Abstract

Backgrounds

   Aspirin has been shown to enhance endometrial receptivity (ER) during
   the window of implantation in patients with polycystic ovary syndrome
   (PCOS). However, the underlying mechanisms remain unclear. This study
   aimed to elucidate the mechanisms by which aspirin improves ER
   through metabolic analysis of uterine lavage fluid.

Methods

   A PCOS rat model was established using letrozole. Body weight and
   estrous cycles were monitored, and the number of implanted embryos was
   assessed across groups. We evaluated endometrial ultrastructure,
   ovarian and endometrial histomorphometry. Serum levels of
   estradiol(E[2]) and progesterone(P)were measured. Moreover, through
   ultra-performance liquid chromatography-mass spectrometry, the study of
   uterine lavage fluid metabolites revealed the potential mechanism of
   action of aspirin.

Results

   Compared with the model group, aspirin treatment significantly
   increased embryo implantation rates, improved endometrial morphology
   and hormone levels. Metabolomic analysis identified 48 differential
   metabolites, among which five—2, 6-dihydroxypurine, gluconolactone,
   Oxaceprol, PC (18:1/18:1), and PC (20:3e/17:1)—were identified as
   potential biomarkers for aspirin-mediated improvement of ER in PCOS
   rats. Pathway analysis revealed that aspirin primarily modulates the
   pentose phosphate pathway, arginine and proline metabolism, and
   glycerophospholipid metabolism.

Conclusions

   Aspirin may enhance glucose metabolism, alleviate insulin resistance,
   promote angiogenesis, and improve vascular permeability and endometrial
   receptivity. These effects are likely mediated through the regulation
   of biomarkers involved in the pentose phosphate pathway, arginine and
   proline metabolism, and glycerophospholipid pathways in uterine lavage
   fluid.

Introduction

   Polycystic ovarian syndrome (PCOS), a prevalent reproductive endocrine
   disorder affecting 10–13% of women in their reproductive age, is
   characterized by hyperandrogenemia, ovulatory dysfunction, polycystic
   ovarian morphology, and often by associated metabolic disorders
   [[32]1]. As is known to all, endometrial receptivity of PCOS was
   regarded as the one of the causes of low pregnancy rate and high
   abortion rate [[33]2]. Despite the high prevalence of PCOS,
   pharmacologic interventions for such a complicated syndrome encounter
   substantial challenges. There are limited options available to improve
   endometrial receptivity in patients with PCOS. Therefore, proven
   therapeutic strategies are urgently needed.

   Aspirin, a widely utilized nonsteroidal anti-inflammatory drug (NSAID)
   [[34]3]. Low-dose aspirin has been employed for its anti-inflammatory,
   vasodilatory, and platelet aggregation-inhibitory effects to enhance
   blood flow, support fertility, and improve the success rates of in
   vitro fertilization (IVF) therapy. These characteristics, combined with
   its low cost, widespread availability, and favorable safety profile at
   low doses, render aspirin a promising adjunct for women undergoing
   ovulation induction therapy [[35]4]. Numerous studies have demonstrated
   that low-dose aspirin significantly enhances uterine perfusion and
   improves pregnancy outcomes in IVF patients by promoting ER
   [[36]5,[37]6]. Therefore, we regard that aspirin may be a potential
   therapeutic agent for improving ER. However, the specific mechanisms
   through which aspirin modulates ER, especially in PCOS, remain poorly
   understood.

   Recent studies suggest that uterine fluid collection may be proposed to
   become a less-invasive routine practice compared to endometrial biopsy,
   avoiding the risk of endometrial damage [[38]7]. Moreover, the
   relationship between uterine lavage fluid metabolites and ER in PCOS
   models remains under-explored. Therefore, in this study, we performed
   an un-targeted metabolomic analysis using ultra-high-performance liquid
   chromatography-tandem mass spectrometry (UHPLC-MS/MS) to investigate
   the effects of aspirin on uterine lavage fluid metabolites in PCOS rats
   during the window of implantation. Our primary objective was to
   identify potential biomarkers associated with aspirin-induced
   improvement in endometrial receptivity (ER) and to elucidate the
   underlying metabolic pathways involved. Based on our findings, we
   hypothesized that aspirin improves ER in PCOS rats by modulating key
   metabolic pathways, such as the pentose phosphate pathway, arginine and
   proline metabolism, and glycerophospholipid metabolism, thereby
   enhancing endometrial morphology, angiogenesis, and pinopodes
   development, and consequently, promotes embryo implantation. Thereby
   providing a foundational understanding of aspirin's role and underlying
   mechanisms in improving ER.

Materials and methods

Chemicals and reagents

   Letrozole (211026KL) was provided by Jiangsu Hengrui Medical Co., Ltd.
   (Jiangsu, China). Aspirin enteric-coated tablets (210503) from Hunan
   Xinhui Pharmaceutical Co. Wright staining (G1040) was purchased from
   Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China).
   Hematoxylin and eosin (HE) staining solution (ZLI-1040、ZLI-9610) was
   bought from Beijing Zhongshan Jinqiao Biotechnology Co. (Beijing,
   China). The standards of HPLC grade ultra-pure water, methanol, formic
   acid, and ammonium acetate (7732-18-5, 67–561, 64-18-6, 631-61-8) were
   bought from Thermo Fisher (USA). Estradiol (E[2]) (ML-E202302731) and
   progesterone (P) (ML-E202302225) of the enzyme-linked immunosorbent
   assay (ELISA) kits were all acquired from Shanghai Fusheng Industrial
   Co. Ltd. (Shanghai, China). The electron microscope fixative and PBS
   were purchased from Wuhan Xavier Biotechnology Co.

Animals and induction of PCOS

   Eighteen female Sprague Dawley (SD) rats (weight 190-210g, 6–8 weeks
   old) and nine sexually mature male SD rats (weight 340-360g, 13–15
   weeks old) were brought from Hunan SJA Laboratory Animal Co, Ltd.
   (Animal certificate number: SCXK (Xiang) 2019–0004). All rats were
   maintained in a light (06:00–18:00)-dark (18:00–6:00) cycle with free
   food. The control temperature was 22 °C ± 2 °C, and humidity was
   50 ± 10% in the Laboratory Animal Center of Hunan University of Chinese
   Medicine. All animal experiments were approved by the Ethics Committee
   of Hunan University of Chinese Medicine (ethics approval number:
   LL2022031603). The disposal of animals complied with The Guiding
   Opinions on Treating Laboratory Animals Kindly published by the
   Ministry of Science and Technology of China.

   The rats were acclimatized and fed for 7 days. Vaginal smears of the
   rats were taken to determine the estrous cycle at 7:00–8:00 a.m. daily
   [[39]8]. Eighteen rats were divided into a control group (n = 6) and a
   model group (n = 12). The control group received a normal diet, whereas
   the model group received letrozole (1 mg kg^−1 day^−1, dissolved in
   0.5% carboxymethyl cellulose (CMC-Na) for 21 consecutive days to
   establish the PCOS model [[40]9]. The rats showed disturbed, prolonged
   or even stagnant estrous cycle, and the histopathological section of
   the ovary showed polycystic changes, suggesting that the modeling was
   successful. The PCOS rats were divided into the model group (n = 6) and
   the Aspirin group (n = 6). The rats in the aspirin group were given
   intragastric administration of aspirin (8 mg kg^−1 day^−1, dissolved in
   0.5% CMC-Na). Meanwhile, the rats of the control and model groups were
   given the same volume of saline as the Aspirin group rats based on
   their individual body weight.

Experimental design

   Pharmacological intervention in rats in the aspirin group lasted for 12
   days. On the day of the last drug administration, the cages were
   combined according to the ratio of male to female of 2:1. Every
   morning, female rats were observed for vaginal plugs and vaginal smears
   for spermatozoa, with the observation of vaginal plugs or vaginal
   smears for spermatozoa recorded as day 1 of gestation, and the samples
   were taken on day 5 of gestation. Deep anesthesia was induced by
   intraperitoneal injection of 2% sodium pentobarbital to ensure that the
   rats remained pain-free throughout the procedure. The depth of
   anesthesia was assessed by the absence of corneal reflexes and pain
   stimulus responses. After confirming complete anesthesia, euthanasia
   was performed by exsanguination via abdominal aorta puncture. Blood was
   collected until confirmed cardiac arrest occurred, which was verified
   by cessation of heartbeat and respiration for at least 5 minutes.Blood
   samples were centrifuged at 3000 r/min for 15 minutes, and the
   supernatants were obtained for sex hormone level tests [[41]10].
   Histopathology was performed on one side of each rat's ovaries and
   treated with a 4% paraformaldehyde buffer [[42]11]. The uterus was
   separated bilaterally and the bicornuate uterus was removed. Repeatedly
   rinse the uterine cavity with 2 ml of sterilized ultra-pure water and
   the fluid was collected and placed in a lyophilization tube. The
   samples were stored in liquid nitrogen and finally in the –80°C
   refrigerator. The uterus was dissected, and the number of follicle
   implantation was recorded. One fourth of the uterine tissue was fixed
   in 4% paraformaldehyde, embedded in paraffin, and sliced for HE
   detection [[43]11]. Another 5 mm^3 of uterine tissue was fixed in the
   electron microscope fixative for scanning electron microscopy [[44]12].

Detection of uterine lavage fluid metabolites by mass spectrometry

Sample pretreatment.

   Remove the sample of uterine lavage fluid from the refrigerator at
   -80°C, thaw at 4°C. Uterine lavage fluid (100 μL) was placed in the
   Eppendorf (EP) tubes and re-suspended with pre-chilled 80% methanol
   (400 μL) by the well vortex. After 5 min on ice, the samples were
   centrifuged at 15,000 × g, 4 °C for 20 mins. The supernatant (400 μL)
   was diluted to a final concentration containing 53% methanol by LC-MS
   grade water. The samples were transferred to a fresh EP tube and
   centrifuged at 15,000 × g, 4°C for 20 mins. Finally, the supernatant
   was injected into the LC-MS/MS system. The quality control (QC) sample
   was made by combining equal quantities of each sample and 30 μL of
   supernatant as part of the quality control and system conditioning
   process [[45]13].

UHPLC-MS/MS conditions.

   The UHPLC separation was performed using a Vanquish UHPLC system
   (Thermo Fisher, Germany) equipped with a Hypersil GOLD column
   (100 × 2.1 mm, 1.9 μm). The positive polarity mode eluents consisted of
   0.1% formic acid in water (A) and methanol (B). The eluents of the
   negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0)
   and eluent B (methanol). Gradient elution of liquid chromatography at
   the flow rate of 0.2 mL/min was set as follows: 2% B, 1.5 min; 2–85% B,
   3 min; 85–100% B, 10 min; 100–2% B, 10.1 min; and 2% B, 12 min. AQ
   Exactive TM HF-X mass spectrometer (Thermo Fisher, Germany) provided
   with an electrospray ionization source was used to obtain the mass
   spectrometric data. Ion source parameters were as follows: spray
   voltage of 3.5 kV, capillary temperature of 320 °C, sheath gas flow
   rate of 35 psi, aux gas flow rate of 10 L/min, S-lens RF level of 60,
   and aux gas heater temperature of 350°C. Polarity: positive, negative.
   negative; MS/MS secondary scans were data-dependent scans [[46]14].

Multivariate data processing and analysis.

   Compound Discoverer 3.1 (CD3.1, Thermo Fisher) was used to align,
   identify, and quantify metabolites from UHPLCMS/MS data. These
   metabolites were annotated using the Kyoto Encyclopedia of Genes and
   Genomes (KEGG) database ([47]https://www.genome.jp/kegg/pathway.html)
   and the Human Metabolome Database (HMDB)
   ([48]https://hmdb.ca/metabolites). For the multivariate statistical
   analysis part, the data were transformed using metabolomics data
   processing software metaX and then subjected to principal component
   analysis (PCA) and partial least squares discriminant analysis (PLS-DA)
   to obtain the VIP value for each metabolite [[49]15]. We applied
   univariate analysis (t-test) to calculate the statistical significance
   (P-value). A threshold value of VIP > 1.0 and P < 0.05 was set for
   screening the metabolite, in which VIP values stand for variable
   influence on projection (VIP) and P-values represent statistical
   significance [[50]16]. Statistical analyses were performed using the
   statistical software R (R version R-3.4.3), Python (Python 2.7.6
   version), and CentOS (CentOS release 6.6). When data were not normally
   distributed, normal transformations were attempted using the area
   normalization method.

Statistical analysis

   All data were expressed as mean ± Standard Deviation (SD) and analyzed
   by SPSS 27.0 statistical analysis software. The one-way ANOVA was used
   for multiple group comparisons, and the least significant difference
   method LSD test was used for two-by-two comparisons; for non-normally
   distributed data, Kruskal-Wallis test was used A value of P < 0.05 is
   considered statistically significant. Graphs were generated using
   GraphPad Prism 9.5.0 software.

Results

Determination of PCOS rat model

   The pre-motility period (proestrus, P) was dominated by expanded
   oval-shaped nucleated epithelial cells; the estrus period (estrus, E)
   was dominated by irregularly shaped anucleate keratinized cells; the
   metestrus period (metestrus, M) was characterized by leukocytes,
   nucleated epithelial cells and keratinized cells in comparable
   quantities; and the inter-estrus period (diestrus, D) was mainly
   dominated by leukocytes. The results of estrous cycle monitoring showed
   that the rats in the control group had a regular estrous cycle, while
   the rats in the model group had a disorganized estrous cycle with a
   prolonged cycle, or even stagnation persisting in the inter-estrous
   phase, and the estrous cycle lost its regularity ([51]Fig 1). Sections
   of ovarian tissue showed that in the control group, the oocyte
   structure was intact and clear, the granulosa cells were multilayered,
   tightly and neatly arranged, and multiple corpus luteum and follicles
   at all levels were visible; in the model group, a large number of
   cystic follicles were visible in the ovary, the number of oocytes and
   corpus luteum was significantly reduced, and the number of granulosa
   cell layers was reduced and loosely arranged ([52]Fig 2). The rats
   showed disturbed, prolonged or even stagnant estrous cycle, and the
   histopathological section of the ovary showed polycystic changes,
   suggesting that the modeling was successful.

Fig 1. Changes in the estrous cycle of PCOS rats (Wright’s staining, scare
bar = 100 μm).

   [53]Fig 1
   [54]Open in a new tab

   (a–d) Vaginal smears corresponding to different phases of the estrous
   cycle: (a) Proestrus, (P) was dominated by expanded oval-shaped
   nucleated epithelial cells (black arrow); (b) Estrus (E), estrus was
   dominated by irregularly shaped anucleate keratinized cells(red arrow);
   (c) Metestrus (M), metestrus was characterized by leukocytes(green
   arrow), nucleated epithelial cells (black arrow) and keratinized
   cells(red arrow) in comparable quantities; (d) Diestrus (D), diestrus
   was mainly dominated by leukocytes(green arrow).

Fig 2. Morphological changes in ovarian tissue of PCOS rats.

   [55]Fig 2
   [56]Open in a new tab

   (a, c) Control group, the oocyte structure was intact and clear, the
   granulosa cells were multilayered, tightly and neatly arranged, and
   multiple corpus luteum and follicles at all levels were visible; (b, d)
   Model group, a large number of cystic follicles were visible in the
   ovary, the number of oocytes and corpus luteum was significantly
   reduced, and the number of granulosa cell layers was reduced and
   loosely arranged. The lower panels (HE staining, scare bar = 200μm)
   show magnified views of the boxed regions in the upper panels (HE
   staining, scare bar = 500μm).

Effect of aspirin on blastocyst implantation in PCOS rats during the window
of implantation

   The number of implanted blastocysts was significantly reduced in the
   model group compared to the control group (P < 0.01). In contrast,
   aspirin treatment led to a significant increase in the number of
   implanted blastocysts compared to the model group (P < 0.05). These
   findings suggest that aspirin effectively enhances blastocyst
   implantation in PCOS rats during the window of implantation ([57]Fig
   3).

Fig 3. Effect of aspirin on the number of blastocysts implanted in PCOS rats
during the window of implantation.

   Fig 3
   [58]Open in a new tab

   (n = 6). **P ＜ 0.01 compared with the control group, and
   ^#P ＜ 0.05compared with the model group.

Effect of aspirin on serum E[2] and P levels in PCOS rats during the window
of implantation

   Serum levels of estradiol (E[2]) and progesterone (P) were
   significantly lower in the model group compared to the control group
   (P < 0.01). In contrast, aspirin treatment significantly increased the
   levels of E[2] and P compared to the model group (P < 0.01), suggesting
   that aspirin effectively promotes the elevation of E[2] and P levels in
   the serum of PCOS rats ([59]Fig 4).

Fig 4. Effect of aspirin on serum E[2] and P levels in PCOS rats during the
window of implantation.

   [60]Fig 4
   [61]Open in a new tab

   (n = 4). (a) Serum E[2] level; (b) Serum P level. **P ＜ 0.01 compared
   with the control group, and ^##P ＜ 0.01 compared with the model group.

Effects of aspirin on endometrial histomorphology in PCOS rats during the
window of implantation

   Compared to the control group, the model group exhibited significantly
   reduced endometrial thickness, along with a marked decrease in the
   number of endometrial glands and blood vessels (P < 0.01). In contrast,
   aspirin treatment significantly increased endometrial thickness and the
   number of endometrial glands and blood vessels compared to the model
   group (P < 0.01). These findings suggest that aspirin effectively
   enhances endometrial thickness, promotes glandular development, and
   stimulates angiogenesis ([62]Figs 5–[63]7).

Fig 5. Effect of aspirin on endometrial histomorphometry in PCOS rats during
the window of implantation.

   [64]Fig 5
   [65]Open in a new tab

   (n = 5). (HE staining, scare bar = 200μm). (a) Control group; (b) Model
   group; (c) Aspirin group.

Fig 7. Effect of aspirin on endometrial histomorphology in PCOS rats during
the window of implantation.

   [66]Fig 7
   [67]Open in a new tab

   (n = 5). (a) Endometrial thickness; (b) Number of endometrial glands;
   (c) Number of endometrial blood vessels. **P ＜ 0.01 compared with the
   control group, and ^##P ＜ 0.01 compared with the model group.

Fig 6. Effect of aspirin on endometrial histomorphometry in PCOS rats during
the window of implantation.

   [68]Fig 6
   [69]Open in a new tab

   (n = 5). (HE staining, scare bar = 50μm). (a) Control group, the
   endometrial stroma is loose, with synchronous development of glands and
   stroma, abundant glands, and a rich vascular supply; (b) Model group,
   endometrial glandular hypoplasia is observed, characterized by small
   glandular lumina and a reduced number of glands, along with a decrease
   in vascularity; (c) Aspirin group, endometrial development shows
   improvement, manifested by larger glandular lumina, loose stroma, and
   an increased number of glands and vessels within the stroma. Black
   arrows indicate glands, and red arrows indicate blood vessels.

Effect of aspirin on the expression of pinopodes in PCOS rats during the
window of implantation

   Scanning electron microscopy was used to observe changes in the
   morphology and number of pinopodes in the endometrium of PCOS rats. In
   the control group, the endometrial structure appeared normal and
   intact, with numerous pinopodes and abundant, uniformly distributed
   microvilli. In contrast, the model group exhibited severe endometrial
   damage, with a significant reduction in the expression of pinopodes.
   Most of these synapses were severely crumpled and collapsed, and
   microvilli were sparse and unevenly distributed. Compared to the model
   group, the aspirin group demonstrated partial repair of endometrial
   damage, with an increased expression of pinopodes and more abundant,
   evenly distributed microvilli. These findings suggest that aspirin can
   improve the expression of pinopodes in the endometrium of PCOS rats
   during the window of implantation, and thus promote embryo adhesion
   ([70]Fig 8).

Fig 8. Effect of aspirin on the expression of pinopodes in PCOS rats during
the window of implantation.

   [71]Fig 8
   [72]Open in a new tab

   (n = 3). (a, d) Control group, the endometrial architecture appeared
   normal and intact, exhibiting a dense and uniform distribution of
   microvilli and numerous pinopodes were observed, evenly distributed
   across the surface; (b, e) Model group, the endometrial structure
   showed significant disruption, characterized by sparse and irregularly
   distributed microvilli and the number of pinopodes was markedly
   reduced, with the majority exhibiting pronounced wrinkling and
   collapse; (c, f) Aspirin group, the endometrial structure was slightly
   damaged, with abundant and evenly distributed microvilli and a large
   number of pinopodes, but some of them were slightly wrinkled. The lower
   panels (scare bar = 10μm) magnify the boxed regions in the upper panels
   (scare bar = 50μm). Red arrows indicate pinopodes.

Effect of aspirin on the metabolic profile of uterine lavage fluid in PCOS
rats during the window of implantation

UHPLC-MS/MS stability analysis.

   Metabolic profiling of uterine lavage fluid from PCOS rats revealed
   comparable total ion chromatograms (TICs) for the control, model, and
   aspirin groups, although differences in the response values of the
   peaks were observed. TICs, including retention time (RT), peak
   intensity, and resolution, were recorded for five quality control (QC)
   samples in both positive and negative ion modes using UHPLC-MS/MS.
   Pearson correlation coefficients between the QC samples were calculated
   based on the relative quantitative values of the metabolites. A higher
   correlation between the QC samples (with R² closer to 1) indicates
   better assay stability and higher data quality.

Multivariate statistical analysis.

   Partial Least Squares Discrimination Analysis (PLS-DA) was employed to
   model the relationship between metabolite expression and sample
   categories, revealing significant metabolic differences between the
   groups. The analysis demonstrated clear distinctions in metabolites
   between the control and model groups, as well as between the model and
   aspirin groups of uterine lavage fluid ([73]Fig 9.a–[74]b). To assess
   whether over-fitting occurred in the PLS-DA model, a 200 × permutation
   test was performed. The results indicated that for the model group vs.
   the control group, the R²Y was 0.90, and Q² was -0.69; for the aspirin
   group vs. the model group, the R² Y was 0.93, and Q² was -0.66. The Q²
   regression lines intercept on the model's longitudinal axis was below
   zero, suggesting that the model was well-constructed
   without over-fitting and is reliable for further analysis ([75]Fig
   9.c–[76]d). These results confirm the validity of the model and its
   suitability for differential metabolite screening. Mars plots displayed
   trends of differential metabolite expression between the model and
   control groups, and between the aspirin and model groups ([77]Fig
   9.e–[78]f). In these plots, each dot represents a specific metabolite,
   with dot size reflecting the Variable Importance in Projection (VIP)
   value. Larger dots indicate higher VIP values, while green dots
   represent down-regulated metabolites, and red dots represent
   up-regulated metabolites.

Fig 9. Multivariate statistical analysis diagram.

   [79]Fig 9
   [80]Open in a new tab

   (a, b) PLS-DA diagrams. The separation pattern was satisfactory, with
   significant differences in principal components among the groups,
   indicating that the metabolic profiles underwent significant
   alterations between different groups. The ellipses represent the 95%
   confidence intervals. (c, d) 200 × permutation test of 7-fold
   cross-validation. The PLS-DA model was validated by a 200 × permutation
   test, and the results were R^2Y = 0.90, Q^2 = -0.69 < 0 and
   R^2Y = 0.93, Q^2 = -0.66 < 0. The model is well established, there is
   no over-fitting. (e, f) Volcano plots. The volcano plots showed the
   changing trend of the differential metabolites expression in the Model
   group vs. Control group and Aspirin group vs. Model group. Each dot
   represents a specific metabolite, and the size of the dot indicates the
   VIP value. Red dots represent upregulated metabolites; green dots
   represent down-regulated metabolites. (g, h) Heat maps of metabolites.
   In the form of a heat map, shows a full range of metabolites in uterine
   lavage fluid samples from the Model group vs. Control group and Aspirin
   group vs. Model group and the relative expression magnitude of
   metabolites in each sample group. Red indicates increased biomarker
   expression; blue indicates decreased biomarker expression. (i) Venn
   diagram of metabolites. Five common cross metabolites were screened by
   intersecting the metabolites in the Model group vs. Control group and
   Aspirin group vs. Model group. (j, k) Metabolite pathways. Large size
   and red color represent major pathway enrichment and high pathway
   effect values, respectively. Statistically significant pathways
   (P < 0.05) have been labelled. (j) Model group vs. Control group; (k)
   Aspirin group vs. Model group.

Identification of potential biomarkers.

   Based on the PLS-DA results, P < 0.05 and VIP > 1.0 were considered as
   significance thresholds, which were used as criteria to screen for
   differential metabolites. A total of 146 significantly different
   metabolites were identified in the comparison between the model group
   and the control group, with notable changes following aspirin
   intervention. In the comparison between the aspirin and model groups,
   48 significantly different metabolites were found. Compared with the
   control group, 48 metabolites were up-regulated and 98 were
   down-regulated in the model group (P < 0.05); in the aspirin group, 25
   metabolites were up-regulated and 23 were down-regulated compared to
   the model group (P < 0.05). The heat maps illustrating the total
   metabolites in uterine lavage samples from the model group versus the
   control group and from the aspirin group versus the model group are
   shown in ([81]Fig 9.g–[82]h). The heat maps represent the relative
   expression of metabolites in each group's samples, with red indicating
   increased biomarker expression and blue indicating decreased biomarker
   expression. To explore the relationship between potential biomarkers,
   metabolic pathways, and the mechanisms by which aspirin improves ER in
   PCOS rats, the metabolites from both the model and control groups, as
   well as the aspirin and model groups, were intersected. This led to the
   identification of five common crossover metabolites:
   2,6-dihydroxypurine, gluconolactone, Oxaceprol, PC (18:1/18:1), and PC
   (20:3e/17:1), which were considered potential biomarkers ([83]Fig 9.i
   and [84]Table 1).

Table 1. Potential biomarkers.

   Metabolism Formula KEGG_ID HMBD_ID Model vs Control Aspirin vs Model
   P^a FC^b Trend^c P FC Trend
   PC (20:3e/17:1) C45H84NO7P -- -- 0.0004^** 0.45 ↓ 0.0105^# 3.15 ↑
   2,6-dihydroxypurine C5H4N4O2 -- HMDB0000292 0.0007^** 0.42 ↓ 0.0235^#
   2.02 ↑
   gluconolactone C6H10O6 -- HMDB0000150 0.0093^** 0.53 ↓ 0.0150^# 1.95 ↑
   Oxaceprol C7H11NO4 -- HMDB0000725 0.0213^* 2.54 ↑ 0.0233^# 0.33 ↓
   PC (18:1/18:1) C44H84NO8P -- -- 0.0364^* 0.6 ↓ 0.0376^# 1.86 ↑
   [85]Open in a new tab

   a We applied univariate analysis (t-test) to calculate the statistical
   significance (P-value).

   b The fold change (FC) of relative amounts of the former group compared
   to the latter group.

   c ↑indicates up-regulation; ↓ indicates down-regulation.

   *P ＜ 0.05; **P ＜ 0.01 vs. Control group.

   # P ＜ 0.05 vs. Model group.

Metabolic pathway results.

   Differential metabolites were imported into the MetaboAnalyst 6.0
   platform for pathway enrichment analysis to identify metabolic pathways
   with P < 0.05. Metabolic pathway analysis was performed on the
   differential metabolites from both the model group compared to the
   control group and the aspirin group compared to the model group. The
   results revealed three major metabolic pathways in the model group
   compared to the control group: the pentose phosphate pathway, arginine
   biosynthesis, and arginine and proline metabolism ([86]Fig 9.j). In the
   aspirin group compared to the model group, four major metabolic
   pathways were identified: purine metabolism, pentose phosphate pathway,
   glutathione metabolism, and arginine and proline metabolism ([87]Fig
   9.k). Notably, the pentose phosphate pathway and arginine and proline
   metabolism were shared between the control, model, and aspirin groups.

Discussion

   Polycystic ovary syndrome (PCOS) is a common, complex, and
   heterogeneous endocrine disorder with a multifactorial pathogenesis
   [[88]15]. It is currently believed to be linked to hyperandrogenemia,
   insulin resistance, chronic inflammation, and oxidative stress. These
   pathophysiological changes may contribute to female infertility
   [[89]2]. PCOS-related infertility accounts for approximately 6%-21% of
   all infertility cases[[90]17]. The spontaneous abortion rate in PCOS
   patients is as high as 20%-45%, with recurrent miscarriage rates
   ranging from 30%-50% [[91]18]. Nearly two-thirds of implantation
   failures are associated with poor ER [[92]6]. The endometrium is a
   unique tissue that undergoes cyclic changes under the influence of
   ovarian steroids, including menstruation, proliferation, secretion, and
   shedding [[93]19]. A normal endometrial structure and function are
   essential for promoting embryo implantation and development. Changes in
   the intrauterine micro-environment can impact ER. ER refers to a series
   of physiological changes in the endometrium that create an optimal
   environment for embryo localization, adhesion, invasion, and
   implantation. A previous study by our team found that aspirin
   significantly reduced the expression of androgen receptor protein and
   mRNA, while the expression of progesterone receptor, estrogen receptor,
   VEGF, and integrin αvβ3 proteins and mRNA were significantly increased
   in PCOS rats during the window of implantation. This suggests that
   aspirin may promote angiogenesis through the regulation of VEGF,
   thereby improving ER [[94]20].

   Aspirin is an antiplatelet agent that prevents microthrombus formation
   by inhibiting platelet aggregation, while simultaneously inhibiting
   vasoconstriction, leading to improved uterine vasospasm and enhanced
   ER. Low-dose aspirin (50–150 mg/day) reduces vascular tone and improves
   tissue perfusion, which results in increased endometrial thickness,
   glandular development, and activation of multiple estrogen and
   progesterone receptors. This enhances the uterine response to ovarian
   hormones, thereby increasing the clinical pregnancy rate [[95]21].
   Administration of 50 mg/day of aspirin has been shown to improve
   endometrial blood perfusion. After two months of treatment with this
   dose, the endometrial blood flow rate in patients with unexplained
   recurrent biochemical pregnancy failure significantly increased and
   even returned to normal levels [[96]22]. Low-dose aspirin also
   significantly increases the levels of endometrial capacitance proteins,
   such as leukemia inhibitory factor (LIF) and integrin αvβ3, during the
   window of implantation [[97]23]. Adequate endometrial blood supply is
   considered essential for successful implantation. Aspirin irreversibly
   inhibits cyclooxygenase, reducing the activity of thromboxane A2 (TXA2)
   and prostaglandins (PGs), which in turn decreases vascular tone by
   preventing vasoconstriction and improving tissue perfusion, including
   uterine blood flow velocity [[98]3]. By improving the environment of
   the endometrium, aspirin facilitates better ER and pregnancy outcomes.

   The transformation of the uterus into a receptive state for embryo
   implantation is primarily regulated by progesterone and estrogen.
   Additionally, the presence of cytosolic synapses in the endometrium at
   the capacitated stage is a prerequisite for successful embryo
   implantation [[99]24]. Numerous studies in rats have demonstrated that
   the development of cytosolic protrusions is progesterone-dependent and
   coincides precisely with the implantation window [[100]25]. Cytosolic
   synapses are critical ultrastructural markers of ER and the
   implantation window [[101]26]. In the present study, we found that,
   compared with the model group, the aspirin group exhibited a
   significant increase in follicle implantation rate, as well as elevated
   serum levels of estradiol (E[2]) and progesterone (P). Furthermore,
   endometrial thickness, glandular development, blood vessel formation,
   increased expression of pinopodes, and an abundance of evenly
   distributed microvilli were observed, suggesting that aspirin improves
   sex hormone levels, pinopodes expression, and endometrial morphology in
   PCOS rats during the window of implantation. These changes contribute
   to improved ER. This indicates that aspirin can enhance the expression
   of pinopodes and improve endometrial morphology during the window of
   implantation in PCOS, which in turn enhances ER and promotes embryo
   implantation, offering therapeutic potential for treating infertility.

   The metabolomics results of the present study indicated that the
   metabolic profile in the uterine lavage fluid of the PCOS rats during
   the window of implantation was altered compared with the control group,
   primarily in the metabolism of metabolites such as 2,6-dihydroxypurine,
   gluconolactone, Oxaceprol, PC (18:1/18:1), and PC (20:3e/17:1). Aspirin
   can reverse the changes in the abundance of these five metabolites.
   Notably, the pentose phosphate pathway and arginine and proline
   metabolism were the two metabolic pathways shared among the control,
   model, and aspirin groups.

   The pentose phosphate pathway (PPP) is a glucose oxidation pathway that
   parallels the upper portion of glycolysis, generating ribose
   5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH)
   [[102]27]. Disruptions in glucose metabolism can impede endometrial
   glucose utilization, as well as the growth and activity of endometrial
   cells, potentially leading to endometrial dysplasia, thinning, impaired
   metaphase, and compromised ER [[103]28]. One critical aspect of
   establishing ER is the progesterone-mediated differentiation of
   endometrial stromal cells (ESCs) into decidual cells, which are
   essential for embryo survival during the early stages of implantation.
   Dehydroepiandrosterone (DHEA) is an endogenous hormone produced by the
   adrenal glands, with serum levels elevated approximately twofold in
   about 50% of patients with PCOS [[104]29]. DHEA is known to be a
   non-competitive inhibitor of glucose-6-phosphate dehydrogenase (G6PDH),
   which is the rate-limiting enzyme in the PPP [[105]30]. Frolova
   proposed that elevated DHEA levels impair implantation by interfering
   with normal endometrial growth, differentiation, and development into
   decidual cells, partly due to the inhibition of G6PDH and the resulting
   blockage of glucose flow through the PPP, which may account for reduced
   decidualization [[106]29]. The differentiation of ESCs into decidual
   cells promotes angiogenesis and the maturation of the endometrial
   vasculature [[107]5]. In the present study, serum metabolites
   associated with the pentose phosphate pathway, such as gluconolactone,
   were upregulated after aspirin intervention. Gluconolactone is a
   polyhydroxy acid (PHA), a lactone or oxidized derivative of glucose. A
   study has demonstrated that gluconolactone primarily activates
   extracellular signal-regulated kinase (ERK) signaling through PKCε
   [[108]31]. ERK, a serine/threonine protein kinase, belongs to the MAPK
   (mitogen-activated protein kinase) family. Several studies have
   demonstrated that ERK1/2 phosphorylation (p-ERK1/2) regulates cell
   proliferation and activity, thereby influencing endometrial
   receptivity. This kinase plays a critical role in both endometrial
   epithelial cells (EECs) and stromal cells (ESCs), promoting endometrial
   cell proliferation, differentiation, and vascular permeability
   [[109]32]. Upon activation of the ERK1/2-AMPK pathway, the expression
   of Forkhead Box Protein O1 (FOXO1) is upregulated. Since glucose
   transporter-1 (GLUT1) is positively regulated by FOXO1,
   the up-regulation of GLUT1 promotes glucose uptake [[110]33]. This
   suggests that enhancing the pentose phosphate pathway (PPP) can improve
   the biosynthesis required for decidualization. Therefore, we
   hypothesize that the pentose phosphate pathway is one of the mechanisms
   through which aspirin improves ER in the PCOS rats during the window of
   implantation.

   The arginine and proline metabolic pathway is one of the potential
   target pathways involved in the development of PCOS [[111]34].
   Endocrine dysregulation resulting from chronic inflammation in PCOS may
   lead to disturbances in vascular homeostasis, contributing to vascular
   endothelial dysfunction. These disturbances are closely linked to
   nitric oxide (NO) synthesis, reactive oxygen species, and insulin
   signaling [[112]35]. Approximately 65%-95% of women with PCOS exhibit
   insulin resistance and compensatory hyperinsulinemia [[113]36].
   L-arginine, a precursor of nitric oxide, is a potent antioxidant that
   exerts anti-inflammatory effects by scavenging reactive oxygen species
   [[114]37]. Nitric oxide plays a critical role in vascularization,
   regulating vascular tone, promoting endometrial metaplasia, mediating
   angiogenesis, and increasing endometrial vascular permeability and
   diastolic function. These processes ensure an adequate endometrial
   blood supply necessary for embryo implantation [[115]38]. The
   significance of nitric oxide in female reproduction is highlighted by
   its involvement in follicular maturation, ovulation, decidualization,
   embryo implantation, and cervical secretion. Increased inducible nitric
   oxide synthase (iNOS) expression in the uterus during the window of
   implantation confirms that low nitric oxide levels are associated with
   reduced uterine tolerance in women with PCOS [[116]39]. Moreover,
   L-arginine’s ability to lower serum testosterone levels is linked to
   the binding of nitric oxide to cytochrome P-450, thereby inhibiting
   steroidogenesis [[117]40]. Arginine, as an intermediate metabolite,
   connects the tricarboxylic acid cycle to arginine and proline
   metabolism, suggesting that disturbances in arginine and nitric oxide
   metabolism may be closely associated with insulin resistance signaling
   pathways. Studies indicate that insulin signaling is impaired in the
   pathogenesis of PCOS, potentially due to disruptions in arginine and
   proline metabolism, leading to nitric oxide production that affects the
   IRS-1-PI3K/Akt signaling pathway. Evidence suggests that the PI3K/Akt
   pathway plays a pivotal role in ER by promoting endometrial
   angiogenesis during the pre-implantation period [[118]41]. Our research
   results show that the number of endometrial blood vessels increased in
   the aspirin group. A study has indicated that the metabolism of
   arginine and proline is reduced in thin endometrial tissues [[119]42].
   Our results demonstrate that the endometrium became thinner in the
   model group, while it thickened in the aspirin group, suggesting that
   aspirin may increase endometrial thickness by regulating this pathway.
   Based on these findings, we hypothesize that the arginine and proline
   metabolic pathways may be the primary metabolic routes through which
   aspirin improves endometrial receptivity (ER). This improvement is
   likely achieved by enhancing endometrial blood supply, increasing
   endometrial thickness, and alleviating insulin resistance, thereby
   improving endometrial receptivity.

   It is reported that PC is predominantly altered in endometrial fluid
   from implanted and non-implanted IVF cycles, suggesting its importance
   for ER [[120]43].In our research, in addition to the pentose phosphate
   pathway and arginine and proline metabolism, two shared metabolites, PC
   (18:1/18:1) and PC (20:3e/17:1), are noteworthy. Studies have shown
   that glycerophospholipid metabolism is one of the primary pathways of
   lipid metabolism. Glycerophospholipids can induce the expression of
   cyclooxygenase-2 (COX-2), which catalyzes the conversion of arachidonic
   acid. The metabolites of arachidonic acid are further involved in
   regulating the gene expression and developmental processes of the
   endometrium [[121]10]. Glycerophospholipids, such as
   phosphatidylcholine (PC), are essential components of cell membranes
   and play pivotal roles in cellular transport, signaling, and the
   regulation of protein function [[122]44]. Phosphatidylcholine is a
   primary source of lysophosphatidylcholine (LPC) [[123]45], a lipid that
   has been linked to inflammatory responses and activation of the
   phospholipid pathway in insulin resistance. Elevated levels of LPCs
   suggest a reduced inflammatory response and an improvement in
   phospholipid metabolism. LPC is an important intermediate in the fatty
   acid-induced insulin resistance pathway [[124]46]. Women with PCOS
   often exhibit localized insulin resistance, particularly in the
   endometrium, where key molecules in the insulin signaling pathway are
   impaired, leading to disrupted signaling and reduced glucose uptake
   [[125]21]. LPCs have been shown to promote glucose uptake in
   adipocytes, with studies on diabetic mouse models suggesting that LPC
   lowers blood glucose levels by upregulating glucose transporter-4
   (GLUT-4) expression on adipocytes, enhancing glucose uptake [[126]47].
   GLUT-4 plays a crucial role in facilitating rapid glucose uptake by
   various cells, maintaining glucose homeostasis. Several studies have
   reported reduced mRNA and protein levels of GLUT-4 in the endometrium
   of women with PCOS compared to control individuals, with an even
   greater reduction observed in women with PCOS and insulin resistance
   (PCOS-IR). This abnormal expression of GLUT-4 may contribute to
   endometrial insulin resistance in PCOS patients [[127]48], impairing
   ER. Relevant studies have shown that abnormal glucose and lipid
   metabolism is an independent risk factor for insulin resistance (IR),
   and IR can lead to an increase in testosterone (T) levels in the body,
   while E[2] and P levels are suppressed and remain low. Additionally,
   the study also indicates that E[2] and P levels are positively
   correlated with phosphatidylcholine, suggesting that enhancing lipid
   transport and metabolic capacity plays an important role in restoring
   hormone levels. The steroid hormones estrogen and progesterone are the
   main factors controlling uterine receptivity. Estrogen promotes the
   thickening of the endometrium and increases the number of glands
   [[128]41], while progesterone significantly enhances the number of
   pinopodes and promotes their structural integrity[[129]28]. These
   findings are highly consistent with our research results. Given the
   importance of the glycerophospholipid metabolic pathway in modulating
   insulin resistance, we hypothesize that aspirin may improve endometrial
   insulin sensitivity by regulating this pathway. Through this mechanism,
   aspirin may enhance ER, potentially contributing to improved fertility
   outcomes in PCOS patients.

   This study focuses on the critical role of glucose metabolism, lipid
   metabolism, and oxidative stress-related metabolic pathways in the
   development and progression of PCOS. Based on this finding, we will
   conduct systematic and in-depth research on the aforementioned three
   metabolic pathways. Furthermore, we are exploring whether uterine fluid
   can predict endometrial tolerance in patients with PCOS. If successful,
   uterine fluid testing may provide a faster and less invasive way for
   predicting the implantation window and endometrial receptivity.
   However, several limitations of this study need to be addressed.
   Firstly, the research lacks experimental validation of key molecules in
   the relevant metabolic pathways, such as molecular biological evidence
   from Western blot analysis and immunohistochemical staining. Secondly,
   due to the small sample size and the fact that only animal experiments
   were conducted, the generalizability of the research findings requires
   further verification. Based on these limitations, we plan to carry out
   follow-up research: on one hand, we will explore the clinical safety
   and efficacy of aspirin in treating patients with polycystic ovary
   syndrome (PCOS); on the other hand, we will determine the optimal
   therapeutic dose of aspirin for improving endometrial receptivity
   through rigorously designed clinical trials.

Conclusions

   In conclusion, this study provides the first evidence that aspirin
   modulates glucose metabolism and ameliorates insulin resistance in a
   PCOS rat model, as revealed by non-targeted metabolic analysis.
   Specifically, aspirin improves the morphology of the endometrium,
   promotes endometrial angiogenesis and the development of pinopodes, and
   ultimately improves embryo implantation rates during the implantation
   window. These effects are mediated through metabolic pathways in the
   uterine cavity fluid, including the pentose phosphate pathway, arginine
   and proline metabolism, and glycerophospholipid metabolism. To further
   validate these findings, we plan to collect human-derived uterine
   lavage fluid for multi-sample, multi-dimensional analysis. This will
   elucidate the mechanisms by which aspirin enhances endometrial
   receptivity, offering a robust scientific foundation for addressing
   clinical reproductive challenges.

Supporting information

   S1 Table. The number of blastocysts.

   (XLSX)
   [130]pone.0324432.s001.xlsx^ (10.2KB, xlsx)
   S2 Table. The levels of E2 and P.

   (XLSX)
   [131]pone.0324432.s002.xlsx^ (10.4KB, xlsx)
   S3 Table. Endometrial thickness.

   (XLSX)
   [132]pone.0324432.s003.xlsx^ (12.2KB, xlsx)
   S4 Table. Aspirin.vs.Model_Differentially expressed metabolites.

   (XLSX)
   [133]pone.0324432.s004.xlsx^ (25.1KB, xlsx)
   S5 Table. Model.vs.Control_Differentially expressed metabolites.

   (XLSX)
   [134]pone.0324432.s005.xlsx^ (57.3KB, xlsx)
   S6 Table. Aspirin.vs.Model_pathway_results.

   (XLSX)
   [135]pone.0324432.s006.xlsx^ (10.2KB, xlsx)
   S7 Table. Model.vs.Control_pathway_results.

   (XLSX)
   [136]pone.0324432.s007.xlsx^ (11.5KB, xlsx)

Data Availability

   All relevant data are within the paper and its [137]Supporting
   Information files.

Funding Statement

   This research was funded by the National Natural Science Foundation of
   China (81503611), the Hunan Provincial Natural Science Foundation
   (2025JJ50716) ,the Key Fund Project of Hunan Provincial Department of
   Education (24A0257); the Changsha Natural Science Fund Project
   (kq2402176) and the Key Project of Hunan University of Chinese Medicine
   (2024XJZA003) (all awarded to S.Y.).The funders had no role in study
   design, data collection and analysis, decision to publish, or
   preparation of the manuscript.

References