Mitochondrial dysfunction involves the impairment of bioenergetic processes inside the mitochondria compartment, resulting in a reduction in energy production and dysregulation of calcium, electron-proton, and substrates balance in the cell, leading to excessive formation of ROS and high rates of mtDNA mutation47, 48. Mitochondrial dysfunction can lead to infertility in several ways. Studies have shown that the capacity of sperm and oocytes in assisted reproductive technologies is determined by mitochondrial function49, 50. Mutations in genes critical for mitochondrial function, particularly those involved in maintaining mtDNA or mitochondrial protein translation, have been increasingly recognized as a contributing factor to infertility20. Variants in any part of these genes can lead to mitochondrial disorders, which can cause infertility. Mitochondrial dysfunction can affect various cell types within the reproductive system, including oocytes, sperm, and somatic cells such as cumulus cells (CCs) and granulosa cells in the ovary51, which can also contribute to infertility.

Approximately, sperm cells contain 70-80 mitochondria40, whereas a normal oocyte has around 100,000 mitochondria135. In female reproductive cells, mitochondria are especially critical for the growth of oocytes, which are the precursor cells of mature eggs29. Oocyte maturation features an increase in mitochondrial activity to supply energy demands for developing cells30. Once the oocyte is fertilized, mitochondria maintain their critical role in supporting embryonic development at the early stages31. Oocyte mitochondria influence oocyte quality, particularly in terms of proper chromosomal segregation during oocyte maturation32. Apart from generating energy for basic cellular demand, mitochondria also act as regulators of calcium homeostasis, which is critical for cell survival and balancing cell function35. This balanced state is achieved through calcium storage and release interdependently, ensuring suitable free intracellular calcium levels throughout numerous stages of cell growth36. Moreover, mitochondria are also involved in controlling the epigenetic alterations in oocytes and embryos by regulating biochemical activities including histone acetylation, histone and DNA methylation-demethylation21. Indeed, mitochondrial malfunction can imperil fetal viability, particularly in older women, and mitochondrial defects can be transferred to the fetus regardless of the mother's age33, 34. The involvement of mitochondria in oocyte function is summarized in Figure 3.

Mitochondrial DNA is uniparentally inherited in nearly all eukaryotes196. After sperm-oocyte interaction, the head of sperm transmits to the axoplasmic, and sperm mitochondria are trapped outside the oocyte plasma membrane. Research on animal embryos has shown the presence of sperm mitochondria197, which are eliminated by the paternal mitochondria selective autophagy mechanism in the cleavage-stage embryos198. However, it has been recently reported that mtDNA can be inherited from both parents199. Indeed, most of the mitochondria are of oocyte origin in the embryo. As mtDNA is unable to undergo replication before the blastocyst stage, the copy number of mtDNA remains at this level for the rest of the cleavage stage200. Therefore, the oocyte must increase mitochondria numbers from several hundred in primordial germ cells (PGCs) to about 100,000 in the metaphase II oocyte135. The embryo requires sufficient mitochondria count to divide into blastomeres and produce ATP, which is a primary source of energy before embryonic implantation148. During preimplantation, the embryo depends mainly on pyruvate for energy, which is produced via oxidative phosphorylation, while glycolysis is limited. Pyruvates are produced by cumulus cells surrounding oocytes; they are absorbed into the mitochondrial matrix oocyte and are oxidized by the pyruvate dehydrogenase to produce acetyl-CoA201. Cellular respiration produces about 15 ATP from each pyruvate molecule, supplying other organelles. The mechanism maintains a low oxidative phosphorylation pathway, allowing limiting oxidative stress202. ROS increase in poor-quality environmental conditions, that activates protein HIF of Krebs cycle. HIFs are key protein transcription genes that regulate cell adaptation in hypoxic conditions203. Mitochondrial dysfunction is correlated to fertilization failure and embryo development200, 204, 205. Indeed, mitochondrial function significantly impacts the sperm-triggered calcium signaling that activates oocytes and embryo development. Mitochondrial inhibitors such as FCCP or antimycin A uncoupler disrupt cytosolic calcium ions, generating Ca²⁺ waves206, 207. This event induces mitochondrial dysfunction, which leads to a sustained increase in the level of cytosolic Ca²⁺, subsequently inducing apoptotic cell death. In addition, both mitosis and meiosis require ATP to promote chromosome cohesion and segregation. Insufficient ATP can block the function of spindle organization, resulting in aneuploidy products. Consequently, this would result in poor-quality embryos and arrested embryo development208. Studies have shown that a different allocation of mitochondria in blastomeres can lead to abnormal cell division209. Mitochondrial dysfunction can be caused by insufficient mtDNA count and damaged mtDNA200. The mtDNA structure lacks histones and is not packed into chromosomes, which makes them more susceptible to damage than nuclear DNA. Without efficient mtDNA repair mechanisms, its mutation rate is approximately 20 times greater than nuclear genomes in vertebrates210. On the other hand, it was shown that cleavage-stage embryonic mtDNA mutation did not affect embryo quality211. Similarly to gametes, perhaps embryonic mitochondria may be damaged by endogenous and exogenous factors such as ROS, apoptosis, and drugs.

ART procedures can improve the reproductive function of infertility patients; however, research on repeated ovarian stimulation has presented an adverse impact on mtDNA. After repeated ovarian stimulation in mice, mtDNA copy number decreased, while oocyte number increased, bringing abnormally distributed mitochondria, which can be suppressed with an antioxidant212. More importantly, mtDNA diminishing increased after using recombinant gonadotropin in primate oocytes213. However, injecting recombinant gonadotropin in golden hamsters raised mitochondria number, leading to insufficient pyruvate, and producing high levels of ROS214. Redox is maintained in equilibrium in normal physiological conditions, which is crucial for embryo development. Specific events during fertilization and bovine embryo development have been associated with redox states. Increased ROS level after fertilization was related to better embryonic development215. For instance, a brief exposure to 50-100 µM hydrogen peroxide resulted in a higher blastocyst rate in comparison with control group216. Although pyruvate is an essential substrate of mitochondrial respiration, high pyruvate levels can scavenge hydrogen peroxide, resulting in reduced oxidative stress and low ATP production217. Thus, low-concentration pyruvate is provided in an in vitro culture medium to limit the effect on redox states. This evidence indicated an undeniable role of mitochondrial homeostasis during embryonic development, where mtDNA optimization and balance of mitochondrial metabolism have significant impacts on the rate of embryo quality and blastocyst formation. Thus, ensuring mitochondrial health and capacity remains critical after fertilization, which is essential to define the birth rate.

The use of chemicals, especially antioxidants, has been proposed in ART to improve mitochondrial function by reducing oxidative stress. Coenzyme Q10 (CoQ10) is a common endogenous quinone present in biological membranes. The critical role of CoQ in the mitochondria includes two functions: bioenergetic and antioxidant. It is an essential cofactor of mitochondrial respiratory complexes contributing to the mitochondrial ATP synthesis by the electron transfer process. The reduced form of coenzyme Q10 (rCoQ10) possesses antioxidant activity218. Patients with varicocele and oligozoospermia frequently have low CoQ10 levels. Both CoQ10 monotherapy and compound combination supplementation have shown positive effects on semen parameters. Strong antioxidant properties have the capacity to protect sperm membranes to stay intact by controlling ROS levels and maintaining efficient energy production219. Similarly, oocytes and embryos have high energy demands in female fertility, and the aging of ovaries is associated with mitochondrial dysfunction. CoQ10 supplementation was shown to improve ART outcomes in females and prevent aging220. Expression of oocyte mitochondrial genes was restored, and mitochondrial activity was enhanced with CoQ10 treatment221, and sufficient energy and reduced ROS levels promoted oocyte maturation, fertilization process, embryo development, and embryo implantation222. However, in a recent study, it was shown that the use of some mitoquinone, such as TPP and MitoQ at the concentration of 0.5 µM and 1 µM, respectively, could reduce cleavage rate, 4-cell embryos, and blastocyst rate223, suggesting that the use of antioxidants may disrupt the balance of mitochondrial bioenergetics, which negatively affects embryonic development. The use of L-carnitine has also been investigated, which was effective in increasing antioxidant glutathione (GSH) levels and reducing ROS production in mouse oocytes224. The data also showed that L-carnitine treatment improved oocyte maturation and blastocyst development in mice224. Other benefits of L-carnitine have also been extensively reviewed elsewhere, with minor warnings of the side effects of the supplement being included225. Another direction is to target calcium homeostasis in oocytes. The use of Ruthenium Red (RR), an inhibitor of Ca²⁺ uptake into mitochondria from the cytosol was shown to effectively reduce Ca²⁺ levels of porcine zygotes 6 hours after in vitro fertilization (IVF), which was associated with increased ATP production and mitochondrial membrane potential, and reduced mitochondrial ROS226. RR treatment, especially at the concentration of 20 µM, also significantly increased blastocyst development and quality of the zygotes226. Despite the effectiveness of pharmacological reagents, the side effects of those supplements in ART remain to be clarified. Future directions may continue with evaluation and examination of their effects on different stages of oocyte and embryonic development.

Given that mitochondrial capacity and function of germ cells determine fertility and healthy embryonic development, ensuring mitochondrial health poses a critical role in the reproductive system and fertility rate. Mitotherapy has been proposed as a potential method in ART, where preclinical and clinical data showed that transferring healthy mitochondria can support oocyte function, fertilization, and live birth rates. As discussed above, since the fetus primarily inherits mtDNA from the mother, mitochondrial replacement is currently the only technique available to help women with primary mitochondrial diseases caused by mtDNA mutations have healthy babies. Research has shown that approximately one in 5,000 people carry either nuclear DNA or mtDNA mutations that can potentially lead to mitochondrial diseases227. In Australia, the prevalence of individuals carrying a disease that could result in mtDNA mutations is one in 250, which may be inherited and negatively impact their lifetime228. Although the mtDNA mutation rate is 25 times higher than that of nuclear DNA, it is generally accepted that mutation levels must exceed 70% to cause clinical symptoms229, 230. Nevertheless, the burden of diseases caused by mtDNA mutations is lifelong and can be inherited across generations.

Figure 3

Mitochondria replacement is a type of mitotherapy in ART, where preclinical and clinical data showed that the transfer of healthy mitochondria can improve oocyte function and fertilization and healthy live birth rates. The transfer of mitochondria into aged oocytes or oocytes with pathogenic mutation of mtDNA has been performed and resulted in the births of “three-parent babies,” providing hopes for women with infertility or inherited mtDNA diseases231. Several technologies have been developed to perform mitochondria transfer into eggs and embryos to improve the outcomes of ART. These techniques include partial or total cytoplasmic transfer in oocytes, germinal vesicle transfer (GCT), maternal spindle transfer (MST), pronuclear transfer (PNT), blastomere transfer (BT), and polar body transfer (PBT), where PNT and MST are two most applicable ones22, 232. In brief, MST involves the transfer of the spindle, where chromosomes are packed in the affected oocyte, into the healthy oocyte of the donor, whose spindle is discarded233. As a result, a healthy oocyte with the mother’s chromosomes will be fertilized and transferred to the mother's uterus. On the other hand, PNT is performed after the oocytes of the mother and donor are fertilized233. The pronuclear of the affected oocytes will then be transferred into the healthy fertilized oocyte of the donor, whose pronuclear is removed. Even though the technology can help with infertile females in general, it must be noted that these techniques are mostly applied to female patients with mitochondrial disorders, in which mutations in mtDNA may affect the health of the offspring. Having newborns with mtDNA disorders is presented as a highly concerned aspect rather than infertility itself since mtDNA mutations are more likely to affect male than female fertility147. The implications associated with these techniques have been thoroughly analyzed but remain controversial from various perspectives, including science, economic, social, ethical, cultural, and possibly religious issues, which warrant further investigation and the involvement of multiple parties and disciplines234, 235.

Consequently, mitochondria replacement therapy requires more research to clarify the mechanism of mitochondria replacement throughout the body, as well as to assess its efficacy and safety regarding the welfare of the offspring. These biological challenges have raised ethical concerns for clinical applications and have limited the legalization of mitochondria transplantation to certain countries and thus, require considerable effort235, 236, 237.

Current advancements in ART have provided hope for infertile patients without mtDNA mutations, whose oocytes of low quality could not be fertilized, or the embryos could not reach the full developmental expectation, making the technology more approachable in terms of ethical and legal issues. Mitochondria transplantation involves the transfer of mitochondria into eggs or embryos of low quality, without the need to completely separate the existing mitochondria. Adding healthy mitochondria to low-quality oocytes was suggested to supplement the cells with fresh and functional mitochondria to meet the developmental requirements. In fact, mitochondria transfer, and transplantation have been utilized to rescue oxidative stress in multiple pathological models, proposing the potential application in human health238, 239, 240. Supplementing reproductive cells with functional mitochondria, either heterologously or autologously, is also discussed elsewhere in the fertility aspect22. However, since mtDNA and nuclear DNA mismatch appears to be the biggest ethical and practical issues in oocyte regeneration, autologous mitochondria transfer has been proposed to minimize these effects. Autologous stem cells were shown as an effective source of mitochondria for oocyte rejuvenation in both animal and human models and the direct injection of mitochondria together with sperm during intracytoplasmic sperm injection (ICSI) has been successfully applied. For instance, direct injection of autologous mitochondria from adipose-derived stem cells (ADSCs) was able to improve mtDNA numbers and quality of mature MII oocytes in aged mice241. The number of pups also increased after mitochondria were co-injected with ICSI into MII oocytes241. The transfer of mitochondria isolated from ADSCs has also been applied to enhance the quality of cryopreserved oocytes in mouse models242. The data showed that injecting isolated mitochondria from ADSCs into cryopreserved oocytes significantly enhanced ATP production in embryos of the 2-cell stage and increased the rates of blastulation in both oocytes and 2-cell embryos242. However, not all types of mitochondria can rescue poor-quality oocytes. In a mouse model, neither fresh (14 hours) nor aged (20-24 hours) post-ovulation oocytes showed any improvement in fertilization rate and embryonic development after the transfer of liver-derived mitochondria, even though oxygen consumption rate significantly increased243. Another study indicated that mitochondria derived from aged ADSCs to oocytes collected from young or aged (10-month-old) mice neither increased fertilization rates nor embryonic development244. These data emphasize the critical importance of mitochondrial selection for oocytes specifically and in mitochondrial transplantation technique in general. The compatibility and competence of donor mitochondria have a great impact on the cells and thus, define the outcomes of the therapy.

In fact, the effort has also been invested into finding the matching mitochondria for oocyte development. Oocyte mitochondria are immature and featured with spherical shape and little cristae formation, while early oocytes show low ROS production and low MMP, most probably to protect DNA integrity. The shape and metabolic profile of oocytes change with oocyte fertilization and embryonic development. Thus, the selection of mitochondria compatible with oocyte mitochondria would be beneficial for the recovery of low-quality oocytes. Based on this, Zhang and colleagues have shown that the transplantation of mitochondria from iPSCs, which contained mitochondria similar to oocytes, were efficient to improve egg quality, embryo quality, and live birth rates in aged mice245. A recent study by Jiang and colleagues has shown that the mitochondria from urine-derived MSCs (USCs) were efficient to improve mitochondria function in early embryonic development of age-oocytes after ICSI procedure246. The data emphasized that mitochondria from USCs were the most representative of oocyte mitochondria with spherical shape and immature cristae, while relatively low ROS and high MMP (mitochondrial membrane potential) and a high metabolic profile identified the competency in metabolism246. Transferring mitochondria purified from endometrial mesenchymal stem cells (EnMSCs), isolated from the endometrium, to improve oocyte quality and fertilization has also been investigated in mouse models247. Mitochondria purified from EnMSCs showed high MMP and low ROS when compared to endometrial stromal cells (ESCs), and positively improved oocyte quality, pregnancy rate, embryonic growth, and number of live births247. Compared to ADSC mitochondria, the isolation of iPSCs, USCs, and EnMSCs appears to be more challenging to perform. Overall, these data indicate the continuous efforts being made in mitotherapy, suggesting that the approach is promising and potential.

Another technique in mitochondrial replacement is autologous germline mitochondrial energy transfer (AUGMENT), introducing a technological advancement for rescuing infertility in aged women and has been tested clinically248. The method is based on the transfer of stem cell mitochondria from the female germline, which were referred to as oogonial stem cells (OSCs), and showed competency and similarity to egg mitochondria. Clinical trials of the method have been reported with positive results in improving fertilization rate and live births in women with repeatedly failed IVF, proposing a new method for ART. Based on this technique, different clinics have successfully reproduced the positive results in women with repeated IVF failure249, 250. However, a randomized pilot study involving infertile women, which employed AUGMENT from egg precursor cells to metaphase oocytes (MII oocytes), failed to improve the outcomes in the subject population251, which was argued to be generated from technical misunderstanding252. These results highlight that the accuracy in selecting cells for mitochondrial transplantation is important to the success of ART in women with repeated IVF failure. Given that different technologies have been shown successful with positive results in infertile women, the effort might also be contributed to make it even better feasible for a broader translation in different clinics, as different clinical setups might be met with difficulty in performing particular procedures.

It must be emphasized that autologous mitochondria supplementary techniques are only suitable for aged and low-quality eggs, where mtDNA is not affected by pathogenic mutations. The current technology in mitochondrial replacement into oocytes or embryos mostly involves the discarding of donor oocytes or fertilized eggs, which are encountered for ethical issues22. However, recent research has shown that this issue may not be a concern in the future as mitoception procedures could effectively transfer isolated mitochondria into both oocytes and zygotes253. In detail, the authors indicated that low temperature centrifugation at 500g for 5 minutes was efficient to transfer 50 ng/µL of isolated human mitochondria from peripheral blood mononuclear cells into mouse oocytes253. Even though the data could not give a conclusion on the retention of human mitochondria in mouse pups, it has proposed a non-invasive technique for transferring healthy mitochondria into oocytes253. Tang and colleagues have developed a non-invasive technique for autologous mitochondria transfer by using umbilical cord-derived mesenchymal stem cells (UC-MSCs)254. In the experiments, UC-MSCs collected since birth of the female mice were cryopreserved. When those female mice were aged, UC-MSCs were thawed and induced to form granulosa cells, which aggregated around aged oocytes. The authors observed mitochondria from the induced granulosa being transferred into oocytes via transzonal filopodia, which significantly improved oocyte quality, embryonic development, and live births in aged mice254. Even though the technique is promising, the need to cryopreserve UC-MSCs at birth might be difficult in application, and further investigations would be warranted to understand the mechanism of mitochondria transfer in those mice.

Table 1

Species	Transplantation methods	Donor cells	Host cells	Outcomes	Reference
Mice	Autologous Mito-ICSI	ADSCs	MII oocytes	-Improved mtDNA number, quality of mature oocytes -Increased blastocyst and birth rate	241
Mice	Autologous Mito-ICSI	ADSC	Cryopreserved oocytes	Improved fertilization rate and embryonic development	242
Mice	Autologous microinjection	iPSC	Fertilized oocytes	Improve blastocyst development, preimplantation rate, and birth rate	245
Mice	Autologous Mito-ICSI	EnMSC	GV oocytes	-Improve oocyte maturation and quality in vivo embryonic development and birth rate	247
Human	Autologous Mito-ICSI	USC	MI oocytes	-Improved mitochondrial function in early embryos	246
Human	Autologous Mito-ICSI	OPCs/ OSCs	Oocytes	-Improve pregnancy and live birth rates	249, 250, 255

Abbreviations: ADSC: Adipose-Derived Stem Cell, EnMSCs: Endometrial Mesenchymal Stem Cells, GV: Germinal vesicle, ICSI: Intracytoplasmic Sperm Injection, iPSC: Induced Pluripotent Stem Cell, MI: Metaphase I, OPCs: Oogonial Progenitor cells, OSCs: Oogonial Stem Cells, USC: Urine-Derived Mesenchymal Stem Cells

Embryo selection is the method to evaluate embryo quality and select embryos with biomarkers potential for development and implantation. The approaches can involve direct analysis of mtDNA blastomeres by biopsy or indirect analysis of somatic cells and cell-free mtDNA. Although embryonic mitochondria have critical embryo functions, analysis of mtDNA blastomeres is still under discussion. A few studies have shown appreciation of mtDNA analysis, which this biomarker may serve as a predictive indicator for embryo development. Some studies investigated the relative mtDNA numbers on oocyte and embryo quality, and treatment outcomes. Oocytes with low mtDNA content are associated with high fertilization failure200, 256, 257. In contrast, lower trophectoderm (TE) mtDNA levels were correlated with higher trophoblast quality. In day-5 transferred embryonic outcomes, higher implantation rate and live birth were associated with low TE mtDNA content258, 259. Nevertheless, no relationship was found between inner cell mass and mtDNA content260. The levels of mtDNA might be dependent on the biopsied date, with higher levels observed on day 5 compared to day 6261. Contrasting results have been reported that the biopsy on day 6 or day 7 was higher than day 5 mtDNA levels262. Furthermore, no correlation was found between TE mtDNA levels and embryonic implantation ability, which disagreed with the ability to use mtDNA content as an embryo selection criterion to transfer263. As a result, TE biopsy might be an advantageous approach for mtDNA analysis, however, biopsy still poses a risk of injury, which can lead to embryonic damage.

Cumulus cells (CCs) originate from granulosa cells, which surround oocytes, creating the oocyte-cumulus complex (OCC). Cumulus cell function is a metabolic cooperation during oocyte development, meiosis, fertilization, and early embryo development264. Besides producing cell cycle signals to oocytes, the cumulus cell glycolysis produces pyruvate to supply oocyte OXPHOS265. Equally, cumulus cell metabolism is incapable in the absence of the oocytes. Several studies have demonstrated factors of cumulus cells that can predict the oocyte quality266, 267. The genes PFKP, HAS2, TNFIP6, PTGS, and PTX3 of CCs have been shown to be correlated with embryo potential development and implantation268, 269. Moreover, the levels of mtDNA in CCs are not significant in predicting embryo development and outcome treatment270. Thus, evaluation of CC metabolism and gene expression could be a potential method for embryo selection.

Cell-free mtDNA (cf-mtDNA) is also a possible non-invasive marker for investigating mitochondrial health. Mitochondria communicate adaptively with stress by signaling cf-mtDNA molecules. Most mtDNA mutations in embryos have the potential to be identified from mtDNA in conditioned media271. The cf-mtDNA collected from media culture positively correlated with morphokinetic parameters from the beginning of blastulation to full blastocyst stage, where higher cf-mtDNA content reflects poorer quality blastocyst272. On the other hand, high cf-mtDNA content from cleavage-stage embryo culture medium is a positive predictive marker of good quality embryos273, 274.