Aspirin enhances endometrial decidualization markers in vitro among women with and without endometriosis

DIVA International provided the menstrual cups for the ROSE study.

Our results demonstrate that ASA enhances ESC decidualization markers and reduces ESC proliferation, without inducing significant cytotoxicity. There was no difference in the effects of ASA on ESC outcomes when comparing those cells obtained from women with vs without endometriosis. Note that limited numbers of endometriosis-ESCs were analyzed ( n = 4). Decidualization assays revealed enhanced levels of ESC decidualization markers IGFBP1 and PRL after ASA treatment at the doses studied ( Fig. 1A, B, C, D ). In particular, ASA (1 mM) pre-treatment of ESCs increased production of decidualization marker IGFBP1 compared to vehicle in both cAMP-treated (median = 1.69 (IQR: 1.39, 2.65), P = 0.002) and cAMP + MPA-treated (median = 1.59 (IQR: 1.24, 3.13), P = 0.0003) ESCs ( Fig. 1A and B ). The addition of ASA (2.5 mM) increased production of decidualization marker IGFBP1 compared to vehicle treatment in both cAMP-treated (median = 2.33 (IQR: 1.62, 3.09), P < 0.0001) and cAMP + MPA-treated (median = 1.57 (IQR: 1.18, 3.41), P = 0.0001) ESCs. Likewise, ASA-enhanced decidualization marker PRL at both 1 and 2.5 mM ((median = 1.45 (IQR: 1.13, 1.64), P = 0.0003) and (median 1.30 (IQR 1.08, 1.57), P = 0.002), respectively) for cAMP induction ( Fig. 1A and B ). Similarly, PRL production following cAMP + MPA stimulation also increased, supporting enhanced decidualization following ASA 1 and 2.5 mM pre-treatment ((median = 1.34 (IQR: 1.25, 1.50), P = 0.0001) and (median = 1.17 (IQR: 0.99, 1.41), P = 0.038), respectively) ( Fig. 1C and D ). There were no statistically significant differences between the responsiveness of participants’ ESCs to ASA regardless of disease state (control vs endometriosis) (see Supplementary Fig. 2A, B, C, D, E, F, G, H). The direct comparisons of the raw data for IGFBP1 and PRL values for vehicle vs ASA 2.5 mM on a per-subject basis show similar results (see Supplementary Fig. 3A, B, C, D, E, F, G, H). (A, B, C, D) Aspirin (ASA) enhances decidualization markers of endometrial stromal cells (ESCs). Treatment of ESCs with ASA (1–2.5 mM) prior to stimulation with either (A) cAMP alone or (B) cAMP + MPA enhances decidualization markers, as determined by IGFBP1 protein levels by ELISA when compared to vehicle (Veh)-treated ESCs. Treatment of ESCs with ASA (1–2.5 mM) prior to stimulation with either (C) cAMP alone or (D) cAMP + MPA enhances decidualization markers, as determined by PRL protein levels by ELISA compared to vehicle (Veh)-treated ESCs. Each dot represents data from ESCs isolated from one participant. Significance was determined by the Kruskal–Wallis test with post hoc Dunn’s multiple comparison test; P -values are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Although limited cytotoxicity was observed following ASA treatment of ESCs ( n = 10 per group), no significant cytotoxicity was found ( Fig. 2A and B ). As shown in Fig. 2A , Kruskal–Wallis testing for the three groups of ESCs treated with cAMP (vehicle, 1 mM ASA (median = 0.97 (IQR: 0.84, 1.06)), and 2.5 mM ASA (median = 0.83 (IQR: 0.74, 0.96))) did not reveal statistically significant results ( P = 0.07). Therefore, post hoc testing to identify specific differences among the three groups in Fig. 2A was not performed. Similarly, as shown in Fig. 2B for ESCs treated with cAMP + MPA, Kruskal–Wallis testing for the three groups (vehicle, 1 mM ASA (median = 1.00 (IQR: 0.88, 1.17)), and 2.5 mM ASA (median = 0.94 (IQR: 0.75, 1.06))) did not reveal statistically significant results ( P = 0.669). Thus, post hoc testing was not performed. (A and B) Effects of aspirin (ASA) on ESC cytotoxicity. Treatment of ESCs with ASA (1–2.5 mM) prior to stimulation with either (A) cAMP alone or (B) cAMP + MPA was not cytotoxic, as determined by neutral red cellular assay when compared to vehicle (Veh)-treated ESCs. Each dot represents data from ESCs isolated from one participant. The Kruskal–Wallis test was used to determine significance; P -values are shown. Note: post hoc testing was not performed in the absence of significant findings. Aspirin-treated ESCs showed no significant cytotoxicity vs vehicle treatment regardless of whether they were obtained from control ( n = 6) or endometriosis participants ( n = 4, a limited sample size) (see Supplementary Fig. 4A, B, C, D). Direct comparisons of the raw data for cytotoxicity for vehicle vs ASA 2.5 mM on a per-participant basis showed no significant cytotoxic effects (see Supplementary Fig. 5A, B, C, D). While ASA (1 mM) treatment of ESCs did not significantly affect their proliferation (median = 1.00 (IQR: 0.91, 1.06), P = 0.546), the higher dose of ASA (2.5 mM) significantly reduced proliferation compared to vehicle treatment (median = 0.75 (IQR: 0.70, 0.87), P = 0.001) ( Fig. 3 ). High-dose aspirin (ASA) reduces ESC proliferation. ASA treatment (2.5 mM) decreased proliferation of ESCs when compared to vehicle (Veh)-treated ESCs. There was no difference in proliferation of ESCs treated with ASA (1 mM) compared to vehicle (Veh)-treated ESCs. Each dot represents data from ESCs isolated from one participant. Significance was determined by the Kruskal–Wallis test with post hoc Dunn’s multiple comparison test; P -values are shown. ** P < 0.01. Although limited numbers of endometriosis-ESCs were tested ( n = 4), there were no statistically significant differences in proliferation at either dose of ASA regardless of disease state (control vs endometriosis) (see Supplementary Fig. 6A and B). See Supplementary Fig. 7, which provides direct comparisons of the raw proliferation data for vehicle vs ASA 2.5 mM on a per-subject basis; 2.5 mM ASA treatment significantly reduced ESC proliferation. Compared to vehicle treatment, ASA (2.5 mM) treatment significantly reduced AKT phosphorylation by ESCs by approximately 30% ( P = 0.03), as determined by western blotting ( Fig. 4 ). See Supplementary Fig. 8 for full blots and density calculations. By contrast, ASA (2.5 mM) treatment had no effect on AKT expression by ESCs ( Fig. 4 ). Neither COX-1 nor COX-2 expression by ESCs was detected. See Supplementary Fig. 9 for COX-1 and COX-2 full western blots, showing the absence of bands of expected molecular weight. (A, B, C) High-dose aspirin (ASA) reduces AKT phosphorylation without affecting total AKT expression. ASA treatment (2.5 mM) of control ESCs decreased AKT phosphorylation (p-AKT) when compared to vehicle (Veh)-treated ESCs, without altering total AKT expression. (A) Representative blots showing phospho-AKT (P-AKT), total AKT, and GAPDH (loading control) from ESCs obtained from two subjects. (B and C) Quantification of band densities for (B) p-AKT and (C) total (AKT). Each dot represents data from ESCs isolated from one participant ( n = 6 participants total). Significance was determined by the Wilcoxon signed-rank test for paired samples; P -values are shown.

8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (cAMP), medroxyprogesterone acetate (MPA), and acetylsalicylic acid (ASA or aspirin) were purchased from Sigma-Aldrich (USA). Women of reproductive age (22–40 years with a mean age of 32.3 years (SD: 5.5)) who were not pregnant, breastfeeding, or taking oral contraceptives and were menstruating and willing to provide ME samples were recruited and consented through the IRB- approved ROSE study at Northwell Health (13-376A, https://feinstein.northwell.edu/institutes-researchers/institute-molecular-medicine/robert-s-boas-center-for-genomics-and-human-genetics/rose-research-outsmarts-endometriosis ). All participants in this study provided written informed consent before donating their ME (including ME-derived cells that were isolated and cultured) for ongoing and future studies related to endometriosis and uterine health. Women with histologically confirmed endometriosis (determined following laparoscopic surgery and documented in a pathology report) were recruited and enrolled as ‘endometriosis’ participants. Control participants who self-reported no history suggestive of a diagnosis of endometriosis were recruited and enrolled. ME was collected, de-identified, and processed to culture and cryopreserve ME-derived ESCs, as previously described ( Warren et al. 2018 , Delenko et al. 2024 ). Briefly, participants collected their ME at home for 4–8 h on the day of their heaviest menstrual flow (typically day 1 or 2 of the cycle) using a menstrual cup provided by DIVA International. After collection, they shipped their ME overnight at 4°C to the laboratory for processing. De-identified ME samples (500 μL per T-75 flask) were plated in growth media (DMEM high-glucose media containing 10% MSC-FBS, Normocin (a novel antibiotic formulation that protects against mycoplasma, bacterial, and fungal contaminations), and penicillin–streptomycin–glutamine (PSQ)) to culture ME-ESCs. ME-ESC cultures were monitored over time by visualization under a light microscope and phenotyping as in Warren et al. (2018) . When mostly confluent, passage 0 and 1 ME-ESCs were cryopreserved. Formal sample size calculations were not performed as this is the first known study to examine the impact of ASA on ME-derived stromal cells, and there are no prior studies to inform effect size calculations. De-identified cryopreserved ME-derived ESCs (ME-ESCs) (passages 2–4) from 12 participants (8 control and 4 endometriosis) were defrosted, cultured, and expanded in growth media at 37°C/5%CO 2 , as previously described ( Warren et al. 2018 , Nayyar et al. 2020 , Delenko et al. 2024 ). Decidualization assays were initiated using confluent ME-ESC cultures collected from controls ( n = 8) and endometriosis participants ( n = 4). For each participant, 1.5 × 10 4 ME-ESCs in 200 μL of growth media (as described above) were plated per well in a 96-well plate and allowed to grow at 37°C/5%CO 2 until confluence (∼1–2 days). Media was replaced with decidualization media (DMEM high-glucose media containing 2% MSC-FBS, Normocin, and PSQ). The next day, confluent ME-ESCs were pre-treated with vehicle or ASA (1–2.5 mM) for 4 h. ME-ESCs were then stimulated with vehicle ( n = 3 wells per participant), 0.5 mM cAMP alone ( n = 3 wells per participant), or 0.5 mM cAMP + 10 −7 M MPA ( n = 3 wells per participant), as previously described ( Nayyar et al. 2020 , Delenko et al. 2024 ). After 48 h, culture supernatants were collected after brief centrifugation and cell-free supernatants were analyzed for decidualization markers, IGFBP1 or PRL, using Human IGFBP1 and PRL DuoSet ELISA Kits (R&D Systems®, USA), respectively, according to the manufacturer’s directions and as previously reported ( Warren et al. 2018 , Nayyar et al. 2020 , Delenko et al. 2024 ). All standards and samples were tested in triplicate (technical replicates) by ELISA following the manufacturer’s instructions. Immediately after adding the stop solution (Fisher Scientific), optical densities/absorbance readings were obtained at 450 nm/570 nm using an ELISA plate reader (MRX plate reader, Dynex/Dynatech), and values were extrapolated using IGFBP1 and PRL standard curves. After determining the average IGFBP1 and PRL protein values for each set of wells, data for cAMP-treated and cAMP + MPA-treated cells were normalized to fold-change using vehicle-treated decidualized cells as 1. ASA doses were chosen based on published studies using stromal or fibroblast cells or differentiation assays ( Sakurada et al. 1996 , Ricchi et al. 1997 , Li et al. 2017 , Funke et al. 2024 ), as well as a dose–response curve obtained by pre-treating ESCs (obtained from three participants) for 4 h prior to decidualization (assessed by IGFBP1 production 48 h later) as described above (Supplementary Fig. 1, see the section on Supplementary materials given at the end of the article). Neutral red uptake assays were used to assess potential cytotoxicity of ASA treatment of ME-ESCs obtained from participants with endometriosis ( n = 4) and without endometriosis ( n = 6), as previously described ( Repetto et al. 2008 ). Proliferation assays were initiated under basal (non-decidualization) conditions using ME-ESCs from 6 control and 4 endometriosis participants. For each participant, 1.5 × 10 3 ME-ESCs in 200 μL of growth media (as described above) were plated per well in a 96-well plate. ME-ESCs were incubated overnight at 37°C/5%CO 2 and then treated with either vehicle or ASA (1–2.5 mM). After 72 h, the cells were analyzed using the CyQUANT TM Cell Proliferation Assay Kit (Thermo Fisher, USA), according to the manufacturer’s instructions. All samples were tested in at least quadruplicate (technical replicates) for proliferation following the manufacturer’s instructions. Data were normalized to fold-change (cell number) using vehicle-treated cells as 1. ESCs (p3-4) from healthy controls were plated at 2.5 × 10 5 cells/mL (2 mL/well) in growth media in a 6-well plate (2 wells/condition). When confluent, growth media was replaced with decidualization media. The next day, ESCs were treated with either vehicle or ASA (2.5 mM). After 4 h, ESCs were washed with cold PBS and lysed with RIPA buffer containing protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher) and lysates were analyzed by western blotting, as in Delenko et al. (2024) and Delenko et al. (2025) . Briefly, pre-stained molecular weight standards or ESC lysates (35–40 μg protein/lane) were separated by electrophoresis, transferred onto Immobilon FL-PVDF membranes, and immunoblotted sequentially with primary antibodies: phospho-AKT (Ser473, S473) monoclonal, AKT polyclonal, COX-1 monoclonal, COX-2 monoclonal, and GAPDH monoclonal antibodies (1:1,000) (Cell Signaling Technology, USA), as per manufacturer’s instructions. Band densities were quantified using NIH ImageJ and normalized to GAPDH protein levels and to specific total protein where applicable (e.g. phospho-AKT:total AKT) on the same blots. GraphPad Prism version 10.2.3 (403) (GraphPad Software Inc., https://www.graphpad.com/scientific-software/prism/ ) was used for all statistical analyses. Because data did not meet the assumptions of normality and equal variance, non-parametric tests were employed. For data sets with three or more groups, we used the Kruskal–Wallis test (a non-parametric ANOVA equivalent), and for all significant results, post hoc testing was performed using Dunn’s multiple comparison test. For groups of two, Mann–Whitney U tests were used. P < 0.05 was considered significant. For western blotting data, groups were compared using the Wilcoxon signed-rank test, a non-parametric test for paired samples.

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

This is the first study reporting the effect of ASA on human ESC decidualization. Our results support that ASA pre-treatment enhances ESC decidualization, as evidenced by the production of two decidualization markers (IGFBP1 and PRL), and reduces ESC proliferation, without inducing significant cytotoxicity. Although limited numbers of endometriosis-ESCs were analyzed, comparable results were observed in ESCs obtained from women with and without endometriosis. Mechanistic studies support that ASA reduces AKT phosphorylation or AKT signaling, consistent with the inhibition of AKT activation observed during decidualization ( Lee et al. 2016 , Fabi et al. 2017 ). Although historically, ASA has been utilized as an adjunct in early pregnancy, data are mixed regarding its effectiveness ( Siristatidis et al. 2016 , Wang et al. 2017 , Glujovsky et al. 2020 ). ASA use is recommended during early pregnancy for many patients to reduce the risk of preeclampsia (ACOG Committee Opinion No. 743. 2018 ). One study supports the initiation of ASA prior to conception to improve pregnancy outcomes ( Naimi et al. 2021 ). This randomized trial demonstrated that preconception ASA treatment was associated with an increased live birth rate and reduced pregnancy loss among a cohort of women with a history of pregnancy loss ( Naimi et al. 2021 ). However, the mechanism(s) by which ASA treatment may improve pregnancy outcomes remains unknown. Likewise, a systematic review and meta-analysis of randomized controlled trials demonstrated that ASA use in IVF ( in vitro fertilization)/ICSI (intracytoplasmic sperm injection) cycles was associated with improved clinical pregnancy rates ( Wang et al. 2017 ). However, other studies have not yielded similar findings ( Davar et al. 2020 , He et al. 2023 ). Despite these reports, mechanistic studies examining how ASA affects human ESCs, in vitro and in vivo , are lacking. In humans, decidualization is an essential, well-regulated differentiation process that precedes successful implantation ( Okada et al. 2018 ), and this process is accompanied by reduced ESC proliferation along with reduced AKT phosphorylation (also known as AKT signaling) ( Lee et al. 2016 , Fabi et al. 2017 ). Decidualized ESCs contribute to the microenvironment at the endometrial–trophoblast interface and play multiple essential roles in the complex interaction between the endometrium and the attaching embryo ( Okada et al. 2018 ). Although improper decidualization has been reported in the settings of infertility and recurrent pregnancy loss ( Teklenburg et al. 2010 , Okada et al. 2018 , Dambaeva et al. 2020 ), endometriosis ( Nayyar et al. 2020 ), and preeclampsia ( Garrido-Gomez et al. 2017 ) and reduced decidualized cells at the implantation site are known to predispose to early pregnancy failure ( Muter et al. 2021 ), this process is still incompletely understood, and much remains unknown. The majority of prior studies of human ESCs have relied on invasive endometrial biopsies for collecting ESCs, and this procedure has been mainly restricted to those patients undergoing biopsies for uterine-related conditions or via hysterectomies, many of which occur outside the reproductive years. Thus, little is known about the functional responses of ESCs in healthy women. Our ‘population-based’ approach using ME-derived ESCs is non-invasive and significantly expands the collection of ESCs to include most menstruating participants with or without uterine health conditions and provides larger samples (and hence, larger yields of ESCs for experiments) than typical endometrial biopsies. Our results indicate significant increases in decidualization markers, IGFBP1 and PRL, following treatment with ASA (1–2.5 mM). IGFBP1 and PRL are two of the most well-established and commonly used biomarkers of human ESC decidualization in vitro ( Gellersen & Brosens 2014 , Okada et al. 2018 , Rodriguez-Caro et al. 2019 ); their relative concentrations measured in the culture supernatants of decidualizing ESCs represent the functional cell-based secretion of nutritive growth factors by decidualizing ESCs required for human embryo implantation and successful pregnancy ( Gellersen & Brosens 2014 ). Assessment of both IGFBP1 and PRL added rigor and certainty to our results. Given previously published findings that, in decidualizing ESCs, IGFBP1 production increases more rapidly than PRL production ( Fluhr et al. 2006 ), it was anticipated that IGFBP1 results may be more robust compared to PRL results given the timing of decidualization marker assessment (48 h after cAMP ± MPA). In addition, our proliferation assay results revealed a statistically significant reduction in ESC proliferation when treated with the higher dose of ASA (2.5 mM, P = 0.001). Evidence supports the reciprocal relationship between cell proliferation and cell differentiation and that cell cycle arrest accompanies cellular differentiation ( Wille & Scott 1986 , Rogers & Abberton 2003 , Kolly et al. 2005 , Logan et al. 2012 ). Accordingly, many factors or agents that reduce ESC proliferation ( Das 2009 , Mestre Citrinovitz et al. 2020 , Lyu et al. 2023 , Delenko et al. 2024 ) promote ESC decidualization (e.g., quercetin). By contrast, inhibitors of decidualization may promote ESC proliferation ( Le et al. 2017 ). High-dose ASA (2.5 mM) significantly inhibited proliferation and showed limited effects on cell viability following cAMP stimulation ( Fig. 2A ). However, the effect on viability was not significant. Moreover, high-dose ASA (2.5 mM) had no significant cytotoxic effect on ESCs treated with cAMP + MPA ( Fig. 2B ), which better reflects the in vivo decidualization response ( Doi-Tanaka et al. 2024 ). There is emerging evidence that endometrial compaction, which can be detected by ultrasound during the secretory phase when decidualization occurs, may predict successful implantation, although data on live birth outcomes remain lacking ( Al-Lamee et al. 2024 ). Our study demonstrated significant increases in decidualization markers following pre-treatment of ESCs with ASA (1–2.5 mM), which could become clinically relevant if replicated in vivo . This assessment of the in vivo effects of ASA on ME-derived ESCs is possible in a clinical study where ESCs are collected and analyzed for decidualization response ex vivo pre- and post-ASA administration in vivo , along with assessment of ASA or its metabolites in ME (representative of the local uterine environment). Regarding the mechanisms underlying the deciduogenic activity of ASA, ASA irreversibly inhibits both COX-1 and COX-2 enzyme activity, with stronger affinity for COX-1 inhibition ( Mitchell et al. 1993 , Qureshi & Dua 2024 ). PGI 2 and PGE 2 production are dependent upon arachidonic acid cleavage by COX-2 ( Gnecco et al. 2019 ), and prostaglandin secretion by ESCs promotes enhanced responsiveness to progesterone via cAMP activation, resulting in increased decidualization ( Gnecco et al. 2019 ). A prior study noted decreased decidualization in vivo when using celecoxib to block COX-2 expression in rats ( Sookvanichsilp & Pulbutr 2002 ). Although other studies have demonstrated COX-1 and COX-2 activity in human ESCs ( Shaw et al. 1994 , Wang et al. 2006 ), we were unable to detect protein expression by western blotting analysis, which precluded further assessing preferential inhibition of COX-1 (or COX-2) protein expression as a mechanism of action to explain our results. ASA has been shown to lead to loss of AKT phosphorylation in both rodent ( Liu et al. 2017 ) and human cell lines ( Cho et al. 2013 , Chen et al. 2020 , Peng et al. 2023 ). Interestingly, ASA-induced loss of AKT phosphorylation occurs in a COX-1-dependent manner in human ovarian cancer cells, which are known to express higher levels of COX-1 rather than COX-2 ( Cho et al. 2013 ). Because our study is the first to examine the impact of ASA on ESC decidualization markers, we hypothesize that the effect of ASA on decidualization may be due to the inhibition of COX-1 and subsequent reduced AKT phosphorylation as described by numerous prior studies ( Cho et al. 2013 , Liu et al. 2017 , Chen et al. 2020 , Peng et al. 2023 ). However, we were unable to detect COX-1 or COX-2 protein expression by ESCs under our culture conditions. Although the mechanism of action for ASA’s impact on ESCs was not the primary focus of our study, our western blotting analysis of signaling proteins implicated in decidualization demonstrated loss of AKT phosphorylation after exposure to ASA. COX-1 inhibitors can also lead to loss of AKT phosphorylation ( Altintop et al. 2023 ), and loss of AKT phosphorylation reduces the expression of collagenI ( Bujor et al. 2008 ). ASA has been shown to reduce expression of collagen 1A1, α-smooth muscle actin (αSMA), and fibronectin and inhibit endometrial fibrosis ( Zhang Z et al. 2020 ) and improve endometrial thickness in some studies ( Lebovitz & Orvieto 2014 , Li et al. 2023 ); however, other studies report no such effects ( Davar et al. 2020 ). Interestingly, COL1A1 mRNA expression is increased in ME-derived ESCs derived from patients with endometriosis when compared to ESCs collected from unaffected controls ( Shih et al. 2022 ). Prior studies demonstrated that downregulation of phospho-Ser473-AKT (loss of AKT signaling) promotes decidualization ( Fabi et al. 2017 ), while persistent expression of phospho-Ser473-AKT inhibits decidualization ( Yin et al. 2012 ). Based on these and other studies showing that loss of AKT phosphorylation enhances decidualization ( Delenko et al. 2024 , 2025 ), our data support that ASA promotes loss of AKT phosphorylation, and this is accompanied by enhanced decidualization biomarker expression. Our findings are consistent with recent studies describing the pro-deciduogenic activity of quercetin mediated through the AKT signaling pathway ( Park et al. 2019 , Delenko et al. 2024 , 2025 ). This study has several notable strengths, including utilizing primary human ESCs and measuring both IGFBP1 and PRL for assessing decidualization, analyzing the outcome measures in primary ESCs obtained from both unaffected controls and women with endometriosis, and being the first of its kind. In addition, our western blot analysis suggests that ASA (2.5 mM) inhibits AKT phosphorylation to promote ESC decidualization. Limitations of our study include the use of an in vitro model system, relatively small sample sizes (which may include some heterogeneity in responses among participants in our study), limited knowledge of participants’ reproductive/fertility and medical histories, and not fully elucidating the underlying mechanism(s) of action. Finally, although considered hallmark features of decidualization, relying on two of the most well-established markers (IGFBP1 and PRL) at the 48 h time point may not fully capture the complete process of decidualization. Despite these limitations, this study is the first to report the enhancing effects of ASA on ESC decidualization markers and the inhibitory effects of ASA on ESC proliferation. Future research should include a larger sample size, include healthy controls and endometriosis patients, including infertility patients with defined fertility and reproductive histories, and aim to further elucidate how ASA regulates AKT phosphorylation in ESCs to better define the mechanism(s) of action of ASA. In conclusion, our results support that ASA, at 1 and 2.5 mM, significantly enhanced decidualization biomarker expression by human ESCs without inducing significant cytotoxicity. In addition, high-dose ASA (2.5 mM) significantly reduced ESC proliferation and reduced AKT phosphorylation. These findings are consistent with prior studies showing that stromal cell cycle arrest is associated with decidualization ( Logan et al. 2012 ), and reduced AKT signaling positively regulates decidualization ( Fabi et al. 2017 ), while overactivation of AKT signaling disrupts decidualization ( Yin et al. 2012 ). We observed no difference in ESC responsiveness based on control vs endometriosis status of the participants. Because endometriosis has been associated with impaired decidualization ( Klemmt et al. 2006 , Minici et al. 2008 , Warren et al. 2018 , Nayyar et al. 2020 ), further in vitro studies assessing ASA’s impact on ESCs are warranted among this patient population. In addition, while our western blot analysis suggests that ASA may act through the AKT pathway to affect decidualization, further research into the precise mechanism of action using pharmacological inhibition or genetic manipulation is warranted. Before utilizing ASA as an adjunct to improve fertility and reproductive outcomes in patients with endometriosis and infertility, future studies are required to explore the effects of ASA in vivo at doses typically utilized in clinical practice. Our results should prompt further mechanistic investigations into ASA’s effect on ESC function and ASA’s potential to enhance decidualization and improve fertility and pregnancy outcomes.

Decidualization of endometrial stromal cells (ESCs) is an essential, well-regulated differentiation process that precedes the successful implantation of a human embryo ( Gellersen & Brosens 2014 , Okada et al. 2018 ). Improper decidualization has been reported in infertility and recurrent pregnancy loss ( Teklenburg et al. 2010 , Gellersen & Brosens 2014 , Okada et al. 2018 , Dambaeva et al. 2020 ), endometriosis ( Barragan et al. 2016 , Warren et al. 2018 , Nayyar et al. 2020 ), and preeclampsia ( Garrido-Gomez et al. 2017 ). Reduced decidualization at the implantation site predisposes to early pregnancy failure ( Muter et al. 2021 ). In 2023, the European Society of Human Reproduction and Embryology prioritized research into decidualization as a therapeutic target for implantation failure ( ESHRE Working Group on Recurrent Implantation Failure 2023 ). Endometriosis is associated with both infertility and defective endometrial decidualization ( Barragan et al. 2016 , Warren et al. 2018 , Nayyar et al. 2020 , Liao et al. 2024 ). Multiple regulatory pathways have been implicated in the impaired decidualization response observed in endometriosis ( Cai et al. 2022 ). Loss of AKT phosphorylation is an essential aspect of ESC decidualization ( Fabi et al. 2017 ). Higher AKT phosphorylation has been reported in human ESCs obtained from women with endometriosis compared to unaffected controls ( Cinar et al. 2009 ). Increased AKT phosphorylation in the setting of endometriosis has been implicated in endometrial dysregulation ( Lee & Kim 2014 ). Aberrant endometrial COX-2 expression has also been reported in endometriosis ( Teague et al. 2010 ). COX-1 and COX-2 have both been implicated in proper decidualization with COX-1 levels decreasing during decidualization ( St-Louis et al. 2010 ). Aspirin (ASA), an inhibitor of COX-1 and COX-2, is safe when taken prior to conception and may increase implantation and live birth in certain patients ( Connell et al. 2017 , Naimi et al. 2021 ). However, administration of ASA in assisted reproduction remains controversial ( Siristatidis et al. 2016 , Wang et al. 2017 , Glujovsky et al. 2020 ). It is hypothesized that ASA improves uterine and ovarian blood flow ( Rubinstein et al. 1999 ) and inhibits thromboxane synthesis by inhibiting the COX-1 pathway ( Walsh & Strauss 2021 , Boelig et al. 2024 ), thereby preventing thrombosis in placental vasculature ( Fishman et al. 1995 ) and reducing placental vasculopathy ( Boelig et al. 2024 ). COX-1 inhibitors can also lead to loss of AKT phosphorylation ( Altintop et al. 2023 ). ESC differentiation into decidual stromal cells that secrete critical growth factors, including IGFBP1 and PRL, is proposed to be the essential component of decidualization ( Gellersen & Brosens 2014 , Ng et al. 2020 ). Much research has focused on the effects of ASA therapy on early pregnancy and trophoblast cells ( Bose et al. 2005 , Panagodage et al. 2016 , Ren et al. 2023 ) and later pregnancy ( ACOG Committee Opinion No. 743, 2018 ). Pre-conception initiation of ASA has been shown to increase live births and reduce pregnancy loss among women with a history of pregnancy loss ( Naimi et al. 2021 ). Nonetheless, there remains a paucity of research on the impact of ASA on ESCs and their function. This study is the first to examine the effect of ASA pre-treatment on ESC decidualization among participants with and without endometriosis.

ERM is an active-duty member of the US Army. RHG is a peer reviewer for UpToDate and a Medical Expert at Roon. XX, PC, NH, RAB, PKG and CNM have nothing to disclose.

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

ERM conceived the study, curated the data, performed formal analysis and investigation, and wrote, reviewed, and edited the manuscript. XX, PC, and H curated the data, performed investigation, and wrote, reviewed, and edited the manuscript. RAB, RHG, PKG supervised the study and wrote, reviewed, and edited the manuscript. CNM conceived the study, designed the methodology, curated the data, performed formal analysis and investigation, supervised the study, and wrote, reviewed, and edited the manuscript.