Abstract Background Age-related diminished ovarian reserve (DOR) leads to declining fertility, miscarriage, and systemic health issues. Cell-free fat extract (CEFFE) has demonstrated therapeutic effects in various tissues. In our previous study, CEFFE successfully improved ovarian function in mice without adverse effects; however whether it can prevent DOR remains unclear. This study aimed to determine if early intervention with CEFFE can delay DOR and extend reproductive lifespan, exploring its potential as a novel clinical approach for women with DOR. Methods The mice in the Prophylactic group received tail vein injections of 200 μL of CEFFE per mouse every 3 days for three months; the Control group were administered an equivalent volume of saline injections. Post-treatment outcomes assessed included body weight, ovarian weight, follicle count, embryo quality, production rates, and levels of serum hormones. Safety was evaluated via organ weights and hematoxylin and eosin staining in parents and offspring. The impact of CEFFE on cell proliferation, hormone synthesis, oxidative stress, and senescence was assessed via Cell counting Kit-8 assays, enzyme-linked immunosorbent assays, and reverse transcription quantitative real-time PCR, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide and Senescence-associated beta-galactosidase staining. Results Compared with the Control group, CEFFE treatment effectively preserved ovarian function in aging mice, improving ovarian weight, hormone levels, and follicular development, and reduced follicular atresia. CEFFE treatment enhanced embryo quality and development, induced a higher pregnancy rate, reduced the number of abnormal pregnancies, and increased the litter size and the number of live births. CEFFE demonstrated favorable safety in the Prophylactic group and their offspring, with no significant adverse effects on organ morphology or coefficients, except for increased liver weight in treated mice. Transcriptomic analysis suggested CEFFE influences cell proliferation, hormone response, and oxidative stress pathways in ovarian granulosa cells, which was verified in primary mouse granulosa cells. In vitro studies demonstrated that CEFFE pre-treatment alleviated the phenotype of Control mice by inhibiting oxidative stress, promoting proliferation, and enhancing hormone secretion. Conclusions Early prophylactic administration of CEFFE in adult mice can prevent DOR and extend their reproductive lifespan by inhibiting granulosa cell senescence. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04383-6. Keywords: Cell-free fat extract, Diminished ovarian reserve, Granulosa cell senescence Background Optimal ovarian reserve is a prerequisite for maintaining normal female fertility. However, under normal physiological conditions, ovarian reserve declines with advancing age. This phenomenon, known as physiological diminished ovarian reserve (DOR), is primarily characterized by elevated basal follicle-stimulating hormone (FSH) levels, decreased anti-Müllerian hormone (AMH) levels, a reduced antral follicle count, and a decline in both the quantity and quality of oocytes. Thus, DOR significantly impacts female reproductive health [[34]1]. Research has indicated that fertility begins to decline at 31 years old, with a marked acceleration after 35 years old [[35]2], accompanied by an increase in chromosomal abnormalities, leading to higher rates of infertility, miscarriage, or severe congenital disorders [[36]3]. By 45 years old, the infertility rate reaches 87% [[37]4, [38]5]. Furthermore, DOR is associated with systemic changes, including a sharp increase in the incidence of cardiovascular, skeletal, and neurological diseases [[39]6–[40]8], and might even affect female longevity [[41]9]. Therefore, early intervention to protect ovarian function, delay ovarian reserve decline, and extend fertility remains a significant challenge in assisted reproductive treatments. Research investigating preventative strategies for DOR remains limited; however, some studies have shown promising results. Long-term nicotinamide mononucleotide administration in young mice was effective in preventing ovarian decline and reducing inflammation; however, its impact on fertility requires further investigation [[42]10]. Melatonin has been shown to improve oocyte quality and ovarian hormone secretion, although clinical studies have not observed significant improvements in pregnancy or live birth rates. Coenzyme Q10, while improving mitochondrial function and oocyte quality, has demonstrated inconsistent results in clinical trials regarding ovulation stimulation [[43]11]. Overall, the clinical efficacy of these substances in preventing DOR remains unsatisfactory. Extensive research has explored the use of stem cells or exosomes in improving ovarian reserve function in mice and rats [[44]12–[45]14]. However, these approaches have limitations, hindering their clinical application. Furthermore, the potential risks associated with stem cell therapy, including tumorigenicity, immunogenicity, and ethical concerns, preclude its use as a preventative measure for DOR [[46]10]. Therefore, prioritizing research focused on preventing physiological ovarian decline and identifying novel, clinically applicable active substances for the effective prevention of DOR is of paramount importance. Cell-free fat extract (CEFFE) is derived from adipose tissue through mechanical emulsification, followed by multiple rounds of centrifugation to remove cellular debris and viable cells, followed by sterilization to obtain the active protein components. There are several advantages to CEFFE, including autologous tissue origin, low immunogenicity, non-tumorigenicity, high clinical safety, and minimal ethical risks. It contains abundant cytokines and tissue factors with pro-angiogenic and proliferative activities [[47]15]. Indeed, CEFFE has demonstrated therapeutic effects in promoting wound healing [[48]16, [49]17], cartilage regeneration, and inhibition of osteocyte apoptosis [[50]18, [51]19]. Our previous studies revealed that CEFFE can also promote endometrial repair [[52]13], and improve both physiologically DOR and chemotherapy-induced pathological premature ovarian insufficiency (POI), effectively restoring fertility in mice without adverse effects on the health of the parent or offspring [[53]20]. The present study aims to further investigate whether early intervention with CEFFE, administered before the onset of ovarian decline, can delay DOR, and extend the reproductive lifespan in mice. The ultimate goal is to explore a novel clinical intervention to prevent ovarian reserve decline and prolong reproductive lifespan in women. Methods Preparation of CEFFE SEME Cell Technology Co., Ltd. (Shanghai, China) provided the CEFFE and the extraction solution was described previously [[54]15]. A stock solution of CEFFE was prepared at 10 μg/μL and stored at −80 °C. Treatment of mice The work has been reported in section with the ARRIVE guidelines 2.0 ([55]https://arriveguidelines.org/), and a completed ARRIVE guidelines 2.0 checklist was included as a supplementary file. Forty-five female and twelve male C57BL/6 J mice were obtained from and housed at the Shanghai Branch of Beijing Vital River Laboratory Animal Technologies (Shanghai, China). Female mice with longitudinal vaginal septum were excluded from the study. All experimental protocols were endorsed by the Animal Research Committee of Vital River Laboratory Animal Technologies (IACUC-P2022002 authorization) in compliance with the Guide for the Care and Use of Laboratory Animals (8th edition, 2011, US National Institutes of Health publication). Female mice were randomly assigned to either the Control or Prophylactic group. The Control and Prophylactic groups comprised 20-week-old female mice. The Prophylactic group received tail vein injections of 200 μL of CEFFE per mouse every 3 days for 3 months. In contrast, the Control group received saline injections of an equal volume at the same period. Prior to the start of the experiment, all mice underwent a 4-week acclimatization period during which their health status was closely monitored to ensure suitability for inclusion in the study. Anesthesia was not administered during the animal procedures in this study. Mouse vaginal smears Vaginal smears were performed daily at 10 am for 2 weeks after the mice had finished CEFFE treatment. The small cotton swab wetted with normal saline was gently inserted into the vagina of mice, and the exfoliated vaginal cells were collected by rotating clockwise for five turns. Smears were made on adherent slides and allowed to dry before hematoxylin and eosin (H&E) staining. The morphology of the cells was observed under a microscope and the stage of the estrous cycle was recorded. Enzyme-linked immunosorbent assay (ELISA) Blood samples were obtained from the retro-orbital sinus and allowed to stand for 30 min at room temperature. Samples were centrifuged at 2000 g for 15 min and the serum supernatant was collected. Subsequently, AMH, Estradiol (E[2]), and FSH levels were measured using ELISA kits (Mlbio, Shanghai, China). In vitro embryo culture Three months after CEFFE or control treatment, each group of mice was administered with 10 IU of pregnant mare serum gonadotropin for superovulation, followed by 10 IU of human chorionic gonadotropin 48 h later. Then, females were mated immediately with 8-week-old, fertile males. Oocytes or fertilized eggs were collected from the ampulla 18 h post-mating and cultured in vitro. The number of total retrieved eggs was recorded. Next, the oocytes or fertilized eggs were cultured in droplets containing 40 μL of K + Simplex Optimised Medium (Millipore, Billerica, MA, USA) under an environment of 37 °C and 5% CO[2,] and covered with mineral oil (Sigma, St. Louis, MO, USA). Blastocysts were observed and recorded approximately 3 days later. Euthanasia of mice Mice were euthanized using carbon dioxide (CO[2]) inhalation. A flow rate of 6.5 L/min of CO[2] was used to fill the euthanasia chamber. Mice were exposed to CO[2] for a minimum of 5–6 min until cessation of breathing. Following respiratory arrest, mice were left in the chamber for an additional 2 min to ensure death. Death was confirmed by thoracotomy, involving bilateral incision of the chest cavity to prevent recovery from asphyxiation. Ovarian follicle quantification Ovaries were collected after three months of CEFFE treatment. After fixation with 4% paraformaldehyde, the ovaries were paraffin-embedded, sectioned at 5 μm, and then subjected to H&E staining. The follicles were counted in every tenth section, employing distinct morphological characteristics to classify them into various stages, including primordial, primary, secondary, antral, and atretic follicles, as described previously [[56]21]. Finally, the relative proportions of these different follicle types within each ovary were further analyzed. Fertility assay After treating the female mice with CEFFE, they were mated with 8-week-old male mice of normal fertility. Long-term cohabitation was maintained at a 1:1 ratio of female to male mice until the female mice became pregnant. At this point, they were individually housed until giving birth and the number of newborn mice and the time of delivery was recorded. If a female mouse did not become pregnant for an extended period, the cohabitation period was prolonged for a total of 3 months from the first day of pairing. Females were individually housed for 21 days to confirm pregnancy status after the males were removed. The pregnancy rate was calculated as the number of female mice that gave birth divided by the total number of female mice paired for mating. The abnormal pregnancy rate was calculated as the sum of stillbirths, dystocia cases, and abortions divided by the total number of pups delivered. Parent and offspring mouse safety assessments Safety assessment was conducted at three months following the completion of CEFFE treatment. After euthanizing the mice, various organs, including the heart, liver, spleen, lungs, kidneys, uterus, and ovaries, were harvested. Organ weights were measured, organ coefficients were calculated, and H&E staining was performed. The same procedure was applied to the offspring mice, and their body weights were monitored weekly from birth up to the 8th week. Immunohistochemistry (IHC) Ovarian sections underwent IHC with the following steps: dewaxing, rehydration, antigen retrieval, peroxidase and serum blocking, incubation with primary and secondary antibodies, 3,3′-Diaminobenzidine staining, nuclear counterstaining, and mounting. Specific details about the primary antibodies can be found in Additional file: Table S1. Cell culture For the in vitro study, the human ovarian granulosa-like tumor (KGN) cell line (Feiya Biotechnology Co., Ltd., Taizhou, China) was utilized. KGN cells were cultured at 37 °C, 5% CO[2] in Dulbecco’s modified Eagle’s medium/F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). Mouse granulosa cell collection The ovarian follicles were punctured using 30-gauge needles, allowing for the precise separation of oocytes from the surrounding granulosa cells under a stereomicroscope. Granulosa cells (GCs) were collected, snap-frozen, and stored at − 80 °C until RNA-seq or RT-qPCR analysis. Bioinformatic analysis of RNA sequencing Total RNA was isolated from mouse GCs using an miRNeasy Mini Kit (Qiagen, Germantown, MD, USA) and sequenced by Shanghai Silver Crown Biomedical Technology Co., Ltd. (Shanghai, China), followed by bioinformatic analysis from the database([57]https://metascape.org/gp/index.html). Three samples constituted each experimental group. Differentially expressed genes (DEGs) were identified between the groups according to a cutoff of |fold change|> 1.5 and P < 0.05. Functional enrichment analysis (gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis) and Volcano plot visualization were performed on the identified DEGs. Cell proliferation assay A Cell Counting Kit 8 (CCK-8, Yeasen, Shanghai, China) was used to evaluate KGN cell proliferation. Briefly, 2000 cells/well were seeded in 96-well plates. After 24 h of treatment, 10 µL of CCK-8 solution was added to each well and absorbance was measured at 450 nm at different time points with a microplate reader (Bio-Tek, Beijing, China). Cell cycle analysis Following fixation in 70% ethanol for 24 h at 4 °C, cells were stained with propidium iodide, reacted with RNase for 30 min at 37 °C in the dark, followed by flow cytometry analysis. ModFit software was used to determine the proportions of cells in G0/G1, S, and G2/M phases. Assessment of mitochondrial membrane potential 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide (JC-1; Beyotime, Haimen, China) was used to assess mitochondrial membrane potential as an indicator of early apoptosis. The red/green fluorescence ratio (JC-1 aggregates/monomers, Phycoerythrin /fluorescein isothiocyanate channels) was measured using flow cytometry and then the apoptosis rate was calculated. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis RNA was extracted using an RNA extraction kit (9767; Takara, Dalian, China). After reverse transcription of the mRNA to cDNA, the cDNA was used as the template for the qPCR step. mRNA expression was normalized to that of Gapdh/GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) and the relative mRNA expression levels were determined utilizing the 2–ΔΔCt method. Detailed information of the primer pairs employed can be found in Additional file: Table S2. Fasting glucose test Fasting blood glucose was measured in mice following an overnight fast (12–16 h). Blood glucose levels were measured from tail vein blood samples (Viva Check, China). Analysis of bone mineral density in mice Femurs were harvested from Control Group and mice following three months of CEFFE treatment. Following euthanasia, femurs were excised and the musculature and adipose tissue surrounding the femoral head were carefully dissected and were fixed in 4% paraformaldehyde. The femoral head was then processed for H&E staining for later analyse. Statistical analysis Experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0 (GraphPad Inc., La Jolla, CA, USA) and are presented as the mean ± the standard deviation (SD). Two-group comparisons were analyzed using unpaired two-tailed Student’s t-tests. Multiple-group comparisons were performed using one-way or two-way analysis of variance. A P-value of less than 0.05 was considered statistically significant. Results CEFFE treatment prevented diminished ovarian function in aged mice To evaluate the preventive effect of CEFFE on DOR in mice, we weighed the mice weekly and assessed ovarian organ coefficients, hormone secretion, and regulatory hormones monthly throughout the treatment period. Starting from week five of CEFFE administration, the weight gain in the Prophylactic group was significantly lower than that in the Control group, suggesting reduced age-related adiposity (Fig. [58]1A). After three months of treatment, the ovarian organ coefficient in the CEFFE Prophylactic group was significantly higher than that in the Control group (Fig. [59]1B). After two months and three months of CEFFE treatment, the secretion of estrogen and AMH in the Prophylactic group was significantly higher, while FSH levels were significantly lower, compared to the Control group (Fig. [60]1D–F). After three months of prevention, the secretion of upstream regulatory hormones gonadotropin-releasing hormone (GnRH) was significantly elevated and gonadotropin-inhibitory hormone (GnIH) were markedly reduced in the Prophylactic group compared to the Control group (Fig. S1). H&E staining revealed that the number of primordial and primary follicles in the Prophylactic group mice was significantly higher than that in the Control group, while the number of atretic follicles was significantly reduced (Fig. [61]1G, [62]H) These results suggests that CEFFE treatment could reduce follicular atresia and promote follicular development. Fig. 1. [63]Fig. 1 [64]Open in a new tab CEFFE treatment prevents ovarian function decrease in aged mice. A Body weight trend of the mice. The red line represents the Control group, the blue line represents the Prophylactic group. B Ovarian weight and C ovarian morphology changes over time (months). D Serum hormone levels measured by ELISA. E The level of E2, AMH, and FSH in the second month and F the third month of CEFFE treatment. G Representative pictures of H&E staining of mouse ovaries, and H statistical analysis of follicle counts at different stages. PMF Primordial follicles, PF Primary follicles, SF Secondary follicles, AnF Antral follicles, ATF atretic follicles. The data are represented as the mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, compared with Control group (n = 5) CEFFE treatment effectively prolonged fertility in aged mice We observed that CEFFE promotes an improvement in ovarian weight and the secretion of sex hormones. To further confirm its impact on ovarian function, we confirmed whether the fertility of mice changed after CEFFE treatment. Marked enhancements were observed in the quality of embryos in the Prophylactic group. The counts of 2 cell, 4–8 cell embryos, and blastocysts were significantly higher following CEFFE treatment compared with those in the Control group (Fig. [65]2A and [66]C). The pregnancy rate in the Prophylactic group surpassed that of the Control group by 10%, accompanied by a marked reduction in the occurrence of abnormal pregnancies. Furthermore, there was a notable enhancement in both the total litter count and the number of live births in the Prophylactic group in comparison with those in the Control group (Fig. [67]2B andD). Fig. 2. [68]Fig. 2 [69]Open in a new tab CEFFE treatment effectively prolongs fertility in control mice. A Representative images of embryos at different developmental stages (2-cell, 4 to 8-cell, morula, and blastocyst) and C statistical analysis of the respective numbers. B Representative images of the litter number and D statistical analysis of pregnancy rates and productivity. The data are represented as the mean ± SD. *P < 0.05, compared with Control group (n = 5) Safety evaluation of CEFFE treatment in parental mice To assess the safety of CEFFE treatment, we collected the heart, liver, kidney, spleen, lung, and uterus from Prophylactic and Control groups of mice. The morphology of each organ and the organ coefficients were recorded, and H&E staining was performed. The experimental results showed that there were no significant differences in organ morphology and H&E staining between the Prophylactic group and the Control group (Fig. [70]3A, [71]B). Most organ coefficients showed no significant intergroup differences, except for the liver, which was significantly heavier in the CEFFE-treated group compared to the Control group (Fig. [72]3C). The liver is essential for both lipid and glucose metabolism [[73]22]. Furthermore, we examined lipid metabolism, glucose metabolism in the two groups of mice. We observed a significant increase in total cholesterol (TC) in mice, while triglyceride (TG) levels remained unchanged. Further investigation into high density lipoprotein (HDL) and low density lipoprotein (LDL) revealed a significant increase in HDL and decrease in LDL, which might suggest a beneficial effect of CEFFE (Fig. [74]3D). However, the results showed that CEFFE administration did not affect glucose metabolism in the mice (Fig. S2A). Given the established interplay between ovarian function and skeletal health [[75]6–[76]8], we further investigated the impact of CEFFE on bone mineral density (BMD) in mice. However, prophylactic CEFFE treatment did not result in a statistically significant improvement in BMD (Fig. S4). Fig. 3. [77]Fig. 3 [78]Open in a new tab Safety Evaluation of CEFFE in parental mice. A Representative images of organs morphology of Control group and Prophylactic group. B Representative pictures of H&E staining of mouse organs. C Statistical analysis of organ weights. D The levels of HDL cholesterol, LDL cholesterol, total cholesterol, and triglyceride in serum measured by ELISA. HDL High-density lipoprotein, LDL Low-density lipoprotein, TC Total cholesterol, TG Triglycerides. The data are represented as the mean ± SD. ns: P > 0.05, **P < 0.01, ***P < 0.001, compared with the Control group (n = 5) Safety evaluation of CEFFE treatment in offspring mice Offspring mice produced by the Prophylactic group and Control group were monitored for body weight from birth until 8 weeks of age. No significant differences in body weight were observed between the two groups of offspring mice (Fig. [79]4A). At 8 weeks of age, offspring mice were euthanized, and the heart, liver, kidney, spleen, lung, testes /uterus were collected from male and female offspring, respectively. Organ weight was recorded, and organ coefficients were calculated. No significant differences in organ coefficients were observed between the two groups, neither in female offspring nor in male offspring (Fig. [80]4B). Fig. 4. [81]Fig. 4 [82]Open in a new tab Safety evaluation of CEFFE treatment in offspring mice. A Weight change curves of female and male mice. B Statistical analysis of the organ weights of the different groups. The data are represented as the mean ± SD. ns: P > 0.05, compared with the Control group (n = 5–8) CEFFE treatment mitigates age-related ovarian decrease in mice by influencing cellular proliferation, hormone synthesis, and oxidative stress. To further analyze the effects of CEFFE prevention on ovarian function in mice, mRNA sequencing was performed on ovarian granulosa cells from the Prophylactic and Control groups. A total of 2458 differential expressed genes (DEGs) were identified, including 1176 upregulated genes and 1282 downregulated genes (Fig. [83]5A). Analyses using GO and KEGG pathway enrichment analysis were performed on these DEGs. The GO pathways were mainly enriched in “regulation of cell population proliferation”, “response to hormone”, “cellular response to oxidative stress”, and other important pathways, suggesting that CEFFE might affect senescence in mice by influencing cell proliferation, hormone synthesis, and oxidative stress (Fig. [84]5B). Further analysis of ovarian glutathione peroxidase 4 (GPX4) staining showed a significant reduction after CEFFE treatment (Fig. [85]5C, [86]D). Additionally, RT-qPCR was performed on ovarian granulosa cells from the Control and Prophylactic group of mice. Compared to the Control group, the mRNA expression levels of oxidative stress-related factors decreased significantly decreased, antioxidant factor mRNA expression increased significantly, pro-proliferation mRNA expression increased significantly, and hormone synthesis gene mRNA expression also increased significantly in the Prophylactic group (Fig. [87]5E–G). Fig. 5. [88]Fig. 5 [89]Open in a new tab CEFFE treatment mitigates age-related ovarian decrease in mice by influencing cellular proliferation, hormone synthesis, and oxidative stress. A The volcano plot of DEGs of the Control group vs. the Prophylactic group. Among the total 2458 genes with an absolute fold change > 1.5, 1282 were upregulated while 1176 were downregulated. B Top 20 GO terms for DEGs. C Representative images of GPX4 staining in mouse ovaries and D statistical analysis (n = 5). The mRNA expression levels of E Ndufs3, Nos2, Sod1, and Cat; F Pcna and Mki67; G Cyp17aA1 and CypP11a1 in granular cells in the different groups. The data are represented as the mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01, compared with Control group (n = 5) CEFFE treatment ameliorated granulosa cell senescence by promoting proliferation and hormone synthesis and inhibiting oxidative stress In vitro studies demonstrated that CEFFE pre-treatment mitigated H[2]O[2]-induced senescence in KGN cells. KGN cells were pre-treated with varying concentrations of CEFFE (1, 2, 5, and 10 μg/μL) for 24 h prior to induction of cellular senescence with H[2]O[2] (200 μM and 400 μM). Cells were then exposed to H[2]O[2] for 2 or 4 h. CEFFE pre-treatment prevented the H[2]O[2]-induced decline in cell vitality in a dose-dependent manner. Specifically, pre-treatment with 1 μg/μL CEFFE resulted in a significant increase in cell vitality (P < 0.05) compared to the corresponding H[2]O[2]-treated controls. Pre-treatment with higher concentrations of CEFFE (2, 5, and 10 μg/μL) conferred even greater protection, leading to a more pronounced increase in cell vitality (P < 0.001) compared to their respective H[2]O[2]-only groups at both the 2 h and 4 h time points. The results showed that CEFFE pre-treatment significantly improved the viability of KGN cells compared to those in the H[2]O[2] group (Fig. [90]6A). Subsequently, KGN cells were induced to undergo senescence using 200 μM H[2]O[2] for 2 h after 10 μg/μL CEFFE pre-treatment for 24 h. This pre-treatment significantly reduced the percentage of Senescence-associated beta-galactosidase (SA-β-GAL)-positive cells compared with those in the H[2]O[2] group (Fig. [91]6B, [92]C). Cell cycle analysis revealed that CEFFE pre-treatment facilitated cell cycle progression in KGN cells, preventing cell cycle arrest in the G0/G1 phase (Fig. [93]6D, [94]E). Furthermore, CEFFE pre-treatment significantly reduced reactive oxygen species (ROS) levels, increased the mRNA expression of superoxide dismutase (SOD) and GPX4, and decreased the expression of HO1 (encoding heme oxygenase 1) in KGN cells, suggesting a protective effect against oxidative stress (Fig. [95]6F). Flow cytometry analysis of JC-1 stained cells demonstrated a significantly higher mitochondrial membrane potential in the CEFFE pre-treatment group compared to the H[2]O[2] group (Fig. [96]6G, H), indicating improved mitochondrial function. Finally, CEFFE pre-treatment resulted in a significant increase in the secretion of E[2] and AMH into the supernatant of KGN cells compared to the H[2]O[2] group (Fig. [97]6I), suggesting enhanced hormone synthesis. Collectively, these findings demonstrated that pre-treatment with CEFFE could delay DOR by promoting cell proliferation, inhibiting oxidative stress, mitigating GC senescence, and improving GC hormone secretion. Fig. 6. [98]Fig. 6 [99]Open in a new tab CEFFE treatment ameliorates granulosa cell senescence by promoting proliferation and hormone synthesis, and inhibiting oxidative stress. A Statistical analysis of the CCK-8 test. B Representative images of SA-β-GAL, and C statistical analysis for SA-β-GAL. D Representative images of the cell cycle, and E statistical analysis for cell cycle. F The level of ROS, SOD, GPX4, HO-1. G JC-1 probe detection of changes in the mitochondrial membrane potential, and H statistical analysis for JC-1. I The levels of hormones E[2] and AMH measured by ELISA in cell supernatants. The data are represented as the mean ± SD. ns: P > 0.05, *P < 0.05, **P < 0.01, compared with the H[2]O[2] group (n = 3) Discussion With advances in medical technology and improvements in quality of life, the life expectancy of women has steadily increased. However, ovarian function begins to decline as early as 31 years old. Additionally, with shifting reproductive attitudes, more women are choosing to delay childbearing, further narrowing the reproductive window [[100]23]. Ovarian functional decline is associated with the onset of various systemic diseases [[101]24, [102]25], and DOR severely impacts women’s physical and mental health. Enhancing ovarian function is a key focus and challenge in the field of assisted reproductive technology. Current research primarily focuses on salvage treatments for already-declining ovarian function; however, their efficacy remains limited. Compared with salvage treatments, preventing DOR is of greater importance. Our previous studies demonstrated that CEFFE treatment in physiologically aging or chemotherapy-induced ovarian insufficiency mouse models effectively improved ovarian function and fertility [[103]19, [104]20]. Building on these findings, this study aimed to shift the intervention window earlier to further explore the role of CEFFE in preventing ovarian reserve decline. The results suggested that CEFFE could delay DOR and extend reproductive lifespan by promoting cell proliferation, inhibiting oxidative stress, mitigating GC senescence, and improving GC hormone secretion, which offers a novel potential protective strategy for preventing ovarian functional decline in women. In women, DOR is the most direct cause of declining fertility, characterized by a decrease in both the quantity and quality of follicles, leading to reduced fertility in older women [[105]26]. In this study, mice were divided into two groups: a CEFFE prophylactic group, and a Control group. The CEFFE prophylactic group received tail vein injections of CEFFE every 72 h for three months. The results showed that, compared with the Control group, the mice in the CEFFE prophylactic group exhibited a significantly increased ovarian organ coefficient and a higher number of growing and antral follicles, improved early embryo development, a significant increase in litter size, and a shorter interval between litters, indicating a marked improvement in fertility. Additionally, we found that serum AMH and E[2]levels in the CEFFE prophylactic group were significantly higher than those in the Control group, while FSH secretion was significantly reduced. This suggested a substantial improvement in ovarian endocrine function. These findings indicated that prolonged CEFFE administration prevents the decline in ovarian function and fertility typically associated with DOR in mice. Furthermore, CEFFE treatment increased HDL and lowered LDL levels, suggesting that CEFFE might also have beneficial effects on the changes in lipid metabolism caused by natural aging. Studies have indicated that adipose-derived stem cell (ADSC) therapy might carry a risk of tumorigenesis, and senescent ADSCs in a pro-inflammatory state can induce ovarian damage [[106]27]. Our research demonstrated that CEFFE effectively prevented ovarian functional decline in mice. Consequently, we investigated whether CEFFE treatment poses any adverse risks to parent and offspring mice. To assess the safety of CEFFE in preventing ovarian functional decline, we measured the body weight and organ coefficients of both parent and offspring mice, and performed H&E staining on these organs. The results revealed that, except for the liver of parent mice, CEFFE treatment did not affect the body weight or organ morphology of either parent or offspring mice, and no histopathological abnormalities were observed. The liver plays a crucial role in both glucose homeostasis, transitioning between glucose consumption and production, and lipid metabolism, encompassing the uptake, synthesis, packaging, and secretion of lipids and lipoproteins [[107]22].We observed a significant increase in liver weight in parental mice after three months of CEFFE treatment, prompting further investigation into lipid metabolism including TC, TG, HDL, LDL, and glucose metabolism. CEFFE treatment did not affect glucose metabolism or serum TG levels. However, CEFFE treatment significantly increased HDL and decreased LDL. Given the strong association between elevated LDL and reduced HDL levels with the development of atherosclerosis [[108]28], these findings suggest that CEFFE, as a lipid extract, may exert beneficial effects on atherosclerosis-related diseases by modulating hepatic lipid metabolism. Further investigation into this mechanism is warranted. Additionally, our previous studies have shown that CEFFE treatment does not induce abnormal expression of tumor markers and has no detrimental effects on parent or offspring mice [[109]19, [110]20]. These findings indicated that CEFFE effectively prevents ovarian function decline and extends reproductive lifespan in mice without compromising the safety of either parent or offspring. Our study clearly demonstrated the efficacy and safety of CEFFE in preventing ovarian functional decline in mice. The next step was to explore the potential molecular mechanisms underlying the protective effects of CEFFE. We conducted mRNA transcriptome sequencing on GCs from the Control group and Prophylactic group of mice, to identify DEGs. Both GO and KEGG pathway analyses suggested that CEFFE affects GC proliferation-related functions. Previous studies on muscle injury in mice, as well as our prior research on CEFFE treatment of ovarian insufficiency, have indicated a role for CEFFE in promoting cell proliferation [[111]15, [112]20]. The sequencing results revealed that anti-aging effects of CEFFE might be achieved by promoting cell proliferation, enhancing the response to hormones, and inhibiting oxidative stress. We validated these findings at the mRNA level in mouse ovaries, observing significant changes in the expression of genes related to proliferation, oxidative stress, and hormone synthesis. Next, we examined the effects of CEFFE on the aging of KGN cells. The results showed that CEFFE treatment maintained KGN cell vitality, suppressed the appearance of senescence phenotypes, and significantly reduced cell cycle arrest. Oxidative stress markers were notably reduced, while antioxidant factors increased, the mitochondrial membrane potential was stabilized, and hormone secretion improved (Fig. [113]6). However, given the complexity of CEFFE, which contains numerous active factors, the specific factors and signaling pathways responsible for promoting GC proliferation remain to be further investigated. During follicular development, under the influence of various hormones and signaling molecules, GCs proliferate and differentiate from an initial single layer of pre-GCs into a specialized secretory cell population. Along with the follicular fluid, these cells create the microenvironment essential for oocyte survival. Through cellular junctions, GCs and oocytes exchange various cytokines, thereby promoting each other’s growth and maintaining normal follicular development. Inhibition of GC proliferation and the appearance of senescence phenotypes can lead to impaired oocyte maturation, meiotic abnormalities, and follicular degeneration, ultimately exacerbating follicular depletion and resulting in DOR [[114]29, [115]30]. Studies suggested that GC proliferation suppression and subsequent cellular aging are critical factors contributing to follicular atresia [[116]31, [117]32]. Based on these findings, we concluded that CEFFE could prevent ovarian function decline by inhibiting oxidative stress, promoting proliferation, and enhancing hormone secretion, thereby promoting GC senescence, enhancing ovarian function, and prolonging fertility. For the first time, we revealed that early prophylactic administration of CEFFE in adult mice could prevent DOR and extend their reproductive lifespan by inhibiting GC senescence. This study offers a novel approach to prevent DOR in women and provides new insights into anti-aging therapies. In future research, we will further investigate the specific molecules or molecular combinations involved in preventing DOR to develop more precise therapeutic strategies. Moreover, given the advantages of CEFFE and our well-established extraction techniques, we have obtained approval from the hospital’s ethics committee to initiate clinical studies on the application of autologous CEFFE therapy for DOR. Conclusions We examined the protective effects of CEFFE against DOR in mice. Our comprehensive analysis revealed that CEFFE prevented GC senescence, mitigated aging-related phenotypes, improved embryo quality in aged mice, and extended their reproductive lifespan, without causing harm to either the treated mice or their offspring. This study introduces a promising new candidate for anti-aging therapy. Future research could investigate the specific components and formulations within CEFFE that underlie its effects. Supplementary Information [118]Additional file 1.^ (2.9MB, docx) Acknowledgements