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The effect of low-dose aspirin on platelet function during pregnancy compared to placebo: An explorative study

  • Author Footnotes
    1 Shared first authorship, both authors contributed equally to this study.
    Jeske M. bij de Weg
    Correspondence
    Corresponding author at: Department of Obstetrics and Gynaecology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Reproduction and Development research institute, P.O. box 7057, 1081 HV Amsterdam, The Netherlands.
    Footnotes
    1 Shared first authorship, both authors contributed equally to this study.
    Affiliations
    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Obstetrics and Gynaecology, Amsterdam, The Netherlands

    Amsterdam Reproduction and Development Research Institute, Amsterdam, The Netherlands
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  • Author Footnotes
    1 Shared first authorship, both authors contributed equally to this study.
    Anadeijda J.E.M.C. Landman
    Footnotes
    1 Shared first authorship, both authors contributed equally to this study.
    Affiliations
    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Obstetrics and Gynaecology, Amsterdam, The Netherlands

    Amsterdam Reproduction and Development Research Institute, Amsterdam, The Netherlands
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  • Johanna I.P. de Vries
    Affiliations
    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Obstetrics and Gynaecology, Amsterdam, The Netherlands

    Amsterdam Reproduction and Development Research Institute, Amsterdam, The Netherlands
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  • Abel Thijs
    Affiliations
    Amsterdam UMC, Vrije Universiteit Amsterdam, Internal Medicine, De Boelelaan 1117, Amsterdam, The Netherlands
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  • Ankie M. Harmsze
    Affiliations
    St. Antonius Hospital, Clinical Pharmacy, Koekoekslaan 1, Nieuwegein, The Netherlands
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  • Martijn A. Oudijk
    Affiliations
    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Obstetrics and Gynaecology, Amsterdam, The Netherlands

    Amsterdam Reproduction and Development Research Institute, Amsterdam, The Netherlands
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  • Marjon A. de Boer
    Affiliations
    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Obstetrics and Gynaecology, Amsterdam, The Netherlands

    Amsterdam Reproduction and Development Research Institute, Amsterdam, The Netherlands
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  • Author Footnotes
    1 Shared first authorship, both authors contributed equally to this study.
Open AccessPublished:September 02, 2022DOI:https://doi.org/10.1016/j.ejogrb.2022.08.025

      Highlights

      • Aspirin is used for prevention of preeclampsia and other obstetric complications.
      • Aspirin treatment failure could be attributable to resistance or non-adherence.
      • Three platelet function tests were performed in women assigned placebo or aspirin.
      • Aspirin 80 mg has a clear inhibitory effect on platelet function during pregnancy.
      • The effect of aspirin on platelet function is similar in second and third trimester.

      Abstract

      Objectives

      To evaluate the effect of aspirin 80 mg compared to placebo on platelet function tests in the second and third trimester of pregnancy.

      Study design

      An explorative study was performed to assess laboratory platelet function in a subpopulation of the APRIL trial: a randomized double-blind trial comparing aspirin 80 mg once daily to placebo for the prevention of recurrent preterm birth. Platelet function was measured between 18 and 22, and between 28 and 32 weeks gestational age with three platelet function tests: VerifyNow®, Chronolog light transmission aggregometry (Chronolog LTA) and serum thromboxane B2 (TxB2). Medication adherence was evaluated by pill counts, self-reported diaries and structured interviews.

      Results

      We included 11 women, six in the aspirin and five in the placebo group. In women receiving aspirin, platelet function was significantly lower compared to women receiving placebo for all three tests: VerifyNow® Aspirin Reaction Units (450.5 vs 648.0, p = 0.017); Chronolog LTA (9.5% vs 94.5%, p = 0.009); serum TxB2 levels (11.9 ng/mL versus 175.9 ng/mL, p = 0.030). For all three tests, platelet function did not differ between the second and third trimester of pregnancy in the aspirin group. In the placebo group, serum TxB2 levels were significantly higher in the third trimester. One non-adherent participant in the aspirin group showed results similar to the placebo group.

      Conclusion

      Aspirin 80 mg has a clear inhibitory effect on laboratory platelet function during pregnancy compared to placebo. This effect is similar in the second and third trimester of pregnancy.

      Introduction

      Low-dose aspirin has been widely implemented for the prevention for preterm preeclampsia [
      • Rolnik D.L.
      • Nicolaides K.H.
      • Poon L.C.
      Prevention of preeclampsia with aspirin.
      ,
      • Rolnik D.L.
      • Wright D.
      • Poon L.C.
      • O'Gorman N.
      • Syngelaki A.
      • de Paco M.C.
      • et al.
      Aspirin versus Placebo in Pregnancies at High Risk for Preterm Preeclampsia.
      ]. It also has the potential to prevent fetal growth restriction, preterm birth and perinatal mortality [
      • Bujold E.
      • Roberge S.
      • Lacasse Y.
      • Bureau M.
      • Audibert F.
      • Marcoux S.
      • et al.
      Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early pregnancy: a meta-analysis.
      ,
      • Roberge S.
      • Nicolaides K.
      • Demers S.
      • Hyett J.
      • Chaillet N.
      • Bujold E.
      The role of aspirin dose on the prevention of preeclampsia and fetal growth restriction: systematic review and meta-analysis.
      ,
      • Duley L.
      • Meher S.
      • Hunter K.E.
      • Seidler A.L.
      • Askie L.M.
      Antiplatelet agents for preventing pre-eclampsia and its complications.
      ,
      • Allshouse A.A.
      • Jessel R.H.
      • Heyborne K.D.
      The impact of low-dose aspirin on preterm birth: secondary analysis of a randomized controlled trial.
      ,
      • Andrikopoulou M.
      • Purisch S.E.
      • Handal-Orefice R.
      • Gyamfi-Bannerman C.
      Low-dose aspirin is associated with reduced spontaneous preterm birth in nulliparous women.
      ,
      • van Vliet E.O.G.
      • Askie L.A.
      • Mol B.W.J.
      • Oudijk M.A.
      Antiplatelet Agents and the Prevention of Spontaneous Preterm Birth: A Systematic Review and Meta-analysis.
      ]. The common underlying mechanism of these pregnancy complications is utero-placental insufficiency.
      Aspirin has a wide range of pharmacological activities, including analgesic, antipyretic, antiplatelet and vasomotor properties [
      • Cadavid A.P.
      Aspirin: The Mechanism of Action Revisited in the Context of Pregnancy Complications.
      ]. The exact mechanism by which aspirin prevents preeclampsia is unknown, but it is thought that antithrombotic and vasodilator effects, and restoring the imbalance of thromboxane and prostacyclin play an important role by improving placentation [
      • Cadavid A.P.
      Aspirin: The Mechanism of Action Revisited in the Context of Pregnancy Complications.
      ,
      • Mirabito Colafella K.M.
      • Neuman R.I.
      • Visser W.
      • Danser A.H.J.
      • Versmissen J.
      Aspirin for the prevention and treatment of pre-eclampsia: A matter of COX-1 and/or COX-2 inhibition?.
      ]. Anti-inflammatory effects have also been thought to contribute [
      • Cadavid A.P.
      Aspirin: The Mechanism of Action Revisited in the Context of Pregnancy Complications.
      ,
      • Mirabito Colafella K.M.
      • Neuman R.I.
      • Visser W.
      • Danser A.H.J.
      • Versmissen J.
      Aspirin for the prevention and treatment of pre-eclampsia: A matter of COX-1 and/or COX-2 inhibition?.
      ].
      The optimal dose of aspirin has not been explored sufficiently. Current international guidelines advise aspirin doses of 75–150 mg once daily to prevent preterm preeclampsia [

      ACOG Committee Opinion No. 743: Low-Dose Aspirin Use During Pregnancy. Obstetrics and gynecology. 2018;132(1):e44-e52.

      ,

      NICE guideline. Hypertension in pregnancy: diagnosis and management. 2019.

      ,

      NVOG. Hypertensieve aandoeningen in de zwangerschap. 2018.

      ,
      • Tranquilli A.L.
      • Dekker G.
      • Magee L.
      • Roberts J.
      • Sibai B.M.
      • Steyn W.
      • et al.
      The classification, diagnosis and management of the hypertensive disorders of pregnancy: A revised statement from the ISSHP.
      ]. Recent meta-analyses suggest that aspirin doses above 100 mg may be more effective than lower doses [
      • Duley L.
      • Meher S.
      • Hunter K.E.
      • Seidler A.L.
      • Askie L.M.
      Antiplatelet agents for preventing pre-eclampsia and its complications.
      ,
      • Seidler A.L.
      • Askie L.
      • Ray J.G.
      Optimal aspirin dosing for preeclampsia prevention.
      ,
      • Roberge S.
      • Bujold E.
      • Nicolaides K.H.
      Aspirin for the prevention of preterm and term preeclampsia: systematic review and metaanalysis.
      ]. However, this hypothesis is based on indirect comparisons.
      To evaluate the inhibitory effect of the frequently used low-dose aspirin 80 mg on platelet function, we performed an explorative study comparing aspirin 80 mg once daily with placebo.

      Methods

      Design and setting

      In this explorative study, we performed platelet function tests in a subset of APRIL study participants. The APRIL study was a multicentre double-blind placebo-controlled randomized trial assessing the effect of aspirin 80 mg once daily on the prevention of recurrent preterm birth [
      • Visser L.
      • de Boer M.A.
      • de Groot C.J.M.
      • Nijman T.A.J.
      • Hemels M.A.C.
      • Bloemenkamp K.W.M.
      • et al.
      Low dose aspirin in the prevention of recurrent spontaneous preterm labour - the APRIL study: a multicenter randomized placebo controlled trial.
      ]. The APRIL study was funded by ZonMw, The Dutch Organization for Health Research and Development (Grant No 836041006), and registered in the Dutch Clinical Trial Registry (NL5553, NTR5675). The study protocol and trial results have been described in detail previously [
      • Visser L.
      • de Boer M.A.
      • de Groot C.J.M.
      • Nijman T.A.J.
      • Hemels M.A.C.
      • Bloemenkamp K.W.M.
      • et al.
      Low dose aspirin in the prevention of recurrent spontaneous preterm labour - the APRIL study: a multicenter randomized placebo controlled trial.
      ,
      • Landman A.J.E.M.C.
      • De Boer M.A.
      • Visser L.
      • Nijman T.A.J.
      • Hemels M.A.C.
      • Naaktgeboren C.
      • et al.
      Evaluation of Low-dose Aspirin for the Prevention of Recurrent Spontaneous Preterm Labour (the APRIL study): a multicentre, randomised, double-blinded, placebo-controlled trial.
      ].
      The present sub-study was performed in the Amsterdam University Medical Centre, and was also approved by the Medical Research Ethics Committee (no. 2015_332). Women gave additional written informed consent for this study. No grant was received from funding agencies in the public, commercial, or not-for-profit sectors.

      Participants

      Our sub-study applied the same in- and exclusion criteria as the APRIL study. The APRIL study included women of 18 years and older with a singleton pregnancy and a history of spontaneous preterm birth between 22 and 37 weeks gestational age. Exclusion criteria were a previous indicated preterm birth for maternal or fetal reasons; other indications for aspirin use; thrombocytopenia or thrombocytopathy; or major fetal malformations in the current pregnancy or in a prior pregnancy ending in spontaneous preterm birth.

      Intervention

      Women were randomized between aspirin 80 mg once daily or placebo. Treatment was initiated between 8 and 16 weeks gestational age and continued until 36 weeks gestational age or delivery, if delivery occurred earlier. Women were instructed to take the medication in the evening.
      Participants, their health care providers, and researchers were blinded for treatment allocation. The technician performing the platelet function tests was blinded for the use of aspirin or placebo, was unaware of the clinical condition and management of the patients, and was not involved in the analysis of the results of the APRIL study. Allocation deblinding of the research team was performed after the completion of data collection.

      Data collection

      Venous blood samples were drawn at regular antenatal outpatient visits in the second trimester (between 18 and 22 weeks gestational age) and the third trimester (between 28 and 32 weeks gestational age) with an interval of at least eight weeks between measurements. Participants were instructed to take their study medication in the evening and to have a light breakfast on the day of blood sampling. Data on maternal characteristics, medical and obstetric history, platelet function tests, and pregnancy outcomes were recorded in an electronic case record form.
      We evaluated overall medication adherence throughout pregnancy as well as medication adherence in the then days prior to blood sampling. Overall medication adherence was calculated by dividing the number of used tablets by the expected number of doses per participant. We considered overall medication adherence to be good if ≥ 80% of tablets were taken. At the time of blood sampling, the sub-study participants completed a structured interview including the number of tablets taken in the ten days prior to testing. If at least eight tablets were taken in the ten days prior to the test, women were considered adherent at the respective time points.

      Laboratory techniques

      Three different platelet function tests were used to analyze the blood samples since no gold standard for aspirin response in pregnancy exists: VerifyNow® point-of-care system (Accumetrics, CA, USA), Chronolog light transmittance aggregometry (LTA) and serum thromboxane B2 (TxB2) levels using an enzyme immunoassay kit (Assay Designs®, Ann Arbor, MI, USA). Blood samples for platelet function tests with VerifyNow® and Chronolog LTA were analyzed within three hours of collection. Maternal serum for TxB2 measurements was prepared and stored within three hours of collection.
      VerifyNow® measures the antiplatelet effect of aspirin along the inhibition of the cyclooxygenase-1 pathway by utilizing 1.0 mM arachidonic acid [

      VerifyNow [cited 2021 1st March]. Available from: https://www.instrumentationlaboratory.com/en/verifynow-system.

      ]. Whole blood of the participant was tested. Change in light transmittance was measured and the results were displayed in Aspirin Reaction Units (ARU).
      Chronolog LTA measures the light transmittance of platelet rich plasma stimulated by arachidonic acid [

      Chronolog [cited 2021 1st March]. Available from: http://www.chronolog.com/index.html.

      ]. Addition of arachidonic acid reduces formation of thromboxane A2 (TxA2) and thereby platelet aggregation. In the case of aspirin resistance, platelets aggregation is not reduced and the light transmittance is increased. Maximal percentage of aggregation was measured.
      Serum TxB2 is a direct measure of the capacity of platelets to synthesize TxA2 and is a direct measure of the pharmacological effect of aspirin on platelets [
      • Wojtowicz A.
      • Undas A.
      • Huras H.
      • Musial J.
      • Rytlewski K.
      • Reron A.
      • et al.
      Aspirin resistance may be associated with adverse pregnancy outcomes.
      ]. After the blood drawn, whole blood samples were stored in a stove at 37 degrees Celsius for one hour. Thereafter, samples were centrifuged with 3000 rotations per minute for ten minutes to create serum. Serum was stored at −80 degrees Celsius. At the end of the data collection, all serum samples were analyzed for TxB2 by enzyme immunoassay in the laboratory for hematology, unit thrombosis and hemostasis of the Radboud University Medical Center in Nijmegen, the Netherlands.

      Outcomes

      The main outcomes of our sub-study were platelet function based on the three platelet function tests VerifyNow®, Chronolog LTA and serum TxB2. Furthermore, secondary outcomes were preterm birth < 37 weeks gestational age, pregnancy-induced hypertension, preeclampsia, birth weight, small-for-gestational age (<10th centile), and gestational age at birth.

      Statistical analysis

      This sub-study started while the RCT was well in progress. We could not perform an accurate sample size calculation due to large variations in measured platelet responses in earlier studies with the same test outcome measures [
      • Bij de Weg J.M.
      • Abheiden C.N.H.
      • Fuijkschot W.W.
      • Harmsze A.M.
      • de Boer M.A.
      • Thijs A.
      • et al.
      Resistance of aspirin during and after pregnancy: A longitudinal cohort study.
      ]. The median and interquartile range (IQR) was calculated for all three platelet function tests for each time point, as well as the median and IQR of the average of the two time points. In case only one time point was available, the single measurement was used. The Wilcoxon signed rank test was performed to compare the measurements at the two time points and a p-value was reported. Differences in platelet function between treatment groups was calculated with the Mann Whitney U test by using the average of the two time points. We reported the differences in medians and the p-values. Statistical analyses were performed in SPSS version 26.0. P-values of <0.05 were considered to be statistically significant.

      Results

      From September 2018 to May 2019, 11 participants of the APRIL study were included in the present study: six in the aspirin group and five in the placebo group. Baseline characteristics were similar between treatment groups (Table 1).
      Table 1Baseline characteristics.
      CharacteristicsAspirin group (n = 6)Placebo group (n = 5)
      Maternal age (years)32.0 (28.3 – 33.0)31.0 (27.5 – 34.5)
      BMI (kg/m2)25.0 (20.4 – 25.3)23.1 (20.8 – 26.4)
      Ethnic origin, n (%)
      White4 (66.7%)5 (100%)
      Other2 (33.3%)0 (0%)
      Smoking, n (%)0 (0%)0 (0%)
      Alcohol, n (%)0 (0%)0 (0%)
      Systolic blood pressure first trimester (mmHg)103 (100 – 106)105 (100 – 113)
      Diastolic blood pressure first trimester (mmHg)70 (64 – 70)60 (54 – 63)
      Obstetric history
      Parity
      13 (50%)5 (100%)
      23 (50%)0 (0%)
      Gestational age of previous spontaneous preterm birth
      22+0 – 29+6 weeks4 (66.7%)3 (60.0%)
      30+0 – 33+6 weeks1 (16.7%)1 (20.0%)
      34+0 – 36+6 weeks1 (16.7%)1 (20.0%)
      Data are presented as medians (IQR) or numbers (%) as appropriate.
      Two women in the aspirin group had one measurement instead of two measurements during pregnancy: one only in the second trimester and one only in the third trimester. The TxB2 measurement in the second trimester of one participant in the aspirin group failed due to blood clotting. The results of the measurements per participant and the median of the measurements according to treatment group are illustrated in Fig. 1. In the aspirin group, the measurements between the second and third trimester were comparable for all three platelet function tests (for all p > 0.05, Table 2). In the placebo group, the serum TxB2 levels were higher in the third trimester compared to the second trimester (p = 0.043). The median differences of platelet function in the second and third trimester between allocation groups are shown in Table 3, all showing statistically significant lower levels in the aspirin group (for all p < 0.05). As seen in Fig. 1, there was one participant in the aspirin group without platelet inhibition in all three tests. She reported to be non-adherent to the study medication in both trimesters.
      Figure thumbnail gr1
      Fig. 1Platelet function tests during pregnancy.
      Table 2Comparison of platelet function in second and third trimester.
      Platelet function testAspirin group (n = 6)Placebo group (n = 5)
      Second trimester

      (18–22 weeks)
      Third trimester

      (28–32 weeks)
      p-value paired samplesSecond trimester

      (18–22 weeks)
      Third trimester

      (28–32 weeks)
      p-value

      paired samples
      VerifyNow® (ARU)461.0

      (431.0–564.5)
      466.0

      (415.0–532.5)
      0.273647.0

      (608.0–666.5)
      629.0

      (589.0–648.0)
      0.345
      Chronolog LTA (%)9.0

      (4.5–55.0)
      9.0

      (1.5–51.3)
      1.00097.0

      (82.5–99.0)
      92.5

      (90.3–94.0)
      0.893
      Serum TxB2 (ng/mL)3.3

      (2.1–103.4)
      3.5

      (2.0–24.7)
      0.593160.1

      (111.1–235.6)
      227.8

      (177.6–254.1)
      0.043
      Data are depicted as medians (IQR).
      Table 3Comparison of platelet function between treatment groups.
      Platelet function testMedian of second and third trimester
      Aspirin

      (n = 6)
      Placebo

      (n = 5)
      Median differencep-value
      VerifyNow® (ARU)450.5

      (437.5–507.0)
      648.0

      (599.0–651.8)
      197.50.017
      Chronolog LTA (%)9.5

      (5.9–30.3)
      94.5

      (87.5–96.0)
      850.009
      Serum TxB2 (ng/mL)11.9

      (2.4–70.3)
      175.9

      (161.6–236.6)
      1640.030
      Data are depicted as medians (IQR).
      Overall medication adherence was good with a median of 99.4% in the aspirin group and 95.9% in the placebo group. Details on medication adherence prior to blood sampling are listed in Table 4. No adverse obstetric outcomes occurred in the aspirin group. In the placebo group, there were three preterm births and one small-for-gestational age neonate. None of the women developed preeclampsia.
      Table 4Medication use and adherence per treatment group.
      Aspirin group (n = 6)Placebo group (n = 5)p-value
      Medication use
      Start medication (gestational age)13+6 (11+5-14+6)14+1 (11+4-15+4)0.690
      Stop medication (gestational age)36+0 (34+6-36+0)35+0 (32+4-36+0)0.190
      Overall medication adherence (%)99.4% (94.2%-100%)95.9% (90.7%-97.2%)0.393
      No. of adherent women 1st measurement3/4 (75.0%)5/5 (100%)0.556
      No. of adherent women 2nd measurement3/5 (60.0%)3/4 (75.0%)0.730
      Data are presented as medians (IQR) or numbers (%) as appropriate.

      Discussion

      Principal findings

      This explorative study evaluated the effect of low-dose aspirin 80 mg on platelet function with three platelet function tests in the second and third trimester of pregnancy in a placebo-controlled setting. Low-dose aspirin 80 mg had a clear inhibitory effect on platelet function as assessed by VerifyNow®, Chronolog LTA and serum TxB2 levels. Platelet function in the aspirin group was comparable in the second and third trimester of pregnancy.

      Interpretation in light of other evidence

      We demonstrated the clear inhibitory effect of aspirin 80 mg on platelet function. These results are in line with a previous placebo-controlled study demonstrating a clear inhibitory effect of aspirin 60 mg on TxB2 levels in a population of 1002 pregnant women [

      Finneran MM, Gonzalez-Brown VM, Smith DD, Landon MB, Rood KM. Obesity and laboratory aspirin resistance in high-risk pregnant women treated with low-dose aspirin. Am. J. Obstetr. Gynecol. 2019;220(4):385 e1- e6.

      ]. A longitudinal study evaluating the effect of aspirin 80 mg on platelet function tests during the three trimesters of pregnancy and > 3 months postpartum showed a more variable platelet response over time in individual women, but lacked a placebo group for comparison [
      • Bij de Weg J.M.
      • Abheiden C.N.H.
      • Fuijkschot W.W.
      • Harmsze A.M.
      • de Boer M.A.
      • Thijs A.
      • et al.
      Resistance of aspirin during and after pregnancy: A longitudinal cohort study.
      ].
      In our study, the levels of the VerifyNow® and Chronolog LTA were comparable between the second and third trimester. However, serum TxB2 levels in the placebo group were higher in the third trimester as compared to the second trimester. The trend of rising TxB2 levels during pregnancy has been reported before [
      • Hui C.
      • Lili M.
      • Libin C.
      • Rui Z.
      • Fang G.
      • Ling G.
      • et al.
      Changes in coagulation and hemodynamics during pregnancy: a prospective longitudinal study of 58 cases.
      ]. In the aspirin group, on the other hand, the TxB2 levels were comparable during the second and third trimester of pregnancy. Aspirin may have diminished the rising TxB2 levels. In the prevention of preeclampsia, it is thought that aspirin corrects the imbalance of prostacyclin and thromboxane [
      • Walsh S.W.
      • Strauss 3rd., J.F.
      The Road to Low-Dose Aspirin Therapy for the Prevention of Preeclampsia Began with the Placenta.
      ].
      In adult cardiology, the VerifyNow®, Chronolog LTA and serum TxB2 assays have proven to be useful for the prediction of recurrent cardiovascular events in aspirin-treated patients [
      • Wisman P.P.
      • Roest M.
      • Asselbergs F.W.
      • de Groot P.G.
      • Moll F.L.
      • van der Graaf Y.
      • et al.
      Platelet-reactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis.
      ]. The present sample size was too small to determine whether the observed platelet response to aspirin 80 mg relates to adverse obstetric outcome. Three previous studies did evaluate this relationship. Two studies found an association between aspirin non-responsiveness and preeclampsia and fetal growth restriction, however, they did not take medication adherence into account [
      • Wojtowicz A.
      • Undas A.
      • Huras H.
      • Musial J.
      • Rytlewski K.
      • Reron A.
      • et al.
      Aspirin resistance may be associated with adverse pregnancy outcomes.
      ,
      • Rey E.
      • Rivard G.E.
      Is testing for aspirin response worthwhile in high-risk pregnancy?.
      ]. Navaratman et al, who did take medication adherence into account, did not find and association between aspirin non-responsiveness and adverse obstetric outcome [
      • Navaratnam K.
      • Alfirevic A.
      • Jorgensen A.
      • Alfirevic Z.
      Aspirin non-responsiveness in pregnant women at high-risk of pre-eclampsia.
      ].
      Obesity might also be a cause of poor platelet response. Finneran et al. have shown that high-risk obese women have less marked decrease of TxB2 levels in response to aspirin 60 mg as compared to non-obese women in pregnancy, suggesting that higher doses of aspirin may be necessary in the obese pregnant patient [

      Finneran MM, Gonzalez-Brown VM, Smith DD, Landon MB, Rood KM. Obesity and laboratory aspirin resistance in high-risk pregnant women treated with low-dose aspirin. Am. J. Obstetr. Gynecol. 2019;220(4):385 e1- e6.

      ].
      The exact threshold for an adequate platelet response is currently unknown. Future studies should evaluate whether platelet function tests correlate with clinical outcomes in the obstetric population. Finding a gold standard with a clinical threshold would be useful to determine the optimal individual aspirin dose. In addition to an antiplatelet response, low-dose aspirin also has anti-inflammatory properties that are thought to contribute to improved pregnancy outcomes [
      • Shanmugalingam R.
      • Wang X.
      • Motum P.
      • Fulcher I.
      • Lee G.
      • Kumar R.
      • et al.
      The 15-Epilipoxin-A4 Pathway with Prophylactic Aspirin in Preventing Preeclampsia: A Longitudinal Cohort Study.
      ]. Low-dose aspirin of 81 mg is known to increase the formation of anti-inflammatory mediator aspirin-triggered 15-epilipoxinA4 (ATL) [
      • Chiang N.
      • Bermudez E.A.
      • Ridker P.M.
      • Hurwitz S.
      • Serhan C.N.
      Aspirin triggers antiinflammatory 15-epi-lipoxin A4 and inhibits thromboxane in a randomized human trial.
      ]. In women at risk for developing preeclampsia, ATL plasma concentration is up to 70% lower than low-risk women [
      • Shanmugalingam R.
      • Wang X.
      • Motum P.
      • Fulcher I.
      • Lee G.
      • Kumar R.
      • et al.
      The 15-Epilipoxin-A4 Pathway with Prophylactic Aspirin in Preventing Preeclampsia: A Longitudinal Cohort Study.
      ]. Aspirin use during pregnancy resulted in higher ATL plasma concentration and lower incidence of preeclampsia, indicating an anti-inflammatory effect of aspirin on the development of preeclampsia [
      • Shanmugalingam R.
      • Wang X.
      • Motum P.
      • Fulcher I.
      • Lee G.
      • Kumar R.
      • et al.
      The 15-Epilipoxin-A4 Pathway with Prophylactic Aspirin in Preventing Preeclampsia: A Longitudinal Cohort Study.
      ]. Our study only addressed aspirin’s effect on platelet function. Anti-inflammatory effects should also be considered when evaluating optimal aspirin dosing.
      We found no evidence of laboratory aspirin resistance, but this could not be ruled out with the present sample size. In a recent study assessing aspirin 100–150 mg in 220 high-risk pregnant women, none of the adherent women showed signs of laboratory aspirin resistance measured by PFA-100 [
      • Shanmugalingam R.
      • Wang X.
      • Motum P.
      • Fulcher I.
      • Lee G.
      • Kumar R.
      • et al.
      Clinical Influence of Nonadherence With Prophylactic Aspirin in Preventing Preeclampsia in High-Risk Pregnancies: A Multicenter, Prospective.
      ]. Also Mone et al. concluded that in case of confirmed biochemical adherence, aspirin non-responsiveness did not exist [
      • Mone F.
      • Gupta J.K.
      • Phelan M.M.
      • Meher S.
      • Lian L.Y.
      • Francis B.
      • et al.
      Platelet response to aspirin in UK and Irish pregnancy cohorts: a genome-wide approach.
      ]. This raises the question whether laboratory aspirin resistance exists, or if a poor platelet response to low-dose aspirin is a result of medication non-adherence.
      Medication adherence may play a more important role in the prophylactic treatment effect than variable platelet responses between individuals. Previous studies have shown that pregnant women with aspirin adherence ≥ 90% have a significantly improved treatment effect as compared to non-adherent women [
      • Shanmugalingam R.
      • Wang X.
      • Motum P.
      • Fulcher I.
      • Lee G.
      • Kumar R.
      • et al.
      Clinical Influence of Nonadherence With Prophylactic Aspirin in Preventing Preeclampsia in High-Risk Pregnancies: A Multicenter, Prospective.
      ,

      Wright D, Poon LC, Rolnik DL, Syngelaki A, Delgado JL, Vojtassakova D, et al. Aspirin for Evidence-Based Preeclampsia Prevention trial: influence of compliance on beneficial effect of aspirin in prevention of preterm preeclampsia. Am. J. Obstetr. Gynecol. 2017;217(6):685 e1- e5.

      ]. Non-adherence to aspirin is reported to occur in up to 46% of pregnant women [
      • Abheiden C.N.
      • van Reuler A.V.
      • Fuijkschot W.W.
      • de Vries J.I.
      • Thijs A.
      • de Boer M.A.
      Aspirin adherence during high-risk pregnancies, a questionnaire study.
      ]. Efforts should be made to enhance medication adherence in pregnancy.

      Strengths and limitations

      This explorative study gives insight in the pharmacodynamic effects of an aspirin dose of 80 mg in the second and third trimester of pregnancy using three platelet function tests. It was performed in an unique setting of a placebo-controlled double blind RCT. The platelet function tests were evaluated as continuous outcomes since there is no evidence supporting previously reported cut-off values for aspirin resistance in the pregnant population. We confirmed the great variety of outcomes between the different platelet function tests, similar to the observations in our prior study [
      • Bij de Weg J.M.
      • Abheiden C.N.H.
      • Fuijkschot W.W.
      • Harmsze A.M.
      • de Boer M.A.
      • Thijs A.
      • et al.
      Resistance of aspirin during and after pregnancy: A longitudinal cohort study.
      ]. The striking difference between the aspirin and the placebo group overcomes the main limitation of the present study, namely the limited sample size. The latter, however, hinders the performance of a next step in the investigation: to relate platelet response to clinical obstetric outcome, although this was known from the start of this sub-study.

      Future

      Despite the clear inhibitory effect of 80 mg of aspirin on platelet function in this study, the pharmacodynamics and pharmacokinetics of aspirin in pregnancy, and the optimal aspirin dose to prevent obstetrics complications remain unknown. Future studies should further explore the pharmacology of different aspirin doses in pregnant women. Such studies should include platelet function tests, measurements of (anti-)inflammatory markers as for instance ATL, and detailed data on medication adherence, preferably determined by biochemical assays. It is also important to determine how factors such as body-mass index influence aspirin’s pharmacokinetics. In addition, it may be evaluated whether a higher dosing frequency might result in improved platelet inhibition. In adult cardiology, there are indications that patients might benefit from a twice daily regimen of low-dose aspirin due to a more stable level of platelet inhibition [
      • van Diemen J.J.
      • Fuijkschot W.W.
      • Wessels T.J.
      • Veen G.
      • Smulders Y.M.
      • Thijs A.
      Evening intake of aspirin is associated with a more stable 24-h platelet inhibition compared to morning intake: a study in chronic aspirin users.
      ]. However, increased dosing frequency may affect medication adherence.

      Conclusion

      Aspirin 80 mg has a clear inhibitory effect on platelet function as assessed by VerifyNow®, Chronolog LTA and serum TxB2. The on-aspirin platelet response appeared comparable in the second and third trimester of pregnancy.

      Funding

      This research did not receive any specific grant from funCcies in the public, commercial, or not-for-profit sectors. The original APRIL study received funding from ZonMw, The Dutch Organization for Health Research and Development (Grant No 836041006).

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      We want to thank the women who participated in this study.

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