Objective
We sought to critically evaluate platelet function in recurrent miscarriage (RM).
Study Design
We conducted a prospective study comparing 30 patients with unexplained recurrent first-trimester pregnancy loss with 30 control subjects matched for age and serum progesterone level. Platelet function was determined using a modified assay of light transmission aggregometry with multiple agonists at different concentrations. Dose-response curves were created and half-maximal effective concentration values were calculated.
Results
At test completion the half-maximal effective concentration values for arachidonic acid in the patients with RM were significantly less than in the control subjects (0.153 vs 0.230; P = .0099). The dose-response curves were tightly matched for the other agonists.
Conclusion
This novel measurement of platelet function has demonstrated that patients with unexplained RM have significantly increased platelet aggregation in response to arachidonic acid. The enhanced response to this agonist provides an empirical rationale for the use of aspirin in management of this clinical condition.
Although many advances in reproductive medicine have been made during the past quarter century, miscarriage remains the most common complication of pregnancy. Recurrent miscarriage (RM) is traditionally defined as ≥3 consecutive losses <20 weeks postmenstruation. This distressing problem may affect as many as 0.5-3% of fertile couples of reproductive age. Despite investigation a significant proportion of cases of RM remain unexplained. A causal factor can be identified in less than half of affected couples. This has led to the term “idiopathic recurrent miscarriage.”
The historical hypothesis behind RM has been that affected women are already in a prothrombotic state before pregnancy begins. Evidence has been accumulating over the years that some cases of RM are the result of exaggerated hemostatic response as shown by investigations of placental thrombosis, thrombin-antithrombin complexes, circulating microparticles, and performance of thromboelastography in the unexplained RM population. Indeed, there is also evidence of long-term prothrombotic risk. It is often concluded that there is a subgroup of RM patients who are in a prothrombotic state and would likely benefit from antithrombotic treatment to improve outcome.
The major limitation of conventional hemostasis tests such as measuring the levels of individual coagulation and anticoagulation proteins are that they ignore the fact that in vivo hemostasis is a dynamic process that involves the interaction of coagulation and fibrinolytic factors with cellular elements such as platelets and the blood vessel wall. One way of determining the thrombotic risk of this cohort of patients would be to assess in vivo platelet function that, to our knowledge, has not been performed previously. We have recently developed a novel platelet assay that uses multiple concentrations of different agonists and thus reflects the complexity of agonist-induced platelet aggregation in vivo. The aim of this study was to critically evaluate platelet function in an idiopathic RM cohort as compared to a matched control group.
Materials and Methods
Ethical approval was obtained from the Rotunda Hospital Ethics Committee and the study complied with the Declaration of Helsinki. Informed consent was obtained from all patients prior to phlebotomy.
Patients
Cases were nonpregnant women recruited from the Rotunda Hospital RM outpatient clinic. Eligibility criteria included patients who had experienced at least 3 consecutive first-trimester miscarriages, who had undergone routine RM investigation with no positive results. Routine investigations included as outlined by the American College of Obstetricians and Gynecologists and European Society of Human Reproduction and Embryology were: full blood cell count (blood sugar level and thyroid function tests), antiphospholipid antibodies (Lupus anticoagulant and anticardiolipin antibody), parental karyotype, and pelvic ultrasound and/or hysterosalpingogram and hysteroscopy. The recruits had no significant medical disorders, in particular, no conditions that directly affect platelet function such as spherocytosis.
The control group was comprised of age- and ethnicity-matched women with no known medical disorders, or personal or family history of venous thromboembolism. The control group all had prior successful obstetric outcomes without a history of >1 miscarriage, stillbirth, intrauterine growth restriction, preeclampsia, or preterm labor. All subjects were provided with written instructions outlining measures such as the avoidance of medications such as aspirin or nonsteroidal antiinflammatories for a minimum period of 14 days prior to the platelet assay, and the avoidance of alcohol, tobacco, and vigorous exercise in the preceding 24 hours; all were fasting. None of the recruits were taking hormonal treatments such as oral contraception.
Phlebotomy
The assay was performed at least 12 weeks since the last miscarriage and during the first half of the menstrual cycle. This allowed us to rule out pregnancy and to ensure low progesterone levels. Blood was drawn from all patients by the same phlebotomist. All samples were obtained uncuffed. Blood was collected through a 19-gauge butterfly needle and the first 5 mL were sent for hormone profile, specifically progesterone level. A further 27 mL was collected into a syringe containing 3.2% sodium citrate. Blood was then centrifuged for 10 minutes at 150 g . Platelet-rich plasma (PRP) aspirated from the supernatant was placed in a reagent reservoir. Using a multichannel pipette, the PRP was dispensed across wells in a 96-well plate (black isoplate with clear flat-bottomed wells; Perkin Elmer, Wellesley, MA) containing different concentrations of the 5 agonists arachidonic acid (AA), collagen (Coll) (type 1 soluble calf skin), adenosine diphosphate (ADP), epinephrine (EPI), and thrombin receptor-activating peptide (TRAP).
Platelet function assay
To assess platelet function, we used a novel platelet function assay based on a modification of light transmission aggregometry, described in detail elsewhere. In brief, 180 μL of PRP was added to each well of a 96-well plate containing the different agonists. Light absorbance was measured at standard times. To characterize platelet aggregation, increasing concentrations of the agonists were tested. Platelet aggregation measured as a percentage of absorbance from baseline, using a 572-nm filter, was assayed at 0, 3, 6, 9, 12, 15, and 18 minutes. Between each of these standardized times, the plate was rotated at 1000 rpm through a 0.1-mm orbit. The concentrations of the agonists used were 500, 375, 188, 83.8, 46.9, 23.4, 11.8, and 5.86 μg/mL for AA; 190, 143, 71.3, 35.6, 17.8, 8.9, 4.45, and 2.23 μg/mL for Coll; 20, 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 μmol/L for ADP and TRAP; and 20, 5, 1.25, 0.313, 0.078, 0.0195, 0.00488, and 0.00122 μmol/L for EPI. The agonist volumes used were 50 μL of AA, 50 μL of Coll, 40 μL of ADP, 40 μL of EPI, and 40 μL of TRAP. The 96-well plate was then read using a Victor 3 multilabel plate reader (Perkin Elmer). Light absorbance values were normalized based on PRP and platelet-poor plasma control absorbance values, which represented 0 and 100% aggregation. Using this, the percentage aggregation response for each concentration of each agonist was calculated. These values were then plotted against the log values of the concentrations of agonist used with Graphprism software (Graphprism, San Diego, CA). To further analyze the aggregatory responses with respect to different agonist concentrations and time points the half-maximal effective concentration (E C 50) results generated from the dose-response curves by Graphprism were also recorded.
Statistical analysis
A 2-way analysis of variance was used to compare the maximum aggregation response to the different agonists for the RM group and control group. The mean of the E C 50 results were compared using Student t tests. The χ 2 tests were also performed to determine the difference between groups with respect to levels of platelet aggregation to each agonist at successive time points. A P value < .05 was used for statistical significance.
Results
There were 30 subjects in each group. Table 1 outlines the baseline characteristics of each group.
| Characteristic | Recurrent miscarriage n = 30 | Controls n = 30 | P value |
|---|---|---|---|
| Mean age, y (range) | 35.0 (25–46) | 36.6 (28–49) | .29 |
| Ethnic origin | 86.7% Caucasian (2 Asian, 2 African) | 86.7% Caucasian (2 Asian, 2 African) | 1.0 |
| No. of miscarriages | 3-12 | 0-1 | |
| Previous live birth | 54% | 100% | |
| Progesterone level, nmol/L (range) | 3.95 (0.4–10.4) | 4.81 (0.2–6.5) | .70 |
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