The role of aspirin and inflammation on reproduction: the EAGeR trial

Abstract

Inflammation has been linked to several complications in pregnancy, including pregnancy loss. Due to its anti-inflammatory properties, aspirin, a widely available and inexpensive therapy, has potential to help mitigate the negative effects of inflammation along the reproductive pathway. Therefore, the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial was designed to elucidate whether preconception-initiated daily low-dose aspirin would increase the live birth rate in women with 1–2 prior pregnancy losses and no infertility diagnosis and attempting unassisted conception. Here, we present an overview of the collected findings. Low-dose aspirin was associated with an increased live birth rate among women with a single loss at <20 weeks gestation within the past year. When stratified by tertile of C-reactive protein (CRP), a biomarker of inflammation, treatment with aspirin restored a decrement in the live birth rate in women in the highest CRP tertile (relative risk 1.35, 95% confidence interval 1.08–1.67), increasing to similar rates as women of the lower and mid-CRP tertiles. The same effect modification by inflammation status was observed when examining the effect of low-dose aspirin on offspring sex ratio. These results suggest that inflammation plays an important role in reproduction, and that chronic, low-grade inflammation may be amenable to aspirin treatment.

Introduction

Aspirin has been utilized as an anti-inflammatory and pain-relieving treatment since the 1800s (Handin 2016), and its plant-based derivatives are documented as having been used as early as the third century B.C. (Choi et al. 2015). Aspirin primarily works by blocking the action of the enzyme cyclooxygenase-1, which thereby prevents the conversion of arachidonic acid into prostaglandins, which further prevents the synthesis of thromboxane. Without thromboxane, platelets are not activated and do not aggregate, mediating aspirin’s anticoagulant and blood-thinning properties (Vane and Botting 2003). Platelets are unable to reverse the cyclooxygenase-1 inhibition, so inhibition remains throughout the 8–10 day lifespan of the platelet (Campbell et al. 2007). Orally ingested aspirin is rapidly absorbed in the stomach and upper intestine; in fact, peak plasma levels are achieved within 30 min, followed by rapid clearance (a half-life of 15–20 min) (Campbell et al. 2007). Low-dose aspirin (LDA) (81 mg/day), as opposed to regular dose (325 mg/day), has been shown to be sufficient to fully inhibit platelet thromboxane production, and confers a lower risk of bleeding compared with higher doses — although dosage has been debated more in the context of cardiovascular disease (Campbell et al. 2007).

Recently, aspirin has been found to be beneficial for the prevention of poor reproductive health outcomes, possibly through reducing inflammation and improving blood flow, both of which seem to play a role at critical timepoints throughout the human reproductive process. LDA has been used to increase uterine blood flow, which may help optimize implantation and prevent preeclampsia, thereby decreasing pregnancy loss (Singh et al. 2012) and preterm birth (Cui et al. 2018). It has been also suggested that LDA initiated prior to 16 weeks gestation is effective in reducing preeclampsia or intrauterine growth restriction (Villa et al. 2013; Xu et al. 2015); in fact, the American College of Obstetricians and Gynecologists recommends daily LDA, initiated prior to 16 weeks gestation, for pregnant women at high risk of preeclampsia (ACOG 2018). However, studies utilizing aspirin as treatment initiated at different timepoints during gestation have yielded mixed results (de Jong et al. 2014; Kaandorp et al. 2010).

Inflammation has been identified as playing a critical role in a variety of reproductive health outcomes. A systemic marker of inflammation, high-sensitivity C-reactive protein (hsCRP), has been associated with preeclampsia (Thilaganathan et al. 2010), preterm delivery (Lohsoonthorn et al. 2007), pregnancy loss (Ahmed et al. 2015), and intrauterine growth restriction (Tjoa et al. 2003). In addition, abnormal uterine and ovarian blood flow was associated with similar reproductive outcomes such as stillbirth (Singh et al. 2012), preeclampsia (Xu et al. 2015), and pregnancy loss (Koo et al. 2015; Mesdaghinia et al. 2017; Thilaganathan et al. 2010). Although the pathophysiological mechanisms surrounding pregnancy loss are not fully understood, LDA has been suggested as a potential treatment to reduce pregnancy loss based on commonly observed symptoms in women who have experienced loss, which include inflammation in the reproductive tract and abnormal uterine, endometrial, ovarian, and placental blood flow (Koo et al. 2015; Lazzarin et al. 2009; Mesdaghinia et al. 2017). Indeed, an association between preconception-initiated LDA and increased clinical pregnancy rates was suggested in women undergoing in vitro fertilization treatment (Ruopp et al. 2008). A potential role of postconception-initiated LDA was studied in women with recurrent pregnancy loss (as defined by at least 2 losses); however, aspirin’s efficacy has not been proven (Di Nisio et al. 2005).

Achieving pregnancy is an inefficient process in humans with approximately a 30% chance of becoming pregnant in any given menstrual cycle (Zinaman et al. 1996). When conception does occur, around 30% of these pregnancies end in a spontaneous loss (Wilcox et al. 1999) due largely to unknown causes, as well as some known causes including chromosomal abnormalities and conditions such as diabetes, uterine fibroids, and thrombophilia (Silver and Branch 2008). Women who experience a pregnancy loss are at higher risk for a subsequent loss and other future adverse pregnancy outcomes such as preterm birth (Risch et al. 1988). Aspirin might be an ideal preventative therapy for pregnancy loss due to its potential role in placentation (Roberge et al. 2018) and endometrial vascularization, as well as its anti-inflammatory properties, safety during pregnancy (Xu et al. 2015), wide availability, and inexpensive cost. However, despite a suggested role of aspirin in reducing pregnancy loss, it is largely unknown whether LDA initiated prior to conception is effective among women with a prior spontaneous loss or among women seeking natural conception.

Given the existing data gaps and aspirin’s potential to positively affect pregnancy outcomes, the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial sought to elucidate whether daily preconception-initiated treatment with LDA, compared with placebo, improved the live birth rate in women with 1 to 2 prior pregnancy losses. Here, we review the trial design and results regarding the role of LDA on pregnancy, live birth, pregnancy loss, fecundability, pregnancy complications, and the role of inflammation in these findings.

Methods — the EAGeR trial

The EAGeR trial (ClinicalTrials.gov: NCT00467363) was designed to assess the efficacy of preconception LDA treatment on live birth in 1228 women from 4 university medical centers in the United States from 2006 to 2012 (Schisterman et al. 2013). Randomization to LDA was stratified by center and by eligibility strata. The original eligibility stratum included women aged 18–40 years who were attempting pregnancy with a single documented pregnancy loss <20 weeks gestation that occurred in the previous 12 months. Eligibility was expanded (i.e., expanded stratum) to include women with 1 or 2 documented pregnancy losses regardless of gestational age or the date of the loss in the past. Women must also have had ≤2 previous live births. Exclusion criteria for both strata included a history of infertility or subfertility, clinical indication for anticoagulant therapy, clinical indication for chronic nonsteroidal anti-inflammatory drug therapy, and presence of major medical disorders, among others. Participants were block randomized 1:1 either to LDA (81 mg) plus 400 [H9262]g folic acid or placebo plus 400 [H9262]g folic acid. Both LDA and placebo were to be taken daily until up to 6 menstrual cycles of attempting pregnancy and through 36 weeks of gestation for women who became pregnant. Participants were instructed to take the study medication at about the same time every day, preferably in the morning. During preconception follow-up, for the first 2 menstrual cycles, study visits occurred twice per cycle and included blood and urine collection. For each menstrual cycle thereafter, study visits occurred once per cycle. At home, women completed daily diary entries, and for 2 cycles of participation women also collected daily first-morning urine specimens. Fertility monitors were utilized throughout all menstrual cycles to assist with timing of intercourse to optimize conception and to assist with the scheduling of study visits. Women who became pregnant were followed throughout gestation with monthly study visits, also including biospecimen collection. Each participating center’s Institutional Review Board approved the study, and participants provided written informed consent.

The primary outcome of the EAGeR trial was live birth. Secondary outcomes included human chorionic gonadotropin pregnancy, as confirmed by a positive urine pregnancy test; clinical pregnancy, as clinically confirmed by ultrasound at approximately 6.5 weeks gestation; pregnancy loss occurring <20 weeks gestation; preterm birth, as defined by delivery at <37 weeks gestation; and other obstetric complications, including gestational diabetes and preeclampsia. A unique aspect of study design included study-specific early pregnancy ultrasounds, as opposed to relying on chart abstraction of clinically indicated ultrasounds. Additionally, augmented, laboratory urine human chorionic gonadotropin testing was utilized on daily first-morning urine samples, which allowed for a more sensitive, very early detection of pregnancy than would be detected by conventional urine pregnancy tests alone. These daily first-morning urine human chorionic gonadotropin tests allowed the study to include pregnancies and very early losses of which the participants may not otherwise have been aware. An additional aim of the study beyond evaluating the efficacy of preconception-initiated LDA was to evaluate the role of inflammation in the preconception period and throughout pregnancy.

Results and discussion

LDA and pregnancy and live birth

Of the 1228 women who were randomized in the study, 1088 women completed the trial. Of the 140 (11%) women who withdrew early, 128 (10%) were not pregnant and 12 (1%) were pregnant at the time of withdrawal. Among the 1088 who completed follow-up, there were 732 (67%) confirmed pregnancies, 133 (12%) clinical pregnancy losses, and 597 (55%) live births (Mumford et al. 2016). Baseline demographics including age, body mass index (BMI), race, educa tion, and other demographics were similar between treatment groups, demonstrating that randomization was effective at balancing these characteristics (Schisterman et al. 2013).

Overall, the trial found a potential benefit of LDA for live birth, but only among a certain group of women (Schisterman et al. 2014). Specifically, among all EAGeR participants, there was no statistically significant difference in live birth rates between LDA and placebo groups (relative risk (RR) 1.10, 95% confidence interval (CI) 0.98–1.22) (Fig. 1). However, there was a significantly higher live birth rate among the LDA group among women enrolled in the “original” stratum, those with a single recent loss (RR 1.17, 95% CI 1.01–1.37), whereas no significant difference was observed in the expanded stratum alone (RR 1.03, 95% CI 0.89–1.20).

Fig. 1.

The effect of low-dose aspirin (LDA, 81 mg) versus placebo on live birth rate in the Effects of Aspirin in Gestation and Reproduction (EAGeR) Trial (2006–2012). Original stratum includes women aged 18–40 years who were attempting pregnancy with 1 documented pregnancy loss <20 weeks gestation that occurred in the previous 12 months. Expanded stratum includes women with 1–2 documented pregnancy losses regardless of gestational age or the date of the loss.

Inflammation and pregnancy and live birth

To investigate how LDA’s effect on inflammation may impact pregnancy rate, pregnancy loss, and live birth rate, an additional secondary analysis was performed. Given that hsCRP is a commonly used biomarker of systemic, chronic inflammation, it was hypothesized that high levels of hsCRP would indicate a systemic, low-grade inflammatory milieu that may be amenable to LDA treatment. Indeed, when the comparison between LDA and placebo was stratified by tertiles of preconception, pre-intervention levels of hsCRP, a significant effect modification was observed. Specifically, the lowest pregnancy and live birth rates (54% clinically confirmed pregnancy, 44% live birth) were observed among women receiving placebo with hsCRP levels in the highest tertile. Treatment with LDA increased live birth rate in women with the highest levels of hsCRP to 59% (RR 1.35, 95% CI 1.08–1.67). Thus, LDA restored the live birth rate in women with the highest level of inflammation to similar rates observed in women of the lower and mid-hsCRP tertiles. LDA did not appear to affect live birth in the low (59% LDA, 54% placebo) or mid-level (59% LDA, 59% placebo) hsCRP tertiles (Sjaarda et al. 2017). Indeed, it appeared that among women with elevated hsCRP levels, LDA therapy restored live birth rates to the rates expected in the women without low-grade inflammation.

Given that aspirin may play a role in increasing endometrial receptivity and implantation, we also conducted an analysis of clinically confirmed pregnancy rates, again, stratified by tertile of hsCRP. Similar to the trends observed for live birth rates, among women in the highest tertile of hsCRP, LDA significantly increased clinically confirmed pregnancy rates by 31% compared with placebo (Sjaarda et al. 2017). Conversely, LDA did not affect clinically confirmed pregnancy rates for women in the lowest or middle hsCRP tertiles. Interestingly, the chance of both clinically confirmed pregnancy and live birth decreased with increasing hsCRP levels and, again, LDA appeared to restore the pregnancy and live birth rates in women with elevated hsCRP to rates similar to women with lower hsCRP levels.

As obesity is considered an inflammatory state (in which case otherwise healthy women would have low-grade inflammation and the corresponding elevated hsCRP), women with hsCRP in the top tertile (≥1.95 mg/L) were further stratified by waist-to-hip ratio, as a marker of central obesity, and by BMI, as an overall measure of obesity. Perhaps counterintuitively, we observed that LDA showed a significant, positive effect on pregnancy and live birth rates only among leaner (waist-to-hip ratio less than the median) women with highest hsCRP (RR 1.60, 95% CI 1.11–2.30) (Sjaarda et al. 2017) (Fig. 2). However, the beneficial effect of LDA on pregnancy and live birth rates was attenuated in women with high hsCRP having higher waist-to-hip ratio (RR 1.18, 95% CI 0.89–1.56). Similar results were observed when we stratified the analysis by normal BMI (BMI ≤ 25 kg/m²) versus overweight or obese BMI (>25 kg/m²). Thus, it appears that the preconception, preintervention inflammation level, and level of adiposity may significantly modify chances of achieving pregnancy and the efficacy of LDA. Moreover, similar to its utility in cardiovascular medicine research, hsCRP may be a useful biomarker to identify the subset of women most likely to benefit from preconception-initiated LDA (i.e., lean women with low-grade inflammation).

Fig. 2.

Live birth rates among women in highest high-sensitivity C-reactive protein (hsCRP) tertile (≥1.95 mg/L), stratified by measures of obesity. BMI, body mass index (kg/m²); CI, confidence interval; LDA, low-dose aspirin; RR, relative risk.

LDA, pregnancy loss, and fecundability

Despite our hypothesis that LDA might decrease pregnancy loss through increased endometrial blood flow and reduced inflammation, LDA was not associated with pregnancy loss (RR 1.07, 95% CI 0.83–1.39) (Mumford et al. 2016). Furthermore, LDA therapy was not found to affect any subtype of pregnancy loss: LDA was not associated with risk of implantation failures (RR 1.06, 95% CI 0.64–1.78), clinically recognized pregnancy losses (RR 1.07, 95% CI 0.78–1.48), or the rate of euploid losses (RR 1.11, 95% CI: 0.99–1.26).

A similar pattern to that observed for live birth emerged for time to pregnancy, a measure of fecundability (Schisterman et al. 2015). Overall, LDA was not associated with time to pregnancy (fecundability odds ratio (FOR) 1.14, 95% CI 0.97–1.33); the average rate of pregnancy per cycle was 11.5% for LDA and 10.5% for placebo (P = 0.08). However, when the analysis was restricted to the original stratum, LDA was associated with a shorter time to pregnancy (FOR 1.28, 95% CI 1.02–1.62), and the average probability of pregnancy per cycle was 12.3% for LDA and 10.5% for placebo (P = 0.02).

To try to elucidate the mechanism of increased fecundability among specific EAGeR participants, an analysis was performed to evaluate the impact of LDA versus placebo on per-cycle risk of sporadic anovulation. For this analysis, a menstrual cycle was considered ovulatory if urinary luteinizing hormone was 2.5 times greater than the mean of the previous 5 days (Park et al. 2007), indicating the mid-cycle luteinizing hormone peak. Among 4340 cycles analyzed, 12% were considered anovulatory (Radin et al. 2017). LDA was not associated with sporadic anovulation in the overall cohort (RR 1.16, 95% CI 0.88–1.52) or in the original stratum (RR 1.06, 95% CI 0.68–1.54).

Taken together, these findings suggest that preconception-initiated LDA may play a role in the establishment of pregnancy and is less important for the maintenance of the pregnancy. The benefit observed in the original stratum with regard to increased fecundability supports the hypothesis that aspirin plays a role in establishing pregnancy — perhaps by enhancing embryo survival and implantation, but not by increasing rates of ovulation. These findings may partially explain why previous aspirin trials, in which aspirin was initiated after conception and during early pregnancy, did not observe a beneficial effect (Di Nisio et al. 2005).

Inflammation and offspring sex ratio

It is believed that inflammatory mediators exchanged between the embryo and the maternal endometrium are important components of the communication that drives the endometrium’s function as a “sensor” of the preimplantation embryo’s viability (Macklon and Brosens 2014). In fact, the endometrial decidua plays an important role in recognizing chromosomally abnormal embryos and creates a stress response to apparently prevent the implantation of an abnormal embryo (Macklon and Brosens 2014). Because a normal response to an abnormal embryo involves inflammatory mediators, it was therefore hypothesized that in the context of a disordered maternal inflammatory milieu, the low-grade inflammation may lead to the rejection of chromosomally normal embryos. To further understand the differential effect of maternal inflammation on male and female embryos, another secondary analysis of the EAGeR trial was conducted to assess LDA use and birth of male versus female offspring. Previous research in animal models have indicated that male embryos may be particularly vulnerable to maternal inflammation, thereby affecting rates of male embryo implantation (Dobbs et al. 2014; Pérez-Crespo et al. 2005). Utilizing genetic data from pregnancy losses (Schisterman et al. 2013), sex at both implantation and birth were assessed. Genetic testing was performed on 84 of 127 clinical pregnancy losses (66%): 55 tests determined sex and 29 had no results due to testing failure (n = 5) or indeterminate results (n = 24).

Of the 1078 women in the EAGeR trial who completed follow-up, women in the LDA group were more likely to give birth to male offspring than women in the placebo group (RR 1.31, 95% CI 1.07–1.59) (Radin et al. 2015). When dividing all women into tertiles of hsCRP, the proportion of male offspring decreased with each increasing tertile of hsCRP in the placebo group. The effect of LDA on the proportion of male offspring increased compared with the placebo group as hsCRP increased (first hsCRP tertile: RR 0.97, 95% CI 0.70–1.35; second hsCRP tertile: RR 1.36, 95% CI 0.98–1.90; third hsCRP tertile: RR 1.70, 95% CI 1.13–2.57; P interaction = 0.03). Similarly, LDA demonstrated a positive effect on male embryo implantation (first hsCRP tertile: RR 1.00, 95% CI 0.74–1.34; second hsCRP tertile: RR 1.46, 95% CI 1.07–2.00; third hsCRP tertile: RR 1.58, 95% CI 1.07–2.33; P interaction = 0.06). Collectively, these data suggest that maternal inflammation is negatively associated with male offspring ratio and that preconception-initiated LDA has the potential to restore this ratio to expected proportions.

LDA and pregnancy complications (preterm birth, preeclampsia, gestational diabetes)

To further investigate the effects of aspirin on adverse pregnancy outcomes in women with proven fecundity, we analyzed risk of preterm birth. By reducing the risk of pre-eclampsia, small for gestational age, and placental insufficiency via reduced uterine contractility and inflammation through cyclooxygenase inhibition, LDA has the potential to reduce rates of medically indicated preterm birth. However, we observed that preconception-initiated LDA was not significantly associated with the overall rate of preterm birth. In the overall cohort, spontaneous preterm birth rates were 1.1% (6/535 LDA) and 2.2% (12/543 placebo) (RR 0.51, 95% CI 0.19–1.34); medically indicated preterm birth rates were 2.6% (14/535 LDA) and 2.9% (16/543 placebo) (RR 0.89, 95% CI 0.44–1.80). When the analysis was restricted to confirmed pregnancies in the original stratum alone, preterm birth occurred in 3.8% of the LDA group and 9.7% in the placebo group (RR 0.39, 95% CI 0.16–0.94). The study was underpowered for this secondary analysis; however, the trends toward a benefit of LDA in protecting against spontaneous preterm birth in this specific population of women with a single, recent early pregnancy loss (i.e., the original stratum) warrant further investigation (Silver et al. 2015).

It was originally thought that aspirin may play a role in preventing medically indicated preterm birth by reducing rates of preeclampsia and intrauterine growth restriction. The EAGeR data are therefore of particular interest because it was not observed that LDA reduced preeclampsia (RR 1.08, 95% CI 0.67–1.76), although there were very few cases (32 LDA versus 30 placebo) (Schisterman et al. 2014). Because preeclampsia predicates medically indicated preterm birth, this finding is consistent with the observation that LDA may decrease spontaneous preterm birth more than medically indicated preterm birth in this cohort. The inflammatory nature of parturition and labor, and in particular aspirin’s ability to inhibit cyclooxygenase, which thereby decreases prostaglandin synthesis, which is known to play a role in both normal and abnormal labor, lends biologic plausibility towards a greater impact of LDA on spontaneous preterm birth.

Further, LDA was not observed to be associated with risk of developing gestational diabetes mellitus (RR 1.01, 95% CI 0.44–2.32 in the overall cohort). However, similarly to preeclampsia, the rates of gestational diabetes mellitus among EAGeR participants was low (11 LDA versus 11 placebo) (Schisterman et al. 2014).

LDA and inflammatory markers during pregnancy

In light of the fact that inflammation has been implicated in many adverse pregnancy outcomes, including preeclampsia and gestational diabetes, an analysis of the effect of LDA on hsCRP levels throughout gestation was also completed (Sjaarda et al. 2017). hsCRP levels were measured at 8, 20, and 36 weeks of gestation among EAGeR participants who achieved pregnancy (N = 732). An assessment of the effect of LDA on hsCRP at those timepoints revealed that women in the highest tertile of hsCRP at baseline and treated with LDA showed a significant reduction in hsCRP levels over the course of pregnancy. Among women with lower baseline hsCRP levels, however, LDA treatment had no effect on hsCRP levels throughout pregnancy. If LDA has the potential to lower hsCRP exacerbation throughout pregnancy in women with elevated levels, then an important potential application of LDA therapy may be to prevent adverse birth outcomes that are associated with inflammation and such applications warrant further investigation.

Conclusions

The collected findings from the EAGeR trial demonstrate that preconception-initiated daily LDA therapy improved the chances of clinically recognized pregnancy and live birth, particularly among lean women with low-grade inflammation and prior pregnancy loss. The effect modification by hsCRP on the efficacy of LDA demonstrates that inflammation may play a critical role in reproduction and pregnancy. EAGeR was the first study to assess the effects of LDA in women without recurrent pregnancy loss; EAGeR participants only had ≤2 prior pregnancy losses, giving us the unique ability to assess the efficacy of aspirin on a less severe — and more common — phenotype. Moreover, we studied the use of preconception-initiated LDA, rather than postconception-initiated LDA, which allowed us to capture the effects of aspirin on fertility, conception, embryo implantation, and very early pregnancy loss. hsCRP is an attractive screening tool because the clinical assays are standardized and relatively inexpensive, and it is an established biomarker of inflammation. This finding of an effect modification by hsCRP and low-grade inflammation status should drive future research to help identify anti-inflammatory treatments to help improve reproductive outcomes for a broader array of women. Future research should focus on elucidating the associations between inflammation and fertility/infertility and pregnancy outcomes, and such research should inform the conversation surrounding the use of aspirin in reproductive medicine.