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.

× 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 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.

× 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. 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 de