Outcomes Among Women Using Low-dose Aspirin and Undergoing Euploid, Autologous, Programmed Frozen Embryo Transfer Cycles: A Retrospective Cohort Study

Abstract

Background:

Low-dose aspirin (LDA) has been used during assisted reproduction; however, the evidence supporting its use remains conflicting.

Aim:

To assess whether LDA improves the clinical pregnancy rate (CPR) in a patient’s first autologous, programmed, single frozen embryo transfer (FET) cycle with an euploid embryo.

Settings and Design:

Retrospective cohort study at an academic center.

Materials and Methods:

A retrospective cohort study of all first autologous, programmed, single FETs of a single euploid embryo performed at our academic center between 2020 and 2022 assessed the association between LDA and CPR and live birth rate (LBR). A subgroup analysis assessed the prevalence of hypertensive disorders of pregnancy (HDP).

Statistical Analysis Used:

Chi-square tests of association and fixed-effects multivariable logistic regression were performed. Among patients with a live birth, Chi-square tests of association were utilised. Binary logistic regression was performed, adjusting for age and body mass index. Data were analysed using SAS software, version 3.8, Enterprise Edition (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at P < 0.05.

Results:

Two hundred seventy FETs resulted in 189 clinical pregnancies, and 153 live births. LDA was not associated with CPR or LBR. LDA was also not associated with HDP, although this outcome was only able to be assessed among one hundred twenty patients (78% of all live births).

Conclusion:

LDA use was associated with neither clinical pregnancy nor live birth among autologous, euploid, programmed, single FET cycles.

INTRODUCTION

Low-dose aspirin (LDA) is safe when taken preconception and may increase live births among certain patients with unassisted conceptions.[1,2] Nevertheless, LDA use in assisted reproduction remains controversial. A meta-analysis of 13 randomised controlled trials (RCTs) found an increased relative risk of clinical pregnancy among in vitro fertilisation (IVF) patients on LDA.[3] Similarly, a randomised controlled trial showed improved clinical pregnancy rate (CPR), implantation rates and live birth rate (LBR) with lower miscarriage rates among patients using LDA while undergoing untested multiple embryo transfers.[4] However, other studies have demonstrated no such impact.[5] After reviewing the totality of the evidence, a Cochrane review concluded that there is very low-quality evidence available to determine whether aspirin improves LBR or CPR among women undergoing frozen embryo transfer (FET).[6]

LDA has also been shown to reduce the risk of pre-eclampsia, small for gestational age babies and preterm birth among all pregnant women.[7] However, a large retrospective study of 4454 patients undergoing programmed FET also found no effect of aspirin on LBR or postpartum complications. This study had several limitations including using an aspirin dose not commonly prescribed in the United States (50 mg), inability to control for aneuploidy, the low mean age of participants which also differed statistically between groups, and the normal mean body mass index (BMI) of participants all of which may affect the generalisability of these results to patients in the United States and Europe.[8] The utilisation of pre-implantation genetic testing for aneuploidy (PGT-A) is increasing in the United States,[9] but data on how LDA impacts patients utilising this technology remains lacking. Currently, it is also unclear how LDA affects HDP risk in women utilising advanced reproductive technology, and a systematic review is currently underway to help guide clinical practice.[10]

We conducted a retrospective cohort study to assess the association between the use of LDA (81 mg) during euploid FET and both CPR and LBR. A subgroup analysis was performed to assess the potential association between LDA and hypertensive disorders of pregnancy (HDP) amongst patients with a live birth in our health system.

MATERIALS AND METHODS

This is a retrospective cohort study including all first autologous, programmed, single FETs of an euploid embryo performed at an academic center between 2020 and 2022. The study was approved by our Institutional Review Board (23-0416) and adhered to the ethical guidelines as outlined in the Helsinki Declaration. Written informed consent was obtained from all patients prior to initiating their IVF cycle and again prior to their FET cycle.

Four hundred and thirty-one FET cycles occurred during the study period (2020–2022). Of these, 270 programmed FETs met the inclusion criteria for our study. Aneuploidy was controlled for by only including patients undergoing transfer of an euploid embryo by PGT-A testing. PGT-A testing was performed electively at the patient’s request to screen for aneuploidy prior to embryo transfer. Embryos were created following IVF cycles using either agonist or antagonist protocols at the discretion of the patient’s attending physician (n = 10). Patients underwent ovarian hyperstimulation with gonadotropins, resulting in superovulation. After achieving three lead follicles of at least 18 mm, patients were triggered with a GnRH agonist, human chorionic gonadotropin or both at the discretion of the patient’s attending physician. Thirty-five hours after trigger, patients underwent transvaginal oocyte retrieval. Cycles were performed in one embryology laboratory with seven embryologists and included fertilisation via both insemination and ICSI. Embryos were cultured with assisted hatching performed at the cleavage stage. Embryo biopsy was performed on day 5–7 by removing 4–7 trophectoderm cells from fully expanded blastocysts through a small opening of the zona pellucida before cryopreservation. After receiving euploid PGT-A results, patients were seen on cycle day three of their menstrual cycle, assessed with a baseline ultrasound and labwork and started on oral estradiol 6 mg daily for at least 10 days. After this duration of estradiol exposure and after achieving an endometrial thickness of at least 7 mm, patients began progesterone starting 5 days preceding the embryo transfer.

The LDA cohort was instructed to begin taking aspirin 81 mg (LDA) once daily prior to undergoing FET. LDA initiation was used as an adjunct only and was either recommended or not recommended based solely on the typical practice pattern of the patient’s attending physician, with some physicians routinely electing to include this treatment in all FET cycles and others routinely electing not to include LDA in their patients’ cycles. All patients instructed to take LDA were informed that this was an adjunct therapy with inconclusive evidence supporting its clinical use, and all patients in this cohort provided verbal consent to take LDA based on this discussion with their attending physician. The timing of LDA initiation varied according to the judgment and typical practice pattern of the patient’s attending physician. Patients initiated LDA after endometrial thickening was noted on transvaginal ultrasonography which typically occurred within 1–2 weeks of starting exogenous estrogen prior to undergoing FET. LDA was continued until a negative pregnancy test, diagnosis of a non-viable pregnancy or through 8 weeks of gestation when the patient’s care was transferred to her OB/GYN. The control cohort was considered to be LDA unexposed because they were not instructed to take LDA during their FET cycle and did not report LDA use to their physician. Patients reported adherence to their prescribed medication regimen on the day of each scheduled patient visit.

The primary outcome of CPR was assessed amongst all recorded FET cycles. The secondary outcome of LBR was assessed amongst all recorded FET cycles. The tertiary outcome of HDP (gestational hypertension and pre-eclampsia) was assessed for all patients who delivered within our health system, using standard definitions provided by the American College of Obstetricians and Gynecologists. Chi-square tests and fixed-effects multivariable logistic regression were performed to evaluate the association between LDA use and FET outcomes, adjusting for age (advanced maternal age), race/ethnicity, BMI (obesity) and endometrial thickness (<8 mm vs. ≥8 mm). For endometrial thickness, <8 mm versus ≥8 mm was selected a priori because 8 mm is commonly used as the threshold for endometrial receptivity in FET cycles,[11] These covariates were selected a priori. The model was limited to these potential confounders, as inclusion of a larger set of covariates in the regression model would have risked overfitting, resulting in unstable estimates. Among the live birth cohort, binary logistic regression was utilised to discern potential associations between LDA use and HDP after adjusting for age and BMI. This model was likewise limited to these potential confounders, as inclusion of a larger set of covariates in the regression model would have risked overfitting, resulting in unstable estimates. Patients with missing demographic data were retained for analyses that did not require those missing variables but were excluded from regression analyses. Data were analysed using SAS software, version 3.8, Enterprise Edition (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at P < 0.05.

RESULTS

Two hundred seventy FETs resulted in 189 clinical pregnancies and 153 live births. The median age amongst the LDA cohort was 37 years (interquartile range [IQR] 34, 39) and 36 years (IQR 33, 39) amongst the non-LDA cohort. Similarly, the median BMI among the LDA cohort was 26.04 kg/m² (IQR 22.27, 31.11) among the LDA cohort and 24.96 kg/m² (IQR 21.96, 28.62) among the non-LDA cohort. The median endometrial thickness among the LDA cohort was 8.00 mm (IQR 7.80, 9.80) and 9.10 mm (IQR 8.00, 10.90) among the non-LDA cohort. Table 1 reports our demographic data which includes patients with all types of underlying infertility diagnoses. One hundred twenty of these patients (78%) delivered within our health system and were included in our tertiary HDP analysis.

Table 1.

Demographic Information

	LDA, n (%)	No LDA, n (%)	P
Demographic dataa
Advanced maternal age	51 (71.83)	128 (64.32)	0.25
Obeseb	17 (27.42)	35 (19.23)	0.17
Endometrial thickness ≥8 mm	48 (67.61)	150 (75.38)	0.20
Race/ethnicityc
Hispanic	3 (4.23)	10 (5.03)	0.64
Non-hispanic white	38 (53.52)	109 (54.77)
Non-hispanic black	4 (5.63)	10 (5.03)
Asian	5 (7.04)	22 (11.06)
Other	14 (19.72)	24 (12.06)
Missing	7 (9.86)	24 (12.06)
Cause of infertilityc
DOR	8 (11.27)	18 (9.05)	0.58
Endometriosis	2 (2.82)	8 (4.02)
Male infertility	13 (18.31)	24 (12.06)
Multiple	10 (14.08)	37 (18.59)
Other	6 (8.45)	21 (10.55)
PCOS	8 (11.27)	29 (14.57)
RPL	7 (9.86)	12 (6.03)
Tubal factor	6 (8.45)	11 (5.53)
Unexplained	11 (15.49)	39 (19.60)

^aA Chi-square test was used to calculate P values based on the categories chosen, a priori, for the multivariable logistic regression model. ^bFor obesity, there were 62/71 patients with a BMI recorded in the LDA group and 182/199 patients with a BMI recorded in the non-LDA group, ^cA Chi-square test was used to calculate P. BMI=Body mass index, LDA=Low-dose aspirin, FET=Frozen embryo transfer, PCOS=Polycystic ovary syndrome, RPL=Recurrent pregnancy loss, DOR=Diminished ovarian reserve

LDA was not found to be statistically significantly associated with either our primary or secondary outcomes of interest, CPR and LBR [Table 2]. CPR and LBR were calculated, including 71 patients in the LDA group and 199 patients not taking LDA. Regarding the primary outcome of interest, in the LDA group, 64.79% of patients had a clinical pregnancy compared to 71.86% of patients not taking LDA (adjusted odds ratio [aOR] 0.81 [95% confidence interval (CI) 0.43–1.55]). For the secondary outcome of interest, in the LDA group, 54.93% of patients had a live birth compared to 57.29% of patients not taking LDA (aOR 0.91 [95% CI 0.50–1.67]).

Table 2.

Patient Outcomes

Outcomesa	LDA, n (%)	No LDA, n (%)	aOR (95% CI)
CPR	46 (64.79)	143 (71.86)	0.81 (0.43–1.55)
LBR	39 (54.93)	114 (57.29)	0.91 (0.50–1.67)

^aCPR and LBR were calculated, including 71 patients in the LDA cohort and 199 patients in the no-LDA cohort. Due to missing demographic information, the aOR for CPR and LBR were calculated, including 62 patients in the LDA cohort and 182 patients in the no-LDA cohort. The unadjusted OR calculated using all patients was also not significant: CPR 0.79 (95% CI=0.45–1.41) and LBR 0.91 (95% CI=0.53–1.57). CPR=Clinical pregnancy rate, LDA=Low-dose aspirin, OR=Odds ratio, aOR=Adjusted OR, CI=Confidence interval, LBR=Live birth rate

Of the patients for whom we were able to assess HDP prevalence, in the LDA group, 21.62% of patients were diagnosed with HDP compared to 22.89% of patients who did not take LDA (Chi-square P = 0.88; aOR 1.46 [95% CI: 0.54–4.00]). The unadjusted analysis was likewise not significant, odds ratio 0.93 (95%CI 0.37–2.37). In the LDA cohort, of the eight cases of HDP, there were three patients with preeclampsia and five patients with GHTN. Two patients in the LDA cohort had chronic hypertension with one developing preeclampsia. In the cohort not taking LDA, of the nineteen cases of HDP there were ten patients with preeclampsia and nine patients with GHTN. The cohort not taking LDA included three patients with chronic hypertension with one developing preeclampsia.

DISCUSSION

LDA was associated with neither CPR nor LBR among autologous, euploid, programmed, single FET cycles. These findings for CPR and LBR are consistent with previously published studies showing no impact[5,8] or insufficient evidence to recommend the use of LDA,[6] but conflict with other studies that have found a potential benefit.[3,4]

There are several proposed mechanisms of action by which LDA may improve early pregnancy success, including improved endometrial receptivity and increased uterine blood flow,[6] improved endometrial growth and vascularisation[12] and reduced inflammation.[2] However, the lack of a clearly demonstrated mechanism of action and clinical studies recommending initiating LDA at various times in the cycle[3,4] creates uncertainty.

While LDA is safe when taken preconception and may increase live births among certain patients with unassisted conceptions,[1,2] it remains unclear if LDA benefits patients undergoing FET. The data is particularly lacking for patients undergoing autologous, euploid, programmed, single FET as examined in our novel study. Unfortunately, while our study is reassuring that LDA did not adversely impact CPR or LBR among our patients, we also did not demonstrate a benefit; however, the study was underpowered to detect small differences between groups.

Although there was no difference in HDP based on LDA use, our HDP incidence was over 20% in both groups, which is more than four times the previously published rate of 5.3% for autologous FET cycles.[13] Our incidence was more similar to a recent study that found a 32.4% prevalence of HDP in programmed cycles.[14] An increased risk of HDP may be due to abnormal placentation related to the freezing and thawing process with a FET,[15] the absence of a corpus luteum in a programmed cycle,[16] and the trophectoderm biopsy when PGT-A is performed.[17] While the incidence of HDP in our study warrants further investigation, this result should be taken with caution, as we could only assess HDP among approximately three-fourths of our cohort study, which leaves these results susceptible to selection bias.

Our study has several strengths, including utilising a dose of LDA typically utilised in clinical practice in the United States, as well as the implementation of stringent inclusion criteria representing a practice pattern that increasingly reflects clinical practice in the United States. Specifically, we limited our study to autologous, programmed, single FETs of euploid embryos performed at our academic center. These design features enhance the specificity of our study population and contribute to the novelty of our findings.

There were several limitations to the study. First, our study is inherently limited by its retrospective design. Compliance with medication protocols was assessed via patient reporting at scheduled care visits rather than utilising an objective measure such as pill counting. In addition, it is possible that patients in the control group were taking LDA without their physician’s knowledge; however, this is exceedingly unlikely due to our patients’ hesitancy to initiate therapy outside of their treatment protocols, as well as the multiple times during treatment that a patient’s medication list was collected and assessed. Due to our small sample size, we were unable to adjust for various potential confounders that may be relevant in clinical practice. Furthermore, our study was inadequately powered to detect small associations for our outcomes of interest (CPR and LBR) as reported in several prior RCTs performed among patients conceiving both with and without assistance.[1,2,3,4] The varying start times of LDA in our study are an additional limitation. Interestingly, prior studies indicating LDA may be beneficial also had variable LDA start times ranging from the 1^st day of the cycle through the day of FET.[3,4] Likewise, in unassisted conception cycles, some studies recommend starting LDA at the start of the menstrual cycle[1,2] while others show benefit only for women taking LDA at the time of implantation.[18] This LDA initiation heterogeneity, as well as the lack of a clear mechanism of action for LDA to improve outcomes, creates clinical uncertainty. Finally, to avoid outcome misclassification, we only included patients who delivered within our health system when assessing HDP which may result in selection bias for this outcome. Therefore, the HDP results may not fully reflect the experience of all patients in our study and are presented only to assist with hypothesis generation for future studies.

CONCLUSION

Our results demonstrated LDA was associated with neither CPR nor LBR among women undergoing autologous, programmed, FET of an euploid embryo. Both in vitro and in vivo human basic science research examining how aspirin affects human endometrial stromal cells is needed to guide the ideal start timing for LDA before future clinical studies are undertaken. Future clinical trials should randomise patients to treatment groups and ensure they are adequately powered.

Author contributions

EM: Writing-original draft, Writing-review and editing, Investigation, Data Curation, Conceptualisation. YC: Writing-review and editing, Investigation, Data Curation. PC: Writing-review and editing, Investigation, Data Curation. AA: Writing-original draft, Writing-review and editing, Data Curation, Formal Analysis. RG: Writing-review and editing, Supervision. RB: Writing-original draft, Writing-review and editing, Investigation, Conceptualisation, Supervision.

Disclaimer

The views and information presented are those of the authors and do not represent the official position of the U.S. Army Medical Center of Excellence, the U.S. Army Training and Doctrine Command, or the Department of the Army, Department of Defense, or U.S. Government.

Conflict of interests

E.M. is an active-duty member of the U.S. Army. Y.C. has nothing to disclose. P.C. has nothing to disclose. A.A. has nothing to disclose. R.G. is a peer reviewer for UpToDate and a Medical Expert at Roon. R.B. has nothing to disclose.

Data availability statement

The data underlying this article will be shared on reasonable request to the corresponding author.

Disclosure on use of Artificial Intelligence

Artificial Intelligence was not utilized in the composition of this manuscript.

Funding Statement

Nil.