Selenium nanoparticles mitigate Cyclophosphamide-Induced premature ovarian failures in mice by activating PI3K/AKT signaling pathway and inhibiting ferroptosis.

As shown in Supplementary Fig. 1, CTX administration significantly impaired ovarian physiology, with reductions in ovarian weight (Supplementary Fig. 1a, p  < 0.05) and ovarian index (Supplementary Fig. 1b, p  < 0.05) compared to the NC group. SeNPs treatment significantly restored both ovarian weight (Supplementary Fig. 1a, p  < 0.05) and index and ovarian index (Supplementary Fig. 1b, p  < 0.05). Serological hormone analysis indicated that CTX markedly increased FSH (Supplementary Fig. 1c, p  < 0.05) and LH (Supplementary Fig. 1d, p  < 0.05), while decreasing AMH (Supplementary Fig. 1e, p  < 0.05) and E 2 (Supplementary Fig. 1f, p  < 0.05), compared to the NC group. SeNPs administration mitigated these disruptions, bringing hormone levels closer to normal. Given the absence of differences in ovarian weight, ovarian index, and serum hormone levels between the MD and HD groups, the medium-dose SeNPs (300 mg/kg) group was selected for subsequent analyses. Histopathological evaluation revealed that CTX exposure induced ovarian damage, including interstitial fibrosis, inflammatory cell infiltration, and vessel dilation (Fig.  1 a and b). SeNPs treatment attenuated these changes and improved ovarian morphology. Follicle quantification confirmed that SeNPs significantly increased the numbers of primordial (Fig.  1 c, p  < 0.05), primary (Fig.  1 d, p  < 0.05), secondary (Fig.  1 e, p  < 0.05), and mature follicles (Fig.  1 f, p  < 0.05), while reducing atretic follicles (Fig.  1 g, i < 0.05). Gene expression and IHC staining analysis of folliculogenesis-related genes ( Bmp15 , Gdf9 , Inhibin α , and Foxl2 ) and proteins (INHIBIN α, FOXO3A, and SIRT1) demonstrated significant alterations in the CTX group (Fig.  1 h-m and Supplementary Fig. 2), which were reversed by SeNPs administration. Fig. 1 Ameliorative Effects of SeNPs Administration on CTX-Induced Folliculogenesis Disorder Ameliorative Effects of SeNPs Administration on CTX-Induced Folliculogenesis Disorder As shown in Fig.  2 , CTX treatment suppressed antioxidant enzyme activities, including SOD (Fig.  2 a, p  < 0.05), GSH-Px (Fig.  2 b, p  < 0.05), GSH (Fig.  2 c, p  < 0.05), and CAT (Fig.  2 d, p  < 0.05), while elevating MDA levels (Fig.  2 e, p  < 0.05). SeNPs administration restored the activities of these enzymes and reduced MDA concentrations. Furthermore, expression profiling of antioxidant-related genes substantiated these findings, as SeNPs modulated the expression of antioxidant-related genes (Fig.  2 f-j, p  < 0.05), further corroborating their role in mitigating CTX-induced oxidative damage. Fig. 2 Protective Effects of SeNPs Against CTX-Induced Oxidative Stress Protective Effects of SeNPs Against CTX-Induced Oxidative Stress As shown in Fig.  3 , CTX exposure significantly increased Tunnel-positive granulosa cells (Fig.  3 a-b, p  < 0.05) and elevated γH2A.X (Fig.  3 c-d, p  < 0.05) and P53 (Fig.  3 e-f, p  < 0.05) staining, suggesting DNA double-strand breaks and activation of DNA damage responses. At the transcriptional level, CTX upregulated Bax (Fig.  3 g, p  < 0.05) and Caspase-3 (Fig.  3 i, p  < 0.05) while downregulating Bcl-2 (Fig.  3 h, p  < 0.05), shifting the balance toward apoptosis. SeNPs treatment reduced Tunnel positivity, decreased γH2A.X and P53 staining, downregulated Bax and Caspase-3 , and upregulated Bcl-2 , thereby protecting granulosa cells from genotoxic stress and apoptosis. Fig. 3 Ameliorative Effects of SeNPs on CTX-Induced DNA Damage and Apoptosis Ameliorative Effects of SeNPs on CTX-Induced DNA Damage and Apoptosis Transcriptomic analysis of ovarian tissues was conducted to explore underlying mechanisms. Quality control through violin plots (Supplementary Fig. 3a), Pearson correlation (Supplementary Fig. 3b), and heatmaps (Supplementary Fig. 3c) confirmed high data reliability. A total of 1,476 DEGs were identified in CTX vs. NC (831 upregulated, 645 downregulated), and 1,535 DEGs in SeNPs vs. CTX (923 upregulated, 612 downregulated) (Supplementary Fig. 4a-b). Hierarchical clustering distinguished transcriptomic signatures between groups (Supplementary Fig. 4c-d). GO enrichment showed that CTX-induced DEGs were mainly involved in cell adhesion, inflammatory response, ERK1/2 cascade regulation, chemokine signaling, immune response, and angiogenesis inhibition (Fig.  4 a). KEGG analysis indicated disruptions in pathways including PPAR signaling, retinol metabolism, steroid hormone biosynthesis, p53, JAK-STAT, TLR, Th17 differentiation, IL-17, and NF-κB signaling (Fig.  4 c). In contrast, SeNPs-regulated DEGs were enriched in processes related to cell differentiation, apoptosis, adhesion, cell cycle, DNA repair, and proliferation control (Fig.  4 b). KEGG analysis revealed protective roles of SeNPs via PI3K/AKT, cell cycle regulation, mitophagy, senescence, TGF-β, p53, glutathione metabolism, ferroptosis, and mTOR signaling (Fig.  4 d). Fig. 4 Molecular Mechanisms Underlying the Ameliorative Effects of SeNPs on CTX-Induced POF Molecular Mechanisms Underlying the Ameliorative Effects of SeNPs on CTX-Induced POF To validate the therapeutic potential in vitro, granulosa cells were exposed to 4-HC, the active in vitro metabolite of CTX. Cell viability was significantly reduced compared to NC (Supplementary Fig. 5a, p  < 0.05), with suppressed secretion of E 2 and P 4 (Supplementary Fig. 5b-c, p  < 0.05). SeNPs improved viability and hormone secretion. Based on dose-response analysis, 200 µg/mL SeNPs were selected for further experiments. 4-HC reduced EdU-positive cells (Fig.  5 a-b, p  < 0.05), induced cell cycle arrest (Fig.  5 c-d, Supplementary Fig. 6), elevated γH2A.X (Fig.  5 e-f, p  < 0.05), and increased apoptosis (Fig.  5 g-h, p  < 0.05). It also altered expression of folliculogenesis- and apoptosis-related genes (Fig.  5 i-n, p  < 0.05). SeNPs supplementation increased proliferation, normalized the cell cycle, reduced DNA damage and apoptosis, and restored gene expression profiles. Fig. 5 Ameliorative Effects of SeNPs on 4-HC-Induced Proliferation Deficiency and Apoptosis in Granulosa Cells Ameliorative Effects of SeNPs on 4-HC-Induced Proliferation Deficiency and Apoptosis in Granulosa Cells According to the results of transcriptomic analysis, the expression levels of proteins associated with the PI3K/AKT signaling pathway in granulosa cells were corroborated by western blot. Western blot analysis showed that 4-HC downregulated phosphorylated PI3K and AKT (Fig. 6 a-c, p < 0.05), indicating pathway suppression. The downregulation of the PI3K/AKT axis was concomitant with reduced granulosa cell proliferation and increased apoptosis, in line with previous studies [ 39 ]. SeNPs partially restored phosphorylation of PI3K and AKT, suggesting that SeNPs activate the PI3K/AKT signaling pathway, thereby mitigating the detrimental effects of 4-HC exposure and protecting granulosa cells from apoptosis and growth inhibition. Fig. 6 Protective Effect of SeNPs Supplementation on 4-HC-Induced Downregulation of the PI3K/AKT Signaling Pathway in Granulosa Cells Protective Effect of SeNPs Supplementation on 4-HC-Induced Downregulation of the PI3K/AKT Signaling Pathway in Granulosa Cells Previous studies have established that the PI3K/AKT signaling pathway and glutathione metabolism play pivotal roles in mediating cell ferroptosis, a process partly consistent with our findings [ 40 , 41 ]. To further validate these results in vivo, we investigated the protective effects of SeNPs on 4-HC-induced ferroptosis in granulosa cells. As shown in Fig.  7 a, amp and b, 4-HC exposure significantly increased the intensity of DCFH-DA staining, a marker of ROS production, in granulosa cells compared to the NC group ( p  < 0.05). SeNPs supplementation attenuated ROS accumulation, as evidenced by a significant reduction in DCFH-DA staining intensity in the SeNPs-treated group relative to the 4-HC group ( p  < 0.05). In addition, exposure to 4-HC led to a marked decrease in MitoTracker staining intensity (Fig.  7 c & d, p  < 0.05) and MMP collapse (Fig.  7 e & f, p  < 0.05), indicative of mitochondrial dysfunction. This was further supported by alterations in mitochondrial complex activity (Fig.  7 j-k, p  < 0.05). SeNPs supplementation restored MitoTracker staining intensity, stabilized MMP, and improved mitochondrial complex activity in SeNPs-treated granulosa cells ( p  < 0.05), demonstrating the protective effect of SeNPs on mitochondrial integrity and function. Fig. 7 Protective Effect of SeNPs Supplementation on 4-HC-Induced Ferroptosis in Granulosa Cells Protective Effect of SeNPs Supplementation on 4-HC-Induced Ferroptosis in Granulosa Cells The results also confirmed that GSH activity was significantly reduced in 4-HC-exposed granulosa cells (Fig.  7 l, p  < 0.05), while MDA levels were significantly elevated (Fig.  7 m, p  < 0.05), both indicative of ferroptosis. Additionally, increased ferrous and total iron levels in the 4-HC group support the occurrence of ferroptosis (Fig.  7 n&o, p  < 0.05). SeNPs supplementation increased GSH activity ( p  < 0.05) and reduced both MDA levels and iron content in the SeNPs-treated group compared to the 4-HC group ( p  < 0.05). These changes, together with the expression patterns of ferroptosis-related proteins (Fig.  7 p-r), confirm that SeNPs mitigate ferroptosis-induced damage in granulosa cells.

All chemicals, reagents, culture media, and supplements were obtained from Sigma-Aldrich and Thermo Fisher Scientific unless otherwise noted. SeNPs were generously provided by Mrs. Mengqi Wang, College of Life Science and Oceanography, Weifang University, Shandong, China. Six-week-old female C57BL/6 mice were sourced from SPF (Beijing) Biotechnology Co., Ltd. and housed under controlled environmental conditions at Inner Mongolia Medical University (temperature: 20–23 °C, humidity: 60 ± 5%, 12-h light/dark cycle), with ad libitum access to food and water. After a 7-day acclimatization period, body weight and vaginal smear analysis were performed to ensure uniformity across experimental groups. To induce POF [ 27 ], 40 mice received a single intraperitoneal injection of 50 mg/kg (C0768, Sigma-Aldrich, Shanghai, China) on Day 1, followed by daily intraperitoneal injections of 8 mg/kg CTX for the subsequent 14 d. Mice receiving an equivalent volume of normal saline served as the negative control (NC) group ( n = 10). Following the final CTX injection, POF mice were randomly assigned to four treatment groups ( n = 10 per group): CTX only group (oral 200 µL normal saline), low-dose (LD) SeNPs group (oral 0.5 mg/kg SeNPs in 200 µL normal saline), medium-dose group (MD) SeNPs group (oral 1 mg/kg SeNPs in 200 µL normal saline), and high-dose (HD) SeNPs group (oral 2 mg/kg SeNPs in 200 µL normal saline) [ 28 ]. SeNPs were administered daily for 30 d. It is important to emphasize that all SeNPs-treated mice had previously received CTX to induce POF, and thus the SeNPs intervention was assessed in the context of chemotherapy-induced ovarian injury. The dosages and treatment durations for CTX and SeNPs applied in the present study were selected based on previous studies with small modifications to optimize consistency and efficacy [ 27 , 28 ]. At 24 h following the final treatment, mice were anesthetized with 500 mg/kg tribromoethanol (T831042, Macklin, Shanghai, China) via intragastric administration [ 29 ]. Following final body weight measurement, terminal blood collection was conducted via retro-orbital puncture under deep anesthesia. The blood sample was permitted to clot at room temperature for 30 min and was then centrifuged at 3,000 × g for 10 min at 4 °C [ 30 ]. The resulting serum supernatant was carefully aliquoted and stored for subsequent hormonal analysis. Mice were then humanely euthanized by cervical dislocation, and bilateral ovaries were excised and weighed. Ovarian tissues were washed in phosphate-buffered saline (PBS, P1010, Solarbio, Beijing, China), and the wet ovarian weight was recorded. The ovarian index was calculated as the ratio of ovarian weight to body weight × 100% [ 28 ]. Serum estradiol (E 2 ), AMH, luteinizing hormone (LH), and FSH levels were measured using commercially available ELISA kits (H102-1-1 for E 2 , H324-1-2 for AMH, H206-1-1 for LH, and H101-1-1 for FSH, Jiancheng, Nanjing, China), following the manufacturer’s instructions. For histology analyses, ovarian tissues were collected and fixed in 10% neutral-buffered formalin (G2161, Solarbio, Beijing, China). Ovarian morphology was evaluated by embedding tissues from the NC, CTX, and SeNPs-treated groups in paraffin, following standard protocols [ 31 ]. Serial 5-µm sections were prepared and stained with hematoxylin and eosin (HE) using a commercial kit (G1120, Solarbio, Beijing, China) [ 32 ]. Follicles at various developmental stages (primordial, primary, secondary, mature, and atretic) were counted for each Sect [ 27 ]. Moreover, ovarian tissues from the NC, CTX, and SeNPs-treated groups were initially fixed in 2.5% glutaraldehyde (G1102, Solarbio, Beijing, China), followed by post-fixation in 1% osmium tetroxide, dehydration in ethanol, embedding, and sectioning. The prepared ultrathin sections were stained with uranyl acetate and lead citrate and then visualized using transmission electron microscopy (TEM) by the Shandong Huiyan Scientific Services Co., Ltd (Qingdao, China) [ 33 ]. Immunohistochemistry (IHC) staining was performed to assess protein expression related to folliculogenesis (INHIBIN α, FOXO3A, and SIRT1) and DNA damage (P53 and γH2A.X) [ 27 ]. Paraffin-embedded ovarian tissue sections were dewaxed, rehydrated, and subjected to antigen retrieval. Sections were blocked with 10% bovine serum albumin (BSA, SW3015, Solarbio, Beijing, China) at room temperature for 30 min, followed by overnight incubation with primary antibodies at 4 °C. After washing, sections were incubated with HRP-conjugated secondary antibodies at 25 °C for 30 min, followed by visualization with 3,3′-diaminobenzidine (DAB, DA1010, Solarbio, Beijing, China) and counterstaining with hematoxylin. Staining intensity was quantified using ImageJ software. The details of the antibodies used in this study have been summarized in Supplementary Table 1. According to the manufacturer’s instructions, oxidative stress in ovarian tissues was assessed by measuring the activities of antioxidant enzymes (SOD, GPH-p x , GSH, CAT, and MDA) using commercial kits (BC0175 for SOD, BC1195 for GPH-p x , BC1175 for GSH, BC0205 for CAT, and BC0025 for MDA, Solarbio, Beijing, China) [ 34 , 35 ]. Apoptosis in ovarian tissues was identified using terminal deoxynucleotidyl transferase dUTP nick-end labeling (Tunnel) staining [ 27 ]. After deparaffinization and rehydration, tissue sections were treated with proteinase K and incubated with TdT reaction solution (G1502, Servicebio, Wuhan, China) at 37 °C for 60 min. After incubation with Streptavidin-Horse-radish peroxidase and DAB solution, sections were counterstained with hematoxylin. Tunnel-positive signals were quantified using ImageJ software [ 34 ]. To explore the molecular mechanisms underlying SeNPs’ effects on ovarian tissues, transcriptomic analysis was performed [ 34 ]. RNA was extracted from ovarian tissues of NC, CTX, and SeNPs-treated groups ( n = 3). Libraries were constructed using the NEBNext ® Ultra™ RNA Library Prep Kit (New England Biolabs, Ipswich) and sequenced on the Illumina NovaSeq 6000 platform at Gene Denovo Biotechnology Co., Ltd. Differentially expressed genes (DEGs) were identified using DESeq2 software, considering genes with false discovery rates (FDR) < 0.05 and absolute fold changes ≥ 2. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify relevant biological functions and signaling pathways. Granulosa cells were isolated from three-week-old female C57BL/6 mice, following established protocols with minor modifications [ 27 ]. Mice were injected intraperitoneally with 10 IU/mL pregnant mare serum gonadotropin (PMSG) and sacrificed 48 h post-injection. Bilateral ovaries were collected, washed with Dulbecco’s phosphate-buffered saline (DPBS, 14190250, Thermo Fisher Scientific, Shanghai, China) containing 100 U/mL penicillin/streptomycin (15070063, Thermo Fisher Scientific, Shanghai, China), and mechanically disrupted using 27-gauge needles. Ovarian tissue was then digested with hyaluronidase (H3605, Sigma-Aldrich, Shanghai, China) to isolate granulosa cells, which were then cultured in DMEM/F12 medium (11320033, Thermo Fisher Scientific, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, 10091148, Thermo Fisher Scientific, Shanghai, China) and 100 U/mL penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO₂. Granulosa cells were treated with 100 µg/mL 4-hydroxycyclophosphamide (4-HC, abs42009947, Absin, Shanghai, China), either alone or in combination with SeNPs at concentrations of 100 (LD group), 200 (MD group), and 300 (HD group) µg/mL in a humidified incubator (37 °C with 5% CO₂) for 24 h. The experimental groups included: NC without any drug treatment, 4-HC only group, and 4-HC combined with SeNPs. It should be clearly noted that all SeNPs treatments in this in vitro model were administered in combination with 4-HC, to evaluate their protective effects against chemotherapy-induced cellular damage. The dosages and treatment durations for 4-HC and SeNPs applied in the present study were selected based on previous studies, with minor adjustments to optimize the experimental conditions [ 36 , 37 ]. Cellular dysfunction was assessed by evaluating the cell viability and the secretion levels of E 2 and progesterone (P 4 ) [ 34 ]. For viability assay, cells of each group were incubated with MTT reagent (C0009S, Beyotime, Shanghai, China) for 4 h, followed by the addition of dimethyl sulfoxide (DMSO; 67-68-5, Coolaber, Beijing, China) solution. The absorbance at 570 nm was measured using a microplate reader. For assessing the secretion levels of E 2 and P 4 , supernatant of each group was collected, with the levels of E 2 and P 4 measured by commercial ELISA kits (H089-1-2 for P 4 , Jiancheng, Nanjing, China) according to the manufacturer’s instructions. These results were used to determine the optimal SeNPs concentration for subsequent analyses. Granulosa cell proliferation was evaluated using the EdU incorporation assay [ 34 ]. Cells post-drug treatment were incubated with 10 µM EdU solution (C0071S, Beyotime, Shanghai, China) for 2 h, followed by fixation in 4% paraformaldehyde (PFA, P1110, Solarbio, Beijing, China) and counterstaining with DAPI (C0065, Solarbio, Shanghai, China). EdU-positive cells were quantified using a fluorescence microscope, and the relative intensity was quantified using ImageJ software. After granulosa cell collection, the cell cycle progression was assessed by flow cytometry (FACS) after fixing granulosa cells in ice-cold 70% ethanol overnight [ 38 ]. Cells were then stained with propidium iodide (PI, MA0334, Meilunbio, Dalian, China) and analyzed by FACS. The distribution of cells across the G0/G1, S, and G2/M phases was determined. DNA damage was assessed using immunofluorescence (IF) staining [ 38 ]. For IF staining, cells post-drug treatment were fixed, permeabilized with 0.5% Triton X-100 solution (T8200, Solarbio, Beijing, China) at 37 °C for 15 min, and incubated with anti-γH2A.X antibody at 4 °C overnight, followed by secondary antibody incubation and counterstaining with DAPI. DNA damage was quantified based on γH2A.X staining intensity using ImageJ software. The details of the antibodies used in this study have been summarized in Supplementary Table 1. Cells post-drug treatment were collected, washed with cold DPBS, and incubated with Annexin V-FITC (CA1020, Solarbio, Beijing, China) at 37 °C for 5 min, followed by PI staining. Subsequently, apoptosis was assessed using FACS [ 38 ]. Cells post-drug treatment were stained with 10 µM dichlorofluorescein diacetate probes (DCFH-DA, S0033, Beyotime, Shanghai, China) at 37 °C for 30 min, followed by DAPI counterstaining. ROS production levels were quantified by fluorescence microscopy and ImageJ software [ 34 , 38 ]. Mitochondrial activity and mitochondrial membrane potential (MMP) collapse were assessed using MitoTracker staining and JC-1 staining. For MitoTracker staining, cells post-drug treatment were incubated with 200 nM MitoTracker Red (C1049, Beyotime, Shanghai, China) at 37 °C for 30 min, followed by DAPI counterstaining. The fluorescence intensity was recorded and analyzed using Image J software. For JC-1 staining, cells post-drug treatment were incubated with 10 µM JC-1 (C2006, Beyotime, Shanghai, China) at 37 °C for 20 min. Fluorescence intensity was measured using a fluorescence microscope, and the ratio of J-aggregate (red) to J-monomer (green) fluorescence was used to quantify the MMP stability. After granulosa cell collection, levels of ferroptosis-related biomarkers (GSH, MDA, ferrous iron, and total iron) and activities of mitochondrial complex were measured using commercial kits according to the manufacturer’s protocols [ 38 ]. Total RNA was extracted from ovarian tissues and granulosa cells using Trizol reagent (79306, Thermo Fisher Scientific, Shanghai, China). cDNA synthesis was performed with a Prime Script™ RT reagent kit (RR047A, Takara, Dalian, China). qRT-PCR was conducted on a Roche Lightcycler 480 II System, with GAPDH as the internal control. Relative gene expression was calculated using the 2 −ΔΔCt method [ 32 ]. The gene primer sequences applied in the present study are added in Supplemental Table 2. Total protein lysates were extracted from granulosa cells using RIPA buffer (R0010, Solarbio, Beijing, China) supplemented with protease inhibitors. Protein concentration was determined using a BCA assay (PC0020, Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes ( PVH00010 , Millipore, Shanghai, China), and blocked with 5% BSA. Membranes were incubated overnight with primary antibodies targeting PI3K/AKT signaling pathway and ferroptosis, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (W1001, Promega, Beijing, China) and imaged with a chemiluminescence imaging system. The details of the antibodies used in this study have been summarized in Supplementary Table 1. Statistical analysis was performed using SPSS software (IBM, version 19.0). Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was conducted to compare differences among groups, and the homogeneity of variances was assessed using Levene’s test. If the assumption of equal variances was met, post hoc comparisons were performed using the least significant difference (LSD) test. For data that did not meet the assumptions of normality or equal variance, the non-parametric Wilcoxon rank-sum test was used. A p -value < 0.05 was considered statistically significant.

In summary, this study establishes SeNPs as a multifaceted therapeutic agent for POF, capable of counteracting CTX-induced ovarian damage through PI3K/AKT signaling pathway activation and ferroptosis inhibition. These findings not only advance applications of SeNPs in reproductive health but also highlight ferroptosis as a novel therapeutic target in ovarian disorders. Future studies should prioritize clinical translation and mechanistic exploration to fully harness SeNPs’ potential in preserving fertility and endocrine function.

Over the past few decades, significant advances in cancer screening, prognosis, and treatment have markedly improved survivorship rates, yet declining cancer-related mortality has unveiled chemotherapy-induced sequelae in reproductive function preservation [ 42 ]. Ovarian tissue exhibits pronounced vulnerability to widely utilized chemotherapeutics, including CTX, cisplatin, doxorubicin, and paclitaxel, which lead to the longer-term side of chemotherapy-associated ovarian damage (CAOD). This pathological process manifests as follicular pool depletion, POF, and early menopause, ultimately compromising fertility [ 43 , 44 ]. Several pharmacological interventions, such as antioxidants and gonadotropin-releasing hormone (GnRH) agonists, have demonstrated partial efficacy in mitigating CTX-induced POF. These agents appear to counteract the alterations in the ovarian microenvironment, such as angiogenesis disturbance, cell senescence, extracellular matrix (ECM) deposition, immune microenvironment disorder, oxidation-reduction imbalance, and ovarian stem cell exhaustion [ 43 – 46 ]. However, their clinical utility remains constrained by transient effects, nonspecific targeting, and incomplete protection against follicular loss [ 43 ]. Emerging strategies prioritize tissue-specific drug delivery, sustained therapeutic activity, and the concurrent targeting of multiple signaling pathways, paving the way for therapies that not only preserve fertility but also restore ovarian function [ 43 ]. To address the limitations of current pharmacological interventions, innovative nanotechnology has revolutionized advanced drug delivery systems (DDSs) in recent years, enabling site-specific and controlled therapeutic administration while expanding treatment options [ 47 , 48 ]. Central to this field is the intriguing application of NPs-structures ranging from 1 to 100 nanometers-which exhibit unique electronic, mechanical, and physicochemical properties distinct from bulk materials [ 49 , 50 ]. Among various NPs, SeNPs, as a type of modern nano-composites above their elemental analogs, have garnered significant attention due to their low systemic toxicity, high permeability, excellent biocompatibility, and selective cytotoxicity toward malignant cells, making them promising candidates for therapeutic applications, particularly in DDSs [ 47 , 50 , 51 ]. Numerous studies have demonstrated that the economical and eco-friendly synthesis of SeNPs using physical, chemical, and biological approaches, offers distinct advantages in particle size, bioactivity, stability, and biocompatibility [ 13 ]. Furthermore, functionalizing SeNPs with specific targeting ligands and genes enables precise site-specific drug delivery, reducing off-target effects, and enhancing therapeutic efficacy [ 13 , 51 ]. Their desirable ability to optimize pharmacokinetics, improve therapeutic specificity, and modulate multiple signaling pathways highlights the transformative potential of SeNPs in precision medicine and nanotherapeutics [ 47 ]. Consistent with previous studies highlighting the protective roles of SeNPs in ovarian health [ 11 , 24 , 26 ], our data show that SeNPs administration effectively ameliorated changes in ovarian weight, ovarian index, serum hormone levels, and folliculogenesis in CTX-treated mice. Furthermore, SeNPs reduced CTX-induced DNA damage and apoptosis in ovarian granulosa cells. Mechanistically, the transcriptomic analysis revealed that SeNPs exert their protective effects against CTX-induced POF primarily by activating the PI3K/AKT signaling pathway and inhibiting ferroptosis. This mechanism was further validated in granulosa cell-based experimental models, where SeNPs reversed the 4-HC-induced granulosa cell dysfunctions by activating the PI3K/AKT signaling pathway and inhibiting ferroptosis. The PI3K/AKT signaling pathway, a conserved eukaryotic network, plays a pivotal role in regulating essential cellular processes, including cell survival, proliferation, differentiation, autophagy, apoptosis, metabolic homeostasis, and stress response adaptation [ 52 , 53 ]. By integrating extracellular signals and engaging in complex cross-talk with other multiple signaling pathways, this complex network modulates cellular function, influences transcriptional activity, and coordinates critical decisions regarding cell fate [ 54 ]. Dysregulation of this pathway is implicated in a range of pathological conditions, including cardiovascular diseases, oncogenesis, neurodegenerative diseases, and metabolic disorders [ 53 , 55 – 57 ]. Previous studies have also highlighted the critical role of the PI3K/AKT signaling pathway in mammalian ovarian development, particularly in regulating steroid hormone synthesis, follicle development, granulosa cell survival, and ovarian reserve maintenance [ 58 – 62 ]. In agreement with prior publications [ 63 , 64 ], our data confirmed that CXT-induced POF is associated with the inhibition of the PI3K/AKT signaling pathway in granulosa cells. Given the critical role of the PI3K/AKT signaling pathway in follicular survival, therapeutic agents such as rapamycin [ 63 ] and metformin [ 64 ] show promise in managing and potentially reversing CXT-induced POF. Notably, our findings demonstrated that SeNPs restore the PI3K/AKT signaling pathway in CTX-treated granulosa cells, offering a novel therapeutic avenue for CXT-induced POF. In 2022, Mi et al. reported that silymarin-functionalized SeNPs suppressed the PI3K/AKT signaling pathway, thereby inducing autophagy and apoptosis in gastric cancer cells [ 65 ]. Similarly, Kaempferia parviflora root extract-modified SeNPs inhibited the PI3K/AKT axis, triggering programmed cell death in AGS cells [ 66 ]. Additionally, zinc oxide (ZnO)-hybrid SeNPs promoted apoptosis and autophagy in Mycobacterium tuberculosis -infected macrophages by inhibiting the PI3K/AKT signaling pathway [ 67 ]. In contrast, lentinan polysaccharides-functionalized SeNPs significantly activated the PI3K/AKT pathway in neuronal cells, promoting their proliferation, differentiation, and survival [ 68 ]. This apparent contradiction likely stems from the context-dependent nature of SeNPs’ biological effects, which are influenced by both the cellular environment and nanoparticle properties. In cancerous or pathogen-infected cells, where the PI3K/AKT pathway is often constitutively overactivated, SeNPs may exert selective cytotoxicity by inhibiting this survival pathway to promote apoptosis or autophagy. Conversely, in non-malignant cells exposed to oxidative or cytotoxic stress, such as CTX-injured granulosa cells, SeNPs may activate suppressed PI3K/AKT signaling to enhance cell survival and antioxidant defenses. Additionally, differences in physicochemical characteristics such as size, charge, and surface functionalization may further shape cellular uptake and signaling responses. These findings underscore the functional versatility of SeNPs as modulators of PI3K/AKT signaling in a cell-type- and context-specific manner. In the present study, we observed that SeNPs not only rescued granulosa cells from CTX-induced damage by activating the PI3K/AKT signaling pathway but also effectively suppressed ferroptosis, suggesting a mechanistic convergence between these two processes. Ferroptosis, a recently characterized form of programmed cell death, is uniquely triggered by iron-dependent lipid peroxidation, distinguishing it from other cell death mechanisms. This process has garnered substantial interest in recent years due to its involvement in various pathological conditions, including those contributing to reproductive dysfunction. Germline cells, such as granulosa cells, oocytes, and trophoblast cells, are particularly vulnerable to ferroptosis, particularly under pathological conditions, owing to their intrinsic vulnerability to oxidative stress and iron dysregulation [ 69 ]. Extensive research has underscored the pathogenic role of ferroptosis in ovarian disorders, including endometriosis, PCOS-associated infertility, and tubal factor infertility [ 69 – 72 ]. In the context of CTX-induced POF, ferroptosis activation has been shown to significantly exacerbate ovarian dysfunction and cellular damage, supporting its critical role in chemotherapy-induced ovarian toxicity [ 73 – 77 ]. Consistent with previous studies highlighting the anti-ferroptotic effects of SeNPs [ 78 – 85 ], our results demonstrate that SeNPs alleviate CTX-induced POF via a dual mechanism: activation of the PI3K/AKT pathway and inhibition of ferroptotic signaling. Numerous studies have established that PI3K/AKT signaling acts as a negative regulator of ferroptosis across various disease models, including cancer, rheumatoid arthritis, osteoporosis, and aplastic anemia [ 86 – 90 ]. Mechanistically, PI3K/AKT activation inhibits ferroptosis by enhancing NRF2 nuclear retention and transcriptional activity, leading to upregulation of ferroptosis-suppressor genes such as SLC7A11 and GPX4. The mTORC1 downstream cascade further promotes GPX4 synthesis and stabilizes the protein by limiting its degradation via m6A RNA modification and chaperone interaction [ 91 , 92 ]. Additionally, PI3K/AKT signaling modulates intracellular iron and lipid metabolism, reducing levels of redox-active iron and lipid peroxides, both hallmarks of ferroptosis [ 92 ]. In alignment with these mechanisms, our data show that SeNPs robustly activate PI3K/AKT signaling, upregulate GPX4 and SLC7A11, suppress ferroptosis, and attenuate oxidative damage in granulosa cells. These findings provide compelling evidence that SeNPs act as a multifaceted therapeutic agent, conferring protection against POF by integrating survival signaling with anti-ferroptotic mechanisms. Nevertheless, the precise regulatory mechanisms and complex signaling pathways underlying these protective effects remain to be fully elucidated. In particular, the use of inhibitor-based rescue experiments, such as LY294002 (a PI3K inhibitor) or erastin (a ferroptosis inducer), would provide more direct evidence confirming the mechanistic involvement of the PI3K/AKT pathway and ferroptosis inhibition in mediating the actions of SeNPs. Given the promising results observed, additional studies are necessary to more comprehensively elucidate the mechanisms by which SeNPs counteract CTX-induced ovarian damage, with a particular focus on the interplay between the PI3K/AKT signaling pathway activation and ferroptosis suppression. Such investigations will be crucial to fully clarify the molecular basis of SeNPs’ protective effects and support their potential for clinical translation.

Premature ovarian failure (POF), also termed premature menopause or premature ovarian insufficiency (POI), is a clinically heterogeneous disorder characterized by the loss of ovarian function before the age of 40, affecting 1–4% of women globally [ 1 , 2 ]. The diagnostic criteria for POF include prolonged amenorrhea or oligomenorrhea (≥ 4 months), elevated follicle-stimulating hormone (FSH) ≥ 25 IU/L on two assessments ≥ 4 weeks apart, and hypoestrogenism. Its prevalence escalates with age, impacting 0.01%, 0.1%, and 1% of women at ages 20, 30, and 40, respectively. Etiologically, POF arises from multifactorial mechanisms, including genetic, autoimmune, iatrogenic, and environmental insults [ 2 ]. Genetic contributors encompass X chromosome abnormalities and mutations in autosomal genes vital for folliculogenesis. Autoimmune pathogenesis involves ovarian autoantibodies targeting steroidogenic enzymes or oocyte antigens, which accelerate follicular depletion. Iatrogenic triggers, particularly chemotherapy and radiation, induce direct ovarian damage, while environmental toxins disrupt follicular development. Beyond infertility, POF confers systemic endocrine disturbances, elevating risks of osteoporosis, cardiovascular disease, and cognitive decline, alongside profound psychosocial sequelae, such as depression and diminished quality of life. Despite these insights, the molecular mechanisms linking these factors to POF remain elusive, hampering therapeutic innovation. Current management prioritizes hormone replacement therapy (HRT) to alleviate hypoestrogenic symptoms and mitigate long-term sequelae. However, HRT fails to restore fertility and may elevate the risks of hormone-sensitive malignancies [ 3 ]. Assisted reproductive technologies (ART), particularly in vitro fertilization using donor oocytes, provide limited success in patients with severe follicular depletion [ 4 ]. Critically, existing interventions do not address ovarian rejuvenation or reversal of follicular attrition, underscoring the urgent need for mechanism-driven interventions. During the past decades, nanotechnology has revolutionized biomedical research, offering innovative solutions for complex reproductive pathologies [ 5 ]. By harnessing the unique physicochemical properties of nanomaterials, researchers have developed targeted therapies that enhance drug delivery, improve bioavailability, and minimize off-target effects. In reproductive medicine, nanotechnology-driven interventions hold significant promise for counteracting oxidative stress, preserving follicular reserves, and modulating key molecular pathways implicated in ovarian dysfunction, including polycystic ovary syndrome (PCOS), endometriosis, and chemotherapy-induced POF [ 5 ]. For instance, cerium oxide nanoparticles (CeO₂ NPs), with intrinsic antioxidant properties, have been shown to alleviate high-fat diet (HFD)-induced ovarian dysfunction by reducing reactive oxygen species (ROS) production, enhancing mitochondrial function, and reducing endoplasmic reticulum (ER) stress [ 6 ]. Similarly, CeO₂@Resveratrol NPs exhibit potent antioxidative and anti-inflammatory properties, promoting granulosa cell proliferation in PCOS models [ 7 ]. Curcumin-encapsulated NPs effectively attenuate ischemia-reperfusion injury in ovarian tissues by boosting antioxidant defenses and suppressing pro-inflammatory cascades, thereby preserving follicular integrity [ 8 ]. Moreover, superparamagnetic iron oxide functionalized with curcumin reduces granulosa cell apoptosis in polycystic ovaries [ 9 ]. Additionally, combinatorial L-carnitine and zinc oxide NPs (ZnO NPs) restore hormonal balance and redox homeostasis in diabetic models [ 10 ], further illustrating the potential of nanotechnology to address iatrogenic fertility impairments and highlighting nanotechnology-driven approaches to combat reproductive dysfunction. Among these, selenium nanoparticles (SeNPs) have emerged as promising candidates in biomedicine, distinguished by their biocompatibility, antioxidant efficacy, and tunable therapeutic delivery [ 11 ]. Selenium, an essential trace element, constitutes the structural core of a diverse family of selenoproteins, collectively termed the selenoproteome, which orchestrate redox homeostasis, inflammation regulation, mitochondrial function, and cellular protection [ 12 , 13 ]. While canonical selenoproteins such as GPXs and TrxRs directly neutralize ROS, other selenoproteins, including Selenoprotein S (SelS), Selenoprotein P (SelP), Selenoprotein M (SelM), Selenoprotein I (SELENOI), Selenoprotein F-Like (SPF-L), and Selenoprotein BthD, critically support ovarian health by regulating ER stress, maintaining follicle integrity, mitigating oxidative damage, delaying age-related decline, and modulating chemoresistance, collectively safeguarding female reproductive function [ 14 – 19 ]. Meanwhile, accumulating evidence also address selenium’s protective role in ovarian health. Experimental studies demonstrate that selenium pretreatment alleviates doxorubicin-induced ovarian toxicity by enhancing antioxidant defenses, suppressing inflammation and apoptosis, and preserving follicular architecture [ 20 ]. Clinically, selenium combined with vitamin E supplementation improves ovarian reserve markers, including anti-Müllerian hormone (AMH), antral follicle count (AFC), and mean ovarian volume (MOV), in women with occult premature ovarian insufficiency (OPOI) [ 21 ]. Compared with these conventional selenium formulations, SeNPs display enhanced stability, bioavailability, and safety, positioning them as superior candidates for targeted ovarian therapies [ 22 , 23 ]. Mechanistically, SeNPs preserve ovarian function by modulating steroidogenesis-related genes and androgen receptor signaling pathways [ 11 ]. Their dual antioxidant and anti-inflammatory properties ameliorate ovarian pathologies, as demonstrated in letrozole-induced PCOS models, where SeNPs restore hormonal equilibrium and promote folliculogenesis [ 24 ]. Selenium-encapsulated chitosan NPs counteract silver NPs-induced ovarian damage by scavenging ROS and suppressing inflammation [ 25 ]. Moreover, synergistic approaches, such as co-administering SeNPs with metformin, alleviate PCOS in rat models by normalizing the PI3K/AKT signaling pathway, mitochondrial dynamics, and redox balance [ 26 ]. Despite these promising advances, the therapeutic potential of SeNPs in POF remains underexplored. In this study, we aim to investigate the therapeutic efficacy of SeNPs in mitigating cyclophosphamide (CTX)-induced POF. By elucidating the mechanisms underlying the protective effects of SeNPs, we seek to provide a scientific basis for the development of SeNPs-based therapies for POF. Our findings will not only advance our understanding of SeNPs but also highlight the potential of nanotechnology in improving ovarian function and supporting fertility preservation.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2.