Mitochondrial dysfunction in oocytes: implications for fertility and ageing

Abstract

The impact of environmental pollution on fertility has become an essential issue in global public health. Maturation, fertilisation, and embryonic development of oocytes depend on the energy provided by mitochondria; however, with increased environmental pollution and ageing, mitochondrial dysfunction and its subsequent functional and metabolic abnormalities have become leading causes of female fertility decline. When mitochondrial dysfunction occurs in the oocyte, reduced metabolic efficiency leads to impaired nuclear and cytoplasmic maturation of the oocyte, affecting the quality of the oocyte, which further contributes to decreased female fertility and increased risk of infertility, miscarriage, and aneuploid foetuses due to ovarian dysfunction. Several factors affect mitochondrial function, including excess reactive oxygen species (ROS)-induced mutations in mitochondrial DNA (mtDNA), changes in mtDNA copy number, oxidative stress (OS), damage to key cellular components and organelles, and changes in metabolic intermediates and byproducts at the cellular level, further affecting oocyte developmental competence. Mitochondrial dysfunction leads to problems such as abnormal spindle formation and chromosome misalignment, reducing fertilisation potential and embryonic developmental capacity. Mitochondrial dysfunction plays a key role in oocyte ageing and the decline in germ cell function, and an in-depth study of its molecular mechanisms and intervention strategies is highly important for slowing oocyte ageing, increasing fertility, and improving the success rate of assisted reproduction techniques.

Clinical trial number

Not applicable.

Introduction

Against the backdrop of rapid socioeconomic development in contemporary society, humanity is facing a crisis in reproductive health caused by multiple factors, such as environmental pollution, life stress, and unhealthy lifestyles. The worsening of environmental pollution, including air, water, and soil pollution, has led to deterioration of the reproductive environment. Fast-paced life and intense work pressure have increased physical and mental burdens, coupled with the prevalence of unhealthy lifestyle habits. Together, these factors have led to a continuous decline in the social birth rate and a significant increase in the incidence of fertility-related diseases. Notably, oocytes, a key determinant of female fertility, are fully formed at birth and are not renewable [1]. Environmental pollutants can enter the human body through respiratory inhalation (PM2.5, benzo[a]pyrene, etc.) [2], food chain ingestion (2,3,7,8-tetrachlorodibenzo-p-dioxin) [3], and dermal contact (phthalate esters) [4], etc. Prolonged exposure to hazardous substances in the environment may lead to accumulation over the long term in female oocytes, which may result in damage to the oocytes and a decrease in their function [5]. In contrast, male spermatozoa can be continuously produced [6]. Thus, female oocytes are at increased risk of long-term environmental pollution and damage. In addition to environmental factors, age also directly affects female fertility. The risk associated with ovarian decline is exacerbated by the gradual postponement of childbearing by women. This physiological deterioration not only significantly reduces fertility but also leads to an increased miscarriage rate in older pregnancies and a higher risk of chromosomal aneuploidy in the embryo. The live birth rate per oocyte has been shown to decrease dramatically with age, decreasing from 26% in women under 35 years to only 1% in women older than 42 years [7]. Age-related fertility decline and ovarian dysfunction are due primarily to natural follicular depletion, which causes oocyte depletion. This depletion results in both a reduced number of oocytes and a decline in their quality, with mitochondrial dysfunction being a central mechanism of oocyte senescence.

Mitochondrial dysfunction may induce OS, mtDNA damage and related mutations, epigenetic alterations, spindle assembly defects, meiotic errors, chromosomal misalignments, and telomere shortening, which may lead to a decrease in oocyte quantity and quality [8–11]. In turn, the number and quality of oocytes a woman has may decrease with age, which may lead to impaired mitochondrial function; these two factors may interact. The key function of mitochondria is to generate ATP for the cellular energy supply through oxidative phosphorylation, and the role of mitochondria in cellular metabolism is multifaceted; mitochondria also regulate apoptosis and the cell cycle [12] and influence the cellular redox state and energy sensing by controlling the production of ROS and the maintenance of the mitochondrial membrane potential [13]. Oocyte maturation and development depend on mitochondrial oxidative phosphorylation to produce the necessary ATP. Mitochondria are therefore required to provide sufficient ATP to support both the smooth progression of intracellular biosynthesis throughout oocyte maturation and the successful segregation of chromosomes during the oocyte maturation stage. This provision is also essential to ensure that the oocyte enters the first cleavage after fertilisation and ultimately supports the rapid division of the embryo during early development. Therefore, the most serious effects of mitochondrial dysfunction are impaired ATP synthesis and mitochondrial DNA damage, which negatively affect chromosome segregation and impair embryonic development.

The potential of the oocyte is reflected in its ability to mature successfully, participate in the fertilisation process, and facilitate the development of the embryo to the first mitotic division and genome activation. This ability is directly linked to the vitality of the embryo and thus affects the chances of a woman conceiving. During its growth, an oocyte interacts closely with surrounding granulosa cells and follicular fluid components; these interactions are critical for oocyte maturation and function. As oocyte quality is a key determinant of embryo viability and fertilisation success, its interaction with the follicular microenvironment during growth has a decisive impact on quality [14, 15]. Mitochondria, the centres of cellular energy metabolism, exhibit unique biological characteristics during oocyte development.Compared with the typical branching morphology and structure of somatic mitochondria containing abundant crista, mitochondria in human oocytes and early embryos (up to the mulberry embryo stage) present a special round/cap-like morphology and poorly developed cristae, which leads to a significant reduction in their oxidative phosphorylation efficiency. To meet the ATP requirements of oocytes during maturation, mammalian oocytes have evolved a specific mitochondrial strategy: mature oocytes contain up to ~ 100,000 mitochondria, which is thousands of times more than the amounts found in spermatozoa (50–75) and somatic cells (~ 2,000). This reserve meets the ATP requirement for oocyte maturation and provides energy security for early embryonic development after fertilisation. Recent studies have revealed that mitochondrial dysfunction is significantly correlated with decreased oocyte quality. This dysfunction involves abnormalities such as the accumulation of mitochondrial DNA mutations, decreased membrane potential, and abnormal autophagy. These defects may interfere with meiotic spindle formation and calcium oscillator signalling, ultimately leading to oocyte senescence and related reproductive disorders. In this paper, we systematically review the substantial research progress in this field in recent years and aim to elucidate the associations between mitochondrial dysfunction and the regulation of oocyte quality and its importance.

Biological characteristics of oocyte senescence

Decline in fertility is due mainly to ovarian ageing, which is characterised by changes in the quantity and quality of oocytes; as women age, the decrease in the number of oocytes is due mainly to natural follicular depletion, and the decline in the quality of oocytes with ageing deserves our attention [16]. The effects of ageing on oocytes primarily manifest as impaired maturation of both the nucleus and cytoplasm. When nuclear or cytoplasmic maturation is compromised, oocytes exhibit increased rates of aneuploidy. This maturation failure further increases the risk of embryonic developmental abnormalities and miscarriages [17]. Next, this paper presents two aspects of cytoplasmic and nuclear maturation disorders in senescent oocytes of decreased quality.

Characterisation of reduced oocyte quality

Impaired maturation of oocyte nuclei

Increased aneuploidy associated with meiotic segregation errors is a significant factor contributing to the reduced fertility of germ cell senescence, characterised by incorrect chromosome numbers, which may be due to chromosome segregation errors during meiosis [18]. Errors in chromosome number in aged oocytes may be due to one of two reasons: (1) meiotic errors caused by premature segregation of sister chromatids during meiosis I, which should have split in meiosis II [19], and (2) Meiotic errors caused by reverse segregation during meiosis [20]. Mechanisms leading to meiotic errors in older oocytes (shown in Fig. 1) include (1) impaired chromosome integrity due to increased DNA damage (e.g., DNA double-strand breaks) [21], (2) weakened chromosome cohesion during chromosome segregation, causing a decrease in the level of chromosomal adhesin subunits, making oocytes susceptible to premature sister chromatid segregation in homologous chromosomes [22, 23], (3) dysfunction of spindle assembly checkpoints, which interferes with chromosome segregation, leading to premature chromosome segregation and chromosome mislocalisation [24], (4) meiotic recombination errors [25], and (5) telomere shortening leading to impaired chromosome integrity [25].

Fig. 1.

Schematic representation of the obstacles to oocyte nucleus and cytoplasm maturation

Impaired cytoplasmic maturation of oocytes

Mitochondria have a significant influence on the cytoplasmic maturation and development of oocytes, and in previous studies, it was shown that mitochondria in older or less fertile oocytes have a greater proportion of electron dense matrix, a lower proportion of cristae, and a lower proportion of coronal mitochondria [26]. In mature oocytes, the distribution pattern of mitochondria shifts from a homogeneous to a heterogeneous pattern [27], and the distribution pattern of mitochondria in the cytoplasm is a key indicator of the quality of mature oocytes [28]. By triggering an imbalance in redox homeostasis, mitochondrial dysfunction not only impairs electron transport chain activity but also disrupts the dynamic balance between organelles, leading to ultrastructural abnormalities (e.g., mitochondrial cristae rupture, endoplasmic reticulum vesiculation) and oxidative damage to key biomolecules in oocytes, which, in turn, significantly reduces the developmental potential of oocytes and the cytoplasm of oocytes. Ageing oocytes often present an abnormally smooth endoplasmic reticulum (SER), as shown in Fig. 1, characterised by the swelling of isolated SER vesicles that fuse to form a multivesicular body (MVB) complex. This SER abnormality is associated with microtubule instability, abnormal spindle assembly, and impaired calcium signalling, further affecting oocyte function and embryo developmental potential [25].

Molecular mechanisms of oocyte senescence due to mitochondrial dysfunction

MtDNA

Mitochondria are semiautonomous organelles within cells [29] with their own DNA, mtDNA, which encodes several proteins. Although most proteins required for mitochondrial function are encoded by nuclear DNA and synthesised by cytoplasmic ribosomes for targeted transport [30], regular respiratory chain activity requires structurally intact and functionally normal mitochondrial genes [31]. mtDNA plays an essential role in the regulation and maintenance of mitochondrial function, and the interactions between nuclear DNA and mtDNA work together to maintain mitochondrial function [32, 33].

MtDNA mutations and oocyte senescence

mtDNA mutations may result from several factors: damage due to environmental factors, ROS-induced damage, polymerase errors during DNA replication, dysfunction of the mtDNA repair system, impaired degradation mechanisms of damaged mtDNA, and age-related accumulation of mutations (as shown in Fig. 2).

Fig. 2.

Molecular mechanisms of mtDNA mutations in oocyte senescence

OS can induce double-strand breaks (DSBs), a highly severe type of DNA damage. When DSBs occur in older oocytes, the chance of chromosomal misalignment is significantly greater than that in younger females [34]. Since mitochondria replicate their DNA independently of the cell cycle, there is a greater chance of these replication errors occurring [8]. Moreover, mtDNA repair capacity is limited and relies mainly on base-excision repair and mismatch repair (MMR). However, the mitochondrial MMR system is weak and ineffective for preventing mutation accumulation. In addition, mitochondria lack more advanced repair pathways, such as nucleotide excision repair and homologous recombination, which makes mtDNA particularly susceptible to the accumulation of mutations [35], which leads to a higher rate of damaged mtDNA in mitochondria and further aggravates oocyte damage. The expression of DNA repair-related genes was significantly downregulated in the oocytes of aged mice (≥ 12 months of age), and their fertilised eggs and two-cell embryos also presented diminished DNA damage response activity, clearly suggesting that the repair capacity decreases with age [36]. This phenomenon is generalisable to mammalian models—the mRNA expression of BRCA1 and H2AX, key DNA damage repair genes, are synchronously reduced when the primordial follicles of the ovaries of young (~ 9-week-old) and old (32-week-old) rats are compared, further validating the systematic decline in DNA repair efficiency during reproductive ageing [37, 38]. This age-dependent decrease in efficiency may lead to an increase in DSBs in the oocytes of older women, which in turn increases the risk of chromosomal mislocalisation, a hypothesis that was simultaneously corroborated by the results of a study by Marilina Raices [39]. In previous studies, according to the oxygen free radical theory, ROS-induced damage may be one of the main drivers of the age-related decline in oocyte function [40]. Elevated levels of ROS increase the incidence of mtDNA mutations [41], and the accumulation of induced mtDNA mutations as well as restricted DNA repair capacity, together with errors during replication, lead to increased DNA damage and chromosome damage [42, 43]. The combination of these three factors leads to increased DNA damage, weakened cohesion, dysfunction of the spindle assembly checkpoint, and meiotic recombination errors, forming a vicious cycle that leads to the generation of chromosomal aneuploidies and, ultimately, to an age-related decline in oocyte function. However, recent studies have revealed the opposite result. Superoxide dismutase 2 (SOD2) is a core antioxidant enzyme in the mitochondrial matrix [44]. Long S et al. knocked down expression of the SOD2 gene in mice; this did not affect the number of mtDNA mutations or the frequency of mutated genes in oocytes from 3-week- and 9-month-old mice, suggesting that an increase in oxidative stress in the mitochondrial matrix of oocytes does not lead to a significant increase in mtDNA mutations [45]. These contradictory results may be due to limitations such as the presence of other SOD isoform-activated antioxidant mechanisms in oocytes or the fact that mutation accumulation may require more prolonged high-dose ROS exposure, which is not triggered by a sufficient experimental period (3–9 months). Alternatively, ROS may indirectly affect mtDNA stability by inducing DSBs rather than directly driving mutations.

Changes in MtDNA copy number in senescent oocytes

The prevalence of infertility, spontaneous abortion, and congenital disabilities has increased in recent years with increasing reproductive age in women, and the core pathologic basis of these reproductive outcomes lies in the decline in developmental potential triggered by damage to the quality of oocytes [46, 47]. Many studies have shown that reproductively senescent oocytes are positively associated with reduced mtDNA copy number [48]. Older oocytes contain fewer mtDNA copies and lower mtDNA content [8]. Moreover, the mtDNA copy number in oocytes has a significant effect on oocyte fertilisation and embryo development, and in this study, it was observed that the embryos of older females presented higher mtDNA copy numbers than did those of younger females did, which may be due to a compensatory mtDNA mechanism [8]. LONG S et al. established a mouse model of ROS overproduction by SOD2 expression knockdown, and in their observations, they reported that a compensatory adaptive metabolic mechanism exists in young mouse oocytes and that this compensatory mechanism is not sufficient to maintain the stability of mtDNA when mitochondrial dysfunction is caused by the accumulation of ROS; this leads to a certain degree of oocyte senescence, resulting in a decrease in mtDNA copy number [45]. Yi, Z. Y. et al. reported a similar phenomenon in mice in which total maternal sleep deprivation led to oocyte OS, with an increase in the ROS concentration in mouse germinal vesicle (GV) and middle-intermediate II (MII) oocytes, which resulted in an increase in the number of mtDNA copies in GV-stage oocytes and a decrease in the number of mtDNA copies in MII oocytes due to a compensatory mechanism [49]. Thus, the mtDNA copy number in oocytes can reflect their functional status. Telomere length shortening leads to a reduction in mtDNA copy number. In a study by BAO L et al., stillborn cloned calves presented lower mtDNA copy numbers with telomere length shortening than did normal neonatal calves and naturally conceived neonatal heifers. Notably, this study also revealed a positive correlation between telomere length and mtDNA copy number. These findings suggest that abnormal telomere shortening and reduced mtDNA copy number may be associated with abnormal embryo development and death [50]. A study by Inoue, Y. et al. further supported the above association experimentally. They explored the correlation between telomere length and mtDNA copy number in bovine embryos and treated them with telomerase inhibitors. The results revealed that the mtDNA copy number of the inhibitor-treated embryos decreased significantly with telomerase activity inhibition. This study confirmed the correlation between telomere length and mtDNA copy number in bovine embryos and suggests that telomere length may be a potential regulator of mtDNA copy number in blastocyst-stage embryos [51]. However, the specific regulatory mechanism involved remains to be elucidated. Regarding assessment of oocyte quality, direct assessment of mtDNA copy number is not feasible because it can lead to oocyte destruction. The mtDNA copy number in cumulus granulosa cells (CGCs) is correlated with embryo quality and implantation in patients undergoing in vitro fertilisation (IVF) procedures, and measuring copy number in CGCs can be used to assess oocyte developmental competence [52]. Furthermore, the mtDNA/genomic DNA (gDNA) ratio in follicular fluid cumulus cells (CCs) was found to be negatively correlated with patient age. On this basis, it can be hypothesised that a low mtDNA copy number in CCs may negatively impact embryo quality during IVF cycles. These findings suggest that the low copy number of mtDNA in CCs may adversely affect the quality of embryos in IVF cycles and indicate that the mtDNA/gDNA ratio of CCs can be used as a biomarker to predict the IVF outcome. Dynamic changes in mtDNA copy number are a core biomarker of reproductive ageing, and the optimisation of the assessment system and the development of intervention strategies (e.g., antioxidant combination, mitochondrial transplantation) will provide new avenues to improve the reproductive outcomes of older women.

Impact of oxidative stress on mitochondrial function

Mitochondrial complexes I-IV perform their electron transfer functions while generating ROS, including oxygen free radicals and hydrogen peroxide. OS, i.e., the normal homeostasis of the body, produces excessive ROS beyond its own scavenging capacity. These substances are highly reactive and capable of causing damage to key cellular components and organelles, including lipids, nucleic acids, and proteins, leading to the onset of senescence [53, 54]. It is a predisposing factor leading to mitochondrial dysfunction.

Effects on meiosis

Several lactation experiments have shown that ROS levels are high in aged oocytes [55], whereas in senescent oocytes in vitro, ROS levels are correlated with meiotic spindle defects [56]. In addition, the exposure of oocytes to hydrogen peroxide (H₂O₂) increases the risk of fragmentation [57] as well as the possibility of meiotic spindle abnormalities [58]. The production of ROS can be induced by exposure to various factors, such as heavy metals, tobacco, smoke, drugs, exogenous substances, pollutants, and radiation [59]. Oocytes are highly susceptible to OS due to environmental factors. Perfluorononanoic acid (PFNA) is widely used in various consumer products as an antifouling agent, water repellent, and grease inhibitor. Recently, PFNA was shown to increase oocyte ROS levels; induce oocyte OS; disrupt spindle assembly and mitochondrial function; induce DNA damage; and induce apoptosis in vitro, impeding oocyte maturation [60]. Methyl parahydroxybenzoate (MP) is used as an antimicrobial preservative in processed foods, pharmaceuticals, and cosmetics and has been detected primarily in baby care products [61]. HUANG H et al. reported that MP inhibited the expansion of porcine oocytes and significantly decreased the expression of the expansion-related genes MAPK1 and ERK1. Glutathione (GSH) levels were considerably reduced, increasing the ROS/GSH ratio, significantly decreasing the expression of the autophagy protein LC3B and related genes, and substantially increasing the expression of the caspase3 protein and apoptosis-related genes in apoptosis assays. MP disrupts redox homeostasis and induces mitochondrial dysfunction during meiosis in porcine oocytes, inhibiting meiotic progression [62]. 3-methyl-4-nitrophenol (PNMC) is a degradation product of organophosphorus insecticides and a byproduct of fuel combustion and was shown in a mouse study by MA C et al. to significantly reduce phosphorylated mitogen-activated protein kinase (p-MAPK) expression and destroy pericentromeric proteins; phosphorylated AMPK disrupts the localisation of pericentromeric proteins and phosphorylated Aurora A (p-Aurora A), thereby interfering with the function of the microtubule tissue centre (MTOC) [63]. In a study by CHEN F et al., PNMC administration decreased spindle density and increased spindle length, thereby disturbing spindle stability, and the spindle pole position of the oocyte microtubule-severing enzyme Fignl1 was weakened in oocytes exposed to PNMC, which may have led to defects in its spindle apparatus [64]. Acetylated α-microtubulin (ac-tubulin) plays a key role in regulating microtubule stability [65]. PNMC exposure also increased α-microtubulin acetylation and decreased microtubule stability. In addition, PNMC exposure impaired mitochondrial function, as evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, the release of cytochrome C into the cytoplasm, and increased ROS levels; in short, PNMC exposure adversely affected the meiotic maturation of mouse oocytes [63]. Fortunately, recent experiments have revealed that melatonin can promote oocyte maturation by removing excess ROS, enhancing mitochondrial function, and protecting against PNMC-induced oocyte damage [63, 66]. Environmental toxins damage key structures (spindles) and functions (mitochondrial energy metabolism, DNA integrity, and apoptosis/autophagy balance) of oocytes by inducing ROS-related oxidative stress, which ultimately impairs their maturation capacity and quality. Most studies have used in vitro exposure to a single, relatively high dose of environmental toxins, which is very different from the actual exposure of humans to the environment, which is usually a mixture of multiple pollutants in chronic low doses. And there are fewer studies on the possible antagonistic, synergistic, and other effects of mixed exposures.

Effects on telomere integrity

Biomarkers that have been used to quantify ovarian ageing include sinus follicle counting (AFC) and anti-Müllerian hormone (AMH) levels in clinical applications [67]. However, these biomarkers have been questioned by researchers in recent years. Some studies have shown no association between markers of oocyte number and the probability of conception [68]. This finding reminds us that we should not rely solely on biomarkers of oocyte quantification but should pay more attention to the study of oocyte quality. Telomeres are located at the ends of chromosomes, and their primary function is to maintain chromosome integrity. When the ends of chromosomes are not replicated during cell division, it leads to gradual shortening of telomeres [69]. OS accelerates telomere shortening and induces a DNA damage response, impaired cell proliferation, oocyte senescence, or apoptosis [70]. Ozturk S. et al. demonstrated that an increase in ROS, continuous cell division, and telomere shortening due to genetic and epigenetic alterations significantly affected oocyte development [71]. Telomere dysfunction and reduced telomerase activity in oocytes are standard features of female subfertility and advanced maternal age [72]. In female germ cells, telomerase ensures the correct replication of DNA sequences, and telomere length and activity are closely related to oocyte quality. Yu, T. et al. reported that telomerase activity was lower in women with ovarian insufficiency [73]. In addition, telomeres in the oocytes of aged female mice appeared to be shortened [74]. Therefore, telomere length and telomerase activity may serve as potential biomarkers for predicting oocyte quality [69]. Translationally controlled tumour protein (TCTP) is a highly conserved and widely expressed protein, and in a recent study, overexpression of TCTP in mouse oocytes was shown to reverse age-associated telomere shortening by increasing telomerase activity [75]. These findings provide new ideas and potential therapeutic targets for improving oocyte quality. However, these emerging markers are still in the basic research stage, lacking large samples to validate their clinical sensitivity, specificity and predictive value, and the refinement of telomere length shortening measurement to the extent that it is substantially associated with the developmental potential of the embryo has yet to be thoroughly investigated. The hypothesis that the telomere system is a marker of oocyte quality has not yet been translated into a practical clinical tool, and the pathway and mechanism of its translation are more complex than initially expected.

Effects of abnormal mitochondrial metabolism on oocyte senescence

Age-related abnormalities in mitochondrial metabolism affect the cellular levels of intermediates and byproducts, thereby affecting oocyte quality. Abnormalities in mitochondrial metabolism are most often reflected in changes in oocyte ATP levels. Senescent oocytes exhibit a decrease in ATP levels, reduced energy production capacity, and decreased mitochondrial function [76–78]. This leads to a reduction in the quality of oocytes, particularly resulting in reduced metabolic activity, which may affect cell cycle regulation, spindle formation during mitosis, chromosome segregation, fertilisation, embryo development, and implantation [79]. The tricarboxylic acid cycle (TCA cycle) is a central process in mitochondrial energy metabolism, and we next summarise the effects of metabolic abnormalities in this area on oocyte senescence.

Effects of abnormal NAD+ metabolism in the TCA cycle on oocytes

As a hub of energy metabolism and ageing-related signalling pathways, the attenuation of TCA cycle activity is closely related to age-related metabolic disorders, thus becoming an important molecular target for antiaging interventions [80]. Studies have shown that TCA cycle activity decreases significantly with age, which directly leads to an imbalance in the NAD+/NADH redox state and a decrease in the intracellular NAD+ level [81]. YANG Q et al. further revealed that functional impairment of the de novo pathway induces premature ovarian ageing: NAD + deficiency triggers ATP synthesis deficiency and ROS overaccumulation by inhibiting OXPHOS complex activity, which in turn induces granulocyte cell cycle arrest (G1/S phase) and mitochondria-dependent apoptotic pathway activation. This study demonstrated for the first time that disturbed NAD+ metabolism accelerates follicular atresia and ovarian reserve depletion by disrupting the microenvironment of oocyte‒somatic cell interactions, ultimately leading to a reproductive ageing phenotype [82]. A recent study revealed that with increasing age, oocyte senescence leads to the downregulation of all the cellular pathways involved in NAD+ synthesis (kynurenine pathway, Preiss-Handler pathway, and NAD+ rescue pathway) and profoundly affects the activity of NAD+-dependent enzymes, i.e., PARPs and SIRTs, with further implications for many cellular functions, including impaired ROS detoxification [83]. NAD+ is also a cofactor for nonredox NAD+-dependent enzymes, including sirtuins, CD38, SARM1, poly (ADP‒ribose) polymerase (PARP), ADP‒ribosyltransferase (ART) and RNA polymerase, which are essential for the maintenance of intracellular homeostasis [84, 85]. Sirtuins are a class of NAD+-dependent enzymes that are vital for maintaining intracellular homeostasis [86]. Studies have shown that SIRT3 expression in ovarian tissue decreases in an age-dependent manner and that its loss of function accelerates the process of reproductive senescence. This is manifested by a decrease in reproductive reserve, a reduction in the expression of oocyte markers (Bmp15 and Gdf9) and an increase in the expression of genes related to senescence and inflammation (p16, p21, Il-1α and Il-1β) [87]. In addition, Sirt3 expression deficiency leads to superoxide accumulation, impaired spindle assembly, and mitochondrial dysfunction, including uneven mitochondrial distribution, decreased mitochondrial potential, and reduced mitochondrial DNA content in senescent oocytes [88]. In conclusion, NAD+ is essential for maintaining redox homeostasis for oocyte development, normal meiosis, oocyte fertilization, and embryonic development.

NADH is the reduced form of NAD+ [89]. Recent experiments have shown that supplementation with nicotinamide mononucleotide (NMN) significantly elevates the intracellular levels of NAD(H) and nicotinamide adenine dinucleotide phosphate during in vitro fertilisation (IVF) of bovine oocytes, enhances the development of oocytes to the blastocyst stage, and regulates the expression of genes related to mitochondrial function. Elevation of NAD+ levels by NMN supplementation restored the function of the sirtuin protein [86, 90]. In another study, supplementation of aged mares with NAD+ precursors during oocyte maturation ameliorated severe spindle defects and corrected chromosome alignment by activating SIRT2 and SIRT4 [91]. NMN supplementation reduces oocyte ROS levels, improves mitochondrial function, reduces late chromosomal lag, and enhances postfertilisation development [90]. In addition, the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, is abnormal in senescent oocytes, and the NAMPT activator P7C3 is able to promote maturation, improve spindle assembly and mitochondrial bioenergetics, and reduce mitochondrial plasma leakage in senescent oocytes, providing an opportunity to study effective NAD+ interventions [83].

Effects of abnormal ATP metabolism in the TCA cycle on oocytes

Healthy mitochondria produce ATP through the TCA cycle and oxidative phosphorylation, and ATP produced by the mitochondrial electron respiratory chain is a major source of energy for cellular biological activity; this bioenergetic basis is essential for cellular homeostasis and organismal viability [92].

A sustained decrease in ATP levels leads to a relative increase in the intracellular AMP content, and blocking ATP production triggers cellular senescence, which is demonstrated by an increase in the expression levels of two proteins, p16INK4A and p21CIP1/WAF1 [93]. A decrease in ATP production also leads to an increase in the ratio of AMP (or ADP) to ATP, causing an imbalance in intracellular bioenergetic metabolism. Several studies have shown that the ratio of AMP to ATP increases during cellular senescence [94]. Increasing the ratio of AMP to ATP, either by decreasing ATP levels or by increasing exogenous AMP levels, can contribute to cell growth arrest and senescence characteristics [95]. When the ratio of AMP (or ADP) to ATP is increased, which in turn triggers the AMP-activated protein kinase pathway, the exact mechanism of action of AMPK in oocyte senescence is not clear, thereby providing a potential direction for future research. Once the AMPK signalling pathway is activated, it regulates key enzymes in several metabolic pathways, including lipid and glucose metabolism, mitochondrial dynamics, autophagy, and protein synthesis through phosphorylation, thereby helping to restore mitochondrial energy homeostasis [96, 97], which, when disrupted, damages mitochondria. Moreover, AMPK is an important energy-sensing enzyme, and AMPK activation induces cell cycle arrest [98], resulting in cellular damage. Studies have shown that AMPK has a regulatory function in mitochondria and can reduce ROS levels to prevent OS in oocytes and that the loss of AMPK activity induces a decrease in ATP production and mitochondrial mtDNA copy number. Altered levels of AMPK and inhibition of AMPK activity induce maturation defects in mouse oocytes, which severely impact oocyte quality [99]. AMPK is also involved in the induction of multiple pathways during the establishment and maintenance of cellular senescence. AMPK and other signalling pathways, such as the insulin/IGF-1 signalling (IIS), rapamycin-targeting protein (mTOR), nuclear factor-κB (NF-κB), and deacetylase (Sirtuins) pathways, interact with each other to form a complex network closely related to longevity and ageing [76, 100]. Recently, it has been shown that docosahexaenoic acid supplementation in porcine oocytes during IVM can improve AMPK regulation, upregulate the relative expression of genes related to mitochondrial potential and lipid metabolism, increase the levels of fatty acids and lipid droplets in mature oocytes, and promote an increase in ATP synthesis, further improving mitochondrial function and lipid metabolism to enhance homeostasis of energy metabolism, thereby improving the quality of mature oocytes and enhancing the embryonic developmental potential of IVP embryos [101]. Although the specific mechanism of the role of AMPK in oocyte senescence is still unclear, it provides a potential direction for future research.

Mitochondrial strategies to delay oocyte senescence

Antioxidants that improve mitochondrial function

Natural antioxidants that directly scavenge free radicals

Antioxidants, which are derived mainly from plant or food extracts, restore the dynamic balance between ROS and the antioxidant system by virtue of their antioxidant properties and ability to scavenge ROS [102], thereby increasing the mtDNA copy number, increasing the ATP content of oocytes, and improving mitochondrial activity and function [102–104]. In addition, this class of antioxidants may improve spindle abnormalities in aged oocytes, enhance their in vitro maturation quality, and promote embryonic development [105]. Resveratrol (Res) is a natural plant antitoxin polyphenol [106]. Recent studies have shown that Res synergises with ascorbic acid to preserve ovarian extracellular matrix components (e.g., collagen and the GAG network) and improve follicular survival [107]. When applied at low concentrations, it improves embryonic development after vitrification of feline oocytes [108]. Moreover, resveratrol enhances granulosa cell mitochondrial function and energy metabolism by activating the SIRT1/PGC-1α pathway [109]. Equol is a metabolite of soybean flavonoids, and recent studies have demonstrated that it regulates the ratio of BCL2/BAX expression, inhibits early apoptosis, and improves mitochondrial function [110]. In addition, equol improves IVM in porcine oocytes by activating the Nrf2/KEAP1 antioxidant pathway, decreasing ROS levels, and increasing the expression of haem oxygenase (HO-1), catalase(CAT), glutathione peroxidase 1 (GPX1) and SOD, and attenuating OS [111]. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, regulates the expression of the cytoskeletal gene Arf6, maintains spindle F-actin assembly, reduces aneuploidy [105]. Oleanolic acid (OA, 3β-hydroxy-olean-12-en-28-oic acid) is a naturally occurring pentacyclic triterpenoid plant compound found in many herbs and fruits. OA activates the Nrf2/HO-1 pathway, increases GSH levels and SOD and CAT activities, and significantly reduces intracellular ROS accumulation [112]. Apedylenolide III (AIII) is a sesquiterpenoid that is a core component of Atractylodes macrocephala. AIII attenuates TM-induced endoplasmic reticulum (ER) stress, and AIII-treated oocytes presented increased rates of oocyte cleavage and blastocyst formation and reduced apoptosis. It prevents OS, improves oocyte quality and developmental potential, and has potential application in assisted reproductive technologies (ART) [113]. Limonin (LIM) and nigrosinin (NOB) are both found in citrus fruits as LIM and polymethoxyflavonoids, respectively [114]. Lycium barbarum polysaccharide (LBP) has antioxidant and anti-inflammatory properties [115]. Recent studies have shown that the three, alone or in combination, significantly upregulate GPX1 expression, effectively scavenging intracellular hydrogen peroxide and organic peroxides, reducing ROS accumulation, and enhancing antioxidant defence. Moreover, they can activate SIRT1, ensure the ATP supply for oocyte meiosis and cytoplasmic reorganisation, and enhance cellular OS tolerance through deacetylation. LIM, NOB, and LBP can reduce the level of OS in porcine oocytes, improve the quality of oocytes, and increase the survival rate of the cells [116]. Above antioxidant applications will be shown in Table 1.

Table 1.

Applications of antioxidants

Category	Antioxidant	Model	Intervention	Outcomes
Natural polyphenolic antioxidants that directly scavenge free radicals	Resveratrol (Res)	Cow	50 µg/mL ascorbic acid, 50 µg/mL ascorbic acid and 20 µM resveratrol	Ascorbic acid or ascorbic acid and resveratrol increased follicle diameter and survival, as well as the number of granulocytes and stromal cells. Improved collagen density and preserved glycosaminoglycan network, and increased sulfhydryl levels and catalase activity
		Cat	Heated vitrified oocytes + 0.2 µM resveratrol, 2 µM resveratrol, 20 µM resveratrol	The 0.2 µM resveratrol-treated oocytes had the highest viability (68.89%), and the cleavage rate of 0.2 µM resveratrol-treated oocytes after fertilisation was 88.34% higher than that of the control group (75%) when incubated together.
		Human	Placebo, 800 mg/day of resveratrol	Res decreased total oxidative status and OS index in follicular fluid, increased SIRT1 and PGC-1α protein expression, increased mtDNA copy number and ATP content in GCs, and resulted in higher oocyte maturation rate and embryo quality in the experimental group.
	Equol	Cow	2.5 µM, 5 µM, 10 µM, 20 µM equol	Equol treatment increased the monopole extrusion rate, egg cleavage rate, blastocyst rate, blastocyst hatching rate, blastocyst cell number, and oocyte membrane potential and ATP level in the experimental group. BAX and KEAP1 expression was downregulated, and BCL2, Nrf2 expression was upregulated Antioxidant genes related to the Nrf2/KEAP1 pathway (HO-1, CAT, GPX1, and SOD) showed upregulated expression.
		Sow	2.5 µM, 5 µM, 10 µM equol	2.5 µM oestradiol treatment significantly reduced ROS levels, improved mitochondrial activity and function, attenuated DNA damage and apoptosis in porcine oocytes, and increased embryonic developmental rate
	Epigallocatechin-3-gallate (EGCG)	Mouse	DMSO treatment, 1 µM, 10 µM, 100 µM EGCG treatment groups	The EGCG-treated group exhibited a significant increase in MMP index and improved spindle abnormalities in aged oocytes. Arf6 expression around the spindle was elevated in aged oocytes in the EGCG-treated group.
	Atractylenolide III (AIII)	Sow	AIII: 0 mg/L, 0.01 mg/L AIII, 0.1 mg/L AIII, 1 mg/L AIII	The oocyte maturation rate was higher in the 1 mg/L group than in the other groups.
	Oleanolic acid (OA)	Sow	1 µM OA, 5 µM OA, 10 µM OA, 25 µM OA treatment groups	OA-treated oocytes showed an increase in the percentage of polar body extrusion, an increase in the mRNA expression of fatty acid β-oxidation-related genes, a decrease in the expression of BAX/BCL2 and caspase-3 proteins, and an increase in the intracellular protein expression of Nrf2 and HO-1, as well as in the activities of GSH, SOD and catalase.
	Limonin (LIM), Lycium barbarum polysaccharide (LBP), nigrosinin (NOB)	Sow	LIM + LBP: 20 + 100 µg/ml LIM + NOB: 20 + 25 µg/ml LBP + NOB: 100 + 25 µg/ml LM + LBP + NOB: 20 µg/ml + 100 µg/ml + 25 µg/ml	The first polar body extrusion rate was highest in the LBP + NOB treatment group, and ROS levels were elevated in the combined antioxidant treatment group. Addition of LIM, LBP, or LIM + NOB to IVM medium upregulated GPX1 mRNA expression and enhanced SIRT1 expression.
Substances that enhance the endogenous antioxidant system	Melatonin	Human	Placebo once daily (3 mg/day), melatonin once daily (3 mg/day)	The melatonin group had an increased number of oocytes, fertilisation rate, and embryo quality, reduced glutathione and total antioxidant capacity, and increased biochemical pregnancy rate.
		Human	Melatonin group: (n = 86, 2 mg/day for ≥ 8 weeks)	The clinical and sustained pregnancy rates were higher in the melatonin group than in the no-treatment group, and the expression of antioxidant proteins Nrf2 and KEAP1 was increased in the melatonin group.
	N-Acetyl-L-cysteine (NAC)	Ewe	0 mM, 1.0 mM NAC, 1.5 mM NAC, 2 mM NAC treatment groups	Increased rate of oocyte maturation and oocyte expansion, increased division and blastocyst rates, increased expression of the IDH3G gene for energy metabolism and the SIRT1, GGCT, and MITF genes, and decreased expression of CASP8, FOS, MMP1, and CCL2, which are associated with apoptosis and cell invasion, in the NAC group
	γ-glutamine	Ewe	5 µM γ-glutamine treatment	The experimental group showed an increase in GSH activity, a decrease in the mean relative level of ROS, and a significant increase in the mitochondrial membrane potential of treated oocytes.
	Betulinic acid(BA)	Cow	0 µM, 0.01 µM , 0.1 µM, 1.0 µM BA treatment groups	The expression levels of SOD1, SOD2, CAT, GPX1, and HO-1 were elevated in the BA-treated group; H2O2-induced decrease in Nrf2 expression and increase in Keap1 expression; Bru-induced decrease in nuclear Nrf2 expression; Bru treatment-induced increase in apoptosis rate, and increase in ROS level.

Antioxidants that enhance the endogenous antioxidant system

This section summarises the roles of melatonin, N-acetyl-L-cysteine (NAC), γ-glutamine, and betulinic acid (BA). These antioxidants mainly protect oocytes indirectly by activating antioxidant enzymes (e.g., SOD and GSH-Px) in vivo or elevating GSH levels [117, 118]. Melatonin significantly elevates the levels of reduced glutathione (rGSH) and total antioxidant capacity (TAC), reduces follicular OS [119], and upregulates expression of the key antioxidant proteins Nrf2 and KEAP1 to enhance the repair of cellular oxidative damage [120]. Melatonin restored the expression of genes involved in glycolysis (HK2, ENO1, PKM1, LDHA) and the TCA cycle (CS, IDH1, SUCLA2, FH, MDH1), reversed H₂O₂-induced metabolic disorders, increased the activity of mitochondrial oxidative phosphorylation complexes (II, III, and V), and increased the efficiency of ATP generation to safeguard the energy requirements of oocytes. Melatonin maintains metal ion homeostasis, inhibiting programmed cell death by upregulating expression of the copper transporter protein ATP7B and that of the iron death inhibitor GPX4 [120]. Melatonin also activates the mitochondrial autophagy pathway: upregulating expression of PINK1/Parkin and the autophagy marker LC3 results in the degradation of damaged mitochondria (decreased P62 expression) and the maintenance of mitochondrial health [121]. NAC acts as a GSH synthesis precursor, and NAC enhances the cellular antioxidant capacity by promoting GSH biosynthesis to effectively alleviate GSH depletion during OS and reduce ROS-mediated cellular damage [122]. It was demonstrated that 1.5 mM NAC significantly increased the oocyte maturation rate, the efficiency of oocyte thalamic cell expansion, and the blastocyst rate of lone female embryos, and upregulated expression of energy metabolism-related genes (SIRT1, GGCT, MITF, and IDH3G) and downregulated expression of apoptosis- and inflammation-related genes (CASP8, FOS, MMP1, CCL2, and CXCL8). Therefore, NAC supplementation significantly reduces OS, increases antioxidant capacity and metabolic activity, promotes oocyte maturation, and improves oocyte quality [123]. γ-glutaredoxin (derived from rice bran oil) significantly improved oocyte maturation and embryonic developmental potential in sheep in vitro [124]. γ-glutaredoxin upregulated the expression of the Nrf2 pathway and its downstream enzymes (CAT, SOD), enhanced endogenous antioxidant defence, increased GSH levels and reduced ROS accumulation [125]. Experiments have shown that betulinic acid (BA) treatment can improve oocyte development and that BA-treated oocytes have lower ROS levels, higher GSH levels, and increased expression of antioxidant genes. BA also regulates the Nrf2/Keap1 signalling pathway, restores H₂O₂-induced impaired nuclear maturation of oocytes, restores the levels of ROS and GSH, and acts as an effective antioxidant [126]. Above antioxidant applications will be shown in Table 1.

Exploration of mitochondrial replacement therapy and the potential of mitochondrial transplantation

Egg mass transfer techniques

Egg mass transfer is an ART in which donor and recipient mitochondrial DNA may coexist in the same oocyte, leading to mitochondrial heterogeneity. Subsequent animal studies have shown that mitochondrial heterogeneity can lead to a metabolic syndrome-like phenotype and adversely affect cognitive function. For example, in some animal models, with growth and development, their metabolic indices gradually show characteristics consistent with metabolic syndrome, such as blood glucose levels, lipid metabolism disorders and other conditions appearing one after another; moreover, these animals show memory, learning ability and other cognitive-behavioural declines [127]. These findings indicate that mitochondrial heterogeneity affects the physiology and metabolism of the body and cognitive functions related to the nervous system, highlighting the importance and urgency of exploring and solving the problem of mitochondrial heterogeneity in ART [46, 128].

Autologous germline mitochondrial energy transfer (AUGMENT)

AUGMENT technology is designed to counteract mitochondrial heterogeneity by utilising autologous mitochondria sourced from a woman’s somatic cells (e.g., granulosa cells) to drive bioenergetic activation of an egg and enhance fertilisation and embryo development. A preliminary study covering two centres in Canada and the United Arab Emirates revealed that AUGMENT treatment resulted in a three- to sixfold increase in clinical pregnancy rates and an 11- to 18-fold increase in clinical pregnancy rates per transferred embryo compared with patients’ previous cycles. However, the study had several limitations, such as the lack of a suitable control group and the possibility of regression to the mean when relying on historical controls in patients with a poor prior prognosis but a good current prognosis [129]. Another randomised controlled study on the prognostic impact of the AUGMENT technique on patients with failed IVF revealed nonsignificant differences between patients who received the AUGMENT technique intervention and those who did not in terms of fertilisation rates and live birth rates of transferred embryos [130]. Currently, the AUGMENT technique is still in the experimental phase and has not been approved for use in many cases. In addition, there is a lack of sufficient evidence to confirm a positive effect on pregnancy outcomes or safety, and the clinical use of this technique has been put on hold [46, 131].

Mitochondrial replacement therapy (MRT)

The two basic methods for performing MRT are maternal spindle transfer (MST) and pronuclear transfer (PNT), which improve reproductive success by transferring the nuclear genome from the affected oocyte (MST) or fertilised egg (PNT) to the donor’s “healthy” cytoplasm [46]. In a study by Tang M et al., the blastocyst formation rate was significantly greater in fertilised eggs from old and young mice that underwent PNT (74.6% and 69.2%, respectively, P < 0.05). Transplantation of NZB/OlaHsd stalled embryos PNT to B6D2F1 nonstunted cytoplasm resulted in a blastocyst rate of 89.7% (P < 0.001) and a blastocyst rate of 89.7% (P < 0.001). Both old and young mice presented significant increases in fertilisation and blastocyst rates after MST. Moreover, PNT/MST blastocysts from young mice presented a normal high ploidy rate, and the rate of chromosomal abnormalities significantly increased when young fertilised PNT eggs or spindle bodies were transplanted into an aged cytoplasm [132]. Nuno Costa-Borges et al. demonstrated that MST was successful in overcoming embryonic developmental blockage in the NZB strain of oocytes (a human infertility model), that normal MST offspring presented no physiological/behavioural abnormalities, and that their fertility and offspring survival rates did not differ from those of the naturally conceived group. mtDNA heterozygosity levels were low in the F1 generation and completely undetectable after the F2 generation, precluding the risk of mitochondrial chimaerism. This study confirmed that the MST technique can circumvent maternal developmental defects and is genetically safe, suggesting its translational potential for the treatment of human infertility [133]. In a subsequent study by Nuno Costa-Borges et al., the technical feasibility of the MST technique was investigated in cases of recurrent IVF failure and idiopathic infertility. The study was conducted in women under 40 years of age without mitochondrial disease, and after 28 cycles of MST, six children were born. Subsequent follow-up revealed that the children’s growth and developmental markers were within the normal range, with no phenotypic abnormalities, and that the mtDNA was inherited exclusively from the intended parents, with no residual oocyte donor genes [134]. Following the intervention of patients with m.8993T > G mutant Leigh syndrome via MST, live-born neonates maintained 2.36–9.23% mtDNA mutation carriers across all tissues [135]. Although the mutation carriage rate was significantly higher than the rates reported in previous MST studies, it was still well below the pathogenic threshold (> 60%), thus giving the child a high probability of circumventing disease development. Long-term follow-up data were not available because the family refused follow-up [135]. Even if the initial maternal mtDNA residue is less than 1%, tissue-specific amplification may still occur during embryonic development or after birth. Seven of the 8 embryonic stem cell (ESC) lines derived via MST did not contain residual mtDNA after long-term culture (> 30 generations); however, the residual amount of one of these lines fluctuated abnormally from 1% in the 1st generation to 53% in the 36th generation and then decreased to 1% in the 59th generation. This suggests that there is amplification potential for residual mtDNA, and although current technologies can significantly reduce the initial carriage rate, there is a lack of data from cross-generation studies in animal models to assess long-term genetic risk [136]. Recently, it has been shown that female pronucleus transfer (FPNT), a modified prokaryotic transplantation technique, significantly reduces the risk of heterozygous transmission of mtDNA by precisely controlling donor cytoplasmic residues. Four healthy offspring of rhesus monkeys were successfully bred by FPNT. The analysis revealed that the mtDNA of the monkeys originated mainly from cytoplasmic donors (93.3–96.2%). The residual mtDNA of the FPNT donors accounted for only 3.8–6.7%, which was significantly lower than that of the traditional PNT strategy. FPNT was much better than the conventional PNT strategy for humans or mice. The traditional PNT method can reduce mtDNA residue in mice [137]. The above preliminary results are promising, but larger clinical trials are needed to fully evaluate the safety and efficacy of MST treatment. Since embryos or infants produced through MRT carry maternal mtDNA, future studies should also focus on combining MRT with mtDNA gene editing to reduce mtDNA carriage to undetectable levels, providing potential directions for future research [138, 139]. Variations in reproductive technology regulations in different countries have led to several ethical and legislative challenges, so there is an urgent need for a more rigorous and clearly defined approach to regulating the field [46].

Ethical considerations for MRT

To date, clinical research on MRT has been completely banned in the United States, and the transfer of germline gene-edited embryos (including MRT embryos) is illegal. In contrast, Japan’s Council for Science and Technology in the Cabinet Office (CSTI) has established three major access principles for human embryo research—scientific rationality, safety, and societal relevance—which allow for the use of gene-edited embryos for basic research on assisted reproduction; research on the kinetics of mitochondrial function in oocytes/fertilised embryos; and the application of nuclear transfer to human embryos for fertilisation [40]. As the first and currently the only country in the world to legislate for the approval of MRT, the UK has implemented differential regulation, allowing the use of spindle or prokaryotic transplantation techniques only if there is a proven genetic risk of severe mitochondrial disease in the offspring. Authorised clinics must develop a long-term follow-up plan for MRT offspring, resulting in a statutory tracking mechanism [140]. During the MRT policy development process in Canada, the country’s expert working group formally advocated for the use of MRT to prevent and control the transgenerational genetic risk of severe mitochondrial diseases while calling for consultations to establish appropriate regulatory mechanisms. The government explicitly proposed a dual restriction: first, the technology is limited to male embryos to block the inheritance of modified genes; and second, if approved for clinical use, it must be restricted to very severe, highly episodic diseases for which there are no alternatives, reducing the target population and controlling the impact of the gene pool [140]. In the United States, although an unsuccessful attempt was made to pass a bill to ban the clinical use of MRT in 2019, the current ban remains legally binding; meanwhile, in Canada, legislation was passed to completely ban the clinical and scientific use of MRT. In contrast, the United Kingdom has established the world’s first regulatory system for the clinical authorisation of MRT. Australia and Singapore are following this path of policy restructuring; both governments and academic institutions have initiated public consultation processes to pave the way for potential legislative legalisation. Notably, at the heart of the regulatory differences between countries is a divergence in the positioning of MRT and germline gene editing: the US and Canada have implemented binary homogeneous regulation (categorising both under the same prohibition), the UK has established binary heterogeneous regulation (clearly distinguishing between the technical attributes of the two), and the emerging regulators are more inclined to construct a split regulatory framework (with separate regulations governing them separately) [141].

MRT via ooplasmic transfer may be a potential treatment for advanced maternal age. Nevertheless, owing to the medical risks involved and ethical controversies, this therapy is currently not suitable as a standard treatment option for women of advanced maternal age [40].

Conclusions and outlook

Childbearing in advanced maternal age poses a challenge related to ovarian function and oocyte quality, and as fertility decreases with age and oocyte senescence, MRT cannot be the primary strategy for addressing mitochondrial disorders. Moreover, there are no accurate diagnostic tools for assessing oocyte quality. This review aimed to elucidate the main molecular mechanisms leading to the mitochondria-related aspects of oocyte senescence. OS and mitochondrial dysfunction are associated with aberrant nuclear and cytoplasmic maturation, and the increased incidence of aneuploidy in advanced maternal age results from ROS-induced DNA damage, loss of chromosome integrity, SAC dysfunction, and telomere shortening. It can be hypothesised that the incidence of DNA damage due to mitochondrial dysfunction in oocytes can be reduced by supplementation with antioxidants, for example. Moreover, adequate levels of mtDNA and healthy mitochondria are essential for normal energy metabolism in oocytes, contributing to oocyte growth and maturation, normal ovarian function, and delayed oocyte senescence. By linking ageing and oocyte quality decline, we will continue to explore ways to address the age-related decline in oocyte capacity and overcome the effects of mitochondrial dysfunction in the future. Additional strategies to continually improve oocyte quality and rejuvenation should be investigated to increase conception and reduce the likelihood of miscarriage at advanced maternal age.

Author contributions

T.W. and PX.X. wrote the main manuscript text and JL.Y. and YR.W. prepared Figs. 1, 2 and 3. All authors reviewed the manuscript.

Fig. 3.

Molecular mechanisms of mitochondrial dysfunction in oocytes

Funding

NationalNaturalScienceFoundationofChina(31960154);Key Research and Development and Achievement Transformation Program of Inner Mongolia Autonomous Region( 2025YFSH0093); Inner Mongolia Science and Technology Project (2025YFSH0089); Inner Mongolia Autonomous Region Provincial Health Commission, Science and Technology Program for Building High-level Clinical Specialties in Public Hospitals in the Capital Region (2024SGGZ071); Science and Technology Project of the Scientific Research Joint Fund for Public Hospitals in Inner Mongolia Autonomous Region (2024GLLH0321); Inner Mongolia Medical University Affiliated Hospital Talent Training Project-Sailing Series; Youth Innovation Talents Training Program of the Inner Mongolia Autonomous Region “Prairie Excellence” Project (Q2022085); Natural Science Foundation of Inner Mongolia (2023QN03047); Scientific Research Project of Inner Mongolia Autonomous Region Colleges and Universities (NJZZ23017); Central Guided Local Science and Technology Development Funds Project (2023ZY0004); Affiliated Hospital of Inner Mongolia Medical University National Nature Cultivation Program (2023NYFYPY006); 2024 Joint Fund for Research in Public Hospitals Science and Technology Projects (2024GLLH0298).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.