Mitochondrial dysfunction in ovarian aging

Abstract

Mitochondria serve as multifunctional powerhouses within cells, coordinating essential biological activities that are critical for cell viability, including material metabolism, signal transduction, and the maintenance of homeostasis. They support cells in adapting to complex and fluctuating environments. Oocytes, being the largest cells in multicellular organisms, contain a high number of mitochondria with unique structural characteristics. Mitochondria play active roles in the development and maturation of oocytes. A decline in mitochondrial function negatively affects both the quality and quantity of oocytes, thereby contributing to ovarian aging. However, the specific mechanisms through which mitochondrial dysfunction influences the progression of ovarian aging and impacts reproductive longevity remain unclear. Furthermore, medical strategies aimed at rejuvenating mitochondria to restore ovarian reserve and improve female reproductive potential may open new avenues for clinical treatment. In this review, we summarize the current understanding and key evidence regarding the role of mitochondrial dysfunction in ovarian aging and present emerging medical approaches targeting mitochondria to alleviate premature ovarian aging and enhance reproductive performance.

Introduction

In recent years, infertility has increasingly affected women of advanced reproductive age (ARA), drawing significant attention from both the public and the medical community.^[1] With continued socioeconomic development, there has been a marked rise in female participation in higher education and the workforce, accompanied by improved access to contraceptive services. As a result, the average age at first childbirth among married women has risen substantially.^[2] Demographic and epidemiological studies have confirmed a strong association between advancing maternal age and reduced fecundity. Correspondingly, both the proportion of women seeking assisted reproductive technology (ART) and the number of treatment cycles have shown a steady upward trend.^[3] The primary cause of impaired fertility in this population is a progressive decline in ovarian function due to physiological aging, which leads to diminished ovarian reserve and suboptimal ovarian response.^[2]

Ovarian senescence is marked by a progressive decline in both the quality and quantity of oocytes housed within follicles in the ovarian cortex.^[4] Multiple factors, including endocrinological, genetic, and microenvironmental influences, contribute to the process of reproductive aging in the ovaries^[5] as outlined in Figure 1. Recent research has highlighted the critical role of mitochondrial dysfunction in accelerating ovarian aging. The proposed mechanisms by which mitochondrial dysfunction contributes to the onset of senescence include qualitative and quantitative alterations in mitochondrial DNA (mtDNA), disruptions in mitochondrial dynamics, increased oxidative stress within mitochondria, reduced mitochondrial membrane potential (MMP), and disturbances in mitochondrial homeostasis.^[6] Despite these advances, the detailed molecular pathways and clinical implications remain incompletely understood.

Figure 1.

A schematic plot about the physiological factors related to conserved ovarian reserve and exacerbating the age-associated decline in female fertility.

In this review, we provide a comprehensive overview of the mechanisms through which mitochondrial dysfunction contributes to ovarian aging and the current clinical advancements in mitochondria-targeted therapies aimed at extending reproductive longevity.

Biology of Mitochondrial Functions

Inherited maternally, mitochondria are enclosed by a double-membrane structure that divides the organelle into four distinct compartments: the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the intermembrane space, and the matrix. These domains are spatially separated yet functionally interconnected.^[7]

Widely recognized as the powerhouses of cells, mitochondria exhibit diverse biological behaviors. Their morphology, number, and distribution vary markedly across different cell types to meet functional demands. This phenomenon is referred to as mitochondrial dynamics, encompassing the processes of mitochondrial fusion and fission. The coordination of key GTPases—including the mitofusin homologues Mfn1 and Mfn2, optic atrophy 1 (OPA1), and dynamin-related protein 1 (DRP1)—is essential for regulating these dynamic changes. Reversible alterations in mitochondrial morphology serve as indicators of shifts in cellular metabolic states.^[6,7]

Oxidative phosphorylation (OXPHOS) is a complex process in which molecules are oxidized within the IMM to drive the conversion of adenosine diphosphate (ADP) into adenosine triphosphate (ATP). This bioenergetic process is initiated by the flow of electrons through the mitochondrial respiratory chain (MRC), which comprises complexes I to IV, two mobile electron carriers, and ATP synthase.^[8,9] When redox reactions proceed incompletely, they can lead to the generation of toxic by-products, most notably reactive oxygen species (ROS).^[10]

In addition to their energy-converting roles, mitochondria function as processing factories for steroidogenesis in granulosa cells (GCs). Two rate-limiting enzymatic steps regulate the core framework of this steroidogenic process. The steroidogenic acute regulatory protein (StAR) interacts with the complex “transduceosome” located in the OMM to facilitate the transfer of cholesterol to the IMM. The cholesterol side-chain cleavage enzyme, P450scc (CYP11A1), processes cholesterol and catalyzes the subsequent reactions necessary for the synthesis of estradiol and progesterone.^[11,12] GCs play a critical role in providing physiological protection and support for oocyte development and maturation.

Mitochondria play a crucial role in maintaining cellular quality control through processes such as mitophagy and apoptosis. Mitophagy, a specialized form of autophagy, involves the elimination of dysfunctional or excessive mitochondria to maintain mitochondrial and cellular homeostasis. Its significance in the aging process and age-related diseases has been well established.^[13,14] There are two primary pathways of programmed cell death: the endogenous (mitochondria-dependent) pathway and the exogenous (death receptor) pathway, as illustrated in Figure 2. In the mitochondrial-dependent pathway, excessive ROS induces changes in MMP and mitochondrial permeability, triggering the release of cytochrome c (Cyt c) from mitochondria. This, in turn, activates the caspase-9/caspase-3/B cell lymphoma 2-associated protein X (BAX)/B cell lymphoma 2 (BCL-2) signaling axis, leading to apoptosis. In the exogenous pathway, elevated ROS levels cause the release of tumor necrosis factor alpha (TNFα), which activates the Fas/Fas ligand (FasL)/caspase-8/caspase-3 pathway. Notably, activated caspase-8 induces Bid-mediated Cyt c release from mitochondria, creating a crosstalk between the intrinsic and extrinsic apoptotic pathways.^[15,16]

Figure 2.

Oxidative pressure induced apoptosis via the endogenous (mitochondrial) pathway and exogenous (death receptor) pathway. Apaf-1: Apoptotic protease activating factor 1; ATP: Adenosine triphosphate; CASP: Caspase; Cyt-c: Cytochrome c; dATP: 2′-deoxy-ATP; DISC: Death-inducing signaling complex; FADD: FAS-associated protein with death domain; MMP: Mitochondrial membrane potential; ROS: Reactive oxygen species; TNFα: Tumor necrosis factor alpha; tBid: Truncated Bid.

The following sections will elaborate on the specific molecular mechanisms underlying mitochondrial dysfunction in mitochondrial dynamics, bioenergetics, steroidogenesis, mitophagy, and apoptosis, and how these alterations contribute to the acceleration of ovarian aging.

Molecular Profiles of Mitochondrial Function in Ovarian Aging

Mitochondrial dynamics

Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that possesses both kinase and GTPase activities.^[17] In mouse oocytes, LRRK2 exhibited a distribution pattern similar to that of mitochondria, accumulating around the spindles. By upregulating the expression of actin-related proteins, including Rho-kinase (ROCK), cofilin, and fascin, LRRK2 regulated spindle migration as well as the temporal and spatial movement of mitochondria during oocyte meiosis. Dysfunction of LRRK2 impaired ATP production and reduced the mtDNA copy number, further compromising the developmental competence of oocytes.^[18]

Sirtuin 2 (SIRT2) belongs to the sirtuin family, which consists of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases and exerts versatile effects on mitochondrial function.^[19] Seven homologous substances (SIRT1-7) have been elucidated in mammals. In bovine oocytes, SIRT2 recovered the balance between mitochondrial fission and fusion by flexibly increasing the expression of mitochondrial fusion-related protein mitofusin 2 (MFN2) and decreasing the expression of mitochondrial fission-related protein DRP1. Besides, SIRT2 supported mitochondrial biology in MMP maintenance and ATP production, through provoking mitochondrial transcription factor A (TFAM)-dependent biogenetic activities. SIRT2-mediated mitochondrial dynamics and biogenesis contributed to meiotic progression and oocyte developmental potential.^[20]

In close proximity to the OMM, mitochondrial Rho GTPase 1 (MIRO1) anchors mitochondria to motor proteins for spatiotemporal distribution, which subsequently drives cellular metabolism.^[21] In mature oocytes of mice, MIRO1 was highly expressed and colocalized with mitochondria. After Miro1 disruption, mitochondria exhibited a larger volume and more complex cristae. The stage-specific patterns of mitochondrial dispersion and aggregation were disrupted during meiotic metaphase I (MI). Mitochondria were heterogeneously redistributed from the cytoplasm to the spindle, disturbing the completion of germinal vesicle breakdown (GVBD) and meiotic maturation of oocytes. In addition, MIRO1 expression stimulated the bioactivities of the mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) to meet the basic energy demands for meiotic progression and subsequent oocyte maturation.^[21,22]

Apart from proteins and genes, some molecular domains also capably impact oocyte physiology. In mature oocytes, genetic transcription remains silent. The preservation of messenger RNAs (mRNAs) for meiosis and early embryogenesis is quite crucial at this stage. In mammalian oocytes, maternal RNAs were docked in a mitochondria-associated ribonucleoprotein domain (MARDO), keeping them in a translationally inactive form. The packaging of MARDO around mitochondria was mediated by zygote arrest 1 (ZAR1), a kind of RNA-binding protein, for mitochondria enrichment. Incompletion of this interaction contributed to the chaotic distribution of mitochondria and premature loss of MARDO-associated mRNAs. This discovery elaborated the significance of maternal compounds in oocyte development potential.^[23]

Mitochondrial fusion and fission were not only the structural basis for its biological functionality but also indispensable for organelle–organelle interaction. The permutation distribution of mitochondria precisely conducts and governs the oocyte meiotic process. By targeting biomolecules involved in mitochondrial dynamics, we could regulate meiotic arrest and resumption in different meiotic stages.

Mitochondrial biogenesis

The process of oocyte growth and maturation is mainly influenced by some hypothalamic, hypophyseal, and peripheral hormones. The roles of several intra-ovarian autocrine or paracrine factors are of equal importance, such as insulin-like growth factors (IGFs) and transforming growth factor β (TGF-β) family members.^[24] In zebrafish oocytes, IGF1 enhanced OXPHOS by upregulating the expression of MRC subunits encoded by nuclear DNA (nDNA) and mtDNA. Enhanced mitochondrial metabolic status was evidenced by increased membrane polarization and ATP generation. Mechanistically, IGF1 mediated the activation of phosphatidylinositol-3-kinases/protein-serine-threonine kinase/nuclear respiratory factor 1 (PI3K/AKT/NRF-1) signaling cascades that consequently induced the phosphorylation of glycogen synthase kinase 3β (GSK3β) and accumulation of peroxisome proliferator-activated receptor gamma coactivator-1β (PGC-1β). Through its functional relationship with mitochondria, IGF1 promoted the resumption of meiotic maturation in oocytes from meiotic prophase I (G2) to the MI transition.^[25] Collectively, IGF1 administration can compensate for the deterioration of mitochondrial bioactivities and morphology, thus rescuing the quality of aging oocytes. As a member of the TGF-β superfamily, growth differentiation factor-9 (GDF-9) is involved in folliculogenesis and steroidogenesis in mammals.^[26] In grown secondary follicles of sheep oocytes, exogenous supplements of GDF-9 provoked mitochondrial activities and increased the proportion of active mitochondria, thus promoting oocyte maturation.^[27]

During the developmental stages of zebrafish oocytes, the expression of oxidation resistance 1a (oxr1a) was increased during the process of follicle activation and oocyte maturation. Deletion of oxr1a exacerbated mitochondrial oxidative damage and reduced mtDNA copy number, contributing to the aging phenotype of oocytes. By upregulating the expression of key antioxidant genes, superoxide dismutase 1 (sod1) and catalase (cat), OXR1A accelerated ROS metabolism and alleviated mitochondrial oxidative pressure, thus providing a favorable microenvironment for the viability and growth of oocytes.^[28]

Situated in the IMM, inner mitochondrial membrane peptidase 2-like (IMMP2L) participates in receiving and cleaving space-sorting signals to trigger cellular activity.^[29,30] In mice with Immp2l knockout, more degenerated and atretic follicles were observed, exhibiting specific characteristics such as marked luteinization and failure of ovulation. Loss-of-function assays indicated that Immp2l deletion perturbed the activity of mitochondrial respiratory complex I+III by targeting the signal peptide sequence processing of glycerol phosphate dehydrogenase 2 and the Cyt c1, which consequently induced mitochondrial hyperpolarization and excess superoxide ion generation. Functional inhibition of mitochondria through IMMP2L deficiency culminated in reproductive defects in mutant mice.^[30]

Located in the OMM, mitochondrial E3 ubiquitin ligase (MARCH5) governs the balance between mitochondrial fusion and fission by ubiquitinating related proteins.^[31] In March5-knockdown oocytes of mice, the bioenergetic capacity of mitochondria was compromised, with lower ATP content, decreased mtDNA copy number, and excessive ROS. Subsequently, energy deficiency and the accumulation of abnormal metabolites disturbed the functional interplay between kinetochores and spindle microtubules, arresting meiotic progress at the MI stage. Follicular growth was arrested, accelerating follicle loss and the depletion of ovarian reserve.^[32]

Nicotinamide phosphoribosyltransferase (NAMPT) is primarily responsible for catalyzing substrates in the NAD⁺ biosynthetic pathway of mammals.^[33] As a transcription factor, Forkhead box O3a (FoxO3a) participates in regulating cell survival and death. In reproductively old mice, NAMPT enzyme activity was essential for mitochondrial thermodynamics and bioenergetics by targeting FoxO3a expression. NAMPT can alleviate mitochondrial malfunction caused by FoxO3a expression, thereby recovering hijacked oocyte meiosis and promoting oocyte proliferation in aging oocytes.^[34]

As the evidence showed, apart from providing a structural basis, the oocyte meiotic process also requires mitochondrial energy supply. Sufficient ATP output from mitochondria ensures the completion of oocyte proliferation and follicular maturation. Proper activation of OXPHOS is important for maintaining mitochondrial and cellular homeostasis. The discovery of effectors related to mitochondrial biogenetics promotes multitarget and multipathway strategies for oocyte rejuvenation.

Mitochondrial biosynthesis

Compared with younger women with normal or diminished ovarian reserve (DOR), the expression of mitochondrial SIRT3 protein in GCs and cumulus cells (CCs) from women with ARA was significantly downregulated, along with the SIRT3-mediated deacetylation activity of glutamate dehydrogenase. Consequently, the reduced deacetylation activity of mitochondrial SIRT3 led to metabolic reprogramming and alterations in steroidogenesis within the oocyte microenvironment, ultimately compromising ovarian function and oocyte quality in aged women.^[35,36]

DNA binding inhibitory factor 3 (ID3) plays a crucial role in the endocrine regulation of mammalian ovaries by modulating mitochondrial protein expression.^[37] In bovine CCs, ID3 downregulated the expression of Parkin and DRP1, while upregulating MFN1 and MFN2, thereby promoting mitochondrial fusion rather than fission or autophagy. These alterations in mitochondrial morphology and motility facilitated steroidogenic processes. Concurrently, ID3 stimulated the expression of key steroidogenic proteins, including StAR, CYP11A1, and 3-beta-hydroxysteroid dehydrogenase 1 (HSD3B1), thereby facilitating the conversion of cholesterol into progesterone. ID3 significantly enhanced mitochondrial-mediated progesterone synthesis, thereby supporting oocyte proliferation and maturation at the molecular level.^[38]

Part of the molecular machinery through which Neuromedin S (NMS) induced steroidogenesis in goat ovarian GCs was similar to that of ID3. NMS treatment facilitated steroid hormone synthesis by altering the dynamic morphological structure of mitochondria, which depended on the fluctuating expression of fusion and fission genes, as well as key steroidogenesis-related genes.^[39,40] In addition, NMS enhanced mitochondrial function in ROS elimination, ATP generation, and the activity of MRC complexes by modulating the expression of mitochondrial unfolded protein response markers. Activation of the neuromedin U receptor type-2/Yes-associated protein isoform 1/PPARG coactivator 1 alpha (NMUR2/YAP1/PPARGC1A) signaling cascades, a component of the classical Hippo pathway, was responsible for the aforementioned cellular alterations. Collectively, NMS played a central role in mitochondrial-dependent steroidogenesis and consequently promoted follicular development and maturation.^[39,40]

The process of mitochondrial steroidogenesis also necessitates coordination with mitochondrial dynamics and biogenesis. Numerous effectors execute enzymatic reactions and material transformations essential for steroid synthesis. These mechanistic insights offer novel perspectives for targeting mitochondria to enhance endocrine regulation of the ovaries and promote long-term female health.

Mitochondrial apoptosis and mitophagy

Growth arrest-specific gene 6 (Gas6) is widely expressed in human tissues and plays a role in cell survival, division, and proliferation.^[41] In aging mice, inhibition of Gas6 impaired cytoplasmic maturation through mitochondrial dysfunction, as evidenced by abnormal mitochondrial accumulation and activation. This may be attributed to mitophagy inhibition, mediated by the activation of mammalian target of rapamycin (mTOR) and downregulation of autophagy-related genes. GAS6 mediated mitochondrial mitophagy to improve cytoplasmic maturation and prepare for subsequent fertilization.^[42] Furthermore, GAS6 restored mitochondrial biogenesis by increasing mtDNA copy number, mtDNA expression, and intracellular levels of ATP and glutathione (GSH). GAS6 attenuated aging-related mitochondrial dysfunction and maintained the competence of aged oocytes.^[43]

In addition to influencing mitochondrial dynamics and biogenesis, SIRT2 knockdown in sheep CCs contributed to increased mitophagy and apoptotic activity by activating the formation of mitophagosomes and lysosomes. Mechanistically, disinhibition of mitogen-activated protein kinase 15 (MAPK15) by SIRT2 silencing upregulated the expression of mitophagy genes and triggered the Parkin-mediated mitophagy pathway. Concurrently, this reciprocal interaction triggered CASP3/9 bioactivities, as well as a massive release of Cyt c and Ca²⁺ into the cytoplasm. SIRT2 regulated mitophagy in CCs by targeting MAPK15, which impaired cytoplasmic maturation, oocyte viability, and quality.^[44,45]

Protein kinase D (PKD), expressed ubiquitously in all tissues, is a member of the mitogen-activated protein kinase family and regulates various cellular states.^[46] In germinal vesicle (GV) oocytes of mice, PKD deficiency altered mitochondrial architecture, resulting in a clustered and homogeneous arrangement. Mitochondrial OXPHOS was incomplete, leading to abnormal accumulation of ROS and oxidative damage to mtDNA. This vicious cycle further exacerbated mitochondrial dysfunction and oocyte apoptosis. Mechanistically, PKD modulated the expression of autophagy-related proteins, microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (p62), by promoting the phosphorylation of UNC-52-like kinase 1 (ULK1). In addition, PKD upregulated the mitophagy effector protein Parkin and accelerated MFN2 degradation to initiate and execute mitophagy. PKD attenuated oxidative stress and maintained mitochondrial homeostasis by promoting mitophagy, thus rescuing age-related deterioration in oocyte quality.^[47]

MicroRNA-484 (miR-484), a novel non-coding RNA, has been shown to impact mitochondrial dynamics and cell apoptosis. MiR-484 levels were elevated in GCs of women with DOR. By binding to the 3′ untranslated region of YAP1, miR-484 negatively regulated YAP1 mRNA transcription, thereby intensifying mitochondria-dependent apoptosis and functional inhibition in human GCs. Mitochondrial dysfunction-induced degeneration in GCs quality ultimately promoted the onset of DOR.^[48] In mouse GCs, miR-484 overexpression contributed to the accumulation of mitochondrial and cellular ROS by impairing the bioactivities of both nonenzymatic and enzymatic antioxidant systems, such as SOD and GSH. Through the close interplay of the LINC00958/miR-484/Sestrin2 (SESN2) signaling loop, miR-484 dynamically reconstructed the mitochondrial network to coordinate cellular trafficking and drive programmed cell death. Several apoptosis-related proteins, including Cyt c, CASP3, BAX, and BCL-2, were activated to execute cell apoptosis. MiR-484 impaired GC function by disrupting the mitochondrial network and inducing a pro-apoptotic microenvironment, ultimately leading to follicular atresia and ovarian disorders.^[49]

As a type of aminoacyl-tRNA synthetase, Leucyl-tRNA synthetase 2 (LARS2) regulates the expression of genes encoded by mtDNA.^[50] In mouse GCs and human GC line (KGN cells), LARS2 deficiency impaired the metabolic status and stress response capacity of mitochondria, as evidenced by abnormal levels of Ca²⁺, ATP, and ROS production. LARS2 inhibition downregulated the expression of mitochondrial fusion protein MFN2 by disrupting the binding of E2 promoter binding factor 1 (E2F1) to the MFN2 promoter, while simultaneously upregulating the expression of apoptotic proteins, including BAX, Cyt c, CASP3, and BCL-2. This established the structural and kinetic basis for mitochondrial-mediated cell death induced by excessive ROS accumulation. Collectively, LARS2 played a pivotal role in the proliferation and apoptosis of GCs and contributed to the pathogenesis of premature ovarian insufficiency (POI).^[51] As an mRNA-binding protein, Lin28a regulates cellular metabolism and the maintenance of stemness. Intriguingly, Lin28a abrogated the promotive effects of LARS2 in KGN cells by directly binding to Lars2 mRNA and promoting its transcript degradation. Lin28a disrupted metabolic homeostasis and mitochondrial biogenesis by interfering with Lars2-mediated expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and nuclear respiratory factor 2 (NRF2), thereby impairing oocyte developmental potential.^[52]

Mitochondrial ATP-dependent Lon protease 1 (LONP1) exerts its proteolytic function by degrading or recycling mitochondrial proteins and enzymes to maintain cellular homeostasis.^[53] The expression of LONP1 was significantly reduced in aged women. Lonp1 disruption increased the expression of numerous apoptosis-related genes and reduced the expression of genes involved in mitochondrial bioenergetics, biogenesis, and dynamics, leading to a pro-apoptotic environment for developing follicles. Anchored in the IMM, apoptosis-inducing factor mitochondria-associated 1 (AIFM1) translocated from the cytoplasm to the nucleus upon Lonp1 ablation, subsequently leading to oocyte death in secondary follicles. These pathological processes induced by Lonp1 deficiency underscore its essential role in oocyte viability, follicular maturation, and early oogenesis.^[54]

As a member of the RAS oncogene family, the small GTPase RAB7 regulates cytoskeletal dynamics and vesicular trafficking involved in endosomal and lysosomal formation. Parkin RBR E3 ubiquitin protein ligase (PRKN) has been preliminarily identified as a mediator of RAB7-induced mitophagy.^[55,56] In MI oocytes of mice, reproductive aging was associated with a significant decrease in RAB7 protein levels and a gradual increase in PRKN expression. To initiate mitophagy and eliminate dysfunctional mitochondria, RAB7 translocated to and anchored on damaged mitochondria, facilitating mitophagosome formation in aged oocytes. However, PRKN colocalized with RAB7 and inhibited its mitophagy signaling cascade. Moreover, RAB7 recruited actin nucleation factors to the mitochondrial surface to promote actin polarization and polymerization, thereby enhancing phosphorylation of DRP1 at the Ser616 site and inducing mitochondrial fission. The coordinated dynamics of actin filaments and mitochondria maintained the spatiotemporal localization and trafficking of the spindle, ensuring proper meiotic maturation. Collectively, RAB7 preserved oocyte quality during aging by regulating mitophagy and mitochondrial movement.^[58]

The balance between apoptosis and cell survival is essential for maintaining cellular quality control. In ovarian oocytes, mitochondria-mediated mitophagy and apoptosis facilitate the selective removal of dysfunctional mitochondria and damaged cells, respectively. Targeting these processes can significantly mitigate the decline in oocyte quality and developmental competence.

Although numerous studies employing gene deletion, overexpression, or mitochondrial perturbation models have demonstrated the pivotal role of mitochondrial regulators in oocyte quality and quantity, direct evidence showing their dysregulation in physiologically aged ovaries remains limited. Therefore, further studies are urgently needed to validate the pathological relevance of these molecules under physiological aging conditions. The genes summarized in this review may serve as potential candidates for future experimental investigations aimed at elucidating the molecular underpinnings of ovarian aging [Table 1].

Table 1.

The underlying molecular mechanisms of mitochondria related to ovarian aging.

Process	Molecule	Experimental model	Target	Efficacy	Reference
Mitochondrial dynamics	LRRK2	GV-stage oocytes of mice	ROCK/cofilin/Fascin	Facilitate spindle migration and mitochondrial function during meiosis	[18]
	SIRT2	Bovine oocytes	TFAM/MFN2/DRP1	Maintain homeostasis of mitochondrial fission–fusion dynamics	[20]
	MIRO1	GV-stage oocytes of mice	Stage-specific dynamic distribution of mitochondria	Ensure spindle length and chromosome arrangement during MI stage	[21,22]
	MARDO	Drosophila oocytes	ZAR1	Maintain maternal mRNA stability and mitochondrial movement	[23]
Mitochondrial biogenesis	IGF1	Zebrafish oocytes	MRC and PI3K/AKT/NRF-1/GSK3β/PGC-1β	Promote oocyte bioenergetics and G2-MI transition	[24,25]
	GDF-9	Grown secondary follicles of sheep	Mitochondrial activities	Contribute to oocyte meiotic resumption	[27]
	OXR1A	Zebrafish oocytes	Sod1/Cat	Reduce mitochondrial oxidative damage and enhance oocyte proliferation	[28]
	IMMP2L	Mice	Glycerol phosphate dehydrogenase 2 and Cyt c1	Influence the oxidative homeostasis of mitochondria and folliculogenesis	[30]
	MARCH5	Mice	ATP generation	Impair kinetochore-microtubule movement and spindle formation during MI	[32]
	NAMPT	Naturally aged mice	FoxO3a	Promote mitochondrial thermodynamics and oocyte meiosis resumption	[34]
Mitochondrial biosynthesis	SIRT3	GCs and CCs of women	Glutamate dehydrogenase	Alter the metabolic and steroidogenic process in aging oocytes.	[35,36]
	ID3	Bovine CCs	MFN1/MFN2/DRP1/Parkin	Promote mitochondrial fusion	[38]
			StAR/CYP11A1/HSD3B1	Enhance oocyte maturation and quality
	NMS	Goat ovarian GCs	OPA1/MFN1/MFN2/ DNM1L/FIS1 and StAR/CYP11A1/3BHSD/CYP19A1	Promote mitochondrial fusion and estrogen synthesis	[39,40]
			Mitochondrial unfolded protein response markers	Enhance MRC activities for energy generation
			NMUR2/YAP1/PPARGC1A	Improve steroidogenesis for follicular development and maturation
Mitochondrial apoptosis and mitophagy	GAS6	Aged mice	mTOR	Trigger mitophagy and improve cytoplasmic maturation	[42,43]
	SIRT2	Sheep CCs	MAPK15	Inhibit CC apoptosis by modifying mitophagy and improving the viability of oocytes	[44,45]
	PKD	GV-stage oocytes of mice	ULK1/ LC3/p62	Maintain mitophagy to preserve oocyte quality	[47]
			Parkin/MF2
	miR-484	GCs of human	YAP1	Induce mitochondria-dependent apoptosis and follicular atresia	[48]
		GCs of mice	LINC00958/miR-484/SESN2	Aggravate apoptosis induced by oxidative damage	[49]
	LARS2	Mice GCs and human granulosa cell line	E2F1-mediated MFN2 translation	Provide the structural and kinetic basis for mitochondrial-mediated cell death	[51]
	Lin28a	Immortal human GCs	Lars2/ Pgc-1α/Nrf2	Alter mitochondrial biogenesis and oocyte metabolic status	[52]
	LONP1	Mice oocytes	AIFM1 translocation	Causes massive apoptosis and compromised oocyte growth	[54]
	RAB7	Mice oocytes	PRKN-RAB7 interaction	Interfere clearance of damaged mitochondria and induce age-related deterioration of oocyte competence	[57]
			DRP1 phosphorylation	Change mitochondrial dynamics for the meiotic spindle arrangement	[58]

3BHSD: 3 Beta-hydroxysteroid dehydrogenase; AIFM1: Apoptosis-inducing factor mitochondria-associated 1; AMPK: AMP-activated protein kinase; ATP: Adenosine triphosphate; BAX: B cell lymphoma 2-associated protein X; BCL-2: B cell lymphoma 2; BCL-XL: B-cell lymphoma-extra-large; Cat: Catalase; CCs: Cumulus cells; Cyt c1: Cytochrome c1; CYP11A1: P450scc; CYP19A1: Cytochrome P450 family 19 subfamily A member 1; DNM1L: Dynamin 1-like gene; DRP1: Dynamin-related protein 1; E2F1: E2 promoter binding factor 1; FIS1: Fission 1 protein; FOXO3A: Forkhead box O3a; G2: Meiotic prophase I; GAS6: Growth arrest-specific gene 6; GCs: Granulose cells; GDF-9: Growth differentiation factor-9; GV: Germinal vesicle; GSK3β: Glycogen synthase kinase 3β; HSD3B1: 3-Beta-hydroxysteroid dehydrogenase 1; ID3: DNA binding inhibitory factor 3; IMMP2L: Inner mitochondrial membrane peptidase 2-like; IGFs: Insulin-like growth factors; LC3: Microtubule-associated protein light chain 3; LARS2: Leucyl-tRNA synthetase 2; LONP1: Mitochondrial ATP-dependent Lon protease 1; LRRK2: Leucine-rich-repeat kinase 2; MAPK15: Mitogen-activated protein kinase 15; MARCH5: Mitochondrial E3 ubiquitin ligase; MARDO: Mitochondria-associated ribonucleoprotein domain; MI: Meiotic metaphase I; MIRO1: Mitochondrial rho GTPase1; MFN1: Mitofusins 1; MFN2: Mitofusins 2; MRC: Mitochondrial respiratory chain; miR-484: microRNA-484; mRNA: messenger RNA; mTOR: Mammalian target of rapamycin; NAMPT: Nicotinamide phosphoribosyltransferase; NMS: Neuromedin S; NMUR2: Neuromedin U receptor type 2; Nrf2: Nuclear respiratory factor2; OPA1: Optic atrophy 1; OXPHOS: Oxidative phosphorylation; OXR1A: Oxidation resistance 1a; p62: Sequestosome 1; PI3K/AKT/NRF-1: Phosphatidylinositol-3-kinases/protein-serine-threonine kinase/nuclear respiratory factor 1; PKD: Protein kinase D; PRKN: Parkin RBR E3 ubiquitin protein ligase; PPARGC1A: PPARG coactivator 1 alpha; Pgc1α: Proliferator-activated receptor gamma coactivator 1 alpha; PGC-1β: Peroxisome proliferator-activated receptor gamma coactivator-1β; ROS: Reactive oxygen species; ROCK: Rho-kinase; SIRT2: Sirtuin 2; SIRT3: Sirtuin 3; SESN2: Sestrin2; StAR: Steroidogenic acute regulatory protein; sod1: Superoxide dismutase 1; TFAM: Mitochondrial transcription factor A; ULK1: UNC-52-like kinase 1; YAP1: Yes-associated protein isoform 1; ZAR1: Zygote arrest 1.

Mitochondria as a Novel Target for Clinical Therapy

Given the pivotal role of mitochondria in maintaining oocyte health and ovarian reserve, mitochondria-targeted medical interventions hold considerable promise in delaying or reversing ovarian aging. Increased oxidative stress disrupts the intracellular microenvironment, thereby accelerating ovarian aging. Antioxidants and nutritional therapies have emerged as potential interventions to mitigate the age-related decline in female fertility. Several agents, including coenzyme Q10, resveratrol, quercetin, growth hormone, dehydroepiandrosterone, and secoisolariciresinol diglucoside, have been extensively investigated for their pharmacological roles in mitochondrial rejuvenation.^[59–62] This review subsequently highlights several emerging therapies and their cytoprotective effects against mitochondrial dysfunction and premature ovarian aging.

Traditional chinese medicine

Traditional Chinese Medicine (TCM) has garnered growing global recognition for its multifaceted therapeutic potential across a wide range of diseases. Accumulating evidence suggests that TCM represents a promising therapeutic option for gynecological and endocrine disorders.^[63,64] This review outlines the critical roles of TCM-mediated mechanisms in addressing mitochondrial dysfunction and premature depletion of ovarian reserve as summarized in Table 2.

Table 2.

The signaling cascades regulated by Traditional Chinese Medicine and relevant influence on ovarian aging.

Cellular process	Active ingredients	Experimental model	Signal pathway	Functional activities	Reference
Mitochondrial-dependent cell apoptosis	ZGP	Cyclophosphamide-induced POF mice models	BAX/BCL-2 and Cyt c	Suppress mitochondria-induced apoptosis of oocytes and delay ovarian aging.	[65]
	DOP	Naturally aged mice with POF	p53/BCL-2 and NF-κB	Inhibit cell apoptosis and the inflammatory cascade	[66]
	HYF	Female mice of POI	quaporin8 and BCL-XL/BAX	Rescue mitochondrial apoptosis and enhance follicular development	[67]
	JPYS	POF models of human GC line	PGC-1α	Improve mitochondrial biogenetics and dynamics	[68]
			OPA1/MFN1/MFN2
			DRP1 and FIS1
			ASK1/JNK	Inhibit cell apoptosis and the etiology of ovarian failure
	TEAS	Radiation-induced POI mice	BCL-2/BAX	Suppress apoptosis and improve ovarian folliculogenesis	[69]
Mitochondrial dynamics or biogenetics	HSYC	Aged mice	Mitochondrial microstructure.	Improves mitochondrial function and oocyte metabolism in aged oocytes.	[70]
	Yu Linzhu	Aged mice			[71]
	Kuntai capsule	Aged mice	Mitochondrial morphology	Rescue defective mitochondria and follicular apoptosis	[72]
			SOD2/BCL-2/BAX
	KYZY	Mice	Polarized mitochondrial distribution	Provoke oocyte competence under physiological stress	[73]
			MRC or ATP synthase

ASK1: Apoptosis signal-regulating kinase 1; ATP: Adenosine triphosphate; BAX: B cell lymphoma 2-associated protein X; BCL-2: B cell lymphoma 2; BCL-XL: B-cell lymphoma-extra-large; Cyt c: Cytochrome c; DOP: Dendrobium officinal polysaccharide; DRP1: Dynamin-related protein 1; FIS1: Fission 1 protein; HSYC: Yangchao Recipe; HYF: Huyang Yangkun Formula; JNK: c-Jun N-terminal kinase; JPYS: Jian-Pi-Yi-Shen; KYZY: Kai Yu Zhong Yu; Mfn1: Mitofusins 1; MFN2: Mitofusins 2; MRC: Mitochondrial respiratory chain; NF-κB: Nuclear factor-κB; OPA1: Optic atrophy 1; PGC-1alpha: Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha; POF: Premature ovarian failure; POI: Primary ovarian insufficiency; SOD2: Superoxide dismutase 2; TEAS: Transcutaneous electrical acupoint stimulation; ZGP: Zuogui pills.

The mitochondria-mediated apoptotic pathway represents a classical signaling cascade involved in ovarian senescence. Several TCM formulations have been shown to target key biomolecules within this pathway to mitigate ovarian aging, including Zuogui pills (ZGP), Dendrobium officinale polysaccharide (DOP), Huyang Yangkun formula (HYF), and Jian-Pi-Yi-Shen (JPYS).^[65–68] In aged mice, administration of ZGP increased the BAX/BCL-2 expression ratio in ovarian tissue. Expression of the Cyt c gene and its mitochondrial release were concurrently suppressed, suggestive of follicular apoptosis hijacked by ZGP treatment.^[65] DOP enhanced mitochondrial structure and function by inhibiting NF-κB-mediated inflammation and p53/BCL-2-regulated apoptosis. DOP administration attenuated the inflammatory response and restored follicular development in aged mice.^[66] HYF attenuated mitochondrial apoptosis and promoted follicular development by downregulating the protein expression of aquaporin 8 and CASP3, accompanied by an increased BCL-XL/BAX ratio.^[67] JPYS exerted multifaceted effects in counteracting age-related ovarian hypofunction. By regulating the expression of mitochondrial biogenesis, fusion, and fission, JPYS improved mitochondrial ultrastructure and enhanced MRC activity for energy production. JPYS treatment inhibited the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK), thereby suppressing mitochondrial apoptosis-related signaling pathways. Notably, JPYS supplementation reversed progressive follicular depletion.^[68] As a noninvasive intervention, transcutaneous electrical acupoint stimulation (TEAS) reduced mitochondrial apoptosis and increased follicular counts in irradiation-treated mice by modulating the expression of BCL-2 and BAX, suggesting that TEAS may alleviate radiotherapy-induced oxidative stress and ovarian dysfunction.^[69]

The distribution and morphology of cellular mitochondria are dynamic and continuously changing. Mitochondria maintain functional interconnectivity to support cellular activities and meet metabolic demands.^[6] Herbal formulations such as He’s Yangchao Recipe (HSYC), Yu Linzhu, Kuntai Capsule, and Kai Yu Zhong Yu (KYZY) have been shown to restore ovarian function and improve reproductive performance by ameliorating disorganized mitochondrial architecture and localization.^[70–73] HSYC and Yu Linzhu exhibited similar pharmacological effects on mitochondria. Administration of HSYC and Yu Linzhu in mice ovaries rescued the vacuolated structures and disrupted cristae of abnormal mitochondria, and improved mitochondrial volume and density, thereby contributing to improved oocyte morphology and metabolism in aged mice.^[70,71] Kuntai Capsule reduced vacuolization and restored the function of mitochondrial cristae and matrix. By modulating the expression of SOD2, BCL-2, and BAX, Kuntai Capsule treatment inhibited follicular atresia and improved ovarian reserve.^[72] Moreover, KYZY not only restored damaged mitochondria and corrected aberrant mitochondrial distribution but also enhanced mitochondrial bioenergetics. Mechanistically, KYZY administration upregulated a cohort of genes involved in oocyte MRC function and ATP synthesis. KYZY enhanced the oocyte stress response and viability by preserving mitochondrial functionality.^[73] Collectively, TCM effectively rejuvenated aging oocytes by modulating mitochondrial dynamics and function.

Following the widespread promotion of TCM under China’s national rejuvenation plan, related scientific research has rapidly expanded. Increasing attention has been focused on the safety and efficacy of TCM treatments. Although compelling evidence supports a strong association between TCM-mediated mitochondrial function and ovarian aging, the complexity and variability of TCM formulations complicate further investigations into the multiple effectors and pathways underlying TCM pharmacodynamics. To date, existing studies remain limited and primarily focus on in vivo experiments. Further investigations, including in vitro and additional animal studies, are required to validate conclusions drawn from current research.

Stem cell transplantation

The application of stem cell-based therapies offers unprecedented opportunities for the medical community to address intractable diseases. Stem cell distribution varies among different organs. Currently, stem cell therapy has been widely applied to multisystem diseases, including neurodegenerative disorders, skin tissue engineering, and hematopoietic system lesions.^[74–76] The therapeutic potential of stem cells in the reproductive system has increasingly attracted scientific interest.

Recent evidence has elucidated the comprehensive effects of mesenchymal stem cells (MSCs) in counteracting age-associated ovarian hypofunction as summarized in Table 3. Human umbilical cord mesenchymal stem cells (hUCMSCs) possess abundant tissue sources, rapid proliferative capacity, and low immunogenicity, indicating their therapeutic potential for natural and premature ovarian aging.^[77] In theca interstitial cells (TICs) of POI rats, phosphorylated nuclear receptor 4A1 (NR4A1) translocated from the nucleus to mitochondria during apoptotic cascades, triggering increased mitochondrial membrane permeabilization and release of apoptosis-related substrates (BAX, Cyt c, and CASP9/3). By modulating NR4A1-mediated mitochondrial interactions, hUCMSCs effectively suppressed apoptosis and restored the endocrine function of TICs.^[78] Furthermore, hUCMSC treatment enhanced testosterone production by inhibiting GSK3β expression and activity.^[79] Thus, hUCMSC treatment ameliorated the ovarian microenvironment and metabolism in aging mice.

Table 3.

The pluripotent activities of stem cells and/or their mitochondria in ovarian physiology.

Stem cells and/or their mitochondria	Experimental model	Signal pathway	Functional activities	Reference
hUCMSCs	In TICs of POI mice	NR4A1 translocation and gene expression (BAX, Cyt c, CASP9, and CASP3)	Inhibits programmed cell death and improves oocyte health.	[72]
	In theca cells of POI mice	GSK3β expression	Enhance mitochondrial dynamics and steroidogenesis	[73,74]
EnMSC mitochondria	Aged mice	Meiotic process	Provoke oocyte developmental potential and mitochondrial activities.	[77]
		MMP and mtDNA copy number
MenSCs and MenSC mitochondria	Aged mice	Genes encoding mitochondrial compounds and functional enzymes.	Reverse age-related mitochondrial dysfunction and enhance oocyte competence	[79,80]
BMSCs	In POF mice	Mitochondrial dynamics	Attenuate age-related deterioration of ovarian quality	[81]
hAMSCs	In DOR mice	AMPK / FoxO3a	Regulate mitophagy and improve oocyte survival and development	[82]
hAMSCs-Exos	In aged mice	miRNA-320a/ SIRT4/OPA1	Enhance oocyte development and follicular maturation	[89,90]

AMPK: AMP-activated protein kinase; BAX: B cell lymphoma 2-associated protein X; BMSCs: Bone-marrow mesenchymal stem cells; CASP3: Aspase-3; CASP9: Caspase-9; Cyt c: Cytochrome c; DOR: Diminished ovarian reserve; EnMSC: Endometrial mesenchymal stem cell; Exos: Exosomes; FoxO3a: Forkhead box O3a; GSK3β: Glycogen Synthase Kinase 3β; hAMSCs: Human amnion-derived mesenchymal stem cells; hUCMSCs: Human umbilical cord mesenchymal stem cells; MenSCs: Menstrual blood-derived mesenchymal stem cells; MMP: Mitochondrial membrane potential; miRNA: microRNA; mtDNA: Mitochondrial DNA; NR4A1: Nuclear receptor 4A 1; OPA1: Optic atrophy 1; POF: Premature ovarian failure; POI: Primary ovarian insufficiency; SIRT4: Sirtuin 4; TICs: Theca interstitial cells.

Endometrial mesenchymal stem cells (EnMSCs), located in the endometrium, are typically isolated from menstrual blood or endometrial tissue.^[80] Following microinjection of EnMSC-derived mitochondria into aged mice, meiotic defects–including increased aneuploidy, chromosome misalignment, and spindle disintegration–were markedly ameliorated. MMP and mtDNA copy numbers were significantly improved. Oocytes successfully progressed to the metaphase II (MII) stage and subsequently developed into viable blastocyst-stage embryos, indicating enhanced developmental potential of aged oocytes conferred by EnMSC mitochondria.^[81]

Similar to EnMSCs, menstrual blood-derived mesenchymal stem cells (MenSCs) exhibit continuous self-renewal throughout the menstrual cycle.^[82] Injection of MenSC-derived mitochondria into aged mice alleviated disrupted ovarian morphology and replenished the follicular pool, accompanied by an increased number of developing follicles. Furthermore, global transcriptomic analysis revealed that incubation with MenSCs or their mitochondria significantly upregulated the expression of numerous genes related to mitochondrial structural components and biosynthetic enzymes.^[82,83]

Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) into aged mice improved the polarized distribution of mitochondria around the spindle during the MII stage and restored mitochondrial ultrastructure, including cristae organization and vacuolated morphology. BMSC transplantation increased follicle numbers and suppressed cell apoptosis, thereby enhancing oocyte quality and ovarian reserve.^[84] Human amnion-derived mesenchymal stem cells (hAMSCs) modulated mitophagy and alleviated DOR in mice by upregulating the AMP-activated protein kinase (AMPK)/FoxO3a signaling pathway. Consequently, ovaries treated with hAMSCs exhibited an increased number of developing follicles and enhanced fertilization capacity.^[85]

Although MSC treatment has been shown to enhance mitochondrial pluripotency and mitigate ovarian aging-related degeneration, the specific signaling molecules involved in MSC-organelle interactions remain unidentified. As a specialized subtype of extracellular vesicles (EVs), exosomes (Exos) are membrane-enclosed structures that carry microRNAs (miRNAs) and proteins.^[86] Several studies have demonstrated that MSCs deliver Exos to target effector cells, thereby promoting cellular repair.^[77,87] In aging mouse models, exosomal miRNA-320a derived from hAMSCs suppressed the SIRT4–OPA1 axis, thereby mitigating oxidative stress and restoring age-associated mitophagy impairment. Thus, hAMSC-derived Exos prevented oocyte degeneration by modulating the balance between proliferation and apoptosis.^[88] Treatment with MSC-derived EVs has been shown to attenuate follicular overactivation and enhance oocyte viability in mouse models of ovarian failure.^[89,90] However, the specific molecular cargos of EVs and their downstream pathways involved in mitochondrial regulation remain largely unexplored and warrant further investigation.

As a rapidly emerging research focus, the therapeutic efficacy of MSCs and MSC-derived mitochondria has garnered significant attention. However, these therapeutic strategies remain controversial concerning safety and ethical considerations. First, the optimal donor selection for mitochondrial extraction and the methods for isolating and culturing functionally intact mitochondria remain inconclusive. Second, current studies remain largely confined to in vitro models owing to the lack of suitable animal systems. These investigations fail to accurately replicate the in vivo intracellular microenvironment and intercellular communication. Moreover, signaling molecules secreted by MSCs may be nonspecifically absorbed by surrounding tissues rather than being delivered to target cells. Several studies have shown that MSCs may promote cancer cell malignancy by enhancing metabolic plasticity and resistance to chemotherapy, thereby compromising the efficacy of MSC transplantation.^[91] Therefore, further research is needed to elucidate the mechanisms by which donor mitochondria specifically recognize and interact with recipient cells. In addition, the molecular mechanisms underlying the various modes of mitochondrial transfer remain poorly understood. Extensive research is further required to determine how these signaling pathways can be coordinated or complemented to enhance the therapeutic efficacy of mitochondrial transfer. Finally, mtDNA also plays essential roles in the normal function of other organelles. The use of xenogeneic mitochondrial supplementation remains ethically controversial due to the maternally inherited nature of mtDNA.

Mitochondrial replacement therapies

Mitochondrial replacement techniques (MRTs), which directly target mitochondria or related cytoplasmic compounds, involve transferring the nDNA of an oocyte or zygote carrying mutated mtDNA to an oocyte or zygote containing healthy mitochondria. The resulting organisms possess nDNA from the biological maternal line and cytoplasmic compounds, including functional mitochondria from donor oocytes.^[92,93] These investigational techniques provide an alternative for reproductively older females to rejuvenate their own oocytes, rather than relying on donor oocytes.

Heterologous mitochondrial transfer

Mitochondrial enrichment during heterologous mitochondrial transfer can be achieved either through the introduction of basic cytoplasmic material into the recipient oocyte (partial ooplasm replacement) or by replacing the compromised cytoplasm with a functionally competent one using nuclear transfer protocols (total ooplasm replacement). Total ooplasm replacement techniques can be further categorized into pronuclear transfer (PNT), spindle transfer (ST), germinal vesicle transfer (GVT), and polar body transfer (PBT).^[94]

Partial ooplasm replacement involves transferring a portion of cytoplasm from healthy donor oocytes into recipient oocytes, concurrently introducing energy substrates, metabolic intermediates, and other cytoplasmic organelles to overcome mitochondrial dysfunction and enhance oocyte competence.^[94,95] This procedure can be performed either by fusing the donor oocyte cytoplasm (membrane-enclosed cytoplasmic fraction) with the recipient oocyte or by microinjecting a small volume of donor cytoplasm.^[94,95] Various sources of donor cytoplasm have been identified for transfer, including tripronuclear zygotes and both fresh and cryopreserved MII oocytes. Depending on the cell cycle stage of the donor and recipient oocytes, procedures can be classified as synchronous transfer (e.g., MII to MII) or asynchronous transfer (e.g., interphase zygote cytoplasm to MII oocyte).^[95] However, several studies have raised concerns that mitochondrial heteroplasmy may disrupt the signaling crosstalk between nDNA and mtDNA, potentially impairing oocyte development and embryogenesis.^[96,97]

In the context of total ooplasm replacement, PNT involves transferring the pronuclei from a zygote carrying mutated mtDNA into an enucleated donor zygote containing healthy mitochondria.^[98,99] The reconstructed zygote retains nDNA from the original parents and inherits functional mtDNA from the donor.^[98] Consequently, the primary controversy surrounding this technique stems from ethical concerns, as it requires the creation and subsequent disposal of surplus embryos. Due to their large size and central location, pronuclei are relatively easy to isolate. However, mitochondria surrounding the pronuclei are inevitably co-transferred during the PNT process, leading to mtDNA contamination in the offspring and an increased risk of mitochondrial carryover.^[95]

ST involves transplanting a karyoplast containing the MII spindle from aged or deficient oocytes into enucleated donor cytoplasm from which the spindle has been removed.^[99,100] Multiple studies have demonstrated the significant therapeutic potential of ST in enhancing fertility. One research group utilized spindles isolated from in vitro-matured MII human oocytes to model age-related aneuploidies in oocytes from reproductively older women. This approach markedly improved oocyte developmental potential by rescuing age-associated chromosomal abnormalities.^[101] Moreover, robust evidence has shown that ST exhibits high efficiency and a low incidence of mitochondrial carryover.^[102,103] Due to their small size and heterogeneous distribution within the cytoplasm, spindles are difficult to visualize, increasing the risk of inaccurate transfer. Furthermore, spindles are highly sensitive to micromanipulation, posing technical challenges for their retrieval.

GVT involves enucleating donor oocytes at the GV stage before they transition to the MII stage, and using them as recipients for GV nuclei from the patient’s primary oocytes. The reconstructed oocytes must undergo in vitro maturation to complete the GV-to-MII transition before fertilization.^[104,105] GVT represents a promising approach for women with DOR or those unable to generate developmentally competent oocytes or viable embryos during ART. GVT facilitates meiotic resumption and promotes the maturation of previously immature oocytes.^[104] As the GV is enclosed by a protective nuclear membrane, it is readily visualized under microscopy, rendering GVT a technically less invasive procedure.

Polar bodies are generated through asymmetric cell divisions during MI and MII. PBT encompasses two distinct procedures—first polar body transfer (PB1T) and second polar body transfer (PB2T)—which can be performed independently or sequentially.^[106] PB1T involves isolating the first polar body (PB1) from compromised MII oocytes of a patient and transferring it into an enucleated donor oocyte that is at the MII stage but lacks the meiotic spindle.^[93,95] This procedure resembles ST, except that PB1 substitutes for the meiotic spindle in the donor enucleated oocyte. PB2T entails transferring the second polar body (PB2) from the patient’s zygote into a healthy donor zygote in which the maternal pronucleus has already been removed. The resulting zygote carries nuclear genetic material from the patient’s polar body and the intended paternal sperm (as pronuclei), along with healthy mtDNA.^[107,108] A key controversy concerns potential epigenetic disparities between the nuclear genome and the polar bodies extracted during meiosis.^[93,107,108]

Autologous mitochondrial transfer

To address concerns and controversies arising from heterologous mitochondrial sources, autologous germline mitochondria have gradually attracted attention. Ovarian germline cells—such as ovarian stem cells/egg precursor cells (OSCs/EggPCs) and GCs—are considered ideal autologous donor sources. Because these cells have undergone the mitochondrial bottleneck, their mtDNA repertoire harbors fewer deletions and mutations.^[109] This procedure—termed autologous germline mitochondrial energy transfer (AUGMENT)—involves injecting mitochondria isolated from OSCs/EggPCs in the ovarian cortex into recipient oocytes at the time of intracytoplasmic sperm injection (ICSI), along with the spermatozoon.^[110] In this protocol, DDX4-positive OSCs are isolated from the patient’s ovarian cortex for subsequent mitochondrial extraction and transfer.^{[103,110,111]} The functional integrity, quality, and quantity of mitochondria provide the foundation for oocyte development, maturation, and fertilization.^[103,112] However, some experts have questioned the reliability of DDX4 as a marker, noting its nonspecific and relatively weak expression in OSCs from mouse and adult human ovaries.^[103,112] Ovarian aging is associated with a decline in mitochondrial competence. Therefore, mitochondrial transfer from OSCs of reproductively aged patients may not achieve optimal therapeutic outcomes for infertility. Moreover, the process of oocyte retrieval may damage the ovarian cortex and further deplete ovarian reserve.

The controversies and challenges surrounding MRT primarily center on ethical and technical issues. It remains unclear whether mtDNA heteroplasmy induces epigenetic anomalies in reconstructed oocytes or embryos. Standardizing the retrieval procedure—to precisely target desired cells, reduce mitochondrial carryover across generations, and minimize mechanical damage to the ovaries—remains an unresolved challenge. Existing in vivo, in vitro, and animal model studies on MRT remain limited and inconclusive. Further investigations are required to comprehensively assess the developmental potential of oocytes derived from OSCs, including cell viability, metabolic status, and genetic profiles. Long-term studies evaluating the safety of mitochondrial transfer across successive generations are also warranted.

Conclusion

This article provides a comprehensive review of the expanding roles of mitochondrial function in ovarian aging and reproductive longevity in humans, highlighting its impact on key physiological processes. Mitochondria-targeted therapies show significant potential in mitigating age-related deterioration of oocytes. The application of TCM, stem cells, or their mitochondria offers novel avenues for restoring mitochondrial function. However, evidence supporting the biological relevance and clinical significance of mitochondrial factors in female oocyte aging remains limited. Further preclinical and clinical studies are needed to validate the therapeutic potential of mitochondrial-based interventions in humans and their offspring.

Funding

This work was supported by grants from the Center for Reproductive Medicine, Shandong University, the National Key Research and Developmental Program of China (No. 2022YFC2703800), and the National Natural Science Foundation of China (Nos. 82125014, 82421004, and U24A20661).

Acknowledgement

The authors acknowledge the technical support from the Center for Reproductive Medicine, Shandong University, and our team for helping us in completing the manuscript.

Conflicts of interest

None.