Potential Roles of mtDNA Mutations in PCOS-IR: A Review.

Because mitochondria are the major sites for ROS generation, overproduction of ROS will lead to OS and consequent imunbalance between the oxidant and antioxidant systems. 39 This imbalance may be caused by several metabolic activities, including obesity, hyperinsulinemia, and dyslipidemia. 40 The predominant ROS in the mitochondria are superoxide anions (O 2 − ), which are produced by the leakage of electrons from the ETC, which can then react with O 2 . 41 Endometrial IR is linked to hyperandrogenemia, obesity, and inflammation and strongly associated with OS, resulting in an upregulation of OS caused by excessive ROS and pregnancy impairment. 42 OS can lead to IR through the impairment of insulin signaling and causing adipokine dysregulation. 43 OS also regulates some classical signaling pathways, such as NFκB and JNK, which in turn phosphorylate insulin-receptor substrate proteins and lead to their degradation. 44 Overproduction of ROS also suppresses GLUT4 translocation in cells via affecting insulin signaling. 45

Insulin is the master regulator of glucose metabolism. This hormone works under the condition of glucose uptake by insulin-sensitive tissue (muscle, liver, and adipose). 15 , 16 IR is caused by defects in insulin signaling, reducing the ability of insulin to stimulate glucose utilization, and can thus lead to high insulin levels (hyperinsulinemia). It has been suggested that >75% of patients have associated IR. 17 At the molecular level, IR and hyperinsulinemia may stimulate P450c17α and influence the activity of 17-hydroxylase and 17,20-lyase. 18 Subsequently, these biochemical processes promote the secretion of androgen, increase free-androgen levels, and inhibit insulin signal transduction and translocation of glucose transporter 4, which affects glucose and lipid metabolism. 19 Androgens can produce IR by directly affecting insulin action in skeletal muscle and adipose tissue, changing adipokine secretion and increasing visceral adiposity. Moreover, insulin and IGF1 20 synergize with luteinizing hormone (LH). 21 Hyperinsulinemia enhances LH binding and androgen-producing response to LH. 22 Hyperinsulinemia also reduces hepatic sex hormone–binding globulin, 23–25 increasing free-testosterone levels in the blood and thus contributing to PCOS phenotypes ( Figures 1 and 2 ). Figure 1 Influence of hyperinsulinemia on various human organs. Figure 2 Summarized scheme of the pathophysiology of PCOS. Abbreviations : GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicule-stimulating hormone; SHBG, sex hormone –binding globulin. Influence of hyperinsulinemia on various human organs. Summarized scheme of the pathophysiology of PCOS.

The well-known m.A14693G mutation occurs at a conserved position of the TΨC loop of tRNA Glu ( Figure 5G ). The nucleotide at position 54 (m.A14693G) of tRNA Glu is often modified, thus having an impact on tRNA functions. 86 It has been proposed that the m.A14693G mutation can cause failure in tRNA Glu metabolism and impair mitochondrial protein synthesis. 87

Polycystic ovary syndrome (PCOS) is the most common endocrine disease occurring during reproductive years. It was a kind of endocrine and metabolic disorders that results in obesity, irregularity of menstruation, OS, hyperinsulinemia, hyperandrogenism, infertility, and sterility. 1 , 2 First identified in 1935, PCOS was also recognized as Stein–Leventhal syndrome. 3 Diagnosis of PCOS in adults can be made when at least two of three criteria are met: impairment of ovarian function, clinical and/or biochemical hyperandrogenism, and polycystic ovaries. 4 , 5 Despite significant progress in diagnostic criteria for PCOS, the syndrome is still underdiagnosed or misunderstood by many practitioners. 6 In the early stage, PCOS is often complicated with infertility and adverse pregnancy outcomes, while in the long term, the incidence of endometrial cancer, type 2 diabetes mellitus (T2DM), and cardiovascular diseases gradually increase, seriously harming women’s physical and mental health. Based on the National Institutes of Health’s diagnostic criteria, the geographical prevalence of this disease is 8.7%, 17.8%, and 12% based on the definition proposed by the Androgen Excess and PCOS Society. 7 In a 2019 study, the prevalence of PCOS in Chinese women of reproductive age was 5.6%, which was consistent with other studies, 8 while its incidence in Indian women was 9.13% according to a recent study. 9 Interestingly, PCOS seems to be more frequent in black women (8.0%) than white women (4.8%), with an incidence of 6.6%. 10 The prevalence of oligoanovulation and hyperandrogenemia is 56.6% and 60% among women with PCOS, respectively. 11 Multiple morbidities are linked to PCOS, such as infertility, impairment of glucose tolerance, T2DM, coronary heart disease, depression, gynecological oncology, and nonalcoholic fatty-liver disease. 12 Despite these well-characterized phenotypes, the pathogenesis of PCOS remains unclear. Increasing evidence suggests that genetic, epigenetic, and environmental factors contribute to PCOS progression. 13 However, it was generally accepted that IR and hyperandrogenism play key roles in its etiology. 14

The D-loop region is where mtDNA replication and transcription occur, and is important for transcription of both heavy and light strands. 58 A recent case–control study by Deng et al suggested that variants m.G207A, m.16036GGins, and m.16049Gins may decrease the risk of PCOS in a Chinese population. 59 The m.G207A substitution was located at the heavy strand, which is critical for mtDNA replication, suggesting that m.G207A may affect the binding affinity and influence the replication of mtDNA. 60 While m.16036GGins and m.16049Gins both occurred at hypervariable region 1, notably they were found to reduce the risk of endometriosis, 61 highlighting the importance of these mutations in maintaining mitochondrial functions.

The 4977-bp deletion is one of the most common deletions of mtDNA, spanning approximately a third of the entire mitochondrial genome (nucleotides 8470–13,447), and is regarded as a pathogenic deletion in PCOS. 62 , 63 The 4977-bp deletion removes five tRNAs and seven genes encoding respiratory chain complexes that are important for normal OxPhos functions. The 4977-bp deletion results in an impairment of protein synthesis and reduces ATP and mtDNA copy number. 64

Mutations in mtDNA have structural and functional consequences, such as affecting OxPhos complexes and influencing mitochondrial protein synthesis. Mmost of these mtDNA mutations occurred with mt-tRNA genes ( Figure 5 and Table 1 ). mt-tRNA mutations may destabilize tRNA tertiary structure, alter RNA processing, and lead to defects in nucleotide modification. Subsequently, these mutations lead to failures in tRNA metabolism. These mitochondrial protein-synthesis defects resulte in decreased in ATP production in granulosa cells or pancreatic cells, thus contributing to PCOS clinical phenotypes. Table 1 Summary of PCOS-IR–associated Mt-tRNA mutations tRNA species Allele Nucleotide position in tRNAs Structure location Homoplasmy/heteroplasmy Aberrant tRNA Biology References tRNA Leu(UUR) C3275T 44 Variable region Homoplasmy Disrupts conserved base-pairing [ 69 ] A3302G 71 Acceptor arm Heteroplasmy Affects 3’-end processing [ 65 ] tRNA Gln T4363C 38 Anticodon stem Homoplasmy Affects tRNA posttranscriptional modification [ 69 ] T4395C 6 Acceptor arm Homoplasmy Creates new base-pairing [ 69 ] tRNA Cys G5821A 6 Acceptor arm Homoplasmy Disrupts conserved base-pairing [ 75 , 76 ] tRNA Ser(UCN) C7492T 26 Anticodon stem Homoplasmy Creates new base-pairing [ 52 ] tRNA Asp A7543G 29 Anticodon stem Heteroplasmy Affects tRNA aminoacylation and steady-state level [ 71 ] tRNA Lys A8343G 54 TψC loop Homoplasmy Affects tRNA posttranscriptional modification [ 69 ] tRNA Glu A14693G 54 TψC loop Homoplasmy Affects tRNA posttranscriptional modification [ 46 , 71 ] Summary of PCOS-IR–associated Mt-tRNA mutations

Mutations in mtDNA important contributors to PCOS-IR. Genetic variants in mitochondrial genomes can perturb OxPhos and are thought to contribute to the clinical pathology of PCOS. mtDNA damage is believed to increase OS and create a proinflammatory state, which could accelerate the progression of PCOS. 88 Therefore, mtDNA may offer a viable alternative target for genetic studies tackling this complex but common disease and attempting to explain the discrepancies in clinical phenotype and progression of PCOS.

mtDNA has a very high sequence-evolution rate, in part because it is exposed to ROS. mtDNA mutations include point mutations, deletions, and insertions. mtDNA mutations can be either homoplasmic or heteroplasmic when just one or more than two variants exist, respectively. 35 , 36 The heteroplasmic level of a certain mtDNA mutation is critical in clinical phenotypes. 36 Heteroplasmic mtDNA mutations are frequently associated with human pathologies because they cause more severe mitochondrial dysfunction than homoplasmic mtDNA mutations. Under normal conditions, mtDNA can “repair” mitochondrial dysfunction. When it comes to a certain heteroplasmic level, nevertheless, such compensation will be insufficient and lead to clinical expression of disease ( Figure 4 ). 37 , 38 Figure 4 Heteroplasmy and the threshold effect. Heteroplasmy and the threshold effect.

Mitochondria are very important organelles consisting an of outer membrane, intermembrane space, and inner membrane that surround the matrix. Structurally, the inner membrane is tightly folded and is the major site for electron-transport chain (ETC) (complexes I–IV), which are essential for oxygen consumption in mammalian cells. 26 Among these, complex I — nicotinamide adenine dinucleotide Q (NADH-Q) oxidoreductase, comprises enzymes consisting of iron sulfur and flavin mononucleotide. 27 Complex II, also known as succinate dehydrogenase (SDH), contains four nuclear encoded subunits: SDHA, SDHB, SDHC, and SDHD. Interestingly, complex II has a dual role, ie, in the ETC. and the tricarboxylic acid cycle, linking the two essential energy-producing processes of the cell. 28 , 29 Complex I and II oxidize NADH and flavin adenine dinucleotide 2, respectively, transferring the resulting electrons to ubiquinol, which carries electrons to complex III. Complex III shunts the electrons across the intermembrane space to cytochrome C, which brings electrons to complex IV. 30 , 31 Complex IV then uses the electrons to reduce oxygen to water. There are many enzymes located within the mitochondrial matrix that are critical for metabolic pathways, including tricarboxylic acid cycle or β-oxidation. Mitochondria are also important for the maintenance of cellular energy homeostasis. They are often called the powerhouses of the cell because of their significant role in the supplementation of ATP via oxidative phosphorylation (OxPhos). In contrast, mitochondria also generate reactive oxygen species (ROS) through ETC complexes, and excess ROS production will induce OS and cause mitochondrial dysfunctions. 32 Mitochondria contain their own genetic material, mtDNA, which encodes seven genes of the ETC complex I: one for ETC complex III, three for ETC complex IV, and two for ETC complex V. 33 The rest of the mitochondrial proteins are encoded by nuclear genes ( Figure 3 ). 34 Figure 3 Genetic map of human mitochondrial genome, which has a 16,569 bp sequence. Red boxes indicated PCOS-IR–associated mtDNA mutations. Genetic map of human mitochondrial genome, which has a 16,569 bp sequence. Red boxes indicated PCOS-IR–associated mtDNA mutations.

The ND1 T3394C (p.Y30H) mutation changes an amino acid (AA) that is extremely conserved in >90% of mammalian mtDNAs and believed to be associated with PCOS-IR. 46 , 47 Functional analysis has revealed that this mutation affects the stability of ND1 mRNA, as well as complex I assembly and activity, decreases ATP levels and mitochondrial membrane potential (MMP), and enhances ROS production. 47 , 48 The m.C5178A mutation causes the alternation of leucine to methionine at position 237 of the corresponding AA, which occurrs within the ND2 gene in complex I and is associated with PCOS-IR, 46 longevity, 49 and acute myocardial infarction. 50 Markedly decreased ATP, MMP, superoxide dismutaseand significantly increased ROS, malondialdehyde, and 8-hydroxydeoxyguanosine have been identified in polymononuclear leukocytes derived from subjects harboring this mutation, suggesting that the m.C5178A mutation may cause OS and result in mitochondrial dysfunction. 51 We previously identified homoplasmic m.T12338C (p.M1T) together with tRNA Ser(UCN) C7492T mutation in a patient with PCOS-IR. 52 At the molecular level, m.T12338C altered well-conserved methionine with threonine; therefore, the ND5 mRNA was expected to be shortened by two AAs. 53 Using cybrid cell models, the m.T12338C mutation decreased the stability of the ND5 polypeptide, affecting the assembly and activity of respiratory chain complexes. 54 Therefore, m.T12338C causes a mitochondrial dysfunction that plays a key role in PCOS-IR. The m.T12811C (p.Y159H) mutation occurs at extremely conserved residues in ND5 , which is essential for the functions of complex I. 55 The alternation of tyrosine to histidine is believed to affect the structure and function of the transmembrane region of the ND5 protein. 56 Since ND5 plays a a putative role in maintening the functions of complex I, the m.T12811C mutation may affect the ND5 polypeptide and influence ETC activities. 57