New insights on mitochondrial heteroplasmy observed in ovarian diseases

Yong Zhou: Conceptualization, Writing – review & editing. Yang Jin: Writing – original draft. Tianyu Wu: Writing – original draft. Yinfeng Wang: Conceptualization. Yuanhang Dong: Validation. Pei Chen: Visualization. Changchang Hu: Visualization. Ningping Pan: Validation. Chaoshuang Ye: Visualization. Li Shen: Investigation. Mengyan Lin: Investigation. Tao Fang: Investigation. Ruijin Wu: Writing – review & editing, Conceptualization, Supervision.

Not applicable.

Mitochondrial heteroplasmy is closely related to ovarian diseases, and the current clinical application of mitochondrial modulators from animal and plant food sources is effective in ameliorating ovarian diseases such as PCOS, POI, EMS, etc. However, mitochondrial modulators exhibit low bioavailability, and nanosystems are being developed for effective drug delivery in the treatment of ovarian diseases. Mitochondrial transplantation or mitochondrial gene editing technology can theoretically fundamentally correct mutated mtDNA and improve mitochondrial function. Nevertheless, its effectiveness and safety still need to be further verified and require basic and evidence-based medical support, as well as in-depth discussions of its ethical issues.

Mitochondria, colloquially referred to as the “energy factory” of cells, are important organelles in eukaryotic cells and are the main place for oxidative phosphorylation (OXPHOS) to produce adenosine triphosphate (ATP) [1] . Over 90 % of the energy required by the human body is produced by the mitochondrial inner membrane respiratory chain [2] . In addition, mitochondria are involved in the process of apoptosis, proliferation, nucleic acid synthesis, free radical generation, and signal transduction regulation [3] , [4] , [5] and play a key role in various cellular functions. Human mitochondrial DNA (mtDNA) is the only genetic material outside the nucleus capable of autonomous replication, transcription, and translation [6] , granting mitochondria to promptly adapt to cellular energy requirements, independently of relying solely on instructions from the nucleus. Therefore, mitochondria possess a high degree of efficiency in the use of genetic material, effectively participating in the production and regulation of cellular energy, thereby maintaining normal cellular functions [7] . As mtDNA lacks the protection of histone proteins and is exposed to high concentrations of reactive oxygen species (ROS), it has to face great oxidative pressure [8] ( Fig. 1 A), which is the reason for the high mutation rate of mtDNA [9] . As mitochondria play a critical role in the energy metabolism of cells [10] , generation of oxygen free radicals, and regulation of apoptosis, changes in mtDNA are likely to cause changes in the encoding of proteins, thus affecting the efficiency of ATP synthesis, damaging the normal function of tissues and organs and causing diseases [11] , [12] . Fig. 1 Human mitochondrial DNA and its heteroplasmy. (A) Human mitochondrial DNA (mtDNA) is a double-stranded circular DNA, which is the only genetic material outside the nucleus. (B) The mtDNA in a cell can be either homogeneous or heterogeneous. The number of mutated mtDNA must reach a certain threshold to cause lesions in the corresponding tissue or organ. (C) The structure of mtDNA can be divided into coding and non-coding regions. The non-coding region, also known as the displacement loop (D-Loop), contains mtDNA heavy- and light-strand replication initiation sites and transcription promoters. It is responsible for the regulation of replication and transcription of the entire mtDNA. Human mitochondrial DNA and its heteroplasmy. (A) Human mitochondrial DNA (mtDNA) is a double-stranded circular DNA, which is the only genetic material outside the nucleus. (B) The mtDNA in a cell can be either homogeneous or heterogeneous. The number of mutated mtDNA must reach a certain threshold to cause lesions in the corresponding tissue or organ. (C) The structure of mtDNA can be divided into coding and non-coding regions. The non-coding region, also known as the displacement loop (D-Loop), contains mtDNA heavy- and light-strand replication initiation sites and transcription promoters. It is responsible for the regulation of replication and transcription of the entire mtDNA. In 1981, Anderson et al. first successfully completed the whole sequence determination of mtDNA and drew a functional map [13] . The structure of mtDNA can be divided into coding and non-coding regions [14] ( Fig. 1 C). The non-coding region, also known as the displacement loop (D-loop) region, contains the start point and transcription promoter of heavy- and light-strand replication of mtDNA, which is responsible for the regulation of the entire mtDNA replication and transcription [15] . The D-loop region of mtDNA has no gene coding function and has a high mutation rate. When a serious mutation occurs in the D-loop region, the whole mitochondrial function is disrupted [16] , which is related to the unique structure of the mtDNA D-loop region: 1) The D-loop region is almost all exons without introns, and mtDNA lacks the protection of histones as well as a complete and effective repair system [17] ; 2) In the mtDNA replication process, the D-loop region forms a three-strand structure at the position where mtDNA contacts the mitochondrial inner membrane, making the region a single-strand form, which is vulnerable to the attack of cellular oxygen free radicals [18] and their products [19] ; 3) The D-loop region is in a state of continuous synthesis during the whole cell cycle, with poor stability and poor proofreading function of DNA polymerase γ (POLG), resulting in high replication mismatch frequency [20] ; 4) mtDNA lacks an effective gene repair system, and mutated mtDNA can replicate and transmit continuously in the cell, producing a remarkable number of duplicate copies [21] . Therefore, the mutation rate of mtDNA is much higher than that of nuclear genes, which is 10–200 times that of nuclear genes. The different forms of variation primarily include point mutation, deletion, insertion, and copy number variation [7] . Even a few mutations in mtDNA result in the coexistence of both wild-type and mutant mtDNA sequences within the cell, known as mtDNA heteroplasmy [22] . However, the number of mtDNA mutations in tissues must surpass a certain threshold value before causing damage to the corresponding tissue or organ, referred to as threshold effect [16] ( Fig. 1 B). Individuals carrying heterogeneous mutations have different mutation loads of mtDNA among tissues [23] . The proportion of mutant mtDNA must exceed the tissue-specific critical threshold level to cause cellular biochemical defects, resulting in biochemical phenotypes with impaired mitochondrial function and symptoms of diverse extent. Variations in the degree of heteroplasmy represent a key contributing factor for differences in clinical phenotypes among patients and within the same family [24] . Mitochondria are essential for living organisms as they play a key role in the fundamental energy conversion processes in cells. One of the key physiological processes within the ovary is the development and maturation of oocytes. The maturation of oocytes entails two rounds of meiotic division, each step of which, such as spindle formation, chromatid separation, and fertilization, necessitates a remarkable energy supply [25] ( Fig. 2 ). Lack of phosphofructokinase (PFK), the energy source of oocyte development mainly relies on pyruvate, amino acids, and intermediate metabolites derived from granulosa cells through the gap junctions of the cumulus-oocyte complex [26] . Amino acids can further be metabolized to produce oxaloacetate and α-ketoglutaric acid, while fatty acids can be converted into acetyl coenzyme A (acetyl-CoA) by β-oxidation in oocytes [27] . These metabolic products enter the mitochondrial tricarboxylic acid cycle (TCA cycle) and generate electron donors, which subsequently undergo OXPHOS, resulting in substantial production of ATP to fulfill the energy requirements of the oocytes [28] . Besides, mitochondria can also collaborate with endoplasmic reticulum (ER) to regulate intracellular calcium homeostasis during oocyte development and maturation, which is vital for redox balance and oocyte mitosis [29] . However, calcium overload may adversely impact ATP production and promote ROS generation [30] . Under normal physiological conditions, intracellular antioxidant capacity and ROS levels are in dynamic equilibrium. Proper levels of ROS within the physiological range play a crucial role in maintaining cellular functions, including regulating metabolism and signal transduction, among other things. When the balance between ROS production and detoxification is disturbed, the relative surplus of ROS leads to oxidative stress, causing severe damage to the cell. Fig. 2 Energy metabolism of the ovary. The energy requirements during normal folliculogenesis are dominated by metabolites derived from granulosa cells through the gap junctions in the cumulus-oocyte complex. Additionally, mitochondria cooperate with endoplasmic reticulum to maintain intracellular calcium homeostasis. Energy metabolism of the ovary. The energy requirements during normal folliculogenesis are dominated by metabolites derived from granulosa cells through the gap junctions in the cumulus-oocyte complex. Additionally, mitochondria cooperate with endoplasmic reticulum to maintain intracellular calcium homeostasis. As the main direct energy source, mitochondria are abundant in oocytes and are dynamically distributed during oocyte maturation. Primordial germ cells (PGCs) contain less than 10 mitochondria, but they can be replicated at all stages of oocyte meiosis, eventually exceeding 100,000 in the mature oocyte [31] . Furthermore, the number of copies of mtDNA also increases from 2,000 copies in PGCs to 150,000–250,000 copies in mature oocytes [32] , [33] . Maintaining the copy number of mtDNA is critical for preserving mitochondrial function in oocytes. The ovary also has high energy demand, thereby requiring optimal mtDNA copy numbers and adequate energy supply for oocyte development and maturation. In addition, it is inevitable that mtDNA mutations will accumulate throughout the cellular lifespan, and when a certain threshold is breached, clinical phenotypes will become evident due to severely compromised cellular energy metabolism as a result of dysfunctional mitochondrial function. Specific types of mtDNA mutations can dictate a variety of ovarian diseases, such as polycystic ovary syndrome, premature ovarian insufficiency, and endometriosis.

Researchers have investigated the application of exogenous mitochondrial modulators in the treatment of ovarian diseases, yielding promising results as some of these modulators have gained widespread application in clinical practice [92] . External mitochondrial modulators that have been proven to be effective in ovarian diseases mainly involve the following two categories: animal-derived antioxidants, such as coenzyme Q10 (CoQ10), melatonin (MT), and L-carnitine (LC), and plant-derived antioxidants, such as resveratrol, quercitrin, and pyrroloquinoline quinone (PQQ). CoQ10 is the only endogenously synthesized lipid-soluble electron carrier and plays an essential role in the mitochondrial electron transport chain in OXPHOS. It functions as an antioxidant to maintain redox homeostasis, inhibiting DNA oxidation and lipid peroxidation and eliminating oxygen free radicals [93] , [94] . Studies have shown that CoQ10 supplementation in aged mice could increase the expression of mtDNA and enhance mitochondrial activity in oocytes, ultimately facilitating the developmental potential of oocytes and increasing the litter size per delivery [95] . A randomized controlled trial indicated that pretreatment with CoQ10 improved clinical pregnancy rates in low-prognosis young women with decreased ovarian reserve, enhancing ovarian function via multiple mechanisms. For instance, CoQ10 directly acts on mitochondria and exerts anti-apoptotic effects in oocytes and granulosa cells, thereby inhibiting follicular atresia [96] . Consequently, CoQ10 has now been incorporated into the treatment guidelines for POI in China. Melatonin is a hormone endowed with antioxidant properties that is primarily synthesized within the mitochondria of oocytes during their maturation. It promotes oocyte maturation and enhances oocyte quality by acting on mitochondria via various mechanisms [97] . For example, melatonin can ameliorate mitochondrial function by optimizing mitochondrial distribution, increasing mtDNA copies, and regulating mtDNA transcription by regulating DNMT1 and mtDNA methylation [97] , [98] . In addition, melatonin could upregulate the expression of spindle assembly checkpoint-related genes and promote mitotic spindle assembly, reducing ROS and protecting mtDNA against oxidative stress in oocytes during maturation [99] . Overall, numerous in vitro and vivo preclinical experiments have demonstrated the beneficial effects of melatonin on oocyte and embryo development in PCOS, POI, and EMS, suggesting the promising potential of melatonin to be a bioactive substance for ameliorating mitochondrial heteroplasmy [47] , [100] , [101] . Relevant clinical trials are also underway. Another animal-derived antioxidant, L-carnitine, is indispensable for the transport of activated fatty acids into the mitochondria where ATP is generated via β-oxidation [102] . A recent randomized clinical trial demonstrated that 12-week L-carnitine supplementation in overweight or obese women with PCOS significantly alleviated insulin resistance [103] . Although only a few reports have described the effect of L-carnitine on improving oocyte quality in humans, some in vitro studies have indicated that the inclusion of L-carnitine in oocyte and embryo growth media significantly improved mitochondrial function in human embryos at the morula stage and embryo outcomes by increasing ATP production, with no effects on mitochondrial copy number [104] , [105] . Simultaneously, L-carnitine has also been reported to eliminate ROS and reduce free radical-induced oxidative stress, thereby stabilizing MMP, improving mitochondrial energy metabolism, and inhibiting mitochondria-related apoptosis [106] . Hence, L-carnitine could be a potentially effective treatment for PCOS. Resveratrol is a naturally polyphenolic compound extracted from grapes, berries, peanuts, and other plants, and has been utilized as an alternative drug to treat different cancers owing to its antioxidant, anti-inflammatory, and anti-angiogenic properties [107] . Although EMS has been historically regarded as a benign condition, it demonstrates certain biological behaviors similar to tumors, such as proliferation, migration, invasion, and angiogenesis. While there is currently no direct evidence that resveratrol intervenes in EMS by targeting mitochondrial heteroplasmy, resveratrol induces damage to mtDNA, triggering the autophagy of dysfunctional mitochondria, activating the caspase cascade, and promoting apoptosis in cancer cells [108] , [109] . Besides, multiple in vitro and in vivo studies have shown that resveratrol contributes to ameliorating symptoms, suppressing lesions, and preventing recurrence in EMS [110] , [111] . A Phase 4 clinical trial on resveratrol and pelvic pain in EMS was completed in 2015 ( NCT02475564 ). Quercetin, a compound that belongs to flavonoids, has many effects such as anti-inflammatory, anti-oxidation, and anti-cancer [112] . It has been reported that the upregulated high mobility group box-1 (HMGB1) contributed to the development of EMS partly through regulating inflammation and autophagy [113] . HMGB1 is involved in nDNA and mtDNA damage repairment and quality control, although it triggers inflammation in the extracellular matrix [114] . There are no clinical studies on quercetin in the treatment of EMS, whereas some studies in tumors have indicated that quercetin induced cancer cell apoptosis and exhibited anti-cancer activities via attenuating the expression of HMGB1, thereby inhibiting DNA injury repairment and promoting the release of cytochrome c [115] . Furthermore, a certain number of preclinical studies have demonstrated that quercetin inhibited lesion proliferation in EMS due to its pro-apoptotic and anti-inflammatory properties [116] . We thus speculate that quercetin may promote apoptosis and constrain the lesion size in EMS by modulating the mitochondrial heteroplasmy through the inhibition of HMGB1. PQQ is a newly identified aromatic tricyclic quinone compound with remarkable water solubility and stability, boasting potent catalytic capabilities in redox reactions. While PQQ cannot be synthesized by plants and animals, it is naturally available in various organisms. Plants acquire PQQ from the environment, mainly through soil absorption, whereas animals predominantly obtain it through dietary intake [117] . Therefore, PQQ is a bioactive substance originating from a distinct dietary source. POI is characterized by diminished mitochondrial mass and high mitochondrial heteroplasmy, which lowers both the quantity and quality of oocytes, resulting in infertility and a rapid decline in ovarian reserve. Therefore, preserving the mitochondrial mass and ameliorating the mitochondrial heteroplasmy are crucial therapeutic strategies for POI. Several in vitro and in vivo studies have demonstrated that PQQ treatment not only lowered oxidative stress levels and mitigated mtDNA damage but also enhanced MMP and the expression of mitochondrial genes (Nd1, Ctyb, Cox1, ATPase6), thus facilitating ATP production. Consequently, the number of ovulated oocytes and pups per delivery was increased [118] . Therefore, PQQ might be particularly well-suited for relieving symptoms and enhancing fertility in older women due to its capacity to heighten both the quantity and quality of mitochondria in aging cells. Conventional drug application is characterized by poor effectiveness, limited solubility, and lack of selectivity. To overcome these limitations, drug delivery systems (DDS) are receiving more and more attention from scientists and engineers. Nano-delivery systems can not only increase the efficacy and bioavailability of drugs, but also orientate the specific sites, either passively by enhanced permeability and retention (EPR) effect or actively by conjugating nanocarriers with molecules that bind to overexpressed antigens or receptors on the target cells [119] . Therefore, nanocarriers enhance drug bioactivity while minimizing undesirable side effects. The development of mitochondrial targeting nano-delivery systems has largely been pioneered by the oncology field [119] . Various types of cancer harbor the ability to ferment glucose into lactate in the presence of oxygen (Warburg effect), producing ATP through aerobic glycolysis instead of OXPHOS, thereby demanding more glucose [120] . Mitochondria are the organelle responsible for energy production in eukaryotes, playing an essential role in supplying tumors with ATP required for their survival by OXPHOS, and contributing to tumor proliferation and treatment resistance [121] . Collectively, the key roles of mitochondria in cancer development highlight their potential as therapeutic targets. The phospholipid bilayer surrounding the mitochondria hinders drug targeting and delivery; hence, surface modification with mitochondrial homing agents represents an essential aspect of mitochondria-targeted nanocarriers enabling NPs to selectively target mitochondria upon cell entry [122] . The mitochondrial targeting agents commonly used at present include mitochondria-targeted small molecule moieties, such as triphenylphosphonium (TPP) and rhodamine, and mitochondria-targeted bioactive substances, such as mitochondrial-penetrating peptides (MPPs) and mitochondria-targeted sequences ( Fig. 6 A). TPP, a frequently applied cationic lipophilic molecule, possesses the capability to cross both the plasma and mitochondrial membranes due to their negative membrane potential, thereby entering the mitochondrial matrix compartment [123] . Zhang et al. developed a nano-delivery system (TTP-TK-CPI-613) for pancreatic cancer therapy in which the anti-mitochondrial metabolism agent CPI-613 accumulated in mitochondria and showed robust therapeutic performances in the presence of TPP [124] . MPPs are water-soluble, biologically compatible, and positively charged, gaining access to the cell interior and delivering bioactive agents into mitochondria efficiently. MPPs inhibit tumor growth by combining with decorating mattes, such as mitochondrial modulators and mtDNA [125] , [126] , [127] , [128] . The delivery of a certain quantity of mtDNA into the mitochondrial matrix in cancer cells would decrease the proportion of mutated mtDNA and repair the mitochondrial function. Fig. 6 Mitochondria-targeted nano-delivery systems. (A) Several agents are used to target mitochondria, including alkyltriphenylphosphonium moiety and bioactive compounds. (B) Conceptualization of the design of our research, including the construction of a novel PQQ-loaded nanosystem targeting the ovarian mitochondria and the functional evaluation in vivo. Mitochondria-targeted nano-delivery systems. (A) Several agents are used to target mitochondria, including alkyltriphenylphosphonium moiety and bioactive compounds. (B) Conceptualization of the design of our research, including the construction of a novel PQQ-loaded nanosystem targeting the ovarian mitochondria and the functional evaluation in vivo. Most animal- or plant-derived mitochondrial modulators are primarily administered orally and undergo the first pass elimination, resulting in lessened bioavailability. Therefore, developing safer and more efficient strategies has great clinical significance. Over the past decades, advances in nanoparticles have significantly spurred the development of biomimetic nanosystems. For instance, erythrocyte-mimicking nanovesicles (EM-NVs) are developed by fusing NP cores with naturally derived erythrocyte membranes. These nanovesicles feature prolonged blood circulation time, immune evasion, and surface modifiability [129] . One primary research direction of our team is to develop a biomimetic nano-delivery system loaded with PQQ for the treatment of POI. Blood serum albumin (BSA) nanocores can be employed to load PQQ, thereby increasing its bioavailability. Additionally, these nanocores could be enveloped with erythrocyte membranes to mitigate the uptake by the reticuloendothelial system (RES) and mononuclear phagocyte system. Anti-Müllerian hormone (AMH) is mainly synthesized by granulosa cells in the ovary [130] . Consequently, peptide molecules targeting the ovary were specifically designed and a novel biomimetic nanoplatform（PQQ@RBC-NPs） was constructed（ Fig. 6 B）. This system could potentially be a candidate for POI treatment and also provide avenues for mitochondria-targeted therapy for other ovarian diseases.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Mitochondrial heteroplasmy constitutes a significant factor in several ovarian diseases, and current clinical practice primarily emphasizes symptomatic treatment rather than addressing the specific biochemical defects triggered by distinct mtDNA mutations. Repairing defective mitochondria by gene editing [136] is a more direct and effective way to treat mitochondrial heteroplasmy, and the following three strategies are commonly used: (1) delivery of wild-type mtDNA to the mitochondria to correct or compensate for gene defects [137] , [138] ; (2) elimination of heterogeneous mutated mtDNA by gene editing [139] , [140] , [141] ; and (3) direct repair of the mutated mtDNA by base editing [142] ( Fig. 8 ). Base editors targeting mitochondria have been developed to provide effective tools for the research and treatment of mitochondrial diseases and the construction of mitochondrial disease models. Currently, clinical mitochondrial gene therapy is mainly focused on the treatment of mitochondrial diseases associated with single-gene mutations [143] , [144] . The etiology of PCOS, POI, EMS, and other diseases remains unclear, and the mtDNA mutations are also complex and diverse, which limits the application of mitochondrial gene editing technology in the treatment of ovarian diseases. However, this therapeutic option should be further explored. Fig. 8 Mitochondrial gene-editing technology. General gene-editing techniques to eliminate mtDNA mutations include delivery of wild-type mtDNA, gene editing technologies with various endonucleases, and base editing technology with high target specificity. Mitochondrial gene-editing technology. General gene-editing techniques to eliminate mtDNA mutations include delivery of wild-type mtDNA, gene editing technologies with various endonucleases, and base editing technology with high target specificity.