Preventing ovarian aging: from redox-targeted strategies to extracellular vesicle-based therapies

The female reproductive lifespan is governed by the hypothalamic-pituitary-ovarian (HPO) axis, which orchestrates cyclical hormonal dynamics essential for ovulation, endometrial remodeling, and fertility (Spaziani et al., 2021). With age, the progressive depletion of the ovarian follicular pool leads to declining levels of estradiol and inhibins, resulting in a compensatory rise in gonadotropins such as FSH (Spaziani et al., 2021; Park et al., 2021; Allshouse et al., 2018). This process ultimately culminates in menopause, the permanent cessation of ovarian endocrine and reproductive function, typically around the age of 51 (Peacock et al., 2025).

Perimenopause precedes this transition and is characterized by fluctuating hormone levels and irregular menstrual cycles, often accompanied by vasomotor symptoms, mood disturbances, and sleep disruption (Coborn et al., 2022; Duralde et al., 2023). While this physiological progression is well-documented, deviations from the expected timeline, particularly in premature presentations, warrant deeper investigation into underlying molecular mechanisms. In such cases, factors like oxidative stress, chronic inflammation, and immune dysregulation may contribute to accelerated follicular depletion and endocrine failure (Hamoda and Sharma, 2024), forming the basis for the mechanisms explored in the following sections.

POI is defined as the loss of ovarian function before age 40, leading to hypoestrogenism, elevated FSH levels, and infertility (Hamoda and Sharma, 2024). Affecting ∼1% of women, its implications extend beyond reproductive concerns, encompassing cardiovascular morbidity, skeletal fragility, and neurocognitive risk (Roeters van Lennep et al., 2016; Gallagher, 2007; Sochocka et al., 2023; Liang C. et al., 2023).

Although hormone replacement therapy (HRT) remains the standard of care, it neither restores ovarian reserve nor addresses the underlying molecular damage. A more comprehensive understanding of POI pathogenesis, particularly the contribution of oxidative stress, could support the development of disease-modifying therapies, moving beyond symptomatic management.

POI can arise from a variety of mechanisms, although idiopathic cases, those with no clearly identifiable cause, represent the vast majority, accounting for 85%–90% of instances (Kapoor, 2023). Among the known causes are genetic abnormalities, such as Turner syndrome (Bollig et al., 2023) and Fragile X (Man et al., 2017) premutation carriers, which can compromise ovarian development and function (Ebrahimi and Akbari Asbagh, 2011). Autoimmune disorders, including autoimmune thyroiditis (Meling Stokland et al., 2022), Addison’s disease, and systemic lupus erythematosus, may also disrupt ovarian activity through immune-mediated damage (Szeliga et al., 2021). Certain metabolic conditions like galactosemia (Sozen et al., 2019; Thakur et al., 2018), which involve enzyme deficiencies, have similarly been implicated. Infectious diseases, particularly viral infections such as mumps oophoritis or chronic infections like tuberculosis, can directly impair ovarian tissue (Panay and Kalu, 2009; Goswami and Conway, 2005). Additionally, iatrogenic factors, most commonly chemotherapy or radiotherapy, frequently result in POI (Sklar, 2005; Tham et al., 2007; Oktem and Oktay, 2007). In many cases, the onset of POI appears to reflect a complex interplay between genetic predisposition and environmental exposures (Sapre and Thakur, 2014; Gold et al., 2013), often culminating in oxidative stress-mediated follicular damage. Elevated ROS can injure oocytes and granulosa cells, accelerating atresia and depleting the ovarian reserve. This redox imbalance, whether triggered by toxins, inflammation, or mitochondrial dysfunction, is increasingly recognized as a prime target for interventions aimed at preserving ovarian function (Yan et al., 2022; Jadeja et al., 2021; Agarwal et al., 2008; Behrman et al., 2001). Recognizing these underlying mechanisms is crucial for effective clinical intervention and management, aiming to preserve fertility and mitigate health risks related to early estrogen depletion.

Among lifestyle factors, cigarette smoking stands out as one of the most consistent accelerators of ovarian aging. Epidemiological studies show that smokers, both current and former, experience menopause up to 4 years earlier than non-smokers, with a clear dose-response pattern in heavy smokers (Yan et al., 2022; Pappas, 2011). But beyond correlations, the mechanistic impact of tobacco smoke is particularly striking: it delivers a toxic cocktail of over 7,000 chemicals, many with gonadotoxic, mutagenic, and endocrine-disrupting effects (Pappas, 2011; Zeng et al., 2023).

Key agents like polycyclic aromatic hydrocarbons (PAHs) bind to aryl hydrocarbon receptors (AhR) in ovarian cells, activating apoptotic cascades and promoting follicular depletion. At the same time, smoking induces systemic and local oxidative stress, impairing mitochondrial integrity and disrupting estrogen synthesis by downregulating aromatase and steroidogenic transport proteins. These mechanisms collectively reduce estradiol levels and destabilize the hypothalamic-pituitary-ovarian axis, resulting in irregular cycles and a more symptomatic perimenopause (Matikainen et al., 2001).

Lower anti-Müllerian hormone (AMH) levels in smokers further support the notion that tobacco use accelerates the decline in ovarian reserve (Plante et al., 2010; Dólleman et al., 2013). In short, cigarette smoking acts as a multifactorial disruptor, combining cytotoxicity, oxidative damage, hormonal imbalance, and apoptotic acceleration, to shorten reproductive lifespan and hasten the transition to menopause.

Exposure to endocrine-disrupting chemicals, such as phthalates, bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), and perfluoroalkyl substances (PFAS), has been associated with accelerated ovarian aging and POI. Several cross-sectional and longitudinal studies report that higher systemic levels of endocrine-disrupting chemicals correlate with reduced antral follicle count, earlier loss of ovarian function, and altered hormone levels (Grindler et al., 2015). Mechanistically, these compounds induce oxidative stress, inflammation, and apoptosis in granulosa cells, while interfering with steroidogenesis and folliculogenesis.

More recently, MNPs, especially polystyrene-derived particles, have emerged as an additional environmental concern. These particles, found in food, water, and air, have been detected in human ovarian follicular fluid, raising alarms over their potential to impair follicle viability and hormone secretion. Experimental models demonstrate that MNPs exposure leads to increased apoptosis, granulosa cell dysfunction, disruption of estrous cyclicity, and impaired ovarian development (Camerano Spelta Rapini et al., 2024). Additionally, MNPs may act as “Trojan horses” by facilitating the delivery of toxic endocrine disruptors chemicals such as phthalates, PFAS, and BPA into sensitive ovarian tissues, further amplifying their detrimental effects.

Together, these findings underscore the critical role of modifiable environmental and behavioral exposures in shaping the trajectory of ovarian aging and reproductive lifespan.

The modern rise in high-fat diet (HFD) consumption has been strongly associated with declining female reproductive health, particularly through its detrimental effects on ovarian function and follicular dynamics. Emerging evidence indicates that excessive dietary lipid intake compromises ovarian resilience by triggering a cascade of metabolic disturbances that impair cellular homeostasis within the follicular environment. Specifically, HFDs contribute to lipid accumulation in non-adipose ovarian cells, promote lipotoxicity, induce chronic low-grade inflammation, and elevate ROS, ultimately disrupting oocyte development and fertility outcomes (Di Berardino et al., 2024; Di Berardino et al., 2022; Gonnella et al., 2022).

Understanding the mechanisms through which HFD undermines ovarian physiology is critical, as they extend beyond simple caloric excess to involve complex metabolic, oxidative, and inflammatory pathways. The following sections explore these mechanisms in detail, focusing on lipid-induced toxicity, inflammation-driven oxidative stress, and dysregulated fatty acid metabolism, and their collective impact on ovarian cell viability, follicular development, and reproductive potential.

Although environmental stressors may differ in origin, ranging from lifestyle factors to pollutants, they tend to converge on a limited number of shared pathophysiological mechanisms that drive reproductive aging. A central feature among these is oxidative stress, which impairs mitochondrial function and contributes to cellular damage within the ovary. In parallel, disruptions in hormonal homeostasis, particularly in steroidogenesis, further compromise follicular development and oocyte quality. These alterations are often accompanied by increased apoptosis of granulosa and other somatic ovarian cells, reducing the ovarian reserve. Additionally, persistent activation of inflammatory pathways, such as NF-κB signaling and the release of proinflammatory cytokines, sustains a chronic inflammatory state within the ovarian microenvironment. Compounding these effects is the disruption of neuroendocrine signaling along the hypothalamic-pituitary-ovarian (HPO) axis, which further destabilizes reproductive function. Collectively, these factors contribute to a hostile intraovarian environment that accelerates follicular atresia and leads to a premature decline in reproductive potential. Understanding these converging mechanisms highlights the importance of identifying and mitigating modifiable environmental risks in order to preserve ovarian function and delay reproductive senescence in modern populations. Figure 4 provides a schematic representation of the cascade of intraovarian events triggered by environmental and lifestyle stressors, ultimately contributing to accelerated ovarian aging.

PCOS is characterized by insulin resistance, hyperandrogenemia, chronic low-grade inflammation, and heightened oxidative stress. ROS production is elevated in granulosa cells and circulating leukocytes, and systemic markers such as malondialdehyde (MDA) and myeloperoxidase (MPO) are increased in PCOS patients, particularly those with insulin resistance (Victor et al., 2016). In this setting, oxidative stress acts primarily as a persistent, low-grade metabolic stressor, arising from mitochondrial dysfunction and NADPH oxidase activation, and amplified by inflammatory cytokines (TNFα, IL-6). This chronic redox imbalance interferes with mitochondrial biogenesis, ATP synthesis, and redox homeostasis, thereby impairing follicular development and oocyte quality (Yan et al., 2024). These pathogenic mechanisms have direct clinical consequences and inform targeted therapeutic strategies. As an example, interventions such as N-acetylcysteine (NAC), a precursor to glutathione, have been shown to partially rescue metabolic imbalance, improve insulin sensitivity, and reduce ovulatory dysfunction (Fang et al., 2024).

In contrast to PCOS, POI is characterized by a more abrupt and severe disruption of redox homeostasis, leading to accelerated follicular depletion. Excessive ROS accumulation, mitochondrial DNA damage, and impaired mitophagy contribute to compromised oocyte and granulosa cell survival. Women with POI exhibit lower mtDNA copy numbers, frequent mitochondrial gene mutations, and defective oxidative phosphorylation (Kakinuma and Kakinuma, 2023). Systemic oxidative stress markers (d-ROMs, OSI), are markedly elevated reflecting a collapse of compensatory antioxidant defenses (Kakinuma and Kakinuma, 2023). Mitochondrial dysfunction, mediated by reduced SIRT1/SIRT3 signaling, leads to apoptotic follicular loss and diminished ovarian reserve (Guo et al., 2022). As treatments beyond conventional hormone replacement therapy, a new generation of experimental regenerative approaches for POI—including mitochondrial activation, in vitro follicle activation, stem cell and exosome therapies, biomaterial-based strategies, and intra-ovarian platelet-rich plasma infusion—holds promise for re-establishing oxidative balance and preserving ovarian function (Huang et al., 2022), although clinical translation remains limited.

Iatrogenic ovarian injury, particularly following chemotherapy with alkylating agents (e.g., cyclophosphamide, cisplatin) or anthracyclines (e.g., doxorubicin), represents an extreme form of acute oxidative insult. These agents induce a sudden surge of ROS within oocyte and granulosa cell mitochondria, triggering lipid peroxidation, DNA damage and activation of apoptotic or ferroptotic pathways (Trujill et al., 2023). Unlike chronic or progressive disorders, this acute oxidative damage precipitates immediate granulosa death and rapid depletion of primordial and growing follicles. Protective interventions such as NAC, metformin, or HO-1 modulation have shown protective effects in preclinical models by enhancing antioxidant defense (e.g., GPX4, Nrf2, HO-1) and reducing follicular loss (Zhang S. et al., 2023).

Taken together, these disorders share oxidative stress as a common pathogenic denominator, yet differ fundamentally in the timing, intensity, and reversibility of redox perturbations. In PCOS, oxidative stress functions as a chronic, low-grade metabolic disturbance that primarily disrupts follicular maturation and endocrine signaling (Rudnicka et al., 2022). In POI, a rapid and severe oxidative collapse accelerates mitochondrial dysfunction and apoptotic follicular depletion (Shi et al., 2023), leading to a swift decline in ovarian reserve (Houeis et al., 2025). In contrast, iatrogenic ovarian injury is driven by acute and overwhelming oxidative damage, resulting in abrupt granulosa cell loss and follicle attrition (Trujill et al., 2023). These distinctions have direct therapeutic implications, indicating that redox-modulating strategies should be tailored to disease kinetics, with preventive approaches more suitable for chronic conditions such as PCOS (Motta, 2025; Mihanfar et al., 2021), and immediate protective or regenerative interventions required in acute toxic insults (Ruan et al., 2024; Lambertini et al., 2018; Ali et al., 2022). An overview of experimental models converging on oxidative stress–mediated ovarian dysfunction is provided in Figure 5.

Several chemically induced animal models have been employed to investigate ovarian oxidative stress, among which D-galactose-induced ovarian aging in rodents is particularly prominent. D-galactose administration accelerates aging processes by increasing oxidative stress, mitochondrial dysfunction, and apoptosis in ovarian tissue. This model reliably recapitulates human POI, characterized by increased follicular atresia, reduced ovarian reserve, impaired steroidogenesis, and disrupted reproductive hormone levels (Liang et al., 2020; Rostami et al., 2019; Bandyopadhyay et al., 2003).

In rodent studies, D-galactose induces notable oxidative damage markers such as elevated ROS, malondialdehyde (MDA), advanced glycation end products (AGEs), and upregulated senescence-associated proteins including p16^INK4a (Liang et al., 2020; Rostami et al., 2019). These findings have provided insight into mechanisms of premature ovarian aging, emphasizing the utility of this model for evaluating antioxidant therapies and molecular pathways involved in ovarian senescence (Liang et al., 2020; Rostami et al., 2019).

Nutritional models using a HFD have extensively elucidated links between metabolic dysfunction, lipotoxicity, and ovarian oxidative stress. Rodents fed with HFD develop increased ovarian lipid accumulation, disrupted mitochondrial function, elevated ROS production, and chronic metabolic inflammation, closely mimicking PCOS and metabolic-related reproductive impairment (Gonnella et al., 2022).

HFD-induced oxidative stress models have demonstrated the causal relationship between systemic metabolic derangements and ovarian damage. Mice on HFD exhibit increased markers of oxidative damage (e.g., 8-OHdG, lipid peroxidation), disrupted follicular growth, and diminished fertility outcomes, providing robust platforms to test therapeutic strategies targeting metabolic inflammation and oxidative pathways (Gonnella et al., 2022; Shen et al., 2023).

Surgical induction of oxidative stress via unilateral or bilateral ovarian ischemia-reperfusion injury has also been employed. Ischemic models induce rapid oxidative damage by reperfusion-induced ROS, allowing detailed investigations into acute oxidative injury mechanisms, tissue-specific antioxidant responses, and therapeutic efficacy of antioxidants and regenerative interventions such as stem cell-derived EVs (Table 1).

Transgenic mouse models, such as Prdx4^−/−, have offered crucial genetic insights into oxidative stress responses. The loss of peroxiredoxin-4 significantly elevates oxidative damage, granulosa cell apoptosis, and accelerated ovarian aging under stress conditions such as D-galactose administration, establishing genetic links between antioxidant defense impairment and ovarian function (Liang et al., 2020).

Chemotherapy-induced ovarian damage is another key experimental model extensively studied. Chemotherapeutic agents (e.g., cyclophosphamide, cisplatin, doxorubicin) generate excessive ROS, mitochondrial dysfunction, and subsequent follicle apoptosis. These models accurately mimic clinical iatrogenic ovarian failure and facilitate studies into mechanisms and protective interventions (Liang et al., 2020; Rostami et al., 2019).

Additionally, environmental toxin exposure models, including polycyclic aromatic hydrocarbons (PAHs), have been instrumental in understanding xenobiotic-induced oxidative damage and its reproductive consequences. These models highlight the ovotoxic effects of environmental contaminants through oxidative pathways, providing frameworks for evaluating environmental risks and protective strategies (Montano et al., 2025; El-Sikaily et al., 2023).

A summary of the main animal models used to study ovarian oxidative stress and their key characteristics is presented in Table 1, providing an integrated overview of experimental strategies and their relevance in mimicking human reproductive pathologies. These models are visually categorized by type and mechanism in Figure 6, which highlights their common outcome: ovarian dysfunction driven by oxidative stress.

Given the central role of oxidative damage to ovarian aging and dysfunction, antioxidant therapy has emerged as a promising pharmacological strategy tissues (Zhang J. et al., 2019). By detoxifying excess of ROS, such interventions aim to restore redox balance, improve mitochondrial function, and ultimately enhance clinical outcomes. Different antioxidant interventions are currently under investigation, and a wide range of treatment conditions has been described. Some examples are presented below.

Melatonin shows potential in delaying ovarian aging through multiple mechanisms, but available evidence highlights a marked variability in dosages, treatment durations, and clinical outcomes across studies. Preclinical models provide the most robust data: in mice, optimal concentrations ranged from 10^–5 mol/L, improving oocyte quality and litter size (Zhang L. et al., 2019), to long-term supplementation up to 43 weeks with 100 μg/mL, which increased follicle number, oocyte yield, and fertilization rates (Tamura et al., 2017). Similarly, a 12-month treatment with 10 mg/kg reduced mitochondrial oxidative damage and improved fertility (Song et al., 2016). However, excessive concentrations (1–2 mM) were shown to be toxic, underscoring the importance of dose selection (Adriaens et al., 2006).

Clinical data in humans are more limited and heterogeneous. In perimenopausal women aged 42–49, nightly supplementation with 3 mg melatonin for 6 months significantly reduced LH levels (Bellipanni et al., 2001). In women aged ≥38 years, 2 mg daily for at least 8 weeks was associated with reduced oxidative damage, improved mitochondrial activity, and better IVF outcomes (Tsui et al., 2024). In contrast, a randomized clinical trial in women with diminished ovarian reserve used 3 mg/day only during the ART cycle (approximately 3–5 weeks), reporting higher estradiol levels and improved oocyte and embryo quality without clear effects on pregnancy rates (Jahromi et al., 2017).

Similarly for NAC, available evidence defines clear dose–duration–outcome relationships across models of ovarian aging and ovarian dysfunction. Preclinical evidence indicates that N-acetylcysteine (NAC) improves oocyte quality and delays ovarian aging. In mice, short-term NAC treatment (0.1–1 mM for 2 months) enhanced oocyte and embryo quality, while long-term low-dose administration (0.1 mM for up to 12 months) delayed age-related fertility decline by preserving telomere length and telomerase activity (Liu et al., 2012). Consistently, NAC exerted protective effects in ovarian transplantation models at doses of 150–1,200 mg/kg over 16 days (Amorim et al., 2014). These effects are primarily mediated by reduced oxidative stress, increased telomerase activity, and preservation of follicular development (Wang et al., 2019). Clinically, a meta-analysis of eight randomized trials in women with polycystic ovary syndrome showed that oral NAC (1,200–1800 mg/day for 2–12 months) improved ovulation and pregnancy rates, and an IVF study further reported improved blastocyst quality and reduced gonadotropin requirements in advanced-age women treated with NAC (Li et al., 2022).

Similarly, coenzyme Q10 (CoQ10) has been widely investigated for its role in preserving ovarian function and counteracting age-related reproductive decline. CoQ10, one key component of the mitochondrial electron transport chain, can be found in dietary-derived and synthetic origins. In preclinical models of ovarian failure induced by VCD, cisplatin, or cyclophosphamide, CoQ10 administration at 150 mg/kg/day for 14 days consistently increased follicle numbers and improved oocyte quality, indicating protection of the ovarian reserve (Lee et al., 2021; Özcan et al., 2016; Delkhosh et al., 2019). Clinically, a meta-analysis of 20 trials demonstrated that CoQ10 supplementation significantly increases the number of retrieved oocytes and high-quality embryos, with the most effective regimen being 30 mg/day for 3 months prior to ovarian stimulation, particularly in younger women with diminished ovarian reserve (Shang et al., 2024). In line with these findings, a randomized controlled trial showed that 60 days of CoQ10 pretreatment improved ovarian responsiveness, reduced gonadotropin requirements, and increased fertilization rates (Xu et al., 2018). These effects are mechanistically linked to enhanced mitochondrial function, reduced oxidative stress, and partial rescue of age-related oocyte dysfunction (Ben-Meir et al., 2015; Nie et al., 2023; Brown and McCarthy, 2023).

Despite strong mechanistic support from experimental models, clinical evidence for antioxidant therapies in ovarian aging remains limited and heterogeneous, with most studies relying on small cohorts and surrogate outcomes (Yan et al., 2022; Shang et al., 2024). Variability in experimental models, dosing regimens, and treatment timing limits reproducibility and the definition of safe therapeutic windows. In this context, multiple studies indicate potential risks associated with high-dose or long-term antioxidant supplementation. For instance, Tarín et al. demonstrated that pharmacological doses of vitamins C and E in mice reduced litter frequency, decreased offspring numbers, and increased fetal resorptions (Tarín et al., 2002), while Rutkowski et al. cautioned that excessive intake of synthetic antioxidant vitamins may result in hypervitaminosis and toxicity (Rutkowski and Grzegorczyk, 2012). Consistently, antioxidants have been described as exerting “double-edged effects,” whereby physiological doses may be beneficial, whereas supraphysiological exposure can elicit detrimental cellular responses. Together, these observations raise concerns regarding redox imbalance with prolonged use and underscore the need for adequately powered randomized trials to define optimal dosing strategies and long-term safety profiles (Yang L. et al., 2020; Li X. et al., 2025; Katz-Jaffe et al., 2020).

Key antioxidant compounds and their effects on ovarian function are summarized in the Table 2 below.

Natural dietary compounds play a key role as alternative or complementary agents in antioxidant therapy. Among these, polyphenols are widely studied for their broad biological activities, including the modulation of oxidative stress and inflammation. These effects are mediated through free radical scavenging, the upregulation of antioxidant gene expression, and the enhancement of mitochondrial function. Resveratrol, a well-characterized polyphenol, exerts antioxidant, anti-inflammatory, and anti-aging effects. In the context of ovarian aging, it activates SIRT1-dependent pathways, which are critical for maintaining genomic stability, mitochondrial efficiency, and cellular survival (Pasquariello et al., 2020). Curcumin, another polyphenol, displays similar properties by activating AMPK/mTOR signaling, inhibiting NF-κB, and promoting the expression of endogenous antioxidant enzymes. These mechanisms contribute to its ability to protect ovarian tissue, restore hormone balance, and attenuate oxidative damage in PCOS and POI models (Yang L. et al., 2020; Cheng and He, 2022; Nasiri Bari et al., 2021; Kamal et al., 2021; Yan et al., 2018; Dhiman and Saini, 2025). Quercetin, a flavonoid, has been shown to reduce oxidative stress by upregulating key antioxidant genes such as SOD1, CAT, and GSS, supporting oocyte integrity and ovarian resilience (Zhang J. et al., 2019). Among water-soluble antioxidants, vitamin C (ascorbic acid) functions as a redox cofactor, neutralizing ROS and RNS and regenerating other antioxidants such as vitamin E. Its ability to terminate radical chain reactions makes it an essential component of the intracellular antioxidant defense system (Zhang J. et al., 2019). Vitamin E (α-tocopherol), a lipid-soluble antioxidant, protects cellular membranes from lipid peroxidation by integrating into phospholipid bilayers and interrupting the propagation of lipid radicals. Additionally, it modulates gene expression and immune responses, contributing to oocyte preservation and improved mitochondrial stability in aging ovaries (Agarwal et al., 2008; Zhang J. et al., 2019). Finally, alpha-lipoic acid (ALA), a mitochondrial cofactor and potent antioxidant, contributes to redox homeostasis by regenerating vitamins C and E, modulating NF-κB signaling, and protecting against inflammatory and metabolic damage. Studies have shown that ALA preserves follicular structure and function in vitro and may mitigate ovarian dysfunction in reproductive disorders such as PCOS and endometriosis (Di Nicuolo et al., 2021; Ñaupas et al., 2024).

Although dietary-derived antioxidants, particularly polyphenols, demonstrate robust antioxidant and anti-inflammatory effects in preclinical models, their clinical translation is constrained by significant pharmacokinetic limitations. Many polyphenols exhibit low oral bioavailability, rapid metabolism, and limited tissue accumulation, which may substantially reduce their efficacy in human ovarian tissue compared with experimental settings (Gong et al., 2025; Bešlo et al., 2023; Manach et al., 2004). These factors complicate direct extrapolation of preclinical benefits to human reproductive outcomes. Furthermore, clinical studies investigating dietary antioxidants are often heterogeneous in design, dosage, and formulation, with limited follow-up and inconsistent reproductive endpoints (Gong et al., 2025; Irmak et al., 2024). As a result, definitive conclusions regarding their effectiveness in preserving ovarian function or delaying reproductive aging cannot yet be drawn. Large-scale randomized clinical trials are required to define optimal dosing strategies, treatment windows, and long-term safety profiles, particularly in populations without overt reproductive pathology (Gong et al., 2025).

Key dietary-derived antioxidants with demonstrated ovarian protective effects are summarized in Table 3, including their molecular mechanisms, experimental models, and reported outcomes.

Stem cell-derived EVs have emerged as functional nanotherapeutics capable of modulating redox balance, inflammation, and tissue repair in the context of ovarian aging (Geng et al., 2023; Zhang et al., 2024). EVs, including exosomes (30–150 nm), microvesicles (100–1,000 nm), and apoptotic bodies (500–5,000 nm), are lipid-based nanocarriers released via distinct biogenetic pathways (Ngo et al., 2025; Fei et al., 2024). Stem cell-derived EVs, particularly those from mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), have gained attention for their ability to modulate inflammation, oxidative stress, and tissue repair (Zhou et al., 2023; Firouzabadi et al., 2024). Rather than acting as passive byproducts, EVs serve as functional mediators of intercellular signaling, delivering nucleic acids, proteins, and metabolites to target ovarian cells. Their regenerative and immunomodulatory properties support their potential as nanotherapeutics in the context of ovarian aging.

While a growing body of preclinical evidence supports the therapeutic potential of redox-targeted antioxidants and stem cell–derived EVs in mitigating oxidative stress and restoring ovarian function, significant translational hurdles remain before these strategies can be applied clinically in women. Most available data derive from rodent models of chemically induced POI, PCOS, high-fat diet exposure, or chemotherapy-related ovarian toxicity. Although these models are instrumental for mechanistic studies, they fail to fully recapitulate the multifactorial and long-term nature of human ovarian aging, which is shaped by decades of environmental exposures, metabolic comorbidities, genetic heterogeneity, and chronic inflamm-aging settings (Wang et al., 2025; Gilmer et al., 2023). Additional limitations include the shorter reproductive lifespan of rodents, the absence of perimenopausal endocrine fluctuations, and species-specific differences in mitochondrial and redox regulation settings (Wang et al., 2025; Gilmer et al., 2023). These factors substantially limit the direct extrapolation of robust preclinical efficacy to human clinical setting as large-scale randomized controlled trials with reproductive endpoints are lacking.

Clinical evidence supporting pharmacological antioxidants such as melatonin, coenzyme Q10, N-acetylcysteine, and dietary polyphenols remains limited and heterogeneous. Existing studies are largely confined to small IVF cohorts or mixed POI/PCOS populations, often characterized by inconsistent dosing regimens, variable bioavailability, short follow-up periods, and the use of surrogate endpoints (e.g., AMH levels) rather than clinically meaningful outcomes such as live birth rates. Consequently, antioxidant interventions have not yet been incorporated into clinical guidelines (Liang J. et al., 2023). Key priorities for translation include the validation of redox and mitochondrial biomarkers linked to reproductive longevity, the design of adequately powered randomized controlled trials with standardized formulations, and the integration of antioxidant strategies with lifestyle or metabolic interventions.

For EV-based therapies, major challenges arise from the lack of standardized isolation and characterization protocols. Widely used methods—including differential ultracentrifugation, precipitation-based approaches, and size-exclusion chromatography—yield EV preparations with distinct purity profiles, size distributions, and molecular cargo. Comprehensive characterization of EV identity, including particle size, concentration, surface markers, and cargo composition (miRNAs, proteins, lipids), is not uniformly implemented across studies. Although the MISEV guidelines have improved transparency and rigor in EV reporting, they primarily address minimal reporting standards and do not yet resolve critical issues related to inter- and intra-batch heterogeneity, potency assessment, or clinical-grade validation (MISEV guidelines; (Welsh et al., 2024)).

From a regulatory and manufacturing perspective, EV heterogeneity—strongly influenced by cell source, culture conditions, vesiculation stimuli, isolation methods, and storage—represents a central obstacle to reproducibility, comparability, and scalability. Regulatory and GMP-focused analyses emphasize the need for rigorous definition of identity, purity, potency, safety, and stability criteria to generate clinical-grade EV products (Thakur and Rai, 2024). Importantly, GMP-compliant experimental studies demonstrate that the transition from research-grade to clinical-grade EVs requires substantial process modifications, including controlled cell sourcing, closed-system manufacturing, scalable purification strategies, and validated quality controls (Humbert et al., 2025). These changes can profoundly alter EV composition and biological activity, underscoring that manufacturing is not a neutral step but a determinant of therapeutic performance.

EV-based therapies offer several theoretical safety advantages over whole-cell approaches, including the absence of proliferative capacity, lower immunogenicity, the ability to cross biological barriers, and reduced tumorigenic risk. Consistent with this, systematic reviews report a very low incidence of serious adverse events in EV-based clinical studies to date (Van Delen et al., 2024; Yo et al., 2022; Gowen et al., 2020). Nevertheless, long-term safety data remain limited, particularly with respect to off-target effects, biodistribution, immunomodulation, and potential pro-tumorigenic signaling. In parallel, scalability remains a key bottleneck, as EV production is constrained by low yields and challenges in preserving vesicle integrity during large-scale manufacturing. Emerging strategies—including optimized culture conditions, alternative EV sources, advanced bioreactor systems, and tangential flow filtration—have shown promise, with some approaches achieving up to 100-fold increases in EV yield (Li Z. et al., 2025; Huang et al., 2025; Ng et al., 2022; Debbi et al., 2022). However, these innovations must be integrated with GMP requirements and validated for consistency and functional potency.

Taken together, both antioxidant-based and EV-based interventions face shared translational limitations rooted in model relevance, standardization, and clinical validation. For antioxidants, inconsistent clinical evidence reflects variability in formulation, dosing, and patient selection. For EVs, the main barriers lie in standardizing isolation, characterization, manufacturing, and regulatory pathways. Bridging these gaps will require the integration of human-relevant models such as ovarian organoids and microphysiological systems, the development of validated biomarkers and potency assays, and the execution of large-scale, well-controlled clinical trials with long-term follow-up. Ultimately, combining redox-modulating strategies with EV-based or nanotherapeutic platforms, guided by patient-specific profiling, may represent a rational path toward extending ovarian healthspan and improving reproductive outcomes.