15].
effect found in a randomized controlled trial (RCT) comparing lowmolecular-weight heparin and aspirin versus aspirin alone on recurrent early onset preeclampsia in women with inheritable thrombophilia could be the result of aspirin resistance [21,22]. A follow-up study of this RCT has been performed many years after the pregnancy and could not demonstrate a relation between HDP and aspirin resistance [23]. The present study investigated whether aspirin resistance changes over time during and after pregnancy in pregnant women with an indication for aspirin. We hypothesize that the prevalence of aspirin resistance changes during the three trimesters of pregnancy and differs between pregnancy and postpartum. To our knowledge, aspirin resistance in pregnant women has never been tested longitudinally during and after pregnancy. A previous study only tested longitudinally during the different trimesters of pregnancy, but not postpartum [13]. This study will gain more insight in the prevalence of aspirin resistance in pregnancy over time and the consistency during and after pregnancy. 2. Material and methods 2.1. Participants This longitudinal cohort study was performed from April 2015 till January 2017 at the Amsterdam UMC, Vrije Universiteit Amsterdam, in Amsterdam, the Netherlands. Pregnant women were included if they had an indication for low-dose aspirin (acetylsalicylic acid 80 mg, nonenteric-coated) usage during pregnancy and had their check-ups at this hospital. Inclusion criteria were age ≥ 18 years, understanding of Dutch or English and an indication for aspirin usage during pregnancy. Exclusion criteria were women diagnosed with Systemic Lupus Erythematosus or anti-phospholipid syndrome, use of drugs that are known to alter platelet function (e.g. NSAID’s, tirofiban, eptifibatide, abciximab, beta-lactam antibiotics, dextran, SSRI’s, clomipramine and amitriptyline, dipyridamole, verapamil, diltiazem, ginkgo biloba, ginseng, St John’s wort), a recent cardiovascular event (< 3 months), abnormal cell count, twin pregnancy, hypercholesterolemia before pregnancy or in the first trimester, impaired renal or liver function before pregnancy or in the first trimester and poorly controlled hypertension before pregnancy or in the first trimester. Written informed consent was obtained from all participants.
2.3. Outcomes Baseline characteristics included age, number of previous pregnancies, body mass index before pregnancy, blood pressure in the first trimester, smoking behaviour, alcohol use, indication for aspirin usage, obstetric history and family history including vascular diseases and HDP. Outcomes are the prevalence of aspirin resistance during the three trimesters of pregnancy and postpartum per device and in any device labelled as ‘resistance by any device’, the difference in prevalence between pregnancy and postpartum, correlation between the different devices and aspirin adherence. Moreover, aspirin resistance was classified into three groups: no resistance at all moments of measurement for all tests, resistance at all moments of measurements in at least one device, and partial resistance in all other outcomes. This classification is based on one used in a previous prospective cohort study on aspirin resistance in pregnant women at risk of HDP [13].
2.2. Measurements Aspirin resistance measurements during the first trimester of pregnancy were scheduled between 10 and 15 weeks of gestation, in the second trimester between 19 and 25 weeks of gestation and in the third trimester between 28 and 34 weeks of gestation. The postpartum measurement was scheduled at least three months after delivery. All measurements were performed after a period of at least 10 days of aspirin usage. Participants were instructed to take aspirin in the evening and during ten days before their appointment strictly at 8 pm. Adherence to aspirin was investigated by questioning the study participant at every visit. Taking less than 10 tablets in the previous 10 days was registered as non-adherence. Venous blood samples were collected after an overnight fast at 8am and participants were instructed not to smoke 30 min prior to the appointment. Besides aspirin resistance measurements, blood sampling was performed for hemoglobin, hematocrit, thrombocytes and leucocyte counts. During the visit in the first trimester, a physical examination including blood pressure, body weight and length measurements was performed and a questionnaire about family history was answered. During the postpartum visit blood pressure and body weight were measured again. Four complementary tests were used to analyse the blood samples [20]: Platelet Function Analyzer 200 (PFA-200, INNOVANCE® PFA-200 System, Siemens Healthcare, Marburg, Germany), VerifyNow® point-ofcare system (Accumetrics, CA, USA), Chronolog light transmittance aggregometry (LTA) and Serum thromboxane B2 (TxB2) level using an
2.4. Statistical analysis This is a pilot study, therefore a power calculation was not performed. The number of participants is based on previous studies measuring aspirin resistance [12,15,32]. Outcomes of the four complementary tests were compared with Mixed Models and when not normally distributed a logarithmic transformation was performed in advance. Dichotomous data were analysed with a Generalized Estimating Equations. Correlation for continuous data were analysed with Pearman’s rho and dichotomous data with Cohen’s kappa. SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses. Results were considered significant at the two-sided 5% level. 2.5. Ethical approval The study was conducted in accordance with the Helsinki II Declaration and was approved by the Institutional Review Board of the 26
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Table 1 Baseline characteristics. (n = 23) Maternal age (years) Number of pregnancy BMI in kg/m2 Blood pressure in mmHg in first trimester Systolic Diastolic Smoking Alcohol usage Indications for aspirin usage* Previous HDP Gestational age HDP IUFD Gestational age IUFD SGA ≥2 moderate risk factors**
33.0 ± 3.9 3.2 ± 1.0 24.1 ± 5.7 110.4 ± 11.6 71.7 ± 9.6 1 (4.3) 0 (0.0) 16 (69.6) 31.6 ± 3.3 3 (13.0) 30.1 ± 6.2 6 (26.1) 2 (8.7)
Fig. 1a. Continuous results of the PFA-200 over time during and after pregnancy. On the horizontal axis the moments of measurement are shown. On the vertical axis the closure time in seconds is shown. Each line represents one participant, n = 23. The dotted line is the mean of the results of all participants per trimester. The broken line is the cut-off value of the PFA-200: 150 s. Aspirin resistance PFA-200: closure-time ≤150 s.
Data are depicted as mean ± SD or numbers (%) as appropriate. *Some women had more than one indication for aspirin usage during pregnancy. **≥2 of the following moderate risk factors indicates aspirin usage during pregnancy: first pregnancy, maternal age ≥40 years, pregnancy interval > 10 years, BMI > 35 kg/m2, family (medical) history of HDP, twin pregnancy and pregnancy after egg donation. BMI, body mass index; HDP, hypertensive disorder of pregnancy; SGA, small for gestational age (< p10); IUFD, intra uterine fetal death.
Amsterdam UMC, Vrije Universiteit Amsterdam, in Amsterdam, the Netherlands. The study was registered at the Dutch Trial Register (Nederlands Trial register; www.trialregister.nl) with number NTR5106. 3. Results Out of 38 invited women, 14 declined participation and 1 had a spontaneous miscarriage, resulting in 23 participants in this study (participation rate 60.5%). There were two drop-outs after the third trimester (1 woman moved and 1 had an indication for the use of lowmolecular weight heparin (LMWH) due to a postpartum sagittal sinus thrombosis). Seven participants also participated in another study in the Amsterdam UMC, Vrije Universiteit Amsterdam to evaluate aspirin adherence [33]. Baseline characteristics are listed in Table 1. Prevalence of aspirin resistance of all four devices over all four moments of measurements are depicted in Table 2. In the continuous data of the VerifyNow®, Chronolog LTA and serum TxB2, as depicted in
Fig. 1b. Continuous results VerifyNow® Aspirin Test over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis Aspirin Reaction Units (ARU) are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value of the VerifyNow® Aspirin Resistance: 550 ARU. Aspirin resistance VerifyNow® Aspirin Test: ≥550 ARU.
Table 2 Aspirin resistance measurements with four devices in first, second and third trimester, and > 3 months postpartum.
PFA-200 Resistant VerifyNow® Resistant Chronolog LTA Resistant Serum TxB2 Resistant Resistance by any device
Trimester 1 (n = 23)
Trimester 2 (n = 23)
Trimester 3 (n = 23)
> 3 months postpartum (n = 21)
P-value
226.35 ± 13.82 5 (21.7) 465[124] 4 (17.4) 11.5[5.0] 2 (8.7) 12.81 ± 2.45 5 (21.7) 14 (60.9)
231.00 ± 12.04 3 (13.0) 420[79] 4 (17.4) 12.5[5.5] 2 (8.7) 12.24 ± 2.45 5 (21.7) 11 (47.8)
207.87 ± 15.14 7 (30.4) 421[76] 2 (8.7) 14.5[14.5] 6 (26.1) 14.16 ± 2.45 4 (17.4) 16 (69.6)
219.48 ± 15.15 4 (19.0) 388[11] 0 (0.0) 9.0[6.0] 1 (4.8) 10.05 ± 2.48 5 (23.8) 8 (38.1)
P = 0.45 P = 0.36 P < =0.01 N/A P < =0.01 P = 0.07 P < =0.05 P = 0.92 P < =0.05
95%-CI
0.02–0.11* 0.08–0.51** 0.30–7.92*** 0.05–0.68****
Data are depicted as mean ± standard deviation, median with [inter quartile range] or numbers (%) as appropriate. N/A; not available. Aspirin resistance cut-off values per device; PFA-200: closure-time ≤150 s; VerifyNow® Aspirin Test: ≥550 ARU; Chronolog LTA: > 22%; TxB2: above the 75th percentile per moment of measurement, respectively 15.3 ng/ml for trimester 1, 15.8 ng/ml for trimester 2, 13.9 ng/ml for trimester 3 and 10.4 ng/ml postpartum. *VerifyNow®: Statistically significant difference between first trimester and postpartum. **Chronolog LTA: Statistically significant difference between third trimester and postpartum. ***Serum TxB2: Statistically significant difference between third trimester and postpartum. **** Resistance by any device: Statistically significant difference between third trimester and postpartum. 27
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Table 3 Pregnancy outcomes. (n = 23) Gestational age of delivery Induction Gestational age Indication induction HDP Elective Obstetric history IUGR Persistent reduced fetal movements HDP PIH Gestational age PIH PE Gestational age PE Eclampsia HELLP Cesarean section Primary cesarean section Secondary cesarean section SGA
Fig. 1c. Continuous results Chronolog LTA over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis the percentages of maximal aggregation are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value of the Chronolog LTA: 22%. Aspirin resistance Chronolog LTA: > 22%.
38.3 ± 1.7 14 (60.9) 38.0 ± 1.7 5 (35.7) 4 (28.6) 3 (21.4) 1 (7.1) 1 (7.1) 12 (52.2) 9 (39.1) 36.5 ± 3.0 3 (13.0) 34.6 ± 3.0 0 (0.0) 0 (0.0) 12 (52.2) 7 (58.3) 5 (41.7) 3 (13.0)
Data are depicted as mean ± standard deviation or numbers (%) as appropriate. HDP, hypertensive disorders of pregnancy; IUGR, intra uterine growth restriction; PIH, pregnancy-induced hypertension; PE, preeclampsia; HELLP, hemolysis elevated liver enzymes low platelets; SGA, small for gestational age (birthweight < p10).
first and second trimester, in 20 participants (87.0%) during the third trimester and 18 participants (85.7%) postpartum. The two participants who were non-adherent in the first and second trimester, did not show aspirin resistance. In the third trimester, all 3 participants who were non-adherent showed aspirin resistance on one single device, one on the VerifyNow® and two on the PFA-200. Post-partum, one out of five participants reporting non-adherence showed aspirin resistance on the PFA-200. Pregnancy outcomes are depicted in Table 3.
Fig. 1d. Continuous results of the serum thromboxane B2 over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis ng/ml are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value defined as above the 75th percentile per moment of measurement, respectively 15.3 ng/ml for trimester 1, 15.8 ng/ml for trimester 2, 13.9 ng/ml for trimester 3 and 10.4 ng/ml postpartum.
4. Discussion In this prospective cohort study we examined aspirin resistance in pregnant women during the three trimesters of pregnancy and more than three months postpartum by four complementary devices. A considerable part of the participants showed aspirin resistance, despite the variation between participants, within participants over time and between the different devices. The prevalence of aspirin resistance during pregnancy measured by the four different devices found in this study varied from 8.7% to 30.4% and aspirin resistance in any device from 47.8% to 69.6%. The resistance rates found in our cohort are similar or slightly higher to a previous study measuring aspirin resistance in women with obstetric history of HDP with different devices [23]. The investigated population in our study differs from the previous study because in our study women were pregnant, did not have inheritable thrombophilia and the mean age was more than 10 years lower: 33.0 versus 44.7 years old. So the same or slightly higher aspirin resistance rates were found in a younger, pregnant population without inheritable thrombophilia. No resistance was found in 13.0% of the participants and resistance in 26.1%. The majority, namely 60.9%, showed partial resistance over time. These rates are lower in comparison to a previous study in which a comparable classification was made [13]. In this previous study aspirin resistance was measured twice during pregnancy by aggregometry, VerifyNow® and urinary TxB2. In addition, urinary aspirin metabolites were determined. Participants were categorized as responsive if two or all platelet function tests were outside normal range at both visits, non-responsive if none or one platelet function test was
Figs. 1a–d, statistical significant differences in aspirin resistance were found between the measurements during and after pregnancy, respectively for the VerifyNow® between the first trimester and postpartum, and for the Chronolog LTA, serum TxB2 and resistance by any device between the third trimester and postpartum. Aspirin resistance was found in one participant on the Chronolog LTA and serum TxB2 during all four measurements. In this participant aspirin resistance was also shown in the PFA-200 in the three trimesters of pregnancy, however not postpartum. Another participant showed aspirin resistance on the VerifyNow® over the three trimesters of pregnancy. One participant showed resistance by serum TxB2 over all four moments of measurement. Three participants (13.1%) showed no resistance, meaning no resistance at any devices during all moments of measurement. Six participants (26.1%) showed resistance, meaning resistance in at least one device during all moments of measurement. The majority showed partial resistance, meaning resistance in at least one device but not at all moments of measurement. Correlation for continuous data in Pearman’s rho showed a weak correlation between the four devices in a range of 0.01 to −0.53. Agreement for dichotomous data in Cohen’s kappa showed poor to fair correlation in a range of −0.24 to 0.33. Aspirin adherence was found in 21 participants (91.3%) during the
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neonates (13.0%) were small for gestational age which is in line with the prevalence in the general population. Unfortunately, the influence of aspirin resistance on the obstetric outcomes could not be examined, since the study is not powered for this question. The present study showed that aspirin resistance rates differ during and after pregnancy and within participants over time. Thus aspirin resistance measurements 9–16 years after pregnancy as performed in the follow-up study of Abheiden et al [23] might not be representative for aspirin resistance during pregnancy. Thereby, the question of Bujold whether a beneficial effect of LMWH in women with adverse obstetric history and inheritable thrombophilia is caused by aspirin resistance could not be answered yet [21,22]. This study elucidates for the first time aspirin resistance during pregnancy using the four complementary devices and the inconsistency of aspirin resistance during and after pregnancy with complementary devices. The strength of this study is its prospective character with multiple moments of measurement. We examined aspirin resistance with the current available tests which are complementary and therefore provide more insight. Factors which could influence the measurements, such as time of aspirin intake, fasting over night the day before the appointment and no smoking more than 30 min before the appointment were minimized with clear instructions to the participants. The limitation of this study is that no well-defined cut-off values for pregnant women exist. Cut-off values of the different devices were based on values of previous studies [12,14,15,23]. Pregnancy-specific cut-off values should be investigated, since these might differ due to the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [14–16] and thereby might influence the proportion of pregnant women being aspirin resistant. Another limitation is that the amount of investigated women is relatively small, so that caution is warranted when interpreting these results. Future studies could investigate the optimal dosage and dosing frequency of aspirin therapy during pregnancy. A possible dose-response effect of aspirin for prevention of preeclampsia was shown in a previous review [35]. An answer to the question which dose is preferred could come from a trial comparing different doses. One could also hypothesize that aspirin resistance might be influenced by body weight and that dosage adjustment for body weight is needed for optimal individualized treatment. A previous systematic review in patients at risk for vascular events showed that the effectiveness of aspirin depends on the dosage of aspirin [36]. Low dose aspirin was effective in patients with low bodyweight and high dose in patients with high bodyweight. A one-dose-fits-all approach might be suboptimal in this population and could be quested in the pregnant population as well. More research is needed about this topic in pregnant women.
outside normal range at both visits and aspirin metabolites were detectable, indeterminate response if none or one platelet function test was outside normal range at both visits and aspirin metabolites were not detectable, and variable response if test results changed between both visits in the same participants. The majority, 59.0%, was categorized as responder, 0% was non-responder, 7.0% as indeterminate responder, and 34.0% as variable responder. The different outcome can partly be explained by a different categorization due to other tests and moments of measurement. Studies which investigated aspirin resistance with one single device (PFA-100 or urinary TxB2) also reported similar resistance rates around 30% [12,14,15]. Despite the variation in aspirin resistance between participants, within participants over time and between devices as shown in Figs. 1a–d, aspirin resistance rates measured by the VerifyNow®, Chronolog LTA, serum TxB2 and resistance by any device were statistical significant higher during pregnancy in comparison to after pregnancy. By the VerifyNow®, this difference was found between the first trimester and postpartum. In case of the Chronolog LTA, serum TxB2 and resistance by any device, this difference was found between the third trimester and postpartum. The differences in aspirin resistance between during and after pregnancy might be explained by the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [16–18]. This possible pregnancy-induced effect has to our knowledge never been demonstrated before. Operationalization of aspirin resistance measurements is complicated. Firstly, due to the enormous variation between the different devices. This confirms the importance of testing with multiple, complementary devices to grasp its various working mechanisms, or indicates that the existence of aspirin resistance is not yet certain and the present tests may be influenced by multiple unknown factors. The accepted gold standard to measure aspirin resistance is the Chronolog LTA. However, this method is limited in practical usage for clinical researchers, because it is a costly, time consuming method and requires platelet rich plasma. Therefore, in previous studies other devices, namely PFA-200, VerifyNow® and serum TxB2, were also used [11–15]. To be able to compare the results of our study with previous performed studies, we measured aspirin resistance with all four available devices. Weak correlation between the devices was found. This was expected due to the different methods of measuring aspirin resistance. The weak correlation between the devices complicates the interpretation of the results of the devices. A powered study for clinical outcome is needed. Secondly, the large variation between the participants makes operationalization of aspirin resistance measurements complicated. Non-optimal adherence could cause non-optimal aspirin influence. We investigated non-adherence by questioning at every visit. We might underestimate the non-adherence of our cohort, since questioning is vulnerable to inaccuracy. Despite this limitation, non-adherence rates in this study are in line with a previous study on aspirin adherence in women with high-risk pregnancies showing non-adherence rates of 21.4% and 46.3%, depending on which questionnaire was used [33]. Our study demonstrated the highest rates of non-adherence postpartum. This might be explained by the fact that participants had to take the aspirin postpartum solely for the purpose of the study, in contrast to their medical indication during pregnancy. Non-adherence can influence the results of the measurements of aspirin resistance [19,20] and thereby are harder to interpret. Finally, operationalization is complicated by the enormous variation in aspirin resistance within participants over time. Intra-individual variability is a well-known phenomenon in other internal medicine-related diagnosis, such as hypertension and hypercholesterolemia. Due to variation over time, these diagnoses can only be made by multiple measurements. We should wonder if repetitive testing is also required for measuring aspirin resistance. The pregnancy outcomes revealed that more than 50% of the pregnant women developed HDP despite aspirin use during pregnancy. Our induction and cesarean rates are above average [34]. Three
5. Conclusions In conclusion, in this prospective cohort study a considerable part of the women showed aspirin resistance, despite the enormous variation between participants, within participants over time and between the different devices. We reveal a difference in prevalence of aspirin resistance during and after pregnancy in three out of the four devices, which might be pregnancy-induced. A powered study for aspirin resistance and clinical obstetric outcome is needed to clarify the relevance of measuring aspirin resistance. Afterwards, trials regarding optimal dosage and dosing frequency could be performed for adjustment and optimizing aspirin therapy. Acknowledgements We would like to express our gratitude to all women who participated in this study. We would like to thank A. Arduc for his help with the measurements. Furthermore, we want to thank W. Rosendal and H. Hopman, laboratory technicians of the hemostasis laboratory of the 29
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Amsterdam UMC, Vrije Universiteit Amsterdam.
[17] B. Allolio, J. Hoffmann, E.A. Linton, W. Winkelmann, M. Kusche, H.M. Schulte, Diurnal salivary cortisol patterns during pregnancy and after delivery - relationship to plasma corticotropin-releasing-hormone, Clin. Endocrinol. 33 (2) (1990) 279–289. [18] A.T. Toriola, M. Vaarasmaki, M. Lehtinen, A. Zeleniuch-Jacquotte, E. Lundin, K.G. Rodgers, et al., Determinants of maternal sex steroids during the first half of pregnancy, Obstet. Gynecol. 118 (5) (2011) 1029–1036. [19] J. Dawson, T. Quinn, M. Rafferty, P. Higgins, G. Ray, K.R. Lees, et al., Aspirin resistance and compliance with therapy, Cardiovasc. Ther. 29 (5) (2011) 301–307. [20] S.M. Knoepp, M. Laposata, Aspirin resistance: moving forward with multiple definitions, different assays, and a clinical imperative, Am. J. Clin. Pathol. 123 (Suppl) (2005) S125–S132. [21] J.I. de Vries, M.G. van Pampus, W.M. Hague, P.D. Bezemer, J.H. Joosten, F. Investigators, Low-molecular-weight heparin added to aspirin in the prevention of recurrent early-onset pre-eclampsia in women with inheritable thrombophilia: the FRUIT-RCT, J. Thromb. Haemost. 10 (1) (2012) 64–72. [22] E. Bujold, K. Gouin, S. Cote, Low-molecular-weight heparin added to aspirin in the prevention of recurrent early-onset pre-eclampsia in women with inheritable thrombophilia: the FRUIT-RCT: a rebuttal, J. Thromb. Haemost. 10 (6) (2012) 1195 author reply 6. [23] C.N.H. Abheiden, W.W. Fuijkschot, A. Arduc, J.J.K. van Diemen, A.M. Harmsze, M.A. de Boer, et al., Post-pregnancy aspirin resistance appears not to be related with recurrent hypertensive disorders of pregnancy, Eur. J. Obstet. Gyn. R B. 210 (2017) 139–143. [24] SIEMENS. INNOVANCE PFA-200 System. 2015. [25] http://www.accumetrics.com/products/verifynow-aspirin. [26] R. Grimaldi, M. Bisi, E. Lonni, E. Beggiato, A. Valpreda, M.F. Lococo, et al., Laboratory aspirin resistance reversibility in diabetic patients: a pilot study using different pharmaceutical formulations, Cardiovasc. Drugs Ther. 28 (4) (2014) 323–329. [27] L. Consuegra-Sanchez, R. Lopez-Palop, P. Cano, P. Carrillo, F. Pico, M. Villegas, et al., Assessment of high on-treatment platelet reactivity in patients with ischemic heart disease: concordance between the Multiplate and VerifyNow assays, J. Thromb. Haemost. 11 (2) (2013) 379–381. [28] Chronolog-Corporation. Chronolog LTA [cited 2016 4th of January]. Available from: http://www.chronolog.com/index.html. [29] O.M. Akay, Z. Canturk, E. Akin, C. Bal, Z. Gulbas, Aspirin-resistance frequency: a prospective study in 280 healthy Turkish volunteers, Clin. Appl. Thromb-Hem. 15 (1) (2009) 98–102. [30] P.A. Gurbel, K.P. Bliden, J. DiChiara, J. Newcomer, W. Weng, N.K. Neerchal, et al., Evaluation of dose-related effects of aspirin on platelet function – RESULTS from the aspirin-induced platelet effect (ASPECT) study, Circulation 115 (25) (2007) 3156–3164. [31] P.A. Gum, K. Kottke-Marchant, E.C. Poggio, H. Gurm, P.A. Welsh, L. Brooks, et al., Profile and prevalence of aspirin resistance in patients with cardiovascular disease, Am. J. Cardiol. 88 (3) (2001) 230–235. [32] J.J.K. van Diemen, W.W. Fuijkschot, T.J. Wessels, G. Veen, Y.M. Smulders, A. Thijs, Evening intake of aspirin is associated with a more stable 24-h platelet inhibition compared to morning intake: a study in chronic aspirin users, Platelets 27 (4) (2016) 351–356. [33] C.N.H. Abheiden, A.V.R. van Reuler, W.W. Fuijkschot, J.I.P. de Vries, A. Thijs, M.A. de Boer, Aspirin adherence during high-risk pregnancies, a questionnaire study, Pregnancy Hypertens.on 6 (4) (2016) 350–355. [34] Perined. Perinatale Zorg in Nederland 2015. 2016. [35] A.L. Seidler, L. Askie, J.G. Ray, Optimal aspirin dosing for preeclampsia prevention, Am. J. Obstet. Gynecol. 219 (1) (2018) 117–118. [36] P.M. Rothwell, N.R. Cook, J.M. Gaziano, J.F. Price, J.F.F. Belch, M.C. Roncaglioni, et al., Effects of aspirin on risks of vascular events and cancer according to bodyweight and dose: analysis of individual patient data from randomised trials, Lancet 392 (10145) (2018) 387–399.
Declaration of interest None. Funding information We had a contract with Siemens and The Surgical Company involving purchase of cartridges with discount. These funding had no influence on the collection and interpretation of the data. References [1] B.W. Mol, C.T. Roberts, S. Thangaratinam, L.A. Magee, C.J. de Groot, G.J. Hofmeyr, Pre-eclampsia, Lancet (2015). [2] K. Sliwa, M. Bohm, Incidence and prevalence of pregnancy-related heart disease, Cardiovasc. Res. 101 (4) (2014) 554–560. [3] I. Brosens, R. Pijnenborg, L. Vercruysse, R. Romero, The, “Great Obstetrical Syndromes” are associated with disorders of deep placentation, Am. J. Obstet. Gynecol. 204 (3) (2011) 193–201. [4] WHO Recommendations for Prevention and Treatment of Pre-Eclampsia and Eclampsia. WHO Guidelines Approved by the Guidelines Review Committee. Geneva 2011. [5] NICE guideline. Hypertension in pregnancy: diagnosis and management. 2010 [updated January 2011; cited 2017 30 August]. Available from: https://www.nice. org.uk/guidance/cg107. [6] E. Bujold, S. Roberge, Y. Lacasse, M. Bureau, F. Audibert, S. Marcoux, et al., Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early pregnancy: a meta-analysis, Obstet. Gynecol. 116 (2 Pt 1) (2010) 402–414. [7] D.L. Rolnik, D. Wright, L.C. Poon, N. O'Gorman, A. Syngelaki, Matallana C de Paco, et al., Aspirin versus placebo in pregnancies at high risk for preterm preeclampsia, N. Engl. J. Med. 377 (7) (2017) 613–622. [8] S.I. Al-Azzam, K.H. Alzoubi, O. Khabour, A. Alowidi, D. Tawalbeh, The prevalence and factors associated with aspirin resistance in patients premedicated with aspirin, Acta Cardiol. 67 (4) (2012) 445–448. [9] R. Fitzgerald, M. Pirmohamed, Aspirin resistance: effect of clinical, biochemical and genetic factors, Pharmacol. Ther. 130 (2) (2011) 213–225. [10] X.F. Liu, J. Cao, L. Fan, L. Liu, J. Li, G.L. Hu, et al., Prevalence of and risk factors for aspirin resistance in elderly patients with coronary artery disease, J. Geriatr. Cardiol. 10 (1) (2013) 21–27. [11] K. Navaratnam, A. Alfirevic, Z. Alfirevic, Low dose aspirin and pregnancy: how important is aspirin resistance? BJOG 123 (9) (2016) 1481–1487. [12] N. Caron, G.E. Rivard, N. Michon, F. Morin, D. Pilon, J.M. Moutquin, et al., Lowdose ASA response using the PFA-100 in women with high-risk pregnancy, J. Obstet. Gynaecol. Can. 31 (11) (2009) 1022–1027. [13] K. Navaratnam, A. Alfirevic, A. Jorgensen, Z. Alfirevic, Aspirin non-responsiveness in pregnant women at high-risk of pre-eclampsia, Eur. J. Obstet. Gynecol. Reprod. Biol. 221 (2018) 144–150. [14] E. Rey, G.E. Rivard, Is testing for aspirin response worthwhile in high-risk pregnancy? Eur. J. Obstet. Gynecol. Reprod. Biol. 157 (1) (2011) 38–42. [15] A. Wojtowicz, A. Undas, H. Huras, J. Musial, K. Rytlewski, A. Reron, et al., Aspirin resistance may be associated with adverse pregnancy outcomes, Neuro Endocrinol. Lett. 32 (3) (2011) 334–339. [16] Maternal cardiovascular and hemodynamic adaptations to pregnancy cited 2017 12th of January Available from: http://www.uptodate.com/contents/maternalcardiovascular-and-hemodynamic-adaptations-to-pregnancy 2015.
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Contents lists available at ScienceDirect
Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Resistance of aspirin during and after pregnancy: A longitudinal cohort study☆
T
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Jeske M. bij de Wega, , Carolien N.H. Abheidena, Wessel W. Fuijkschotb, Ankie M. Harmszec, Marjon A. de Boera, Abel Thijsb, Johanna I.P. de Vriesa a
Amsterdam UMC, location VUmc, Department of Obstetrics and Gynaecology, Reproduction and Development, De Boelelaan 1117, Amsterdam, the Netherlands Amsterdam UMC, location VUmc, Department of Internal Medicine, Amsterdam Cardiovascular Sciences, De Boelelaan 1117, Amsterdam, the Netherlands c Amsterdam UMC, location VUmc, Department of Clinical Pharmacology and Pharmacy, De Boelelaan 1117, Amsterdam, the Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspirin Drug resistance HELLP Pre-eclampsia Pregnancy Preventive therapy
Objectives: The objective of this study is to investigate possible changes in aspirin resistance during and after pregnancy over time. Study design: A longitudinal cohort study in obstetric high risk women with an indication for aspirin usage during pregnancy to prevent placenta mediated pregnancy complications. Main outcome measures: Aspirin resistance measured in the first, second and third trimester of pregnancy and at least three months postpartum by four complementary test: PFA-200, VerifyNow®, Chronolog light transmission aggregometry (Chronolog LTA) and serum thromboxane B2 (TxB2) level measurements. Correlation between the devices was investigated. Results: In total, 23 pregnant women participated in the present study. Aspirin resistance according to the PFA200, VerifyNow®, Chronolog LTA and serum TxB2, was 30.4%, 17.4%, 26.1% and 23.8% respectively. Resistance by any device was 69.6%. Aspirin resistance measured by the VerifyNow®, Chronolog LTA, serum TxB2 and aspirin resistance by any device during pregnancy was demonstrated more frequently than aspirin resistance after pregnancy. Correlation between the different devices was weak. Conclusion: Aspirin resistance was found in a considerable part of the participants. Considerable variation between participants, within participants over time and between the different devices was found. Prevalence of aspirin resistance during pregnancy differs from after pregnancy. More research on aspirin resistance and clinical obstetric outcome is needed.
1. Introduction Hypertensive disorders of pregnancy (HDP) are an important cause of maternal and neonatal morbidity and mortality [1,2]. It is thought that poor spiral artery remodelling contributes to the onset of HDP [3]. Poor spiral artery remodelling is primary linked to early-onset HDP. Women at risk of early-onset HDP have an indication to use aspirin during pregnancy [4,5] which reduces the risk for recurrent early onset HDP [6,7]. Aspirin resistance is the phenomenon of a less effective response to aspirin in some patients. Prevalence of aspirin resistance in populations of men and non-pregnant women with cardiovascular diseases of about 5 up to 65% is reported, depending on which test and cut-off values are used [8–11]. Aspirin resistance during pregnancy is investigated in four
cohort studies and one review showing that around one third is resistant to aspirin, depending on the cut-off values and definition used [11–15]. In three studies the timing of measurement of aspirin resistance was not standardized which might be a possible confounder [12,14,15]. Aspirin resistance may change during pregnancy by the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [16–18]. All these factors can have an effect on pharmacokinetics and/or pharmacodynamics and therefore lead to possible differences in occurrence of aspirin resistance over time during pregnancy. Finally, non-adherence to aspirin can influence results of aspirin resistance measurements [19,20]. Aspirin resistance could be an explanation of (recurrent) HDP despite the use of aspirin. Bujold et al commented that the beneficial
☆ ⁎
The work has been carried out in the Amsterdam UMC, location VUmc, Amsterdam, the Netherlands. Corresponding author at: Department of Obstetrics and Gynaecology, Amsterdam UMC, location VUmc, P.O. Box 7057, 1007 MB Amsterdam, the Netherlands. E-mail address: [email protected] (J.M. bij de Weg).
https://doi.org/10.1016/j.preghy.2019.11.008 Received 28 June 2019; Received in revised form 23 October 2019; Accepted 25 November 2019 2210-7789/ © 2019 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
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enzyme immunoassay kit (Assay Designs®, Ann Arbor, MI, USA). PFA-200 measures the process of primary hemostasis [24]. The system measures platelet plug formation in a capillary through which citrated whole blood is passed. The closure time (CT) is measured: the time needed for complete obstruction of the capillary. A CT of ≤150 s is used for the dichotomous definition of aspirin resistance [12,14]. PFA has a theoretical maximum of 300 s, therefore any CT > 300 s was reported as 301 s. The Collagen/Epinephrine cartridge is used. VerifyNow® utilizes arachidonic acid as an agonist to measure the antiplatelet effect of aspirin specifically along the pathway of inhibiting cyclooxygenase-1. Change in light transmittance in patient’s whole blood sample is measured in an aspirin cartridge. Results are displayed in Aspirin Reactions Units (ARU) [25]. An ARU of ≥550 is used for the dichotomous definition of aspirin resistance [26,27]. Chronolog LTA measures the light transmittance of platelet rich plasma which is influenced by platelet aggregation stimulated by arachidonic acid [28]. Platelet aggregation is reduced by lesser production of TxA2 through addition of arachidonic acid. In women resistant to aspirin, more aggregation occurs and the light transmittance is increased. The percentage of maximal aggregation is measured and a value > 22% is defined as aspirin resistant [29–31]. Serum TxB2 is a direct measure of the capacity of platelets to synthesize thromboxane A2 (TxA2) and a specific measure of the pharmacological effect of aspirin on platelets. Blood samples are placed in a stove for one hour at 37 degrees Celsius, directly after collection. Samples are spinned with 3000 rotations per minute for 10 min and serum samples were stored at −80 degrees Celsius. Thereafter, samples were analyzed by enzyme immunoassay in the laboratory for hematology, unit thrombosis and hemostasis of the Radboud University Medical Center in Nijmegen, the Netherlands. A TxB2 concentration in the highest quartile is defined as aspirin resistant [15].
effect found in a randomized controlled trial (RCT) comparing lowmolecular-weight heparin and aspirin versus aspirin alone on recurrent early onset preeclampsia in women with inheritable thrombophilia could be the result of aspirin resistance [21,22]. A follow-up study of this RCT has been performed many years after the pregnancy and could not demonstrate a relation between HDP and aspirin resistance [23]. The present study investigated whether aspirin resistance changes over time during and after pregnancy in pregnant women with an indication for aspirin. We hypothesize that the prevalence of aspirin resistance changes during the three trimesters of pregnancy and differs between pregnancy and postpartum. To our knowledge, aspirin resistance in pregnant women has never been tested longitudinally during and after pregnancy. A previous study only tested longitudinally during the different trimesters of pregnancy, but not postpartum [13]. This study will gain more insight in the prevalence of aspirin resistance in pregnancy over time and the consistency during and after pregnancy. 2. Material and methods 2.1. Participants This longitudinal cohort study was performed from April 2015 till January 2017 at the Amsterdam UMC, Vrije Universiteit Amsterdam, in Amsterdam, the Netherlands. Pregnant women were included if they had an indication for low-dose aspirin (acetylsalicylic acid 80 mg, nonenteric-coated) usage during pregnancy and had their check-ups at this hospital. Inclusion criteria were age ≥ 18 years, understanding of Dutch or English and an indication for aspirin usage during pregnancy. Exclusion criteria were women diagnosed with Systemic Lupus Erythematosus or anti-phospholipid syndrome, use of drugs that are known to alter platelet function (e.g. NSAID’s, tirofiban, eptifibatide, abciximab, beta-lactam antibiotics, dextran, SSRI’s, clomipramine and amitriptyline, dipyridamole, verapamil, diltiazem, ginkgo biloba, ginseng, St John’s wort), a recent cardiovascular event (< 3 months), abnormal cell count, twin pregnancy, hypercholesterolemia before pregnancy or in the first trimester, impaired renal or liver function before pregnancy or in the first trimester and poorly controlled hypertension before pregnancy or in the first trimester. Written informed consent was obtained from all participants.
2.3. Outcomes Baseline characteristics included age, number of previous pregnancies, body mass index before pregnancy, blood pressure in the first trimester, smoking behaviour, alcohol use, indication for aspirin usage, obstetric history and family history including vascular diseases and HDP. Outcomes are the prevalence of aspirin resistance during the three trimesters of pregnancy and postpartum per device and in any device labelled as ‘resistance by any device’, the difference in prevalence between pregnancy and postpartum, correlation between the different devices and aspirin adherence. Moreover, aspirin resistance was classified into three groups: no resistance at all moments of measurement for all tests, resistance at all moments of measurements in at least one device, and partial resistance in all other outcomes. This classification is based on one used in a previous prospective cohort study on aspirin resistance in pregnant women at risk of HDP [13].
2.2. Measurements Aspirin resistance measurements during the first trimester of pregnancy were scheduled between 10 and 15 weeks of gestation, in the second trimester between 19 and 25 weeks of gestation and in the third trimester between 28 and 34 weeks of gestation. The postpartum measurement was scheduled at least three months after delivery. All measurements were performed after a period of at least 10 days of aspirin usage. Participants were instructed to take aspirin in the evening and during ten days before their appointment strictly at 8 pm. Adherence to aspirin was investigated by questioning the study participant at every visit. Taking less than 10 tablets in the previous 10 days was registered as non-adherence. Venous blood samples were collected after an overnight fast at 8am and participants were instructed not to smoke 30 min prior to the appointment. Besides aspirin resistance measurements, blood sampling was performed for hemoglobin, hematocrit, thrombocytes and leucocyte counts. During the visit in the first trimester, a physical examination including blood pressure, body weight and length measurements was performed and a questionnaire about family history was answered. During the postpartum visit blood pressure and body weight were measured again. Four complementary tests were used to analyse the blood samples [20]: Platelet Function Analyzer 200 (PFA-200, INNOVANCE® PFA-200 System, Siemens Healthcare, Marburg, Germany), VerifyNow® point-ofcare system (Accumetrics, CA, USA), Chronolog light transmittance aggregometry (LTA) and Serum thromboxane B2 (TxB2) level using an
2.4. Statistical analysis This is a pilot study, therefore a power calculation was not performed. The number of participants is based on previous studies measuring aspirin resistance [12,15,32]. Outcomes of the four complementary tests were compared with Mixed Models and when not normally distributed a logarithmic transformation was performed in advance. Dichotomous data were analysed with a Generalized Estimating Equations. Correlation for continuous data were analysed with Pearman’s rho and dichotomous data with Cohen’s kappa. SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses. Results were considered significant at the two-sided 5% level. 2.5. Ethical approval The study was conducted in accordance with the Helsinki II Declaration and was approved by the Institutional Review Board of the 26
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Table 1 Baseline characteristics. (n = 23) Maternal age (years) Number of pregnancy BMI in kg/m2 Blood pressure in mmHg in first trimester Systolic Diastolic Smoking Alcohol usage Indications for aspirin usage* Previous HDP Gestational age HDP IUFD Gestational age IUFD SGA ≥2 moderate risk factors**
33.0 ± 3.9 3.2 ± 1.0 24.1 ± 5.7 110.4 ± 11.6 71.7 ± 9.6 1 (4.3) 0 (0.0) 16 (69.6) 31.6 ± 3.3 3 (13.0) 30.1 ± 6.2 6 (26.1) 2 (8.7)
Fig. 1a. Continuous results of the PFA-200 over time during and after pregnancy. On the horizontal axis the moments of measurement are shown. On the vertical axis the closure time in seconds is shown. Each line represents one participant, n = 23. The dotted line is the mean of the results of all participants per trimester. The broken line is the cut-off value of the PFA-200: 150 s. Aspirin resistance PFA-200: closure-time ≤150 s.
Data are depicted as mean ± SD or numbers (%) as appropriate. *Some women had more than one indication for aspirin usage during pregnancy. **≥2 of the following moderate risk factors indicates aspirin usage during pregnancy: first pregnancy, maternal age ≥40 years, pregnancy interval > 10 years, BMI > 35 kg/m2, family (medical) history of HDP, twin pregnancy and pregnancy after egg donation. BMI, body mass index; HDP, hypertensive disorder of pregnancy; SGA, small for gestational age (< p10); IUFD, intra uterine fetal death.
Amsterdam UMC, Vrije Universiteit Amsterdam, in Amsterdam, the Netherlands. The study was registered at the Dutch Trial Register (Nederlands Trial register; www.trialregister.nl) with number NTR5106. 3. Results Out of 38 invited women, 14 declined participation and 1 had a spontaneous miscarriage, resulting in 23 participants in this study (participation rate 60.5%). There were two drop-outs after the third trimester (1 woman moved and 1 had an indication for the use of lowmolecular weight heparin (LMWH) due to a postpartum sagittal sinus thrombosis). Seven participants also participated in another study in the Amsterdam UMC, Vrije Universiteit Amsterdam to evaluate aspirin adherence [33]. Baseline characteristics are listed in Table 1. Prevalence of aspirin resistance of all four devices over all four moments of measurements are depicted in Table 2. In the continuous data of the VerifyNow®, Chronolog LTA and serum TxB2, as depicted in
Fig. 1b. Continuous results VerifyNow® Aspirin Test over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis Aspirin Reaction Units (ARU) are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value of the VerifyNow® Aspirin Resistance: 550 ARU. Aspirin resistance VerifyNow® Aspirin Test: ≥550 ARU.
Table 2 Aspirin resistance measurements with four devices in first, second and third trimester, and > 3 months postpartum.
PFA-200 Resistant VerifyNow® Resistant Chronolog LTA Resistant Serum TxB2 Resistant Resistance by any device
Trimester 1 (n = 23)
Trimester 2 (n = 23)
Trimester 3 (n = 23)
> 3 months postpartum (n = 21)
P-value
226.35 ± 13.82 5 (21.7) 465[124] 4 (17.4) 11.5[5.0] 2 (8.7) 12.81 ± 2.45 5 (21.7) 14 (60.9)
231.00 ± 12.04 3 (13.0) 420[79] 4 (17.4) 12.5[5.5] 2 (8.7) 12.24 ± 2.45 5 (21.7) 11 (47.8)
207.87 ± 15.14 7 (30.4) 421[76] 2 (8.7) 14.5[14.5] 6 (26.1) 14.16 ± 2.45 4 (17.4) 16 (69.6)
219.48 ± 15.15 4 (19.0) 388[11] 0 (0.0) 9.0[6.0] 1 (4.8) 10.05 ± 2.48 5 (23.8) 8 (38.1)
P = 0.45 P = 0.36 P < =0.01 N/A P < =0.01 P = 0.07 P < =0.05 P = 0.92 P < =0.05
95%-CI
0.02–0.11* 0.08–0.51** 0.30–7.92*** 0.05–0.68****
Data are depicted as mean ± standard deviation, median with [inter quartile range] or numbers (%) as appropriate. N/A; not available. Aspirin resistance cut-off values per device; PFA-200: closure-time ≤150 s; VerifyNow® Aspirin Test: ≥550 ARU; Chronolog LTA: > 22%; TxB2: above the 75th percentile per moment of measurement, respectively 15.3 ng/ml for trimester 1, 15.8 ng/ml for trimester 2, 13.9 ng/ml for trimester 3 and 10.4 ng/ml postpartum. *VerifyNow®: Statistically significant difference between first trimester and postpartum. **Chronolog LTA: Statistically significant difference between third trimester and postpartum. ***Serum TxB2: Statistically significant difference between third trimester and postpartum. **** Resistance by any device: Statistically significant difference between third trimester and postpartum. 27
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Table 3 Pregnancy outcomes. (n = 23) Gestational age of delivery Induction Gestational age Indication induction HDP Elective Obstetric history IUGR Persistent reduced fetal movements HDP PIH Gestational age PIH PE Gestational age PE Eclampsia HELLP Cesarean section Primary cesarean section Secondary cesarean section SGA
Fig. 1c. Continuous results Chronolog LTA over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis the percentages of maximal aggregation are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value of the Chronolog LTA: 22%. Aspirin resistance Chronolog LTA: > 22%.
38.3 ± 1.7 14 (60.9) 38.0 ± 1.7 5 (35.7) 4 (28.6) 3 (21.4) 1 (7.1) 1 (7.1) 12 (52.2) 9 (39.1) 36.5 ± 3.0 3 (13.0) 34.6 ± 3.0 0 (0.0) 0 (0.0) 12 (52.2) 7 (58.3) 5 (41.7) 3 (13.0)
Data are depicted as mean ± standard deviation or numbers (%) as appropriate. HDP, hypertensive disorders of pregnancy; IUGR, intra uterine growth restriction; PIH, pregnancy-induced hypertension; PE, preeclampsia; HELLP, hemolysis elevated liver enzymes low platelets; SGA, small for gestational age (birthweight < p10).
first and second trimester, in 20 participants (87.0%) during the third trimester and 18 participants (85.7%) postpartum. The two participants who were non-adherent in the first and second trimester, did not show aspirin resistance. In the third trimester, all 3 participants who were non-adherent showed aspirin resistance on one single device, one on the VerifyNow® and two on the PFA-200. Post-partum, one out of five participants reporting non-adherence showed aspirin resistance on the PFA-200. Pregnancy outcomes are depicted in Table 3.
Fig. 1d. Continuous results of the serum thromboxane B2 over time during and after pregnancy. On the horizontal axis moments of measurement are shown. On the vertical axis ng/ml are shown. Each line represents one participant, n = 23. The dotted line is the median of the results of all participants per trimester. The broken line is the cut-off value defined as above the 75th percentile per moment of measurement, respectively 15.3 ng/ml for trimester 1, 15.8 ng/ml for trimester 2, 13.9 ng/ml for trimester 3 and 10.4 ng/ml postpartum.
4. Discussion In this prospective cohort study we examined aspirin resistance in pregnant women during the three trimesters of pregnancy and more than three months postpartum by four complementary devices. A considerable part of the participants showed aspirin resistance, despite the variation between participants, within participants over time and between the different devices. The prevalence of aspirin resistance during pregnancy measured by the four different devices found in this study varied from 8.7% to 30.4% and aspirin resistance in any device from 47.8% to 69.6%. The resistance rates found in our cohort are similar or slightly higher to a previous study measuring aspirin resistance in women with obstetric history of HDP with different devices [23]. The investigated population in our study differs from the previous study because in our study women were pregnant, did not have inheritable thrombophilia and the mean age was more than 10 years lower: 33.0 versus 44.7 years old. So the same or slightly higher aspirin resistance rates were found in a younger, pregnant population without inheritable thrombophilia. No resistance was found in 13.0% of the participants and resistance in 26.1%. The majority, namely 60.9%, showed partial resistance over time. These rates are lower in comparison to a previous study in which a comparable classification was made [13]. In this previous study aspirin resistance was measured twice during pregnancy by aggregometry, VerifyNow® and urinary TxB2. In addition, urinary aspirin metabolites were determined. Participants were categorized as responsive if two or all platelet function tests were outside normal range at both visits, non-responsive if none or one platelet function test was
Figs. 1a–d, statistical significant differences in aspirin resistance were found between the measurements during and after pregnancy, respectively for the VerifyNow® between the first trimester and postpartum, and for the Chronolog LTA, serum TxB2 and resistance by any device between the third trimester and postpartum. Aspirin resistance was found in one participant on the Chronolog LTA and serum TxB2 during all four measurements. In this participant aspirin resistance was also shown in the PFA-200 in the three trimesters of pregnancy, however not postpartum. Another participant showed aspirin resistance on the VerifyNow® over the three trimesters of pregnancy. One participant showed resistance by serum TxB2 over all four moments of measurement. Three participants (13.1%) showed no resistance, meaning no resistance at any devices during all moments of measurement. Six participants (26.1%) showed resistance, meaning resistance in at least one device during all moments of measurement. The majority showed partial resistance, meaning resistance in at least one device but not at all moments of measurement. Correlation for continuous data in Pearman’s rho showed a weak correlation between the four devices in a range of 0.01 to −0.53. Agreement for dichotomous data in Cohen’s kappa showed poor to fair correlation in a range of −0.24 to 0.33. Aspirin adherence was found in 21 participants (91.3%) during the
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neonates (13.0%) were small for gestational age which is in line with the prevalence in the general population. Unfortunately, the influence of aspirin resistance on the obstetric outcomes could not be examined, since the study is not powered for this question. The present study showed that aspirin resistance rates differ during and after pregnancy and within participants over time. Thus aspirin resistance measurements 9–16 years after pregnancy as performed in the follow-up study of Abheiden et al [23] might not be representative for aspirin resistance during pregnancy. Thereby, the question of Bujold whether a beneficial effect of LMWH in women with adverse obstetric history and inheritable thrombophilia is caused by aspirin resistance could not be answered yet [21,22]. This study elucidates for the first time aspirin resistance during pregnancy using the four complementary devices and the inconsistency of aspirin resistance during and after pregnancy with complementary devices. The strength of this study is its prospective character with multiple moments of measurement. We examined aspirin resistance with the current available tests which are complementary and therefore provide more insight. Factors which could influence the measurements, such as time of aspirin intake, fasting over night the day before the appointment and no smoking more than 30 min before the appointment were minimized with clear instructions to the participants. The limitation of this study is that no well-defined cut-off values for pregnant women exist. Cut-off values of the different devices were based on values of previous studies [12,14,15,23]. Pregnancy-specific cut-off values should be investigated, since these might differ due to the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [14–16] and thereby might influence the proportion of pregnant women being aspirin resistant. Another limitation is that the amount of investigated women is relatively small, so that caution is warranted when interpreting these results. Future studies could investigate the optimal dosage and dosing frequency of aspirin therapy during pregnancy. A possible dose-response effect of aspirin for prevention of preeclampsia was shown in a previous review [35]. An answer to the question which dose is preferred could come from a trial comparing different doses. One could also hypothesize that aspirin resistance might be influenced by body weight and that dosage adjustment for body weight is needed for optimal individualized treatment. A previous systematic review in patients at risk for vascular events showed that the effectiveness of aspirin depends on the dosage of aspirin [36]. Low dose aspirin was effective in patients with low bodyweight and high dose in patients with high bodyweight. A one-dose-fits-all approach might be suboptimal in this population and could be quested in the pregnant population as well. More research is needed about this topic in pregnant women.
outside normal range at both visits and aspirin metabolites were detectable, indeterminate response if none or one platelet function test was outside normal range at both visits and aspirin metabolites were not detectable, and variable response if test results changed between both visits in the same participants. The majority, 59.0%, was categorized as responder, 0% was non-responder, 7.0% as indeterminate responder, and 34.0% as variable responder. The different outcome can partly be explained by a different categorization due to other tests and moments of measurement. Studies which investigated aspirin resistance with one single device (PFA-100 or urinary TxB2) also reported similar resistance rates around 30% [12,14,15]. Despite the variation in aspirin resistance between participants, within participants over time and between devices as shown in Figs. 1a–d, aspirin resistance rates measured by the VerifyNow®, Chronolog LTA, serum TxB2 and resistance by any device were statistical significant higher during pregnancy in comparison to after pregnancy. By the VerifyNow®, this difference was found between the first trimester and postpartum. In case of the Chronolog LTA, serum TxB2 and resistance by any device, this difference was found between the third trimester and postpartum. The differences in aspirin resistance between during and after pregnancy might be explained by the plasma volume expansion, change in leukocyte and thrombocyte count, changes in coagulation factors and in steroid hormones during pregnancy [16–18]. This possible pregnancy-induced effect has to our knowledge never been demonstrated before. Operationalization of aspirin resistance measurements is complicated. Firstly, due to the enormous variation between the different devices. This confirms the importance of testing with multiple, complementary devices to grasp its various working mechanisms, or indicates that the existence of aspirin resistance is not yet certain and the present tests may be influenced by multiple unknown factors. The accepted gold standard to measure aspirin resistance is the Chronolog LTA. However, this method is limited in practical usage for clinical researchers, because it is a costly, time consuming method and requires platelet rich plasma. Therefore, in previous studies other devices, namely PFA-200, VerifyNow® and serum TxB2, were also used [11–15]. To be able to compare the results of our study with previous performed studies, we measured aspirin resistance with all four available devices. Weak correlation between the devices was found. This was expected due to the different methods of measuring aspirin resistance. The weak correlation between the devices complicates the interpretation of the results of the devices. A powered study for clinical outcome is needed. Secondly, the large variation between the participants makes operationalization of aspirin resistance measurements complicated. Non-optimal adherence could cause non-optimal aspirin influence. We investigated non-adherence by questioning at every visit. We might underestimate the non-adherence of our cohort, since questioning is vulnerable to inaccuracy. Despite this limitation, non-adherence rates in this study are in line with a previous study on aspirin adherence in women with high-risk pregnancies showing non-adherence rates of 21.4% and 46.3%, depending on which questionnaire was used [33]. Our study demonstrated the highest rates of non-adherence postpartum. This might be explained by the fact that participants had to take the aspirin postpartum solely for the purpose of the study, in contrast to their medical indication during pregnancy. Non-adherence can influence the results of the measurements of aspirin resistance [19,20] and thereby are harder to interpret. Finally, operationalization is complicated by the enormous variation in aspirin resistance within participants over time. Intra-individual variability is a well-known phenomenon in other internal medicine-related diagnosis, such as hypertension and hypercholesterolemia. Due to variation over time, these diagnoses can only be made by multiple measurements. We should wonder if repetitive testing is also required for measuring aspirin resistance. The pregnancy outcomes revealed that more than 50% of the pregnant women developed HDP despite aspirin use during pregnancy. Our induction and cesarean rates are above average [34]. Three
5. Conclusions In conclusion, in this prospective cohort study a considerable part of the women showed aspirin resistance, despite the enormous variation between participants, within participants over time and between the different devices. We reveal a difference in prevalence of aspirin resistance during and after pregnancy in three out of the four devices, which might be pregnancy-induced. A powered study for aspirin resistance and clinical obstetric outcome is needed to clarify the relevance of measuring aspirin resistance. Afterwards, trials regarding optimal dosage and dosing frequency could be performed for adjustment and optimizing aspirin therapy. Acknowledgements We would like to express our gratitude to all women who participated in this study. We would like to thank A. Arduc for his help with the measurements. Furthermore, we want to thank W. Rosendal and H. Hopman, laboratory technicians of the hemostasis laboratory of the 29
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Amsterdam UMC, Vrije Universiteit Amsterdam.
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