Mitochondria in the reproduction system and mitotherapy in assisted reproductive technology: the importance of mitochondria selection

Abstract

Mitochondria are central to energy production and are crucial for the proper operation of the reproductive system. Mitochondrial function and capacity determine whether germ cells are fertilizable, and embryos can safely reach developmental expectations. Impaired mitochondrial biogenesis and defects in mitochondrial DNA result in low birth rates, infertility, and, more seriously, unhealthy offspring with inherited and irreversible metabolic diseases. In recent years, mitochondrial transfer and transplantation have contributed greatly to the field of assisted reproductive technology, especially with advances in biotechnology. Much effort has been invested in refining mitotherapy techniques, aiming at improving the safety, efficacy, and accessibility of the treatment, while reducing costs, labor, and ethical issues. Recent research has shown numerous changes in the approaches with innovative ideas and new materials. This review highlights the role of mitochondria in the reproductive system and the current efforts to improve the outcomes in ART cases with mitochondrial issues. We also summarize different types of mitochondrial transplantation techniques and emphasize the importance of mitochondria selection for reproductive purposes.

Introduction

A diagnosis of infertility is made if a couple cannot achieve or maintain a pregnancy after one year of attempting to conceive naturally. The World Health Organisation reported in 2023 that infertility affects about 17.5% of couples worldwide, threatening the stability of global population growth and the economy. Moreover, the age of first childbearing is reported to increase in various countries worldwide. According to the UNECE (United Nations Economic Commission for Europe) data, the mean age of women at the birth of their first child has increased by one year in the 10-year period from 2012 to 2022. In recent years, women have tended to delay their first childbearing plan, while their fertility age is limited, making the issue even more challenging1, 2. Infertility treatments can differ between men and women, which may include medications and surgery. However, with unexplained infertility, these treatments appear to be less effective. In that context, assisted reproductive technology (ART) is meeting increasing demand, contributing greatly to the world population, making up around 8% of the total population in some regions3, 4. The rates of success in ART are controversial and are correlated with the quality of the medical systems in different regions. Furthermore, the success rates also rely on different clinics, which may not always wish to share their data. Generally, depending on the patient's age, the indications could vary. For example, the use of intrauterine insemination (IUI) is suggested to be as effective as in vitro fertilization (IVF) in patients under 40, while IVF may be more effective for older patients5, 6, 7. In fact, both treatments may require several cycles, and even with failed attempts to conceive in the first three cycles in IVF, it was suggested that the success rate may increase, reaching near-natural rates with more repeated cycles8, 9, 10. However, it must be noted that the treatment of infertility can be costly and prolonged, putting an extra burden on the existing stress of the patients. Infertile individuals undergoing therapeutic support or ART often face mental health issues such as emotional adjustment, anxiety, and depression. Although these issues may not be directly related to the outcomes, they can sometimes prolong the time it takes to achieve success11, 12, 13, 14, 15, 16. Even though interventions that provide psychological support were shown to reduce depression in these women, they could not alleviate their anxiety levels and were not correlated with success rates17. Thus, the key to overcoming these challenges greatly relies on the innovation of new technology, as advancements in therapeutic development would provide better options and solutions for current biological and technical difficulties and narrow the gaps in approaching the treatments.

Infertility has various causes, such as genetic, hormonal, anatomical, immunological, infectious, environmental, and lifestyle factors18, 19. Among these factors, mitochondrial dysfunction or abnormality has been proposed as a potential factor contributing to infertility in both males and females20, 21. Mitochondria are responsible for producing and maintaining energy (or ATP) that is required for cellular activities via several metabolic pathways such as oxidative phosphorylation (OXPHOS), amino acid metabolism, beta-oxidation, and calcium homeostasis. Mitochondria contain their own DNA in a ring shape, which is generally referred to as mitochondrial DNA (mtDNA). Even though mtDNA is directly in contact with reactive oxygen species (ROS) produced by nearby metabolic processes, they are less efficiently repaired and thus, are more vulnerable to mutation. Mitochondria are essential for various aspects of reproductive function and processes, such as gamete maturation, fertilization, implantation, placental function, and embryonic development21. Mitochondrial dysfunction can result from mutations or deletions occurring in mtDNA or in nuclear DNA sequences that encode or regulate mitochondrial proteins, as a consequence of oxidative stress, inflammation, toxins, and aging. Mitochondrial dysfunction can affect different cell types involved in reproduction, including germ cells (sperm and oocytes), and embryonic cells, which can result in infertility. Given the importance of the mitochondria in the reproductive system, understanding the role of mitochondrial damage in the pathogenic progression of infertility is necessary for developing effective treatments and interventions. Various types of treatments have been clinically applied and developed, and the growth of different scientific fields has greatly improved the effectiveness of assisted reproductive technology (ART). One way to approach the issues with mitochondrial dysfunction in infertility is mitochondrial therapy (or mitotherapy), in which healthy mitochondria are transferred or transplanted into defective cells, which would be oocytes or zygotes22. The data from preclinical and clinical applications have shown that mitotherapy is a promising and potential method for treating infertility, raising hopes and opportunities for infertile patients; however, it is met with numerous concerns both technically and ethically23, 24, 25. In this review, we cover the biological causes and consequences of mitochondrial damage in various cells and tissues of the reproductive system and their impacts on fertility. We also discuss the current methods to improve the outcomes for mitochondria-related issues and the development of mitochondrial transplantation in ART. We also give examples of how the techniques have been refined over time and highlight the critical importance of mitochondrial selection, specifically for improving poor-quality oocytes and low fertilization rates.

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Figure 1 . The roles of mitochondria in oocytes . ( A ) Functional mitochondria play a vital role in the development and maturation of oocytes and early embryos. Calcium equilibrium is achieved by the storing and releasing of calcium ions between the mitochondria and the endoplasmic reticulum. Oocyte homeostasis is maintained according to the demands of each cellular metabolic activity. In addition, mitochondria help produce ATP to provide the energy required for the cell at each maturation stage, especially during chromosome condensation. During fertilization, ATP synthesis from the mitochondria activates the phosphoinositide pathway, which is essential to achieve successful fertilization. ( B ) Mitochondrial malfunction includes abnormal metabolic activities, loss of mitochondrial membrane potential, changes in mitochondrial dynamics, and oxidative stress response. These abnormalities reduce the homeostatic regulation of mitochondria and the ability to synthesize energy, maintaining Ca 2+ balance and redox status, leading to telomere shortening, disrupted meiosis and mtDNA mutations. These effects consequently result in incomplete oocyte maturation or cell aging, with adverse effects on spindle formation, chromosomal segregation, and fertilization

MITOCHONDRIA IN THE REPRODUCTIVE SYSTEM

In reproductive cells, mitochondria serve a vital function in providing the energy necessary for various processes such as oocyte maturation, sperm motility, and fertilization26. Mitochondria in reproductive cells have unique characteristics, including differences in morphology, size, and distribution compared to those found in other cell types27. Additionally, mitochondrial DNA (mtDNA) is inherited maternally, with the egg providing the majority of the mtDNA in the developing embryo, which is a unique feature of mitochondria in reproductive cells28.

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 meet the energy demands for developing cells30. Once the oocyte is fertilized, mitochondria maintain their critical role in supporting embryonic development in the early stages31. Oocyte mitochondria influence oocyte quality, particularly in terms of proper chromosomal segregation during oocyte maturation32. 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. Apart from generating energy for basic cellular demands, mitochondria also act as regulators of calcium homeostasis, which is critical for cell survival and function35. This balanced state is achieved through calcium storage and release interdependently, ensuring suitable free intracellular calcium levels throughout various 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, and histone and DNA methylation-demethylation21. The involvement of mitochondria in oocyte function is summarized in Figure 1.

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Figure 2 . Mitochondria function in sperm biology and function . ( A ) Mitochondria activity through spermatozoa formation . Spermatogenesis consists of various stages, starting from spermatogonial stem cells and progressing through mitosis and meiosis to form spermatids and eventually mature sperm cells. In the early stages, mitochondria are sparse and immature, and limited ATP is produced from OXPHOS activities. During these stages, spermatogenesis is supported by the energy primarily produced via glycolysis. Mitochondrial activity increases from low OXPHOS in early spermatogonial cells to elongated, clustered mitochondria with higher activity in later stages to meet energy demands of meiosis and sperm formation. In mature sperms, the packed midpiece mitochondria exhibit high OXPHOS for ATP generation, crucial for flagellar motility and fertilization. ( B ) Energy production in spermatozoa movement . Mitochondria found in the midpiece of sperm are elongated and spiral-shaped. They provide energy for capacitation (preparing sperm for fertilization), flagellum propulsion (via high OXPHOS activity for sperm motility), and the acrosome reaction (enabling sperm to penetrate the egg's outer layers). ATP is generated in spermatozoa by two metabolic mechanisms: Glycolysis and OXPHOS, which takes place in distinguished part of the spermatozoa.

In male reproductive cells, mitochondria are mainly located in the midpiece (also called the neck) of the sperm, where they supply energy for the flagellum to propel the sperm forward37. This is critical for successful fertilization because sperm must be able to reach the egg by swimming through the female reproductive tract21. Furthermore, the quantity of active mitochondria in spermatogonia stem cells (SSCs) can vary depending on the development stage38. SSCs are a type of cell found in the testes responsible for the production of sperm39. The specific changes in the quantity of active mitochondria in SSCs during development stages can have implications for the energy requirements and metabolic activities of these cells. It could potentially affect their ability to divide, differentiate, and ultimately contribute to the production of sperm40. Mitochondria in spermatogonia are typically small and spherical, located in the basal structure of the seminiferous epithelium, and have access to the vasculature and interstitial fluid41, which supports low oxidative phosphorylation (OXPHOS) activity at this stage. When mitochondria cross the blood-testis barrier and enter the adluminal compartment, they undergo a process known as intermitochondrial cement (IMC), where they become elongated and cluster around the nuage42. Mitochondrial fragmentation occurs in post-meiotic spermatids42. Finally, as spermiogenesis progresses, mitochondria pack closely around the sperm midpiece43. Only a fraction of mitochondria line the sperm midpiece during elongation; the rest, along with other cellular components, are collected into residual bodies just before spermiation (sperm release) for phagocytic destruction by Sertoli cells44. Furthermore, the presence of exogenous mitochondria in male germ cells emphasizes the relevance of mitochondria in testicular metabolism45. Germ cell survival in the adult testis relies entirely on energy from carbohydrate metabolism, which is produced by both glycolytic and OXPHOS processes46. This is particularly relevant in germ cells undergoing complex and energy-demanding processes such as meiosis and spermatogenesis. Mitochondrial function in sperm biology is summarized in Figure 2.

Mitochondrial dysfunction and the effects on the male reproductive system

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.

Debilitated mtDNA integrity and sperm motility

Mitochondrial dysfunction impacts the integrity of sperm DNA. Alterations affecting the mitochondrial genome can impair male reproductive potential52. Large deletions or single-nucleotide polymorphisms are two examples of mutations that influence sperm mtDNA53, 54. As a result, sperm with mutant mtDNA may have respiratory issues, which impact how energy is produced, as well as motility issues that affect how active they normally are55. Studies suggest a strong link between the alterations or deletions in mtDNA and male infertility56. Deletions in mtDNA that affect cellular equilibrium and energy production have been shown to impair sperm motility56. Moreover, mtDNA copy number and sperm DNA fragmentation (SDF) are both correlated with semen quality52. The study found that asthenozoospermic semen samples contained a higher mtDNA copy number, which was associated with reduced sperm concentration, low sperm number, and decreased motile spermatozoa. Thus, adequate mitochondrial genome content is required for efficient energy metabolism and hence, facilitates sperm motility. In addition, SDF was found to increase in asthenozoospermic samples, which resulted in abnormal forms, even though SDF and mtDNA copy number were not correlated57. The decrease in mtDNA copy number naturally occurring during the process of sperm development could imply alterations in the maturation process of sperm41. This increase in mitochondrial genome content could also be a result of a compensatory response triggered to counteract the mitochondrial dysfunction, which is highly probable to impact sperm quality of infertile males40. Overall, these findings underline the importance of mitochondrial integrity and mtDNA content in maintaining efficient energy metabolism, which ensures proper sperm function, motility, and ultimately, male reproductive potential.

The mitochondrial genome quantity is controlled by various molecules and factors to regulate the genetic material cloning process. The mitochondrial transcription factor A (TFAM) protein is involved in the creation of primers needed for mitochondrial DNA copy number, was found actively in lower total motile sperm and this high TFAM activity also coincided with a rise in abnormally shaped sperm, SDF, and mtDNA replication58. Another study found that sperm cells not only lack intact mtDNA, but they also lack TFAM protein, suggesting that TFAM gene expression is positively correlated with sperm motility59. Therefore, it is impossible to rule out the idea that a post-transcriptional regulatory mechanism underlies the distinct expression of the transcript and protein60. It must be noted that reduced sperm function and male infertility can also be a result of molecular modifications to the mtDNA, which affect sperm movement and shape51. For instance, asthenozoospermia has been linked to large mtDNA deletions, with sizes ranging from 4,977 to 7,599 base pairs61, 62. The typical 4977-bp mtDNA loss has been suggested as an efficient indicator for mtDNA damage because it increases in many organs with aging63 . The 4977-bp deletion in sperm mtDNA occurs more frequently in patients with asthenospermia and oligospermia than in healthy individuals64. Additionally, patients with primary infertility were more likely to experience the 4977-bp mtDNA loss in sperm than were those with secondary infertility65. Seven genes and five transfer RNAs are removed as a result of this loss, which affects the region of the mtDNA between 8483 and 13459 base pairs66. In infertile males with severe or ongoing and unexplained asthenozoospermia, abnormalities might be observed in the structure of the mitochondria within the middle section of their sperm67, 68. Asthenozoospermia may have an underlying etiology related to disruptions in energy synthesis and mitochondrial activity in sperm69. The quality of mtDNA and mitochondrial function are critically important in the male reproduction system, which ensures healthy maturation of sperm and their movement during fertilization. Thus, maintaining mitochondrial function and integrity is essential in male fertility.

Sperm apoptosis

Apoptosis is a cell death program, in which cells with compromised genetic materials are eliminated70. Apoptotic activation may occur in the absence of specific cell surface receptors as substances can enter the cell directly and modify the apoptotic cascade71. Heat shock, stressors, ROS, UV radiation, drugs, synthetic peptides, and poisons are a few examples of such variables72. Nowadays, it is acknowledged that human sperm exhibits and activates apoptotic signals in response to different types of stimuli73. A class of proteases known as caspases is crucial for controlling apoptosis. The mitochondria are crucial in the apoptotic cascade by providing key elements, such as those that activate caspase activity and DNA fragmentation74. Cytochrome c, a significant apoptosis component, facilitates caspase 9 and caspase 3 initiation, which results in cell apoptosis75. Disruptions in cellular homeostasis are known to induce the permeability transition pore (PTP), which is located in the outer membrane of mitochondria to open, which is involved in cell death signaling76. Mitochondrial ATP synthase dimers are in charge of PTP production77. The removal of cytochrome c via PTP activates the caspase cascade and apoptotic program76. Caspases 3 and 9 activity was detected in the midpiece of human sperm after ejaculation78, 79. When the apoptotic program was triggered in spermatozoa, caspase 9 and 3 activity increased, while mitochondrial membrane potential (MMP) decreased, which was associated with low sperm motility79, 80. Studies have established a detrimental link between caspase activation, sperm quality, and the absence of integrity in the plasma membrane81. This condition is evidenced by the presence of externalized phosphatidylserine, which is a marker of programmed cell death82. Understanding the complexities of sperm apoptosis is paramount for developing novel therapeutic interventions and improving assisted reproductive techniques. Furthermore, identifying the key regulators of sperm apoptosis may make way for innovative diagnostic tools and targeted therapies to address male infertility.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is critical in regulating sperm apoptosis. This intracellular signaling system promotes a variety of cellular functions, which include cell survival, cell growth, proliferation, and migration83. AKT (the main protein in the PI3K pathway) activation improves sperm survival, especially under stress conditions84. Spermatozoa viability and function are dependent on the PI3K enzyme phosphorylating AKT1 (also known as RAC-alpha serine/threonine-protein kinase), which inhibits downstream effectors of the apoptotic pathway including the Bcl-2-associated death promoter85. Conversely, spermatozoa are driven toward the apoptosis process as a result of the inactivation of AKT1, when PI3K activity is suppressed86. This activates the caspase cascade in the cytosol, increases ROS production in the mitochondria, resulting in a considerable reduction of sperm motility and oxidative DNA damage73. The active endonucleases, however, are unable to cleave the nuclear DNA because the nucleus in the sperm head is well separated from the sperm midpiece, which contains the mitochondria and cytoplasm87. As a result, although DNA can be affected by oxidative reactions, apoptosis may not lead to DNA fragmentation in human spermatozoa73. The PI3K/AKT pathway expresses a critical role in controlling sperm function and quality, as evidenced by the complex interactions it has with the apoptotic apparatus.

Sperm apoptosis also comes from the dysregulation of mitochondrial dynamics, promoting oxidative stress and cell death. Mitochondrial fission and fusion are essential for maintaining mitochondrial function; excessive fission can lead to apoptosis by promoting mitochondrial outer membrane permeabilization (MOMP)88. Normal expression in fusion and fission genes allows spermatozoa to function successfully89. However, MOMP is regulated by the protein Bcl-2, which is an important factor influencing the release of apoptotic factors like cytochrome c, which activates caspases and initiates cell death90. Another study has shown that Hexavalent chromium-treated rat testis presents a decline in Sirtuin 1 (Sirt1), resulting in an increase in mitochondrial fission91. Moreover, Sirt1 then downregulates nuclear factor-erythroid-2-related factor 2 (Nrf2). These stages greatly activate OS and the expression of apoptotic genes such as Bcl-2, cytochrome c, and promote sperm apoptosis91. The overproduction of ROS could damage organelle structure and exacerbate downstream factors, leading to cellular apoptotic signs, meanwhile, those apoptotic features induced by exogenous factors could induce OS and disrupt mitochondrial dynamic homeostasis88, 92.

Oxidative stress

Sperm cells rely mainly on mitochondria to produce ATP in demand for energy to support their motility, capacitation, acrosome reaction, and fertilization ability through OXPHOS93. Mitochondrial dysfunction in sperm cells can cause male infertility by impairing sperm quality and function94. However, sperm mitochondria are also the main site of ROS production, which can damage cellular components if not scavenged by antioxidants95. Furthermore, ROS induces oxidative stress, negatively affecting the sperm membrane, DNA, proteins, and lipids96, 97.

Mitochondrial metabolism provides the energy required for sperm function98. Sperm movement requires an abundance of energy generated from OXPHOS. This process includes oxidative reactions and the generation of ROS99. According to Munro and Treberg (2017), ROS, namely superoxide radicals and hydrogen peroxide, are produced as a byproduct of the aerobic synthesis of ATP via OXPHOS100. Hydrogen peroxide (H₂O₂) generation in the mitochondrial matrix was increased through processes unrelated to MMP as a result of rotenone-induced suppression of Complex I101. Because of this, the sperm midpiece's lipids started to oxidize, which caused sperm motility to decline102. Nevertheless, using antioxidants such as tocopherol was found to reverse the harmful effects of rotenone103. In oligoasthenozoospermic patients, the co-incubation of spermatozoa with myoinositol improved sperm mobility and increased the number of spermatozoa with high MMP104. Lower MMP is also correlated with poor sperm mobility in infertile men105. When exposed to spermicidal agents, human sperm showed a significant reduction in motility and MMP106. Uncoupling the electron transport chain can lead to abundant ROS release, which is associated with reduced MMP and sperm mobility107. Finally, low sperm MMP can be an indicator of poor sperm quality, which results in lower fertilization rates in in vitro fertilization (IVF)108.

Sperm motility can significantly decrease due to the lack of energetic donation. There are two sources of energy providing ATP for sperm, including OXPHOS and glycolysis, primarily contributing ATP to sperm flagella for movement109. Meanwhile, the energy produced by mitochondrial OXPHOS is used for gluconeogenesis, which in turn produces raw sugar for glycolysis110. Studies have shown that disruption of glycolysis reduces sperm motility; in contrast, inhibition of oxidative phosphorylation does not significantly impair human sperm motility111. Additionally, exogenous pyruvate enhances glycolytic ATP production, motility, hyperactivation, and capacitation in human sperm, indicating a reliance on glycolysis112. Therefore, abnormalities in mitochondrial energy production do not directly affect sperm motility113. Although mitochondria are not the primary ATP donors for human spermatozoa, studies have suggested that mitochondrial activities are a significant source of ROS114. ROS exists in spermatozoa at a certain low controlled concentration, participating in several physiological processes of sperm. However, when ROS production becomes abnormal, the level of intrinsic ROS increases, thereby affecting sperm health115, 116. ROS in semen includes oxygen-centered radicals (e.g., superoxide anion (O2^●−), hydroxyl radical(^●OH)) and non-radical derivatives like hydrogen peroxide (H₂O₂)117. H₂O₂ and O₂^●− are two significant oxidants with key physiological roles118. They are continuously generated by mitochondrial NADH-dependent processes (located in the inner mitochondrial membrane) and extramitochondrial NADPH-dependent systems (located in the plasma membrane)119. O₂^●− can be converted into H₂O₂ through superoxide dismutase (SOD); furthermore, it can react with H₂O₂ to form highly reactive radicals (^●OH), triggered by the presence of iron (Fe²⁺)120, 121. The overall reaction is simply described below:

O₂^●− + H₂O₂ → ^●OH + OH⁻ (hydroxide ion) + O₂

When the process of mitochondrial ROS generation is disrupted, leading to an increase in O₂^●− level122, 123. Such conditions can result in abnormal concentrations of the most highly reactive molecule, hydroxyl radicals124. ^●OH can cause damage to cellular components, including lipids, proteins, and DNA, due to their unpaired electron125. Removing a hydrogen atom from membrane fatty acids leads to the oxidation of the spermatozoa membrane, called membrane lipid peroxidation115. This process produces an unstable lipid peroxyl radical, then initiate a series of reactions and generate harmful compounds, disrupting the membrane flexibility and fusion ability119. These properties are crucial for sperm movement, acrosome release, and successful fertilization119. Therefore, while mitochondria may not be the primary energy source for sperm motility, their role in ROS generation can significantly impact sperm quality and fertility potential.

Oxidative stress can also induce DNA fragmentation, chromosomal abnormalities126, and sperm epigenetic modifications, which can affect gene expression127. Moreover, sperm cells can transmit mtDNA mutations or deletions to the offspring through paternal inheritance128. These mutations or deletions can impair OXPHOS function and cause mitochondrial diseases that affect various organs and systems129. Oxidative imbalance can cause male infertility by the following mechanisms: (i) damaging sperm membrane, thereby reducing sperm motility and ability to fertilize; (ii) damaging sperm DNA leading to reduced fertilization ability and affecting embryonic development after fertilization; and (iii) increasing the process of sperm degradation130, 131. The increase of polyunsaturated fatty acid concentration in the sperm plasma membrane exposes them to oxidative stress, which results in lipid peroxidation and sperm membrane damage132. Oxidative stress-induced caspase activation can also cause cytochrome c to be released from the mitochondria, leading to the apoptosis of spermatozoa133. Various intrinsic and extrinsic factors can contribute to the development of SDF, including varicocele, infection, aging, heat stress, lifestyle, environmental toxins, and ionizing and non-ionizing radiation21. Antioxidants have been found to effectively reduce both ROS and SDF in infertile men with various conditions134. In conclusion, oxidative stress can compromise sperm health by damaging sperm components such as DNA and membrane, potentially leading to fertilization failure. Additional investigation is essential to elucidate the underlying pathways involved and to explore potential therapeutic strategies targeting OS in male infertility.

Mitochondria functions in oocyte development and aging

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.

Oocyte aging and mitochondria DNA mutation

The accumulation of somatic mtDNA mutations, compromised quality control processes, and disrupted mitochondrial biogenesis are among the aspects of mitochondrial dysfunction that are related to aging136, 137. Ovarian aging manifests as a decline in ovarian reserve, a concept encompassing both the quality and oocyte number available for ovulation138. The reduction in primary follicle number indicates an ovarian quantitative decline whereas a qualitative decline is associated with decreased mtDNA content27, 139. Women's age is negatively correlated with mtDNA replication139. This can also be observed in women of similar age groups with diminished ovarian reserve140. Oocytes carrying mutations within their mtDNA can compromise mitochondrial function and potentially lead to reduced oocyte quality. Thus, mtDNA content is crucial to female fertilization. Not only mutations in mtDNA damage mitochondria, but mutations in genetic materials of the nucleus can also cause loss of mitochondrial function. Some mitochondrial proteins are translated from the nuclear genome when expressed abnormally. Allelic mutations of the nuclear-encoded catalytic subunit of the enzyme mtDNA polymerase gamma (PolgA), which encodes its catalytic components, have been linked to a variety of mitochondrial diseases affecting mtDNA stability141. POLG variants can lead to a variety of illnesses such as autosomal dominant and recessive progressive external ophthalmoplegia (PEO), which are defined by mtDNA deletions or depletion142. On the other hand, POLG recessive and dominant mutations are also linked to other conditions such as parkinsonism, and premature menopause143. Many women listed as having this illness had shown hypergonadotropic hypogonadism144. This condition was found to be related to a heterozygous p.Y955C mutation in three families with autosomal dominant inheritance20. Additionally, a family with a heterozygous Y955C mutation also showed cases of testicular atrophy145. A heterozygous POLG variation A was also reported in cases with ovarian dysgenesis, which could be associated with cataracts, muscle atrophy, and 3-methylglutaconic aciduria in females145. More studies on POLG should be undertaken to broaden the knowledge of the interaction between nuclear DNA and mtDNA, and prerequisite research could be conducted to develop disease treatment measures through genetic agents. It must be noted that in female mice with POLG mutator, fertility is reduced by mtDNA mutations that disrupt oocyte NADH/NAD⁺ redox state146, 147, which is an important factor to maintain mtDNA integrity.

Stress response pathways

Like sperm, oocyte mitochondria are susceptible to damage by reactive oxygen species (ROS). Maintained equilibrium of ROS level is necessary for normal physiology of the oocyte. This balance is disrupted by endogenous and exogenous factors such as apoptosis induction, cryoinjury, or exposure to disadvantageous factors, leading to mitochondrial dysfunction and impaired oocyte development148, 149. Oxidative stress affects oocyte development and survival, reducing oocyte quality and ability to fertilize. Particularly, oxidative stress can cause telomere shortening in oocytes, which consequently causes cell cycle arrest, DNA instability and abnormal spindle formation150, 151, 152. Oxidative stress is also correlated with ER stress, disrupting the import and exchange of calcium ion (Ca²⁺), which is important for calcium homeostasis in oocytes, supporting oocytes over maturation stages, especially during meiosis153, 154, 155, 156. Thus, preventing mitochondrial oxidative stress is necessary in early oocytes. In this context, early oocytes exquisitely retain low oxidative stress by repressing the expression of ETC complexes, especially complex I149. It was found that subunit assembling in Complex I was also significantly reduced, leading to the formation of inactive Complex I149. The suppression of Complex I was effective in maintaining low ROS production and redox state in early oocytes until oogenesis occurred and Complex I was activated149. These data indicate that low ROS and reduced redox status are important for oocyte function, as a high oxidative profile contributes to oocyte declining activity at age. Redox imbalance is also affected by aging in oocytes, in which the production of ROS increases while antioxidant levels decrease, disturbing the meiotic process and increasing aneuploidy in mature eggs157, 158, 159. It was shown in porcine oocytes that to overcome the damage from ROS, the cells activate Sirtuin 3 (SIRT3) protein expression, which in turn promotes the deacetylation of superoxide dismutase 2 (SOD2)160, 161. SOD2 is a strong antioxidant, responsible for eliminating ROS accumulation in the mitochondria, indicating that the SIRT3/SOD3 pathway is a mechanism for oocytes to retain low ROS levels160, 161. Therefore, strategies to maintain redox balance are crucial in oocyte function.

Additionally, maintaining a healthy proteome, especially those of long-lived proteins, is an important factor in ensuring the quality and development of oocytes during reproductive life162. Many of these long-lived proteins in women's oocytes are mitochondrial162. It has been shown that oocyte mitochondrial unfolded protein response (mtUPR) stands as a mechanism of cellular defense that helps resolve proteostatic stress163. mtUPR is activated to restore proteostasis and rescue mitochondrial function, whenever an increase in misfolded or unfolded proteins disrupts mitochondrial homeostasis163. This process includes a cascade of nuclear gene transcription to upregulate the expression of genes encoding specialized mitochondrial chaperones164. These molecular chaperones, such as heat shock protein (HSP) family, help misfolded proteins achieve their proper three-dimensional structure, crucial for protein function165. Furthermore, mtUPR also implements a negative feedback mechanism to limit the influx of newly synthesized mitochondrial proteins166. This serves to alleviate the burden on the already stressed protein folding machinery within the mitochondria167. Maintaining a healthy mitochondrial proteome necessitates the coordinated action of chaperones and mitochondrial proteases. Misfolded proteins beyond the capacity of the chaperone system are targeted for degradation by specialized mitochondrial proteases168. mtUPR ensures the proper function of mitochondria169. Any disruption to this quality control system can result in mitochondrial dysfunction169.

The primary regulator of mtUPR, mitochondrial matrix caseinolytic peptidase (Clpp), was discovered to be increased during mitochondrial stress produced by unfolded proteins170. This rise in Clpp protease activity promotes the degradation of misfolded proteins, generating peptide fragments that act as signaling molecules, which activate transcription factor ATFS1. ATFS1 then translocates from the mitochondrial matrix to the nucleus and interacts with two proteins: UBL5 (ubiquitin-like 5) and DVE1 (defective proventriculus in Drosophila homolog 1). This tripartite complex binds to specific nuclear DNA sequences, which regulate gene expression of essential mitochondrial chaperones, such as HSP60 and HSP1027. Some mitochondrial abnormal states such as increased mtDNA replication and ROS level, as well as disrupted MMP, are also determined in animals with Clpp deficiency, resulting in profound female infertility171. Furthermore, metabolic imaging employing fluorescence lifetime imaging (FLI) microscope revealed significant changes in Clpp-deficient oocyte metabolism, including those in the lifetimes of FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide) and the protein-bound percentage of NADH, reflecting disruptions in mitochondrial electron transport chain activity172. Clpp-deficiency in animals was found to induce female infertility with poor oocyte maturation, low two-cell embryonic development, and inability to form blastocysts173. The data were correlated with increased spindle defects in eggs, which led to increased depletion of follicles in mice, which was linked to the initiation of the mammalian target of rapamycin (mTOR) pathway, which could be partially reversed by rapamycin, an inhibitor of mTOR174. In conclusion, it is essential to maintain the quality control mechanism of mitochondrial proteostasis, including mtUPR, to ensure reproductive capability. Elucidating the complicated regulatory networks that govern mitochondrial function has potential implications for understanding infertility and designing treatments.

Mitochondrial dynamics

Mitochondrial ATP production is critical during oocyte and early embryonic development175. These early stages of development exhibit a pronounced dependence on mitochondrial ATP (adenosine triphosphate) production to fuel vital cellular processes176. Beyond ATP generation, mitochondria orchestrate a multitude of cellular functions essential for proper development. These functions encompass the maintenance of mtDNA integrity, mitosis, and apoptosis177. It must be noted that immature oocytes rely on cumulus cells for energy and metabolic supply via gap junctions178. The ATP levels in oocytes fluctuate during the maturation and meiosis processes. ATP levels in oocytes increase during the resumption of meiosis I, then decrease after GV breakdown and reach maximal levels when the first polar body extrusion occurs179. The consumption of ATP also significantly increased during MI and MII179, indicating the need for ATP during oocyte maturation.

Any damaged mitochondria that are beyond repairing capacity or unnecessary will be removed via mitophagy180. To maintain the proper function of both mitochondria and cells, well-coordinated regulations must be in place to balance the formation of the new and the removal of damaged mitochondria181. Additionally, a recent study has shown that mitochondria redistribute during oocyte maturation and development, with mitochondria cluster area increased in the later stages (i.e. MI and MII oocytes)182. The increase in oocyte mitochondria number and clustering has also been shown in oocytes undergoing maturation, which was associated with an increase in ATP production183, most probably to prepare oocytes for energy demand through meiosis as deficiency in ATP production could lead to oocyte spindle disassembling in mice, resulting in apoptosis and arrested growth184. These data suggest that mitochondrial dynamics play an important role in regulating mitochondria biogenesis and energy distribution during oocyte development.

Furthermore, alterations in mitochondrial dynamics can result in mitochondrial dysfunction and cellular malfunction, which is linked to aging conditions and a variety of age-related illnesses185. The dynamin GTPase protein family mediates mitochondrial dynamics to fusion and fission186. Mitochondrial fusion is the process where two or more mitochondria join to form a single interconnected network, while fission is the division of one mitochondrion into separate units186. The balanced interaction between these two opposing processes of fusion and fission is important in maintaining mitochondrial function, inheritance, and quality control within cells187. In mammals, dynamin-related protein (DRP1), which can bind to various mitochondrial receptors, is the main protein involved in regulating the fission process188. Two proteins need to collaborate to run the fusion process, including mitofusin 1 (MFN1), which supports outer mitochondria membrane fusion, and optic atrophy 1 (OPA1), which assists the fusion of inner mitochondria membrane189. Mitochondrial dynamic-related proteins have been examined in germ cells and embryos using knockout mouse models190. DRP1 deletion in oocytes causes a cascade of detrimental effects, including defective mitochondrial dynamics and abnormalities in oocyte function191. During a state of stress in cells, mitochondrial fusion may provide additional cristae to maintain ATP production and hinder autophagy192. Deletion of the fusion protein Mfn1 is reported to cause female infertility in mice due to the developmental arrest of follicles at the secondary stage, leading to oocyte immaturity193. Furthermore, the lack of MFN1 caused ceramide buildup in oocytes and enhanced apoptosis, resulting in rapid follicular depletion138. Similarly, MFN2 deletion in oocytes results in oocyte immaturity, resulting in infertility194. Interestingly, these MFN2-deficient oocytes exhibited shorter telomeres suggesting accelerated cellular aging within the ovary27. Shorter telomeres are associated with a reduced oocyte pool available for ovulation, which contributes to the observed infertility phenotype195. These findings suggest that targeting mitochondrial dynamics could be a promising avenue for future research on female fertility treatments, particularly in cases associated with diminished ovarian reserve and age-related infertility.

Mitochondria and embryo

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.

EFFORTS TO IMPROVE OUTCOMES OF ART TREATMENT

The mutation of mtDNA and mitochondrial dysfunction can lead to infertility and can be inherited by offspring21. ART is an effective tool for the treatment of infertility, including those caused by abnormal mitochondria. Several procedures involve the combination of different ART methods to improve the success rate and limit inherited mtDNA mutation.

Pharmacological Agents

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.

Mitochondrial Replacement

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.

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Figure 3 . Several mitochondria replacement therapies have been successfully applied . ( A ) Maternal Spindle Transfer (MST) involves transferring the nuclear genetic material of a patient oocyte with defective mitochondria to a donor oocyte with its nuclear material removed. Donor oocyte nucleus, including the spindle is removed using micromanipulation techniques. The nucleus from patient oocyte is then transfered into enucleated donor oocyte within competent mitochondria. After that, the reformed oocyte is fertilized and cultured in appropriate conditions. ( B ) Pronuclear Transfer (PNT) is a technique where the pronuclear from a fertilized oocyte with abnormal mitochondria is transferred to a donor zygote with its pronuclear discarded. After being retrieved from the body, both the patient and donor oocytes are fertilized. Two zygotes are developed from fertilized oocytes, and then their pronuclear is extracted. The pronuclear from the patient zygote is transferred into the donor zygote. Finally, the reconstructed embryo is cultured and transplanted to the mother’s uterus. ( C ) Mitochondrial Transplantation involves directly injecting or centrifugation healthy mitochondria isolated from donor cells into recipient oocytes. Donor cells are autologous, such as stem cell and germline.

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.

Mitochondria Transplantation

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.

Autologous mitotherapy in ART

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

Embryo selection

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.

CONCLUSION

Mitotherapy has been applied in ART and infertility for decades, and the innovation in research and technology is contributing greatly to the development of better and more effective procedures and approaches. Some of the techniques have been shown successful in clinical application, providing chances for women with low fertility. It is shown that ART with repeated effort, may potentially replace the natural birth rate, solving the issues of declining population worldwide [8-10]. With the current advancement in mitotherapy, a broader range of patients and customers will benefit, especially women with mtDNA disorders, or with aged, low-quality, and unknown infertility. The development of mitochondrial transplantation techniques has shifted ART toward a less invasive and more ethical approach, where the isolation and removal of the donor’s oocyte and embryonic nuclei are no longer required. The selection of cell sources for mitochondria isolation has been extensively investigated, and the combination of mitotherapy with stem cell therapy has shown positive results in tested procedures. However, there is still room for further refinement of ART employing mitotherapy. Exploring different aspects of mitochondria selection, which involves new insights into mitochondrial biology and metabolism, will lead to further improvements.

Current data showed that mitochondria transplantation might increase the risk of heart defects and other morphological concerns; however, it must be noted that ART, and particularly IVF technology itself, has been shown to raise health issues in IVF babies, including cardiovascular disease (CHD)275, 276. Perhaps the issues related to mito-nuclear mismatch are additional to the already-present risks of exposing germ cells to the in vitro environment. Therefore, future research should focus on examining various factors and chemicals in the in vitro environment, as well as the effects of the therapeutic procedures used in the current technologies. Furthermore, it should be distinguished between the investigation of additional mitochondria in germ cells and the current challenges. A deeper understanding of the mechanisms underlying mitochondrial transplantation and the involvement of donor mitochondria in host cell biology and function should be highlighted. Whether the safety and effectiveness of mitotherapy can be sustained over time throughout a human lifetime are critical questions. Thus, a longitudinal study of individuals conceived with mitotherapy is essential. This will require long-term commitment and collaboration from research, biological, and medical communities. Although concerns regarding the identification and acceptance of parenthood or religious beliefs are important and may be related to the ethics and accessibility of mitotherapy, they are beyond the scope of this work and should be thoroughly elaborated in collaboration with other fields such as humanities or philosophy. Overall, these questions should not hinder the progress in therapeutic development, with the focus remaining on safety, efficacy, and innovation.

Other potential directions have also been considered for age-related infertility, such as using a cell-free scaffold seeded with stem cells for ovarian restoration, which can be possibly performed in vivo277. It must be noted that the majority of the present mitochondria transplantation and transfer technologies are focused on female rather than male reproduction, most probably because sperms carry a limited number of mitochondria, which are strictly removed at the fertilization stages, and are difficult to manipulate when compared to eggs. The targeting of mitochondrial issues in male infertility is currently directed to the use of chemicals and medicines to improve mitochondrial function278, 279. Other directions involve the regeneration of testicular tissues and supporting spermatogenesis such as stem cell therapy280 and making testicular organoids281. With the continuous effort being made in research and medicine, it is promising that more refined treatments will be accessible to infertile patients.

Abbreviations

ADSCs - Adipose-Derived Stem Cells, AKT - Protein Kinase B (also known as RAC-alpha serine/threonine-protein kinase), ART - Assisted Reproductive Technology, ATP - Adenosine Triphosphate, AUGMENT - Autologous Germline Mitochondrial Energy Transfer, BT - Blastomere Transfer, CCs - Cumulus Cells, cf-mtDNA - Cell-Free Mitochondrial DNA, CHD - Congenital Heart Disease, CoQ10 - Coenzyme Q10, EnMSCs - Endometrial Mesenchymal Stem Cells, Fe2+ - Ferrous Iron, GCT - Germinal Vesicle Transfer, GSH - Glutathione, H₂O₂ - Hydrogen Peroxide, ICSI - Intracytoplasmic Sperm Injection, iPSCs - Induced Pluripotent Stem Cells, IUI - Intrauterine Insemination, IVF - In Vitro Fertilization, MMP - Mitochondrial Membrane Potential, MOMP - Mitochondrial Outer Membrane Permeabilization, MST - Maternal Spindle Transfer, mtDNA - Mitochondrial DNA, NADH - Nicotinamide Adenine Dinucleotide (Reduced Form), NADPH - Nicotinamide Adenine Dinucleotide Phosphate (Reduced Form), Nrf2 - Nuclear Factor Erythroid 2-Related Factor 2, O₂^●− - Superoxide Anion, OCC - Oocyte-Cumulus Complex, OSCs - Oogonial Stem Cells, OXPHOS - Oxidative Phosphorylation, PBT - Polar Body Transfer, PI3K - Phosphatidylinositol 3-Kinase, PNT - Pronuclear Transfer, PTP - Permeability Transition Pore, rCoQ10 - Reduced Coenzyme Q10, ROS - Reactive Oxygen Species, RR - Ruthenium Red, SDF - Sperm DNA Fragmentation, Sirt1 - Sirtuin 1, SOD - Superoxide Dismutase, TE - Trophectoderm, TFAM - Mitochondrial Transcription Factor A, UC-MSCs - Umbilical Cord-Derived Mesenchymal Stem Cells, USCs - Urine-Derived Mesenchymal Stem Cells, ^●OH - Hydroxyl Radical

Acknowledgments

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Author’s contributions

Oanh TH Phan: Conceptualization, Visualization, Writing - Original draft. Tran TC Trinh: Conceptualization, Writing- Original draft, Writing- Review and editing. Van TT Nguyen: Conceptualization, Supervision, Visualization, Writing -original draft, Writing- Review and editing. All authors read and approved the final manuscript.

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Competing interests

The authors declare that they have no competing interests.