Dear Editor,
With the global trend toward delayed childbearing, pregnancy at advanced maternal age (>35 years) has become a major clinical challenge. Although poor reproductive outcomes in older women have traditionally been attributed primarily to age-related ovarian decline, increasing evidence indicates that the endometrium plays a critical and independent role in implantation failure and pregnancy loss in this population—an aspect that has long been underestimated (
Pathare et al., 2023;
Vitagliano et al., 2023;
Zhao et al., 2023). Notably, even when euploid embryos are transferred, women of advanced maternal age exhibit reduced biochemical pregnancy, clinical pregnancy, and live birth rates compared with younger counterparts (
Wang et al., 2025), underscoring the pivotal contribution of impaired endometrial receptivity to age-related fertility decline.
Age-associated endometrial dysfunction is increasingly recognized as a multifactorial process involving cellular senescence, mitochondrial impairment, and exacerbated oxidative stress (
Lu et al., 2022;
Chemerinski et al., 2024). Excessive accumulation of reactive oxygen species (ROS) disrupts cellular homeostasis, promotes DNA damage and cell-cycle arrest, and contributes to the development of a senescence-associated secretory phenotype, thereby creating a microenvironment that is unfavorable for embryo implantation (
Palomba et al., 2021;
Sharma, 2022). Accordingly, therapeutic strategies aimed at alleviating oxidative stress and preserving mitochondrial function may represent promising approaches to mitigate age-related endometrial decline.
Astragalus membranaceus (AM) is a widely used traditional medicinal herb that has been reported to exert antioxidant, anti-inflammatory, and cytoprotective effects in multiple aging-related contexts (
Borowicz and Jach, 2025;
Ma et al., 2025). These properties suggest that AM may contribute to the maintenance of tissue homeostasis under conditions of aging-driven stress. However, whether AM can modulate endometrial function and thereby support embryo implantation in the setting of advanced maternal age has not been systematically investigated.
Here, we report that AM treatment alleviates aging-linked endometrial senescence and partially restores uterine receptivity in aged mice. Using a combination of in vivo aging models, in vitro senescence systems, and supportive network pharmacology analyses, our findings suggest that the protective effects of AM are associated with reduced oxidative stress and preservation of mitochondrial function. Together, these observations support a potential role for AM as a non-hormonal intervention for mitigating age-related endometrial dysfunction.
In an established aged mouse model, we first examined whether AM treatment could alleviate age-associated endometrial senescence in vivo, while also performing a preliminary assessment of uterine safety (Fig. S1). Compared with young controls, aged uteri exhibited a pronounced increase in senescence-related markers, consistent with an accelerated senescent phenotype. Quantitative RT-PCR analysis showed that the mRNA levels of Cdkn2a, Cdkn1a, and Tp53 were significantly elevated in the Aging group, whereas AM treatment markedly attenuated their expression (Fig. 1A). Consistently, Western blot analysis revealed increased P21 and P53 protein abundance in aged uteri, which was partially reversed following AM administration (Fig. 1B and 1C).
These molecular changes were accompanied by concordant histological and cellular alterations. Senescence-associated β-galactosidase (SA-β-gal) staining demonstrated a substantial accumulation of senescent cells in aged uterine tissues, which was markedly reduced after AM treatment (Fig. S2A). Immunohistochemical and immunofluorescence analyses further confirmed enhanced P21 and P53 signals in the endometrium of aged mice and their attenuation upon AM intervention (Figs. S2C–F, S3A, and S3B). Consistently, hematoxylin–eosin staining revealed disruption of endometrial architecture in aged mice, characterized by reduced glandular density and a loosely organized stromal compartment, whereas AM treatment partially restored endometrial integrity and tissue organization (Fig. S3E). Together, these findings indicate that AM treatment mitigates multiple molecular and histopathological features of age-related endometrial senescence in vivo.
To determine whether the alleviation of endometrial senescence was accompanied by functional improvement, we next examined markers associated with uterine receptivity. Compared with young controls, aged uteri exhibited a broad reduction in the expression of receptivity-related genes, including Hand2, Hoxa10, Esr1, Areg, Pgr, and Ltf, whereas AM treatment partially restored their mRNA levels (Fig. 1D). Consistently, Western blot analysis showed that the protein abundance of key receptivity regulators, including LIF, HOXA10, and HAND2, was markedly decreased in aged uteri and significantly increased following AM administration (Fig. 1E and 1F). Furthermore, immunohistochemical staining confirmed reduced HAND2 expression in the aged endometrium and its restoration after AM treatment (Fig. S3C, D). Immunofluorescence staining further showed decreased LIF expression and increased MUC1 expression in the aged endometrium, both of which were partially normalized following AM treatment (Fig. S4B, D, F). Concordantly, Ki67 staining revealed excessive epithelial-stromal proliferation in aged uteri, indicative of dysregulated endometrial dynamics, which was attenuated upon AM intervention (Fig. 1G and 1J). Moreover, aged endometria displayed an imbalance in steroid receptor expression, characterized by elevated ESR and reduced PGR levels; this aberrant pattern was partially corrected by AM treatment (Fig. 1H, 1I, 1K, and 1L). Immunofluorescence analyses of MSX1, MSX2, HOXA10, and COX2 further demonstrated that AM restored the expression levels of these key functional markers in aged endometrium (Fig. 1M, N; Fig. S4A, C, D). In summary, these results indicate that AM treatment not only mitigates endometrial senescence but also promotes functional recovery of uterine receptivity in aged mice.
To provide supportive mechanistic context for the observed effects of AM, we performed network pharmacology and bioinformatic analyses using a public transcriptomic dataset (GSE58144). Differential expression analysis identified 524 upregulated and 571 downregulated genes in aged endometrium (Fig. 2A). In parallel, putative targets corresponding to 22 bioactive compounds of AM were retrieved from public pharmacology databases (Table S1). Intersection of these targets with senescence-associated genes from the GSE58144 dataset yielded 55 overlapping genes (Fig. 2B), suggesting their potential relevance to AM-mediated regulation of endometrial aging. Functional enrichment analyses indicated that these overlapping targets were primarily associated with aging-related biological processes. KEGG pathway analysis highlighted cellular senescence-related signaling pathways, while GO enrichment analysis identified response to oxidative stress as a prominently enriched biological process (Fig. 2C and 2D). Detailed results from KEGG and GO enrichment analysis can be found in the Supplementary Materials (see “GO and KEGG enrichment analysis of intersection targets”). Consistent with these findings, protein–protein interaction analysis revealed several highly connected nodes, including ESR1, HIF1A, CCNB1, KIT, MET, and PTGS2, suggesting their potential roles within a broader regulatory network (Fig. S6). Collectively, these analyses support the notion that AM-associated targets converge on pathways related to oxidative stress regulation and senescence-related processes, providing complementary context for the experimental observations.
To complement the in vivo findings, we established in vitro senescence models in endometrial stromal and epithelial cells using D-galactose (D-gal) treatment. Based on preliminary optimization, exposure to 40 mg/mL D-gal for 24 h was selected to induce cellular senescence (Fig. S5). CCK-8 assays showed that AM treatment at the tested concentrations did not significantly affect cell viability under basal conditions, indicating minimal cytotoxicity (Fig. 2E). In contrast, D-gal exposure markedly reduced cell viability, which was dose-dependently improved by AM treatment. Among the concentrations examined, 1.25 mg/mL AM exhibited the most pronounced protective effect and was therefore used in subsequent experiments (Fig. 2F). We next assessed oxidative stress and senescence-associated features in vitro. D-gal treatment led to a significant increase in intracellular ROS levels, whereas AM intervention markedly attenuated ROS accumulation (Fig. 2G and 2H). Consistent with these changes, D-gal markedly upregulated the expression of senescence-associated markers (CDKN2A, CDKN1A, and TP53), which were suppressed following AM treatment (Figs. 2I and S2B). In parallel, mitochondrial function analysis revealed a loss of mitochondrial membrane potential after D-gal exposure, as indicated by JC-1 staining, which was partially restored by AM treatment (Fig. 2J). Cell-cycle analysis further showed an increased proportion of cells arrested at the G0/G1 phase in the D-gal group, whereas AM intervention significantly normalized cell-cycle distribution (Fig. 2K). These regulatory effects were further supported by Western blot analysis at the protein level (Fig. 2L–N). Together, these in vitro observations are consistent with the in vivo findings and provide supportive evidence that AM alleviates oxidative stress-associated senescence phenotypes in endometrial cells. Notably, these protective effects were observed in both stromal and epithelial cells, indicating that AM exerts broadly comparable anti-senescent effects across distinct endometrial cell types under stress conditions.
In summary, this study demonstrates that AM intervention alleviates age-associated endometrial decline and promotes the restoration of uterine receptivity in aged mice. Given that impaired endometrial function represents a critical bottleneck in reproductive outcomes at advanced maternal age (
Wang et al., 2025), our findings underscore the importance of targeting endometrial aging as a complementary strategy beyond conventional hormone-based approaches. We observed that AM treatment attenuated key senescence-associated features, including reduced SA-β-gal activity and downregulation of p53/p21 expression, while restoring the expression of essential receptivity-related markers and partially rebalancing ESR and PGR signaling. Together, these results suggest that AM may represent a potential non-hormonal approach for modulating age-related endometrial dysfunction, warranting further investigation in translational and clinical settings.
From a mechanistic perspective, our study integrates network pharmacology with experimental observations to provide supportive insight into pathways potentially involved in AM-mediated effects. Aging is widely recognized as a process characterized by the accumulation of molecular damage, which can be driven by mitochondrial dysfunction and excessive ROS production (
Srivastava, 2017). In our models, AM treatment was associated with reduced ROS accumulation and partial restoration of mitochondrial membrane potential, accompanied by attenuation of p53/p21-associated senescence features in endometrial stromal cells. These experimentally validated alterations provided the primary rationale for focusing the discussion on oxidative stress–mitochondrial pathways, which were consistently supported across both
in vivo aging models and
in vitro senescence assays. These findings indicate that mitochondrial dysfunction and oxidative stress represent important cellular features accompanying aging-dependent endometrial senescence. Given the multifactorial nature of reproductive aging, mitochondrial alterations may act in concert with other aging-related pathways to shape the senescent endometrial phenotype, and their precise causal contribution remains to be further clarified through targeted interventions focusing on mitochondrial function. In this context, the comparable protective effects observed in both stromal and epithelial cells are more likely to reflect shared cellular responses to age-associated stress conditions rather than preferential targeting of a specific cell type. Given that both compartments are highly sensitive to oxidative stress and senescence-related signals, AM may act by modulating stress-adaptive processes that are commonly operative across different endometrial cell populations.
Although validation in human clinical samples will be required to account for species-specific differences, the present findings provide supportive experimental evidence for the potential relevance of AM in the context of aging-linked endometrial dysfunction. Importantly, the observed protective effects in this study are attributed to the standardized whole extract of AM, reflecting the multi-component nature of this herbal medicine. We acknowledge that the biological activity of AM is likely mediated through the coordinated actions of multiple bioactive constituents, which may represent both a therapeutic advantage and a challenge for reproducibility and clinical translation. Future studies will therefore focus on identifying key active components of AM and elucidating their molecular targets, with the aim of refining mechanistic understanding and facilitating translational development. In conclusion, this study provides a conceptual and experimental framework supporting the potential of AM as a non-hormonal strategy for modulating age-related endometrial dysfunction and reproductive aging.
The Author(s) 2026. Published by Oxford University Press on behalf of Higher Education Press.
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